Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study

PubMed Central

Li, Wan-Ju; Chiang, Hongsen; Kuo, Tzong-Fu; Lee, Hsuan-Shu; Jiang, Ching-Chuan; Tuan, Rocky S.

2013-01-01

The aim of this study was to evaluate a cell-seeded nanofibrous scaffold for cartilage repair in vivo. We used a biodegradable poly(ε-caprolactone) (PCL) nanofibrous scaffold seeded with allogeneic chondrocytes or xenogeneic human mesenchymal stem cells (MSCs), or acellular PCL scaffolds, with no implant as a control to repair iatrogenic, 7 mm full-thickness cartilage defects in a swine model. Six months after implantation, MSC-seeded constructs showed the most complete repair in the defects compared to other groups. Macroscopically, the MSC-seeded constructs regenerated hyaline cartilage-like tissue and restored a smooth cartilage surface, while the chondrocyte-seeded constructs produced mostly fibrocartilage-like tissue with a discontinuous superficial cartilage contour. Incomplete repair containing fibrocartilage or fibrous tissue was found in the acellular constructs and the no-implant control group. Quantitative histological evaluation showed overall higher scores for the chondrocyte- and MSC-seeded constructs than the acellular construct and the no-implant groups. Mechanical testing showed the highest equilibrium compressive stress of 1.5 MPa in the regenerated cartilage produced by the MSC-seeded constructs, compared to 1.2 MPa in the chondrocyte-seeded constructs, 1.0 MPa in the acellular constructs and 0.2 MPa in the no-implant group. No evidence of immune reaction to the allogeneically- and xenogeneically-derived regenerated cartilage was observed, possibly related to the immunosuppressive activities of MSCs, suggesting the feasibility of allogeneic or xenogeneic transplantation of MSCs for cell-based therapy. Taken together, our results showed that biodegradable nanofibrous scaffolds seeded with MSCs effectively repair cartilage defects in vivo, and that the current approach is promising for cartilage repair. PMID:19004029

Influence of serum percentage on the behavior of Wharton's jelly mesenchymal stem cells in culture.

PubMed

Harmouch, C; El-Omar, R; Labrude, P; Decot, V; Menu, P; Kerdjoudj, H

2013-01-01

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several lineages with valuable applications in regenerative medicine. MSCs differentiation is highly dependent on physicochemical properties of the culture substrate, cell density and on culture medium composition. In this study, we assessed the influence of fetal bovine serum (FBS) level on Wharton's jelly (WJ)-MSCs behavior seeded on polyelectrolyte multilayer films (PEMF) made of four bilayers of poly-allylamine hydrochloride (PAH) as polycation and poly-styrene sulfonate (PSS) as polyanion. MSCs isolated from WJ by explants method were amplified until the third passage. Their phenotypic characterization was performed by flow cytometry analyses. MSCs were seeded on PEMF, in Endothelial growth medium-2 (EGM-2) supplemented by either 5% or 2% FBS. Cell's behavior was monitored for 20 days by optical microscopy and immunofluorescence. Until 2 weeks on glass slides, no difference was observed whatever the FBS percentage. Then with 5% FBS, MSCs formed three-dimensional spheroids on PSS/PAH after 20 days of culture with a nuclear aggregate. Whereas, with 2% FBS, these spheroids did not appear and cells grown in 2D conserved the fibroblast-like morphology. The decrease of FBS percentage from 5% to 2% avoids 3D cell spheroids formation on PAH/PSS. Such results could guide bioengineering towards building 2D structures like cell layers or 3D structures by increasing the osteogenic or chondrogenic differentiation potential of MSCs.

Maxillary sinus floor elevation using a tissue-engineered bone with calcium-magnesium phosphate cement and bone marrow stromal cells in rabbits.

PubMed

Zeng, Deliang; Xia, Lunguo; Zhang, Wenjie; Huang, Hui; Wei, Bin; Huang, Qingfeng; Wei, Jie; Liu, Changsheng; Jiang, Xinquan

2012-04-01

The objective of this study was to assess the effects of maxillary sinus floor elevation with a tissue-engineered bone constructed with bone marrow stromal cells (bMSCs) and calcium-magnesium phosphate cement (CMPC) material. The calcium (Ca), magnesium (Mg), and phosphorus (P) ions released from calcium phosphate cement (CPC), magnesium phosphate cement (MPC), and CMPC were detected by inductively coupled plasma atomic emission spectroscopy (ICP-AES), and the proliferation and osteogenic differentiation of bMSCs seeded on CPC, MPC, and CMPC or cultured in CPC, MPC, and CMPC extracts were measured by MTT analysis, alkaline phosphatase (ALP) activity assay, alizarin red mineralization assay, and real-time PCR analysis of the osteogenic genes ALP and osteocalcin (OCN). Finally, bMSCs were combined with CPC, MPC, and CMPC and used for maxillary sinus floor elevation in rabbits, while CPC, MPC, or CMPC without cells served as control groups. The new bone formation in each group was detected by histological finding and fluorochrome labeling at weeks 2 and 8 after surgical operation. It was observed that the Ca ion concentrations of the CMPC and CPC scaffolds was significantly higher than that of the MPC scaffold, while the Mg ions concentration of CMPC and MPC was significantly higher than that of CPC. The bMSCs seeded on CMPC and MPC or cultured in their extracts proliferated more quickly than the cells seeded on CPC or cultured in its extract, respectively. The osteogenic differentiation of bMSCs seeded on CMPC and CPC or cultured in the corresponding extracts was significantly enhanced compared to that of bMSCs seeded on MPC or cultured in its extract; however, there was no significant difference between CMPC and CPC. As for maxillary sinus floor elevation in vivo, CMPC could promote more new bone formation and mineralization compared to CPC and MPC, while the addition of bMSCs could further enhance its new bone formation ability significantly. Our data suggest that CMPC possesses moderate biodegradability and excellent osteoconductivity, which may be attributed to its Ca and Mg ion composition, and the tissue-engineered bone constructed of CMPC and bMSCs might be a potential alterative graft for maxillofacial bone regeneration.

Fabrication of viable and functional pre-vascularized modular bone tissues by coculturing MSCs and HUVECs on microcarriers in spinner flasks.

PubMed

Zhang, Songjie; Zhou, Min; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song

2017-08-01

Slow vascularization often impedes the viability and function of engineered bone replacements. Prevascularization is a promising way to solve this problem. In this study, a new process was developed by integrating microcarrier culture and coculture to fabricate pre-vascularized bone microtissues with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs). Initially, coculture medium and cell ratio between MSCs and HUVECs were optimized in tissue culture plates concerning cell proliferation, osteogenesis and angiogenesis. Subsequently, cells were seeded onto CultiSpher S microcarriers in spinner flasks and subjected to a two-stage (proliferative-osteogenic) culture process for four weeks. Both cells proliferated and functioned well in chosen medium and a 1 : 1 ratio between MSCs and HUVECs was chosen for better angiogenesis. After four weeks of culture in spinner flasks, the microtissues were formed with high cellularity, evenly distributed cells and tube formation ability. While coculture with HUVECs exerted an inhibitory effect on osteogenic differentiation of MSCs, with downregulated alkaline phosphatase activity, mineralization and gene expression of COLI, RUNX2 and OCN, this could be attenuated by employing a delayed seeding strategy of HUVECs against MSCs during the microtissue fabrication process. Collectively, this work established an effective method to fabricate pre-vascularized bone microtissues, which would lay a solid foundation for subsequent development of vascularized tissue grafts for bone regeneration. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

PubMed

Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

2012-03-01

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

Umbilical cord-derived mesenchymal stem cells inhibit growth and promote apoptosis of HepG2 cells.

PubMed

Tang, Ying-Mei; Bao, Wei-Min; Yang, Jin-Hui; Ma, Lin-Kun; Yang, Jing; Xu, Ying; Yang, Li-Hong; Sha, Feng; Xu, Zhi-Yuan; Wu, Hua-Mei; Zhou, Wei; Li, Yan; Li, Yu-Hua

2016-09-01

Hepatocellular carcinoma is the fifth most common type of cancer worldwide and remains difficult to treat. The aim of this study was to investigate the effects of mesenchymal stem cells (MSCs) derived from the umbilical cord (UC‑MSCs) on HepG2 hepatocellular carcinoma cells. UC‑MSCs were co‑cultured with HepG2 cells and biomarkers of UC‑MSCs were analyzed by flow cytometry. mRNA and protein expression of genes were determined by reverse transcription‑polymerase chain reaction and flow cytometry, respectively. Passage three and seven UC‑MSCs expressed CD29, CD44, CD90 and CD105, whereas CD34 and CD45 were absent on these cells. Co‑culture with UC‑MSCs inhibited proliferation and promoted apoptosis of HepG2 cells in a time‑dependent manner. The initial seeding density of UC‑MSCs also influenced the proliferation and apoptosis of HepG2 cells, with an increased number of UC‑MSCs causing enhanced proliferation inhibition and cell apoptosis. Co‑culture with UC‑MSCs downregulated mRNA and protein expression of α‑fetoprotein (AFP), Bcl‑2 and Survivin in HepG2 cells. Thus, UC‑MSCs may inhibit growth and promote apoptosis of HepG2 cells through downregulation of AFP, Bcl‑2 and Survivin. US-MSCs may be used as a novel therapy for treating hepatocellular carcinoma in the future.

Compared to the amniotic membrane, Wharton's jelly may be a more suitable source of mesenchymal stem cells for cardiovascular tissue engineering and clinical regeneration.

PubMed

Pu, Lei; Meng, Mingyao; Wu, Jian; Zhang, Jing; Hou, Zongliu; Gao, Hui; Xu, Hui; Liu, Boyu; Tang, Weiwei; Jiang, Lihong; Li, Yaxiong

2017-03-21

The success of developing cardiovascular tissue engineering (CTE) grafts greatly needs a readily available cell substitute for endothelial and interstitial cells. Perinatal annexes have been proposed as a valuable source of mesenchymal stem cells (MSCs) for tissue engineering and regenerative medicine. The objective of the present study is to evaluate the potential of human Wharton's jelly MSCs (WJ-MSCs) and amniotic membrane MSCs (AM-MSCs) as a seeding cell in CTE and cardiovascular regenerative medicine. WJ-MSCs/AM-MSCs were isolated and characterized in vitro according to their morphology, proliferation, self-renewal, phenotype, and multipotency. More importantly, the characteristics of hemocompatibility, extracellular matrix deposition, and gene expression and viability of both MSCs were investigated. Fibroblast-like human WJ-MSCs and AM-MSCs were successfully isolated and positively expressed the characteristic markers CD73, CD90, and CD105 but were negative for CD34, CD45, and HLA-DR. Both MSCs shared trilineage differentiation toward the adipogenic, osteogenic, and chondrogenic lineages. The proliferative and self-renewal capacity of WJ-MSCs was significantly higher than that of AM-MSCs (P < 0.001). WJ-MSCs provided comparable properties of antiplatelet adhesion and did not activate the coagulation cascade to endothelial cells. However, aggregated platelets were visualized on the surface of AM-MSCs-derived cell sheets and the intrinsic pathway was activated. Furthermore, WJ-MSCs have superior properties of collagen deposition and higher viability than AM-MSCs during cell sheet formation. This study highlights that WJ-MSCs could act as a functional substitute of endothelial and interstitial cells, which could serve as an appealing and practical single-cell source for CTE and regenerative therapy.

The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds.

PubMed

Ren, Tianbin; Ren, Jie; Jia, Xiaozhen; Pan, Kefeng

2005-09-15

Highly porous scaffolds of poly(lactide-co-glycolide) (PLGA) were prepared by solution-casting/salt-leaching method. The in vitro degradation behavior of PLGA scaffold was investigated by measuring the change of normalized weight, water absorption, pH, and molecular weight during degradation period. Mesenchymal stem cells (MSCs) were seeded and cultured in three-dimensional PLGA scaffolds to fabricate in vitro tissue engineering bone, which was investigated by cell morphology, cell number and deposition of mineralized matrix. The proliferation of seeded MSCs and their differentiated function were demonstrated by experimental results. To compare the reconstructive functions of different groups, mandibular defect repair of rabbit was made with PLGA/MSCs tissue engineering bone, control PLGA scaffold, and blank group without scaffold. Histopathologic methods were used to estimate the reconstructive functions. The result suggests that it is feasible to regenerate bone tissue in vitro using PLGA foams with pore size ranging from 100-250 microm as scaffolding for the transplantation of MSCs, and the PLGA/MSCs tissue engineering bone can greatly promote cell growth and have better healing functions for mandibular defect repair. The defect can be completely recuperated after 3 months with PLGA/MSCs tissue engineering bone, and the contrastive experiments show that the defects could not be repaired with blank PLGA scaffold. PLGA/MSCs tissue engineering bone has great potential as appropriate replacement for successful repair of bone defect. (c) 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005.

Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells.

PubMed

Yang, Shu; Sun, Hai-Mei; Yan, Ji-Hong; Xue, Hong; Wu, Bo; Dong, Fang; Li, Wen-Shuai; Ji, Feng-Qing; Zhou, De-Shan

2013-07-01

Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD. Copyright © 2013 Wiley Periodicals, Inc.

Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels.

PubMed

Richardson, Stephen M; Hughes, Nesta; Hunt, John A; Freemont, Anthony J; Hoyland, Judith A

2008-01-01

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan-glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.

Intra-hydrogel culture prevents transformation of mesenchymal stem cells induced by monolayer expansion.

PubMed

Jiang, Tongmeng; Liu, Junting; Ouyang, Yiqiang; Wu, Huayu; Zheng, Li; Zhao, Jinmin; Zhang, Xingdong

2018-05-01

In this study, we report that the intra-hydrogel culture system mitigates the transformation of mesenchymal stem cells (MSCs) induced by two-dimensional (2D) expansion. MSCs expanded in monolayer culture prior to encapsulation in collagen hydrogels (group eMSCs-CH) featured impaired stemness in chondrogenesis, comparing with the freshly isolated bone marrow mononuclear cells seeded directly in collagen hydrogels (group fMSCs-CH). The molecular mechanism of the in vitro expansion-triggered damage to MSCs was detected through genome-wide microarray analysis. Results indicated that pathways such as proteoglycans in cancer and pathways in cancer expansion were highly enriched in eMSCs-CH. And multiple up-regulated oncoma-associated genes were verified in eMSCs-CH compared with fMSCs-CH, indicating that expansion in vitro triggered cellular transformation was associated with signaling pathways related to tumorigenicity. Besides, focal adhesion (FA) and mitogen-activated protein kinase (MAPK) signaling pathways were also involved in in vitro expansion, indicating restructuring of the cell architecture. Thus, monolayer expansion in vitro may contribute to vulnerability of MSCs through the regulation of FA and MAPK. This study indicates that intra-hydrogel culture can mitigate the monolayer expansion induced transformation of MSCs and maintain the uniformity of the stem cells, which is a viable in vitro culture system for stem cell therapy.

Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium.

PubMed

Pustlauk, W; Paul, B; Brueggemeier, S; Gelinsky, M; Bernhardt, A

2017-06-01

Studies on tissue-engineering approaches for the regeneration of traumatized cartilage focus increasingly on multipotent human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes. The present study applied porous scaffolds made of collagen from the jellyfish Rhopilema esculentum for the in vitro chondrogenic differentiation of hMSCs. Culture conditions in those scaffolds differ from conditions in high-density pellet cultures, making a re-examination of these data necessary. We systematically investigated the influence of seeding density, basic culture media [Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM)] with varying glucose content and supplementation with fetal calf serum (FCS) or bovine serum albumin (BSA) on the chondrogenic differentiation of hMSCs. Gene expression analyses of selected markers for chondrogenic differentiation and hypertrophic development were conducted. Furthermore, the production of cartilage extracellular matrix (ECM) was analysed by quantification of sulphated glycosaminoglycan and collagen type II contents. The strongest upregulation of chondrogenic markers, along with the highest ECM deposition was observed in scaffolds seeded with 2.4 × 10 6 cells/cm 3 after cultivation in high-glucose DMEM and 0.125% BSA. Lower seeding densities compared to high-density pellet cultures were sufficient to induce in vitro chondrogenic differentiation of hMSCs in collagen scaffolds, which reduces the amount of cells required for the seeding of scaffolds and thus the monolayer expansion period. Furthermore, examination of the impact of FCS and α-MEM on chondrogenic MSC differentiation is an important prerequisite for the development of an osteochondral medium for simultaneous osteogenic and chondrogenic differentiation in biphasic scaffolds for osteochondral tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Bone Marrow–Derived Mesenchymal Stem Cells Enhance Bacterial Clearance and Preserve Bioprosthetic Integrity in a Model of Mesh Infection

PubMed Central

Criman, Erik T.; Kurata, Wendy E.; Matsumoto, Karen W.; Aubin, Harry T.; Campbell, Carmen E.

2016-01-01

Background: The reported incidence of mesh infection in contaminated operative fields is as high as 30% regardless of the material used. Recently, mesenchymal stem cells (MSCs) have been shown to possess favorable immunomodulatory properties and improve tissue incorporation when seeded onto bioprosthetics. The aim of this study was to evaluate whether seeding noncrosslinked bovine pericardium (Veritas Collagen Matrix) with allogeneic bone marrow–derived MSCs improves infection resistance in vivo after inoculation with Escherichia coli (E. coli). Methods: Rat bone marrow–derived MSCs at passage 3 were seeded onto bovine pericardium and cultured for 7 days before implantation. Additional rats (n = 24) were implanted subcutaneously with MSC-seeded or unseeded mesh and inoculated with 7 × 105 colony-forming units of E. coli or saline before wound closure (group 1, unseeded mesh/saline; group 2, unseeded mesh/E. coli; group 3, MSC-seeded mesh/E. coli; 8 rats per group). Meshes were explanted at 4 weeks and underwent microbiologic and histologic analyses. Results: MSC-seeded meshes inoculated with E. coli demonstrated superior bacterial clearance and preservation of mesh integrity compared with E. coli–inoculated unseeded meshes (87.5% versus 0% clearance; p = 0.001). Complete mesh degradation concurrent with abscess formation was observed in 100% of rats in the unseeded/E. coli group, which is in contrast to 12.5% of rats in the MSC-seeded/E. coli group. Histologic evaluation determined that remodeling characteristics of E. coli–inoculated MSC-seeded meshes were similar to those of uninfected meshes 4 weeks after implantation. Conclusions: Augmenting a bioprosthetic material with stem cells seems to markedly enhance resistance to bacterial infection in vivo and preserve mesh integrity. PMID:27482490

Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons

PubMed Central

Mao, Mao; Zhou, Liang

2017-01-01

We aim to identify levels of signal factors secreted by MSCs cultured in 2D monolayers (2D-MSCs), spheroids (spheroids MSCs), and cocultures of microvesicles (MVs) derived from 2D-MSCs or spheroid MSCs and retinal photoreceptor neurons. We seeded 2D-MSCs, spheroid MSCs, and cells derived from spheroids MSCs at equal numbers. MVs isolated from all 3 culture conditions were incubated with 661W cells. Levels of 51 signal factors in conditioned medium from those cultured conditions were quantified with bead-based assay. We found that IL-8, IL-6, and GROα were the top three most abundant signal factors. Moreover, compared to 2D-MSCs, levels of 11 cytokines and IL-2Rα were significantly increased in conditioned medium from spheroid MSCs. Finally, to test if enhanced expression of these factors reflects altered immunomodulating activities, we assessed the effect of 2D-MSC-MVs and 3D-MSC-MVs on CD14+ cell chemoattraction. Compared to 2D-MSC-MVs, 3D-MSC-MVs significantly decreased the chemotactic index of CD14+ cells. Our results suggest that spheroid culture conditions improve the ability of MSCs to selectively secrete signal factors. Moreover, 3D-MSC-MVs also possessed an enhanced capability to promote signal factors secretion compared to 2D-MSC-MVs and may possess enhanced immunomodulating activities and might be a better regenerative therapy for retinal degenerative diseases. PMID:28194184

Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernemann, Inga, E-mail: bernemann@imp.uni-hannover.de; Mueller, Thomas; Blasczyk, Rainer

Highlights: {yields} Marmoset bone marrow-derived MSCs differentiate in suspension into adipogenic, osteogenic and chondrogenic lineages. {yields} Marmoset MSCs integrate in collagen type I scaffolds and differentiate excellently into adipogenic cells. {yields} Common marmoset monkey is a suitable model for soft tissue engineering in human regenerative medicine. -- Abstract: In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 {mu}m were optimal for cell seeding and cultivating. However, before clinical application andmore » transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human. Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells.« less

[Marrow stromal cells cultured in N2-supplemented medium: implications on the generation of neural cells].

PubMed

Castillo-Díaz, L; de la Cuétara-Bernal, K; García-Varona, A Y

Most of the culture system for in vitro maintenance and neural differentiation of marrow stromal cells (MSCs) use synthetic media supplemented with 10 or 20% fetal bovine serum (FBS). Serum, however, is comprised of unknown quantities of undefined substances which could interfere the effect of exogenous substances on neural differentiation of MSCs. AIM. Here we describe survival of MSCs cultured in culture conditions where serum was reduced at 0.5 and 1% using Bottenstein and Sato's N2 formula (1979) and poly-L-lysine (PLL)-coated substrate. Stromal cells isolated from rat femurs were cultivated in Dulbecco's modified Eagle medium at 10, 1, 0.5% FBS or in serum free medium containing N2 formula. In serum free medium or at low serum concentration culture surface was coated with PLL. Cell survival was determined by MTT method or by counting viable cells. Survival of MSCs cultured in N2 supplement was reduced at about 40% of that observed in 10% FBS containing medium. Under these conditions cell morphology was also affected. When N2 containing medium was supplemented with FBS at 0.5 or 1% a significant increase of survival with respect to that observed in N2-supplemented cultures was observed. Cells seeded on PLL-coated surface increased their survival by contrast with their homologous cultures seeded on uncoated surface. The culture system which combines N2 formula with FBS 1% and PLL-coated surface is useful for the maintenance of MSCs. These conditions offer advantages for the study of differentiation of these cells because they reduce the confounding influence of serum. The possible implication of this culture system for the study of neural differentiation by these cells is discussed.

Decellularized cartilage-derived matrix as substrate for endochondral bone regeneration.

PubMed

Gawlitta, Debby; Benders, Kim E M; Visser, Jetze; van der Sar, Anja S; Kempen, Diederik H R; Theyse, Lars F H; Malda, Jos; Dhert, Wouter J A

2015-02-01

Following an endochondral approach to bone regeneration, multipotent stromal cells (MSCs) can be cultured on a scaffold to create a cartilaginous callus that is subsequently remodeled into bone. An attractive scaffold material for cartilage regeneration that has recently regained attention is decellularized cartilage-derived matrix (CDM). Since this material has shown potential for cartilage regeneration, we hypothesized that CDM could be a potent material for endochondral bone regeneration. In addition, since decellularized matrices are known to harbor bioactive cues for tissue formation, we evaluated the need for seeded MSCs in CDM scaffolds. In this study, ectopic bone formation in rats was evaluated for CDM scaffolds seeded with human MSCs and compared with unseeded controls. The MSC-seeded samples were preconditioned in chondrogenic medium for 37 days. After 8 weeks of subcutaneous implantation, the extent of mineralization was significantly higher in the MSC-seeded constructs versus unseeded controls. The mineralized areas corresponded to bone formation with bone marrow cavities. In addition, rat-specific bone formation was confirmed by collagen type I immunohistochemistry. Finally, fluorochrome incorporation at 3 and 6 weeks revealed that the bone formation had an inwardly directed progression. Taken together, our results show that decellularized CDM is a promising biomaterial for endochondral bone regeneration when combined with MSCs at ectopic locations. Modification of current decellularization protocols may lead to enhanced functionality of CDM scaffolds, potentially offering the prospect of generation of cell-free off-the-shelf bone regenerative substitutes.

Closure of a Recurrent Bronchopleural Fistula Using a Matrix Seeded With Patient-Derived Mesenchymal Stem Cells.

PubMed

Aho, Johnathon M; Dietz, Allan B; Radel, Darcie J; Butler, Greg W; Thomas, Mathew; Nelson, Timothy J; Carlsen, Brian T; Cassivi, Stephen D; Resch, Zachary T; Faubion, William A; Wigle, Dennis A

2016-10-01

: Management of recurrent bronchopleural fistula (BPF) after pneumonectomy remains a challenge. Although a variety of devices and techniques have been described, definitive management usually involves closure of the fistula tract through surgical intervention. Standard surgical approaches for BPF incur significant morbidity and mortality and are not reliably or uniformly successful. We describe the first-in-human application of an autologous mesenchymal stem cell (MSC)-seeded matrix graft to repair a multiply recurrent postpneumonectomy BPF. Adipose-derived MSCs were isolated from patient abdominal adipose tissue, expanded, and seeded onto bio-absorbable mesh, which was surgically implanted at the site of BPF. Clinical follow-up and postprocedural radiological and bronchoscopic imaging were performed to ensure BPF closure, and in vitro stemness characterization of patient-specific MSCs was performed. The patient remained clinically asymptomatic without evidence of recurrence on bronchoscopy at 3 months, computed tomographic imaging at 16 months, and clinical follow-up of 1.5 years. There is no evidence of malignant degeneration of MSC populations in situ, and the patient-derived MSCs were capable of differentiating into adipocytes, chondrocytes, and osteocytes using established protocols. Isolation and expansion of autologous MSCs derived from patients in a malnourished, deconditioned state is possible. Successful closure and safety data for this approach suggest the potential for an expanded study of the role of autologous MSCs in regenerative surgical applications for BPF. Bronchopleural fistula is a severe complication of pulmonary resection. Current management is not reliably successful. This work describes the first-in-human application of an autologous mesenchymal stem cell (MSC)-seeded matrix graft to the repair of a large, multiply recurrent postpneumonectomy BPF. Clinical follow-up of 1.5 years without recurrence suggests initial safety and feasibility of this approach. Further assessment of MSC grafts in these difficult clinical scenarios requires expanded study. ©AlphaMed Press.

Ultrastructural evaluation of mesenchymal stem cells from inflamed periodontium in different in vitro conditions.

PubMed

Zaganescu, Raluca; Barbu Tudoran, Lucian; Pall, Emoke; Florea, Adrian; Roman, Alexandra; Soanca, Andrada; Mihaela Mihu, Carmen

2015-09-01

This research aimed to observe the behavior of mesenchymal stem cells (MSCs) isolated from periodontal granulation tissue (gt) when manipulated ex vivo to induce three-dimensional (3D) spheroid (aggregates) formation as well as when seeded on two bone scaffolds of animal origin. Periodontal gt was chosen as a MSC source because of its availability, considering that it is eliminated as a waste material during conventional surgical therapies. 3D aggregates of cells were generated; they were grown for 3 and 7 days, respectively, and then prepared for transmission electron microscopic analysis. The two biomaterials were seeded for 72 h with gtMSCs and prepared for scanning electronic microscopic observation. The ultrastructural analysis of 3D spheroids remarked some differences between the inner and the outer cell layers, with a certain commitment observed at the inner cells. Both scaffolds showed a relatively smooth surface at low magnification. Macro- and micropores having a scarce distribution were observed on both bone substitutes. gtMSCs grew with relative difficulty on the biomaterials. After 72 h of proliferation, gtMSCs scarcely covered the surface of bovine bone scaffolds, demonstrating fibroblast-like or star-like shapes with elongated filiform extensions. Our results add other data on the possible usefulness of gtMSC and could question the current paradigm regarding the complete removal of chronically inflamed gts from the defects during periodontal surgeries. Until optimal protocols for ex vivo manipulation of MSCs are available for clinical settings, it is advisable to use biocompatible bone substitutes that allow the development of progenitor cells. © 2015 Wiley Periodicals, Inc.

Fabrication, vascularization and osteogenic properties of a novel synthetic biomimetic induced membrane for the treatment of large bone defects

PubMed Central

Browne, Christopher; Bishop, Julius; Yang, Yunzhi

2014-01-01

The induced membrane has been widely used in the treatment of large bone defects but continues to be limited by a relatively lengthy healing process and a requisite two stage surgical procedure. Here we report the development and characterization of a synthetic biomimetic induced membrane (BIM) consisting of an inner highly pre-vascularized cell sheet and an outer osteogenic layer using cell sheet engineering. The pre-vascularized inner layer was formed by seeding human umbilical vein endothelial cells (HUVECs) on a cell sheet comprised of a layer of undifferentiated human bone marrow-derived mesenchymal stem cells (hMSCs). The outer osteogenic layer was formed by inducing osteogenic differentiation of hMSCs. In vitro results indicated the undifferentiated hMSCs cell sheet facilitated the alignment of HUVECs and significantly promoted the formation of vascular-like networks. Furthermore, seeded HUVECs rearranged the extracellular matrix produced by hMSCs sheet. After subcutaneously implantation, the composite constructs showed rapid vascularization and anastomosis with the host vascular system, forming functional blood vessels in vivo. Osteogenic potential of the BIM was evidenced by immunohistochemistry staining of osteocalcin, tartrate-resistant acid phosphatase (TRAP) staining, and alizarin red staining. In summary, the synthetic BIM showed rapid vascularization, significant anastomoses, and osteogenic potential in vivo. This synthetic BIM has the potential for treatment of large bone defects in the absence of infection. PMID:24747351

Mesenchymal stem cell-based repair of articular cartilage with polyglycolic acid-hydroxyapatite biphasic scaffold.

PubMed

Zhou, X Z; Leung, V Y; Dong, Q R; Cheung, K M; Chan, D; Lu, W W

2008-06-01

This study investigates the capacity of a composite scaffold composed of polyglycolic acid-hydroxyapatite (PGA-HA) and autologous mesenchymal stem cells (MSCs) to promote repair of osteochondral defects. MSCs from culture-expanded rabbits were seeded onto a PGA and HA scaffold. After a 72-hour co-culture period, the cell-adhered PGA and HA were joined together, forming an MSCs-PGA-HA composite. Full-thickness cartilage defects in the intercondylar fossa of the femur were then implanted with the MSC-PGA-HA composite, the PGA-HA scaffold only, or they were left empty (n=20). Animals were sacrificed 16 or 32 weeks after surgery and the gross appearance of the defects was evaluated. The specimens were examined histologically for morphologic features, and stained immunohistochemically for type 2 collagen. Specimens of the MSCs-PGA-HA composite implantation group demonstrated hyaline cartilage and a complete subchondral bone formation. At 16 weeks post-implantation, significant integration of the newly formed tissue with surrounding normal cartilage and subchondral bone was observed when compared to the two control groups. At 32 weeks, no sign of progressive degeneration of the newly formed tissue was found. A significant difference in histological grading score was found compared with the control groups. The novel MSCs-seeded, PGA-HA biphasic graft facilitated both articular cartilage and subchondral bone regeneration in an animal model and might serve as a new approach for clinical applications.

Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells.

PubMed

Mitra, Debika; Whitehead, Jacklyn; Yasui, Osamu W; Leach, J Kent

2017-11-01

Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs in vitro to boost their bone forming capacity in vivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks in vivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days in vitro improves the quantity and quality of bone formation in vivo. This study highlights the need for optimizing in vitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tenogenesis of bone marrow-, adipose-, and tendon-derived stem cells in a dynamic bioreactor.

PubMed

Youngstrom, Daniel W; LaDow, Jade E; Barrett, Jennifer G

2016-11-01

Tendons are frequently damaged and fail to regenerate, leading to pain, loss of function, and reduced quality of life. Mesenchymal stem cells (MSCs) possess clinically useful tissue-regenerative properties and have been exploited for use in tendon tissue engineering and cell therapy. However, MSCs exhibit phenotypic heterogeneity based on the donor tissue used, and the efficacy of cell-based treatment modalities may be improved by optimizing cell source based on relative differentiation capacity. Equine MSCs were isolated from bone marrow (BM), adipose (AD), and tendon (TN), expanded in monolayer prior to seeding on decellularized tendon scaffolds (DTS), and cell-laden constructs were placed in a bioreactor designed to mimic the biophysical environment of the tendon. It was hypothesized that TN MSCs would differentiate toward a tendon cell phenotype better than BM and AD MSCs in response to a conditioning period involving cyclic mechanical stimulation for 1 hour per day at 3% strain and 0.33 Hz. All cell types integrated into DTS adopted an elongated morphology similar to tenocytes, expressed tendon marker genes, and improved tissue mechanical properties after 11 days. TN MSCs expressed the greatest levels of scleraxis, collagen type-I, and cartilage oligomeric matrix protein. Major histocompatibility class-II protein mRNA expression was not detected in any of the MSC types, suggesting low immunogenicity for allogeneic transplantation. The results suggest that TN MSCs are the ideal cell type for regenerative medicine therapies for tendinopathies, exhibiting the most mature tendon-like phenotype in vitro. When TN MSCs are unavailable, BM or AD MSCs may serve as robust alternatives.

Overexpression of Insulin-Like Growth Factor 1 Enhanced the Osteogenic Capability of Aging Bone Marrow Mesenchymal Stem Cells.

PubMed

Chen, Ching-Yun; Tseng, Kuo-Yun; Lai, Yen-Liang; Chen, Yo-Shen; Lin, Feng-Huei; Lin, Shankung

2017-01-01

Many studies have indicated that loss of the osteoblastogenic potential in bone marrow mesenchymal stem cells (bmMSCs) is the major component in the etiology of the aging-related bone deficit. But how the bmMSCs lose osteogenic capability in aging is unclear. Using 2-dimentional cultures, we examined the dose response of human bmMSCs, isolated from adult and aged donors, to exogenous insulin-like growth factor 1 (IGF-1), a growth factor regulating bone formation. The data showed that the mitogenic activity and the osteoblastogenic potential of bmMSCs in response to IGF-1 were impaired with aging, whereas higher doses of IGF-1 increased the proliferation rate and osteogenic potential of aging bmMSCs. Subsequently, we seeded IGF-1-overexpressing aging bmMSCs into calcium-alginate scaffolds and incubated in a bioreactor with constant perfusion for varying time periods to examine the effect of IGF-1 overexpression to the bone-forming capability of aging bmMSCs. We found that IGF-1 overexpression in aging bmMSCs facilitated the formation of cell clusters in scaffolds, increased the cell survival inside the cell clusters, induced the expression of osteoblast markers, and enhanced the biomineralization of cell clusters. These results indicated that IGF-1 overexpression enhanced cells' osteogenic capability. Thus, our data suggest that the aging-related loss of osteogenic potential in bmMSCs can be attributed in part to the impairment in bmMSCs' IGF-1 signaling, and support possible application of IGF-1-overexpressing autologous bmMSCs in repairing bone defect of the elderly and in producing bone graft materials for repairing large scale bone injury in the elderly.

Prospect of Stem Cells in Bone Tissue Engineering: A Review

PubMed Central

Yousefi, Azizeh-Mitra; James, Paul F.; Akbarzadeh, Rosa; Subramanian, Aswati; Flavin, Conor; Oudadesse, Hassane

2016-01-01

Mesenchymal stem cells (MSCs) have been the subject of many studies in recent years, ranging from basic science that looks into MSCs properties to studies that aim for developing bioengineered tissues and organs. Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) have been the focus of most studies due to the inherent potential of these cells to differentiate into various cell types. Although, the discovery of induced pluripotent stem cells (iPSCs) represents a paradigm shift in our understanding of cellular differentiation. These cells are another attractive stem cell source because of their ability to be reprogramed, allowing the generation of multiple cell types from a single cell. This paper briefly covers various types of stem cell sources that have been used for tissue engineering applications, with a focus on bone regeneration. Then, an overview of some recent studies making use of MSC-seeded 3D scaffold systems for bone tissue engineering has been presented. The emphasis has been placed on the reported scaffold properties that tend to improve MSCs adhesion, proliferation, and osteogenic differentiation outcomes. PMID:26880976

The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns.

PubMed

Cejka, Cestmir; Holan, Vladimir; Trosan, Peter; Zajicova, Alena; Javorkova, Eliska; Cejkova, Jitka

2016-01-01

The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties.

Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

PubMed

Allen, Ashley B; Butts, Emily B; Copland, Ian B; Stevens, Hazel Y; Guldberg, Robert E

2017-10-01

Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Neonatal Desensitization Supports Long-Term Survival and Functional Integration of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells in Rat Joint Cartilage Without Immunosuppression

PubMed Central

Zhang, Shufang; Jiang, Yang Zi; Zhang, Wei; Chen, Longkun; Tong, Tong; Liu, Wanlu; Mu, Qin; Liu, Hua; Ji, Junfeng; Ouyang, Hong Wei

2013-01-01

Immunological response hampers the investigation of human embryonic stem cells (hESCs) or their derivates for tissue regeneration in vivo. Immunosuppression is often used after surgery, but exhibits side effects of significant weight loss and allows only short-term observation. The purpose of this study was to investigate whether neonatal desensitization supports relative long-term survival of hESC-derived mesenchymal stem cells (hESC-MSCs) and promotes cartilage regeneration. hESC-MSCs were injected on the day of birth in rats. Six weeks after neonatal injection, a full-thickness cylindrical cartilage defect was created and transplanted with a hESC-MSC-seeded collagen bilayer scaffold (group d+s+c) or a collagen bilayer scaffold (group d+s). Rats without neonatal injection were transplanted with the hESC-MSC-seeded collagen bilayer scaffold to serve as controls (group s+c). Cartilage regeneration was evaluated by histological analysis, immunohistochemical staining, and biomechanical test. The role of hESC-MSCs in cartilage regeneration was analyzed by CD4 immunostaining, cell death detection, and visualization of human cells in regenerated tissues. hESC-MSCs expressed CD105, CD73, CD90, CD29, and CD44, but not CD45 and CD34, and possessed trilineage differentiation potential. Group d+s+c exhibited greater International Cartilage Repair Society (ICRS) scores than group d+s or group s+c. Abundant collagen type II and improved mechanical properties were detected in group d+s+c. There were less CD4+ inflammatory cell infiltration and cell death at week 1, and hESC-MSCs were found to survive as long as 8 weeks after transplantation in group d+s+c. Our study suggests that neonatal desensitization before transplantation may be an efficient way to develop a powerful tool for preclinical study of human cell-based therapies in animal models. PMID:22788986

Neonatal desensitization supports long-term survival and functional integration of human embryonic stem cell-derived mesenchymal stem cells in rat joint cartilage without immunosuppression.

PubMed

Zhang, Shufang; Jiang, Yang Zi; Zhang, Wei; Chen, Longkun; Tong, Tong; Liu, Wanlu; Mu, Qin; Liu, Hua; Ji, Junfeng; Ouyang, Hong Wei; Zou, Xiaohui

2013-01-01

Immunological response hampers the investigation of human embryonic stem cells (hESCs) or their derivates for tissue regeneration in vivo. Immunosuppression is often used after surgery, but exhibits side effects of significant weight loss and allows only short-term observation. The purpose of this study was to investigate whether neonatal desensitization supports relative long-term survival of hESC-derived mesenchymal stem cells (hESC-MSCs) and promotes cartilage regeneration. hESC-MSCs were injected on the day of birth in rats. Six weeks after neonatal injection, a full-thickness cylindrical cartilage defect was created and transplanted with a hESC-MSC-seeded collagen bilayer scaffold (group d+s+c) or a collagen bilayer scaffold (group d+s). Rats without neonatal injection were transplanted with the hESC-MSC-seeded collagen bilayer scaffold to serve as controls (group s+c). Cartilage regeneration was evaluated by histological analysis, immunohistochemical staining, and biomechanical test. The role of hESC-MSCs in cartilage regeneration was analyzed by CD4 immunostaining, cell death detection, and visualization of human cells in regenerated tissues. hESC-MSCs expressed CD105, CD73, CD90, CD29, and CD44, but not CD45 and CD34, and possessed trilineage differentiation potential. Group d+s+c exhibited greater International Cartilage Repair Society (ICRS) scores than group d+s or group s+c. Abundant collagen type II and improved mechanical properties were detected in group d+s+c. There were less CD4+ inflammatory cell infiltration and cell death at week 1, and hESC-MSCs were found to survive as long as 8 weeks after transplantation in group d+s+c. Our study suggests that neonatal desensitization before transplantation may be an efficient way to develop a powerful tool for preclinical study of human cell-based therapies in animal models.

Encapsulation of mesenchymal stem cells in chitosan/β-glycerophosphate hydrogel for seeding on a novel calcium phosphate cement scaffold.

PubMed

Liu, Tao; Li, Jian; Shao, Zengwu; Ma, Kaige; Zhang, Zhicai; Wang, Baichuan; Zhang, Yannan

2018-06-01

Due to its moldability, biocompatibility, osteoconductivity and resorbability, calcium phosphate cement (CPC) is a highly promising scaffold material for orthopedic applications. However, pH changes and ionic activity during the CPC setting reaction may adversely affect cells seeded directly on CPC. Moreover, a lack of macropores in CPC limits ingrowth of new bone. The objectives of this study were to prepare macroporous CPC scaffolds via porogen leaching, using mannitol crystals as the porogen and to evaluate the in vitro proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) encapsulated in chitosan/β-glycerophosphate (C/GP) hydrogel prior to exposure to the novel CPC scaffold. MSCs were found to be adhered to the surfaces of CPC macropores via scanning electron microscopy. The viability and osteogenic differentiation of MSCs in C/GP hydrogel with or without exposure to CPC constructs containing mannitol crystals indicated that coating with C/GP hydrogel protected the cells during cement mixing and setting. In conclusion, novel, macroporous CPC scaffolds were prepared, and our data indicate that a hydrogel encapsulation-based strategy can be used to protect cells during scaffold formation. Thus, the MSC-laden CPC scaffolds show promise for the delivery of stem cells to promote bone regeneration. Copyright © 2018 IPEM. Published by Elsevier Ltd. All rights reserved.

Autologous mesenchymal stem cells or meniscal cells: what is the best cell source for regenerative meniscus treatment in an early osteoarthritis situation?

PubMed

Zellner, Johannes; Pattappa, Girish; Koch, Matthias; Lang, Siegmund; Weber, Johannes; Pfeifer, Christian G; Mueller, Michael B; Kujat, Richard; Nerlich, Michael; Angele, Peter

2017-10-10

Treatment of meniscus tears within the avascular region represents a significant challenge, particularly in a situation of early osteoarthritis. Cell-based tissue engineering approaches have shown promising results. However, studies have not found a consensus on the appropriate autologous cell source in a clinical situation, specifically in a challenging degenerative environment. The present study sought to evaluate the appropriate cell source for autologous meniscal repair in a demanding setting of early osteoarthritis. A rabbit model was used to test autologous meniscal repair. Bone marrow and medial menisci were harvested 4 weeks prior to surgery. Bone marrow-derived mesenchymal stem cells (MSCs) and meniscal cells were isolated, expanded, and seeded onto collagen-hyaluronan scaffolds before implantation. A punch defect model was performed on the lateral meniscus and then a cell-seeded scaffold was press-fit into the defect. Following 6 or 12 weeks, gross joint morphology and OARSI grade were assessed, and menisci were harvested for macroscopic, histological, and immunohistochemical evaluation using a validated meniscus scoring system. In conjunction, human meniscal cells isolated from non-repairable bucket handle tears and human MSCs were expanded and, using the pellet culture model, assessed for their meniscus-like potential in a translational setting through collagen type I and II immunostaining, collagen type II enzyme-linked immunosorbent assay (ELISA), and gene expression analysis. After resections of the medial menisci, all knees showed early osteoarthritic changes (average OARSI grade 3.1). However, successful repair of meniscus punch defects was performed using either meniscal cells or MSCs. Gross joint assessment demonstrated donor site morbidity for meniscal cell treatment. Furthermore, human MSCs had significantly increased collagen type II gene expression and production compared to meniscal cells (p < 0.05). The regenerative potential of the meniscus by an autologous cell-based tissue engineering approach was shown even in a challenging setting of early osteoarthritis. Autologous MSCs and meniscal cells were found to have improved meniscal healing in an animal model, thus demonstrating their feasibility in a clinical setting. However, donor site morbidity, reduced availability, and reduced chondrogenic differentiation of human meniscal cells from debris of meniscal tears favors autologous MSCs for clinical use for cell-based meniscus regeneration.

Feasibility of human hair follicle-derived mesenchymal stem cells/CultiSpher(®)-G constructs in regenerative medicine.

PubMed

Li, Pengdong; Liu, Feilin; Wu, Chunling; Jiang, Wenyue; Zhao, Guifang; Liu, Li; Bai, Tingting; Wang, Li; Jiang, Yixu; Guo, Lili; Qi, Xiaojuan; Kou, Junna; Fan, Ruirui; Hao, Deshun; Lan, Shaowei; Li, Yulin; Liu, Jin Yu

2015-10-01

The use of human mesenchymal stem cells (hMSCs) in cell therapies has increased the demand for strategies that allow efficient cell scale-up. Preliminary data on the three-dimensional (3D) spinner culture describing the potential use of microcarriers for hMSCs culture scale-up have been reported. We exploited a rich source of autologous stem cells (human hair follicle) and demonstrated the robust in vitro long-term expansion of human hair follicle-derived mesenchymal stem cells (hHF-MSCs) by using CultiSpher(®)-G microcarriers. We analyzed the feasibility of 3D culture by using hHF-MSCs/CultiSpher(®)-G microcarrier constructs for its potential applicability in regenerative medicine by comparatively analyzing the performance of hHF-MSCs adhered to the CultiSpher(®)-G microspheres in 3D spinner culture and those grown on the gelatin-coated plastic dishes (2D culture), using various assays. We showed that the hHF-MSCs seeded at various densities quickly adhered to and proliferated well on the microspheres, thus generating at least hundreds of millions of hHF-MSCs on 1 g of CultiSpher(®)-G within 12 days. This resulted in a cumulative cell expansion of greater than 26-fold. Notably, the maximum and average proliferation rates in 3D culture were significantly greater than that of the 2D culture. However, the hHF-MSCs from both the cultures retained surface marker and nestin expression, proliferation capacity and differentiation potentials toward adipocytes, osteoblasts and smooth muscle cells and showed no significant differences as evidenced by Edu incorporation, cell cycle, colony formation, apoptosis, biochemical quantification and qPCR assays.

Structure-function relationships in the stem cell's mechanical world A: seeding protocols as a means to control shape and fate of live stem cells.

PubMed

Zimmermann, Joshua A; Knothe Tate, Melissa L

2011-12-01

Shape and fate are intrinsic manifestations of form and function at the cell scale. Here we hypothesize that seeding density and protocol affect the form and function of live embryonic murine mesenchymal stem cells (MSCs) and their nuclei. First, the imperative for study of live cells was demonstrated in studies showing changes in cell nucleus shape that were attributable to fixation per se. Hence, we compared live cell and nuclear volume and shape between groups of a model MSC line (C3H10T1/2) seeded at, or proliferated from 5,000 cells/cm2 to one of three target densities to achieve targeted development contexts. Cell volume was shown to be dependent on initial seeding density whereas nucleus shape was shown to depend on developmental context but not seeding density. Both smaller cell volumes and flatter nuclei were found to correlate with increased expression of markers for mesenchymal condensation as well as chondrogenic and osteogenic differentiation but a decreased expression of pre-condensation and adipogenic markers. Considering the data presented here, both seeding density and protocol significantly alter the morphology of mesenchymal stem cells even at very early stages of cell culture. Thus, these design parameters may play a critical role in the success of tissue engineering strategies seeking to recreate condensation events. However, a better understanding of how these changes in cell volume and nucleus shape relate to the differentiation of MSCs is important for prescribing precise seeding conditions necessary for the development of the desired tissue type. In a companion study (Part B, following), we address the effect of concomitant volume and shape changing stresses on spatiotemporal distribution of the cytoskeletal proteins actin and tubulin. Taken together, these studies bring us one step closer to our ultimate goal of elucidating the dynamics of nucleus and cell shape change as tissue templates grow (cell proliferation) and specialize (cell differentiation).

Extracellular matrix components and culture regimen selectively regulate cartilage formation by self-assembling human mesenchymal stem cells in vitro and in vivo.

PubMed

Ng, Johnathan; Wei, Yiyong; Zhou, Bin; Burapachaisri, Aonnicha; Guo, Edward; Vunjak-Novakovic, Gordana

2016-12-09

Cartilage formation from self-assembling mesenchymal stem cells (MSCs) in vitro recapitulate important cellular events during mesenchymal condensation that precedes native cartilage development. The goal of this study was to investigate the effects of cartilaginous extracellular matrix (ECM) components and culture regimen on cartilage formation by self-assembling human MSCs in vitro and in vivo. Human bone marrow-derived MSCs (hMSCs) were seeded and compacted in 6.5-mm-diameter transwell inserts with coated (type I, type II collagen) or uncoated (vehicle) membranes, at different densities (0.5 × 10 6 , 1.0 × 10 6 , 1.5 × 10 6 per insert). Pellets were formed by aggregating hMSCs (0.25 × 10 6 ) in round-bottomed wells. All tissues were cultured for up to 6 weeks for in vitro analyses. Discs (cultured for 6, 8 or 10 weeks) and pellets (cultured for 10 weeks) were implanted subcutaneously in immunocompromised mice to evaluate the cartilage stability in vivo. Type I and type II collagen coatings enabled cartilage disc formation from self-assembling hMSCs. Without ECM coating, hMSCs formed dome-shaped tissues resembling the pellets. Type I collagen, expressed in the prechondrogenic mesenchyme, improved early chondrogenesis versus type II collagen. High seeding density improved cartilage tissue properties but resulted in a lower yield of disc formation. Discs and pellets exhibited compositional and organizational differences in vitro and in vivo. Prolonged chondrogenic induction of the discs in vitro expedited endochondral ossification in vivo. The outcomes of cartilage tissues formed from self-assembling MSCs in vitro and in vivo can be modulated by the control of culture parameters. These insights could motivate new directions for engineering cartilage and bone via a cartilage template from self-assembling MSCs.

Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

PubMed Central

Schmal, Olga; Seifert, Jan; Schäffer, Tilman E.; Walter, Christina B.; Aicher, Wilhelm K.; Klein, Gerd

2016-01-01

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard. PMID:26839560

Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture.

PubMed

Schmal, Olga; Seifert, Jan; Schäffer, Tilman E; Walter, Christina B; Aicher, Wilhelm K; Klein, Gerd

2016-01-01

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34(+) hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

Engineering Adipose-like Tissue in vitro and in vivo Utilizing Human Bone Marrow and Adipose-derived Mesenchymal Stem Cells with Silk Fibroin 3D Scaffolds

PubMed Central

Mauney, Joshua R; Nguyen, Trang; Gillen, Kelly; Kirker-Head, Carl; Gimble, Jeffrey M.; Kaplan, David L.

2009-01-01

Biomaterials derived from silk fibrion prepared by aqueous (AB) and organic (HFIP) solvent based processes, along with collagen (COL) and poly-lactic acid (PLA) based scaffolds were studied in vitro and in vivo for their utility in adipose tissue engineering strategies. For in vitro studies, human bone marrow and adipose-derived mesenchymal stem cells (hMSCs and hASCs) were seeded on the various biomaterials and cultured for 21 days in the presence of adipogenic stimulants (AD) or maintained as noninduced controls. Alamar Blue analysis revealed each biomaterial supported initial attachment of hMSCs and hASCs to similar levels for all matrices except COL in which higher levels were observed. hASCs and hMSCs cultured on all biomaterials in the presence of AD showed significant upregulation of adipogenic mRNA transcript levels (LPL, GLUT4, FABP4, PPARγ, adipsin, ACS) to similar extents when compared to noninduced controls. Similarly Oil-Red O analysis of hASC or hMSC-seeded scaffolds displayed substantial amounts of lipid accumulating adipocytes following cultivation with AD. The data revealed AB and HFIP scaffolds supported similar extents of lipid accumulating cells while PLA and COL scaffolds qualitatively displayed lower and higher extents by comparison, respectively. Following a 4 week implantation period in a rat muscle pouch defect model, both AB and HFIP scaffolds supported in vivo adipogenesis either alone or seeded with hASCs or hMSCs as assessed by Oil-Red O analysis, however the presence of exogenous cell sources substantially increased the extent and frequency of adipogenesis observed. In contrast, COL and PLA scaffolds underwent rapid scaffold degradation and were irretrievable following the implantation period. The results suggest that macroporous 3D AB and HFIP silk fibroin scaffolds offer an important platform for cell-based adipose tissue engineering applications, and in particular, provide longer-term structural integrity to promote the maintenance of soft tissue in vivo. PMID:17765303

Chondrogenesis and integration of mesenchymal stem cells within an in vitro cartilage defect repair model.

PubMed

Vinardell, T; Thorpe, S D; Buckley, C T; Kelly, D J

2009-12-01

Integration of repair tissue is a key indicator of the long-term success of cell-based therapies for cartilage repair. The objective of this study was to compare the in vitro chondrogenic differentiation and integration of agarose hydrogels seeded with either chondrocytes or bone marrow-derived mesenchymal stem cells (MSCs) in defects created in cartilage explants. Chondrocytes and MSCs were isolated from porcine donors, suspended in 2% agarose and then injected into cylindrical defects within the explants. These constructs were maintained in a chemically defined medium supplemented with 10 ng/mL of TGF-beta3. Cartilage integration was assessed by histology and mechanical push-out tests. After 6 weeks in culture, chondrocyte-seeded constructs demonstrated a higher integration strength (64.4 +/- 8.3 kPa) compared to MSC-seeded constructs (22.7 +/- 5.9 kPa). Glycosaminoglycan (GAG) (1.27 +/- 0.3 vs. 0.19 +/- 0.03 kPa) and collagen (0.31 +/- 0.08 vs. 0.09 +/- 0.01 kPa) accumulation in chondrocyte-seeded constructs was greater than that measured in the MSC-seeded group. The GAG, collagen, and DNA content of both chondrocyte- and MSC-seeded hydrogels cultured in cartilage explants was significantly lower than control constructs cultured in free swelling conditions. The results of this study suggest that the explant model may constitute a more rigorous in vitro test to assess MSC therapies for cartilage defect repair.

MSC/ECM Cellular Complexes Induce Periodontal Tissue Regeneration.

PubMed

Takewaki, M; Kajiya, M; Takeda, K; Sasaki, S; Motoike, S; Komatsu, N; Matsuda, S; Ouhara, K; Mizuno, N; Fujita, T; Kurihara, H

2017-08-01

Transplantation of mesenchymal stem cells (MSCs), which possess self-renewing properties and multipotency, into a periodontal defect is thought to be a useful option for periodontal tissue regeneration. However, developing more reliable and predictable implantation techniques is still needed. Recently, we generated clumps of an MSC/extracellular matrix (ECM) complex (C-MSC), which consisted of cells and self-produced ECM. C-MSCs can regulate their cellular functions in vitro and can be grafted into a defect site, without any artificial scaffold, to induce bone regeneration. Accordingly, this study aimed to evaluate the effect of C-MSC transplantation on periodontal tissue regeneration in beagle dogs. Seven beagle dogs were employed to generate a premolar class III furcation defect model. MSCs isolated from dog ilium were seeded at a density of 7.0 × 10 4 cells/well into 24-well plates and cultured in growth medium supplemented with 50 µg/mL ascorbic acid for 4 d. To obtain C-MSCs, confluent cells were scratched using a micropipette tip and were then torn off as a cellular sheet. The sheet was rolled up to make round clumps of cells. C-MSCs were maintained in growth medium or osteoinductive medium (OIM) for 5 or 10 d. The biological properties of C-MSCs were evaluated in vitro, and their periodontal tissue regenerative activity was tested by using a dog class III furcation defect model. Immunofluorescence analysis revealed that type I collagen fabricated the form of C-MSCs. OIM markedly elevated calcium deposition in C-MSCs at day 10, suggesting its osteogenic differentiation capacity. Both C-MSCs and C-MSCs cultured with OIM transplantation without an artificial scaffold into the dog furcation defect induced periodontal tissue regeneration successfully compared with no graft, whereas osteogenic-differentiated C-MSCs led to rapid alveolar bone regeneration. These findings suggested that the use of C-MSCs refined by self-produced ECM may represent a novel predictable periodontal tissue regenerative therapy.

Distinct Effects of RGD-glycoproteins on Integrin-Mediated Adhesion and Osteogenic Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schwab, Elisabeth H.; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A.

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α5-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β3-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation. PMID:24324361

Distinct effects of RGD-glycoproteins on Integrin-mediated adhesion and osteogenic differentiation of human mesenchymal stem cells.

PubMed

Schwab, Elisabeth H; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.

Mesenchymal Stem Cells for Cartilage Regeneration of TMJ Osteoarthritis

PubMed Central

Li, Hongyu; Xu, Xin; Ye, Ling; Zhou, Xuedong

2017-01-01

Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease, characterized by progressive cartilage degradation, subchondral bone remodeling, synovitis, and chronic pain. Due to the limited self-healing capacity in condylar cartilage, traditional clinical treatments have limited symptom-modifying and structure-modifying effects to restore impaired cartilage as well as other TMJ tissues. In recent years, stem cell-based therapy has raised much attention as an alternative approach towards tissue repair and regeneration. Mesenchymal stem cells (MSCs), derived from the bone marrow, synovium, and even umbilical cord, play a role as seed cells for the cartilage regeneration of TMJ OA. MSCs possess multilineage differentiation potential, including chondrogenic differentiation as well as osteogenic differentiation. In addition, the trophic modulations of MSCs exert anti-inflammatory and immunomodulatory effects under aberrant conditions. Furthermore, MSCs combined with appropriate scaffolds can form cartilaginous or even osseous compartments to repair damaged tissue and impaired function of TMJ. In this review, we will briefly discuss the pathogenesis of cartilage degeneration in TMJ OA and emphasize the potential sources of MSCs and novel approaches for the cartilage regeneration of TMJ OA, particularly focusing on the MSC-based therapy and tissue engineering. PMID:29123550

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing.

PubMed

Chu, Jing; Shi, Panpan; Deng, Xiaoyuan; Jin, Ying; Liu, Hao; Chen, Maosheng; Han, Xue; Liu, Hanping

2018-03-25

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP-labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full-thickness cutaneous wound site in streptozotocin-induced diabetic mice. Wounds treated with MSC-ADM demonstrated an increased percentage of wound closure. The treatment of MSC-ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col-I) fibers synthesis via second harmonic generation imaging. The synthesis of Col-I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP-labeled MSCs during wound healing was simultaneously traced via two-photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo-multiplier tube. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

PubMed

Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

2015-03-13

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Single-Crystalline, Nanoporous Gallium Nitride Films With Fine Tuning of Pore Size for Stem Cell Engineering.

PubMed

Han, Lin; Zhou, Jing; Sun, Yubing; Zhang, Yu; Han, Jung; Fu, Jianping; Fan, Rong

2014-11-01

Single-crystalline nanoporous gallium nitride (GaN) thin films were fabricated with the pore size readily tunable in 20-100 nm. Uniform adhesion and spreading of human mesenchymal stem cells (hMSCs) seeded on these thin films peak on the surface with pore size of 30 nm. Substantial cell elongation emerges as pore size increases to ∼80 nm. The osteogenic differentiation of hMSCs occurs preferentially on the films with 30 nm sized nanopores, which is correlated with the optimum condition for cell spreading, which suggests that adhesion, spreading, and stem cell differentiation are interlinked and might be coregulated by nanotopography.

Transplantation of mesenchymal stem cells cultured on biomatrix support induces repairing of digestive tract defects, in animal model.

PubMed

Sîrbu-Boeţi, Mirela-Patricia; Chivu, Mihaela; Pâslaru, Liliana Livia; Efrimescu, C; Herlea, V; Pecheanu, C; Moldovan, Lucia; Dragomir, Laura; Bleotu, Coralia; Ciucur, Elena; Vidulescu, Cristina; Vasilescu, Mihaela; Boicea, Anişoara; Mănoiu, S; Ionescu, M I; Popescu, I

2009-01-01

Transplanted mesenchymal stem cells (MSCs) appear to play a significant role in adult tissue repair. The aim of this research was to obtain MSCs enriched, three dimensional (3D) patches for transplant, and to test their ability to induce repair of iatrogenic digestive tract defects in rats. MSCs were obtained from human and rat bone marrow, cultured in vitro, and seeded in a collagen-agarose scaffold, where they showed enhanced viability and proliferation. The phenotype of the cultured cells was representative for MSCs (CD105+, CD90+, and CD34-, CD45-, CD3-, CD14-). The 3D patch was obtained by laying the MSCs enriched collagen-agarose scaffold on a human or swine aortic fragment. After excision of small portions of the rat digestive tract, the 3D patches were sutured at the edge of the defect using micro-surgical techniques. The rats were sacrificed at time-points and the regeneration of the digestive wall was investigated by immunofluorescence, light and electron microscopy. The MSCs enriched 3D patches were biocompatible, biodegradable, and prompted the regeneration of the four layers of the stomach and intestine wall in rats. Human cells were identified in the rat regenerated digestive wall as a hallmark of the transplanted MSCs. For the first time we constructed 3D patches made of cultured bone marrow MSCs, embedded into a collagen-rich biomatrix, on vascular bio-material support, and transplanted them in order to repair iatrogenic digestive tract defects. The result was a complete repair with preservation of the four layered structure of the digestive wall.

Investigating the effect of peptide agonists on the chondrogenic differentiation of human mesenchymal stem cells using design of experiments.

PubMed

Renner, Julie N; Liu, Julie C

2013-01-01

Human mesenchymal stem cells (MSCs) are attractive for use in cartilage tissue engineering. Cells are often seeded in a structural scaffold containing growth factors. Peptide mimics of full-length growth factors are a promising alternative because they are less expensive and easier to manufacture. We investigated four short peptides for their effect on the chondrogenesis of human MSCs. The peptides were originally designed to mimic bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta 1 (TGF-β1), and insulin, all of which have been shown to affect MSC chondrogenesis. Previous studies demonstrated that the peptides elicited bioactivity in other cell types, but the peptides have not been investigated for their effect on chondrogenesis in human MSCs. In a preliminary investigation, peptides were added to a pellet culture of human MSCs and assayed for their effect on glycosaminoglycan (GAG) production. These experiments determined peptide concentrations used in a full-factorial experiment to investigate any interactions. The experiment revealed the BMP peptide as a robust stimulant for GAG production. . © 2013 American Institute of Chemical Engineers.

3D Non-Woven Polyvinylidene Fluoride Scaffolds: Fibre Cross Section and Texturizing Patterns Have Impact on Growth of Mesenchymal Stromal Cells

PubMed Central

Schellenberg, Anne; Ross, Robin; Abagnale, Giulio; Joussen, Sylvia; Schuster, Philipp; Arshi, Annahit; Pallua, Norbert; Jockenhoevel, Stefan; Gries, Thomas; Wagner, Wolfgang

2014-01-01

Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds. PMID:24728045

Phage nanofibers induce vascularized osteogenesis in 3D printed bone scaffolds.

PubMed

Wang, Jianglin; Yang, Mingying; Zhu, Ye; Wang, Lin; Tomsia, Antoni P; Mao, Chuanbin

2014-08-06

A virus-activated matrix is developed to overcome the challenge of forming vascularized bone tissue. It is generated by filling a 3D printed bioceramic scaffold with phage nanofibers displaying high-density RGD peptide. After it is seeded with mesenchymal stem cells (MSCs) and implanted into a bone defect, the phage nanofibers induce osteogenesis and angiogenesis by activating endothelialization and osteogenic differentiation of MSCs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

miR-10a restores human mesenchymal stem cell differentiation by repressing KLF4

PubMed Central

Li, Jiao; Dong, Jun; Zhang, Zhen-hui; Zhang, Dong-Cheng; You, Xiang-Yu; Zhong, Yun; Chen, Min-Sheng; Liu, Shi-Ming

2013-01-01

miRNAs have recently been shown to play a significant role in human aging. However, data demonstrating the effects of aging-related miRNAs in human mesenchymal stem cells (hMSCs) are limited. We observed that hMSC differentiation decreased with aging. We also identified that miR-10a expression was significantly decreased with age by comparing the miRNA expression of hMSCs derived from young and aged individuals. Therefore, we hypothesized that the downregulation of miR-10a may be associated with the decreased differentiation capability of hMSCs from aged individuals. Lentiviral constructs were used to up- or downregulate miR-10a in young and old hMSCs. Upregulation of miR-10a resulted in increased differentiation to adipogenic, osteogenic, and chondrogenic lineages and in reduced cell senescence. Conversely, downregulation of miR-10a resulted in decreased cell differentiation and increased cell senescence. A chimeric luciferase reporter system was generated, tagged with the full-length 3′-UTR region of KLF4 harboring the seed-matched sequence with or without four nucleotide mutations. These constructs were cotransfected with the miR-10a mimic into cells. The luciferase activity was significantly repressed by the miR-10a mimic, proving the direct binding of miR-10a to the 3′-UTR of KLF4. Direct suppression of KLF4 in aged hMSCs increased cell differentiation and decreased cell senescence. In conclusion, miR-10a restores the differentiation capability of aged hMSCs through repression of KLF4. Aging-related miRNAs may have broad applications in the restoration of cell dysfunction caused by aging. J. Cell. Physiol. 228: 2324–2336, 2013. © The Authors. Published by Wiley Periodicals, Inc. PMID:23696417

Research of osteoblastic induced rat bone marrow mesenchymal stem cells cultured on β-TCP/PLLA porous scaffold.

PubMed

Yang, Yi; Wu, Jiang; Jin, Gele; Li, Liang; Li, Zhongwei; Li, Cao

2015-01-01

Ceramic and polymer composite scaffolds are widely used in tissue engineering for bone tissue regeneration. Composite of β-tricalcium phosphate (β-TCP) and poly L-lactic acid (PLLA), due to its biocompatibility and biodegradability, is widely used in bioengineering. However, optimal ratio, porosity and pore size of this kind of scaffolds were not very clear yet. We cultured osteoblastic induced rMSCs on β-TCP/PLLA scaffolds to investigate the optimum construction, which owned better properties for supporting cells growth, proliferation and differentiation. A total of 24 mice were divided into three groups: rMSCs + β-TCP/PLLA, osteoblastic rMSCs + β-TCP/PLLA and β-TCP/PLLA without cells. 8 rude mice were implanted with rMSCs + β-TCP/PLLA in the left thighs and β-TCP/PLLA without cells in the right thighs. 8 rude mice were implanted with osteoblastic rMSCs + β-TCP/PLLA in the left thighs and the same treatments in the right thighs as the above. After 8 and 12 weeks, the mice were sacrificed and implants with the surrounding tissues were harvested together. Paraffin sections were got and HE stain and Masson-Goldner stain were employed to observe the ectopic bone formation. The scaffolds of β-TCP/PLLA = 2:1 significantly increased osteocalcin production of the cells. In addition, scaffolds with NaCl = 70 wt%, pore size 200~450 μm showed better compatibility to these seeding cells. A significantly larger area of bone formation in the osteoblastic rMSCs and β-TCP/PLLA composite than that in rMSCs/scaffold and in the scaffold without cells in vivo. compounds of osteoblastic induced rMSCs and the scaffold with β-TCP/PLLA = 2:1, NaCl = 70 wt%, pore size = 200-450 μm had good properties as a kind of bone substitute.

Composition-function relations of cartilaginous tissues engineered from chondrocytes and mesenchymal stem cells isolated from bone marrow and infrapatellar fat pad.

PubMed

Vinardell, T; Buckley, C T; Thorpe, S D; Kelly, D J

2011-10-01

The objective of this study was to determine the functional properties of cartilaginous tissues generated by porcine MSCs isolated from different tissue sources, and to compare these properties to those derived from chondrocytes (CCs). MSCs were isolated from bone marrow (BM) and infrapatellar fat pad (FP), while CCs were harvested from the articular surface of the femoro-patellar joint. Culture-expanded CCs and MSCs were encapsulated in agarose hydrogels and cultured in the presence of TGFβ3. Samples were analysed biomechanically, biochemically and histologically at days 0, 21 and 42. After 42 days in free swelling culture, mean GAG content was 1.50% w/w in CC-seeded constructs, compared to 0.95% w/w in FP- and 0.43% w/w in BM-seeded constructs. Total collagen accumulation was highest in FP constructs. DNA content increased with time for all the groups. The mechanical functionality of cartilaginous tissues engineered using CCs was superior to that generated from either source of MSCs. Differences were also observed in the spatial distribution of matrix components in tissues engineered using CCs and MSCs, which appears to have a strong influence on the apparent mechanical properties of the constructs. Therefore, while functional cartilaginous tissues can be engineered using MSCs isolated from different sources, the spatial composition of these tissues is unlike that generated using chondrocytes, suggesting that MSCs and chondrocytes respond differently to the regulatory factors present within developing cartilaginous constructs. Copyright © 2010 John Wiley & Sons, Ltd.

Comparison of Human Denuded Amniotic Membrane and Porcine Small Intestine Submucosa as Scaffolds for Limbal Mesenchymal Stem Cells.

PubMed

Sous Naasani, Liliana I; Rodrigues, Cristiano; Azevedo, Jéssica Gonçalves; Damo Souza, Aline F; Buchner, Silvio; Wink, Márcia R

2018-04-29

Blinding corneal scarring is usually treated with allogeneic graft tissue. Nevertheless, the global shortage of donors leaves millions of patients in need of therapy. Traditional tissue engineering strategies involves the combination of cells, growth factors, and scaffolds that can supply cellular biological components allowing to restore the tissue function. The mesenchymal stem cells found in the limbal stroma (L-MSCs) have a self-renewal potential for multilineage differentiation. Thus, in this work we compared the potential of human amniotic membrane (hAM) and porcine small intestine submucosa (SIS) as scaffolds for L-MSCs, aiming at potential applications in corneal regeneration. For that, L-MSCs were seeded on hAM and SIS and we analyzed their viability, actin cytoskeleton, nuclei morphology, cell density, adhesion and surface markers. Our results showed that cells adhered and integrated into both membranes with a high cell density, an important characteristic for cell therapy. However, due to its transparency, the hAM allowed a better observation of L-MSCs. In addition, the analysis of surface markers expression on L-MSCs after two weeks showed a slight increase in the percentages of negative markers for MSCs grown on SIS membrane. Thus, considering a long-term culture, the hAM was considered better in maintaining the MSCs phenotype. Regarding the function as scaffolds, SIS was as efficient as the amniotic membrane, considering that these two types of biological matrices maintained the cell viability, actin cytoskeleton, nuclei morphology and mesenchymal phenotype, without causing cell death. Therefore, our data in vitro provides evidence for future pre-clinical studies were these membranes can be used as a support to transport mesenchymal stem cells to the injured area, creating a kind of temporary curative, allowing the release of bioactive molecules, such as cytokines and growth factors and then promoting the tissue regeneration, both in human and veterinary medicine.

Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

PubMed

Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

2015-01-01

Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

Characterization of mesenchymal stem cells and fibrochondrocytes in three-dimensional co-culture: analysis of cell shape, matrix production, and mechanical performance.

PubMed

McCorry, Mary Clare; Puetzer, Jennifer L; Bonassar, Lawrence J

2016-03-12

Bone marrow mesenchymal stem cells (MSCs) have shown positive therapeutic effects for meniscus regeneration and repair. Preliminary in vitro work has indicated positive results for MSC applications for meniscus tissue engineering; however, more information is needed on how to direct MSC behavior. The objective of this study was to examine the effect of MSC co-culture with primary meniscal fibrochondrocytes (FCCs) in a three-dimensional collagen scaffold in fibrochondrogenic media. Co-culture of MSCs and FCCs was hypothesized to facilitate the transition of MSCs to a FCC cell phenotype as measured by matrix secretion and morphology. MSCs and FCCs were isolated from bovine bone marrow and meniscus, respectively. Cells were seeded in a 20 mg/mL high-density type I collagen gel at MSC:FCC ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs were cultured for up to 2 weeks and then analyzed for cell morphology, glycosaminoglycan content, collagen content, and production of collagen type I, II, and X. Cells were homogeneously mixed throughout the scaffold and cells had limited direct cell-cell contact. After 2 weeks in culture, MSCs transitioned from a spindle-like morphology toward a rounded phenotype, while FCCs remained rounded throughout culture. Although MSC shape changed with culture, the overall size was significantly larger than FCCs throughout culture. While 75:25 and 100:0 (MSC mono-culture) culture groups produced significantly more glycosaminoglycan (GAG)/DNA than FCCs in mono-culture, GAG retention was highest in 50:50 co-cultures. Similarly, the aggregate modulus was highest in 100:0 and 50:50 co-cultures. All samples contained both collagen types I and II after 2 weeks, and collagen type X expression was evident only in MSC mono-culture gels. MSCs shift to a FCC morphology in both mono- and co-culture. Co-culture reduced hypertrophy by MSCs, indicated by collagen type X. This study shows that MSC phenotype can be influenced by indirect homogeneous cell culture in a three-dimensional gel, demonstrating the applicability of MSCs in meniscus tissue engineering applications.

Mesenchymal stem cells delivered in a microsphere-based engineered skin contribute to cutaneous wound healing and sweat gland repair.

PubMed

Huang, Sha; Lu, Gang; Wu, Yan; Jirigala, Enhe; Xu, Yongan; Ma, Kui; Fu, Xiaobing

2012-04-01

Bone-marrow-derived mesenchymal stem cells (BM-MSCs) can contribute to wound healing after skin injury. However, the role of BM-MSCs on repairing skin appendages in renewal tissues is incompletely explored. Moreover, most preclinical studies suggest that the therapeutic effects afforded by BM-MSCs transplantation are short-lived and relatively unstable. To assess whether engrafted bone-marrow-derived mesenchymal stem cells via a delivery system can participate in cutaneous wound healing and sweat-gland repair in mice. For safe and effective delivery of BM-MSCs to wounds, epidermal growth factor (EGF) microspheres were firstly developed to both support cells and maintain appropriate stimuli, then cell-seeded microspheres were incorporated with biomimetic scaffolds and thus fabricated an engineered skin construct with epithelial differentiation and proliferative potential. The applied efficacy was examined by implanting them into excisional wounds on both back and paws of hind legs in mice. After 3 weeks, BM-MSC-engineered skin (EGF loaded) treated wounds exhibited accelerated healing with increased re-epithelialization rates and less skin contraction. Furthermore, histological and immunofluorescence staining analysis revealed sweat glands-like structures became more apparent in BM-MSC-engineered skin (EGF loaded) treated wounds but the number of implanted BM-MSCs were decreased gradually in later phases of healing progression. Our study suggests that BM-MSCs delivered by this EGF microspheres-based engineered skin model may be a promising strategy to repair sweat glands and improve cutaneous wound healing after injury and success in this study might provide a potential benefit for BM-MSCs administration clinically. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Cyclic tension promotes fibroblastic differentiation of human MSCs cultured on collagen-fibre scaffolds.

PubMed

Qiu, Yongzhi; Lei, Jennifer; Koob, Thomas J; Temenoff, Johnna S

2016-12-01

Mesenchymal stem cells (MSCs) have been suggested as a potential cell source for tendon/ligament tissue engineering. Extrinsic cues, such as the chemical and physical properties of scaffolds, as well as external forces, play an important role in fibroblastic differentiation of these cells. In this study, we employed a collagen-fibre scaffold that mimics the chemical and fibrous structure and mechanical properties of tendon/ligament, and studied how imparting cyclic tension to these fibrous collagen scaffolds affects tendon/ligament fibroblastic differentiation of MSCs. Human MSCs attached and spread on the surface of the scaffolds, and appeared aligned along the fibres 24 h after seeding. Cyclic tension was then applied to cell-laden scaffolds over a period of 14 days (10% strain, 1 Hz, 3 h on/3 h off). Real time RT-PCR analysis indicated that scleraxis, a transcription factor associated with the tendon fibroblast phenotype, was found to be significantly upregulated only under cyclic tension. Immunohistochemical staining demonstrated that MSCs cultured under cyclic tension after 14 days secreted more extracellular matrix, including collagen I, collagen III and tenascin-C, compared to constructs in static culture, after 14 days in vitro. Our data indicate that cyclic tension can promote fibroblastic differentiation of MSCs in these fibrous collagen-based scaffolds, which may have significant applications in the development of tissue-engineered graft alternatives for tendon and ligament injuries. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

In vivo bone formation by human marrow stromal cells in biodegradable scaffolds that release dexamethasone and ascorbate-2-phosphate.

PubMed

Kim, Hyongbum; Suh, Hwal; Jo, Sangmee Ahn; Kim, Hyun Woo; Lee, Jung Min; Kim, Eun Hae; Reinwald, Yvonne; Park, Sang-Hyug; Min, Byoung-Hyun; Jo, Inho

2005-07-15

An unsolved problem with stem cell-based engineering of bone tissue is how to provide a microenvironment that promotes the osteogenic differentiation of multipotent stem cells. Previously, we fabricated porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds that released biologically active dexamethasone (Dex) and ascorbate-2-phosphate (AsP), and that acted as osteogenic scaffolds. To determine whether these osteogenic scaffolds can be used for bone formation in vivo, we seeded multipotent human marrow stromal cells (hMSCs) onto the scaffolds and implanted them subcutaneously into athymic mice. Higher alkaline phosphatase expression was observed in hMSCs in the osteogenic scaffolds compared with that of hMSCs in control scaffolds. Furthermore, there was more calcium deposition and stronger von Kossa staining in the osteogenic scaffolds, which suggested that there was enhanced mineralized bone formation. We failed to detect cartilage in the osteogenic scaffolds (negative Safranin O staining), which implied that there was intramembranous ossification. This is the first study to demonstrate the successful formation of mineralized bone tissue in vivo by hMSCs in PLGA scaffolds that release Dex and AsP.

Bacterial nanocellulose stimulates mesenchymal stem cell expansion and formation of stable collagen-I networks as a novel biomaterial in tissue engineering.

PubMed

Vielreicher, Martin; Kralisch, Dana; Völkl, Simon; Sternal, Fabian; Arkudas, Andreas; Friedrich, Oliver

2018-06-20

Biomimetic scaffolds are of great interest to tissue engineering (TE) and tissue repair as they support important cell functions. Scaffold coating with soluble collagen-I has been used to achieve better tissue integration in orthopaedy, however, as collagen persistence was only temporary such efforts were limited. Adequate coverage with cell-derived ECM collagen-I would promise great success, in particular for TE of mechanically challenged tissues. Here, we have used label-free, non-invasive multiphoton microscopy (MPM) to characterise bacterial nanocellulose (BNC) - a promising biomaterial for bone TE - and their potency to stimulate collagen-I formation by mesenchymal stem cells (MSCs). BNC fleeces were investigated by Second Harmonic Generation (SHG) imaging and by their characteristic autofluorescence (AF) pattern, here described for the first time. Seeded MSCs adhered fast, tight and very stable, grew to multilayers and formed characteristic, wide-spread and long-lasting collagen-I. MSCs used micron-sized lacunae and cracks on the BNC surface as cell niches. Detailed analysis using a collagen-I specific binding protein revealed a highly ordered collagen network structure at the cell-material interface. In addition, we have evidence that BNC is able to stimulate MSCs towards osteogenic differentiation. These findings offer new options for the development of engineered tissue constructs based on BNC.

Tissue-engineered tracheal reconstruction using three-dimensionally printed artificial tracheal graft: preliminary report.

PubMed

Chang, Jae Won; Park, Su A; Park, Ju-Kyeong; Choi, Jae Won; Kim, Yoo-Suk; Shin, Yoo Seob; Kim, Chul-Ho

2014-06-01

Three-dimensional printing has come into the spotlight in the realm of tissue engineering. We intended to evaluate the plausibility of 3D-printed (3DP) scaffold coated with mesenchymal stem cells (MSCs) seeded in fibrin for the repair of partial tracheal defects. MSCs from rabbit bone marrow were expanded and cultured. A half-pipe-shaped 3DP polycaprolactone scaffold was coated with the MSCs seeded in fibrin. The half-pipe tracheal graft was implanted on a 10 × 10-mm artificial tracheal defect in four rabbits. Four and eight weeks after the operation, the reconstructed sites were evaluated bronchoscopically, radiologically, histologically, and functionally. None of the four rabbits showed any sign of respiratory distress. Endoscopic examination and computed tomography showed successful reconstruction of trachea without any collapse or blockage. The replaced tracheas were completely covered with regenerated respiratory mucosa. Histologic analysis showed that the implanted 3DP tracheal grafts were successfully integrated with the adjacent trachea without disruption or granulation tissue formation. Neo-cartilage formation inside the implanted graft was sufficient to maintain the patency of the reconstructed trachea. Scanning electron microscope examination confirmed the regeneration of the cilia, and beating frequency of regenerated cilia was not different from those of the normal adjacent mucosa. The shape and function of reconstructed trachea using 3DP scaffold coated with MSCs seeded in fibrin were restored successfully without any graft rejection. Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Osteogenesis and Trophic Factor Secretion are Influenced by the Composition of Hydroxyapatite/Poly(Lactide-Co-Glycolide) Composite Scaffolds

PubMed Central

He, Jiawei; Genetos, Damian C.

2010-01-01

The use of composite biomaterials for bone repair capitalizes on the beneficial aspects of individual materials while tailoring the mechanical properties of the composite. We hypothesized that substrate composition would modulate the osteogenic response and secretion of potent trophic factors by human mesenchymal stem cells (hMSCs). Composite scaffolds were prepared by combining nanosized hydroxyapatite (HA) and microspheres formed of poly(lactic-co-glycolic acid) (PLG) at varying ratios between 0:1 and 5:1. Scaffolds were seeded with hMSCs for culture in osteogenic conditions or subcutaneous implantation into nude rats. Compressive moduli increased with HA content in a near-linear fashion. The osteogenic differentiation of hMSCs increased in a dose-dependent manner as determined by alkaline phosphatase activity and osteopontin secretion after 4 weeks of culture. Further, endogenous secretion of vascular endothelial growth factor was sustained at significantly higher levels over 28 days for hMSCs seeded in 2.5:1 and 5:1 HA:PLG scaffolds. Eight weeks after implantation, scaffolds with higher HA:PLG ratios exhibited greater vascularization and more mineralized tissue. These data demonstrate that the mechanical properties, osteogenic differentiation, as well as the timing and duration of trophic factor secretion by hMSCs can be tailored through controlling the composition of the polymer–bioceramic composite. PMID:19642853

Repair of traumatic skeletal muscle injury with bone-marrow-derived mesenchymal stem cells seeded on extracellular matrix.

PubMed

Merritt, Edward K; Cannon, Megan V; Hammers, David W; Le, Long N; Gokhale, Rohit; Sarathy, Apurva; Song, Tae J; Tierney, Matthew T; Suggs, Laura J; Walters, Thomas J; Farrar, Roger P

2010-09-01

Skeletal muscle injury resulting in tissue loss poses unique challenges for surgical repair. Despite the regenerative potential of skeletal muscle, if a significant amount of tissue is lost, skeletal myofibers will not grow to fill the injured area completely. Prior work in our lab has shown the potential to fill the void with an extracellular matrix (ECM) scaffold, resulting in restoration of morphology, but not functional recovery. To improve the functional outcome of the injured muscle, a muscle-derived ECM was implanted into a 1 x 1 cm(2), full-thickness defect in the lateral gastrocnemius (LGAS) of Lewis rats. Seven days later, bone-marrow-derived mesenchymal stem cells (MSCs) were injected directly into the implanted ECM. Partial functional recovery occurred over the course of 42 days when the LGAS was repaired with an MSC-seeded ECM producing 85.4 +/- 3.6% of the contralateral LGAS. This was significantly higher than earlier recovery time points (p < 0.05). The specific tension returned to 94 +/- 9% of the contralateral limb. The implanted MSC-seeded ECM had more blood vessels and regenerating skeletal myofibers than the ECM without cells (p < 0.05). The data suggest that the repair of a skeletal muscle defect injury by the implantation of a muscle-derived ECM seeded with MSCs can improve functional recovery after 42 days.

Osteogenic Response to BMP-2 of hMSCs Grown on Apatite-Coated Scaffolds

PubMed Central

Davis, Hillary E.; Case, Erin M.; Miller, Stephanie L.; Genetos, Damian C.; Leach, J. Kent

2011-01-01

Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to nonmineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration. PMID:21656707

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

PubMed Central

Altaie, Ala; Owston, Heather; Jones, Elena

2016-01-01

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells (MSCs). Although MSCs are most commonly grown in media containing fetal calf serum, human platelet lysate (PL) offers an effective alternative. Bone marrow - derived MSCs grown in PL-containing media display faster proliferation whilst maintaining good osteogenic differentiation capacity. Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo. In an alternative approach, nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices. Even though methods to coat scaffolds with PL vary, in vitro studies suggest that PL allows for MSC adhesion, migration and differentiation inside these scaffolds. Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo. This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration. To minimise inconsistency between the studies, further work is required towards standardisation of PL preparation in terms of the starting material, platelet concentration, leukocyte depletion, and the method of platelet lysis. PL quality control procedures and its “potency” assessment are urgently needed, which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation. Furthermore, different PL formulations could be tailor-made for specific bone repair indications. Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration. PMID:26981170

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

PubMed

Altaie, Ala; Owston, Heather; Jones, Elena

2016-02-26

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells (MSCs). Although MSCs are most commonly grown in media containing fetal calf serum, human platelet lysate (PL) offers an effective alternative. Bone marrow - derived MSCs grown in PL-containing media display faster proliferation whilst maintaining good osteogenic differentiation capacity. Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo. In an alternative approach, nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices. Even though methods to coat scaffolds with PL vary, in vitro studies suggest that PL allows for MSC adhesion, migration and differentiation inside these scaffolds. Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo. This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration. To minimise inconsistency between the studies, further work is required towards standardisation of PL preparation in terms of the starting material, platelet concentration, leukocyte depletion, and the method of platelet lysis. PL quality control procedures and its "potency" assessment are urgently needed, which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation. Furthermore, different PL formulations could be tailor-made for specific bone repair indications. Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.

Development and characterization of hybrid tubular structure of PLCL porous scaffold with hMSCs/ECs cell sheet.

PubMed

Pangesty, Azizah Intan; Arahira, Takaaki; Todo, Mitsugu

2017-09-15

Tissue engineering offers an alternate approach to providing vascular graft with potential to grow similar with native tissue by seeding autologous cells into biodegradable scaffold. In this study, we developed a combining technique by layering a sheet of cells onto a porous tubular scaffold. The cell sheet prepared from co-culturing human mesenchymal stem cells (hMSCs) and endothelial cells (ECs) were able to infiltrate through porous structure of the tubular poly (lactide-co-caprolactone) (PLCL) scaffold and further proliferated on luminal wall within a week of culture. Moreover, the co-culture cell sheet within the tubular scaffold has demonstrated a faster proliferation rate than the monoculture cell sheet composed of MSCs only. We also found that the co-culture cell sheet expressed a strong angiogenic marker, including vascular endothelial growth factor (VEGF) and its receptor (VEGFR), as compared with the monoculture cell sheet within 2 weeks of culture, indicating that the co-culture system could induce differentiation into endothelial cell lineage. This combined technique would provide cellularization and maturation of vascular construct in relatively short period with a strong expression of angiogenic properties.

Can the outcomes of mesenchymal stem cell-based therapy for myocardial infarction be improved? Providing weapons and armour to cells.

PubMed

Karpov, Andrey A; Udalova, Daria V; Pliss, Michael G; Galagudza, Michael M

2017-04-01

Use of mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI) has been found to have infarct-limiting effects in numerous experimental and clinical studies. However, recent meta-analyses of randomized clinical trials on MSC-based MI therapy have highlighted the need for improving its efficacy. There are two principal approaches for increasing therapeutic effect of MSCs: (i) preventing massive MSC death in ischaemic tissue and (ii) increasing production of cardioreparative growth factors and cytokines with transplanted MSCs. In this review, we aim to integrate our current understanding of genetic approaches that are used for modification of MSCs to enable their improved survival, engraftment, integration, proliferation and differentiation in the ischaemic heart. Genetic modification of MSCs resulting in increased secretion of paracrine factors has also been discussed. In addition, data on MSC preconditioning with physical, chemical and pharmacological factors prior to transplantation are summarized. MSC seeding on three-dimensional polymeric scaffolds facilitates formation of both intercellular connections and contacts between cells and the extracellular matrix, thereby enhancing cell viability and function. Use of genetic and non-genetic approaches to modify MSC function holds great promise for regenerative therapy of myocardial ischaemic injury. © 2016 John Wiley & Sons Ltd.

Circumferential Esophageal Replacement by a Tissue-engineered Substitute Using Mesenchymal Stem Cells

PubMed Central

Catry, Jonathan; Luong-Nguyen, Minh; Arakelian, Lousineh; Poghosyan, Tigran; Bruneval, Patrick; Domet, Thomas; Michaud, Laurent; Sfeir, Rony; Gottrand, Frederic; Larghero, Jerome; Vanneaux, Valerie

2018-01-01

Tissue engineering appears promising as an alternative technique for esophageal replacement. Mesenchymal stem cells (MSCs) could be of interest for esophageal regeneration. Evaluation of the ability of an acellular matrix seeded with autologous MSCs to promote tissue remodeling toward an esophageal phenotype after circumferential replacement of the esophagus in a mini pig model. A 3 cm long circumferential replacement of the abdominal esophagus was performed with an MSC-seeded matrix (MSC group, n = 10) versus a matrix alone (control group, n = 10), which has previously been matured into the great omentum. The graft area was covered with an esophageal removable stent. A comparative histological analysis of the graft area after animals were euthanized sequentially is the primary outcome of the study. Histological findings after maturation, overall animal survival, and postoperative morbidity were also compared between groups. At postoperative day 45 (POD 45), a mature squamous epithelium covering the entire surface of the graft area was observed in all the MSC group specimens but in none of the control group before POD 95. Starting at POD 45, desmin positive cells were seen in the graft area in the MSC group but never in the control group. There were no differences between groups in the incidence of surgical complications and postoperative death. In this model, MSCs accelerate the mature re-epitheliazation and early initiation of muscle cell colonization. Further studies will focus on the use of cell tracking tools in order to analyze the becoming of these cells and the mechanisms involved in this tissue regeneration. PMID:29390879

Oxygen tension differentially regulates the functional properties of cartilaginous tissues engineered from infrapatellar fat pad derived MSCs and articular chondrocytes.

PubMed

Buckley, C T; Vinardell, T; Kelly, D J

2010-10-01

For current tissue engineering or regenerative medicine strategies, chondrocyte (CC)- or mesenchymal stem cell (MSC)-seeded constructs are typically cultured in normoxic conditions (20% oxygen). However, within the knee joint capsule a lower oxygen tension exists. The objective of this study was to investigate how CCs and infrapatellar fad pad derived MSCs will respond to a low oxygen (5%) environment in 3D agarose culture. Our hypothesis was that culture in a low oxygen environment (5%) will enhance the functional properties of cartilaginous tissues engineered using both cell sources. Cell-encapsulated agarose hydrogel constructs (seeded with CCs or infrapatellar fat pad (IFP) derived MSCs) were prepared and cultured in a chemically defined serum-free medium in the presence (CCs and MSCs) or absence (CCs only) of transforming growth factor-beta3 (TGF-β3) in normoxic (20%) or low oxygen (5%) conditions for 42 days. Constructs were assessed at days 0, 21 and 42 in terms of mechanical properties, biochemical content and histologically. Low oxygen tension (5%) was observed to promote extracellular matrix (ECM) production by CCs cultured in the absence of TGF-β3, but was inhibitory in the presence of TGF-β3. In contrast, a low oxygen tension enhanced chondrogenesis of IFP constructs in the presence of TGF-β3, leading to superior mechanical functionality compared to CCs cultured in identical conditions. Extrapolating the results of this study to the in vivo setting, it would appear that joint fat pad derived MSCs may possess a superior potential to generate a functional repair tissue in low oxygen tensions. However, in the context of in vitro cartilage tissue engineering, CCs maintained in normoxic conditions in the presence of TGF-β3 generate the most mechanically functional tissue. Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Development of scaffold architectures and heterotypic cell systems for hepatocyte transplantation

NASA Astrophysics Data System (ADS)

Alzebdeh, Dalia Abdelrahim

In vitro assembly of functional liver tissue is needed to enable the transplantation of tissue-engineered livers. In addition, there is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of sizable implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability, an issue which is closely related to the high metabolic rate of hepatocytes. In this study, we first compared two designs of highly porous chitosan-heparin scaffolds seeded with hepatocytes in dynamic perfusion bioreactor systems. The aim was to promote cell seeding efficiency by effectively entrapping 100 million hepatocytes at high density. We found that scaffolds with radially tapering pore architecture had highly efficient cell entrapment that maximized donor hepatocyte utilization, compared to alternate pore structures. Hepatocytes showed higher seeding efficiency and metabolic function when seeded as single cell suspensions as opposed to pre-formed, 100microm aggregates. Seeding efficiency was found to increase with flow rate, with single cell and aggregate suspension exhibiting different optimal flow rates. However, metabolic performance results indicated significant shear damage to cells at high efficiency flow rates. To better maintain hepatocyte basement membrane and cell polarity, spheroid co-cultures with mesenchymal stem cells (MSC) were investigated. Hepatocytes and MSCs were seeded in three different architectures in an effort to optimize the spatial arrangement of the two cell types. MSC co-culture greatly enhanced hepatocyte metabolic function in agitated cultures. Interestingly, the effects of diffusion limitations in spheroid culture, coupled with shear damage and subsequent removal of outer hepatocyte layers produced a defined oscillation of urea production rates in certain co-culture arrangements. A mathematical model of urea synthesis in shear-exposed, co-culture spheroids reproduced the metabolic oscillations observed. This result together with culture observations suggests that MSCs can provide both physiological support and some direct shear protection to hepatocytes in perfused or shear-exposed culture environments. Finally, in order to reduce hepatocyte exposure to excessive shear forces in perfused scaffolds, a modular scaffold design based on polyelectrolyte fiber encapsulation was explored. Scaffolds with uniformly distributed, shear protected cells were achieved.

Functional collagen conduits combined with human mesenchymal stem cells promote regeneration after sciatic nerve transection in dogs.

PubMed

Cui, Yi; Yao, Yao; Zhao, Yannan; Xiao, Zhifeng; Cao, Zongfu; Han, Sufang; Li, Xing; Huan, Yong; Pan, Juli; Dai, Jianwu

2018-05-01

Numerous studies have focused on the development of novel and innovative approaches for the treatment of peripheral nerve injury using artificial nerve guide conduits. In this study, we attempted to bridge 3.5-cm defects of the sciatic nerve with a longitudinally oriented collagen conduit (LOCC) loaded with human umbilical cord mesenchymal stem cells (hUC-MSCs). The LOCC contains a bundle of longitudinally aligned collagenous fibres enclosed in a hollow collagen tube. Our previous studies showed that an LOCC combined with neurotrophic factors enhances peripheral nerve regeneration. However, it remained unknown whether an LOCC seeded with hUC-MSCs could also promote regeneration. In this study, using various histological and electrophysiological analyses, we found that an LOCC provides mechanical support to newly growing nerves and functions as a structural scaffold for cells, thereby stimulating sciatic nerve regeneration. The LOCC and hUC-MSCs synergistically promoted regeneration and improved the functional recovery in a dog model of sciatic nerve injury. Therefore, the combined use of an LOCC and hUC-MSCs might have therapeutic potential for the treatment of peripheral nerve injury. Copyright © 2018 John Wiley & Sons, Ltd.

Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous beta-TCP ceramic scaffolds.

PubMed

Guo, Xiaodong; Zheng, Qixin; Kulbatski, Iris; Yuan, Quan; Yang, Shuhua; Shao, Zengwu; Wang, Hong; Xiao, Baojun; Pan, Zhengqi; Tang, Shuo

2006-09-01

Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focused on combining gene transfer with tissue engineering techniques. Basic fibroblast growth factor (bFGF) is one of the most prominent osteogenic growth factors that has the potential to accelerate bone healing by promoting the proliferation and differentiation of mesenchymal stem cells (MSCs) and the regeneration of capillary vasculature. However, the short biological half-lives of growth factors may impose severe restraints on their clinical usefulness. Gene-based delivery systems provide a better way of achieving a sustained high concentration of growth factors locally in the defect and delivering a more biologically active product than that achieved by exogenous application of recombinant proteins. The objective of this experimental study was to investigate whether the bFGF gene modified MSCs could enhance the repair of large segmental bone defects. The pcDNA3-bFGF gene transfected MSCs were seeded on biodegradable porous beta tricalcium phosphate (beta-TCP) ceramics and allografted into the 15 mm critical-sized segmental bone defects in the radius of 18 New Zealand White rabbits. The pcDNA3 vector gene transfected MSCs were taken as the control. The follow-up times were 2, 4, 6, 8, 10 and 12 weeks. Scanning electron microscopic, roentgenographic, histologic and immunohistological studies were used to assess angiogenesis and bone regeneration. In vitro, the proliferation and differentiation of bFGF gene transfected MSCs were more active than that of the control groups. In vivo, significantly more new bone formation accompanied by abundant active capillary regeneration was observed in pores of the ceramics loaded with bFGF gene transfected MSCs, compared with control groups. Transfer of gene encoding bFGF to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation. This new bFGF gene enhanced tissue engineering strategy could be of potential benefit to accelerate bone healing, especially in defects caused by atrophic nonunion and avascular necrosis of the femoral head.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

PubMed

Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

2013-01-01

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

PubMed

Song, Yang; Tang, Yinhong; Song, Jinlin; Lei, Mingxing; Liang, Panpan; Fu, Tiwei; Su, Xudong; Zhou, Pengfei; Yang, Li; Huang, Enyi

2018-04-01

The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

Engineering Vascularized Bone Grafts by Integrating a Biomimetic Periosteum and β-TCP Scaffold

PubMed Central

2015-01-01

Treatment of large bone defects using synthetic scaffolds remain a challenge mainly due to insufficient vascularization. This study is to engineer a vascularized bone graft by integrating a vascularized biomimetic cell-sheet-engineered periosteum (CSEP) and a biodegradable macroporous beta-tricalcium phosphate (β-TCP) scaffold. We first cultured human mesenchymal stem cells (hMSCs) to form cell sheet and human umbilical vascular endothelial cells (HUVECs) were then seeded on the undifferentiated hMSCs sheet to form vascularized cell sheet for mimicking the fibrous layer of native periosteum. A mineralized hMSCs sheet was cultured to mimic the cambium layer of native periosteum. This mineralized hMSCs sheet was first wrapped onto a cylindrical β-TCP scaffold followed by wrapping the vascularized HUVEC/hMSC sheet, thus generating a biomimetic CSEP on the β-TCP scaffold. A nonperiosteum structural cell sheets-covered β-TCP and plain β-TCP were used as controls. In vitro studies indicate that the undifferentiated hMSCs sheet facilitated HUVECs to form rich capillary-like networks. In vivo studies indicate that the biomimetic CSEP enhanced angiogenesis and functional anastomosis between the in vitro preformed human capillary networks and the mouse host vasculature. MicroCT analysis and osteocalcin staining show that the biomimetic CSEP/β-TCP graft formed more bone matrix compared to the other groups. These results suggest that the CSEP that mimics the cellular components and spatial configuration of periosteum plays a critical role in vascularization and osteogenesis. Our studies suggest that a biomimetic periosteum-covered β-TCP graft is a promising approach for bone regeneration. PMID:24858072

Electrospun scaffold containing TGF-β1 promotes human mesenchymal stem cell differentiation towards a nucleus pulposus-like phenotype under hypoxia.

PubMed

Cui, Xiang; Liu, Minghan; Wang, Jiaxu; Zhou, Yue; Xiang, Qiang

2015-04-01

The study was aimed at evaluating the effect of electrospun scaffold containing TGF-β1 on promoting human mesenchymal stem cells (MSCs) differentiation towards a nucleus pulposus-like phenotype under hypoxia. Two kinds of nanofibrous scaffolds containing TGF-β1 were fabricated using uniaxial electrospinning (Group I) and coaxial electrospinning (Group II). Human MSCs were seeded on both kinds of scaffolds and cultured in a hypoxia chamber (2% O2), and then the scaffolds were characterised. Cell proliferation and differentiation were also evaluated after 3 weeks of cell culture. Results showed that both kinds of scaffolds shared similar diameter distributions and protein release. However, Group I scaffolds were more hydrophilic than that of Group II. Both kinds of scaffolds induced the MSCs to differentiate towards the nucleus pulposus-type phenotype in vitro. In addition, the expression of nucleus pulposus-associated genes (aggrecan, type II collagen, HIF-1α and Sox-9) in Group I increased more than that of Group II. These results indicate that electrospinning nanofibrous scaffolds containing TGF-β1 supports the differentiation of MSCs towards the pulposus-like phenotype in a hypoxia chamber, which would be a more appropriate choice for nucleus pulposus regeneration.

Peptide modified nanofibrous scaffold promotes human mesenchymal stem cell proliferation and long-term passaging.

PubMed

Mobasseri, Rezvan; Tian, Lingling; Soleimani, Masoud; Ramakrishna, Seeram; Naderi-Manesh, Hossein

2018-03-01

Long-term culture, passage and proliferation of human mesenchymal stem cells (hMSCs) cause loss of their stemness properties including self-renewal and multipotency. By optimizing the MSCs environment in vitro, maintaining the stemness state and better controlling the cell fate might be possible. We have recently reported the significant effects of bioactive Tat protein-derived peptide named R-peptide on hMSC adhesion, morphology and proliferation, which has demonstrated R-peptide enhanced MSC early adhesion and proliferation in comparison to other bioactive molecules including RGD peptide, fibronectin and collagen. In this study, R-peptide was used to evaluate stemness properties of MSCs after long-term passaging. R-peptide conjugated poly caprolactone (PCL) nanofibrous scaffold and unmodified nanofibrous scaffold were used to study the impact of R-peptide modified PCL nanofibers and PCL nanofibers on cell behavior. The results showed early formation of focal adhesion (FA) complex on R-peptide modified scaffolds at 30min after cell seeding. The rate of cell proliferation was significantly increased due to presence of R-peptide, and the MSCs marker analyses using flow cytometry and immunocytochemistry staining proved the ability of R-peptide to maintain mesenchymal stem cell properties (high proliferation, expression of multipotent markers and differentiation capacity) even after long-term passage culturing. Accordingly, our (The) results concluded that bioactive R-peptide in combination with nanofibrous scaffold can mimic the native ECM comprising micro/nano architecture and biochemical molecules in a best way. The designed scaffold can link extracellular matrix (ECM) to nucleus via formation of FA and organization of cytoskeleton, causing fast and strong attachment of MSCs and allowing integrin-mediated signaling to start. Copyright © 2017 Elsevier B.V. All rights reserved.

Human fibroblast-derived extracellular matrix constructs for bone tissue engineering applications.

PubMed

Tour, Gregory; Wendel, Mikael; Tcacencu, Ion

2013-10-01

We exploited the biomimetic approach to generate constructs composed of synthetic biphasic calcium phosphate ceramic and extracellular matrix (SBC-ECM) derived from adult human dermal fibroblasts in complete xeno-free culture conditions. The construct morphology and composition were assessed by scanning electron microscopy, histology, immunohistochemistry, Western blot, glycosaminoglycan, and hydroxyproline assays. Residual DNA quantification, endotoxin testing, and local inflammatory response after implantation in a rat critical-sized calvarial defect were used to access the construct biocompatibility. Moreover, in vitro interaction of human mesenchymal stem cells (hMSCs) with the constructs was studied. The bone marrow- and adipose tissue-derived mesenchymal stem cells were characterized by flow cytometry and tested for osteogenic differentiation capacity prior seeding onto SBC-ECM, followed by alkaline phosphatase, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and real-time quantitative polymerase chain reaction to assess the osteogenic differentiation of hMSCs after seeding onto the constructs at different time intervals. The SBC-ECM constructs enhanced osteogenic differentiation of hMSCs in vitro and exhibited excellent handling properties and high biocompatibility in vivo. Our results highlight the ability to generate in vitro fibroblast-derived ECM constructs in complete xeno-free conditions as a step toward clinical translation, and the potential use of SBC-ECM in craniofacial bone tissue engineering applications. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Pore size and LbL chitosan coating influence mesenchymal stem cell in vitro fibrosis and biomineralization in 3D porous poly(epsilon-caprolactone) scaffolds.

PubMed

Mehr, Nima Ghavidel; Li, Xian; Chen, Gaoping; Favis, Basil D; Hoemann, Caroline D

2015-07-01

Poly(epsilon-caprolactone) (PCL) is a hydrophobic bioplastic under development for bone tissue engineering applications. Limited information is available on the role of internal geometry and cell-surface attachment on osseous integration potential. We tested the hypothesis that human bone marrow mesenchymal stem cells (MSCs) deposit more mineral inside porous 3D PCL scaffolds with fully interconnected 84 or 141 µm pores, when the surfaces are coated with chitosan via Layer-by-Layer (LbL)-deposited polyelectrolytes. Freshly trypsinized MSCs were seeded on PCL 3D cylinders using a novel static cold seeding method in 2% serum to optimally populate all depths of the scaffold discs, followed by 10 days of culture in proliferation medium and 21 additional days in osteogenic medium. MSCs were observed by SEM and histology to spread faster and to proliferate more on chitosan-coated pore surfaces. Most pores, with or without chitosan, became filled by collagen networks sparsely populated with fibroblast-like cells. After 21 days of culture in osteogenic medium, sporadic matrix mineralization was detected histologically and by micro-CT in highly cellular surface layers that enveloped all scaffolds and in cell aggregates in 141 µm pores near the edges. LbL-chitosan promoted punctate mineral deposition on the surfaces of 84 µm pores (p < 0.05 vs. PCL-only) but not the 141 µm pores. This study revealed that LbL-chitosan coatings are sufficient to promote MSC attachment to PCL but only enhance mineral formation in 84 µm pores, suggesting a potential inhibitory role for MSC-derived fibroblasts in osteoblast terminal differentiation. © 2014 Wiley Periodicals, Inc.

Chondrogenic Differentiation of Mesenchymal Stem Cells in Three-Dimensional Chitosan Film Culture

PubMed Central

Lu, Tsai-Jung; Chiu, Fang-Yao; Chiu, Hsiao-Ying; Chang, Ming-Chau; Hung, Shih-Chieh

2017-01-01

Articular cartilage has a very limited capacity for self-repair, and mesenchymal stem cells (MSCs) have the potential to treat cartilage defects and osteoarthritis. However, in-depth mechanistic studies regarding their applications are required. Here we demonstrated the use of chitosan film culture for promoting chondrogenic differentiation of MSCs. We found that MSCs formed spheres 2 days after seeding on dishes coated with chitosan. When MSCs were induced in a chondrogenic induction medium on chitosan films, the size of the spheres continuously increased for up to 21 days. Alcian blue staining and immunohistochemistry demonstrated the expression of chondrogenic proteins, including aggrecan, type II collagen, and type X collagen at 14 and 21 days of differentiation. Importantly, chitosan, with a medium molecular weight (size: 190–310 kDa), was more suitable than other sizes for inducing chondrogenic differentiation of MSCs in terms of sphere size and expression of chondrogenic proteins and endochondral markers. We identified that the mechanistic target of rapamycin (mTOR) signaling and its downstream S6 kinase (S6K)/S6 were activated in chitosan film culture compared to that of monolayer culture. The activation of mTOR/S6K was continuously upregulated from days 2 to 7 of differentiation. Furthermore, we found that mTOR/S6K signaling was required for chondrogenic differentiation of MSCs in chitosan film culture through rapamycin treatment and mTOR knockdown. In conclusion, we showed the suitability of chitosan film culture for promoting chondrogenic differentiation of MSCs and its potential in the development of new strategies in cartilage tissue engineering. PMID:27737727

Is Graphene a Promising Nano-Material for Promoting Surface Modification of Implants or Scaffold Materials in Bone Tissue Engineering?

PubMed Central

Gu, Ming; Liu, Yunsong; Chen, Tong; Du, Feng; Zhao, Xianghui; Xiong, Chunyang

2014-01-01

Bone tissue engineering promises to restore bone defects that are caused by severe trauma, congenital malformations, tumors, and nonunion fractures. How to effectively promote the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) or seed cells has become a hot topic in this field. Many researchers are studying the ways of conferring a pro-osteodifferentiation or osteoinductive capability on implants or scaffold materials, where osteogenesis of seed cells is promoted. Graphene (G) provides a new kind of coating material that may confer the pro-osteodifferentiation capability on implants and scaffold materials by surface modification. Here, we review recent studies on the effects of graphene on surface modifications of implants or scaffold materials. The ability of graphene to improve the mechanical and biological properties of implants or scaffold materials, such as nitinol and carbon nanotubes, and its ability to promote the adhesion, proliferation, and osteogenic differentiation of MSCs or osteoblasts have been demonstrated in several studies. Most previous studies were performed in vitro, but further studies will explore the mechanisms of graphene's effects on bone regeneration, its in vivo biocompatibility, its ability to promote osteodifferentiation, and its potential applications in bone tissue engineering. PMID:24447041

Laminated electrospun nHA/PHB-composite scaffolds mimicking bone extracellular matrix for bone tissue engineering.

PubMed

Chen, Zhuoyue; Song, Yue; Zhang, Jing; Liu, Wei; Cui, Jihong; Li, Hongmin; Chen, Fulin

2017-03-01

Electrospinning is an effective means to generate nano- to micro-scale polymer fibers resembling native extracellular matrix for tissue engineering. However, a major problem of electrospun materials is that limited pore size and porosity may prevent adequate cellular infiltration and tissue ingrowth. In this study, we first prepared thin layers of hydroxyapatite nanoparticle (nHA)/poly-hydroxybutyrate (PHB) via electrospinning. We then laminated the nHA/PHB thin layers to obtain a scaffold for cell seeding and bone tissue engineering. The results demonstrated that the laminated scaffold possessed optimized cell-loading capacity. Bone marrow mesenchymal stem cells (MSCs) exhibited better adherence, proliferation and osteogenic phenotypes on nHA/PHB scaffolds than on PHB scaffolds. Thereafter, we seeded MSCs onto nHA/PHB scaffolds to fabricate bone grafts. Histological observation showed osteoid tissue formation throughout the scaffold, with most of the scaffold absorbed in the specimens 2months after implantation, and blood vessels ingrowth into the graft could be observed in the graft. We concluded that electrospun and laminated nanoscaled biocomposite scaffolds hold great therapeutic potential for bone regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Is graphene a promising nano-material for promoting surface modification of implants or scaffold materials in bone tissue engineering?

PubMed

Gu, Ming; Liu, Yunsong; Chen, Tong; Du, Feng; Zhao, Xianghui; Xiong, Chunyang; Zhou, Yongsheng

2014-10-01

Bone tissue engineering promises to restore bone defects that are caused by severe trauma, congenital malformations, tumors, and nonunion fractures. How to effectively promote the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) or seed cells has become a hot topic in this field. Many researchers are studying the ways of conferring a pro-osteodifferentiation or osteoinductive capability on implants or scaffold materials, where osteogenesis of seed cells is promoted. Graphene (G) provides a new kind of coating material that may confer the pro-osteodifferentiation capability on implants and scaffold materials by surface modification. Here, we review recent studies on the effects of graphene on surface modifications of implants or scaffold materials. The ability of graphene to improve the mechanical and biological properties of implants or scaffold materials, such as nitinol and carbon nanotubes, and its ability to promote the adhesion, proliferation, and osteogenic differentiation of MSCs or osteoblasts have been demonstrated in several studies. Most previous studies were performed in vitro, but further studies will explore the mechanisms of graphene's effects on bone regeneration, its in vivo biocompatibility, its ability to promote osteodifferentiation, and its potential applications in bone tissue engineering.

Spatially patterned matrix elasticity directs stem cell fate

NASA Astrophysics Data System (ADS)

Yang, Chun; DelRio, Frank W.; Ma, Hao; Killaars, Anouk R.; Basta, Lena P.; Kyburz, Kyle A.; Anseth, Kristi S.

2016-08-01

There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.

Reconstruction of cartilage with clonal mesenchymal stem cell-acellular dermal matrix in cartilage defect model in nonhuman primates.

PubMed

Ma, Anlun; Jiang, Li; Song, Lijun; Hu, Yanxin; Dun, Hao; Daloze, Pierre; Yu, Yonglin; Jiang, Jianyuan; Zafarullah, Muhammad; Chen, Huifang

2013-07-01

Articular cartilage defects are commonly associated with trauma, inflammation and osteoarthritis. Mesenchymal stem cell (MSC)-based therapy is a promising novel approach for repairing articular cartilage. Direct intra-articular injection of uncommitted MSCs does not regenerate high-quality cartilage. This study explored utilization of a new three-dimensional, selected chondrogenic clonal MSC-loaded monkey acellular dermal matrix (MSC-ADM) scaffold to repair damaged cartilage in an experimental model of knee joint cartilage defect in Cynomolgus monkeys. MSCs were characterized for cell size, cell yield, phenotypes, proliferation and chondrogenic differentiation capacity. Chondrogenic differentiation assays were performed at different MSC passages by sulfated glycosaminoglycans (sGAG), collagen, and fluorescence activated cell sorter (FACS) analysis. Selected chondrogenic clonal MSCs were seeded onto ADM scaffold with the sandwich model and MSC-loaded ADM grafts were analyzed by confocal microscopy and scanning electron microscopy. Cartilage defects were treated with normal saline, clonal MSCs and clonal MSC-ADM grafts, respectively. The clinical parameters, and histological and immunohistochemical examinations were evaluated at weeks 8, 16, 24 post-treatment, respectively. Polyclonal and clonal MSCs could differentiate into the chondrogenic lineage after stimulation with suitable chondrogenic factors. They expressed mesenchymal markers and were negative for hematopoietic markers. Articular cartilage defects were considerably improved and repaired by selected chondrogenic clonal MSC-based treatment, particularly, in MSC-ADM-treated group. The histological scores in MSC-ADM-treated group were consistently higher than those of other groups. Our results suggest that selected chondrogenic clonal MSC-loaded ADM grafts could improve the cartilage lesions in Cynomolgus monkey model, which may be applicable for repairing similar human cartilage defects. Copyright © 2013 Elsevier B.V. All rights reserved.

Human mesenchymal stem cell behavior on femtosecond laser-textured Ti-6Al-4V surfaces.

PubMed

Cunha, Alexandre; Zouani, Omar Farouk; Plawinski, Laurent; Botelho do Rego, Ana Maria; Almeida, Amélia; Vilar, Rui; Durrieu, Marie-Christine

2015-01-01

The aim of the present work was to investigate ultrafast laser surface texturing as a surface treatment of Ti-6Al-4V alloy dental and orthopedic implants to improve osteoblastic commitment of human mesenchymal stem cells (hMSCs). Surface texturing was carried out by direct writing with an Yb:KYW chirped-pulse regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. The surface topography and chemical composition were investigated by scanning electron microscopy and x-ray photoelectron spectroscopy, respectively. Three types of surface textures with potential interest to improve implant osseointegration can be produced by this method: laser-induced periodic surface structures (LIPSSs); nanopillars (NPs); and microcolumns covered with LIPSSs, forming a bimodal roughness distribution. The potential of the laser treatment in improving hMSC differentiation was assessed by in vitro study of hMSCs spreading, adhesion, elongation and differentiation using epifluorescence microscopy at different times after cell seeding, after specific stainings and immunostainings. Cell area and focal adhesion area were lower on the laser-textured surfaces than on a polished reference surface. Obviously, the laser-textured surfaces have an impact on cell shape. Osteoblastic commitment was observed independently of the surface topography after 2 weeks of cell seeding. When the cells were cultured (after 4 weeks of seeding) in osteogenic medium, LIPSS- and NP- textured surfaces enhanced matrix mineralization and bone-like nodule formation as compared with polished and microcolumn-textured surfaces. The present work shows that surface nanotextures consisting of LIPSSs and NPs can, potentially, improve hMSC differentiation into an osteoblastic lineage.

Stem cell engineered bone with calcium-phosphate coated porous titanium scaffold or silicon hydroxyapatite granules for revision total joint arthroplasty.

PubMed

García-Gareta, Elena; Hua, Jia; Rayan, Faizal; Blunn, Gordon W

2014-06-01

Aseptic loosening in total joint replacements (TJRs) is mainly caused by osteolysis which leads to a reduction of the bone stock necessary for implant fixation in revision TJRs. Our aim was to develop bone tissue-engineered constructs based on scaffolds of clinical relevance in revision TJRs to reconstitute the bone stock at revision operations by using a perfusion bioreactor system (PBRS). The hypothesis was that a PBRS will enhance mesenchymal stem cells (MSCs) proliferation and osteogenic differentiation and will provide an even distribution of MSCs throughout the scaffolds when compared to static cultures. A PBRS was designed and implemented. Scaffolds, silicon substituted hydroxyapatite granules and calcium-phosphate coated porous TiAl6V4 cylinders, were seeded with MSCs and cultured either in static conditions or in the PBRS at 0.75 mL/min. Statistically significant increased cell proliferation and alkaline phosphatase activity was found in samples cultured in the PBRS. Histology revealed a more even cell distribution in the perfused constructs. SEM showed that cells arranged in sheets. Long cytoplasmic processes attached the cells to the scaffolds. We conclude that a novel tissue engineering approach to address the issue of poor bone stock at revision operations is feasible by using a PBRS.

Crosstalk between immune cells and mesenchymal stromal cells in a 3D bioreactor system.

PubMed

Seifert, Martina; Lubitz, Annika; Trommer, Jeanne; Könnig, Darja; Korus, Gabriela; Marx, Uwe; Volk, Hans-Dieter; Duda, Georg; Kasper, Grit; Lehmann, Kerstin; Stolk, Meaghan; Giese, Christoph

2012-11-01

Mesenchymal stromal cells (MSC), known for their high immune modulatory capacity are promising tools for several cell-based therapies. To better mimic the in vivo situation of MSC interactions with immune cells, we applied an artificial lymph node (ALN)-bioreactor culture system combining a miniaturized perfusion bioreactor with a 3D matrix-based cell culture of immune competent cells forming micro-organoids. Rat lymph node cells and allogeneic bone marrow-derived MSCs were seeded in a 20:1 ratio within the agarose matrix of the ALN-reactor. Lymphocytes were pre-incubated with Concanavalin A (ConA) and then co-cultured with MSC in the matrix with additional ConA in the perfusing medium. Live/dead staining showed survival of the co-cultures during the 8-day ALN-reactor run. Paraffin sections of bioreactor matrices were analyzed by proliferating cell nuclear antigen (PCNA)-specific stai-ning to determine MSC proliferation. Immune modulatory capacity was defined by daily analysis of cytokine secretion profiles (TNFa, IFNy, IL-1a, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12p40/p70, GM-CSF). Cytokine peak secretion at day 2 was significantly inhibited by MSCs for TNFa (96.8 ± 4.8%) and IFNy (88.7 ± 12.0%) in 3D co-cultures. In contrast, other cytokines (IL-1, IL-6, IL-12) were induced. Furthermore, we detected a significantly higher (58.8%) fraction of proliferating MSCs in the presence of immune cells compared to control bioreactors loaded with MSCs only. In the future, this system might be an excellent tool to investigate the mechanisms of MSC-mediated immune modulation during simulated in vivo conditions.

Homologous structure-function relationships between native fibrocartilage and tissue engineered from MSC-seeded nanofibrous scaffolds.

PubMed

Nerurkar, Nandan L; Han, Woojin; Mauck, Robert L; Elliott, Dawn M

2011-01-01

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of tissues to replace diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials. Copyright © 2010 Elsevier Ltd. All rights reserved.

HOMOLOGOUS STRUCTURE-FUNCTION RELATIONSHIPS BETWEEN NATIVE FIBROCARTILAGE AND TISSUE ENGINEERED FROM MSC-SEEDED NANOFIBROUS SCAFFOLDS

PubMed Central

Nerurkar, Nandan L.; Han, Woojin; Mauck, Robert L.; Elliott, Dawn M.

2010-01-01

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of replacement tissues for diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials. PMID:20880577

Effect of doping in carbon nanotubes on the viability of biomimetic chitosan-carbon nanotubes-hydroxyapatite scaffolds.

PubMed

Fonseca-García, Abril; Mota-Morales, Josué D; Quintero-Ortega, Iraís A; García-Carvajal, Zaira Y; Martínez-López, V; Ruvalcaba, Erika; Landa-Solís, Carlos; Solis, Lilia; Ibarra, Clemente; Gutiérrez, María C; Terrones, Mauricio; Sanchez, Isaac C; del Monte, Francisco; Velasquillo, María C; Luna-Bárcenas, G

2014-10-01

This work describes the preparation and characterization of biomimetic chitosan/multiwall carbon nanotubes/nano-hydroxyapatite (CTS/MWCNT/nHAp) scaffolds and their viability for bone tissue engineering applications. The cryogenic process ice segregation-induced self-assembly (ISISA) was used to fabricate 3D biomimetic CTS scaffolds. Proper combination of cryogenics, freeze-drying, nature and molecular ratio of solutes give rise to 3D porous interconnected scaffolds with clusters of nHAp distributed along the scaffold surface. The effect of doping in CNT (e.g. with oxygen and nitrogen atoms) on cell viability was tested. Under the same processing conditions, pore size was in the range of 20-150 μm and irrespective on the type of CNT. Studies on cell viability with scaffolds were carried out using human cells from periosteum biopsy. Prior to cell seeding, the immunophenotype of mesenchymal periosteum or periosteum-derived stem cells (MSCs-PCs) was characterized by flow cytometric analysis using fluorescence-activated and characteristic cell surface markers for MSCs-PCs. The characterized MSCs-PCs maintained their periosteal potential in cell cultures until the 2nd passage from primary cell culture. Thus, the biomimetic CTS/MWCNT/nHAp scaffolds demonstrated good biocompatibility and cell viability in all cases such that it can be considered as promising biomaterials for bone tissue engineering. © 2013 Wiley Periodicals, Inc.

Application of a novel sorting system for equine mesenchymal stem cells (MSCs)

PubMed Central

Radtke, Catherine L.; Nino-Fong, Rodolfo; Esparza Gonzalez, Blanca P.; McDuffee, Laurie A.

2014-01-01

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 × 103 MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of < 0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and veterinary medicine. PMID:25355998

PVA-chitosan composite hydrogel versus alginate beads as a potential mesenchymal stem cell carrier for the treatment of focal cartilage defects.

PubMed

Dashtdar, Havva; Murali, Malliga Raman; Abbas, Azlina Amir; Suhaeb, Abdulrazzaq Mahmod; Selvaratnam, Lakshmi; Tay, Liang Xin; Kamarul, Tunku

2015-05-01

To investigate whether mesenchymal stem cells (MSCs) seeded in novel polyvinyl alcohol (PVA)-chitosan composite hydrogel can provide comparable or even further improve cartilage repair outcomes as compared to previously established alginate-transplanted models. Medial femoral condyle defect was created in both knees of twenty-four mature New Zealand white rabbits, and the animals were divided into four groups containing six animals each. After 3 weeks, the right knees were transplanted with PVA-chitosan-MSC, PVA-chitosan scaffold alone, alginate-MSC construct or alginate alone. The left knee was kept as untreated control. Animals were killed at the end of 6 months after transplantation, and the cartilage repair was assessed through Brittberg morphological score, histological grading by O'Driscoll score and quantitative glycosaminoglycan analysis. Morphological and histological analyses showed significant (p < 0.05) tissue repair when treated with PVA-chitosan-MSC or alginate MSC as compared to the scaffold only and untreated control. In addition, safranin O staining and the glycosaminoglycan (GAG) content were significantly higher (p < 0.05) in MSC treatment groups than in scaffold-only or untreated control group. No significant difference was observed between the PVA-chitosan-MSC- and alginate-MSC-treated groups. PVA-chitosan hydrogel seeded with mesenchymal stem cells provides comparable treatment outcomes to that of previously established alginate-MSC construct implantation. This study supports the potential use of PVA-chitosan hydrogel seeded with MSCs for clinical use in cartilage repair such as traumatic injuries.

Clinical efficacy of stem cell mediated osteogenesis and bioceramics for bone tissue engineering.

PubMed

Neman, Josh; Hambrecht, Amanda; Cadry, Cherie; Goodarzi, Amir; Youssefzadeh, Jonathan; Chen, Mike Y; Jandial, Rahul

2012-01-01

Lower back pain is a common disorder that often requires bony spinal fusion for long-term relief. Current arthrodesis procedures use bone grafts from autogenous bone, allogenic backed bone or synthetic materials. Autogenous bone grafts can result in donor site morbidity and pain at the donor site, while allogenic backed bone and synthetic materials have variable effectiveness. Given these limitations, researchers have focused on new treatments that will allow for safe and successful bone repair and regeneration. Mesenchymal stem cells (MSCs) have received attention for their ability to differentiate into osteoblasts, cells that synthesize the extracellular matrix and regulate matrix mineralization. Successful bone regeneration requires three elements: MSCs that serve as osteoblastic progenitors, osteoinductive growth factors and their pathways that promote development and differentiation of the cells as well as an osteoconductive scaffold that allows for the formation of a vascular network. Future treatments should strive to combine mesenchymal stem cells, cell-seeded scaffolds and gene therapy to optimize the efficiency and safety of tissue repair and bone regeneration.

Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor.

PubMed

Tsai, Ang-Chen; Ma, Teng

2016-01-01

Human mesenchymal stem cells (hMSCs) are considered as a primary candidate in cell therapy owing to their self-renewability, high differentiation capabilities, and secretions of trophic factors. In clinical application, a large quantity of therapeutically competent hMSCs is required that cannot be produced in conventional petri dish culture. Bioreactors are scalable and have the capacity to meet the production demand. Microcarrier suspension culture in stirred-tank bioreactors is the most widely used method to expand anchorage dependent cells in a large scale. Stirred-tank bioreactors have the potential to scale up and microcarriers provide the high surface-volume ratio. As a result, a spinner flask bioreactor with microcarriers has been commonly used in large scale expansion of adherent cells. This chapter describes a detailed culture protocol for hMSC expansion in a 125 mL spinner flask using microcarriers, Cytodex I, and a procedure for cell seeding, expansion, metabolic sampling, and quantification and visualization using microculture tetrazolium (MTT) reagent.

In Vitro Studies on the Degradability, Bioactivity, and Cell Differentiation of PRP/AZ31B Mg Alloys Composite Scaffold.

PubMed

Zou, Jian; Shi, Zhongmin; Xu, Hongwei; Li, Xiaolin

2017-01-01

In recent years, more and more methods have been developed to improve the bioactivity of the biodegradable materials in bone tissue regeneration. In present study, we used rat mesenchymal stem cells (rMSCs) to evaluate the outcomes of Mg alloys (AZ31B, Magnesium, and Aluminum) and Platelet-rich plasma (PRP)/Mg alloys on rMSCs biocompatibility and osteogenic differentiation. Water absorption experiments indicated that both bare AZ31B and PRP/AZ31B were capable of absorbing large amounts of water. But the water absorption ratio for PRP/AZ31B was significantly higher than that for bare AZ31B. The degradability experiments implied that both samples degraded at same speed. rMSCs on the surface of AZ31B distributed more and better than those on the AZ31B scaffold. In ALP activity experiment, the activity of rMSCs on the PRP/AZ31B was markedly higher than that on the AZ31B scaffolds on the 7th day and 14th day. qRT-PCR also showed that OPN and OCN were expressed in both samples. OPN and OCN expression in PRP/AZ31B sample were higher than those in bare AZ31B samples. In summary, the in vitro study implied that AZ31B combined with PRP could remarkably improve cell seeding, attachment, proliferation, and differentiation.

Cartilage matrix formation by bovine mesenchymal stem cells in three-dimensional culture is age-dependent.

PubMed

Erickson, Isaac E; van Veen, Steven C; Sengupta, Swarnali; Kestle, Sydney R; Mauck, Robert L

2011-10-01

Cartilage degeneration is common in the aged, and aged chondrocytes are inferior to juvenile chondrocytes in producing cartilage-specific extracellular matrix. Mesenchymal stem cells (MSCs) are an alternative cell type that can differentiate toward the chondrocyte phenotype. Aging may influence MSC chondrogenesis but remains less well studied, particularly in the bovine system. The objectives of this study were (1) to confirm age-related changes in bovine articular cartilage, establish how age affects chondrogenesis in cultured pellets for (2) chondrocytes and (3) MSCs, and (4) determine age-related changes in the biochemical and biomechanical development of clinically relevant MSC-seeded hydrogels. Native bovine articular cartilage from fetal (n = 3 donors), juvenile (n = 3 donors), and adult (n = 3 donors) animals was analyzed for mechanical and biochemical properties (n = 3-5 per donor). Chondrocyte and MSC pellets (n = 3 donors per age) were cultured for 6 weeks before analysis of biochemical content (n = 3 per donor). Bone marrow-derived MSCs of each age were also cultured within hyaluronic acid hydrogels for 3 weeks and analyzed for matrix deposition and mechanical properties (n = 4 per age). Articular cartilage mechanical properties and collagen content increased with age. We observed robust matrix accumulation in three-dimensional pellet culture by fetal chondrocytes with diminished collagen-forming capacity in adult chondrocytes. Chondrogenic induction of MSCs was greater in fetal and juvenile cell pellets. Likewise, fetal and juvenile MSCs in hydrogels imparted greater matrix and mechanical properties. Donor age and biochemical microenvironment were major determinants of both bovine chondrocyte and MSC functional capacity. In vitro model systems should be evaluated in the context of age-related changes and should be benchmarked against human MSC data.

Safety Evaluation of a Bioglass–Polylactic Acid Composite Scaffold Seeded with Progenitor Cells in a Rat Skull Critical-Size Bone Defect

PubMed Central

El-Kady, Abeer M.; Arbid, Mahmoud S.; Abd El-Hady, Bothaina M.; Marzi, Ingo; Seebach, Caroline

2014-01-01

Treating large bone defects represents a major challenge in traumatic and orthopedic surgery. Bone tissue engineering provides a promising therapeutic option to improve the local bone healing response. In the present study tissue biocompatibility, systemic toxicity and tumorigenicity of a newly developed composite material consisting of polylactic acid (PLA) and 20% or 40% bioglass (BG20 and BG40), respectively, were analyzed. These materials were seeded with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) and tested in a rat calvarial critical size defect model for 3 months and compared to a scaffold consisting only of PLA. Serum was analyzed for organ damage markers such as GOT and creatinine. Leukocyte count, temperature and free radical indicators were measured to determine the degree of systemic inflammation. Possible tumor occurrence was assessed macroscopically and histologically in slides of liver, kidney and spleen. Furthermore, the concentrations of serum malondialdehyde (MDA) and sodium oxide dismutase (SOD) were assessed as indicators of tumor progression. Qualitative tissue response towards the implants and new bone mass formation was histologically investigated. BG20 and BG40, with or without progenitor cells, did not cause organ damage, long-term systemic inflammatory reactions or tumor formation. BG20 and BG40 supported bone formation, which was further enhanced in the presence of EPCs and MSCs. This investigation reflects good biocompatibility of the biomaterials BG20 and BG40 and provides evidence that additionally seeding EPCs and MSCs onto the scaffold does not induce tumor formation. PMID:24498345

Design of experiments approach to engineer cell-secreted matrices for directing osteogenic differentiation.

PubMed

Decaris, Martin L; Leach, J Kent

2011-04-01

The presentation of extracellular matrix (ECM) proteins provides an opportunity to instruct the phenotype and behavior of responsive cells. Decellularized cell-secreted matrix coatings (DM) represent a biomimetic culture surface that retains the complexity of the natural ECM. Microenvironmental culture conditions alter the composition of these matrices and ultimately the ability of DMs to direct cell fate. We employed a design of experiments (DOE) multivariable analysis approach to determine the effects and interactions of four variables (culture duration, cell seeding density, oxygen tension, and media supplementation) on the capacity of DMs to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DOE analysis revealed that matrices created with extended culture duration, ascorbate-2-phosphate supplementation, and in ambient oxygen tension exhibited significant correlations with enhanced hMSC differentiation. We validated the DOE model results using DMs predicted to have superior (DM1) or lesser (DM2) osteogenic potential for naïve hMSCs. Compared to cells on DM2, hMSCs cultured on DM1 expressed 2-fold higher osterix levels and deposited 3-fold more calcium over 3 weeks. Cells on DM1 coatings also exhibited greater proliferation and viability compared to DM2-coated substrates. This study demonstrates that DOE-based analysis is a powerful tool for optimizing engineered systems by identifying significant variables that have the greatest contribution to the target output.

Attachment and spatial organisation of human mesenchymal stem cells on poly(ethylene glycol) hydrogels.

PubMed

Chahal, Aman S; Schweikle, Manuel; Heyward, Catherine A; Tiainen, Hanna

2018-08-01

Strategies that enable hydrogel substrates to support cell attachment typically incorporate either entire extracellular matrix proteins or synthetic peptide fragments such as the RGD (arginine-glycine-aspartic acid) motif. Previous studies have carefully analysed how material characteristics can affect single cell morphologies. However, the influence of substrate stiffness and ligand presentation on the spatial organisation of human mesenchymal stem cells (hMSCs) have not yet been examined. In this study, we assessed how hMSCs organise themselves on soft (E = 7.4-11.2 kPa) and stiff (E = 27.3-36.8 kPa) poly(ethylene glycol) (PEG) hydrogels with varying concentrations of RGD (0.05-2.5 mM). Our results indicate that hMSCs seeded on soft hydrogels clustered with reduced cell attachment and spreading area, irrespective of RGD concentration and isoform. On stiff hydrogels, in contrast, cells spread with high spatial coverage for RGD concentrations of 0.5 mM or higher. In conclusion, we identified that an interplay of hydrogel stiffness and the availability of cell attachment motifs are important factors in regulating hMSC organisation on PEG hydrogels. Understanding how cells initially interact and colonise the surface of this material is a fundamental prerequisite for the design of controlled platforms for tissue engineering and mechanobiology studies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Braided and Stacked Electrospun Nanofibrous Scaffolds for Tendon and Ligament Tissue Engineering

PubMed Central

Rothrauff, Benjamin B.; Lauro, Brian B.; Yang, Guang; Debski, Richard E.; Musahl, Volker

2017-01-01

Tendon and ligament injuries are a persistent orthopedic challenge given their poor innate healing capacity. Nonwoven electrospun nanofibrous scaffolds composed of polyesters have been used to mimic the mechanics and topographical cues of native tendons and ligaments. However, nonwoven nanofibers have several limitations that prevent broader clinical application, including poor cell infiltration, as well as tensile and suture-retention strengths that are inferior to native tissues. In this study, multilayered scaffolds of aligned electrospun nanofibers of two designs–stacked or braided–were fabricated. Mechanical properties, including structural and mechanical properties and suture-retention strength, were determined using acellular scaffolds. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on scaffolds for up to 28 days, and assays for tenogenic differentiation, histology, and biochemical composition were performed. Braided scaffolds exhibited improved tensile and suture-retention strengths, but reduced moduli. Both scaffold designs supported expression of tenogenic markers, although the effect was greater on braided scaffolds. Conversely, cell infiltration was superior in stacked constructs, resulting in enhanced cell number, total collagen content, and total sulfated glycosaminoglycan content. However, when normalized against cell number, both designs modulated extracellular matrix protein deposition to a similar degree. Taken together, this study demonstrates that multilayered scaffolds of aligned electrospun nanofibers supported tenogenic differentiation of seeded MSCs, but the macroarchitecture is an important consideration for applications of tendon and ligament tissue engineering. PMID:28071988

Braided and Stacked Electrospun Nanofibrous Scaffolds for Tendon and Ligament Tissue Engineering.

PubMed

Rothrauff, Benjamin B; Lauro, Brian B; Yang, Guang; Debski, Richard E; Musahl, Volker; Tuan, Rocky S

2017-05-01

Tendon and ligament injuries are a persistent orthopedic challenge given their poor innate healing capacity. Nonwoven electrospun nanofibrous scaffolds composed of polyesters have been used to mimic the mechanics and topographical cues of native tendons and ligaments. However, nonwoven nanofibers have several limitations that prevent broader clinical application, including poor cell infiltration, as well as tensile and suture-retention strengths that are inferior to native tissues. In this study, multilayered scaffolds of aligned electrospun nanofibers of two designs-stacked or braided-were fabricated. Mechanical properties, including structural and mechanical properties and suture-retention strength, were determined using acellular scaffolds. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on scaffolds for up to 28 days, and assays for tenogenic differentiation, histology, and biochemical composition were performed. Braided scaffolds exhibited improved tensile and suture-retention strengths, but reduced moduli. Both scaffold designs supported expression of tenogenic markers, although the effect was greater on braided scaffolds. Conversely, cell infiltration was superior in stacked constructs, resulting in enhanced cell number, total collagen content, and total sulfated glycosaminoglycan content. However, when normalized against cell number, both designs modulated extracellular matrix protein deposition to a similar degree. Taken together, this study demonstrates that multilayered scaffolds of aligned electrospun nanofibers supported tenogenic differentiation of seeded MSCs, but the macroarchitecture is an important consideration for applications of tendon and ligament tissue engineering.

Engineering cartilaginous grafts using chondrocyte-laden hydrogels supported by a superficial layer of stem cells.

PubMed

Mesallati, Tariq; Buckley, Conor T; Kelly, Daniel J

2017-05-01

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co-cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self-assembled on top of CC-laden agarose gels (structured co-culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co-culture). Porcine infrapatellar fat pad-derived stem cells (FPSCs) and bone marrow-derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co-culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co-culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co-culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co-culture for both MSC sources. In conclusion, assembling MSCs onto CC-laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

The role of calcium signalling in the chondrogenic response of mesenchymal stem cells to hydrostatic pressure.

PubMed

Steward, A J; Kelly, D J; Wagner, D R

2014-10-28

The object of this study was to elucidate the role of Ca++ signalling in the chondrogenic response of mesenchymal stem cells (MSCs) to hydrostatic pressure (HP). MSCs were seeded into agarose hydrogels, subjected to HP or kept in free swelling conditions, and were cultured either with or without pharmacological inhibitors of Ca++ mobility and downstream targets. Chelating free Ca++, inhibiting voltage-gated calcium channels, and depleting intracellular calcium storessuppressed the beneficial effect of HP on chondrogenesis, indicating that Ca++ mobility may play an important role in the mechanotransduction of HP. However, inhibition of stretch-activated calcium channels in the current experiment yielded similar results to the control group, suggesting that mechanotransduction of HP is distinct from loads that generate cell deformations. Inhibition of the downstream targets calmodulin, calmodulin kinase II, and calcineurin all knocked down the effect of HP on chondrogenesis, implicating these targets in MSCs response to HP. All of the pharmacological inhibitors that abolished the chondrogenic response to HP also maintained a punctate vimentin organisation in the presence of HP, as opposed to the mechanoresponsive groups where the vimentin structure became more diffuse. These results suggest that Ca++ signalling may transduce HP via vimentin adaptation to loading.

Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc.

PubMed

Bhunia, Bibhas K; Kaplan, David L; Mandal, Biman B

2018-01-16

Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col I α 1 , sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.

In vitro cartilage construct generation from silk fibroin- chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition.

PubMed

Agrawal, Parinita; Pramanik, Krishna; Biswas, Amit; Ku Patra, Ranjan

2018-02-01

Cartilage construct generation includes a scaffold with appropriate composition to mimic matrix of the damaged tissue on which the stem cells grow and differentiate. In this study, umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) were seeded on freeze dried porous silk-fibroin (SF)/chitosan (CS) scaffolds. Influence of static and dynamic (spinner flask bioreactor) culture conditions on the developing cartilage construct were studied by in-vitro characterization for viability, proliferation, distribution, and chondrogenic differentiation of hMSCs over the scaffold. Constructs developed in spinner flask consisted of 62% live cells, and exhibited 543% more cell density at the core than constructs cultured in static system. Quantification of DNA and glycosaminoglycans accumulation after 21 days showed the progression of chondrogenic differentiation of hMSCs was higher in dynamic culture compared to static one. In constructs generated under dynamic condition, histology staining for proteoglycan matrix, and fluorescence staining for collagen-II and aggrecan showed positive correlation between early and late stage chondrogenic markers, which was further confirmed by quantitative PCR analysis, showing low collagen-I expression and highly expressed Sox9, collagen-II and aggrecan. The present study demonstrated that construct generated by combining 3D SF/CS scaffold with UCB-hMSCs under dynamic condition using spinner flask bioreactor can be used for cartilage tissue regeneration for future medical treatments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 397-407, 2018. © 2017 Wiley Periodicals, Inc.

Photostimulation of osteogenic differentiation on silk scaffolds by plasma arc light source.

PubMed

Çakmak, Anıl Sera; Çakmak, Soner; Vatansever, H Seda; Gümüşderelioğlu, Menemşe

2018-05-01

Low-level laser therapy (LLLT) has been used for more than 30 years to heal wounds. In recent years, LLLT or photostimulation has been indicated as an effective tool for regenerative and dental medicine by using monochromatic light. The aim of this study is to indicate the usability of plasma arc light source for bone regeneration. This is why we used polychromatic light source providing effective wavelengths in the range of 590-1500 nm for cellular response and investigated photostimulation effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D silk scaffolds. Cellular responses were examined by using cell culture methods in terms of proliferation, differentiation, and morphological analyses. The results showed that photostimulation with a polychromatic light source (applied for 5 min from the 3rd day after seeding up to the 28th day in 2-day intervals with 92-mW/cm 2 power from 10-cm distance to the cells) enhanced osteogenic differentiation of hMSCs according to higher alkaline phosphatase (ALP) activity, collagen and calcium content, osteogenic gene expressions, and matrix mineralization. In conclusion, we suggest that the plasma arc light source that was used here has a great potential for bone regeneration.

Tissue-engineered composite scaffold of poly(lactide-co-glycolide) and hydroxyapatite nanoparticles seeded with autologous mesenchymal stem cells for bone regeneration*

PubMed Central

Zhang, Bing; Zhang, Pei-biao; Wang, Zong-liang; Lyu, Zhong-wen; Wu, Han

2017-01-01

Objective: A new therapeutic strategy using nanocomposite scaffolds of grafted hydroxyapatite (g-HA)/poly(lactide-co-glycolide) (PLGA) carried with autologous mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) was assessed for the therapy of critical bone defects. At the same time, tissue response and in vivo mineralization of tissue-engineered implants were investigated. Methods: A composite scaffold of PLGA and g-HA was fabricated by the solvent casting and particulate-leaching method. The tissue-engineered implants were prepared by seeding the scaffolds with autologous bone marrow MSCs in vitro. Then, mineralization and osteogenesis were observed by intramuscular implantation, as well as the repair of the critical radius defects in rabbits. Results: After eight weeks post-surgery, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) revealed that g-HA/PLGA had a better interface of tissue response and higher mineralization than PLGA. Apatite particles were formed and varied both in macropores and micropores of g-HA/PLGA. Computer radiographs and histological analysis revealed that there were more and more quickly formed new bone formations and better fusion in the bone defect areas of g-HA/PLGA at 2–8 weeks post-surgery. Typical bone synostosis between the implant and bone tissue was found in g-HA/PLGA, while only fibrous tissues formed in PLGA. Conclusions: The incorporation of g-HA mainly improved mineralization and bone formation compared with PLGA. The application of MSCs can enhance bone formation and mineralization in PLGA scaffolds compared with cell-free scaffolds. Furthermore, it can accelerate the absorption of scaffolds compared with composite scaffolds. PMID:29119734

Tissue-engineered composite scaffold of poly(lactide-co-glycolide) and hydroxyapatite nanoparticles seeded with autologous mesenchymal stem cells for bone regeneration.

PubMed

Zhang, Bing; Zhang, Pei-Biao; Wang, Zong-Liang; Lyu, Zhong-Wen; Wu, Han

A new therapeutic strategy using nanocomposite scaffolds of grafted hydroxyapatite (g-HA)/ poly(lactide-co-glycolide) (PLGA) carried with autologous mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) was assessed for the therapy of critical bone defects. At the same time, tissue response and in vivo mineralization of tissue-engineered implants were investigated. A composite scaffold of PLGA and g-HA was fabricated by the solvent casting and particulate-leaching method. The tissue-engineered implants were prepared by seeding the scaffolds with autologous bone marrow MSCs in vitro. Then, mineralization and osteogenesis were observed by intramuscular implantation, as well as the repair of the critical radius defects in rabbits. After eight weeks post-surgery, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) revealed that g-HA/PLGA had a better interface of tissue response and higher mineralization than PLGA. Apatite particles were formed and varied both in macropores and micropores of g-HA/PLGA. Computer radiographs and histological analysis revealed that there were more and more quickly formed new bone formations and better fusion in the bone defect areas of g-HA/PLGA at 2-8 weeks post-surgery. Typical bone synostosis between the implant and bone tissue was found in g-HA/PLGA, while only fibrous tissues formed in PLGA. The incorporation of g-HA mainly improved mineralization and bone formation compared with PLGA. The application of MSCs can enhance bone formation and mineralization in PLGA scaffolds compared with cell-free scaffolds. Furthermore, it can accelerate the absorption of scaffolds compared with composite scaffolds.

Three-Dimensional Bio-Printed Scaffold Sleeves With Mesenchymal Stem Cells for Enhancement of Tendon-to-Bone Healing in Anterior Cruciate Ligament Reconstruction Using Soft-Tissue Tendon Graft.

PubMed

Park, Sin Hyung; Choi, Yeong-Jin; Moon, Sang Won; Lee, Byung Hoon; Shim, Jin-Hyung; Cho, Dong-Woo; Wang, Joon Ho

2018-01-01

To investigate the efficacy of the insertion of 3-dimensional (3D) bio-printed scaffold sleeves seeded with mesenchymal stem cells (MSCs) to enhance osteointegration between the tendon and tunnel bone in anterior cruciate ligament (ACL) reconstruction in a rabbit model. Scaffold sleeves were fabricated by 3D bio-printing. Before ACL reconstruction, MSCs were seeded into the scaffold sleeves. ACL reconstruction with hamstring tendon was performed on both legs of 15 adult rabbits (aged 12 weeks). We implanted 15 bone tunnels with scaffold sleeves with MSCs and implanted another 15 bone tunnels with scaffold sleeves without MSCs before passing the graft. The specimens were harvested at 4, 8, and 12 weeks. H&E staining, immunohistochemical staining of type II collagen, and micro-computed tomography of the tunnel cross-sectional area were evaluated. Histologic assessment was conducted with a histologic scoring system. In the histologic assessment, a smooth bone-to-tendon transition through broad fibrocartilage formation was identified in the treatment group, and the interface zone showed abundant type II collagen production on immunohistochemical staining. Bone-tendon healing histologic scores were significantly higher in the treatment group than in the control group at all time points. Micro-computed tomography at 12 weeks showed smaller tibial (control, 9.4 ± 0.9 mm 2 ; treatment, 5.8 ± 2.9 mm 2 ; P = .044) and femoral (control, 9.6 ± 2.9 mm 2 ; treatment, 6.0 ± 1.0 mm 2 ; P = .03) bone-tunnel areas in the treated group than in the control group. The 3D bio-printed scaffold sleeve with MSCs exhibited excellent results in osteointegration enhancement between the tendon and tunnel bone in ACL reconstruction in a rabbit model. If secure biological healing between the tendon graft and tunnel bone can be induced in the early postoperative period, earlier, more successful rehabilitation may be facilitated. Three-dimensional bio-printed scaffold sleeves with MSCs have the potential to accelerate bone-tendon healing in ACL reconstruction. Copyright © 2017 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Repair of Torn Avascular Meniscal Cartilage Using Undifferentiated Autologous Mesenchymal Stem Cells: From In Vitro Optimization to a First-in-Human Study.

PubMed

Whitehouse, Michael R; Howells, Nicholas R; Parry, Michael C; Austin, Eric; Kafienah, Wael; Brady, Kyla; Goodship, Allen E; Eldridge, Jonathan D; Blom, Ashley W; Hollander, Anthony P

2017-04-01

Meniscal cartilage tears are common and predispose to osteoarthritis (OA). Most occur in the avascular portion of the meniscus where current repair techniques usually fail. We described previously the use of undifferentiated autologous mesenchymal stem cells (MSCs) seeded onto a collagen scaffold (MSC/collagen-scaffold) to integrate meniscal tissues in vitro. Our objective was to translate this method into a cell therapy for patients with torn meniscus, with the long-term goal of delaying or preventing the onset of OA. After in vitro optimization, we tested an ovine-MSC/collagen-scaffold in a sheep meniscal cartilage tear model with promising results after 13 weeks, although repair was not sustained over 6 months. We then conducted a single center, prospective, open-label first-in-human safety study of patients with an avascular meniscal tear. Autologous MSCs were isolated from an iliac crest bone marrow biopsy, expanded and seeded into the collagen scaffold. The resulting human-MSC/collagen-scaffold implant was placed into the meniscal tear prior to repair with vertical mattress sutures and the patients were followed for 2 years. Five patients were treated and there was significant clinical improvement on repeated measures analysis. Three were asymptomatic at 24 months with no magnetic resonance imaging evidence of recurrent tear and clinical improvement in knee function scores. Two required subsequent meniscectomy due to retear or nonhealing of the meniscal tear at approximately 15 months after implantation. No other adverse events occurred. We conclude that undifferentiated MSCs could provide a safe way to augment avascular meniscal repair in some patients. Registration: EU Clinical Trials Register, 2010-024162-22. Stem Cells Translational Medicine 2017;6:1237-1248. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling.

PubMed

Marsano, Anna; Medeiros da Cunha, Carolina M; Ghanaati, Shahram; Gueven, Sinan; Centola, Matteo; Tsaryk, Roman; Barbeck, Mike; Stuedle, Chiara; Barbero, Andrea; Helmrich, Uta; Schaeren, Stefan; Kirkpatrick, James C; Banfi, Andrea; Martin, Ivan

2016-12-01

: Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-β pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues. Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype. ©AlphaMed Press.

Design and efficacy of a single-use bioreactor for heart valve tissue engineering.

PubMed

Converse, Gabriel L; Buse, Eric E; Neill, Kari R; McFall, Christopher R; Lewis, Holley N; VeDepo, Mitchell C; Quinn, Rachael W; Hopkins, Richard A

2017-02-01

Heart valve tissue engineering offers the promise of improved treatments for congenital heart disorders; however, widespread clinical availability of a tissue engineered heart valve (TEHV) has been hindered by scientific and regulatory concerns, including the lack of a disposable, bioreactor system for nondestructive valve seeding and mechanical conditioning. Here we report the design for manufacture and the production of full scale, functional prototypes of such a system. To evaluate the efficacy of this bioreactor as a tool for seeding, ovine aortic valves were decellularized and subjected to seeding with human mesenchymal stem cells (hMSC). The effects of pulsatile conditioning using cyclic waveforms tuned to various negative and positive chamber pressures were evaluated, with respect to the seeding of cells on the decellularized leaflet and the infiltration of seeded cells into the interstitium of the leaflet. Infiltration of hMSCs into the aortic valve leaflet was observed following 72 h of conditioning under negative chamber pressure. Additional conditioning under positive pressure improved cellular infiltration, while retaining gene expression within the MSC-valve interstitial cell phenotype lineage. This protocol resulted in a subsurface pilot population of cells, not full tissue recellularization. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 249-259, 2017. © 2015 Wiley Periodicals, Inc.

Functional cartilage repair capacity of de-differentiated, chondrocyte- and mesenchymal stem cell-laden hydrogels in vitro

PubMed Central

Rackwitz, Lars; Djouad, Farida; Janjanin, Sasa; Nöth, Ulrich; Tuan, Rocky S.

2017-01-01

Objective The long-term performance of cell seeded matrix based cartilage constructs depends on (1) the development of sufficient biomechanical properties, and (2) lateral integration with host tissues, both of which require cartilage specific matrix deposition within the scaffold. In this study, we have examined the potential of tissue-engineered cartilage analogs developed using different cell types, i.e., MSCs versus chondrocytes and de-differentiated chondrocytes, in an established “construct in cartilage ring” model. Design Cell-laden constructs of differentiated chondrocytes, de-differentiated chondrocytes after 2, 5 or 8 population doublings, and MSCs were either implanted into a native cartilage ring immediately after fabrication (immature group) or pretreated for 21 days in a transforming growth factor-β3 (TGF-β3) containing medium prior to implantation. After additional culture for 28 days in a serum-free, chemically defined medium, the extent of lateral integration, and biochemical and biomechanical characteristics of the implants as hybrid constructs were assessed. Results The quality of integration, the amount of accumulated cartilage-specific matrix components and associated biomechanical properties were found to be highest when using differentiated chondrocytes. De-differentiation of chondrocytes negatively impacted the properties of the implants, as even two population doublings of the chondrocytes in culture significantly lowered cartilage repair capacity. In contrast, MSCs showed chondrogenic differentiation with TGF-β3 pre-treatment and superior integrational behavior. Conclusions Chondrocyte expansion and de-differentiation impaired the cell response, resulting in inferior cartilage repair in vitro. With TGF-β3 pre-treatment, MSCs were able to undergo sustained chondrogenic differentiation and exhibited superior matrix deposition and integration compared to de-differentiated chondrocytes. PMID:24887551

Comparison of human mesenchymal stromal cells from four neonatal tissues: Amniotic membrane, chorionic membrane, placental decidua and umbilical cord.

PubMed

Araújo, Anelise Bergmann; Salton, Gabrielle Dias; Furlan, Juliana Monteiro; Schneider, Natália; Angeli, Melissa Helena; Laureano, Álvaro Macedo; Silla, Lúcia; Passos, Eduardo Pandolfi; Paz, Ana Helena

2017-05-01

Mesenchymal stromal cells (MSCs) are being investigated as a potential alternative for cellular therapy. This study was designed to compare the biological characteristics of MSCs isolated from amniotic membrane (A-MSCs), chorionic membrane (C-MSCs), placental decidua (D-MSCs) and umbilical cord (UC-MSCs) to ascertain whether any one of these sources is superior to the others for cellular therapy purposes. MSCs were isolated from amniotic membrane, chorionic membrane, umbilical cord and placental decidua. Immunophenotype, differentiation ability, cell size, cell complexity, polarity index and growth kinetics of MSCs isolated from these four sources were analyzed. MSCs were successfully isolated from all four sources. Surface marker profile and differentiation ability were consistent with human MSCs. C-MSCs in suspension were the smallest cells, whereas UC-MSCs presented the greatest length and least width. A-MSCs had the lowest polarity index and UC-MSCs, as more elongated cells, the highest. C-MSCs, D-MSCs and UC-MSCs exhibited similar growth capacity until passage 8 (P8); C-MSCs presented better lifespan, whereas insignificant proliferation was observed in A-MSCs. Neonatal and maternal tissues can serve as sources of multipotent stem cells. Some characteristics of MSCs obtained from four neonatal tissues were compared and differences were observed. Amniotic membrane was the least useful source of MSCs, whereas chorionic membrane and umbilical cord were considered good options for future use in cell therapy because of the known advantages of immature cells. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel.

PubMed

Cochis, A; Grad, S; Stoddart, M J; Farè, S; Altomare, L; Azzimonti, B; Alini, M; Rimondini, L

2017-03-23

Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes' poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na 2 SO 4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis.

Biomineralized hydroxyapatite nanoclay composite scaffolds with polycaprolactone for stem cell-based bone tissue engineering.

PubMed

Ambre, Avinash H; Katti, Dinesh R; Katti, Kalpana S

2015-06-01

Nanoclay modified with unnatural amino acid was used to design a nanoclay-hydroxyapatite (HAP) hybrid by mineralizing HAP in the nanoclay galleries mimicking biomineralization. This hybrid (in situ HAPclay) was used to fabricate polycaprolactone (PCL)/in situ HAPclay films and scaffolds for bone regeneration. Cell culture assays and imaging were used to study interactions between human mesenchymal stem cells (hMSCs) and PCL/in situ HAPclay composites (films and scaffolds). SEM imaging indicated MSC attachment, formation of mineralized extracellular (ECM) on PCL/in situ HAPclay films, and infiltration of MSCs to the interior of PCL/in situ HAPclay scaffolds. Mineralized ECM was formed by MSCs without use of osteogenic supplements. AFM imaging performed on this in vitro generated mineralized ECM on PCL/in situ HAPclay films revealed presence of components (collagen and mineral) of hierarchical organization reminiscent of natural bone. Cellular events observed during two-stage seeding experiments on PCL/in situ HAPclay films indicated similarities with events occurring during in vivo bone formation. PCL/in situ HAPclay films showed significantly increased (100-595% increase in elastic moduli) nanomechanical properties and PCL/in situ HAPclay scaffolds showed increased degradation. This work puts forth PCL/in situ HAPclay composites as viable biomaterials for bone tissue engineering. © 2014 Wiley Periodicals, Inc.

Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement.

PubMed

Shieh, Hester F; Graham, Christopher D; Brazzo, Joseph A; Zurakowski, David; Fauza, Dario O

2017-06-01

We sought to examine amniotic fluid mesenchymal stem cell (afMSC) viability within two FDA-approved collagen-based scaffolds, as a prerequisite to clinical translation of afMSC-based engineered diaphragmatic repair. Human afMSCs were seeded in a human-derived collagen hydrogel and in a bovine-derived collagen sheet at 3 matching densities. Cell viability was analyzed at 1, 3, and 5days using an ATP-based 3D bioluminescence assay. Statistical comparisons were by ANOVA (P<0.05). There was a highly significant 3-way interaction between scaffold type, seeding density, and time in 3D culture as determinants of cell viability, clearly favoring the human hydrogel (P<0.001). In both scaffolds, cell viability was highest at the highest seeding density of 150,000 cells/mL. Time in 3D culture impacted cell viability at the optimal seeding density in the human hydrogel, with the highest levels on days 1 (P<0.001) and 5 (P=0.05) with no significant effect in the bovine sheet (P=0.39-0.96). Among clinically-approved cell delivery vehicles, mesenchymal stem cell viability is significantly enhanced in a collagen hydrogel when compared with a collagen sheet. Cell viability can be further optimized by seeding density and time in 3D culture. These data further support the regulatory viability of clinical trials of engineered diaphragmatic repair. N/A (animal and laboratory study). Copyright © 2017 Elsevier Inc. All rights reserved.

Embryonic Stem Cell-Derived Mesenchymal Stem Cells (MSCs) Have a Superior Neuroprotective Capacity Over Fetal MSCs in the Hypoxic-Ischemic Mouse Brain.

PubMed

Hawkins, Kate E; Corcelli, Michelangelo; Dowding, Kate; Ranzoni, Anna M; Vlahova, Filipa; Hau, Kwan-Leong; Hunjan, Avina; Peebles, Donald; Gressens, Pierre; Hagberg, Henrik; de Coppi, Paolo; Hristova, Mariya; Guillot, Pascale V

2018-05-01

Human mesenchymal stem cells (MSCs) have huge potential for regenerative medicine. In particular, the use of pluripotent stem cell-derived mesenchymal stem cells (PSC-MSCs) overcomes the hurdle of replicative senescence associated with the in vitro expansion of primary cells and has increased therapeutic benefits in comparison to the use of various adult sources of MSCs in a wide range of animal disease models. On the other hand, fetal MSCs exhibit faster growth kinetics and possess longer telomeres and a wider differentiation potential than adult MSCs. Here, for the first time, we compare the therapeutic potential of PSC-MSCs (ES-MSCs from embryonic stem cells) to fetal MSCs (AF-MSCs from the amniotic fluid), demonstrating that ES-MSCs have a superior neuroprotective potential over AF-MSCs in the mouse brain following hypoxia-ischemia. Further, we demonstrate that nuclear factor (NF)-κB-stimulated interleukin (IL)-13 production contributes to an increased in vitro anti-inflammatory potential of ES-MSC-conditioned medium (CM) over AF-MSC-CM, thus suggesting a potential mechanism for this observation. Moreover, we show that induced pluripotent stem cell-derived MSCs (iMSCs) exhibit many similarities to ES-MSCs, including enhanced NF-κB signaling and IL-13 production in comparison to AF-MSCs. Future studies should assess whether iMSCs also exhibit similar neuroprotective potential to ES-MSCs, thus presenting a potential strategy to overcome the ethical issues associated with the use of embryonic stem cells and providing a potential source of cells for autologous use against neonatal hypoxic-ischemic encephalopathy in humans. Stem Cells Translational Medicine 2018;7:439-449. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Macromolecular Crowding Amplifies Adipogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells by Enhancing the Pro-Adipogenic Microenvironment

PubMed Central

Ang, Xiu Min; Lee, Michelle H.C.; Blocki, Anna; Chen, Clarice; Ong, L.L. Sharon; Asada, H. Harry; Sheppard, Allan

2014-01-01

The microenvironment plays a vital role in both the maintenance of stem cells in their undifferentiated state (niche) and their differentiation after homing into new locations outside this niche. Contrary to conventional in-vitro culture practices, the in-vivo stem cell microenvironment is physiologically crowded. We demonstrate here that re-introducing macromolecular crowding (MMC) at biologically relevant fractional volume occupancy during chemically induced adipogenesis substantially enhances the adipogenic differentiation response of human bone marrow-derived mesenchymal stem cells (MSCs). Both early and late adipogenic markers were significantly up-regulated and cells accumulated 25–40% more lipid content under MMC relative to standard induction cocktails. MMC significantly enhanced deposition of extracellular matrix (ECM), notably collagen IV and perlecan, a heparan sulfate proteoglycan. As a novel observation, MMC also increased the presence of matrix metalloproteinase −2 in the deposited ECM, which was concomitant with geometrical ECM remodeling typical of adipogenesis. This suggested a microenvironment that was richer in both matrix components and associated ligands and was conducive to adipocyte maturation. This assumption was confirmed by seeding undifferentiated MSCs on decellularized ECM deposited by adipogenically differentiated MSCs, Adipo-ECM. On Adipo-ECM generated under crowding, MSCs differentiated much faster under a classical differentiation protocol. This was evidenced throughout the induction time course, by a significant up-regulation of both early and late adipogenic markers and a 60% higher lipid content on MMC-generated Adipo-ECM in comparison to standard induction on tissue culture plastic. This suggests that MMC helps build and endow the nascent microenvironment with adipogenic cues. Therefore, MMC initiates a positive feedback loop between cells and their microenvironment as soon as progenitor cells are empowered to build and shape it, and, in turn, are informed by it to respond by attaining a stable differentiated phenotype if so induced. This work sheds new light on the utility of MMC to tune the microenvironment to augment the generation of adipose tissue from differentiating human MSCs. PMID:24147829

MOLD-SHAPED, NANOFIBER SCAFFOLD-BASED CARTILAGE ENGINEERING USING HUMAN MESENCHYMAL STEM CELLS AND BIOREACTOR

PubMed Central

Janjanin, Sasa; Li, Wan-Ju; Morgan, Meredith T.; Shanti, Rabie M.; Tuan, Rocky S.

2008-01-01

Background Mesenchymal stem cell (MSC)-based tissue engineering is a promising future alternative to autologous cartilage grafting. This study evaluates the potential of using MSCs, seeded into electrospun, biodegradable polymeric nanofibrous scaffolds, to engineer cartilage with defined dimensions and shape, similar to grafts used for subcutaneous implantation in plastic and reconstructive surgery. Materials and methods Human bone marrow derived MSCs seeded onto nanofibrous scaffolds and placed in custom-designed molds were cultured for up to 42 days in bioreactors. Chondrogenesis was induced with either transforming growth factor-β1 (TGF-β1) alone or in combination with insulin-like growth factor-I (IGF-I). Results Constructs exhibited hyaline cartilage histology with desired thickness and shape as well as favorable tissue integrity and shape retention, suggesting the presence of elastic tissue. Time-dependent increase in cartilage matrix gene expression was seen in both types of culture; at Day 42, TGF-β1/IGF-I treated cultures showed higher collagen type II and aggrecan expression. Both culture conditions showed significant time-dependent increase in sulfated glycosaminoglycan and hydroxyproline contents. TGF-β1/IGF-I treated samples were significantly stiffer; with equilibrium compressive Young’s modulus values reaching 17 kPa by Day 42. Conclusions The successful ex vivo development of geometrically defined cartilaginous construct using customized molding suggests the potential of cell-based cartilage tissue for reconstructive surgery. PMID:18316094

A novel albumin-based tissue scaffold for autogenic tissue engineering applications.

PubMed

Li, Pei-Shan; Lee, I-Liang; Yu, Wei-Lin; Sun, Jui-Sheng; Jane, Wann-Neng; Shen, Hsin-Hsin

2014-07-18

Tissue scaffolds provide a framework for living tissue regeneration. However, traditional tissue scaffolds are exogenous, composed of metals, ceramics, polymers, and animal tissues, and have a defined biocompatibility and application. This study presents a new method for obtaining a tissue scaffold from blood albumin, the major protein in mammalian blood. Human, bovine, and porcine albumin was polymerised into albumin polymers by microbial transglutaminase and was then cast by freeze-drying-based moulding to form albumin tissue scaffolds. Scanning electron microscopy and material testing analyses revealed that the albumin tissue scaffold possesses an extremely porous structure, moderate mechanical strength, and resilience. Using a culture of human mesenchymal stem cells (MSCs) as a model, we showed that MSCs can be seeded and grown in the albumin tissue scaffold. Furthermore, the albumin tissue scaffold can support the long-term osteogenic differentiation of MSCs. These results show that the albumin tissue scaffold exhibits favourable material properties and good compatibility with cells. We propose that this novel tissue scaffold can satisfy essential needs in tissue engineering as a general-purpose substrate. The use of this scaffold could lead to the development of new methods of artificial fabrication of autogenic tissue substitutes.

[Alterations in expression of F-actin and DNA of fluid shear stress treated-mesenchymal stem cells affected by titanium particles loading].

PubMed

Wu, Jiang; Chen, Huiqing; Cao, Hui; Zhou, Jiang; Zhang, Li; Sung, K L

2004-02-01

Particulate wear debris within the bone-prosthesis microenvironment generated by normal wear and corrosion of orthopaedic implants is considered to be one of the main factors responsible for chronic aseptic inflammation and development of osteolysis in the long-term instability and failure of total joint arthroplasty. While the decrease in bone volume caused by wear debris-induced osteolysis could have been compensated by enough new bone matrix secreted by osteoblasts. Actually, the normal osteoblastic population depend on the regular differentiation and proliferation of their progenitor cells--bone marrow mesenchymal stem cells (MSCs). This study aims to investigate the potential mechanism for the rat MSCs cytotoxicity upon exposure to Titanium (Ti) particles. Rat mesenchymal stem cells (rMSCs) isolated from 3-month-old male Sprague-Dawley rats by Percoll intensity gradient method were cultured in DMEM medium (low glucose) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 micrograms/ml streptomycin in a humidified incubator with 5% CO2 at 37 degrees C. In order to gain the homogenous cell population, rMSCs were passaged to 3-4th subpassage which were used in all the experiment groups. Then rMSCs were seeded in the 6 well culture plates and exposed to three different circle diameters (mean size, TD1: 0.9 micron, TD2: 2.7 microns, TD3: 6.9 microns) with three different concentrations (0.1 wt%, 0.05 wt%, 0.01 wt%, W/V) at different durations (8 h, 16 h, 24 h,), respectively. Unexposed rMSCs were used as control. In the given periods of Ti loading, fluid shear stress (FSS) was applied to each group cells. The expression of F-actin and DNA of the rMSCs at the indicated time were determined with laser confocal scanning microscopy and image analysis software. The results showed that there was up-regulation expression of F-actin in the rMSCs without Ti particles loading but in the presence of FSS. Ti particles loading can suppress the expression of F-action and DNA of rMSCs, but this down-regulation response varied with the three circle diameter, concentrations and durations of Ti particles. Among three kinds of diametrically different Ti particles, submicron Ti particles (0.9 micron) had the greatest suppressive response on rMSCs, together with some apoptosis bodies. Under the same diameter condition, the inhibition induced by Ti particles loading was in a manner dependent on the particles concentration and exposure duration. The reductive effects produced from 0.1 wt% Ti was the greatest and earliest among the responses from Ti particles at three different concentrations; and the lower the concentration, the weaker the repressive influence. Furthermore, with the elongation of exposure to Ti particles, the expression of F-actin and DNA decreased gradually, the lowest level was at 32 h. These findings demonstrated that Ti particles loading can attenuate rMSCs' viability in a manner dependent on the circle diameter, particles concentration, treatment period, suggesting that a reduction in the number of viable MSCs together with a compromise of the their differentiation into functional osteoblast may exacerbate aseptic loosening of total joint implant. Further investigation into particles-mediated suppression of MCSs viability may reveal novel mechanism of implant loosening and aid in development and application of osteolytic drug therapy and the optimization of design and selection of future orthopaedic biomaterials, thereby improving long-term compatibility and stability for arthroplasty patients.

Silk fibroin/chitosan scaffold with tunable properties and low inflammatory response assists the differentiation of bone marrow mesenchymal stem cells.

PubMed

Li, Da-Wei; Lei, Xiaohua; He, Feng-Li; He, Jin; Liu, Ya-Li; Ye, Ya-Jing; Deng, Xudong; Duan, Enkui; Yin, Da-Chuan

2017-12-01

The physical and chemical properties of the scaffold are known to play important roles in three-dimensional (3D) cell culture, which always determine the cellular fate or the results of implantation. To control these properties becomes necessary for meeting the requirements of a variety of tissue engineering applications. In this study, a series of silk fibroin/chitosan (SF/CS) scaffolds with tunable properties were prepared using freeze-drying method, and the rat bone marrow-derived mesenchymal stem cells (BM-MSCs) were seeded in these scaffolds to evaluate their availability of use in tissue engineering. The 3D structure, mechanical properties and degradation ability of SF/CS scaffold can be tuned by changing the total concentration of the precursor solution and the blending ratio between SF and CS. BM-MSCs cultured in the SF/CS scaffold exhibited excellent proliferation and multiple morphologies. The induction of osteogenic and adipogenic differentiation of BM-MSCs were successful in this scaffold when cultured in vitro. Subcutaneous implantation of the SF/CS scaffolds did not cause any inflammatory response within four weeks, which revealed good compatibility. Moreover, the implanted scaffold allowed host cells to invade, adhere, grow and form new blood vessels. With these excellent performance, SF/CS scaffold has great potential in preparing implants for tissue engineering applications. Copyright © 2017. Published by Elsevier B.V.

Adipose Tissue-Derived Pericytes for Cartilage Tissue Engineering.

PubMed

Zhang, Jinxin; Du, Chunyan; Guo, Weimin; Li, Pan; Liu, Shuyun; Yuan, Zhiguo; Yang, Jianhua; Sun, Xun; Yin, Heyong; Guo, Quanyi; Zhou, Chenfu

2017-01-01

Mesenchymal stem cells (MSCs) represent a promising alternative source for cartilage tissue engineering. However, MSC culture is labor-intensive, so these cells cannot be applied immediately to regenerate cartilage for clinical purposes. Risks during the ex vivo expansion of MSCs, such as infection and immunogenicity, can be a bottleneck in their use in clinical tissue engineering. As a novel stem cell source, pericytes are generally considered to be the origin of MSCs. Pericytes do not have to undergo time-consuming ex vivo expansion because they are uncultured cells. Adipose tissue is another optimal stem cell reservoir. Because adipose tissue is well vascularized, a considerable number of pericytes are located around blood vessels in this accessible and dispensable tissue, and autologous pericytes can be applied immediately for cartilage regeneration. Thus, we suggest that adipose tissue-derived pericytes are promising seed cells for cartilage regeneration. Many studies have been performed to develop isolation methods for the adipose tissuederived stromal vascular fraction (AT-SVF) using lipoaspiration and sorting pericytes from AT-SVF. These methods are useful for sorting a large number of viable pericytes for clinical therapy after being combined with automatic isolation using an SVF device and automatic magnetic-activated cell sorting. These tools should help to develop one-step surgery for repairing cartilage damage. However, the use of adipose tissue-derived pericytes as a cell source for cartilage tissue engineering has not drawn sufficient attention and preclinical studies are needed to improve cell purity, to increase sorting efficiency, and to assess safety issues of clinical applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering.

PubMed

Kim, In Gul; Ko, Jaehoon; Lee, Hye Rim; Do, Sun Hee; Park, Kwideok

2016-04-01

Mesenchymal cells condensation is crucial in chondrogenic development. However current tissue-engineered scaffolds for chondrogenesis pay little attention to this phenomenon. In this study, we fabricate poly(l-lactide-co-glycolide) (PLGA)/poly(l-lactide) (PLLA) microfiber scaffolds and coat them with human fibroblast-derived matrix (hFDM) that is a decellularized extracellular matrix (ECM) obtained from in vitro cultured human lung fibroblasts (WI-38). Those scaffolds were then conjugated with heparin via EDC chemistry and subsequently immobilized with transforming growth factor (TGF)-β1. The amount of TGF-β1 was quantitatively measured and the release profile showed a continuous release of TGF-β1 for 4 weeks. Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were seeded in four different scaffolds; control, fibronectin (FN)-coated, hFDM-coated, hFDM/TGF-β1 and subjected to chondrogenic differentiation in vitro for up to 28 days. Both hFDM and hFDM/TGF-β1 groups exhibited significantly more synthesis of glycosaminoglycan (GAG) and much better upregulation of chondrogenic markers expression. Interestingly, MSCs condensation that led to cell aggregates was clearly observed with time in the two hFDM-coated groups and the quantitative difference was obvious compared to the control and FN group. A mechanistic study in gene and protein level indicated that focal adhesion kinase (FAK) was involved at the early stage of cell adhesion and cell-cell contact-related markers, N-cadherin and neural cell adhesion molecule (NCAM), were highly up-regulated at later time point. In addition histological analysis proved that hFDM/TGF-β1 group was the most effective in forming neocartilage tissue in a rabbit articular cartilage defect model. Taken together, this study demonstrates not only the positive effect of hFDM on chondrogenesis of MSCs and cartilage repair but also provides an important insight toward the significance of in vitro mesenchymal condensation on chondrogenic development of MSCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative analysis of the immunomodulatory capacities of human bone marrow- and adipose tissue-derived mesenchymal stromal cells from the same donor.

PubMed

Valencia, Jaris; Blanco, Belén; Yáñez, Rosa; Vázquez, Miriam; Herrero Sánchez, Carmen; Fernández-García, María; Rodríguez Serrano, Concepción; Pescador, David; Blanco, Juan F; Hernando-Rodríguez, Miriam; Sánchez-Guijo, Fermín; Lamana, María Luisa; Segovia, José Carlos; Vicente, Ángeles; Del Cañizo, Consuelo; Zapata, Agustín G

2016-10-01

The immunomodulatory properties of mesenchymal stromal cells (MSCs), together with their tissue regenerative potential, make them interesting candidates for clinical application. In the current study, we analyzed the in vitro immunomodulatory effects of MSCs derived from bone marrow (BM-MSCs) and from adipose tissue (AT-MSCs) obtained from the same donor on both innate and acquired immunity cells. BM-MSCs and AT-MSCs were expanded to fourth or fifth passage and co-cultured with T cells, monocytes or natural killer (NK) cells isolated from human peripheral blood and stimulated in vitro. The possible differing impact of MSCs obtained from distinct sources on phenotype, cell proliferation and differentiation, cytokine production and function of these immune cells was comparatively analyzed. BM-MSCs and AT-MSCs induced a similar decrease in NK-cell proliferation, cytokine secretion and expression of both activating receptors and cytotoxic molecules. However, only BM-MSCs significantly reduced NK-cell cytotoxic activity, although both MSC populations showed the same susceptibility to NK-cell-mediated lysis. AT-MSCs were more potent in inhibiting dendritic-cell (DC) differentiation than BM-MSC, but both MSC populations similarly reduced the ability of DCs to induce CD4(+) T-cell proliferation and cytokine production. BM-MSCs and AT-MSCs induced a similar decrease in T-cell proliferation and production of inflammatory cytokines after activation. AT-MSCs and BM-MSCs from the same donor had similar immunomodulatory capacity on both innate and acquired immunity cells. Thus, other variables, such as accessibility of samples or the frequency of MSCs in the tissue should be considered to select the source of MSC for cell therapy. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Production of hyaline-like cartilage by bone marrow mesenchymal stem cells in a self-assembly model.

PubMed

Elder, Steven H; Cooley, Avery J; Borazjani, Ali; Sowell, Brittany L; To, Harrison; Tran, Scott C

2009-10-01

A scaffoldless or self-assembly approach to cartilage tissue engineering has been used to produce hyaline cartilage from bone marrow-derived mesenchymal stem cells (bMSCs), but the mechanical properties of such engineered cartilage and the effects the transforming growth factor (TGF) isoform have not been fully explored. This study employs a cell culture insert model to produce tissue-engineered cartilage using bMSCs. Neonatal pig bMSCs were isolated by plastic adherence and expanded in monolayer before being seeded into porous transwell inserts and cultured for 4 or 8 weeks in defined chondrogenic media containing either TGF-beta1 or TGF-beta3. Following biomechanical evaluation in confined compression, colorimetric dimethyl methylene blue and Sircol dye-binding assays were used to analyze glycosaminoglycan (GAG) and collagen contents, respectively. Histological sections were stained with toluidine blue for proteoglycans and with picrosirius red to reveal collagen orientation, and immunostained for detection of collagen types I and II. Neocartilage increased in thickness, collagen, and GAG content between 4 and 8 weeks. Proteoglycan concentration increased with depth from the top surface. The tissue contained much more collagen type II than type I, and there was a consistent pattern of collagen alignment. TGF-beta1-treated and TGF-beta3-treated constructs were similar at 4 weeks, but 8-week TGF-beta1 constructs had a higher aggregate modulus and GAG content compared to TGF-beta3. These results demonstrate that bMSCs can generate functional hyaline-like cartilage through a self-assembling process.

Vesicular delivery of crystalline calcium minerals to ECM in biomineralized nanoclay composites

NASA Astrophysics Data System (ADS)

Katti, Kalpana S.; Ambre, Avinash H.; Payne, Scott; Katti, Dinesh R.

2015-04-01

The mechanisms of mineralization and new bone formation were explored in newly formed extracellular matrix in a nanoclay based composite. Nanoclay films were prepared by intercalating the clays with amino acids and using the amino acids for mineralization of hydroxyapatite. The biomineralized hydroxyapatite (HAP) inside nanoclay galleries or in situ HAP/clay was further used to make films (substrates) using polycaprolactone (PCL) that were seeded with mesenchymal stem cells in a two-stage seeding process. SEM imaging experiments performed on PCL/in situ HAPclay composite films seeded with human MSCs indicated formation of matrix vesicles. The vesicles appear to emerge from the cells that are adhered to the nanoclay HAP films and also deposited in the extracellular space. Vesicles are also observed to be embedded in the cells or under the surface of cells. Crystalline structures with Ca and P were found inside vesicles. The Ca/P ratios obtained using energy dispersive spectroscopy indicate values ranging from below 0.7 to the stoichiometric HAP value of 1.67. The Ca/P ratios were obtained to be closer to the stoichiometric value for single seeding experiments as compared to the double seeding experiments indicating more new bone formation in double seeding experiments. New bone formation with bone mimetic mineralization is thus observed on the in situ HAP nanoclay PCL samples. Hence the PCL/in situ HAPclay composites besides being osteoinductive are also capable of providing a favorable micro-environment for cell dependent processes involved in bone mineral formation.

Human Mesenchymal Stromal Cells from Adult and Neonatal Sources: A Comparative In Vitro Analysis of Their Immunosuppressive Properties Against T Cells

PubMed Central

Castro-Manrreza, Marta E.; Mayani, Hector; Monroy-García, Alberto; Flores-Figueroa, Eugenia; Chávez-Rueda, Karina; Legorreta-Haquet, Victoria; Santiago-Osorio, Edelmiro

2014-01-01

Bone marrow-mesenchymal stromal cells (BM-MSCs) have immunosuppressive properties and have been used in cell therapies as immune regulators for the treatment of graft-versus-host disease. We have previously characterized several biological properties of MSCs from placenta (PL) and umbilical cord blood (UCB), and compared them to those of BM—the gold standard. In the present study, we have compared MSCs from BM, UCB, and PL in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3+ T cells. Our results confirm the immunosuppressive potential of BM-MSCs, and demonstrate that MSCs from UCB and, to a lesser extent PL, also have immunosuppressive potential. In contrast to PL-MSCs, BM-MSCs and UCB-MSCs significantly inhibited the proliferation of both CD4+ and CD8+ activated T cells in a cell–cell contact-dependent manner. Such a reduced proliferation in cell cocultures correlated with upregulation of programmed death ligand 1 on MSCs and cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) on T cells, and increased production of interferon-γ, interleukin-10, and prostaglandin E2. Importantly, and in contrast to PL-MSCs, both BM-MSCs and UCB-MSCs favored the generation of T-cell subsets displaying a regulatory phenotype CD4+CD25+CTLA-4+. Our results indicate that, besides BM-MSCs, UCB-MSCs might be a potent and reliable candidate for future therapeutic applications. PMID:24428376

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

PubMed Central

2014-01-01

Introduction Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA. Results The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied. Conclusions The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ‘‘in vitro’’ differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes. PMID:24559797

TOPICAL REVIEW: Stem cell technology using bioceramics: hard tissue regeneration towards clinical application

NASA Astrophysics Data System (ADS)

Ohnishi, Hiroe; Oda, Yasuaki; Ohgushi, Hajime

2010-02-01

Mesenchymal stem cells (MSCs) are adult stem cells which show differentiation capabilities toward various cell lineages. We have already used MSCs for treatments of osteoarthritis, bone necrosis and bone tumor. For this purpose, culture expanded MSCs were combined with various ceramics and then implanted. Because of rejection response to allogeneic MSC implantation, we have utilized patients' own MSCs for the treatment. Bone marrow is a good cell source of MSCs, although the MSCs also exist in adipose tissue. When comparing osteogenic differentiation of these MSCs, bone marrow MSCs show more extensive bone forming capability than adipose MSCs. Thus, the bone marrow MSCs are useful for bone tissue regeneration. However, the MSCs show limited proliferation and differentiation capabilities that hindered clinical applications in some cases. Recent advances reveal that transduction of plural transcription factors into human adult cells results in generation of new type of stem cells called induced pluripotent stem cells (iPS cells). A drawback of the iPS cells for clinical applications is tumor formation after their in vivo implantation; therefore it is difficult to use iPS cells for the treatment. To circumvent the problem, we transduced a single factor of either SOX2 or NANOG into the MSCs and found high proliferation as well as osteogenic differentiation capabilities of the MSCs. The stem cells could be combined with bioceramics for clinical applications. Here, we summarize our recent technologies using adult stem cells in viewpoints of bone tissue regeneration.

Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita

2010-03-12

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activitymore » in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.« less

Chorion Mesenchymal Stem Cells Show Superior Differentiation, Immunosuppressive, and Angiogenic Potentials in Comparison With Haploidentical Maternal Placental Cells

PubMed Central

González, Paz L.; Carvajal, Catalina; Cuenca, Jimena; Alcayaga-Miranda, Francisca; Figueroa, Fernando E.; Bartolucci, Jorge; Salazar-Aravena, Lorena

2015-01-01

Mesenchymal stem cells (MSCs) of placental origin have become increasingly translational owing to their abundance and accessibility. MSCs of different origin share several features but also present biological differences that might point to distinct clinical properties. Hence, mixing fetal and maternal cells from the same placenta can lead to contradicting results. We analyzed the biological characteristics of haploidentical MSCs isolated from fetal sources, including the umbilical cord (UC-MSCs) and chorion (Ch-MSCs), compared with maternal decidua MSCs (Dc-MSCs). All MSCs were analyzed for general stem cell properties. In addition, immunosuppressive capacity was assessed by the inhibition of T-cell proliferation, and angiogenic potential was evaluated in a Matrigel transplantation assay. The comparison between haploidentical MSCs displayed several distinct features, including (a) marked differences in the expression of CD56, (b) a higher proliferative capacity for Dc-MSCs and UC-MSCs than for Ch-MSCs, (c) a diversity of mesodermal differentiation potential in favor of fetal MSCs, (d) a higher capacity for Ch-MSCs to inhibit T-cell proliferation, and (e) superior angiogenic potential of Ch-MSCs evidenced by a higher capability to form tubular vessel-like structures and an enhanced release of hepatocyte growth factor and vascular endothelial growth factor under hypoxic conditions. Our results suggest that assessing the prevalence of fetomaternal contamination within placental MSCs is necessary to increase robustness and limit side effects in their clinical use. Finally, our work presents evidence positioning fetoplacental cells and notably Ch-MSCs in the forefront of the quest for cell types that are superior for applications in regenerative medicine. Significance This study analyzed the biological characteristics of mesenchymal stem cells (MSCs) isolated from fetal and maternal placental origins. The findings can be summarized as follows: (a) important differences were found in the expression of CD56, (b) a different mesodermal differentiation potential was found in favor of fetal MSCs, (c) a higher immunosuppressive capacity for chorion MSCs was noted, and (d) superior angiogenic potential of Ch-MSCs was observed. These results suggest that assessing the prevalence of fetomaternal contamination within placental MSCs is necessary to increase robustness and limit side effects in their clinical use. The evidence should allow clinicians to view fetoplacental cells, notably Ch-MSCs, favorably as candidates for use in regenerative medicine. PMID:26273064

Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells.

PubMed

Kadekar, Darshana; Rangole, Sonal; Kale, Vaijayanti; Limaye, Lalita

2016-01-01

The limited cell dose in umbilical cord blood (UCB) necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS). Conditioned medium from mesenchymal stem cells (MSCs-CM) has been shown to alleviate the oxidative stress during wound healing, Alzheimer's disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells. CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM). UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them. Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability.

Umbilical Cord Blood-Derived Mesenchymal Stem Cells Inhibit, But Adipose Tissue-Derived Mesenchymal Stem Cells Promote, Glioblastoma Multiforme Proliferation

PubMed Central

Akimoto, Keiko; Kimura, Kenichi; Nagano, Masumi; Takano, Shingo; To'a Salazar, Georgina; Yamashita, Toshiharu

2013-01-01

Mesenchymal stem cells (MSCs) possess self-renewal and multipotential differentiation abilities, and they are thought to be one of the most reliable stem cell sources for a variety of cell therapies. Recently, cell therapy using MSCs has been studied as a novel therapeutic approach for cancers that show refractory progress and poor prognosis. MSCs from different tissues have different properties. However, the effect of different MSC properties on their application in anticancer therapies has not been thoroughly investigated. In this study, to characterize the anticancer therapeutic application of MSCs from different sources, we established two different kinds of human MSCs: umbilical cord blood-derived MSCs (UCB-MSCs) and adipose-tissue-derived MSCs (AT-MSCs). We used these MSCs in a coculture assay with primary glioblastoma multiforme (GBM) cells to analyze how MSCs from different sources can inhibit GBM growth. We found that UCB-MSCs inhibited GBM growth and caused apoptosis, but AT-MSCs promoted GBM growth. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling assay clearly demonstrated that UCB-MSCs promoted apoptosis of GBM via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL was expressed more highly by UCB-MSCs than by AT-MSCs. Higher mRNA expression levels of angiogenic factors (vascular endothelial growth factor, angiopoietin 1, platelet-derived growth factor, and insulin-like growth factor) and stromal-derived factor-1 (SDF-1/CXCL12) were observed in AT-MSCs, and highly vascularized tumors were developed when AT-MSCs and GBM were cotransplanted. Importantly, CXCL12 inhibited TRAIL activation of the apoptotic pathway in GBM, suggesting that AT-MSCs may support GBM development in vivo by at least two distinct mechanisms—promoting angiogenesis and inhibiting apoptosis. The opposite effects of AT-MSCs and UCB-MSCs on GBM clearly demonstrate that differences must be considered when choosing a stem cell source for safety in clinical application. PMID:23231075

Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSCs) Engraft In Vivo and Support Hematopoiesis without Suppressing Immune Function: Implications for Off-The Shelf ES-MSC Therapies

PubMed Central

Li, Ou; Tormin, Ariane; Sundberg, Berit; Hyllner, Johan; Le Blanc, Katarina; Scheding, Stefan

2013-01-01

Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function. PMID:23383153

Growth Inhibitory Effect of Palatine Tonsil-derived Mesenchymal Stem Cells on Head and Neck Squamous Cell Carcinoma Cells

PubMed Central

Lim, Yun-Sung; Lee, Jin-Choon; Lee, Yoon Se; Wang, Soo-Geun

2012-01-01

Objectives Mesenchymal stem cells (MSCs) play an important role in the development and growth of tumor cells. However, the effect of human MSCs on the growth of human tumors is not well understood. The purpose of this study is to confirm the growth effect of palatine tonsil-derived MSCs (TD-MSCs) on head and neck squamous cell carcinoma (HNSCC) cell lines and to elucidate the mechanism of their action. Methods TD-MSCs were isolated from patient with chronic tonsillitis and tonsillar hypertrophy. Two human HNSCC cell lines (PNUH-12 and SNU-899) were studied and cocultured with isolated palatine tonsil-derived MSC. The growth inhibitory effect of MSCs on HNSCC cell lines was tested through methylthiazolyldiphenyl-tetrazolium (MTT) assay. The apoptosis induction effect of MSCs on cell lines was assessed with flow cytometry and reverse transcriptase (RT)-PCR. Results Palatine tonsil-derived MSCs exhibited a growth inhibitory effect on both cell lines. Cell cycle analysis showed an accumulation of tumor cells predominantly in G0/G1 phase with an increase in concentration of TD-MSCs, which was confirmed by increased mRNA expression of cell cycle negative regulator p21. Apoptosis of tumor cells increased significantly as concentration of cocultured TD-MSCs increased. Additionally, mRNA expression of caspase 3 was upregulated with increased concentration of TD-MSCs. Conclusion TD-MSCs have a potential growth inhibitory effect on HNSCC cell lines in vitro by inducing apoptotic cell death and G1 phase arrest of cell lines. PMID:22737289

In Vitro Evidence of the Presence of Mesenchymal Stromal Cells in Cervical Cancer and Their Role in Protecting Cancer Cells from Cytotoxic T Cell Activity

PubMed Central

Montesinos, Juan J.; Mora-García, María de L.; Mayani, Héctor; Flores-Figueroa, Eugenia; García-Rocha, Rosario; Fajardo-Orduña, Guadalupe R.; Castro-Manrreza, Marta E.; Weiss-Steider, Benny

2013-01-01

Mesenchymal stromal cells (MSCs) have been isolated from different tumors and it has been suggested that they support tumor growth through immunosuppression processes that favor tumor cell evasion from the immune system. To date, however, the presence of MSCs in cervical cancer (CeCa) and their possible role in tumor growth remains unknown. Herein we report on the presence of MSCs in cervical tissue, both in normal conditions (NCx-MSCs) and in CeCa (CeCa-MSCs), and described several biological properties of such cells. Our study showed similar patterns of cell surface antigen expression, but distinct differentiation potentials, when we compared both cervical MSC populations to MSCs from normal bone marrow (BM-MSCs, the gold standard). Interestingly, CeCa-MSCs were negative for the presence of human papiloma virus, indicating that these cells are not infected by such a viral agent. Also, interestingly, and in contrast to NCx-MSCs, CeCa-MSCs induced significant downregulation of surface HLA class I molecules (HLA-A*0201) on CaSki cells and other CeCa cell lines. We further observed that CeCa-MSCs inhibited antigen-specific T cell recognition of CaSki cells by cytotoxic T lymphocytes (CTLs). HLA class I downregulation on CeCa cells correlated with the production of IL-10 in cell cocultures. Importantly, this cytokine strongly suppressed recognition of CeCa cells by CTLs. In summary, this study demonstrates the presence of MSCs in CeCa and suggests that tumor-derived MSCs may provide immune protection to tumor cells by inducing downregulation of HLA class I molecules. This mechanism may have important implications in tumor growth. PMID:23656504

Bone tissue engineering with human mesenchymal stem cell sheets constructed using magnetite nanoparticles and magnetic force.

PubMed

Shimizu, Kazunori; Ito, Akira; Yoshida, Tatsuro; Yamada, Yoichi; Ueda, Minoru; Honda, Hiroyuki

2007-08-01

An in vitro reconstruction of three-dimensional (3D) tissues without the use of scaffolds may be an alternative strategy for tissue engineering. We have developed a novel tissue engineering strategy, termed magnetic force-based tissue engineering (Mag-TE), in which magnetite cationic liposomes (MCLs) with a positive charge at the liposomal surface, and magnetic force were used to construct 3D tissue without scaffolds. In this study, human mesenchymal stem cells (MSCs) magnetically labeled with MCLs were seeded onto an ultra-low attachment culture surface, and a magnet (4000 G) was placed on the reverse side. The MSCs formed multilayered sheet-like structures after a 24-h culture period. MSCs in the sheets constructed by Mag-TE maintained an in vitro ability to differentiate into osteoblasts, adipocytes, or chondrocytes after a 21-day culture period using each induction medium. Using an electromagnet, MSC sheets constructed by Mag-TE were harvested and transplanted into the bone defect in the crania of nude rats. Histological observation revealed that new bone surrounded by osteoblast-like cells was formed in the defect area 14 days after transplantation with MSC sheets, whereas no bone formation was observed in control rats without the transplant. These results indicated that Mag-TE could be used for the transplantation of MSC sheets using magnetite nanoparticles and magnetic force, providing novel methodology for bone tissue engineering.

Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.

PubMed

Wagner, Diane R; Lindsey, Derek P; Li, Kelvin W; Tummala, Padmaja; Chandran, Sheena E; Smith, R Lane; Longaker, Michael T; Carter, Dennis R; Beaupre, Gary S

2008-05-01

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel

PubMed Central

Cochis, A.; Grad, S.; Stoddart, M. J.; Farè, S.; Altomare, L.; Azzimonti, B.; Alini, M.; Rimondini, L.

2017-01-01

Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes’ poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na2SO4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis. PMID:28332587

Effects of mesenchymal stem cells from human induced pluripotent stem cells on differentiation, maturation, and function of dendritic cells.

PubMed

Gao, Wen-Xiang; Sun, Yue-Qi; Shi, Jianbo; Li, Cheng-Lin; Fang, Shu-Bin; Wang, Dan; Deng, Xue-Quan; Wen, Weiping; Fu, Qing-Ling

2017-03-02

Mesenchymal stem cells (MSCs) have potent immunomodulatory effects on multiple immune cells and have great potential in treating immune disorders. Induced pluripotent stem cells (iPSCs) serve as an unlimited and noninvasive source of MSCs, and iPSC-MSCs have been reported to have more advantages and exhibit immunomodulation on T lymphocytes and natural killer cells. However, the effects of iPSC-MSCs on dendritic cells (DCs) are unclear. The aim of this study is to investigate the effects of iPSC-MSCs on the differentiation, maturation, and function of DCs. Human monocyte-derived DCs were induced and cultured in the presence or absence of iPSC-MSCs. Flow cytometry was used to analyze the phenotype and functions of DCs, and enzyme-linked immunosorbent assay (ELISA) was used to study cytokine production. In this study, we successfully induced MSCs from different clones of human iPSCs. iPSC-MSCs exhibited a higher proliferation rate with less cell senescence than BM-MSCs. iPSC-MSCs inhibited the differentiation of human monocyte-derived DCs by both producing interleukin (IL)-10 and direct cell contact. Furthermore, iPSC-MSCs did not affect immature DCs to become mature DCs, but modulated their functional properties by increasing their phagocytic ability and inhibiting their ability to stimulate proliferation of lymphocytes. More importantly, iPSC-MSCs induced the generation of IL-10-producing regulatory DCs in the process of maturation, which was mostly mediated by a cell-cell contact mechanism. Our results indicate an important role for iPSC-MSCs in the modulation of DC differentiation and function, supporting the clinical application of iPSC-MSCs in DC-mediated immune diseases.

Purinergic Signaling Regulates the Transforming Growth Factor-β3-Induced Chondrogenic Response of Mesenchymal Stem Cells to Hydrostatic Pressure.

PubMed

Steward, Andrew J; Kelly, Daniel J; Wagner, Diane R

2016-06-01

Although hydrostatic pressure (HP) is known to regulate chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs), improved insight into the mechanotransduction of HP may form the basis for novel tissue engineering strategies. Previously, we demonstrated that matrix stiffness and calcium ion (Ca(++)) mobility regulate the mechanotransduction of HP; however, the mechanisms, by which these Ca(++) signaling pathways are initiated, are currently unknown. The purinergic pathway, in which adenosine triphosphate (ATP) is released and activates P-receptors to initiate Ca(++) signaling, plays a key role in the mechanotransduction of compression, but has yet to be investigated with regard to HP. Therefore, the objective of this study was to investigate the interplay between purinergic signaling, matrix stiffness, and the chondrogenic response of MSCs to HP. Porcine bone marrow-derived MSCs were seeded into soft or stiff agarose hydrogels and subjected to HP (10 MPa at 1 Hz for 4 h/d for 21 days) or kept in free swelling conditions. Stiff constructs were incubated with pharmacological inhibitors of extracellular ATP, P2 receptors, or hemichannels, or without any inhibitors as a control. As with other loading modalities, HP significantly increased ATP release in the control group; however, inhibition of hemichannels completely abrogated this response. The increase in sulfated glycosaminoglycan (sGAG) synthesis and vimentin reorganization observed in the control group in response to HP was suppressed in the presence of all three inhibitors, suggesting that purinergic signaling is involved in the mechanoresponse of MSCs to HP. Interestingly, ATP was released from both soft and stiff hydrogels in response to HP, but HP only enhanced chondrogenesis in the stiff hydrogels, indicating that matrix stiffness may act downstream of purinergic signaling to regulate the mechanoresponse of MSCs to HP. Addition of exogenous ATP did not replicate the effects of HP on chondrogenesis, suggesting that mechanisms other than purinergic signaling also regulate the response of MSCs to HP.

Dose-response of superparamagnetic iron oxide labeling on mesenchymal stem cells chondrogenic differentiation: a multi-scale in vitro study.

PubMed

Roeder, Emilie; Henrionnet, Christel; Goebel, Jean Christophe; Gambier, Nicolas; Beuf, Olivier; Grenier, Denis; Chen, Bailiang; Vuissoz, Pierre-André; Gillet, Pierre; Pinzano, Astrid

2014-01-01

The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues. MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity. No influence of various SPIO concentrations was noted on human bone marrow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations. This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5-25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.

Secreted factors from metastatic prostate cancer cells stimulate mesenchymal stem cell transition to a pro-tumourigenic 'activated' state that enhances prostate cancer cell migration.

PubMed

Ridge, Sarah M; Bhattacharyya, Dibyangana; Dervan, Eoin; Naicker, Serika D; Burke, Amy J; Murphy, J M; O'leary, Karen; Greene, John; Ryan, Aideen E; Sullivan, Francis J; Glynn, Sharon A

2018-05-15

Mesenchymal stem cells (MSCs) are a heterogeneous population of multipotent cells that are capable of differentiating into osteocytes, chondrocytes and adipocytes. Recently, MSCs have been found to home to the tumour site and engraft in the tumour stroma. However, it is not yet known whether they have a tumour promoting or suppressive function. We investigated the interaction between prostate cancer cell lines 22Rv1, DU145 and PC3, and bone marrow-derived MSCs. MSCs were 'educated' for extended periods in prostate cancer cell conditioned media and PC3-educated MSCs were found to be the most responsive with a secretory profile rich in pro-inflammatory cytokines. PC3-educated MSCs secreted increased osteopontin (OPN), interleukin-8 (IL-8) and fibroblast growth factor-2 (FGF-2) and decreased soluble fms-like tyrosine kinase-1 (sFlt-1) compared to untreated MSCs. PC3-educated MSCs showed a reduced migration and proliferation capacity that was dependent on exposure to PC3-conditioned medium. Vimentin and α-smooth muscle actin (αSMA) expression was decreased in PC3-educated MSCs compared to untreated MSCs. PC3 and DU145 education of healthy donor and prostate cancer patient-derived MSCs led to a reduced proportion of FAP+ αSMA+ cells contrary to characteristics commonly associated with cancer associated fibroblasts (CAFs). The migration of PC3 cells was increased toward both PC3-educated and DU145-educated MSCs compared to untreated MSCs, while DU145 migration was only enhanced toward patient-derived MSCs. In summary, MSCs developed an altered phenotype in response to prostate cancer conditioned medium which resulted in increased secretion of pro-inflammatory cytokines, modified functional activity and the chemoattraction of prostate cancer cells. © 2017 UICC.

Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor

NASA Technical Reports Server (NTRS)

Sikavitsas, Vassilios I.; Bancroft, Gregory N.; Mikos, Antonios G.; McIntire, L. V. (Principal Investigator)

2002-01-01

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds. Copyright 2002 Wiley Periodicals, Inc.

Opposite Effects of Coinjection and Distant Injection of Mesenchymal Stem Cells on Breast Tumor Cell Growth.

PubMed

Zheng, Huilin; Zou, Weibin; Shen, Jiaying; Xu, Liang; Wang, Shu; Fu, Yang-Xin; Fan, Weimin

2016-09-01

: Mesenchymal stem cells (MSCs) usually promote tumor growth and metastasis. By using a breast tumor 4T1 cell-based animal model, this study determined that coinjection and distant injection of allogeneic bone marrow-derived MSCs with tumor cells could exert different effects on tumor growth. Whereas the coinjection of MSCs with 4T1 cells promoted tumor growth, surprisingly, the injection of MSCs at a site distant from the 4T1 cell inoculation site suppressed tumor growth. We further observed that, in the distant injection model, MSCs decreased the accumulation of myeloid-derived suppressor cells and regulatory T cells in tumor tissues by enhancing proinflammatory factors such as interferon-γ, tumor necrosis factor-α, Toll-like receptor (TLR)-3, and TLR-4, promoting host antitumor immunity and inhibiting tumor growth. Unlike previous reports, this is the first study reporting that MSCs may exert opposite roles on tumor growth in the same animal model by modulating the host immune system, which may shed light on the potential application of MSCs as vehicles for tumor therapy and other clinical applications. Mesenchymal stem cells (MSCs) have been widely investigated for their potential roles in tissue engineering, autoimmune diseases, and tumor therapeutics. This study explored the impact of coinjection and distant injection of allogeneic bone marrow-derived MSCs on mouse 4T1 breast cancer cells. The results showed that the coinjection of MSCs and 4T1 cells promoted tumor growth. MSCs might act as the tumor stromal precursors and cause immunosuppression to protect tumor cells from immunosurveillance, which subsequently facilitated tumor metastasis. Interestingly, the distant injection of MSCs and 4T1 cells suppressed tumor growth. Together, the results of this study revealed the dual functions of MSCs in immunoregulation. ©AlphaMed Press.

A biological approach to assembling tissue modules through endothelial capillary network formation.

PubMed

Riesberg, Jeremiah J; Shen, Wei

2015-09-01

To create functional tissues having complex structures, bottom-up approaches to assembling small tissue modules into larger constructs have been emerging. Most of these approaches are based on chemical reactions or physical interactions at the interface between tissue modules. Here we report a biological assembly approach to integrate small tissue modules through endothelial capillary network formation. When adjacent tissue modules contain appropriate extracellular matrix materials and cell types that support robust endothelial capillary network formation, capillary tubules form and grow across the interface, resulting in assembly of the modules into a single, larger construct. It was shown that capillary networks formed in modules of dense fibrin gels seeded with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs); adjacent modules were firmly assembled into an integrated construct having a strain to failure of 117 ± 26%, a tensile strength of 2208 ± 83 Pa and a Young's modulus of 2548 ± 574 Pa. Under the same culture conditions, capillary networks were absent in modules of dense fibrin gels seeded with either HUVECs or MSCs alone; adjacent modules disconnected even when handled gently. This biological assembly approach eliminates the need for chemical reactions or physical interactions and their associated limitations. In addition, the integrated constructs are prevascularized, and therefore this bottom-up assembly approach may also help address the issue of vascularization, another key challenge in tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Involvement of WNT Signaling in the Regulation of Gestational Age-Dependent Umbilical Cord-Derived Mesenchymal Stem Cell Proliferation

PubMed Central

Shono, Akemi; Yoshida, Makiko; Yamana, Keiji; Thwin, Khin Kyae Mon; Kuroda, Jumpei; Kurokawa, Daisuke; Koda, Tsubasa; Nishida, Kosuke; Ikuta, Toshihiko; Mizobuchi, Masami; Taniguchi-Ikeda, Mariko

2017-01-01

Mesenchymal stem cells (MSCs) are a heterogeneous cell population that is isolated initially from the bone marrow (BM) and subsequently almost all tissues including umbilical cord (UC). UC-derived MSCs (UC-MSCs) have attracted an increasing attention as a source for cell therapy against various degenerative diseases due to their vigorous proliferation and differentiation. Although the cell proliferation and differentiation of BM-derived MSCs is known to decline with age, the functional difference between preterm and term UC-MSCs is poorly characterized. In the present study, we isolated UC-MSCs from 23 infants delivered at 22–40 weeks of gestation and analyzed their gene expression and cell proliferation. Microarray analysis revealed that global gene expression in preterm UC-MSCs was distinct from term UC-MSCs. WNT signaling impacts on a variety of tissue stem cell proliferation and differentiation, and its pathway genes were enriched in differentially expressed genes between preterm and term UC-MSCs. Cell proliferation of preterm UC-MSCs was significantly enhanced compared to term UC-MSCs and counteracted by WNT signaling inhibitor XAV939. Furthermore, WNT2B expression in UC-MSCs showed a significant negative correlation with gestational age (GA). These results suggest that WNT signaling is involved in the regulation of GA-dependent UC-MSC proliferation. PMID:29138639

Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells.

PubMed

Lian, Qizhou; Zhang, Yuelin; Liang, Xiaoting; Gao, Fei; Tse, Hung-Fat

2016-01-01

Multipotent stromal cells, also known as mesenchymal stem cells (MSCs), possess great potential to generate a wide range of cell types including endothelial cells, smooth muscle cells, bone, cartilage, and lipid cells. This protocol describes in detail how to perform highly efficient, lineage-specific differentiation of human-induced pluripotent stem cells (iPSCs) with an MSCs fate. The approach uses a clinically compliant protocol with chemically defined media, feeder-free conditions, and a CD105 positive and CD24 negative selection to achieve a single cell-based MSCs derivation from differentiating human pluripotent cells in approximately 20 days. Cells generated with this protocol express typical MSCs surface markers and undergo adipogenesis, osteogenesis, and chondrogenesis similar to adult bone marrow-derived MSCs (BM-MSCs). Nonetheless, compared with adult BM-MSCs, iPSC-MSCs display a higher proliferative capacity, up to 120 passages, without obvious loss of self-renewal potential and constitutively express MSCs surface antigens. MSCs generated with this protocol have numerous applications, including expansion to large scale cell numbers for tissue engineering and the development of cellular therapeutics. This approach has been used to rescue limb ischemia, allergic disorders, and cigarette smoke-induced lung damage and to model mesenchymal and vascular disorders of Hutchinson-Gilford progeria syndrome (HGPS).

Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

PubMed

Kim, So-Yeon; Kim, Ye-Ryung; Park, Woo-Jae; Kim, Han Su; Jung, Sung-Chul; Woo, So-Youn; Jo, Inho; Ryu, Kyung-Ha; Park, Joo-Won

2015-01-01

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on β-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges.

PubMed

Yong, Kar Wey; Wan Safwani, Wan Kamarul Zaman; Xu, Feng; Wan Abas, Wan Abu Bakar; Choi, Jane Ru; Pingguan-Murphy, Belinda

2015-08-01

Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.

Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells

PubMed Central

Kadekar, Darshana; Rangole, Sonal; Kale, Vaijayanti; Limaye, Lalita

2016-01-01

Background The limited cell dose in umbilical cord blood (UCB) necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS). Conditioned medium from mesenchymal stem cells (MSCs-CM) has been shown to alleviate the oxidative stress during wound healing, Alzheimer’s disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells. Methodology/Principle findings CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM). UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them. Conclusions/Significance Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability. PMID:27780236

CD44 fucosylation on mesenchymal stem cell enhances homing and macrophage polarization in ischemic kidney injury.

PubMed

Chou, Kang-Ju; Lee, Po-Tsang; Chen, Chien-Liang; Hsu, Chih-Yang; Huang, Wei-Chieh; Huang, Chien-Wei; Fang, Hua-Chang

2017-01-01

The lack of homing ability possibly reduces the healing potential of bone-marrow-derived mesenchymal stem cells (MSCs). Therefore, transforming native CD44 on MSCs into a hematopoietic cell E-/L-selectin ligand (HCELL) that possesses potent E-selectin affinity might enhance the homing and regenerative abilities of MSCs. Through fucosyltransferase VI (FTVI) transfection, MSCs were fucosylated on N-glycans of CD44 to become HCELL positive, thus interacting with E-selectin on injured endothelial cells. HCELL expression facilitated MSC homing in kidneys within 24h after injury and reduced lung stasis. An in vitro adhesion assay revealed that transfection enhanced the association between MSCs and hypoxic endothelial cells. In mice treated with HCELL-positive MSCs, the injured kidneys exhibited clusters of homing MSCs, whereas MSCs were rarely observed in mouse kidneys treated with HCELL-negative MSCs. Most MSCs were initially localized at the renal capsule, and some MSCs later migrated inward between tubules. Most homing MSCs were in close contact with inflammatory cells without tubular transdifferentiation. Furthermore, HCELL-positive MSCs substantially alleviated renal injury, partly by enhancing the polarization of infiltrating macrophages. In conclusion, engineering the glycan of CD44 on MSCs through FTVI transfection might enhance renotropism and the regenerating ability of MSCs in ischemic kidney injury. Copyright © 2016 Elsevier Inc. All rights reserved.

Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugino, Noriko; Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507; Miura, Yasuo, E-mail: ym58f5@kuhp.kyoto-u.ac.jp

Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansionmore » of CD34{sup +} hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation. - Highlights: • Human BM-MSCs in an early osteoinductive stage (e-MSCs) support hematopoiesis. • Adhesion- and chemotaxis-associated gene signatures are altered in e-MSCs. • Expression of CXCL12 and VCAM1 is remarkably decreased in e-MSCs. • e-MSCs are at differentiation stage between MSCs and osteoprogenitor cells. • Osteoinductive treatment favors hematopoietic recovery after BMT in mice.« less

Optimizing surface characteristics for cell adhesion and proliferation on titanium plasma spray coatings on polyetheretherketone.

PubMed

Yoon, Byung Jo Victor; Xavier, Fred; Walker, Brendon R; Grinberg, Samuel; Cammisa, Frank P; Abjornson, Celeste

2016-10-01

Titanium plasma spray coating on polyetheretherketone (PEEK) is a recent innovation to interbody spacer technology. The inherent hydrophobic properties of standard, uncoated PEEK implants can hamper cell attachment and bone healing during fusion. The addition of titanium coating not only offers initial stability due to increased surface roughness but also long-term stability due to bony ongrowth created from osteoconductive microenvironment on the device surface. The previously established hydrophilic and osteophilic properties of commercially pure titanium (CPTi) can potentially provide an ideal environment promoting cell attachment and bony ongrowth when applied at the end plate level of the fusion site. Because the surface material composition and topography is what seems to directly affect cell adhesion, it is important to determine the ideal titanium coating for the highest effectiveness. The purpose of the study is to determine whether there is an optimal surface roughness for the titanium coatings and whether different polishing methods have a greater effect than roughness or topography in mediating cell adhesion to the surface. The study was divided into two phases. In Phase 1, the effects of varying surface roughnesses on identical polishing method were compared. In Phase 2, the effect of varying polishing methods was compared on identical surface roughnesses. Coating thickness, porosity, and surface roughness were characterized using an optical microscope as per ASTM F 1854 standards. For both phases, PEEK coupons with plasma-sprayed CPTi were used, and human mesenchymal stem cells (hMSCs) at an initial density of 25,000 cells/cm 2 were seeded and cultured for 24 hours before fixation in 10% formalin. The cultured hMSCs were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining, a fluorescent stain that binds to the DNA of living cells. Samples were imaged using an environmental scanning electron microscope (eSEM) (Carl Zeiss Microscopy, Thornwood, NY, USA) using a backscattered detector. Image analysis of the CPTi coatings showed uniform and rough surfaces. For Phase 1, roughness was evaluated as fine, medium, and coarse. The eSEM image analysis and cell counting by DAPI demonstrated that hMSCs have a tendency to form stronger adhesion and greater pseudopodia extensions on fine roughness surfaces. Individual hMSCs were seen forming cytoplasmic processes extending across the width of a pore. There was a 4- and 20-fold reduction in adhered hMSCs with an increase to medium and coarse roughnesses, respectively. For Phase 2, studied groups are (1) medium CPTi coating with zirconia polishing, (2) medium CPTi coating with CPTi polishing, and (3) fine CPTi coating with CPTi polishing. The eSEM image analysis and cell counting by DAPI demonstrated that hMSCs have a tendency to form stronger adhesion and greater pseudopodia extensions on Group 3 over the other two groups. There was a twofold reduction in adhered hMSCs on medium roughness relative to fine. No difference in cell adhesion was found between Groups 1 and 2. Individual hMSCs were seen forming cytoplasmic processes extending across the width of a pore. Previously, it was accepted without much scrutiny that surface coatings were beneficial. This study begins to discover that surface topography directly affects the potential for cells to adhere and proliferate and lead to greater surgical efficacy. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhanced Healing of Rat Calvarial Defects with MSCs Loaded on BMP-2 Releasing Chitosan/Alginate/Hydroxyapatite Scaffolds

PubMed Central

He, Xiaoning; Liu, Yang; Yuan, Xue; Lu, Li

2014-01-01

In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2), and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs) by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy. PMID:25084008

Isolation and Characterisation of Mesenchymal Stem Cells from Rat Bone Marrow and the Endosteal Niche: A Comparative Study

PubMed Central

Yusop, Norhayati; Battersby, Paul; Alraies, Amr; Moseley, Ryan

2018-01-01

Within bone, mesenchymal stromal cells (MSCs) exist within the bone marrow stroma (BM-MSC) and the endosteal niche, as cells lining compact bone (CB-MSCs). This study isolated and characterised heterogeneous MSC populations from each niche and subsequently investigated the effects of extensive cell expansion, analysing population doublings (PDs)/cellular senescence, colony-forming efficiencies (CFEs), MSC cell marker expression, and osteogenic/adipogenic differentiation. CB-MSCs and BM-MSCs demonstrated similar morphologies and PDs, reaching 100 PDs. Both populations exhibited consistent telomere lengths (12–17 kb), minimal senescence, and positive telomerase expression. CB-MSCs (PD15) had significantly lower CFEs than PD50. CB-MSCs and BM-MSCs both expressed MSC (CD73/CD90/CD105); embryonic (Nanog) and osteogenic markers (Runx2, osteocalcin) but no hematopoietic markers (CD45). CB-MSCs (PD15) strongly expressed Oct4 and p16INK4A. At early PDs, CB-MSCs possessed a strong osteogenic potency and low potency for adipogenesis, whilst BM-MSCs possessed greater overall bipotentiality for osteogenesis and adipogenesis. At PD50, CB-MSCs demonstrated reduced potency for both osteogenesis and adipogenesis, compared to BM-MSCs at equivalent PDs. This study demonstrates similarities in proliferative and mesenchymal cell characteristics between CB-MSCs and BM-MSCs, but contrasting multipotentiality. Such findings support further comparisons of human CB-MSCs and BM-MSCs, facilitating selection of optimal MSC populations for regenerative medicine purposes. PMID:29765418

Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

PubMed

Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

2017-09-29

Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK cell lysis and, hence, their potential in vivo lifespan. Our results further support the use of HIF-1 alpha-expressing dental MSCs for cell therapy in tissue injury and immune disorders.

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Yanfu; Chai, Jiake, E-mail: cjk304@126.com; Sun, Tianjun

2011-10-07

Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. Inmore » this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.« less

Apatite-coated Silk Fibroin Scaffolds to Healing Mandibular Border Defects in Canines

PubMed Central

Zhao, Jun; Zhang, Zhiyuan; Wang, Shaoyi; Sun, Xiaojuan; Zhang, Xiuli; Chen, Jake; Kaplan, David L.; Jiang, Xinquan

2010-01-01

Tissue engineering has become a new approach for repairing bony defects. Highly porous osteoconductive scaffolds perform the important role for the success of bone regeneration. By biomimetic strategy, apatite-coated porous biomaterial based on silk fibroin scaffolds (SS) might provide an enhanced osteogenic environment for bone-related outcomes. To assess the effects of apatite-coated silk fibroin (mSS) biomaterials for bone healing as a tissue engineered bony scaffold, we explored a tissue engineered bony graft using mSS seeded with osteogenically induced autologous bone marrow stromal cells (bMSCs) to repair inferior mandibular border defects in a canine model. The results were compared with those treated with bMSCs/SS constructs, mSS alone, SS alone, autologous mandibular grafts and untreated blank defects. According to radiographic and histological examination, new bone formation was observed from 4 weeks post-operation, and the defect site was completely repaired after 12 months for the bMSCs/mSS group. In the bMSCs/SS group, new bone formation was observed with more residual silk scaffold remaining at the center of the defect compared with the bMSCs/mSS group. The engineered bone with bMSCs/mSS achieved satisfactory bone mineral densities (BMD) at 12 months post-operation close to those of normal mandible (p>0.05). The quantities of newly formed bone area for the bMSCs/mSS group was higher than the bMSCs/SS group (p<0.01), but no significant differences were found when compared with the autograft group (p>0.05). In contrast, bony defects remained in the center with undegraded silk fibroin scaffold and fibrous connective tissue, and new bone only formed at the periphery in the groups treated with mSS or SS alone. The results suggested apatite-coated silk fibroin scaffolds combined with bMSCs could be successfully used to repair mandibular critical size border defects and the premineralization of these porous silk fibroin protein scaffolds provided an increased osteoconductive environment for bMSCs to regenerate sufficient new bone tissue. PMID:19505603

Impact of adipose tissue or umbilical cord derived mesenchymal stem cells on the immunogenicity of human cord blood derived endothelial progenitor cells

PubMed Central

Tan, Kefang; Zheng, Ke; Li, Daiye; Lu, Haiyuan; Wang, Siqi; Sun, Xuan

2017-01-01

The application of autologous endothelial progenitor cell (EPC) transplantation is a promising approach in therapeutic cardiovascular diseases and ischemic diseases. In this study, we compared the immunogenicity of EPCs, adipose tissue (AD)-derived mesenchymal stem cells (MSCs) and umbilical cord (UC)-derived MSCs by flow cytometry and the mixed lymphocyte reaction. The impact of AD-MSCs and UC-MSCs on the immunogenicity of EPCs was analyzed by the mixed lymphocyte reaction and cytokine secretion in vitro and was further tested by allogenic peripheral blood mononuclear cell (PBMC) induced immuno-rejection on a cell/matrigel graft in an SCID mouse model. EPCs and AD-MSCs express higher levels of MHC class I than UC-MSCs. All three kinds of cells are negative for MHC class II. UC-MSCs also express lower levels of IFN-γ receptor mRNA when compared with EPCs and AD-MSCs. EPCs can stimulate higher rates of proliferation of lymphocytes than AD-MSCs and UC-MSCs. Furthermore, AD-MSCs and UC-MSCs can modulate immune response and inhibit lymphocyte proliferation induced by EPCs, mainly through inhibition of the proliferation of CD8+ T cells. Compared with UC-MSCs, AD-MSCs can significantly improve vessel formation and maintain the integrity of neovascular structure in an EPC+MSC/matrigel graft in SCID mice, especially under allo-PBMC induced immuno-rejection. In conclusion, our study shows that AD-MSC is a powerful candidate to minimize immunological rejection and improve vessel formation in EPC transplantation treatment. PMID:28562647

Enhanced gastric cancer growth potential of mesenchymal stem cells derived from gastric cancer tissues educated by CD4+ T cells.

PubMed

Xu, Rongman; Zhao, Xiangdong; Zhao, Yuanyuan; Chen, Bin; Sun, Li; Xu, Changgen; Shen, Bo; Wang, Mei; Xu, Wenrong; Zhu, Wei

2018-04-01

Gastric cancer mesenchymal stem cells (GC-MSCs) can promote the development of tumour growth. The tumour-promoting role of tumour-associated MSCs and T cells has been demonstrated. T cells as the major immune cells may influence and induce a pro-tumour phenotype in MSCs. This study focused on whether CD4 + T cells can affect GC-MSCs to promote gastric cancer growth. CD4 + T cells upregulation of programmed death ligand 1 (PD-L1) expression in GC-MSCs through the phosphorylated signal transducer and activator of transcription (p-STAT3) signalling pathway was confirmed by immunofluorescence, western blotting and RT-PCR. Migration of GC cells was detected by Transwell migration assay, and apoptosis of GC cells was measured by flow cytometry using annexin V/propidium iodide double staining. CD4 + T cell-primed GC-MSCs promoted GC growth in a subcutaneously transplanted tumour model in BALB/c nu/nu mice. Gastric cancer mesenchymal stem cells stimulated by activated CD4 + T cells promoted migration of GC cells and enhanced GC growth potential in BALB/c nu/nu xenografts. PD-L1 upregulation of GC-MSCs stimulated by CD4 + T cells was mediated through the p-STAT3 signalling pathway. CD4 + T cells-primed GC-MSCs have greater GC volume and growth rate-promoting role than GC-MSCs, with cancer cell-intrinsic PD-1/mammalian target of rapamycin (mTOR) signalling activation. This study showed that GC-MSCs are plastic. The immunophenotype of GC-MSCs stimulated by CD4 + T cells has major changes that may influence tumour cell growth. This research was based on the interaction between tumour cells, MSCs and immune cells, providing a new understanding of the development and immunotherapy of GC. © 2017 John Wiley & Sons Ltd.

Recovery of Donor Hematopoiesis after Graft Failure and Second Hematopoietic Stem Cell Transplantation with Intraosseous Administration of Mesenchymal Stromal Cells

PubMed Central

Sats, Natalia; Risinskaya, Natalya; Sudarikov, Andrey; Dubniak, Daria; Kraizman, Alina

2018-01-01

Multipotent mesenchymal stromal cells (MSCs) participate in the formation of bone marrow niches for hematopoietic stem cells. Donor MSCs can serve as a source of recovery for niches in patients with graft failure (GF) after allogeneic bone marrow (BM) transplantation. Since only few MSCs reach the BM after intravenous injection, MSCs were implanted into the iliac spine. For 8 patients with GF after allo-BMT, another hematopoietic stem cell transplantation with simultaneous implantation of MSCs from their respective donors into cancellous bone was performed. BM was aspirated from the iliac crest of these patients at 1-2, 4-5, and 9 months after the intraosseous injection of donor MSCs. Patients' MSCs were cultivated, and chimerism was determined. In 6 out of 8 patients, donor hematopoiesis was restored. Donor cells (9.4 ± 3.3%) were detected among MSCs. Thus, implanted MSCs remain localized at the site of administration and do not lose the ability to proliferate. These results suggest that MSCs could participate in the restoration of niches for donor hematopoietic cells or have an immunomodulatory effect, preventing repeated rejection of the graft. Perhaps, intraosseous implantation of MSCs contributes to the success of the second transplantation of hematopoietic stem cells and patient survival. PMID:29760731

Recovery of Donor Hematopoiesis after Graft Failure and Second Hematopoietic Stem Cell Transplantation with Intraosseous Administration of Mesenchymal Stromal Cells.

PubMed

Petinati, Nataliya; Drize, Nina; Sats, Natalia; Risinskaya, Natalya; Sudarikov, Andrey; Drokov, Michail; Dubniak, Daria; Kraizman, Alina; Nareyko, Maria; Popova, Natalia; Firsova, Maya; Kuzmina, Larisa; Parovichnikova, Elena; Savchenko, Valeriy

2018-01-01

Multipotent mesenchymal stromal cells (MSCs) participate in the formation of bone marrow niches for hematopoietic stem cells. Donor MSCs can serve as a source of recovery for niches in patients with graft failure (GF) after allogeneic bone marrow (BM) transplantation. Since only few MSCs reach the BM after intravenous injection, MSCs were implanted into the iliac spine. For 8 patients with GF after allo-BMT, another hematopoietic stem cell transplantation with simultaneous implantation of MSCs from their respective donors into cancellous bone was performed. BM was aspirated from the iliac crest of these patients at 1-2, 4-5, and 9 months after the intraosseous injection of donor MSCs. Patients' MSCs were cultivated, and chimerism was determined. In 6 out of 8 patients, donor hematopoiesis was restored. Donor cells (9.4 ± 3.3%) were detected among MSCs. Thus, implanted MSCs remain localized at the site of administration and do not lose the ability to proliferate. These results suggest that MSCs could participate in the restoration of niches for donor hematopoietic cells or have an immunomodulatory effect, preventing repeated rejection of the graft. Perhaps, intraosseous implantation of MSCs contributes to the success of the second transplantation of hematopoietic stem cells and patient survival.

Human adipose tissue-derived mesenchymal stem cells inhibit T-cell lymphoma growth in vitro and in vivo.

PubMed

Ahn, Jin-Ok; Chae, Ji-Sang; Coh, Ye-Rin; Jung, Woo-Sung; Lee, Hee-Woo; Shin, Il-Seob; Kang, Sung-Keun; Youn, Hwa-Young

2014-09-01

Human mesenchymal stem cells (hMSCs) are thought to be one of the most reliable stem cell sources for a variety of cell therapies. This study investigated the anti-tumor effect of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) on EL4 murine T-cell lymphoma in vitro and in vivo. The growth-inhibitory effect of hAT-MSCs on EL4 tumor cells was evaluated using a WST-1 cell proliferation assay. Cell-cycle arrest and apoptosis were investigated by flow cytometry and western blot. To evaluate an anti-tumor effect of hAT-MSCs on T-cell lymphoma in vivo, CM-DiI-labeled hAT-MSCs were circumtumorally injected in tumor-bearing nude mice, and tumor size was measured. hAT-MSCs inhibited T-cell lymphoma growth by altering cell-cycle progression and inducing apoptosis in vitro. hAT-MSCs inhibited tumor growth in tumor-bearing nude mice and prolonged survival time. Immunofluorescence analysis showed that hAT-MSCs migrated to tumor sites. hAT-MSCs suppress the growth of T-cell lymphoma, suggesting a therapeutic option for T-cell lymphoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Improved osteogenesis and upregulated immunogenicity in human placenta-derived mesenchymal stem cells primed with osteogenic induction medium.

PubMed

Fu, Xuejie; Yang, Huilin; Zhang, Hui; Wang, Guichao; Liu, Ke; Gu, Qiaoli; Tao, Yunxia; Chen, Guangcun; Jiang, Xiaohua; Li, Gang; Gu, Yanzheng; Shi, Qin

2016-09-20

Mesenchymal stem cells (MSCs) are widely used in cell-based therapy owing to their multilineage potential and low immunogenicity. However, low differentiation efficiency and unpredictable immunogenicity of allogeneic MSCs in vivo limit their success in therapeutic treatment. Herein, we evaluated the differentiation potential and immunogenicity of human placenta-derived MSCs manipulated with osteogenic priming and dedifferentiation process. MSCs from human placentas were subjected to osteogenic induction and then cultivated in osteogenic factor-free media; the obtained cell population was termed dedifferentiated mesenchymal stem cells (De-MSCs). De-MSCs were induced into osteo-, chondro- and adipo-differentiation in vitro. Cell proliferation was quantified by a Cell-Counting Kit-8 or tritiated thymidine ([(3)H]-TdR) incorporation. Meanwhile, the osteogenesis of De-MSCs in vivo was assayed by real-time PCR and histological staining. The expressions of stem cell markers and co-stimulatory molecules on De-MSCs and lymphocytes from primed BALB/c mouse with De-MSCs were determined by flow cytometry. De-MSCs exhibited some properties similar to MSCs including multiple differentiation potential and hypoimmunogenicity. Upon re-osteogenic induction, De-MSCs exhibited higher differentiation capability than MSCs both in vitro and in vivo. Of note, De-MSCs had upregulated immunogenicity in association with their osteogenesis, reflected by the alternated expressions of co-stimulatory molecules on the surface and decreased suppression on T cell activation. Functionally, De-MSC-derived osteoblasts could prime lymphocytes of peripheral blood and spleen in BALB/c mice in vivo. These data are of great significance for the potential application of De-MSCs as an alternative resource for regenerative medicine and tissue engineering. In order to avoid being rejected by the host during allogeneic De-MSC therapy, we suggest that immune intervention should be considered to boost the immune acceptance and integration because of the upregulated immunogenicity of De-MSCs with redifferentiation in clinical applications.

Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival

PubMed Central

Facco, Monica; Chiodin, Giorgia; Frezzato, Federica; Martini, Veronica; Gattazzo, Cristina; Lessi, Federica; Giorgi, Carlo Alberto; Visentin, Andrea; Castelli, Monica; Severin, Filippo; Zambello, Renato; Piazza, Francesco; Semenzato, Gianpietro; Trentin, Livio

2015-01-01

Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines. As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients. PMID:26517523

Transplanted Umbilical Cord Mesenchymal Stem Cells Modify the In Vivo Microenvironment Enhancing Angiogenesis and Leading to Bone Regeneration

PubMed Central

Todeschi, Maria Rosa; El Backly, Rania; Capelli, Chiara; Daga, Antonio; Patrone, Eugenio; Introna, Martino; Cancedda, Ranieri

2015-01-01

Umbilical cord mesenchymal stem cells (UC-MSCs) show properties similar to bone marrow mesenchymal stem cells (BM-MSCs), although controversial data exist regarding their osteogenic potential. We prepared clinical-grade UC-MSCs from Wharton's Jelly and we investigated if UC-MSCs could be used as substitutes for BM-MSCs in muscoloskeletal regeneration as a more readily available and functional source of MSCs. UC-MSCs were loaded onto scaffolds and implanted subcutaneously (ectopically) and in critical-sized calvarial defects (orthotopically) in mice. For live cell-tracking experiments, UC-MSCs were first transduced with the luciferase gene. Angiogenic properties of UC-MSCs were tested using the mouse metatarsal angiogenesis assay. Cell secretomes were screened for the presence of various cytokines using an array assay. Analysis of implanted scaffolds showed that UC-MSCs, contrary to BM-MSCs, remained detectable in the implants for 3 weeks at most and did not induce bone formation in an ectopic location. Instead, they induced a significant increase of blood vessel ingrowth. In agreement with these observations, UC-MSC-conditioned medium presented a distinct and stronger proinflammatory/chemotactic cytokine profile than BM-MSCs and a significantly enhanced angiogenic activity. When UC-MSCs were orthotopically transplanted in a calvarial defect, they promoted increased bone formation as well as BM-MSCs. However, at variance with BM-MSCs, the new bone was deposited through the activity of stimulated host cells, highlighting the importance of the microenvironment on determining cell commitment and response. Therefore, we propose, as therapy for bone lesions, the use of allogeneic UC-MSCs by not depositing bone matrix directly, but acting through the activation of endogenous repair mechanisms. PMID:25685989

Mesenchymal Stem Cell Preparation and Transfection-free Ferumoxytol Labeling for MRI Cell Tracking.

PubMed

Liu, Li; Ho, Chien

2017-11-15

Mesenchymal stem cells (MSCs) are multipotent cells and are the most widely studied cell type for stem cell therapies. In vivo cell tracking of MSCs labeled with an FDA-approved superparamagnetic iron-oxide (SPIO) particle by magnetic resonance imaging (MRI) provides essential information, e.g., MSC engraftment, survival, and fate, thus improving cell therapy accuracy. However, current methodology for labeling MSCs with Ferumoxytol (Feraheme ® ), the only FDA-approved SPIO particle, needs transfection agents. This unit describes a new "bio-mimicry" protocol to prepare more native MSCs by using more "in vivo environment" of MSCs, so that the phagocytic activity of cultured MSCs is restored and expanded MSCs can be labeled with Ferumoxytol, without the need for transfection agents and/or electroporation. Moreover, MSCs re-size to a more native size, reducing from 32.0 to 19.5 μm. The MSCs prepared from this protocol retain more native properties and would be useful for biomedical applications and MSC-tracking studies by MRI. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Isolation and characterization of human mesenchymal stem cells derived from synovial fluid by magnetic-activated cell sorting (MACS).

PubMed

Jia, Zhaofeng; Liang, Yujie; Xu, Xiao; Li, Xingfu; Liu, Qisong; Ou, Yangkan; Duan, Li; Zhu, Weimin; Lu, Wei; Xiong, Jianyi; Wang, Daping

2018-03-01

Mesenchymal stem cells (MSCs) are the primary source of cells used for cell-based therapy in tissue engineering. MSCs are found in synovial fluid, a source that could be conveniently used for cartilage tissue engineering. However, the purification and characterization of SF-MSCs has been poorly documented in the literature. Here, we outline an easy-to-perform approach for the isolation and culture of MSCs derived from human synovial fluid (hSF-MSCs). We have successfully purified hSF-MSCs using magnetic-activated cell sorting (MACS) using the MSC surface marker, CD90. Purified SF-MSCs demonstrate significant renewal capacity following several passages in culture. Furthermore, we demonstrated that MACS-sorted CD90 + cells could differentiated into osteoblasts, adipocytes, and chondrocytes in vitro. In addition, we show that these cells can generate cartilage tissue in micromass culture as well. This study demonstrates that MACS is a useful tool that can be used for the purification of hSF-MSCs from synovial fluid. The proliferation properties and ability to differentiate into chondrocytes make these hSF-MSCs a promising source of stem cells for applications in cartilage repair. © 2017 International Federation for Cell Biology.

Effect of Priming of Multipotent Mesenchymal Stromal Cells with Interferon γ on Their Immunomodulating Properties.

PubMed

Kapranov, N M; Davydova, Y O; Galtseva, I V; Petinati, N A; Drize, N I; Kuzmina, L A; Parovichnikova, E N; Savchenko, V G

2017-10-01

Multipotent mesenchymal stromal cells (MSCs) are widely used for cell therapy, in particular for prophylaxis and treatment of graft-versus-host disease. Due to their immunomodulatory properties, MSCs affect the composition of lymphocyte subpopulations, which depends on the immunological state of the organism and can change in different diseases and during treatment. Administration of MSCs is not always effective. Treatment of MSCs with different cytokines (in particular IFN-γ) leads to enhancement of their immunomodulatory properties. The aim of this study was to investigate subpopulational alterations and activation markers in lymphocytes (activated and non-activated) after interaction with MSCs and MSCs pretreated with IFN-γ (γMSCs) in vitro. Lymphocytes were co-cultured with MSCs or γMSCs for 4 days. The proportion of CD4+ and CD8 + expressing CD25, CD38, CD69, HLA-DR, and PD-1 and distribution of memory and effector subsets were measured by flow cytometry after co-cultivation of lymphocytes with MSCs or γMSCs. The distribution of lymphocyte subpopulations changes during culturing. In non-activated lymphocytes cultured without MSCs, decrease in the proportion of naïve cells and increase in the number of effector cells was observed. That could be explained as activation of lymphocytes in the presence of serum in culturing medium. Co-culturing of lymphocytes with MSCs and γMSCs leads to retention of their non-activated state. Activation of lymphocytes with phytohemagglutinin increases the number of central memory cells and activates marker expression. Interaction with MSCs and γMSCs prevents activation of lymphocytes and keeps their naïve state. Priming with IFN-γ did not induce MSCs inhibitory effect on activation of lymphocytes.

Injectable calcium phosphate with hydrogel fibers encapsulating induced pluripotent, dental pulp and bone marrow stem cells for bone repair

PubMed Central

Wang, Lin; Zhang, Chi; Li, Chunyan; Weir, Michael D.; Wang, Ping; Reynolds, Mark A.; Zhao, Liang; Xu, Hockin H.K.

2017-01-01

Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs), dental pulp stem cells (hDPSCs) and bone marrow MSCs (hBMSCs) are exciting cell sources in regenerative medicine. However, there has been no report comparing hDPSCs, hBMSCs and hiPSC-MSCs for bone engineering in an injectable calcium phosphate cement (CPC) scaffold. The objectives of this study were to: (1) develop a novel injectable CPC containing hydrogel fibers encapsulating stem cells for bone engineering, and (2) compare cell viability, proliferation and osteogenic differentiation of hDPSCs, hiPSC-MSCs from bone marrow (BM-hiPSC-MSCs) and from foreskin (FS-hiPSC-MSCs), and hBMSCs in CPC for the first time. The results showed that the injection did not harm cell viability. The porosity of injectable CPC was 62%. All four types of cells proliferated and differentiated down the osteogenic lineage inside hydrogel fibers in CPC. hDPSCs, BM-hiPSC-MSCs, and hBMSCs exhibited high alkaline phosphatase, runt-related transcription factor, collagen I, and osteocalcin gene expressions. Cell-synthesized minerals increased with time (p < 0.05), with no significant difference among hDPSCs, BM-hiPSC-MSCs and hBMSCs (p > 0.1). Mineralization by hDPSCs, BM-hiPSC-MSCs, and hBMSCs inside CPC at 14 d was 14-fold that at 1 d. FS-hiPSC-MSCs were inferior in osteogenic differentiation compared to the other cells. In conclusion, hDPSCs, BM-hiPSC-MSCs and hBMSCs are similarly and highly promising for bone tissue engineering; however, FS-hiPSC-MSCs were relatively inferior in osteogenesis. The novel injectable CPC with cell-encapsulating hydrogel fibers may enhance bone regeneration in dental, craniofacial and orthopedic applications. PMID:27612810

Hydrogel fibers encapsulating hiPSC-MSCs, hESC-MSCs and hUCMSCs in injectable calcium phosphate scaffold for bone tissue engineering

PubMed Central

Wang, Lin; Wang, Ping; Weir, Michael D.; Reynolds, Mark A.; Zhao, Liang; Xu, Hockin H. K.

2016-01-01

Human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs) and human umbilical cord MSCs (hUCMSCs) are exciting cell sources for use in regenerative medicine. There has been no report on long hydrogel fibers encapsulating stem cells inside injectable calcium phosphate cement (CPC) scaffold for bone tissue engineering. The objectives of this study were to: (1) develop a novel injectable CPC construct containing hydrogel fibers encapsulating cells for bone engineering, and (2) investigate and compare cell viability, proliferation and osteogenic differentiation of hiPSC-MSCs, hESC-MSCs and hUCMSCs in injectable CPC. The stem cell-encapsulating pastes were fully injectable under a small injection force, and the injection did not harm the cells, compared to cells without injection (p > 0.1). Mechanical properties of stem cell-CPC construct were much higher than previous injectable polymers and hydrogels for cell delivery. hiPSC-MSCs, hESC-MSCs and hUCMSCs in hydrogel fibers in CPC had excellent proliferation and osteogenic differentiation. All three cells yielded high alkaline phosphatase, runt-related transcription factor, collagen I, and osteocalcin expressions (mean ± sd; n = 6). Cell-synthesized minerals increased substantially with time (p < 0.05), with no significant difference among the three types of cells (p > 0.1). Mineralization by hiPSC-MSCs, hESC-MSCs and hUCMSCs in CPC at 14 d was 13-fold that at 1 d. In conclusion, all three types of cells (hiPSC-MSCs, hESC-MSCs and hUCMSCs) in CPC scaffold showed high potential for bone tissue engineering, and the novel injectable CPC construct with cell-encapsulating hydrogel fibers is promising to enhance bone regeneration in dental, craniofacial and orthopedic applications. PMID:27811389

Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells' therapeutic efficacy for myocardial infarction.

PubMed

Han, Jin; Kim, Bokyoung; Shin, Jung-Youn; Ryu, Seungmi; Noh, Myungkyung; Woo, Jongsu; Park, Jin-Sil; Lee, Youjin; Lee, Nohyun; Hyeon, Taeghwan; Choi, Donghoon; Kim, Byung-Soo

2015-03-24

Electrophysiological phenotype development and paracrine action of mesenchymal stem cells (MSCs) are the critical factors that determine the therapeutic efficacy of MSCs for myocardial infarction (MI). In such respect, coculture of MSCs with cardiac cells has windowed a platform for cardiac priming of MSCs. Particularly, active gap junctional crosstalk of MSCs with cardiac cells in coculture has been known to play a major role in the MSC modification through coculture. Here, we report that iron oxide nanoparticles (IONPs) significantly augment the expression of connexin 43 (Cx43), a gap junction protein, of cardiomyoblasts (H9C2), which would be critical for gap junctional communication with MSCs in coculture for the generation of therapeutic potential-improved MSCs. MSCs cocultured with IONP-harboring H9C2 (cocultured MSCs: cMSCs) showed active cellular crosstalk with H9C2 and displayed significantly higher levels of electrophysiological cardiac biomarkers and a cardiac repair-favorable paracrine profile, both of which are responsible for MI repair. Accordingly, significantly improved animal survival and heart function were observed upon cMSC injection into rat MI models compared with the injection of unmodified MSCs. The present study highlights an application of IONPs in developing gap junctional crosstalk among the cells and generating cMSCs that exceeds the reparative potentials of conventional MSCs. On the basis of our finding, the potential application of IONPs can be extended in cell biology and stem cell-based therapies.

Mesenchymal stem cells from the Wharton's jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture.

PubMed

Bakhshi, Tiki; Zabriskie, Ryan C; Bodie, Shamanique; Kidd, Shannon; Ramin, Susan; Paganessi, Laura A; Gregory, Stephanie A; Fung, Henry C; Christopherson, Kent W

2008-12-01

Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow-derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton's jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated. Umbilical cord-derived MSCs (UC-MSCs) were cultured from the Wharton's jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay. Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays. These data suggest the potential therapeutic application of Wharton's jelly-derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation.

Tumor Associated Mesenchymal Stromal Cells Show Higher Immunosuppressive and Angiogenic Properties Compared to Adipose Derived MSCs.

PubMed

Langroudi, Ladan; Hassan, Zuhair Muhammad; Soleimani, Masoud; Hashemi, Seyed Mahmoud

2015-12-01

Differentiation, migratory properties and availability of Mesenchymal Stromal Cells (MSC) have become an important part of biomedical research. However, the functional heterogeneity of cells derived from different tissues has hampered providing definitive phenotypic markers for these cells. To characterize and compare the phenotype and cytokines of adipose derived MSCs (AD-MSCs) and tumoral-MSCs (T-MSCs) isolated from mammary tumors of BALB/c mice. Immunophenotyping and in vitro differentiation tests were used for MSC characterization. Cytokine and enzyme profiles were assessed using ELISA and Real-time PCR, respectively. T-MSCs expressed significantly higher levels of HLA-DR (p=0.04). Higher levels of PGE2 and COX-2 enzyme were also observed in T-MSCs (p=0.07 and p=0.00, respectively). Additionally, T-MSCs expressed higher levels of iNOS and MMP9 (p=0.01 and p=0.01, respectively). T-MSCs were also able to induce higher levels of proliferation and migration of HUVEC endothelial cells in wound scratch assay compared to AD-MSCs (p=0.015). Functional differences showed by the surface markers of MSCs, cytokine and enzyme production indicate the effect of different microenvironments on MSCs phenotype and function.

Subretinal transplantation of rat MSCs and erythropoietin gene modified rat MSCs for protecting and rescuing degenerative retina in rats.

PubMed

Guan, Y; Cui, L; Qu, Z; Lu, L; Wang, F; Wu, Y; Zhang, J; Gao, F; Tian, H; Xu, L; Xu, G; Li, W; Jin, Y; Xu, G-T

2013-11-01

For degenerative retinal diseases, like the acquired form exemplified by age-related macular degeneration (AMD), there is currently no cure. This study was to explore a stem cell therapy and a stem cell based gene therapy for sodium iodate (SI)-induced retinal degeneration in rats. Three cell types, i.e., rat mesenchymal stem cells (rMSCs) alone, erythropoietin (EPO) gene modified rMSCs (EPO-rMSCs) or doxycycline (DOX) inducible EPO expression rMSCs (Tet-on EPO-rMSCs), were transplanted into the subretinal spaces of SI-treated rats. The rMSCs were prepared for transplantation after 3 to 5 passages or modified with EPO gene. During the 8 weeks after the transplantation, the rats treated with rMSCs alone or with two types of EPO-rMSCs were all monitored with fundus examination, fundus fluorescein angiography (FFA) and electroretinogram. The transplantation efficiency of donor cells was examined for their survival, integration and differentiation. Following the transplantation, labeled donor cells were observed in subretinal space and adopted RPE morphology. EPO concentration in vitreous and retina of SI-treated rats which were transplanted with EPO-rMSCs or Tet-on EPO-rMSCs was markedly increased, in parallel with the improvement of retinal morphology and function. These findings suggest that rMSCs transplantation could be a new therapy for degenerative retinal diseases since it can protect and rescue RPE and retinal neurons, while EPO gene modification to rMSCs could be an even better option.

Increased apoptosis and peripheral blood mononuclear cell suppression of bone marrow mesenchymal stem cells in severe aplastic anemia.

PubMed

Chao, Yu-Hua; Lin, Chiao-Wen; Pan, Hui-Hsien; Yang, Shun-Fa; Weng, Te-Fu; Peng, Ching-Tien; Wu, Kang-Hsi

2018-06-05

Although immune-mediated pathogenesis is considered an important aspect of severe aplastic anemia (SAA), its underlying mechanisms remain unclear. Mesenchymal stem cells (MSCs) are essential to the formation of specialized microenvironments in the bone marrow (BM), and MSC insufficiency can trigger the development of SAA. To find MSC alterations in the SAA BM, we compared BM MSCs from five children with SAA and five controls. Peripheral blood mononuclear cells (PBMCs) were cocultured with MSCs to evaluate the supportive effects of MSCs on hematopoiesis. Cytometric bead array immunoassay was used to determine cytokine excretion by MSCs. The immune functions of MSCs and their conditioned medium (CM) were evaluated by PBMC proliferation assays. SAA MSCs were characterized by a high percentage of cells in the abnormal sub-G1 phase of the cell cycle, which suggests an increased rate of apoptosis in SAA MSCs. In comparison with control MSCs, PBMCs cocultured with SAA MSCs displayed significantly reduced PBMC proliferation (P = 0.009). Aberrant cytokine profiles were secreted by SAA MSCs, with increased concentrations of interleukin-6, interferon-γ, tumor necrosis factor-α, and interleukin-1β in the CM. PBMC proliferation assays demonstrated additional immunosuppressive effects of SAA MSCs (P = 0.016) and their CM (P = 0.013). Our data revealed increased apoptosis and PBMC suppression of SAA MSCs. The alterations of MSCs may contribute to the formation of functionally abnormal microenvironments in SAA BM. © 2018 Wiley Periodicals, Inc.

Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone.

PubMed

Smith, Christopher A; Board, Tim N; Rooney, Paul; Eagle, Mark J; Richardson, Stephen M; Hoyland, Judith A

2017-01-01

To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC) osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC). BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP) enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1) concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors.

Student Award for Outstanding Research Winner in the Ph.D. Category for the 9th World Biomaterials Congress, Chengdu, China, June 1-5, 2012: The interplay of bone-like extracellular matrix and TNF-α signaling on in vitro osteogenic differentiation of mesenchymal stem cells.

PubMed

Mountziaris, Paschalia M; Tzouanas, Stephanie N; Mikos, Antonios G

2012-05-01

As an initial step in the development of a bone tissue engineering strategy to rationally control inflammation, we investigated the interplay of bone-like extracellular matrix (ECM) and varying doses of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) on osteogenically differentiating mesenchymal stem cells (MSCs) cultured in vitro on 3D poly(ε-caprolactone) (PCL) microfiber scaffolds containing pregenerated bone-like ECM. To generate the ECM, PCL scaffolds were seeded with MSCs and cultured in medium containing the typically required osteogenic supplement dexamethasone. However, since dexamethasone antagonizes TNF-α, the interplay of ECM and TNF-α was investigated by culturing naïve MSCs on the decellularized scaffolds in the absence of dexamethasone. MSCs cultured on ECM-coated scaffolds continued to deposit mineralized matrix, a late stage marker of osteogenic differentiation. Mineralized matrix deposition was not adversely affected by exposure to TNF-α for 4-8 days, but was significantly reduced after continuous exposure to TNF-α over 16 days, which simulates the in vivo response, where brief TNF-α signaling stimulates bone regeneration, while prolonged exposure has damaging effects. This underscores the exciting potential of PCL/ECM constructs as a more clinically realistic in vitro culture model to facilitate the design of new bone tissue engineering strategies that rationally control inflammation to promote regeneration. Copyright © 2012 Wiley Periodicals, Inc.

Mesenchymal Stem Cells Modulate Differentiation of Myeloid Progenitor Cells During Inflammation.

PubMed

Amouzegar, Afsaneh; Mittal, Sharad K; Sahu, Anuradha; Sahu, Srikant K; Chauhan, Sunil K

2017-06-01

Mesenchymal stem cells (MSCs) possess distinct immunomodulatory properties and have tremendous potential for use in therapeutic applications in various inflammatory diseases. MSCs have been shown to regulate pathogenic functions of mature myeloid inflammatory cells, such as macrophages and neutrophils. Intriguingly, the capacity of MSCs to modulate differentiation of myeloid progenitors (MPs) to mature inflammatory cells remains unknown to date. Here, we report the novel finding that MSCs inhibit the expression of differentiation markers on MPs under inflammatory conditions. We demonstrate that the inhibitory effect of MSCs is dependent on direct cell-cell contact and that this intercellular contact is mediated through interaction of CD200 expressed by MSCs and CD200R1 expressed by MPs. Furthermore, using an injury model of sterile inflammation, we show that MSCs promote MP frequencies and suppress infiltration of inflammatory cells in the inflamed tissue. We also find that downregulation of CD200 in MSCs correlates with abrogation of their immunoregulatory function. Collectively, our study provides unequivocal evidence that MSCs inhibit differentiation of MPs in the inflammatory environment via CD200-CD200R1 interaction. Stem Cells 2017;35:1532-1541. © 2017 AlphaMed Press.

Isolation and culture of human multipotent stromal cells from the pancreas.

PubMed

Seeberger, Karen L; Eshpeter, Alana; Korbutt, Gregory S

2011-01-01

Mesenchymal stem cells, also termed multipotent mesenchymal stromal cells (MSCs), can be isolated from most adult tissues. Although the exact origin of MSCs expanded from the human pancreas has not been resolved, we have developed protocols to isolate and expand MSCs from human pancreatic tissue that remains after islet procurement. Similar to techniques used to isolate MSCs from bone marrow, pancreatic MSCs are isolated based on their cell adherence, expression of several cell surface antigens, and multilineage differentiation. The protocols for isolating, characterizing, and differentiating MSCs from the pancreas are presented in this chapter.

Concise review: adult multipotent stromal cells and cancer: risk or benefit?

PubMed Central

Lazennec, Gwendal; Jorgensen, Christian

2008-01-01

This review will focus on the interaction between multipotent stromal cells (MSCs) and carcinoma and the possible use of MSCs in cell-based anti-cancer therapies. MSCs are present in multiple tissues and are defined as cells displaying the ability to differentiate in multiple lineages including chondrocytes, osteoblasts and adipocytes. Recent evidence suggests also that they could play a role in the progression of carcinogenesis and that MSCs could migrate towards primary tumors and metastatic sites. It is possible that MSCs could be also involved in the early stages of carcinogenesis through spontaneous transformation. In addition, it is thought that MSCs can modulate tumor growth and metastasis, although this issue remains controversial and not well understood. The immuno-suppressive properties and pro-angiogenic properties of MSCs account, at least in part, for their effects on cancer development. On the other hand, cancer cells also have the ability to enhance MSC migration. This complex dialog between MSCs and cancer cells is certainly critical for the outcome of tumor development. Interestingly, several studies have shown that MSCs engineered to express anti-tumor factors could be an innovative choice as a cell-mediated gene therapy to counteract tumor growth. More evidence will be needed to understand how MSCs positively or negatively modulate carcinogenesis and to evaluate the safety of MSCs use in cell-mediated gene strategies. PMID:18388305

Differential effects of culture senescence and mechanical stimulation on the proliferation and leiomyogenic differentiation of MSC from different sources: implications for engineering vascular grafts.

PubMed

Koobatian, Maxwell T; Liang, Mao-Shih; Swartz, Daniel D; Andreadis, Stelios T

2015-04-01

We examined the effects of senescence on the proliferation and leiomyogenic differentiation potential of mesenchymal stem cells (MSCs) isolated from bone marrow (BM-MSCs) or hair follicles (HF-MSCs). To this end, we compared ovine HF-MSCs and BM-MSCs in terms of their proliferation and differentiation potential to the smooth muscle cell lineage. We discovered that HF-MSCs are less susceptible to culture senescence compared with BM-MSCs. We hypothesized that application of mechanical forces may enhance the contractility and mechanical properties of vascular constructs prepared from senescent MSCs. Interestingly, HF-MSCs and BM-MSCs responded differently to changes in the mechanical microenvironment, suggesting that despite phenotypic similarities, MSCs from different anatomic locations may activate different pathways in response to the same microenvironmental factors. In turn, this may also suggest that cell-based tissue regeneration approaches may need to be tailored to the stem cell origin, donor age, and culture time for optimal results.

Curcumin Inhibits Chondrocyte Hypertrophy of Mesenchymal Stem Cells through IHH and Notch Signaling Pathways.

PubMed

Cao, Zhen; Dou, Ce; Dong, Shiwu

2017-01-01

Using tissue engineering technique to repair cartilage damage caused by osteoarthritis is a promising strategy. However, the regenerated tissue usually is fibrous cartilage, which has poor mechanical characteristics compared to hyaline cartilage. Chondrocyte hypertrophy plays an important role in this process. Thus, it is very important to find out a suitable way to maintain the phenotype of chondrocytes and inhibit chondrocyte hypertrophy. Curcumin deriving from turmeric was reported with anti-inflammatory and anti-tumor pharmacological effects. However, the role of curcumin in metabolism of chondrocytes, especially in the chondrocyte hypertrophy remains unclear. Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering as seed cells. So we investigated the effect of curcumin on chondrogenesis and chondrocyte hypertrophy in MSCs through examination of cell viability, glycosaminoglycan synthesis and specific gene expression. We found curcumin had no effect on expression of chondrogenic markers including Sox9 and Col2a1 while hypertrophic markers including Runx2 and Col10a1 were down-regulated. Further exploration showed that curcumin inhibited chondrocyte hypertrophy through Indian hedgehog homolog (IHH) and Notch signalings. Our results indicated curcumin was a potential agent in modulating cartilage homeostasis and maintaining chondrocyte phenotype.

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

PubMed Central

Kim, Eun Young; Lee, Kyung-Bon; Kim, Min Kyu

2014-01-01

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140] PMID:24499672

Transcriptomic profile induced in bone marrow mesenchymal stromal cells after interaction with multiple myeloma cells: implications in myeloma progression and myeloma bone disease

PubMed Central

Garcia-Gomez, Antonio; Las Rivas, Javier De; Ocio, Enrique M.; Díaz-Rodríguez, Elena; Montero, Juan C.; Martín, Montserrat; Blanco, Juan F.; Sanchez-Guijo, Fermín M.; Pandiella, Atanasio; San Miguel, Jesús F.; Garayoa, Mercedes

2014-01-01

Despite evidence about the implication of the bone marrow (BM) stromal microenvironment in multiple myeloma (MM) cell growth and survival, little is known about the effects of myelomatous cells on BM stromal cells. Mesenchymal stromal cells (MSCs) from healthy donors (dMSCs) or myeloma patients (pMSCs) were co-cultured with the myeloma cell line MM.1S, and the transcriptomic profile of MSCs induced by this interaction was analyzed. Deregulated genes after co-culture common to both d/pMSCs revealed functional involvement in tumor microenvironment cross-talk, myeloma growth induction and drug resistance, angiogenesis and signals for osteoclast activation and osteoblast inhibition. Additional genes induced by co-culture were exclusively deregulated in pMSCs and predominantly associated to RNA processing, the ubiquitine-proteasome pathway, cell cycle regulation, cellular stress and non-canonical Wnt signaling. The upregulated expression of five genes after co-culture (CXCL1, CXCL5 and CXCL6 in d/pMSCs, and Neuregulin 3 and Norrie disease protein exclusively in pMSCs) was confirmed, and functional in vitro assays revealed putative roles in MM pathophysiology. The transcriptomic profile of pMSCs co-cultured with myeloma cells may better reflect that of MSCs in the BM of myeloma patients, and provides new molecular insights to the contribution of these cells to MM pathophysiology and to myeloma bone disease. PMID:25268740

Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b{sup +} myeloid cells to the lungs and facilitates B16-F10 melanoma colonization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Souza, Lucas E.B., E-mail: lucasebsouza@usp.br; Hemotherapy Center of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP; Almeida, Danilo C., E-mail: gudaalmeida@gmail.com

The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice weremore » subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b{sup +} myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize “premetastatic niches” in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b{sup +} myeloid cells and tumor cells. - Highlights: • BM-MSCs enhanced B16-F10 proliferation in a dose-dependent manner in vitro. • BM-MSCs facilitated lung colonization by B16-F10 melanoma cells. • BM-MSCs administration did not alter the number of endothelial cells and T lymphocytes in the lungs. • BM-MSCs enhanced the recruitment of CD11b{sup +} myeloid cells during tumor colonization.« less

CXCR4-mediated osteosarcoma growth and pulmonary metastasis is promoted by mesenchymal stem cells through VEGF.

PubMed

Zhang, Peng; Dong, Ling; Yan, Kang; Long, Hua; Yang, Tong-Tao; Dong, Ming-Qing; Zhou, Yong; Fan, Qing-Yu; Ma, Bao-An

2013-10-01

Chemokines and chemokine receptor 4 (CXCR4) play an important role in metastasis. CXCR4 is also expressed in the human osteosarcoma cell line 9607-F5M2 (F5M2), which has a high tumorigenic ability and potential for spontaneous pulmonary metastasis. Mesenchymal stem cells (MSCs) contribute to the formation of the tumor stroma and promote metastasis. However, mechanisms underlying the promotion of osteosarcoma growth and pulmonary metastasis by MSCs are still elusive. Our study co-injected the human MSCs and F5M2 cells into the caudal vein of nude mice. The total number of tumor nodules per lung was significantly increased in the F5M2+MSC group compared to the other groups (control, F5M2 cells alone and MSCs alone) at week six. Moreover, a high number of Dil-labeled MSCs was present also at the osteosarcoma metastasis sites in the lung. Using Transwell assays, we found that F5M2 cells migrate towards MSCs, while the CXCR4 inhibitor AMD3100 decreased the migration potential of F5M2 cells towards MSCs. Furthermore, upon treatment with F5M2-conditioned medium, MSCs expressed and secreted higher levels of VEGF as determined by immunohistochemistry, western blotting and ELISA, respectively. Importantly, co-cultured with F5M2 cells, MSCs expressed and secreted higher VEGF levels, while AMD3100 dramatically decreased the VEGF secretion by MSCs. However, CXCR4 expression on F5M2 cells was not significantly increased in the co-culture system. Additionally, VEGF increased the proliferation of both MSCs and F5M2 cells. These findings suggest that CXCR4-mediated osteosarcoma growth and pulmonary metastasis are promoted by MSCs through VEGF.

Human bone marrow-derived clonal mesenchymal stem cells inhibit inflammation and reduce acute pancreatitis in rats.

PubMed

Jung, Kyung Hee; Song, Sun U; Yi, Tacghee; Jeon, Myung-Shin; Hong, Sang-Won; Zheng, Hong-Mei; Lee, Hee-Seung; Choi, Myung-Joo; Lee, Don-Haeng; Hong, Soon-Sun

2011-03-01

Acute pancreatitis (AP) has a high mortality rate; repetitive AP induces chronic AP and pancreatic adenocarcinoma. Mesenchymal stem cells (MSCs) have immunoregulatory effects and reduce inflammation. We developed a protocol to isolate human bone marrow-derived clonal MSCs (hcMSCs) from bone marrow aspirate and investigated the effects of these cells in rat models of mild and severe AP. Mild AP was induced in Sprague-Dawley rats by 3 intraperitoneal injections of cerulein (100 μg/kg), given at 2-hour intervals; severe AP was induced by intraparenchymal injection of 3% sodium taurocholate solution. hcMSCs were labeled with CM-1,1'-dioctadecyl-3,3,3'-tetramethylindo-carbocyanine perchloride and administered to rats through the tail vein. hcMSCs underwent self-renewal and had multipotent differentiation capacities and immunoregulatory functions. Greater numbers of infused hcMSCs were detected in pancreas of rats with mild and severe AP than of control rats. Infused hcMSCs reduced acinar-cell degeneration, pancreatic edema, and inflammatory cell infiltration in each model of pancreatitis. The hcMSCs reduced expression of inflammation mediators and cytokines in rats with mild and severe AP. hcMSCs suppressed the mixed lymphocyte reaction and increased expression of Foxp3(+) (a marker of regulatory T cells) in cultured rat lymph node cells. Rats with mild or severe AP that were given infusions of hcMSCs had reduced numbers of CD3(+) T cells and increased expression of Foxp3(+) in pancreas tissues. hcMSCs reduced inflammation and damage to pancreatic tissue in a rat model of AP; they reduced levels of cytokines and induced numbers of Foxp3(+) regulatory T cells. hcMSCs might be developed as a cell therapy for pancreatitis. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Mesenchymal stem cells with overexpression of midkine enhance cell survival and attenuate cardiac dysfunction in a rat model of myocardial infarction.

PubMed

Zhao, Shu-Li; Zhang, Yao-Jun; Li, Ming-Hui; Zhang, Xin-Lei; Chen, Shao-Liang

2014-03-17

Elevated midkine (MK) expression may contribute to ventricular remodeling and ameliorate cardiac dysfunction after myocardial infarction (MI). Ex vivo modification of signaling mechanisms in mesenchymal stem cells (MSCs) with MK overexpression may improve the efficacy of cell-based therapy. This study sought to assess the safety and efficacy of MSCs with MK overexpression transplantation in a rat model of MI. A pLenO-DCE vector lentivirus encoding MK was constructed and infected in MSCs. MSC migration activity and cytoprotection was examined in hypoxia-induced H9C2 cells using transwell insert in vitro. Rats were randomized into five groups: sham, MI plus injection of phosphate buffered saline (PBS), MSCs, MSCs-green fluorescent protein (MSCs-GFP) and MSCs-MK, respectively. Survival rates were compared among groups using log-rank test and left ventricular function was measured by echocardiography at baseline, 4, 8 and 12 weeks. Overexpression of MK partially prevented hypoxia-induced MSC apoptosis and exerted MSC cytoprotection to anoxia induced H9C2 cells. The underlying mechanisms may be associated with the increased mRNA and protein levels of vascular endothelial growth factor (VEGF), transformation growth factor-β (TGF-β), insulin-like growth factor 1 (IGF-1) and stromal cell-derived factor 1 (SDF-1a) in MSCs-MK compared with isolated MSCs and MSCs-GFP. Consistent with the qPCR results, the culture supernatant of MSCs-MK had more SDF-1a (9.23 ng/ml), VEGF (8.34 ng/ml) and TGF-β1 (17.88 ng/ml) expression. In vivo, a greater proportion of cell survival was observed in the MSCs-MK group than in the MSCs-GFP group. Moreover, MSCs-MK administration was related to a significant improvement of cardiac function compared with other control groups at 12 weeks. Therapies employing MSCs with MK overexpression may represent an effective treatment for improving cardiac dysfunction and survival rate after MI.

Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.

PubMed

Morgan, Joshua T; Wood, Joshua A; Walker, Naomi J; Raghunathan, Vijay Krishna; Borjesson, Dori L; Murphy, Christopher J; Russell, Paul

2014-01-01

To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

Human Trabecular Meshwork Cells Exhibit Several Characteristics of, but Are Distinct from, Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Morgan, Joshua T.; Wood, Joshua A.; Walker, Naomi J.; Raghunathan, Vijay Krishna; Borjesson, Dori L.; Murphy, Christopher J.

2014-01-01

Abstract Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities. PMID:24456002

A living thick nanofibrous implant bifunctionalized with active growth factor and stem cells for bone regeneration.

PubMed

Eap, Sandy; Keller, Laetitia; Schiavi, Jessica; Huck, Olivier; Jacomine, Leandro; Fioretti, Florence; Gauthier, Christian; Sebastian, Victor; Schwinté, Pascale; Benkirane-Jessel, Nadia

2015-01-01

New-generation implants focus on robust, durable, and rapid tissue regeneration to shorten recovery times and decrease risks of postoperative complications for patients. Herein, we describe a new-generation thick nanofibrous implant functionalized with active containers of growth factors and stem cells for regenerative nanomedicine. A thick electrospun poly(ε-caprolactone) nanofibrous implant (from 700 μm to 1 cm thick) was functionalized with chitosan and bone morphogenetic protein BMP-7 as growth factor using layer-by-layer technology, producing fish scale-like chitosan/BMP-7 nanoreservoirs. This extracellular matrix-mimicking scaffold enabled in vitro colonization and bone regeneration by human primary osteoblasts, as shown by expression of osteocalcin, osteopontin, and bone sialoprotein (BSPII), 21 days after seeding. In vivo implantation in mouse calvaria defects showed significantly more newly mineralized extracellular matrix in the functionalized implant compared to a bare scaffold after 30 days' implantation, as shown by histological scanning electron microscopy/energy dispersive X-ray microscopy study and calcein injection. We have as well bifunctionalized our BMP-7 therapeutic implant by adding human mesenchymal stem cells (hMSCs). The activity of this BMP-7-functionalized implant was again further enhanced by the addition of hMSCs to the implant (living materials), in vivo, as demonstrated by the analysis of new bone formation and calcification after 30 days' implantation in mice with calvaria defects. Therefore, implants functionalized with BMP-7 nanocontainers associated with hMSCs can act as an accelerator of in vivo bone mineralization and regeneration.

Comparative analysis of human UCB and adipose tissue derived mesenchymal stem cells for their differentiation potential into brown and white adipocytes.

PubMed

Rashnonejad, Afrooz; Ercan, Gulinnaz; Gunduz, Cumhur; Akdemir, Ali; Tiftikcioglu, Yigit Ozer

2018-06-01

The differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) into brown and white adipocytes in comparison to Adipose tissue derived MSCs (AD-MSCs) were investigated in order to characterize their potency for future cell therapies. MSCs were isolated from ten UCB samples and six liposuction materials. MSCs were differentiated into white and brown adipocytes after characterization by flow cytometry. Differentiated adipocytes were stained with Oil Red O and hematoxylin/eosin. The UCP1 protein levels in brown adipocytes were investigated by immunofluoresence and western blot analysis. Cells that expressed mesenchymal stem cells markers (CD34-, CD45-, CD90+ and CD105+) were successfully isolated from UCB and adipose tissue. Oil Red O staining demonstrated that white and brown adipocytes obtained from AD-MSCs showed 85 and 61% of red pixels, while it was 3 and 1.9%, respectively for white and brown adipocytes obtained from UCB-MSCs. Fluorescence microscopy analysis showed strong uncoupling protein 1 (UCP1) signaling in brown adipocytes, especially which were obtained from AD-MSCs. Quantification of UCP1 protein amount showed 4- and 10.64-fold increase in UCP1 contents of brown adipocytes derived from UCB-MSCs and AD-MSCs, respectively in comparison to undifferentiated MSCs (P < 0.004). UCB-MSCs showed only a little differentiation tendency into adipocytes means it is not an appropriate stem cell type to be differentiated into these cell types. In contrast, high differentiation efficiency of AD-MSCs into brown and white adipocytes make it appropriate stem cell type to use in future regenerative medicine of soft tissue disorders or fighting with obesity and its related disorders.

Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells.

PubMed

Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Juritz, Stephanie; Birk, Richard; Goessler, Ulrich Reinhart; Bieback, Karen; Bugert, Peter; Schultz, Johannes; Hörmann, Karl; Kinscherf, Ralf; Faber, Anne

2014-01-01

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium.

Mesenchymal stem cells derived from bone marrow of diabetic patients portrait unique markers influenced by the diabetic microenvironment.

PubMed

Phadnis, Smruti M; Ghaskadbi, Surendra M; Hardikar, Anandwardhan A; Bhonde, Ramesh R

2009-01-01

Cellular microenvironment is known to play a critical role in the maintenance of human bone marrow-derived mesenchymal stem cells (BM-MSCs). It was uncertain whether BM-MSCs obtained from a 'diabetic milieu' (dBM-MSCs) offer the same regenerative potential as those obtained from healthy (non-diabetic) individuals (hBM-MSCs). To investigate the effect of diabetic microenvironment on human BM-MSCs, we isolated and characterized these cells from diabetic patients (dBM-MSCs). We found that dBM-MSCs expressed mesenchymal markers such as vimentin, smooth muscle actin, nestin, fibronectin, CD29, CD44, CD73, CD90, and CD105. These cells also exhibited multilineage differentiation potential, as evident from the generation of adipocytes, osteocytes, and chondrocytes when exposed to lineage specific differentiation media. Although the cells were similar to hBM-MSCs, 6% (3/54) of dBM-MSCs expressed proinsulin/C-peptide. Emanating from the diabetic microenvironmental milieu, we analyzed whether in vitro reprogramming could afford the maturation of the islet-like clusters (ICAs) derived from dBM-MSCs. Upon mimicking the diabetic hyperglycemic niche and the supplementation of fetal pancreatic extract, to differentiate dBM-MSCs into pancreatic lineage in vitro, we observed rapid differentiation and maturation of dBM-MSCs into islet-like cell aggregates. Thus, our study demonstrated that diabetic hyperglycemic microenvironmental milieu plays a major role in inducing the differentiation of human BM-MSCs in vivo and in vitro.

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation.

PubMed

Cuerquis, Jessica; Romieu-Mourez, Raphaëlle; François, Moïra; Routy, Jean-Pierre; Young, Yoon Kow; Zhao, Jing; Eliopoulos, Nicoletta

2014-02-01

Mesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α-pretreated human bone marrow-derived MSCs on resting or activated T cells. MSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed. Unprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions. Unprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

[Differentiation of mesenchymal stem cells into cardiomyocytes induced by cardiomyocytes].

PubMed

Wang, Ting-Zhong; Ma, Ai-Qun; Xu, Zheng-Yun; Jiang, Wen-Hui; Du, Yuan

2005-06-01

To investigate the role of adult cardiomyocytes in the differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. Rat MSCs were isolated by a Percoll's gradient solution and cultured in low-glucose Dulbecco' s modified Eagle' s medium (DMEM). After 2 passages, cell-surface antigen CD34, CD71 and CD90 for rat MSCs were determined by flow cytometry, and these MSCs were transfected with pEGFP-N3 by Lipofectamine2000. Then those MSCs labeled with GFP, were cultured in contacted, nocontacted and conditioned with adult rat myocardiocytes. Immunofluorescence staining against alpha-actin, desmin, and troponin-T were performed after 1 week. Immunofluorescence staining was positive against alpha-actin, desmin, and troponin-T on MSCs in contacted culture group. In contrast, no alpha-actin, desmin, and troponin-T expression on MSCs were observed in the noncontacted culture group and the conditioned culture group. Direct cell-to-cell contact between MSCs and adult cardiomyocytes may induce differentiation of MSCs into cardiomyocytes.

Differential efficacy of human mesenchymal stem cells based on source of origin

PubMed Central

Collins, Erin; Gu, Fei; Qi, Maosong; Molano, Ivan; Ruiz, Phillip; Sun, Linyun; Gilkeson, Gary S.

2014-01-01

Mesenchymal stem cells (MSCs) are useful in tissue repair, but also possess immunomodulatory properties. Murine and uncontrolled human trials suggest efficacy of MSCs in treating lupus. Autologous cells are preferable, however, recent studies suggest that lupus derived MSCs lack efficacy in treating disease. Thus, the optimum derivation of MSCs for use in lupus is unknown. It is also unknown which in vitro assays of MSC function predict in vivo efficacy. The objectives for this study were to provide insight into the optimum source of MSCs and to identify in vitro assays that predict in vivo efficacy. We derived MSCs from four umbilical cords (UC), four healthy bone marrows (HBM) and four lupus bone marrows (LBM). In diseased MRL/lpr mice, MSCs from HBM and UC significantly decreased renal disease, while LBM-MSCs only delayed disease. Current in vitro assays did not differentiate efficacy of the different MSCs. Inhibition of B cell proliferation did differentiate based on efficacy. Our results suggest that autologous MSCs from lupus patients are not effective in treating disease. Furthermore, standard in vitro assays for MSC licensing are not predictive of in vivo efficacy, while inhibiting B cell proliferation appears to differentiate effective from ineffective MSCs. PMID:25274529

Enhanced cell survival and paracrine effects of mesenchymal stem cells overexpressing hepatocyte growth factor promote cardioprotection in myocardial infarction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Liyan; Liu, Xiaolin; Zhang, Yuelin

Poor cell survival post transplantation compromises the therapeutic benefits of mesenchymal stem cells (MSCs) in myocardial infarction (MI). Hepatocyte growth factor (HGF) is an important cytokine for angiogenesis, anti-inflammation and anti-apoptosis. This study aimed to evaluate the cardioprotective effects of MSCs overexpressing HGF in a mouse model of MI. The apoptosis of umbilical cord-derived MSCs (UC-MSCs) and HGF-UC-MSCs under normoxic and hypoxic conditions was detected. The conditioned medium (CdM) of UC-MSCs and HGF-UC-MSCs under a hypoxic condition was harvested and its protective effect on neonatal cardiomyocytes (NCMs) exposed to a hypoxic challenge was examined. UC-MSCs and HGF-UC-MSCs were transplanted intomore » the peri-infarct region in mice following MI and heart function assessed 4 weeks post transplantation. The apoptosis of HGF-UC-MSCs under hypoxic conditions was markedly decreased compared with that of UC-MSCs. NCMs treated with HGF-UC-MSC hypoxic CdM (HGF-UC-MSCs-hy-CdM) exhibited less cell apoptosis in response to hypoxic challenge than those treated with UC-MSC hypoxic CdM (UC-MSCs-hy-CdM). HGF-UC-MSCs-hy-CdM released the inhibited p-Akt and lowered the enhanced ratio of Bax/Bcl-2 induced by hypoxia in the NCMs. HGF-UC-MSCs-hy-CdM expressed higher levels of HGF, EGF, bFGF and VEGF than UC-MSCs-hy-CdM. Transplantation of HGF-UC-MSCs or UC-MSCs greatly improved heart function in the mouse model of MI. Compared with UC-MSCs, transplantation of HGF-UC-MSCs was associated with less cardiomyocyte apoptosis, enhanced angiogenesis and increased proliferation of cardiomyocytes. This study may provide a novel therapeutic strategy for MSC-based therapy in cardiovascular disease.« less

Anatomic Mesenchymal Stem Cell-Based Engineered Cartilage Constructs for Biologic Total Joint Replacement

PubMed Central

Saxena, Vishal; Kim, Minwook; Keah, Niobra M.; Neuwirth, Alexander L.; Stoeckl, Brendan D.; Bickard, Kevin; Restle, David J.; Salowe, Rebecca; Wang, Margaret Ye; Steinberg, David R.

2016-01-01

Cartilage has a poor healing response, and few viable options exist for repair of extensive damage. Hyaluronic acid (HA) hydrogels seeded with mesenchymal stem cells (MSCs) polymerized through UV crosslinking can generate functional tissue, but this crosslinking is not compatible with indirect rapid prototyping utilizing opaque anatomic molds. Methacrylate-modified polymers can also be chemically crosslinked in a cytocompatible manner using ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED). The objectives of this study were to (1) compare APS/TEMED crosslinking with UV crosslinking in terms of functional maturation of MSC-seeded HA hydrogels; (2) generate an anatomic mold of a complex joint surface through rapid prototyping; and (3) grow anatomic MSC-seeded HA hydrogel constructs using this alternative crosslinking method. Juvenile bovine MSCs were suspended in methacrylated HA (MeHA) and crosslinked either through UV polymerization or chemically with APS/TEMED to generate cylindrical constructs. Minipig porcine femoral heads were imaged using microCT, and anatomic negative molds were generated by three-dimensional printing using fused deposition modeling. Molded HA constructs were produced using the APS/TEMED method. All constructs were cultured for up to 12 weeks in a chemically defined medium supplemented with TGF-β3 and characterized by mechanical testing, biochemical assays, and histologic analysis. Both UV- and APS/TEMED-polymerized constructs showed increasing mechanical properties and robust proteoglycan and collagen deposition over time. At 12 weeks, APS/TEMED-polymerized constructs had higher equilibrium and dynamic moduli than UV-polymerized constructs, with no differences in proteoglycan or collagen content. Molded HA constructs retained their hemispherical shape in culture and demonstrated increasing mechanical properties and proteoglycan and collagen deposition, especially at the edges compared to the center of these larger constructs. Immunohistochemistry showed abundant collagen type II staining and little collagen type I staining. APS/TEMED crosslinking can be used to produce MSC-seeded HA-based neocartilage and can be used in combination with rapid prototyping techniques to generate anatomic MSC-seeded HA constructs for use in filling large and anatomically complex chondral defects or for biologic joint replacement. PMID:26871863

OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells.

PubMed

Yannarelli, Gustavo; Pacienza, Natalia; Montanari, Sonia; Santa-Cruz, Diego; Viswanathan, Sowmya; Keating, Armand

2017-01-01

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment.

OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells

PubMed Central

Montanari, Sonia; Santa-Cruz, Diego; Viswanathan, Sowmya; Keating, Armand

2017-01-01

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment. PMID:29216265

Chondrogenic Differentiation Increases Antidonor Immune Response to Allogeneic Mesenchymal Stem Cell Transplantation

PubMed Central

Ryan, Aideen E; Lohan, Paul; O'Flynn, Lisa; Treacy, Oliver; Chen, Xizhe; Coleman, Cynthia; Shaw, Georgina; Murphy, Mary; Barry, Frank; Griffin, Matthew D; Ritter, Thomas

2014-01-01

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3+ and CD68+ infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use. PMID:24184966

Mesenchymal Stem/Stromal Cells under Stress Increase Osteosarcoma Migration and Apoptosis Resistance via Extracellular Vesicle Mediated Communication.

PubMed

Vallabhaneni, Krishna C; Hassler, Meeves-Yoni; Abraham, Anu; Whitt, Jason; Mo, Yin-Yuan; Atfi, Azeddine; Pochampally, Radhika

2016-01-01

Studies have shown that mesenchymal stem/stromal cells (MSCs) from bone marrow are involved in the growth and metastasis of solid tumors but the mechanism remains unclear in osteosarcoma (OS). Previous studies have raised the possibility that OS cells may receive support from associated MSCs in the nutrient deprived core of the tumors through the release of supportive macromolecules and growth factors either in vesicular or non-vesicular forms. In the present study, we used stressed mesenchymal stem cells (SD-MSCs), control MSCs and OS cells to examine the hypothesis that tumor-associated MSCs in nutrient deprived core provide pro-proliferative, anti-apoptotic, and metastatic support to nearby tumor cells. Assays to study of the effects of SD-MSC conditioned media revealed that OS cells maintained proliferation when compared to OS cells grown under serum-starved conditions alone. Furthermore, OS cells in MSCs and SD-MSC conditioned media were significantly resistant to apoptosis and an increased wound healing rate was observed in cells exposed to either conditioned media or EVs from MSCs and SD-MSCs. RT-PCR assays of OS cells incubated with extracellular vesicles (EVs) from SD-MSCs revealed microRNAs that could potentially target metabolism and metastasis associated genes as predicted by in silico algorithms, including monocarboxylate transporters, bone morphogenic receptor type 2, fibroblast growth factor 7, matrix metalloproteinase-1, and focal adhesion kinase-1. Changes in the expression levels of focal adhesion kinase, STK11 were confirmed by quantitative PCR assays. Together, these data indicate a tumor supportive role of MSCs in osteosarcoma growth that is strongly associated with the miRNA content of the EVs released from MSCs under conditions that mimic the nutrient deprived core of solid tumors.

Mesenchymal stem cells promote pancreatic adenocarcinoma cells invasion by transforming growth factor-β1 induced epithelial-mesenchymal transition.

PubMed

Zhou, Hai-Sen; Su, Xiao-Fang; Fu, Xing-Li; Wu, Guo-Zhong; Luo, Kun-Lun; Fang, Zheng; Yu, Feng; Liu, Hong; Hu, Hong-Juan; Chen, Liu-Sheng; Cai, Bing; Tian, Zhi-Qiang

2016-07-05

Mesenchymal stem cells (MSCs) could be ideal delivery vehicles for antitumor biological agents in pancreatic adenocarcinoma (PA). While the role of MSCs in tumor growth is elusive. Inflammation is an important feature of PA. In this study, we reported that MSCs pre-stimulated with the combination of TNF-α and IFN-γ promote PA cells invasion. The invasion of PA cell lines were evaluate by wound healing assay and transwell assay in vitro and liver metastasis in nude mice. We observed MSCs pre-stimulated with the combination of TNF-α and IFN-γ promoted PA cells invasion in vitro and in vivo. Consistent with MSCs promoting PA cells invasion, PA cells were found undergo epithelial-mesenchymal transition (EMT). We demonstrated that MSCs pre-stimulated with both of TNF-α and IFN-γ provoked expression transforming growth factor-β1 (TGF-β1). MSCs promoting EMT-mediated PA cells invasion could be reversed by short interfering RNA of TGF-β1. Our results suggest that MSCs could promote PA cells invasion in inflammation microenvironment and should be cautious as delivery vehicles in molecular target therapy.

Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration.

PubMed

Inoue, Yuji; Iriyama, Aya; Ueno, Shuji; Takahashi, Hidenori; Kondo, Mineo; Tamaki, Yasuhiro; Araie, Makoto; Yanagi, Yasuo

2007-08-01

Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.

Human mesenchymal stem cells - current trends and future prospective

PubMed Central

Ullah, Imran; Subbarao, Raghavendra Baregundi; Rho, Gyu Jin

2015-01-01

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials. PMID:25797907

Concise review: adult multipotent stromal cells and cancer: risk or benefit?

PubMed

Lazennec, Gwendal; Jorgensen, Christian

2008-06-01

This review focuses on the interaction between multipotent stromal cells (MSCs) and carcinoma and the possible use of MSCs in cell-based anticancer therapies. MSCs are present in multiple tissues and are defined as cells displaying the ability to differentiate in multiple lineages, including chondrocytes, osteoblasts, and adipocytes. Recent evidence also suggests that they could play a role in the progression of carcinogenesis and that MSCs could migrate toward primary tumors and metastatic sites. It is possible that MSCs could also be involved in the early stages of carcinogenesis through spontaneous transformation. In addition, it is thought that MSCs can modulate tumor growth and metastasis, although this issue remains controversial and not well understood. The immunosuppressive properties and proangiogenic properties of MSCs account, at least in part, for their effects on cancer development. On the other hand, cancer cells also have the ability to enhance MSC migration. This complex dialog between MSCs and cancer cells is certainly critical for the outcome of tumor development. Interestingly, several studies have shown that MSCs engineered to express antitumor factors could be an innovative choice as a cell-mediated gene therapy to counteract tumor growth. More evidence will be needed to understand how MSCs positively or negatively modulate carcinogenesis and to evaluate the safety of MSC use in cell-mediated gene strategies. Disclosure of potential conflicts of interest is found at the end of this article.

Involvement of Wnt/β-catenin signaling in the mesenchymal stem cells promote metastatic growth and chemoresistance of cholangiocarcinoma.

PubMed

Wang, Weiwei; Zhong, Wei; Yuan, Jiahui; Yan, Congcong; Hu, Shaoping; Tong, Yinping; Mao, Yubin; Hu, Tianhui; Zhang, Bing; Song, Gang

2015-12-08

Mesenchymal stem cells (MSCs) are multi-potent progenitor cells with ability to differentiate into multiple lineages, including bone, cartilage, fat, and muscles. Recent research indicates that MSCs can be efficiently recruited to tumor sites, modulating tumor growth and metastasis. However, the underlying molecular mechanisms are not fully understood. Here, we first demonstrated that human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), when mixed with human cholangiocarcinoma cell lines QBC939 in a xenograft tumor model, significantly increased the cancer cells proliferation and metastatic potency. MSCs and their conditioned media (MSC-CM) could improve the drug resistance of tumor when the compound K (CK) as an anti-cancer drug, a major intestinal bacterial metabolite of panaxoside, was administered to xenograft tumor mice. Furthermore, MSCs greatly increased the colony formation and invasion of cholangiocarcinoma cells QBC939 and Mz-ChA-1. Immunochemistry studies of cholangiocarcinoma tissue chips and transplantation tumor from nude mice showed that the expression of β-catenin was important for cholangiocarcinoma development. We further demonstrated that MSCs and MSCs-CM could promote proliferation and migration of cholangiocarcinoma cells through targeting the Wnt/β-catenin signaling pathway. hUC-MSCs or MSCs-CM stimulated Wnt activity by promoting the nuclear translocation of β-catenin, and up-regulated Wnt target genes MMPs family, cyclin D1 and c-Myc. Together, our studies highlight a critical role for MSCs on cancer metastasis and indicate MSCs promote metastatic growth and chemoresistance of cholangiocarcinoma cells via activation of Wnt/β-catenin signaling.

Involvement of Wnt/β-catenin signaling in the mesenchymal stem cells promote metastatic growth and chemoresistance of cholangiocarcinoma

PubMed Central

Yuan, Jiahui; Yan, Congcong; Hu, Shaoping; Tong, Yinping; Mao, Yubin; Hu, Tianhui; Zhang, Bing; Song, Gang

2015-01-01

Mesenchymal stem cells (MSCs) are multi-potent progenitor cells with ability to differentiate into multiple lineages, including bone, cartilage, fat, and muscles. Recent research indicates that MSCs can be efficiently recruited to tumor sites, modulating tumor growth and metastasis. However, the underlying molecular mechanisms are not fully understood. Here, we first demonstrated that human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), when mixed with human cholangiocarcinoma cell lines QBC939 in a xenograft tumor model, significantly increased the cancer cells proliferation and metastatic potency. MSCs and their conditioned media (MSC-CM) could improve the drug resistance of tumor when the compound K (CK) as an anti-cancer drug, a major intestinal bacterial metabolite of panaxoside, was administered to xenograft tumor mice. Furthermore, MSCs greatly increased the colony formation and invasion of cholangiocarcinoma cells QBC939 and Mz-ChA-1. Immunochemistry studies of cholangiocarcinoma tissue chips and transplantation tumor from nude mice showed that the expression of β-catenin was important for cholangiocarcinoma development. We further demonstrated that MSCs and MSCs-CM could promote proliferation and migration of cholangiocarcinoma cells through targeting the Wnt/β-catenin signaling pathway. hUC-MSCs or MSCs-CM stimulated Wnt activity by promoting the nuclear translocation of β-catenin, and up-regulated Wnt target genes MMPs family, cyclin D1 and c-Myc. Together, our studies highlight a critical role for MSCs on cancer metastasis and indicate MSCs promote metastatic growth and chemoresistance of cholangiocarcinoma cells via activation of Wnt/β-catenin signaling. PMID:26474277

Microenvironmental cues enhance mesenchymal stem cell-mediated immunomodulation and regulatory T-cell expansion.

PubMed

Kadle, Rohini L; Abdou, Salma A; Villarreal-Ponce, Alvaro P; Soares, Marc A; Sultan, Darren L; David, Joshua A; Massie, Jonathan; Rifkin, William J; Rabbani, Piul; Ceradini, Daniel J

2018-01-01

Mesenchymal stem cells (MSCs) are known to both have powerful immunosuppressive properties and promote allograft tolerance. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are optimally primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. Of particular interest is the mechanisms governing the interaction between MSCs and regulatory T cells (Tregs), which is relatively unknown. We performed our experiments utilizing rat bone marrow derived MSCs. We observed that priming MSCs in hypoxia promotes maintenance of stem-like characteristics, with greater expression of typical MSC cell-surface markers, increased proliferation, and maintenance of differentiation potential. Addition of autologous MSCs to CD4+/allogeneic endothelial cell (EC) co-culture increases regulatory T cell (Treg) proliferation, which is further enhanced when MSCs are primed in hypoxia. Furthermore, MSC-mediated Treg expansion does not require direct cell-cell contact. The expression of indolamine 2,3-dioxygenase, a mediator of MSC immunomodulation, increases when MSCs are primed in hypoxia, and inhibition of IDO significantly decreases the expansion of Tregs. Priming with inflammatory cytokines IFNγ and TNFα increases also expression of markers associated with MSC immunomodulatory function, but decreases MSC proliferation. The expression of IDO also increases when MSCs are primed with inflammatory cytokines. However, there is no increase in Treg expansion when MSCs are primed with IFNγ, suggesting an alternate mechanism for inflammatory-stimulated MSC immunomodulation. Overall, these results suggest that MSCs primed in hypoxia or inflammatory conditions are optimally primed for immunosuppressive function. These results provide a clearer picture of how to enhance MSC immunomodulation for clinical use.

Microenvironmental cues enhance mesenchymal stem cell-mediated immunomodulation and regulatory T-cell expansion

PubMed Central

Abdou, Salma A.; Villarreal-Ponce, Alvaro P.; Soares, Marc A.; Sultan, Darren L.; David, Joshua A.; Massie, Jonathan; Rabbani, Piul

2018-01-01

Mesenchymal stem cells (MSCs) are known to both have powerful immunosuppressive properties and promote allograft tolerance. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are optimally primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. Of particular interest is the mechanisms governing the interaction between MSCs and regulatory T cells (Tregs), which is relatively unknown. We performed our experiments utilizing rat bone marrow derived MSCs. We observed that priming MSCs in hypoxia promotes maintenance of stem-like characteristics, with greater expression of typical MSC cell-surface markers, increased proliferation, and maintenance of differentiation potential. Addition of autologous MSCs to CD4+/allogeneic endothelial cell (EC) co-culture increases regulatory T cell (Treg) proliferation, which is further enhanced when MSCs are primed in hypoxia. Furthermore, MSC-mediated Treg expansion does not require direct cell-cell contact. The expression of indolamine 2,3-dioxygenase, a mediator of MSC immunomodulation, increases when MSCs are primed in hypoxia, and inhibition of IDO significantly decreases the expansion of Tregs. Priming with inflammatory cytokines IFNγ and TNFα increases also expression of markers associated with MSC immunomodulatory function, but decreases MSC proliferation. The expression of IDO also increases when MSCs are primed with inflammatory cytokines. However, there is no increase in Treg expansion when MSCs are primed with IFNγ, suggesting an alternate mechanism for inflammatory-stimulated MSC immunomodulation. Overall, these results suggest that MSCs primed in hypoxia or inflammatory conditions are optimally primed for immunosuppressive function. These results provide a clearer picture of how to enhance MSC immunomodulation for clinical use. PMID:29513756

Effect of heme oxygenase-1 transduced bone marrow mesenchymal stem cells on damaged intestinal epithelial cells in vitro.

PubMed

Cao, Yi; Wu, Ben-Juan; Zheng, Wei-Ping; Yin, Ming-Li; Liu, Tao; Song, Hong-Li

2017-07-01

In this study, we explored the effects of mesenchymal stem cells (MSCs) from bone marrow overexpressing heme oxygenase-1 (HO-1) on the damaged human intestinal epithelial barrier in vitro. Rat MSCs were isolated from bone marrow and transduced with rat HO-1 recombinant adenovirus (HO-MSCs) for stable expression of HO-1. Colorectal adenocarinoma 2 (Caco2) cells were treated with tumor necrosis factor-α (TNF-α) to establish a damaged colon epithelial model. Damaged Caco2 were cocultured with MSCs, Ad-MSCs, Ad-HO + MSCs or HO-MSCs. mRNA and protein expression of Zona occludens-1 (ZO-1) and human HO-1 and the release of cytokines were measured. ZO-1 and human HO-1 in Caco2 were significantly decreased after treatment with TNF-α; and this effect was reduced when coculture with MSCs from bone marrow. Expression of ZO-1 was not significantly affected by Caco2 treatment with TNF-α, Ad-HO, and MSCs. In contrast, ZO-1 and human HO-1 increased significantly when the damaged Caco2 was treated with HO-MSCs. HO-MSCs showed the strongest effect on the expression of ZO-1 in colon epithelial cells. Coculture with HO-MSCs showed the most significant effects on reducing the expression of IL-2, IL-6, IFN-γ and increasing the expression of IL-10. HO-MSCs protected the intestinal epithelial barrier, in which endogenous HO-1 was involved. HO-MSCs play an important role in the repair process by reducing the release of inflammatory cytokines and increasing the release of anti-inflammatory factors. These results suggested that HO-MSCs from bone marrow were more effective in repairing the damaged intestinal epithelial barrier, and the effectiveness of MSCs was improved by HO-1 gene transduction, which provides favorable support for the application of stem cell therapy in the intestinal diseases. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

Usage of Human Mesenchymal Stem Cells in Cell-based Therapy: Advantages and Disadvantages.

PubMed

Kim, Hee Jung; Park, Jeong-Soo

2017-03-01

The use of human mesenchymal stem cells (hMSCs) in cell-based therapy has attracted extensive interest in the field of regenerative medicine, and it shows applications to numerous incurable diseases. hMSCs show several superior properties for therapeutic use compared to other types of stem cells. Different cell types are discussed in terms of their advantages and disadvantages, with focus on the characteristics of hMSCs. hMSCs can proliferate readily and produce differentiated cells that can substitute for the targeted affected tissue. To maximize the therapeutic effects of hMSCs, a substantial number of these cells are essential, requiring extensive ex vivo cell expansion. However, hMSCs have a limited lifespan in an in vitro culture condition. The senescence of hMSCs is a double-edged sword from the viewpoint of clinical applications. Although their limited cell proliferation potency protects them from malignant transformation after transplantation, senescence can alter various cell functions including proliferation, differentiation, and migration, that are essential for their therapeutic efficacy. Numerous trials to overcome the limited lifespan of mesenchymal stem cells are discussed.

Usage of Human Mesenchymal Stem Cells in Cell-based Therapy: Advantages and Disadvantages

PubMed Central

Kim, Hee Jung; Park, Jeong-Soo

2017-01-01

ABSTRACT The use of human mesenchymal stem cells (hMSCs) in cell-based therapy has attracted extensive interest in the field of regenerative medicine, and it shows applications to numerous incurable diseases. hMSCs show several superior properties for therapeutic use compared to other types of stem cells. Different cell types are discussed in terms of their advantages and disadvantages, with focus on the characteristics of hMSCs. hMSCs can proliferate readily and produce differentiated cells that can substitute for the targeted affected tissue. To maximize the therapeutic effects of hMSCs, a substantial number of these cells are essential, requiring extensive ex vivo cell expansion. However, hMSCs have a limited lifespan in an in vitro culture condition. The senescence of hMSCs is a double-edged sword from the viewpoint of clinical applications. Although their limited cell proliferation potency protects them from malignant transformation after transplantation, senescence can alter various cell functions including proliferation, differentiation, and migration, that are essential for their therapeutic efficacy. Numerous trials to overcome the limited lifespan of mesenchymal stem cells are discussed. PMID:28484739

In Vitro Characterization of a Stem-Cell-Seeded Triple-Interpenetrating-Network Hydrogel for Functional Regeneration of the Nucleus Pulposus

PubMed Central

Smith, Lachlan J.; Gorth, Deborah J.; Showalter, Brent L.; Chiaro, Joseph A.; Beattie, Elizabeth E.; Elliott, Dawn M.; Mauck, Robert L.; Chen, Weiliam

2014-01-01

Intervertebral disc degeneration is implicated as a major cause of low-back pain. There is a pressing need for new regenerative therapies for disc degeneration that restore native tissue structure and mechanical function. To that end we investigated the therapeutic potential of an injectable, triple-interpenetrating-network hydrogel comprised of dextran, chitosan, and teleostean, for functional regeneration of the nucleus pulposus (NP) of the intervertebral disc in a series of biomechanical, cytotoxicity, and tissue engineering studies. Biomechanical properties were evaluated as a function of gelation time, with the hydrogel reaching ∼90% of steady-state aggregate modulus within 10 h. Hydrogel mechanical properties evaluated in confined and unconfined compression were comparable to native human NP properties. To confirm containment within the disc under physiological loading, toluidine-blue-labeled hydrogel was injected into human cadaveric spine segments after creation of a nucleotomy defect, and the segments were subjected to 10,000 cycles of loading. Gross analysis demonstrated no implant extrusion, and further, that the hydrogel interdigitated well with native NP. Constructs were next surface-seeded with NP cells and cultured for 14 days, confirming lack of hydrogel cytotoxicity, with the hydrogel maintaining NP cell viability and promoting proliferation. Next, to evaluate the potential of the hydrogel to support cell-mediated matrix production, constructs were seeded with mesenchymal stem cells (MSCs) and cultured under prochondrogenic conditions for up to 42 days. Importantly, the hydrogel maintained MSC viability and promoted proliferation, as evidenced by increasing DNA content with culture duration. MSCs differentiated along a chondrogenic lineage, evidenced by upregulation of aggrecan and collagen II mRNA, and increased GAG and collagen content, and mechanical properties with increasing culture duration. Collectively, these results establish the therapeutic potential of this novel hydrogel for functional regeneration of the NP. Future work will confirm the ability of this hydrogel to normalize the mechanical stability of cadaveric human motion segments, and advance the material toward human translation using preclinical large-animal models. PMID:24410394

IL17/IL17RA as a Novel Signaling Axis Driving Mesenchymal Stem Cell Therapeutic Function in Experimental Autoimmune Encephalomyelitis.

PubMed

Kurte, Mónica; Luz-Crawford, Patricia; Vega-Letter, Ana María; Contreras, Rafael A; Tejedor, Gautier; Elizondo-Vega, Roberto; Martinez-Viola, Luna; Fernández-O'Ryan, Catalina; Figueroa, Fernando E; Jorgensen, Christian; Djouad, Farida; Carrión, Flavio

2018-01-01

The therapeutic effect of mesenchymal stem cells (MSCs) in multiple sclerosis (MS) and the experimental autoimmune encephalomyelitis (EAE) model has been well described. This effect is, in part, mediated through the inhibition of IL17-producing cells and the generation of regulatory T cells. While proinflammatory cytokines such as IFNγ, TNFα, and IL1β have been shown to enhance MSCs immunosuppressive function, the role of IL17 remains poorly elucidated. The aim of this study was, therefore, to investigate the role of the IL17/IL17R pathway on MSCs immunoregulatory effects focusing on Th17 cell generation in vitro and on Th17-mediated EAE pathogenesis in vivo . In vitro , we showed that the immunosuppressive effect of MSCs on Th17 cell proliferation and differentiation is partially dependent on IL17RA expression. This was associated with a reduced expression level of MSCs immunosuppressive mediators such as VCAM1, ICAM1, and PD-L1 in IL17RA -/- MSCs as compared to wild-type (WT) MSCs. In the EAE model, we demonstrated that while WT MSCs significantly reduced the clinical scores of the disease, IL17RA -/- MSCs injected mice exhibited a clinical worsening of the disease. The disability of IL17RA -/- MSCs to reduce the progression of the disease paralleled the inability of these cells to reduce the frequency of Th17 cells in the draining lymph node of the mice as compared to WT MSCs. Moreover, we showed that the therapeutic effect of MSCs was correlated with the generation of classical Treg bearing the CD4 + CD25 + Foxp3 + signature in an IL17RA-dependent manner. Our findings reveal a novel role of IL17RA on MSCs immunosuppressive and therapeutic potential in EAE and suggest that the modulation of IL17RA in MSCs could represent a novel method to enhance their therapeutic effect in MS.

Mesenchymal stem cells from the Wharton’s jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture

PubMed Central

Bakhshi, Tiki; Zabriskie, Ryan C.; Bodie, Shamanique; Kidd, Shannon; Ramin, Susan; Paganessi, Laura A.; Gregory, Stephanie A.; Fung, Henry C.; Christopherson, Kent W.

2012-01-01

BACKGROUND Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow–derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton’s jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated. STUDY DESIGN AND METHODS Umbilical cord–derived MSCs (UC-MSCs) were cultured from the Wharton’s jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay. RESULTS Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays. CONCLUSION These data suggest the potential therapeutic application of Wharton’s jelly–derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation. PMID:18798803

EphA2 is a biomarker of hMSCs derived from human placenta and umbilical cord.

PubMed

Shen, Shih-Pei; Liu, Wei-Ting; Lin, Yun; Li, Yuan-Tsung; Chang, Chih-Hao; Chang, Fung-Wei; Wang, Le-Ming; Teng, Sen-Wen; Hsuan, Yogi

2015-12-01

The heterogeneous nature of mesenchymal stem cells (MSCs) and the absence of known MSC-specific biomarkers make it challenging to define MSC phenotypes and characteristics. In this study, we compared the phenotypic and functional features of human placenta-derived MSCs with those of human dermal fibroblasts in vitro in order to identify a biomarker that can be used to increase the purity of MSCs in a primary culture of placenta-derived cells. Liquid chromatography-tandem mass spectrometry analysis was used to analyze and compare the proteome of human placenta-derived MSCs with that of fibroblasts. Quantitative real-time polymerase chain reaction, immunofluorescence, and flow cytometry were used to determine expression levels of EphA2 in placenta-derived MSCs. EphA2-positive cells were enriched by magnetic-activated cell sorting or with a cell sorter. An shRNA-mediated EphA2 knockdown was used to assess the role of EphA2 in MSC response to Tumor necrosis factor (TNF)-α stimulation. Analysis of proteomics data from MSCs and fibroblasts resulted in the identification of the EphA2 surface protein biomarker, which could reliably distinguish MSCs from fibroblasts. EphA2 was significantly upregulated in placenta-derived MSCs when compared to fibroblasts. EphA2 played an important role in MSC migration in response to inflammatory stimuli, such as TNF-α. EphA2-enriched MSCs were also more responsive to inflammatory stimuli in vitro when compared to unsorted MSCs, indicating a role for EphA2 in the immunomodulatory functionality of MSCs. EphA2 can be used to distinguish and isolate MSCs from a primary culture of placenta-derived cells. EphA2-sorted MSCs exhibited superior responsiveness to TNF-α signaling in an inflammatory environment compared with unsorted MSCs or MSC-like cells. Copyright © 2015. Published by Elsevier B.V.

Mesenchymal Stromal Cells Engineered to Produce IGF-I by Recombinant Adenovirus Ameliorate Liver Fibrosis in Mice

PubMed Central

Fiore, Esteban J.; Bayo, Juan M.; Garcia, Mariana G.; Malvicini, Mariana; Lloyd, Rodrigo; Piccioni, Flavia; Rizzo, Manglio; Peixoto, Estanislao; Sola, M. Beatriz; Atorrasagasti, Catalina; Alaniz, Laura; Camilletti, María A.; Enguita, Mónica; Prieto, Jesús; Aquino, Jorge B.

2015-01-01

Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis. PMID:25315017

Isolation and characterization of equine endometrial mesenchymal stromal cells.

PubMed

Rink, B Elisabeth; Amilon, Karin R; Esteves, Cristina L; French, Hilari M; Watson, Elaine; Aurich, Christine; Donadeu, F Xavier

2017-07-12

Equine mesenchymal stromal/stem cells (MSCs) are most commonly harvested from bone marrow (BM) or adipose tissue, requiring the use of surgical procedures. By contrast, the uterus can be accessed nonsurgically, and may provide a more readily available cell source. While human endometrium is known to harbor mesenchymal precursor cells, MSCs have not been identified in equine endometrium. This study reports the isolation, culture, and characterization of MSCs from equine endometrium. The presence of MSC and pericyte markers in endometrial sections was determined using immunohistochemistry. Stromal cells were harvested and cultured after separation of epithelial cells from endometrial fragments using Mucin-1-bound beads. For comparison, MSCs were also harvested from BM. The expression of surface markers in endometrial and BM-derived MSCs was characterized using flow cytometry and quantitative polymerase chain reaction. MSCs were differentiated in vitro into adipogenic, chondrogenic, osteogenic, and smooth muscle lineages. Typical markers of MSCs (CD29, CD44, CD90, and CD105) and pericytes (NG2 and CD146) were localized in the equine endometrium. Both endometrial and BM MSCs grew clonally and robustly expressed MSC and pericyte markers in culture while showing greatly reduced or negligible expression of hematopoietic markers (CD45, CD34) and MHC-II. Additionally, both endometrial and BM MSCs differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro, and endometrial MSCs had a distinct ability to undergo smooth muscle differentiation. We have demonstrated for the first time the presence of cells in equine endometrium that fulfill the definition of MSCs. The equine endometrium may provide an alternative, easily accessible source of MSCs, not only for therapeutic regeneration of the uterus, but also for other tissues where MSCs from other sources are currently being used therapeutically.

Identification of Galectin-1 as a Critical Factor in Function of Mouse Mesenchymal Stromal Cell-Mediated Tumor Promotion

PubMed Central

Blazsó, Péter; Katona, Róbert László; Novák, Julianna; Szabó, Enikő; Czibula, Ágnes; Fajka-Boja, Roberta; Hegyi, Beáta; Uher, Ferenc; Krenács, László; Joó, Gabriella; Monostori, Éva

2012-01-01

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development. These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression. PMID:22844466

Human mesenchymal stromal cells enhance the immunomodulatory function of CD8+CD28− regulatory T cells

PubMed Central

Liu, Qiuli; Zheng, Haiqing; Chen, Xiaoyong; Peng, Yanwen; Huang, Weijun; Li, Xiaobo; Li, Gang; Xia, Wenjie; Sun, Qiquan; Xiang, Andy Peng

2015-01-01

One important aspect of mesenchymal stromal cells (MSCs)-mediated immunomodulation is the recruitment and induction of regulatory T (Treg) cells. However, we do not yet know whether MSCs have similar effects on the other subsets of Treg cells. Herein, we studied the effects of MSCs on CD8+CD28− Treg cells and found that the MSCs could not only increase the proportion of CD8+CD28− T cells, but also enhance CD8+CD28−T cells' ability of hampering naive CD4+ T-cell proliferation and activation, decreasing the production of IFN-γ by activated CD4+ T cells and inducing the apoptosis of activated CD4+ T cells. Mechanistically, the MSCs affected the functions of the CD8+CD28− T cells partially through moderate upregulating the expression of IL-10 and FasL. The MSCs had no distinct effect on the shift from CD8+CD28+ T cells to CD8+CD28− T cells, but did increase the proportion of CD8+CD28− T cells by reducing their rate of apoptosis. In summary, this study shows that MSCs can enhance the regulatory function of CD8+CD28− Treg cells, shedding new light on MSCs-mediated immune regulation. PMID:25482073

Osteogenically differentiated mesenchymal stem cells and ceramics for bone tissue engineering.

PubMed

Ohgushi, Hajime

2014-02-01

In the human body, cells having self-renewal and multi-differentiation capabilities reside in many tissues and are called adult stem cells. In bone marrow tissue, two types of stem cells are well known: hematopoietic stem cells and mesenchymal stem cells (MSCs). Though the number of MSCs in bone marrow tissue is very low, it can be increased by in vitro culture of the marrow, and culture-expanded MSCs are available for various tissue regeneration. The culture-expanded MSCs can further differentiate into osteogenic cells such as bone forming osteoblasts by culturing the MSCs in an osteogenic medium. This paper discusses osteogenically differentiated MSCs derived from the bone marrow of patients. Importantly, the differentiation can be achieved on ceramic surfaces which demonstrate mineralized bone matrix formation as well as appearance of osteogenic cells. The cell/matrix/ceramic constructs could show immediate in vivo bone formation and are available for bone reconstruction surgery. Currently, MSCs are clinically available for the regeneration of various tissues due to their high proliferation/differentiation capabilities. However, the capabilities are still limited and thus technologies to improve or recover the inherent capabilities of MSCs are needed.

Key Transcription Factors in the Differentiation of Mesenchymal Stem Cells

PubMed Central

Almalki, Sami G.; Agrawal, Devendra K.

2016-01-01

Mesenchymal stem cells (MSCs) are multipotent cells that represent a promising source for regenerative medicine. MSCs are capable of osteogenic, chondrogenic, adipogenic and myogenic differentiation. Efficacy of differentiated MSCs to regenerate cells in the injured tissues requires the ability to maintain the differentiation toward the desired cell fate. Since MSCs represent an attractive source for autologous transplantation, cellular and molecular signaling pathways and micro-environmental changes have been studied in order to understand the role of cytokines, chemokines, and transcription factors on the differentiation of MSCs. The differentiation of MSC into a mesenchymal lineage is genetically manipulated and promoted by specific transcription factors associated with a particular cell lineage. Recent studies have explored the integration of transcription factors, including Runx2, Sox9, PPARγ, MyoD, GATA4, and GATA6 in the differentiation of MSCs. Therefore, the overexpression of a single transcription factor in MSCs may promote trans-differentiation into specific cell lineage, which can be used for treatment of some diseases. In this review, we critically discussed and evaluated the role of transcription factors and related signaling pathways that affect the differentiation of MSCs toward adipocytes, chondrocytes, osteocytes, skeletal muscle cells, cardiomyocytes, and smooth muscle cells. PMID:27012163

Feline bone marrow-derived mesenchymal stromal cells (MSCs) show similar phenotype and functions with regards to neuronal differentiation as human MSCs.

PubMed

Munoz, Jessian L; Greco, Steven J; Patel, Shyam A; Sherman, Lauren S; Bhatt, Suresh; Bhatt, Rekha S; Shrensel, Jeffrey A; Guan, Yan-Zhong; Xie, Guiqin; Ye, Jiang-Hong; Rameshwar, Pranela; Siegel, Allan

2012-09-01

Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimer's disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin βIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimer's disease and stroke. Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells.

PubMed

Pourgholaminejad, Arash; Aghdami, Nasser; Baharvand, Hossein; Moazzeni, Seyed Mohammad

2016-09-01

Mesenchymal stem cells (MSCs), as cells with potential clinical utilities, have demonstrated preferential incorporation into inflammation sites. Immunophenotype and immunomodulatory functions of MSCs could alter by inflamed-microenvironments due to the local pro-inflammatory cytokine milieu. A major cellular mediator with specific function in promoting inflammation and pathogenicity of autoimmunity are IL-17-producing T helper 17 (Th17) cells that polarize in inflamed sites in the presence of pro-inflammatory cytokines such as Interleukin-1β (IL-1β), IL-6 and IL-23. Since MSCs are promising candidate for cell-based therapeutic strategies in inflammatory and autoimmune diseases, Th17 cell polarizing factors may alter MSCs phenotype and function. In this study, human bone-marrow-derived MSCs (BM-MSC) and adipose tissue-derived MSCs (AD-MSC) were cultured with or without IL-1β, IL-6 and IL-23 as pro-inflammatory cytokines. The surface markers and their differentiation capacity were measured in cytokine-untreated and cytokine-treated MSCs. MSCs-mediated immunomodulation was analyzed by their regulatory effects on mixed lymphocyte reaction (MLR) and the level of IL-10, TGF-β, IL-4, IFN-γ and TNF-α production as immunomodulatory cytokines. Pro-inflammatory cytokines showed no effect on MSCs morphology, immunophenotype and co-stimulatory molecules except up-regulation of CD45. Adipogenic and osteogenic differentiation capacity increased in CD45+ MSCs. Moreover, cytokine-treated MSCs preserved the suppressive ability of allogeneic T cell proliferation and produced higher level of TGF-β and lower level of IL-4. We concluded pro-inflammatory cytokines up-regulate the efficacy of MSCs in cell-based therapy of degenerative, inflammatory and autoimmune disorders. Copyright © 2016. Published by Elsevier Ltd.

Structure-function relationships in the stem cell's mechanical world B: emergent anisotropy of the cytoskeleton correlates to volume and shape changing stress exposure.

PubMed

Chang, Hana; Knothe Tate, Melissa L

2011-12-01

In the preceding study (Part A), we showed that prescribed seeding conditions as well as seeding density can be used to subject multipotent stem cells (MSCs) to volume changing stresses and that changes in volume of the cell are associated with changes in shape, but not volume, of the cell nucleus. In the current study, we aim to control the mechanical milieu of live cells using these prescribed seeding conditions concomitant to delivery of shape changing stresses via fluid flow, while observing adaptation of the cytoskeleton, a major cellular transducer that modulates cell shape, stiffness and remodeling. We hypothesize that the spatiotemporal organization of tubulin and actin elements of the cytoskeleton changes in response to volume and shape changing stresses emulating those during development, prior to the first beating of the heart or twitching of muscle. Our approach was to quantify the change over baseline in spatiotemporal distribution of actin and tubulin in live C3H/10T1/2 model stem cells subjected to volume changing stresses induced by seeding at density as well as low magnitude, short duration, shape changing (shear) stresses induced by fluid flow (0.5 or 1.0 dyne/cm2 for 30/60/90 minutes). Upon exposure to fluid flow, both tubulin thickness (height) and concentration (fluorescence intensity) change significantly over baseline, as a function of proximity to neighboring cells (density) and the substrate (apical-basal height). Given our recently published studies showing amplification of stress gradients (flow velocity) with increasing distance to nearest neighbors and the substrate, i.e. with decreasing density and toward the apical side of the cell, tubulin adaptation appears to depend significantly on the magnitude of the stress to which the cell is exposed locally. In contrast, adaptation of actin to the changing mechanical milieu is more global, exhibiting less significant differences attributable to nearest neighbors or boundaries than differences attributable to magnitude of the stress to which the cell is exposed globally (0.5 versus 1.0 dyne/cm2). Furthermore, changes in the actin cytoskeletal distribution correlate positively with one pre-mesenchymal condensation marker (Msx2) and negatively with early markers of chondrogenesis (ColIIaI alone, indicative of pre-hypertrophic chondrogenesis) and osteogenesis (Runx2). Changes in the tubulin cytoskeletal distribution correlate positively with a marker of pericondensation (Sox9 alone), negatively with chondrogenesis (ColIIaI) and positively with adipogenesis (Ppar-gamma 2). Taken as a whole, exposure of MSCs to volume and shape changing stresses results in emergent anisotropy of cytoskeletal architecture (structure), which relate to emergent cell fate (function).

Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

PubMed Central

El Haddad, Najib; Heathcote, Dean; Moore, Robert; Yang, Sunmi; Azzi, Jamil; Mfarrej, Bechara; Atkinson, Mark; Sayegh, Mohamed H.; Lee, Jeng-Shin; Ashton-Rickardt, Philip G.

2011-01-01

Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases. PMID:21076046

Immune modulatory mesenchymal stem cells derived from human embryonic stem cells through a trophoblast-like stage.

PubMed

Wang, Xiaofang; Lazorchak, Adam S; Song, Li; Li, Enqin; Zhang, Zhenwu; Jiang, Bin; Xu, Ren-He

2016-02-01

Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells "T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T-MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T-MSCs constitutively express a high level of the immune inhibitory ligand PD-L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC-based therapies. © 2015 AlphaMed Press.

Wnt5a-Ror2 signaling in mesenchymal stem cells promotes proliferation of gastric cancer cells by activating CXCL16-CXCR6 axis.

PubMed

Takiguchi, Gosuke; Nishita, Michiru; Kurita, Kana; Kakeji, Yoshihiro; Minami, Yasuhiro

2016-03-01

Wnt5a-Ror2 signaling has been shown to play important roles in promoting aggressiveness of various cancer cells in a cell-autonomous manner. However, little is known about its function in cancer-associated stromal cells, including mesenchymal stem cells (MSCs). Thus, we examined the role of Wnt5a-Ror2 signaling in bone marrow-derived MSCs in regulating proliferation of undifferentiated gastric cancer cells. Coculture of a gastric cancer cell line, MKN45, with MSCs either directly or indirectly promotes proliferation of MKN45 cells, and suppressed expression of Ror2 in MSCs prior to coculture inhibits enhanced proliferation of MKN45 cells. In addition, conditioned media from MSCs, treated with control siRNA, but not siRNAs against Ror2, can enhance proliferation of MKN45 cells. Interestingly, it was found that expression of CXCL16 in MSCs is augmented by Wnt5a-Ror2 signaling, and that recombinant chemokine (C-X-C motif) ligand (CXCL)16 protein can enhance proliferation of MKN45 cells in the absence of MSCs. In fact, suppressed expression of CXCL16 in MSCs or an addition of a neutralizing antibody against CXCL16 fails to promote proliferation of MKN45 cells in either direct or indirect coculture with MSCs. Importantly, we show that MKN45 cells express chemokine (C-X-C motif) receptor (CXCR)6, a receptor for CXCL16, and that suppressed expression of CXCR6 in MKN45 cells results in a failure of its enhanced proliferation in either direct or indirect coculture with MSCs. These findings indicate that Wnt5a-Ror2 signaling enhances expression of CXCL16 in MSCs and, as a result, enhanced secretion of CXCL16 from MSCs might act on CXCR6 expressed on MKN45, leading to the promotion of its proliferation. © 2015 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells.

PubMed

Barbet, Romain; Peiffer, Isabelle; Hatzfeld, Antoinette; Charbord, Pierre; Hatzfeld, Jacques A

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1(low), BMPR1B(low), FLT4(low), LRRC32(low), and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

Serum-deprived human multipotent mesenchymal stromal cells (MSCs) are highly angiogenic

PubMed Central

Oskowitz, Adam; McFerrin, Harris; Gutschow, Miriam; Carter, Mary Leita; Pochampally, Radhika

2016-01-01

Recent reports have indicated that mesenchymal stromal cells (MSCs) from bone marrow have a potential in vascular remodeling and angiogenesis. Here, we report a unique phenomenon that under serum-deprived conditions MSCs survive and replicate. Secretome analysis of MSCs grown under serum-deprived conditions (SD-MSCs) identified a significant upregulation of prosurvival and angiogenic factors including VEGF-A, ANGPTs, IGF-1, and HGF. An ex vivo rat aortic assay demonstrated longer neovascular sprouts generated from rat aortic rings cultured in SD-MSC-conditioned media compared to neovascular sprouts from aortas grown in MSC-conditioned media. With prolonged serum deprivation, a subpopulation of SD-MSCs began to exhibit an endothelial phenotype. This population expressed endothelial-specific proteins including VEGFR2, Tie2/TEK, PECAM/CD31, and eNOS and also demonstrated the ability to uptake acetylated LDL. SD-MSCs also exhibited enhanced microtubule formation in an in vitro angiogenesis assay. Modified chick chorioallantoic membrane (CAM) angiogenesis assays showed significantly higher angiogenic potential for SD-MSCs compared to MSCs. Analysis of CAMs grown with SD-MSCs identified human-specific CD31-positive cells in vascular structures. We conclude that under the stress of serum deprivation MSCs are highly angiogenic and a population of these cells has the potential to differentiate into endothelial-like cells. PMID:21421339

Comparative analysis of rat mesenchymal stem cells derived from slow and fast skeletal muscle in vitro.

PubMed

Okumachi, Etsuko; Lee, Sang Yang; Niikura, Takahiro; Iwakura, Takashi; Dogaki, Yoshihiro; Waki, Takahiro; Takahara, Shunsuke; Ueha, Takeshi; Sakai, Yoshitada; Kuroda, Ryosuke; Kurosaka, Masahiro

2015-03-01

Skeletal muscle comprises different kinds of muscle fibres that can be classified as slow and fast fibres. The purpose of this study was to compare the yield, proliferation, and multi-potentiality of rat mesenchymal stem cells (MSCs) from the tibialis anterior (TA; fast muscle) and soleus (SO; slow muscle) in vitro. The TA and SO muscles were harvested, and isolated cells were plated. After two hours, the cells were washed extensively to remove any cell that did not adhere to the cell culture plate. The adherent cells, namely MSCs, were then cultured. Both types of MSCs were differentiated toward the osteogenic, chondrogenic and adipogenic lineages using lineage specific induction factors. The colony-forming unit fibroblast (CFU-F) assay revealed that the SO contained significantly higher quantities of MSCs than the TA. The self-renewal capacity of MSCs derived from the TA was significantly higher at later passages (passage 9-11). Both types of MSCs exhibited similar cell surface antigens to bone marrow (BM)-derived MSCs and were positive for CD29, CD44, and CD90 and negative for CD11b, CD34, and CD45. TA-derived MSCs were superior in terms of osteogenic differentiation capacity, but there was no significant difference in chondrogenic and adipogenic differentiation capacity. Our results demonstrated significant differences in the properties of muscle-derived MSCs from different muscle types (i.e. fast or slow muscles). The greater expandability and osteogenic differentiation ability of TA-derived MSCs suggests that fast muscle may be a better source for generating large numbers of MSCs for bone regeneration.

Intraperitoneal but not intravenous cryopreserved mesenchymal stromal cells home to the inflamed colon and ameliorate experimental colitis.

PubMed

Castelo-Branco, Morgana T L; Soares, Igor D P; Lopes, Daiana V; Buongusto, Fernanda; Martinusso, Cesonia A; do Rosario, Alyson; Souza, Sergio A L; Gutfilen, Bianca; Fonseca, Lea Mirian B; Elia, Celeste; Madi, Kalil; Schanaider, Alberto; Rossi, Maria Isabel D; Souza, Heitor S P

2012-01-01

Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1β decreased, while VEGF and TGF-β did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis.

Mesenchymal stem cells with overexpression of midkine enhance cell survival and attenuate cardiac dysfunction in a rat model of myocardial infarction

PubMed Central

2014-01-01

Introduction Elevated midkine (MK) expression may contribute to ventricular remodeling and ameliorate cardiac dysfunction after myocardial infarction (MI). Ex vivo modification of signaling mechanisms in mesenchymal stem cells (MSCs) with MK overexpression may improve the efficacy of cell-based therapy. This study sought to assess the safety and efficacy of MSCs with MK overexpression transplantation in a rat model of MI. Methods A pLenO-DCE vector lentivirus encoding MK was constructed and infected in MSCs. MSC migration activity and cytoprotection was examined in hypoxia-induced H9C2 cells using transwell insert in vitro. Rats were randomized into five groups: sham, MI plus injection of phosphate buffered saline (PBS), MSCs, MSCs-green fluorescent protein (MSCs-GFP) and MSCs-MK, respectively. Survival rates were compared among groups using log-rank test and left ventricular function was measured by echocardiography at baseline, 4, 8 and 12 weeks. Results Overexpression of MK partially prevented hypoxia-induced MSC apoptosis and exerted MSC cytoprotection to anoxia induced H9C2 cells. The underlying mechanisms may be associated with the increased mRNA and protein levels of vascular endothelial growth factor (VEGF), transformation growth factor-β (TGF-β), insulin-like growth factor 1 (IGF-1) and stromal cell-derived factor 1 (SDF-1a) in MSCs-MK compared with isolated MSCs and MSCs-GFP. Consistent with the qPCR results, the culture supernatant of MSCs-MK had more SDF-1a (9.23 ng/ml), VEGF (8.34 ng/ml) and TGF-β1 (17.88 ng/ml) expression. In vivo, a greater proportion of cell survival was observed in the MSCs-MK group than in the MSCs-GFP group. Moreover, MSCs-MK administration was related to a significant improvement of cardiac function compared with other control groups at 12 weeks. Conclusions Therapies employing MSCs with MK overexpression may represent an effective treatment for improving cardiac dysfunction and survival rate after MI. PMID:24635859

Adenosinergic Immunosuppression by Human Mesenchymal Stromal Cells Requires Co-Operation with T cells.

PubMed

Kerkelä, Erja; Laitinen, Anita; Räbinä, Jarkko; Valkonen, Sami; Takatalo, Maarit; Larjo, Antti; Veijola, Johanna; Lampinen, Milla; Siljander, Pia; Lehenkari, Petri; Alfthan, Kaija; Laitinen, Saara

2016-03-01

Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5'-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5'-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs. © 2016 AlphaMed Press.

A CD8 T Cell/Indoleamine 2,3-Dioxygenase Axis Is Required for Mesenchymal Stem Cell Suppression of Human Systemic Lupus Erythematosus

PubMed Central

Wang, Dandan; Feng, Xuebing; Lu, Lin; Konkel, Joanne E; Zhang, Huayong; Chen, Zhiyong; Li, Xia; Gao, Xiang; Lu, Liwei; Shi, Songtao; Chen, Wanjun; Sun, Lingyun

2014-01-01

Objective Allogeneic mesenchymal stem cells (MSCs) exhibit therapeutic effects in human autoimmune diseases such as systemic lupus erythematosus (SLE), but the underlying mechanisms remain largely unknown. The aim of this study was to investigate how allogeneic MSCs mediate immunosuppression in lupus patients. Methods The effects of allogeneic umbilical cord–derived MSCs (UC-MSCs) on inhibition of T cell proliferation were determined. MSC functional molecules were stimulated with peripheral blood mononuclear cells from healthy controls and SLE patients and examined by real-time polymerase chain reaction. CD4+ and CD8+ T cells were purified using microbeads to stimulate MSCs in order to determine cytokine expression by MSCs and to further determine which cell subset(s) or which molecule(s) is involved in inhibition of MSC–mediated T cell proliferation. The related signaling pathways were assessed. We determined levels of serum cytokines in lupus patients before and after UC-MSC transplantation. Results Allogeneic UC-MSCs suppressed T cell proliferation in lupus patients by secreting large amounts of indoleamine 2,3-dioxygenase (IDO). We further found that interferon-γ (IFNγ), which is produced predominantly by lupus CD8+ T cells, is the key factor that enhances IDO activity in allogeneic MSCs and that it is associated with IFNGR1/JAK-2/STAT signaling pathways. Intriguingly, bone marrow–derived MSCs from patients with active lupus demonstrated defective IDO production in response to IFNγ and allogeneic CD8+ T cell stimulation. After allogeneic UC-MSC transplantation, serum IDO activity increased in lupus patients. Conclusion We found a previously unrecognized CD8+ T cell/IFNγ/IDO axis that mediates the therapeutic effects of allogeneic MSCs in lupus patients. PMID:24756936

[Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers].

PubMed

Wang, Yue-Chun; Zhang, Yuan

2008-06-25

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

Canine mesenchymal stem cells treated with TNF-α and IFN-γ enhance anti-inflammatory effects through the COX-2/PGE2 pathway.

PubMed

Yang, Hye-Mi; Song, Woo-Jin; Li, Qiang; Kim, Su-Yeon; Kim, Hyeon-Jin; Ryu, Min-Ok; Ahn, Jin-Ok; Youn, Hwa-Young

2018-05-14

Mesenchymal stem cells (MSCs) have been used in studies on treatment of various diseases, and their application to immune-mediated diseases has garnered interest. Various methods for enhancing the immunomodulation effect of human MSCs have been used; however, similar approaches for canine MSCs are relatively unexplored. Accordingly, we evaluated immunomodulatory effects and mechanisms in canine MSCs treated with TNF-α and IFN-γ. Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were incubated with the conditioned media (CM) from canine MSCs for 48 h. Expression of RNA was assessed by quantitative reverse transcription PCR (qRT-PCR), and protein levels were assessed by western blot. Expression of inducible nitric oxide synthase (iNOS), IL-6 and IL-1β was significantly (one-way ANOVA) decreased in LPS-stimulated RAW 264.7 cells incubated with CM from canine MSCs compared to that in LPS-stimulated RAW 264.7 cells alone. Furthermore, anti-inflammatory effects of TNF-α- and IFN-γ-primed canine MSCs were significantly increased compared with those of naïve canine MSCs. Expression of cyclooxygenase 2 (COX-2) and prostaglandin E 2 (PGE 2 ) were likewise significantly increased in primed canine MSCs. The level of iNOS protein in LPS-stimulated RAW 264.7 cells incubated with CM from the primed canine MSCs was decreased, but it increased when the cells were treated with NS-398(PGE 2 inhibitor). In conclusion, compared with naïve canine MSCs, cells primed with TNF-α and IFN-γ cause a greater reduction in release of anti-inflammatory cytokines from LPS-stimulated RAW 264.7 cells; the mechanism is upregulation of the COX-2/PGE 2 pathway. Copyright © 2018. Published by Elsevier Ltd.

Gastric cancer-derived MSC-secreted PDGF-DD promotes gastric cancer progression.

PubMed

Huang, Feng; Wang, Mei; Yang, Tingting; Cai, Jie; Zhang, Qiang; Sun, Zixuan; Wu, Xiaodan; Zhang, Xu; Zhu, Wei; Qian, Hui; Xu, Wenrong

2014-11-01

This study was designed to investigate the role of PDGF-DD secreted by gastric cancer-derived mesenchymal stem cells (GC-MSCs) in human gastric cancer progression. Gastric cancer cells were indirectly co-cultured with GC-MSCs in a transwell system. The growth and migration of gastric cancer cells were evaluated by cell colony formation assay and transwell migration assay, respectively. The production of PDGF-DD in GC-MSCs was determined by using Luminex and ELISA. Neutralization of PDGFR-β by su16f and siRNA interference of PDGF-DD in GC-MSCs was used to demonstrate the role of PDGF-DD produced by GC-MSCs in gastric cancer progression. GC-MSC conditioned medium promoted gastric cancer cell proliferation and migration in vitro and in vivo. Co-culture with GC-MSCs increased the phosphorylation of PDGFR-β in SGC-7901 cells. Neutralization of PDGFR-β by su16f blocked the promoting role of GC-MSC conditioned medium in gastric cancer cell proliferation and migration. Recombinant PDGF-DD duplicated the effects of GC-MSC conditioned medium on gastric cancer cells. Knockdown of PDGF-DD in GC-MSCs abolished its effects on gastric cancer cells in vitro and in vivo. PDGF-DD secreted by GC-MSCs is capable of promoting gastric cancer cell progression in vitro and in vivo. Targeting the PDGF-DD/PDGFR-β interaction between MSCs and gastric cancer cells may represent a novel strategy for gastric cancer therapy.

Mesenchymal Stem Cells Retain Their Defining Stem Cell Characteristics After Exposure to Ionizing Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nicolay, Nils H., E-mail: n.nicolay@dkfz.de; Department of Molecular and Radiation Oncology, German Cancer Research Center, Heidelberg; Sommer, Eva

2013-12-01

Purpose: Mesenchymal stem cells (MSCs) have the ability to migrate to lesion sites and undergo differentiation into functional tissues. Although this function may be important for tissue regeneration after radiation therapy, the influence of ionizing radiation (IR) on cellular survival and the functional aspects of differentiation and stem cell characteristics of MSCs have remained largely unknown. Methods and Materials: Radiation sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells was examined, and cellular morphology, cell cycle effects, apoptosis, and differentiation potential after exposure to IR were assessed. Stem cell gene expression patterns after exposure to IRmore » were studied using gene arrays. Results: MSCs were not more radiosensitive than human primary fibroblasts, whereas there were considerable differences regarding radiation sensitivity within individual MSCs. Cellular morphology, cytoskeletal architecture, and cell motility were not markedly altered by IR. Even after high radiation doses up to 10 Gy, MSCs maintained their differentiation potential. Compared to primary fibroblast cells, MSCs did not show an increase in irradiation-induced apoptosis. Gene expression analyses revealed an upregulation of various genes involved in DNA damage response and DNA repair, but expression of established MSC surface markers appeared only marginally influenced by IR. Conclusions: These data suggest that human MSCs are not more radiosensitive than differentiated primary fibroblasts. In addition, upon photon irradiation, MSCs were able to retain their defining stem cell characteristics both on a functional level and regarding stem cell marker expression.« less

Human Umbilical Cord Mesenchymal Stem Cells Inhibit the Function of Allogeneic Activated Vγ9Vδ2 T Lymphocytes In Vitro

PubMed Central

Liu, Xiaohuan; Feng, Ting; Gong, Tianxiang; Shen, Chongyang; Zhu, Tingting; Wu, Qihong; Li, Qiang; Li, Hong

2015-01-01

Background. Human umbilical cord mesenchymal stem cells (UC-MSCs) can regulate the function of immune cells. However, whether and how UC-MSCs can modulate the function of Vγ9Vδ2 T cells has not been fully understood. Methods. The PBMCs or Vγ9Vδ2 T cells were activated and expanded with pamidronate (PAM) and interleukin-2 (IL-2) with or without the presence UC-MSCs. The effects of UC-MSCs on the proliferation, cytokine expression, and cytotoxicity of Vγ9Vδ2 T cells were determined by flow cytometry. The effects of UC-MSCs on Fas-L, TRAIL-expressing Vγ9Vδ2 T cells, and Vγ9Vδ2 T cell apoptosis were determined by flow cytometry. Results. UC-MSCs inhibited Vγ9Vδ2 T cell proliferation in a dose-dependent but cell-contact independent manner. Coculture with UC-MSCs reduced the frequency of IFNγ+ but increased granzyme B+ Vγ9Vδ2 T cells. UC-MSCs inhibited the cytotoxicity of Vγ9Vδ2 T cells against influenza virus H1N1 infected A549 cells and also reduced the frequency of Fas-L+, TRAIL+ Vγ9Vδ2 T cells but failed to modulate the apoptosis of Vγ9Vδ2 T cells. Conclusions. These results indicated that UC-MSCs efficiently suppressed the proliferation and cytotoxicity of Vγ9Vδ2 T cells and modulated their cytokine production. Fas-L and TRAIL were involved in the regulation. Cell contact and apoptosis of Vγ9Vδ2 T cells were not necessary for the inhibition. PMID:25984529

β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype.

PubMed

Azizi, Zahra; Lange, Claudia; Paroni, Federico; Ardestani, Amin; Meyer, Anke; Wu, Yonghua; Zander, Axel R; Westenfelder, Christof; Maedler, Kathrin

2016-08-02

Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow-derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes.Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment.With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP.After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes.

Differentiation of Mesenchymal Stem Cells Towards Nephrogenic Lineage and Their Enhanced Resistance to Oxygen Peroxide-induced Oxidative Stress.

PubMed

Tayyeb, Asima; Shahzad, Naveed; Ali, Gibran

2017-07-01

Mesenchymal stem cells (MSCs) have been publicized to ameliorate kidney injury both in vitro and in vivo. However, very less is known if MSCs can be differentiated towards renal lineages and their further application potential in kidney injuries. The present study developed a conditioning system of growth factors fibroblast growth factor 2, transforming growth factor-β2, and leukemia inhibitory factor for in vitro differentiation of MSCs isolated from different sources towards nephrogenic lineage. Less invasively isolated adipose-derived MSCs were also compared to bone marrow-derived MSCs for their differentiation potential to induce renal cell. Differentiated MSCs were further evaluated for their resistance to oxidative stress induced by oxygen peroxide. A combination of growth factors successfully induced differentiation of MSCs. Both types of differentiated cells showed significant expression of pronephrogenic markers (Wnt4, Wt1, and Pax2) and renal epithelial markers (Ecad and ZO1). In contrast, expression of mesenchymal stem cells marker Oct4 and Vim were downregulated. Furthermore, differentiated adipose-derived MSCs and bone marrow-derived MSCs showed enhanced and comparable resistance to oxygen peroxide-induced oxidative stress. Adipose-derived MSC provides a promising alternative to bone marrow-derived MSC as a source of autologous stem cells in human kidney injuries. In addition, differentiated MSCs with further in vivo investigations may serve as a cell source for tissue engineering or cell therapy in different renal ailments.

Cell culture density affects the proliferation activity of human adipose tissue stem cells.

PubMed

Kim, Dae Seong; Lee, Myoung Woo; Ko, Young Jong; Chun, Yong Hoon; Kim, Hyung Joon; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2016-01-01

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.

Long-Term Cultured Human Term Placenta-Derived Mesenchymal Stem Cells of Maternal Origin Displays Plasticity

PubMed Central

Sabapathy, Vikram; Ravi, Saranya; Srivastava, Vivi; Srivastava, Alok; Kumar, Sanjay

2012-01-01

Mesenchymal stem cells (MSCs) are an alluring therapeutic resource because of their plasticity, immunoregulatory capacity and ease of availability. Human BM-derived MSCs have limited proliferative capability, consequently, it is challenging to use in tissue engineering and regenerative medicine applications. Hence, placental MSCs of maternal origin, which is one of richest sources of MSCs were chosen to establish long-term culture from the cotyledons of full-term human placenta. Flow analysis established bonafied MSCs phenotypic characteristics, staining positively for CD29, CD73, CD90, CD105 and negatively for CD14, CD34, CD45 markers. Pluripotency of the cultured MSCs was assessed by in vitro differentiation towards not only intralineage cells like adipocytes, osteocytes, chondrocytes, and myotubules cells but also translineage differentiated towards pancreatic progenitor cells, neural cells, and retinal cells displaying plasticity. These cells did not significantly alter cell cycle or apoptosis pattern while maintaining the normal karyotype; they also have limited expression of MHC-II antigens and are Naive for stimulatory factors CD80 and CD 86. Further soft agar assays revealed that placental MSCs do not have the ability to form invasive colonies. Taking together all these characteristics into consideration, it indicates that placental MSCs could serve as good candidates for development and progress of stem-cell based therapeutics. PMID:22550499

The Immunomodulatory and Neuroprotective Effects of Mesenchymal Stem Cells (MSCs) in Experimental Autoimmune Encephalomyelitis (EAE): A Model of Multiple Sclerosis (MS)

PubMed Central

Al Jumah, Mohammed A.; Abumaree, Mohamed H.

2012-01-01

Mesenchymal stem cells (MSCs) are multipotent cells that differentiate into the mesenchymal lineages of adipocytes, osteocytes and chondrocytes. MSCs can also transdifferentiate and thereby cross lineage barriers, differentiating for example into neurons under certain experimental conditions. MSCs have anti-proliferative, anti-inflammatory and anti-apoptotic effects on neurons. Therefore, MSCs were tested in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), for their effectiveness in modulating the pathogenic process in EAE to develop effective therapies for MS. The data in the literature have shown that MSCs can inhibit the functions of autoreactive T cells in EAE and that this immunomodulation can be neuroprotective. In addition, MSCs can rescue neural cells via a mechanism that is mediated by soluble factors, which provide a suitable environment for neuron regeneration, remyelination and cerebral blood flow improvement. In this review, we discuss the effectiveness of MSCs in modulating the immunopathogenic process and in providing neuroprotection in EAE. PMID:22942767

Actin Cytoskeletal Disruption following Cryopreservation Alters the Biodistribution of Human Mesenchymal Stromal Cells In Vivo

PubMed Central

Chinnadurai, Raghavan; Garcia, Marco A.; Sakurai, Yumiko; Lam, Wilbur A.; Kirk, Allan D.; Galipeau, Jacques; Copland, Ian B.

2014-01-01

Summary Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and human endothelial cells (80% reduction), respectively. This reduction correlated with a reduced cytoskeletal F-actin content in post-thaw MSCs (60% reduction). In vivo, live human MSCs could be detected in murine lung tissues for up to 24 hr, whereas thawed MSCs were undetectable. Similarly, live MSCs whose actin cytoskeleton was chemically disrupted were undetectable at 24 hr postinfusion. Our data suggest that post-thaw cryopreserved MSCs are distinct from live MSCs. This distinction could significantly affect the utility of MSCs as a cellular therapeutic. PMID:25068122

Comparison of in vitro developmental competence of cloned caprine embryos using donor karyoplasts from adult bone marrow mesenchymal stem cells vs ear fibroblast cells.

PubMed

Kwong, P J; Nam, H Y; Wan Khadijah, W E; Kamarul, T; Abdullah, R B

2014-04-01

The aim of this study was to produce cloned caprine embryos using either caprine bone marrow-derived mesenchymal stem cells (MSCs) or ear fibroblast cells (EFCs) as donor karyoplasts. Caprine MSCs were isolated from male Boer goats of an average age of 1.5 years. To determine the pluripotency of MSCs, the cells were induced to differentiate into osteocytes, chondrocytes and adipocytes. Subsequently, MSCs were characterized through cell surface antigen profiles using specific markers, prior to their use as donor karyoplasts for nuclear transfer. No significant difference (p > 0.05) in fusion rates was observed between MSCs (87.7%) and EFCs (91.3%) used as donor karyoplasts. The cleavage rate of cloned embryos derived with MSCs (87.0%) was similar (p > 0.05) to those cloned using EFCs (84.4%). However, the in vitro development of MSCs-derived cloned embryos (25.3%) to the blastocyst stage was significantly higher (p < 0.05) than those derived with EFCs (20.6%). In conclusion, MSCs could be reprogrammed by caprine oocytes, and production of cloned caprine embryos with MSCs improved their in vitro developmental competence, but not in their fusion and cleavage rate as compared to cloning using somatic cells such as EFCs. © 2014 Blackwell Verlag GmbH.

In Vivo Immunogenic Response to Allogeneic Mesenchymal Stem Cells and the Role of Preactivated Mesenchymal Stem Cells Cotransplanted with Allogeneic Islets

PubMed Central

Chagastelles, Pedro Cesar; Sesterheim, Patrícia

2017-01-01

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into cells from the mesenchymal lineage. The hypoimmunogenic characteristic of MSCs has encouraged studies using allogeneic MSCs for the treatment of autoimmune diseases and inflammatory conditions. Promising preclinical results and the safety of allogeneic MSC transplantation have created the possibility of “off-the-shelf” clinical application of allogeneic cells. This study has aimed to evaluate the survival of untreated and IFN-γ- and TNF-α-treated (preactivated) allogeneic MSCs transplanted under the kidney capsule of immunocompetent mice together with the role of preactivated MSCs after cotransplantation with allogeneic islets. The preactivation of MSCs upregulated the gene expression of anti-inflammatory molecules and also enhanced their immunomodulatory capacity in vitro. In vivo, allogeneic MSCs provoked an immunogenic response, with the infiltration of inflammatory cells at the transplant site and full graft rejection in both the untreated and preactivated groups. Allogeneic islets cotransplanted with preactivated MSCs prolonged graft survival for about 6 days, compared with islet alone. The present results corroborate the hypothesis that allogeneic MSCs are not immune-privileged and that after playing their therapeutic role they are rejected. Strategies that reduce allogeneic MSC immunogenicity can potentially prolong their in vivo persistence and improve the therapeutic effects. PMID:28553360

Niche matters: The comparison between bone marrow stem cells and endometrial stem cells and stromal fibroblasts reveal distinct migration and cytokine profiles in response to inflammatory stimulus

PubMed Central

Sorjamaa, Anna; Kangasniemi, Marika; Sutinen, Meeri; Salo, Tuula; Liakka, Annikki; Lehenkari, Petri; Tapanainen, Juha S.; Vuolteenaho, Olli; Chen, Joseph C.; Lehtonen, Siri; Piltonen, Terhi T.

2017-01-01

Objective Intrinsic inflammatory characteristics play a pivotal role in stem cell recruitment and homing through migration where the subsequent change in niche has been shown to alter these characteristics. The bone marrow mesenchymal stem cells (bmMSCs) have been demonstrated to migrate to the endometrium contributing to the stem cell reservoir and regeneration of endometrial tissue. Thus, the aim of the present study was to compare the inflammation-driven migration and cytokine secretion profile of human bmMSCs to endometrial mesenchymal stem cells (eMSCs) and endometrial fibroblasts (eSFs). Materials and methods The bmMSCs were isolated from bone marrow aspirates through culturing, whereas eMSCs and eSFs were FACS-isolated. All cell types were tested for their surface marker, proliferation profiles and migration properties towards serum and inflammatory attractants. The cytokine/chemokine secretion profile of 35 targets was analysed in each cell type at basal level along with lipopolysaccharide (LPS)-induced state. Results Both stem cell types, bmMSCs and eMSCs, presented with similar stem cell surface marker profiles as well as possessed high proliferation and migration potential compared to eSFs. In multiplex assays, the secretion of 16 cytokine targets was detected and LPS stimulation expanded the cytokine secretion pattern by triggering the secretion of several targets. The bmMSCs exhibited higher cytokine secretion of vascular endothelial growth factor (VEGF)-A, stromal cell-derived factor-1 alpha (SDF)-1α, interleukin-1 receptor antagonist (IL-1RA), IL-6, interferon-gamma inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)1α and RANTES compared to eMSCs and/or eSFs after stimulation with LPS. The basal IL-8 secretion was higher in both endometrial cell types compared to bmMSCs. Conclusion Our results highlight that similar to bmMSCs, the eMSCs possess high migration activity while the differentiation process towards stromal fibroblasts seemed to result in loss of stem cell surface markers, minimal migration activity and a subtler cytokine profile likely contributing to normal endometrial function. PMID:28419140

Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.

PubMed

Hsu, Yi-Chao; Wu, Yu-Ting; Yu, Ting-Hsien; Wei, Yau-Huei

2016-04-01

Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying mechanisms in the regulation of mitochondrial metabolism of MSCs may ultimately improve therapeutic outcomes of stem cell therapy in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Osteogenic potential of bone marrow mesenchymal stem cells from ovariectomied osteoporotic rat].

PubMed

Li, Dong-ju; Ge, Dong-xia; Wu, Wen-chao; Wu, Jiang; Li, Liang

2005-05-01

To investigate the difference of osteogenic potential of bone marrow mesenchymal stem cells (MSCs) between healthy rats and osteoporotic rats. We established the animal model of osteoporosis by performing ovariectom on the 3-month-old female Sprague-Dawley rats. Bone marrow mesenchymal stem cells(MSCs) were isolated from the rats of control group and of ovariectomized (ovx) group by means of the density-gradient centrifugation method, and the 3rd-4th passage MSCs were used in all the experiments. The experiments comprised 4 groups: (1) Marrow mesenchymal stem cells control group (MSCs control group); (2) Marrow mesenchymal stem cells ovx group (MSCs ovx group); (3) Osteogenesis induction control group (OSI control group); (4) Osteogenesis induction ovx group (OSI ovx group). Cell cycle and proliferation index (PI) of MSCs were detected by flow cytometry. The expression of alkaline phosphatase (ALP) was detected by dynamics method with substrate of phosphoric acid para-Nitro benzene. The levels of osteocalcin were detected with the isotope labelling method. (1) PI of MSCs was lower in MSCs ovx group than in MSCs control group. (2) The expression of alkaline phosphatase (ALP) was much higher in OSI control group than in the MSCs control group; the expression of alkaline phosphatase (ALP) was much higher in the OSI control group than in OSI ovx group after 7-day and 14-day osteogenic induction. (3) The level of osteocalcin was much higher in the OSI control group than in the MSCs control group after 14-day, 21-day, 28-day osteogenic induction. The level of osteocalcin was much higher in the OSI control group than in the OSI ovx group. Both the proliferative potential and the osteogenic potential of bone marrow mesenchymal stem cells (MSCs) from the ovariectomized osteoporotic rat are decreased.

Transplantated mesenchymal stem cells derived from embryonic stem cells promote muscle regeneration and accelerate functional recovery of injured skeletal muscle.

PubMed

Ninagawa, Nana Takenaka; Isobe, Eri; Hirayama, Yuri; Murakami, Rumi; Komatsu, Kazumi; Nagai, Masataka; Kobayashi, Mami; Kawabata, Yuka; Torihashi, Shigeko

2013-08-01

We previously established that mesenchymal stem cells originating from mouse embryonic stem (ES) cells (E-MSCs) showed markedly higher potential for differentiation into skeletal muscles in vitro than common mesenchymal stem cells (MSCs). Further, the E-MSCs exhibited a low risk for teratoma formation. Here we evaluate the potential of E-MSCs for differentiation into skeletal muscles in vivo and reveal the regeneration and functional recovery of injured muscle by transplantation. E-MSCs were transplanted into the tibialis anterior (TA) muscle 24 h following direct clamping. After transplantation, the myogenic differentiation of E-MSCs, TA muscle regeneration, and re-innervation were morphologically analyzed. In addition, footprints and gaits of each leg under spontaneous walking were measured by CatWalk XT, and motor functions of injured TA muscles were precisely analyzed. Results indicate that >60% of transplanted E-MSCs differentiated into skeletal muscles. The cross-sectional area of the injured TA muscles of E-MSC-transplanted animals increased earlier than that of control animals. E-MSCs also promotes re-innervation of the peripheral nerves of injured muscles. Concerning function of the TA muscles, we reveal that transplantation of E-MSCs promotes the recovery of muscles. This is the first report to demonstrate by analysis of spontaneous walking that transplanted cells can accelerate the functional recovery of injured muscles. Taken together, the results show that E-MSCs have a high potential for differentiation into skeletal muscles in vivo as well as in vitro. The transplantation of E-MSCs facilitated the functional recovery of injured muscles. Therefore, E-MSCs are an efficient cell source in transplantation.

Transplantated Mesenchymal Stem Cells Derived from Embryonic Stem Cells Promote Muscle Regeneration and Accelerate Functional Recovery of Injured Skeletal Muscle

PubMed Central

Ninagawa, Nana Takenaka; Isobe, Eri; Hirayama, Yuri; Murakami, Rumi; Komatsu, Kazumi; Nagai, Masataka; Kobayashi, Mami; Kawabata, Yuka

2013-01-01

Abstract We previously established that mesenchymal stem cells originating from mouse embryonic stem (ES) cells (E-MSCs) showed markedly higher potential for differentiation into skeletal muscles in vitro than common mesenchymal stem cells (MSCs). Further, the E-MSCs exhibited a low risk for teratoma formation. Here we evaluate the potential of E-MSCs for differentiation into skeletal muscles in vivo and reveal the regeneration and functional recovery of injured muscle by transplantation. E-MSCs were transplanted into the tibialis anterior (TA) muscle 24 h following direct clamping. After transplantation, the myogenic differentiation of E-MSCs, TA muscle regeneration, and re-innervation were morphologically analyzed. In addition, footprints and gaits of each leg under spontaneous walking were measured by CatWalk XT, and motor functions of injured TA muscles were precisely analyzed. Results indicate that >60% of transplanted E-MSCs differentiated into skeletal muscles. The cross-sectional area of the injured TA muscles of E-MSC–transplanted animals increased earlier than that of control animals. E-MSCs also promotes re-innervation of the peripheral nerves of injured muscles. Concerning function of the TA muscles, we reveal that transplantation of E-MSCs promotes the recovery of muscles. This is the first report to demonstrate by analysis of spontaneous walking that transplanted cells can accelerate the functional recovery of injured muscles. Taken together, the results show that E-MSCs have a high potential for differentiation into skeletal muscles in vivo as well as in vitro. The transplantation of E-MSCs facilitated the functional recovery of injured muscles. Therefore, E-MSCs are an efficient cell source in transplantation. PMID:23914336

Microvesicles derived from Alde-Low EPCs support the wound healing capacity of AT-MSCs.

PubMed

Tu, Tran Cam; Yamashita, Toshiharu; Kato, Toshiki; Nagano, Masumi; Trinh, Nhu Thuy; Hamada, Hiromi; Sato, Fujio; Ohneda, Kinuko; Matsuo-Takasaki, Mami; Ohneda, Osamu

2016-08-12

Mesenchymal stem cells (MSCs) are defined as multipotent cells that can give rise to various kinds of differentiated mesenchymal cells, and are thus considered to be useful for clinical therapy. However, the big hurdles of MSC therapy are the inability of MSCs to reach the appropriate tissues or sites with high efficiency and engraftment after transplantation. In this study, we investigated how adipose tissue-derived MSCs (AT-MSCs) improve their homing ability after intravenous injection. We previously found that human endothelial progenitor cells with low aldehyde dehydrogenase activity (Alde-Low EPCs) are suitable for the treatment of ischemic tissues. In addition, we demonstrated that microvesicles (MVs) derived from Alde-Low EPCs possessed the ability to improve the homing ability of non-functional Alde-High EPCs, resulting in wound healing. We initially transfected MVs derived from Alde-Low EPCs (EMVs) to human AT-MSCs, which were originally unable to cure ischemic tissues by intravenous transplantation. Remarkably, AT-MSC transfected EMVs dramatically repaired the ischemic skin flap compared with AT-MSC derived-MV (MMVs) transfected AT-MSCs or control AT-MSCs. We then found that the expression of CXCR4, an important chemokine receptor for cell migration, was highly elevated in EMV-transfected AT-MSCs. Moreover, AT-MSCs transfected with EMVs, but not control AT-MSCs, migrated to wound sites after intravenous injection. Consequently, CD45(+) inflammatory cells were successfully recruited at the wound sites after the injection of EMV-transfected AT-MSCs. These results demonstrate that EMVs are a useful source to improve the homing ability and wound healing ability of MSCs at the wound sites. Copyright © 2016 Elsevier Inc. All rights reserved.

Osteogenic differentiation of mesenchymal progenitor cells in computer designed fibrin-polymer-ceramic scaffolds manufactured by fused deposition modeling.

PubMed

Schantz, Jan-Thorsten; Brandwood, Arthur; Hutmacher, Dietmar Werner; Khor, Hwei Ling; Bittner, Katharina

2005-09-01

Biomimetic scaffolds offer great potentials in the development of bone analogs for tissue engineering. The studies presented in this paper focus specifically on the osteogenic potential of the novel PCL/CaP matrices and its degradation behavior. Biodegradable Polymer-ceramic Scaffolds were fabricated using the solid free form fabrication technology: Fused Deposition Modeling (FDM). The scaffold architecture was characterized by a honeycomb-like design and a complete interconnectivity of the pores. Human mesenchymal stem cells (MSCs) were seeded together with fibrin glue into PCL/CaP scaffolds and cultured in vitro for periods of up to eight weeks. Cellular adhesion, proliferation and osteogenic differentiation were assessed in these constructs using a range of histological and microscopic techniques. In additional experiments, degradation was assessed by measuring mass loss, diameter change, molecular weight change and by scanning electron micrographs. MSCs were able to adhere, migrate, and differentiate along the osteogenic lineage with in these scaffolds. The PCL/CaP scaffolds showed up to 27 fold increased degradation of compared to PCL scaffolds.

Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone

PubMed Central

Smith, Christopher A.; Board, Tim N.; Rooney, Paul; Eagle, Mark J.; Richardson, Stephen M.

2017-01-01

To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC) osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC). BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP) enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1) concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors. PMID:28505164

Adult mesenchymal stem cells and cell-based tissue engineering

PubMed Central

Tuan, Rocky S; Boland, Genevieve; Tuli, Richard

2003-01-01

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions. PMID:12716446

Efficient generation of hepatic cells from mesenchymal stromal cells by an innovative bio-microfluidic cell culture device.

PubMed

Yen, Meng-Hua; Wu, Yuan-Yi; Liu, Yi-Shiuan; Rimando, Marilyn; Ho, Jennifer Hui-Chun; Lee, Oscar Kuang-Sheng

2016-08-19

Mesenchymal stromal cells (MSCs) are multipotent and have great potential in cell therapy. Previously we reported the differentiation potential of human MSCs into hepatocytes in vitro and that these cells can rescue fulminant hepatic failure. However, the conventional static culture method neither maintains growth factors at an optimal level constantly nor removes cellular waste efficiently. In addition, not only is the duration of differentiating hepatocyte lineage cells from MSCs required to improve, but also the need for a large number of hepatocytes for cell therapy has not to date been addressed fully. The purpose of this study is to design and develop an innovative microfluidic device to overcome these shortcomings. We designed and fabricated a microfluidic device and a culture system for hepatic differentiation of MSCs using our protocol reported previously. The microfluidic device contains a large culture chamber with a stable uniform flow to allow homogeneous distribution and expansion as well as efficient induction of hepatic differentiation for MSCs. The device enables real-time observation under light microscopy and exhibits a better differentiation efficiency for MSCs compared with conventional static culture. MSCs grown in the microfluidic device showed a higher level of hepatocyte marker gene expression under hepatic induction. Functional analysis of hepatic differentiation demonstrated significantly higher urea production in the microfluidic device after 21 days of hepatic differentiation. The microfluidic device allows the generation of a large number of MSCs and induces hepatic differentiation of MSCs efficiently. The device can be adapted for scale-up production of hepatic cells from MSCs for cellular therapy.

Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells.

PubMed

Shu, Sai-Nan; Wei, Lai; Wang, Jiang-Hua; Zhan, Yu-Tao; Chen, Hong-Song; Wang, Yu

2004-10-01

To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before differentiation. Albumin mRNA and protein were expressed positively in MSCs, without detection of alpha-fetoprotein (AFP). HSCs were polygonal in shape after differentiation. The expression of albumin signal decreased and AFP signal increased. The expression of CK18 was continuous in MSCs and HSCs both before and after induction. Both MSCs and HSCs have hepatic differentiation capabilities. However, their capabilities are not the same. MSCs can differentiate into mature hepatocyte-like cells, never expressing early hepatic specific genes, while Thy-1.1(+) cells are inclined to differentiate into hepatic stem cell-like cells, with an increasing AFP expression and a decreasing albumin signal. CK18 mRNA is positive in Thy-1.1(+) cells and MSCs, negative in Thy-1.1(-) cells. It seems that CK18 has some relationship with Thy-1.1 antigen, and CK18 may be a predictive marker of hepatic differentiation capability.

Biotechnological and biomedical applications of mesenchymal stem cells as a therapeutic system.

PubMed

Rahimzadeh, Amirbahman; Mirakabad, Fatemeh Sadat Tabatabaei; Movassaghpour, Aliakbar; Shamsasenjan, Karim; Kariminekoo, Saber; Talebi, Mehdi; Shekari, Abolfazl; Zeighamian, Vahideh; Ghalhar, Masoud Gandomkar; Akbarzadeh, Abolfazl

2016-01-01

Mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent progenitor cells which reside in bone marrow (BM), support homing of hematopoietic stem cells (HSCs) and self-renewal in the BM. These cells have the potential to differentiate into tissues of mesenchymal origin, such as fibroblasts, adipocytes, cardiomyocytes, and stromal cells. MSCs can express surface molecules like CD13, CD29, CD44, CD73, CD90, CD166, CXCL12 and toll-like receptors (TLRs). Different factors, such as TGF-β, IL-10, IDO, PGE-2, sHLA-G5, HO, and Galectin-3, secreted by MSCs, induce interaction in cell to cell immunomodulatory effects on innate and adaptive cells of the immune system. Furthermore, these cells can stimulate and increase the TH2 and regulatory T-cells through inhibitory effects on the immune system. MSCs originate from the BM and other tissues including the brain, adipose tissue, peripheral blood, cornea, thymus, spleen, fallopian tube, placenta, Wharton's jelly and umbilical cord blood. Many studies have focused on two significant features of MSC therapy: (I) MSCs can modulate T-cell-mediated immunological responses, and (II) systemically administered MSCs home in to sites of ischemia or injury. In this review, we describe the known mechanisms of immunomodulation and homing of MSCs. As a result, this review emphasizes the functional role of MSCs in modulating immune responses, their capability in homing to injured tissue, and their clinical therapeutic potential.

Comparison of Adipose-Derived and Bone Marrow Mesenchymal Stromal Cells in a Murine Model of Crohn's Disease.

PubMed

Xie, Minghao; Qin, Huabo; Luo, Qianxin; He, Xiaosheng; He, Xiaowen; Lan, Ping; Lian, Lei

2017-01-01

Mesenchymal stromal cells (MSCs) have been used in the treatment of Crohn's disease (CD) because of the immunomodulatory ability. The aim of this study was to investigate the therapeutic effect of adipose-derived MSCs (AD-MSCs) and to compare the therapeutic effect of AD-MSCs with that of bone marrow MSCs (BM-MSCs) in a murine model of CD. Murine colitis model of CD was created by trinitrobenzene sulfonic acid (TNBS). Twelve hours after treatment with TNBS, the mouse model was injected with MSCs intraperitoneally. Real-time polymerase chain reaction and immunohistochemistry staining were used to measure the expression levels of inflammatory cytokines in colonic tissues to investigate the therapeutic effect of AD-MSCs. The ten-day survival was recorded after infusion of MSCs. Intraperitoneal injection of MSCs alleviated the clinical and histopathologic severity of intestinal inflammation, and improved the survival of the TNBS-induced mouse model of CD. AD-MSCs could effectively increase the expression of interleukin-10 and reduce the secretion of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-12, and vascular endothelial growth factor. The mucosal injury was repaired by AD-MSCs. These effects were comparable between AD-MSCs and BM-MSCs. The therapeutic effect appears similar between AD-MSCs and BM-MSCs in treating CD. AD-MSCs may be a potential alternative of cell-based therapy for CD.

Differential expression of cell cycle and WNT pathway-related genes accounts for differences in the growth and differentiation potential of Wharton's jelly and bone marrow-derived mesenchymal stem cells.

PubMed

Batsali, Aristea K; Pontikoglou, Charalampos; Koutroulakis, Dimitrios; Pavlaki, Konstantia I; Damianaki, Athina; Mavroudi, Irene; Alpantaki, Kalliopi; Kouvidi, Elisavet; Kontakis, George; Papadaki, Helen A

2017-04-26

In view of the current interest in exploring the clinical use of mesenchymal stem cells (MSCs) from different sources, we performed a side-by-side comparison of the biological properties of MSCs isolated from the Wharton's jelly (WJ), the most abundant MSC source in umbilical cord, with bone marrow (BM)-MSCs, the most extensively studied MSC population. MSCs were isolated and expanded from BM aspirates of hematologically healthy donors (n = 18) and from the WJ of full-term neonates (n = 18). We evaluated, in parallel experiments, the MSC immunophenotypic, survival and senescence characteristics as well as their proliferative potential and cell cycle distribution. We also assessed the expression of genes associated with the WNT- and cell cycle-signaling pathway and we performed karyotypic analysis through passages to evaluate the MSC genomic stability. The hematopoiesis-supporting capacity of MSCs from both sources was investigated by evaluating the clonogenic cells in the non-adherent fraction of MSC co-cultures with BM or umbilical cord blood-derived CD34 + cells and by measuring the hematopoietic cytokines levels in MSC culture supernatants. Finally, we evaluated the ability of MSCs to differentiate into adipocytes and osteocytes and the effect of the WNT-associated molecules WISP-1 and sFRP4 on the differentiation potential of WJ-MSCs. Both ex vivo-expanded MSC populations showed similar morphologic, immunophenotypic, survival and senescence characteristics and acquired genomic alterations at low frequency during passages. WJ-MSCs exhibited higher proliferative potential, possibly due to upregulation of genes that stimulate cell proliferation along with downregulation of genes related to cell cycle inhibition. WJ-MSCs displayed inferior lineage priming and differentiation capacity toward osteocytes and adipocytes, compared to BM-MSCs. This finding was associated with differential expression of molecules related to WNT signaling, including WISP1 and sFRP4, the respective role of which in the differentiation potential of WJ-MSCs was specifically investigated. Interestingly, treatment of WJ-MSCs with recombinant human WISP1 or sFRP4 resulted in induction of osteogenesis and adipogenesis, respectively. WJ-MSCs exhibited inferior hematopoiesis-supporting potential probably due to reduced production of stromal cell-Derived Factor-1α, compared to BM-MSCs. Overall, these data are anticipated to contribute to the better characterization of WJ-MSCs and BM-MSCs for potential clinical applications.

Long noncoding RNA H19 upregulates vascular endothelial growth factor A to enhance mesenchymal stem cells survival and angiogenic capacity by inhibiting miR-199a-5p.

PubMed

Hou, Jingying; Wang, Lingyun; Wu, Quanhua; Zheng, Guanghui; Long, Huibao; Wu, Hao; Zhou, Changqing; Guo, Tianzhu; Zhong, Tingting; Wang, Lei; Chen, Xuxiang; Wang, Tong

2018-04-19

Currently, the overall therapeutic efficiency of mesenchymal stem cells (MSCs) transplantation for the treatment of cardiovascular disease is not satisfactory. The low viability and angiogenic capacity of the implanted cells in the local infarct tissues restrict their further application. Evidence shows that long noncoding RNA H19 (lncRNA-H19) mediates cell survival and angiogenesis. Additionally, it is also involved in MSCs biological activities. This study aimed to explore the functional role of lncRNA-H19 in MSCs survival and angiogenic capacity as well as the underlying mechanism. MSCs were obtained from C57BL/6 mice and cultured in vitro. Cells at the third passage were divided into the following groups: MSCs+H19, MSCs+H19 NC, MSCs+si-H19, MSCs+si-H19 NC and MSCs. The MSCs+H19 and MSCs+H19 NC groups were transfected with lncRNA-H19 and lncRNA-H19 scramble RNA respectively. The MSCs+si-H19 and MSCs+si-H19 NC groups were transfected with lncRNA-H19 siRNA and lncRNA-H19 siRNA scramble respectively. MSCs were used as the blank control. All groups were exposed to normoxia (20% O 2 ) and hypoxia (1% O 2 )/serum deprivation (H/SD) conditions for 24 h. Cell proliferation, apoptosis and vascular densities were assessed. Bioinformatics and dual luciferase reporter assay were performed. Relevant biomarkers were detected in different experimental groups. Overexpression of lncRNA-H19 improved survival and angiogenic capacity of MSCs under both normoxia and H/SD conditions, whereas its knockdown impaired cell viability and their angiogenic potential. MicroRNA-199a-5p (miR-199a-5p) targeted and downregulated vascular endothelial growth factor A (VEGFA). MiR-199a-5p was a target of lncRNA-H19. LncRNA-H19 transfection led to a decreased level of miR-199a-5p, accompanied with an elevated expression of VEGFA. However, both miR-199a-5p and VEGFA presented inverse alterations in the condition of lncRNA-H19 knockdown. LncRNA-H19 enhanced MSCs survival and their angiogenic potential in vitro. It could directly upregulate VEGFA expression by inhibiting miR-199a-5p as a competing endogenous RNA. This mechanism contributes to a better understanding of MSCs biological activities and provides new insights for cell therapy based on MSCs transplantation.

Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells.

PubMed

Kim, Dae Seong; Lee, Myoung Woo; Lee, Tae-Hee; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2017-03-01

The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm 2 . After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox ( Nanog ), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog ( c-Myc ), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc , were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent). These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

Inflammation by Breast Implants and Adenocarcinoma: Not Always a Bad Company.

PubMed

Orciani, Monia; Sorgentoni, Giulia; Olivieri, Fabiola; Mattioli-Belmonte, Monica; Di Benedetto, Giovanni; Di Primio, Roberto

2017-07-01

Inflammation and tumor are now an inseparable binomial. Inflammation may also derive by the use of breast implants followed by the formation of a periprosthetic capsule. It is known that tumor cells, in an inflamed microenvironment, can profit by the paracrine effect exerted also by mesenchymal stem cells (MSCs). Here we evaluated the role of inflammation on the immunobiology of MSCs before and after cocultures with cells derived from breast adenocarcinoma. MSCs derived from both inflamed (I-MSCs) and control (C-MSCs) tissues were isolated and cocultured with MCF7 cells derived from breast adenocarcinoma. Before and after cocultures, the proliferation rate of MCF7 cells and the expression/secretion of cytokines related to inflammation were tested. Before cocultures, higher levels of cytokine related to chronic inflammation were detected in I-MSCs than in C-MSCs. After cocultures with MCF7, C- and I-MSCs show a variation in cytokine production. In detail, IL-2, IL-4, IL-5, IL-10, IL-13, TGF-β and G-CSF were decreased, whereas IL-6, IL-12, IFN-γ, and IL-17 were oversecreted. Proliferation of MCF7 was significantly increased after cocultures with I-MSCs. Inflammation at the site of origin of MSCs affects their immunobiology. Even if tumor cells increased their proliferation rate after cocultures with I-MSCs, the analysis of the cytokines, known to play a role in the interference of tumor cells with the host immune system, absolves completely the breast implants from the insult to enforce the risk of adenocarcinoma. Copyright © 2017 Elsevier Inc. All rights reserved.

[Differentiation of bone marrow derived from mesenchymal stem cells into cardiomyocyte-like cells induced by co-culture with rat myocardial cells].

PubMed

Zhang, Rong-Li; Jiang, Er-Lie; Wang, Mei; Zhou, Zheng; Zhai, Wen-Jing; Zhai, Wei-Hua; Wang, Hua; Wang, Zhi-Yong; Bao, Yu-Shi; DU, Hong; Han, Ming-Zhe

2008-10-01

The study was purposed to investigate the differentiation ability of mesenchymal stem cells (MSCs) into myocardial cells in vitro. Rat bone marrow-derived MSCs were labeled and co-cultured with neonatal rat cardiomyocytes (CM) for 5 - 7 days. The expression of cell surface antigens was detected by flow cytometry, and the expression of muscle-specific marker myosin and troponin T in labeled cells was detected by immunofluorescence. The results showed that in vitro cultured MSCs expressed CD90, CD44, CD105, CD54, not expressed CD34, CD45, CD31. After co-cultured with neonatal rat CM, labeled MSCs differentiated into cardiomyocyte-like cells expressing myosin and troponin T. It is concluded that MSCs can differentiate into cardiomyocyte-like cells when co-cultured with neonatal myocardial cells in vitro. In co-culture of two kind of cells in ratio of four to one showed obvious efficacy differentiating MSCs into CMs.

Hsp20-Engineered Mesenchymal Stem Cells Are Resistant to Oxidative Stress via Enhanced Activation of Akt and Increased Secretion of Growth Factors

PubMed Central

Wang, Xiaohong; Zhao, Tiemin; Huang, Wei; Wang, Tao; Qian, Jiang; Xu, Meifeng; Kranias, Evangelia G.; Wang, Yigang; Fan, Guo-Chang

2009-01-01

Although heat-shock preconditioning has been shown to promote cell survival under oxidative stress, the nature of heat-shock response from different cells is variable and complex. Therefore, it remains unclear whether mesenchymal stem cells (MSCs) modified with a single heat-shock protein (Hsp) gene are effective in the repair of a damaged heart. In this study, we genetically engineered rat MSCs with Hsp20 gene (Hsp20-MSCs) and examined cell survival, revascularization, and functional improvement in rat left anterior descending ligation (LAD) model via intracardial injection. We observed that overexpression of Hsp20 protected MSCs against cell death triggered by oxidative stress in vitro. The survival of Hsp20-MSCs was increased by approximately twofold by day 4 after transplantation into the infarcted heart, compared with that of vector-MSCs. Furthermore, Hsp20-MSCs improved cardiac function of infarcted myocardium as compared with vector-MSCs, accompanied by reduction of fibrosis and increase in the vascular density. The mechanisms contributing to the beneficial effects of Hsp20 were associated with enhanced Akt activation and increased secretion of growth factors (VEGF, FGF-2, and IGF-1). The paracrine action of Hsp20-MSCs was further validated in vitro by cocultured adult rat cardiomyocytes with a stress-conditioned medium from Hsp20-MSCs. Taken together, these data support the premise that genetic modification of MSCs before transplantation could be salutary for treating myocardial infarction. PMID:19816949

Comprehensive characterization of mesenchymal stromal cells from patients with Fanconi anaemia.

PubMed

Mantelli, Melissa; Avanzini, Maria Antonia; Rosti, Vittorio; Ingo, Daniela M; Conforti, Antonella; Novara, Francesca; Arrigo, Giulia; Boni, Marina; Zappatore, Rita; Lenta, Elisa; Moretta, Antonia; Acquafredda, Gloria; de Silvestri, Annalisa; Cirillo, Valentina; Cicchetti, Elisa; Algeri, Mattia; Strocchio, Luisa; Vinti, Luciana; Starc, Nadia; Biagini, Simone; Sirleto, Pietro; Bernasconi, Paolo; Zuffardi, Orsetta; Maserati, Emanuela; Maccario, Rita; Zecca, Marco; Locatelli, Franco; Bernardo, Maria Ester

2015-09-01

Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony-forming unit (CFU-F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long-term haematopoiesis and immunomodulatory properties of FA-MSCs were analysed and compared with those of MSCs expanded from 15 age-matched healthy donors (HD-MSCs). FA-MSCs were genetically characterized through conventional karyotyping, diepoxybutane-test and array-comparative genomic hybridization. FA-MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability in vitro to inhibit mitogen-induced T-cell proliferation and to support long-term haematopoiesis did not differ between FA-MSCs and HD-MSCs. CFU-F ability and proliferative capacity of FA-MSCs isolated after HSCT were significantly lower than those of HD-MSCs. FA-MSCs reached senescence significantly earlier than HD-MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA-MSCs are defective in their ability to survive in vitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation. © 2015 John Wiley & Sons Ltd.

The mechanosensor of mesenchymal stem cells: mechanosensitive channel or cytoskeleton?

PubMed

Xiao, E; Chen, Chider; Zhang, Yi

2016-09-20

Mesenchymal stem cells (MSCs) are multipotent adult stem cells. MSCs and their potential for use in regenerative medicine have been investigated extensively. Recently, the mechanisms by which MSCs detect mechanical stimuli have been described in detail. As in other cell types, both mechanosensitive channels, such as transient receptor potential melastatin 7 (TRPM7), and the cytoskeleton, including actin and actomyosin, have been implicated in mechanosensation in MSCs. This review will focus on discussing the precise role of TRPM7 and the cytoskeleton in mechanosensation in MSCs.

Cholinergic and dopaminergic neuronal differentiation of human adipose tissue derived mesenchymal stem cells.

PubMed

Marei, Hany El Sayed; El-Gamal, Aya; Althani, Asma; Afifi, Nahla; Abd-Elmaksoud, Ahmed; Farag, Amany; Cenciarelli, Carlo; Thomas, Caceci; Anwarul, Hasan

2018-02-01

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various cell types such as cartilage, bone, and fat cells. Recent studies have shown that induction of MSCs in vitro by growth factors including epidermal growth factor (EGF) and fibroblast growth factor (FGF2) causes them to differentiate into neural like cells. These cultures also express ChAT, a cholinergic marker; and TH, a dopaminergic marker for neural cells. To establish a protocol with maximum differentiation potential, we examined MSCs under three experimental culture conditions using neural induction media containing FGF2, EGF, BMP-9, retinoic acid, and heparin. Adipose-derived MSCs were extracted and expanded in vitro for 3 passages after reaching >80% confluency, for a total duration of 9 days. Cells were then characterized by flow cytometry for CD markers as CD44 positive and CD45 negative. MSCs were then treated with neural induction media and were characterized by morphological changes and Q-PCR. Differentiated MSCs expressed markers for immature and mature neurons; β Tubulin III (TUBB3) and MAP2, respectively, showing the neural potential of these cells to differentiate into functional neurons. Improved protocols for MSCs induction will facilitate and ensure the reproducibility and standard production of MSCs for therapeutic applications in neurodegenerative diseases. © 2017 Wiley Periodicals, Inc.

The role of mmu-miR-155-5p-NF-κB signaling in the education of bone marrow-derived mesenchymal stem cells by gastric cancer cells.

PubMed

Wang, Mei; Yang, Fang; Qiu, Rong; Zhu, Mengchu; Zhang, Huiling; Xu, Wenrong; Shen, Bo; Zhu, Wei

2018-03-01

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are important precursors of tumor stromal cells. Previously, we have demonstrated that miR-155-5p inhibition directly induced transition of BM-MSCs into gastric cancer-associated MSCs. Whether miR-155-5p is involved in the education of BM-MSCs by gastric cancer cells has not been established. Murine BM-MSCs (mMSCs) were isolated and grown in conditioned medium derived from gastric cancer cell line MFC (MFC-CM). The tumor-promoting phenotype and function of mMSCs were detected by immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cell colony formation assay, transwell migration, and invasion assays. Luciferase reporter assays and western blot analyses were conducted to reveal the relationship between nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 and mmu-miR-155-5p. miRNA mimics, inhibitor, and the NF-κB inhibitor pyrrolidine dithiocarbamic acid (PDTC) were used to evaluate the role of miR-155-5p-NF-κB signaling in the education of mMSCs by MFC-CM. We successfully established the education model of mMSCs by MFC-CM and found that mmu-miR-155-5p expression levels were reduced in mMSCs. Mimicking this deregulation by transfecting miRNA inhibitor into mMSCs produced a similar effect as that of MFC-CM on mMSCs. NF-κB p65 was validated as a target of mmu-miR-155-5p, which also negatively regulated NF-κB activation. Inhibition of NF-κB activation by PDTC abolished the effect of the miRNA inhibitor on mMSCs. mmu-miR-155-5p overexpression partially blocked the effect of MFC-CM in educating mMSCs, while PDTC treatment completely eliminated MFC-CM activity. These results indicate that miR-155-5p is not the sole miRNA mediating the education of BM-MSCs by gastric cancer cells, but downstream NF-κB signaling is indispensable for this process. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

PubMed Central

2012-01-01

Introduction Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration. Methods Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined. Results Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs. Conclusions Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment. PMID:22676383

Interferon-γ regulates the function of mesenchymal stem cells from oral lichen planus via indoleamine 2,3-dioxygenase activity.

PubMed

Zhang, Zhihui; Han, Ying; Song, Jiangyuan; Luo, Ruxi; Jin, Xin; Mu, Dongdong; Su, Sha; Ji, Xiaoli; Ren, Yan-Fang; Liu, Hongwei

2015-01-01

Little is known about mesenchymal stem cells (MSCs) in normal or inflammatory oral mucosal tissues, such as in oral lichen planus (OLP). Our objectives were to identify, isolate, and characterize MSCs from normal human oral mucosa and OLP lesions, and to evaluate indoleamine 2,3 dioxygenase (IDO) activity in mediating immunomodulation of MSCs from these tissues. Expressions of MSCs-related markers were examined in isolated cells by flow cytometry. Self-renewal and multilineage differentiations were studied to characterize these MSCs. Interferon-γ (IFN-γ), IDO, and STRO-1 were assessed by immunofluorescence. MSCs from oral mucosa and OLP or IFN-γ-pretreated MSCs were co-cultured with allogeneic mixed lymphocyte reaction assays (MLR). Proliferation and apoptosis of MLR or MSCs were detected by CCK8 and the annexin V-FITC apoptosis detection kit, respectively. IDO expression and activity were measured by real-time PCR, Western blotting, and high-performance liquid chromatography. Isolated cells from oral mucosa and OLP expressed MSC-related markers STRO-1, CD105, and CD90 but were absent for hematopoietic stem cell markers CD34. Besides, they all showed self-renewal and multilineage differentiation capacities. MSCs in OLP presented STRO-1/IDO+ phenotype by immunofluorescence. MSCs and IFN-γ-pretreated MSCs could inhibit lymphocyte proliferation via IDO activity, but not via cell apoptosis. Long-term IFN-γ could also inhibit MSC proliferation via IDO activity. Mesenchymal stem cells can be isolated from human oral mucosa and OLP tissues. Besides self-renewal and multilineage differentiation properties, these cells may participate in immunomodulation mediated by IFN-γ via IDO activity in human OLP. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b(+) myeloid cells to the lungs and facilitates B16-F10 melanoma colonization.

PubMed

Souza, Lucas E B; Almeida, Danilo C; Yaochite, Juliana N U; Covas, Dimas T; Fontes, Aparecida M

2016-07-15

The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b(+) myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize "premetastatic niches" in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b(+) myeloid cells and tumor cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Reprogramming of single-cell derived mesenchymal stem cells into hair cell-like cells

PubMed Central

Lin, Zhaoyu; Perez, Philip; Sun, Zhenyu; Liu, Jan-Jan; Shin, June Ho; Hyrc, Krzysztof L.; Samways, Damien; Egan, Terry; Holley, Matthew C.; Bao, Jianxin

2012-01-01

Hypothesis Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination. Background Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells. Methods Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed by RT-PCR, and cell structures were examined by immunocytochemistry. Results Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells prior to overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model. Conclusion We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells. PMID:23111404

Bone-derived mesenchymal stromal cells from HIV transgenic mice exhibit altered proliferation, differentiation capacity and paracrine functions along with impaired therapeutic potential in kidney injury

PubMed Central

Cheng, Kang; Rai, Partab; Lan, Xiqian; Plagov, Andrei; Malhotra, Ashwani; Gupta, Sanjeev; Singhal, Pravin C

2013-01-01

Mesenchymal stem cells (MSCs) secrete paracrine factors that could be cytoprotective and serve roles in immunoregulation during tissue injury. Although MSCs express HIV receptors, and co-receptors, and are susceptible to HIV infection, whether HIV-1 may affect biological properties of MSCs needs more study. We evaluated cellular proliferation, differentiation and paracrine functions of MSCs isolated from compact bones of healthy control mice and Tg26 HIV-1 transgenic mice. The ability of MSCs to protect against cisplatin toxicity was studied in cultured renal tubular cells as well as in intact mice. We successfully isolated MSCs from healthy mice and Tg26 HIV-1 transgenic mice and found the latter expressed viral Nef, Vpu, NL4-3 and Vif genes. The proliferation and differentiation of Tg26 HIV-1 MSCs was inferior to MSCs from healthy mice. Moreover, transplantation of Tg26 HIV-1 MSCs less effectively improved outcomes compared with healthy MSCs in mice with acute kidney injury. Also, Tg26 HIV-1 MSCs secreted multiple cytokines, but at significantly lower levels than healthy MSCs, which resulted in failure of conditioned medium from these MSCs to protect cultured renal tubular cells from cisplatin toxicity. Therefore, HIV-1 had adverse biological effects on MSCs extending to their proliferation, differentiation, function, and therapeutic potential. These findings will help in advancing mechanistical insight in renal injury and repair in the setting of HIV-1 infection. PMID:23806280

Bone marrow-derived human mesenchymal stem cells express cardiomyogenic proteins but do not exhibit functional cardiomyogenic differentiation potential.

PubMed

Siegel, Georg; Krause, Petra; Wöhrle, Stefanie; Nowak, Patrick; Ayturan, Miriam; Kluba, Torsten; Brehm, Bernhard R; Neumeister, Birgid; Köhler, David; Rosenberger, Peter; Just, Lothar; Northoff, Hinnak; Schäfer, Richard

2012-09-01

Despite their paracrine activites, cardiomyogenic differentiation of bone marrow (BM)-derived mesenchymal stem cells (MSCs) is thought to contribute to cardiac regeneration. To systematically evaluate the role of differentiation in MSC-mediated cardiac regeneration, the cardiomyogenic differentiation potential of human MSCs (hMSCs) and murine MSCs (mMSCs) was investigated in vitro and in vivo by inducing cardiomyogenic and noncardiomyogenic differentiation. Untreated hMSCs showed upregulation of cardiac tropopin I, cardiac actin, and myosin light chain mRNA and protein, and treatment of hMSCs with various cardiomyogenic differentiation media led to an enhanced expression of cardiomyogenic genes and proteins; however, no functional cardiomyogenic differentiation of hMSCs was observed. Moreover, co-culturing of hMSCs with cardiomyocytes derived from murine pluripotent cells (mcP19) or with murine fetal cardiomyocytes (mfCMCs) did not result in functional cardiomyogenic differentiation of hMSCs. Despite direct contact to beating mfCMCs, hMSCs could be effectively differentiated into cells of only the adipogenic and osteogenic lineage. After intramyocardial transplantation into a mouse model of myocardial infarction, Sca-1(+) mMSCs migrated to the infarcted area and survived at least 14 days but showed inconsistent evidence of functional cardiomyogenic differentiation. Neither in vitro treatment nor intramyocardial transplantation of MSCs reliably generated MSC-derived cardiomyocytes, indicating that functional cardiomyogenic differentiation of BM-derived MSCs is a rare event and, therefore, may not be the main contributor to cardiac regeneration.

The ability of multipotent mesenchymal stromal cells from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells.

PubMed

Sorokina, Tamara; Shipounova, Irina; Bigildeev, Alexey; Petinati, Nataliya; Drize, Nina; Turkina, Anna; Chelysheva, Ekaterina; Shukhov, Oleg; Kuzmina, Larisa; Parovichnikova, Elena; Savchenko, Valery

2016-09-01

The development of leukemia impairs normal hematopoiesis and marrow stromal microenvironment. The aim of the investigation was to study the ability of multipotent mesenchymal stromal cells (MSCs) derived from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells. MSCs were obtained from the bone marrow of 14 patients with acute lymphoblastic (ALL), 25 with myeloid (AML), and 15 with chronic myeloid (CML) leukemia. As a control, MSCs from 22 healthy donors were used. The incidence of cobblestone area forming cells (CAFC 7-8 d) in the bone marrow of healthy donor cultivated on the supportive layer of patients MSCs was measured. The ability of MSCs from AML and ALL patients at the moment of diagnosis to maintain normal CAFC was significantly decreased when compared to donors. After chemotherapy, the restoration of ALL patients' MSCs functions was slower than that of AML. CML MSCs maintained CAFC better than donors' at the moment of diagnosis and this ability increased with treatment. The ability of patients' MSCs to maintain normal hematopoietic progenitor cells was shown to change in comparison with MSCs from healthy donors and depended on nosology. During treatment, the functional capacity of patients' MSCs had been partially restored. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Comparative Analysis of Human Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue, and Umbilical Cord Blood as Sources of Cell Therapy

PubMed Central

Jin, Hye Jin; Bae, Yun Kyung; Kim, Miyeon; Kwon, Soon-Jae; Jeon, Hong Bae; Choi, Soo Jin; Kim, Seong Who; Yang, Yoon Sun; Oh, Wonil; Chang, Jong Wook

2013-01-01

Various source-derived mesenchymal stem cells (MSCs) have been considered for cell therapeutics in incurable diseases. To characterize MSCs from different sources, we compared human bone marrow (BM), adipose tissue (AT), and umbilical cord blood-derived MSCs (UCB-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, and paracrine activity. Although MSCs from different tissues have similar levels of surface antigen expression, immunosuppressive activity, and differentiation ability, UCB-MSCs had the highest rate of cell proliferation and clonality, and significantly lower expression of p53, p21, and p16, well known markers of senescence. Since paracrine action is the main action of MSCs, we examined the anti-inflammatory activity of each MSC under lipopolysaccharide (LPS)-induced inflammation. Co-culture of UCB-MSCs with LPS-treated rat alveolar macrophage, reduced expression of inflammatory cytokines including interleukin-1α (IL-1α), IL-6, and IL-8 via angiopoietin-1 (Ang-1). Using recombinant Ang-1 as potential soluble paracrine factor or its small interference RNA (siRNA), we found that Ang-1 secretion was responsible for this beneficial effect in part by preventing inflammation. Our results demonstrate that primitive UCB-MSCs have biological advantages in comparison to adult sources, making UCB-MSCs a useful model for clinical applications of cell therapy. PMID:24005862

IL17/IL17RA as a Novel Signaling Axis Driving Mesenchymal Stem Cell Therapeutic Function in Experimental Autoimmune Encephalomyelitis

PubMed Central

Kurte, Mónica; Luz-Crawford, Patricia; Vega-Letter, Ana María; Contreras, Rafael A.; Tejedor, Gautier; Elizondo-Vega, Roberto; Martinez-Viola, Luna; Fernández-O’Ryan, Catalina; Figueroa, Fernando E.; Jorgensen, Christian; Djouad, Farida; Carrión, Flavio

2018-01-01

The therapeutic effect of mesenchymal stem cells (MSCs) in multiple sclerosis (MS) and the experimental autoimmune encephalomyelitis (EAE) model has been well described. This effect is, in part, mediated through the inhibition of IL17-producing cells and the generation of regulatory T cells. While proinflammatory cytokines such as IFNγ, TNFα, and IL1β have been shown to enhance MSCs immunosuppressive function, the role of IL17 remains poorly elucidated. The aim of this study was, therefore, to investigate the role of the IL17/IL17R pathway on MSCs immunoregulatory effects focusing on Th17 cell generation in vitro and on Th17-mediated EAE pathogenesis in vivo. In vitro, we showed that the immunosuppressive effect of MSCs on Th17 cell proliferation and differentiation is partially dependent on IL17RA expression. This was associated with a reduced expression level of MSCs immunosuppressive mediators such as VCAM1, ICAM1, and PD-L1 in IL17RA−/− MSCs as compared to wild-type (WT) MSCs. In the EAE model, we demonstrated that while WT MSCs significantly reduced the clinical scores of the disease, IL17RA−/− MSCs injected mice exhibited a clinical worsening of the disease. The disability of IL17RA−/− MSCs to reduce the progression of the disease paralleled the inability of these cells to reduce the frequency of Th17 cells in the draining lymph node of the mice as compared to WT MSCs. Moreover, we showed that the therapeutic effect of MSCs was correlated with the generation of classical Treg bearing the CD4+CD25+Foxp3+ signature in an IL17RA-dependent manner. Our findings reveal a novel role of IL17RA on MSCs immunosuppressive and therapeutic potential in EAE and suggest that the modulation of IL17RA in MSCs could represent a novel method to enhance their therapeutic effect in MS. PMID:29760692

Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells.

PubMed

Fujii, Sumie; Miura, Yasuo; Iwasa, Masaki; Yoshioka, Satoshi; Fujishiro, Aya; Sugino, Noriko; Kaneko, Hitomi; Nakagawa, Yoko; Hirai, Hideyo; Takaori-Kondo, Akifumi; Ichinohe, Tatsuo; Maekawa, Taira

2017-07-05

Umbilical cord blood (UCB) has advantages over other tissues because it can be obtained without an invasive procedure and complex processing. We explored the availability of cryopreserved UCB cells as a source of mesenchymal stromal/stem cells (MSCs). MSCs were successfully isolated from six of 30 UCB units (median volume, 34.0 mL; median nucleated cell number, 4.4×10 8 ) that were processed and cryopreserved using CP-1/human serum albumin. This isolation rate was lower than that (57%) from non-cryopreserved UCB cells. The number of nucleated cells before and after hydroxyethyl starch separation, UCB unit volume, and cell viability after thawing did not significantly differ between UCB units from which MSCs were successfully isolated and those from which they were not. When CryoSure-DEX40 was used as a cryoprotectant, MSCs were isolated from two of ten UCB units. Logistic regression analysis demonstrated that the cryopreservation method was not significantly associated with the success of MSC isolation. The isolated MSCs had a similar morphology and surface marker expression profile as bone marrow-derived MSCs and were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. In summary, MSCs can be isolated from cryopreserved UCB cells. However, the cryopreservation process reduces the isolation rate; therefore, freshly donated UCB cells are preferable for the isolation of MSCs.

Mesenchymal Stem/Stromal Cells: A New "Cells as Drugs" Paradigm. Efficacy and Critical Aspects in Cell Therapy

PubMed Central

de Girolamo, Laura; Lucarelli, Enrico; Alessandri, Giulio; Avanzini, Maria Antonietta; Bernardo, Maria Ester; Biagi, Ettore; Brini, Anna Teresa; D’Amico, Giovanna; Fagioli, Franca; Ferrero, Ivana; Locatelli, Franco; Maccario, Rita; Marazzi, Mario; Parolini, Ornella; Pessina, Augusto; Torre, Maria Luisa

2013-01-01

Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects. PMID:23278600

Bone Marrow-Derived Mesenchymal Stem Cells Attenuate Immune-Mediated Liver Injury and Compromise Virus Control During Acute Hepatitis B Virus Infection in Mice.

PubMed

Qu, Mengmeng; Yuan, Xu; Liu, Dan; Ma, Yuhong; Zhu, Jun; Cui, Jun; Yu, Mengxue; Li, Changyong; Guo, Deyin

2017-06-01

Mesenchymal stem cells (MSCs) have been used as therapeutic tools not only for their ability to differentiate toward different cells, but also for their unique immunomodulatory properties. However, it is still unknown how MSCs may affect immunity during hepatitis B virus (HBV) infection. This study was designed to explore the effect of bone marrow-derived MSCs (BM-MSCs) on hepatic natural killer (NK) cells in a mouse model of acute HBV infection. Mice were injected with 1 × 10 6 BM-MSCs, which stained with chloromethyl derivatives of fluorescein diacetate fluorescent probe, 24 h before hydrodynamic injection of viral DNA (pHBV1.3) through the tail vein. In vivo imaging system revealed that BM-MSCs were accumulated in the injured liver, and they attenuated immune-mediated liver injury during HBV infection, as shown by lower alanine aminotransferase levels, reduced proinflammatory cytokine production, and decreased inflammatory cell infiltration in the liver. Importantly, administration of BM-MSCs restrained the increased expression of natural-killer group 2, member D (NKG2D), an important receptor required for NK cell activation in the liver from HBV-infected mice. BM-MSCs also reduced NKG2D expression on NK cells and suppressed the cytotoxicity of NK cells in vitro. Furthermore, BM-MSC-derived transforming growth factor-β1 suppressed NKG2D expression on NK cells. As a consequence, BM-MSC treatment enhanced HBV gene expression and replication in vivo. These results demonstrate that adoptive transfer of BM-MSCs influences innate immunity and limits immune-mediated liver injury during acute HBV infection by suppressing NK cell activity. Meanwhile, the effect of BM-MSCs on prolonging virus clearance needs to be considered in the future.

Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

PubMed

Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

2010-06-01

Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

Targeted inhibition of osteosarcoma tumor growth by bone marrow-derived mesenchymal stem cells expressing cytosine deaminase/5-fluorocytosine in tumor-bearing mice.

PubMed

NguyenThai, Quynh-Anh; Sharma, Neelesh; Luong, Do Huynh; Sodhi, Simrinder Singh; Kim, Jeong-Hyun; Kim, Nameun; Oh, Sung-Jong; Jeong, Dong Kee

2015-01-01

Mesenchymal stem cells (MSCs) are considered as an attractive approach for gene or drug delivery in cancer therapy. In the present study, the ability of human bone marrow-derived MSCs expressing the cytosine deaminase/5-fluorocytosine prodrug (CD/5-FC MSCs) to target the human osteosarcoma cell line Cal72 was evaluated. The stable CD/5-FC MSC cell line was established by transfection of pEGFP containing the cytosine deaminase gene into MSCs with G418 selection. The anti-tumor effect was verified by a bystander effect assay in vitro and co-injection of Cal72 and CD/5-FC MSCs in cancer-bearing mice. The therapeutic CD/5-FC MSCs retained the characteristics of multipotent cells, such as differentiation into adipocytes/osteocytes and expression of mesenchymal markers (CD90 and CD44), and showed migration toward Cal72 cells to a greater extent than the native MSCs. The bystander effect assay showed that the CD/5-FC MSCs significantly augmented Cal72 cytotoxicity in direct co-culture and in the presence of 5-FC through the application of conditioned medium. In osteosarcoma-bearing mice, the CD/5-FC MSCs inhibited tumor growth compared to control mice subcutaneously injected with only Cal72 cells. Taken together, these findings suggest that CD/5-FC MSCs may be suitable for targeting human osteosarcoma. Copyright © 2015 John Wiley & Sons, Ltd.

Potential Use of Human Periapical Cyst-Mesenchymal Stem Cells (hPCy-MSCs) as a Novel Stem Cell Source for Regenerative Medicine Applications

PubMed Central

Tatullo, Marco; Codispoti, Bruna; Pacifici, Andrea; Palmieri, Francesca; Marrelli, Massimo; Pacifici, Luciano; Paduano, Francesco

2017-01-01

Mesenchymal stem cells (MSCs) are attracting growing interest by the scientific community due to their huge regenerative potential. Thus, the plasticity of MSCs strongly suggests the utilization of these cells for regenerative medicine applications. The main issue about the clinical use of MSCs is related to the complex way to obtain them from healthy tissues; this topic has encouraged scientists to search for novel and more advantageous sources of these cells in easily accessible tissues. The oral cavity hosts several cell populations expressing mesenchymal stem cell like-features, furthermore, the access to oral and dental tissues is simple and isolation of cells is very efficient. Thus, oral-derived stem cells are highly attractive for clinical purposes. In this context, human periapical cyst mesenchymal stem cells (hPCy-MSCs) exhibit characteristics similar to other dental-derived MSCs, including their extensive proliferative potential, cell surface marker profile and the ability to differentiate into various cell types such as osteoblasts, adipocytes and neurons. Importantly, hPCy-MSCs are easily collected from the surgically removed periapical cysts; this reusing of biological waste guarantees a smart source of stem cells without any impact on the surrounding healthy tissues. In this review, we report the most interesting research topics related to hPCy-MSCs with a newsworthy discussion about the future insights. This newly discovered cell population exhibits interesting and valuable potentialities that could be of high impact in the future regenerative medicine applications. PMID:29259970

Potential Use of Human Periapical Cyst-Mesenchymal Stem Cells (hPCy-MSCs) as a Novel Stem Cell Source for Regenerative Medicine Applications.

PubMed

Tatullo, Marco; Codispoti, Bruna; Pacifici, Andrea; Palmieri, Francesca; Marrelli, Massimo; Pacifici, Luciano; Paduano, Francesco

2017-01-01

Mesenchymal stem cells (MSCs) are attracting growing interest by the scientific community due to their huge regenerative potential. Thus, the plasticity of MSCs strongly suggests the utilization of these cells for regenerative medicine applications. The main issue about the clinical use of MSCs is related to the complex way to obtain them from healthy tissues; this topic has encouraged scientists to search for novel and more advantageous sources of these cells in easily accessible tissues. The oral cavity hosts several cell populations expressing mesenchymal stem cell like-features, furthermore, the access to oral and dental tissues is simple and isolation of cells is very efficient. Thus, oral-derived stem cells are highly attractive for clinical purposes. In this context, human periapical cyst mesenchymal stem cells (hPCy-MSCs) exhibit characteristics similar to other dental-derived MSCs, including their extensive proliferative potential, cell surface marker profile and the ability to differentiate into various cell types such as osteoblasts, adipocytes and neurons. Importantly, hPCy-MSCs are easily collected from the surgically removed periapical cysts; this reusing of biological waste guarantees a smart source of stem cells without any impact on the surrounding healthy tissues. In this review, we report the most interesting research topics related to hPCy-MSCs with a newsworthy discussion about the future insights. This newly discovered cell population exhibits interesting and valuable potentialities that could be of high impact in the future regenerative medicine applications.

Bone marrow mesenchymal stem cells are an attractive donor cell type for production of cloned pigs as well as genetically modified cloned pigs by somatic cell nuclear transfer.

PubMed

Li, Zicong; He, Xiaoyan; Chen, Liwen; Shi, Junsong; Zhou, Rong; Xu, Weihua; Liu, Dewu; Wu, Zhenfang

2013-10-01

The somatic cell nuclear transfer (SCNT) technique has been widely applied to clone pigs or to produce genetically modified pigs. Currently, this technique relies mainly on using terminally differentiated fibroblasts as donor cells. To improve cloning efficiency, only partially differentiated multipotent mesenchymal stem cells (MSCs), thought to be more easily reprogrammed to a pluripotent state, have been used as nuclear donors in pig SCNT. Although in vitro-cultured embryos cloned from porcine MSCs (MSCs-embryos) were shown to have higher preimplantation developmental ability than cloned embryos reconstructed from fibroblasts (Fs-embryos), the difference in in vivo full-term developmental rate between porcine MSCs-embryos and Fs-embryos has not been investigated so far. In this study, we demonstrated that blastocyst total cell number and full-term survival abilities of MSCs-embryos were significantly higher than those of Fs-embryos cloned from the same donor pig. The enhanced developmental potential of MSCs-embryos may be associated with their nuclear donors' DNA methylation profile, because we found that the methylation level of imprinting genes and repeat sequences differed between MSCs and fibroblasts. In addition, we showed that use of transgenic porcine MSCs generated from transgene plasmid transfection as donor cells for SCNT can produce live transgenic cloned pigs. These results strongly suggest that porcine bone marrow MSCs are a desirable donor cell type for production of cloned pigs and genetically modified cloned pigs via SCNT.

Knockdown of OY-TES-1 by RNAi causes cell cycle arrest and migration decrease in bone marrow-derived mesenchymal stem cells.

PubMed

Cen, Yan-Hui; Guo, Wen-Wen; Luo, Bin; Lin, Yong-Da; Zhang, Qing-Mei; Zhou, Su-Fang; Luo, Guo-Rong; Xiao, Shao-Wen; Xie, Xiao-Xun

2012-10-01

OY-TES-1 is a member of the CTA (cancer-testis antigen) group expressed in a variety of cancer and restrictedly expressed in adult normal tissues, except for testis. To determine whether MSCs (mesenchymal stem cells) express OY-TES-1 and its possible roles on MSCs, OY-TES-1 expression in MSCs isolated from human bone marrow was tested with RT (reverse transcription)-PCR, immunocytochemistry and Western blot. Using RNAi (RNA interference) technology, OY-TES-1 expression was knocked down followed by analysing cell viability, cell cycle, apoptosis and migration ability. MSCs expressed OY-TES-1 at both mRNA and protein levels. The down-regulation of OY-TES-1 expression in these MSCs caused cell growth inhibition, cell cycle arrest, apoptosis induction and migration ability attenuation. Through these primary results it was suggested that OY-TES-1 may influence the biological behaviour of MSCs.

Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells

PubMed Central

Kim, Hyung-Sik; Kang, Kyung-Sun

2012-01-01

Recent studies have shown that amniotic membrane tissue is a rich source of stem cells in humans. In clinical applications, the amniotic membrane tissue had therapeutic effects on wound healing and corneal surface reconstruction. Here, we successfully isolated and identified multipotent stem cells (MSCs) from canine amniotic membrane tissue. We cultured the canine amniotic membrane-derived multipotent stem cells (cAM-MSCs) in low glucose DMEM medium. cAM-MSCs have a fibroblast-like shape and adhere to tissue culture plastic. We characterized the immunophenotype of cAM-MSCs by flow cytometry and measured cell proliferation by the cumulative population doubling level (CPDL). We performed differentiation studies for the detection of trilineage multipotent ability, under the appropriate culture conditions. Taken together, our results show that cAM-MSCs could be a rich source of stem cells in dogs. Furthermore, cAM-MSCs may be useful as a cell therapy application for veterinary regenerative medicine. PMID:23024756

Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: role of the S100A4-miR155-SOCS1-MMP9 axis.

PubMed

Yan, Xin-Long; Jia, Ya-Li; Chen, Lin; Zeng, Quan; Zhou, Jun-Nian; Fu, Chun-Jiang; Chen, Hai-Xu; Yuan, Hong-Feng; Li, Zhi-Wei; Shi, Lei; Xu, Ying-Chen; Wang, Jing-Xue; Zhang, Xiao-Mei; He, Li-Juan; Zhai, Chao; Yue, Wen; Pei, Xue-Tao

2013-06-01

Cancer-associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer-associated MSCs (LC-MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC-MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC-MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC-MSCs compared with liver normal MSCs (LN-MSCs) from adjacent cancer-free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC-MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)-155 in HCC cells was significantly up-regulated by coculture with LC-MSCs and by S100A4 ectopic overexpression. The invasion-promoting effects of S100A4 were significantly attenuated by a miR-155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR-155 expression in HCC cells. We demonstrate that S100A4 secreted from LC-MSCs promotes the expression of miR-155, which mediates the down-regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. S100A4 secreted from LC-MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013). Copyright © 2013 American Association for the Study of Liver Diseases.

Mesenchymal stem cell-based NK4 gene therapy in nude mice bearing gastric cancer xenografts

PubMed Central

Zhu, Yin; Cheng, Ming; Yang, Zhen; Zeng, Chun-Yan; Chen, Jiang; Xie, Yong; Luo, Shi-Wen; Zhang, Kun-He; Zhou, Shu-Feng; Lu, Nong-Hua

2014-01-01

Mesenchymal stem cells (MSCs) have been recognized as promising delivery vehicles for gene therapy of tumors. Gastric cancer is the third leading cause of worldwide cancer mortality, and novel treatment modalities are urgently needed. NK4 is an antagonist of hepatocyte growth factor receptors (Met) which are often aberrantly activated in gastric cancer and thus represent a useful candidate for targeted therapies. This study investigated MSC-delivered NK4 gene therapy in nude mice bearing gastric cancer xenografts. MSCs were transduced with lentiviral vectors carrying NK4 complementary DNA or enhanced green fluorescent protein (GFP). Such transduction did not change the phenotype of MSCs. Gastric cancer xenografts were established in BALB/C nude mice, and the mice were treated with phosphate-buffered saline (PBS), MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tropism of MSCs toward gastric cancer cells was determined by an in vitro migration assay using MKN45 cells, GES-1 cells and human fibroblasts and their presence in tumor xenografts. Tumor growth, tumor cell apoptosis and intratumoral microvessel density of tumor tissue were measured in nude mice bearing gastric cancer xenografts treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 via tail vein injection. The results showed that MSCs migrated preferably to gastric cancer cells in vitro. Systemic MSCs-NK4 injection significantly suppressed the growth of gastric cancer xenografts. MSCs-NK4 migrated and accumulated in tumor tissues after systemic injection. The microvessel density of tumor xenografts was decreased, and tumor cellular apoptosis was significantly induced in the mice treated with MSCs-NK4 compared to control mice. These findings demonstrate that MSC-based NK4 gene therapy can obviously inhibit the growth of gastric cancer xenografts, and MSCs are a better vehicle for NK4 gene therapy than lentiviral vectors. Further studies are warranted to explore the efficacy and safety of the MSC-based NK4 gene therapy in animals and cancer patients. PMID:25525335

Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype

PubMed Central

Zanini, Cristina; Bruno, Stefania; Mandili, Giorgia; Baci, Denisa; Cerutti, Francesco; Cenacchi, Giovanna; Izzi, Leo; Camussi, Giovanni; Forni, Marco

2011-01-01

Background Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. Methodology/Principal Findings In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. Conclusions/Significance Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes. PMID:22194812

Mesenchymal stromal cells of osteosarcoma patients do not show evidence of neoplastic changes during long-term culture.

PubMed

Buddingh, Emilie P; Ruslan, S Eriaty N; Reijnders, Christianne M A; Szuhai, Karoly; Kuijjer, Marieke L; Roelofs, Helene; Hogendoorn, Pancras C W; Maarten Egeler, R; Cleton-Jansen, Anne-Marie; Lankester, Arjan C

2015-01-01

In vitro expanded mesenchymal stromal cells (MSCs) are increasingly used as experimental cellular therapy. However, there have been concerns regarding the safety of their use, particularly with regard to possible oncogenic transformation. MSCs are the hypothesized precursor cells of high-grade osteosarcoma, a tumor with often complex karyotypes occurring mainly in adolescents and young adults. To determine if MSCs from osteosarcoma patients could be predisposed to malignant transformation we cultured MSCs of nine osteosarcoma patients and five healthy donors for an average of 649 days (range 601-679 days). Also, we compared MSCs derived from osteosarcoma patients at diagnosis and from healthy donors using genome wide gene expression profiling. Upon increasing passage, increasing frequencies of binucleate cells were detected, but no increase in proliferation suggestive of malignant transformation occurred in MSCs from either patients or donors. Hematopoietic cell specific Lyn substrate 1 (HLCS1) was differentially expressed (fold change 0.25, P value 0.0005) between MSCs of osteosarcoma patients (n = 14) and healthy donors (n = 9). This study shows that although HCLS1 expression was downregulated in MSCs of osteosarcoma patients and binucleate cells were present in both patient and donor derived MSCs, there was no evidence of neoplastic changes to occur during long-term culture.

Hepatocyte Growth Factor Improves the Therapeutic Efficacy of Human Bone Marrow Mesenchymal Stem Cells via RAD51.

PubMed

Lee, Eun Ju; Hwang, Injoo; Lee, Ji Yeon; Park, Jong Nam; Kim, Keun Cheon; Kim, Gi-Hwan; Kang, Chang-Mo; Kim, Irene; Lee, Seo-Yeon; Kim, Hyo-Soo

2018-03-07

Human embryonic stem cell-derived mesenchymal stem cells (hE-MSCs) have greater proliferative capacity than other human mesenchymal stem cells (hMSCs), suggesting that they may have wider applications in regenerative cellular therapy. In this study, to uncover the anti-senescence mechanism in hE-MSCs, we compared hE-MSCs with adult bone marrow (hBM-MSCs) and found that hepatocyte growth factor (HGF) was more abundantly expressed in hE-MSCs than in hBM-MSCs and that it induced the transcription of RAD51 and facilitated its SUMOylation at K70. RAD51 induction/modification by HGF not only increased telomere length but also increased mtDNA replication, leading to increased ATP generation. Moreover, HGF-treated hBM-MSCs showed significantly better therapeutic efficacy than naive hBM-MSCs. Together, the data suggest that the RAD51-mediated effects of HGF prevent hMSC senescence by promoting telomere lengthening and inducing mtDNA replication and function, which opens the prospect of developing novel therapies for liver disease. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Adipose tissue-derived mesenchymal stem cells in long-term dialysis patients display downregulation of PCAF expression and poor angiogenesis activation.

PubMed

Yamanaka, Shuichiro; Yokote, Shinya; Yamada, Akifumi; Katsuoka, Yuichi; Izuhara, Luna; Shimada, Yohta; Omura, Nobuo; Okano, Hirotaka James; Ohki, Takao; Yokoo, Takashi

2014-01-01

We previously demonstrated that mesenchymal stem cells (MSCs) differentiate into functional kidney cells capable of urine and erythropoietin production, indicating that they may be used for kidney regeneration. However, the viability of MSCs from dialysis patients may be affected under uremic conditions. In this study, we isolated MSCs from the adipose tissues of end-stage kidney disease (ESKD) patients undergoing long-term dialysis (KD-MSCs; mean: 72.3 months) and from healthy controls (HC-MSCs) to compare their viability. KD-MSCs and HC-MSCs were assessed for their proliferation potential, senescence, and differentiation capacities into adipocytes, osteoblasts, and chondrocytes. Gene expression of stem cell-specific transcription factors was analyzed by PCR array and confirmed by western blot analysis at the protein level. No significant differences of proliferation potential, senescence, or differentiation capacity were observed between KD-MSCs and HC-MSCs. However, gene and protein expression of p300/CBP-associated factor (PCAF) was significantly suppressed in KD-MSCs. Because PCAF is a histone acetyltransferase that mediates regulation of hypoxia-inducible factor-1α (HIF-1α), we examined the hypoxic response in MSCs. HC-MSCs but not KD-MSCs showed upregulation of PCAF protein expression under hypoxia. Similarly, HIF-1α and vascular endothelial growth factor (VEGF) expression did not increase under hypoxia in KD-MSCs but did so in HC-MSCs. Additionally, a directed in vivo angiogenesis assay revealed a decrease in angiogenesis activation of KD-MSCs. In conclusion, long-term uremia leads to persistent and systematic downregulation of PCAF gene and protein expression and poor angiogenesis activation of MSCs from patients with ESKD. Furthermore, PCAF, HIF-1α, and VEGF expression were not upregulated by hypoxic stimulation of KD-MSCs. These results suggest that the hypoxic response may be blunted in MSCs from ESKD patients.

Adipose Tissue-Derived Mesenchymal Stem Cells in Long-Term Dialysis Patients Display Downregulation of PCAF Expression and Poor Angiogenesis Activation

PubMed Central

Yamanaka, Shuichiro; Yokote, Shinya; Yamada, Akifumi; Katsuoka, Yuichi; Izuhara, Luna; Shimada, Yohta; Omura, Nobuo; Okano, Hirotaka James; Ohki, Takao; Yokoo, Takashi

2014-01-01

We previously demonstrated that mesenchymal stem cells (MSCs) differentiate into functional kidney cells capable of urine and erythropoietin production, indicating that they may be used for kidney regeneration. However, the viability of MSCs from dialysis patients may be affected under uremic conditions. In this study, we isolated MSCs from the adipose tissues of end-stage kidney disease (ESKD) patients undergoing long-term dialysis (KD-MSCs; mean: 72.3 months) and from healthy controls (HC-MSCs) to compare their viability. KD-MSCs and HC-MSCs were assessed for their proliferation potential, senescence, and differentiation capacities into adipocytes, osteoblasts, and chondrocytes. Gene expression of stem cell-specific transcription factors was analyzed by PCR array and confirmed by western blot analysis at the protein level. No significant differences of proliferation potential, senescence, or differentiation capacity were observed between KD-MSCs and HC-MSCs. However, gene and protein expression of p300/CBP-associated factor (PCAF) was significantly suppressed in KD-MSCs. Because PCAF is a histone acetyltransferase that mediates regulation of hypoxia-inducible factor-1α (HIF-1α), we examined the hypoxic response in MSCs. HC-MSCs but not KD-MSCs showed upregulation of PCAF protein expression under hypoxia. Similarly, HIF-1α and vascular endothelial growth factor (VEGF) expression did not increase under hypoxia in KD-MSCs but did so in HC-MSCs. Additionally, a directed in vivo angiogenesis assay revealed a decrease in angiogenesis activation of KD-MSCs. In conclusion, long-term uremia leads to persistent and systematic downregulation of PCAF gene and protein expression and poor angiogenesis activation of MSCs from patients with ESKD. Furthermore, PCAF, HIF-1α, and VEGF expression were not upregulated by hypoxic stimulation of KD-MSCs. These results suggest that the hypoxic response may be blunted in MSCs from ESKD patients. PMID:25025381

Natural stimulus responsive scaffolds/cells for bone tissue engineering: influence of lysozyme upon scaffold degradation and osteogenic differentiation of cultured marrow stromal cells induced by CaP coatings.

PubMed

Martins, Ana M; Pham, Quynh P; Malafaya, Patrícia B; Raphael, Robert M; Kasper, F Kurtis; Reis, Rui L; Mikos, Antonios G

2009-08-01

This work proposes the use of nonporous, smart, and stimulus responsive chitosan-based scaffolds for bone tissue engineering applications. The overall vision is to use biodegradable scaffolds based on chitosan and starch that present properties that will be regulated by bone regeneration, with the capability of gradual in situ pore formation. Biomimetic calcium phosphate (CaP) coatings were used as a strategy to incorporate lysozyme at the surface of chitosan-based materials with the main objective of controlling and tailoring their degradation profile as a function of immersion time. To confirm the concept, degradation tests with a lysozyme concentration similar to that incorporated into CaP chitosan-based scaffolds were used to study the degradation of the scaffolds and the formation of pores as a function of immersion time. Degradation studies with lysozyme (1.5 g/L) showed the formation of pores, indicating an increase of porosity ( approximately 5-55% up to 21 days) resulting in porous three-dimensional structures with interconnected pores. Additional studies investigated the influence of a CaP biomimetic coating on osteogenic differentiation of rat marrow stromal cells (MSCs) and showed enhanced differentiation of rat MSCs seeded on the CaP-coated chitosan-based scaffolds with lysozyme incorporated. At all culture times, CaP-coated chitosan-based scaffolds with incorporated lysozyme demonstrated greater osteogenic differentiation of MSCs, bone matrix production, and mineralization as demonstrated by calcium deposition measurements, compared with controls (uncoated scaffolds). The ability of these CaP-coated chitosan-based scaffolds with incorporated lysozyme to create an interconnected pore network in situ coupled with the demonstrated positive effect of these scaffolds upon osteogenic differentiation of MSCs and mineralized matrix production illustrates the strong potential of these scaffolds for application in bone tissue engineering strategies.

Analgesia for neuropathic pain by dorsal root ganglion transplantation of genetically engineered mesenchymal stem cells: initial results.

PubMed

Yu, Hongwei; Fischer, Gregory; Ebert, Allison D; Wu, Hsiang-En; Bai, Xiaowen; Hogan, Quinn H

2015-02-12

Cell-based therapy may hold promise for treatment of chronic pain. Mesenchymal stem cells (MSCs) are readily available and robust, and their secretion of therapeutic peptides can be enhanced by genetically engineering. We explored the analgesic potential of transplanting bone marrow-derived MSCs that have been transduced with lentivectors. To optimize efficacy and safety, primary sensory neurons were targeted by MSC injection into the dorsal root ganglia (DRGs). MSCs were transduced using lentivectors to express enhanced green fluorescent protein (EGFP) or to co-express the analgesic peptide glial cell line-derived neurotrophic factor (GDNF) and EGFP by a viral 2A bicistronic transgene cassette. Engineered MSCs were injected into the 4(th) lumbar (L4) and L5 DRGs of adult allogeneic rats to evaluate survival in the DRGs. MSCs were detected by immunofluorescence staining up to 2-3 weeks after injection, distributed in the extracellular matrix space without disrupting satellite glial cell apposition to sensory neurons, suggesting well-tolerated integration of engrafted MSCs into DRG tissue. To examine their potential for inhibiting development of neuropathic pain, MSCs were injected into the L4 and L5 DRGs ipsilateral to a spinal nerve ligation injury. Animals injected with GDNF-engineered MSCs showed moderate but significant reduction in mechanical allodynia and hyperalgesia compared to controls implanted with MSCs expressing EGFP alone. We also observed diminished long-term survival of allografted MSCs at 3 weeks, and the development of a highly-proliferating population of MSCs in 12% of DRGs after transplantation. These data indicate that genetically modified MSCs secreting analgesic peptides could potentially be developed as a novel DRG-targeted cell therapy for treating neuropathic pain. However, further work is needed to address the challenges of MSC survival and excess proliferation, possibly with trials of autologous MSCs, evaluation of clonally selected populations of MSCs, and investigation of regulation of MSC proliferation.

Bone-derived mesenchymal stromal cells from HIV transgenic mice exhibit altered proliferation, differentiation capacity and paracrine functions along with impaired therapeutic potential in kidney injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Kang; Rai, Partab; Lan, Xiqian

Mesenchymal stem cells (MSCs) secrete paracrine factors that could be cytoprotective and serve roles in immunoregulation during tissue injury. Although MSCs express HIV receptors, and co-receptors, and are susceptible to HIV infection, whether HIV-1 may affect biological properties of MSCs needs more study. We evaluated cellular proliferation, differentiation and paracrine functions of MSCs isolated from compact bones of healthy control mice and Tg26 HIV-1 transgenic mice. The ability of MSCs to protect against cisplatin toxicity was studied in cultured renal tubular cells as well as in intact mice. We successfully isolated MSCs from healthy mice and Tg26 HIV-1 transgenic micemore » and found the latter expressed viral Nef, Vpu, NL4-3 and Vif genes. The proliferation and differentiation of Tg26 HIV-1 MSCs was inferior to MSCs from healthy mice. Moreover, transplantation of Tg26 HIV-1 MSCs less effectively improved outcomes compared with healthy MSCs in mice with acute kidney injury. Also, Tg26 HIV-1 MSCs secreted multiple cytokines, but at significantly lower levels than healthy MSCs, which resulted in failure of conditioned medium from these MSCs to protect cultured renal tubular cells from cisplatin toxicity. Therefore, HIV-1 had adverse biological effects on MSCs extending to their proliferation, differentiation, function, and therapeutic potential. These findings will help in advancing mechanistical insight in renal injury and repair in the setting of HIV-1 infection. -- Highlights: •MSCs isolated from HIV mice displayed HIV genes. •MSCs isolated from HIV mice exhibited attenuated growth and paracrine functions. •AKI mice with transplanted HIV-MSC displayed poor outcome. •HIV-1 MSC secreted multiple cytokines but at a lower level.« less

Human mesenchymal stem cells inhibit osteoclastogenesis through osteoprotegerin production.

PubMed

Oshita, Koichi; Yamaoka, Kunihiro; Udagawa, Nobuyuki; Fukuyo, Shunsuke; Sonomoto, Koshiro; Maeshima, Keisuke; Kurihara, Ryuji; Nakano, Kazuhisa; Saito, Kazuyoshi; Okada, Yosuke; Chiba, Kenji; Tanaka, Yoshiya

2011-06-01

Mesenchymal stem cells (MSCs) have been proposed to be a useful tool for treatment of rheumatoid arthritis (RA), not only because of their multipotency but also because of their immunosuppressive effect on lymphocytes, dendritic cells, and other proinflammatory cells. Since bone destruction caused by activated osteoclasts occurs in RA, we undertook the present study to investigate the effect of MSCs on osteoclast function and differentiation in order to evaluate their potential use in RA therapy. Human MSCs and peripheral blood mononuclear cells were cultured under cell-cell contact-free conditions with osteoclast induction medium. Differentiation into osteoclast-like cells was determined by tartrate-resistant acid phosphatase staining and expression of osteoclast differentiation markers. The number of osteoclast-like cells was decreased and expression of cathepsin K and nuclear factor of activated T cells c1 (NF-ATc1) was down-regulated by the addition of either MSCs or a conditioned medium obtained from MSCs. Osteoprotegerin (OPG) was constitutively produced by MSCs and inhibited osteoclastogenesis. However, osteoclast differentiation was not fully recovered upon treatment with either anti-OPG antibody or OPG small interfering RNA, suggesting that OPG had only a partial role in the inhibitory effect of MSCs. Moreover, bone-resorbing activity of osteoclast-like cells was partially recovered by addition of anti-OPG antibody into the conditioned medium. The present results indicate that human MSCs constitutively produce OPG, resulting in inhibition of osteoclastogenesis and expression of NF-ATc1 and cathepsin K in the absence of cell-cell contact. Therefore, we conclude that human MSCs exert a suppressive effect on osteoclastogenesis, which may be beneficial in inhibition of joint damage in RA. Copyright © 2011 by the American College of Rheumatology.

Mesenchymal stem cells-derived MFG-E8 accelerates diabetic cutaneous wound healing.

PubMed

Uchiyama, Akihiko; Motegi, Sei-Ichiro; Sekiguchi, Akiko; Fujiwara, Chisako; Perera, Buddhini; Ogino, Sachiko; Yokoyama, Yoko; Ishikawa, Osamu

2017-06-01

Diabetic wounds are intractable due to complex factors, such as the inhibition of angiogenesis, dysfunction of phagocytosis by macrophages and abnormal inflammatory responses. It is recognized that mesenchymal stem cells (MSCs) promote wound healing in diabetic mice. We previously demonstrated that MSCs produce large amounts of MFG-E8. The objective was to ascertain the role of MSCs-derived MFG-E8 in murine diabetic wounds. MFG-E8 WT/KO MSCs or rMFG-E8 were subcutaneously injected around the wound in diabetic db/db mice, and wound areas were analyzed. Quantification of angiogenesis, infiltrating inflammatory cells, apoptotic cells at the wound area was performed by immunofluorescence staining and real-time PCR. Phagocytosis assay was performed using peritoneal macrophages from WT or db/db mice. MFG-E8 expression in granulation tissue in diabetic mice was significantly reduced compared with that in non-diabetic mice. We next examined the effect of subcutaneous injection of MFG-E8 WT/KO MSCs around the wound. Diabetic wound healing was significantly accelerated by the injection of MSCs. Diabetic wound healing in MFG-E8 KO MSCs-injected wounds was significantly delayed compared to that in WT MSCs-injected wounds. The numbers of CD31 + EC and NG2 + pericytes, as well as M2 macrophages in wounds in KO MSCs-injected mice were significantly decreased. MFG-E8 WT MSCs treatment suppressed the number of apoptotic cells and TNF-α + cells in wounds. In an in vitro assay, MFG-E8 WT MSCs-conditioned medium enhanced phagocytosis of apoptotic cells by peritoneal macrophages from diabetic mice. MSCs-derived MFG-E8 might accelerate diabetic wound healing by promoting angiogenesis, the clearance of apoptotic cells, and the infiltration of M2 macrophages, and by suppressing inflammatory cytokines in wound area. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

A living thick nanofibrous implant bifunctionalized with active growth factor and stem cells for bone regeneration

PubMed Central

Eap, Sandy; Keller, Laetitia; Schiavi, Jessica; Huck, Olivier; Jacomine, Leandro; Fioretti, Florence; Gauthier, Christian; Sebastian, Victor; Schwinté, Pascale; Benkirane-Jessel, Nadia

2015-01-01

New-generation implants focus on robust, durable, and rapid tissue regeneration to shorten recovery times and decrease risks of postoperative complications for patients. Herein, we describe a new-generation thick nanofibrous implant functionalized with active containers of growth factors and stem cells for regenerative nanomedicine. A thick electrospun poly(ε-caprolactone) nanofibrous implant (from 700 μm to 1 cm thick) was functionalized with chitosan and bone morphogenetic protein BMP-7 as growth factor using layer-by-layer technology, producing fish scale-like chitosan/BMP-7 nanoreservoirs. This extracellular matrix-mimicking scaffold enabled in vitro colonization and bone regeneration by human primary osteoblasts, as shown by expression of osteocalcin, osteopontin, and bone sialoprotein (BSPII), 21 days after seeding. In vivo implantation in mouse calvaria defects showed significantly more newly mineralized extracellular matrix in the functionalized implant compared to a bare scaffold after 30 days’ implantation, as shown by histological scanning electron microscopy/energy dispersive X-ray microscopy study and calcein injection. We have as well bifunctionalized our BMP-7 therapeutic implant by adding human mesenchymal stem cells (hMSCs). The activity of this BMP-7-functionalized implant was again further enhanced by the addition of hMSCs to the implant (living materials), in vivo, as demonstrated by the analysis of new bone formation and calcification after 30 days’ implantation in mice with calvaria defects. Therefore, implants functionalized with BMP-7 nanocontainers associated with hMSCs can act as an accelerator of in vivo bone mineralization and regeneration. PMID:25709432

Immunogenicity of allogeneic mesenchymal stem cells

PubMed Central

Schu, Sabine; Nosov, Mikhail; O'Flynn, Lisa; Shaw, Georgina; Treacy, Oliver; Barry, Frank; Murphy, Mary; O'Brien, Timothy; Ritter, Thomas

2012-01-01

Mesenchymal stem cells (MSCs) inhibit proliferation of allogeneic T cells and express low levels of major histocompatibility complex class I (MHCI), MHCII and vascular adhesion molecule-1 (VCAM-1). We investigated whether their immunosuppressive properties and low immunophenotype protect allogeneic rat MSCs against cytotoxic lysis in vitro and result in a reduced immune response in vivo. Rat MSCs were partially protected against alloantigen-specific cytotoxic T cells in vitro. However, after treatment with IFN-γ and IL-1β, MSCs upregulated MHCI, MHCII and VCAM-1, and cytotoxic lysis was significantly increased. In vivo, allogeneic T cells but not allogeneic MSCs induced upregulation of the activation markers CD25 and CD71 as well as downregulation of CD62L on CD4+ T cells from recipient rats. However, intravenous injection of allo-MSCs in rats led to the formation of alloantibodies with the capacity to facilitate complement-mediated lysis, although IgM levels were markedly decreased compared with animals that received T cells. The allo-MSC induced immune response was sufficient to lead to significantly reduced survival of subsequently injected allo-MSCs. Interestingly, no increased immunogenicity of IFN-γ stimulated allo-MSCs was observed in vivo. Both the loss of protection against cytotoxic lysis under inflammatory conditions and the induction of complement-activating antibodies will likely impact the utility of allogeneic MSCs for therapeutic applications. PMID:22151542

Cultured Human Adipose Tissue Pericytes and Mesenchymal Stromal Cells Display a Very Similar Gene Expression Profile

PubMed Central

Malta, Tathiane Maistro; de Deus Wagatsuma, Virgínia Mara; Palma, Patrícia Viana Bonini; Araújo, Amélia Goes; Ribeiro Malmegrim, Kelen Cristina; Morato de Oliveira, Fábio; Panepucci, Rodrigo Alexandre; Silva, Wilson Araújo; Kashima Haddad, Simone; Covas, Dimas Tadeu

2015-01-01

Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3+ lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols. PMID:26192741

Influence of bone marrow-derived mesenchymal stem cells pre-implantation differentiation approach on periodontal regeneration in vivo.

PubMed

Cai, Xinjie; Yang, Fang; Yan, Xiangzhen; Yang, Wanxun; Yu, Na; Oortgiesen, Daniel A W; Wang, Yining; Jansen, John A; Walboomers, X Frank

2015-04-01

The implantation of bone marrow-derived mesenchymal stem cells (MSCs) has previously been shown successful to achieve periodontal regeneration. However, the preferred pre-implantation differentiation strategy (e.g. maintenance of stemness, osteogenic or chondrogenic induction) to obtain optimal periodontal regeneration is still unknown. This in vivo study explored which differentiation approach is most suitable for periodontal regeneration. Mesenchymal stem cells were obtained from Fischer rats and seeded onto poly(lactic-co-glycolic acid)/poly(ɛ-caprolactone) electrospun scaffolds, and then pre-cultured under different in vitro conditions: (i) retention of multilineage differentiation potential; (ii) osteogenic differentiation approach; and (iii) chondrogenic differentiation approach. Subsequently, the cell-scaffold constructs were implanted into experimental periodontal defects of Fischer rats, with empty scaffolds as controls. After 6 weeks of implantation, histomorphometrical analyses were applied to evaluate the regenerated periodontal tissues. The chondrogenic differentiation approach showed regeneration of alveolar bone and ligament tissues. The retention of multilineage differentiation potential supported only ligament regeneration, while the osteogenic differentiation approach boosted alveolar bone regeneration. Chondrogenic differentiation of MSCs before implantation is a useful strategy for regeneration of alveolar bone and periodontal ligament, in the currently used rat model. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Comparative Analysis of the In Vitro Effects of Pulsed Electromagnetic Field Treatment on Osteogenic Differentiation of Two Different Mesenchymal Cell Lineages

PubMed Central

Ceccarelli, Gabriele; Bloise, Nora; Mantelli, Melissa; Gastaldi, Giulia; Fassina, Lorenzo; De Angelis, Maria Gabriella Cusella; Ferrari, Davide; Imbriani, Marcello

2013-01-01

Abstract Human mesenchymal stem cells (MSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications. Exposure of MSCs to physical stimuli favors early and rapid activation of the tissue repair process. In this study we investigated the in vitro effects of pulsed electromagnetic field (PEMF) treatment on the proliferation and osteogenic differentiation of bone marrow MSCs (BM-MSCs) and adipose-tissue MSCs (ASCs), to assess if both types of MSCs could be indifferently used in combination with PEMF exposure for bone tissue healing. We compared the cell viability, cell matrix distribution, and calcified matrix production in unstimulated and PEMF-stimulated (magnetic field: 2 mT, amplitude: 5 mV) mesenchymal cell lineages. After PEMF exposure, in comparison with ASCs, BM-MSCs showed an increase in cell proliferation (p<0.05) and an enhanced deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p<0.05). Calcium deposition was 1.5-fold greater in BM-MSC–derived osteoblasts (p<0.05). The immunofluorescence related to the deposition of bone matrix proteins and calcium showed their colocalization to the cell-rich areas for both types of MSC-derived osteoblast. Alkaline phosphatase activity increased nearly 2-fold (p<0.001) and its protein content was 1.2-fold higher in osteoblasts derived from BM-MSCs. The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed up-regulated transcription specific for bone sialoprotein, osteopontin, osteonectin, and Runx2, but at a higher level for cells differentiated from BM-MSCs. All together these results suggest that PEMF promotion of bone extracellular matrix deposition is more efficient in osteoblasts differentiated from BM-MSCs. PMID:23914335

Interleukin-3 enhances the migration of human mesenchymal stem cells by regulating expression of CXCR4.

PubMed

Barhanpurkar-Naik, Amruta; Mhaske, Suhas T; Pote, Satish T; Singh, Kanupriya; Wani, Mohan R

2017-07-14

Mesenchymal stem cells (MSCs) represent an important source for cell therapy in regenerative medicine. MSCs have shown promising results for repair of damaged tissues in various degenerative diseases in animal models and also in human clinical trials. However, little is known about the factors that could enhance the migration and tissue-specific engraftment of exogenously infused MSCs for successful regenerative cell therapy. Previously, we have reported that interleukin-3 (IL-3) prevents bone and cartilage damage in animal models of rheumatoid arthritis and osteoarthritis. Also, IL-3 promotes the differentiation of human MSCs into functional osteoblasts and increases their in-vivo bone regenerative potential in immunocompromised mice. However, the role of IL-3 in migration of MSCs is not yet known. In the present study, we investigated the role of IL-3 in migration of human MSCs under both in-vitro and in-vivo conditions. MSCs isolated from human bone marrow, adipose and gingival tissues were used for in-vitro cell migration, motility and wound healing assays in the presence or absence of IL-3. The effect of IL-3 preconditioning on expression of chemokine receptors and integrins was examined by flow cytometry and real-time PCR. The in-vivo migration of IL-3-preconditioned MSCs was investigated using a subcutaneous matrigel-releasing stromal cell-derived factor-1 alpha (SDF-1α) model in immunocompromised mice. We observed that human MSCs isolated from all three sources express IL-3 receptor-α (IL-3Rα) both at gene and protein levels. IL-3 significantly enhances in-vitro migration, motility and wound healing abilities of MSCs. Moreover, IL-3 preconditioning upregulates expression of chemokine (C-X-C motif) receptor 4 (CXCR4) on MSCs, which leads to increased migration of cells towards SDF-1α. Furthermore, CXCR4 antagonist AMD3100 decreases the migration of IL-3-treated MSCs towards SDF-1α. Importantly, IL-3 also induces in-vivo migration of MSCs towards subcutaneously implanted matrigel-releasing-SDF-1α in immunocompromised mice. The present study demonstrates for the first time that IL-3 has an important role in enhancing the migration of human MSCs through regulation of the CXCR4/SDF-1α axis. These findings suggest a potential role of IL-3 in improving the efficacy of MSCs in regenerative cell therapy.

Comparison of Immunological Characteristics of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells and Bone Marrow

PubMed Central

Fu, Xin; Chen, Yao; Xie, Fang-Nan; Dong, Ping; Liu, Wen-bo; Cao, Yilin

2015-01-01

Mesenchymal stem cell (MSC) has great potential for both regenerative medicine and immunotherapy due to its multipotency and immunomodulatory property. The derivation of MSCs from human tissues involves an invasive procedure and the obtained MSCs often suffer from inconsistent quality. To overcome these issues, the approaches of deriving a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs) were established. However, few studies compared the immunological characteristics of MSCs derived from hESCs with tissue-derived MSCs or demonstrated differences and the underlying mechanisms. Here, we differentiated H9 hESCs into MSC-like cells (H9-MSCs) through an embryoid body outgrowth method and compared the immunological characteristics of H9-MSCs with bone marrow-derived MSCs (BMSCs). Both sources of derived cells exhibited typical MSC morphologies and surface marker expressions, as well as multipotency to differentiate into osteogenic and adipogenic lineages. A immunological characterization study showed that H9-MSCs and BMSCs had similar immunoprivileged properties without triggering allogeneic lymphocyte proliferation as well as equivalent immunosuppressive effects on T-cell proliferation induced by either cellular or mitogenic stimuli. Flow cytometry analysis revealed a lower expression of human major histocompatability complex class II molecule human lymphocyte antigen (HLA)-DR and a higher expression of coinhibitory molecule B7-H1 in H9-MSCs than in BMSCs. Interferon gamma (IFN-γ) is a proinflammatory cytokine that can induce the expression of HLA class II molecules in many cell types. Our results showed that pretreatment of H9-MSCs and BMSCs with IFN-γ did not change their immunogenicity and immunosuppressive abilities, but increased the difference between H9-MSCs and BMSCs for their expression of HLA-DR. Further detection of expression of molecules involved in IFN-γ signaling pathways suggested that the lower expression of HLA-DR in H9-MSCs could be partially attributed to the lower expression and the less nuclear translocation of its transcriptional factor CIITA. The present study provides evidence that the hESC-derived MSCs share similar immunogenicity and immunosuppressive abilities with BMSCs, but differ in the expression profile of immunological markers and the responsiveness to certain inflammatory cytokines, which suggests that H9-MSCs could be a safe and efficient candidate for MSC treatment in patients with inflammatory disorders. PMID:25256849

Fibrin matrices enhance the transplant and efficacy of cytotoxic stem cell therapy for post-surgical cancer

PubMed Central

Bagó, Juli R.; Pegna, Guillaume J.; Okolie, Onyi; Hingtgen, Shawn D.

2016-01-01

Tumor-homing cytotoxic stem cell (SC) therapy is a promising new approach for treating the incurable brain cancer glioblastoma (GBM). However, problems of retaining cytotoxic SCs within the post-surgical GBM resection cavity are likely to significantly limit the clinical utility of this strategy. Here, we describe a new fibrin-based transplant approach capable of increasing cytotoxic SC retention and persistence within the resection cavity, yet remaining permissive to tumoritropic migration. This fibrin-based transplant can effectively treat both solid and post-surgical human GBM in mice. Using our murine model of image-guided model of GBM resection, we discovered that suspending human mesenchymal stem cells (hMSCS) in a fibrin matrix increased initial retention in the surgical resection cavity 2-fold and prolonged persistence in the cavity 3-fold compared to conventional delivery strategies. Time-lapse motion analysis revealed that cytotoxic hMSCs in the fibrin matrix remain tumoritropic, rapidly migrating from the fibrin matrix to co-localize with cultured human GBM cells. We encapsulated hMSCs releasing the cytotoxic agent TRAIL (hMSC-sTR) in fibrin, and found hMSC-sTR/fibrin therapy reduced the viability of multiple 3-D human GBM spheroids and regressed established human GBM xenografts 3-fold in 11 days. Mimicking clinical therapy of surgically resected GBM, intra-cavity seeding of therapeutic hMSC-sTR encapsulated in fibrin reduced post-surgical GBM volumes 6-fold, increased time to recurrence 4-fold, and prolonged median survival from 15 to 36 days compared to control-treated animals. Fibrin-based SC therapy could represent a clinically compatible, viable treatment to suppress recurrence of post-surgical GBM and other lethal cancer types. PMID:26803410

Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine

PubMed Central

Bae, Yoo-Jin; Kwon, Yong-Rim; Kim, Hye Joung; Lee, Seok

2017-01-01

Background Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. Methods 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. Results AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. Conclusion 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs. PMID:28401097

Mesenchymal Stem Cells from Human Amniotic Membrane and Umbilical Cord Can Diminish Immunological Response in an in vitro Allograft Model.

PubMed

Dabrowski, Filip A; Burdzinska, Anna; Kulesza, Agnieszka; Chlebus, Marcin; Kaleta, Beata; Borysowski, Jan; Zolocinska, Aleksandra; Paczek, Leszek; Wielgos, Miroslaw

2017-01-01

Mesenchymal stem cells (MSCs) are gaining rising interest in gynecology and obstetrics. MSCs immunomodulatory properties are suitable enough to reduce perinatal morbidity caused by inflammation in premature neonates. The aim of this study was to evaluate and compare the ability to inhibit allo-activated lymphocytes proliferation by MSCs derived from different sources: amniotic membrane (AM), umbilical cord (UC) and adipose tissue (AT). MSCs were isolated from AM (n = 7) and UC (n = 6) and AT (n = 6) of healthy women. Cells were characterized by flow cytometry and differentiation assay. To evaluate the potential of fetal and adult MSCs to diminish immunological response, mixed lymphocytes reaction (MLR) was performed. Amnion and UC-derived cells displayed typical MSCs characteristics. Addition of MSCs to MLR significantly inhibited the proliferation of stimulated lymphocytes. The effect was observed regardless of the MSCs type used (p < 0.01 in all groups). Comparative analysis revealed no significant differences in this action between tested MSCs types. Additionally, no type of MSCs significantly stimulated allogeneic lymphocytes. The results prove the immunosuppressive capacities of fetal-derived MSCs in vitro. In the future, they may be potentially used to treat premature newborn as well as in immunomodulation in post-transplant therapy. © 2016 S. Karger AG, Basel.

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

PubMed Central

Lee, Janet; Baek, Jeong-Hwa; Choi, Kyu-Sil; Kim, Hyun-Soo; Park, Hye-Young; Ha, Geun-Hyoung; Park, Ho; Lee, Kyo-Won; Lee, Chang Geun; Yang, Dong-Yun; Moon, Hyo Eun; Paek, Sun Ha; Lee, Chang-Woo

2013-01-01

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs. PMID:23324348

Potency of umbilical cord blood- and Wharton's jelly-derived mesenchymal stem cells for scarless wound healing.

PubMed

Doi, Hanako; Kitajima, Yuriko; Luo, Lan; Yan, Chan; Tateishi, Seiko; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Mori, Ryoichi; Masuzaki, Hideaki; Shimokawa, Isao; Hirano, Akiyoshi; Li, Tao-Sheng

2016-01-05

Postnatally, scars occur as a consequence of cutaneous wound healing. Scarless wound healing is highly desired for patients who have undergone surgery or trauma, especially to exposed areas. Based on the properties of mesenchymal stem cells (MSCs) for tissue repair and immunomodulation, we investigated the potential of MSCs for scarless wound healing. MSCs were expanded from umbilical cord blood (UCB-MSCs) and Wharton's jelly (WJ-MSCs) from healthy donors who underwent elective full-term pregnancy caesarean sections. UCB-MSCs expressed lower levels of the pre-inflammatory cytokines IL1A and IL1B, but higher levels of the extracellular matrix (ECM)-degradation enzymes MMP1 and PLAU compared with WJ-MSCs, suggesting that UCB-MSCs were more likely to favor scarless wound healing. However, we failed to find significant benefits for stem cell therapy in improving wound healing and reducing collagen deposition following the direct injection of 1.0 × 10(5) UCB-MSCs and WJ-MSCs into 5 mm full-thickness skin defect sites in nude mice. Interestingly, the implantation of UCB-MSCs tended to increase the expression of MMP2 and PLAU, two proteases involved in degradation of the extracellular matrix in the wound tissues. Based on our data, UCB-MSCs are more likely to be a favorable potential stem cell source for scarless wound healing, although a better experimental model is required for confirmation.

PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact.

PubMed

Lee, Byung-Chul; Kim, Hyung-Sik; Shin, Tae-Hoon; Kang, Insung; Lee, Jin Young; Kim, Jae-Jun; Kang, Hyun Kyoung; Seo, Yoojin; Lee, Seunghee; Yu, Kyung-Rok; Choi, Soon Won; Kang, Kyung-Sun

2016-05-27

Mesenchymal stem cells (MSCs) possess unique immunomodulatory abilities. Many studies have elucidated the clinical efficacy and underlying mechanisms of MSCs in immune disorders. Although immunoregulatory factors, such as Prostaglandin E2 (PGE2), and their mechanisms of action on immune cells have been revealed, their effects on MSCs and regulation of their production by the culture environment are less clear. Therefore, we investigated the autocrine effect of PGE2 on human adult stem cells from cord blood or adipose tissue, and the regulation of its production by cell-to-cell contact, followed by the determination of its immunomodulatory properties. MSCs were treated with specific inhibitors to suppress PGE2 secretion, and proliferation was assessed. PGE2 exerted an autocrine regulatory function in MSCs by triggering E-Prostanoid (EP) 2 receptor. Inhibiting PGE2 production led to growth arrest, whereas addition of MSC-derived PGE2 restored proliferation. The level of PGE2 production from an equivalent number of MSCs was down-regulated via gap junctional intercellular communication. This cell contact-mediated decrease in PGE2 secretion down-regulated the suppressive effect of MSCs on immune cells. In conclusion, PGE2 produced by MSCs contributes to maintenance of self-renewal capacity through EP2 in an autocrine manner, and PGE2 secretion is down-regulated by cell-to-cell contact, attenuating its immunomodulatory potency.

Neural differentiation of mesenchymal stem cells influences chemotactic responses to HGF.

PubMed

Zheng, Bing; Wang, Chunyan; He, Lihong; Xu, Xiaojing; Qu, Jing; Hu, Jun; Zhang, Huanxiang

2013-01-01

Recently, mesenchymal stem cells (MSCs) have been extensively used for cell-based therapies in neuronal degenerative disease. Although much effort has been devoted to the delineation of factors involved in the migration of MSCs, the relationship between the chemotactic responses and the differentiation status of these cells remains elusive. Here, we report that MSCs in varying neural differentiation states display different chemotactic responses to hepatocyte growth factor (HGF): first, the number of chemotaxing MSCs and the optimal concentrations of HGF that induced the peak migration varied greatly; second, time-lapse video analysis showed that MSCs in certain differentiation state migrated more efficiently toward HGF; third, the phosphorylation levels of Akt, ERK1/2, SAPK/JNK, and p38MAPK were closely related to the differentiation levels of MSCs subjected to HGF; and finally, although inhibition of ERK1/2 signaling significantly attenuated HGF-stimulated transfilter migration of both undifferentiated and differentiating MSCs, abolishment of PI3K/Akt, p38MAPK, or SAPK/JNK signaling only decreased the number of migrated cells in certain differentiation state(s). Blocking of PI3K/Akt or MAPK signaling impaired the migration efficiency and/or speed, the extent of which depends on the cell differentiation states. Meanwhile, F-actin rearrangement, which is essential for MSCs chemotaxis, was induced by HGF, and the time points of cytoskeletal reorganization were different among these cells. Collectively, these results demonstrate that neural differentiation of MSCs influences their chemotactic responses to HGF: MSCs in varying differentiation states possess different migratory capacities, thereby shedding light on optimization of the therapeutic potential of MSCs to be employed for neural regeneration after injury. Copyright © 2012 Wiley Periodicals, Inc.

Mesenchymal stem cells are efficiently transduced with adenoviruses bearing type 35-derived fibers and the transduced cells with the IL-28A gene produces cytotoxicity to lung carcinoma cells co-cultured.

PubMed

Suzuki, Takeo; Kawamura, Kiyoko; Li, Quanhai; Okamoto, Shinya; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Yamaguchi, Naoto; Tagawa, Masatoshi

2014-09-25

Transduction of human mesenchymal stem cells (MSCs) with type 5 adenoviruses (Ad5) is limited in the efficacy because of the poor expression level of the coxsackie adenovirus receptor (CAR) molecules. We examined a possible improvement of Ad-mediated gene transfer in MSCs by substituting the fiber region of type 5 Ad with that of type 35 Ad. Expression levels of CAR and CD46 molecules, which are the major receptors for type 5 and type 35 Ad, respectively, were assayed with flow cytometry. We constructed vectors expressing the green fluorescent protein gene with Ad5 or modified Ad5 bearing the type 35 fiber region (AdF35), and examined the infectivity to MSCs with flow cytometry. We investigated anti-tumor effects of MSCs transduced with interleukin (IL)-28A gene on human lung carcinoma cells with a colorimetric assay. Expression of IL-28A receptors was tested with the polymerase chain reaction. A promoter activity of transcriptional regulatory regions in MSCs was determined with a luciferase assay and a tumor growth-promoting ability of MSCs was tested with co-injection of human tumor cells in nude mice. MSCs expressed CD46 but scarcely CAR molecules, and subsequently were transduced with AdF35 but not with Ad5. Growth of MSCs transduced with the IL-28A gene remained the same as that of untransduced cells since MSCs were negative for the IL-28A receptors. The IL-28A-transduced MSCs however suppressed growth of lung carcinoma cells co-cultured, whereas MSCs transduced with AdF35 expressing the β-galactosidase gene did not. A regulatory region of the cyclooygenase-2 gene possessed transcriptional activities greater than other tumor promoters but less than the cytomegalovirus promoter, and MSCs themselves did not support tumor growth in vivo. AdF35 is a suitable vector to transduce MSCs that are resistant to Ad5-mediated gene transfer. MSCs infected with AdF35 that activate an exogenous gene by the cytomegalovirus promoter can be a vehicle to deliver the gene product to targeted cells.

N-acetylcysteine-pretreated human embryonic mesenchymal stem cell administration protects against bleomycin-induced lung injury.

PubMed

Wang, Qiao; Zhu, Hong; Zhou, Wu-Gang; Guo, Xiao-Can; Wu, Min-Juan; Xu, Zhen-Yu; Jiang, Jun-feng; Shen, Ce; Liu, Hou-Qi

2013-08-01

The transplantation of mesenchymal stem cells (MSCs) has been reported to be a promising approach in the treatment of acute lung injury. However, the poor efficacy of transplanted MSCs is one of the serious handicaps in the progress of MSC-based therapy. Therefore, the purpose of this study was to investigate whether the pretreatment of human embryonic MSCs (hMSCs) with an antioxidant, namely N-acetylcysteine (NAC), can improve the efficacy of hMSC transplantation in lung injury. In vitro, the antioxidant capacity of NAC-pretreated hMSCs was assessed using intracellular reactive oxygen species (ROS) and glutathione assays and cell adhesion and spreading assays. In vivo, the therapeutic potential of NAC-pretreated hMSCs was assessed in a bleomycin-induced model of lung injury in nude mice. The pretreatment of hMSCs with NAC improved antioxidant capacity to defend against redox imbalances through the elimination of cellular ROS, increasing cellular glutathione levels, and the enhancement of cell adhesion and spreading when exposed to oxidative stresses in vitro. In addition, the administration of NAC-pretreated hMSCs to nude mice with bleomycin-induced lung injury decreased the pathological grade of lung inflammation and fibrosis, hydroxyproline content and numbers of neutrophils and inflammatory cytokines in bronchoalveolar lavage fluid and apoptotic cells, while enhancing the retention and proliferation of hMSCs in injured lung tissue and improving the survival rate of mice compared with results from untreated hMSCs. The pretreatment of hMSCs with NAC could be a promising therapeutic approach to improving cell transplantation and, therefore, the treatment of lung injury.

Bcl-xL Genetic Modification Enhanced the Therapeutic Efficacy of Mesenchymal Stem Cell Transplantation in the Treatment of Heart Infarction

PubMed Central

Xue, Xiaodong; Liu, Yu; Zhang, Jian; Liu, Tao; Yang, Zhonglu; Wang, Huishan

2015-01-01

Objectives. Low survival rate of mesenchymal stem cells (MSCs) severely limited the therapeutic efficacy of cell therapy in the treatment of myocardial infarction (MI). Bcl-xL genetic modification might enhance MSC survival after transplantation. Methods. Adult rat bone marrow MSCs were modified with human Bcl-xL gene (hBcl-xL-MSCs) or empty vector (vector-MSCs). MSC apoptosis and paracrine secretions were characterized using flow cytometry, TUNEL, and ELISA in vitro. In vivo, randomized adult rats with MI received myocardial injections of one of the three reagents: hBcl-xL-MSCs, vector-MSCs, or culture medium. Histochemistry, TUNEL, and echocardiography were carried out to evaluate cell engraftment, apoptosis, angiogenesis, scar formation, and cardiac functional recovery. Results. In vitro, cell apoptosis decreased 43%, and vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and plate-derived growth factor (PDGF) increased 1.5-, 0.7-, and 1.2-fold, respectively, in hBcl-xL-MSCs versus wild type and vector-MSCs. In vivo, cell apoptosis decreased 40% and 26% in hBcl-xL-MSC group versus medium and vector-MSC group, respectively. Similar results were observed in cell engraftment, angiogenesis, scar formation, and cardiac functional recovery. Conclusions. Genetic modification of MSCs with hBcl-xL gene could be an intriguing strategy to improve the therapeutic efficacy of cell therapy in the treatment of heart infarction. PMID:26074971

Bcl-xL Genetic Modification Enhanced the Therapeutic Efficacy of Mesenchymal Stem Cell Transplantation in the Treatment of Heart Infarction.

PubMed

Xue, Xiaodong; Liu, Yu; Zhang, Jian; Liu, Tao; Yang, Zhonglu; Wang, Huishan

2015-01-01

Objectives. Low survival rate of mesenchymal stem cells (MSCs) severely limited the therapeutic efficacy of cell therapy in the treatment of myocardial infarction (MI). Bcl-xL genetic modification might enhance MSC survival after transplantation. Methods. Adult rat bone marrow MSCs were modified with human Bcl-xL gene (hBcl-xL-MSCs) or empty vector (vector-MSCs). MSC apoptosis and paracrine secretions were characterized using flow cytometry, TUNEL, and ELISA in vitro. In vivo, randomized adult rats with MI received myocardial injections of one of the three reagents: hBcl-xL-MSCs, vector-MSCs, or culture medium. Histochemistry, TUNEL, and echocardiography were carried out to evaluate cell engraftment, apoptosis, angiogenesis, scar formation, and cardiac functional recovery. Results. In vitro, cell apoptosis decreased 43%, and vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and plate-derived growth factor (PDGF) increased 1.5-, 0.7-, and 1.2-fold, respectively, in hBcl-xL-MSCs versus wild type and vector-MSCs. In vivo, cell apoptosis decreased 40% and 26% in hBcl-xL-MSC group versus medium and vector-MSC group, respectively. Similar results were observed in cell engraftment, angiogenesis, scar formation, and cardiac functional recovery. Conclusions. Genetic modification of MSCs with hBcl-xL gene could be an intriguing strategy to improve the therapeutic efficacy of cell therapy in the treatment of heart infarction.

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Zhenhua; Key Laboratory of Neurodegeneration, Ministry of Education, Beijing; Department of Anatomy, Anhui Medical University, Hefei, 230032

2011-12-10

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristicsmore » of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy. -- Highlights: Black-Right-Pointing-Pointer Spontaneous transformation of cynomolgus monkey MSCs in vitro. Black-Right-Pointing-Pointer Transformed mesenchymal cells lack multipotency. Black-Right-Pointing-Pointer Transformed mesenchymal cells are highly tumorigenic. Black-Right-Pointing-Pointer Transformed mesenchymal cells do not have the characteristics of cancer stem cells.« less

Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Yiting; State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193; Liu, Lan

2015-06-10

Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest bymore » reducing cyclinB1 expression. Compared with WT-MSCs, Wip1{sup −/−} MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1{sup −/−} MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1{sup −/−} MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs. - Highlights: • Wip1 knockout inhibited MSCs proliferation through reducing cyclinB1 expression. • Wip1{sup −/−} MSCs displayed premature growth arrest in vitro after six passages. • Knocking out Wip1 increases the migratory capacity of MSCs. • The PI3K/AKT/Rac1 pathway mediates the Wip1-knockout-induced migration of MSCs. • Overexpression of Wip1 reversed premature senescence and migration of Wip1{sup −/−} MSCs.« less

Cancer cell-soluble factors reprogram mesenchymal stromal cells to slow cycling, chemoresistant cells with a more stem-like state.

PubMed

El-Badawy, Ahmed; Ghoneim, Mohamed A; Gabr, Mahmoud M; Salah, Radwa Ayman; Mohamed, Ihab K; Amer, Marwa; El-Badri, Nagwa

2017-11-07

Mesenchymal stem cells (MSCs) play different roles in modulating tumor progression, growth, and metastasis. MSCs are recruited to the tumor site in large numbers and subsequently have an important microenvironmental role in modulating tumor progression and drug sensitivity. However, the effect of the tumor microenvironment on MSC plasticity remains poorly understood. Herein, we report a paracrine effect of cancer cells, in which they secrete soluble factors that promote a more stem-like state in bone marrow mesenchymal stem cells (BM-MSCs). The effect of soluble factors secreted from MCF7, Hela, and HepG2 cancer cell lines on BM-MSCs was assessed using a Transwell indirect coculture system. After 5 days of coculture, BM-MSCs were characterized by flow cytometry for surface marker expression, by qPCR for gene expression profile, and by confocal immunofluorescence for marker expression. We then measured the sensitivity of cocultured BM-MSCs to chemotherapeutic agents, their cell cycle profile, and their response to DNA damage. The sphere formation, invasive properties, and in-vivo performance of BM-MSCs after coculture with cancer cells were also measured. Indirect coculture of cancer cells and BM-MSCs, without direct cell contact, generated slow cycling, chemoresistant spheroid stem cells that highly expressed markers of pluripotency, cancer cells, and cancer stem cells (CSCs). They also displayed properties of a side population and enhanced sphere formation in culture. Accordingly, these cells were termed cancer-induced stem cells (CiSCs). CiSCs showed a more mesenchymal phenotype that was further augmented upon TGF-β stimulation and demonstrated a high expression of the β-catenin pathway and ALDH1A1. These findings demonstrate that MSCs, recruited to the tumor microenvironment in large numbers, may display cellular plasticity, acquire a more stem-like state, and acquire some properties of CSCs upon exposure to cancer cell-secreted factors. These acquired characteristics may contribute to tumor progression, survival, and metastasis. Our findings provide new insights into the interactions between MSCs and cancer cells, with the potential to identify novel molecular targets for cancer therapy.

Comparison of the paracrine activity of mesenchymal stem cells derived from human umbilical cord, amniotic membrane and adipose tissue.

PubMed

Dabrowski, Filip A; Burdzinska, Anna; Kulesza, Agnieszka; Sladowska, Anna; Zolocinska, Aleksandra; Gala, Kamila; Paczek, Leszek; Wielgos, Miroslaw

2017-11-01

The study was conducted to investigate secretory activity and define the paracrine potential of mesenchymal stem cells from human umbilical cord and amniotic membrane (UC-MSCs and AM-MSCs, respectively). UC-MSCs (n = 6) were obtained from tissue explants using an adherent method after two weeks of incubation. AM-MSCs (n = 6) were obtained by digestion with tripsin and collagenase. MSC phenotype was confirmed in vitro by performing flow cytometry, differentiation assays and vimentin staining. Supernatants were collected after 48 h culturing in serum-free conditions and the following concentrations were determined: epidermal growth factor (EGF), interleukin (IL)-6, IL-10, tumor necrosis factor-α, transforming growth factor-β (TGF-β), vascular endothelial growth factor-α (VEGF-α) and metalloproteinase (MMP) 1, 8 and 13, using multiplex supernatant cytokine assay. Data were compared with adipose tissue derived MSCs (AD-MSCs, n = 6). Both UC-MSC and AM-MSC populations were positively identified as MSCs by flow cytometry and differentiation potential into bone, cartilage and adipose tissue. Using a multiple cytokine detection assay, we proved that both UC-MSCs and AM-MSCs show high secretive capacity. However, the secretion profile differed between cells from various sources. UC-MSCs showed significantly higher production of TGF-β and lower production of VEGF-α, compared to AD-MSCs (P = 0.004) and AM-MSCs (P = 0.039) and lower levels of EGF (P = 0005). AM-MSCs showed significantly lower levels of MMP-8 than UC-MSCs (P = 0.024); however, there was no difference in levels of released cytokines compared to AD-MSCs. AM-MSCs show similar IL production as AD-MSCs, while UC-MSCs have a significantly different profile, which suggests diverse biological potential of both cell types for immunomodulative and regenerative therapy. © 2017 Japan Society of Obstetrics and Gynecology.

Cells Isolated from Human Periapical Cysts Express Mesenchymal Stem Cell-like Properties

PubMed Central

Marrelli, Massimo; Paduano, Francesco; Tatullo, Marco

2013-01-01

We provide a detailed description of mesenchymal stem cells (MSCs) isolated from human periapical cysts, which we have termed hPCy-MSCs. These cells have a fibroblast-like shape and adhere to tissue culture plastic surfaces. hPCy-MSCs possess high proliferative potential and self-renewal capacity properties. We characterised the immunophenotype of hPCy-MSCs (CD73+, CD90+, CD105+, CD13+, CD29+, CD44+, CD45-, STRO-1+, CD146+) by flow cytometry and immunofluorescence. hPCy-MSCs possess the potential to differentiate into osteoblast- and adipocyte-like cells in vitro. Multi-potentiality was evaluated with culture-specific staining and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis for osteo/odontogenic and adipogenic markers. This is the first report to indicate that human periapical cysts contain cells with MSC-like properties. Taken together, our findings indicate that human periapical cysts could be a rich source of MSCs. PMID:24250252

Cells isolated from human periapical cysts express mesenchymal stem cell-like properties.

PubMed

Marrelli, Massimo; Paduano, Francesco; Tatullo, Marco

2013-01-01

We provide a detailed description of mesenchymal stem cells (MSCs) isolated from human periapical cysts, which we have termed hPCy-MSCs. These cells have a fibroblast-like shape and adhere to tissue culture plastic surfaces. hPCy-MSCs possess high proliferative potential and self-renewal capacity properties. We characterised the immunophenotype of hPCy-MSCs (CD73(+), CD90(+), CD105(+), CD13(+), CD29(+), CD44(+), CD45(-), STRO-1(+), CD146(+)) by flow cytometry and immunofluorescence. hPCy-MSCs possess the potential to differentiate into osteoblast- and adipocyte-like cells in vitro. Multi-potentiality was evaluated with culture-specific staining and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis for osteo/odontogenic and adipogenic markers. This is the first report to indicate that human periapical cysts contain cells with MSC-like properties. Taken together, our findings indicate that human periapical cysts could be a rich source of MSCs.

Mesenchymal stem cells-derived exosomes are more immunosuppressive than microparticles in inflammatory arthritis

PubMed Central

Cosenza, Stella; Toupet, Karine; Maumus, Marie; Luz-Crawford, Patricia; Blanc-Brude, Olivier; Jorgensen, Christian; Noël, Danièle

2018-01-01

Objectives: Mesenchymal stem cells (MSCs) release extracellular vesicles (EVs) that display a therapeutic effect in inflammatory disease models. Although MSCs can prevent arthritis, the role of MSCs-derived EVs has never been reported in rheumatoid arthritis. This prompted us to compare the function of exosomes (Exos) and microparticles (MPs) isolated from MSCs and investigate their immunomodulatory function in arthritis. Methods: MSCs-derived Exos and MPs were isolated by differential ultracentrifugation. Immunosuppressive effects of MPs or Exos were investigated on T and B lymphocytes in vitro and in the Delayed-Type Hypersensitivity (DTH) and Collagen-Induced Arthritis (CIA) models. Results: Exos and MPs from MSCs inhibited T lymphocyte proliferation in a dose-dependent manner and decreased the percentage of CD4+ and CD8+ T cell subsets. Interestingly, Exos increased Treg cell populations while parental MSCs did not. Conversely, plasmablast differentiation was reduced to a similar extent by MSCs, Exos or MPs. IFN-γ priming of MSCs before vesicles isolation did not influence the immunomodulatory function of isolated Exos or MPs. In DTH, we observed a dose-dependent anti-inflammatory effect of MPs and Exos, while in the CIA model, Exos efficiently decreased clinical signs of inflammation. The beneficial effect of Exos was associated with fewer plasmablasts and more Breg-like cells in lymph nodes. Conclusions: Both MSCs-derived MPs and Exos exerted an anti-inflammatory role on T and B lymphocytes independently of MSCs priming. However, Exos were more efficient in suppressing inflammation in vivo. Our work is the first demonstration of the therapeutic potential of MSCs-derived EVs in inflammatory arthritis. PMID:29507629

Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta.

PubMed

Mareschi, Katia; Castiglia, Sara; Sanavio, Fiorella; Rustichelli, Deborah; Muraro, Michela; Defedele, Davide; Bergallo, Massimiliano; Fagioli, Franca

2016-02-01

Mesenchymal stromal cells (MSCs) are a promising tool in cell therapies because of their multipotent, bystander, and immunomodulatory properties. Although bone marrow represents the main source of MSCs, there remains a need to identify a stem cell source that is safe and easily accessible and yields large numbers of cells without provoking debates over ethics. In this study, MSCs isolated from amniotic fluid and placenta were compared with bone marrow MSCs. Their immunomodulatory properties were studied in total activated T cells (peripheral blood mononuclear cells) stimulated with phytohemagglutinin (PHA-PBMCs). In particular, an in vitro co-culture system was established to study: (i) the effect on T-lymphocyte proliferation; (ii) the presence of T regulatory lymphocytes (Treg); (iii) the immunophenotype of various T subsets (Th1 and Th2 naïve, memory, effector lymphocytes); (iv) cytokine release and master gene expression to verify Th1, Th2, and Th17 polarization; and (v) IDO production. Under all co-culture conditions with PHA-PBMCs and MSCs (independently of tissue origin), data revealed: (i) T proliferation inhibition; (ii) increase in naïve T and decrease in memory T cells; (iii) increase in T regulatory lymphocytes; (iv) strong Th2 polarization associated with increased interleukin-10 and interleukin-4 levels, Th1 inhibition (significant decreases in interleukin-2, tumor necrosis factor-α, interferon-γ, and interleukin-12) and Th17 induction (production of high concentrations of interleukins-6 and -17); (v) indoleamine-2,3-dioxygenase mRNA induction in MSCs co-cultured with PHA-PBMCs. AF-MSCs had a more potent immunomodulatory effect on T cells than BM-MSCs, only slightly higher than that of placenta MSCs. This study indicates that MSCs isolated from fetal tissues may be considered a good alternative to BM-MSCs for clinical applications. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Production of Pigs by Hand-Made Cloning Using Mesenchymal Stem Cells and Fibroblasts.

PubMed

Yang, Zhenzhen; Vajta, Gábor; Xu, Ying; Luan, Jing; Lin, Mufei; Liu, Cong; Tian, Jianing; Dou, Hongwei; Li, Yong; Liu, Tianbin; Zhang, Yijie; Li, Lin; Yang, Wenxian; Bolund, Lars; Yang, Huanming; Du, Yutao

2016-08-01

Mesenchymal stem cells (MSCs) exhibited self-renewal and less differentiation, making the MSCs promising candidates for adult somatic cell nuclear transfer (SCNT). In this article, we tried to produce genome identical pigs through hand-made cloning (HMC), with MSCs and adult skin fibroblasts as donor cells. MSCs were derived from either adipose tissue or peripheral blood (aMSCs and bMSCs, respectively). MSCs usually showed the expression pattern of CD29, CD73, CD90, and CD105 together with lack of expression of the hematopoietic markers CD34and CD45. Flow cytometry results demonstrated high expression of CD29 and CD90 in both MSC lines, while CD73, CD34, and CD45 expression were not detected. In contrary, in reverse transcription-polymerase chain reaction (RT-PCR) analysis, CD73 and CD34 were detected indicating that human antibodies CD73 and CD34 were not suitable to identify porcine cell surface markers and porcine MSC cellular surface markers of CD34 might be different from other species. MSCs also had potential to differentiate successfully into chondrocytes, osteoblasts, and adipocytes. After HMC, embryos reconstructed with aMSCs had higher blastocyst rate on day 5 and 6 than those reconstructed with bMSCs and fibroblasts (29.6% ± 1.3% and 41.1% ± 1.4% for aMSCs vs. 23.9% ± 1.2% and 35.5% ± 1.6% for bMSCs and 22.1% ± 0.9% and 33.3% ± 1.1% for fibroblasts, respectively). Live birth rate per transferred blastocyst achieved with bMSCs (1.59%) was the highest among the three groups. This article was the first report to compare the efficiency among bMSCs, aMSCs, and fibroblasts for boar cloning, which offered a realistic perspective to use the HMC technology for commercial breeding.

“Ins” and “Outs” of mesenchymal stem cell osteogenesis in regenerative medicine

PubMed Central

Yamaguchi, Dean T

2014-01-01

Repair and regeneration of bone requires mesenchymal stem cells that by self-renewal, are able to generate a critical mass of cells with the ability to differentiate into osteoblasts that can produce bone protein matrix (osteoid) and enable its mineralization. The number of human mesenchymal stem cells (hMSCs) diminishes with age and ex vivo replication of hMSCs has limited potential. While propagating hMSCs under hypoxic conditions may maintain their ability to self-renew, the strategy of using human telomerase reverse transcriptase (hTERT) to allow for hMSCs to prolong their replicative lifespan is an attractive means of ensuring a critical mass of cells with the potential to differentiate into various mesodermal structural tissues including bone. However, this strategy must be tempered by the oncogenic potential of TERT-transformed cells, or their ability to enhance already established cancers, the unknown differentiating potential of high population doubling hMSCs and the source of hMSCs (e.g., bone marrow, adipose-derived, muscle-derived, umbilical cord blood, etc.) that may provide peculiarities to self-renewal, differentiation, and physiologic function that may differ from non-transformed native cells. Tissue engineering approaches to use hMSCs to repair bone defects utilize the growth of hMSCs on three-dimensional scaffolds that can either be a base on which hMSCs can attach and grow or as a means of sequestering growth factors to assist in the chemoattraction and differentiation of native hMSCs. The use of whole native extracellular matrix (ECM) produced by hMSCs, rather than individual ECM components, appear to be advantageous in not only being utilized as a three-dimensional attachment base but also in appropriate orientation of cells and their differentiation through the growth factors that native ECM harbor or in simulating growth factor motifs. The origin of native ECM, whether from hMSCs from young or old individuals is a critical factor in “rejuvenating” hMSCs from older individuals grown on ECM from younger individuals. PMID:24772237

Proteomic analysis of porcine mesenchymal stem cells derived from bone marrow and umbilical cord: implication of the proteins involved in the higher migration capability of bone marrow mesenchymal stem cells.

PubMed

Huang, Lei; Niu, Chenguang; Willard, Belinda; Zhao, Weimin; Liu, Lan; He, Wei; Wu, Tianwen; Yang, Shulin; Feng, Shutang; Mu, Yulian; Zheng, Lemin; Li, Kui

2015-04-15

Mesenchymal stem cells (MSCs) have the ability to proliferate in vivo with a large variety of differentiation potentials and therefore are widely used as an ideal material for cell therapy. MSCs derived from pig and human sources are similar in many aspects, such as cell immunophenotype and functional characteristics. However, differences in proteomics and the molecular mechanisms of cell functions between porcine bone marrow MSCs (BM-MSCs) and umbilical cord MSCs (UC-MSCs) are largely unknown. To the best of our knowledge, MSCs collected from different tissue have specific phenotype and differentiation ability in response to microenvironment, known as a niche. Porcine BM-MSCs and UC-MSCs were evaluated with flow cytometric and adipogenic and osteogenic differentiation analyses. We used isobaric tagging for relative and absolute quantitation (iTRAQ), combined with liquid chromatography-tandem mass spectrometry, to identify differentially expressed proteins (DEPs) between these two types of MSCs. Kyoto Encyclopedia of Genes and Genomes pathway and phenotype analyses were used to understand the links between cell migration ability and DEPs. Two separate iTRAQ experiments were conducted, identifying 95 DEPs (95% confidence interval). Five of these proteins were verified by Western blotting. These 95 DEPs were classified in terms of biological regulation, metabolic process, developmental process, immune system process, reproduction, death, growth, signaling, localization, response to stimulus, biological adhesion, and cellular component organization. Our study is the first to show results indicating that porcine BM-MSCs have a higher migration capability than UC-MSCs. Finally, one of the DEPs, Vimentin, was verified to have a positive role in MSC migration. These results represent the first attempt to use proteomics specifically targeted to porcine MSCs of different tissues. The identified components should help reveal a variety of tissue-specific functions in tissue-derived MSC populations and could serve as important tools for the regeneration of particular tissues in future stem cell-based tissue engineering studies using animal models.

Sca-1(+) mesenchymal stromal cells inhibit splenic marginal zone B lymphocytes commitment through Caspase-3.

PubMed

Chen, Yaozhen; Yang, Jialei; Zhang, Hui-Jie; Fan, Hong; An, Ning; Xin, Jiajia; Li, Na; Xu, Jinmei; Yin, Wen; Wu, Zhongliang; Hu, Xingbin

2016-05-01

Mesenchymal stromal cells (MSCs) have been characterized as an important component of hematopoietic niche, which are capable of modulating the immune system through interaction with a wide range of immune cells. Marginal zone B cells, one main type of mature B lymphocytes, play a central role in eliciting antibody response against pathogens. However, how MSCs and its subpopulations regulate marginal zone B cells commitment is unknown yet. In this study, we assessed the contribution of Sca-1(+) MSCs on marginal zone B cells commitment. Our results showed that Sca-1(+) MSCs inhibit the commitment of marginal zone B lymphocytes. The inhibition was exerted through lowered Caspase-3 expression. Furthermore, we found marginal zone B lymphocytes in spleen of Caspase-3 knockout mice decreased and Caspase-3 knockout Sca-1(+) MSCs accounted for the MZB lymphocytes decrease. In conclusion, our investigation provided clues about Sca-1(+) MSCs regulation on the commitment of marginal zone B cells through Caspase-3 gene. © 2016 International Federation for Cell Biology.

Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance

PubMed Central

Kurtova, Antonina V.; Balakrishnan, Kumudha; Chen, Rong; Ding, Wei; Schnabl, Susanne; Quiroga, Maite P.; Sivina, Mariela; Wierda, William G.; Estrov, Zeev; Keating, Michael J.; Shehata, Medhat; Jäger, Ulrich; Gandhi, Varsha; Kay, Neil E.; Plunkett, William

2009-01-01

Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL–MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL–MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell–cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC–CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures. PMID:19762485

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

PubMed Central

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M.; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2018-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4+ FoxP3+ regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3+-Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance. PMID:29422893

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo.

PubMed

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2017-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4 + FoxP3 + regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3 + -Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo . They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells.

PubMed

Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Abdel-Rahman, Engy A; Reda, Asmaa M; Ali, Sameh S; Khater, Sherry M; Ashamallah, Sylvia A; Ismail, Amani M; Ismail, Hossam El-Din A; El-Badri, Nagwa; Ghoneim, Mohamed A

2017-01-01

The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs), for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs), was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion . BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine.

From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells

PubMed Central

Abdel-Rahman, Engy A.; Reda, Asmaa M.; Ashamallah, Sylvia A.; Ismail, Amani M.; Ismail, Hossam El-Din A.; El-Badri, Nagwa

2017-01-01

The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs), for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs), was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion. BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine. PMID:28584815

Emerging role of mesenchymal stem cells during tuberculosis: The fifth element in cell mediated immunity.

PubMed

Khan, Arshad; Hunter, Robert L; Jagannath, Chinnaswamy

2016-12-01

Mesenchymal stem cells (MSCs) are non-hematopoietic cells that occur in almost all human tissues and can be cultured and expanded to large numbers in vitro. They secrete growth factors, cytokines, and chemokines and express Toll-like receptors on their surface, although multiple cell biological mechanisms remain unclear. MSCs are multi-potent and can differentiate into many cell types including adipocytes, neuronal cells and osteoclasts. Despite gaps in cell biology, because of their immunomodulatory and regenerative capacity, several hundred clinical trials have used MSCs for therapy of cancer, autoimmune diseases and control of inflammation during organ transplantation. MSCs secrete immune-modulatory factors and are able to skew T cell responses and shift M1 to M2 differentiation of macrophages. We review the emerging role of MSCs to act as phagocytes for Mycobacterium tuberculosis and its role during the persistence of M. tuberculosis and spread of infection. Paradoxically, MSCs use innate defense mechanisms of autophagy and nitric oxide to inhibit the growth of intracellular M. tuberculosis. In addition, transplantation with autologous MSCs improved the clinical condition of patients with multi-drug resistant tuberculosis. Thus, in addition to the well-known immune defense played by macrophages, DCs, classical T cells and non-classical immune cells, MSCs have emerged as a fifth element capable of regulating immune responses during tuberculosis. We discuss their immunomodulatory properties and innate defense mechanisms in the context of developing immunotherapeutic strategies for tuberculosis. Published by Elsevier Ltd.

In vitro migration and proliferation ("wound healing") potential of mesenchymal stromal cells generated from human CD271(+) bone marrow mononuclear cells.

PubMed

Latifi-Pupovci, Hatixhe; Kuçi, Zyrafete; Wehner, Sibylle; Bönig, Halvard; Lieberz, Ralf; Klingebiel, Thomas; Bader, Peter; Kuçi, Selim

2015-09-25

Emerging evidence indicates that mesenchymal stromal cells (MSCs) isolated from different tissue sources may be used in vivo as tissue restorative agents. To date, there is no evidence, however, on migration and proliferation ("wound healing") potential of different subsets of MSCs. The main goal of this study was therefore to compare the in vitro "wound healing" capacity of MSCs generated from positively selected CD271(+) bone marrow mononuclear cells (CD271-MSCs) and MSCs generated by plastic adherence (PA-MSCs). The in vitro model of wound healing (CytoSelect™ 24-Well Wound Healing Assay) was used in order to compare the migration and proliferation potential of CD271-MSCs and PA-MSCs of passage 2 and 4 cultured in presence or absence of growth factors or cytokines. CD271-MSCs of both passages when compared to PA-MSCs demonstrated a significantly higher potential to close the wound 12 and 24 h after initiation of the wound healing assay (P < 0.003 and P < 0.002, respectively). Noteworthy, the migration capacity of PA-MSCs of second passage was significantly improved after stimulation with FGF-2 (P < 0.02), PDGF-BB (P < 0.006), MCP-1 (P < 0.002) and IL-6 (P < 0.03), whereas only TGF-β enhanced significantly migration process of PA-MSCs of P4 12 h after the treatment (P < 0.02). Interestingly, treatment of CD271-MSCs of both passages with growth factors or cytokines did not affect their migratory potential. Our in vitro data provide the first evidence that CD271-MSCs are significantly more potent in "wound healing" than their counterparts PA-MSCs.

Comparison of Preterm and Term Wharton's Jelly-Derived Mesenchymal Stem Cell Properties in Different Oxygen Tensions.

PubMed

Balgi-Agarwal, Saloni; Winter, Caitlyn; Corral, Alexis; Mustafa, Shamimunisa B; Hornsby, Peter; Moreira, Alvaro

2018-06-27

Mesenchymal stem cells (MSCs) have shown promise as therapeutic agents in treating morbidities associated with premature birth. MSCs derived from the human umbilical cord are easy to isolate and have low immunogenicity and a robust ability to secrete paracrine factors. To date, there are no studies evaluating preterm versus term umbilical cord tissue-derived MSCs. Therefore, our aim was twofold: (1) to compare stem cell properties in preterm versus term MSCs and (2) to examine the impact of oxygen tension on stem cell behavior. Umbilical cord tissue was obtained from 5 preterm and 5 term neonates. The cells were isolated and characterized as MSCs in accordance with the International Society for Cellular Therapy. We exposed MSCs to different oxygen tensions to examine the impact of environmental factors on cell performance. We studied the following stem cell properties: (i) motility, (ii) proliferation, (iii) senescence, (iv) cell viability, (v) colony-forming unit efficiency, and (vi) inflammatory cytokine expression. Under normoxia (21% O2), cells from preterm and term infants had similar properties. Under hypoxic conditions (1% O2), term MSCs had better cell proliferation; however, cells exposed to hyperoxia (90% O2) had the slowest motility and lowest cell viability (p < 0.05). There was no difference in the expression of senescence or cytokine expression between the groups. The term cells demonstrated more colony-forming efficiency than the preterm cells. In sum, our preliminary findings suggest that MSCs derived from term and preterm umbilical cords have similar characteristics, offering the potential of future autologous/allogeneic MSC transplants in neonates. © 2018 S. Karger AG, Basel.

Genetic Engineering of Mesenchymal Stem Cells for Regenerative Medicine.

PubMed

Nowakowski, Adam; Walczak, Piotr; Janowski, Miroslaw; Lukomska, Barbara

2015-10-01

Mesenchymal stem cells (MSCs), which can be obtained from various organs and easily propagated in vitro, are one of the most extensively used types of stem cells and have been shown to be efficacious in a broad set of diseases. The unique and highly desirable properties of MSCs include high migratory capacities toward injured areas, immunomodulatory features, and the natural ability to differentiate into connective tissue phenotypes. These phenotypes include bone and cartilage, and these properties predispose MSCs to be therapeutically useful. In addition, MSCs elicit their therapeutic effects by paracrine actions, in which the metabolism of target tissues is modulated. Genetic engineering methods can greatly amplify these properties and broaden the therapeutic capabilities of MSCs, including transdifferentiation toward diverse cell lineages. However, cell engineering can also affect safety and increase the cost of therapy based on MSCs; thus, the advantages and disadvantages of these procedures should be discussed. In this review, the latest applications of genetic engineering methods for MSCs with regenerative medicine purposes are presented.

Effects of heme oxygenase-1 gene modulated mesenchymal stem cells on vasculogenesis in ischemic swine hearts.

PubMed

Jiang, Yi-Bo; Zhang, Xiao-Li; Tang, Yao-Liang; Ma, Gen-Shan; Shen, Cheng-Xing; Wei, Qin; Zhu, Qi; Yao, Yu-Yu; Liu, Nai-Feng

2011-02-01

Mesenchymal stem cells (MSCs) transplantation may partially restore heart function in the treatment of acute myocardial infarction (AMI). The aim of this study was to explore the beneficial effects of MSCs modified with heme xygenase-1 (HO-1) on post-infarct swine hearts to determine whether the induction of therapeutic angiogenesis is modified by the angiogenic cytokines released from the implanted cells. In vitro, MSCs were divided into four groups: (1) non-transfected MSCs (MSCs group), (2) MSCs transfected with the pcDNA3.1-Lacz plasmid (Lacz-MSCs group), (3) MSCs transfected with pcDNA3.1-hHO-1 (HO-1-MSCs group), and (4) MSCs transfected with pcDNA3.1-hHO-1 and pretreatment with an HO inhibitor, tin protoporphyrin (SnPP) (HO-1-MSCs + SnPP group). Cells were cultured in an airtight incubation bottle for 24 hours, in which the oxygen concentration was maintained at < 1%, followed by 12 hours of reoxygenation. After hypoxia/reoxygen treatment, ELISA was used to measure transforming growth factor (TGF-β) and fibroblast growth factor (FGF-2) in the supernatant. In vivo, 28 Chinese mini-pigs were randomly allocated to the following treatment groups: (1) control group (saline), (2) Lacz-MSCs group, (3) HO-1-MSCs group, and (4) HO-1-MSCs + SnPP group. About 1 × 10(7) of autologous stem cells or an identical volume of saline was injected intracoronary into porcine hearts 1 hour after MI. Magnetic resonance imaging (MRI) assay and postmortem analysis were assessed four weeks after stem cell transplantation. Post hypoxia/reoxygenation in vitro, TGF-β in the supernatant was significantly increased in the HO-1-MSCs ((874.88 ± 68.23) pg/ml) compared with Lacz-MSCs ((687.81 ± 57.64) pg/ml, P < 0.001). FGF-2 was also significantly increased in the HO-1-MSCs ((1106.48 ± 107.06) pg/ml) compared with the Lacz-MSCs ((853.85 ± 74.44) pg/ml, P < 0.001). In vivo, at four weeks after transplantation, HO-1 gene transfer increased the capillary density in the peri-infarct area compared with the Lacz-MSCs group (14.24 ± 1.66/HPFs vs. 11.51 ± 1.34/HPFs, P < 0.001). Arteriolar density was also significantly higher in HO-1-MSCs group than in the Lacz-MSCs group (7.86 ± 2.00/HPFs vs. 6.45 ± 1.74/HPFs, P = 0.001). At the same time, the cardiac function was significantly improved in the HO-1-MSCs group compared with the Lacz-MSCs group ((53.17 ± 3.55)% vs. (48.82 ± 2.98)%, P < 0.05). However, all these effects were significantly abrogated by SnPP. MSCs provided a beneficial effect on cardiac function after ischemia/reperfusion by the induction of therapeutic angiogenesis, and this effect was amplified by HO-1 overexpression.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smirnov, Sergey V.; Harbacheuski, Ryhor; Lewis-Antes, Anita

Mesenchymal stem cells (MSCs) in bone marrow (BM) regulate the differentiation and proliferation of adjacent hematopoietic precursor cells and contribute to the regeneration of mesenchymal tissues, including bone, cartilage, fat and connective tissue. BM is an important site for the pathogenesis of human cytomegalovirus (HCMV) where the virus establishes latency in hematopoietic progenitors and can transmit after reactivation to neighboring cells. Here we demonstrate that BM-MSCs are permissive to productive HCMV infection, and that HCMV alters the function of MSCs: (i) by changing the repertoire of cell surface molecules in BM-MSCs, HCMV modifies the pattern of interaction between BM-MSCs andmore » hematopoietic cells; (ii) HCMV infection of BM-MSCs undergoing adipogenic or osteogenic differentiation impaired the process of differentiation. Our results suggest that by altering BM-MSC biology, HCMV may contribute to the development of various diseases.« less

MicroRNA-133 overexpression promotes the therapeutic efficacy of mesenchymal stem cells on acute myocardial infarction.

PubMed

Chen, Yueqiu; Zhao, Yunfeng; Chen, Weiqian; Xie, Lincen; Zhao, Zhen-Ao; Yang, Junjie; Chen, Yihuan; Lei, Wei; Shen, Zhenya

2017-11-25

Our study aim was to evaluate the therapeutic efficacy and mechanisms of miR-133-overexpressing mesenchymal stem cells (MSCs) on acute myocardial infarction. Rat MSCs were isolated and purified by whole bone marrow adherent culturing. After transfection with the agomir or antagomir of miR-133, MSCs were collected for assay of cell vitality, apoptosis, and cell cycle progression. At the same time, exosomes were isolated from the supernatant to analyze the paracrine miR-133. For in-vivo studies, constitutive activation of miR-133 in MSCs was achieved by lentivirus-mediated miR-133 overexpression. A rat myocardial infarction model was created by ligating the left anterior descending coronary artery, while control MSCs (vector-MSCs) or miR-133-overexpressed MSCs (miR-133-MSCs) were injected into the zone around the myocardial infarction. Subsequently, myocardial function was evaluated by echocardiography on days 7 and 28 post infarction. Finally the infarcted hearts were collected on days 7 and 28 for myocardial infarct size measurement and detection of snail 1 expression. Hypoxia-induced apoptosis of MSCs obviously reduced, along with enhanced expression of total poly ADP-ribose polymerase protein, after miR-133 agomir transfection, while the apoptosis rate increased in MSCs transfected with miR-133 antagomir. However, no change in cell viability and cell-cycle distribution was observed in control, miR-133-overexpressed, and miR-133-interfered MSCs. Importantly, rats transplanted with miR-133-MSCs displayed more improved cardiac function after acute myocardial infarction, compared with those that received vector-MSC injection. Further studies indicated that cardiac expression of snail 1 was significantly repressed by adjacent miR-133-overexpressing MSCs, and both the inflammatory level and the infarct size decreased in miR-133-MSC-injected rat hearts. miR-133-MSCs obviously improved cardiac function in a rat model of myocardial infarction. Transplantation of miR-133-overexpressing MSCs provides an effective strategy for cardiac repair and modulation of cardiac-related diseases.

Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance.

PubMed

Li, Yi; Guo, Gang; Li, Li; Chen, Fei; Bao, Ji; Shi, Yu-Jun; Bu, Hong

2015-05-01

Mesenchymal stem cell (MSC) transplantation is a promising treatment of many diseases. However, conventional techniques with cells being cultured as a monolayer result in slow cell proliferation and insufficient yield to meet clinical demands. Three-dimensional (3D) culture systems are gaining attention with regard to recreating a complex microenvironment and to understanding the conditions experienced by cells. Our aim is to establish a novel 3D system for the culture of human umbilical cord MSCs (hUC-MSCs) within a real 3D microenvironment but with no digestion or passaging. Primary hUC-MSCs were isolated and grown in serum-free medium (SFM) on a suspension Rocker system. Cell characteristics including proliferation, phenotype and multipotency were recorded. The therapeutic effects of 3D-cultured hUC-MSCs on carbon tetrachloride (CCl4)-induced acute liver failure in mouse models were examined. In the 3D Rocker system, hUC-MSCs formed spheroids in SFM and maintained high viability and active proliferation. Compared with monolayer culture, the 3D-culture system yielded more hUC-MSCs cells within the same volume. The spheroids expressed higher levels of stem cell markers and displayed stronger multipotency. After transplantation into mouse, 3D hUC-MSCs significantly promoted the secretion of interferon-γ and interleukin-6 but inhibited that of tumor necrosis factor-α, thereby alleviating liver necrosis and promoting regeneration following CCl4 injury. The 3D culture of hUC-MSCs thus promotes cell yield and stemness maintenance and represents a promising strategy for hUC-MSCs expansion on an industrial scale with great potential for cell therapy and biotechnology.

Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

PubMed Central

Li, Zicong; He, Xiaoyan; Chen, Liwen; Shi, Junsong; Zhou, Rong; Xu, Weihua

2013-01-01

Abstract The somatic cell nuclear transfer (SCNT) technique has been widely applied to clone pigs or to produce genetically modified pigs. Currently, this technique relies mainly on using terminally differentiated fibroblasts as donor cells. To improve cloning efficiency, only partially differentiated multipotent mesenchymal stem cells (MSCs), thought to be more easily reprogrammed to a pluripotent state, have been used as nuclear donors in pig SCNT. Although in vitro–cultured embryos cloned from porcine MSCs (MSCs-embryos) were shown to have higher preimplantation developmental ability than cloned embryos reconstructed from fibroblasts (Fs-embryos), the difference in in vivo full-term developmental rate between porcine MSCs-embryos and Fs-embryos has not been investigated so far. In this study, we demonstrated that blastocyst total cell number and full-term survival abilities of MSCs-embryos were significantly higher than those of Fs-embryos cloned from the same donor pig. The enhanced developmental potential of MSCs-embryos may be associated with their nuclear donors' DNA methylation profile, because we found that the methylation level of imprinting genes and repeat sequences differed between MSCs and fibroblasts. In addition, we showed that use of transgenic porcine MSCs generated from transgene plasmid transfection as donor cells for SCNT can produce live transgenic cloned pigs. These results strongly suggest that porcine bone marrow MSCs are a desirable donor cell type for production of cloned pigs and genetically modified cloned pigs via SCNT. PMID:24033142

Mesenchymal Stem/Stromal Cells in Liver Fibrosis: Recent Findings, Old/New Caveats and Future Perspectives.

PubMed

Fiore, Esteban J; Mazzolini, Guillermo; Aquino, Jorge B

2015-08-01

Mesenchymal stem/stromal cells (MSCs) are progenitors which share plastic-adherence capacity and cell surface markers but have different properties according to their cell and tissue sources and to culture conditions applied. Many recent publications suggest that MSCs can differentiate into hepatic-like cells, which can be a consequence of either a positive selection of rare in vivo pluripotent cells or of the original plasticity of some cells contributing to MSC cultures. A possible role of MSCs in hereditary transmission of obesity and/or diabetes as well as properties of MSCs regarding immunomodulation, cell fusion and exosome release capacities are discussed according to recent literature. Limitations in methods used to track MSCs in vivo especially in the context of liver cirrhosis are addressed as well as strategies explored to enhance their migratory, survival and proliferation properties, which are known to be relevant for their future clinical use. Current knowledge regarding mechanisms involved in liver cirrhosis amelioration mediated by naïve and genetically modified MSCs as well as the effects of applying preconditioning and combined strategies to improve their therapeutic effects are evaluated. Finally, first reports of GMP guidelines and biosafety issues in MSCs applications are discussed.

Mesenchymal stem cells support neuronal fiber growth in an organotypic brain slice co-culture model.

PubMed

Sygnecka, Katja; Heider, Andreas; Scherf, Nico; Alt, Rüdiger; Franke, Heike; Heine, Claudia

2015-04-01

Mesenchymal stem cells (MSCs) have been identified as promising candidates for neuroregenerative cell therapies. However, the impact of different isolation procedures on the functional and regenerative characteristics of MSC populations has not been studied thoroughly. To quantify these differences, we directly compared classically isolated bulk bone marrow-derived MSCs (bulk BM-MSCs) to the subpopulation Sca-1(+)Lin(-)CD45(-)-derived MSCs(-) (SL45-MSCs), isolated by fluorescence-activated cell sorting from bulk BM-cell suspensions. Both populations were analyzed with respect to functional readouts, that are, frequency of fibroblast colony forming units (CFU-f), general morphology, and expression of stem cell markers. The SL45-MSC population is characterized by greater morphological homogeneity, higher CFU-f frequency, and significantly increased nestin expression compared with bulk BM-MSCs. We further quantified the potential of both cell populations to enhance neuronal fiber growth, using an ex vivo model of organotypic brain slice co-cultures of the mesocortical dopaminergic projection system. The MSC populations were cultivated underneath the slice co-cultures without direct contact using a transwell system. After cultivation, the fiber density in the border region between the two brain slices was quantified. While both populations significantly enhanced fiber outgrowth as compared with controls, purified SL45-MSCs stimulated fiber growth to a larger degree. Subsequently, we analyzed the expression of different growth factors in both cell populations. The results show a significantly higher expression of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor in the SL45-MSCs population. Altogether, we conclude that MSC preparations enriched for primary MSCs promote neuronal regeneration and axonal regrowth, more effectively than bulk BM-MSCs, an effect that may be mediated by a higher BDNF secretion.

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro.

PubMed

Griffiths, Sarah; Baraniak, Priya R; Copland, Ian B; Nerem, Robert M; McDevitt, Todd C

2013-12-01

Multipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown. MSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to "rescue" the proliferative capacity of MSCs. hPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS. hPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

PubMed

de Soure, António M; Fernandes-Platzgummer, Ana; Moreira, Francisco; Lilaia, Carla; Liu, Shi-Hwei; Ku, Chen-Peng; Huang, Yi-Feng; Milligan, William; Cabral, Joaquim M S; da Silva, Cláudia L

2017-05-01

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGRO TM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO TM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO TM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Mesenchymal stem cells induce epithelial proliferation within the inflamed stomach.

PubMed

Donnelly, Jessica M; Engevik, Amy; Feng, Rui; Xiao, Chang; Boivin, Gregory P; Li, Jing; Houghton, JeanMarie; Zavros, Yana

2014-06-15

Bone marrow-derived mesenchymal stem cells (MSCs) sustain cancer cells by creating a microenvironment favorable for tumor growth. In particular, MSCs have been implicated in gastric cancer development. There is extensive evidence suggesting that Hedgehog signaling regulates tumor growth. However, very little is known regarding the precise roles of Hedgehog signaling and MSCs in tumor development within the stomach. The current study tests that hypothesis that Sonic Hedgehog (Shh), secreted from MSCs, provides a proliferative stimulus for the gastric epithelium in the presence of inflammation. Red fluorescent protein-expressing MSCs transformed in vitro (stMSCs) were transduced with lentiviral constructs containing a vector control (stMSC(vect)) or short hairpin RNA (shRNA) targeting the Shh gene (stMSC(ShhKO)). Gastric submucosal transplantation of wild-type MSCs (wtMSCs), wild-type MSCs overexpressing Shh (wtMSC(Shh)), stMSC(vect), or stMSC(ShhKO) cells in C57BL/6 control (BL/6) or gastrin-deficient (GKO) mice was performed and mice analyzed 30 and 60 days posttransplantation. Compared with BL/6 mice transplanted with wtMSC(Shh) and stMSC(vect) cells, inflamed GKO mice developed aggressive gastric tumors. Tumor development was not observed in mouse stomachs transplanted with wtMSC or stMSC(ShhKO) cells. Compared with stMSC(ShhKO)-transplanted mice, within the inflamed GKO mouse stomach, Shh-expressing stMSC(vect)- and wtMSC(Shh)-induced proliferation of CD44-positive cells. CD44-positive cells clustered in gland-like structures within the tumor stroma and were positive for Patched (Ptch) expression. We conclude that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium that is associated with tumor development, a response that is sustained by chronic inflammation. Copyright © 2014 the American Physiological Society.

Mesenchymal stem cells induce epithelial proliferation within the inflamed stomach

PubMed Central

Donnelly, Jessica M.; Engevik, Amy; Feng, Rui; Xiao, Chang; Boivin, Gregory P.; Li, Jing; Houghton, JeanMarie

2014-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) sustain cancer cells by creating a microenvironment favorable for tumor growth. In particular, MSCs have been implicated in gastric cancer development. There is extensive evidence suggesting that Hedgehog signaling regulates tumor growth. However, very little is known regarding the precise roles of Hedgehog signaling and MSCs in tumor development within the stomach. The current study tests that hypothesis that Sonic Hedgehog (Shh), secreted from MSCs, provides a proliferative stimulus for the gastric epithelium in the presence of inflammation. Red fluorescent protein-expressing MSCs transformed in vitro (stMSCs) were transduced with lentiviral constructs containing a vector control (stMSCvect) or short hairpin RNA (shRNA) targeting the Shh gene (stMSCShhKO). Gastric submucosal transplantation of wild-type MSCs (wtMSCs), wild-type MSCs overexpressing Shh (wtMSCShh), stMSCvect, or stMSCShhKO cells in C57BL/6 control (BL/6) or gastrin-deficient (GKO) mice was performed and mice analyzed 30 and 60 days posttransplantation. Compared with BL/6 mice transplanted with wtMSCShh and stMSCvect cells, inflamed GKO mice developed aggressive gastric tumors. Tumor development was not observed in mouse stomachs transplanted with wtMSC or stMSCShhKO cells. Compared with stMSCShhKO-transplanted mice, within the inflamed GKO mouse stomach, Shh-expressing stMSCvect- and wtMSCShh-induced proliferation of CD44-positive cells. CD44-positive cells clustered in gland-like structures within the tumor stroma and were positive for Patched (Ptch) expression. We conclude that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium that is associated with tumor development, a response that is sustained by chronic inflammation. PMID:24789207

Quantification of Mesenchymal Stem Cells (MSCs) at sites of human prostate cancer.

PubMed

Brennen, W Nathaniel; Chen, Shuangling; Denmeade, Samuel R; Isaacs, John T

2013-01-01

Circulating bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. The prostate is bombarded by numerous infectious and inflammatory insults over a lifetime. Chronic inflammation is associated with CXCL12, CCL5, and CCL2, which are highly overexpressed in prostate cancer. Among other cell types, these chemoattractant stimuli recruit BM-MSCs to the tumor. MSCs are minimally defined as plastic-adhering cells characterized by the expression of CD90, CD73, and CD105 in the absence of hematopoietic markers, which can differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs are immunoprivileged and have been implicated in tumorigenesis through multiple mechanisms, including promoting proliferation, angiogenesis, and metastasis, in addition to the generation of an immunosuppressive microenvironment. We have demonstrated that MSCs represent 0.01-1.1% of the total cells present in core biopsies from primary human prostatectomies. Importantly, these analyses were performed on samples prior to expansion in tissue culture. MSCs in these prostatectomy samples are FAP-, CD90-, CD73-, and CD105-positive, and CD14-, CD20-, CD34-, CD45-, and HLA-DR-negative. Additionally, like BM-MSCs, these prostate cancer-derived stromal cells (PrCSCs) were shown to differentiate into osteoblasts, adipocytes and chondrocytes. In contrast to primary prostate cancer-derived epithelial cells, fluorescently-labeled PrCSCs and BM-MSCs were both shown to home to CWR22RH prostate cancer xenografts following IV injection. These studies demonstrate that not only are MSCs present in sites of prostate cancer where they may contribute to carcinogenesis, but these cells may also potentially be used to deliver cytotoxic or imaging agents for therapeutic and/or diagnostic purposes.

Mesenchymal stem cells: The roles and functions in cutaneous wound healing and tumor growth.

PubMed

Motegi, Sei-Ichiro; Ishikawa, Osamu

2017-05-01

Mesenchymal stem cells (MSCs) are bone marrow-derived non-hematopoietic progenitor cells. MSCs are able to differentiate into various types of cells, including chondrocytes, adipocytes, osteocytes, myocytes, endothelial cells, and keratinocytes. There is increasing evidence that MSCs might be located external to the vasculature, and that perivascular cells in the skin, generally called as "pericytes", might include MSCs. It has been suggested that MSCs localized around blood vessels might migrate into wounds and contribute to the restoration of injured tissues. Many studies have demonstrated that intravenous or intradermal administration of MSCs enhanced cutaneous wound healing, such as acute incisional and excisional wounds, diabetic ulcers, radiation ulcers, and burns in animals and humans. Several mechanisms of the acceleration of wound healing by MSCs have been identified, including the enhancement of angiogenesis by secretion of pro-angiogenic factors and the differentiation into endothelial cells and/or pericytes, M2 macrophages polarization, the recruitment of endogenous stem/progenitor cells, extracellular matrix production and remodeling, and immunosuppressive effects. Since the microenvironments of wounds and/or injured tissues are similar to those of tumors, MSCs also play similar roles in malignant tumors, such as the enhancement of angiogenesis, M2 macrophages polarization, and immunosuppressive effects. In addition, the mechanisms of homing of MSCs might have a commonality in the pathogenesis of wound healing and tumors. Thus, the regulating factors of MSCs, including MFG-E8, could be a therapeutic target and lead to the establishment of new therapeutic approaches for both intractable wound healing and tumors. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Restoring the quantity and quality of elderly human mesenchymal stem cells for autologous cell-based therapies.

PubMed

Block, Travis J; Marinkovic, Milos; Tran, Olivia N; Gonzalez, Aaron O; Marshall, Amanda; Dean, David D; Chen, Xiao-Dong

2017-10-27

Degenerative diseases are a major public health concern for the aging population and mesenchymal stem cells (MSCs) have great potential for treating many of these diseases. However, the quantity and quality of MSCs declines with aging, limiting the potential efficacy of autologous MSCs for treating the elderly population. Human bone marrow (BM)-derived MSCs from young and elderly donors were obtained and characterized using standard cell surface marker criteria (CD73, CD90, CD105) as recommended by the International Society for Cellular Therapy (ISCT). The elderly MSC population was isolated into four subpopulations based on size and stage-specific embryonic antigen-4 (SSEA-4) expression using fluorescence-activated cell sorting (FACS), and subpopulations were compared to the unfractionated young and elderly MSCs using assays that evaluate MSC proliferation, quality, morphology, intracellular reactive oxygen species, β-galactosidase expression, and adenosine triphosphate (ATP) content. The ISCT-recommended cell surface markers failed to detect any differences between young and elderly MSCs. Here, we report that elderly MSCs were larger in size and displayed substantially higher concentrations of intracellular reactive oxygen species and β-galactosidase expression and lower amounts of ATP and SSEA-4 expression. Based on these findings, cell size and SSEA-4 expression were used to separate the elderly MSCs into four subpopulations by FACS. The original populations (young and elderly MSCs), as well as the four subpopulations, were then characterized before and after culture on tissue culture plastic and BM-derived extracellular matrix (BM-ECM). The small SSEA-4-positive subpopulation representing ~ 8% of the original elderly MSC population exhibited a "youthful" phenotype that was similar to that of young MSCs. The biological activity of this elderly subpopulation was inhibited by senescence-associated factors produced by the unfractionated parent population. After these "youthful" cells were isolated and expanded (three passages) on a "young microenvironment" (i.e., BM-ECM produced by BM cells from young donors), the number of cells increased ≈ 17,000-fold to 3 × 10 9 cells and retained their "youthful" phenotype. These results suggest that it is feasible to obtain large numbers of high-quality autologous MSCs from the elderly population and establish personal stem cell banks that will allow serial infusions of "rejuvenated" MSCs for treating age-related diseases.

Effect of Transplanting Various Concentrations of a Composite of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronic Acid Hydrogel on Articular Cartilage Repair in a Rabbit Model

PubMed Central

Ha, Chul-Won; Kim, Jin-A; Rhim, Ji-Heon; Park, Yong-Geun; Chung, Jun Young; Lee, Han-Jun

2016-01-01

Background Mesenchymal stem cells (MSCs) are known to have therapeutic potential for cartilage repair. However, the optimal concentration of MSCs for cartilage repair remains unclear. Therefore, we aimed to explore the feasibility of cartilage repair by human umbilical cord blood-derived MSCs (hUCB-MSCs) and to determine the optimal concentrations of the MSCs in a rabbit model. Methods Osteochondral defects were created in the trochlear groove of femur in 55 rabbits. Four experimental groups (11 rabbits/group) were treated by transplanting the composite of hUCB-MSCs and HA with various MSCs concentrations (0.1, 0.5, 1.0, and 1.5 x 107 cells/ml). One control group was left untreated. At 4, 8, and 16 weeks post-transplantation, the degree of cartilage repair was evaluated grossly and histologically. Findings Overall, transplanting hUCB-MSCs and HA hydrogel resulted in cartilage repair tissue with better quality than the control without transplantation (P = 0.015 in 0.1, P = 0.004 in 0.5, P = 0.004 in 1.0, P = 0.132 in 1.5 x 107 cells/ml). Interestingly, high cell concentration of hUCB-MSCs (1.5×107 cells/ml) was inferior to low cell concentrations (0.1, 0.5, and 1.0 x 107 cells/ml) in cartilage repair (P = 0.394,P = 0.041, P = 0.699, respectively). The 0.5 x 107 cells/ml group showed the highest cartilage repair score at 4, 8 and 16 weeks post transplantation, and followed by 0.1x107 cells/ml group or 1.0 x 107 cell/ml group. Conclusions The results of this study suggest that transplantation of the composite of hUCB-MSCs and HA is beneficial for cartilage repair. In addition, this study shows that optimal MSC concentration needs to be determined for better cartilage repair. PMID:27824874

Effect of Transplanting Various Concentrations of a Composite of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronic Acid Hydrogel on Articular Cartilage Repair in a Rabbit Model.

PubMed

Park, Yong-Beom; Ha, Chul-Won; Kim, Jin-A; Rhim, Ji-Heon; Park, Yong-Geun; Chung, Jun Young; Lee, Han-Jun

2016-01-01

Mesenchymal stem cells (MSCs) are known to have therapeutic potential for cartilage repair. However, the optimal concentration of MSCs for cartilage repair remains unclear. Therefore, we aimed to explore the feasibility of cartilage repair by human umbilical cord blood-derived MSCs (hUCB-MSCs) and to determine the optimal concentrations of the MSCs in a rabbit model. Osteochondral defects were created in the trochlear groove of femur in 55 rabbits. Four experimental groups (11 rabbits/group) were treated by transplanting the composite of hUCB-MSCs and HA with various MSCs concentrations (0.1, 0.5, 1.0, and 1.5 x 107 cells/ml). One control group was left untreated. At 4, 8, and 16 weeks post-transplantation, the degree of cartilage repair was evaluated grossly and histologically. Overall, transplanting hUCB-MSCs and HA hydrogel resulted in cartilage repair tissue with better quality than the control without transplantation (P = 0.015 in 0.1, P = 0.004 in 0.5, P = 0.004 in 1.0, P = 0.132 in 1.5 x 107 cells/ml). Interestingly, high cell concentration of hUCB-MSCs (1.5×107 cells/ml) was inferior to low cell concentrations (0.1, 0.5, and 1.0 x 107 cells/ml) in cartilage repair (P = 0.394,P = 0.041, P = 0.699, respectively). The 0.5 x 107 cells/ml group showed the highest cartilage repair score at 4, 8 and 16 weeks post transplantation, and followed by 0.1x107 cells/ml group or 1.0 x 107 cell/ml group. The results of this study suggest that transplantation of the composite of hUCB-MSCs and HA is beneficial for cartilage repair. In addition, this study shows that optimal MSC concentration needs to be determined for better cartilage repair.

Effect of human mesenchymal stem cells on the growth of HepG2 and Hela cells.

PubMed

Long, Xiaohui; Matsumoto, Rena; Yang, Pengyuan; Uemura, Toshimasa

2013-01-01

Human mesenchymal stem cells (hMSCs) accumulate at carcinomas and have a great impact on cancer cell's behavior. Here we demonstrated that hMSCs could display both the promotional and inhibitive effects on growth of HepG2 and Hela cells by using the conditioned media, indirect co-culture, and cell-to-cell co-culture. Cell growth was increased following the addition of lower proportion of hMSCs while decreased by treatment of higher proportion of hMSCs. We also established a novel noninvasive label way by using internalizing quantum dots (i-QDs) for study of cell-cell contact in the co-culture, which was effective and sensitive for both tracking and distinguishing different cells population without the disturbance of cells. Furthermore, we investigated the role of hMSCs in regulation of cell growth and showed that mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways were involved in hMSC-mediated cell inhibition and proliferation. Our findings suggested that hMSCs regulated cancer cell function by providing a suitable environment, and the discovery from the study would provide some clues for development of effective strategy for hMSC-based cancer therapies.

Interactions between human mesenchymal stem cells and natural killer cells.

PubMed

Sotiropoulou, Panagiota A; Perez, Sonia A; Gritzapis, Angelos D; Baxevanis, Constantin N; Papamichail, Michael

2006-01-01

Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing an attractive therapeutic tool for regenerative medicine. They possess unique immunomodulatory properties, being capable of suppressing T-cell responses and modifying dendritic cell differentiation, maturation, and function, whereas they are not inherently immunogenic, failing to induce alloreactivity to T cells and freshly isolated natural killer (NK) cells. To clarify the generation of host immune responses to implanted MSCs in tissue engineering and their potential use as immunosuppressive elements, the effect of MSCs on NK cells was investigated. We demonstrate that at low NK-to-MSC ratios, MSCs alter the phenotype of NK cells and suppress proliferation, cytokine secretion, and cyto-toxicity against HLA-class I- expressing targets. Some of these effects require cell-to-cell contact, whereas others are mediated by soluble factors, including transforming growth factor-beta1 and prostaglandin E2, suggesting the existence of diverse mechanisms for MSC-mediated NK-cell suppression. On the other hand, MSCs are susceptible to lysis by activated NK cells. Overall, these data improve our knowledge of interactions between MSCs and NK cells and consequently of their effect on innate immune responses and their contribution to the regulation of adaptive immunity, graft rejection, and cancer immunotherapy.

Bone marrow mesenchymal stem cells ameliorate inflammatory factor-induced dysfunction of INS-1 cells on chip.

PubMed

Sun, Yu; Yao, Zhina; Lin, Peng; Hou, Xinguo; Chen, Li

2014-05-01

Using a microfluidic chip, we have investigated whether bone marrow mesenchymal stem cells (BM-MSCs) could ameliorate IL-1β/IFN-γ-induced dysfunction of INS-1 cells. BM-MSCs were obtained from diabetes mellitus patients and their cell surface antigen expression profiles were analyzed by flow cytometric. INS-1 cells were cocultured with BM-MSCs on a microfluidic chip with persistent perfusion of medium containing 1 ng/mL IL-1β and 2.5 U/mL IFN-γ for 72 h. BM-MSCs could partially rescue INS-1 cells from cytokine-induced dysfunction and ameliorate the expression of insulin and PDX-1 gene in INS-1 cells. Thus BM-MSCs can be viewed as a promising stem cell source to depress inflammatory factor-induced dysfunction of pancreatic β cells in diabetic patients. © 2014 International Federation for Cell Biology.

Generation of inner ear sensory cells from bone marrow-derived human mesenchymal stem cells.

PubMed

Durán Alonso, M Beatriz; Feijoo-Redondo, Ana; Conde de Felipe, Magnolia; Carnicero, Estela; García, Ana Sánchez; García-Sancho, Javier; Rivolta, Marcelo N; Giráldez, Fernando; Schimmang, Thomas

2012-11-01

Hearing loss is the most common sensory disorder in humans, its main cause being the loss of cochlear hair cells. We studied the potential of human mesenchymal stem cells (hMSCs) to differentiate towards hair cells and auditory neurons. hMSCs were first differentiated to neural progenitors and subsequently to hair cell- or auditory neuron-like cells using in vitro culture methods. Differentiation of hMSCs to an intermediate neural progenitor stage was critical for obtaining inner ear sensory lineages. hMSCs generated hair cell-like cells only when neural progenitors derived from nonadherent hMSC cultures grown in serum-free medium were exposed to EGF and retinoic acid. Auditory neuron-like cells were obtained when treated with retinoic acid, and in the presence of defined growth factor combinations containing Sonic Hedgehog. The results show the potential of hMSCs to give rise to inner ear sensory cells.

Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

PubMed

Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

2016-12-30

This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

IGF-1-overexpressing mesenchymal stem cells accelerate bone marrow stem cell mobilization via paracrine activation of SDF-1alpha/CXCR4 signaling to promote myocardial repair.

PubMed

Haider, Husnain Kh; Jiang, Shujia; Idris, Niagara M; Ashraf, Muhammad

2008-11-21

We hypothesized that mesenchymal stem cells (MSCs) overexpressing insulin-like growth factor (IGF)-1 showed improved survival and engraftment in the infarcted heart and promoted stem cell recruitment through paracrine release of stromal cell-derived factor (SDF)-1alpha. Rat bone marrow-derived MSCs were used as nontransduced ((Norm)MSCs) or transduced with adenoviral-null vector ((Null)MSCs) or vector encoding for IGF-1 ((IGF-1)MSCs). (IGF-1)MSCs secreted higher IGF-1 until 12 days of observation (P<0.001 versus (Null)MSCs). Molecular studies revealed activation of phosphoinositide 3-kinase, Akt, and Bcl.xL and inhibition of glycogen synthase kinase 3beta besides release of SDF-1alpha in parallel with IGF-1 expression in (IGF-1)MSCs. For in vivo studies, 70 muL of DMEM without cells (group 1) or containing 1.5x10(6) (Null)MSCs (group 2) or (IGF-1)MSCs (group 3) were implanted intramyocardially in a female rat model of permanent coronary artery occlusion. One week later, immunoblot on rat heart tissue (n=4 per group) showed elevated myocardial IGF-1 and phospho-Akt in group 3 and higher survival of (IGF-1)MSCs (P<0.06 versus (Null)MSCs) (n=6 per group). SDF-1alpha was increased in group 3 animal hearts (20-fold versus group 2), with massive mobilization and homing of ckit(+), MDR1(+), CD31(+), and CD34(+) cells into the infarcted heart. Infarction size was significantly reduced in cell transplanted groups compared with the control. Confocal imaging after immunostaining for myosin heavy chain, actinin, connexin-43, and von Willebrand factor VIII showed extensive angiomyogenesis in the infarcted heart. Indices of left ventricular function, including ejection fraction and fractional shortening, were improved in group 3 as compared with group 1 (P<0.05). In conclusion, the strategy of IGF-1 transgene expression induced massive stem cell mobilization via SDF-1alpha signaling and culminated in extensive angiomyogenesis in the infarcted heart.

Hydrogen peroxide preconditioning enhances the therapeutic efficacy of Wharton's Jelly mesenchymal stem cells after myocardial infarction.

PubMed

Zhang, Jin; Chen, Guang-Hui; Wang, Yong-Wei; Zhao, Jing; Duan, Hai-Feng; Liao, Lian-Ming; Zhang, Xiao-Zhong; Chen, Yun-Dai; Chen, Hu

2012-10-01

Exposure of cells to sublethal concentrations of hydrogen peroxide (H2O2) can alleviate subsequent oxidative stress-induced apoptosis. We assessed the effects of H2O2 preconditioning on the therapeutic potential of human umbilical cord Wharton's Jelly mesenchymal stem cells (WJ-MSCs) in a murine model of myocardial infarction. WJ-MSCs were incubated in the media for 2 hours with or without 200 µmol/L H2O2. Mice underwent left anterior descending coronary artery ligation, and received injection of phosphate buffered saline, 1×10(6) WJ-MSCs, or 1×10(6) H2O2 preconditioned WJ-MSCs 3 hours later via tail vein. Echocardiography was performed 0, 7, 14 and 28 days after surgery, and the mice were euthanized on day 28 for histological analysis. In vitro cytokine concentrations in the WJ-MSC cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The effect of WJ-MSC cell supernatant on the migration and proliferation of endothelial cells were observed by transwell migration and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assays. Echocardiographic measurements revealed a significant improvement in the left ventricular contractility of the WJ-MSCs-H2O2 group compared to the WJ-MSCs group. Histological analysis revealed increased neovascularization and reduced myocardial fibrosis in the WJ-MSCs-H2O2 group compared to the WJ-MSCs group. Pretreatment of WJ-MSCs with H2O2 increased the secretion of interleukin-6 (IL-6) into the cell culture supernatant by approximately 25-fold. The culture supernatant from WJ-MSCs-H2O2 significantly increased the migration and proliferation of endothelial cells; these effects could be blocked using an anti-IL-6 antibody. This study demonstrates that H2O2 preconditioning significantly enhanced the therapeutic potential of WJ-MSCs, possibly by stimulating the production of IL-6 by WJ-MSCs, which may cause migration and proliferation of endothelial cells and increase neovascularization.

Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2.

PubMed

Kim, Hyung-Sik; Shin, Tae-Hoon; Lee, Byung-Chul; Yu, Kyung-Rok; Seo, Yoojin; Lee, Seunghee; Seo, Min-Soo; Hong, In-Sun; Choi, Soon Won; Seo, Kwang-Won; Núñez, Gabriel; Park, Jong-Hwan; Kang, Kyung-Sun

2013-12-01

Decreased levels or function of nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease. NOD2 regulates intestinal inflammation, and also is expressed by human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation. We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis. Colitis was induced in mice by administration of dextran sulfate sodium or trinitrobenzene sulfonic acid. Mice then were given intraperitoneal injections of NOD2-activated hUCB-MSCs; colon tissues and mesenteric lymph nodes were collected for histologic analyses. A bromodeoxyuridine assay was used to determine the ability of hUCB-MSCs to inhibit proliferation of human mononuclear cells in culture. Administration of hUCB-MSCs reduced the severity of colitis in mice. The anti-inflammatory effects of hUCB-MSCs were greatly increased by activation of NOD2 by its ligand, muramyl dipeptide (MDP). Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin (IL)-10 and infiltration by T regulatory cells, and reduced production of inflammatory cytokines. Proliferation of mononuclear cells was inhibited significantly by co-culture with hUCB-MSCs that had been stimulated with MDP. MDP induced prolonged production of prostaglandin (PG)E2 in hUCB-MSCs via the NOD2-RIP2 pathway, which suppressed proliferation of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs in response to MDP increased production of IL-10 and T regulatory cells. In mice, production of PGE2 by MSCs and subsequent production of IL-10 were required to reduce the severity of colitis. Activation of NOD2 is required for the ability of hUCB-MSCs to reduce the severity of colitis in mice. NOD2 signaling increases the ability of these cells to suppress mononuclear cell proliferation by inducing production of PGE2. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Won-Jae; Hah, Young-Sool; Ock, Sun-A.

The in vitro differentiation and immunosuppressive capacity of mesenchymal stem cells (MSCs) derived from synovial fluid (SF-MSCs) and bone marrow extract (BM-MSCs) in an isogenic background of minipigs were comparatively analyzed in a collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis (RA). The proliferation capacity and expression of pluripotent transcription factors (Oct3/4 and Sox2) were significantly (P<0.05) higher in SF-MSCs than in BM-MSCs. The differentiation capacity of SF-MSCs into adipocytes, osteocytes and neurocytes was significantly (P<0.05) lower than that of BM-MSCs, and the differentiation capacity of SF-MSCs into chondrocytes was significantly (P<0.05) higher than that of BM-MSCs. Systemic injection ofmore » BM- and SF-MSCs significantly (P<0.05) ameliorated the clinical symptoms of CIA mice, with SF-MSCs having significantly (P<0.05) higher clinical and histopathological recovery scores than BM-MSCs. Furthermore, the immunosuppressive properties of SF-MSCs in CIA mice were associated with increased levels of the anti-inflammatory cytokine interleukin (IL)-10, and decreased levels of the pro-inflammatory cytokine IL-1β and osteoclast-related sRANKL. In conclusion, SF-MSCs exhibited eminent pluripotency and differentiation capacity into chondrocytes, addition to substantial in vivo immunosuppressive capacity by elevating IL-10 and reducing IL-1β levels in CIA mice. - Highlights: • Immunosuppressive capacity of BM-, SM-, and SF-MSCs was evaluated in an RA model. • Proliferation, pluripotency and chondrogenic differentiation capacity were higher in SF-MSCs. • SF-MSCs exhibited improved therapeutic effects than BM-MSCs. • SF-MSCs may have applications as immunosuppressive therapy in autoimmune diseases.« less

Surfactant effects on the viability and function of human mesenchymal stem cells: in vitro and in vivo assessment.

PubMed

Chen, Chung-Ming; Chou, Hsiu-Chu; Lin, Willie; Tseng, Chris

2017-08-03

Surfactant therapy has become the standard of care for preterm infants with respiratory distress syndrome. Preclinical studies have reported the therapeutic benefits of mesenchymal stem cells (MSCs) in experimental bronchopulmonary dysplasia. This study investigated the effects of a surfactant on the in vitro viability and in vivo function of human MSCs. The viability, phenotype, and mitochondrial membrane potential (MMP) of MSCs were assessed through flow cytometry. The in vivo function was assessed after intratracheal injection of human MSCs (1 × 10 5 cells) diluted in 30 μl of normal saline (NS), 10 μl of a surfactant diluted in 20 μl of NS, and 10 μl of a surfactant and MSCs (1 × 10 5 cells) diluted in 20 μl of NS in newborn rats on postnatal day 5. The pups were reared in room air (RA) or an oxygen-enriched atmosphere (85% O 2 ) from postnatal days 1 to 14; eight study groups were examined: RA + NS, RA + MSCs, RA + surfactant, RA + surfactant + MSCs, O 2  + NS, O 2  + MSCs, O 2  + surfactant, and O 2  + surfactant + MSCs. The lungs were excised for histological and cytokine analysis on postnatal day 14. Compared with the controls, surfactant-treated MSCs showed significantly reduced viability and MMP after exposure to 1:1 and 1:2 of surfactant:MSCs for 15 and 60 minutes. All human MSC samples exhibited similar percentages of CD markers, regardless of surfactant exposure. The rats reared in hyperoxia and treated with NS exhibited a significantly higher mean linear intercept (MLI) than did those reared in RA and treated with NS, MSCs, surfactant, or surfactant + MSCs. Treatment with MSCs, surfactant, or surfactant + MSCs significantly reduced the hyperoxia-induced increase in MLI. The O 2  + surfactant + MSCs group exhibited a significantly higher MLI than did the O 2  + MSCs group. Furthermore, treatment with MSCs and MSCs + surfactant significantly reduced the hyperoxia-induced increase in apoptotic cells. Combination therapy involving a surfactant and MSCs does not exert additive effects on lung development in hyperoxia-induced lung injury.

Spatial and temporal characterization of endometrial mesenchymal stem-like cells activity during the menstrual cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shan, Xu; Chan, Rachel W.S., E-mail: rwschan@hku.hk; Centre of Reproduction, Development of Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR

The human endometrium is a highly dynamic tissue with the ability to cyclically regenerate during the reproductive life. Endometrial mesenchymal stem-like cells (eMSCs) located throughout the endometrium have shown to functionally contribute to endometrial regeneration. In this study we examine whether the menstrual cycle stage and the location in the endometrial bilayer (superficial and deep portions of the endometrium) has an effect on stem cell activities of eMSCs (CD140b{sup +}CD146{sup +} cells). Here we show the percentage and clonogenic ability of eMSCs were constant in the various stages of the menstrual cycle (menstrual, proliferative and secretory). However, eMSCs from themore » menstrual endometrium underwent significantly more rounds of self-renewal and enabled a greater total cell output than those from the secretory phase. Significantly more eMSCs were detected in the deeper portion of the endometrium compared to the superficial layer but their clonogenic and self-renewal activities remained similar. Our findings suggest that eMSCs are activated in the menstrual phase for the cyclical regeneration of the endometrium. - Highlights: • The percentages of endometrial mesenchymal-like stem cells (eMSCs) were constant across the menstrual cycle. • Menstruation eMSCs display superior self-renewal and long-term proliferative activities. • More eMSCs reside in the deeper portion of the endometrium than the superficial layer.« less

Musculoskeletal tissue engineering with human umbilical cord mesenchymal stromal cells

PubMed Central

Wang, Limin; Ott, Lindsey; Seshareddy, Kiran; Weiss, Mark L; Detamore, Michael S

2011-01-01

Multipotent mesenchymal stromal cells (MSCs) hold tremendous promise for tissue engineering and regenerative medicine, yet with so many sources of MSCs, what are the primary criteria for selecting leading candidates? Ideally, the cells will be multipotent, inexpensive, lack donor site morbidity, donor materials should be readily available in large numbers, immunocompatible, politically benign and expandable in vitro for several passages. Bone marrow MSCs do not meet all of these criteria and neither do embryonic stem cells. However, a promising new cell source is emerging in tissue engineering that appears to meet these criteria: MSCs derived from Wharton’s jelly of umbilical cord MSCs. Exposed to appropriate conditions, umbilical cord MSCs can differentiate in vitro along several cell lineages such as the chondrocyte, osteoblast, adipocyte, myocyte, neuronal, pancreatic or hepatocyte lineages. In animal models, umbilical cord MSCs have demonstrated in vivo differentiation ability and promising immunocompatibility with host organs/tissues, even in xenotransplantation. In this article, we address their cellular characteristics, multipotent differentiation ability and potential for tissue engineering with an emphasis on musculoskeletal tissue engineering. PMID:21175290

Equine bone marrow-derived mesenchymal stromal cells are heterogeneous in MHC class II expression and capable of inciting an immune response in vitro

PubMed Central

2014-01-01

Introduction The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSCs) in regenerative treatments. No studies to date have examined recipient response to major histocompatibility complex (MHC)-mismatched equine MSCs. The purposes of this study were to immunophenotype MSCs from horses of known MHC haplotype and to compare the immunogenicity of MSCs with differing MHC class II expression. Methods MSCs and peripheral blood leukocytes (PBLs) were obtained from Thoroughbred horses (n = 10) of known MHC haplotype (ELA-A2, -A3, and -A9 homozygotes). MSCs were cultured through P8; cells from each passage (P2 to P8) were cryopreserved until used. Immunophenotyping of MHC class I and II, CD44, CD29, CD90, LFA-1, and CD45RB was performed by using flow cytometry. Tri-lineage differentiation assays were performed to confirm MSC multipotency. Recombinant equine IFN-γ was used to stimulate MHC class II negative MSCs in culture, after which expression of MHC class II was re-examined. To assess the ability of MHC class II negative or positive MSCs to stimulate an immune response, modified one-way mixed leukocyte reactions (MLRs) were performed by using MHC-matched and mismatched responder PBLs and stimulator PBLs or MSCs. Proliferation of gated CFSE-labeled CD3+ responder T cells was evaluated via CFSE attenuation by using flow cytometry and reported as the number of cells in the proliferating T-cell gate. Results MSCs varied widely in MHC class II expression despite being homogenous in terms of “stemness” marker expression and ability to undergo trilineage differentiation. Stimulation of MHC class II negative MSCs with IFN-γ resulted in markedly increased expression of MHC class II. MLR results revealed that MHC-mismatched MHC class II-positive MSCs caused significantly increased responder T-cell proliferation compared with MHC-mismatched MHC class II-negative and MHC-matched MSCs, and equivalent to that of the positive control of MHC-mismatched leukocytes. Conclusions The results of this study suggest that MSCs should be confirmed as MHC class II negative before allogeneic application. Additionally, it must be considered that even MHC class II-negative MSCs could upregulate MHC class II expression if implanted into an area of active inflammation, as demonstrated with in vitro stimulation with IFN-γ. PMID:24461709

Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

PubMed

Jin, Hye Jin; Kwon, Ji Hye; Kim, Miyeon; Bae, Yun Kyung; Choi, Soo Jin; Oh, Wonil; Yang, Yoon Sun; Jeon, Hong Bae

2016-04-01

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated β-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs. One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required. However, long-term growth inevitably induces cellular senescence, which potentially causes poor clinical outcomes by inducing growth arrest and the loss of stem cell properties. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This study provides strong evidence that CD146 is a novel and useful marker for predicting senescence in human umbilical cord blood-derived MSCs (hUCB-MSCs), and CD146 can potentially be applied in quality-control assessments of hUCB-MSC-based therapy. ©AlphaMed Press.

Improvement of cardiac function by placenta-derived mesenchymal stem cells does not require permanent engraftment and is independent of the insulin signaling pathway.

PubMed

Passipieri, Juliana A; Kasai-Brunswick, Tais H; Suhett, Grazielle; Martins, Andreza B; Brasil, Guilherme V; Campos, Dilza B; Rocha, Nazareth N; Ramos, Isalira P; Mello, Debora B; Rodrigues, Deivid C; Christie, Beatriz B; Silva-Mendes, Bernardo J; Balduíno, Alex; Sá, Renato M; Lopes, Laudelino M; Goldenberg, Regina C; Campos de Carvalho, Antonio C; Carvalho, Adriana B

2014-08-21

The objective of this work was to evaluate the efficacy of placenta-derived mesenchymal stem cell (MSC) therapy in a mouse model of myocardial infarction (MI). Since MSCs can be obtained from two different regions of the human term placenta (chorionic plate or villi), cells obtained from both these regions were compared so that the best candidate for cell therapy could be selected. For the in vitro studies, chorionic plate MSCs (cp-MSCs) and chorionic villi MSCs (cv-MSCs) were extensively characterized for their genetic stability, clonogenic and differentiation potential, gene expression, and immunophenotype. For the in vivo studies, C57Bl/6 mice were submitted to MI and, after 21 days, received weekly intramyocardial injections of cp-MSCs for 3 weeks. Cells were also stably transduced with a viral construct expressing luciferase, under the control of the murine stem cell virus (MSCV) promoter, and were used in a bioluminescence assay. The expression of genes associated with the insulin signaling pathway was analyzed in the cardiac tissue from cp-MSCs and placebo groups. Morphology, differentiation, immunophenotype, and proliferation were quite similar between these cells. However, cp-MSCs had a greater clonogenic potential and higher expression of genes related to cell cycle progression and genome stability. Therefore, we considered that the chorionic plate was preferable to the chorionic villi for the isolation of MSCs. Sixty days after MI, cell-treated mice had a significant increase in ejection fraction and a reduction in end-systolic volume. This improvement was not caused by a reduction in infarct size. In addition, tracking of cp-MSCs transduced with luciferase revealed that cells remained in the heart for 4 days after the first injection but that the survival period was reduced after the second and third injections. Quantitative reverse transcription-polymerase chain reaction revealed similar expression of genes involved in the insulin signaling pathway when comparing cell-treated and placebo groups. Improvement of cardiac function by cp-MSCs did not require permanent engraftment and was not mediated by the insulin signaling pathway.

Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow.

PubMed

Lee, Junmin; Abdeen, Amr A; Tang, Xin; Saif, Taher A; Kilian, Kristopher A

2016-09-15

Mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical and biochemical properties of the extracellular matrix. In this work we conduct a combinatorial study of matrix properties that influence adipogenesis and neurogenesis including: adhesion proteins, stiffness, and cell geometry, for mesenchymal stem cells derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs). We uncover distinct differences in integrin expression, the magnitude of traction stress, and lineage specification to adipocytes and neuron-like cells between cell sources. In the absence of media supplements, adipogenesis in AT-MSCs is not significantly influenced by matrix properties, while the converse is true in BM-MSCs. Both cell types show changes in the expression of neurogenesis markers as matrix cues are varied. When cultured on laminin conjugated microislands of the same adhesive area, BM-MSCs display elevated adipogenesis markers, while AT-MSCs display elevated neurogenesis markers; integrin analysis suggests neurogenesis in AT-MSCs is guided by adhesion through integrin αvβ3. Overall, the properties of the extracellular matrix guides MSC adhesion and lineage specification to different degrees and outcomes, in spite of their similarities in general characteristics. This work will help guide the selection of MSCs and matrix components for applications where high fidelity of differentiation outcome is desired. Mesenchymal stem cells (MSCs) are an attractive cell type for stem cell therapies; however, in order for these cells to be useful in medicine, we need to understand how they respond to the physical and chemical environments of tissue. Here, we explore how two promising sources of MSCs-those derived from bone marrow and from adipose tissue-respond to the compliance and composition of tissue using model extracellular matrices. Our results demonstrate a source-specific propensity to undergo adipogenesis and neurogenesis, and uncover a role for adhesion, and the degree of traction force exerted on the substrate in guiding these lineage outcomes. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Adaptive Regulation of Osteopontin Production by Dendritic Cells Through the Bidirectional Interaction With Mesenchymal Stromal Cells.

PubMed

Scutera, Sara; Salvi, Valentina; Lorenzi, Luisa; Piersigilli, Giorgia; Lonardi, Silvia; Alotto, Daniela; Casarin, Stefania; Castagnoli, Carlotta; Dander, Erica; D'Amico, Giovanna; Sozzani, Silvano; Musso, Tiziana

2018-01-01

Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β). OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E 2 (PGE 2 ). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE 2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c + ) are a small percentage of BM cells, we demonstrated colocalization of CD271 + MSCs with CD1c + DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c + cells in the proximity of CD271 + MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.

Characterisation of synovial fluid and infrapatellar fat pad derived mesenchymal stromal cells: The influence of tissue source and inflammatory stimulus

PubMed Central

Garcia, John; Wright, Karina; Roberts, Sally; Kuiper, Jan Herman; Mangham, Chas; Richardson, James; Mennan, Claire

2016-01-01

The infrapatellar fat pad (FP) and synovial fluid (SF) in the knee serve as reservoirs of mesenchymal stromal cells (MSCs) with potential therapeutic benefit. We determined the influence of the donor on the phenotype of donor matched FP and SF derived MSCs and examined their immunogenic and immunomodulatory properties before and after stimulation with the pro-inflammatory cytokine interferon-gamma (IFN-γ). Both cell populations were positive for MSC markers CD73, CD90 and CD105, and displayed multipotency. FP-MSCs had a significantly faster proliferation rate than SF-MSCs. CD14 positivity was seen in both FP-MSCs and SF-MSCs, and was positively correlated to donor age but only for SF-MSCs. Neither cell population was positive for the co-stimulatory markers CD40, CD80 and CD86, but both demonstrated increased levels of human leukocyte antigen-DR (HLA-DR) following IFN-γ stimulation. HLA-DR production was positively correlated with donor age for FP-MSCs but not SF-MSCs. The immunomodulatory molecule, HLA-G, was constitutively produced by both cell populations, unlike indoleamine 2, 3-dioxygenase which was only produced following IFN-γ stimulation. FP and SF are accessible cell sources which could be utilised in the treatment of cartilage injuries, either by transplantation following ex-vivo expansion or endogenous targeting and mobilisation of cells close to the site of injury. PMID:27073003

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium.

PubMed

Seeberger, Karen L; Dufour, Jannette M; Shapiro, Andrew M James; Lakey, Jonathan R T; Rajotte, Ray V; Korbutt, Gregory S

2006-02-01

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.

Repair of diaphyseal bone defects with calcitriol-loaded PLGA scaffolds and marrow stromal cells.

PubMed

Yoon, Sun Jung; Park, Ki Suk; Kim, Moon Suk; Rhee, John M; Khang, Gilson; Lee, Hai Bang

2007-05-01

Calcitriol (1,25(OH)2D3)-loaded porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds prepared by solvent casting/salt leaching method were used to repair a 1.5 cm diaphyseal segmental bone defect as a fully absorbable osteogenic biomaterial. The in vitro release of sulforhodamine B (SRB) from PLGA scaffold was measured using spectrophotometer, considering SRB as a model drug. The SRB released from SRB-incorporated PLGA scaffold during 3 months was with relatively low initial burst. The calcitriol-loaded PLGA scaffolds with or without marrow stromal cells (MSCs) were implanted in a critical-sized intercalated bone defect in rabbit femur. Defects were assessed by radiographs until 9 weeks. The bony union of the defect was observed only in the calcitriol-loaded groups. RT-PCR results indicated that MSCs, which were seeded into calcitriol-loaded scaffold, expressed an increased level of alkaline phosphatase, osteonectin, and type I collagen mRNA at day 10. After 2 and 4 weeks, the implanted scaffolds were evaluated by histology. New osteoid matrix and direct calcium deposits were more evident in calcitriol/PLGA/MSC group. Three-dimensional computed tomography and frontal tomographic images of repaired femur showed that normal femur anatomy had been restored with cortical bone with no implanted PLGA remnants at 20 weeks. It can be concluded that the porous calcitriol-loaded PLGA scaffold combined with MSCs may be a novel method for repairing the large loaded bone defect.

Biofabrication of soft tissue templates for engineering the bone-ligament interface.

PubMed

Harris, Ella; Liu, Yurong; Cunniffe, Grainne; Morrissey, David; Carroll, Simon; Mulhall, Kevin; Kelly, Daniel J

2017-10-01

Regenerating damaged tissue interfaces remains a significant clinical challenge, requiring recapitulation of the structure, composition, and function of the native enthesis. In the ligament-to-bone interface, this region transitions from ligament to fibrocartilage, to calcified cartilage and then to bone. This gradation in tissue types facilitates the transfer of load between soft and hard structures while minimizing stress concentrations at the interface. Previous attempts to engineer the ligament-bone interface have utilized various scaffold materials with an array of various cell types and/or biological cues. The primary goal of this study was to engineer a multiphased construct mimicking the ligament-bone interface by driving differentiation of a single population of mesenchymal stem cells (MSCs), seeded within blended fibrin-alginate hydrogels, down an endochondral, fibrocartilaginous, or ligamentous pathway through spatial presentation of growth factors along the length of the construct within a custom-developed, dual-chamber culture system. MSCs within these engineered constructs demonstrated spatially distinct regions of differentiation, adopting either a cartilaginous or ligamentous phenotype depending on their local environment. Furthermore, there was also evidence of spatially defined progression toward an endochondral phenotype when chondrogenically primed MSCs within this construct were additionally exposed to hypertrophic cues. The study demonstrates the feasibility of engineering spatially complex soft tissues within a single MSC laden hydrogel through the defined presentation of biochemical cues. This novel approach represents a new strategy for engineering the ligament-bone interface. Biotechnol. Bioeng. 2017;114: 2400-2411. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Safety Profile of Good Manufacturing Practice Manufactured Interferon γ-Primed Mesenchymal Stem/Stromal Cells for Clinical Trials.

PubMed

Guess, Adam J; Daneault, Beth; Wang, Rongzhang; Bradbury, Hillary; La Perle, Krista M D; Fitch, James; Hedrick, Sheri L; Hamelberg, Elizabeth; Astbury, Caroline; White, Peter; Overolt, Kathleen; Rangarajan, Hemalatha; Abu-Arja, Rolla; Devine, Steven M; Otsuru, Satoru; Dominici, Massimo; O'Donnell, Lynn; Horwitz, Edwin M

2017-10-01

Mesenchymal stem/stromal cells (MSCs) are widely studied by both academia and industry for a broad array of clinical indications. The collective body of data provides compelling evidence of the clinical safety of MSC therapy. However, generally accepted proof of therapeutic efficacy has not yet been reported. In an effort to generate a more effective therapeutic cell product, investigators are focused on modifying MSC processing protocols to enhance the intrinsic biologic activity. Here, we report a Good Manufacturing Practice-compliant two-step MSC manufacturing protocol to generate MSCs or interferon γ (IFNγ) primed MSCs which allows freshly expanded cells to be infused in patients on a predetermined schedule. This protocol eliminates the need to infuse cryopreserved, just thawed cells which may reduce the immune modulatory activity. Moreover, using (IFNγ) as a prototypic cytokine, we demonstrate the feasibility of priming the cells with any biologic agent. We then characterized MSCs and IFNγ primed MSCs prepared with our protocol, by karyotype, in vitro potential for malignant transformation, biodistribution, effect on engraftment of transplanted hematopoietic cells, and in vivo toxicity in immune deficient mice including a complete post-mortem examination. We found no evidence of toxicity attributable to the MSC or IFNγ primed MSCs. Our data suggest that the clinical risk of infusing MSCs or IFNγ primed MSCs produced by our two-step protocol is not greater than MSCs currently in practice. While actual proof of safety requires phase I clinical trials, our data support the use of either cell product in new clinical studies. Stem Cells Translational Medicine 2017;6:1868-1879. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Effects of Passage Number and Differentiation Protocol on the Generation of Dopaminergic Neurons from Rat Bone Marrow-Derived Mesenchymal Stem Cells.

PubMed

Shall, Gabrielle; Menosky, Megan; Decker, Sarah; Nethala, Priya; Welchko, Ryan; Leveque, Xavier; Lu, Ming; Sandstrom, Michael; Hochgeschwender, Ute; Rossignol, Julien; Dunbar, Gary

2018-03-02

Multiple studies have demonstrated the ability of mesenchymal stem cells (MSCs) to differentiate into dopamine-producing cells, in vitro and in vivo, indicating their potential to be used in the treatment of Parkinson's disease (PD). However, there are discrepancies among studies regarding the optimal time (i.e., passage number) and method for dopaminergic induction, in vitro. In the current study, we compared the ability of early (P4) and later (P40) passaged bone marrow-derived MSCs to differentiate into dopaminergic neurons using two growth-factor-based approaches. A direct dopaminergic induction (DDI) was used to directly convert MSCs into dopaminergic neurons, and an indirect dopaminergic induction (IDI) was used to direct MSCs toward a neuronal lineage prior to terminal dopaminergic differentiation. Results indicate that both early and later passaged MSCs exhibited positive expression of neuronal and dopaminergic markers following either the DDI or IDI protocols. Additionally, both early and later passaged MSCs released dopamine and exhibited spontaneous neuronal activity following either the DDI or IDI. Still, P4 MSCs exhibited significantly higher spiking and bursting frequencies as compared to P40 MSCs. Findings from this study provide evidence that early passaged MSCs, which have undergone the DDI, are more efficient at generating dopaminergic-like cells in vitro, as compared to later passaged MSCs or MSCs that have undergone the IDI.

Mesenchymal Stem Cells Adopt Lung Cell Phenotype in Normal and Radiation-induced Lung Injury Conditions.

PubMed

Maria, Ola M; Maria, Ahmed M; Ybarra, Norma; Jeyaseelan, Krishinima; Lee, Sangkyu; Perez, Jessica; Shalaby, Mostafa Y; Lehnert, Shirley; Faria, Sergio; Serban, Monica; Seuntjens, Jan; El Naqa, Issam

2016-04-01

Lung tissue exposure to ionizing irradiation can invariably occur during the treatment of a variety of cancers leading to increased risk of radiation-induced lung disease (RILD). Mesenchymal stem cells (MSCs) possess the potential to differentiate into epithelial cells. However, cell culture methods of primary type II pneumocytes are slow and cannot provide a sufficient number of cells to regenerate damaged lungs. Moreover, effects of ablative radiation doses on the ability of MSCs to differentiate in vitro into lung cells have not been investigated yet. Therefore, an in vitro coculture system was used, where MSCs were physically separated from dissociated lung tissue obtained from either healthy or high ablative doses of 16 or 20 Gy whole thorax irradiated rats. Around 10±5% and 20±3% of cocultured MSCs demonstrated a change into lung-specific Clara and type II pneumocyte cells when MSCs were cocultured with healthy lung tissue. Interestingly, in cocultures with irradiated lung biopsies, the percentage of MSCs changed into Clara and type II pneumocytes cells increased to 40±7% and 50±6% at 16 Gy irradiation dose and 30±5% and 40±8% at 20 Gy irradiation dose, respectively. These data suggest that MSCs to lung cell differentiation is possible without cell fusion. In addition, 16 and 20 Gy whole thorax irradiation doses that can cause varying levels of RILD, induced different percentages of MSCs to adopt lung cell phenotype compared with healthy lung tissue, providing encouraging outlook for RILD therapeutic intervention for ablative radiotherapy prescriptions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xingbing, E-mail: wangxingbing91@hotmail.com; Cheng, Qiansong; Li, Lailing

Bone marrow derived-mesenchymal stromal cells (BM-MSCs) are multipotent, nonhematopoietic progenitors in a hematopoietic microenvironment and indispensable for regulating hematopoiesis. Several studies have reported that toll-like receptors (TLRs) are expressed in mesenchymal stromal cells (MSCs) to modulate their biological functions. In this study, we investigated the possible role(s) of TLRs in mediating the hematopoiesis-supporting role of human BM-MSCs. Human BM-MSCs were analyzed for mRNA expression of TLR1-10 by reverse transcription-polymerase chain reaction. TLR1-6, but not TLR7-10 were expressed by BM-MSCs. The protein expression of TLR2 and TLR4 was also confirmed by flow cytometry. We further explored the role of TLR2 andmore » TLR4 in mediating the capacity of BM-MSCs to support the proliferation and differentiation of CD34{sup +} hematopoietic stem/progenitor cells obtained from cord blood. BM-MSCs increased proliferation of CD34{sup +} cells and promoted the differentiation towards the myeloid lineage 7 or 14 days after co-culture, as well as colony formation by those cells and the production of interleukin 1 (IL-1), IL-8, IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF, if MSCs had been stimulated with TLR2 agonist (PAM{sub 3}CSK{sub 4}) or TLR4 agonist (LPS). Interestingly, although these effects were elevated in a different degree, a synergistic effect was not observed in BM-MSCs co-stimulated with PAM{sub 3}CSK{sub 4} and LPS. Together, our findings suggest that TLR2 and TLR4 signaling may indirectly regulate hematopoiesis by modulating BM-MSCs' functions. The increased hematopoietic proliferation and differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production of BM-MSCs.« less

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype

PubMed Central

Sheldrake, Tara A.; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

2017-01-01

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs. PMID:28376684

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

PubMed

Esteves, Cristina L; Sheldrake, Tara A; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

2017-07-01

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

Inducible indoleamine 2,3-dioxygenase 1 and programmed death ligand 1 expression as the potency marker for mesenchymal stromal cells.

PubMed

Guan, Qingdong; Li, Yun; Shpiruk, Tanner; Bhagwat, Swaroop; Wall, Donna A

2018-05-01

Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs. Clinical-grade bone marrow-derived MSCs were manufactured. The phenotype and immunosuppressive functions of the MSCs were evaluated based on the International Society for Cellular Therapy guidelines. Resting MSCs licensed by interferon (IFN)-γ exposure overnight were evaluated for changes in immune suppression and immune-relevant proteins. The relationship of immune-relevant protein expression with immunosuppression of MSCs was analyzed. MSC supressed third-party T-lymphocyte proliferation with high inter-donor and inter-test variability. The suppression of T-lymphocyte proliferation by IFN-γ-licensed MSCs correlated with that by resting MSCs. Many cellular proteins were up-regulated after IFN-γ exposure, including indoleamine 2,3-dioxygenase 1 (IDO-1), programmed death ligand 1 (PD-L1), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and bone marrow stromal antigen 2 (BST-2). The expression levels of IDO-1 and PD-L1 on licensed MSCs, not VCAM-1, ICAM-1 or BST-2 on licensed MSCs, correlated with MSC suppression of third-party T-cell proliferation. A flow cytometry-based assay of MSCs post-IFN-γ exposure measuring expression of intracellular protein IDO-1 and cell surface protein PD-L1 captures two mechanisms of suppression and offers the potential of a relevant, rapid assay for MSC-mediated immune suppression that would fit with the manufacturing process. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

PubMed

de Lourdes Mora-García, María; García-Rocha, Rosario; Morales-Ramírez, Omar; Montesinos, Juan José; Weiss-Steider, Benny; Hernández-Montes, Jorge; Ávila-Ibarra, Luis Roberto; Don-López, Christian Azucena; Velasco-Velázquez, Marco Antonio; Gutiérrez-Serrano, Vianey; Monroy-García, Alberto

2016-10-26

In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

MSCs: The Sentinel and Safe-Guards of Injury.

PubMed

Caplan, Arnold I

2016-07-01

Mesenchymal stem cells (MSCs) were originally named because they could differentiate in a variety of mesenchymal phenotypes in culture. Evidence indicates that MSCs arise from perivascular cells, pericytes, when the blood vessels are broken or inflamed. These pericyte/MSCs are situated on every blood vessel in the body. The MSCs sense the micro-environment of the injury site and secrete site-specific factors that serve several important reparative functions: first, a curtain of molecules from the front of the MSCs provide a barrier from the interrogation of the over-aggressive immune system. Second, from the back of the MSCs, a different set of bioactive agents inhibit scar formation and establish a regenerative micro-environment. Third, if bacteria are sensed by the MSCs, they produce powerful protein antibiotics that kill the bacteria on contact. Last, the MSCs surround and encyst intruding solid objects like a piece of wood (a "splinter") or other foreign objects. The MSCs act as a combination paramedic and emergency room (ER) staff to survey the damage, isolate foreign components, stabilize the injured tissues, provide antibiotics and encysting protection before a slower, medicinal sequence can be initiated to regenerate the damaged tissue. The MSCs, thus, act as sentinels to safeguard the individual from intrusion and chronic injury. A societal treatment system has evolved, paramedics and ER procedures, which mirror in a macro-sense what MSCs orchestrate in a micro-sense. Key to this new understanding is that MSCs are not "stem cells," but rather as Medicinal Signaling Cells as the therapeutic agents. © 2015 Wiley Periodicals, Inc.

Increased expression of pigment epithelium-derived factor in aged mesenchymal stem cells impairs their therapeutic efficacy for attenuating myocardial infarction injury‡

PubMed Central

Liang, Hongliang; Hou, Huiyuan; Yi, Wei; Yang, Guodong; Gu, Chunhu; Lau, Wayne Bond; Gao, Erhe; Ma, Xinliang; Lu, Zifan; Wei, Xufeng; Pei, Jianming; Yi, Dinghua

2013-01-01

Aims Mesenchymal stem cells (MSCs) can ameliorate myocardial infarction (MI) injury. However, older-donor MSCs seem less efficacious than those from younger donors, and the contributing underlying mechanisms remain unknown. Here, we determine how age-related expression of pigment epithelium-derived factor (PEDF) affects MSC therapeutic efficacy for MI. Methods and results Reverse transcriptase–polymerized chain reaction  and enzyme-linked immunosorbent assay analyses revealed dramatically increased PEDF expression in MSCs from old mice compared to young mice. Morphological and functional experiments demonstrated significantly impaired old MSC therapeutic efficacy compared with young MSCs in treatment of mice subjected to MI. Immunofluorescent staining demonstrated that administration of old MSCs compared with young MSCs resulted in an infarct region containing fewer endothelial cells, vascular smooth muscle cells, and macrophages, but more fibroblasts. Pigment epithelium-derived factor overexpression in young MSCs impaired the beneficial effects against MI injury, and induced cellular profile changes in the infarct region similar to administration of old MSCs. Knocking down PEDF expression in old MSCs improved MSC therapeutic efficacy, and induced a cellular profile similar to young MSCs administration. Studies in vitro showed that PEDF secreted by MSCs regulated the proliferation and migration of cardiac fibroblasts. Conclusions This is the first evidence that paracrine factor PEDF plays critical role in the regulatory effects of MSCs against MI injury. Furthermore, the impaired therapeutic ability of aged MSCs is predominantly caused by increased PEDF secretion. These findings indicate PEDF as a promising novel genetic modification target for improving aged MSC therapeutic efficacy. PMID:21606086

Uremic Toxins Affect the Imbalance of Redox State and Overexpression of Prolyl Hydroxylase 2 in Human Adipose Tissue-Derived Mesenchymal Stem Cells Involved in Wound Healing.

PubMed

Khanh, Vuong Cat; Ohneda, Kinuko; Kato, Toshiki; Yamashita, Toshiharu; Sato, Fujio; Tachi, Kana; Ohneda, Osamu

2017-07-01

Chronic kidney disease (CKD) results in a delay in wound healing because of its complications such as uremia, anemia, and fluid overload. Mesenchymal stem cells (MSCs) are considered to be a candidate for wound healing because of the ability to recruit many types of cells. However, it is still unclear whether the CKD-adipose tissue-derived MSCs (CKD-AT-MSCs) have the same function in wound healing as healthy donor-derived normal AT-MSCs (nAT-MSCs). In this study, we found that uremic toxins induced elevated reactive oxygen species (ROS) expression in nAT-MSCs, resulting in the reduced expression of hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions. Consistent with the uremic-treated AT-MSCs, there was a definite imbalance of redox state and high expression of ROS in CKD-AT-MSCs isolated from early-stage CKD patients. In addition, a transplantation study clearly revealed that nAT-MSCs promoted the recruitment of inflammatory cells and recovery from ischemia in the mouse flap model, whereas CKD-AT-MSCs had defective functions and the wound healing process was delayed. Of note, the expression of prolyl hydroxylase domain 2 (PHD2) is selectively increased in CKD-AT-MSCs and its inhibition can restore the expression of HIF-1α and the wound healing function of CKD-AT-MSCs. These results indicate that more studies about the functions of MSCs from CKD patients are required before they can be applied in the clinical setting.

IL-1β produced by aggressive breast cancer cells is one of the factors that dictate their interactions with mesenchymal stem cells through chemokine production.

PubMed

Escobar, Pauline; Bouclier, Céline; Serret, Julien; Bièche, Ivan; Brigitte, Madly; Caicedo, Andres; Sanchez, Elodie; Vacher, Sophie; Vignais, Marie-Luce; Bourin, Philippe; Geneviève, David; Molina, Franck; Jorgensen, Christian; Lazennec, Gwendal

2015-10-06

The aim of this work was to understand whether the nature of breast cancer cells could modify the nature of the dialog of mesenchymal stem cells (MSCs) with cancer cells. By treating MSCs with the conditioned medium of metastatic Estrogen-receptor (ER)-negative MDA-MB-231, or non-metastatic ER-positive MCF-7 breast cancer cells, we observed that a number of chemokines were produced at higher levels by MSCs treated with MDA-MB-231 conditioned medium (CM). MDA-MB-231 cells were able to induce NF-κB signaling in MSC cells. This was shown by the use of a NF-kB chemical inhibitor or an IκB dominant negative mutant, nuclear translocation of p65 and induction of NF-κB signature. Our results suggest that MDA-MB-231 cells exert their effects on MSCs through the secretion of IL-1β, that activates MSCs and induces the same chemokines as the MDA-MB-231CM. In addition, inhibition of IL-1β secretion in the MDA-MB-231 cells reduces the induced production of a panel of chemokines by MSCs, as well the motility of MDA-MB-231 cells. Our data suggest that aggressive breast cancer cells secrete IL-1β, which increases the production of chemokines by MSCs.

IL-1β produced by aggressive breast cancer cells is one of the factors that dictate their interactions with mesenchymal stem cells through chemokine production

PubMed Central

Serret, Julien; Bièche, Ivan; Brigitte, Madly; Caicedo, Andres; Sanchez, Elodie; Vacher, Sophie; Vignais, Marie-Luce; Bourin, Philippe; Geneviève, David; Molina, Franck; Jorgensen, Christian; Lazennec, Gwendal

2015-01-01

The aim of this work was to understand whether the nature of breast cancer cells could modify the nature of the dialog of mesenchymal stem cells (MSCs) with cancer cells. By treating MSCs with the conditioned medium of metastatic Estrogen-receptor (ER)-negative MDA-MB-231, or non-metastatic ER-positive MCF-7 breast cancer cells, we observed that a number of chemokines were produced at higher levels by MSCs treated with MDA-MB-231 conditioned medium (CM). MDA-MB-231 cells were able to induce NF-κB signaling in MSC cells. This was shown by the use of a NF-kB chemical inhibitor or an IκB dominant negative mutant, nuclear translocation of p65 and induction of NF-κB signature. Our results suggest that MDA-MB-231 cells exert their effects on MSCs through the secretion of IL-1β, that activates MSCs and induces the same chemokines as the MDA-MB-231CM. In addition, inhibition of IL-1β secretion in the MDA-MB-231 cells reduces the induced production of a panel of chemokines by MSCs, as well the motility of MDA-MB-231 cells. Our data suggest that aggressive breast cancer cells secrete IL-1β, which increases the production of chemokines by MSCs. PMID:26362269

Iron overload promotes mitochondrial fragmentation in mesenchymal stromal cells from myelodysplastic syndrome patients through activation of the AMPK/MFF/Drp1 pathway.

PubMed

Zheng, Qingqing; Zhao, Youshan; Guo, Juan; Zhao, Sida; Fei, Chengming; Xiao, Chao; Wu, Dong; Wu, Lingyun; Li, Xiao; Chang, Chunkang

2018-05-03

Iron overload (IO) has been reported to contribute to mesenchymal stromal cell (MSC) damage, but the precise mechanism has yet to be clearly elucidated. In this study, we found that IO increased cell apoptosis and lowered cell viability in MSCs, accompanied by extensive mitochondrial fragmentation and autophagy enhancement. All these effects were reactive oxygen species (ROS) dependent. In MSCs with IO, the ATP concentrations were significantly reduced due to high ROS levels and low electron respiratory chain complex (ETC) II/III activity. Reduced ATP phosphorylated AMP-activated protein kinase (AMPK). Activation of AMPK kinase complexes triggered mitochondrial fission. Moreover, gene knockout of AMPK via CRISPR/Cas9 reduced cell apoptosis, enhanced cell viability and attenuated mitochondrial fragmentation and autophagy caused by IO in MSCs. Further, AMPK-induced mitochondrial fragmentation of MSCs with IO was mediated via phosphorylation of mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for the GTPase dynamin-related protein 1 (Drp1). Gene knockdown of MFF reversed AMPK-induced mitochondrial fragmentation in MSCs with IO. In addition, MSCs from IO patients with myelodysplastic syndrome (MDS) showed increased cell apoptosis, decreased cell viability, higher ROS levels, lower ATP concentrations and increased mitochondrial fragmentation compared with MSCs from non-IO patients. In addition, iron chelation or antioxidant weakened the activity of the AMPK/MFF/Drp1 pathway in MDS-MSCs with IO from several patients, accompanied by attenuation of mitochondrial fragmentation and autophagy. Taken together, the AMPK/MFF/Drp1 pathway has an important role in the damage to MDS-MSCs caused by IO.

Study of the quantitative, functional, cytogenetic, and immunoregulatory properties of bone marrow mesenchymal stem cells in patients with B-cell chronic lymphocytic leukemia.

PubMed

Pontikoglou, Charalampos; Kastrinaki, Maria-Christina; Klaus, Mirjam; Kalpadakis, Christina; Katonis, Pavlos; Alpantaki, Kalliopi; Pangalis, Gerassimos A; Papadaki, Helen A

2013-05-01

The bone marrow (BM) microenvironment has clearly been implicated in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL). However, the potential involvement of BM stromal progenitors, the mesenchymal stem cells (MSCs), in the pathophysiology of the disease has not been extensively investigated. We expanded in vitro BM-MSCs from B-CLL patients (n=11) and healthy individuals (n=16) and comparatively assessed their reserves, proliferative potential, differentiation capacity, and immunoregulatory effects on T- and B-cells. We also evaluated the anti-apoptotic effect of patient-derived MSCs on leukemic cells and studied their cytogenetic characteristics in comparison to BM hematopoietic cells. B-CLL-derived BM MSCs exhibit a similar phenotype, differentiation potential, and ability to suppress T-cell proliferative responses as compared with MSCs from normal controls. Furthermore, they do not carry the cytogenetic abnormalities of the leukemic clone, and they exert a similar anti-apoptotic effect on leukemic cells and healthy donor-derived B-cells, as their normal counterparts. On the other hand, MSCs from B-CLL patients significantly promote normal B-cell proliferation and IgG production, in contrast to healthy-donor-derived MSCs. Furthermore, they have impaired reserves, defective cellular growth due to increased apoptotic cell death and exhibit aberrant production of stromal cell-derived factor 1, B-cell activating factor, a proliferation inducing ligand, and transforming growth factor β1, cytokines that are crucial for the survival/nourishing of the leukemic cells. We conclude that ex vivo expanded B-CLL-derived MSCs harbor intrinsic qualitative and quantitative abnormalities that may be implicated in disease development and/or progression.

Nkx2.5 enhances the efficacy of mesenchymal stem cells transplantation in treatment heart failure in rats.

PubMed

Deng, Bo; Wang, Jin Xin; Hu, Xing Xing; Duan, Peng; Wang, Lin; Li, Yang; Zhu, Qing Lei

2017-08-01

The aim of this study is to determine whether Nkx2.5 transfection of transplanted bone marrow mesenchymal stem cells (MSCs) improves the efficacy of treatment of adriamycin-induced heart failure in a rat model. Nkx2.5 was transfected in MSCs by lentiviral vector transduction. The expressions of Nkx2.5 and cardiac specific genes in MSCs and Nkx2.5 transfected mesenchymal stem cells (MSCs-Nkx2.5) were analyzed with quantitative real-time PCR and Western blot in vitro. Heart failure models of rats were induced by adriamycin and were then randomly divided into 3 groups: injected saline, MSCs or MSCs-Nkx2.5 via the femoral vein respectively. Four weeks after injection, the cardiac function, expressions of cardiac specific gene, fibrosis formation and collagen volume fraction in the myocardium as well as the expressions of GATA4 and MEF2 in rats were analyzed with echocardiography, immunohistochemistry, Masson staining, quantitative real-time PCR and Western blot, respectively. Nkx2.5 enhanced cardiac specific gene expressions including α-MHC, TNI, CKMB, connexin-43 in MSCs-Nkx2.5 in vitro. Both MSCs and MSCs-Nkx2.5 improved cardiac function, promoted the differentiation of transplanted MSCs into cardiomyocyte-like cells, decreased fibrosis formation and collagen volume fraction in the myocardium, as well as increased the expressions of GATA4 and MEF2 in adriamycin-induced rat heart failure models. Moreover, the effect was much more remarkable in MSCs-Nkx2.5 than in MSCs group. This study has found that Nkx2.5 enhances the efficacy of MSCs transplantation in treatment adriamycin-induced heart failure in rats. Nkx2.5 transfected to transplanted MSCs provides a potential effective approach to heart failure. Copyright © 2017 Elsevier Inc. All rights reserved.

Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rattigan, Yanique I.; Patel, Brijesh B.; Department of Pharmacology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901

2012-02-15

Human mesenchymal stem cells (hMSCs) are bone marrow-derived stromal cells, which play a role in tumor progression. We have shown earlier that breast cancer cells secrete higher levels of interleukin-6 (IL-6) under hypoxia, leading to the recruitment of hMSCs towards hypoxic tumor cells. We found that (i) MDA-MB-231 cells secrete significantly higher levels of lactate (3-fold more) under hypoxia (1% O{sub 2}) than under 20% O{sub 2} and (ii) lactate recruits hMSCs towards tumor cells by activating signaling pathways to enhance migration. The mRNA and protein expression of functional MCT1 in hMSCs is increased in response to lactate exposure. Thus,more » we hypothesized that hMSCs and stromal carcinoma associated fibroblasts (CAFs) in the tumor microenvironment have the capacity to take up lactate expelled from tumor cells and use it as a source of energy. Our {sup 13}C NMR spectroscopic measurements indicate that {sup 13}C-lactate is converted to {sup 13}C-alpha ketoglutarate in hMSCs and CAFs supporting this hypothesis. To our knowledge this is the first in vitro model system demonstrating that hMSCs and CAFs can utilize lactate produced by tumor cells.« less

Mesenchymal Stem Cells: Rising Concerns over Their Application in Treatment of Type One Diabetes Mellitus

PubMed Central

Hashemian, Seyed Jafar; Kouhnavard, Marjan; Nasli-Esfahani, Ensieh

2015-01-01

Type 1 diabetes mellitus (T1DM) is an autoimmune disorder that leads to beta cell destruction and lowered insulin production. In recent years, stem cell therapies have opened up new horizons to treatment of diabetes mellitus. Among all kinds of stem cells, mesenchymal stem cells (MSCs) have been shown to be an interesting therapeutic option based on their immunomodulatory properties and differentiation potentials confirmed in various experimental and clinical trial studies. In this review, we discuss MSCs differential potentials in differentiation into insulin-producing cells (IPCs) from various sources and also have an overview on currently understood mechanisms through which MSCs exhibit their immunomodulatory effects. Other important issues that are provided in this review, due to their importance in the field of cell therapy, are genetic manipulations (as a new biotechnological method), routes of transplantation, combination of MSCs with other cell types, frequency of transplantation, and special considerations regarding diabetic patients' autologous MSCs transplantation. At the end, utilization of biomaterials either as encapsulation tools or as scaffolds to prevent immune rejection, preparation of tridimensional vascularized microenvironment, and completed or ongoing clinical trials using MSCs are discussed. Despite all unresolved concerns about clinical applications of MSCs, this group of stem cells still remains a promising therapeutic modality for treatment of diabetes. PMID:26576437

Cord-Blood-Derived Mesenchymal Stromal Cells Downmodulate CD4+ T-Cell Activation by Inducing IL-10-Producing Th1 Cells

PubMed Central

Selleri, Silvia; Dieng, Mame Massar; Nicoletti, Simon; Louis, Isabelle; Beausejour, Christian; Le Deist, Françoise

2013-01-01

The mechanisms by which mesenchymal stromal cells (MSCs) induce immunomodulation are still poorly understood. In the current work, we show by a combination of polymerase chain reaction (PCR) array, flow cytometry, and multiplex cytokine data analysis that during the inhibition of an alloantigen-driven CD4+ T-cell response, MSCs induce a fraction of CD4+ T-cells to coexpress interferon-γ (IFNγ) and interleukin-10 (IL-10). This CD4+ IFNγ+ IL-10+ cell population shares properties with recently described T-cells originating from switched Th1 cells that start producing IL-10 and acquire a regulatory function. Here we report that IL-10-producing Th1 cells accumulated with time during T-cell stimulation in the presence of MSCs. Moreover, MSCs caused stimulated T-cells to downregulate the IFNγ receptor (IFNγR) without affecting IL-10 receptor expression. Further, the inhibitory effect of MSCs could be reversed by an anti-IFNγR-blocking antibody, indicating that IFNγ is one of the major players in MSC-induced T-cell suppression. Stimulated (and, to a lesser extent, resting) CD4+ T-cells treated with MSCs were able to inhibit the proliferation of autologous CD4+ T-cells, demonstrating their acquired regulatory properties. Altogether, our results suggest that the generation of IL-10-producing Th1 cells is one of the mechanisms by which MSCs can downmodulate an immune response. PMID:23167734

Human mesenchymal stem cells: a bank perspective on the isolation, characterization and potential of alternative sources for the regeneration of musculoskeletal tissues.

PubMed

Moroni, Lorenzo; Fornasari, Pier Maria

2013-04-01

The continuous discovery of human mesenchymal stem cells (hMSCs) in different tissues is stirring up a tremendous interest as a cell source for regenerative medicine therapies. Historically, hMSCs have been always considered a sub-population of mononuclear cells present in the bone marrow (BM). Although BM-hMSCs are still nowadays considered as the most promising mesenchymal stem cell population to reach the clinics due to their capacity to differentiate into multiple tissues, hMSCs derived from other adult and fetal tissues have also demonstrated to possess similar differentiation capacities. Furthermore, different reports have highlighted a higher recurrence of hMSCs in some of these tissues as compared to BM. This offer a fascinating panorama for cell banking, since the creation of a stem cell factory could be envisioned where hMSCs are stocked and used for ad hoc clinical applications. In this review, we summarize the main findings and state of the art in hMSCs isolation, characterization, and differentiation from alternative tissue sources and we attempt to compare their potency for musculoskeletal regeneration. Copyright © 2012 Wiley Periodicals, Inc.

Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses.

PubMed

Kol, Amir; Wood, Joshua A; Carrade Holt, Danielle D; Gillette, Jessica A; Bohannon-Worsley, Laurie K; Puchalski, Sarah M; Walker, Naomi J; Clark, Kaitlin C; Watson, Johanna L; Borjesson, Dori L

2015-04-15

Intravenous (IV) injection of mesenchymal stem cells (MSCs) is used to treat systemic human diseases and disorders but is not routinely used in equine therapy. In horses, MSCs are isolated primarily from adipose tissue (AT) or bone marrow (BM) and used for treatment of orthopedic injuries through one or more local injections. The objective of this study was to determine the safety and lymphocyte response to multiple allogeneic IV injections of either AT-derived MSCs (AT-MSCs) or BM-derived MSCs (BM-MSCs) to healthy horses. We injected three doses of 25 × 10(6) allogeneic MSCs from either AT or BM (a total of 75 × 10(6) MSCs per horse) into five and five, respectively, healthy horses. Horses were followed up for 35 days after the first MSC infusion. We evaluated host inflammatory and immune response, including total leukocyte numbers, serum cytokine concentration, and splenic lymphocyte subsets. Repeated injection of allogeneic AT-MSCs or BM-MSCs did not elicit any clinical adverse effects. Repeated BM-MSC injection resulted in increased blood CD8(+) T-cell numbers. Multiple BM-MSC injections also increased splenic regulatory T cell numbers compared with AT-MSC-injected horses but not controls. These data demonstrate that multiple IV injections of allogeneic MSCs are well tolerated by healthy horses. No clinical signs or clinico-pathologic measurements of organ toxicity or systemic inflammatory response were recorded. Increased numbers of circulating CD8(+) T cells after multiple IV injections of allogeneic BM-MSCs may indicate a mild allo-antigen-directed cytotoxic response. Safety and efficacy of allogeneic MSC IV infusions in sick horses remain to be determined.

In utero transplantation of human bone marrow-derived multipotent mesenchymal stem cells in mice.

PubMed

Chou, Shiu-Huey; Kuo, Tom K; Liu, Ming; Lee, Oscar K

2006-03-01

Mesenchymal stem cells (MSCs) are multipotent cells that can be isolated from human bone marrow and possess the potential to differentiate into progenies of embryonic mesoderm. However, current evidence is based predominantly on in vitro experiments. We used a murine model of in utero transplantation (IUT) to study the engraftment capabilities of human MSCs. MSCs were obtained from bone marrow by negative immunoselection and limiting dilution, and were characterized by flow cytometry and by in vitro differentiation into osteoblasts, chondrocytes, and adipocytes. MSCs were transplanted into fetal mice at a gestational age of 14 days. Engraftment of human MSCs was determined by flow cytometry, polymerase chain reaction, and fluorescence in situ hybridization (FISH). MSCs engrafted into tissues originating from all three germ layers and persisted for up to 4 months or more after delivery, as evidenced by the expression of the human-specific beta-2 microglobulin gene and by FISH for donor-derived cells. Donor-derived CD45+ cells were detectable in the peripheral blood of recipients, suggesting the participation of MSCs in hematopoiesis at the fetal stage. This model can further serve to evaluate possible applications of MSCs. Copyright 2006 Orthopaedic Research Society.

Cellular Reparative Mechanisms of Mesenchymal Stem Cells for Retinal Diseases.

PubMed

Ding, Suet Lee Shirley; Kumar, Suresh; Mok, Pooi Ling

2017-07-28

The use of multipotent mesenchymal stem cells (MSCs) has been reported as promising for the treatment of numerous degenerative disorders including the eye. In retinal degenerative diseases, MSCs exhibit the potential to regenerate into retinal neurons and retinal pigmented epithelial cells in both in vitro and in vivo studies. Delivery of MSCs was found to improve retinal morphology and function and delay retinal degeneration. In this review, we revisit the therapeutic role of MSCs in the diseased eye. Furthermore, we reveal the possible cellular mechanisms and identify the associated signaling pathways of MSCs in reversing the pathological conditions of various ocular disorders such as age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma. Current stem cell treatment can be dispensed as an independent cell treatment format or with the combination of other approaches. Hence, the improvement of the treatment strategy is largely subjected by our understanding of MSCs mechanism of action.

Cellular Reparative Mechanisms of Mesenchymal Stem Cells for Retinal Diseases

PubMed Central

Ding, Suet Lee Shirley; Kumar, Suresh; Mok, Pooi Ling

2017-01-01

The use of multipotent mesenchymal stem cells (MSCs) has been reported as promising for the treatment of numerous degenerative disorders including the eye. In retinal degenerative diseases, MSCs exhibit the potential to regenerate into retinal neurons and retinal pigmented epithelial cells in both in vitro and in vivo studies. Delivery of MSCs was found to improve retinal morphology and function and delay retinal degeneration. In this review, we revisit the therapeutic role of MSCs in the diseased eye. Furthermore, we reveal the possible cellular mechanisms and identify the associated signaling pathways of MSCs in reversing the pathological conditions of various ocular disorders such as age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma. Current stem cell treatment can be dispensed as an independent cell treatment format or with the combination of other approaches. Hence, the improvement of the treatment strategy is largely subjected by our understanding of MSCs mechanism of action. PMID:28788088

Immunoregulation by Mesenchymal Stem Cells: Biological Aspects and Clinical Applications

PubMed Central

Castro-Manrreza, Marta E.; Montesinos, Juan J.

2015-01-01

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into mesenchymal lineages and that can be isolated from various tissues and easily cultivated in vitro. Currently, MSCs are of considerable interest because of the biological characteristics that confer high potential applicability in the clinical treatment of many diseases. Specifically, because of their high immunoregulatory capacity, MSCs are used as tools in cellular therapies for clinical protocols involving immune system alterations. In this review, we discuss the current knowledge about the capacity of MSCs for the immunoregulation of immunocompetent cells and emphasize the effects of MSCs on T cells, principal effectors of the immune response, and the immunosuppressive effects mediated by the secretion of soluble factors and membrane molecules. We also describe the mechanisms of MSC immunoregulatory modulation and the participation of MSCs as immune response regulators in several autoimmune diseases, and we emphasize the clinical application in graft versus host disease (GVHD). PMID:25961059

Doxorubicin, mesenchymal stem cell toxicity and antitumour activity: implications for clinical use.

PubMed

Baxter-Holland, Mia; Dass, Crispin R

2018-03-01

The use of doxorubicin, an antineoplastic medication used for the treatment of cancers via mechanisms that prevent replication of cells or lead to their death, can result in damage to healthy cells as well as malignant. Among the affected cells are mesenchymal stem cells (MSCs), which are involved in the maintenance and repair of tissues in the body. This review explores the mechanisms of biological effects and damage attributed to doxorubicin on MSCs. The PubMed database was used as a source of literature for this review. Doxorubicin has the potential to lead to significant and irreversible damage to the human bone marrow environment, including MSCs. The primary known mechanism of these changes is through free radical damage and activation of apoptotic pathways. The presence of MSCs in culture or in vivo appears to either suppress or promote tumour growth. Interactions between doxorubicin and MSCs have the potential to increase chemotherapy resistance. Doxorubicin-induced damage to MSCs is of concern clinically. However, MSCs also have been associated with resistance of tumour cells to drugs including doxorubicin. Further studies, particularly in vivo, are needed to provide consistent results of how the doxorubicin-induced changes to MSCs affect treatment and patient health. © 2018 Royal Pharmaceutical Society.

Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

PubMed

Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

2017-08-01

Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture.

PubMed

Yoon, D S; Kim, Y H; Jung, H S; Paik, S; Lee, J W

2011-10-01

This study has aimed to repopulate 'primitive' cells from late-passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Effects of low density culture compared t high density culture on late-passage bone marrow (BM)-derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2. © 2011 Blackwell Publishing Ltd.

Improvement of mesh recolonization in abdominal wall reconstruction with adipose vs. bone marrow mesenchymal stem cells in a rodent model.

PubMed

van Steenberghe, M; Schubert, T; Guiot, Y; Goebbels, R M; Gianello, P

2017-08-01

Reconstruction of muscle defects remains a challenge. Our work assessed the potential of an engineered construct made of a human acellular collagen matrix (HACM) seeded with porcine mesenchymal stem cells (MSCs) to reconstruct abdominal wall muscle defects in a rodent model. This study compared 2 sources of MSCs (bone-marrow, BMSCs, and adipose, ASCs) in vitro and in vivo for parietal defect reconstruction. Cellular viability and growth factor release (VEGF, FGF-Beta, HGF, IGF-1, TGF-Beta) were investigated under normoxic/hypoxic culture conditions. Processed and recellularized HACMs were mechanically assessed. The construct was tested in vivo in full thickness abdominal wall defect treated with HACM alone vs. HACM+ASCs or BMSCs (n=14). Tissue remodeling was studied at day 30 for neo-angiogenesis and muscular reconstruction. A significantly lower secretion of IGF was observed with ASCs vs. BMSCs under hypoxic conditions (-97.6%, p<0.005) whereas significantly higher VEGF/FGF secretions were found with ASCs (+92%, p<0.001 and +72%, p<0.05, respectively). Processing and recellularization did not impair the mechanical properties of the HACM. In vivo, angiogenesis and muscle healing were significantly improved by the HACM+ASCs in comparison to BMSCs (p<0.05) at day 30. A composite graft made of an HACM seeded with ASCs can improve muscle repair by specific growth factor release in hypoxic conditions and by in vivo remodeling (neo-angiogenesis/graft integration) while maintaining mechanical properties. Copyright © 2017 Elsevier Inc. All rights reserved.

Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells.

PubMed

Lange, Claudia; Bruns, Helge; Kluth, Dietrich; Zander, Axel-R; Fiegel, Henning-C

2006-04-21

To investigate the hepatocytic differentiation of mesenchymal stem cells (MSCs) in co-cultures with fetal liver cells (FLC) and the possibility to expand differentiated hepatocytic cells. MSCs were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor (SCF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor 4 (FGF-4) alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction (RT-PCR) for the liver specific markers cytokeratin-18 (CK-18), albumin, and alpha-fetoprotein (AFP) was performed in different cell populations. Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential. The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also provides a beneficial environment for expansion and differentiation of FLC.

MicroRNA-Mediated Down-Regulation of Apoptosis Signal-Regulating Kinase 1 (ASK1) Attenuates the Apoptosis of Human Mesenchymal Stem Cells (MSCs) Transplanted into Infarcted Heart.

PubMed

Lee, Chang Youn; Shin, Sunhye; Lee, Jiyun; Seo, Hyang-Hee; Lim, Kyu Hee; Kim, Hyemin; Choi, Jung-Won; Kim, Sang Woo; Lee, Seahyung; Lim, Soyeon; Hwang, Ki-Chul

2016-10-20

Stem cell therapy using adult stem cells, such as mesenchymal stem cells (MSCs) has produced some promising results in treating the damaged heart. However, the low survival rate of MSCs after transplantation is still one of the crucial factors that limit the therapeutic effect of stem cells. In the damaged heart, oxidative stress due to reactive oxygen species (ROS) production can cause the death of transplanted MSCs. Apoptosis signal-regulating kinase 1 (ASK1) has been implicated in the development of oxidative stress-related pathologic conditions. Thus, we hypothesized that down-regulation of ASK1 in human MSCs (hMSCs) might attenuate the post-transplantation death of MSCs. To test this hypothesis, we screened microRNAs (miRNAs) based on a miRNA-target prediction database and empirical data and investigated the anti-apoptotic effect of selected miRNAs on human adipose-derived stem cells (hASCs) and on rat myocardial infarction (MI) models. Our data indicated that miRNA-301a most significantly suppressed ASK1 expression in hASCs. Apoptosis-related genes were significantly down-regulated in miRNA-301a-enriched hASCs exposed to hypoxic conditions. Taken together, these data show that miRNA-mediated down-regulation of ASK1 protects MSCs during post-transplantation, leading to an increase in the efficacy of MSC-based cell therapy.

Induced pluripotent stem cells have similar immunogenic and more potent immunomodulatory properties compared with bone marrow-derived stromal cells in vitro

PubMed Central

Schnabel, Lauren V; Abratte, Christian M; Schimenti, John C; Felippe, M Julia Bevilaqua; Cassano, Jennifer M; Southard, Teresa L; Cross, Jessica A; Fortier, Lisa A

2015-01-01

Aim To evaluate the in vitro immunogenic and immunomodulatory properties of induced pluripotent stem cells (iPSCs) compared with bone marrow-derived mesenchymal stromal cells (MSCs). Materials & methods Mouse embryonic fibroblasts (MEFs) were isolated from C3HeB/FeJ and C57BL/6J mice, and reprogrammed to generate iPSCs. Mixed leukocyte reactions were performed using MHC-matched and -mismatched responder leukocytes and stimulator leukocytes, iPSCs or MSCs. To assess immunogenic potential, iPSCs and MSCs were used as stimulator cells for responder leukocytes. To assess immunomodulatory properties, iPSCs and MSCs were cultured in the presence of stimulator and responder leukocytes. MEFs were used as a control. Results iPSCs had similar immunogenic properties but more potent immunomodulatory effects than MSCs. Co-culture of MHC-mismatched leukocytes with MHC-matched iPSCs resulted in significantly less responder T-cell proliferation than observed for MHC-mismatched leukocytes alone and at more responder leukocyte concentrations than with MSCs. In addition, MHC-mismatched iPSCs significantly reduced responder T-cell proliferation when co-cultured with MHC-mismatched leukocytes, while MHC-mismatched MSCs did not. Conclusion These results provide important information when considering the use of iPSCs in place of MSCs in both regenerative and transplantation medicine. PMID:24773530

Mitochondrial transfer of mesenchymal stem cells effectively protects corneal epithelial cells from mitochondrial damage.

PubMed

Jiang, Dan; Gao, Fei; Zhang, Yuelin; Wong, David Sai Hung; Li, Qing; Tse, Hung-Fat; Xu, Goufeng; Yu, Zhendong; Lian, Qizhou

2016-11-10

Recent studies have demonstrated that mesenchymal stem cells (MSCs) can donate mitochondria to airway epithelial cells and rescue mitochondrial damage in lung injury. We sought to determine whether MSCs could donate mitochondria and protect against oxidative stress-induced mitochondrial dysfunction in the cornea. Co-culturing of MSCs and corneal epithelial cells (CECs) indicated that the efficiency of mitochondrial transfer from MSCs to CECs was enhanced by Rotenone (Rot)-induced oxidative stress. The efficient mitochondrial transfer was associated with increased formation of tunneling nanotubes (TNTs) between MSCs and CECs, tubular connections that allowed direct intercellular communication. Separation of MSCs and CECs by a transwell culture system revealed no mitochiondrial transfer from MSCs to CECs and mitochondrial function was impaired when CECs were exposed to Rot challenge. CECs with or without mitochondrial transfer from MSCs displayed a distinct survival capacity and mitochondrial oxygen consumption rate. Mechanistically, increased filopodia outgrowth in CECs for TNT formation was associated with oxidative inflammation-activated NFκB/TNFαip2 signaling pathways that could be attenuated by reactive oxygen species scavenger N-acetylcysteine (NAC) treatment. Furthermore, MSCs grown on a decellularized porcine corneal scaffold were transplanted onto an alkali-injured eye in a rabbit model. Enhanced corneal wound healing was evident following healthy MSC scaffold transplantation. And transferred mitochondria was detected in corneal epithelium. In conclusion, mitochondrial transfer from MSCs provides novel protection for the cornea against oxidative stress-induced mitochondrial damage. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

Characterization of Canine Adipose-Derived Mesenchymal Stromal/Stem Cells in Serum-Free Medium.

PubMed

Liu, Zhuoming; Screven, Rudell; Boxer, Lynne; Myers, Michael J; Devireddy, Lax R

2018-06-20

In this article, we report on the development of a defined serum-free medium capable of supporting the culture expansion of mesenchymal stromal/stem cells (MSCs) from canine adipose tissue (canine Ad-MSCs). The potential benefits of serum-free media can only be utilized if cells cultured in serum-free media maintain the same functional characteristics as cells cultured in serum-containing media. Therefore, we analyze the characteristics of canine Ad-MSCs cultured in this serum-free medium or in serum-containing medium through evaluation of growth kinetics, clonogenic capacity, senescence, and differentiation capacity. Both, serum-containing medium and our serum-free medium, supported efficient growth and colony formation of canine Ad-MSCs. In addition, canine Ad-MSCs cultured in both media demonstrated similar viability after freeze/thaw, similar cell surface marker expression, and were capable of trilineage differentiation. While canine Ad-MSCs cultured in both media were generally similar, under the conditions of our study, canine Ad-MSCs cultured in serum-free medium demonstrated a shorter lag phase and higher colony-forming capacity, accelerated population doubling, maintained multipotentiality at higher passage numbers, and underwent senescence at higher passage numbers compared to canine Ad-MSCs cultured in conventional serum-containing medium. These results suggest that canine Ad-MSCs cultured in serum-free medium retain the basic characteristics associated with canine Ad-MSCs cultured in serum-containing medium, although some differences in growth kinetics were observed.

Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.

PubMed

Moscoso, I; Centeno, A; López, E; Rodriguez-Barbosa, J I; Santamarina, I; Filgueira, P; Sánchez, M J; Domínguez-Perles, R; Peñuelas-Rivas, G; Domenech, N

2005-01-01

Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, characterization, and immortalization of porcine MSCs (pMSCs) to study their potential differentiation "in vitro" into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90(pos), CD29(pos), CD44(pos), SLA-I(pos), CD106(pos), CD46(pos) and CD45(neg), CD14(neg), CD31(neg), and CD11b(neg), similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After "in vitro" stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%-50%) of cells with cardiomyocyte characteristics, namely, positive for alpha-Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate "ex vivo" into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model.

Inhibition of IKK/NF-κB Signaling Enhances Differentiation of Mesenchymal Stromal Cells from Human Embryonic Stem Cells.

PubMed

Deng, Peng; Zhou, Chenchen; Alvarez, Ruth; Hong, Christine; Wang, Cun-Yu

2016-04-12

Embryonic stem cell-derived mesenchymal stromal cells (MSCs; also known as mesenchymal stem cells) represent a promising source for bone regenerative medicine. Despite remarkable advances in stem cell biology, the molecular mechanism regulating differentiation of human embryonic stem cells (hESCs) into MSCs remains poorly understood. Here, we report that inhibition of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling enhances differentiation of hESCs into MSCs by expediting the loss of pluripotent markers and increasing the expression of MSC surface markers. In addition, a significantly higher quantity of MSCs was produced from hESCs with IKK/NF-κB suppression. These isolated MSCs displayed evident multipotency with capacity to terminally differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and to form bone in vivo. Collectively, our data provide important insights into the role of NF-κB in mesenchymal lineage specification during hESC differentiation, suggesting that IKK inhibitors could be utilized as an adjuvant in generating MSCs for cell-mediated therapies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Preconditioning mesenchymal stem cells with the mood stabilizers lithium and valproic acid enhances therapeutic efficacy in a mouse model of Huntington's disease.

PubMed

Linares, Gabriel R; Chiu, Chi-Tso; Scheuing, Lisa; Leng, Yan; Liao, Hsiao-Mei; Maric, Dragan; Chuang, De-Maw

2016-07-01

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by CAG repeat expansions in the huntingtin gene. Although, stem cell-based therapy has emerged as a potential treatment for neurodegenerative diseases, limitations remain, including optimizing delivery to the brain and donor cell loss after transplantation. One strategy to boost cell survival and efficacy is to precondition cells before transplantation. Because the neuroprotective actions of the mood stabilizers lithium and valproic acid (VPA) induce multiple pro-survival signaling pathways, we hypothesized that preconditioning bone marrow-derived mesenchymal stem cells (MSCs) with lithium and VPA prior to intranasal delivery to the brain would enhance their therapeutic efficacy, and thereby facilitate functional recovery in N171-82Q HD transgenic mice. MSCs were treated in the presence or absence of combined lithium and VPA, and were then delivered by brain-targeted single intranasal administration to eight-week old HD mice. Histological analysis confirmed the presence of MSCs in the brain. Open-field test revealed that ambulatory distance and mean velocity were significantly improved in HD mice that received preconditioned MSCs, compared to HD vehicle-control and HD mice transplanted with non-preconditioned MSCs. Greater benefits on motor function were observed in HD mice given preconditioned MSCs, while HD mice treated with non-preconditioned MSCs showed no functional benefits. Moreover, preconditioned MSCs reduced striatal neuronal loss and huntingtin aggregates in HD mice. Gene expression profiling of preconditioned MSCs revealed a robust increase in expression of genes involved in trophic effects, antioxidant, anti-apoptosis, cytokine/chemokine receptor, migration, mitochondrial energy metabolism, and stress response signaling pathways. Consistent with this finding, preconditioned MSCs demonstrated increased survival after transplantation into the brain compared to non-preconditioned cells. Our results suggest that preconditioning stem cells with the mood stabilizers lithium and VPA before transplantation may serve as an effective strategy for enhancing the therapeutic efficacy of stem cell-based therapies. Copyright © 2016. Published by Elsevier Inc.

Boron nitride nanotube-enhanced osteogenic differentiation of mesenchymal stem cells.

PubMed

Li, Xia; Wang, Xiupeng; Jiang, Xiangfen; Yamaguchi, Maho; Ito, Atsuo; Bando, Yoshio; Golberg, Dmitri

2016-02-01

The interaction between boron nitride nanotubes (BNNTs) layer and mesenchymal stem cells (MSCs) is evaluated for the first time in this study. BNNTs layer supports the attachment and growth of MSCs and exhibits good biocompatibility with MSCs. BNNTs show high protein adsorption ability, promote the proliferation of MSCs and increase the secretion of total protein by MSCs. Especially, BNNTs enhance the alkaline phosphatase (ALP) activity as an early marker of osteoblasts, ALP/total protein and osteocalcin (OCN) as a late marker of osteogenic differentiation, which shows that BNNTs can enhance osteogenesis of MSCs. The release of trace boron and the stress on cells exerted by BNNTs with a fiber structure may account for the enhanced differentiation of MSCs into osteoblasts. Therefore BNNTs are potentially useful for bone regeneration in orthopedic applications. © 2015 Wiley Periodicals, Inc.

The neurotrophic effects of different human dental mesenchymal stem cells.

PubMed

Kolar, Mallappa K; Itte, Vinay N; Kingham, Paul J; Novikov, Lev N; Wiberg, Mikael; Kelk, Peyman

2017-10-03

The current gold standard treatment for peripheral nerve injury is nerve grafting but this has disadvantages such as donor site morbidity. New techniques focus on replacing these grafts with nerve conduits enhanced with growth factors and/or various cell types such as mesenchymal stem cells (MSCs). Dental-MSCs (D-MSCs) including stem cells obtained from apical papilla (SCAP), dental pulp stem cells (DPSC), and periodontal ligament stem cells (PDLSC) are potential sources of MSCs for nerve repair. Here we present the characterization of various D-MSCs from the same human donors for peripheral nerve regeneration. SCAP, DPSC and PDLSC expressed BDNF, GDNF, NGF, NTF3, ANGPT1 and VEGFA growth factor transcripts. Conditioned media from D-MSCs enhanced neurite outgrowth in an in vitro assay. Application of neutralizing antibodies showed that brain derived neurotrophic factor plays an important mechanistic role by which the D-MSCs stimulate neurite outgrowth. SCAP, DPSC and PDLSC were used to treat a 10 mm nerve gap defect in a rat sciatic nerve injury model. All the stem cell types significantly enhanced axon regeneration after two weeks and showed neuroprotective effects on the dorsal root ganglia neurons. Overall the results suggested SCAP to be the optimal dental stem cell type for peripheral nerve repair.

Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

PubMed

Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

2016-10-01

Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.

Mesenchymal stem cells: immune evasive, not immune privileged

PubMed Central

Ankrum, James A.; Ong, Joon Faii; Karp, Jeffrey M.

2014-01-01

The diverse immunomodulatory properties of mesenchymal stem/stromal cells (MSCs) may be exploited for treatment of a multitude of inflammatory conditions. MSCs have long been reported to be hypoimmunogenic or ‘immune privileged’; this property is thought to enable MSC transplantation across major histocompatibility barriers and the creation of off-the-shelf therapies consisting of MSCs grown in culture. However, recent studies describing generation of antibodies against and immune rejection of allogeneic donor MSCs suggest that MSCs may not actually be immune privileged. Nevertheless, whether rejection of donor MSCs influences the efficacy of allogeneic MSC therapies is not known, and no definitive clinical advantage of autologous MSCs over allogeneic MSCs has been demonstrated to date. Although MSCs may exert therapeutic function through a brief ‘hit and run’ mechanism, protecting MSCs from immune detection and prolonging their persistence in vivo may improve clinical outcomes and prevent patient sensitization toward donor antigens. PMID:24561556

Journey of Mesenchymal Stem Cells for Homing: Strategies to Enhance Efficacy and Safety of Stem Cell Therapy

PubMed Central

Kang, Sung Keun; Shin, Il Seob; Ko, Myung Soon; Jo, Jung Youn; Ra, Jeong Chan

2012-01-01

Human mesenchymal stem cells (MSCs) communicate with other cells in the human body and appear to “home” to areas of injury in response to signals of cellular damage, known as homing signals. This review of the state of current research on homing of MSCs suggests that favorable cellular conditions and the in vivo environment facilitate and are required for the migration of MSCs to the site of insult or injury in vivo. We review the current understanding of MSC migration and discuss strategies for enhancing both the environmental and cellular conditions that give rise to effective homing of MSCs. This may allow MSCs to quickly find and migrate to injured tissues, where they may best exert clinical benefits resulting from improved homing and the presence of increased numbers of MSCs. PMID:22754575

Wharton's Jelly Mesenchymal Stem Cells Protect the Immature Brain in Rats and Modulate Cell Fate.

PubMed

Mueller, Martin; Oppliger, Byron; Joerger-Messerli, Marianne; Reinhart, Ursula; Barnea, Eytan; Paidas, Michael; Kramer, Boris W; Surbek, Daniel V; Schoeberlein, Andreina

2017-02-15

The development of a mammalian brain is a complex and long-lasting process. Not surprisingly, preterm birth is the leading cause of death in newborns and children. Advances in perinatal care reduced mortality, but morbidity still represents a major burden. New therapeutic approaches are thus desperately needed. Given that mesenchymal stem/stromal cells (MSCs) emerged as a promising candidate for cell therapy, we transplanted MSCs derived from the Wharton's Jelly (WJ-MSCs) to reduce the burden of immature brain injury in a murine animal model. WJ-MSCs transplantation resulted in protective activity characterized by reduced myelin loss and astroglial activation. WJ-MSCs improved locomotor behavior as well. To address the underlying mechanisms, we tested the key regulators of responses to DNA-damaging agents, such as cyclic AMP-dependent protein kinase/calcium-dependent protein kinase (PKA/PKC), cyclin-dependent kinase (CDK), ataxia-telangiectasia-mutated/ATM- and Rad3-related (ATM/ATR) substrates, protein kinase B (Akt), and 14-3-3 binding protein partners. We characterized WJ-MSCs using a specific profiler polymerase chain reaction array. We provide evidence that WJ-MSCs target pivotal regulators of the cell fate such as CDK/14-3-3/Akt signaling. We identified leukemia inhibitory factor as a potential candidate of WJ-MSCs' induced modifications as well. We hypothesize that WJ-MSCs may exert adaptive responses depending on the type of injury they are facing, making them prominent candidates for cell therapy in perinatal injuries.

Secretion of immunoregulatory cytokines by mesenchymal stem cells

PubMed Central

Kyurkchiev, Dobroslav; Bochev, Ivan; Ivanova-Todorova, Ekaterina; Mourdjeva, Milena; Oreshkova, Tsvetelina; Belemezova, Kalina; Kyurkchiev, Stanimir

2014-01-01

According to the minimal criteria of the International Society of Cellular Therapy, mesenchymal stem cells (MSCs) are a population of undifferentiated cells defined by their ability to adhere to plastic surfaces when cultured under standard conditions, express a certain panel of phenotypic markers and can differentiate into osteogenic, chondrogenic and adipogenic lineages when cultured in specific inducing media. In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contacts, secretion of cytokines and/or by a combination of both mechanisms. There are no convincing data about a principal difference in the profile of cytokines secreted by MSCs isolated from different tissue sources, although some papers report some quantitative but not qualitative differences in cytokine secretion. The present review focuses on the basic cytokines secreted by MSCs as described in the literature by which the MSCs exert immunodulatory effects. It should be pointed out that MSCs themselves are objects of cytokine regulation. Hypothetical mechanisms by which the MSCs exert their immunoregulatory effects are also discussed in this review. These mechanisms may either influence the target immune cells directly or indirectly by affecting the activities of predominantly dendritic cells. Chemokines are also discussed as participants in this process by recruiting cells of the immune systems and thus making them targets of immunosuppression. This review aims to present and discuss the published data and the personal experience of the authors regarding cytokines secreted by MSCs and their effects on the cells of the immune system. PMID:25426252

Activation of β-adrenergic receptors is required for elevated α1A-adrenoreceptors expression and signaling in mesenchymal stromal cells

PubMed Central

Tyurin-Kuzmin, Pyotr A.; Fadeeva, Julia I.; Kanareikina, Margarita A.; Kalinina, Natalia I.; Sysoeva, Veronika Yu.; Dyikanov, Daniyar T.; Stambolsky, Dmitriy V.; Tkachuk, Vsevolod A.

2016-01-01

Sympathetic neurons are important components of mesenchymal stem cells (MSCs) niche and noradrenaline regulates biological activities of these cells. Here we examined the mechanisms of regulation of MSCs responsiveness to noradrenaline. Using flow cytometry, we demonstrated that α1A adrenergic receptors isoform was the most abundant in adipose tissue-derived MSCs. Using calcium imaging in single cells, we demonstrated that only 6.9 ± 0.8% of MSCs responded to noradrenaline by intracellular calcium release. Noradrenaline increases MSCs sensitivity to catecholamines in a transitory mode. Within 6 hrs after incubation with noradrenaline the proportion of cells responding by Ca2+ release to the fresh noradrenaline addition has doubled but declined to the baseline after 24 hrs. Increased sensitivity was due to the elevated quantities of α1A-adrenergic receptors on MSCs. Such elevation depended on the stimulation of β-adrenergic receptors and adenylate cyclase activation. The data for the first time clarify mechanisms of regulation of MSCs sensitivity to noradrenaline. PMID:27596381

A New Method for Preparing Mesenchymal Stem Cells and Labeling with Ferumoxytol for Cell Tracking by MRI.

PubMed

Liu, Li; Tseng, Lanya; Ye, Qing; Wu, Yijen L; Bain, Daniel J; Ho, Chien

2016-05-18

Mesenchymal stem cells (MSCs) are among the major stem cells used for cell therapy and regenerative medicine. In-vivo cell-tracking by magnetic resonance imaging (MRI) is crucial for regenerative medicine, allowing verification that the transplanted cells reach the targeted sites. Cellular MRI combined with superparamagnetic iron-oxide (SPIO) contrast agents is an effective cell-tracking method. Here, we are reporting a new "bio-mimicry" method by making use of the "in-vivo environment" of MSCs to prepare native MSCs, so that (i) the phagocytic activity of cultured MSCs can be recovered and expanded MSCs can be ex-vivo labeled with Ferumoxytol, which is currently the only FDA approved SPIO nanoparticles for human use. Using our new method, 7-day cultured MSCs regain the capability to take up Ferumoxytol and exhibit an intracellular iron concentration of 2.50 ± 0.50 pg/MSC, comparable to that obtained by using Ferumoxytol-heparin-protamine nanocomplex; and (ii) cells can be re-sized to more native size, reducing from 32.0 ± 7.2 μm to 19.5 ± 5.2 μm. Our method can be very useful for expanding MSCs and labeling with Ferumoxytol, without the need for transfection agents and/or electroporation, allowing cell-tracking by MRI in both pre-clinical and clinical studies.

A New Method for Preparing Mesenchymal Stem Cells and Labeling with Ferumoxytol for Cell Tracking by MRI

PubMed Central

Liu, Li; Tseng, Lanya; Ye, Qing; Wu, Yijen L.; Bain, Daniel J.; Ho, Chien

2016-01-01

Mesenchymal stem cells (MSCs) are among the major stem cells used for cell therapy and regenerative medicine. In-vivo cell-tracking by magnetic resonance imaging (MRI) is crucial for regenerative medicine, allowing verification that the transplanted cells reach the targeted sites. Cellular MRI combined with superparamagnetic iron-oxide (SPIO) contrast agents is an effective cell-tracking method. Here, we are reporting a new “bio-mimicry” method by making use of the “in-vivo environment” of MSCs to prepare native MSCs, so that (i) the phagocytic activity of cultured MSCs can be recovered and expanded MSCs can be ex-vivo labeled with Ferumoxytol, which is currently the only FDA approved SPIO nanoparticles for human use. Using our new method, 7-day cultured MSCs regain the capability to take up Ferumoxytol and exhibit an intracellular iron concentration of 2.50 ± 0.50 pg/MSC, comparable to that obtained by using Ferumoxytol-heparin-protamine nanocomplex; and (ii) cells can be re-sized to more native size, reducing from 32.0 ± 7.2 μm to 19.5 ± 5.2 μm. Our method can be very useful for expanding MSCs and labeling with Ferumoxytol, without the need for transfection agents and/or electroporation, allowing cell-tracking by MRI in both pre-clinical and clinical studies. PMID:27188664

Differentiation of Rat bone marrow Mesenchymal stem cells into Adipocytes and Cardiomyocytes after treatment with platelet lysate.

PubMed

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Barzegar, Kazem

2016-01-01

Mesenchymal stem cells (MSCs) are multipotential cells and their therapeutic potency is under intense investigation. Studying the effect of different induction factors on MSCs could increase our knowledge about the differentiation potency of these cells. One of the most important sources of these factors in mammalian body is platelet. Platelet lysate (PL) contains many growth factors and therefore, it can be used as a differentiation inducer. In the present study, the effect of PL on differentiation of rat bone marrow MSCs into cardiomyocytes was studied. To study the differentiation-inducing effect of PL, MSCs were treated with 2.5, 5 and 10% PL. Early results of this study showed that PL in high concentrations (10%) induces adipogenic differentiation of MSCs. Therefore, to evaluate differentiation to cardiomyocytes, MSCs were cultured in media containing lower levels of PL (2.5% and 5%) and then cardiomyogenic differentiation was induced by treatment with 5-azacytidine. Differentiation of MSCs was evaluated using direct observation of beating cells, immunostaining and real-time PCR techniques. The results of qPCR showed that treatment with PL alone increased the expression of cardiac alpha actinin (CAA) being predictable by earlier observation of beating cells in PL-treated groups. The results of staining assays against cardiac alpha actinin also showed that there were stained cells in PL-treated groups. The results of the present study showed that PL is a powerful induction factor for differentiation of MSCs into different cell lines such as cardiomyocytes and adipocytes.

Effect of Secreted Molecules of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells on Acute Hepatic Failure Model.

PubMed

Lotfinia, Majid; Kadivar, Mehdi; Piryaei, Abbas; Pournasr, Behshad; Sardari, Soroush; Sodeifi, Niloofar; Sayahpour, Forugh-Azam; Baharvand, Hossein

2016-12-15

Adult tissue-derived mesenchymal stem cells (MSCs) show tremendous promise for a wide array of therapeutic applications predominantly through paracrine activity. Recent reports showed that human embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative cellular therapy due to manufacturing large quantities of MSCs from a single donor. However, no study has been reported to uncover the secretome of human ESC-MSCs as treatment of an acute liver failure (ALF) mouse model. We demonstrated that human ESC-MSCs showed similar morphology and cell surface markers compared with bone marrow-derived MSCs. ESC-MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic differentiation potential. Treatment with ESC-MSC-conditioned medium (CM) led to statistically significant enhancement of primary hepatocyte viability and increased immunomodulatory interleukin-10 secretion from lipopolysaccharide-induced human blood mononuclear cells. Analysis of the MSCs secretome by a protein array screen showed an association between higher frequencies of secretory proteins such as vascular endothelial growth factor (VEGF) and regulation of cell proliferation, cell migration, the development process, immune system process, and apoptosis. In this thioacetamide-induced mouse model of acute liver injury, we observed that systemic infusion of VEGF led to significant survival. These data have provided the first experimental evidence of the therapeutic potential of human ESC-MSC-derived molecules. These molecules show trophic support to hepatocytes, which potentially creates new avenues for the treatment of ALF, as an inflammatory condition.

Mesenchymal stem cell-derived microparticles: a promising therapeutic strategy.

PubMed

Tan, Xi; Gong, Yong-Zhen; Wu, Ping; Liao, Duan-Fang; Zheng, Xi-Long

2014-08-18

Mesenchymal stem cells (MSCs) are multipotent stem cells that give rise to various cell types of the mesodermal germ layer. Because of their unique ability to home in on injured and cancerous tissues, MSCs are of great potential in regenerative medicine. MSCs also contribute to reparative processes in different pathological conditions, including cardiovascular diseases and cancer. However, many studies have shown that only a small proportion of transplanted MSCs can actually survive and be incorporated into host tissues. The effects of MSCs cannot be fully explained by their number. Recent discoveries suggest that microparticles (MPs) derived from MSCs may be important for the physiological functions of their parent. Though the physiological role of MSC-MPs is currently not well understood, inspiring results indicate that, in tissue repair and anti-cancer therapy, MSC-MPs have similar pro-regenerative and protective properties as their cellular counterparts. Thus, MSC-MPs represent a promising approach that may overcome the obstacles and risks associated with the use of native or engineered MSCs.

Mesenchymal Stem Cell-Derived Microparticles: A Promising Therapeutic Strategy

PubMed Central

Tan, Xi; Gong, Yong-Zhen; Wu, Ping; Liao, Duan-Fang; Zheng, Xi-Long

2014-01-01

Mesenchymal stem cells (MSCs) are multipotent stem cells that give rise to various cell types of the mesodermal germ layer. Because of their unique ability to home in on injured and cancerous tissues, MSCs are of great potential in regenerative medicine. MSCs also contribute to reparative processes in different pathological conditions, including cardiovascular diseases and cancer. However, many studies have shown that only a small proportion of transplanted MSCs can actually survive and be incorporated into host tissues. The effects of MSCs cannot be fully explained by their number. Recent discoveries suggest that microparticles (MPs) derived from MSCs may be important for the physiological functions of their parent. Though the physiological role of MSC-MPs is currently not well understood, inspiring results indicate that, in tissue repair and anti-cancer therapy, MSC-MPs have similar pro-regenerative and protective properties as their cellular counterparts. Thus, MSC-MPs represent a promising approach that may overcome the obstacles and risks associated with the use of native or engineered MSCs. PMID:25196436

L-Glutamine in vitro Modulates some Immunomodulatory Properties of Bone Marrow Mesenchymal Stem Cells.

PubMed

Dos Santos, Guilherme Galvão; Hastreiter, Araceli Aparecida; Sartori, Talita; Borelli, Primavera; Fock, Ricardo Ambrósio

2017-08-01

Glutamine (GLUT) is a nonessential amino acid that can become conditionally essential under stress conditions, being able to act in the modulation of the immune responses. Mesenchymal stem cells (MSCs) are known to their capability in the modulation of immune responses through cell-cell contact and by the secretion of soluble factors. Considering that GLUT is an immunonutrient and little is known about the influence of GLUT on the capability of MSCs to modulate immune cells, this work aims to investigate how variations in GLUT concentrations in vitro could affect some immunomodulatory properties of MSCs. In order to evaluate the effects of GLUT on MSCs immunomodulatory properties, cell proliferation rates, the expression of NFκB and STAT-3, and the production of IL-1β, IL-6, IL-10, TGF-β and TNF-α by MSCs were assessed. Based on our findings, GLUT at high doses (10 mM) augmented the proliferation of MSCs and modulated immune responses by decreasing levels of pro-inflammatory cytokines, such as IL-1β and IL-6, and by increasing levels of anti-inflammatory cytokines IL-10 and TGF-β. In addition, MSCs cultured in higher GLUT concentrations (10 mM) expressed lower levels of NF-κB and higher levels of STAT-3. Furthermore, conditioned media from MSCs cultured at higher GLUT concentrations (10 mM) reduced lymphocyte and macrophage proliferation, increased IL-10 production by both cells types, and decreased IFN-γ production by lymphocytes. Overall, this study showed that 10 mM of GLUT is able to modify immunomodulatory properties of MSCs.

Effect of Mesenchymal Stromal Cells on T Cells in a Septic Context: Immunosuppression or Immunostimulation?

PubMed

Le Burel, Sébastien; Thepenier, Cédric; Boutin, Laetitia; Lataillade, Jean-Jacques; Peltzer, Juliette

2017-10-15

Sepsis is a complex process, including a first wave of damage partially due to the body's response to pathogens, followed by a phase of immune cell dysfunction. The efficacy of a pharmacological approach facing a rapidly evolving system implies a perfect timing of administration-this difficulty could explain the recent failure of clinical trials. Mesenchymal stromal cells (MSCs) are usually defined as immunosuppressive and their beneficial effects in preclinical models of acute sepsis have been shown to rely partly on such ability. If nonregulated, this phenotype could be harmful in the immunosuppressed context arising hours after sepsis onset. However, MSCs being environment sensitive, we hypothesized that they could reverse their immunosuppressive properties when confronted with suffering immune cells. Our objective was to evaluate the effect of human MSCs on activated human lymphocytes in an in vitro endotoxemia model. Peripheral blood mononuclear cells (PBMCs) underwent a 24-h lipopolysaccharide (LPS) intoxication and were stimulated with phytohemagglutinin (PHA) in contact with MSCs. MSCs induced a differential effect on lymphocytes depending on PBMC intoxication with LPS. Unintoxicated lymphocytes were highly proliferative with PHA and were inhibited by MSCs, whereas LPS-intoxicated lymphocytes showed a low proliferation rate, but were supported by MSCs, even when monocytes were depleted. These data, highlighting MSC plasticity in their immunomodulatory activity, pave the way for further studies investigating the mechanisms of mutual interactions between MSCs and immune cells in sepsis. Thus, MSCs might be able to fight against both early sepsis-induced hyperinflammatory response and later time points of immune dysfunction.

Isolation and Characterization of Human Mesenchymal Stem Cells From Facet Joints and Interspinous Ligaments.

PubMed

Kristjánsson, Baldur; Limthongkul, Worawat; Yingsakmongkol, Wicharn; Thantiworasit, Pattarawat; Jirathanathornnukul, Napaphat; Honsawek, Sittisak

2016-01-01

A descriptive in vitro study on isolation and differentiation of human mesenchymal stem cells (MSCs) derived from the facet joints and interspinous ligaments. To isolate cells from the facet joints and interspinous ligaments and investigate their surface marker profile and differentiation potentials. Lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament are progressive conditions characterized by the hypertrophy and ossification of ligaments and joints within the spinal canal. MSCs are believed to play a role in the advancement of these diseases and the existence of MSCs has been demonstrated within the ligamentum flavum and posterior longitudinal ligament. The aim of this study was to investigate whether these cells could also be found within facet joints and interspinous ligaments. Samples were harvested from 10 patients undergoing spinal surgery. The MSCs from facet joints and interspinous ligaments were isolated using direct tissue explant technique. Cell surface antigen profilings were performed via flow cytometry. Their lineage differentiation potentials were analyzed. The facet joints and interspinous ligaments-derived MSCs have the tri-lineage potential to be differentiated into osteogenic, adipogenic, and chondrogenic cells under appropriate inductions. Flow cytometry analysis revealed both cell lines expressed MSCs markers. Both facet joints and interspinous ligaments-derived MSCs expressed marker genes for osteoblasts, adipocytes, and chondrocytes. The facet joints and interspinous ligaments may provide alternative sources of MSCs for tissue engineering applications. The facet joints and interspinous ligaments-derived MSCs are part of the microenvironment of the human ligaments of the spinal column and might play a crucial role in the development and progression of degenerative spine conditions.

hMSCs suppress neutrophil-dominant airway inflammation in a murine model of asthma

PubMed Central

Hong, Gyong Hwa; Kwon, Hyouk-Soo; Lee, Kyoung Young; Ha, Eun Hee; Moon, Keun-Ai; Kim, Seong Who; Oh, Wonil; Kim, Tae-Bum; Moon, Hee-Bom; Cho, You Sook

2017-01-01

Although chronic eosinophilic inflammation is a common feature in patients with asthma, some patients have neutrophil-dominant inflammation, which is known to be associated with severe asthma.Human mesenchymal stem cells (hMSCs) have shown promise in treating various refractory immunological diseases. Thus, hMSCs may represent an alternative therapeutic option for asthma patients with neutrophil-dominant inflammation, in whom current treatments are ineffective. BALB/c mice exposed to ovalbumin and polyinosinic:polycytidylic acid (Poly I:C) to induce neutrophilic airway inflammation were systemically treated with hMSCs to examine whether the hMSCs can modulate neutrophilic airway inflammation. In addition, cytokine production was evaluated in co-cultures of hMSCs with either anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asthmatic patients or cells of the human bronchial epithelial cell line BEAS-2B to assess the response to hMSC treatment. The total number of immune cells in bronchoalveolar lavage fluid (BALF) showed a dramatic decrease in hMSC-treated asthmatic mice, and, in particular, neutrophilic infiltration was significantly attenuated. This phenomenon was accompanied by reduced CXCL15 production in the BALF. BEAS-2B cells co-cultured with hMSCs showed reduced secretion of IL-8. Moreover, decreased secretion of IL-4, IL-13 and IFN-γ was observed when human PBMCs were cultured with hMSCs, whereas IL-10 production was greatly enhanced. Our data imply that hMSCs may have a role in reducing neutrophilic airway inflammation by downregulating neutrophil chemokine production and modulating T-cell responses. PMID:28127050

A biomaterial-assisted mesenchymal stromal cell therapy alleviates colonic radiation-induced damage.

PubMed

Moussa, Lara; Pattappa, Girish; Doix, Bastien; Benselama, Sarra-Louiza; Demarquay, Christelle; Benderitter, Marc; Sémont, Alexandra; Tamarat, Radia; Guicheux, Jérôme; Weiss, Pierre; Réthoré, Gildas; Mathieu, Noëlle

2017-01-01

Healthy tissues surrounding abdomino-pelvic tumours can be impaired by radiotherapy, leading to chronic gastrointestinal complications with substantial mortality. Adipose-derived Mesenchymal Stromal Cells (Ad-MSCs) represent a promising strategy to reduce intestinal lesions. However, systemic administration of Ad-MSCs results in low cell engraftment within the injured tissue. Biomaterials, able to encapsulate and withstand Ad-MSCs, can overcome these limitations. A silanized hydroxypropylmethyl cellulose (Si-HPMC) hydrogel has been designed and characterized for injectable cell delivery using the operative catheter of a colonoscope. We demonstrated that hydrogel loaded-Ad-MSCs were viable, able to secrete trophic factors and responsive to the inflammatory environment. In a rat model of radiation-induced severe colonic damage, Ad-MSC + Si-HPMC improve colonic epithelial structure and hyperpermeability compared with Ad-MSCs injected intravenously or locally. This therapeutic benefit is associated with greater engraftment of Si-HPMC-embedded Ad-MSCs in the irradiated colonic mucosa. Moreover, macrophage infiltration near the injection site was less pronounced when Ad-MSCs were embedded in the hydrogel. Si-HPMC induces modulation of chemoattractant secretion by Ad-MSCs that could contribute to the decrease in macrophage infiltrate. Si-HPMC is suitable for cell delivery by colonoscopy and induces protection of Ad-MSCs in the tissue potentiating their therapeutic effect and could be proposed to patients suffering from colon diseases. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into Ewing sarcoma tumors.

PubMed

Duan, Xiaoping; Guan, Hui; Cao, Ying; Kleinerman, Eugenie S

2009-01-01

This study evaluated the therapeutic efficacy of interleukin 12 (IL-12) gene therapy in Ewing sarcoma and whether murine mesenchymal stem cells (MSCs) could serve as vehicles for IL-12 gene delivery. MSCs were isolated from murine bone marrow cells. Cells were phenotyped using flow cytometry. Cultured MSCs differentiated into osteocytes and adipocytes using the appropriate media. Freshly isolated MSCs were transfected with adenoviral vectors containing either the beta-galactosidase (Ad:beta-gal) or the IL-12 (Ad:IL-12) gene. Expression of IL-12 was confirmed using reverse transcription polymerase chain reaction. Mice with TC71 Ewing sarcoma tumors were then treated intravenously with MSCs transfected with Ad:beta-gal or Ad:IL-12. Tumors were measured and analyzed by immunohistochemical analysis for expression of IL-12 protein. Expression of both p35 and p40 IL-12 subunits was demonstrated in MSCs transfected in vitro with Ad:IL-12. IL-12 expression was seen in tumors from mice treated with MSCs transfected with Ad:IL-12. Tumor growth was also significantly inhibited compared with that in mice treated with MSCs transfected with Ad:beta-gal. MSCs can be transfected with the IL-12 gene. These transfected cells localize to tumors after intravenous injection and induce local IL-12 protein production and the regression of established tumors. Copyright (c) 2008 American Cancer Society.

RIA fractions contain mesenchymal stroma cells with high osteogenic potency.

PubMed

Kuehlfluck, Pamela; Moghaddam, Arash; Helbig, Lars; Child, Christopher; Wildemann, Britt; Schmidmaier, Gerhard

2015-12-01

The gold standard for treatment of non-union is the transplantation of autologous bone from iliac crest. As an alternative, material can be harvested by femoral reaming with the Reamer-Irrigator-Aspirator(®) (RIA)-System. This material might be a source for human mesenchymal stroma cells (MSCs) with osteogenic potency. The aim of this study was the characterisation of cells harvested with the RIA system and the comparison of their properties with cells isolated from bone marrow ("BM") and fat tissue ("adipose"). The RIA material was separated into the liquid aspiration fraction ("liquid") and the solid RIA fraction. From the solid RIA fraction the cells were cultured either directly ("native") or after collagenase digestion and filtration ("filtrate"). Stem cell characteristics were analysed and the osteogenic potential was investigated in vitro and in vivo. Fat tissue and bone marrow were harvested from nine patients (three women, six males, with a mean of 48.1 years) with atrophic non-union RIA material. The cells were isolated and characterised by flow cytometry, three lineage differentiation capacities and colony-forming unit fibroblast assay. Gene expression profiles were performed and osteogenic differentiation in vivo was analysed. All three RIA fractions contained mesenchymal stromal cells (MSCs) as demonstrated by CFU-F assay, three linage differentiation and surface marker analysis. The RIA-MSCs exhibited a significantly higher osteogenic potential in vitro compared to adipose-MSCs, whereas no difference was seen compared to BM-MSCs. Quantitative RT-PCR analysis revealed an expression of osteogenic markers in all isolated cells. The implantation of MSCs with β-TCP scaffolds into the mice muscle showed significantly higher bone formation for the filtrate RIA-MSC, native RIA-MSC and BM-MSC groups compared to the adipose-MSC group. The filtrate RIA-MSCs formed twice as much new bone in vivo compared to BM-MSCs. The present study showed high potency of cells isolated by reaming. Even in the irrigation fluid, which is normally discarded, cells with the characteristics of stromal stem cells were isolated. In comparison to adipose-MSCs and BM-MSCs, the RIA-MSCs showed a similar or even better osteogenic potential in vitro and in vivo and this supports their usability in orthopaedic surgery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Adipogenesis of the mesenchymal stromal cells and bone oedema in rheumatoid arthritis.

PubMed

Okada, A; Yamasaki, S; Koga, T; Kawashiri, S Y; Tamai, M; Origuchi, T; Nakamura, H; Eguchi, K; Kawakami, A

2012-01-01

Bone oedema is a pathological change in rheumatoid arthritis (RA) that is detectable by magnetic resonance imaging (MRI). Recent histological analyses revealed that a prominent feature of bone oedema is the replacement of adipose tissue with inflammatory cells. Here, we demonstrate the possible roles of mesenchymal stromal cells (MSCs) in bone oedema formation and the pathogenic potential of the cells in RA. Adipogenesis of bone marrow-derived human MSCs was induced by a standard adipogenic induction medium in the presence or absence of cytokines. The cytokine productions from MSCs were screened by an antibody array system and confirmed by ELISA. The migration assay was performed to determine the locomotive abilities of undifferentiated MSCs or MSCs after adipogenesis. The expression of α smooth muscle actin (SMA) and F-actin was examined by immunostaining and phalloidin staining, respectively. TNF-α, interleukin (IL)-1β, IL-6, and TGF-β clearly inhibited the adipogenesis of MSCs. Production of IL-6 was markedly reduced, and IL-8 secretion was augmented in MSCs after adipogenesis. The mobility of MSCs after adipogenesis was clearly reduced in migration assays compared to that of undifferentiated MSCs. Consistent with these findings, SMA and F-actin expressions were clearly suppressed in MSCs committed to adipogenesis. Our data suggest that the inflammatory milieu promotes bone oedema by blocking adipogenesis of MSCs. In bone oedema, the enhanced IL-6 production and the increased mobility of MSCs may contribute to the progression of RA. Therefore, bone oedema may be an important target lesion in the treatment of RA.

Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun

2014-08-08

Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuousmore » TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.« less

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

PubMed

Fazzina, R; Iudicone, P; Fioravanti, D; Bonanno, G; Totta, P; Zizzari, I G; Pierelli, L

2016-08-25

Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking and for cell-based therapy settings.

Hepatocyte Growth Factor Gene-Modified Mesenchymal Stem Cells Augment Sinonasal Wound Healing

PubMed Central

Li, Jing; Li, Yong; Yang, Chen; Lin, Hai; Duan, Hong-Gang

2015-01-01

This study was designed to investigate the effects of hepatocyte growth factor (HGF) transgenic mesenchymal stem cells (HGF-MSCs) on wound healing in the sinonasal mucosa and nasal epithelial cells (NECs). We also sought to determine whether HGF-MSCs and MSCs can migrate into the injured mucosa and differentiate into ciliated cells. Human HGF-overexpressing umbilical cord MSCs (hHGF-UCMSCs) were established, and upregulation of hHGF expression was confirmed by real-time PCR (RT-PCR) and enzyme-linked immunosorbant assay (ELISA). To investigate the paracrine effect of human MSCs (hMSCs) on nasal epithelial repair, hMSC- and HGF-MSC-conditioned media (CM) were used in NEC proliferation assays and in an in vitro scratch-wound repair model. The in vivo sinonasal wound-healing model was established, and all enrolled rabbits were randomly assigned to four groups: the GFP-MSC group, the HGF-MSC group, the Ad-HGF group, and the surgery control group. The average decreased diameter was recorded, and the medial wall of the maxillary sinus was removed for histological analysis and scanning electron microscopy. Collagen deposition in the wound tissue was detected via Masson trichrome (M&T) staining. The distribution of MSCs and HGF-MSCs was observed by immunofluorescence. MSCs improved nasal wound healing both in vivo and in vitro. HGF overexpression in MSCs augmented the curative effects. Reduced collagen deposition and transforming growth factor beta1 (TGF-β1) expression were detected in the HGF-MSC group compared with the MSC-, Ad-HGF-, and phosphate-buffered saline-treated groups based on M&T staining and ELISA. The enhanced therapeutic effects of HGF-MSCs were accompanied by decreased level of the fibrogenic cytokine TGF-β1. In addition, both HGF-MSCs and MSCs can migrate to the injured mucosa and epithelial layer. PMID:25835956

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression ofmore » surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.« less

Hepatocyte Growth Factor Gene-Modified Mesenchymal Stem Cells Augment Sinonasal Wound Healing.

PubMed

Li, Jing; Zheng, Chun-Quan; Li, Yong; Yang, Chen; Lin, Hai; Duan, Hong-Gang

2015-08-01

This study was designed to investigate the effects of hepatocyte growth factor (HGF) transgenic mesenchymal stem cells (HGF-MSCs) on wound healing in the sinonasal mucosa and nasal epithelial cells (NECs). We also sought to determine whether HGF-MSCs and MSCs can migrate into the injured mucosa and differentiate into ciliated cells. Human HGF-overexpressing umbilical cord MSCs (hHGF-UCMSCs) were established, and upregulation of hHGF expression was confirmed by real-time PCR (RT-PCR) and enzyme-linked immunosorbant assay (ELISA). To investigate the paracrine effect of human MSCs (hMSCs) on nasal epithelial repair, hMSC- and HGF-MSC-conditioned media (CM) were used in NEC proliferation assays and in an in vitro scratch-wound repair model. The in vivo sinonasal wound-healing model was established, and all enrolled rabbits were randomly assigned to four groups: the GFP-MSC group, the HGF-MSC group, the Ad-HGF group, and the surgery control group. The average decreased diameter was recorded, and the medial wall of the maxillary sinus was removed for histological analysis and scanning electron microscopy. Collagen deposition in the wound tissue was detected via Masson trichrome (M&T) staining. The distribution of MSCs and HGF-MSCs was observed by immunofluorescence. MSCs improved nasal wound healing both in vivo and in vitro. HGF overexpression in MSCs augmented the curative effects. Reduced collagen deposition and transforming growth factor beta1 (TGF-β1) expression were detected in the HGF-MSC group compared with the MSC-, Ad-HGF-, and phosphate-buffered saline-treated groups based on M&T staining and ELISA. The enhanced therapeutic effects of HGF-MSCs were accompanied by decreased level of the fibrogenic cytokine TGF-β1. In addition, both HGF-MSCs and MSCs can migrate to the injured mucosa and epithelial layer.

Mesenchymal stem cells and myoblast differentiation under HGF and IGF-1 stimulation for 3D skeletal muscle tissue engineering.

PubMed

Witt, R; Weigand, A; Boos, A M; Cai, A; Dippold, D; Boccaccini, A R; Schubert, D W; Hardt, M; Lange, C; Arkudas, A; Horch, R E; Beier, J P

2017-02-28

Volumetric muscle loss caused by trauma or after tumour surgery exceeds the natural regeneration capacity of skeletal muscle. Hence, the future goal of tissue engineering (TE) is the replacement and repair of lost muscle tissue by newly generating skeletal muscle combining different cell sources, such as myoblasts and mesenchymal stem cells (MSCs), within a three-dimensional matrix. Latest research showed that seeding skeletal muscle cells on aligned constructs enhance the formation of myotubes as well as cell alignment and may provide a further step towards the clinical application of engineered skeletal muscle. In this study the myogenic differentiation potential of MSCs upon co-cultivation with myoblasts and under stimulation with hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) was evaluated. We further analysed the behaviour of MSC-myoblast co-cultures in different 3D matrices. Primary rat myoblasts and rat MSCs were mono- and co-cultivated for 2, 7 or 14 days. The effect of different concentrations of HGF and IGF-1 alone, as well as in combination, on myogenic differentiation was analysed using microscopy, multicolour flow cytometry and real-time PCR. Furthermore, the influence of different three-dimensional culture models, such as fibrin, fibrin-collagen-I gels and parallel aligned electrospun poly-ε-caprolacton collagen-I nanofibers, on myogenic differentiation was analysed. MSCs could be successfully differentiated into the myogenic lineage both in mono- and in co-cultures independent of HGF and IGF-1 stimulation by expressing desmin, myocyte enhancer factor 2, myosin heavy chain 2 and alpha-sarcomeric actinin. An increased expression of different myogenic key markers could be observed under HGF and IGF-1 stimulation. Even though, stimulation with HGF/IGF-1 does not seem essential for sufficient myogenic differentiation. Three-dimensional cultivation in fibrin-collagen-I gels induced higher levels of myogenic differentiation compared with two-dimensional experiments. Cultivation on poly-ε-caprolacton-collagen-I nanofibers induced parallel alignment of cells and positive expression of desmin. In this study, we were able to myogenically differentiate MSC upon mono- and co-cultivation with myoblasts. The addition of HGF/IGF-1 might not be essential for achieving successful myogenic differentiation. Furthermore, with the development of a biocompatible nanofiber scaffold we established the basis for further experiments aiming at the generation of functional muscle tissue.

Extrinsic and intrinsic mechanisms by which mesenchymal stem cells suppress the immune system

PubMed Central

Coulson-Thomas, Vivien J.; Coulson-Thomas, Yvette M.; Gesteira, Tarsis F.; Kao, Winston W.-Y.

2016-01-01

Mesenchymal stem cells (MSCs) are a group of fibroblast-like multipotent mesenchymal stromal cells that have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Recent studies have demonstrated that MSCs possess a unique ability to exert suppressive and regulatory effects on both adaptive and innate immunity in an autologous and allogeneic manner. A vital step in stem cell transplantation is overcoming the potential graft-versus-host disease, which is a limiting factor to transplantation success. Given that MSCs attain powerful differentiation capabilities and also present immunosuppressive properties, which enable them to survive host immune rejection, MSCs are of great interest. Due to their ability to differentiate into different cell types and to suppress and modulate the immune system, MSCs are being developed for treating a plethora of diseases, including immune disorders. Moreover, in recent years, MSCs have been genetically engineered to treat and sometimes even cure some diseases, and the use of MSCs for cell therapy presents new perspectives for overcoming tissue rejection. In this review, we discuss the potential extrinsic and intrinsic mechanisms that underlie MSCs’ unique ability to modulate inflammation, and both innate and adaptive immunity. PMID:26804815

Treatment of inflammatory diseases with mesenchymal stem cells.

PubMed

Newman, Robert E; Yoo, Dana; LeRoux, Michelle A; Danilkovitch-Miagkova, Alla

2009-06-01

Human mesenchymal stem cells (hMSCs) are rare progenitor cells present in adult bone marrow that have the capacity to differentiate into a variety of tissue types, including bone, cartilage, tendon, fat, and muscle. In addition to multilineage differentiation capacity, MSCs regulate immune and inflammatory responses, providing therapeutic potential for treating diseases characterized by the presence of an inflammatory component. The availability of bone marrow and the ability to isolate and expand hMSCs ex vivo make these cells an attractive candidate for drug development. The low immunogenicity of these cells suggests that hMSCs can be transplanted universally without matching between donors and recipients. MSCs universality, along with the ability to manufacture and store these cells long-term, present a unique opportunity to produce an "off-the-shelf" cellular drug ready for treatment of diseases in acute settings. Accumulated animal and human data support MSC therapeutic potential for inflammatory diseases. Several phase III clinical trials for treatment of acute Graft Versus Host Disease (GVHD) and Crohn's disease are currently in progress. The current understanding of cellular and molecular targets underlying the mechanisms of MSCs action in inflammatory settings as well as clinical experience with hMSCs is summarized in this review.

Augmenting the bioactivity of polyetheretherketone using a novel accelerated neutral atom beam technique.

PubMed

Ajami, S; Coathup, M J; Khoury, J; Blunn, G W

2017-08-01

Polyetheretherketone (PEEK) is an alternative to metallic implants in orthopedic applications; however, PEEK is bioinert and does not osteointegrate. In this study, an accelerated neutral atom beam technique (ANAB) was employed to improve the bioactivity of PEEK. The aim was to investigate the growth of human mesenchymal stem cells (hMSCs), human osteoblasts (hOB), and skin fibroblasts (BR3G) on PEEK and ANAB PEEK. The surface roughness and contact angle of PEEK and ANAB PEEK was measured. Cell metabolic activity, proliferation and alkaline phosphatase (ALP) was measured and cell attachment was determined by quantifying adhesion plaques with cells. ANAB treatment increased the surface hydrophilicity [91.74 ± 4.80° (PEEK) vs. 74.82 ± 2.70° (ANAB PEEK), p < 0.001] but did not alter the surface roughness. Metabolic activity and proliferation for all cell types significantly increased on ANAB PEEK compared to PEEK (p < 0.05). Significantly increased cell attachment was measured on ANAB PEEK surfaces. MSCs seeded on ANAB PEEK in the presence of osteogenic media, expressed increased levels of ALP compared to untreated PEEK (p < 0.05) CONCLUSION: Our results demonstrated that ANAB treatment increased the cell attachment, metabolic activity, and proliferation on PEEK. ANAB treatment may improve the osteointegration of PEEK implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1438-1446, 2017. © 2016 Wiley Periodicals, Inc.

Mesenchymal stem cells cancel azoxymethane-induced tumor initiation.

PubMed

Nasuno, Masanao; Arimura, Yoshiaki; Nagaishi, Kanna; Isshiki, Hiroyuki; Onodera, Kei; Nakagaki, Suguru; Watanabe, Shuhei; Idogawa, Masashi; Yamashita, Kentaro; Naishiro, Yasuyoshi; Adachi, Yasushi; Suzuki, Hiromu; Fujimiya, Mineko; Imai, Kohzoh; Shinomura, Yasuhisa

2014-04-01

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O(6)-methylguanine (O(6) MeG) adducts through O(6) MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-β signaling because such properties were completely abrogated by absorption of TGF-β under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-β-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease. © AlphaMed Press.

Bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art.

PubMed

Malgieri, Arianna; Kantzari, Eugenia; Patrizi, Maria Patrizia; Gambardella, Stefano

2010-09-07

Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in all tissues, as part of the perivascular population. As multipotent cells, MSCs can differentiate into different tissues originating from mesoderm ranging from bone and cartilage, to cardiac muscle. MSCs are an excellent candidate for cell therapy because they are easily accessible, their isolation is straightforward, they can be bio-preserved with minimal loss of potency, and they have shown no adverse reactions to allogeneic versus autologous MSCs transplants. Therefore, MSCs are being explored to regenerate damaged tissue and treat inflammation, resulting from cardiovascular disease and myo-cardial infarction (MI), brain and spinal cord injury, stroke, diabetes, cartilage and bone injury, Crohn's disease and graft versus host disease (GvHD). Most of the application and clinical trials involve MSCs from bone marrow (BMMSCs). Transplantation of MSCs from bone marrow is considered safe and has been widely tested in clinical trials of cardiovascular, neurological, and immunological disease with encouraging results. There are examples of MSCs utilization in the repair of kidney, muscle and lung. The cells were also found to promote angiogenesis, and were used in chronic skin wound treatment. Recent studies involve also mesenchymal stem cell transplant from umbilical cord (UCMSCt). One of these demonstrate that UCMSCt may improve symptoms and biochemical values in patients with severe refractory systemic lupus erythematosus (SLE), and therefore this source of MSCs need deeper studies and require more attention. However, also if there are 79 registered clinical trial sites for evaluating MSC therapy throughout the world, it is still a long way to go before using these cells as a routinely applied therapy in clinics.

Bone marrow mesenchymal stem cells could acquire the phenotypes of epithelial cells and accelerate vaginal reconstruction combined with small intestinal submucosa.

PubMed

Li, Yanan; Liu, Fangfang; Zhang, Zhiqiang; Zhang, Mingle; Cao, Shanjin; Li, Yachai; Zhang, Lin; Huang, Xianghua; Xu, Yanfang

2015-11-01

Grafting material for vaginal reconstruction commonly includes the bowel, peritoneum, skin, and amniotic membrane. Bone marrow mesenchymal stem cells (MSCs) have the potential of multilineage differentiation into a variety of cells and have been widely explored in tissue engineering. In the current study, we examined whether MSCs could be differentiated to vaginal epithelial cells (VECs) upon co-culturing with VECs. We also examined whether Wnt/β-catenin signaling pathway is implicated in such differentiation. Co-culture of MSCs with VECs using a transwell insert system (with no direct contact) induced the expression of VECs marker AE1/AE3 in MSCs. MSCs combined with small intestinal submucosa (SIS) scaffold were implanted in place of the native vagina in rats to observe the implications for vaginal reconstruction in vivo. Anatomic repair of neovagina was assessed by histological staining for H/E and Masson's Trichrome. GSK-3β and β-catenin, main members of Wnt/β-catenin signaling pathway, in MSCs were increased upon co-culturing with VECs. Exposure of co-cultured MSCs to a Wnt/β-catenin signaling activator, lithium chloride (LiCl, 20 µM) increased phosphorylated GSK-3β and β-catenin and enhanced expression of AE1/AE3. In vivo-grafted cells displayed significant matrix infiltration and expressed epithelial markers in neovagina. These findings suggest that MSCs could acquire the phenotype of VECs when co-cultured with VECs, possibly via activation of Wnt/β-catenin signaling. MSCs provide an alternative cell source for potential use in vaginal tissue engineering. © 2015 International Federation for Cell Biology.

Comparison of Osteogenic and Chondrogenic Differentiation Ability of Buccal Fat Pad Derived Mesenchymal Stem Cells and Gingival Derived Cells.

PubMed

Ghaderi, Hamid; Razmkhah, Mahboobeh; Kiany, Farin; Chenari, Nooshafarin; Haghshenas, Mohammad Reza; Ghaderi, Abbas

2018-06-01

One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells (MSCs) with higher differentiation ability. In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs (BFP-MSCs) with gingival derived cells (GDCs) were compared. BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I (COLL), BGLA and bone morphogenetic protein 2 (BMP2) using qRT-PCR. Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls. Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries.

Cell Toxicity in Fibroblasts, Tenocytes, and Human Mesenchymal Stem Cells-A Comparison of Necrosis and Apoptosis-Inducing Ability in Ropivacaine, Bupivacaine, and Triamcinolone.

PubMed

Zhang, Anja Z; Ficklscherer, Andreas; Gülecyüz, Mehmet F; Paulus, Alexander C; Niethammer, Thomas R; Jansson, Volkmar; Müller, Peter E

2017-04-01

To analyze the ability of ropivacaine, bupivacaine, and triamcinolone to induce apoptosis and necrosis in fibroblasts, tenocytes, and human mesenchymal stem cells. Human dermal fibroblasts, adipose-derived human mesenchymal stem cells (hMSCs), and tenocytes gained from the rotator cuff tendon were seeded with a cell density of 0.5 × 10 4 /cm 2 . One specimen of ropivacaine, bupivacaine, and triamcinolone was tested separately on the cells with separate concentrations of 0.5%, 0.25%, and 0.125% for each specimen. The negative control received no agent, only a change of medium. The incubation period for each agent was 30 minutes. After a change of medium and 1 hour, 24 hours, and 7 days of incubation, 10 4  cells were harvested and analyzed via fluorescence-activated cell sorting with double-staining with annexin V and propidium iodide. Statistical analysis to determine significant difference (P < .05) between the groups with SPSS statistics 23 through one-way analysis of variance with a univariate general linear model was performed. Bupivacaine showed necrosis-inducing effects on fibroblasts and tenocytes, with the necrotic effect peaking at 0.5% and 0.25%. Ropivacaine and triamcinolone caused no significant necrosis. Compared with fibroblasts and tenocytes, hMSCs did not show significant necrotic or apoptotic effects after exposure to bupivacaine. Overall, no significant differences in apoptosis were detected between different cell lines, varying concentrations, or time measurements. Bupivacaine 0.5% and 0.25% have the most necrosis-inducing effects on fibroblasts and tenocytes. Ropivacaine caused less necrosis than bupivaine. Compared with fibroblasts and tenocytes, hMSCs were not affected by necrosis using any of the tested agents. A significant apoptosis-inducing effect could not be detected for the different cell lines. Possible cell toxicity raises questions of concern for intra-articular injections using local anesthetics and corticosteroids. The present study demonstrates the necrotic and apoptotic effects of ropivacaine, bupivacaine, and triamcinolone and may give recommendations for intra-articular use of local anesthetics and corticosteroids. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Impairment in immuno-modulatory function of Flk1(+)CD31(-)CD34(-) MSCs from MDS-RA patients.

PubMed

Han, Qin; Sun, Zhao; Liu, Lihui; Chen, Bin; Cao, Ying; Li, Kanghua; Zhao, Robert Chunhua

2007-11-01

Myelodysplastic syndromes are a group of hematopoietic disorders characterized by hematopoietic stem cell dysregulation and abnormalities in the immune system. Mesenchymal stem cells (MSCs) and their derived stromal cells constitute a bone marrow microenvironment, which is the niche for hematopoiesis and a key compartment for immune development and regulation. Existing evidence has shown that MSCs from MDS patients have impaired capacity in supporting hematopoiesis. Here, we conducted an investigation to determine whether the immuno-modulatory function of MSCs is also impaired in MDS-RA (refractory anemia) patients. Flk1(+)CD31(-)CD34(-) MSCs were isolated from 15 MDS-RA patients and cultured for testing biological and immunological characteristics. MDS-RA patient-derived Flk1(+)CD31(-)CD34(-) MSCs showed normal morphology, phenotype and karyotype but appeared impaired in immuno-modulatory function. The capacity of patient Flk1(+)CD31(-)CD34(-) MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro. In conclusion, MDS-RA patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than HSCs. MSCs might be a potential target for developing efficacious cures for MDS.

Impaired redox environment modulates cardiogenic and ion-channel gene expression in cardiac-resident and non-resident mesenchymal stem cells.

PubMed

Subramani, Baskar; Subbannagounder, Sellamuthu; Ramanathanpullai, Chithra; Palanivel, Sekar; Ramasamy, Rajesh

2017-03-01

Redox homeostasis plays a crucial role in the regulation of self-renewal and differentiation of stem cells. However, the behavioral actions of mesenchymal stem cells in redox imbalance state remain elusive. In the present study, the effect of redox imbalance that was induced by either hydrogen peroxide (H 2 O 2 ) or ascorbic acid on human cardiac-resident (hC-MSCs) and non-resident (umbilical cord) mesenchymal stem cells (hUC-MSCs) was evaluated. Both cells were sensitive and responsive when exposed to either H 2 O 2 or ascorbic acid at a concentration of 400 µmol/L. Ascorbic acid pre-treated cells remarkably ameliorated the reactive oxygen species level when treated with H 2 O 2 . The endogenous antioxidative enzyme gene (Sod1, Sod2, TRXR1 and Gpx1) expressions were escalated in both MSCs in response to reactive oxygen species elevation. In contrast, ascorbic acid pre-treated hUC-MSCs attenuated considerable anti-oxidative gene (TRXR1 and Gpx1) expressions, but not the hC-MSCs. Similarly, the cardiogenic gene (Nkx 2.5, Gata4, Mlc2a and β-MHC) and ion-channel gene ( I KDR , I KCa , I to and I Na.TTX ) expressions were significantly increased in both MSCs on the oxidative state. On the contrary, reduced environment could not alter the ion-channel gene expression and negatively regulated the cardiogenic gene expressions except for troponin-1 in both cells. In conclusion, redox imbalance potently alters the cardiac-resident and non-resident MSCs stemness, cardiogenic, and ion-channel gene expressions. In comparison with cardiac-resident MSC, non-resident umbilical cord-MSC has great potential to tolerate the redox imbalance and positively respond to cardiac regeneration. Impact statement Human mesenchymal stem cells (h-MSCs) are highly promising candidates for tissue repair in cardiovascular diseases. However, the retention of cells in the infarcted area has been a major challenge due to its poor viability and/or low survival rate after transplantation. The regenerative potential of mesenchymal stem cells (MSCs) repudiate and enter into premature senescence via oxidative stress. Thus, various strategies have been attempted to improve the MSC survival in 'toxic' conditions. Similarly, we investigated the response of cardiac resident MSC (hC-MSCs) and non-resident MSCs against the oxidative stress induced by H 2 O 2 . Supplementation of ascorbic acid (AA) into MSCs culture profoundly rescued the stem cells from oxidative stress induced by H 2 O 2 . Our data showed that the pre-treatment of AA is able to inhibit the cell death and thus preserving the viability and differentiation potential of MSCs.

Electrochemical Detection of Human Mesenchymal Stem Cell Differentiation on Fabricated Gold Nano-Dot Cell Chips.

PubMed

An, Jeung Hee; Kim, Seung U; Park, Mi-Kyung; Choi, Jeong Woo

2015-10-01

Human mesenchymal stem cells (MSCs) have the capacity for self-renewal and maintain pluripotency, which is defined by their ability to differentiate into cells such as osteoblasts, neurons, and glial cells. In this study, we report a method for defining the status of human MSCs based on electrochemical detection systems. Gold nano-dot structures were fabricated using a nanoporous alumina mask, and the structural formations were confirmed by scanning electron microscopy (SEM). Human MSCs were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to the MSCs attached on the chip surface. The cultured MSCs were shown to differentiate into neural cell types, as indicated by immunocytochemical staining for tyrosine hydroxylase and beta tubulin III. Following treatment with basic fibroblast growth factor (bFGF) for 14 days, most of the B10 cells exhibited bipolar or multipolar morphology with branched processes, and the proportion of B10 cells expressing neuronal cell markers considerably increased. Electrophysiological recordings from MSCs treated with bFGF for 5-14 days were examined with cyclic voltammetry, and the electrochemical signals were shown to increase during differentiation from MSCs to neuronal cells. This human MSC cell line is a useful tool for studying organogenesis, specifically neurogenesis, and in addition, the cell line provides a valuable source of cells for cell therapy. The electrochemical measurement system proposed here could be utilized in electrical cell chips for numerous applications, including cell differentiation, disease diagnosis, drug detection, and on-site monitoring.

Multipotent Mesenchymal Stromal Stem Cell Expansion by Plating Whole Bone Marrow at a Low Cellular Density: A More Advantageous Method for Clinical Use

PubMed Central

Mareschi, Katia; Rustichelli, Deborah; Calabrese, Roberto; Gunetti, Monica; Sanavio, Fiorella; Castiglia, Sara; Risso, Alessandra; Ferrero, Ivana; Tarella, Corrado; Fagioli, Franca

2012-01-01

Mesenchymal stem cells (MSCs) are a promising source for cell therapy due to their pluripotency and immunomodulant proprieties. As the identification of “optimal” conditions is important to identify a standard procedure for clinical use. Percoll, Ficoll and whole bone marrow directly plated were tested from the same sample as separation methods. The cells were seeded at the following densities: 100 000, 10 000, 1000, 100, 10 cells/cm2. After reaching confluence, the cells were detached, pooled and re-plated at 1000, 500, 100, and 10 cells/cm2. Statistical analyses were performed. Cumulative Population Doublings (PD) did not show significant differences for the separation methods and seeding densities but only for the plating density. Some small quantity samples plated in T25 flasks at plating densities of 10 and 100 cells/cm2 did not produce any expansion. However, directly plated whole bone marrow resulted in a more advantageous method in terms of CFU-F number, cellular growth and minimal manipulation. No differences were observed in terms of gross morphology, differentiation potential or immunophenotype. These data suggest that plating whole bone marrow at a low cellular density may represent a good procedure for MSC expansion for clinical use. PMID:23715383

The role of bone marrow mesenchymal stromal cell derivatives in skin wound healing in diabetic mice.

PubMed

de Mayo, Tomas; Conget, Paulette; Becerra-Bayona, Silvia; Sossa, Claudia L; Galvis, Virgilio; Arango-Rodríguez, Martha L

2017-01-01

Mesenchymal stromal cells (MSCs) have shown to be a promising tool in cell therapies to treat different conditions. Several pre-clinical and clinical studies have proved that the transplantation of MSCs improves wound healing. Here, we compare the beneficial effects of mouse bone marrow-derived allogeneic MSCs (allo-mBM-MSCs) and their acelullar derivatives (allo-acd-mMSCs) on skin wound healing in Non-Obese Diabetic (NOD) mice. One dose of allo-mBM-MSCs (1×106 cells) or one dose of allo-acd-mMSCs (1X) were intradermally injected around wounds in 8-10 week old female NOD mice. Wound healing was evaluated macroscopically (wound closure) every two days, and microscopically (reepithelialization, dermoepidermal junction, skin appendage regeneration, leukocyte infiltration, vascularization, granulation tissue formation, and density of collagen fibers in the dermis) after 16 days of MSC injection. In addition, we measured growth factors and specific proteins that were present in the allo-acd-mMSCs. Results showed significant differences in the wound healing kinetics of lesions that received allo-acd-mMSCs compared to lesions that received vehicle or allo-mBM-MSCs. In particular, mice treated with allo-acd-mMSCs reached significantly higher percentages of wound closure at day 4, 6 and 8, relative to the allo-mBM-MSCs and vehicle groups (p < 0.05), while wound closure percentages could not be statistically distinguished between the allo-mBM-MSCs and vehicle groups. Also, allo-acd-mMSCs had a greater influence in the skin would healing process. Specifically, they caused a less pronounced inflammatory severe response (p < 0.0001), more granulation tissue formation at an advanced stage (p < 0.0001), and higher density of collagen fibers (p < 0.05) compared to the other groups. Nevertheless, at day 16, both allo-mBM-MSCs and allo-acd-mMSCs revealed a higher effect on the recovery of the quality skin (continuous epidermis; regular dermoepidermal junction and skin appendages) relative to untreated lesions (p < 0.0001), but not between them. On the other hand, ELISA analyses indicated that the allo-acd-mMSCs contained growth factors and proteins relevant to wound healing such as IGF-1, KGF, HGF, VEGF, ANG-2, MMP-1, CoL-1 and PGE2. Compared to allo-acd-mMSCs, the administration of allo-mBM-MSCs is insufficient for wound healing in diabetic mice and delays the therapeutic effect, which maybe explained by the fact that trophic factors secreted by MSCs are critical for skin regeneration, and not the cells per se, suggesting that MSCs may require some time to secrete these factors after their administration.

Mechanisms of T-Cell Immunosuppression by Mesenchymal Stromal Cells: What Do We Know So Far?

PubMed Central

Haddad, Rodrigo; Saldanha-Araujo, Felipe

2014-01-01

Mesenchymal stromal cells (MSCs) are multipotent cells, which can give rise to several cell types including osteoblasts, adipocytes, and chondroblasts. These cells can be found in a variety of adult and fetal tissues, such as bone marrow, adipose tissue, cord blood, and placenta. In recent years, the biological properties of MSCs have attracted the attention of researchers worldwide due to their potential application for treating a series of clinical situations. Among these properties, special attention should be given to the immunoregulatory potential of those cells. MSCs are able to act on all cells of the immune system, which includes the capacity to inhibit the proliferation and function of T-cells. This feature renders them natural candidates to treat several diseases in which cellular immune response is exacerbated. In this review, we outline the main mechanisms by which MSCs immunosuppress T-cell response, focusing on cell-cell contact, secretion of soluble factors, and regulatory T-cell generation. The influence of surface markers in the immunosuppression process and features of MSCs isolated from different sources are also discussed. Finally, the influences of toll-like receptors and cytokines on the inflammatory microenvironment are highlighted regarding the activation of MSCs to exert their immunoregulatory function. PMID:25025040

Tracing the destiny of mesenchymal stem cells from embryo to adult bone marrow and white adipose tissue via Pdgfrα expression.

PubMed

Miwa, Hiroyuki; Era, Takumi

2018-01-29

Mesenchymal stem cells (MSCs) are somatic stem cells that can be derived from adult bone marrow (BM) and white adipose tissue (WAT), and that display multipotency and self-renewal capacity. Although MSCs are essential for tissue formation and have already been used in clinical therapy, the origins and markers of these cells remain unknown. In this study, we first investigated the developmental process of MSCs in mouse embryos using the gene encoding platelet-derived growth factor receptor α ( Pdgfra ) as a marker. We then traced cells expressing Pdgfra and other genes (brachyury, Sox1 and Pmx1 ) in various mutant mouse embryos until the adult stage. This tracing of MSC origins and destinies indicates that embryonic MSCs emerge in waves and that almost all adult BM MSCs and WAT MSCs originate from mesoderm and embryonic Pdgfrα-positive cells. Furthermore, we demonstrate that adult Pdgfrα-positive cells are involved in some pathological conditions. © 2018. Published by The Company of Biologists Ltd.

Real-Time H2 O2 Measurements in Bone Marrow Mesenchymal Stem Cells (MSCs) Show Increased Antioxidant Capacity in Cells From Osteoporotic Women.

PubMed

Román, Flavia; Urra, Carla; Porras, Omar; Pino, Ana María; Rosen, Clifford J; Rodríguez, Juan Pablo

2017-03-01

Oxidative stress (OS) derived from an increase in intracellular reactive oxygen species (ROS) is a major determinant of aging and lifespan. It has also been associated with several age-related disorders, like postmenopausal osteoporosis of Mesenchymal stem cells (MSCs). MSCs are the common precursors for osteoblasts and adipocytes; appropriate commitment and differentiation of MSCs into a specific phenotype is modulated, among other factors, by ROS balance. MSCs have shown more resistance to ROS than differentiated cells, and their redox status depends on complex and abundant anti-oxidant mechanisms. The purpose of this work was to analyze in real time, H 2 O 2 signaling in individual h-MSCs, and to compare the kinetic parameters of H 2 O 2 management by cells derived from both control (c-) and osteoporotic (o-) women. For these purposes, cells were infected with a genetically encoded fluorescent biosensor named HyPer, which is specific for detecting H 2 O 2 inside living cells. Subsequently, cells were sequentially challenged with 50 and 500 μM H 2 O 2 pulses, and the cellular response was recorded in real time. The results demonstrated adequate expression of the biosensor allowing registering fluorescence from HyPer at a single cell level. Comparison of the response of c- and o-MSCs to the oxidant challenges demonstrated improved antioxidant activity in o-MSCs. This was further corroborated by measuring the relative expression of mRNAs for catalase, superoxide dismutase-1, thioredoxine, and peroxiredoxine, as well as by cell-surviving capacity under short-term H 2 O 2 treatment. We conclude that functional differences exist between healthy and osteoporotic human MSCs. The mechanism for these differences requires further study. J. Cell. Biochem. 118: 585-593, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Ex vivo identification and characterization of a population of CD13(high) CD105(+) CD45(-) mesenchymal stem cells in human bone marrow.

PubMed

Muñiz, Carmen; Teodosio, Cristina; Mayado, Andrea; Amaral, Ana Teresa; Matarraz, Sergio; Bárcena, Paloma; Sanchez, Maria Luz; Alvarez-Twose, Iván; Diez-Campelo, María; García-Montero, Andrés C; Blanco, Juan F; Del Cañizo, Maria Consuelo; del Pino Montes, Javier; Orfao, Alberto

2015-09-07

Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs. Multiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13(high) CD105(+) CD45(-) cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expression profile of these cells was further assessed on (paired) primary and in vitro expanded BM MSCs, and their adipogenic, chondrogenic and osteogenic differentiation potential was also determined. Our results show that the CD13(high) CD105(+) CD45(-) immunophenotype defines a minor subset of cells that are systematically present ex vivo in normal/reactive BM (n = 65) and that display immunophenotypic features, plastic adherence ability, and osteogenic, adipogenic and chondrogenic differentiation capacities fully compatible with those of MSCs. In addition, we also show that in vitro expansion of these cells modulates their immunophenotypic characteristics, including changes in the expression of markers currently used for the definition of MSCs, such as CD105, CD146 and HLA-DR. BM MSCs can be identified ex vivo in normal/reactive BM, based on a robust CD13(high) CD105(+) and CD45(-) immunophenotypic profile. Furthermore, in vitro expansion of these cells is associated with significant changes in the immunophenotypic profile of MSCs.

Human mesenchymal stromal cells attenuate graft-versus-host disease and maintain graft-versus-leukemia activity following experimental allogeneic bone marrow transplantation1

PubMed Central

Auletta, Jeffery J.; Eid, Saada K.; Wuttisarnwattana, Patiwet; Silva, Ines; Metheny, Leland; Keller, Matthew D.; Guardia-Wolff, Rocio; Liu, Chen; Wang, Fangjing; Bowen, Theodore; Lee, Zhenghong; Solchaga, Luis A.; Ganguly, Sudipto; Tyler, Megan; Wilson, David L.; Cooke, Kenneth R.

2014-01-01

We sought to define the effects and underlying mechanisms of human, marrow-derived mesenchymal stromal cells (hMSCs) on graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T-cells and treated with hMSCs had reduced systemic GvHD, donor T-cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72h; and target tissues harvested from hMSC-treated alloBMT mice had less GvHD than untreated controls. Cryo-imaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T-cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T-cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T-cell proliferation, reduced TNFα, IFNγ, and IL-10, but increased PGE2 levels. Indomethacin and E-prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC-mediated in vitro T-cell suppression, confirming the role for PGE2. Furthermore, cyclo-oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T-cell proliferation likely through PGE2 induction. PMID:25336340

Self-assembling nanoparticles encapsulating zoledronic acid inhibit mesenchymal stromal cells differentiation, migration and secretion of proangiogenic factors and their interactions with prostate cancer cells

PubMed Central

Pivetta, Eliana; Colombatti, Alfonso; Boccellino, Mariarosaria; Amler, Evzen; Normanno, Nicola; Caraglia, Michele; De Rosa, Giuseppe; Aldinucci, Donatella

2017-01-01

Zoledronic Acid (ZA) rapidly concentrates into the bone and reduces skeletal-related events and pain in bone metastatic prostate cancer (PCa), but exerts only a limited or absent impact as anti-cancer activity. Recently, we developed self-assembling nanoparticles (NPS) encapsulating zoledronic acid (NZ) that allowed a higher intratumor delivery of the drug compared with free zoledronic acid (ZA) in in vivo cancer models of PCa. Increasing evidence suggests that Bone Marrow (BM) Mesenchymal stromal cells (BM-MSCs) are recruited into the stroma of developing tumors where they contribute to progression by enhancing tumor growth and metastasis. We demonstrated that treatment with NZ decreased migration and differentiation into adipocytes and osteoblasts of MSCs and inhibited osteoclastogenesis. Treatment with NZ reduced the capability of MSCs to promote the migration and the clonogenic growth of the prostate cancer cell lines PC3 and DU145. The levels of Interleukin-6 and of the pro-angiogenic factors VEGF and FGF-2 were significantly reduced in MSC-CM derived from MSCs treated with NZ, and CCL5 secretion was almost totally abolished. Moreover, treatment of MSCs with supernatants from PC3 cells, leading to tumor-educated MSCs (TE-MSCs), increased the secretion of IL-6, CCL5, VEGF and FGF-2 by MSCs and increased their capability to increase PC3 cells clonogenic growth. Treatment with NZ decreased cytokine secretion and the pro-tumorigenic effects also of TE-MSCS. In conclusion, demonstrating that NZ is capable to inhibit the cross talk between MSCs and PCa, this study provides a novel insight to explain the powerful anticancer activity of NZ on PCa. PMID:28477013

Interleukin 7 Plays a Role in T Lymphocyte Apoptosis Inhibition Driven by Mesenchymal Stem Cell without Favoring Proliferation and Cytokines Secretion

PubMed Central

Normanton, Marilia; Alvarenga, Heliene; Hamerschlak, Nelson; Ribeiro, Andreza; Kondo, Andrea; Rizzo, Luiz Vicente; Marti, Luciana Cavalheiro

2014-01-01

Since 2004, when a case report describing the use of human mesenchymal stem cells (hMSCs) infusion as a therapy for GVHD after bone marrow transplantation, a new perspective in MSC function emerged. Since then hMSCs immunomodulatory potential became the target of several studies. Although great progress has been made in our understanding of hMSCs, their effect on T cell remains obscure. Our study has confirmed the already described effect of hMSCs on lymphocytes proliferation and survival. We also show that the impairment of lymphocyte proliferation and apoptosis is contact-independent and occurs in a prostaglandin-independent manner. A potential correlation between IL-7 and hMSCs effect is suggested, as we observed an increase in IL-7 receptors (CD127) on lymphocyte membrane in MSC presence. Additionally, blocking IL-7 in hMSCs-lymphocytes co-cultures increased lymphocytes apoptosis and we also have demonstrated that hMSCs are able to produce this interleukin. Moreover, we found that during Th1/Th17 differentiation in vitro, hMSCs presence leads to Th1/Th17 cells with reduced capacity of INF-y and IL-17 secretion respectively, regardless of having several pro-inflammatory cytokines in culture. We did not confirm an increment of Treg in these cultures, but a reduced percentage of INF-y/IL-17 secreting cells was observed, suggesting that the ratio between anti and pro-inflammatory cells changed. This changed ratio is very important to GvHD therapy and links hMSCs to an anti-inflammatory role. Taken together, our findings provide important preliminary results on the lymphocyte pathway modulated by MSCs and may contribute for developing novel treatments and therapeutic targets for GvHD and others autoimmune diseases. PMID:25184791

Human amniotic fluid-derived mesenchymal stem cells as therapeutic vehicles: a novel approach for the treatment of bladder cancer.

PubMed

Bitsika, Vasiliki; Roubelakis, Maria G; Zagoura, Dimitra; Trohatou, Ourania; Makridakis, Manousos; Pappa, Kalliopi I; Marini, Frank C; Vlahou, Antonia; Anagnou, Nicholas P

2012-05-01

Recent studies support cell-based therapies for cancer treatment. An advantageous cell type for such therapeutic schemes are the mesenchymal stem cells (MSCs) that can be easily propagated in culture, genetically modified to express therapeutic proteins, and exhibit an innate tropism to solid tumors in vivo. Recently, we successfully isolated and expanded MSCs from second-trimester amniotic fluid (AF-MSCs). The main characteristic of AF-MSCs is their efficient and rapid expansion in vitro. Herein, we investigated the AF-MSCs tropism and capability to transport interferon beta (IFNβ) to the region of neoplasia in a bladder tumor model. To this end, we used the T24M bladder cancer cell line, previously generated from our studies, and developed a disease progression model in immunosuppressed mice, that can recapitulate the molecular events of bladder carcinogenesis. Our results documented that AF-MSCs exhibited high motility, when migrated either to T24M cells or to T24M-conditioned medium, and we further identified and studied the secreted factors which may trigger these enhanced migratory properties. Further, lentivirus-transduced AF-MSCs, expressing green fluorescent protein (GFP) or IFNβ, were intravenously administered to T24M tumor-bearing animals at multiple doses to examine their therapeutic effect. GFP- and IFNβ-AF-MSCs successfully migrated and colonized at the tumor site. Notably, significant inhibition of tumor growth as well as prolonged survival of mice were observed in the presence of IFNβ-AF-MSCs. Collectively, these results document the great potential of AF-MSCs as anti-cancer vehicles, implemented by the targeting of the tumor site and further facilitated by their high proliferation rate and expansion efficiency in culture.

MSCs ameliorates DPN induced cellular pathology via [Ca2+ ]i homeostasis and scavenging the pro-inflammatory cytokines.

PubMed

Chandramoorthy, Harish C; Bin-Jaliah, Ismaeel; Karari, Hussian; Rajagopalan, Prasanna; Ahmed Shariff, Mohammed Eajaz; Al-Hakami, Ahmed; Al-Humayad, Suliman M; Baptain, Fawzi A; Ahmed, Humeda Suekit; Yassin, Hanaa Z; Haidara, Mohamed A

2018-02-01

The MSCs of various origins are known to ameliorate or modulate cell survival strategies. We investigated, whether UCB MSCs could improve the survival of the human neuronal cells and/or fibroblast assaulted with DPN sera. The results showed, the co-culture of UCB MSCs with human neuronal cells and/or fibroblasts could effectively scavenge the pro-inflammatory cytokines TNF-α, IL-1β, IFN-ɤ and IL - 12 and control the pro-apoptotic expression of p53/Bax. Further co-culture of UCB MSCs have shown to induce anti-inflammatory cytokines like IL-4, IL-10 and TGF-β and anti-apoptotic Bclxl/Bcl2 expression in the DPN sera stressed cells. Amelioration of elevated [Ca 2+ ] i and cROS, the portent behind the NFκB/Caspase-3 mediated inflammation in DPN rescued the cells from apoptosis. The results of systemic administration of BM MSCs improved DPN pathology in rat as extrapolated from human cell model. The BM MSCs ameliorated prolonged distal motor latency (control: 0.70 ± 0.06, DPN: 1.29 ± 0.13 m/s DPN + BM MSCs: 0.89 ± 0.02 m/s, p < 0.05) and lowered high amplitude of compound muscle action potentials (CMAPs) (control: 12.36 ± 0.41, DPN: 7.52 ± 0.61 mV, DPN + MSCs: 8.79 ± 0.53 mV, p < 0.05), while slowly restoring the plasma glucose levels. Together, all these results showed that administration of BM or UCB MSCs improved the DPN via ameliorating pro-inflammatory cytokine signaling and [Ca 2+ ] i homeostasis. © 2017 Wiley Periodicals, Inc.

Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-β/Smad2/3 pathway.

PubMed

Tu, Bing; Peng, Zhao-Xiang; Fan, Qi-Ming; Du, Lin; Yan, Wei; Tang, Ting-Ting

2014-01-01

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-β (TGF-β) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-β in tumor CM was proved to be an important factor. The TGF-β neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of β-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of β-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-β to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells. © 2013 Published by Elsevier Inc.

Wound-healing potential of human umbilical cord blood-derived mesenchymal stromal cells in vitro--a pilot study.

PubMed

You, Hi-Jin; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

2015-11-01

Our previous studies demonstrated that human bone marrow-derived mesenchymal stromal cells have great potential for wound healing. However, it is difficult to clinically utilize cultured stem cells. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialized for cartilage repair as a first cell therapy product that uses allogeneic stem cells. Should hUCB-MSCs have a superior effect on wound healing as compared with fibroblasts, which are the main cell source in current cell therapy products for wound healing, they may possibly replace fibroblasts. The purpose of this in vitro study was to compare the wound-healing activity of hUCB-MSCs with that of fibroblasts. This study was particularly designed to compare the effect of hUCB-MSCs on diabetic wound healing with those of allogeneic and autologous fibroblasts. Healthy (n = 5) and diabetic (n = 5) fibroblasts were used as the representatives of allogeneic and autologous fibroblasts for diabetic patients in the control group. Human UCB-MSCs (n = 5) were used in the experimental group. Cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-β) production were compared among the three cell groups. Human UCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor when compared with both fibroblast groups. Human UCB-MSCs were superior to diabetic fibroblasts but not to healthy fibroblasts in collagen synthesis. There were no significant differences in cell proliferation and transforming growth factor-β production. Human UCB-MSCs may have greater capacity for diabetic wound healing than allogeneic or autologous fibroblasts, especially in angiogenesis. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Optimized labeling of bone marrow mesenchymal cells with superparamagnetic iron oxide nanoparticles and in vivo visualization by magnetic resonance imaging

PubMed Central

2011-01-01

Background Stem cell therapy has emerged as a promising addition to traditional treatments for a number of diseases. However, harnessing the therapeutic potential of stem cells requires an understanding of their fate in vivo. Non-invasive cell tracking can provide knowledge about mechanisms responsible for functional improvement of host tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used to label and visualize various cell types with magnetic resonance imaging (MRI). In this study we performed experiments designed to investigate the biological properties, including proliferation, viability and differentiation capacity of mesenchymal cells (MSCs) labeled with clinically approved SPIONs. Results Rat and mouse MSCs were isolated, cultured, and incubated with dextran-covered SPIONs (ferumoxide) alone or with poly-L-lysine (PLL) or protamine chlorhydrate for 4 or 24 hrs. Labeling efficiency was evaluated by dextran immunocytochemistry and MRI. Cell proliferation and viability were evaluated in vitro with Ki67 immunocytochemistry and live/dead assays. Ferumoxide-labeled MSCs could be induced to differentiate to adipocytes, osteocytes and chondrocytes. We analyzed ferumoxide retention in MSCs with or without mitomycin C pretreatment. Approximately 95% MSCs were labeled when incubated with ferumoxide for 4 or 24 hrs in the presence of PLL or protamine, whereas labeling of MSCs incubated with ferumoxide alone was poor. Proliferative capacity was maintained in MSCs incubated with ferumoxide and PLL for 4 hrs, however, after 24 hrs it was reduced. MSCs incubated with ferumoxide and protamine were efficiently visualized by MRI; they maintained proliferation and viability for up to 7 days and remained competent to differentiate. After 21 days MSCs pretreated with mitomycin C still showed a large number of ferumoxide-labeled cells. Conclusions The efficient and long lasting uptake and retention of SPIONs by MSCs using a protocol employing ferumoxide and protamine may be applicable to patients, since both ferumoxides and protamine are approved for human use. PMID:21542946

Mesenchymal stem cells alleviate oxidative stress-induced mitochondrial dysfunction in the airways.

PubMed

Li, Xiang; Michaeloudes, Charalambos; Zhang, Yuelin; Wiegman, Coen H; Adcock, Ian M; Lian, Qizhou; Mak, Judith C W; Bhavsar, Pankaj K; Chung, Kian Fan

2018-05-01

Oxidative stress-induced mitochondrial dysfunction can contribute to inflammation and remodeling in patients with chronic obstructive pulmonary disease (COPD). Mesenchymal stem cells protect against lung damage in animal models of COPD. It is unknown whether these effects occur through attenuating mitochondrial dysfunction in airway cells. We sought to examine the effect of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) on oxidative stress-induce mitochondrial dysfunction in human airway smooth muscle cells (ASMCs) in vitro and in mouse lungs in vivo. ASMCs were cocultured with iPSC-MSCs in the presence of cigarette smoke medium (CSM), and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis were measured. Conditioned medium from iPSC-MSCs and transwell cocultures were used to detect any paracrine effects. The effect of systemic injection of iPSC-MSCs on airway inflammation and hyperresponsiveness in ozone-exposed mice was also investigated. Coculture of iPSC-MSCs with ASMCs attenuated CSM-induced mitochondrial ROS, apoptosis, and ΔΨm loss in ASMCs. iPSC-MSC-conditioned medium or transwell cocultures with iPSC-MSCs reduced CSM-induced mitochondrial ROS but not ΔΨm or apoptosis in ASMCs. Mitochondrial transfer from iPSC-MSCs to ASMCs was observed after direct coculture and was enhanced by CSM. iPSC-MSCs attenuated ozone-induced mitochondrial dysfunction, airway hyperresponsiveness, and inflammation in mouse lungs. iPSC-MSCs offered protection against oxidative stress-induced mitochondrial dysfunction in human ASMCs and in mouse lungs while reducing airway inflammation and hyperresponsiveness. These effects are, at least in part, dependent on cell-cell contact, which allows for mitochondrial transfer, and paracrine regulation. Therefore iPSC-MSCs show promise as a therapy for oxidative stress-dependent lung diseases, such as COPD. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

PubMed Central

Ahmed, Atique U; Rolle, Cleo E; Tyler, Matthew A; Han, Yu; Sengupta, Sadhak; Wainwright, Derek A; Balyasnikova, Irina V; Ulasov, Ilya V; Lesniak, Maciej S

2010-01-01

Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow–derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. PMID:20588259

Multipotent human stromal cells isolated from cord blood, term placenta and adult bone marrow show distinct differences in gene expression pattern

PubMed Central

Matigian, Nicholas; Brooke, Gary; Zaibak, Faten; Rossetti, Tony; Kollar, Katarina; Pelekanos, Rebecca; Heazlewood, Celena; Mackay-Sim, Alan; Wells, Christine A.; Atkinson, Kerry

2014-01-01

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues. PMID:26484151

Proteomic Definitions of Mesenchymal Stem Cells

PubMed Central

Maurer, Martin H.

2011-01-01

Mesenchymal stem cells (MSCs) are pluripotent cells isolated from the bone marrow and various other organs. They are able to proliferate and self-renew, as well as to give rise to progeny of at least the osteogenic, chondrogenic, and adipogenic lineages. Despite this functional definition, MSCs can also be defined by their expression of a distinct set of cell surface markers. In the current paper, studies investigating the proteome of human MSCs are reviewed with the aim to identify common protein markers of MSCs. The proteomic analysis of MSCs revealed a distinct set of proteins representing the basic molecular inventory, including proteins for (i) cell surface markers, (ii) the responsiveness to growth factors, (iii) the reuse of developmental signaling cascades in adult stem cells, (iv) the interaction with molecules of the extracellular matrix, (v) the expression of genes regulating transcription and translation, (vi) the control of the cell number, and (vii) the protection against cellular stress. PMID:21437194

Mesenchymal stem cells do not suppress lymphoblastic leukemic cell line proliferation.

PubMed

Mousavi Niri, Neda; Jaberipour, Mansooreh; Razmkhah, Mahboobeh; Ghaderi, Abbas; Habibagahi, Mojtaba

2009-12-01

Several studies have demonstrated the immunosuppresive effects of mesenchymal stem cells (MSCs) in allogeneic or mitogenic interactions. Cell-cell contact inhibition and secretion of suppressive soluble factors have been suggested in this regard. To investigate if adipose derived MSCs could inhibit Jurkat lymphoblastic leukemia T cell proliferation during coculture. Adherent cells with the ability of cellular growth were isolated from normal adipose tissues. Initial characterization of growing cells by flow cytometry suggested their mesenchymal stem cell characteristics. Cells were maintained in culture and used during third to fifth culture passages. Jurkat or allogeneic peripheral blood mononuclear cells (PBMCs) were labeled with carboxy fluorescein diacetate succinimidyl ester and cocultured with increasing doses of MSCs or MSC culture supernatant. Proliferation of PBMCs or Jurkat cells under these conditions was assessed by flow cytometry after 2 and 3 days of coculture, respectively. Results showed the expression of CD105, CD166 and CD44, and the absence of CD45, CD34 and CD14 on the surface of MSC like cells. Moreover, initial differentiation studies showed the potential of cell differentiation into hepatocytes. Comparison of Jurkat cell proliferation in the presence and absence of MSCs showed no significant difference, with 70% of cells displaying signs of at least one cell division. Similarly, the highest concentration of MSC culture supernatant (50% vol/vol) had no significant effect on Jurkat cell proliferation (p>0.6). The same MSC lots significantly suppressed the allogeneic PHA activated PBMCs under similar culture conditions. Using Jurkat cells as a model of leukemia T cells, our results indicated an uncertainty about the suppressive effect of MSCs and their inhibitory metabolites on tumor or leukemia cell proliferation. Additional systematic studies with MSCs of different sources are needed to fully characterize the immunological properties of MSCs before planning clinical applications.