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.

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.

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.

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.

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

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.

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.

The fate of autologous endometrial mesenchymal stromal cells after application in the healthy equine uterus.

PubMed

Rink, Elisabeth; Beyer, Teresa; French, Hilari; Watson, Elaine; Aurich, Christine; Donadeu, Xavier

2018-05-23

Because of their distinct differentiation, immunomodulatory and migratory capacities, endometrial mesenchymal stromal cells (MSCs) may provide an optimum source of therapeutic cells not only in relation to the uterus but also for regeneration of other tissues. This study reports the fate of endometrial MSCs following intrauterine application in mares. Stromal cell fractions were isolated from endometrial biopsies taken from seven reproductively healthy mares, expanded and fluorescence-labeled in culture. MSCs (15 x 106) or PBS were autologously infused into each uterine horn during early diestrus and subsequently tracked by fluorescence microscopy and flow cytometry of endometrial biopsies and blood samples taken periodically after infusion. The inflammatory response to cell infusion was monitored in endometrial cytology samples. MSCs were detected in endometrial sections at 6, 12 and 24 hours but not later (7 or 14 days) after cell infusion. Cells were in all cases located in the uterine lumen, never within endometrial tissue. No fluorescence signal was detected in blood samples at any time point after infusion. Cytology analyses showed an increase in %PMN between 1 and 3 hours after uterine infusion with either MSCs or PBS, and a further increase by 6 hours only in mares infused with PBS. In summary, endometrial MSCs were detected in the uterine lumen for up to 24 h after infusion but did not migrate into healthy endometrium. Moreover, MSCs effectively attenuated the inflammatory response to uterine infusion. We conclude that endometrial MSCs obtained from routine uterine biopsies could provide a safe and effective cell source for treatment of inflammatory conditions of the uterus and potentially other tissues.

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

Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs)

PubMed Central

Bartosh, Thomas J.; Ullah, Mujib; Zeitouni, Suzanne; Beaver, Joshua; Prockop, Darwin J.

2016-01-01

Patients with breast cancer often develop malignant regrowth of residual drug-resistant dormant tumor cells years after primary treatment, a process defined as cancer relapse. Deciphering the causal basis of tumor dormancy therefore has obvious therapeutic significance. Because cancer cell behavior is strongly influenced by stromal cells, particularly the mesenchymal stem/stromal cells (MSCs) that are actively recruited into tumor-associated stroma, we assessed the impact of MSCs on breast cancer cell (BCC) dormancy. Using 3D cocultures to mimic the cellular interactions of an emerging tumor niche, we observed that MSCs sequentially surrounded the BCCs, promoted formation of cancer spheroids, and then were internalized/degraded through a process resembling the well-documented yet ill-defined clinical phenomenon of cancer cell cannibalism. This suspected feeding behavior was less appreciable in the presence of a rho kinase inhibitor and in 2D monolayer cocultures. Notably, cannibalism of MSCs enhanced survival of BCCs deprived of nutrients but suppressed their tumorigenicity, together suggesting the cancer cells entered dormancy. Transcriptome profiles revealed that the resulting BCCs acquired a unique molecular signature enriched in prosurvival factors and tumor suppressors, as well as inflammatory mediators that demarcate the secretome of senescent cells, also referred to as the senescence-associated secretory phenotype. Overall, our results provide intriguing evidence that cancer cells under duress enter dormancy after cannibalizing MSCs. Importantly, our practical 3D coculture model could provide a valuable tool to understand the antitumor activity of MSCs and cell cannibalism further, and therefore open new therapeutic avenues for the prevention of cancer recurrence. PMID:27698134

Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs).

PubMed

Bartosh, Thomas J; Ullah, Mujib; Zeitouni, Suzanne; Beaver, Joshua; Prockop, Darwin J

2016-10-18

Patients with breast cancer often develop malignant regrowth of residual drug-resistant dormant tumor cells years after primary treatment, a process defined as cancer relapse. Deciphering the causal basis of tumor dormancy therefore has obvious therapeutic significance. Because cancer cell behavior is strongly influenced by stromal cells, particularly the mesenchymal stem/stromal cells (MSCs) that are actively recruited into tumor-associated stroma, we assessed the impact of MSCs on breast cancer cell (BCC) dormancy. Using 3D cocultures to mimic the cellular interactions of an emerging tumor niche, we observed that MSCs sequentially surrounded the BCCs, promoted formation of cancer spheroids, and then were internalized/degraded through a process resembling the well-documented yet ill-defined clinical phenomenon of cancer cell cannibalism. This suspected feeding behavior was less appreciable in the presence of a rho kinase inhibitor and in 2D monolayer cocultures. Notably, cannibalism of MSCs enhanced survival of BCCs deprived of nutrients but suppressed their tumorigenicity, together suggesting the cancer cells entered dormancy. Transcriptome profiles revealed that the resulting BCCs acquired a unique molecular signature enriched in prosurvival factors and tumor suppressors, as well as inflammatory mediators that demarcate the secretome of senescent cells, also referred to as the senescence-associated secretory phenotype. Overall, our results provide intriguing evidence that cancer cells under duress enter dormancy after cannibalizing MSCs. Importantly, our practical 3D coculture model could provide a valuable tool to understand the antitumor activity of MSCs and cell cannibalism further, and therefore open new therapeutic avenues for the prevention of cancer recurrence.

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.

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

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

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.

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

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.

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.

Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling

PubMed Central

Murphy, Kaitlin C.; Hoch, Allison I.; Harvestine, Jenna N.; Zhou, Dejie

2016-01-01

The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone-forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone-forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone-forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer-cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2β1 integrin binding to cell-secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral-producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. Significance Despite the promise of mesenchymal stem cells (MSCs) for cell-based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell-secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair. PMID:27365484

[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.

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

Detection of Osteogenic Differentiation by Differential Mineralized Matrix Production in Mesenchymal Stromal Cells by Raman Spectroscopy

PubMed Central

Chen, He-Guei; Chiang, Hui-Hua Kenny; Lee, Oscar Kuang-Sheng

2013-01-01

Mesenchymal stromal cells (MSCs) hold great potential in skeletal tissue engineering and regenerative medicine. However, conventional methods that are used in molecular biology to evaluate osteogenic differentiation of MSCs require a relatively large amount of cells. Cell lysis and cell fixation are also required and all these steps are time-consuming. Therefore, it is imperative to develop a facile technique which can provide real-time information with high sensitivity and selectivity to detect the osteogenic maturation of MSCs. In this study, we use Raman spectroscopy as a biosensor to monitor the production of mineralized matrices during osteogenic induction of MSCs. In summary, Raman spectroscopy is an excellent biosensor to detect the extent of maturation level during MSCs-osteoblast differentiation with a non-disruptive, real-time and label free manner. We expect that this study will promote further investigation of stem cell research and clinical applications. PMID:23734254

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.

Quantitative Raman spectral changes of the differentiation of mesenchymal stem cells into islet-like cells by biochemical component analysis and multiple peak fitting

NASA Astrophysics Data System (ADS)

Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; He, Yingtian; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-12-01

Mesenchymal stem cells (MSCs) differentiate into islet-like cells, providing a possible solution for type I diabetes treatment. To search for the precise molecular mechanism of the directional differentiation of MSC-derived islet-like cells, biomolecular composition, and structural conformation information during MSC differentiation, is required. Because islet-like cells lack specific surface markers, the commonly employed immunostaining technique is not suitable for their identification, physical separation, and enrichment. Combining Raman spectroscopic data, a fitting accuracy-improved biochemical component analysis, and multiple peaks fitting approach, we identified the quantitative biochemical and intensity change of Raman peaks that show the differentiation of MSCs into islet-like cells. Along with increases in protein and glycogen content, and decreases in deoxyribonucleic acid and ribonucleic acid content, in islet-like cells relative to MSCs, it was found that a characteristic peak of insulin (665 cm-1) has twice the intensity in islet-like cells relative to MSCs, indicating differentiation of MSCs into islet-like cells was successful. Importantly, these Raman signatures provide useful information on the structural and pathological states during MSC differentiation and help to develop noninvasive and label-free Raman sorting methods for stem cells and their lineages.

Immunomodulatory function of regulatory dendritic cells induced by mesenchymal stem cells.

PubMed

Zhao, Zhi-Gang; Xu, Wen; Sun, Li; You, Yong; Li, Fang; Li, Qiu-Bai; Zou, Ping

2012-01-01

Mesenchymal stem cells (MSCs) provide an excellent model for development of stem cell therapeutics, and their potential treatment in the immunopathogenic diseases have gained further interest after demonstration of immunomodulatory effects on complicated interactions between T cells and even dendritic cells (DCs). However, the mechanisms underlying these immunoregulatory effects of MSCs are poorly understood. In this study, we show that bone marrow derived MSCs can differentiate mature DCs (mDCs) into a distinct regulatory DC population. Compared with mDCs, they have lower expression of CD1a, CD80, CD86 and CD40, but higher expression of CD11b. MSCs induced DCs (MSC-DCs) can hardly stimulate T-cell proliferation even when MSC-DCs are stimulated by LPS. In addition, high endocytosic capacity, low immunogenicity, and strong immunoregulatory function of MSC-DCs are also observed. Moreover, MSC-DCs can efficiently generate CD4+CD25+Foxp3+ Treg cells from CD4+CD25-Foxp3-T cells. The inhibitory function of MSC-DCs is mediated not only through TGF-β1, but also by inducing the production of Treg cells or T-cell anergy. These results demonstrate that the immunomodulatory effects of regulatory DCs induced by MSCs provide efficacious treatment for immunopathogenic diseases.

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.

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.

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.

Autophagy promotes apoptosis of mesenchymal stem cells under inflammatory microenvironment.

PubMed

Dang, Shipeng; Yu, Zhi-Ming; Zhang, Chang-Ying; Zheng, Jie; Li, Ku-Lin; Wu, Ying; Qian, Ling-Ling; Yang, Zhen-Yu; Li, Xiao-Rong; Zhang, Yanyun; Wang, Ru-Xing

2015-12-15

Mesenchymal stem cells (MSCs) have been widely applied to treat various inflammatory diseases. Inflammatory cytokines can induce both apoptosis and autophagy in MSCs. However, whether autophagy plays a pro- or con-apoptosis effect on MSCs in an inflammatory microenvironment has not been clarified. We inhibited autophagy by constructing MSCs with lentivirus containing small hairpin RNA to knockdown Beclin-1 and applied these MSCs to a model of sepsis to evaluate therapeutic effect of MSCs. Here we show that inhibition of autophagy in MSCs increases the survival rate of septic mice more than control MSCs, and autophagy promotes apoptosis of MSCs during application to septic mice. Further study demonstrated that autophagy aggravated tumor necrosis factor alpha plus interferon gamma-induced apoptosis of MSCs. Mechanically, autophagy inhibits the expression of the pro-survival gene Bcl-2 via suppressing reactive oxygen species/mitogen-activated protein kinase 1/3 pathway. Our findings indicate that an inflammatory microenvironment-induced autophagy promotes apoptosis of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel approach to improve MSC survival during immunotherapy.

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.

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.

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

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 stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs

PubMed Central

Phinney, Donald G.; Di Giuseppe, Michelangelo; Njah, Joel; Sala, Ernest; Shiva, Sruti; St Croix, Claudette M.; Stolz, Donna B.; Watkins, Simon C.; Di, Y. Peter; Leikauf, George D.; Kolls, Jay; Riches, David W. H.; Deiuliis, Giuseppe; Kaminski, Naftali; Boregowda, Siddaraju V.; McKenna, David H.; Ortiz, Luis A.

2015-01-01

Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs. PMID:26442449

Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs.

PubMed

Phinney, Donald G; Di Giuseppe, Michelangelo; Njah, Joel; Sala, Ernest; Shiva, Sruti; St Croix, Claudette M; Stolz, Donna B; Watkins, Simon C; Di, Y Peter; Leikauf, George D; Kolls, Jay; Riches, David W H; Deiuliis, Giuseppe; Kaminski, Naftali; Boregowda, Siddaraju V; McKenna, David H; Ortiz, Luis A

2015-10-07

Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs.

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.

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.

Activated Tissue-Resident Mesenchymal Stromal Cells Regulate Natural Killer Cell Immune and Tissue-Regenerative Function.

PubMed

Petri, Robert Michael; Hackel, Alexander; Hahnel, Katrin; Dumitru, Claudia Alexandra; Bruderek, Kirsten; Flohe, Stefanie B; Paschen, Annette; Lang, Stephan; Brandau, Sven

2017-09-12

The interaction of mesenchymal stromal cells (MSCs) with natural killer (NK) cells is traditionally thought of as a static inhibitory model, whereby resting MSCs inhibit NK cell effector function. Here, we use a dynamic in vitro system of poly(I:C) stimulation to model the interaction of NK cells and tissue-resident MSCs in the context of infection or tissue injury. The experiments suggest a time-dependent system of regulation and feedback, where, at early time points, activated MSCs secrete type I interferon to enhance NK cell effector function, while at later time points TGF-β and IL-6 limit NK cell effector function and terminate inflammatory responses by induction of a regulatory senescent-like NK cell phenotype. Importantly, feedback of these regulatory NK cells to MSCs promotes survival, proliferation, and pro-angiogenic properties. Our data provide additional insight into the interaction of stromal cells and innate immune cells and suggest a model of time-dependent MSC polarization and licensing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

[Basics and clinical application of human mesenchymal stromal/stem cells].

PubMed

Miura, Yasuo

2015-10-01

Human mesenchymal stromal/stem cells (MSCs) show a variety of biological characteristics. The clinical trials database provided by the National Institutes of Health, USA, contains about 400 clinical trials of MSCs for a wide range of therapeutic applications internationally (http://www.clinicaltrials.gov, key words "mesenchymal stem cells", as of April, 2015). Encouraging results from these clinical trials include evidence of efficacy against graft versus host disease (GVHD) in hematopoietic stem cell transplantation. Treatment for and/or prevention of engraftment failure and insufficient hematopoietic recovery have also been explored. Herein, we will address the basic principles of MSCs and the current status of clinical studies using MSCs. Future prospects for MSC-based therapy will also be discussed.

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.

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.

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.

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.

Inhibition of Bone Marrow-Derived Mesenchymal Stem Cells Homing Towards Triple-Negative Breast Cancer Microenvironment Using an Anti-PDGFRβ Aptamer

PubMed Central

Camorani, Simona; Hill, Billy Samuel; Fontanella, Raffaela; Greco, Adelaide; Gramanzini, Matteo; Auletta, Luigi; Gargiulo, Sara; Albanese, Sandra; Lucarelli, Enrico; Cerchia, Laura; Zannetti, Antonella

2017-01-01

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are shown to participate in tumor progression by establishing a favorable tumor microenvironment (TME) that promote metastasis through a cytokine networks. However, the mechanism of homing and recruitment of BM-MSCs into tumors and their potential role in malignant tissue progression is poorly understood and controversial. Here we show that BM-MSCs increase aggressiveness of triple-negative breast cancer (TNBC) cell lines evaluated as capability to migrate, invade and acquire stemness markers. Importantly, we demonstrate that the treatment of BM-MSCs with a nuclease-resistant RNA aptamer against platelet-derived growth factor receptor β (PDGFRβ) causes the inhibition of receptor-dependent signaling pathways thus drastically hampering BM-MSC recruitment towards TNBC cell lines and BM-MSCs trans-differentiation into carcinoma-associated fibroblast (CAF)-like cells. Moreover, in vivo molecular imaging analysis demonstrated the aptamer ability to prevent BM-MSCs homing to TNBC xenografts. Collectively, our results indicate the anti-PDGFRβ aptamer as a novel therapeutic tool to interfere with BM-MSCs attraction to TNBC providing the rationale to further explore the aptamer in more complex pre-clinical settings. PMID:28912898

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.

“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

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

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.

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.

Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro.

PubMed

Bussche, Leen; Harman, Rebecca M; Syracuse, Bethany A; Plante, Eric L; Lu, Yen-Chun; Curtis, Theresa M; Ma, Minglin; Van de Walle, Gerlinde R

2015-04-11

The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro. MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules. Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors. Our results demonstrate that the use of CM from MSCs might be a promising new therapy for impaired cutaneous wounds and that encapsulation may be a suitable way to effectively deliver CM to wounded cells in vivo.

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

Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism

PubMed Central

Harvey, Adam; Yen, Ten-Yang; Aizman, Irina; Tate, Ciara; Case, Casey

2013-01-01

Mesenchymal stromal cells (MSCs) transiently transfected with notch1 intracellular domain (NICD) are beneficial for neurological disorders as observed in several preclinical studies. Extracellular matrix (ECM) derived from NICD-transfected MSCs has been previously shown to support in vitro neural cell growth and survival better than that of un-transfected MSCs. To understand the underlying mechanism(s) by which NICD-transfected MSC-derived ECM supports neural cell growth and survival, we investigated the differences in NICD-transfected MSC- and MSC-derived ECM protein quantity and composition. To compare the ECM derived from MSCs and NICD-transfected MSCs, the proteins were sequentially solubilized using sodium dodecyl sulfate (SDS) and urea, quantified, and compared across four human donors. We then analyzed ECM proteins using either in-gel digests or in-solution surfactant-assisted trypsin digests (SAISD) coupled with reverse phase nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Analyses using nLC-MS/MS identified key components of ECM from NICD-transfected MSCs and un-transfected MSCs and revealed significant differences in their respective compositions. This work provides a reproducible method for identifying and comparing in vitro cell-derived ECM proteins, which is crucial for exploring the mechanisms underlying cellular therapy. PMID:24244468

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.

Characteristics, applications and prospects of mesenchymal stem cells in cell therapy.

PubMed

Guadix, Juan A; Zugaza, José L; Gálvez-Martín, Patricia

2017-05-10

Recent advances in the field of cell therapy and regenerative medicine describe mesenchymal stem cells (MSCs) as potential biological products due to their ability to self-renew and differentiate. MSCs are multipotent adult cells with immunomodulatory and regenerative properties, and, given their therapeutic potential, they are being widely studied in order to evaluate their viability, safety and efficacy. In this review, we describe the main characteristics and cellular sources of MSCs, in addition to providing an overview of their properties and current clinical applications, as well offering updated information on the regulatory aspects that define them as somatic cell therapy products. Cell therapy based on MSCs is offered nowadays as a pharmacological alternative, although there are still challenges to be addressed in this regard. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

3,3′-Diindolylmethane stimulates exosomal Wnt11 autocrine signaling in human umbilical cord mesenchymal stem cells to enhance wound healing

PubMed Central

Shi, Hui; Xu, Xiao; Zhang, Bin; Xu, Jiahao; Pan, Zhaoji; Gong, Aihua; Zhang, Xu; Li, Rong; Sun, Yaoxiang; Yan, Yongmin; Mao, Fei; Qian, Hui; Xu, Wenrong

2017-01-01

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) are suggested as a promising therapeutic tool in regenerative medicine, however, their efficacy requires improvement. Small molecules and drugs come up to be a convenient strategy in regulating stem cells fate and function. Here, we evaluated 3,3′-diindolylmethane (DIM), a natural small-molecule compound involved in the repairing effects of hucMSCs on a deep second-degree burn injury rat model. HucMSCs primed with 50 μM of DIM exhibited desirable repairing effects compared with untreated hucMSCs. DIM enhanced the stemness of hucMSCs, which was related to the activation of Wnt/β-catenin signaling. β-catenin inhibition impaired the healing effects of DIM-primed hucMSCs (DIM-hucMSCs) in vivo. Moreover, we demonstrated that DIM upregulated Wnt11 expression in hucMSC-derived exosomes. Wnt11 knockdown inhibited β-catenin activation and stemness induction in DIM-hucMSCs and abrogated their therapeutic effects in vivo. Thus, our findings indicate that DIM promotes the stemness of hucMSCs through increased exosomal Wnt11 autocrine signaling, which provides a novel strategy for improving the therapeutic effects of hucMSCs on wound healing. PMID:28529644

Intrapleural delivery of mesenchymal stem cells: a novel potential treatment for pleural diseases

PubMed Central

Qin, Zhao-hui; Qu, Jie-ming; Xu, Jin-fu; Zhang, Jing; Summah, Hanssa; Sai-Yin, He-xi Ge; Chen, Chun-mei; Yu, Long

2011-01-01

Aim: To develop a method to deliver mesenchymal stem cells (MSCs) into the pleural cavity for the treatment of pleural diseases. Methods: MSCs were isolated from rat bone marrow of rats and labeled with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) or green fluorescent protein (GFP) using a lentiviral vector. Eighteen Sprague-Dawley (SD) rats were inoculated intrapleurally with 1×106 MSCs-DAPI. The distribution of the fluorescent cells was observed using fluorescent microscopy for the following 30 d. Another 12 rats inoculated intrapleurally with 1×106 MSCs-GFP were observed for 14 d. Results: The isolated cells were typical MSC phenotypes and could differentiate into adipocytes, osteoblasts, and chondroblasts in vitro. Microscopic analysis revealed that the labeled cells adhered to the surface of the pleural cavity. The highest number of the labeled cells was found to be adhered to all specimens from the mediastinal pleura, but no labeled cells were detected in the lung parenchyma or other tissues/organs, such as the liver, kidney, spleen, and mesenterium. Incidentally, stomas were found in the mediastinal pleura. The recovered MSCs-GFP from the pleural cavity retained their ability to adhere and proliferate. Conclusion: We have established a novel method for intrapleural delivery of MSCs. The distribution of intrapleurally delivered MSCs was found to be limited to the pleurae and the pleural cavity, thereby providing us with a new approach to further investigation of the therapeutic roles of MSCs in pleural diseases. PMID:21532612

In Vivo MR Imaging of Dual MRI Reporter Genes and Deltex-1 Gene-modified Human Mesenchymal Stem Cells in the Treatment of Closed Penile Fracture.

PubMed

Guo, Ruomi; Li, Qingling; Yang, Fei; Hu, Xiaojun; Jiao, Ju; Guo, Yu; Wang, Jin; Zhang, Yong

2018-06-01

The purpose of this study was to investigate the feasibility of dual magnetic resonance imaging (MRI) reporter genes, including ferritin heavy subunit (Fth) and transferrin receptor (TfR), which provide sufficient MRI contrast for in vivo MRI tracking, and the Deltex-1 (DTX1) gene, which promotes human mesenchymal stem cell (hMSC) differentiation to smooth muscle cells (SMCs), to treat closed penile fracture (CPF). Multi-gene co-expressing hMSCs were generated. The expression of mRNA and proteins was assessed, and the original biological properties of hMSCs were determined and compared. The intracellular uptake of iron was evaluated, and the ability to differentiate into SMCs was detected. Fifty rabbits with CPF were randomly transplanted with PBS, hMSCs, Fth-TfR-hMSCs, DTX1-hMSCs, and Fth-TfR-DTX1-hMSCs. In vivo MRI was performed to detect the distribution and migration of the grafted cells and healing progress of CPF, and the results were correlated with histology. The mRNA and proteins of the multi-gene were highly expressed. The transgenes could not influence the original biological properties of hMSCs. The dual MRI reporter genes increased the iron accumulation capacity, and the DTX1 gene promoted hMSC differentiation into SMCs. The distribution and migration of the dual MRI reporter gene-modified hMSCs, and the healing state of CPF could be obviously detected by MRI and confirmed by histology. The dual MRI reporter genes could provide sufficient MRI contrast, and the distribution and migration of MSCs could be detected in vivo. The DTX1 gene can promote MSC differentiation into SMCs for the treatment of CPF and effectively inhibit granulation tissue formation.

Breast Tissue Stromal Cells Preferentially Promote Generation of M2 Macrophages: A Novel Mechanism for Tumor Supportive Properties of Breast Microenvironment

DTIC Science & Technology

2011-08-01

macrophages (MQs), on growth of breast tumor cells, and (2) to test the hypothesis that MSCs of non -breast adipose tissues, in contrast to MSCs of...macrophages in normal and malignant tissues. In contrast to all studies focused on the role of breast tissue microenvironment in growth of primary breast...the phenotype of macrophages, provide an immune environment suitable for growth of breast cancer cells, but MSCs present in non -breast adipose

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

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.

Transplantation of BDNF-Secreting Mesenchymal Stem Cells Provides Neuroprotection in Chronically Hypertensive Rat Eyes

PubMed Central

Harper, Matthew M.; Grozdanic, Sinisa D.; Blits, Bas; Kuehn, Markus H.; Zamzow, Daniel; Buss, Janice E.; Kardon, Randy H.; Sakaguchi, Donald S.

2011-01-01

Purpose. To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH). Methods. COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells (RGCs) and evaluating optic nerve cross-sections. Results. After transplantation into COH eyes, BDNF-MSCs preserved significantly more retina and optic nerve function than GFP-MSC–treated eyes when pupil light reflex (PLR) and ERG function were evaluated. PLR analysis showed significantly better function (P = 0.03) in BDNF-MSC–treated eyes (operated/control ratio = 63.00% ± 11.39%) than GFP-MSC–treated eyes (operated/control ratio = 31.81% ± 9.63%) at 42 days after surgery. The BDNF-MSC–transplanted eyes also displayed a greater level of RGC preservation than eyes that received the GFP-MSCs only (RGC cell counts: BDNF-MSC–treated COH eyes, 112.2 ± 19.39 cells/section; GFP-MSC–treated COH eyes, 52.21 ± 11.54 cells/section; P = 0.01). Conclusions. The authors have demonstrated that lentiviral-transduced BDNF-producing MSCs can survive in eyes with chronic hypertension and can provide retina and optic nerve functional and structural protection. Transplantation of BDNF-producing stem cells may be a viable treatment strategy for glaucoma. PMID:21498611

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

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.

Bone Morphogenetic Protein-2 Promotes Human Mesenchymal Stem Cell Survival and Resultant Bone Formation When Entrapped in Photocrosslinked Alginate Hydrogels.

PubMed

Ho, Steve S; Vollmer, Nina L; Refaat, Motasem I; Jeon, Oju; Alsberg, Eben; Lee, Mark A; Leach, J Kent

2016-10-01

There is a substantial need to prolong cell persistence and enhance functionality in situ to enhance cell-based tissue repair. Bone morphogenetic protein-2 (BMP-2) is often used at high concentrations for osteogenic differentiation of mesenchymal stem cells (MSCs) but can induce apoptosis. Biomaterials facilitate the delivery of lower doses of BMP-2, reducing side effects and localizing materials at target sites. Photocrosslinked alginate hydrogels (PAHs) can deliver osteogenic materials to irregular-sized bone defects, providing improved control over material degradation compared to ionically cross-linked hydrogels. It is hypothesized that the delivery of MSCs and BMP-2 from a PAH increases cell persistence by reducing apoptosis, while promoting osteogenic differentiation and enhancing bone formation compared to MSCs in PAHs without BMP-2. BMP-2 significantly decreases apoptosis and enhances survival of photoencapsulated MSCs, while simultaneously promoting osteogenic differentiation in vitro. Bioluminescence imaging reveals increased MSC survival when implanted in BMP-2 PAHs. Bone defects treated with MSCs in BMP-2 PAHs demonstrate 100% union as early as 8 weeks and significantly higher bone volumes at 12 weeks, while defects with MSC-entrapped PAHs alone do not fully bridge. This study demonstrates that transplantation of MSCs with BMP-2 in PAHs achieves robust bone healing, providing a promising platform for bone repair. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human mesenchymal stem cells suppress chronic airway inflammation in the murine ovalbumin asthma model

PubMed Central

Koloze, Mary; Lennon, Donald P.; Zuchowski, Brandon; Yang, Sung Eun; Caplan, Arnold I.

2010-01-01

Allogeneic human mesenchymal stem cells (hMSCs) introduced intravenously can have profound anti-inflammatory activity resulting in suppression of graft vs. host disease as well as regenerative events in the case of stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of these diseases. hMSCs produce bioactive factors that provide molecular cuing for: 1) immunosuppression of T cells; 2) antiscarring; 3) angiogenesis; 4) antiapoptosis; and 5) regeneration (i.e., mitotic for host-derived progenitor cells). Studies have shown that hMSCs have profound effects on the immune system and are well-tolerated and therapeutically active in immunocompetent rodent models of multiple sclerosis and stroke. Furthermore, intravenous administration of MSCs results in pulmonary localization. Asthma is a major debilitating pulmonary disease that impacts in excess of 150 million people in the world with uncontrolled asthma potentially leading to death. In addition, the socioeconomic impact of asthma-associated illnesses at the pediatric and adult level are in the millions of dollars in healthcare costs and lost days of work. hMSCs may provide a viable multiaction therapeutic for this inflammatory lung disease by secreting bioactive factors or directing cellular activity. Our studies show the effectiveness and specificity of the hMSCs on decreasing chronic airway inflammation associated with the murine ovalbumin model of asthma. In addition, the results from these studies verify the in vivo immunoeffectiveness of hMSCs in rodents and support the potential therapeutic use of hMSCs for the treatment of airway inflammation associated with chronic asthma. PMID:20817776

Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis

PubMed Central

Dang, Shipeng; Xu, Huanbai; Xu, Congfeng; Cai, Wei; Li, Qian; Cheng, Yiji; Jin, Min; Wang, Ru-Xing; Peng, Yongde; Zhang, Yi; Wu, Changping; He, Xiaozhou; Wan, Bing; Zhang, Yanyun

2014-01-01

Mesenchymal stem cell (MSC)-based therapy is a promising approach to treat various inflammatory disorders including multiple sclerosis. However, the fate of MSCs in the inflammatory microenvironment is largely unknown. Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal model of multiple sclerosis. We demonstrated that autophagy occurred in MSCs during their application for EAE treatment. Inflammatory cytokines, e.g., interferon gamma and tumor necrosis factor, induced autophagy in MSCs synergistically by inducing expression of BECN1/Beclin 1. Inhibition of autophagy by knockdown of Becn1 significantly improved the therapeutic effects of MSCs on EAE, which was mainly attributable to enhanced suppression upon activation and expansion of CD4+ T cells. Mechanistically, inhibition of autophagy increased reactive oxygen species generation and mitogen-activated protein kinase 1/3 activation in MSCs, which were essential for PTGS2 (prostaglandin-endoperoxide synthase 2 [prostaglandin G/H synthase and cyclooxygenase]) and downstream prostaglandin E2 expression to exert immunoregulatory function. Furthermore, pharmacological treatment of MSCs to inhibit autophagy increased their immunosuppressive effects on T cell-mediated EAE. Our findings indicate that inflammatory microenvironment-induced autophagy downregulates the immunosuppressive function of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel strategy to improve MSC-based immunotherapy. PMID:24905997

Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis.

PubMed

Dang, Shipeng; Xu, Huanbai; Xu, Congfeng; Cai, Wei; Li, Qian; Cheng, Yiji; Jin, Min; Wang, Ru-Xing; Peng, Yongde; Zhang, Yi; Wu, Changping; He, Xiaozhou; Wan, Bing; Zhang, Yanyun

2014-07-01

Mesenchymal stem cell (MSC)-based therapy is a promising approach to treat various inflammatory disorders including multiple sclerosis. However, the fate of MSCs in the inflammatory microenvironment is largely unknown. Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal model of multiple sclerosis. We demonstrated that autophagy occurred in MSCs during their application for EAE treatment. Inflammatory cytokines, e.g., interferon gamma and tumor necrosis factor, induced autophagy in MSCs synergistically by inducing expression of BECN1/Beclin 1. Inhibition of autophagy by knockdown of Becn1 significantly improved the therapeutic effects of MSCs on EAE, which was mainly attributable to enhanced suppression upon activation and expansion of CD4(+) T cells. Mechanistically, inhibition of autophagy increased reactive oxygen species generation and mitogen-activated protein kinase 1/3 activation in MSCs, which were essential for PTGS2 (prostaglandin-endoperoxide synthase 2 [prostaglandin G/H synthase and cyclooxygenase]) and downstream prostaglandin E2 expression to exert immunoregulatory function. Furthermore, pharmacological treatment of MSCs to inhibit autophagy increased their immunosuppressive effects on T cell-mediated EAE. Our findings indicate that inflammatory microenvironment-induced autophagy downregulates the immunosuppressive function of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel strategy to improve MSC-based immunotherapy.

Interactions between MSCs and Immune Cells: Implications for Bone Healing

PubMed Central

Kovach, Tracy K.; Dighe, Abhijit S.; Lobo, Peter I.; Cui, Quanjun

2015-01-01

It is estimated that, of the 7.9 million fractures sustained in the United States each year, 5% to 20% result in delayed or impaired healing requiring therapeutic intervention. Following fracture injury, there is an initial inflammatory response that plays a crucial role in bone healing; however, prolonged inflammation is inhibitory for fracture repair. The precise spatial and temporal impact of immune cells and their cytokines on fracture healing remains obscure. Some cytokines are reported to be proosteogenic while others inhibit bone healing. Cell-based therapy utilizing mesenchymal stromal cells (MSCs) is an attractive option for augmenting the fracture repair process. Osteoprogenitor MSCs not only differentiate into bone, but they also exert modulatory effects on immune cells via a variety of mechanisms. In this paper, we review the current literature on both in vitro and in vivo studies on the role of the immune system in fracture repair, the use of MSCs in the enhancement of fracture healing, and interactions between MSCs and immune cells. Insight into this paradigm can provide valuable clues in identifying cellular and noncellular targets that can potentially be modulated to enhance both natural bone healing and bone repair augmented by the exogenous addition of MSCs. PMID:26000315

Culturing on Wharton's jelly extract delays mesenchymal stem cell senescence through p53 and p16INK4a/pRb pathways.

PubMed

Hao, Haojie; Chen, Guanghui; Liu, Jiejie; Ti, Dongdong; Zhao, Yali; Xu, Shenjun; Fu, Xiaobing; Han, Weidong

2013-01-01

Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Wharton's jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Wharton's jelly would be beneficial for in vitro expansion of MSCs. Wharton's jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.

The Roles of Mesenchymal Stromal/Stem Cells in Tumor Microenvironment Associated with Inflammation

PubMed Central

Krstić, Jelena; Djordjević, Ivana Okić; Jauković, Aleksandra

2016-01-01

State of tumor microenvironment (TME) is closely linked to regulation of tumor growth and progression affecting the final outcome, refractoriness, and relapse of disease. Interactions of tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. Due to their outstanding features, stem cell-like properties, capacity to regulate immune response, and dynamic functional phenotype dependent on microenvironmental stimuli, MSCs have been perceived as important players in TME. Signals provided by tumor-associated chronic inflammation educate MSCs to alter their phenotype and immunomodulatory potential in favor of tumor-biased state of MSCs. Adjustment of phenotype to TME and acquisition of tumor-promoting ability by MSCs help tumor cells in maintenance of permissive TME and suppression of antitumor immune response. Potential utilization of MSCs in treatment of tumor is based on their inherent ability to home tumor tissue that makes them suitable delivery vehicles for immune-stimulating factors and vectors for targeted antitumor therapy. Here, we review data regarding intrusive effects of inflammatory TME on MSCs capacity to affect tumor development through modification of their phenotype and interactions with immune system. PMID:27630452

Indoleamine 2,3-dioxygenase and regulatory T cells in acute myeloid leukemia.

PubMed

Mansour, Iman; Zayed, Rania A; Said, Fadwa; Latif, Lamyaa Abdel

2016-09-01

The microenvironment of acute myeloid leukemia (AML) is suppressive for immune cells. Regulatory T cells (Tregs) have been recognized to play a role in helping leukemic cells to evade immunesurveillance. The mesenchymal stem cells (MSCs) are essential contributors in immunomodulation of the microenvironment as they can promote differentiation of Tregs via the indoleamine 2,3-dioxygenase (IDO) pathway. The aim of the present work was to evaluate the expression of IDO in bone marrow derived MSCs and to study its correlation to percentage of Tregs. Thirty-seven adult bone marrow samples were cultured in appropriate culture medium to isolate MSCs. Successful harvest of MSCs was determined by plastic adherence, morphology, and positive expression of CD271 and CD105; negative expression of CD34 and CD45 using flowcytometry. MSCs were examined for IDO expression by immunocytochemistry using anti-IDO monoclonal antibody. CD4+ CD25+ cells (Tregs) were measured in bone marrow samples by flowcytometry. MSCs were successfully isolated from 20 of the 37 bone marrow samples cultured. MSCs showed higher expression of IDO and Tregs percentage was higher in AML patients compared to control subjects (P = 0.002 and P < 0.001, respectively). A positive correlation was found between IDO expression and Tregs percentage (P value = 0.012, r = 0.5). In this study, we revealed an association between high IDO expression in MSCs and elevated levels of Tregs which could have an important role in the pathogenesis of AML, providing immunosuppressive microenvironment.

Overexpression of Gremlin1 in Mesenchymal Stem Cells Improves Hindlimb Ischemia in Mice by Enhancing Cell Survival.

PubMed

Xiang, Qiuling; Hong, Dongxi; Liao, Yan; Cao, Yong; Liu, Muyun; Pang, Jun; Zhou, Junjie; Wang, Guang; Yang, Renhao; Wang, Maosheng; Xiang, Andy Peng

2017-05-01

Mesenchymal stem cells (MSCs) are a promising cell resource for the treatment of ischemic diseases, partially through paracrine effects. One of the major obstacles of MSC treatment is the poor survival rate and low efficiency of transplanted stem cells due to ischemic or inflammatory environments. Gremlin1 (GREM1), a regulator of growth, differentiation and development, has been identified as a novel proangiogenic factor. However, the role and mechanism of GREM1 in MSCs remains unclear. Therefore, we assessed the putative beneficial effects of GREM1 on MSC-based therapy for hindlimb ischemia. The lentiviral vector, EF1a-GREM1, was constructed using the Multisite Gateway System and used to transduce MSCs. In vitro studies demonstrated increased survival of GREM1-MSCs exposed to H 2 O 2 , which is consistent with the activation of caspase-3. Conditional medium from GREM1-MSCs (GREM1-MSC-CM) increased the anti-apoptotic effects of human umbilical vein endothelial cells (HUVECs), and this effect was attenuated by treatment with the PI3K/Akt pathway inhibitor LY294002. MSCs modified with GREM1 could significantly increase blood perfusion of the ischemic hindlimb in vivo in a mouse model, which was correlated to improved MSC survival. This study demonstrates that overexpression of GREM1 in MSCs have greater therapeutic effects against ischemia compared with wild-type MSCs by enhancing the survival of MSCs and ECs, which may provide new tools for studies investigating the treatment of ischemic diseases. J. Cell. Physiol. 232: 996-1007, 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.

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.

Differentiation within autologous fibrin scaffolds of porcine dermal cells with the mesenchymal stem cell phenotype

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

Puente, Pilar de la, E-mail: pilardelapuentegarcia@gmail.com; Ludeña, Dolores; López, Marta

2013-02-01

Porcine mesenchymal stem cells (pMSCs) are an attractive source of cells for tissue engineering because their properties are similar to those of human stem cells. pMSCs can be found in different tissues but their dermal origin has not been studied in depth. Additionally, MSCs differentiation in monolayer cultures requires subcultured cells, and these cells are at risk of dedifferentiation when implanting them into living tissue. Following this, we attempted to characterize the MSCs phenotype of porcine dermal cells and to evaluate their cellular proliferation and differentiation in autologous fibrin scaffolds (AFSs). Dermal biopsies and blood samples were obtained from 12more » pigs. Dermal cells were characterized by flow cytometry. Frozen autologous plasma was used to prepare AFSs. pMSC differentiation was studied in standard structures (monolayers and pellets) and in AFSs. The pMSCs expressed the CD90 and CD29 markers of the mesenchymal lineage. AFSs afforded adipogenic, osteogenic and chondrogenic differentiation. The porcine dermis can be proposed to be a good source of MSCs with adequate proliferative capacity and a suitable expression of markers. The pMSCs also showed optimal proliferation and differentiation in AFSs, such that these might serve as a promising autologous and implantable material for use in tissue engineering. -- Highlights: ► Low fibrinogen concentration provides a suitable matrix for cell migration and differentiation. ► Autologous fibrin scaffolds is a promising technique in tissue engineering. ► Dermal cells are an easily accessible mesenchymal stem cell source. ► Fibrin scaffolds afforded adipogenic, osteogenic and chondrogenic differentiation.« less

Hepatocyte growth factor enhances the inflammation-alleviating effect of umbilical cord-derived mesenchymal stromal cells in a bronchiolitis obliterans model.

PubMed

Cao, Xiao-Pei; Han, Dong-Mei; Zhao, Li; Guo, Zi-Kuan; Xiao, Feng-Jun; Zhang, Yi-Kun; Zhang, Xiao-Yan; Wang, Li-Sheng; Wang, Heng-Xiang; Wang, Hua

2016-03-01

Specific and effective therapy for prevention or reversal of bronchiolitis obliterans (BO) is lacking. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF) gene modified mesenchymal stromal cells (MSCs) on BO. A mouse model of experimental BO was established by subcutaneously transplanting the tracheas from C57BL/6 mice into Balb/C recipients, which were then administered saline, Ad-HGF-modified human umbilical cord-MSCs (MSCs-HGF) or Ad-Null-modified MSCs (MSCs-Null). The therapeutic effects of MSCs-Null and MSCs-HGF were evaluated by using fluorescence-activated cell sorting (FACS) for lymphocyte immunophenotype of spleen, enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (rt-PCR) for cytokine expression, and histopathological analysis for the transplanted trachea. The histopathologic recovery of allograft tracheas was improved significantly after MSCs-Null and MSCs-HGF treatment and the beneficial effects were particularly observed in MSCs-HGF-treated mice. Furthermore, the allo-transplantation-induced immunophenotype disorders of the spleen, including regulatory T (Treg), T helper (Th)1, Th2 and Th17, were attenuated in both cell-treated groups. MSCs-HGF treatment reduced expression and secretion of inflammation cytokines interferon-gamma (IFN-γ), and increased expression of anti-inflammatory cytokine interleukin (IL)-4 and IL-10. It also decreased the expression level of the profibrosis factor transforming growth factor (TGF)-β. Treatment of BO with HGF gene modified MSCs results in reduction of local inflammation and promotion in recovery of allograft trachea histopathology. These findings might provide an effective therapeutic strategy for BO. 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

Comparative study of adipose-derived stem cells and bone marrow-derived stem cells in similar microenvironmental conditions

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

Guneta, Vipra; Tan, Nguan Soon; KK Research Centre, KK Women's and Children Hospital, 100 Bukit Timah Road, Singapore 229899

Mesenchymal stem cells (MSCs), which were first isolated from the bone marrow, are now being extracted from various other tissues in the body, including the adipose tissue. The current study presents systematic evidence of how the adipose tissue-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (Bm-MSCs) behave when cultured in specific pro-adipogenic microenvironments. The cells were first characterized and identified as MSCs in terms of their morphology, phenotypic expression, self-renewal capabilities and multi-lineage potential. Subsequently, the proliferation and gene expression profiles of the cell populations cultured on two-dimensional (2D) adipose tissue extracellular matrix (ECM)-coated tissue culture plastic (TCP)more » and in three-dimensional (3D) AlgiMatrix® microenvironments were analyzed. Overall, it was found that adipogenesis was triggered in both cell populations due to the presence of adipose tissue ECM. However, in 3D microenvironments, ASCs and Bm-MSCs were predisposed to the adipogenic and osteogenic lineages respectively. Overall, findings from this study will contribute to ongoing efforts in adipose tissue engineering as well as provide new insights into the role of the ECM and cues provided by the immediate microenvironment for stem cell differentiation. - Highlights: • Native adipose tissue ECM coated on 2D TCP triggers adipogenesis in both ASCs and Bm-MSCs. • A 3D microenvironment with similar stiffness to adipose tissue induces adipogenic differentiation of ASCs. • ASCs cultured in 3D alginate scaffolds exhibit predisposition to adipogenesis. • Bm-MSCs cultured in 3D alginate scaffolds exhibit predisposition to osteogenesis. • The native microenvironment of the cells affects their differentiation behaviour in vitro.« less

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.

Transplantation of CX3CL1-expressing mesenchymal stem cells provides neuroprotective and immunomodulatory effects in a rat model of retinal degeneration.

PubMed

Huang, Libin; Xu, Wei; Xu, Guoxing

2013-08-01

To investigate the neuroprotective and immunomodulatory effects of mesenchymal stem cells (MSCs) engineered to secrete CX3CL1 on the light-injured retinal structure and function. Normal MSCs and CX3CL1-expressing MSCs (CX3CL1-MSCs) were transplanted into the subretinal space of light-injured rats. By ERG and TUNEL methods, their rescue effect of the host retina was compared with untreated light-injured and vehicle-injected rats. Activated microglia in the retina were stained by ED-1 antibody, and Western blot was performed to quantify cytokines secreted by the retina post-transplantation. ERG analysis showed better function in CX3CL1-MSC-injected group than other groups at 21 days after transplantation (p < 0.05). CX3CL1-MSCs inhibited apoptosis of the retinal cells and microglial activation. Neurotrophic factors expression in host retina that received CX3CL1-MSCs was stronger than in the retina that received normal MSCs. Conversely, the expression of proinflammatory factors was downregulated. CX3CL1-MSCs subretinal transplantation may enhance protective effect against light-induced retinal degeneration.

Mesenchymal stem cells: a double-edged sword in regulating immune responses

PubMed Central

Li, W; Ren, G; Huang, Y; Su, J; Han, Y; Li, J; Chen, X; Cao, K; Chen, Q; Shou, P; Zhang, L; Yuan, Z-R; Roberts, A I; Shi, S; Le, A D; Shi, Y

2012-01-01

Mesenchymal stem cells (MSCs) have been employed successfully to treat various immune disorders in animal models and clinical settings. Our previous studies have shown that MSCs can become highly immunosuppressive upon stimulation by inflammatory cytokines, an effect exerted through the concerted action of chemokines and nitric oxide (NO). Here, we show that MSCs can also enhance immune responses. This immune-promoting effect occurred when proinflammatory cytokines were inadequate to elicit sufficient NO production. When inducible nitric oxide synthase (iNOS) production was inhibited or genetically ablated, MSCs strongly enhance T-cell proliferation in vitro and the delayed-type hypersensitivity response in vivo. Furthermore, iNOS−/− MSCs significantly inhibited melanoma growth. It is likely that in the absence of NO, chemokines act to promote immune responses. Indeed, in CCR5−/−CXCR3−/− mice, the immune-promoting effect of iNOS−/− MSCs is greatly diminished. Thus, NO acts as a switch in MSC-mediated immunomodulation. More importantly, the dual effect on immune reactions was also observed in human MSCs, in which indoleamine 2,3-dioxygenase (IDO) acts as a switch. This study provides novel information about the pathophysiological roles of MSCs. PMID:22421969

Isolation, Characterization and Growth Kinetic Comparison of Bone Marrow and Adipose Tissue Mesenchymal Stem Cells of Guinea Pig.

PubMed

Aliborzi, Ghaem; Vahdati, Akbar; Mehrabani, Davood; Hosseini, Seyed Ebrahim; Tamadon, Amin

2016-05-30

Mesenchymal stem cells (MSCs) from different sources have different characteristics. Moreover, MSCs are not isolated and characterized in Guinea pig for animal model of cell therapy. was the isolating of bone marrow MSCs (BM-MSCs) and adipose tissue MSCs (AT-MSCs) from Guinea pig and assessing their characteristics. In this study, bone marrow and adipose tissue were collected from three Guinea pigs and cultured and expanded through eight passages. BM-MSCs and AT-MSCs at passages 2, 5 and 8 were seeded in 24-well plates in triplicate. Cells were counted from each well 1~7 days after seeding to determine population doubling time (PDT) and cell growth curves. Cells of passage 3 were cultured in osteogenic and adipogenic differentiation media. BM-MSCs and AT-MSCs attached to the culture flask and displayed spindle-shaped morphology. Proliferation rate of AT-MSCs in the analyzed passages was more than BM-MSCs. The increase in the PDT of MSCs occurs with the increase in the number of passages. Moreover, after culture of BM-MSCs and AT-MSCs in differentiation media, the cells differentiated toward osteoblasts and adipocytes as verified by Alizarin Red staining and Oil Red O staining, respectively. BM-MSCs and AT-MSCs of Guinea pig could be valuable source of multipotent stem cells for use in experimental and preclinical studies in animal models.

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.

Rationale of mesenchymal stem cell therapy in kidney injury.

PubMed

Cantaluppi, Vincenzo; Biancone, Luigi; Quercia, Alessandro; Deregibus, Maria Chiara; Segoloni, Giuseppe; Camussi, Giovanni

2013-02-01

Numerous preclinical and clinical studies suggest that mesenchymal stem cells, also known as multipotent mesenchymal stromal cells (MSCs), may improve pathologic conditions involving different organs. These beneficial effects initially were ascribed to the differentiation of MSCs into organ parenchymal cells. However, at least in the kidney, this is a very rare event and the kidney-protective effects of MSCs have been attributed mainly to paracrine mechanisms. MSCs release a number of trophic, anti-inflammatory, and immune-modulatory factors that may limit kidney injury and favor recovery. In this article, we provide an overview of the biologic activities of MSCs that may be relevant for the treatment of kidney injury in the context of a case vignette concerning a patient at high immunologic risk who underwent a second kidney transplantation followed by the development of ischemia-reperfusion injury and acute allograft rejection. We discuss the possible beneficial effect of MSC treatment in the light of preclinical and clinical data supporting the regenerative and immunomodulatory potential of MSCs. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Mesenchymal Stem Cells as Therapeutics Agents: Quality and Environmental Regulatory Aspects

PubMed Central

Sabata, Roger; Verges, Josep; Zugaza, José L.; Ruiz, Adolfina; Clares, Beatriz

2016-01-01

Mesenchymal stem cells (MSCs) are one of the main stem cells that have been used for advanced therapies and regenerative medicine. To carry out the translational clinical application of MSCs, their manufacturing and administration in human must be controlled; therefore they should be considered as medicine: stem cell-based medicinal products (SCMPs). The development of MSCs as SCMPs represents complicated therapeutics due to their extreme complex nature and rigorous regulatory oversights. The manufacturing process of MSCs needs to be addressed in clean environments in compliance with requirements of Good Manufacturing Practice (GMP). Facilities should maintain these GMP conditions according to international and national medicinal regulatory frameworks that introduce a number of specifications in order to produce MSCs as safe SCMPs. One of these important and complex requirements is the environmental monitoring. Although a number of environmental requirements are clearly defined, some others are provided as recommendations. In this review we aim to outline the current issues with regard to international guidelines which impact environmental monitoring in cleanrooms and clean areas for the manufacturing of MSCs. PMID:27999600

Premature exhaustion of mesenchymal stromal cells from myelodysplastic syndrome patients.

PubMed

Pang, Yanbin; Deng, Chengxin; Geng, Suxia; Weng, Jianyu; Lai, Peilong; Liao, Pengjun; Zeng, Lingji; Lu, Zesheng; Zhang, Jing; Du, Xin

2017-01-01

Myelodysplastic syndrome (MDS) predominantly occurs in aging people. Over the past decades, the cellular and molecular pathologies of MDS cells have been intensively investigated. However, how the bone marrow stromal niches are altered during MDS development remains elusive. In this study, we attempted to isolate and characterize mesenchymal stromal cells (MSCs) from 30 MDS patients. We observed that only 9/30 bone marrow aspirations from MDS patients successfully formed a monolayer in vitro, while 17/17 bone marrow aspirations from normal donors (median age 45 years, range: 22-73 years) succeeded in this process. Compared to normal MSCs, the MDS MSCs showed premature exhaustion, including reduced osteogenic differentiation ability, slower passage rate, and extremely limited passage times. These functional defects were associated with downregulation of Osterix and Runx2 genes and increased cell cycle arrest and apoptosis. However, the premature exhaustion of MDS MSCs did not correlate with patients' ages, indicating that natural aging is not the cause of dysfunction in MDS MSCs. Our result provides a strong rational to target prematurely exhausting MSCs in future MDS treatment.

Imaging modalities for the in vivo surveillance of mesenchymal stromal cells.

PubMed

Hossain, Mohammad Ayaz; Chowdhury, Tina; Bagul, Atul

2015-11-01

Bone marrow stromal cells exist as mesenchymal stromal cells (MSCs) and have the capacity to differentiate into multiple tissue types when subjected to appropriate culture conditions. This property of MSCs creates therapeutic opportunities in regenerative medicine for the treatment of damage to neural, cardiac and musculoskeletal tissues or acute kidney injury. The prerequisite for successful cell therapy is delivery of cells to the target tissue. Assessment of therapeutic outcomes utilize traditional methods to examine cell function of MSC populations involving routine biochemical or histological analysis for cell proliferation, protein synthesis and gene expression. However, these methods do not provide sufficient spatial and temporal information. In vivo surveillance of MSC migration to the site of interest can be performed through a variety of imaging modalities such as the use of radiolabelling, fluc protein expression bioluminescence imaging and paramagnetic nanoparticle magnetic resonance imaging. This review will outline the current methods of in vivo surveillance of exogenously administered MSCs in regenerative medicine while addressing potential technological developments. Furthermore, nanoparticles and microparticles for cellular labelling have shown that migration of MSCs can be spatially and temporally monitored. In vivo surveillance therefore permits time-stratified assessment in animal models without disruption of the target organ. In vivo tracking of MSCs is non-invasive, repeatable and non-toxic. Despite the excitement that nanoparticles for tracking MSCs offer, delivery methods are difficult because of the challenges with imaging three-dimensional systems. The current advances and growth in MSC research, is likely to provide a wealth of evidence overcoming these issues. Copyright © 2014 John Wiley & Sons, Ltd.

14S,21R-dihydroxy-docosahexaenoic acid treatment enhances mesenchymal stem cell amelioration of renal ischemia/reperfusion injury.

PubMed

Tian, Haibin; Lu, Yan; Shah, Shraddha P; Wang, Quansheng; Hong, Song

2012-05-01

Bone marrow mesenchymal stem cells (MSCs) have shown potential to improve treatment of renal failure. The prohealing functions of MSCs have been found to be enhanced by treatment with the lipid mediator, 14S,21R-dihydroxy-docosa4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (14S,21R-diHDHA). In this article, using a murine model of renal ischemia/reperfusion (I/R) injury, we found that treatment with 14S,21R-diHDHA enhanced MSC amelioration of renal I/R injury. Treated MSCs more efficiently inhibited I/R-induced elevation of serum creatinine levels, reduced renal tubular cell death, and inhibited infiltration of neutrophils, macrophages, and dendritic cells in kidneys. Conditioned medium from treated MSCs reduced the generation of tumor necrosis factor-α and reactive oxygen species by macrophages under I/R conditions. Infusion of treated MSCs more efficiently reduced I/R-damage to renal histological structures compared with untreated MSCs (injury score: 7.9±0.4 vs. 10.5±0.5). Treated MSCs were resistant to apoptosis in vivo when transplanted under capsules of I/R-injured kidneys (active caspase-3+ MSCs: 4.2%±2.8% vs. 11.7%±2.4% of control) and in vitro when cultured under I/R conditions. Treatment with 14S,21R-diHDHA promoted viability of MSCs through a mechanism involving activation of the phosphoinositide 3-kinase -Akt signaling pathway. Additionally, treatment of MSCs with 14S,21R-diHDHA promoted secretion of renotrophic hepatocyte growth factor and insulin growth factor-1. Similar results were obtained when 14S,21RdiHDHA was used to inhibit apoptosis of human MSCs (hMSCs) and to increase the generation of renotrophic cytokines from hMSCs. These findings provide a lead for new strategies in the treatment of acute kidney injury with MSCs.

Intrinsic and extrinsic mechanical properties related to the differentiation of mesenchymal stem cells.

PubMed

Lee, Jin-Ho; Park, Hun-Kuk; Kim, Kyung Sook

2016-05-06

Diverse intrinsic and extrinsic mechanical factors have a strong influence on the regulation of stem cell fate. In this work, we examined recent literature on the effects of mechanical environments on stem cells, especially on differentiation of mesenchymal stem cells (MSCs). We provide a brief review of intrinsic mechanical properties of single MSC and examined the correlation between the intrinsic mechanical property of MSC and the differentiation ability. The effects of extrinsic mechanical factors relevant to the differentiation of MSCs were considered separately. The effect of nanostructure and elasticity of the matrix on the differentiation of MSCs were summarized. Finally, we consider how the extrinsic mechanical properties transfer to MSCs and then how the effects on the intrinsic mechanical properties affect stem cell differentiation. Copyright © 2015 Elsevier Inc. All rights reserved.

Canine Mesenchymal Stem Cell Potential and the Importance of Dog Breed: Implication for Cell-Based Therapies.

PubMed

Bertolo, Alessandro; Steffen, Frank; Malonzo-Marty, Cherry; Stoyanov, Jivko

2015-01-01

The study of canine bone marrow-derived mesenchymal stem cells (MSCs) has a prominent position in veterinary cell-based applications. Yet the plethora of breeds, their different life spans, and interbreed variations provide unclearness on what can be achieved specifically by such therapies. In this study, we compared a set of morphological, physiological, and genetic markers of MSCs derived from large dog breeds, namely, Border collie, German shepherd, Labrador, Malinois, Golden retriever, and Hovawart. We compared colony-forming units (CFUs) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-gal) activity, telomere length, and gene expression of MSCs, as well as the ability of cells to differentiate to osteogenic, adipogenic, and chondrogenic phenotypes. The influence of the culture media α-MEM, low-glucose DMEM, and high-glucose DMEM, used in cell isolation and expansion, was investigated in the presence and absence of basic fibroblast growth factor (bFGF). Initial cell yield was not affected by culturing medium, but MSCs expanded best in α-MEM supplemented with bFGF. After isolation, the number of MSCs was similar among breeds--as shown by equivalent CFUs--except in the Hovawart samples, which had fivefold less CFU. Telomere lengths were similar among breeds. MSCs divided actively only for 4 weeks in culture (PDT = ∼50 h/division), except Border collie cells divided for a longer time than cells from other groups. The percentage of senescent cells increased linearly in all breeds with time, with a faster rate in German shepherd, Labrador, and Golden retriever. Border collie cells underwent efficient osteogenic differentiation, Hovawart cells performed the best in chondrogenic differentiation, and Labrador cells in both, while German shepherd cells had the lower differentiation potential. MSCs from all breeds preserved the same adipogenic differentiation potential. In conclusion, despite variations, isolated MSCs can be expanded and differentiated in vitro, and all breeds are eligible for MSC-based therapies.

In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

PubMed

Amari, Afshin; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Soleimani, Masoud; Mohammadi Amirabad, Leila; Tahoori, Mohammad Taher; Massumi, Mohammad

2015-08-01

Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

Mesenchymal Stem Cells in the Musculoskeletal System: From Animal Models to Human Tissue Regeneration?

PubMed

Čamernik, Klemen; Barlič, Ariana; Drobnič, Matej; Marc, Janja; Jeras, Matjaž; Zupan, Janja

2018-06-01

The musculoskeletal system includes tissues that have remarkable regenerative capabilities. Bone and muscle sustain micro-damage throughout the lifetime, yet they continue to provide the body with the support that is needed for everyday activities. Our current understanding is that the regenerative capacity of the musculoskeletal system can be attributed to the mesenchymal stem/ stromal cells (MSCs) that reside within its different anatomical compartments. These MSCs can replenish various tissues with progenitor cells to form functional cells, such as osteoblasts, chondrocytes, myocytes, and others. However, with aging and in certain disorders of the musculoskeletal system such as osteoarthritis or osteoporosis, this regenerative capacity of MSCs appears to be lost or diverted for the production of other non-functional cell types, such as adipocytes and fibroblasts. In this review, we shed light on the tissue sources and subpopulations of MSCs in the musculoskeletal system that have been identified in animal models, discuss the mechanisms of their anti-inflammatory action as a prerequisite for their tissue regeneration and their current applications in regenerative medicine. While providing up-to-date evidence of the role of MSCs in different musculoskeletal pathologies, in particular in osteoporosis and osteoarthritis, we share some thoughts on their potential as diagnostic markers in musculoskeletal health and disease.

Hypoxia preconditioning of mesenchymal stromal cells enhances PC3 cell lymphatic metastasis accompanied by VEGFR-3/CCR7 activation.

PubMed

Huang, Xin; Su, Kunkai; Zhou, Limin; Shen, Guofang; Dong, Qi; Lou, Yijia; Zheng, Shu

2013-12-01

Mesenchymal stromal cells (MSCs) in bone marrow may enhance tumor metastases through the secretion of chemokines. MSCs have been reported to home toward the hypoxic tumor microenvironment in vivo. In this study, we investigated prostate cancer PC3 cell behavior under the influence of hypoxia preconditioned MSCs and explored the related mechanism of prostate cancer lymphatic metastases in mice. Transwell assays revealed that VEGF-C receptor, VEGFR-3, as well as chemokine CCL21 receptor, CC chemokine receptor 7 (CCR7), were responsible for the migration of PC3 cells toward hypoxia preconditioned MSCs. Knock-in Ccr7 in PC3 cells also improved cell migration in vitro. Furthermore, when PC3 cells were labeled using the hrGfp-lentiviral vector, and were combined with hypoxia preconditioned MSCs for xenografting, it resulted in an enhancement of lymph node metastases accompanied by up-regulation of VEGFR-3 and CCR7 in primary tumors. Both PI3K/Akt/IκBα and JAK2/STAT3 signaling pathways were activated in xenografts in the presence of hypoxia-preconditioned MSCs. Unexpectedly, the p-VEGFR-2/VEGFR-2 ratio was attenuated accompanied by decreased JAK1 expression, indicating a switching-off of potential vascular signal within xenografts in the presence of hypoxia-preconditioned MSCs. Unlike results from other studies, VEGF-C maintained a stable expression in both conditions, which indicated that hypoxia preconditioning of MSCs did not influence VEGF-C secretion. Our results provide the new insights into the functional molecular events and signalings influencing prostate tumor metastases, suggesting a hopeful diagnosis and treatment in new approaches. © 2013 Wiley Periodicals, Inc.

The Wnt5a Receptor, Receptor Tyrosine Kinase-Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation.

PubMed

Dickinson, Sally C; Sutton, Catherine A; Brady, Kyla; Salerno, Anna; Katopodi, Theoni; Williams, Rhys L; West, Christopher C; Evseenko, Denis; Wu, Ling; Pang, Suzanna; Ferro de Godoy, Roberta; Goodship, Allen E; Péault, Bruno; Blom, Ashley W; Kafienah, Wael; Hollander, Anthony P

2017-11-01

Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2-ve or unfractionated MSCs. In a sheep cartilage-repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis-derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280-2291. © 2017 AlphaMed Press.

The Wnt5a Receptor, Receptor Tyrosine Kinase‐Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation

PubMed Central

Dickinson, Sally C.; Sutton, Catherine A.; Brady, Kyla; Salerno, Anna; Katopodi, Theoni; Williams, Rhys L.; West, Christopher C.; Evseenko, Denis; Wu, Ling; Pang, Suzanna; Ferro de Godoy, Roberta; Goodship, Allen E.; Péault, Bruno; Blom, Ashley W.; Kafienah, Wael

2017-01-01

Abstract Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase‐like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2–ve or unfractionated MSCs. In a sheep cartilage‐repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis‐derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280–2291 PMID:28833807

Allogeneic MSCs and Recycled Autologous Chondrons Mixed in a One-Stage Cartilage Cell Transplantion: A First-in-Man Trial in 35 Patients.

PubMed

de Windt, Tommy S; Vonk, Lucienne A; Slaper-Cortenbach, Ineke C M; Nizak, Razmara; van Rijen, Mattie H P; Saris, Daniel B F

2017-08-01

MSCs are known as multipotent mesenchymal stem cells that have been found capable of differentiating into various lineages including cartilage. However, recent studies suggest MSCs are pericytes that stimulate tissue repair through trophic signaling. Aimed at articular cartilage repair in a one-stage cell transplantation, this study provides first clinical evidence that MSCs stimulate autologous cartilage repair in the knee without engrafting in the host tissue. A phase I (first-in-man) clinical trial studied the one-stage application of allogeneic MSCs mixed with 10% or 20% recycled defect derived autologous chondrons for the treatment of cartilage defects in 35 patients. No treatment-related serious adverse events were found and statistically significant improvement in clinical outcome shown. Magnetic resonance imaging and second-look arthroscopies showed consistent newly formed cartilage tissue. A biopsy taken from the center of the repair tissue was found to have hyaline-like features with a high concentration of proteoglycans and type II collagen. DNA short tandem repeat analysis delivered unique proof that the regenerated tissue contained patient-DNA only. These findings support the hypothesis that allogeneic MSCs stimulate a regenerative host response. This first-in-man trial supports a paradigm shift in which MSCs are applied as augmentations or "signaling cells" rather than differentiating stem cells and opens doors for other applications. Stem Cells 2017;35:1984-1993. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

SDF-1 improves wound healing ability of glucocorticoid-treated adipose tissue-derived mesenchymal stem cells.

PubMed

Kato, Toshiki; Khanh, Vuong Cat; Sato, Kazutoshi; Takeuchi, Kosuke; Carolina, Erica; Yamashita, Toshiharu; Sugaya, Hisashi; Yoshioka, Tomokazu; Mishima, Hajime; Ohneda, Osamu

2017-11-18

Glucocorticoids cause the delayed wound healing by suppressing inflammation that is required for wound healing process. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) play an important role for wound healing by their cytokine productions including stromal derived factor 1 (SDF-1). However, it has not been clear how glucocorticoids affect the wound healing ability of AT-MSCs. In this study, we found that glucocorticoid downregulated SDF-1 expression in AT-MSCs. In addition, glucocorticoid-treated AT-MSCs induced less migration of inflammatory cells and impaired wound healing capacity compared with glucocorticoid-untreated AT-MSCs. Of note, prostaglandin E2 (PGE2) synthesis-related gene expression was downregulated by glucocorticoid and PGE2 treatment rescued not only SDF-1 expression in the presence of glucocorticoid but also their wound healing capacity in vivo. Furthermore, we found SDF-1-overexpressed AT-MSCs restored wound healing capacity even after treatment of glucocorticoid. Consistent with the results obtained from glucocorticoid-treated AT-MSCs, we found that AT-MSCs isolated from steroidal osteonecrosis donors (sAT-MSCs) who received chronic glucocorticoid therapy showed less SDF-1 expression and impaired wound healing capacity compared with traumatic osteonecrosis donor-derived AT-MSCs (nAT-MSCs). Moreover, the SDF-1 level was also reduced in plasma derived from steroidal osteonecrosis donors compared with traumatic osteonecrosis donors. These results provide the evidence that concomitant application of AT-MSCs with glucocorticoid shows impaired biological modulatory effects that induce impaired wound healing. Copyright © 2017 Elsevier Inc. All rights reserved.

Pro-osteogenic effects of fibrin glue in treatment of avascular necrosis of the femoral head in vivo by hepatocyte growth factor-transgenic mesenchymal stem cells

PubMed Central

2014-01-01

Background Autologous transplantation of modified mesenchymal stem cells (MSCs) is a promising candidate for the treatment of the refractory clinical disease, avascular necrosis of the femoral head (ANFH). Our previous attempts by compounding MSCs with medical fibrin glue to treat ANFH in animal model have achieved excellent effects. However, the underlying molecular mechanism is unclear, especially on the transgenic gene expression. Methods Rabbit MSCs were isolated and compounded with fibrin glue. Following degrading of fibrin glue, proliferation, viability, expression of transgenic hepatocyte growth factor gene as well as osteogenic differentiation of MSCs were evaluated together with that of uncompounded MSCs. Fibrin glue-compounded MSCs were transplanted into the lesion of ANFH model, and the therapeutic efficacy was compared with uncompounded MSCs. One-Way ANOVA was used to determine the statistical significance among treatment groups. Results Fibrin glue compounding will not affect molecular activities of MSCs, including hepatocyte growth factor (HGF) secretion, cell proliferation and viability, and osteogenic differentiation in vitro. When applying fibrin glue-compounded MSCs for the therapy of ANFH in vivo, fibrin glue functioned as a drug delivery system and provided a sustaining microenvironment for MSCs which helped the relatively long-term secretion of HGF in the femoral head lesion and resulted in improved therapeutic efficacy when compared with uncompounded MSCs as indicated by hematoxylin-eosin staining and immunohistochemistry of osteocalcin, CD105 and HGF. Conclusion Transplantation of fibrin glue-compounding MSCs is a promising novel method for ANFH therapy. PMID:24885252

Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

PubMed

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2015-10-15

Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils. Specifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces. Collagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space. These findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.

Fibrin Glue Improves the Therapeutic Effect of MSCs by Sustaining Survival and Paracrine Function

PubMed Central

Kim, Inok; Lee, Sung Koo; Yoon, Jung In; Kim, Da Eun

2013-01-01

Fibrin glue has been widely investigated as a cell delivery vehicle for improving the therapeutic effects of mesenchymal stem cells (MSCs). Implanted MSCs produce their therapeutic effects by secreting paracrine factors and by replacing damaged tissues after differentiation. While the influence of fibrin glue on the differentiation potential of MSCs has been well documented, its effect on paracrine function of MSCs is largely unknown. Herein we investigated the influence of fibrin glue on the paracrine effects of MSCs. MSCs were isolated from human adipose tissue. The effects of fibrin glue on survival, migration, secretion of growth factors, and immune suppression of MSCs were investigated in vitro. MSCs in fibrin glue survived and secreted growth factors such as the vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) over 14 days. VEGF and immune modulators, including the transforming growth factor (TGF)-β1 and prostaglandin E2, secreted from MSCs in fibrin glue significantly increased under inflammatory conditions. Thus, MSCs in fibrin glue effectively suppressed immune reactions. In addition, fibrin glue protected the MSCs from oxidative stress and prevented human dermal fibroblast death induced by exposure to extreme stress. In contrast, MSCs within fibrin glue hardly migrated. These results suggest that fibrin glue may sustain survival of implanted MSCs and their paracrine function. Our results provide a mechanistic data to allow further development of MSCs with fibrin glue as a clinical treatment. PMID:23701237

In vitro and in vivo study of the application of volvox spheres to co-culture vehicles in liver tissue engineering.

PubMed

Chang, Siou Han; Huang, Han Hsiang; Kang, Pei Leun; Wu, Yu Chian; Chang, Ming-Huang; Kuo, Shyh Ming

2017-11-01

Volvox sphere is a biomimetic concept of a natural Volvox, wherein a large outer sphere contains smaller inner spheres, which can encapsulate cells and provide a double-layer three-dimensional environment for culturing cells. This study simultaneously encapsulated rat mesenchymal stem cells (MSCs) and AML12 hepatocytes in volvox spheres and extensively evaluated the effects of various culturing modes on cell functions and fates. The results showed that compared with a static flask culture, MSCs encapsulated in volvox spheres differentiated into hepatocyte-like cells with a 2-fold increase in albumin (ALB) expression and a 2.5-fold increase in cytokeratin 18 expression in a dynamic bioreactor. Moreover, the restorative effects of volvox spheres encapsulating cells on retrorsine-exposed CCl 4 -induced liver injuries in rats were evaluated. The data presented significant reductions in AST and ALT levels after the implantation of volvox spheres encapsulating both MSCs and AML12 hepatocytes in vivo. In contrast to the negative control group, histopathological analysis demonstrated liver repair and formation of the new liver tissue in groups implanted with volvox spheres containing cells. These results demonstrate that liver cells implanted with volvox spheres encapsulating both MSCs and AML12 hepatocytes promote liver repair and liver tissue regeneration in liver failure caused by necrotizing agents such as retrorsine and CCl 4 . Hence, volvox spheres encapsulating MSCs and liver cells can be a promising and clinically effective therapy for liver injury. In this study, we used a volvox sphere, which is a unique design that mimics the natural Volvox, that consists of a large outer sphere that contains smaller inner spheres, which provide a three-dimensional environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in volvox spheres and evaluate two different culture methods, dynamic bioreactor and static culture flask,on the cultured cells. In addition, we aimed to evaluate the restorative effects of volvox spheres encapsulating MSCs and/or AML12 liver cells on rats with retrorsine-exposed CCl 4 -induced liver injuries. The results showed that MSCs encapsulated in volvox spheres differentiated into hepatocyte-like cells with a 2-fold increase in albumin expression and a 2.5-fold increase in cytokeratin 18 expression ina dynamic bioreactor. Moreover, the data presented significant reductions in AST and ALT levels after the implantation of volvox spheres encapsulating both MSCs and AML12 hepatocytes in vivo. In contrast to the negative control group, histopathological analysis demonstrated liver repair and formation of new liver tissue in groups implanted with volvox spheres containing cells. These results demonstrate that liver cells implanted with volvox spheres encapsulating both MSCs and AML12 hepatocytes promote liver repair and liver tissue regeneration in liver failure caused by necrotizing agents such as retrorsine and CCl 4 . Hence, volvox spheres encapsulating MSCs and liver cells can be a promising and clinically effective therapy for liver injury. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

Genetic modification of mesenchymal stem cells overexpressing CCR1 increases cell viability, migration, engraftment, and capillary density in the injured myocardium.

PubMed

Huang, Jing; Zhang, Zhiping; Guo, Jian; Ni, Aiguo; Deb, Arjun; Zhang, Lunan; Mirotsou, Maria; Pratt, Richard E; Dzau, Victor J

2010-06-11

Although mesenchymal stem cell (MSC) transplantation has been shown to promote cardiac repair in acute myocardial injury in vivo, its overall restorative capacity appears to be restricted mainly because of poor cell viability and low engraftment in the ischemic myocardium. Specific chemokines are upregulated in the infarcted myocardium. However the expression levels of the corresponding chemokine receptors (eg, CCR1, CXCR2) in MSCs are very low. We hypothesized that this discordance may account for the poor MSC engraftment and survival. To determine whether overexpression of CCR1 or CXCR2 chemokine receptors in MSCs augments their cell survival, migration and engraftment after injection in the infarcted myocardium. Overexpression of CCR1, but not CXCR2, dramatically increased chemokine-induced murine MSC migration and protected MSC from apoptosis in vitro. Moreover, when MSCs were injected intramyocardially one hour after coronary artery ligation, CCR1-MSCs accumulated in the infarcted myocardium at significantly higher levels than control-MSCs or CXCR2-MSCs 3 days postmyocardial infarction (MI). CCR1-MSC-injected hearts exhibited a significant reduction in infarct size, reduced cardiomyocytes apoptosis and increased capillary density in injured myocardium 3 days after MI. Furthermore, intramyocardial injection of CCR1-MSCs prevented cardiac remodeling and restored cardiac function 4 weeks after MI. Our results demonstrate the in vitro and in vivo salutary effects of genetic modification of stem cells. Specifically, overexpression of chemokine receptor enhances the migration, survival and engraftment of MSCs, and may provide a new therapeutic strategy for the injured myocardium.

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

Role of mesenchymal stem cells versus angiotensin converting enzyme inhibitor in kidney repair.

PubMed

Ahmed, Hanaa H; Toson, Elshahat A; El-Mezayen, Hatem A; Rashed, Laila A; Elsherbiny, Eslam S

2017-07-01

The current study sought to clarify the role of bone marrow derived mesenchymal stem cells (BM-MSCs) and adipose tissue derived mesenchymal stem cells (AD-MSCs) in repressing nephropathy in the experimental model. Moreover, the aim of this work was extended to compare between stem cells role and angiotensin converting enzyme inhibitor in kidney repair. Isolation and preparation of MSCs culture, flow cytometry using CD34, CD44 and CD105 cell surface markers, biochemical analyses for determination of serum creatinine, urea, transforming growth factor β (TGF-β), cystatin C (CYS-C) and urinary N-Acetyl-ß-D-Glucosaminidase (UNAG), and histopathological investigation of kidney tissue sections were performed. The results of the present study revealed that single intravenous infusion of MSCs either derived from bone marrow or adipose tissue was able to enhance renal reparative processes through significantly decreased serum creatinine, urea, TGF-β and CYS-C levels as well as UNAG level and significantly increase glomerular filtration rate. Additionally, the histopathological investigations of kidney tissues showed that MSCs have significant regenerative effects as evidenced by the decrease in focal inflammatory cells infiltration, focal interstitial nephritis and congested glomeruli as well as degenerated tubules. The current data provided distinct evidence about the favourable impact of AD-MSCs and BM-MSCs in attenuation of cyclosporine-induced nephropathy in rats through their ability to promote functional and structural kidney repair via transdifferentiation. © 2016 Asian Pacific Society of Nephrology.

Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFβ/Activin Signaling Pathway

PubMed Central

Frith, Jessica E.; Frith, Thomas J.R.; Ovchinnikov, Dmitry A.; Cooper-White, Justin J.; Wolvetang, Ernst J.

2014-01-01

In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases. PMID:25055193

2,5-hexanedione induces bone marrow mesenchymal stem cell apoptosis via inhibition of Akt/Bad signal pathway.

PubMed

Sun, Jingsong; Shi, Xiaoxia; Li, Shuangyue; Piao, Fengyuan

2018-04-01

2,5-Hexanedione (HD) is an important bioactive metabolite of n-hexane and mediates the neurotoxicity of parent compound. Studies show that HD induces apoptotic death of neural progenitor cells. However, its underlying mechanism remains unknown. Mesenchymal stem cells (MSCs) are multipotential stem cells with the ability to differentiate into various cell types and have been used as cell model for studying the toxic effects of chemicals on stem cells. In this study, we exposed rat bone marrow MSCs to 0, 10, 20, and 40 mM HD in vitro. Apoptosis and disruption of mitochondrial transmembrane potential were estimated by immunochemistry staining. The expression of Akt, Bad, phosphorylated Akt (p-Akt), and Bad (p-Bad) as well as cytochrome c in mitochondria and cytosol were examined by Western blot. Moreover, caspase 3 activity, viability, and death of cells were measured by spectrophotometry. Our results showed that HD induced cell apoptosis and increased caspase 3 activity. HD down-regulated the expression levels of p-Akt, p-Bad and induced MMP depolarization, followed by cytochrome c release. Moreover, HD led to a concentration-dependent increase in the MSCs death, which was relative to MSCs apoptosis. However, these toxic effects of HD on the MSCs were significantly mitigated in the presence of IGF, which could activate PI3 K/Akt pathway. These results indicated that HD induced mitochondria-mediated apoptosis in the MSCs via inhibiting Akt/Bad signaling pathway and apoptotic death of MSCs via the signaling pathway. These results might provide some clues for studying further the mechanisms of HD-induced stem cell apoptosis and adverse effect on neurogenesis. © 2017 Wiley Periodicals, Inc.

HGF Gene Modification in Mesenchymal Stem Cells Reduces Radiation-Induced Intestinal Injury by Modulating Immunity.

PubMed

Wang, Hua; Sun, Rui-Ting; Li, Yang; Yang, Yue-Feng; Xiao, Feng-Jun; Zhang, Yi-Kun; Wang, Shao-Xia; Sun, Hui-Yan; Zhang, Qun-Wei; Wu, Chu-Tse; Wang, Li-Sheng

2015-01-01

Effective therapeutic strategies to address intestinal complications after radiation exposure are currently lacking. Mesenchymal stem cells (MSCs), which display the ability to repair the injured intestine, have been considered as delivery vehicles for repair genes. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF)-gene-modified MSCs on radiation-induced intestinal injury (RIII). Female 6- to 8-week-old mice were radiated locally at the abdomen with a single 13-Gy dose of radiation and then treated with saline control, Ad-HGF or Ad-Null-modified MSCs therapy. The transient engraftment of human MSCs was detected via real-time PCR and immunostaining. The therapeutic effects of non- and HGF-modified MSCs were evaluated via FACS to determine the lymphocyte immunophenotypes; via ELISA to measure cytokine expression; via immunostaining to determine tight junction protein expression; via PCNA staining to examine intestinal epithelial cell proliferation; and via TUNEL staining to detect intestinal epithelial cell apoptosis. The histopathological recovery of the radiation-injured intestine was significantly enhanced following non- or HGF-modified MSCs treatment. Importantly, the radiation-induced immunophenotypic disorders of the mesenteric lymph nodes and Peyer's patches were attenuated in both MSCs-treated groups. Treatment with HGF-modified MSCs reduced the expression and secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ), increased the expression of the anti-inflammatory cytokine IL-10 and the tight junction protein ZO-1, and promoted the proliferation and reduced the apoptosis of intestinal epithelial cells. Treatment of RIII with HGF-gene-modified MSCs reduces local inflammation and promotes the recovery of small intestinal histopathology in a mouse model. These findings might provide an effective therapeutic strategy for RIII.

Composite cell sheet for periodontal regeneration: crosstalk between different types of MSCs in cell sheet facilitates complex periodontal-like tissue regeneration.

PubMed

Zhang, Hao; Liu, Shiyu; Zhu, Bin; Xu, Qiu; Ding, Yin; Jin, Yan

2016-11-14

Tissue-engineering strategies based on mesenchymal stem cells (MSCs) and cell sheets have been widely used for periodontal tissue regeneration. However, given the complexity in periodontal structure, the regeneration methods using a single species of MSC could not fulfill the requirement for periodontal regeneration. We researched the interaction between the periodontal ligament stem cells (PDLSCs) and jaw bone marrow-derived mesenchymal stem cells (JBMMSCs), and constructed a composite cell sheet comprising both of the above MSCs to regenerate complex periodontium-like structures in nude mice. Our results show that by co-culturing PDLSCs and JBMMSCs, the expressions of bone and extracellular matrix (ECM)-related genes and proteins were significantly improved in both MSCs. Further investigations showed that, compared to the cell sheet using PDLSCs or JBMMSCs, the composite stem cell sheet (CSCS), which comprises these two MSCs, expressed higher levels of bone- and ECM-related genes and proteins, and generated a composite structure more similar to the native periodontal tissue physiologically in vivo. In conclusion, our results demonstrate that the crosstalk between PDLSCs and JBMMSCs in cell sheets facilitate regeneration of complex periodontium-like structures, providing a promising new strategy for physiological and functional regeneration of periodontal tissue.

Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines

PubMed Central

2010-01-01

To overcome loss of stem-like properties and spontaneous differentiation those hinder the expansion and application of human mesenchymal stem cells (hMSCs), we have clonally isolated permanent and stable human MSC lines by ectopic overexpression of primary cell cultures of hMSCs with HPV 16 E6E7 and human telomerase reverse transcriptase (hTERT) genes. These cells were found to have a differentiation potential far beyond the ordinary hMSCs. They expressed trophoectoderm and germline specific markers upon differentiation with BMP4 and retinoic acid, respectively. Furthermore, they displayed higher osteogenic and neural differentiation efficiency than primary hMSCs or hMSCs expressed HPV16 E6E7 alone with a decrease in methylation level as proven by a global CpG island methylation profile analysis. Notably, the demethylated CpG islands were highly associated with development and differentiation associated genes. Principal component analysis further pointed out the expression profile of the cells converged toward embryonic stem cells. These data demonstrate these cells not only are a useful tool for the studies of cell differentiation both for the mesenchymal and neurogenic lineages, but also provide a valuable source of cells for cell therapy studies in animal models of skeletal and neurological disorders. PMID:20670406

Anti-apoptotic effects of adipose-derived adherent stromal cells in mesenchymal stem cells exposed to oxidative stress.

PubMed

Shin, Sunhye; Choi, Jung-Won; Lim, Soyeon; Lee, Seahyoung; Jun, Eun-Young; Sun, Hyun-Min; Kim, Il-Kwon; Lee, Hoon-Bum; Kim, Sang Woo; Hwang, Ki-Chul

2018-06-19

Adipose-derived stromal vascular fractions (SVFs) are a heterogeneous collection of cells, and their regenerative modality has been applied in various animal experiments and clinical trials. Despite the attractive advantages of SVFs in clinical interventions, the recent status of clinical studies involving the application of SVFs in many diseases has not been fully evaluated. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types despite their low numbers in heart tissue. Here, we sought to determine if SVF implantation into impaired heart tissue affected endogenous MSCs in the heart. Therefore, we investigated the expression levels of proteins associated with oxidation, inflammation, and apoptosis in MSCs co-cultured with adipose-derived adherent stromal cells (ADASs) from 6 donors' SVFs under oxidative stress conditions for their roles in many physiological processes in the heart. Interestingly, p53 pathway proteins and mitogen-activated protein kinase (MAPK) signalling pathway components were up-regulated by H 2 O 2 but exhibited a downward trend in MSCs co-cultured with ADASs. These data suggest that ADASs may inhibit oxidative stress-induced apoptosis in MSCs via the p53 and MAPK pathways. Our findings also suggest that the positive effects of SVF implantation into damaged heart tissue may be attributed to the various responses of MSCs. This finding may provide new insights for the clinical application of adipose-derived SVF transplantation in cardiac diseases. We investigated the expression levels of proteins associated with oxidation, inflammation, and apoptosis in MSCs co-cultured with isolated ADASs from 6 donors' SVFs under oxidative stress conditions. Our results imply that isolated ADASs from SVFs may inhibit oxidative stress-induced cell cycle arrest and/or apoptosis in MSCs via a p53-dependent pathway. Furthermore, we identified an anti-apoptotic mechanism involving oxidative stress-induced apoptosis by adipose-derived ADASs in MSCs for the first time. Our findings suggest that the positive effects of SVF implantation into damaged heart tissue may be attributed to the various actions of MSCs. © 2018 John Wiley & Sons, Ltd.

Paracrine action of mesenchymal stromal cells delivered by microspheres contributes to cutaneous wound healing and prevents scar formation in mice.

PubMed

Huang, Sha; Wu, Yan; Gao, Dongyun; Fu, Xiaobing

2015-07-01

Accumulating evidence suggests that mesenchymal stromal cells (MSCs) participate in wound healing to favor tissue regeneration and inhibit fibrotic tissue formation. However, the evidence of MSCs to suppress cutaneous scar is extremely rare, and the mechanism remains unidentified. This study aimed to demonstrate whether MSCs-as the result of their paracrine actions on damaged tissues-would accelerate wound healing and prevent cutaneous fibrosis. For efficient delivery of MSCs to skin wounds, microspheres were used to maintain MSC potency. Whether MSCs can accelerate wound healing and alleviate cutaneous fibrosis through paracrine action was investigated with the use of a Transwell co-culture system in vitro and a murine model in vivo. MSCs cultured on gelatin microspheres fully retained their cell surface marker expression profile, proliferation, differentiation and paracrine potential. Co-cultures of MSCs and fibroblasts indicated that the benefits of MSCs on suppressing fibroblast proliferation and its fibrotic behavior induced by inflammatory cytokines probably were caused by paracrine actions. Importantly, microspheres successfully delivered MSCs into wound margins and significantly accelerated wound healing and concomitantly reduced the fibrotic activities of cells within the wounds and excessive accumulation of extracellular matrix as well as the transforming growth factor-β1/transforming growth factor-β3 ratio. This study provides insight into what we believe to be a previously undescribed, multifaceted role of MSC-released protein in reducing cutaneous fibrotic formation. Paracrine action of MSCs delivered by microspheres may thus qualify as a promising strategy to enhance tissue repair and to prevent excessive fibrosis during cutaneous wound healing. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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.

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.

Optimization of Pre-transplantation Conditions to Enhance the Efficacy of Mesenchymal Stem Cells

PubMed Central

Haque, Nazmul; Kasim, Noor Hayaty Abu; Rahman, Mohammad Tariqur

2015-01-01

Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits. PMID:25678851

Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells☆

PubMed Central

Nalluri, Sandeep M.; Krishnan, G. Rajesh; Cheah, Calvin; Arzumand, Ayesha; Yuan, Yuan; Richardson, Caley A.; Yang, Shuying; Sarkar, Debanjan

2016-01-01

Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell–matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell–cell and cell–matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell–matrix interaction, and cell–cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration. PMID:26046282

Progress of mesenchymal stem cell therapy for neural and retinal diseases

PubMed Central

Ng, Tsz Kin; Fortino, Veronica R; Pelaez, Daniel; Cheung, Herman S

2014-01-01

Complex circuitry and limited regenerative power make central nervous system (CNS) disorders the most challenging and difficult for functional repair. With elusive disease mechanisms, traditional surgical and medical interventions merely slow down the progression of the neurodegenerative diseases. However, the number of neurons still diminishes in many patients. Recently, stem cell therapy has been proposed as a viable option. Mesenchymal stem cells (MSCs), a widely-studied human adult stem cell population, have been discovered for more than 20 years. MSCs have been found all over the body and can be conveniently obtained from different accessible tissues: bone marrow, blood, and adipose and dental tissue. MSCs have high proliferative and differentiation abilities, providing an inexhaustible source of neurons and glia for cell replacement therapy. Moreover, MSCs also show neuroprotective effects without any genetic modification or reprogramming. In addition, the extraordinary immunomodulatory properties of MSCs enable autologous and heterologous transplantation. These qualities heighten the clinical applicability of MSCs when dealing with the pathologies of CNS disorders. Here, we summarize the latest progress of MSC experimental research as well as human clinical trials for neural and retinal diseases. This review article will focus on multiple sclerosis, spinal cord injury, autism, glaucoma, retinitis pigmentosa and age-related macular degeneration. PMID:24772238

Progress of mesenchymal stem cell therapy for neural and retinal diseases.

PubMed

Ng, Tsz Kin; Fortino, Veronica R; Pelaez, Daniel; Cheung, Herman S

2014-04-26

Complex circuitry and limited regenerative power make central nervous system (CNS) disorders the most challenging and difficult for functional repair. With elusive disease mechanisms, traditional surgical and medical interventions merely slow down the progression of the neurodegenerative diseases. However, the number of neurons still diminishes in many patients. Recently, stem cell therapy has been proposed as a viable option. Mesenchymal stem cells (MSCs), a widely-studied human adult stem cell population, have been discovered for more than 20 years. MSCs have been found all over the body and can be conveniently obtained from different accessible tissues: bone marrow, blood, and adipose and dental tissue. MSCs have high proliferative and differentiation abilities, providing an inexhaustible source of neurons and glia for cell replacement therapy. Moreover, MSCs also show neuroprotective effects without any genetic modification or reprogramming. In addition, the extraordinary immunomodulatory properties of MSCs enable autologous and heterologous transplantation. These qualities heighten the clinical applicability of MSCs when dealing with the pathologies of CNS disorders. Here, we summarize the latest progress of MSC experimental research as well as human clinical trials for neural and retinal diseases. This review article will focus on multiple sclerosis, spinal cord injury, autism, glaucoma, retinitis pigmentosa and age-related macular degeneration.

Establishing criteria for human mesenchymal stem cell potency.

PubMed

Samsonraj, Rebekah M; Rai, Bina; Sathiyanathan, Padmapriya; Puan, Kia Joo; Rötzschke, Olaf; Hui, James H; Raghunath, Michael; Stanton, Lawrence W; Nurcombe, Victor; Cool, Simon M

2015-06-01

This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age- and sex-matched donors. Adherence to plastic was not indicative of potency, yet capacity for long-term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high-growth capacity or low-growth capacity. Using this grouping strategy, high-growth capacity MSCs were smaller in size, had greater colony-forming efficiency, and had longer telomeres. Cell-surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high-growth capacity and low-growth capacity MSCs, whereas STRO-1 and platelet-derived growth factor receptor alpha were preferentially expressed on high-growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST-1 and DERMO-1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high-growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low-growth capacity MSCs when assessed for ectopic bone-forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application. © 2015 AlphaMed Press.

Establishing Criteria for Human Mesenchymal Stem Cell Potency

PubMed Central

Samsonraj, Rebekah M.; Rai, Bina; Sathiyanathan, Padmapriya; Puan, Kia Joo; Rötzschke, Olaf; Hui, James H.; Raghunath, Michael; Stanton, Lawrence W.; Nurcombe, Victor

2015-01-01

Abstract This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age‐ and sex‐matched donors. Adherence to plastic was not indicative of potency, yet capacity for long‐term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high‐growth capacity or low‐growth capacity. Using this grouping strategy, high‐growth capacity MSCs were smaller in size, had greater colony‐forming efficiency, and had longer telomeres. Cell‐surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high‐growth capacity and low‐growth capacity MSCs, whereas STRO‐1 and platelet‐derived growth factor receptor alpha were preferentially expressed on high‐growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST‐1 and DERMO‐1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high‐growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low‐growth capacity MSCs when assessed for ectopic bone‐forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application. Stem Cells 2015;33:1878–1891 PMID:25752682

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.

Neuroprotective effect of mesenchymal stem cell through complement component 3 downregulation after transient focal cerebral ischemia in mice.

PubMed

Jung, Hye-Seon; Jeong, Si-Yeon; Yang, Jiwon; Kim, So-Dam; Zhang, Baojin; Yoo, Hyun Seung; Song, Sun U; Jeon, Myung-Shin; Song, Yun Seon

2016-10-28

Bone marrow-derived mesenchymal stem cells (MSCs) are used in stroke treatment despite the poor understanding of its mode of action. The immune suppressive and anti-inflammatory properties of MSCs possibly play important roles in regulating neuroinflammation after stroke. We investigated whether MSCs reduce the inflammatory complement component 3 (C3) levels, thus, providing neuroprotection during stroke. Mice were subjected to transient focal cerebral ischemia (tFCI), after which MSCs were intravenously injected. The infarct volume of the brain was reduced in MSC-injected tFCI mice, and C3 expression was significantly reduced in both the brain and the blood. Additionally, the profiles of other inflammatory mediators demonstrated neuroprotective changes in the MSCs-treated group. In order to analyze the effect of MSCs on neurons during cerebral ischemia, primary cortical neurons were co-cultured with MSCs under oxygen-glucose deprivation (OGD). Primary neurons co-cultured with MSCs exhibited reduced levels of C3 expression and increased protection against OGD, indicating that treatment with MSCs reduces excessive C3 expression and rescues ischemia-induced neuronal damage. Our finding suggests that reduction of C3 expression by MSCs can help to ameliorate ischemic brain damage, offering a new neuroprotective strategy in stroke therapy. Copyright © 2016. Published by Elsevier Ireland Ltd.

ET-33PLACENTA-DERIVED MESENCHYMAL STEM CELLS AND THEIR SECRETED EXOSOMES INHIBIT THE SELF-RENEWAL AND STEMNESS OF GLIOMA STEM CELLS IN VITRO AND IN VIVO

PubMed Central

Lee, Hae Kyung; Buchris, Efrat; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila; Brodie, Chaya

2014-01-01

Mesenchymal stromal cells (MSCs) are multipotent stem cells that can be obtained from bone marrow and adipose tissues or from other sources such as placenta and umbilical cord. The latter allow the potential use of universal, allogeneic cell therapy because to reduced antigenicity due to low expression of MHC class II molecules. MSCs can be easily expanded in vitro for therapeutic applications and their safety and therapeutic impact have been demonstrated in various pre-clinical and clinical studies. MSCs have been shown to cross the blood brain barrier and migrate to sites of experimental GBM and can deliver cytotoxic compounds that exert anti-tumor effects. In this study we examined the effects of placenta-derived MSCs and their secreted exosomes on GSCs in vitro and in vivo. Conditioned medium of placenta MSCs or their derived exosomes decreased the self-renewal, stemness markers, Sox2 and Oct4 and the migration of these cells. Similarly, intracranial administration of the MSCs decreased the tumor volume of GSC-derived xenografts and prolonged animal survival. miRNA sequencing analysis of placenta MSC-derived exosomes revealed a set of specific miRNAs that were downregulated in GSCs and that acted as tumor suppressor in these cells. We demonstrated delivery of some of these miRNAs to GSCs following treatments with MSC-derived exosomes. We further demonstrated that MSCs or exosomes that were loaded with exogenous miR-124 delivered high levels of this miRNA into glioma cells as detected by a novel quantitative miRNA reporter. Moreover, administration of placenta MSCs loaded with exogenous miR-124 exerted a strong inhibitory effect on GSC-derived xenograft growth. These results demonstrate that placenta-derived MSCs may have important clinical applications in stem cell-based glioma therapeutics. Moreover, these studies provide a novel approach for the targeted delivery of endogenous and exogenous anti-tumor miRNAs to glioma cells as a miRNA replacement therapy for GBM.

Stimulation of Skin and Wound Fibroblast Migration by Mesenchymal Stem Cells Derived from Normal Donors and Chronic Wound Patients

PubMed Central

Rodriguez-Menocal, Luis; Salgado, Marcela; Ford, Dwayne

2012-01-01

Chronic wounds continue to be a major cause of morbidity for patients and an economic burden on the health care system. Novel therapeutic approaches to improved wound healing will need, however, to address cellular changes induced by a number of systemic comorbidities seen in chronic wound patients, such as diabetes, chronic renal failure, and arterial or venous insufficiency. These effects likely include impaired inflammatory cell migration, reduced growth factor production, and poor tissue remodeling. The multifunctional properties of bone marrow-derived mesenchymal stem cells (MSCs), including their ability to differentiate into various cell types and capacity to secrete factors important in accelerating healing of cutaneous wounds, have made MSCs a promising agent for tissue repair and regeneration. In this study we have used an in vitro scratch assay procedure incorporating labeled MSCs and fibroblasts derived from normal donors and chronic wound patients in order to characterize the induction of mobilization when these cells are mixed. A modified Boyden chamber assay was also used to examine the effect of soluble factors on fibroblast migration. These studies suggest that MSCs play a role in skin wound closure by affecting dermal fibroblast migration in a dose-dependent manner. Deficiencies were noted, however, in chronic wound patient fibroblasts and MSCs as compared with those derived from normal donors. These findings provide a foundation to develop therapies targeted specifically to the use of bone marrow-derived MSCs in wound healing and may provide insight into why some wounds do not heal. PMID:23197781

Stimulation of skin and wound fibroblast migration by mesenchymal stem cells derived from normal donors and chronic wound patients.

PubMed

Rodriguez-Menocal, Luis; Salgado, Marcela; Ford, Dwayne; Van Badiavas, Evangelos

2012-03-01

Chronic wounds continue to be a major cause of morbidity for patients and an economic burden on the health care system. Novel therapeutic approaches to improved wound healing will need, however, to address cellular changes induced by a number of systemic comorbidities seen in chronic wound patients, such as diabetes, chronic renal failure, and arterial or venous insufficiency. These effects likely include impaired inflammatory cell migration, reduced growth factor production, and poor tissue remodeling. The multifunctional properties of bone marrow-derived mesenchymal stem cells (MSCs), including their ability to differentiate into various cell types and capacity to secrete factors important in accelerating healing of cutaneous wounds, have made MSCs a promising agent for tissue repair and regeneration. In this study we have used an in vitro scratch assay procedure incorporating labeled MSCs and fibroblasts derived from normal donors and chronic wound patients in order to characterize the induction of mobilization when these cells are mixed. A modified Boyden chamber assay was also used to examine the effect of soluble factors on fibroblast migration. These studies suggest that MSCs play a role in skin wound closure by affecting dermal fibroblast migration in a dose-dependent manner. Deficiencies were noted, however, in chronic wound patient fibroblasts and MSCs as compared with those derived from normal donors. These findings provide a foundation to develop therapies targeted specifically to the use of bone marrow-derived MSCs in wound healing and may provide insight into why some wounds do not heal.

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).

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.

Transplantation of Human Skin-Derived Mesenchymal Stromal Cells Improves Locomotor Recovery After Spinal Cord Injury in Rats.

PubMed

Melo, Fernanda Rosene; Bressan, Raul Bardini; Forner, Stefânia; Martini, Alessandra Cadete; Rode, Michele; Delben, Priscilla Barros; Rae, Giles Alexander; Figueiredo, Claudia Pinto; Trentin, Andrea Gonçalves

2017-07-01

Spinal cord injury (SCI) is a devastating neurologic disorder with significant impacts on quality of life, life expectancy, and economic burden. Although there are no fully restorative treatments yet available, several animal and small-scale clinical studies have highlighted the therapeutic potential of cellular interventions for SCI. Mesenchymal stem cells (MSCs)-which are conventionally isolated from the bone marrow-recently emerged as promising candidates for treating SCI and have been shown to provide trophic support, ameliorate inflammatory responses, and reduce cell death following the mechanical trauma. Here we evaluated the human skin as an alternative source of adult MSCs suitable for autologous cell transplantation strategies for SCI. We showed that human skin-derived MSCs (hSD-MSCs) express a range of neural markers under standard culture conditions and are able to survive and respond to neurogenic stimulation in vitro. In addition, using histological analysis and behavioral assessment, we demonstrated as a proof-of-principle that hSD-MSC transplantation reduces the severity of tissue loss and facilitates locomotor recovery in a rat model of SCI. Altogether, the study provides further characterization of skin-derived MSC cultures and indicates that the human skin may represent an attractive source for cell-based therapies for SCI and other neurological disorders. Further investigation is needed to elucidate the mechanisms by which hSD-MSCs elicit tissue repair and/or locomotor recovery.

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

Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors.

PubMed

Sart, Sébastien; Agathos, Spiros N; Li, Yan; Ma, Teng

2016-01-01

Human mesenchymal stem cells (hMSCs) have emerged as an important cell type in cell therapy and tissue engineering. In these applications, maintaining the therapeutic properties of hMSCs requires tight control of the culture environments and the structural cell organizations. Bioreactor systems are essential tools to achieve these goals in the clinical-scale expansion and tissue engineering applications. This review summarizes how different bioreactors provide cues to regulate the structure and the chemico-mechanical microenvironment of hMSCs with a focus on 3D organization. In addition to conventional bioreactors, recent advances in microfluidic bioreactors as a novel approach to better control the hMSC microenvironment are also discussed. These advancements highlight the key role of bioreactor systems in preserving hMSC's functional properties by providing dynamic and temporal regulation of in vitro cellular microenvironment. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Oxygen Tension Regulates Human Mesenchymal Stem Cell Paracrine Functions

PubMed Central

Deschepper, Mickael; Moya, Adrien; Logeart-Avramoglou, Delphine; Boisson-Vidal, Catherine; Petite, Hervé

2015-01-01

Mesenchymal stem cells (MSCs) have captured the attention and research endeavors of the scientific world because of their differentiation potential. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly due to the multitude of bioactive mediators secreted by these cells. Because the paracrine potential of MSCs is closely related to their microenvironment, the present study investigated and characterized select aspects of the human MSC (hMSC) secretome and assessed its in vitro and in vivo bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. In contrast to supernatant conditioned media (CM) obtained from hMSCs cultured at either 5% or 21% of O2, CM from hMSCs cultured under near anoxia exhibited significantly (p < .05) enhanced chemotactic and proangiogenic properties and a significant (p < .05) decrease in the inflammatory mediator content. An analysis of the hMSC secretome revealed a specific profile under near anoxia: hMSCs increase their paracrine expression of the angiogenic mediators vascular endothelial growth factor (VEGF)-A, VEGF-C, interleukin-8, RANTES, and monocyte chemoattractant protein 1 but significantly decrease expression of several inflammatory/immunomodulatory mediators. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine and could contribute to improving the efficacy of such therapies. Significance The present study investigated and characterized select aspects of the human mesenchymal stem cell (hMSC) secretome and assessed its in vitro and in vivo biological bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. The present study provided the first evidence of a shift of the hMSC cytokine signature induced by oxygen tension, particularly near anoxia (0.1% O2). Conditioned media obtained from hMSCs cultured under near anoxia exhibited significantly enhanced chemotactic and proangiogenic properties and a significant decrease in the inflammatory mediator content. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine, could contribute to improving the efficacy of such therapies, and most importantly highlighted the interest in using conditioned media in therapeutic modalities. PMID:25979862

Oxygen Tension Regulates Human Mesenchymal Stem Cell Paracrine Functions.

PubMed

Paquet, Joseph; Deschepper, Mickael; Moya, Adrien; Logeart-Avramoglou, Delphine; Boisson-Vidal, Catherine; Petite, Hervé

2015-07-01

: Mesenchymal stem cells (MSCs) have captured the attention and research endeavors of the scientific world because of their differentiation potential. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly due to the multitude of bioactive mediators secreted by these cells. Because the paracrine potential of MSCs is closely related to their microenvironment, the present study investigated and characterized select aspects of the human MSC (hMSC) secretome and assessed its in vitro and in vivo bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. In contrast to supernatant conditioned media (CM) obtained from hMSCs cultured at either 5% or 21% of O2, CM from hMSCs cultured under near anoxia exhibited significantly (p < .05) enhanced chemotactic and proangiogenic properties and a significant (p < .05) decrease in the inflammatory mediator content. An analysis of the hMSC secretome revealed a specific profile under near anoxia: hMSCs increase their paracrine expression of the angiogenic mediators vascular endothelial growth factor (VEGF)-A, VEGF-C, interleukin-8, RANTES, and monocyte chemoattractant protein 1 but significantly decrease expression of several inflammatory/immunomodulatory mediators. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine and could contribute to improving the efficacy of such therapies. The present study investigated and characterized select aspects of the human mesenchymal stem cell (hMSC) secretome and assessed its in vitro and in vivo biological bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. The present study provided the first evidence of a shift of the hMSC cytokine signature induced by oxygen tension, particularly near anoxia (0.1% O2). Conditioned media obtained from hMSCs cultured under near anoxia exhibited significantly enhanced chemotactic and proangiogenic properties and a significant decrease in the inflammatory mediator content. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine, could contribute to improving the efficacy of such therapies, and most importantly highlighted the interest in using conditioned media in therapeutic modalities. ©AlphaMed Press.

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

Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells.

PubMed

Nalluri, Sandeep M; Krishnan, G Rajesh; Cheah, Calvin; Arzumand, Ayesha; Yuan, Yuan; Richardson, Caley A; Yang, Shuying; Sarkar, Debanjan

2015-09-01

Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell-matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell-cell and cell-matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell-matrix interaction, and cell-cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration. Copyright © 2015 Elsevier B.V. All rights reserved.

Umbilical Cord Tissue-Derived Mesenchymal Stem Cells Induce T Lymphocyte Apoptosis and Cell Cycle Arrest by Expression of Indoleamine 2, 3-Dioxygenase

PubMed Central

Li, Xiuying; Xu, Zhuo; Bai, Jinping; Yang, Shuyuan; Zhao, Shuli; Zhang, Yingjie; Chen, Xiaodong

2016-01-01

It has been reported that human mesenchymal stem cells are able to inhibit T lymphocyte activation; however, the discrepancy among different sources of MSCs is not well documented. In this study, we have compared the MSCs from bone marrow (BM), adipose tissue (AT), placenta (PL), and umbilical cord (UC) to determine which one displayed the most efficient immunosuppressive effects on phytohemagglutinin-induced T cell proliferation. Among them we found that hUC-MSC has the strongest effects on inhibiting T cell proliferation and is chosen to do the further study. We observed that T lymphocyte spontaneously released abundant IFN-γ. And IFN-γ secreted by T lymphocyte could induce the expression of indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs. IDO was previously reported to induce T lymphocyte apoptosis and cell cycle arrest in S phase. When cocultured with hUC-MSCs, T lymphocyte expression of caspase 3 was significantly increased, while Bcl2 and CDK4 mRNA expression decreased dramatically. Addition of 1-methyl tryptophan (1-MT), an IDO inhibitor, restored T lymphocyte proliferation, reduced apoptosis, and induced resumption of the cell cycle. In addition, the changes in caspase 3, CDK4, and Bcl2 expression were reversed by 1-MT. These findings demonstrate that hUC-MSCs induce T lymphocyte apoptosis and cell cycle arrest by expressing abundant IDO and provide an explanation for some of the immunomodulatory effects of MSCs. PMID:27418932

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.

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

Human Mesenchymal Stem Cells Provide Protection against Radiation-Induced Liver Injury by Antioxidative Process, Vasculature Protection, Hepatocyte Differentiation, and Trophic Effects

PubMed Central

Francois, Sabine; Mouiseddine, Moubarak; Allenet-Lepage, Bénédicte; Voswinkel, Jan; Douay, Luc; Benderitter, Marc; Chapel, Alain

2013-01-01

To evaluate the potential therapeutic effect of the infusion of hMSCs for the correction of liver injuries, we performed total body radiation exposure of NOD/SCID mice. After irradiation, mir-27b level decreases in liver, increasing the directional migration of hMSCs by upregulating SDF1α. A significant increase in plasmatic transaminases levels, apoptosis process in the liver vascular system, and in oxidative stress were observed. hMSC injection induced a decrease in transaminases levels and oxidative stress, a disappearance of apoptotic cells, and an increase in Nrf2, SOD gene expression, which might reduce ROS production in the injured liver. Engrafted hMSCs expressed cytokeratin CK18 and CK19 and AFP genes indicating possible hepatocyte differentiation. The presence of hMSCs expressing VEGF and Ang-1 in the perivascular region, associated with an increased expression of VEGFr1, r2 in the liver, can confer a role of secreting cells to hMSCs in order to maintain the endothelial function. To explain the benefits to the liver of hMSC engraftment, we find that hMSCs secreted NGF, HGF, and anti-inflammatory molecules IL-10, IL1-RA contributing to prevention of apoptosis, increasing cell proliferation in the liver which might correct liver dysfunction. MSCs are potent candidates to repair and protect healthy tissues against radiation damages. PMID:24369528

Ultrasound and photoacoustic imaging to monitor ocular stem cell delivery and tissue regeneration (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kubelick, Kelsey; Snider, Eric; Yoon, Heechul; Ethier, C. Ross; Emelianov, Stanislav Y.

2017-03-01

Glaucoma is associated with dysfunction of the trabecular meshwork (TM), a fluid drainage tissue in the anterior eye. A promising treatment involves delivery of stem cells to the TM to restore tissue function. Currently histology is the gold standard for tracking stem cell delivery and differentiation. To expedite clinical translation, non-invasive longitudinal monitoring in vivo is desired. Our current research explores a technique combining ultrasound (US) and photoacoustic (PA) imaging to track mesenchymal stem cells (MSCs) after intraocular injection. Adipose-derived MSCs were incubated with gold nanospheres to label cells (AuNS-MSCs) for PA imaging. Successful labeling was first verified with in vitro phantom studies. Next, MSC delivery was imaged ex vivo in porcine eyes, while intraocular pressure was hydrostatically clamped to maintain a physiological flow rate through the TM. US/PA imaging was performed before, during, and after AuNS-MSC delivery. Additionally, spectroscopic PA imaging was implemented to isolate PA signals from AuNS-MSCs. In vitro cell imaging showed AuNS-MSCs produce strong PA signals, suggesting that MSCs can be tracked using PA imaging. While the cornea, sclera, iris, and TM region can be visualized with US imaging, pigmented tissues also produce PA signals. Both modalities provide valuable anatomical landmarks for MSC localization. During delivery, PA imaging can visualize AuNS-MSC motion and location, creating a unique opportunity to guide ocular cell delivery. Lastly, distinct spectral signatures of AuNS-MSCs allow unmixing, with potential for quantitative PA imaging. In conclusion, results show proof-of-concept for monitoring MSC ocular delivery, raising opportunities for in vivo image-guided cell delivery.

Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow.

PubMed

Liao, L; Yang, X; Su, X; Hu, C; Zhu, X; Yang, N; Chen, X; Shi, S; Shi, S; Jin, Y

2013-04-18

During the process of aging, especially for postmenopausal females, the cell lineage commitment of mesenchymal stem cells (MSCs) shift to adipocyte in bone marrow, resulting in osteoporosis. However, the cell-intrinsic mechanism of this cell lineage commitment switch is poorly understood. As the post-transcription regulation by microRNAs (miRNAs) has a critical role in MSCs differentiation and bone homeostasis, we performed comprehensive miRNAs profiling and found miR-705 and miR-3077-5p were significantly enhanced in MSCs from osteoporosis bone marrow. Both miR-705 and miR-3077-5p acted as inhibitors of MSCs osteoblast differentiation and promoters of adipocyte differentiation, by targeting on the 3'untranslated region (3'UTR) of HOXA10 and RUNX2 mRNA separately. Combined inhibition of miR-705 and miR-3077-5p rescued the cell lineage commitment disorder of MSCs through restoring HOXA10 and RUNX2 protein level. Furthermore, we found excessive TNFα and reactive oxygen species caused by estrogen deficiency led to the upregulation of both miRNAs through NF-κB pathway. In conclusion, our findings showed that redundant miR-705 and miR-3077-5p synergistically mediated the shift of MSCs cell lineage commitment to adipocyte in osteoporosis bone marrow, providing new insight into the etiology of osteoporosis at the post-transcriptional level. Moreover, the rescue of MSCs lineage commitment disorder by regulating miRNAs expression suggested a novel potential therapeutic target for osteoporosis as well as stem cell-mediated regenerative medicine.

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.

Nicotine and caffeine alter the effects of the LPS- primed mesenchymal stem cells on the co-cultured neutrophils.

PubMed

Abbasi, Ardeshir; Kukia, Nasim Rahmani; Froushani, Seyyed Meysam Abtahi; Hashemi, Seyed Mahmoud

2018-04-15

Mesenchymal stem cells (MSCs) express some of the nicotinic receptor subunits and adenosine receptors. The communication between tissue MSCs with neutrophils has been shown in previous studies. The aim of the present study is to determine the role of nicotine or caffeine on MSCs and its effects on neutrophils. After the isolation, MSCs were pulsed with LPS (10 ng/ml) for 1 h. Then, MSCs were incubated with different concentrations of caffeine (0.1, 0.5 and 1 mM) and or with different concentrations of nicotine (0.1, 0.5, and 1 μM) for 48 h. Afterwards, the medium was aspirated and the cells were used for co-culture experiment with neutrophil. The obtained data showed that LPS primed MSCs could decrease neutrophil vitality, whereas the treatment of MSCs with nicotine and/or especially a treatment with caffeine reverse this effect. Obtained data showed that when the LPS-primed MSCs were treated with nicotine or caffeine, the vitality of co-cultured neutrophils was significantly increased. The rate of the respiratory burst of neutrophils after co-culture by LPS-primed MSCs was decreased compared to the respiratory burst of neutrophil alone. Nicotine and/or caffeine treatment could reverse this reduction. Generally, these findings provide a new insight into understanding the anti-inflammatory and immunomodulatory effects of nicotine and caffeine. Copyright © 2018 Elsevier Inc. All rights reserved.

Human adipose-derived mesenchymal stem cells for osteoarthritis: a pilot study with long-term follow-up and repeated injections.

PubMed

Song, Yang; Du, Hui; Dai, Chengxiang; Zhang, Li; Li, Suke; Hunter, David J; Lu, Liangjing; Bao, Chunde

2018-04-01

This study aimed to evaluate the safety and therapeutic potential of autologous human adipose-derived mesenchymal stem cells (haMSCs) in patients with osteoarthritis. Safety and efficacy of haMSCs were preclinically assessed in vitro and in BALB/c-nu nude mice. 18 patients were enrolled and divided into three dose groups: the low-dose, mid-dose and high-dose group (1 × 10 7 , 2 × 10 7 and 5 × 10 7 cells, respectively), provided three injections and followed up for 96 weeks. The preclinical study established the safety and efficacy of haMSCs. Intra-articular injections of haMSCs were safe and improved pain, function and cartilage volume of the knee joint, rendering them a promising novel treatment for knee osteoarthritis. The dosage of 5 × 10 7 haMSCs exhibited the highest improvement (ClinicalTrials.gov Identifier: NCT01809769).

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

Human periapical cyst-mesenchymal stem cells differentiate into neuronal cells.

PubMed

Marrelli, M; Paduano, F; Tatullo, M

2015-06-01

It was recently reported that human periapical cysts (hPCys), a commonly occurring odontogenic cystic lesion of inflammatory origin, contain mesenchymal stem cells (MSCs) with the capacity for self-renewal and multilineage differentiation. In this study, periapical inflammatory cysts were compared with dental pulp to determine whether this tissue may be an alternative accessible tissue source of MSCs that retain the potential for neurogenic differentiation. Flow cytometry and immunofluorescence analysis indicated that hPCy-MSCs and dental pulp stem cells spontaneously expressed the neuron-specific protein β-III tubulin and the neural stem-/astrocyte-specific protein glial fibrillary acidic protein (GFAP) in their basal state before differentiation occurs. Furthermore, undifferentiated hPCy-MSCs showed a higher expression of transcripts for neuronal markers (β-III tubulin, NF-M, MAP2) and neural-related transcription factors (MSX-1, Foxa2, En-1) as compared with dental pulp stem cells. After exposure to neurogenic differentiation conditions (neural media containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], and retinoic acid), the hPCy-MSCs showed enhanced expression of β-III tubulin and GFAP proteins, as well as increased expression of neurofilaments medium, neurofilaments heavy, and neuron-specific enolase at the transcript level. In addition, neurally differentiated hPCy-MSCs showed upregulated expression of the neural transcription factors Pitx3, Foxa2, Nurr1, and the dopamine-related genes tyrosine hydroxylase and dopamine transporter. The present study demonstrated for the first time that hPCy-MSCs have a predisposition toward the neural phenotype that is increased when exposed to neural differentiation cues, based on upregulation of a comprehensive set of proteins and genes that define neuronal cells. In conclusion, these results provide evidence that hPCy-MSCs might be another optimal source of neural/glial cells for cell-based therapies to treat neurologic diseases. © International & American Associations for Dental Research 2015.

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

Local application of IGFBP5 protein enhanced periodontal tissue regeneration via increasing the migration, cell proliferation and osteo/dentinogenic differentiation of mesenchymal stem cells in an inflammatory niche.

PubMed

Han, Nannan; Zhang, Fengqiu; Li, Guoqing; Zhang, Xiuli; Lin, Xiao; Yang, Haoqing; Wang, Lijun; Cao, Yangyang; Du, Juan; Fan, Zhipeng

2017-09-29

Periodontitis is a widespread infectious disease ultimately resulting in tooth loss. The number of mesenchymal stem cells (MSCs) in patients with periodontitis is decreased, and MSC functions are impaired. Rescuing the impaired function of MSCs in periodontitis is the key for treatment, especially in a manner independent of exogenous MSCs. Our previous study found that overexpressed insulin-like growth factor binding protein 5 (IGFBP5) could promote exogenous MSC-mediated periodontal tissue regeneration. Here, we investigate the role of IGFBP5 protein in MSCs and periodontal tissue regeneration independent of exogenous MSCs in an inflammatory niche. TNFα was used to mimic the inflammatory niche. Lentiviral IGFBP5 shRNA was used to silence IGFBP5 and recombinant human IGFBP5 protein (rhIGFBP5) was used to stimulate the periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs). The effects of IGFBP5 on PDLSCs were evaluated using the scratch-simulated wound migration, Transwell chemotaxis, alkaline phosphatase (ALP) activity, Alizarin red staining, Cell Counting Kit-8, Western blot, Real-time PCR, Co-IP and ChIP assays. The swine model of periodontitis was used to investigate the functions of IGFBP5 for periodontal regeneration and its anti-inflammation effect. We discovered that 0.5 ng/ml rhIGFBP5 protein enhanced the migration, chemotaxis, osteo/dentinogenic differentiation and cell proliferation of MSCs under the inflammatory condition. Moreover, 0.5 ng/ml rhIGFBP5 application could rescue the impaired functions of IGFBP5-silenced-MSCs in the inflammatory niche. Furthermore, local injection of rhIGFBP5 could promote periodontal tissue regeneration and relieve the local inflammation in a minipig model of periodontitis. Mechanistically, we found that BCOR negatively regulated the expression of IGFBP5 in MSCs. BCOR formed a protein complex with histone demethylase KDM6B and raised histone K27 methylation in the IGFBP5 promoter. This study revealed that rhIGFBP5 could activate the functions of MSCs in an inflammatory niche, provided insight into the mechanism underlying the activated capacities of MSCs, and identified IGFBP5 as a potential cytokine for improving tissue regeneration and periodontitis treatment independent of exogenous MSCs and its potential application in dental clinic.

Genetic modification to induce CXCR2 overexpression in mesenchymal stem cells enhances treatment benefits in radiation-induced oral mucositis.

PubMed

Shen, Zongshan; Wang, Jiancheng; Huang, Qiting; Shi, Yue; Wei, Zhewei; Zhang, Xiaoran; Qiu, Yuan; Zhang, Min; Wang, Yi; Qin, Wei; Huang, Shuheng; Huang, Yinong; Liu, Xin; Xia, Kai; Zhang, Xinchun; Lin, Zhengmei

2018-02-14

Radiation-induced oral mucositis affects patient quality of life and reduces tolerance to cancer therapy. Unfortunately, traditional treatments are insufficient for the treatment of mucositis and might elicit severe side effects. Due to their immunomodulatory and anti-inflammatory properties, the transplantation of mesenchymal stem cells (MSCs) is a potential therapeutic strategy for mucositis. However, systemically infused MSCs rarely reach inflamed sites, impacting their clinical efficacy. Previous studies have demonstrated that chemokine axes play an important role in MSC targeting. By systematically evaluating the expression patterns of chemokines in radiation/chemical-induced oral mucositis, we found that CXCL2 was highly expressed, whereas cultured MSCs negligibly express the CXCL2 receptor CXCR2. Thus, we explored the potential therapeutic benefits of the transplantation of CXCR 2 -overexpressing MSCs (MSCs CXCR2 ) for mucositis treatment. Indeed, MSCs CXCR2 exhibited enhanced targeting ability to the inflamed mucosa in radiation/chemical-induced oral mucositis mouse models. Furthermore, we found that MSC CXCR2 transplantation accelerated ulcer healing by suppressing the production of pro-inflammatory chemokines and radiogenic reactive oxygen species (ROS). Altogether, these findings indicate that CXCR2 overexpression in MSCs accelerates ulcer healing, providing new insights into cell-based therapy for radiation/chemical-induced oral mucositis.

Stem cell-based gene therapy activated using magnetic hyperthermia to enhance the treatment of cancer

PubMed Central

Yin, Perry T.; Shah, Shreyas; Pasquale, Nicholas J.; Garbuzenko, Olga B.; Minko, Tamara; Lee, Ki-Bum

2015-01-01

Stem cell-based gene therapies, wherein stem cells are genetically engineered to express therapeutic molecules, have shown tremendous potential for cancer applications owing to their innate ability to home to tumors. However, traditional stem cell-based gene therapies are hampered by our current inability to control when the therapeutic genes are actually turned on, thereby resulting in detrimental side effects. Here, we report the novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes TNF-related apoptosis-inducing ligand (TRAIL) in adipose-derived mesenchymal stem cells (AD-MSCs). By combining the tumor tropism of the AD-MSCs with the spatiotemporal MCNP-based delivery and activation of TRAIL expression, this platform provides an attractive means with which to enhance our control over the activation of stem cell-based gene therapies. In particular, we found that these engineered AD-MSCs retained their innate ability to proliferate, differentiate, and, most importantly, home to tumors, making them ideal cellular carriers. Moreover, exposure of the engineered AD-MSCS to mild magnetic hyperthermia resulted in the selective expression of TRAIL from the engineered AD-MSCs and, as a result, induced significant ovarian cancer cell death in vitro and in vivo. PMID:26720500

Stem cell-based gene therapy activated using magnetic hyperthermia to enhance the treatment of cancer.

PubMed

Yin, Perry T; Shah, Shreyas; Pasquale, Nicholas J; Garbuzenko, Olga B; Minko, Tamara; Lee, Ki-Bum

2016-03-01

Stem cell-based gene therapies, wherein stem cells are genetically engineered to express therapeutic molecules, have shown tremendous potential for cancer applications owing to their innate ability to home to tumors. However, traditional stem cell-based gene therapies are hampered by our current inability to control when the therapeutic genes are actually turned on, thereby resulting in detrimental side effects. Here, we report the novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes TNF-related apoptosis-inducing ligand (TRAIL) in adipose-derived mesenchymal stem cells (AD-MSCs). By combining the tumor tropism of the AD-MSCs with the spatiotemporal MCNP-based delivery and activation of TRAIL expression, this platform provides an attractive means with which to enhance our control over the activation of stem cell-based gene therapies. In particular, we found that these engineered AD-MSCs retained their innate ability to proliferate, differentiate, and, most importantly, home to tumors, making them ideal cellular carriers. Moreover, exposure of the engineered AD-MSCS to mild magnetic hyperthermia resulted in the selective expression of TRAIL from the engineered AD-MSCs and, as a result, induced significant ovarian cancer cell death in vitro and in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

Arterially Delivered Mesenchymal Stem Cells Prevent Obstruction-Induced Renal Fibrosis

PubMed Central

Asanuma, Hiroshi; Vanderbrink, Brian A.; Campbell, Matthew T.; Hile, Karen L.; Zhang, Hongji; Meldrum, Daniel R.; Meldrum, Kirstan K.

2010-01-01

Purpose Mesenchymal stem cells (MSCs) hold promise for the treatment of renal disease. While MSCs have been shown to accelerate recovery and prevent acute renal failure in multiple disease models, the effect of MSC therapy on chronic obstruction-induced renal fibrosis has not previously been evaluated. Materials and Methods Male Sprague-Dawley rats underwent renal artery injection of vehicle or fluorescent-labeled human bone marrow-derived MSCs immediately prior to sham operation or induction of left ureteral obstruction (UUO). One or 4 weeks later, the kidneys were harvested and the renal cortex analyzed for evidence of stem cell infiltration, epithelial-mesenchymal transition (EMT) as evidenced by E-cadherin/α-smooth muscle actin (α-SMA) expression and fibroblast specific protein (FSP+) staining, renal fibrosis (collagen content, Masson’s trichrome staining), and cytokine and growth factor activity (ELISA and real time RT-PCR). Results Fluorescent-labeled MSCs were detected in the interstitium of the kidney up to 4 weeks post-obstruction. Arterially delivered MSCs significantly reduced obstruction-induced α-SMA expression, FSP+ cell accumulation, total collagen content, and tubulointerstitial fibrosis, while simultaneously preserving E-cadherin expression, suggesting that MSCs prevent obstruction-induced EMT and renal fibrosis. Exogenous MSCs reduced obstruction-induced tumor necrosis factor-α (TNF-α) levels, but did not alter transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), fibroblast growth factor (FGF), or hepatocyte growth factor (HGF) expression. Conclusions Human bone marrow-derived MSCs remain viable several weeks after delivery into the kidney and provide protection against obstruction-induced EMT and chronic renal fibrosis. While the mechanism of MSCs-induced renal protection during obstruction remains unclear, our results demonstrate that alterations in TNF-α production may be involved. PMID:20850784

A Systematic Study of the Effect of Different Molecular Weights of Hyaluronic Acid on Mesenchymal Stromal Cell-Mediated Immunomodulation

PubMed Central

Gómez-Aristizábal, Alejandro; Kim, Kyung-Phil; Viswanathan, Sowmya

2016-01-01

Introduction Osteoarthritis (OA) is associated with chronic inflammation, and mesenchymal stromal cells (MSCs) have been shown to provide pain relief and reparative effects in clinical investigations. MSCs are often delivered with hyaluronic acid (HA), although the combined mechanism of action is not fully understood; we thus investigated the immunomodulatory effects of combining MSCs with different molecular weights (MW) of HA. Methods HAs with MWs of 1.6 MDa (hHA), 150 kDa or 7.5 kDa, were added to MSCs alone or MSC-immune cell co-cultures. Gene expression analyses, flow cytometry and cytokine measurements were assessed to determine the effect of HAs on the MSC interactions with immune cells. Results MSCs in the presence of HAs, in both normal and lymphocyte-conditioned medium, showed negligible changes in gene expression. While addition of hHA resulted in increased proliferation of activated lymphocytes, both in the presence and absence of MSCs, the overall combined effect was a more regulated, homeostatic one; this was supported by higher ratios of secreted IL10/IFNγ and IL10/IL2, in lymphocyte cultures, than with lower MW HAs or no HA, both in the presence and absence of MSCs. In addition, examination of monocyte-derived macrophages showed an increased M2 macrophage frequency (CD14+CD163+CD206+) in the presence of hHA, both with and without MSCs. Conclusions hHA produces a less pro-inflammatory environment than lower MW HAs. Moreover, combining hHA with MSCs has an additive effect on the MSC-mediated immunomodulation, suggestive of a more potent combination treatment modality for OA. PMID:26820314

Suppressive effect of compact bone-derived mesenchymal stem cells on chronic airway remodeling in murine model of asthma.

PubMed

Ogulur, Ismail; Gurhan, Gulben; Aksoy, Ayca; Duruksu, Gokhan; Inci, Cigdem; Filinte, Deniz; Kombak, Faruk Erdem; Karaoz, Erdal; Akkoc, Tunc

2014-05-01

New therapeutic strategies are needed in the treatment of asthma besides vaccines and pharmacotherapies. For the development of novel therapies, the use of mesenchymal stem cells (MSCs) is a promising approach in regenerative medicine. Delivery of compact bone (CB) derived MSCs to the injured lungs is an alternative treatment strategy for chronic asthma. In this study, we aimed to isolate highly enriched population of MSCs from mouse CB with regenerative capacity, and to investigate the impact of these cells in airway remodeling and inflammation in experimental ovalbumin-induced mouse model of chronic asthma. mCB-MSCs were isolated, characterized, labeled with GFP and then transferred into mice with chronic asthma developed by ovalbumin (OVA) provocation. Histopathological changes including basement membrane, epithelium, subepithelial smooth thickness and goblet cell hyperplasia, and MSCs migration to lung tissues were evaluated. These histopathological alterations were increased in ovalbumin-treated mice compared to PBS group (P<0.001). Intravenous administration of mCB-MSC significantly reduced these histopathological changes in both distal and proximal airways (P<0.001). We showed that GFP-labeled MSCs were located in the lungs of OVA group 2weeks after intravenous induction. mCB-MSCs also significantly promoted Treg response in ovalbumin-treated mice (OVA+MSC group) (P<0.037). Our studies revealed that mCB-MSCs migrated to lung tissue and suppressed histopathological changes in murine model of asthma. The results reported here provided evidence that mCB-MSCs may be an alternative strategy for the treatment of remodeling and inflammation associated with chronic asthma. Copyright © 2014 Elsevier B.V. All rights reserved.

Applications of Mesenchymal Stem Cells in Sinus Lift Augmentation as a Dental Implant Technology.

PubMed

Parnia, Feridoun; Yazdani, Javad; Maleki Dizaj, Solmaz

2018-01-01

The potential application of stem cell biology in human dentistry is a new and emerging field of research. The objective of the current review was to study the efficiency of mesenchymal stem cells (MSCs) in sinus lift augmentation (SLA). A literature review was performed in PubMed Central using MeSH keywords such as sinus lift, MSCs, dental implants, and augmentation. The searches involved full-text papers written in English, published in the past 10 years (2007-2017). The review included in vitro and in vivo studies on the use of MSCs in SLA. Electronic searching provided 45 titles, and among them, 8 papers were chosen as suitable based on the inclusion requirements of this review. The reviewed studies have revealed the potential of MSCs in SLA. According to these papers, stem cell therapy combined with different biomaterials may considerably improve bone regeneration in previous steps of dental implantation and may veritably lead to efficient clinical usages in the recent future. However, the identification of an ideal source of stem cells as well as long-term studies is vital to assess the success rate of this technology. Further clinical trials are also needed to approve the potential of MSCs in SLA.

Engineered Fibrin Gels for Parallel Stimulation of Mesenchymal Stem Cell Proangiogenic and Osteogenic Potential

PubMed Central

Murphy, Kaitlin C.; Hughbanks, Marissa L.; Binder, Bernard Y.K.; Vissers, Caroline B.; Leach, J. Kent

2014-01-01

Mesenchymal stem/stromal cells (MSCs) are under examination for use in cell therapies to repair bone defects resulting from trauma or disease. MSCs secrete proangiogenic cues and can be induced to differentiate into bone-forming osteoblasts, yet there is limited evidence that these events can be achieved in parallel. Manipulation of the cell delivery vehicle properties represents a candidate approach for directing MSC function in bone healing. We hypothesized that the biophysical properties of a fibrin gel could simultaneously regulate the proangiogenic and osteogenic potential of entrapped MSCs. Fibrin gels were formed by supplementation with NaCl (1.2, 2.3, and 3.9% w/v) to modulate gel biophysical properties without altering protein concentrations. MSCs entrapped in 1.2% w/v NaCl gels were the most proangiogenic in vitro, yet cells in 3.9% w/v gels exhibited the greatest osteogenic response. Compared to the other groups, MSCs entrapped in 2.3% w/v gels provided the best balance between proangiogenic potential, osteogenic potential, and gel contractility. The contribution of MSCs to bone repair was then examined when deployed in 2.3% w/v NaCl gels and implanted into an irradiated orthotopic bone defect. Compared to acellular gels after 3 weeks of implantation, defects treated with MSC-loaded fibrin gels exhibited significant increases in vessel density, early osteogenesis, superior morphology, and increased cellularity of repair tissue. Defects treated with MSC-loaded gels exhibited increased bone formation after 12 weeks compared to blank gels. These results confirm that fibrin gel properties can be modulated to simultaneously promote both the proangiogenic and osteogenic potential of MSCs, and fibrin gels modified by supplementation with NaCl are promising carriers for MSCs to stimulate bone repair in vivo. PMID:25527322

Hypoxic Preconditioning Promotes the Bioactivities of Mesenchymal Stem Cells via the HIF-1α-GRP78-Akt Axis.

PubMed

Lee, Jun Hee; Yoon, Yeo Min; Lee, Sang Hun

2017-06-21

Mesenchymal stem cells (MSC) are ideal materials for stem cell-based therapy. As MSCs reside in hypoxic microenvironments (low oxygen tension of 1% to 7%), several studies have focused on the beneficial effects of hypoxic preconditioning on MSC survival; however, the mechanisms underlying such effects remain unclear. This study aimed to uncover the potential mechanism involving 78-kDa glucose-regulated protein (GRP78) to explain the enhanced MSC bioactivity and survival in hindlimb ischemia. Under hypoxia (2% O₂), the expression of GRP78 was significantly increased via hypoxia-inducible factor (HIF)-1α. Hypoxia-induced GRP78 promoted the proliferation and migration potential of MSCs through the HIF-1α-GRP78-Akt signal axis. In a murine hind-limb ischemia model, hypoxic preconditioning enhanced the survival and proliferation of transplanted MSCs through suppression of the cell death signal pathway and augmentation of angiogenic cytokine secretion. These effects were regulated by GRP78. Our findings indicate that hypoxic preconditioning promotes survival, proliferation, and angiogenic cytokine secretion of MSCs via the HIF-1α-GRP78-Akt signal pathway, suggesting that hypoxia-preconditioned MSCs might provide a therapeutic strategy for MSC-based therapies and that GRP78 represents a potential target for the development of functional MSCs.

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

Mesenchymal stromal cells (MSCs) and colorectal cancer: a troublesome twosome for the anti-tumour immune response?

PubMed Central

O'Malley, Grace; Heijltjes, Madelon; Houston, Aileen M.; Rani, Sweta; Ritter, Thomas; Egan, Laurence J.; Ryan, Aideen E.

2016-01-01

The tumour microenvironment (TME) is an important factor in determining the growth and metastasis of colorectal cancer, and can aid tumours by both establishing an immunosuppressive milieu, allowing the tumour avoid immune clearance, and by hampering the efficacy of various therapeutic regimens. The tumour microenvironment is composed of many cell types including tumour, stromal, endothelial and immune cell populations. It is widely accepted that cells present in the TME acquire distinct functional phenotypes that promote tumorigenesis. One such cell type is the mesenchymal stromal cell (MSC). Evidence suggests that MSCs exert effects in the colorectal tumour microenvironment including the promotion of angiogenesis, invasion and metastasis. MSCs immunomodulatory capacity may represent another largely unexplored central feature of MSCs tumour promoting capacity. There is considerable evidence to suggest that MSCs and their secreted factors can influence the innate and adaptive immune responses. MSC-immune cell interactions can skew the proliferation and functional activity of T-cells, dendritic cells, natural killer cells and macrophages, which could favour tumour growth and enable tumours to evade immune cell clearance. A better understanding of the interactions between the malignant cancer cell and stromal components of the TME is key to the development of more specific and efficacious therapies for colorectal cancer. Here, we review and explore MSC- mediated mechanisms of suppressing anti-tumour immune responses in the colon tumour microenvironment. Elucidation of the precise mechanism of immunomodulation exerted by tumour-educated MSCs is critical to inhibiting immunosuppression and immune evasion established by the TME, thus providing an opportunity for targeted and efficacious immunotherapy for colorectal cancer growth and metastasis. PMID:27542276

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

GDNF-secreting mesenchymal stem cells provide localized neuroprotection in an inflammation-driven rat model of Parkinson's disease.

PubMed

Hoban, D B; Howard, L; Dowd, E

2015-09-10

Constraints involving the delivery method of glial cell line-derived neurotrophic factor (GDNF) have hampered its efficacy as a neuroprotectant in Parkinson's disease. Ex vivo gene therapy, in which suitable cells, such as bone marrow-derived mesenchymal stem cells (MSCs), are genetically engineered to overexpress GDNF (GDNF-MSCs) prior to transplantation may be more beneficial than direct brain infusion of the neurotrophin. Previously, GDNF-MSCs have been assessed in the commonly employed 6-hydroxydopamine neurotoxic model of Parkinson's disease. In this study however, we used an emerging inflammatory model of Parkinson's disease (the lipopolysaccharide (LPS) model) to assess the ability of transplanted GDNF-MSCs to protect against LPS-induced neuroinflammation, neurodegeneration and behavioral impairment. Thirty male Sprague-Dawley rats were used in this experiment. Rats were performance matched based on baseline motor function tests into three groups (LPS lesion only, LPS lesion+GFP-MSCs, LPS lesion+GDNF-MSCs; n=10/group). Both cell groups received a unilateral intra-striatal transplant of either 200,000 GDNF-MSCs or 200,000 GFP-MSCs (as a control). One day post-transplantation, all rats received a unilateral intra-nigral infusion of LPS (10 μg in 2 μl sterile saline). Rats were sacrificed by transcardial perfusion-fixation and their brains were used for post mortem quantitative immunohistochemistry. Injection of LPS into the substantia nigra induced a pronounced local inflammatory response which resulted in 20% loss of nigrostriatal dopaminergic neurons and impaired contralateral motor function. Following transplantation of GDNF-MSCs to the striatum, dense areas of TH-positive staining directly proximal to the transplant site were observed. Most importantly, this effect was observed only in the GDNF-MSC transplanted group and not the GFP-MSC transplanted group demonstrating protection and/or sprouting of the dopaminergic terminals induced by the secreted GDNF. This study is the first to highlight the neurotrophic capability of GDNF in the inflammation-driven LPS model and, while future studies will endeavor to improve this approach by increasing cell survival, this work highlights the potential of GDNF delivery by ex vivo gene therapy using MSCs. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease?

PubMed Central

Broekman, Winifred; Khedoe, Padmini P S J; Schepers, Koen; Roelofs, Helene; Stolk, Jan; Hiemstra, Pieter S

2018-01-01

COPD is characterised by tissue destruction and inflammation. Given the lack of curative treatments and the progressive nature of the disease, new treatments for COPD are highly relevant. In vitro cell culture and animal studies have demonstrated that mesenchymal stromal cells (MSCs) have the capacity to modify immune responses and to enhance tissue repair. These properties of MSCs provided a rationale to investigate their potential for treatment of a variety of diseases, including COPD. Preclinical models support the hypothesis that MSCs may have clinical efficacy in COPD. However, although clinical trials have demonstrated the safety of MSC treatment, thus far they have not provided evidence for MSC efficacy in the treatment of COPD. In this review, we discuss the rationale for MSC-based cell therapy in COPD, the main findings from in vitro and in vivo preclinical COPD model studies, clinical trials in patients with COPD and directions for further research. PMID:29653970

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.

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.

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.

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

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.

Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use.

PubMed

Altaie, Ala; Baboolal, Thomas G; Wall, Owen; Jones, Elena; McGonagle, Dennis

2018-03-01

Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid-derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit-fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca ++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-β3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

PubMed

Brun, Juliane; Lutz, Katrin A; Neumayer, Katharina M H; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K; Hart, Melanie L

2015-01-01

The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications.

Clinical follow-up of horses treated with allogeneic equine mesenchymal stem cells derived from umbilical cord blood for different tendon and ligament disorders.

PubMed

Van Loon, Vic J F; Scheffer, Carmen J W; Genn, Herman J; Hoogendoorn, Arie C; Greve, Jan W

2014-01-01

Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon and ligament disorders in sport horses. Equine allogeneic MSCs derived from umbilical cord blood (eUCB-MSCs) can be obtained in a minimally invasive fashion with successful propagation of MSCs. The objective of this study was to determine the applicability and therapeutic effect of eUCB-MSCs on tendinitis of the superficial digital flexor tendon, desmitis of the suspensory ligament, tendinitis of the deep digital flexor tendon, and desmitis of the inferior check ligament in clinical cases. A retrospective clinical study was performed. At two equine clinics, 52 warmblood horses were treated with cultured eUCB-MSCs between 2009 and 2012. About 2-10 × 10(6) cells per lesion were administered. When a lesion was treated twice, the total amount could run up to 20 × 10(6) cells. Pearson's chi-squared test was used to compare the effect of the injured structure on the success rate, as well as the effect of the age of the horse. Based on repeated examinations, 40 horses (77%) returned to work on the same or a higher level based on information provided by the owner. Neither the injured structure nor the age of the horse had a statistically significant influence on the result. Overall, the results of treatment of some tendon and ligament injuries with eUCB-MSCs in clinical cases are promising.

Early Passage Dependence of Mesenchymal Stem Cell Mechanics Influences Cellular Invasion and Migration.

PubMed

Spagnol, Stephen T; Lin, Wei-Chun; Booth, Elizabeth A; Ladoux, Benoit; Lazarus, Hillard M; Dahl, Kris Noel

2016-07-01

The cellular structures and mechanical properties of human mesenchymal stem cells (hMSCs) vary significantly during culture and with differentiation. Previously, studies to measure mechanics have provided divergent results using different quantitative parameters and mechanical models of deformation. Here, we examine hMSCs prepared for clinical use and subject them to mechanical testing conducive to the relevant deformability associated with clinical injection procedures. Micropipette aspiration of hMSCs shows deformation as a viscoelastic fluid, with little variation from cell to cell within a population. After two passages, hMSCs deform as viscoelastic solids. Further, for clinical applicability during stem cell migration in vivo, we investigated the ability of hMSCs to invade into micropillar arrays of increasing confinement from 12 to 8 μm spacing between adjacent micropillars. We find that hMSC samples with reduced deformability and cells that are more solid-like with passage are more easily able to enter the micropillar arrays. Increased cell fluidity is an advantage for injection procedures and optimization of cell selection based on mechanical properties may enhance efficacy of injected hMSC populations. However, the ability to invade and migrate within tight interstitial spaces appears to be increased with a more solidified cytoskeleton, likely from increased force generation and contractility. Thus, there may be a balance between optimal injection survival and in situ tissue invasion.

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.

Preconditioning of mesenchymal stromal cells toward nucleus pulposus-like cells by microcryogels-based 3D cell culture and syringe-based pressure loading system.

PubMed

Zeng, Yang; Feng, Siyu; Liu, Wei; Fu, Qinyouen; Li, Yaqian; Li, Xiaokang; Chen, Chun; Huang, Chenyu; Ge, Zigang; Du, Yanan

2017-04-01

To precondition mesenchymal stromal/stem cells (MSCs) with mechanical stimulation may enhance cell survival and functions following implantation in load bearing environment such as nucleus pulposus (NP) in intervertebral disc (IVD). In this study, preconditioning of MSCs toward NP-like cells was achieved in previously developed poly (ethylene glycol) diacrylate (PEGDA) microcryogels (PMs) within a syringe-based three-dimensional (3D) culture system which provided a facile and cost-effective pressure loading approach. PMs loaded with alginate and MSCs could be incubated in a sealable syringe which could be air-compressed to apply pressure loading through a programmable syringe pump. Expression levels of chondrogenic marker genes SOX9, COL II, and ACAN were significantly upregulated in MSCs when pressure loading of 0.2 MPa or 0.8 MPa was implemented. Expression levels of COL I and COL X were downregulated when pressure loading was applied. In a nude mouse model, MSCs loaded in PMs mechanically stimulated for three days were subcutaneously injected using the same culture syringe. Three weeks postinjection, more proteoglycans (PGs) were deposited and more SOX9 and COL II but less COL I and COL X were stained in 0.2 MPa group. Furthermore, injectable MSCs-loaded PMs were utilized in an ex vivo rabbit IVD organ culture model that demonstrated the leak-proof function and enhanced cell retention of PMs assisted cell delivery to a load bearing environment for potential NP regeneration. This microcryogels-based 3D cell culture and syringe-based pressure loading system represents a novel method for 3D cell culture with mechanical stimulation for better function. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 507-520, 2017. © 2015 Wiley Periodicals, Inc.

Heparan Sulfate Proteoglycans as Drivers of Neural Progenitors Derived From Human Mesenchymal Stem Cells.

PubMed

Okolicsanyi, Rachel K; Oikari, Lotta E; Yu, Chieh; Griffiths, Lyn R; Haupt, Larisa M

2018-01-01

Background: Due to their relative ease of isolation and their high ex vivo and in vitro expansive potential, human mesenchymal stem cells (hMSCs) are an attractive candidate for therapeutic applications in the treatment of brain injury and neurological diseases. Heparan sulfate proteoglycans (HSPGs) are a family of ubiquitous proteins involved in a number of vital cellular processes including proliferation and stem cell lineage differentiation. Methods: Following the determination that hMSCs maintain neural potential throughout extended in vitro expansion, we examined the role of HSPGs in mediating the neural potential of hMSCs. hMSCs cultured in basal conditions (undifferentiated monolayer cultures) were found to co-express neural markers and HSPGs throughout expansion with modulation of the in vitro niche through the addition of exogenous HS influencing cellular HSPG and neural marker expression. Results: Conversion of hMSCs into hMSC Induced Neurospheres (hMSC IN) identified distinctly localized HSPG staining within the spheres along with altered gene expression of HSPG core protein and biosynthetic enzymes when compared to undifferentiated hMSCs. Conclusion: Comparison of markers of pluripotency, neural self-renewal and neural lineage specification between hMSC IN, hMSC and human neural stem cell (hNSC H9) cultures suggest that in vitro generated hMSC IN may represent an intermediary neurogenic cell type, similar to a common neural progenitor cell. In addition, this data demonstrates HSPGs and their biosynthesis machinery, are associated with hMSC IN formation. The identification of specific HSPGs driving hMSC lineage-specification will likely provide new markers to allow better use of hMSCs in therapeutic applications and improve our understanding of human neurogenesis.

Recent advances in endocrine, metabolic and immune disorders: mesenchymal stem cells (MSCs) and engineered scaffolds.

PubMed

Cantore, Stefania; Crincoli, Vito; Boccaccio, Antonio; Uva, Antonio Emmanuele; Fiorentino, Michele; Monno, Giuseppe; Bollero, Patrizio; Derla, Chiara; Fabiano, Francesca; Ballini, Andrea; Santacroce, Luigi

2018-04-22

New sources of stem cells in adult organisms are constantly emerging. Postnatal Mesenchymal Stem Cells (MSCs), are the most promising support to perform an effective regenerative medicine: such cells have the ability to differentiate into several lineages, such as osteoblasts and chondroblasts, providing novel strategies to improve different complex treatments, during bone regeneration. 3D-printed biomaterials can be designed with geometry aimed to induce stem cells to differentiate towards specific lineage. The interaction between stem cells easy to isolate and engineered 3D-printed scaffolds can translate the tissue bio-engineering into bone regenerative surgery. For those reasons, to better identify the complexity represented by the activities and responses of MSCs requires the advance of new target therapies which are not current in endocrine, metabolic and immune disorders and yet to be developed. This topical review briefly focuses on the new approaches of translational medicine with the use of MSCs and scaffolds engineered with the aid of 3D-printing technology, highlights the osteogenic functions then addressing their applications across the breadth of regenerative medicine. The application of bone constructs consisting of engineered scaffold and MSCs as well as the aspects related to the optimal scaffold geometry that favours the best MSCs differentiation and the improvement of concepts as "sensing surface" were also discussed. Regenerative surgery is largely growing in the field of translational medicine. The use of new sources of MSCs and the improvement of new concepts of bio-engineered scaffolds will certainly be the next step of customized medicine. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Bioreactors to Influence Stem Cell Fate: Augmentation of Mesenchymal Stem Cell Signaling Pathways via Dynamic Culture Systems

PubMed Central

Yeatts, Andrew B.; Choquette, Daniel T.; Fisher, John P.

2012-01-01

Background Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro. Scope of Review This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems. Major Conclusions The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased. General Significance Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. PMID:22705676

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.

Allogeneic guinea pig mesenchymal stem cells ameliorate neurological changes in experimental colitis.

PubMed

Stavely, Rhian; Robinson, Ainsley M; Miller, Sarah; Boyd, Richard; Sakkal, Samy; Nurgali, Kulmira

2015-12-30

The use of mesenchymal stem cells (MSCs) to treat inflammatory bowel disease (IBD) is of great interest because of their immunomodulatory properties. Damage to the enteric nervous system (ENS) is implicated in IBD pathophysiology and disease progression. The most commonly used model to study inflammation-induced changes to the ENS is 2,4,6-trinitrobenzene-sulfonate acid (TNBS)-induced colitis in guinea pigs; however, no studies using guinea pig MSCs in colitis have been performed. This study aims to isolate and characterise guinea pig MSCs and then test their therapeutic potential for the treatment of enteric neuropathy associated with intestinal inflammation. MSCs from guinea pig bone marrow and adipose tissue were isolated and characterised in vitro. In in vivo experiments, guinea pigs received either TNBS for the induction of colitis or sham treatment by enema. MSCs were administered at a dose of 1 × 10(6) cells via enema 3 h after the induction of colitis. Colon tissues were collected 24 and 72 h after TNBS administration to assess the level of inflammation and damage to the ENS. The secretion of transforming growth factor-β1 (TGF-β1) was analysed in MSC conditioned medium by flow cytometry. Cells isolated from both sources were adherent to plastic, multipotent and expressed some human MSC surface markers. In vitro characterisation revealed distinct differences in growth kinetics, clonogenicity and cell morphology between MSC types. In an in vivo model of TNBS-induced colitis, guinea pig bone marrow MSCs were comparatively more efficacious than adipose tissue MSCs in attenuating weight loss, colonic tissue damage and leukocyte infiltration into the mucosa and myenteric plexus. MSCs from both sources were equally neuroprotective in the amelioration of enteric neuronal loss and changes to the neurochemical coding of neuronal subpopulations. MSCs from both sources secreted TGF-β1 which exerted neuroprotective effects in vitro. This study is the first evaluating the functional capacity of guinea pig bone marrow and adipose tissue-derived MSCs and providing evidence of their neuroprotective value in an animal model of colitis. In vitro characteristics of MSCs cannot be extrapolated to their therapeutic efficacy. TGF-β1 released by both types of MSCs might have contributed to the attenuation of enteric neuropathy associated with colitis.

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

Aging Reduces an ERRalpha-Directed Mitochondrial Glutaminase Expression Suppressing Glutamine Anaplerosis and Osteogenic Differentiation of Mesenchymal Stem Cells.

PubMed

Huang, Tongling; Liu, Renzhong; Fu, Xuekun; Yao, Dongsheng; Yang, Meng; Liu, Qingli; Lu, William W; Wu, Chuanyue; Guan, Min

2017-02-01

Aging deteriorates osteogenic capacity of mesenchymal stem/stromal cells (MSCs), contributing to imbalanced bone remodeling and osteoporosis. Glutaminase (Gls) catabolizes glutamine into glutamate at the first step of mitochondrial glutamine (Gln)-dependent anaplerosis which is essential for MSCs upon osteogenic differentiation. Estrogen-related receptor α (ERRα) regulates genes required for mitochondrial function. Here, we found that ERRα and Gls are upregulated by osteogenic induction in human MSCs (hMSCs). In contrast, osteogenic differentiation capacity and glutamine consumption of MSCs, as well as ERRα, Gls and osteogenic marker genes are significantly reduced with age. We demonstrated that ERRα binds to response elements on Gls promoter and affects glutamine anaplerosis through transcriptional induction of Gls. Conversely, mTOR inhibitor rapamycin, ERRα inverse agonist compound 29 or Gls inhibitor BPTES leads to reduced Gln anaplerosis and deteriorated osteogenic differentiation of hMSCs. Importantly, overexpression of ERRα or Gls restored impairment by these inhibitors. Finally, we proved that compensated ERRα or Gls expression indeed potentiated Gln anaplerosis and osteogenic capability of elderly mice MSCs in vitro. Together, we establish that Gls is a novel ERRα target gene and ERRα/Gls signaling pathway plays an important role in osteogenic differentiation of MSCs, providing new sights into novel regenerative therapeutics development. Our findings suggest that restoring age-related mitochondrial Gln-dependent anaplerosis may be beneficial for degenerative bone disorders such as osteoporosis. Stem Cells 2017;35:411-424. © 2016 AlphaMed Press.

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.

Isolation and Characterization of Exosome from Human Embryonic Stem Cell-Derived C-Myc-Immortalized Mesenchymal Stem Cells.

PubMed

Lai, Ruenn Chai; Yeo, Ronne Wee Yeh; Padmanabhan, Jayanthi; Choo, Andre; de Kleijn, Dominique P V; Lim, Sai Kiang

2016-01-01

Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular between 2006 and 2012. However, defined mechanisms of action underpinning the therapeutic efficacy of MSCs are lacking, but they are increasingly attributed to MSC trophic secretion rather than their differentiation potential. A promising secreted therapeutic candidate is an extracellular vesicle (EV) known as the exosome. The use of exosomes instead of cells as a therapeutic agent provides several advantages. A critical advantage is the prospect of a conventional pharmaceutical manufacturing process that is highly scalable and amenable to the stringent manufacturing process. For example, MSCs used as producers of therapeutics, and not as therapeutics per se, could be immortalized to generate infinitely expansible clonal lines to enhance the reproducible production of therapeutic exosomes. In this chapter, we will describe the immortalization of MSCs, and the production, isolation, and characterization of exosomes from immortalized MSC.

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.

Priming integrin α5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis

PubMed Central

Hamidouche, Zahia; Fromigué, Olivia; Ringe, Jochen; Häupl, Thomas; Vaudin, Pascal; Pagès, Jean-Christophe; Srouji, Samer; Livne, Erella; Marie, Pierre J.

2009-01-01

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin α5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised. PMID:19843692

Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis.

PubMed

Hamidouche, Zahia; Fromigué, Olivia; Ringe, Jochen; Häupl, Thomas; Vaudin, Pascal; Pagès, Jean-Christophe; Srouji, Samer; Livne, Erella; Marie, Pierre J

2009-11-03

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin alpha5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

Long Noncoding RNAs: New Players in the Osteogenic Differentiation of Bone Marrow- and Adipose-Derived Mesenchymal Stem Cells.

PubMed

Yang, Qiaolin; Jia, Lingfei; Li, Xiaobei; Guo, Runzhi; Huang, Yiping; Zheng, Yunfei; Li, Weiran

2018-06-01

Mesenchymal stem cells (MSCs) are an important population of multipotent stem cells that differentiate into multiple lineages and display great potential in bone regeneration and repair. Although the role of protein-coding genes in the osteogenic differentiation of MSCs has been extensively studied, the functions of noncoding RNAs in the osteogenic differentiation of MSCs are unclear. The recent application of next-generation sequencing to MSC transcriptomes has revealed that long noncoding RNAs (lncRNAs) are associated with the osteogenic differentiation of MSCs. LncRNAs are a class of non-coding transcripts of more than 200 nucleotides in length. Noncoding RNAs are thought to play a key role in osteoblast differentiation through various regulatory mechanisms including chromatin modification, transcription factor binding, competent endogenous mechanism, and other post-transcriptional mechanisms. Here, we review the roles of lncRNAs in the osteogenic differentiation of bone marrow- and adipose-derived stem cells and provide a theoretical foundation for future research.

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

Generation of insulin-producing cells from rat mesenchymal stem cells using an aminopyrrole derivative XW4.4.

PubMed

Ouyang, Jingfeng; Huang, Wei; Yu, Wanwan; Xiong, Wei; Mula, Ramanjaneya V R; Zou, Hongbin; Yu, Yongping

2014-02-05

Type 1 diabetes mellitus (T1DM), a multisystem disease with both biochemical and anatomical/structural consequences, is a major health concern worldwide. Pancreatic islet transplantation provides a promising treatment for T1DM. However, the limited availability of islet tissue or new sources of insulin producing cells (IPCs) that are responsive to glucose hinder this promising approach. Though slow, the development of pancreatic beta-cell lines from rodent or human origin has been steadily progressing. Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, culture-expanded, non-hematopoietic cells that are currently being investigated as a novel cellular therapy. The in vitro differentiation potential of IPCs has raised hopes for a treatment of clinical diseases associated with autoimmunity. We screened for small molecules that induce pancreatic differentiation of IPCs. There are some compounds which showed positive effects on the DTZ staining. The aminopyrrole derivative compound XW4.4 which shows the best activity among them was found to induce pancreatic differentiation of rat MSCs (rMSCs). The in vitro studies indicated that treatment of rMSCs with compound XW4.4 resulted in differentiated cells with characteristics of IPCs including islet-like clusters, spherical, grape-like morphology, insulin secretion, positive for dithizone, glucose stimulation and expression of pancreatic endocrine cell marker genes. The data has also suggested that hepatocyte nuclear factor 3β (HNF 3β) may be involved in pancreatic differentiation of rMSCs when treated with XW4.4. Results indicate that XW4.4 induced rMSCs support the efforts to derive functional IPCs and serve as a means to alleviate limitations surrounding islet cell transplantation in the treatment of T1DM. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

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.

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.

Different Effects of Human Umbilical Cord Mesenchymal Stem Cells on Glioblastoma Stem Cells by Direct Cell Interaction or Via Released Soluble Factors

PubMed Central

Bajetto, Adriana; Pattarozzi, Alessandra; Corsaro, Alessandro; Barbieri, Federica; Daga, Antonio; Bosio, Alessia; Gatti, Monica; Pisaturo, Valerio; Sirito, Rodolfo; Florio, Tullio

2017-01-01

Glioblastoma (GBM), the most common primary brain tumor in adults, is an aggressive, fast-growing and highly vascularized tumor, characterized by extensive invasiveness and local recurrence. In GBM and other malignancies, cancer stem cells (CSCs) are believed to drive invasive tumor growth and recurrence, being responsible for radio- and chemo-therapy resistance. Mesenchymal stem cells (MSCs) are multipotent progenitors that exhibit tropism for tumor microenvironment mediated by cytokines, chemokines and growth factors. Initial studies proposed that MSCs might exert inhibitory effects on tumor development, although, to date, contrasting evidence has been provided. Different studies reported either MSC anti-tumor activity or their support to tumor growth. Here, we examined the effects of umbilical cord (UC)-MSCs on in vitro GBM-derived CSC growth, by direct cell-to-cell interaction or indirect modulation, via the release of soluble factors. We demonstrate that UC-MSCs and CSCs exhibit reciprocal tropism when co-cultured as 3D spheroids and their direct cell interaction reduces the proliferation of both cell types. Contrasting effects were obtained by UC-MSC released factors: CSCs, cultured in the presence of conditioned medium (CM) collected from UC-MSCs, increased proliferation rate through transient ERK1/2 and Akt phosphorylation/activation. Analysis of the profile of the cytokines released by UC-MSCs in the CM revealed a strong production of molecules involved in inflammation, angiogenesis, cell migration and proliferation, such as IL-8, GRO, ENA-78 and IL-6. Since CXC chemokine receptor 2 (CXCR2), a receptor shared by several of these ligands, is expressed in GBM CSCs, we evaluated its involvement in CSC proliferation induced by UC-MSC-CM. Using the CXCR2 antagonist SB225002, we observed a partial but statistically significant inhibition of CSC proliferation and migration induced by the UC-MSC-released cytokines. Conversely, CXCR2 blockade did not reduce the reciprocal tropism between CSCs and UC-MSCs grown as spheroids. In conclusion, we show that direct (cell-to-cell contact) or indirect (via the release of soluble factors) interactions between GBM CSCs and UC-MSCs in co-culture produce divergent effects on cell growth, invasion and migration, with the former mainly causing an inhibitory response and the latter a stimulatory one, involving a paracrine activation of CXCR2. PMID:29081734

Different Effects of Human Umbilical Cord Mesenchymal Stem Cells on Glioblastoma Stem Cells by Direct Cell Interaction or Via Released Soluble Factors.

PubMed

Bajetto, Adriana; Pattarozzi, Alessandra; Corsaro, Alessandro; Barbieri, Federica; Daga, Antonio; Bosio, Alessia; Gatti, Monica; Pisaturo, Valerio; Sirito, Rodolfo; Florio, Tullio

2017-01-01

Glioblastoma (GBM), the most common primary brain tumor in adults, is an aggressive, fast-growing and highly vascularized tumor, characterized by extensive invasiveness and local recurrence. In GBM and other malignancies, cancer stem cells (CSCs) are believed to drive invasive tumor growth and recurrence, being responsible for radio- and chemo-therapy resistance. Mesenchymal stem cells (MSCs) are multipotent progenitors that exhibit tropism for tumor microenvironment mediated by cytokines, chemokines and growth factors. Initial studies proposed that MSCs might exert inhibitory effects on tumor development, although, to date, contrasting evidence has been provided. Different studies reported either MSC anti-tumor activity or their support to tumor growth. Here, we examined the effects of umbilical cord (UC)-MSCs on in vitro GBM-derived CSC growth, by direct cell-to-cell interaction or indirect modulation, via the release of soluble factors. We demonstrate that UC-MSCs and CSCs exhibit reciprocal tropism when co-cultured as 3D spheroids and their direct cell interaction reduces the proliferation of both cell types. Contrasting effects were obtained by UC-MSC released factors: CSCs, cultured in the presence of conditioned medium (CM) collected from UC-MSCs, increased proliferation rate through transient ERK1/2 and Akt phosphorylation/activation. Analysis of the profile of the cytokines released by UC-MSCs in the CM revealed a strong production of molecules involved in inflammation, angiogenesis, cell migration and proliferation, such as IL-8, GRO, ENA-78 and IL-6. Since CXC chemokine receptor 2 (CXCR2), a receptor shared by several of these ligands, is expressed in GBM CSCs, we evaluated its involvement in CSC proliferation induced by UC-MSC-CM. Using the CXCR2 antagonist SB225002, we observed a partial but statistically significant inhibition of CSC proliferation and migration induced by the UC-MSC-released cytokines. Conversely, CXCR2 blockade did not reduce the reciprocal tropism between CSCs and UC-MSCs grown as spheroids. In conclusion, we show that direct (cell-to-cell contact) or indirect (via the release of soluble factors) interactions between GBM CSCs and UC-MSCs in co-culture produce divergent effects on cell growth, invasion and migration, with the former mainly causing an inhibitory response and the latter a stimulatory one, involving a paracrine activation of CXCR2.

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

A gene delivery system with a human artificial chromosome vector based on migration of mesenchymal stem cells towards human glioblastoma HTB14 cells.

PubMed

Kinoshita, Yusuke; Kamitani, Hideki; Mamun, Mahabub Hasan; Wasita, Brian; Kazuki, Yasuhiro; Hiratsuka, Masaharu; Oshimura, Mitsuo; Watanabe, Takashi

2010-05-01

Mesenchymal stem cells (MSCs) have been expected to become useful gene delivery vehicles against human malignant gliomas when coupled with an appropriate vector system, because they migrate towards the lesion. Human artificial chromosomes (HACs) are non-integrating vectors with several advantages for gene therapy, namely, no limitations on the size and number of genes that can be inserted. We investigated the migration of human immortalized MSCs bearing a HAC vector containing the herpes simplex virus thymidine kinase gene (HAC-tk-hiMSCs) towards malignant gliomas in vivo. Red fluorescence protein-labeled human glioblastoma HTB14 cells were implanted into a subcortical region in nude mice. Four days later, green fluorescence protein-labeled HAC-tk-hiMSCs were injected into a contralateral subcortical region (the HTB14/HAC-tk-hiMSC injection model). Tropism to the glioma mass and the route of migration were visualized by fluorescence microscopy and immunohistochemical staining. HAC-tk-hiMSCs began to migrate toward the HTB14 glioma area via the corpus callosum on day 4, and gathered around the HTB14 glioma mass on day 7. To test whether the delivered gene could effectively treat glioblastoma in vivo, HTB14/HAC-tk-hiMSC injected mice were treated with ganciclovir (GCV) or PBS. The HTB14 glioma mass was significantly reduced by GCV treatment in mice injected with HAC-tk-hiMSCs. It was confirmed that gene delivery by our HAC-hiMSC system was effective after migration of MSCs to the glioma mass in vivo. Therefore, MSCs containing HACs carrying an anticancer gene or genes may provide a new tool for the treatment of malignant gliomas and possibly of other tumor types.

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

Clinical application prospect of umbilical cord-derived mesenchymal stem cells on clearance of advanced glycation end products through autophagy on diabetic wound.

PubMed

Han, Yanfu; Sun, Tianjun; Tao, Ran; Han, Yanqing; Liu, Jing

2017-03-24

Nowadays, wound healing delay due to diabetes is considered to be closely related to the accumulation of advanced glycation end products (AGEs). Although mesenchymal stem cells (MSCs) exhibit positive effects on diabetic wound healing, related mechanisms are still not fully elucidated. It has been reported that MSCs can improve the activity of autophagy in injured tissues, thereby playing an important role in wound healing. The autophagy induced by MSCs may be beneficial to diabetic wound healing via removing AGEs, which provide new ideas for clinical treatment of diabetic wounds with the potential of broad application prospects. In this study, the current research situation and application prospect of umbilical cord-derived MSCs on the clearance of AGEs in diabetic wound were reviewed.

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.

Genetically modified mesenchymal stem cells (MSCs) promote axonal regeneration and prevent hypersensitivity after spinal cord injury.

PubMed

Kumagai, Gentaro; Tsoulfas, Pantelis; Toh, Satoshi; McNiece, Ian; Bramlett, Helen M; Dietrich, W Dalton

2013-10-01

Neurotrophins and the transplantation of bone marrow-derived stromal cells (MSCs) are both candidate therapies targeting spinal cord injury (SCI). While some studies have suggested the ability of MSCs to transdifferentiate into neural cells, other SCI studies have proposed anti-inflammatory and other mechanisms underlying established beneficial effects. We grafted rat MSCs genetically modified to express MNTS1, a multineurotrophin that binds TrkA, TrkB and TrkC, and p75(NTR) receptors or MSC-MNTS1/p75(-) that binds mainly to the Trk receptors. Seven days after contusive SCI, PBS-only, GFP-MSC, MSC-MNTS1/GFP or MSC-MNTS1/p75(-)/GFP were delivered into the injury epicenter. All transplanted groups showed reduced inflammation and cystic cavity size compared to control SCI rats. Interestingly, transplantation of the MSC-MNTS1 and MSC-MNTS1/p75(-), but not the naïve MSCs, enhanced axonal growth and significantly prevented cutaneous hypersensitivity after SCI. Moreover, transplantation of MSC-MNTS1/p75(-) promoted angiogenesis and modified glial scar formation. These findings suggest that MSCs transduced with a multineurotrophin are effective in promoting cell growth and improving sensory function after SCI. These novel data also provide insight into the neurotrophin-receptor dependent mechanisms through which cellular transplantation leads to functional improvement after experimental SCI. © 2013.

Mesenchymal stem cell differentiation in an experimental cartilage defect: restriction of hypertrophy to bone-close neocartilage.

PubMed

Steck, Eric; Fischer, Jennifer; Lorenz, Helga; Gotterbarm, Tobias; Jung, Martin; Richter, Wiltrud

2009-09-01

Mesenchymal stem cells (MSCs) are promising for the treatment of articular cartilage defects; however, common protocols for in vitro chondrogenesis induce typical features of hypertrophic chondrocytes reminiscent of endochondral bone formation. Aim of the study was to compare chondrogenic differentiation of MSCs in vitro and in vivo in experimental full-thickness cartilage defects, asking whether MSCs can differentiate into collagen type X-negative chondrocytes in an orthotopic environment. Cartilage defects in knees of minipigs were covered with a collagen type I/III membrane, and half of them received transplantation of expanded autologous MSCs. At 1, 3, and 8 weeks, morphological and molecular aspects of repair were assessed. The orthotopic environment triggered a spatially organized repair tissue with upper fibrous, intermediate chondrogenic, and low layer hypertrophic differentiation of cells and a trend to more safranin-O and collagen type II-positive samples after MSC transplantation at 8 weeks. Compared to in vitro chondrogenesis, significant lower COL10A1/COL2A1 and MMP13/COL2A1 ratios were obtained for in vivo differentiation. This indicates that, as opposed to in vitro chondrogenic induction of MSCs, the in vivo signaling molecules and biomechanical stimuli provide an appropriate environment for progenitor cells to differentiate into collagen type X-negative chondrocytes. Thus, until better in vitro induction protocols become available for chondrogenesis of MSCs, their predifferentiation before transplantation may be unfavorable.

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

Gene Profiles in a Smoke-Induced COPD Mouse Lung Model Following Treatment with Mesenchymal Stem Cells.

PubMed

Kim, You-Sun; Kokturk, Nurdan; Kim, Ji-Young; Lee, Sei Won; Lim, Jaeyun; Choi, Soo Jin; Oh, Wonil; Oh, Yeon-Mok

2016-10-01

Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The Hbb and Hba, genes with oxygen transport and antioxidant functions, were increased on days 1 and 14. A serine protease inhibitor was also increased at a similar time point after injection of hCB-MSCs. Gene Ontology analysis indicated that the levels of genes related to immune responses, metabolic processes, and blood vessel development were altered, indicating host responses after hCB-MSC injection. These gene expression changes suggest that MSCs induce a regeneration mechanism against COPD induced by cigarette smoke. These analyses provide basic data for understanding the regeneration mechanisms promoted by hCB-MSCs in cigarette smoke-induced COPD.

Effect of in vitro chondrogenic differentiation of autologous mesenchymal stem cells on cartilage and subchondral cancellous bone repair in osteoarthritis of temporomandibular joint.

PubMed

Chen, K; Man, C; Zhang, B; Hu, J; Zhu, S S

2013-02-01

This study investigated the effects of in vitro chondrogenic differentiated mesenchymal stem cells (MSCs) on cartilage and subchondral cancellous bone in temporomandibular joint osteoarthritis (TMJOA). Four weeks after induction of osteoarthritis (OA), the joints received hylartin solution, non-chondrogenic MSCs or in vitro chondrogenic differentiated MSCs. The changes in cartilage and subchondral cancellous bone were evaluated by histology, reverse transcription polymerase chain reaction and micro-computed tomography (CT). Implanted cells were tracked using Adeno-LacZ labelling. The differentiated MSC-treated group had better histology than the MSC-treated group at 4 and 12 weeks, but no difference at 24 weeks. Increased mRNA expression of collegan II, aggeran, Sox9 and decreased matrix metalloproteinase 13 (MMP13) were observed in differentiated MSC-treated groups compared to the undifferentiated MSC-treated group at 4 weeks. The differentiated MSC-treated group had decreased bone volume fraction, trabecular thickness and bone surface density, and increased trabecular spacing in the subchondral cancellous bone than the undifferentiated MSC-treated group. Transplanted cells were observed at cartilage, subchondral bone, and the synovial membrane lining at 4 weeks. Intra-articular injection of MSCs could delay the progression of TMJOA, and in vitro chondrogenic induction of MSCs could enhance the therapeutic effects. This provides new insights into the role of MSCs in cell-based therapies for TMJOA. Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Impact of mesenchymal stem cells' secretome on glioblastoma pathophysiology.

PubMed

Vieira de Castro, Joana; Gomes, Eduardo D; Granja, Sara; Anjo, Sandra I; Baltazar, Fátima; Manadas, Bruno; Salgado, António J; Costa, Bruno M

2017-10-02

Glioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells. The effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton's jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses. We found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. These findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.

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

Effects of the Endocrine-Disrupting Chemical DDT on Self-Renewal and Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Strong, Amy L.; Shi, Zhenzhen; Strong, Michael J.; Miller, David F.B.; Rusch, Douglas B.; Buechlein, Aaron M.; Flemington, Erik K.; McLachlan, John A.; Nephew, Kenneth P.

2014-01-01

Background: Although the global use of the endocrine-disrupting chemical DDT has decreased, its persistence in the environment has resulted in continued human exposure. Accumulating evidence suggests that DDT exposure has long-term adverse effects on development, yet the impact on growth and differentiation of adult stem cells remains unclear. Objectives: Human mesenchymal stem cells (MSCs) exposed to DDT were used to evaluate the impact on stem cell biology. Methods: We assessed DDT-treated MSCs for self-renewal, proliferation, and differentiation potential. Whole genome RNA sequencing was performed to assess gene expression in DDT-treated MSCs. Results: MSCs exposed to DDT formed fewer colonies, suggesting a reduction in self-renewal potential. DDT enhanced both adipogenic and osteogenic differentiation, which was confirmed by increased mRNA expression of glucose transporter type 4 (GLUT4), lipoprotein lipase (LpL), peroxisome proliferator-activated receptor gamma (PPARγ), leptin, osteonectin, core binding factor 1 (CBFA1), and FBJ murine osteosarcoma viral oncogene homolog (c-Fos). Expression of factors in DDT-treated cells was similar to that in estrogen-treated MSCs, suggesting that DDT may function via the estrogen receptor (ER)-mediated pathway. The coadministration of ICI 182,780 blocked the effects of DDT. RNA sequencing revealed 121 genes and noncoding RNAs to be differentially expressed in DDT-treated MSCs compared with controls cells. Conclusion: Human MSCs provide a powerful biological system to investigate and identify the molecular mechanisms underlying the effects of environmental agents on stem cells and human health. MSCs exposed to DDT demonstrated profound alterations in self-renewal, proliferation, differentiation, and gene expression, which may partially explain the homeostatic imbalance and increased cancer incidence among those exposed to long-term EDCs. Citation: Strong AL, Shi Z, Strong MJ, Miller DF, Rusch DB, Buechlein AM, Flemington EK, McLachlan JA, Nephew KP, Burow ME, Bunnell BA. 2015. Effects of the endocrine-disrupting chemical DDT on self-renewal and differentiation of human mesenchymal stem cells. Environ Health Perspect 123:42–48; http://dx.doi.org/10.1289/ehp.1408188 PMID:25014179

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.

Tumor-associated mesenchymal stem cells inhibit naïve T cell expansion by blocking cysteine export from dendritic cells.

PubMed

Ghosh, Tithi; Barik, Subhasis; Bhuniya, Avishek; Dhar, Jesmita; Dasgupta, Shayani; Ghosh, Sarbari; Sarkar, Madhurima; Guha, Ipsita; Sarkar, Koustav; Chakrabarti, Pinak; Saha, Bhaskar; Storkus, Walter J; Baral, Rathindranath; Bose, Anamika

2016-11-01

Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4(+) and CD8(+) T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune-mediated tumor eradication. Notably, tumor MSCs fail to prevent DC-mediated early T cell activation events or the ability of responder T cells to produce IL-2. The immunoregulatory activity of tumor MSCs is IL-10- and STAT3-dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine-to-cysteine. Under cysteine-deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS-like motif within the cystathionase promoter (-269 to -261) leading to IL-10-STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC-mediated T cell inhibition within tumor microenvironment. © 2016 UICC.

Differentiation of neural crest stem cells from nasal mucosa into motor neuron-like cells.

PubMed

Bagher, Zohreh; Kamrava, Seyed Kamran; Alizadeh, Rafieh; Farhadi, Mohammad; Absalan, Moloud; Falah, Masoumeh; Faghihi, Faezeh; Zare-Sadeghi, Arash; Komeili, Ali

2018-05-25

Cell transplantation is a potential therapeutic approach for repairing neuropathological and neurodegenerative disorders of central nervous system by replacing the degenerated cells with new ones. Among a variety of stem cell candidates to provide these new cells, olfactory ectomesenchymal stem cells (OE-MSCs) have attracted a great attention due to their neural crest origin, easy harvest, high proliferation, and autologous transplantation. Since there is no report on differentiation potential of these cells into motor neuron-like cells, we evaluated this potential using Real-time PCR, flowcytometry and immunocytochemistry after the treatment with differentiation cocktail containing retinoic acid and Sonic Hedgehog. Immunocytochemistry staining of the isolated OE-MSCs demonstrated their capability to express nestin and vimentin, as the two markers of primitive neuroectoderm. The motor neuron differentiation of OE-MSCs resulted in changing their morphology into bipolar cells with high expression of motor neuron markers of ChAT, Hb-9 and Islet-1 at the level of mRNA and protein. Consequently, we believe that the OE-MSCs have great potential to differentiate into motor neuron-like cells and can be an ideal stem cell source for the treatment of motor neuron-related disorders of central nervous system. Copyright © 2018 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.

New insights into the cellular makeup and progenitor potential of palatal connective tissues.

PubMed

Pall, Emoke; Cenariu, Mihai; Kasaj, Adrian; Florea, Adrian; Soancă, Andrada; Roman, Alexandra; Georgiu, Carmen

2017-12-01

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco-gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de-epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de-epithelialised MSCs (deMSCs) and re-entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony-forming unit- fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA-DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA-, CD31-) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,-in the pericapillary regions and in remote regions of the lamina propria- and pericytes-surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision-making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue. © 2017 Wiley Periodicals, Inc.

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.

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.

Platelet-Derived Growth Factor-BB Protects Mesenchymal Stem Cells (MSCs) Derived From Immune Thrombocytopenia Patients Against Apoptosis and Senescence and Maintains MSC-Mediated Immunosuppression

PubMed Central

Zhang, Jia-min; Feng, Fei-er; Wang, Qian-ming; Zhu, Xiao-lu; Fu, Hai-xia; Xu, Lan-ping; Liu, Kai-yan

2016-01-01

Immune thrombocytopenia (ITP) is characterized by platelet destruction and megakaryocyte dysfunction. Mesenchymal stem cells (MSCs) from ITP patients (MSC-ITP) do not exhibit conventional proliferative abilities and thus exhibit defects in immunoregulation, suggesting that MSC impairment might be a mechanism involved in ITP. Platelet-derived growth factor (PDGF) improves growth and survival in various cell types. Moreover, PDGF promotes MSC proliferation. The aim of the present study was to analyze the effects of PDGF-BB on MSC-ITP. We showed that MSC-ITP expanded more slowly and appeared flattened and larger. MSC-ITP exhibited increased apoptosis and senescence compared with controls. Both the intrinsic and extrinsic pathways account for the enhanced apoptosis. P53 and p21 expression were upregulated in MSC-ITP, but inhibition of p53 with pifithrin-α markedly inhibited apoptosis and senescence. Furthermore, MSCs from ITP patients showed a lower capacity for inhibiting the proliferation of activated T cells inducing regulatory T cells (Tregs) and suppressing the synthesis of anti-glycoprotein (GP)IIb-IIIa antibodies. PDGF-BB treatment significantly decreased the expression of p53 and p21 and increased survivin expression in MSC-ITP. In addition, the apoptotic rate and number of senescent cells in ITP MSCs were reduced. Their impaired ability for inhibiting activated T cells, inducing Tregs, and suppressing the synthesis of anti-GPIIb-IIIa antibodies was restored after PDGF-BB treatment. In conclusion, we have demonstrated that PDGF-BB protects MSCs derived from ITP patients against apoptosis, senescence, and immunomodulatory defects. This protective effect of PDGF-BB is likely mediated via the p53/p21 pathway, thus potentially providing a new therapeutic approach for ITP. Significance Immune thrombocytopenia (ITP) is characterized by platelet destruction and megakaryocyte dysfunction. Platelet-derived growth factor (PDGF) improves growth and survival in various cell types and promotes mesenchymal stem cell (MSC) proliferation. PDGF-BB protects MSCs derived from ITP patients against apoptosis, senescence, and immunomodulatory defects. This protective effect of PDGF-BB is likely mediated via the p53/p21 pathway, thus potentially providing a new therapeutic approach for ITP. PMID:27471307

Intra-articular delivery of purified mesenchymal stem cells from C57BL/6 or MRL/MpJ superhealer mice prevents posttraumatic arthritis.

PubMed

Diekman, Brian O; Wu, Chia-Lung; Louer, Craig R; Furman, Bridgette D; Huebner, Janet L; Kraus, Virginia B; Olson, Steven A; Guilak, Farshid

2013-01-01

Joint injury dramatically enhances the onset of osteoarthritis (OA) and is responsible for an estimated 12% of OA. Posttraumatic arthritis (PTA) is especially common after intra-articular fracture, and no disease-modifying therapies are currently available. We hypothesized that the delivery of mesenchymal stem cells (MSCs) would prevent PTA by altering the balance of inflammation and regeneration after fracture of the mouse knee. Additionally, we examined the hypothesis that MSCs from the MRL/MpJ (MRL) "superhealer" mouse strain would show increased multilineage and therapeutic potentials as compared to those from C57BL/6 (B6) mice, as MRL mice have shown exceptional in vivo regenerative abilities. A highly purified population of MSCs was prospectively isolated from bone marrow using cell surface markers (CD45-/TER119-/PDGFRα+/Sca-1+). B6 MSCs expanded greater than 100,000-fold in 3 weeks when cultured at 2% oxygen and displayed greater adipogenic, osteogenic, and chondrogenic differentiation as compared to MRL MSCs. Mice receiving only a control saline injection after fracture demonstrated PTA after 8 weeks, but the delivery of 10,000 B6 or MRL MSCs to the joint prevented the development of PTA. Cytokine levels in serum and synovial fluid were affected by treatment with stem cells, including elevated systemic interleukin-10 at several time points. The delivery of MSCs did not reduce the degree of synovial inflammation but did show increased bone volume during repair. This study provides evidence that intra-articular stem cell therapy can prevent the development of PTA after fracture and has implications for possible clinical interventions after joint injury before evidence of significant OA.

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.

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.

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.

Relevance of HCN2-expressing human mesenchymal stem cells for the generation of biological pacemakers.

PubMed

Bruzauskaite, Ieva; Bironaite, Daiva; Bagdonas, Edvardas; Skeberdis, Vytenis Arvydas; Denkovskij, Jaroslav; Tamulevicius, Tomas; Uvarovas, Valentinas; Bernotiene, Eiva

2016-04-30

The transfection of human mesenchymal stem cells (hMSCs) with the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene has been demonstrated to provide biological pacing in dogs with complete heart block. The mechanism appears to be the generation of the ion current (If) by the HCN2-expressing hMSCs. However, it is not clear how the transfection process and/or the HCN2 gene affect the growth functions of the hMSCs. Therefore, we investigated survival, proliferation, cell cycle, and growth on a Kapton® scaffold of HCN2-expressing hMSCs. hMSCs were isolated from the bone marrow of healthy volunteers applying a selective cell adhesion procedure and were identified by their expression of specific surface markers. Cells from passages 2-3 were transfected by electroporation using commercial transfection kits and a pIRES2-EGFP vector carrying the pacemaker gene, mouse HCN2 (mHCN2). Transfection efficiency was confirmed by enhanced green fluorescent protein (EGFP) fluorescence, quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). After hMSCs were transfected, their viability, proliferation, If generation, apoptosis, cell cycle, and expression of transcription factors were measured and compared with non-transfected cells and cells transfected with pIRES2-EGFP vector alone. Intracellular mHCN2 expression after transfection increased from 22.14 to 62.66 ng/mg protein (p < 0.05). Transfection efficiency was 45 ± 5 %. The viability of mHCN2-transfected cells was 82 ± 5 %; they grew stably for more than 3 weeks and induced If current. mHCN2-transfected cells had low mitotic activity (10.4 ± 1.24 % in G2/M and 83.6 ± 2.5 % in G1 phases) as compared with non-transfected cells (52-53 % in G2/M and 31-35 % in G1 phases). Transfected cells showed increased activation of nine cell cycle-regulating transcription factors: the most prominent upregulation was of AMP-dependent transcription factor ATF3 (7.11-fold, p = 0.00056) which regulates the G1 phase. mHCN2-expressing hMSCs were attached and made anchorage-dependent connection with other cells without transmigration through a 12.7-μm thick Kapton® HN film with micromachined 1-3 μm diameter pores. mHCN2-expressing hMSCs preserved the major cell functions required for the generation of biological pacemakers: high viability, functional activity, but low proliferation rate through the arrest of cell cycle in the G1 phase. mHCN2-expressing hMSCs attached and grew on a Kapton® scaffold without transmigration, confirming the relevance of these cells for the generation of biological pacemakers.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production.

PubMed

Camilleri, Emily T; Gustafson, Michael P; Dudakovic, Amel; Riester, Scott M; Garces, Catalina Galeano; Paradise, Christopher R; Takai, Hideki; Karperien, Marcel; Cool, Simon; Sampen, Hee-Jeong Im; Larson, A Noelle; Qu, Wenchun; Smith, Jay; Dietz, Allan B; van Wijnen, Andre J

2016-08-11

Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria. Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007 . Registered August 26, 2013. MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540 . Registered April 23, 2013. Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027 . Registered October 31, 2014. Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145 . Registered August 17, 2006. A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283 . Registered May 18, 2012.

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

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.

Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

PubMed Central

Verdanova, Martina; Pytlik, Robert

2014-01-01

A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBS-free medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO. PMID:24749876

Generation of functional islets from human umbilical cord and placenta derived mesenchymal stem cells.

PubMed

Kadam, Sachin; Govindasamy, Vijayendran; Bhonde, Ramesh

2012-01-01

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been used for allogeneic application in tissue engineering but have certain drawbacks. Therefore, mesenchymal stem cells (MSCs) derived from other adult tissue sources have been considered as an alternative. The human umbilical cord and placenta are easily available noncontroversial sources of human tissue, which are often discarded as biological waste, and their collection is noninvasive. These sources of MSCs are not subjected to ethical constraints, as in the case of embryonic stem cells. MSCs derived from umbilical cord and placenta are multipotent and have the ability to differentiate into various cell types crossing the lineage boundary towards endodermal lineage. The aim of this chapter is to provide a detailed reproducible cookbook protocol for the isolation, propagation, characterization, and differentiation of MSCs derived from human umbilical cord and placenta with special reference to harnessing their potential towards pancreatic/islet lineage for utilization as a cell therapy product. We show here that mesenchymal stromal cells can be extensively expanded from umbilical cord and placenta of human origin retaining their multilineage differentiation potential in vitro. Our report indicates that postnatal tissues obtained as delivery waste represent a rich source of mesenchymal stromal cells, which can be differentiated into functional islets employing three-stage protocol developed by our group. These islets could be used as novel in vitro model for screening hypoglycemics/insulin secretagogues, thus reducing animal experimentation for this purpose and for the future human islet transplantation programs to treat diabetes.

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

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

Type 1 Diabetes Mellitus Donor Mesenchymal Stromal Cells Exhibit Comparable Potency to Healthy Controls In Vitro.

PubMed

Davies, Lindsay C; Alm, Jessica J; Heldring, Nina; Moll, Guido; Gavin, Caroline; Batsis, Ioannis; Qian, Hong; Sigvardsson, Mikael; Nilsson, Bo; Kyllonen, Lauri E; Salmela, Kaija T; Carlsson, Per-Ola; Korsgren, Olle; Le Blanc, Katarina

2016-11-01

: Bone marrow mesenchymal stromal cells (BM-MSCs) have been characterized and used in many clinical studies based on their immunomodulatory and regenerative properties. We have recently reported the benefit of autologous MSC systemic therapy in the treatment of type 1 diabetes mellitus (T1D). Compared with allogeneic cells, use of autologous products reduces the risk of eliciting undesired complications in the recipient, including rejection, immunization, and transmission of viruses and prions; however, comparable potency of autologous cells is required for this treatment approach to remain feasible. To date, no analysis has been reported that phenotypically and functionally characterizes MSCs derived from newly diagnosed and late-stage T1D donors in vitro with respect to their suitability for systemic immunotherapy. In this study, we used gene array in combination with functional in vitro assays to address these questions. MSCs from T1D donors and healthy controls were expanded from BM aspirates. BM mononuclear cell counts and growth kinetics were comparable between the groups, with equivalent colony-forming unit-fibroblast capacity. Gene microarrays demonstrated differential gene expression between healthy and late-stage T1D donors in relation to cytokine secretion, immunomodulatory activity, and wound healing potential. Despite transcriptional differences, T1D MSCs did not demonstrate a significant difference from healthy controls in immunosuppressive activity, migratory capacity, or hemocompatibility. We conclude that despite differential gene expression, expanded MSCs from T1D donors are phenotypically and functionally similar to healthy control MSCs with regard to their immunomodulatory and migratory potential, indicating their suitability for use in autologous systemic therapy. The potential for mesenchymal stromal cells (MSCs) as a cell-based therapy in the treatment of immunologic disorders has been well established. Recent studies reported the clinical potential for autologous MSCs as a systemic therapy in the treatment of type I diabetes mellitus (T1D). The current study compared the genotypic and phenotypic profiles of bone marrow-derived MSCs from T1D and healthy donors as autologous (compared with allogeneic) therapy provides distinct advantages, such as reduced risk of immune reaction and transmission of infectious agents. The findings of the current study demonstrate that despite moderate differences in T1D MSCs at the gene level, these cells can be expanded in culture to an extent corresponding to that of MSCs derived from healthy donors. No functional difference in terms of immunosuppressive activity, blood compatibility, or migratory capacity was evident between the groups. The study findings also show that autologous MSC therapy holds promise as a T1D treatment and should be evaluated further in clinical trials. ©AlphaMed Press.

[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

New MSC: MSCs as pericytes are Sentinels and gatekeepers.

PubMed

Caplan, Arnold I

2017-06-01

Human Mesenchymal Stem Cells, hMSCs, were first named over 25 years ago with the "stem cell" nomenclature derived from the fact that we and others could cause these cells to differentiate into a number of different mesodermal phenotypes in cell culture. The capacity to form skeletal tissue in vitro encouraged the use of hMSCs for the fabrication of tissue engineered skeletal repair tissue with subsequent transplantation to in vivo sites. With the current realization that MSCs are derived from perivascular cells, pericytes, and the immunomodulatory and trophic capabilities of MSCs in both in vitro and in vivo test systems, a complete re-evaluation of the role and functions of MSCs in the body was required. Additionally, the skeleton is a preferred organ for cancer dissemination from various tumor malignancies. To date, most efforts to understand skeletal metastasis have focused on the invasive and digestive capability of disseminated tumor cells (DTCs). The contribution of the target organ-specific microvascular structure influencing extravasation is less well understood. Current targeted cancer therapies are designed to alter not only biological functions in DTCs, but also components of the tumor stroma/microenvironment such as blood vessels. We now have a comprehensive image of the critical role of the host vasculature as an instructive niche for DTCs. The focus of this manuscript is to present the current information about MSC function in situ and to emphasize how these new observations provide insight into understanding the role of the pericyte/MSC in skeletal activities including our new hypothesis for how these cells act as a gatekeeper for metastasis of melanoma into bone. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1151-1159, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Mesenchymal stromal cells and rheumatic diseases: new tools from pathogenesis to regenerative therapies.

PubMed

Cipriani, Paola; Ruscitti, Piero; Di Benedetto, Paola; Carubbi, Francesco; Liakouli, Vasiliki; Berardicurti, Onorina; Ciccia, Francesco; Triolo, Giovanni; Giacomelli, Roberto

2015-07-01

In recent years, mesenchymal stromal cells (MSCs) have been largely investigated and tested as a new therapeutic tool for several clinical applications, including the treatment of different rheumatic diseases. MSCs are responsible for the normal turnover and maintenance of adult mesenchymal tissues as the result of their multipotent differentiation abilities and their secretion of a variety of cytokines and growth factors. Although initially derived from bone marrow, MSCs are present in many different tissues such as many peri-articular tissues. MSCs may exert immune-modulatory properties, modulating different immune cells in both in vitro and in vivo models, and they are considered immune-privileged cells. At present, these capacities are considered the most intriguing aspect of their biology, introducing the possibility that these cells may be used as effective therapy in autoimmune diseases. Therefore, stem cell therapies may represent an innovative approach for the treatment of rheumatic diseases, especially for the forms that are not responsive to standard treatments or alternatively still lacking a definite therapy. At present, although the data from scientific literature appear to suggest that such treatments might be more effective whether administered as soon as possible, the use of MSCs in clinical practice is likely to be restricted to patients with a long history of a severe refractory disease. Further results from larger clinical trials are needed to corroborate preclinical findings and human non-controlled studies, and advancement in the knowledge of MSCs might provide information about the therapeutic role of these cells in the treatment of many rheumatic diseases. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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.

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.

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

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

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.

Tunable Surface Repellency Maintains Stemness and Redox Capacity of Human Mesenchymal Stem Cells.

PubMed

Balikov, Daniel A; Crowder, Spencer W; Boire, Timothy C; Lee, Jung Bok; Gupta, Mukesh K; Fenix, Aidan M; Lewis, Holley N; Ambrose, Caitlyn M; Short, Philip A; Kim, Chang Soo; Burnette, Dylan T; Reilly, Matthew A; Murthy, N Sanjeeva; Kang, Mi-Lan; Kim, Won Shik; Sung, Hak-Joon

2017-07-12

Human bone marrow derived mesenchymal stem cells (hMSCs) hold great promise for regenerative medicine due to their multipotent differentiation capacity and immunomodulatory capabilities. Substantial research has elucidated mechanisms by which extracellular cues regulate hMSC fate decisions, but considerably less work has addressed how material properties can be leveraged to maintain undifferentiated stem cells. Here, we show that synthetic culture substrates designed to exhibit moderate cell-repellency promote high stemness and low oxidative stress-two indicators of naïve, healthy stem cells-in commercial and patient-derived hMSCs. Furthermore, the material-mediated effect on cell behavior can be tuned by altering the molar percentage (mol %) and/or chain length of poly(ethylene glycol) (PEG), the repellant block linked to hydrophobic poly(ε-caprolactone) (PCL) in the copolymer backbone. Nano- and angstrom-scale characterization of the cell-material interface reveals that PEG interrupts the adhesive PCL domains in a chain-length-dependent manner; this prevents hMSCs from forming mature focal adhesions and subsequently promotes cell-cell adhesions that require connexin-43. This study is the first to demonstrate that intrinsic properties of synthetic materials can be tuned to regulate the stemness and redox capacity of hMSCs and provides new insight for designing highly scalable, programmable culture platforms for clinical translation.

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.

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.

Expansion in the presence of FGF-2 enhances the functional development of cartilaginous tissues engineered using infrapatellar fat pad derived MSCs.

PubMed

Buckley, C T; Kelly, D J

2012-07-01

MSCs from non-cartilaginous knee joint tissues such as the infrapatellar fat pad (IFP) and synovium possess significant chondrogenic potential and provide a readily available and clinically feasible source of chondroprogenitor cells. Fibroblast growth factor-2 (FGF-2) has been shown to be a potent mitotic stimulator during ex vivo expansion of MSCs, as well as regulating their subsequent differentiation potential. The objective of this study was to investigate the longer term effects of FGF-2 expansion on the functional development of cartilaginous tissues engineered using MSCs derived from the IFP. IFP MSCs were isolated and expanded to passage 2 in a standard media formulation with or without FGF-2 (5 ng/ml) supplementation. Expanded cells were encapsulated in agarose hydrogels, maintained in chondrogenic media for 42 days and analysed to determine their mechanical properties and biochemical composition. Culture media, collected at each feed, was also analysed for biochemical constituents. MSCs expanded in the presence of FGF-2 proliferated more rapidly, with higher cell yields and lower population doubling times. FGF-2 expanded MSCs generated the most mechanically functional tissue. Matrix accumulation was dramatically higher after 21 days for FGF-2 expanded MSCs, but decreased between day 21 and 42. By day 42, FGF-2 expanded MSCs had still accumulated ∼1.4 fold higher sGAG and ∼1.7 fold higher collagen compared to control groups. The total amount of sGAG synthesised (retained in hydrogels and released into the media) was ∼2.4 fold higher for FGF-2 expanded MSCs, with only ∼25% of the total amount generated being retained within the constructs. Further studies are required to investigate whether IFP derived MSCs have a diminished capacity to synthesise other matrix components important in the aggregation, assembly and retention of proteoglycans. In conclusion, expanding MSCs in the presence of FGF-2 rapidly accelerates chondrogenesis in 3D agarose cultures resulting in superior mechanical functionality. Copyright © 2011 Elsevier Ltd. All rights reserved.

Interleukin-17A increases leptin production in human bone marrow mesenchymal stem cells.

PubMed

Noh, Minsoo

2012-03-01

Lineage commitment of human bone marrow mesenchymal stem cells (hBM-MSCs) to adipocytes or osteoblasts has been suggested as a model system to study the relationship between type II diabetes and abnormal bone metabolism. Leptin and IL-17A inhibit adipogenesis whereas they promote osteogenesis in MSCs. Due to pathophysiologic roles of IL-17A in human metabolic diseases and bone metabolism, it was evaluated whether IL-17A-dependent inverse regulation on adipogenesis and osteogenesis was related to endogenous leptin production in hBM-MSCs. In the analysis of adiponectin and leptin secretion profiles of hBM-MSCs in response to various combinations of differentiation inducing factors, it was found that dexamethasone, a common molecule used for both adipogenesis and osteogenesis, increased leptin production in hBM-MSCs. Importantly, the level of leptin production during osteogenesis in hBM-MSCs was higher than that during adipogenesis, implicating a significant leptin production in extra-adipose tissues. IL-17A increased leptin production in hBM-MSCs and also under the condition of osteogenesis. In spite of direct inhibition on adipogenesis, IL-17A up-regulated leptin production in hBM-MSC-derived adipocytes. Anti-leptin antibody treatment partially antagonized the IL-17A dependent inhibition of adipogenesis in hBM-MSCs, suggesting a role of leptin in mediating the inverse regulation of IL-17A on osteogenesis and adipogenesis in hBM-MSCs. Therefore, the IL-17A-induced leptin production may provide a key clue to understand a molecular mechanism on the lineage commitment of hBM-MSCs into adipocytes or osteoblasts. In addition, leptin production in extra-adipose tissues like MSCs and osteoblasts should be considered in future studies on leptin-associated human diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

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

In vitro proliferation and osteogenic differentiation of mesenchymal stem cells on nanoporous alumina

PubMed Central

Song, Yuanhui; Ju, Yang; Song, Guanbin; Morita, Yasuyuki

2013-01-01

Cell adhesion, migration, and proliferation are significantly affected by the surface topography of the substrates on which the cells are cultured. Alumina is one of the most popular implant materials used in orthopedics, but few data are available concerning the cellular responses of mesenchymal stem cells (MSCs) grown on nanoporous structures. MSCs were cultured on smooth alumina substrates and nanoporous alumina substrates to investigate the interaction between surface topographies of nanoporous alumina and cellular behavior. Nanoporous alumina substrates with pore sizes of 20 nm and 100 nm were used to evaluate the effect of pore size on MSCs as measured by proliferation, morphology, expression of integrin β1, and osteogenic differentiation. An MTT assay was used to measure cell viability of MSCs on different substrates, and determined that cell viability decreased with increasing pore size. Scanning electron microscopy was used to investigate the effect of pore size on cell morphology. Extremely elongated cells and prominent cell membrane protrusions were observed in cells cultured on alumina with the larger pore size. The expression of integrin β1 was enhanced in MSCs cultured on porous alumina, revealing that porous alumina substrates were more favorable for cell growth than smooth alumina substrates. Higher levels of osteoblastic differentiation markers such as alkaline phosphatase, osteocalcin, and mineralization were detected in cells cultured on alumina with 100 nm pores compared with cells cultured on alumina with either 20 nm pores or smooth alumina. This work demonstrates that cellular behavior is affected by variation in pore size, providing new insight into the potential application of this novel biocompatible material for the developing field of tissue engineering. PMID:23935364

Polysaccharide Hydrogel Combined with Mesenchymal Stem Cells Promotes the Healing of Corneal Alkali Burn in Rats

PubMed Central

Liu, Xun; Yu, Min; Yang, Chunbo; Li, Xiaorong

2015-01-01

Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection of mesenchymal stem cells (MSCs) have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid and mesenchymal stem cells (MSCs) were derived from Sprague-Dawley rats. Supplementation of the polysaccharide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs application on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduction in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-β), antiangiogenic cytokine (TSP-1) and decrease those promoting inflammation (TNF-α), chemotaxis (MIP-1α and MCP-1) and angiogenesis (VEGF and MMP-2). This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treatments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder. PMID:25789487

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

PubMed

Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha

2018-05-01

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Potential use of mesenchymal stem cells in human meniscal repair: current insights

PubMed Central

Pak, Jaewoo; Lee, Jung Hun; Park, Kwang Seung; Jeon, Jeong Ho; Lee, Sang Hee

2017-01-01

The menisci of the human knee play an important role in maintaining normal functions to provide stability and nutrition to the articular cartilage, and to absorb shock. Once injured, these important structures have very limited natural healing potential. Unfortunately, the traditional arthroscopic meniscectomy performed on these damaged menisci may predispose the joint toward early development of osteoarthritis. Although a very limited number of studies are available, mesenchymal stem cells (MSCs) have been investigated as an alternative therapeutic modality to repair human knee meniscal tears. This review summarizes the results of published applications of MSCs in human patients, which showed that the patients who received MSCs (autologous adipose tissue-derived stem cells or culture-expanded bone marrow-derived stem cells) presented symptomatic improvements, along with magnetic resonance imaging evidences of the meniscal repair. PMID:28356779

Cell type dependent morphological adaptation in polyelectrolyte hydrogels governs chondrogenic fate.

PubMed

Raghothaman, Deepak; Leong, Meng Fatt; Lim, Tze Chiun; Wan, Andrew C A; Ser, Zheng; Lee, Eng Hin; Yang, Zheng

2016-04-04

Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell-cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.

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

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.

Focal Adhesion Kinase Signaling Mediated the Enhancement of Osteogenesis of Human Mesenchymal Stem Cells Induced by Extracorporeal Shockwave

NASA Astrophysics Data System (ADS)

Hu, Jun; Liao, Haojie; Ma, Zebin; Chen, Hongjiang; Huang, Zhonglian; Zhang, Yuantao; Yu, Menglei; Chen, Youbin; Xu, Jiankun

2016-02-01

Extracorporeal shockwave (ESW) has been shown of great potential in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but it is unknown whether this osteogenic promotion effect can also be achieved in other MSCs (i.e., tendon-derived stem cells (TDSCs) and adipose-derived stem cells (ADSCs)). In the current study, we aimed not only to compare the osteogenic effects of BMSCs induced by ESW to those of TDSCs and ADSCs; but also to investigate the underlying mechanisms. We show here that ESW (0.16 mj/mm2) significantly promoted the osteogenic differentiation in all the tested types of MSCs, accompanied with the downregulation of miR-138, but the activation of FAK, ERK1/2, and RUNX2. The enhancement of osteogenesis in these MSCs was consistently abolished when the cells were pretreated with one of the following conditions: overexpression of miR-138, FAK knockdown using specific siRNA, and U0126, implying that all of these elements are indispensable for mediating the effect of ESW. Moreover, our study provides converging genetic and molecular evidence that the miR-138-FAK-ERK1/2-RUNX2 machinery can be generally activated in ESW-preconditioned MSCs, suggesting that ESW may be a promising therapeutic strategy for the enhancement of osteogenesis of MSCs, regardless of their origins.

Biological performance of cell-encapsulated methacrylated gellan gum-based hydrogels for nucleus pulposus regeneration.

PubMed

Tsaryk, Roman; Silva-Correia, Joana; Oliveira, Joaquim Miguel; Unger, Ronald E; Landes, Constantin; Brochhausen, Christoph; Ghanaati, Shahram; Reis, Rui L; Kirkpatrick, C James

2017-03-01

Limitations of current treatments for intervertebral disc (IVD) degeneration have promoted interest in the development of tissue-engineering approaches. Injectable hydrogels loaded with cells can be used as a substitute material for the inner IVD part, the nucleus pulposus (NP), and provide an opportunity for minimally invasive treatment of IVD degeneration. The NP is populated by chondrocyte-like cells; therefore, chondrocytes and mesenchymal stem cells (MSCs), stimulated to differentiate along the chondrogenic lineage, could be used to promote NP regeneration. In this study, the in vitro and in vivo response of human bone marrow-derived MSCs and nasal chondrocytes (NCs) to modified gellan gum-based hydrogels was investigated. Both ionic- (iGG-MA) and photo-crosslinked (phGG-MA) methacrylated gellan gum hydrogels show no cytotoxicity in extraction assays with MSCs and NCs. Furthermore, the materials do not induce pro-inflammatory responses in endothelial cells. Moreover, MSCs and NCs can be encapsulated into the hydrogels and remain viable for at least 2 weeks, although apoptosis is observed in phGG-MA. Importantly, encapsulated MSCs and NCs show signs of in vivo chondrogenesis in a subcutaneous implantation of iGG-MA. Altogether, the data endorse the potential use of modified gellan gum-based hydrogel as a suitable material in NP tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

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.

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

Bone marrow mesenchymal stem cells suppress IL-9 in adjuvant-induced arthritis.

PubMed

Abd Elhalem, Sahar Sobhy; Haggag, Nawal Zakaria; El-Shinnawy, Nashwa Ahmed

2018-02-01

Interleukin-9 (IL-9) has been shown to be upregulated in rheumatoid arthritis (RA). The exact role of IL-9 has not yet been effectively studied. Mesenchymal stem cells (MSCs) have shown a promising immunomodulatory role towards repairing cartilage and restoring joint function. One of the key problems influencing the therapeutic efficacy of stem cell therapy is the poor cell survival following transplantation. This is attributed to oxidative and inflammatory stresses at the injured sites. Hesperidin (Hsd), a flavanone present in citrus fruits, has been studied as potential therapeutic agents that have anti-oxidant and anti-inflammatory activities. The objective of this study is to evaluate the therapeutic paracrine action of bone marrow MSCs on the IL-9 level in adjuvant-induced arthritis (AIA) and the enhancement effect of Hsd on transplanted MSCs. Articular tissue inflammation and cartilage damage were assessed by histological scoring. Antinuclear autoantibodies, tumour necrosis factor-alpha (TNF-α), IL-9, IL-4, interferon gamma (IFN-δ), and transforming growth factor-beta1 (TGF-β1), as well as malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) levels, were assessed in spleen tissue homogenates after treatment with MSCs either alone or combined with Hsd for 4 weeks in an AIA rat model. Results of this study confirmed that MSCs decreased IL-9 levels in AIA and provide novel insights into the application of Hsd on MSC-based treatments. Highlights Adjuvant-induced arthritis (AIA) is one of the most widely used models that has a great similarity to rheumatoid arthritis (RA). Few studies in recent years have estimated IL-9 in rheumatic diseases and it remains an understudied cytokine. For the first time, bone marrow mesenchymal stem cells (MSCs) therapy has a vital role in splenocytes IL-9 level and further studies are required. Combined therapy of MSCs with antioxidants as hesperidin (Hsd) can alleviate oxidative stress and enhance stem cells immunomodulatory action.

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

An In Vitro Comparison of the Incorporation, Growth, and Chondrogenic Potential of Human Bone Marrow versus Adipose Tissue Mesenchymal Stem Cells in Clinically Relevant Cell Scaffolds Used for Cartilage Repair

PubMed Central

Kohli, Nupur; Wright, Karina T.; Sammons, Rachel L.; Jeys, Lee; Snow, Martyn

2015-01-01

Aim To compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair. Methods Human BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively. Results A greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro-Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source. Conclusion Chondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue. PMID:26425263

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.

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.

Cytoskeletal proteins and stem cell markers gene expression in human bone marrow mesenchymal stromal cells after different periods of simulated microgravity

NASA Astrophysics Data System (ADS)

Gershovich, P. M.; Gershovich, J. G.; Zhambalova, A. P.; Romanov, Yu. A.; Buravkova, L. B.

2012-01-01

Mesenchymal stem (stromal) cells (MSCs) are present in a variety of tissues during prenatal and postnatal human development. In adult organism, they are prevalent in bone marrow and supposed to be involved in space-flight induced osteopenia. We studied expression of various genes in human bone marrow MSCs after different terms of simulated microgravity (SMG) provided by Random Positioning Machine. Simulated microgravity induced transient changes in expression level of genes associated with actin cytoskeleton, especially after 48 h of SMG. However, after 120 h exposure in SMG partial restoration of gene expression levels (relative to the control) was found. Similar results were obtained with bmMSCs subjected to 24 h readaptation in static state after 24 h in SMG. Analysis of 84 genes related to identification, growth and differentiation of stem cells revealed that expression of nine genes was changed slightly after 48 h in SMG. More pronounced changes in gene expression of "stem cells markers" were observed after 120 h of simulated microgravity. Among 84 investigated genes, 30 were up-regulated and 24 were down-regulated. Finally, MSCs osteogenesis induced by long-term (10-20 days) simulation of microgravity was accompanied by down-regulation of gene expression of the main osteogenic differentiation markers ( ALPL, OMD) and master transcription osteogenic factor of MSCs ( Runx2). Thus, our study demonstrated that changes in expression level of some genes associated with actin cytoskeleton and stem cell markers are supposed to be one of the mechanisms, which contribute to precursor's cellular adaptation to the microgravity conditions. These results can clarify genomic mechanisms through which SMG reduces osteogenic differentiation of bmMSCs.

Combined use of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and platelet rich plasma (PRP) stimulates proliferation and differentiation of myoblasts in vitro: new therapeutic perspectives for skeletal muscle repair/regeneration.

PubMed

Sassoli, Chiara; Vallone, Larissa; Tani, Alessia; Chellini, Flaminia; Nosi, Daniele; Zecchi-Orlandini, Sandra

2018-06-01

Satellite cell-mediated skeletal muscle repair/regeneration is compromised in cases of extended damage. Bone marrow mesenchymal stromal cells (BM-MSCs) hold promise for muscle healing but some criticisms hamper their clinical application, including the need to avoid animal serum contamination for expansion and the scarce survival after transplant. In this context, platelet-rich plasma (PRP) could offer advantages. Here, we compare the effects of PRP or standard culture media on C2C12 myoblast, satellite cell and BM-MSC viability, survival, proliferation and myogenic differentiation and evaluate PRP/BM-MSC combination effects in promoting myogenic differentiation. PRP induced an increase of mitochondrial activity and Ki67 expression comparable or even greater than that elicited by standard media and promoted AKT signaling activation in myoblasts and BM-MSCs and Notch-1 pathway activation in BM-MSCs. It stimulated MyoD, myogenin, α-sarcomeric actin and MMP-2 expression in myoblasts and satellite cell activation. Notably, PRP/BM-MSC combination was more effective than PRP alone. We found that BM-MSCs influenced myoblast responses through a paracrine activation of AKT signaling, contributing to shed light on BM-MSC action mechanisms. Our results suggest that PRP represents a good serum substitute for BM-MSC manipulation in vitro and could be beneficial towards transplanted cells in vivo. Moreover, it might influence muscle resident progenitors' fate, thus favoring the endogenous repair/regeneration mechanisms. Finally, within the limitations of an in vitro experimentation, this study provides an experimental background for considering the PRP/BM-MSC combination as a potential therapeutic tool for skeletal muscle damage, combining the beneficial effects of BM-MSCs and PRP on muscle tissue, while potentiating BM-MSC functionality.

Canine and Equine Mesenchymal Stem Cells Grown in Serum Free Media Have Altered Immunophenotype.

PubMed

Clark, Kaitlin C; Kol, Amir; Shahbenderian, Salpi; Granick, Jennifer L; Walker, Naomi J; Borjesson, Dori L

2016-04-01

Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE2, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE2. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions.

Development of lacrimal gland spheroids for lacrimal gland tissue regeneration.

PubMed

Massie, Isobel; Spaniol, Kristina; Barbian, Andreas; Geerling, Gerd; Metzger, Marco; Schrader, Stefan

2018-04-01

Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit. Epithelial cells and MSCs were isolated from porcine LG; endothelial cells were isolated from human foreskin. MSCs were characterised (flow cytometry and differentiation potential assays). All 3 cell types were combined on Matrigel and spheroid formation observed. Spheroids were characterised [immunohistochemistry (IHC) and transmission electron microscopy] and function assessed (β-hexosaminidase assay). Spheroids were transferred to decellularised jejunum (SIS-Muc) in dynamic cultures for 1 week before further characterisation. MSCs did not express CD31 but expressed CD44 and CD105 and differentiated towards osteogenic and adipogenic lineages. Spheroids formed on Matrigel within 18 hr, contracting to ~10% of the well area (p < .005). IHC revealed presence of all 3 cells within spheroids. Transmission electron microscopy revealed cell-cell contacts and polarisation at the apical surface. In static cultures, function was increased in spheroids cf. monolayer controls (p < .05) but over 72 hr, spheroid function (p < .05), viability (p < .05), and proliferation decreased, whilst apoptosis increased. On SIS-Muc under dynamic culture, however, spheroids continued to proliferate to repopulate SIS-Muc. IHC revealed LG epithelial cells coexpressing pan-cytokeratin and lysozyme, as well as endothelial cells and MSCs and cells remained capable of responding to carbachol (p < .05). These spheroids could form the basis of a regenerative medicine treatment approach for dry eye syndrome. In vivo studies are required to evaluate this further. Copyright © 2017 John Wiley & Sons, Ltd.

Therapeutic Effect of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Experimental Corneal Failure Due to Limbal Stem Cell Niche Damage.

PubMed

Galindo, Sara; Herreras, José M; López-Paniagua, Marina; Rey, Esther; de la Mata, Ana; Plata-Cordero, María; Calonge, Margarita; Nieto-Miguel, Teresa

2017-10-01

Limbal stem cells are responsible for the continuous renewal of the corneal epithelium. The destruction or dysfunction of these stem cells or their niche induces limbal stem cell deficiency (LSCD) leading to visual loss, chronic pain, and inflammation of the ocular surface. To restore the ocular surface in cases of bilateral LSCD, an extraocular source of stem cells is needed to avoid dependence on allogeneic limbal stem cells that are difficult to obtain, isolate, and culture. The aim of this work was to test the tolerance and the efficacy of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) to regenerate the ocular surface in two experimental models of LSCD that closely resemble different severity grades of the human pathology. hAT-MSCs transplanted to the ocular surface of the partial and total LSCD models developed in rabbits were well tolerated, migrated to inflamed tissues, reduced inflammation, and restrained the evolution of corneal neovascularization and corneal opacity. The expression profile of the corneal epithelial cell markers CK3 and E-cadherin, and the limbal epithelial cell markers CK15 and p63 was lost in the LSCD models, but was partially recovered after hAT-MSC transplantation. For the first time, we demonstrated that hAT-MSCs improve corneal and limbal epithelial phenotypes in animal LSCD models. These results support the potential use of hAT-MSCs as a novel treatment of ocular surface failure due to LSCD. hAT-MSCs represent an available, non-immunogenic source of stem cells that may provide therapeutic benefits in addition to reduce health care expenses. Stem Cells 2017;35:2160-2174. © 2017 AlphaMed Press.

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.

Biomaterials that promote cell-cell interactions enhance the paracrine function of MSCs.

PubMed

Qazi, Taimoor H; Mooney, David J; Duda, Georg N; Geissler, Sven

2017-09-01

Mesenchymal stromal cells (MSCs) secrete paracrine factors that play crucial roles during tissue regeneration. Whether this paracrine function is influenced by the properties of biomaterials in general, and those used for cell delivery in particular, largely remains unexplored. Here, we investigated if three-dimensional culture in distinct microenvironments - nanoporous hydrogels (mean pore size ∼5 nm) and macroporous scaffolds (mean pore size ∼120 μm) - affects the secretion pattern of MSCs, and consequently leads to differential paracrine effects on target progenitor cells such as myoblasts. We report that compared to MSCs encapsulated in hydrogels, scaffold seeded MSCs show an enhanced secretion profile and exert beneficial paracrine effects on various myoblast functions including migration and proliferation. Additionally, we show that the heightened paracrine effects of scaffold seeded cells can in part be attributed to N-cadherin mediated cell-cell interactions during culture. In hydrogels, this physical interaction between cells is prevented by the encapsulating matrix. Functionally blocking N-cadherin negatively affected the secretion profile and paracrine effects of MSCs on myoblasts, with stronger effects observed for scaffold seeded compared to hydrogel encapsulated cells. Together, these findings demonstrate that the therapeutic potency of MSCs can be enhanced by biomaterials that promote cell-cell interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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.

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.

Effective combination of human bone marrow mesenchymal stem cells and minocycline in experimental autoimmune encephalomyelitis mice

PubMed Central

2013-01-01

Introduction Multiple sclerosis (MS) is the most common inflammatory demyelinating disorder of the central nervous system (CNS). Minocycline ameliorates the clinical severity of MS and exhibits antiinflammatory, neuroprotective activities, and good tolerance for long-term use, whereas it is toxic to the CNS. Recently, the immunomodulation and neuroprotection capabilities of human bone marrow mesenchymal stem cells (hBM-MSCs) were shown in experimental autoimmune encephalomyelitis (EAE). In this study, we evaluated whether the combination of hBM-MSCs and a low-dose minocycline could produce beneficial effects in EAE mice. Methods The sensitivity of hBM-MSCs to minocycline was determined by an established cell-viability assay. Minocycline-treated hBM-MSCs were also characterized with flow cytometry by using MSC surface markers and analyzed for their multiple differentiation capacities. EAE was induced in C57BL/6 mice by using immunization with MOG35-55. Immunopathology assays were used to detect the inflammatory cells, demyelination, and neuroprotection. Interferon gamma (IFN-γ)/tumor necrosis factor alpha (TNF-α) and interleukin-4 (IL-4)/interleukin-10 (IL-10), the hallmark cytokines that direct Th1 and Th2 development, were detected with enzyme-linked immunosorbent assay (ELISA). terminal dUTP nick-end labeling (TUNEL) staining was performed to elucidate the cell apoptosis in the spinal cords of EAE mice. Results Minocycline did not affect the viability, surface phenotypes, or differentiation capacity of hBM-MSCs, while minocycline affected the viability of astrocytes at a high dose. In vivo efficacy experiments showed that combined treatment, compared to the use of minocycline or hBM-MSCs alone, resulted in a significant reduction in clinical scores, along with attenuation of inflammation, demyelination, and neurodegeneration. Moreover, the combined treatment with hBM-MSCs and minocycline enhanced the immunomodulatory effects, which suppressed proinflammatory cytokines (IFN-γ, TNF-α) and conversely increased anti-inflammatory cytokines (IL-4, IL-10). In addition, TUNEL staining also demonstrated a significant decrease of the number of apoptotic cells in the combined treatment compared with either treatment alone. Conclusions The combination of hBM-MSCs and minocycline provides a novel experimental protocol to enhance the therapeutic effects in MS. PMID:23826999

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.

Tissue Engineering in Osteoarthritis: Current Status and Prospect of Mesenchymal Stem Cell Therapy.

PubMed

Im, Gun-Il

2018-04-27

Osteoarthritis (OA) is the most common form of arthritis. Over the last 20 years, attempts have been made to regenerate articular cartilage to overcome the limitations of conventional treatments. As OA is generally associated with larger and diffuse involvement of articular surfaces and alteration of joint homeostasis, a tissue engineering approach for cartilage regeneration is more difficult than in simple chondral defects. Autologous and allogeneic mesenchymal stem cells (MSCs) have rapidly emerged as investigational products for cartilage regeneration. This review outlines points to consider in MSC-based approaches for OA treatment, including allogeneic MSCs, sources of MSCs, dosages, feasibility of multiple injections, indication according to severity of OA lesion and patient age, and issues regarding implantation versus injection. We introduce possible mechanisms of action of implanted or injected MSCs as well as the immunological aspects of MSC therapy and provide a summary of clinical trials of MSCs in the treatment of OA. Given current knowledge, it is too early to draw conclusions on the ultimate effectiveness of intra-articular application of MSCs in terms of regenerative effects. Further radiological and histological data will be needed, with a larger pool of patients, before this question can be answered.

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

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.

In Vivo Tracking of Mesechymal Stem Cells Using Fluorescent Nanoparticles in an Osteochondral Repair Model

PubMed Central

Lee, Jong Min; Kim, Byung-Soo; Lee, Haeshin; Im, Gun-Il

2012-01-01

We devised and tested an in vivo system to monitor the migration of mesenchymal stem cells (MSCs) within the marrow cavity. In vitro studies confirmed that platelet-derived growth factor (PDGF)-AA had the most potent chemotactic effect of the tested factors, and possessed the greatest number of receptors in MSCs. MSCs were labeled with fluorescent nanoparticles and injected into the marrow cavity of nude rats through osteochondral defects created in the distal femur. The defects were sealed with HCF (heparin-conjugated fibrin) or PDGF-AA-loaded HCF. In the HCF-only group, the nanoparticle-labeled MSCs dispersed outside the marrow cavity within 3 days after injection. In the PDGF-AA-loaded HCF group, the labeled cells moved time-dependently for 14 days toward the osteochondral defect. HCF-PDGF in low dose (LD; 8.5 ng/µl) was more effective than HCF-PDGF in high dose (HD; 17 ng/µl) in recruiting the MSCs to the osteochondral defect. After 21 days, the defects treated with PDGF and transforming growth factor (TGF)-β1-loaded HCF showed excellent cartilage repair compared with other groups. Further studies confirmed that this in vivo osteochondral MSCs tracking system (IOMTS) worked for other chemoattractants (chemokine (C-C motif) ligand 2 (CCL2) and PDGF-BB). IOMTS can provide a useful tool to examine the effect of growth factors or chemokines on endogenous cartilage repair. PMID:22491215

Influence of mesenchymal stem cells on stomach tissue engineering using small intestinal submucosa.

PubMed

Nakatsu, Hiroki; Ueno, Tomio; Oga, Atsunori; Nakao, Mitsuhiro; Nishimura, Taku; Kobayashi, Sei; Oka, Masaaki

2015-03-01

Small intestinal submucosa (SIS) is a biodegradable collagen-rich matrix containing functional growth factors. We have previously reported encouraging outcomes for regeneration of an artificial defect in the rodent stomach using SIS grafts, although the muscular layer was diminutive. In this study, we investigated the feasibility of SIS in conjunction with mesenchymal stem cells (MSCs) for regeneration of the gastrointestinal tract. MSCs from the bone marrow of green fluorescence protein (GFP)-transgenic Sprague-Dawley (SD) rats were isolated and expanded ex vivo. A 1 cm whole-layer stomach defect in SD rats was repaired using: a plain SIS graft without MSCs (group 1, control); a plain SIS graft followed by intravenous injection of MSCs (group 2); a SIS graft co-cultured with MSCs (group 3); or a SIS sandwich containing an MSC sheet (group 4). Pharmacological, electrophysiological and immunohistochemical examination was performed to evaluate the regenerated stomach tissue. Contractility in response to a muscarinic receptor agonist, a nitric oxide precursor or electrical field stimulation was observed in all groups. SIS grafts seeded with MSCs (groups 3 and 4) appeared to support improved regeneration compared with SIS grafts not seeded with MSCs (groups 1 and 2), by enabling the development of well-structured smooth muscle layers of significantly increased length. GFP expression was detected in the regenerated interstitial tissue, with fibroblast-like cells in the seeded-SIS groups. SIS potently induced pharmacological and electrophysiological regeneration of the digestive tract, and seeded MSCs provided an enriched environment that supported tissue regeneration by the SIS graft in the engineered stomach. © 2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

SIRT1 directly regulates SOX2 to maintain self-renewal and multipotency in bone marrow-derived mesenchymal stem cells.

PubMed

Yoon, Dong Suk; Choi, Yoorim; Jang, Yeonsue; Lee, Moses; Choi, Woo Jin; Kim, Sung-Hwan; Lee, Jin Woo

2014-12-01

SOX2 is crucial for the maintenance of the self-renewal capacity and multipotency of mesenchymal stem cells (MSCs); however, the mechanism by which SOX2 is regulated remains unclear. Here, we report that RNA interference of sirtuin 1 (SIRT1) in human bone marrow (BM)-derived MSCs leads to a decrease of SOX2 protein, resulting in the deterioration of the self-renewal and differentiation capacities of BM-MSCs. Using immunoprecipitation, we demonstrated direct binding between SIRT1 and SOX2 in HeLa cells overexpressing SOX2. We further discovered that the RNA interference of SIRT1 induces the acetylation, nuclear export, and ubiquitination of SOX2, leading to proteasomal degradation in BM-MSCs. SOX2 suppression by trichostatin A (TSA), a known histone deacetylase inhibitor, was reverted by treatment with resveratrol (0.1 and 1 µM), a known activator of SIRT1 in BM-MSCs. Furthermore, 0.1 and 1 µM resveratrol reduced TSA-mediated acetylation and ubiquitination of SOX2 in BM-MSCs. SIRT1 activation by resveratrol enhanced the colony-forming ability and differentiation potential to osteogenic and adipogenic lineages in a dose-dependent manner. However, the enhancement of self-renewal and multipotency by resveratrol was significantly decreased to basal levels by RNA interference of SOX2. These results strongly suggest that the SIRT1-SOX2 axis plays an important role in maintaining the self-renewal capability and multipotency of BM-MSCs. In conclusion, our findings provide evidence for positive SOX2 regulation by post-translational modification in BM-MSCs through the inhibition of nuclear export and subsequent ubiquitination, and demonstrate that SIRT1-mediated deacetylation contributes to maintaining SOX2 protein in the nucleus. © 2014 AlphaMed Press.

Influence of mesenchymal stem cells on stomach tissue engineering using small intestinal submucosa

PubMed Central

Nakatsu, Hiroki; Ueno, Tomio; Oga, Atsunori; Nakao, Mitsuhiro; Nishimura, Taku; Kobayashi, Sei; Oka, Masaaki

2015-01-01

Small intestinal submucosa (SIS) is a biodegradable collagen-rich matrix containing functional growth factors. We have previously reported encouraging outcomes for regeneration of an artificial defect in the rodent stomach using SIS grafts, although the muscular layer was diminutive. In this study, we investigated the feasibility of SIS in conjunction with mesenchymal stem cells (MSCs) for regeneration of the gastrointestinal tract. MSCs from the bone marrow of green fluorescence protein (GFP)-transgenic Sprague–Dawley (SD) rats were isolated and expanded ex vivo. A 1 cm whole-layer stomach defect in SD rats was repaired using: a plain SIS graft without MSCs (group 1, control); a plain SIS graft followed by intravenous injection of MSCs (group 2); a SIS graft co-cultured with MSCs (group 3); or a SIS sandwich containing an MSC sheet (group 4). Pharmacological, electrophysiological and immunohistochemical examination was performed to evaluate the regenerated stomach tissue. Contractility in response to a muscarinic receptor agonist, a nitric oxide precursor or electrical field stimulation was observed in all groups. SIS grafts seeded with MSCs (groups 3 and 4) appeared to support improved regeneration compared with SIS grafts not seeded with MSCs (groups 1 and 2), by enabling the development of well-structured smooth muscle layers of significantly increased length. GFP expression was detected in the regenerated interstitial tissue, with fibroblast-like cells in the seeded-SIS groups. SIS potently induced pharmacological and electrophysiological regeneration of the digestive tract, and seeded MSCs provided an enriched environment that supported tissue regeneration by the SIS graft in the engineered stomach. © 2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd. PMID:23913876

Human Adipose-derived Mesenchymal Stem Cells Attenuate Early Stage of Bleomycin Induced Pulmonary Fibrosis: Comparison with Pirfenidone

PubMed Central

Reddy, Manoj; Fonseca, Lyle; Gowda, Shashank; Chougule, Basavraj; Hari, Aarya; Totey, Satish

2016-01-01

Background and Objectives Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, invariably fatal fibrotic lung disease with no lasting option for therapy. Mesenchymal stem cells (MSCs) could be a promising modality for the treatment of IPF. Aim of the study was to investigate improvement in survivability and anti-fibrotic efficacy of human adipose-derived mesenchymal stem cells (AD-MSCs) in comparison with pirfenidone in the bleomycin-induced pulmonary fibrosis model. Methods Human AD-MSCs were administered intravenously on day 3, 6 and 9 after an intra-tracheal challenge with bleomycin, whereas, pirfenidone was given orally in drinking water at the rate of 100 mg/kg body weight three times a day daily from day 3 onward. AD-MSCs were labelled with PKH-67 before administration to detect engraftment. Disease severity and improvement was assessed and compared between sham control and vehicle control groups using Kaplan-Meier survival analysis, biochemical and molecular analysis, histopathology and high resolution computed tomography (HRCT) parameters at the end of study. Results Results demonstrated that AD-MSCs significantly increase survivability; reduce organ weight and collagen deposition better than pirfenidone group. Histological analyses and HRCT of the lung revealed that AD-MSCs afforded protection against bleomycin induced fibrosis and protect architecture of the lung. Gene expression analysis revealed that AD-MSCs potently suppressed pro-fibrotic genes induced by bleomycin. More importantly, AD-MSCs were found to inhibit pro-inflammatory related transcripts. Conclusions Our results provided direct evidence that AD-MSC-mediated immunomodulation and anti-fibrotic effect in the lungs resulted in marked protection in pulmonary fibrosis, but at an early stage of disease. PMID:27871152

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

Mesenchymal Stem Cell-Conditioned Medium Reduces Disease Severity and Immune Responses in Inflammatory Arthritis.

PubMed

Kay, Alasdair G; Long, Grace; Tyler, George; Stefan, Andrei; Broadfoot, Stephen J; Piccinini, Anna M; Middleton, Jim; Kehoe, Oksana

2017-12-21

We evaluated the therapeutic potential of mesenchymal stem cell-conditioned medium (CM-MSC) as an alternative to cell therapy in an antigen-induced model of arthritis (AIA). Disease severity and cartilage loss were evaluated by histopathological analysis of arthritic knee joints and immunostaining of aggrecan neoepitopes. Cell proliferation was assessed for activated and naïve CD4+ T cells from healthy mice following culture with CM-MSC or co-culture with MSCs. T cell polarization was analysed in CD4+ T cells isolated from spleens and lymph nodes of arthritic mice treated with CM-MSC or MSCs. CM-MSC treatment significantly reduced knee-joint swelling, histopathological signs of AIA, cartilage loss and suppressed TNFα induction. Proliferation of CD4+ cells from spleens of healthy mice was not affected by CM-MSC but reduced when cells were co-cultured with MSCs. In the presence of CM-MSC or MSCs, increases in IL-10 concentration were observed in culture medium. Finally, CD4+ T cells from arthritic mice treated with CM-MSC showed increases in FOXP3 and IL-4 expression and positively affected the Treg:Th17 balance in the tissue. CM-MSC treatment reduces cartilage damage and suppresses immune responses by reducing aggrecan cleavage, enhancing Treg function and adjusting the Treg:Th17 ratio. CM-MSC may provide an effective cell-free therapy for inflammatory arthritis.

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.

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.

The selection of light emitting diode irradiation parameters for stimulation of human mesenchymal stem cells proliferation

NASA Astrophysics Data System (ADS)

Lewandowski, Rafał; Trafny, ElŻbieta A.; Stepińska, Małgorzata; Gietka, Andrzej; Kotowski, Paweł; Dobrzyńska, Monika; Łapiński, Mariusz P.

2016-12-01

Human mesenchymal stem cells (hMSCs) with their vast differentiation potential are very useful for cell-based regenerative medicine. To achieve sufficient numbers of cells for tissue engineering, many different methods have been used to reach the effective increase of cell proliferation. Low-energy red light provided by light emitting diodes (LEDs) have been recently introduced as a method that promoted biomodulation and proliferation of hMSCs in vitro. The purpose of this study was to find the optimum stimulatory dosimetric parameters of LED (630 nm) irradiation on the hMSCs proliferation. The energy density was 2, 3, 4, 10, 20 J/cm2 and the power density used was 7, 17 or 30 mW/cm2. Human MSCs were irradiated with single or triple exposures daily at room temperature and the cell proliferation rate was evaluated during nine days after irradiation. The results showed that after irradiation 4 J/cm2 and 17 mW/cm2 at a single dose the proliferation rate of hMSCs increased on day 5 and 9 (13% and 7%, respectively) when compared to nonirradiated cells. However, triple LED irradiation under the same parameters resulted in the decline in the cell proliferation rate on day 5, but the proliferation rate was at the same level on day 9, when compared with the cell proliferation after irradiation with a single dose. The effect of a single dose irradiation with 4 J/cm2 and 17 mW/cm2 on the proliferation of cells was the highest when the cells were irradiated in phosphate-buffered saline (PBS) instead of MSCGM culture medium.

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.

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.

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

Comparison of the neuropoietic activity of gene-modified versus parental mesenchymal stromal cells and the identification of soluble and extracellular matrix-related neuropoietic mediators

PubMed Central

2014-01-01

Introduction Transplanting mesenchymal stromal cells (MSCs) or their derivatives into a neurodegenerative environment is believed to be beneficial because of the trophic support, migratory guidance, immunosuppression, and neurogenic stimuli they provide. SB623, a cell therapy for the treatment of chronic stroke, currently in a clinical trial, is derived from bone marrow MSCs by using transient transfection with a vector encoding the human Notch1 intracellular domain. This creates a new phenotype, which is effective in experimental stroke, exhibits immunosuppressive and angiogenic activity equal or superior to parental MSCs in vitro, and produces extracellular matrix (ECM) that is exceptionally supportive for neural cell growth. The neuropoietic activity of SB623 and parental MSCs has not been compared, and the SB623-derived neuropoietic mediators have not been identified. Methods SB623 or parental MSCs were cocultured with rat embryonic brain cortex cells on cell-derived ECM in a previously characterized quantitative neuropoiesis assay. Changes in expression of rat neural differentiation markers were quantified by using rat-specific qRT-PCR. Human mediators were identified by using expression profiling, an enzymatic crosslinking activity, and functional interference studies by means of blocking antibodies, biologic inhibitors, and siRNA. Cocultures were immunolabeled for presynaptic vesicular transporters to assess neuronal specialization. Results Among six MSC/SB623 pairs, SB623 induced expression of rat neural precursor, oligodendrocyte, and astrocyte markers on average 2.6 to 3 times stronger than did their parental MSCs. SB623 expressed significantly higher FGF2, FGF1, and BMP4, and lower FGFR1 and FGFR2 levels; and human FGF1, FGF2, BMPs, and HGF were implicated as neuropoietic mediators. Neural precursors grew faster on SB623- than on MSC-derived ECM. SB623 exhibited higher expression levels and crosslinking activity of tissue transglutaminase (TGM2). TGM2 silencing reduced neural precursor growth on SB623-ECM. SB623 also promoted the induction of GABA-ergic, but not glutamatergic, neurons more effectively than did MSCs. Conclusions These data demonstrate that SB623 cells tend to support neural cell growth more effectively than their parental MSCs and identify both soluble and insoluble mediators responsible, at least in part, for enhanced neuropoietic potency of SB623. The neuropoiesis assay is a useful tool for identifying beneficial factors produced by MSCs and their derivatives. PMID:24572070

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

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.

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.

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.

Characteristics of Labial Gland Mesenchymal Stem Cells of Healthy Individuals and Patients with Sjögren's Syndrome: A Preliminary Study.

PubMed

Wang, Shi-Qin; Wang, Yi-Xiang; Hua, Hong

2017-08-15

Sjögren's syndrome (SS) is a systemic autoimmune disease that is characterized by focal lymphocytic infiltration into exocrine organs such as salivary and lacrimal glands, resulting in dry mouth and eyes, and other systemic injuries. There is no curative clinical therapy for SS, and stem cell therapy has shown great potential in this area. The mesenchymal stem cells (MSCs) in the salivary glands of healthy individuals and in patients with SS have not been extensively studied. The aim of this study was to elucidate the characteristics of MSCs from the labial glands of healthy controls and of those from patients with SS to elucidate the related pathogenesis and to uncover potential avenues for novel clinical interventions. Labial glands from patients with SS and healthy subjects were obtained, and MSCs were isolated and cultured by using the tissue adherent method. The MSC characteristics of the cultured cells were confirmed by using morphology, proliferation, colony forming-unit (CFU) efficiency, and multipotentiality, including osteogenic, adipogenic, and salivary gland differentiation. The MSCs from the healthy controls and SS patients expressed characteristic MSC markers, including CD29, CD44, CD73, CD90, and CD105; they were negative for CD34, CD45, and CD106, and also negative for the salivary gland epithelium markers (CD49f and CD117). Labial gland MSCs from both groups were capable of osteogenic and adipogenic differentiation. The CFU efficiency and adipogenic differentiation potential of MSCs were significantly lower in the SS group compared with the healthy controls. Cells from both groups could also be induced into salivary gland-like cells. Real-time polymerase chain reaction and immunofluorescence staining showed that the gene and protein expression of AMY1, AQP5, and ZO-1 in cells from the SS group was lower than that in cells from the healthy group. Thus, MSCs from the labial glands in patients with SS could lack certain characteristics and functions, especially related to salivary secretion. These preliminary data provided insights that could lead to the development of novel therapeutic strategies for the treatment of SS.

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.

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

Treatment with platelet lysate induces endothelial differentation of bone marrow mesenchymal stem cells under fluid shear stress

PubMed Central

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Moradi, Alireza; Nadri, Hamid; Barzegar, Kazem; Eslami, Gilda

2014-01-01

By considering stem cell-based therapies as a new hope for the treatment of some tragic diseases, marrow stromal cells or marrow mesenchymal stem cells (MSCs) were considered as a suitable and safe multipotential cell source for this new therapeutic approach. For this purpose, many investigations have been performed on differentiation of MSCs toward specific cell lines to overcome the demand for providing the organ specific cells for cell therapy or preparation of engineered tissues. In the present study, differentiation of MSCs to endothelial cells (ECs) by mechanical and chemical stimulation was evaluated. Fluid shear stress (FSS) was used as mechanical inducer, while platelet lysate (PL) and estradiol (E) were used as chemical induction factors. MSCs were placed under FSS with different forces (2, 5 and 10dyn/cm2) for different periods (6, 12 and 24 hours). In some groups, PL and E were added to the culture media to evaluate their effect on expression of EC specific markers. This investigation revealed that FSS with low tension (2.5-5 dyn/cm2) for a long time (24 hours) or high tension (10 dyn/cm2) in short time (6 hours) in the presence of PL could differentiate MSCs toward ECs. The presence of PL was necessary for initiation of endothelial differentiation, and in the absence of PL, there was not any expression of CD34 and Cadherin5 (Cdh5) among cells. Adding E to the culture medium did not change the rate of endothelial differentiation under FSS. Generated endothelial progenitors could produce von Willebrand factor (vWF) after two weeks culture and also they formed tubular structures after culture on matrigel. PMID:26417289

Treatment with platelet lysate induces endothelial differentation of bone marrow mesenchymal stem cells under fluid shear stress.

PubMed

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Moradi, Alireza; Nadri, Hamid; Barzegar, Kazem; Eslami, Gilda

2014-01-01

By considering stem cell-based therapies as a new hope for the treatment of some tragic diseases, marrow stromal cells or marrow mesenchymal stem cells (MSCs) were considered as a suitable and safe multipotential cell source for this new therapeutic approach. For this purpose, many investigations have been performed on differentiation of MSCs toward specific cell lines to overcome the demand for providing the organ specific cells for cell therapy or preparation of engineered tissues. In the present study, differentiation of MSCs to endothelial cells (ECs) by mechanical and chemical stimulation was evaluated. Fluid shear stress (FSS) was used as mechanical inducer, while platelet lysate (PL) and estradiol (E) were used as chemical induction factors. MSCs were placed under FSS with different forces (2, 5 and 10dyn/cm(2)) for different periods (6, 12 and 24 hours). In some groups, PL and E were added to the culture media to evaluate their effect on expression of EC specific markers. This investigation revealed that FSS with low tension (2.5-5 dyn/cm(2)) for a long time (24 hours) or high tension (10 dyn/cm(2)) in short time (6 hours) in the presence of PL could differentiate MSCs toward ECs. The presence of PL was necessary for initiation of endothelial differentiation, and in the absence of PL, there was not any expression of CD34 and Cadherin5 (Cdh5) among cells. Adding E to the culture medium did not change the rate of endothelial differentiation under FSS. Generated endothelial progenitors could produce von Willebrand factor (vWF) after two weeks culture and also they formed tubular structures after culture on matrigel.

The interaction of adipose-derived human mesenchymal stem cells and polyether ether ketone.

PubMed

Wang, Weiwei; Kratz, Karl; Behl, Marc; Yan, Wan; Liu, Yue; Xu, Xun; Baudis, Stefan; Li, Zhengdong; Kurtz, Andreas; Lendlein, Andreas; Ma, Nan

2015-01-01

Polyether ether ketone (PEEK) as a high-performance, thermoplastic implant material entered the field of medical applications due to its structural function and commercial availability. In bone tissue engineering, the combination of mesenchymal stem cells (MSCs) with PEEK implants may accelerate the bone formation and promote the osseointegration between the implant and the adjacent bone tissue. In this concept the question how PEEK influences the behaviour and functions of MSCs is of great interest. Here the cellular response of human adipose-derived MSCs to PEEK was evaluated and compared to tissue culture plate (TCP) as the reference material. Viability and morphology of cells were not altered when cultured on the PEEK film. The cells on PEEK presented a high proliferation activity in spite of a relatively lower initial cell adhesion rate. There was no significant difference on cell apoptosis and senescence between the cells on PEEK and TCP. The inflammatory cytokines and VEGF secreted by the cells on these two surfaces were at similar levels. The cells on PEEK showed up-regulated BMP2 and down-regulated BMP4 and BMP6 gene expression, whereas no conspicuous differences were observed in the committed osteoblast markers (BGLAP, COL1A1 and Runx2). With osteoinduction the cells on PEEK and TCP exhibited a similar osteogenic differentiation potential. Our results demonstrate the biofunctionality of PEEK for human MSC cultivation and differentiation. Its clinical benefits in bone tissue engineering may be achieved by combining MSCs with PEEK implants. These data may also provide useful information for further modification of PEEK with chemical or physical methods to regulate the cellular processes of MSCs and to consequently improve the efficacy of MSC-PEEK based therapies.

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

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.

Gold nanoparticle size and shape influence on osteogenesis of mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Li, Jingchao; Li, Jia'en Jasmine; Zhang, Jing; Wang, Xinlong; Kawazoe, Naoki; Chen, Guoping

2016-04-01

Gold nanoparticles (AuNPs) have been extensively explored for biomedical applications due to their advantages of facile synthesis and surface functionalization. Previous studies have suggested that AuNPs can induce differentiation of stem cells into osteoblasts. However, how the size and shape of AuNPs affect the differentiation response of stem cells has not been elucidated. In this work, a series of bovine serum albumin (BSA)-coated Au nanospheres, Au nanostars and Au nanorods with different diameters of 40, 70 and 110 nm were synthesized and their effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) were investigated. All the AuNPs showed good cytocompatibility and did not influence proliferation of hMSCs at the studied concentrations. Osteogenic differentiation of hMSCs was dependent on the size and shape of AuNPs. Sphere-40, sphere-70 and rod-70 significantly increased the alkaline phosphatase (ALP) activity and calcium deposition of cells while rod-40 reduced the ALP activity and calcium deposition. Gene profiling revealed that the expression of osteogenic marker genes was down-regulated after incubation with rod-40. However, up-regulation of these genes was found in the sphere-40, sphere-70 and rod-70 treatment. Moreover, it was found that the size and shape of AuNPs affected the osteogenic differentiation of hMSCs through regulating the activation of Yes-associated protein (YAP). These results indicate that the size and shape of AuNPs had an influence on the osteogenic differentiation of hMSCs, which should provide useful guidance for the preparation of AuNPs with defined size and shape for their biomedical applications.Gold nanoparticles (AuNPs) have been extensively explored for biomedical applications due to their advantages of facile synthesis and surface functionalization. Previous studies have suggested that AuNPs can induce differentiation of stem cells into osteoblasts. However, how the size and shape of AuNPs affect the differentiation response of stem cells has not been elucidated. In this work, a series of bovine serum albumin (BSA)-coated Au nanospheres, Au nanostars and Au nanorods with different diameters of 40, 70 and 110 nm were synthesized and their effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) were investigated. All the AuNPs showed good cytocompatibility and did not influence proliferation of hMSCs at the studied concentrations. Osteogenic differentiation of hMSCs was dependent on the size and shape of AuNPs. Sphere-40, sphere-70 and rod-70 significantly increased the alkaline phosphatase (ALP) activity and calcium deposition of cells while rod-40 reduced the ALP activity and calcium deposition. Gene profiling revealed that the expression of osteogenic marker genes was down-regulated after incubation with rod-40. However, up-regulation of these genes was found in the sphere-40, sphere-70 and rod-70 treatment. Moreover, it was found that the size and shape of AuNPs affected the osteogenic differentiation of hMSCs through regulating the activation of Yes-associated protein (YAP). These results indicate that the size and shape of AuNPs had an influence on the osteogenic differentiation of hMSCs, which should provide useful guidance for the preparation of AuNPs with defined size and shape for their biomedical applications. Electronic supplementary information (ESI) available: Additional experimental results. See DOI: 10.1039/c5nr08808a

Three-dimensional graphene foams loaded with bone marrow derived mesenchymal stem cells promote skin wound healing with reduced scarring.

PubMed

Li, Zhonghua; Wang, Haiqin; Yang, Bo; Sun, Yukai; Huo, Ran

2015-12-01

The regeneration of functional skin remains elusive, due to poor engraftment, deficient vascularization, and excessive scar formation. Aiming to overcome these issues, the present study proposed the combination of a three-dimensional graphene foam (GF) scaffold loaded with bone marrow derived mesenchymal stem cells (MSCs) to improve skin wound healing. The GFs demonstrated good biocompatibility and promoted the growth and proliferation of MSCs. Meanwhile, the GFs loaded with MSCs obviously facilitated wound closure in animal model. The dermis formed in the presence of the GF structure loaded with MSCs was thicker and possessed a more complex structure at day 14 post-surgery. The transplanted MSCs correlated with upregulation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which may lead to neo-vascularization. Additionally, an anti-scarring effect was observed in the presence of the 3D-GF scaffold and MSCs, as evidenced by a downregulation of transforming growth factor-beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) together with an increase of TGF-β3. Altogether, the GF scaffold could guide the wound healing process with reduced scarring, and the MSCs were crucial to enhance vascularization and provided a better quality neo-skin. The GF scaffold loaded with MSCs possesses necessary bioactive cues to improve wound healing with reduced scarring, which may be of great clinical significance for skin wound healing. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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.

Functional Tissue Engineering: A Prevascularized Cardiac Muscle Construct for Validating Human Mesenchymal Stem Cells Engraftment Potential In Vitro

PubMed Central

Fuseler, John W.; Potts, Jay D.; Davis, Jeffrey M.; Price, Robert L.

2018-01-01

The influence of somatic stem cells in the stimulation of mammalian cardiac muscle regeneration is still in its early stages, and so far, it has been difficult to determine the efficacy of the procedures that have been employed. The outstanding question remains whether stem cells derived from the bone marrow or some other location within or outside of the heart can populate a region of myocardial damage and transform into tissue-specific differentiated progenies, and also exhibit functional synchronization. Consequently, this necessitates the development of an appropriate in vitro three-dimensional (3D) model of cardiomyogenesis and prompts the development of a 3D cardiac muscle construct for tissue engineering purposes, especially using the somatic stem cell, human mesenchymal stem cells (hMSCs). To this end, we have created an in vitro 3D functional prevascularized cardiac muscle construct using embryonic cardiac myocytes (eCMs) and hMSCs. First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were cocultured onto a 3D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions; hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed dense vascular networks. Next, the eCMs and hMSCs were cocultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were characterized at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated progenies revealed neo-cardiomyogenesis and neo-vasculogenesis. In this milieu, for instance, not only were hMSCs able to couple electromechanically with developing eCMs but were also able to contribute to the developing vasculature as mural cells, respectively. Hence, our unique 3D coculture system provides us a reproducible and quintessential in vitro 3D model of cardiomyogenesis and a functioning prevascularized 3D cardiac graft that can be utilized for personalized medicine. PMID:28457188

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.

Adipose-derived mesenchymal stem cells reduce MMP-1 expression in UV-irradiated human dermal fibroblasts: therapeutic potential in skin wrinkling.

PubMed

Son, Woo-Chan; Yun, Jun-Won; Kim, Bae-Hwan

2015-01-01

Adipose-derived mesenchymal stem cells (AdMSCs) have been reported to have therapeutic benefit in skin. The aim of this study was to examine the effects of AdMSCs in UV-irradiated human dermal fibroblasts (HDFs) for therapeutic potential in skin wrinkling. UV irradiation, a model naturally mimic skin wrinkle formation, is known to increase matrix metalloproteinase-1 (MMP-1), making MMP-1 a target for skin photoaging. Our findings identified that AdMSCs reduce MMP-1 level in UV-irradiated HDFs and increase type 1 procollagen in HDFs. A dose-dependent increase in type 1 procollagen was confirmed by AdMSC-conditioned medium. Importantly, our current findings showing the effects of AdMSCs on the induction of MMP-1 in UV-radiated HDFs and the expression of collagen in HDFs can provide an evidence of relationship between MMP-1 and procollagen production for the protection against wrinkle formation. Collectively, AdMSCs may contribute to anti-wrinkle effects in skin but further experiments are needed to identify the mechanism.

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

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

Gene therapy with mesenchymal stem cells expressing IFN-ß ameliorates neuroinflammation in experimental models of multiple sclerosis.

PubMed

Marin-Bañasco, C; Benabdellah, K; Melero-Jerez, C; Oliver, B; Pinto-Medel, M J; Hurtado-Guerrero, I; de Castro, F; Clemente, D; Fernández, O; Martin, F; Leyva, L; Suardíaz, M

2017-02-01

Recombinant IFN-ß is one of the first-line treatments in multiple sclerosis (MS), despite its lack of efficacy in some patients. In this context, mesenchymal stem cells (MSCs) represent a promising therapeutic alternative due to their immunomodulatory properties and multipotency. Moreover, by taking advantage of their pathotropism, these cells can be genetically modified to be used as carriers for delivering or secreting therapeutic drugs into injured tissues. Here, we report the therapeutic effect of systemic delivery of adipose-derived MSCs (AdMSCs), transduced with the IFN-β gene, into mice with experimental autoimmune encephalomyelitis (EAE). Relapsing-remitting and chronic progressive EAE were induced in mice. Cells were injected i.v. Disease severity, inflammation and tissue damage were assessed clinically, by flow cytometry of spleens and histopathological evaluation of the CNS respectively. Genetic engineering did not modify the biological characteristics of these AdMSCs (morphology, growth rate, immunophenotype and multipotency). Furthermore, the transduction of IFN-ß to AdMSCs maintained and, in some cases, enhanced the functional properties of AdMSCs by ameliorating the symptoms of MS in EAE models and by decreasing indications of peripheral and central neuro-inflammation. Gene therapy was found to be more effective than cell therapy in ameliorating several clinical parameters in both EAE models, presumably due to the continuous expression of IFN-β. Furthermore, it has significant advantages over AdMSC therapy, and also over systemic IFN-ß treatment, by providing long-term expression of the cytokine at therapeutic concentrations and reducing the frequency of injections, while minimizing dose-limiting side effects. © 2016 The British Pharmacological Society.

Gene therapy with mesenchymal stem cells expressing IFN‐ß ameliorates neuroinflammation in experimental models of multiple sclerosis

PubMed Central

Marin‐Bañasco, C; Benabdellah, K; Melero‐Jerez, C; Oliver, B; Pinto‐Medel, M J; Hurtado‐Guerrero, I; de Castro, F; Clemente, D; Fernández, O; Martin, F; Leyva, L

2017-01-01

Background and Purpose Recombinant IFN‐ß is one of the first‐line treatments in multiple sclerosis (MS), despite its lack of efficacy in some patients. In this context, mesenchymal stem cells (MSCs) represent a promising therapeutic alternative due to their immunomodulatory properties and multipotency. Moreover, by taking advantage of their pathotropism, these cells can be genetically modified to be used as carriers for delivering or secreting therapeutic drugs into injured tissues. Here, we report the therapeutic effect of systemic delivery of adipose‐derived MSCs (AdMSCs), transduced with the IFN‐β gene, into mice with experimental autoimmune encephalomyelitis (EAE). Experimental Approach Relapsing–remitting and chronic progressive EAE were induced in mice. Cells were injected i.v. Disease severity, inflammation and tissue damage were assessed clinically, by flow cytometry of spleens and histopathological evaluation of the CNS respectively. Key Results Genetic engineering did not modify the biological characteristics of these AdMSCs (morphology, growth rate, immunophenotype and multipotency). Furthermore, the transduction of IFN‐ß to AdMSCs maintained and, in some cases, enhanced the functional properties of AdMSCs by ameliorating the symptoms of MS in EAE models and by decreasing indications of peripheral and central neuro‐inflammation. Conclusion and Implications Gene therapy was found to be more effective than cell therapy in ameliorating several clinical parameters in both EAE models, presumably due to the continuous expression of IFN‐β. Furthermore, it has significant advantages over AdMSC therapy, and also over systemic IFN‐ß treatment, by providing long‐term expression of the cytokine at therapeutic concentrations and reducing the frequency of injections, while minimizing dose‐limiting side effects. PMID:27882538

Human umbilical blood mononuclear cell-derived mesenchymal stem cells serve as interleukin-21 gene delivery vehicles for epithelial ovarian cancer therapy in nude mice.

PubMed

Hu, Weihua; Wang, Jing; He, Xiangfeng; Zhang, Hongyi; Yu, Fangliu; Jiang, Longwei; Chen, Dengyu; Chen, Junsong; Dou, Jun

2011-01-01

Ovarian cancer causes more deaths than any other cancer of the female reproductive system, and its overall cure rate remains low. The present study investigated human umbilical blood mononuclear cell (UBMC)-derived mesenchymal stem cells (UBMC-MSCs) as interleukin-21 (IL-21) gene delivery vehicles for ovarian cancer therapy in nude mice. MSCs were isolated from UBMCs and the expanded cells were phenotyped by flow cytometry. Cultured UBMCs were differentiated into osteocytes and adipocytes using appropriate media and then the UBMC-MSCs were transfected with recombinant pIRES2-IL-21-enhancement green fluorescent protein. UBMC-MSCs expressing IL-21 were named as UBMC-MSC-IL-21. Mice with A2780 ovarian cancer were treated with UBMC-MSC-IL-21 intravenously, and the therapeutic efficacy was evaluated by the tumor volume and mouse survival. To address the mechanism of UBMC-MSC-IL-21 against ovarian cancer, the expression of IL-21, natural killer glucoprotein 2 domain and major histocompatibility complex class I chain-related molecules A/B were detected in UBMC-MSC-IL-21 and in the tumor sites. Interferon-γ-secreting splenocyte numbers and natural killer cytotoxicity were significantly increased in the UBMC-MSC-IL-21-treated mice as compared with the UBMC-MSCs or the UBMC-MSC-mock plasmid-treated mice. Most notably, tumor growth was delayed and survival was prolonged in ovarian-cancer-bearing mice treated with UBMC-MSC-IL-21. Our data provide important evidence that UBMC-MSCs can serve as vehicles for IL-21 gene delivery and inhibit the established tumor. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

PubMed Central

Hosogane, Naobumi; Huang, Zhiping; Rawlins, Bernard A.; Liu, Xia; Boachie-Adjei, Oheneba; Boskey, Adele L.; Zhu, Wei

2010-01-01

Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations. PMID:20362069

Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

PubMed Central

Ali, Dalia; Hamam, Rimi; Alfayez, Musaed; Kassem, Moustapha; Aldahmash, Abdullah

2016-01-01

The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering. Significance This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways controlling adipocyte and osteoblast differentiation of hMSCs. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies for tissue engineering, bone disease, obesity, and metabolic-disorders. PMID:27194745

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.

Syngeneic Transplantation of Olfactory Ectomesenchymal Stem Cells Restores Learning and Memory Abilities in a Rat Model of Global Cerebral Ischemia.

PubMed

Veron, Antoine D; Bienboire-Frosini, Cécile; Girard, Stéphane D; Sadelli, Kevin; Stamegna, Jean-Claude; Khrestchatisky, Michel; Alexis, Jennifer; Pageat, Patrick; Asproni, Pietro; Mengoli, Manuel; Roman, François S

2018-01-01

Stem cells are considered as promising tools to repair diverse tissue injuries. Among the different stem cell types, the "olfactory ectomesenchymal stem cells" (OE-MSCs) located in the adult olfactory mucosa stand as one of the best candidates. Here, we evaluated if OE-MSC grafts could decrease memory impairments due to ischemic injury. OE-MSCs were collected from syngeneic F344 rats. After a two-step global cerebral ischemia, inducing hippocampal lesions, learning abilities were evaluated using an olfactory associative discrimination task. Cells were grafted into the hippocampus 5 weeks after injury and animal's learning abilities reassessed. Rats were then sacrificed and the brains collected for immunohistochemical analyses. We observed significant impairments in learning and memory abilities following ischemia. However, 4 weeks after OE-MSC grafts, animals displayed learning and memory performances similar to those of controls, while sham rats did not improve them. Immunohistochemical analyses revealed that grafts promoted neuroblast and glial cell proliferation, which could permit to restore cognitive functions. These results demonstrated, for the first time, that syngeneic transplantations of OE-MSCs in rats can restore cognitive abilities impaired after brain injuries and provide support for the development of clinical studies based on grafts of OE-MSCs in amnesic patients following brain injuries.

Effects of iron oxide contrast agent in combination with various transfection agents during mesenchymal stem cells labelling: An in vitro toxicological evaluation.

PubMed

Mishra, Sushanta Kumar; Khushu, Subash; Gangenahalli, Gurudutta

2018-03-22

The use of iron oxide nanoparticles for different biomedical applications, hold immense promise to develop negative tissue contrast in magnetic resonance imaging (MRI). Previously, we have optimized the labelling of mesenchymal stem cells (MSCs) with iron oxide nanoparticles complexed to different transfection agents like poly-l-lysine (IO-PLL) and protamine sulfate (Fe-Pro) on the basis of relaxation behaviour and its biological expressions. However, there is a distinct need to investigate the biocompatibility and biosafety concerns coupled with its cytotoxicity and genotoxicity. This study was prepared to evaluate the viability of cells, generation of ROS, changes in actin cytoskeleton, investigation of cell death, level of GSH and TAC, activities of SOD and GPx, and stability of DNA in MSCs after labelling. Results demonstrated a marginal alteration in toxicological parameters like ROS generation, cell length, actin cytoskeleton, total apoptosis and DNA damage was detected after stem cell labelling. Insignificant depletion of GSH and SOD level, and increase in GPx and TAC level in MSCs were measured after labelling with IO-PLL and Fe-Pro complexes, which later on recovered and normalized to its baseline. This MSCs labelling could provide a reference guideline for toxicological analysis and relaxometry based in vivo MRI detection. Copyright © 2018. Published by Elsevier Ltd.

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.

Mesenchymal stromal cell-derived extracellular vesicles attenuate lung ischemia-reperfusion injury and enhance reconditioning of donor lungs after circulatory death.

PubMed

Stone, Matthew L; Zhao, Yunge; Robert Smith, J; Weiss, Mark L; Kron, Irving L; Laubach, Victor E; Sharma, Ashish K

2017-12-21

Lung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs. C57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs. Lungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix. These results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs.

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.

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.

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.

Gastric cancer tissue-derived mesenchymal stem cells impact peripheral blood mononuclear cells via disruption of Treg/Th17 balance to promote gastric cancer progression.

PubMed

Wang, Mei; Chen, Bin; Sun, Xiao-Xian; Zhao, Xiang-Dong; Zhao, Yuan-Yuan; Sun, Li; Xu, Chang-Gen; Shen, Bo; Su, Zhao-Liang; Xu, Wen-Rong; Zhu, Wei

2017-12-01

Gastric cancer tissue-derived mesenchymal stem cells (GC-MSCs) are important resident stromal cells in the tumor microenvironment (TME) and have been shown to play a key role in gastric cancer progression. Whether GC-MSCs exert a tumor-promoting function by affecting anti-tumor immunity is still unclear. In this study, we used GC-MSC conditioned medium (GC-MSC-CM) to pretreat peripheral blood mononuclear cells (PBMCs) from healthy donors. We found that GC-MSC-CM pretreatment markedly reversed the inhibitory effect of PBMCs on gastric cancer growth in vivo, but did not affect functions of PBMCs on gastric cancer cell proliferation, cell cycle and apoptosis in vitro. PBMCs pretreated with GC-MSC-CM significantly promoted gastric cancer migration and epithelial-mesenchymal transition in vitro and liver metastases in vivo. Flow cytometry analysis showed that GC-MSC-CM pretreatment increased the proportion of Treg cells and reduced that of Th17 cells in PBMCs. CFSE labeling and naïve CD4 + T cells differentiation analysis revealed that GC-MSC-CM disrupted the Treg/Th17 balance in PBMCs by suppressing Th17 cell proliferation and inducing differentiation of Treg cells. Overall, our collective results indicate that GC-MSCs impair the anti-tumor immune response of PBMCs through disruption of Treg/Th17 balance, thus providing new evidence that gastric cancer tissue-derived MSCs contribute to the immunosuppressive TME. Copyright © 2017 Elsevier Inc. All rights reserved.

Adsorption force of fibronectin controls transmission of cell traction force and subsequent stem cell fate.

PubMed

Lin, Manping; Mao, Shilong; Wang, Jinfeng; Xing, Juan; Wang, Yuanliang; Cai, Kaiyong; Luo, Yanfeng

2018-04-01

The transmission of cell traction force (CTF) to underlying biomaterials is essential for adhered cells to measure and respond to their mechanical microenvironment. Given that the protein layer adsorbed on materials lies between the cells and materials, we hypothesize that the interfacial strength of protein-material interfaces (i.e., the adsorption force of proteins, F ad ) should have an important role in regulating the transmission of CTF. To test this hypothesis, rat mesenchymal stem cells (rMSCs) were cultured on poly(dimethyl siloxane) (PDMS) substrates with different F ad of fibronectin (FN), and the transmission of CTF was observed by immunofluorescence staining of FN and deformation of PDMS. As revealed, FN on substrates with low F ad is more liable to be desorbed by CTF, which prevents the transmission of CTF to substrates. In contrast, high F ad facilitates the transmission of CTF from rMSCs to the FN layer and PDMS substrates so that rMSCs can perceive the mechanical properties of substrates. We further demonstrated that the divergent transmission of CTF on low and high F ad substrates regulates the lineage specifications of rMSCs. Our study confirms the important role of F ad in CTF transmission and provides a new perspective to gain insights into cell-material interactions and cell fates, which may help to guide the design of better biomaterials. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering.

PubMed

Jiang, Wei-Cheng; Cheng, Yu-Hao; Yen, Meng-Hua; Chang, Yin; Yang, Vincent W; Lee, Oscar K

2014-04-01

Liver transplantation is the ultimate treatment for severe hepatic failure to date. However, the limited supply of donor organs has severely hampered this treatment. So far, great potentials of using mesenchymal stem cells (MSCs) to replenish the hepatic cell population have been shown; nevertheless, there still is a lack of an optimal three-dimensional scaffold for generation of well-transplantable hepatic tissues. In this study, we utilized a cryo-chemical decellularization method which combines physical and chemical approach to generate acellular liver scaffolds (ALS) from the whole liver. The produced ALS provides a biomimetic three-dimensional environment to support hepatic differentiation of MSCs, evidenced by expression of hepatic-associated genes and marker protein, glycogen storage, albumin secretion, and urea production. It is also found that hepatic differentiation of MSCs within the ALS is much more efficient than two-dimensional culture in vitro. Importantly, the hepatic-like tissues (HLT) generated by repopulating ALS with MSCs are able to act as functional grafts and rescue lethal hepatic failure after transplantation in vivo. In summary, the cryo-chemical method used in this study is suitable for decellularization of liver and create acellular scaffolds that can support hepatic differentiation of MSCs and be used to fabricate functional tissue-engineered liver constructs. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

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.

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.

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.

Comparative Analysis of Mouse-Induced Pluripotent Stem Cells and Mesenchymal Stem Cells During Osteogenic Differentiation In Vitro

PubMed Central

Kayashima, Hiroki; Miura, Jiro; Uraguchi, Shinya; Wang, Fangfang; Okawa, Hiroko; Sasaki, Jun-Ichi; Saeki, Makio; Matsumoto, Takuya; Yatani, Hirofumi

2014-01-01

Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and are, therefore, expected to be useful for bone regenerative medicine; however, the characteristics of iPSC-derived osteogenic cells remain unclear. Here, we provide a direct in vitro comparison of the osteogenic differentiation process in mesenchymal stem cells (MSCs) and iPSCs from adult C57BL/6J mice. After 30 days of culture in osteogenic medium, both MSCs and iPSCs produced robustly mineralized bone nodules that contained abundant calcium phosphate with hydroxyapatite crystal formation. Mineral deposition was significantly higher in iPSC cultures than in MSC cultures. Scanning electron microscopy revealed budding matrix vesicles in early osteogenic iPSCs; subsequently, the vesicles propagated to exhibit robust mineralization without rich fibrous structures. Early osteogenic MSCs showed deposition of many matrix vesicles in abundant collagen fibrils that became solid mineralized structures. Both cell types demonstrated increased expression of osteogenic marker genes, such as runx2, osterix, dlx5, bone sialoprotein (BSP), and osteocalcin, during osteogenesis; however, real-time reverse transcription–polymerase chain reaction array analysis revealed that osteogenesis-related genes encoding mineralization-associated molecules, bone morphogenetic proteins, and extracellular matrix collagens were differentially expressed between iPSCs and MSCs. These data suggest that iPSCs are capable of differentiation into mature osteoblasts whose associated hydroxyapatite has a crystal structure similar to that of MSC-associated hydroxyapatite; however, the transcriptional differences between iPSCs and MSCs could result in differences in the mineral and matrix environments of the bone nodules. Determining the biological mechanisms underlying cell-specific differences in mineralization during in vitro iPSC osteogenesis may facilitate the development of clinically effective engineered bone. PMID:24625139

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.

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.

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

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.

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