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Sample records for placenta-derived mesenchymal stromal

  1. Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential.

    PubMed

    Ulrich, Christine; Abruzzese, Tanja; Maerz, Jan K; Ruh, Manuel; Amend, Bastian; Benz, Karin; Rolauffs, Bernd; Abele, Harald; Hart, Melanie L; Aicher, Wilhelm K

    2015-07-01

    Mesenchymal stromal cells (MSCs) are multipotent cells that can be differentiated in vitro into a variety of cell types, including adipocytes or osteoblasts. Our recent studies indicated that a high expression of CD146 on MSCs from bone marrow correlates with their robust osteogenic differentiation potential. We therefore investigated if expression of CD146 on MSCs from the placenta correlates with a similar osteogenic differentiation potential. The MSCs were isolated specifically from the endometrial and fetal parts of human term placenta and expanded in separate cultures and compared with MSCs from bone marrow as controls. The expression of cell surface antigens was investigated by flow cytometry. Differentiation of MSCs was documented by cytochemistry and analysis of typical lineage marker genes. CD146-positive MSCs were separated from CD146-negative cells by magnet-assisted cell sorts (MACS). We report that the expression of CD146 is associated with a higher osteogenic differentiation potential in human placenta-derived MSCs (pMSCs) and the CD146(pos) pMSCs generated a mineralized extracellular matrix, whereas the CD146(neg) pMSCs failed to do so. In contrast, adipogenic and chondrogenic differentiation of pMSCs was not different in CD146(pos) compared with CD146(neg) pMSCs. Upon enrichment of pMSCs by MACS, the CD146(neg) and CD146(pos) populations maintained their expression levels for this antigen for several passages in vitro. We conclude that CD146(pos) pMSCs either respond to osteogenic stimuli more vividly or, alternatively, CD146(pos) pMSCs present a pMSC subset that is predetermined to differentiate into osteoblasts. PMID:25743703

  2. Human term placenta-derived mesenchymal stromal cells are less prone to osteogenic differentiation than bone marrow-derived mesenchymal stromal cells.

    PubMed

    Pilz, Gregor A; Ulrich, Christine; Ruh, Manuel; Abele, Harald; Schäfer, Richard; Kluba, Torsten; Bühring, Hans-Jörg; Rolauffs, Bernd; Aicher, Wilhelm K

    2011-04-01

    Mesenchymal stromal cells (MSC) can be isolated from different tissues. They are capable of differentiating in vitro, for example, to osteoblasts, chondrocytes, or adipocytes. In contrast to CD34 for hematopoietic stem cells, a distinct MSC-defining antibody is not available. Further, for hematopoietic cells lineage-defining antigens such as CD3 or CD20 are known. In contrast, for MSC-derived cells lineage-associated cell surface markers are far from being established. We therefore investigated expression of cell surface antigens on human term placenta-derived MSC (pMSC) in more detail and correlated expression pattern to the osteogenic differentiation capacity of the MSC. We report that pMSC expressed the typical cell surface antigens at levels comparable to bone marrow-derived MSC (bmMSC), including CD73, CD90, and CD105, but did not express CD11b, CD34, and CD45. Further, CD164, TNAP, and the W5C5 antigens were detected on pMSC, whereas CD349 was not observed. Some pMSC expressed CD146 at low or moderate levels, and their osteogenic differentiation potential was weak. In contrast, bmMSC expressed CD146 at high levels, expression of alkaline phosphatase was significantly higher, and they presented a pronounced osteogenic differentiation potential. We conclude that MSC from different sources differ in their expression of distinct markers, and that this may correlate in part with their lineage determination. Thus, a higher percentage of bmMSC expressed CD146 at prominent levels and such cells may be better suited for bone repair. In contrast, many pMSC expressed CD146 at low or moderate levels. They, therefore, may be suitable for applications in which osteogenic differentiation is undesirable. PMID:21047215

  3. Low osteogenic differentiation potential of placenta-derived mesenchymal stromal cells correlates with low expression of the transcription factors Runx2 and Twist2.

    PubMed

    Ulrich, Christine; Rolauffs, Bernd; Abele, Harald; Bonin, Michael; Nieselt, Kay; Hart, Melanie L; Aicher, Wilhelm K

    2013-11-01

    Recent studies indicated that mesenchymal stromal cells from bone marrow (bmMSC) differ in their osteogenic differentiation capacity compared to MSC from term placenta (pMSC). We extended these studies and investigated the expression of factors involved in regulation of bone metabolism in both cell types. To this end, MSC were expanded in vitro and characterized. The total transcriptome was investigated by microarrays, and for selected genes, the differences in gene expression were explored by quantitative reverse transcriptase-polymerase chain reaction, immunocytochemistry, and flow cytometry. We report that bmMSC and pMSC share expression of typical lineage surface markers, including CD73, CD90, CD105, and lack of CD14, CD34, and CD45. However, according to transcriptome analyses, they differ significantly in their expression of more than 590 genes. Factors involved in bone metabolism, including alkaline phosphatase (P<0.05), osteoglycin (P<0.05), osteomodulin (P<0.05), runt-related transcription factor 2 (Runx2) (P<0.04), and WISP2 (P<0.05), were expressed at significantly lower levels in pMSC, but twist-related protein 2 (Twist2) (P<0.0002) was expressed at significantly higher levels. The osteogenic differentiation capacity of pMSC was very low. The adipogenic differentiation was somewhat more prominent in bmMSC, while the chondrogenic differentiation seemed not to differ between bmMSC and pMSC, as determined by histochemical staining. However, expression and induction of peroxisome proliferator-activated receptor gamma-2 (PPARγ2) and Sox9, factors involved in early adipogenesis and chondrogenesis, respectively, were higher in bmMSC. We conclude that despite many similarities between bmMSC and pMSC, when expanded under identical conditions, they vary considerably with respect to their in vitro differentiation potential. For regenerative purposes, the choice of MSC may therefore influence the outcome of a treatment considerably. PMID:23763516

  4. Low Osteogenic Differentiation Potential of Placenta-Derived Mesenchymal Stromal Cells Correlates with Low Expression of the Transcription Factors Runx2 and Twist2

    PubMed Central

    Ulrich, Christine; Rolauffs, Bernd; Abele, Harald; Bonin, Michael; Nieselt, Kay; Hart, Melanie L.

    2013-01-01

    Recent studies indicated that mesenchymal stromal cells from bone marrow (bmMSC) differ in their osteogenic differentiation capacity compared to MSC from term placenta (pMSC). We extended these studies and investigated the expression of factors involved in regulation of bone metabolism in both cell types. To this end, MSC were expanded in vitro and characterized. The total transcriptome was investigated by microarrays, and for selected genes, the differences in gene expression were explored by quantitative reverse transcriptase-polymerase chain reaction, immunocytochemistry, and flow cytometry. We report that bmMSC and pMSC share expression of typical lineage surface markers, including CD73, CD90, CD105, and lack of CD14, CD34, and CD45. However, according to transcriptome analyses, they differ significantly in their expression of more than 590 genes. Factors involved in bone metabolism, including alkaline phosphatase (P<0.05), osteoglycin (P<0.05), osteomodulin (P<0.05), runt-related transcription factor 2 (Runx2) (P<0.04), and WISP2 (P<0.05), were expressed at significantly lower levels in pMSC, but twist-related protein 2 (Twist2) (P<0.0002) was expressed at significantly higher levels. The osteogenic differentiation capacity of pMSC was very low. The adipogenic differentiation was somewhat more prominent in bmMSC, while the chondrogenic differentiation seemed not to differ between bmMSC and pMSC, as determined by histochemical staining. However, expression and induction of peroxisome proliferator-activated receptor gamma-2 (PPARγ2) and Sox9, factors involved in early adipogenesis and chondrogenesis, respectively, were higher in bmMSC. We conclude that despite many similarities between bmMSC and pMSC, when expanded under identical conditions, they vary considerably with respect to their in vitro differentiation potential. For regenerative purposes, the choice of MSC may therefore influence the outcome of a treatment considerably. PMID:23763516

  5. Transplantation of placenta-derived mesenchymal stem cells enhances angiogenesis after ischemic limb injury in mice.

    PubMed

    Xie, Nanzi; Li, Zhihong; Adesanya, Timothy M; Guo, Weixin; Liu, Yang; Fu, Minghuan; Kilic, Ahmet; Tan, Tao; Zhu, Hua; Xie, Xiaoyun

    2016-01-01

    Mesenchymal stem cell-based therapy has emerged as a promising approach for the treatment of peripheral arterial disease. The purpose of this study was to examine the potential effects of human placenta-derived mesenchymal stem cells (PMSCs) on mouse hindlimb ischemia. PMSCs were isolated from human placenta tissue and characterized by flow cytometry. An in vivo surgical ligation-induced murine limb ischemia model was generated with fluorescent dye (CM-DiI) labelled PMSCs delivered via intramuscular injection. Our data show that PMSCs treatment significantly enhanced microvessel density, improved blood perfusion and diminished pathologies in ischemic mouse hindlimbs as compared to those in the control group. Further immunostaining studies suggested that injected PMSCs can incorporate into the vasculature and differentiate into endothelial and smooth muscle cells to enhance angiogenesis in ischemic hind limbs. This may in part explain the beneficial effects of PMSCs treatment. Taken together, we found that PMSCs treatment might be an effective treatment modality for treatment of ischemia-induced injury to mouse hind limbs by enhancement of angiogenesis. PMID:26282458

  6. Osteogenic differentiation of human placenta-derived mesenchymal stem cells (PMSCs) on electrospun nanofiber meshes.

    PubMed

    Zhang, Dongmei; Tong, Aiping; Zhou, Liangxue; Fang, Fang; Guo, Gang

    2012-12-01

    Numerous challenges remain in the successful clinical translation of cell-based therapeutic studies for skeletal tissue repair, including appropriate cell sources and viable cell delivery systems. Poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) amphiphilic block copolymers have been extensively explored in microspheres preparation. Due to the introduction of hydrophilic PEG segments into PCL backbones, these copolymers have shown much more potentials in carrying protein, lipophilic drugs or genes than commonly used poly (ε-caprolactone) (PCL) and poly (lactic acid). The aim of this study is to investigate the attachment and osteogenic differentiation of human placenta derived mesenchymal stem cells (PMSCs) on PEG-PCL triblock copolymers nanofiber scaffolds. Here we demonstrated that PMSCs proliferate robustly and can be effectively differentiated into osteogenic-like cells on nanofiber scaffolds. This study provides evidence for the use of nanofiber scaffolds as an ideal supporting material for in vitro PMSCs culture and an in vivo cell delivery vehicle for bone repair. PMID:22526490

  7. Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

    PubMed

    Chatterjee, Piyali; Chiasson, Valorie L; Pinzur, Lena; Raveh, Shani; Abraham, Eytan; Jones, Kathleen A; Bounds, Kelsey R; Ofir, Racheli; Flaishon, Liat; Chajut, Ayelet; Mitchell, Brett M

    2016-04-01

    Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during

  8. 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. PMID:22610566

  9. Antitumor activity of placenta-derived mesenchymal stem cells producing pigment epithelium-derived factor in a mouse melanoma model.

    PubMed

    Chen, Qiaoling; Cheng, Ping; Song, Na; Yin, Tao; He, Hong; Yang, Li; Chen, Xiancheng; Wei, Yuquan

    2012-09-01

    Mesenchymal stem cells (MSCs) are a new tool that can be used for the delivery of therapeutic agents to tumor cells. Among the various types of MSCs, placenta-derived MSCs (PDMSCs) have emerged as one of the most attractive vehicles for gene therapy due to their high throughput, lack of ethical concerns, non-invasive procedure for their harvesting and ease of isolation. In this study, we evaluated the antitumor activity of human PDMSCs loaded with recombinant adenoviruses expressing pigment epithelium-derived factor (PEDF). PDMSCs were transduced with adenovirus PEDF and the expression of PEDF was confirmed by western blotting and ELISA. The inhibition of angiogenesis mediated by PEDF-expressing PDMSCs (PDMSC-PEDF) was determined using human umbilical vein endothelial cell (HUVEC) proliferation inhibition assay and migration inhibition assay in vitro. In in vivo experiments, C57BL/6 mice bearing B16-F10 melanoma were treated with intratumoral injection of PDMSC-PEDF twice at a 4-day interval. The tumor volume and weight were recorded. The results demonstrated that the administration of PDMSC-PEDF resulted in marked suppression of tumor growth in an established melanoma model, which was associated with a decreased number of microvessels and increased apoptosis of tumor cells compared with the controls. The results suggest that human PDMSCs have potential use as effective delivery vehicles for cancer gene therapy. PMID:23741242

  10. Transplantation of placenta-derived mesenchymal stem cells in the EAE mouse model of MS.

    PubMed

    Fisher-Shoval, Yonit; Barhum, Yael; Sadan, Ofer; Yust-Katz, Shlomit; Ben-Zur, Tali; Lev, Nirit; Benkler, Chen; Hod, Moshe; Melamed, Eldad; Offen, Daniel

    2012-09-01

    Stem cell-based regenerative medicine raises great hope for the treatment of multiple sclerosis (MS). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are being tested in clinical trials. Bone marrow is the traditional source of human MSCs, but human term placenta appears to be an excellent alternative because of its availability, without ethical issues. In this study, the therapeutic effect of human placental MSCs (PL-MSCs) was evaluated in experimental autoimmune encephalomyelitis (EAE), the mice model of MS. EAE mice were transplanted intra-cerebrally with PL-MSCs or with the vehicle saline 5 or 10 days after first MOG injection. The mice were monitored for a month after therapy. A daily EAE score revealed a decrease in disease severity in the transplanted animals when compared to saline. Survival was significantly higher in the transplanted animals. In vitro experiments demonstrated that conditioned media from LPS-activated astrocytes stimulated PL-MSCs to express the gene TNF-α-stimulated gene/protein 6 (TSG-6). The same mRNA expression was obtained when PL-MSCs were exposed to TNF-α or IL1-β. These results demonstrate that PL-MSCs have a therapeutic effect in the EAE mice model. We assume that this effect is caused by reduction of the anti-inflammatory protein, TSG-6, of the inflammatory damage. PMID:22638856

  11. The effects of dan-shen root on cardiomyogenic differentiation of human placenta-derived mesenchymal stem cells

    SciTech Connect

    Li, Kun; Li, Shi-zheng; Zhang, Yun-li; Wang, Xue-zhe

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Conditional medium and dan-shen root were used for cardiomyogenic differentiation. Black-Right-Pointing-Pointer They all could induce hPDMSCs to differentiate into cardiomyocytes. Black-Right-Pointing-Pointer The induction effect of the latter was slightly higher compared to that of the former. Black-Right-Pointing-Pointer Dan-shen root could be a good inducer for cardiomyogenic differentiation. -- Abstract: The aim of this study was to search for a good inducer agent using for cardiomyogenic differentiation of stem cells. Human placenta-derived mesenchymal stem cells (hPDMSCs) were isolated and incubated in enriched medium. Fourth passaged cells were treated with 10 mg/L dan-shen root for 20 days. Morphologic characteristics were analyzed by confocal and electron microscopy. Expression of {alpha}-sarcomeric actin was analyzed by immunohistochemistry. Expression of cardiac troponin-I (TnI) was analyzed by immunohistofluorescence. Atrial natriuretic factor (ANF) and beta-myocin heavy chain ({beta}-MHC) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). hPDMSCs treated with dan-shen root gradually formed a stick-like morphology and connected with adjoining cells. On the 20th day, most of the induced cells stained positive with {alpha}-sarcomeric actin and TnI antibody. ANF and {beta}-MHC were also detected in the induced cells. Approximately 80% of the cells were successfully transdifferentiated into cardiomyocytes. In conclusion, dan-shen root is a good inducer agent used for cardiomyogenic differentiation of hPDMSCs.

  12. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    PubMed Central

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L. H.

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  13. Genetically modified human placenta-derived mesenchymal stem cells with FGF-2 and PDGF-BB enhance neovascularization in a model of hindlimb ischemia

    PubMed Central

    YIN, TAO; HE, SISI; SU, CHAO; CHEN, XIANCHENG; ZHANG, DONGMEI; WAN, YANG; YE, TINGHONG; SHEN, GUOBO; WANG, YONGSHENG; SHI, HUASHAN; YANG, LI; WEI, YUQUAN

    2015-01-01

    Ischemic diseases represent a challenging worldwide health burden. The current study investigated the therapeutic potential of genetically modified human placenta-derived mesenchymal stem cells (hPDMSCs) with basic fibroblast growth factor (FGF2) and platelet-derived growth factor-BB (PDGF-BB) genes in hindlimb ischemia. Mesenchymal stem cells obtained from human term placenta were transfected ex vivo with adenoviral bicistronic vectors carrying the FGF2 and PDGF-BB genes (Ad-F-P). Unilateral hindlimb ischemia was surgically induced by excision of the right femoral artery in New Zealand White rabbits. Ad-F-P genetically modified hPDMSCs, Ad-null (control vector)-modified hPDMSCs, unmodified hPDMSCs or media were intramuscularly implanted into the ischemic limbs 7 days subsequent to the induction of ischemia. Four weeks after cell therapy, angiographic analysis revealed significantly increased collateral vessel formation in the Ad-F-P-hPDMSC group compared with the control group. Histological examination revealed markedly increased capillary and arteriole density in the Ad-F-P-hPDMSC group. The xenografted hPDMSCs survived in the ischemic tissue for at least 4 weeks subsequent to cell therapy. The current study demonstrated that the combination of hPDMSC therapy with FGF2 and PDGF-BB gene therapy effectively induced collateral vessel formation and angiogenesis, suggesting a novel strategy for therapeutic angiogenesis. PMID:26239842

  14. Are mesenchymal stromal cells immune cells?

    PubMed

    Hoogduijn, Martin J

    2015-01-01

    Mesenchymal stromal cells (MSCs) are considered to be promising agents for the treatment of immunological disease. Although originally identified as precursor cells for mesenchymal lineages, in vitro studies have demonstrated that MSCs possess diverse immune regulatory capacities. Pre-clinical models have shown beneficial effects of MSCs in multiple immunological diseases and a number of phase 1/2 clinical trials carried out so far have reported signs of immune modulation after MSC infusion. These data indicate that MSCs play a central role in the immune response. This raises the academic question whether MSCs are immune cells or whether they are tissue precursor cells with immunoregulatory capacity. Correct understanding of the immunological properties and origin of MSCs will aid in the appropriate and safe use of the cells for clinical therapy. In this review the whole spectrum of immunological properties of MSCs is discussed with the aim of determining the position of MSCs in the immune system. PMID:25880839

  15. Lymphoid Tissue Mesenchymal Stromal Cells in Development and Tissue Remodeling

    PubMed Central

    2016-01-01

    Secondary lymphoid organs (SLOs) are sites that facilitate cell-cell interactions required for generating adaptive immune responses. Nonhematopoietic mesenchymal stromal cells have been shown to play a critical role in SLO function, organization, and tissue homeostasis. The stromal microenvironment undergoes profound remodeling to support immune responses. However, chronic inflammatory conditions can promote uncontrolled stromal cell activation and aberrant tissue remodeling including fibrosis, thus leading to tissue damage. Despite recent advancements, the origin and role of mesenchymal stromal cells involved in SLO development and remodeling remain unclear. PMID:27190524

  16. Lymphoid Tissue Mesenchymal Stromal Cells in Development and Tissue Remodeling.

    PubMed

    Genovese, Luca; Brendolan, Andrea

    2016-01-01

    Secondary lymphoid organs (SLOs) are sites that facilitate cell-cell interactions required for generating adaptive immune responses. Nonhematopoietic mesenchymal stromal cells have been shown to play a critical role in SLO function, organization, and tissue homeostasis. The stromal microenvironment undergoes profound remodeling to support immune responses. However, chronic inflammatory conditions can promote uncontrolled stromal cell activation and aberrant tissue remodeling including fibrosis, thus leading to tissue damage. Despite recent advancements, the origin and role of mesenchymal stromal cells involved in SLO development and remodeling remain unclear. PMID:27190524

  17. Ex vivo expansion of mesenchymal stromal cells.

    PubMed

    Bernardo, Maria Ester; Cometa, Angela Maria; Pagliara, Daria; Vinti, Luciana; Rossi, Francesca; Cristantielli, Rosaria; Palumbo, Giuseppe; Locatelli, Franco

    2011-03-01

    Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical application. Despite being efficacious, the use of fetal calf serum for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on platelet lysate, are discussed in this paper. Moreover, this review focuses on the search of novel markers for the prospective identification/isolation of MSCs and on the potential risks connected with ex vivo expansion of MSCs, in particular that of their malignant transformation. Available tests to study the genetic stability of ex vivo expanded MSCs are also analyzed. PMID:21396595

  18. Benign Mesenchymal Stromal Cells in Human Sarcomas

    PubMed Central

    Morozov, Alexei; Downey, Robert J.; Healey, John; Moreira, Andre L.; Lou, Emil; Leung, Roland; Edgar, Mark; Singer, Samuel; LaQuaglia, Michael; Maki, Robert G.; Moore, Malcolm A.S.

    2010-01-01

    Purpose Recent evidence suggests that at least some sarcomas arise through aberrant differentiation of mesenchymal stromal cells (MSCs), but MSCs have never been isolated directly from human sarcoma specimens. Experimental Design We examined human sarcoma cell lines and primary adherent cultures derived from human sarcoma surgical samples for features of MSCs. We further characterized primary cultures as either benign or malignant by the presence of tumor-defining genetic lesions and tumor formation in immunocompromised mice. Results We show that a dedifferentiated liposarcoma cell line DDLS8817 demonstrates fat, bone and cartilage trilineage differentiation potential characteristic of MSCs. Primary sarcoma cultures have the morphology, surface immunophenotype and differentiation potential characteristic of MSCs. Surprisingly, many of these cultures are benign as they do not form tumors in mice and lack sarcoma-defining genetic lesions. Consistent with the recently proposed pericyte origin of MSCs in normal human tissues, sarcoma-derived benign MSCs express markers of pericytes and cooperate with endothelial cells in tube formation assays. In human sarcoma specimens, a subset of CD146-positive microvascular pericytes express CD105, an MSC marker, while malignant cells largely do not. In an in vitro co-culture model, sarcoma-derived benign MSCs as well as normal human pericytes markedly stimulate the growth of sarcoma cell lines. Conclusions Sarcoma-derived benign MSCs/pericytes represent a previously undescribed stromal cell type in sarcoma which may contribute to tumor formation. PMID:21138865

  19. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology

    PubMed Central

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-01-01

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated. PMID:26986509

  20. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

    PubMed

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-01-01

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated. PMID:26986509

  1. Different Procoagulant Activity of Therapeutic Mesenchymal Stromal Cells Derived from Bone Marrow and Placental Decidua.

    PubMed

    Moll, Guido; Ignatowicz, Lech; Catar, Rusan; Luecht, Christian; Sadeghi, Behnam; Hamad, Osama; Jungebluth, Philipp; Dragun, Duska; Schmidtchen, Artur; Ringdén, Olle

    2015-10-01

    While therapeutic mesenchymal stromal/stem cells (MSCs) have usually been obtained from bone marrow, perinatal tissues have emerged as promising new sources of cells for stromal cell therapy. In this study, we present a first safety follow-up on our clinical experience with placenta-derived decidual stromal cells (DSCs), used as supportive immunomodulatory and regenerative therapy for patients with severe complications after allogeneic hematopoietic stem cell transplantation (HSCT). We found that DSCs are smaller, almost half the volume of MSCs, which may favor microvascular passage. DSCs also show different hemocompatibility, with increased triggering of the clotting cascade after exposure to human blood and plasma in vitro. After infusion of DSCs in HSCT patients, we observed a weak activation of the fibrinolytic system, but the other blood activation markers remained stable, excluding major adverse events. Expression profiling identified differential levels of key factors implicated in regulation of hemostasis, such as a lack of prostacyclin synthase and increased tissue factor expression in DSCs, suggesting that these cells have intrinsic blood-activating properties. The stronger triggering of the clotting cascade by DSCs could be antagonized by optimizing the cell graft reconstitution before infusion, for example, by use of low-dose heparin anticoagulant in the cell infusion buffer. We conclude that DSCs are smaller and have stronger hemostatic properties than MSCs, thus triggering stronger activation of the clotting system, which can be antagonized by optimizing the cell graft preparation before infusion. Our results highlight the importance of hemocompatibility safety testing for every novel cell therapy product before clinical use, when applied using systemic delivery. PMID:26192403

  2. Mesenchymal stromal cells for sphincter regeneration.

    PubMed

    Klein, Gerd; Hart, Melanie L; Brinchmann, Jan E; Rolauffs, Bernd; Stenzl, Arnulf; Sievert, Karl-Dietrich; Aicher, Wilhelm K

    2015-03-01

    Stress urinary incontinence (SUI), defined as the involuntary loss of considerable amounts of urine during increased abdominal pressure (exertion, effort, sneezing, coughing, etc.), is a severe problem to the individuals affected and a significant medical, social and economic challenge. SUI is associated with pelvic floor debility, absence of detrusor contraction, or a loss of control over the sphincter muscle apparatus. The pathology includes an increasing loss of muscle cells, replacement of muscular tissue with fibrous tissue, and general aging associated processes of the sphincter complex. When current therapies fail to cure or improve SUI, application of regeneration-competent cells may be an alternative therapeutic option. Here we discuss different aspects of the biology of mesenchymal stromal cells, which are relevant to their clinical applications and for regenerating the sphincter complex. However, there are reports in favor of and against cell-based therapies. We therefore summarize the potential and the risks of cell-based therapies for the treatment of SUI. PMID:25451135

  3. Epigenetic Classification of Human Mesenchymal Stromal Cells.

    PubMed

    de Almeida, Danilo Candido; Ferreira, Marcelo R P; Franzen, Julia; Weidner, Carola I; Frobel, Joana; Zenke, Martin; Costa, Ivan G; Wagner, Wolfgang

    2016-02-01

    Standardization of mesenchymal stromal cells (MSCs) is hampered by the lack of a precise definition for these cell preparations; for example, there are no molecular markers to discern MSCs and fibroblasts. In this study, we followed the hypothesis that specific DNA methylation (DNAm) patterns can assist classification of MSCs. We utilized 190 DNAm profiles to address the impact of tissue of origin, donor age, replicative senescence, and serum supplements on the epigenetic makeup. Based on this, we elaborated a simple epigenetic signature based on two CpG sites to classify MSCs and fibroblasts, referred to as the Epi-MSC-Score. Another two-CpG signature can distinguish between MSCs from bone marrow and adipose tissue, referred to as the Epi-Tissue-Score. These assays were validated by site-specific pyrosequencing analysis in 34 primary cell preparations. Furthermore, even individual subclones of MSCs were correctly classified by our epigenetic signatures. In summary, we propose an alternative concept to use DNAm patterns for molecular definition of cell preparations, and our epigenetic scores facilitate robust and cost-effective quality control of MSC cultures. PMID:26862701

  4. Epigenetic Classification of Human Mesenchymal Stromal Cells

    PubMed Central

    de Almeida, Danilo Candido; Ferreira, Marcelo R.P.; Franzen, Julia; Weidner, Carola I.; Frobel, Joana; Zenke, Martin; Costa, Ivan G.; Wagner, Wolfgang

    2016-01-01

    Summary Standardization of mesenchymal stromal cells (MSCs) is hampered by the lack of a precise definition for these cell preparations; for example, there are no molecular markers to discern MSCs and fibroblasts. In this study, we followed the hypothesis that specific DNA methylation (DNAm) patterns can assist classification of MSCs. We utilized 190 DNAm profiles to address the impact of tissue of origin, donor age, replicative senescence, and serum supplements on the epigenetic makeup. Based on this, we elaborated a simple epigenetic signature based on two CpG sites to classify MSCs and fibroblasts, referred to as the Epi-MSC-Score. Another two-CpG signature can distinguish between MSCs from bone marrow and adipose tissue, referred to as the Epi-Tissue-Score. These assays were validated by site-specific pyrosequencing analysis in 34 primary cell preparations. Furthermore, even individual subclones of MSCs were correctly classified by our epigenetic signatures. In summary, we propose an alternative concept to use DNAm patterns for molecular definition of cell preparations, and our epigenetic scores facilitate robust and cost-effective quality control of MSC cultures. PMID:26862701

  5. Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells--potential and pitfalls.

    PubMed

    Aicher, Wilhelm K; Bühring, Hans-Jörg; Hart, Melanie; Rolauffs, Bernd; Badke, Andreas; Klein, Gerd

    2011-04-30

    Mesenchymal stromal cells, also referred to as mesenchymal stem cells, can be obtained from various tissues. Today the main source for isolation of mesenchymal stromal cells in mammals is the bone marrow. Mesenchymal stromal cells play an important role in tissue formation and organogenesis during embryonic development. Moreover, they provide the cellular and humoral basis for many processes of tissue regeneration and wound healing in infancy, adolescence and adulthood as well. There is increasing evidence that mesenchymal stromal cells from bone marrow and other sources including term placenta or adipose tissue are not a homogenous cell population. Only a restricted number of appropriate stem cells markers have been explored so far. But routine preparations of mesenchymal stromal cells contain phenotypically and functionally distinct subsets of stromal cells. Knowledge on the phenotypical characteristics and the functional consequences of such subsets will not only extend our understanding of stem cell biology, but might allow to develop improved regimen for regenerative medicine and wound healing and novel protocols for tissue engineering as well. In this review we will discuss novel strategies for regenerative medicine by specific selection or separation of subsets of mesenchymal stromal cells in the context of osteogenesis and bone regeneration. Mesenchymal stromal cells, which express the specific cell adhesion molecule CD146, also known as MCAM or MUC18, are prone for bone repair. Other cell surface proteins may allow the selection of chondrogenic, myogenic, adipogenic or other pre-determined subsets of mesenchymal stromal cells for improved regenerative applications as well. PMID:21184789

  6. Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia.

    PubMed

    Hamzic, Edita; Whiting, Karen; Gordon Smith, Edward; Pettengell, Ruth

    2015-06-01

    In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross-over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability is significantly reduced. PMID:25819548

  7. Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation.

    PubMed

    Calabrese, Giovanna; Giuffrida, Raffaella; Lo Furno, Debora; Parrinello, Nunziatina Laura; Forte, Stefano; Gulino, Rosario; Colarossi, Cristina; Schinocca, Luciana Rita; Giuffrida, Rosario; Cardile, Venera; Memeo, Lorenzo

    2015-01-01

    The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271+, CD271- mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271+ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271+ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate. PMID:26184166

  8. Mesenchymal Stem/Stromal Cells in Stromal Evolution and Cancer Progression

    PubMed Central

    Cammarota, Francesca; Laukkanen, Mikko O.

    2016-01-01

    The study of cancer biology has mainly focused on malignant epithelial cancer cells, although tumors also contain a stromal compartment, which is composed of stem cells, tumor-associated fibroblasts (TAFs), endothelial cells, immune cells, adipocytes, cytokines, and various types of macromolecules comprising the extracellular matrix (ECM). The tumor stroma develops gradually in response to the needs of epithelial cancer cells during malignant progression initiating from increased local vascular permeability and ending to remodeling of desmoplastic loosely vascularized stromal ECM. The constant bidirectional interaction of epithelial cancer cells with the surrounding microenvironment allows damaged stromal cell usage as a source of nutrients for cancer cells, maintains the stroma renewal thus resembling a wound that does not heal, and affects the characteristics of tumor mesenchymal stem/stromal cells (MSCs). Although MSCs have been shown to coordinate tumor cell growth, dormancy, migration, invasion, metastasis, and drug resistance, recently they have been successfully used in treatment of hematopoietic malignancies to enhance the effect of total body irradiation-hematopoietic stem cell transplantation therapy. Hence, targeting the stromal elements in combination with conventional chemotherapeutics and usage of MSCs to attenuate graft-versus-host disease may offer new strategies to overcome cancer treatment failure and relapse of the disease. PMID:26798356

  9. [Therapeutic Effects of Multipotent Mesenchymal Stromal Cells after Irradiation].

    PubMed

    Kalmykova, N V; Alexandrova, S A

    2016-01-01

    Multipotent mesenchymal stromal cells (MSC) are now considered to be a perspective multifunctional treatment option for radiation side effects. At present.a great number of sufficient evidence has been collected in favor of therapeutic effects of MSCs in acute radiation reactions. It has been shown that MSC-based products injected locally or systemically have therapeutic effects on irradiated organs and tissues. This review presents summarized experimental and clinical data about protective and regenerative effects of MSCs on different radiation-injured organs and tissues; the main probable therapeutic mechanisms of their action are also discussed. PMID:27534063

  10. Manufacturing mesenchymal stromal cells for phase I clinical trials.

    PubMed

    Hanley, Patrick J; Mei, Zhuyong; da Graca Cabreira-Hansen, Maria; Klis, Mariola; Li, Wei; Zhao, Yali; Durett, April G; Zheng, Xingwu; Wang, Yongping; Gee, Adrian P; Horwitz, Edwin M

    2013-04-01

    Mesenchymal stromal cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytes, osteoblasts and chondroblasts as well as secreting a vast array of soluble mediators. This potentially makes MSCs important mediators of a variety of therapeutic applications. They are actively under evaluation for immunomodulatory purposes such as graft-versus-host disease and Crohn's disease as well as regenerative applications such as stroke and congestive heart failure. We report our method of generating clinical-grade MSCs together with suggestions gathered from manufacturing experience in our Good Manufacturing Practices facility. PMID:23480951

  11. Low Reactive Level Laser Therapy for Mesenchymal Stromal Cells Therapies

    PubMed Central

    Kushibiki, Toshihiro; Hirasawa, Takeshi; Okawa, Shinpei; Ishihara, Miya

    2015-01-01

    Low reactive level laser therapy (LLLT) is mainly focused on the activation of intracellular or extracellular chromophore and the initiation of cellular signaling by using low power lasers. Over the past forty years, it was realized that the laser therapy had the potential to improve wound healing and reduce pain and inflammation. In recent years, the term LLLT has become widely recognized in the field of regenerative medicine. In this review, we will describe the mechanisms of action of LLLT at a cellular level and introduce the application to mesenchymal stem cells and mesenchymal stromal cells (MSCs) therapies. Finally, our recent research results that LLLT enhanced the MSCs differentiation to osteoblast will also be described. PMID:26273309

  12. Functional Characteristics of Multipotent Mesenchymal Stromal Cells from Pituitary Adenomas

    PubMed Central

    Megnis, Kaspars; Mandrika, Ilona; Petrovska, Ramona; Stukens, Janis; Rovite, Vita; Balcere, Inga; Jansone, Laima Sabine; Peculis, Raitis; Pirags, Valdis

    2016-01-01

    Pituitary adenomas are one of the most common endocrine and intracranial neoplasms. Although they are theoretically monoclonal in origin, several studies have shown that they contain different multipotent cell types that are thought to play an important role in tumor initiation, maintenance, and recurrence after therapy. In the present study, we isolated and characterized cell populations from seven pituitary somatotroph, nonhormonal, and lactotroph adenomas. The obtained cells showed characteristics of multipotent mesenchymal stromal cells as observed by cell morphology, cell surface marker CD90, CD105, CD44, and vimentin expression, as well as differentiation to osteogenic and adipogenic lineages. They are capable of growth and passaging under standard laboratory cell culture conditions and do not manifest any hormonal cell characteristics. Multipotent mesenchymal stromal cells are present in pituitary adenomas regardless of their clinical manifestation and show no considerable expression of somatostatin 1–5 and dopamine 2 receptors. Most likely obtained cells are a part of tissue-supportive cells in pituitary adenoma microenvironment. PMID:27340409

  13. Functional Characteristics of Multipotent Mesenchymal Stromal Cells from Pituitary Adenomas.

    PubMed

    Megnis, Kaspars; Mandrika, Ilona; Petrovska, Ramona; Stukens, Janis; Rovite, Vita; Balcere, Inga; Jansone, Laima Sabine; Peculis, Raitis; Pirags, Valdis; Klovins, Janis

    2016-01-01

    Pituitary adenomas are one of the most common endocrine and intracranial neoplasms. Although they are theoretically monoclonal in origin, several studies have shown that they contain different multipotent cell types that are thought to play an important role in tumor initiation, maintenance, and recurrence after therapy. In the present study, we isolated and characterized cell populations from seven pituitary somatotroph, nonhormonal, and lactotroph adenomas. The obtained cells showed characteristics of multipotent mesenchymal stromal cells as observed by cell morphology, cell surface marker CD90, CD105, CD44, and vimentin expression, as well as differentiation to osteogenic and adipogenic lineages. They are capable of growth and passaging under standard laboratory cell culture conditions and do not manifest any hormonal cell characteristics. Multipotent mesenchymal stromal cells are present in pituitary adenomas regardless of their clinical manifestation and show no considerable expression of somatostatin 1-5 and dopamine 2 receptors. Most likely obtained cells are a part of tissue-supportive cells in pituitary adenoma microenvironment. PMID:27340409

  14. Regenerative Potential of Mesenchymal Stromal Cells: Age-Related Changes

    PubMed Central

    Bruna, Flavia; Contador, David; Conget, Paulette; Erranz, Benjamín; Sossa, Claudia L.; Arango-Rodríguez, Martha L.

    2016-01-01

    Preclinical and clinical studies have shown that a therapeutic effect results from mesenchymal stromal cells (MSCs) transplant. No systematic information is currently available regarding whether donor age modifies MSC regenerative potential on cutaneous wound healing. Here, we evaluate whether donor age influences this potential. Two different doses of bone marrow MSCs (BM-MSCs) from young, adult, or old mouse donors or two doses of their acellular derivatives mesenchymal stromal cells (acd-MSCs) were intradermally injected around wounds in the midline of C57BL/6 mice. Every two days, wound healing was macroscopically assessed (wound closure) and microscopically assessed (reepithelialization, dermal-epidermal junction, skin appendage regeneration, granulation tissue, leukocyte infiltration, and density dermal collagen fibers) after 12 days from MSC transplant. Significant differences in the wound closure kinetic, quality, and healing of skin regenerated were observed in lesions which received BM-MSCs from different ages or their acd-MSCs compared to lesions which received vehicle. Nevertheless, our data shows that adult's BM-MSCs or their acd-MSCs were the most efficient for recovery of most parameters analyzed. Our data suggest that MSC efficacy was negatively affected by donor age, where the treatment with adult's BM-MSCs or their acd-MSCs in cutaneous wound promotes a better tissue repair/regeneration. This is due to their paracrine factors secretion. PMID:27247575

  15. Isolation and characterization of primary bone marrow mesenchymal stromal cells.

    PubMed

    Li, Hongzhe; Ghazanfari, Roshanak; Zacharaki, Dimitra; Lim, Hooi Ching; Scheding, Stefan

    2016-04-01

    Bone marrow (BM) contains a rare population of mesenchymal stromal cells (MSCs), which have been characterized as nonhematopoietic skeletal progenitor cells with central importance for the hematopoietic microenvironment. Classically, MSCs are isolated by plastic adherence and subsequent culture. However, as cultured stromal cells differ from their in vivo progenitors, it is important to identify the phenotype of the primary MSCs to study these cells in more detail. In the past years, several surface markers have been reported to be suitable for effective enrichment of BM-MSCs, and recent data indicate that the putative MSC stem/progenitor cell population in human adult BM is highly enriched in Lin(-) CD45(-) CD271(+) CD140a (PDGFRα)(low/-) cells. Moreover, surface marker combinations have been described for the isolation of MSCs from murine BM. On the basis of these findings, the role of primary MSCs can now be studied in normal and, importantly, diseased BM. Furthermore, genetically engineered mouse models have been developed as powerful tools to investigate well-defined BM stromal cell populations in vivo. Our discussion aims to provide a concise overview of the current state of the art in BM-MSC isolation in humans and briefly present murine MSC isolation approaches and genetic models. PMID:27270495

  16. Mesenchymal stem cells/multipotent mesenchymal stromal cells (MSCs): potential role in healing cutaneous chronic wounds.

    PubMed

    Zou, Ji-Ping; Huang, Sha; Peng, Yan; Liu, Hong-Wei; Cheng, Biao; Fu, Xiao-Bing; Xiang, Xiao-Fei

    2012-12-01

    Chronic wounds remain a major challenge in modern medicine and represent a significant health care burden. Several treatments have been suggested, but without a full understanding of the exact mechanism by which chronic wound occurs. Numerous studies have shown that mesenchymal stem cells/multipotent mesenchymal stromal cells (MSCs) may have therapeutic potential in healing cutaneous chronic wounds through various mechanisms. So far, a series of hypotheses have been proposed, but a holistic image of them is lacking. This review provides a systematic analysis of recent research in animal models and preclinical or clinic trails to evaluate the potential role of MSCs in chronic cutaneous wound healing. Most important, we highlight how mesenchymal stem cells could potentially revolutionize our approach to treating cutaneous chronic wounds. Special attention should be focused on ongoing research regarding the challenges in using and prospects of MSCs in clinical settings. PMID:23222159

  17. Isolation of Multipotent Mesenchymal Stromal Cells from Cryopreserved Human Umbilical Cord Tissue.

    PubMed

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2016-02-01

    Umbilical cord stroma is an easily available, convenient, and promising source of multipotent mesenchymal stromal cells for regenerative medicine. Cryogenic storage of umbilical cord tissue provides more possibilities for further isolation of multipotent mesenchymal stromal cells for autologous transplantation or scientific purposes. Here we developed a protocol for preparation of the whole umbilical cord tissue for cryogenic storage that in combination with the previously described modified method of isolation of multipotent mesenchymal stromal cells allowed us to isolate cells with high proliferative potential, typical phenotype, and preserved differentiation potencies. PMID:26902359

  18. A molecular classification of human mesenchymal stromal cells.

    PubMed

    Rohart, Florian; Mason, Elizabeth A; Matigian, Nicholas; Mosbergen, Rowland; Korn, Othmar; Chen, Tyrone; Butcher, Suzanne; Patel, Jatin; Atkinson, Kerry; Khosrotehrani, Kiarash; Fisk, Nicholas M; Lê Cao, Kim-Anh; Wells, Christine A

    2016-01-01

    Mesenchymal stromal cells (MSC) are widely used for the study of mesenchymal tissue repair, and increasingly adopted for cell therapy, despite the lack of consensus on the identity of these cells. In part this is due to the lack of specificity of MSC markers. Distinguishing MSC from other stromal cells such as fibroblasts is particularly difficult using standard analysis of surface proteins, and there is an urgent need for improved classification approaches. Transcriptome profiling is commonly used to describe and compare different cell types; however, efforts to identify specific markers of rare cellular subsets may be confounded by the small sample sizes of most studies. Consequently, it is difficult to derive reproducible, and therefore useful markers. We addressed the question of MSC classification with a large integrative analysis of many public MSC datasets. We derived a sparse classifier (The Rohart MSC test) that accurately distinguished MSC from non-MSC samples with >97% accuracy on an internal training set of 635 samples from 41 studies derived on 10 different microarray platforms. The classifier was validated on an external test set of 1,291 samples from 65 studies derived on 15 different platforms, with >95% accuracy. The genes that contribute to the MSC classifier formed a protein-interaction network that included known MSC markers. Further evidence of the relevance of this new MSC panel came from the high number of Mendelian disorders associated with mutations in more than 65% of the network. These result in mesenchymal defects, particularly impacting on skeletal growth and function. The Rohart MSC test is a simple in silico test that accurately discriminates MSC from fibroblasts, other adult stem/progenitor cell types or differentiated stromal cells. It has been implemented in the www.stemformatics.org resource, to assist researchers wishing to benchmark their own MSC datasets or data from the public domain. The code is available from the CRAN

  19. A molecular classification of human mesenchymal stromal cells

    PubMed Central

    Rohart, Florian; Mason, Elizabeth A.; Matigian, Nicholas; Mosbergen, Rowland; Korn, Othmar; Chen, Tyrone; Butcher, Suzanne; Patel, Jatin; Atkinson, Kerry; Khosrotehrani, Kiarash; Fisk, Nicholas M.; Lê Cao, Kim-Anh

    2016-01-01

    Mesenchymal stromal cells (MSC) are widely used for the study of mesenchymal tissue repair, and increasingly adopted for cell therapy, despite the lack of consensus on the identity of these cells. In part this is due to the lack of specificity of MSC markers. Distinguishing MSC from other stromal cells such as fibroblasts is particularly difficult using standard analysis of surface proteins, and there is an urgent need for improved classification approaches. Transcriptome profiling is commonly used to describe and compare different cell types; however, efforts to identify specific markers of rare cellular subsets may be confounded by the small sample sizes of most studies. Consequently, it is difficult to derive reproducible, and therefore useful markers. We addressed the question of MSC classification with a large integrative analysis of many public MSC datasets. We derived a sparse classifier (The Rohart MSC test) that accurately distinguished MSC from non-MSC samples with >97% accuracy on an internal training set of 635 samples from 41 studies derived on 10 different microarray platforms. The classifier was validated on an external test set of 1,291 samples from 65 studies derived on 15 different platforms, with >95% accuracy. The genes that contribute to the MSC classifier formed a protein-interaction network that included known MSC markers. Further evidence of the relevance of this new MSC panel came from the high number of Mendelian disorders associated with mutations in more than 65% of the network. These result in mesenchymal defects, particularly impacting on skeletal growth and function. The Rohart MSC test is a simple in silico test that accurately discriminates MSC from fibroblasts, other adult stem/progenitor cell types or differentiated stromal cells. It has been implemented in the www.stemformatics.org resource, to assist researchers wishing to benchmark their own MSC datasets or data from the public domain. The code is available from the CRAN

  20. [Multipotent mesenchymal stromal and immune cells interaction: reciprocal effects].

    PubMed

    Andreeva, E R; Buravkova, L B

    2012-12-01

    Adult multipotent mesenchymal stromal cells (MMSCs) are considered now as one of the key players in physiological and pathological tissue remodeling. Clarification of the mechanisms that mediate MMSC functions, is one of the most intriguing issues in modern cell physiology. Present Review summarizes current understanding of the MMSC effects on different types of immune cells. The realization of MMSC immunomodulatory capacity is considered as a contribution of direct cell-to-cell contacts, soluble mediators and of local microenvironmental factors, the most important of which is the partial pressure of oxygen. MMSCs and immune cells interaction is discussed in the terms of reciprocal effects, modifying properties of all "partner cells". Special attention is paid to the influence of immune cells on the MMSCs. "Immunosuppressive" phenomenon of MMSCs is considered as the integral part of the "response to injury" mechanism. PMID:23461191

  1. Mesenchymal stromal cells and chronic inflammatory bowel disease.

    PubMed

    Algeri, M; Conforti, A; Pitisci, A; Starc, N; Tomao, L; Bernardo, M E; Locatelli, F

    2015-12-01

    Recent experimental findings have shown the ability of mesenchymal stromal cells (MSCs) to home to damaged tissues and to produce paracrine factors with anti-inflammatory properties, potentially resulting in reduction of inflammation and functional recovery of the damaged tissues. Prompted by these intriguing properties and on the basis of encouraging preclinical data, MSCs are currently being studied in several immune-mediated disorders. Inflammatory bowel diseases (IBD) represent a setting in which MSCs-based therapy has been extensively investigated. Phase I and II studies have documented the safety and feasibility of MSCs. However, efficacy results have so far been conflicting. In this review, we will discuss the biologic rationale that makes MSCs a promising therapeutic tool for IBD, and analyze recent experimental and clinical findings, highlighting current limitations and future perspectives of MSCs-related immunotherapy for IBD. PMID:26170204

  2. Mesenchymal stromal cells and fibroblasts: a case of mistaken identity?

    PubMed

    Hematti, Peiman

    2012-05-01

    The plastic-adherent fibroblast-looking cells that can be isolated and culture-expanded from bone marrow and many other tissues are widely known as mesenchymal stromal cells (MSC). In addition to their fibroblast-like morphology, they are characterized by a panel of cell-surface markers and their potential to differentiate into bone, fat and cartilage. Based on their intriguing immunomodulatory and regenerative properties, MSC are being investigated as cellular therapeutics for a variety of clinical indications. However, many questions regarding the true identity and functionality of these cells in vivo remain unanswered. Fibroblasts, known for a much longer time but still poorly characterized, are also considered to be a ubiquitous stromal element of almost all tissues and are believed to play a role in tissue homeostasis. Despite the presence of MSC and fibroblasts in almost all tissues, similar morphology and other shared characteristics, the exact relationship between MSC and fibroblasts has remained undetermined. In this review, based on recent and old, but often neglected, literature it is suggested that ex vivo culture-expanded MSC and fibroblasts are indistinguishable by morphology, cell-surface markers, differentiation potential and immunologic properties. PMID:22458957

  3. Melanoma educates mesenchymal stromal cells towards vasculogenic mimicry

    PubMed Central

    VARTANIAN, AMALIA; KARSHIEVA, SAIDA; DOMBROVSKY, VLADISLAV; BELYAVSKY, ALEXANDER

    2016-01-01

    Accumulating evidence suggests that mesenchymal stromal cells (MSCs) are recruited to the tumor, and promote tumor development and growth. The present study was performed to investigate the communication between aggressive melanoma and MSCs in vasculogenic mimicry (VM). Normal human MSCs plated on Matrigel were unable to form capillary-like structures (CLSs). By contrast, MSCs co-cultured with aggressive melanoma cell lines, namely, Mel Cher, Mel Kor and Mel P, generated CLSs. Significantly, MSCs co-cultured with poorly aggressive melanoma cells, namely, Mel Me, failed to form CLSs. To identify factors responsible for VM, the effects of vascular endothelial growth factor A (VEGFA), pro-epidermal growth factor, basic fibroblast growth factor and stromal cell-derived factor 1α on the formation of CLSs by MSCs were tested. VM was induced by the addition of VEGFA, whereas other cytokines were inefficient. To confirm the hypothesis that aggressive tumor cells can increase the vasculogenic ability of MSCs, a standard B16/F10 mouse melanoma test system was used. MSCs isolated from the adipose tissues of C57BL/6 mice with melanoma formed a vascular-like network on Matrigel, whereas MSCs from healthy mice failed to form such structures. This study provides the first direct evidence that melanoma tumors educate MSCs to engage in VM. The education may occur distantly. These findings offer promise for novel therapeutic directions in the treatment of metastatic melanoma. PMID:27313776

  4. Multipotent Mesenchymal Stromal Cells: Possible Culprits in Solid Tumors?

    PubMed Central

    Johann, Pascal David; Müller, Ingo

    2015-01-01

    The clinical use of bone marrow derived multipotent mesenchymal stromal cells (BM-MSCs) in different settings ranging from tissue engineering to immunotherapies has prompted investigations on the properties of these cells in a variety of other tissues. Particularly the role of MSCs in solid tumors has been the subject of many experimental approaches. While a clear phenotypical distinction of tumor associated fibroblasts (TAFs) and MSCs within the tumor microenvironment is still missing, the homing of bone marrow MSCs in tumor sites has been extensively studied. Both, tumor-promoting and tumor-inhibiting effects of BM-MSCs have been described in this context. This ambiguity requires a reappraisal of the different studies and experimental methods employed. Here, we review the current literature on tumor-promoting and tumor-inhibiting effects of BM-MSCs with a particular emphasis on their interplay with components of the immune system and also highlight a potential role of MSCs as cell of origin for certain mesenchymal tumors. PMID:26273308

  5. Mesenchymal stromal cells for cartilage repair in osteoarthritis.

    PubMed

    Mamidi, M K; Das, A K; Zakaria, Z; Bhonde, R

    2016-08-01

    Treatment for articular cartilage damage is quite challenging as it shows limited repair and regeneration following injury. Non-operative and classical surgical techniques are inefficient in restoring normal anatomy and function of cartilage in osteoarthritis (OA). Thus, investigating new and effective strategies for OA are necessary to establish feasible therapeutic solutions. The emergence of the new discipline of regenerative medicine, having cell-based therapy as its primary focus, may enable us to achieve repair and restore the damaged articular cartilage. This review describes progress and development of employing mesenchymal stromal cell (MSC)-based therapy as a promising alternative for OA treatment. The objective of this review is to first, discuss how in vitro MSC chondrogenic differentiation mimics in vivo embryonic cartilage development, secondly, to describe various chondrogenic differentiation strategies followed by pre-clinical and clinical studies demonstrating their feasibility and efficacy. However, several challenges need to be tackled before this research can be translated to the clinics. In particular, better understanding of the post-transplanted cell behaviour and learning to enhance their potency in the disease microenvironment is essential. Final objective is to underscore the importance of isolation, storage, cell shipment, route of administration, optimum dosage and control batch to batch variations to realise the full potential of MSCs in OA clinical trials. PMID:26973328

  6. Mesenchymal stromal cell characteristics vary depending on their origin.

    PubMed

    Wegmeyer, Heike; Bröske, Ann-Marie; Leddin, Mathias; Kuentzer, Karin; Nisslbeck, Anna Katharina; Hupfeld, Julia; Wiechmann, Kornelius; Kuhlen, Jennifer; von Schwerin, Christoffer; Stein, Carsten; Knothe, Saskia; Funk, Jürgen; Huss, Ralf; Neubauer, Markus

    2013-10-01

    Mesenchymal stromal cells (MSCs) are rare progenitor cells that can be isolated from various tissues. They exhibit multilineage differentiation potential, support regenerative processes, and interact with various immune cells. Therefore, MSCs represent a promising tool for regenerative medicine. However, source-dependent and donor-dependent differences of MSC properties, including implications on their clinical application are still largely unknown. We evaluated MSCs derived from perinatal tissues umbilical cord (UC) and amniotic membrane (AM) in comparison to adult MSCs from bone marrow (BM), which were used as gold standard. We found genetic background-independent differences between MSCs from UC and AM. While AM- and UC-MSCs were closer to each other than to BM-MSCs, they also exhibited differences between each other. AM-MSCs from different donors but not UC-MSCs displayed high interdonor variability. In addition, we show that although all MSCs expressed similar surface markers, MSC populations from UC and AM showed differential profiles of gene expression and paracrine factor secretion to BM-derived MSCs. Notably, pathway analysis of gene expression data revealed intriguing differences between MSCs suggesting that MSCs from UC and AM possess in general a higher potential of immunomodulatory capacity, whereas BM-MSCs showed a higher potential of supporting regenerative processes as exemplified by neuronal differentiation and development. These differences between perinatal and BM-derived MSCs may be relevant for clinical applications. PMID:23676112

  7. A relativity concept in mesenchymal stromal cell manufacturing.

    PubMed

    Martin, Ivan; De Boer, Jan; Sensebe, Luc

    2016-05-01

    Mesenchymal stromal cells (MSCs) are being experimentally tested in several biological systems and clinical settings with the aim of verifying possible therapeutic effects for a variety of indications. MSCs are also known to be heterogeneous populations, with phenotypic and functional features that depend heavily on the individual donor, the harvest site, and the culture conditions. In the context of this multidimensional complexity, a recurrent question is whether it is feasible to produce MSC batches as "standard" therapeutics, possibly within scalable manufacturing systems. Here, we provide a short overview of the literature on different culture methods for MSCs, including those employing innovative technologies, and of some typically assessed functional features (e.g., growth, senescence, genomic stability, clonogenicity, etc.). We then offer our perspective of a roadmap on how to identify and refine manufacturing systems for MSCs intended for specific clinical indications. We submit that the vision of producing MSCs according to a unique standard, although commercially attractive, cannot yet be scientifically substantiated. Instead, efforts should be concentrated on standardizing methods for characterization of MSCs generated by different groups, possibly covering a vast gamut of functionalities. Such assessments, combined with hypotheses on the therapeutic mode of action and associated clinical data, should ultimately allow definition of in-process controls and measurable release criteria for MSC manufacturing. These will have to be validated as predictive of potency in suitable pre-clinical models and of therapeutic efficacy in patients. PMID:27059199

  8. Mesenchymal Stromal Cells Affect Disease Outcomes via Macrophage Polarization

    PubMed Central

    Zheng, Guoping; Ge, Menghua; Qiu, Guanguan; Shu, Qiang; Xu, Jianguo

    2015-01-01

    Mesenchymal stromal cells (MSCs) are multipotent and self-renewable cells that reside in almost all postnatal tissues. In recent years, many studies have reported the effect of MSCs on the innate and adaptive immune systems. MSCs regulate the proliferation, activation, and effector function of T lymphocytes, professional antigen presenting cells (dendritic cells, macrophages, and B lymphocytes), and NK cells via direct cell-to-cell contact or production of soluble factors including indoleamine 2,3-dioxygenase, prostaglandin E2, tumor necrosis factor-α stimulated gene/protein 6, nitric oxide, and IL-10. MSCs are also able to reprogram macrophages from a proinflammatory M1 phenotype toward an anti-inflammatory M2 phenotype capable of regulating immune response. Because of their capacity for differentiation and immunomodulation, MSCs have been used in many preclinical and clinical studies as possible new therapeutic agents for the treatment of autoimmune, degenerative, and inflammatory diseases. In this review, we discuss the central role of MSCs in macrophage polarization and outcomes of diseases such as wound healing, brain/spinal cord injuries, and diseases of heart, lung, and kidney in animal models. PMID:26257791

  9. Mesenchymal Stromal Cells Affect Disease Outcomes via Macrophage Polarization.

    PubMed

    Zheng, Guoping; Ge, Menghua; Qiu, Guanguan; Shu, Qiang; Xu, Jianguo

    2015-01-01

    Mesenchymal stromal cells (MSCs) are multipotent and self-renewable cells that reside in almost all postnatal tissues. In recent years, many studies have reported the effect of MSCs on the innate and adaptive immune systems. MSCs regulate the proliferation, activation, and effector function of T lymphocytes, professional antigen presenting cells (dendritic cells, macrophages, and B lymphocytes), and NK cells via direct cell-to-cell contact or production of soluble factors including indoleamine 2,3-dioxygenase, prostaglandin E2, tumor necrosis factor-α stimulated gene/protein 6, nitric oxide, and IL-10. MSCs are also able to reprogram macrophages from a proinflammatory M1 phenotype toward an anti-inflammatory M2 phenotype capable of regulating immune response. Because of their capacity for differentiation and immunomodulation, MSCs have been used in many preclinical and clinical studies as possible new therapeutic agents for the treatment of autoimmune, degenerative, and inflammatory diseases. In this review, we discuss the central role of MSCs in macrophage polarization and outcomes of diseases such as wound healing, brain/spinal cord injuries, and diseases of heart, lung, and kidney in animal models. PMID:26257791

  10. Characterization of mesenchymal stromal cells: potency assay development.

    PubMed

    Hematti, Peiman

    2016-04-01

    Based on their many different mechanisms of action, presumed immune-privileged status, and relative ease of production, mesenchymal stromal cells (MSCs) are under intensive clinical investigation for treating a wide range of degenerative, inflammatory, and immunologic disorders. Identification of relevant and robust potency assays is not only a regulatory requirement, but it is also the basis for producing and delivering a product that is consistent, safe, and ultimately an effective therapy. Although development of an appropriate potency assay is one of the most challenging issues in cell-based therapies, it is of paramount importance in the process of developing and testing cellular products. Regardless of the many different tissue sources and methods used in culture expansion of MSCs, they possess many of the same morphologic, cell surface markers, and differentiation characteristics. However, MSC products with similar phenotypic characteristics could still have major differences in their biologic and functional attributes. Understanding the different mechanisms of action and establishment of relevant potency assays is of pivotal importance in allowing investigators and regulatory agencies to compare MSCs used in different clinical trials. PMID:27079322

  11. Management of Fibrosis: The Mesenchymal Stromal Cells Breakthrough

    PubMed Central

    Usunier, Benoît; Benderitter, Marc; Tamarat, Radia; Chapel, Alain

    2014-01-01

    Fibrosis is the endpoint of many chronic inflammatory diseases and is defined by an abnormal accumulation of extracellular matrix components. Despite its slow progression, it leads to organ malfunction. Fibrosis can affect almost any tissue. Due to its high frequency, in particular in the heart, lungs, liver, and kidneys, many studies have been conducted to find satisfactory treatments. Despite these efforts, current fibrosis management therapies either are insufficiently effective or induce severe adverse effects. In the light of these facts, innovative experimental therapies are being investigated. Among these, cell therapy is regarded as one of the best candidates. In particular, mesenchymal stromal cells (MSCs) have great potential in the treatment of inflammatory diseases. The value of their immunomodulatory effects and their ability to act on profibrotic factors such as oxidative stress, hypoxia, and the transforming growth factor-β1 pathway has already been highlighted in preclinical and clinical studies. Furthermore, their propensity to act depending on the microenvironment surrounding them enhances their curative properties. In this paper, we review a large range of studies addressing the use of MSCs in the treatment of fibrotic diseases. The results reported here suggest that MSCs have antifibrotic potential for several organs. PMID:25132856

  12. Translating Research into Clinical Scale Manufacturing of Mesenchymal Stromal Cells

    PubMed Central

    Bieback, Karen; Kinzebach, Sven; Karagianni, Marianna

    2010-01-01

    It sounds simple to obtain sufficient numbers of cells derived from fetal or adult human tissues, isolate and/or expand the stem cells, and then transplant an appropriate number of these cells into the patient at the correct location. However, translating basic research into routine therapies is a complex multistep process which necessitates product regulation. The challenge relates to managing the expected therapeutic benefits with the potential risks and to balance the fast move to clinical trials with time-consuming cautious risk assessment. This paper will focus on the definition of mesenchymal stromal cells (MSCs), and challenges and achievements in the manufacturing process enabling their use in clinical studies. It will allude to different cellular sources, special capacities of MSCs, but also to current regulations, with a special focus on accessory material of human or animal origin, like media supplements. As cellular integrity and purity, formulation and lot release testing of the final product, validation of all procedures, and quality assurance are of utmost necessity, these topics will be addressed. PMID:21318154

  13. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    SciTech Connect

    Aanei, Carmen Mariana; Eloae, Florin Zugun; Flandrin-Gresta, Pascale; Tavernier, Emmanuelle; Carasevici, Eugen; Guyotat, Denis; Campos, Lydia

    2011-11-01

    Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90{alpha}/{beta} client protein, these results suggest the utility of HSP90{alpha}/{beta} inhibition as a target for adjuvant therapy for myelodysplasia.

  14. Mechanisms of mesenchymal stem/stromal cell function.

    PubMed

    Spees, Jeffrey L; Lee, Ryang Hwa; Gregory, Carl A

    2016-01-01

    The past decade has seen an explosion of research directed toward better understanding of the mechanisms of mesenchymal stem/stromal cell (MSC) function during rescue and repair of injured organs and tissues. In addition to delineating cell-cell signaling and molecular controls for MSC differentiation, the field has made particular progress in defining several other mechanisms through which administered MSCs can promote tissue rescue/repair. These include: 1) paracrine activity that involves secretion of proteins/peptides and hormones; 2) transfer of mitochondria by way of tunneling nanotubes or microvesicles; and 3) transfer of exosomes or microvesicles containing RNA and other molecules. Improved understanding of MSC function holds great promise for the application of cell therapy and also for the development of powerful cell-derived therapeutics for regenerative medicine. Focusing on these three mechanisms, we discuss MSC-mediated effects on immune cell responses, cell survival, and fibrosis and review recent progress with MSC-based or MSC-derived therapeutics. PMID:27581859

  15. 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

  16. Cryopreservation and Revival of Human Mesenchymal Stromal Cells.

    PubMed

    Haack-Sørensen, Mandana; Ekblond, Annette; Kastrup, Jens

    2016-01-01

    Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities.The establishment of methods for large-scale expansion of clinical-grade MSCs in vitro has paved the way for their therapeutic use in clinical trials. However, the clinical application of MSCs also requires cryopreservation and banking of the cell products. To preserve autologous or allogeneic MSCs for future clinical applications, a reliable and effective cryopreservation method is required.Developing a successful cryopreservation protocol for clinical stem cell products, cryopreservation media, cryoprotectant agents (CPAs), the freezing container, the freezing temperature, and the cooling and warming rate are all aspects which should be considered.A major challenge is the selection of a suitable cryoprotectant which is able to penetrate the cells and yet has low toxicity.This chapter focuses on recent technological developments relevant for the cryopreservation of MSCs using the most commonly used cryopreservation medium containing DMSO and animal serum or human-derived products for research use and the animal protein-free cryopreservation media CryoStor (BioLife Solutions) for clinical use. PMID:27236683

  17. CONCISE REVIEW Micro RNA Expression in Multipotent Mesenchymal Stromal Cells

    PubMed Central

    Lakshmipathy, Uma; Hart, Ronald P.

    2009-01-01

    Multipotent mesenchymal stromal cells (MSC) isolated from various adult tissue sources have the capacity to self-renew and to differentiate into multiple lineages. Both of these processes are tightly regulated by genetic and epigenetic mechanisms. Emerging evidence indicates that the class of single-stranded non-coding RNAs known as “microRNAs” also plays a critical role in this process. First described in nematodes and plants, microRNAs have been shown to modulate major regulatory mechanisms in eukaryotic cells involved in a broad array of cellular functions. Studies with various types of embryonic as well as adult stem cells indicate an intricate network of microRNAs regulating key transcription factors and other genes which in turn determine cell fate. In addition, expression of unique microRNAs in specific cell types serves as a useful diagnostic marker to define a particular cell type. MicroRNAs are also found to be regulated by extracellular signaling pathways that are important for differentiation into specific tissues, suggesting that they play a role in specifying tissue identity. In this review we describe the importance of microRNAs in stem cells focusing on our current understanding of microRNAs in MSC and their derivatives. PMID:17991914

  18. [Effects of growth factors on multipotent bone marrow mesenchymal stromal cells].

    PubMed

    Molchanova, E A; Paiushina, O V; Starostin, V I

    2008-01-01

    Multipotent bone marrow mesenchymal stromal cells are progenitors of various cell types capable of long-term self-maintenance. These cells are an adequate model for studying the most important problems in cell biology, such as self-maintenance of stem cells and regulation of their differentiation. Moreover, these cells are a promising resource for regenerative medicine. In this context, isolation of the earliest multipotent mesenchymal stromal cells, their in vitro maintenance in an undifferentiated state, and stimulation of their differentiation in a desired direction appear to be most important. To successfully use the multipotent mesenchymal stromal cells both in fundamental studies and in therapy, it is necessary to modify and standardize the composition of culture medium, replacing blood serum with certain growth factors. These factors have influence on the proliferation and differentiation of most cell types, including multipotent mesenchymal stromal cells. This paper is a review of available data concerning the effects of some growth factors on the multipotent mesenchymal stromal cells of the bone marrow. PMID:19198070

  19. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential.

    PubMed

    Pipino, Caterina; Pandolfi, Assunta

    2015-05-26

    In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine. PMID:26029340

  20. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

    PubMed Central

    Pipino, Caterina; Pandolfi, Assunta

    2015-01-01

    In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine. PMID:26029340

  1. The influence of hypoxia on the differentiation capacities and immunosuppressive properties of clonal mouse mesenchymal stromal cell lines.

    PubMed

    Prado-Lòpez, Sonia; Duffy, Michelle M; Baustian, Claas; Alagesan, Senthilkumar; Hanley, Shirley A; Stocca, Alessia; Griffin, Matthew D; Ceredig, Rhodri

    2014-08-01

    Multipotent mesenchymal stromal cells are multipotent cells capable of differentiating into different mesodermal cell types. Enigmatically, mesenchymal stromal cells present in the bone marrow support early lymphopoiesis yet can inhibit mature lymphocyte growth. Critical features of the bone marrow microenvironment, such as the level of oxygen, play an important role in mesenchymal stromal cell biology. Herein, we show that a panel of continuously growing mouse mesenchymal stromal cell lines, namely OP9, MS5, PA6, ST2 and B16-14, exhibit mesenchymal stromal cell characteristic phenotypes and respond physiologically to oxygen deprivation. Culturing freshly isolated bone marrow-derived mesenchymal stromal cells or cell lines at 5% O2 resulted in a dramatic increase in expression of hypoxia-inducible factor family members and of key genes involved in their differentiation. Phenotypically, their osteogenic and adipogenic differentiation capacity was generally improved in hypoxia, whereas their inhibitory effects on in vitro T-cell proliferation were preserved. Taken together, we conclude that these continuously growing mouse cell lines behave as canonical mesenchymal stromal cells and respond physiologically to hypoxia, thereby providing a potent tool for the study of different aspects of mesenchymal stromal cell biology. PMID:24777310

  2. Human placenta-derived adherent cells induce tolerogenic immune responses.

    PubMed

    Liu, Wei; Morschauser, Andrew; Zhang, Xin; Lu, Xiaohua; Gleason, Joseph; He, Shuyang; Chen, Hong-Jung; Jankovic, Vladimir; Ye, Qian; Labazzo, Kristen; Herzberg, Uri; Albert, Vivian R; Abbot, Stewart E; Liang, Bitao; Hariri, Robert

    2014-05-01

    Human placenta-derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory and anti-inflammatory properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T-cell proliferation in an OT-II T-cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T-cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell-treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow-derived DC. Similarly, human DC differentiated from CD14(+) monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T-cell proliferation and differentiation toward Th1, but skewed T-cell differentiation toward Th2. Inhibition of cyclo-oxygenase-2 activity resulted in a significant, but not complete, abrogation of PDAC cells' effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC-mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells. PMID:25505962

  3. Essential components for ex vivo proliferation of mesenchymal stromal cells.

    PubMed

    Fekete, Natalie; Rojewski, Markus Thomas; Lotfi, Ramin; Schrezenmeier, Hubert

    2014-02-01

    Mesenchymal stromal cells (MSCs) are highly interesting candidates for clinical applications in regenerative medicine. Due to their low occurrence in human tissues, extensive in vitro expansion is necessary to obtain sufficient cell numbers applicable as a clinical dose in the context of cellular therapy. Current cell culture media formulations for the isolation and expansion of MSCs include fetal calf serum (FCS), human AB serum (ABS), or human platelet lysate (PL) as a supplement. However, these established supplements are inherently ill-defined formulations that contain a variety of bioactive molecules in varying batch-to-batch compositions and the risk of transmitting pathogens that escape routine screening procedures. In this study, we have comparatively characterized the capacity of commonly used basal media, such as the Minimum Essential Medium alpha (αMEM), Dulbecco's modified Eagle's medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), and RPMI 1640 as well as human- and animal-derived supplements, that is, PL, ABS, and FCS to stimulate cell proliferation. MSC proliferation was observed to be optimal in the PL-supplemented αMEM. Using a combinatorial approach, we then assessed a library of soluble factors, including mitogens (TGF-β1, Activin A, bFGF, EGF, IGF-I, PDGF-BB, and VEGF), chemokines (CCL21, CCL25, CXCL12, and RANTES), proteins (human serum albumin), lipids (e.g., oleic acid, linoleic acid, and arachidonic acid), and hormones (dexamethasone, insulin, and TSH), to create a defined medium as well as coating of cell culture surfaces to promote robust MSC proliferation in vitro. A combination of recombinant human factors partially met the nutritional requirements of bone marrow-derived MSCs, and was able to promote cell proliferation comparable to about 5% PL if supplemented with auxiliary 0.6%-1.2% PL. Maximal MSC proliferation was achieved by combining 5% PL with a cocktail of recombinant factors and did not depend on coating of cell

  4. Immunomodulatory effects of mesenchymal stromal cells-derived exosome.

    PubMed

    Chen, Wancheng; Huang, Yukai; Han, Jiaochan; Yu, Lili; Li, Yanli; Lu, Ziyuan; Li, Hongbo; Liu, Zenghui; Shi, Chenyan; Duan, Fengqi; Xiao, Yang

    2016-08-01

    The mechanisms underlying immunomodulatory ability of mesenchymal stromal cells (MSCs) remain unknown. Recently, studies suggested that the immunomodulatory activity of MSCs is largely mediated by paracrine factors. Among which, exosome is considered to play a major role in the communication between MSCs and target tissue. The aim of our study is to investigate the effect of MSCs-derived exosome on peripheral blood mononuclear cells (PBMCs), especially T cells. We find that the MSCs-derived exosome extracted from healthy donors' bone marrow suppressed the secretion of pro-inflammatory factor TNF-α and IL-1β, but increased the concentration of anti-inflammatory factor TGF-β during in vitro culture. In addition, exosome may induce conversion of T helper type 1 (Th1) into T helper type 2 (Th2) cells and reduced potential of T cells to differentiate into interleukin 17-producing effector T cells (Th17). Moreover, the level of regulatory T cells (Treg) and cytotoxic T lymphocyte-associated protein 4 were also increased. These results suggested that MSC-derived exosome possesses the immunomodulatory properties. However, it showed no effects on the proliferation of PBMCs or CD3+ T cells, but increases the apoptosis of them. In addition, indoleamine 2, 3-dioxygenase (IDO) was previously shown to mediate the immunoregulation of MSCs, which was increased in PBMCs co-cultured with MSCs. In our study, IDO showed no significant changes in PBMCs exposed to MSCs-derived exosome. We conclude that exosome and MSCs might differ in their immune-modulating activities and mechanisms. PMID:27115513

  5. Simvastatin modulates mesenchymal stromal cell proliferation and gene expression.

    PubMed

    Zanette, Dalila Lucíola; Lorenzi, Julio Cesar Cetrulo; Panepucci, Rodrigo Alexandre; Palma, Patricia Vianna Bonini; Dos Santos, Daiane Fernanda; Prata, Karen Lima; Silva, Wilson Araújo

    2015-01-01

    Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate in vitro into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages in vitro. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential. PMID:25874574

  6. Mesenchymal Stromal Cells Improve Renovascular Function in Polycystic Kidney Disease.

    PubMed

    Franchi, Federico; Peterson, Karen M; Xu, Rende; Miller, Brent; Psaltis, Peter J; Harris, Peter C; Lerman, Lilach O; Rodriguez-Porcel, Martin

    2015-01-01

    Polycystic kidney disease (PKD) is a common cause of end-stage renal failure, for which there is no accepted treatment. Progenitor and stem cells have been shown to restore renal function in a model of renovascular disease, a disease that shares many features with PKD. The objective of this study was to examine the potential of adult stem cells to restore renal structure and function in PKD. Bone marrow-derived mesenchymal stromal cells (MSCs, 2.5 × 10(5)) were intrarenally infused in 6-week-old PCK rats. At 10 weeks of age, PCK rats had an increase in systolic blood pressure (SBP) versus controls (126.22 ± 2.74 vs. 116.45 ± 3.53 mmHg, p < 0.05) and decreased creatinine clearance (3.76 ± 0.31 vs. 6.10 ± 0.48 µl/min/g, p < 0.01), which were improved in PKD animals that received MSCs (SBP: 114.67 ± 1.34 mmHg, and creatinine clearance: 4.82 ± 0.24 µl/min/g, p = 0.001 and p = 0.003 vs. PKD, respectively). MSCs preserved vascular density and glomeruli diameter, measured using microcomputed tomography. PCK animals had increased urine osmolality (843.9 ± 54.95 vs. 605.6 ± 45.34 mOsm, p < 0.01 vs. control), which was improved after MSC infusion and not different from control (723.75 ± 56.6 mOsm, p = 0.13 vs. control). Furthermore, MSCs reduced fibrosis and preserved the expression of proangiogenic molecules, while cyst size and number were unaltered by MSCs. Delivery of exogenous MSCs improved vascular density and renal function in PCK animals, and the benefit was observed up to 4 weeks after a single infusion. Cell-based therapy constitutes a novel approach in PKD. PMID:25290249

  7. Mesenchymal stromal cells in renal transplantation: opportunities and challenges.

    PubMed

    Casiraghi, Federica; Perico, Norberto; Cortinovis, Monica; Remuzzi, Giuseppe

    2016-04-01

    Lifelong immunosuppressive therapy is essential to prevent allograft rejection in transplant recipients. Long-term, nonspecific immunosuppression can, however, result in life-threatening complications and fail to prevent chronic graft rejection. Bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) have emerged as a potential candidate for cell-based therapy to modulate the immune response in organ transplantation. These cells can repair tissue after injury and downregulate many of the effector functions of immune cells that participate in the alloimmune response, converting them into regulatory cells. The findings of preclinical and initial clinical studies support the potential tolerance-inducing effects of MSCs and highlight the unanticipated complexity of MSC therapy in kidney transplantation. In animal models of transplantation MSCs promote donor-specific tolerance through the generation of regulatory T cells and antigen-presenting cells. In some settings, however, MSCs can acquire proinflammatory properties and contribute to allograft dysfunction. The available data from small clinical studies suggest that cell infusion is safe and well tolerated by kidney transplant recipients. Ongoing and future trials will provide evidence regarding the long-term safety of MSC therapy and determine the optimum cell source (either autologous or allogeneic) and infusion protocol to achieve operational tolerance in kidney transplant recipients. These studies will also provide additional evidence regarding the risks and benefits of MSC infusion and will hopefully offer definitive answers to the important questions of when, where, how many and which types of MSCs should be infused to fully exploit their immunomodulatory, pro-tolerogenic and tissue-repairing properties. PMID:26853275

  8. Identification of Factors Produced and Secreted by Mesenchymal Stromal Cells with the SILAC Method.

    PubMed

    Rocha, Beatriz; Calamia, Valentina; Blanco, Francisco J; Ruiz-Romero, Cristina

    2016-01-01

    Mesenchymal stromal cells (MSCs) secrete a large variety of proteins and factors, which shape the secretome. These proteins participate in multiple cellular functions, including the promotion of regenerative processes in the damaged tissue. Secretomes derived from either undifferentiated MSCs or these cells undergoing osteogenic, chondrogenic, or adipogenic differentiation have been characterized using different liquid chromatography tandem mass spectrometry (LC-MS/MS)-based quantitative proteomic approaches. In this chapter, we describe the use of the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) strategy for the identification and relative quantification of the mesenchymal stromal cell secretome, specifically during chondrogenesis. PMID:27236695

  9. Mesenchymal stromal cells support the viability and differentiation of thymocytes through direct contact in autologous co-cultures.

    PubMed

    Azghadi, Seyed Mohammad Reza; Suciu, Maria; Gruia, Alexandra Teodora; Barbu-Tudoran, Lucian; Cristea, Mirabela Iustina; Mic, Ani Aurora; Muntean, Danina; Nica, Dragos Vasile; Mic, Felix Aurel

    2016-08-01

    The development of thymocytes and generation of mature T cells is a complex process that requires spatio-temporal interactions of thymocytes with the other cells of the thymus microenvironment. Recently, mesenchymal stromal cells were isolated from the neonatal human thymus and differentiated into chondrogenic, osteogenic, and adipogenic lineages, just like their bone marrow counterparts. However, their function in thymocyte homeostasis is unknown. In our autologous co-cultures of rat mesenchymal stromal cells and thymocytes, the stromal cells preserve the viability of cultured thymocytes and stimulate the development of CD4-CD8- double-negative and the maturation of mainly CD4+ single-positive thymocytes. Thymocytes also influence the stemness of bone marrow mesenchymal stromal cells, as their expression of CD44, a marker associated with cellular proliferation and migration, is reduced in co-cultures. Mesenchymal stromal cells' influence on thymocyte development requires direct physical contact between the two cells and is not mediated by a soluble factor. When the two types of cells were physically separated, the stimulative effects of mesenchymal stromal cells on thymocytes did not occur. Electron microscopy confirmed the close contact between the membranes of thymocytes and mesenchymal stromal cells. Our experiments suggest that membrane exchanges could occur between mesenchymal stromal cells and thymocytes, such as the transfer of CD44 from mesenchymal stromal cells to the thymocytes, but its functional significance for thymocytes development remains to be established. These results suggest that mesenchymal stromal cells could normally be a part of the in vivo thymic microenvironment and form a niche that could sustain and guide the development of thymocytes. PMID:27085705

  10. Response of hemopoietic, progenitor, and multipotent mesenchymal stromal cells to administration of ketanserin during pulmonary fibrosis.

    PubMed

    Dygai, A M; Skurikhin, E G; Pershina, O V; Stepanova, I E; Khmelevskaya, E S; Ermakova, N N; Reztsova, A M; Krupin, V A; Reikhart, D V; Goldberg, V E

    2014-11-01

    We studied the effect of ketanserin on hemopoietic progenitor cells (Lin(-)Sca-1(+)c-Kit(+)CD34- and Lin(-)Sca-1(+)c-Kit(+)CD34(+)), progenitor hemopoietic cells (Lin(-)Sca-1(+)c-kit(+)), and multipotent mesenchymal stromal cells (CD45(-)CD73(+)CD106(+)) in C57Bl/6 mice during pulmonary fibrosis. It was shown that the blocker of 5-HT2A receptors lowers the activity of bleomycin-induced inflammation in the lungs and prevents the infiltration of alveolar interstitium and alveolar ducts by hemopoietic stem and hemopoietic progenitor cells; in this case, they are more numerous in the bone marrow of sick animals. Ketanserin reduces the capacity for self-renewal of lung multipotent mesenchymal stromal cells in the fibrotic phase of the disease and inhibits their differentiation into stromal cell lines (adipocytes, chondrocytes, and fibroblasts) simultaneously with the decrease in the percentage of connective tissue in the lung parenchyma. PMID:25403389

  11. Mesenchymal stromal cells transiently alter the inflammatory milieu post-transplant to delay graft-versus-host disease

    PubMed Central

    Christensen, Melinda E.; Turner, Brie E.; Sinfield, Laura J.; Kollar, Katarina; Cullup, Hannah; Waterhouse, Nigel J.; Hart, Derek N.J.; Atkinson, Kerry; Rice, Alison M.

    2010-01-01

    Background Multipotent mesenchymal stromal cells suppress T-cell function in vitro, a property that has underpinned their use in treating clinical steroid-refractory graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. However the potential of mesenchymal stromal cells to resolve graft-versus-host disease is confounded by a paucity of pre-clinical data delineating their immunomodulatory effects in vivo. Design and Methods We examined the influence of timing and dose of donor-derived mesenchymal stromal cells on the kinetics of graft-versus-host disease in two murine models of graft-versus-host disease (major histocompatibility complex-mismatched: UBI-GFP/BL6 [H-2b]→BALB/c [H-2d] and the sibling transplant mimic, UBI-GFP/BL6 [H-2b]→BALB.B [H-2b]) using clinically relevant conditioning regimens. We also examined the effect of mesenchymal stromal cell infusion on bone marrow and spleen cellular composition and cytokine secretion in transplant recipients. Results Despite T-cell suppression in vitro, mesenchymal stromal cells delayed but did not prevent graft-versus-host disease in the major histocompatibility complex-mismatched model. In the sibling transplant model, however, 30% of mesenchymal stromal cell-treated mice did not develop graft-versus-host disease. The timing of administration and dose of the mesenchymal stromal cells influenced their effectiveness in attenuating graft-versus-host disease, such that a low dose of mesenchymal stromal cells administered early was more effective than a high dose of mesenchymal stromal cells given late. Compared to control-treated mice, mesenchymal stromal cell-treated mice had significant reductions in serum and splenic interferon-γ, an important mediator of graft-versus-host disease. Conclusions Mesenchymal stromal cells appear to delay death from graft-versus-host disease by transiently altering the inflammatory milieu and reducing levels of interferon-γ. Our data suggest that both the

  12. Nkx2-5+Islet1+ mesenchymal precursors generate distinct spleen stromal cell subsets and participate in restoring stromal network integrity

    PubMed Central

    Castagnaro, Laura; Lenti, Elisa; Maruzzelli, Sara; Spinardi, Laura; Migliori, Edoardo; Farinello, Diego; Sitia, Giovanni; Harrelson, Zachary; Evans, Sylvia; Guidotti, Luca G.; Harvey, Richard P.; Brendolan, Andrea

    2013-01-01

    Lymphoid organ stromal cells comprise different subsets whose origin remains unknown. Herein, we exploit a genetic lineage-tracing approach to show that splenic fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), marginal reticular cells (MRCs), and mural cells, but not endothelial cells, originated from embryonic mesenchymal progenitors of the Nkx2-5+Islet1+ lineage. This lineage included embryonic mesenchymal cells with lymphoid tissue organizer (LTo) activity capable of supporting ectopic lymphoid-like structures, and a subset of resident spleen stromal cells that proliferated and regenerated the splenic stromal microenvironment following resolution of a viral infection. These findings identify progenitor cells that generate stromal diversity in spleen development and repair, and suggest the existence of multipotent stromal progenitors in the adult spleen with regenerative capacity. PMID:23601687

  13. Could cancer and infection be adverse effects of mesenchymal stromal cell therapy?

    PubMed Central

    Arango-Rodriguez, Martha L; Ezquer, Fernando; Ezquer, Marcelo; Conget, Paulette

    2015-01-01

    Multipotent mesenchymal stromal cells [also referred to as mesenchymal stem cells (MSCs)] are a heterogeneous subset of stromal cells. They can be isolated from bone marrow and many other types of tissue. MSCs are currently being tested for therapeutic purposes (i.e., improving hematopoietic stem cell engraftment, managing inflammatory diseases and regenerating damaged organs). Their tropism for tumors and inflamed sites and their context-dependent potential for producing trophic and immunomodulatory factors raises the question as to whether MSCs promote cancer and/or infection. This article reviews the effect of MSCs on tumor establishment, growth and metastasis and also susceptibility to infection and its progression. Data published to date shows a paradoxical effect regarding MSCs, which seems to depend on isolation and expansion, cells source and dose and the route and timing of administration. Cancer and infection may thus be adverse or therapeutic effects arising form MSC administration. PMID:25815124

  14. Effect of Vascular Endothelial Growth Factor and Erythropoietin on Functional Activity of Fibroblasts and Multipotent Mesenchymal Stromal Cells.

    PubMed

    Bondarenko, N A; Nikonorova, Yu V; Surovtseva, M A; Lykov, A P; Poveshchenko, O V; Poveshchenko, A F; Pokushalov, E A; Romanov, A B; Konenkov, V I

    2016-02-01

    The study examined the effect of VEGF and erythropoietin on proliferative and migratory activities of skin fibroblasts and multipotent mesenchymal stromal cells of human adipose tissue. VEGF stimulated proliferation and migration of fi broblasts, but produced no significant effect on functional activity of multipotent mesenchymal stem cells. Erythropoietin stimulated proliferation of both cell types, but did not affect their migration. PMID:26899850

  15. Understanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: the Mesenkillers

    PubMed Central

    Grisendi, Giulia; Bussolari, Rita; Veronesi, Elena; Piccinno, Serena; Burns, Jorge S; De Santis, Giorgio; Loschi, Pietro; Pignatti, Marco; Di Benedetto, Fabrizio; Ballarin, Roberto; Di Gregorio, Carmela; Guarneri, Valentina; Piccinini, Lino; Horwitz, Edwin M; Paolucci, Paolo; Conte, PierFranco; Dominici, Massimo

    2011-01-01

    A tumor represents a complex structure containing malignant cells strictly coupled with a large variety of surrounding cells constituting the tumor stroma (TS). In recent years, the importance of TS for cancer initiation, development, local invasion and metastases has become increasingly clear allowing the identification of TS as one of the possible ways to indirectly target tumors. Inside the heterogeneous stromal cell population, tumor associated fibroblasts (TAF) play a crucial role providing both functional and supportive environments. During both tumor and stroma development, several findings suggest that TAF could be recruited from different sources such as locally derived host fibroblasts, via epithelial/endothelial mesenchymal transitions or from circulating pools of fibroblasts deriving form mesenchymal progenitors, namely mesenchymal stem/stromal cells (MSC). These insights prompted scientists to identify multimodal approaches to target TS by biomolecules, monoclonal antibodies, and more recently via cell based strategies. These latter strategies appear extremely promising, although still associated with debated and unclear findings. This review discusses crosstalk between cancers and their stroma, dissecting specific tumor types, such as sarcoma, pancreatic and breast carcinoma, where stroma plays distinct paradigmatic roles. The recognition of these distinct stromal functions may help in planning effective and safer approaches aimed either to eradicate or to substitute TS by novel compounds and/or MSC having specific killing activities. PMID:22016827

  16. Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes☆

    PubMed Central

    Augusto, Lívia Maria Mendonça; Aguiar, Diego Pinheiro; Bonfim, Danielle Cabral; dos Santos Cavalcanti, Amanda; Casado, Priscila Ladeira; Duarte, Maria Eugênia Leite

    2016-01-01

    Objective This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. Methods Bovine tendons were used for preparation of the extract and were stored at −80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. Results The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. Conclusion Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes. PMID:26962503

  17. Multiple Functions of MSCA-1/TNAP in Adult Mesenchymal Progenitor/Stromal Cells

    PubMed Central

    Estève, David; Galitzky, Jean; Bouloumié, Anne; Fonta, Caroline; Buchet, René; Magne, David

    2016-01-01

    Our knowledge about mesenchymal stem cells has considerably grown in the last years. Since the proof of concept of the existence of such cells in the 70s by Friedenstein et al., a growing mass of reports were conducted for a better definition of these cells and for the reevaluation from the term “mesenchymal stem cells” to the term “mesenchymal stromal cells (MSCs).” Being more than a semantic shift, concepts behind this new terminology reveal the complexity and the heterogeneity of the cells grouped in MSC family especially as these cells are present in nearly all adult tissues. Recently, mesenchymal stromal cell antigen-1 (MSCA-1)/tissue nonspecific alkaline phosphatase (TNAP) was described as a new cell surface marker of MSCs from different tissues. The alkaline phosphatase activity of this protein could be involved in wide range of MSC features described below from cell differentiation to immunomodulatory properties, as well as occurrence of pathologies. The present review aims to decipher and summarize the role of TNAP in progenitor cells from different tissues focusing preferentially on brain, bone marrow, and adipose tissue. PMID:26839555

  18. O-GlcNAcylation Negatively Regulates Cardiomyogenic Fate in Adult Mouse Cardiac Mesenchymal Stromal Cells.

    PubMed

    Zafir, Ayesha; Bradley, James A; Long, Bethany W; Muthusamy, Senthilkumar; Li, Qianhong; Hill, Bradford G; Wysoczynski, Marcin; Prabhu, Sumanth D; Bhatnagar, Aruni; Bolli, Roberto; Jones, Steven P

    2015-01-01

    In both preclinical and clinical studies, cell transplantation of several cell types is used to promote repair of damaged organs and tissues. Nevertheless, despite the widespread use of such strategies, there remains little understanding of how the efficacy of cell therapy is regulated. We showed previously that augmentation of a unique, metabolically derived stress signal (i.e., O-GlcNAc) improves survival of cardiac mesenchymal stromal cells; however, it is not known whether enhancing O-GlcNAcylation affects lineage commitment or other aspects of cell competency. In this study, we assessed the role of O-GlcNAc in differentiation of cardiac mesenchymal stromal cells. Exposure of these cells to routine differentiation protocols in culture increased markers of the cardiomyogenic lineage such as Nkx2.5 and connexin 40, and augmented the abundance of transcripts associated with endothelial and fibroblast cell fates. Differentiation significantly decreased the abundance of O-GlcNAcylated proteins. To determine if O-GlcNAc is involved in stromal cell differentiation, O-GlcNAcylation was increased pharmacologically during the differentiation protocol. Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited. In addition, increasing O-GlcNAcylation further elevated smooth muscle actin expression. In addition to lineage commitment, we also evaluated proliferation and migration, and found that increasing O-GlcNAcylation did not significantly affect either; however, we found that O-GlcNAc transferase--the protein responsible for adding O-GlcNAc to proteins--is at least partially required for maintaining cellular proliferative and migratory capacities. We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function. O-GlcNAcylation and pathological conditions that may affect O-GlcNAc levels (such as diabetes) should be

  19. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

    PubMed Central

    Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

    2009-01-01

    Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA

  20. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles

    PubMed Central

    Chiabotto, Giulia; Bruno, Stefania; Collino, Federica

    2016-01-01

    Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs. PMID:27409796

  1. Rapid Selection of Mesenchymal Stem and Progenitor Cells in Primary Prostate Stromal Cultures

    PubMed Central

    Brennen, W. Nathaniel; Kisteman, L. Nelleke; Isaacs, John T.

    2016-01-01

    BACKGROUND Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (>25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. RESULTS MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 μm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when

  2. Leptin Receptor Promotes Adipogenesis and Reduces Osteogenesis by Regulating Mesenchymal Stromal Cells in Adult Bone Marrow.

    PubMed

    Yue, Rui; Zhou, Bo O; Shimada, Issei S; Zhao, Zhiyu; Morrison, Sean J

    2016-06-01

    Skeletal stem cells (SSCs) that are the major source of osteoblasts and adipocytes in adult bone marrow express leptin receptor (LepR). To test whether LepR regulates SSC function, we conditionally deleted Lepr from limb bone marrow stromal cells, but not from the axial skeleton or hypothalamic neurons, using Prx1-Cre. Prx1-Cre;Lepr(fl/fl) mice exhibited normal body mass and normal hematopoiesis. However, limb bones from Prx1-Cre;Lepr(fl/fl) mice exhibited increased osteogenesis, decreased adipogenesis, and accelerated fracture healing. Leptin increased adipogenesis and reduced osteogenesis by activating Jak2/Stat3 signaling in bone marrow stromal cells. A high-fat diet increased adipogenesis and reduced osteogenesis in limb bones from wild-type mice, but not from Prx1-Cre;Lepr(fl/fl) mice. This reflected local effects of LepR on osteogenesis and adipogenesis by bone marrow stromal cells and systemic effects on bone resorption. Leptin/LepR signaling regulates adipogenesis and osteogenesis by mesenchymal stromal cells in the bone marrow in response to diet and adiposity. PMID:27053299

  3. Identification of Meflin as a Potential Marker for Mesenchymal Stromal Cells

    PubMed Central

    Maeda, Keiko; Enomoto, Atsushi; Hara, Akitoshi; Asai, Naoya; Kobayashi, Takeshi; Horinouchi, Asuka; Maruyama, Shoichi; Ishikawa, Yuichi; Nishiyama, Takahiro; Kiyoi, Hitoshi; Kato, Takuya; Ando, Kenju; Weng, Liang; Mii, Shinji; Asai, Masato; Mizutani, Yasuyuki; Watanabe, Osamu; Hirooka, Yoshiki; Goto, Hidemi; Takahashi, Masahide

    2016-01-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) in culture are derived from BM stromal cells or skeletal stem cells. Whereas MSCs have been exploited in clinical medicine, the identification of MSC-specific markers has been limited. Here, we report that a cell surface and secreted protein, Meflin, is expressed in cultured MSCs, fibroblasts and pericytes, but not other types of cells including epithelial, endothelial and smooth muscle cells. In vivo, Meflin is expressed by immature osteoblasts and chondroblasts. In addition, Meflin is found on stromal cells distributed throughout the BM, and on pericytes and perivascular cells in multiple organs. Meflin maintains the undifferentiated state of cultured MSCs and is downregulated upon their differentiation, consistent with the observation that Meflin-deficient mice exhibit increased number of osteoblasts and accelerated bone development. In the bone and BM, Meflin is more highly expressed in primitive stromal cells that express platelet-derived growth factor receptor α and Sca-1 than the Sca-1-negative adipo-osteogenic progenitors, which create a niche for hematopoiesis. Those results are consistent with a decrease in the number of clonogenic colony-forming unit-fibroblasts within the BM of Meflin-deficient mice. These preliminary data suggest that Meflin is a potential marker for cultured MSCs and their source cells in vivo. PMID:26924503

  4. Identification of Meflin as a Potential Marker for Mesenchymal Stromal Cells.

    PubMed

    Maeda, Keiko; Enomoto, Atsushi; Hara, Akitoshi; Asai, Naoya; Kobayashi, Takeshi; Horinouchi, Asuka; Maruyama, Shoichi; Ishikawa, Yuichi; Nishiyama, Takahiro; Kiyoi, Hitoshi; Kato, Takuya; Ando, Kenju; Weng, Liang; Mii, Shinji; Asai, Masato; Mizutani, Yasuyuki; Watanabe, Osamu; Hirooka, Yoshiki; Goto, Hidemi; Takahashi, Masahide

    2016-01-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) in culture are derived from BM stromal cells or skeletal stem cells. Whereas MSCs have been exploited in clinical medicine, the identification of MSC-specific markers has been limited. Here, we report that a cell surface and secreted protein, Meflin, is expressed in cultured MSCs, fibroblasts and pericytes, but not other types of cells including epithelial, endothelial and smooth muscle cells. In vivo, Meflin is expressed by immature osteoblasts and chondroblasts. In addition, Meflin is found on stromal cells distributed throughout the BM, and on pericytes and perivascular cells in multiple organs. Meflin maintains the undifferentiated state of cultured MSCs and is downregulated upon their differentiation, consistent with the observation that Meflin-deficient mice exhibit increased number of osteoblasts and accelerated bone development. In the bone and BM, Meflin is more highly expressed in primitive stromal cells that express platelet-derived growth factor receptor α and Sca-1 than the Sca-1-negative adipo-osteogenic progenitors, which create a niche for hematopoiesis. Those results are consistent with a decrease in the number of clonogenic colony-forming unit-fibroblasts within the BM of Meflin-deficient mice. These preliminary data suggest that Meflin is a potential marker for cultured MSCs and their source cells in vivo. PMID:26924503

  5. [Nonadhesive populations in cultures of mesenchymal stromal cells from hematopoietic organs in mouse and rat].

    PubMed

    Byeverova, E I; Bragina, E V; Molchanova, E A

    2008-01-01

    The study of adhesive properties of multipotent mesenchymal stromal cells evaluated from fibroblast colony-forming units in the bone marrow of adult mice and rats in populations of cells attached and unattached to plastic substrate after 2 h to 7 days in culture demonstrated both similarities and differences. The increase in the fibroblast colony-forming units in the adhesive population peaked on day 7 of in vitro culture in both cases; however, nearly no fibroblast colony-forming units were observed in the nonadhesive population from the mouse bone marrow in this period. Conversely, the number of colonies from the rat bone marrow nonadhesive population on day 7 of culture considerably increased, and this nonadhesive population in long-term culture became the source for subsequent nonadhesive subpopulations containing fibroblast colony-forming units. After 7 days of in vitro culture, the suspension of cells isolated from the liver of 17-day-old rat fetuses also contained a fraction of unattached fibroblast colony-forming units. In the nonadhesive subpopulations from the bone marrow and fetal liver, fibroblast colony-forming units were observed up to day 48 and 30, respectively. Stromal cell precursors of nonadhesive subpopulations from the rat bone marrow featured a period of colony formation reduced to 7 days (i.e., they were formed 1.5-2 times faster compared to the primary culture). The total number of fibroblast colony-forming units from all nonadhesive subpopulations was roughly 6 and 7.4 times that of the adhesive population of the primary culture from the bone marrow and fetal liver, respectively. Considering that the mammalian bone marrow remains the preferred source of mesenchymal stromal cells, using nonadhesive subpopulations in the presented culture system can considerably increase the yield of stromal precursor cells. PMID:19137707

  6. Concise Review: Different Mesenchymal Stromal/Stem Cell Populations Reside in the Adult Kidney

    PubMed Central

    Bruno, Stefania; Chiabotto, Giulia

    2014-01-01

    During fetal life, mesenchymal stromal/stem cells (MSCs) surround glomeruli and tubules and contribute to the development of the renal interstitium by secretion of growth factors that drive nephron differentiation. In the adult, an MSC-like population has been demonstrated in different compartments of human and murine nephrons. After injury, these cells might provide support for kidney regeneration by recapitulating the role they have in embryonic life. In this short review, we discuss the evidence of an MSC presence within the adult kidney and their potential contribution to the turnover of renal cells and injury repair. PMID:25355731

  7. Isolation and Manufacture of Clinical-Grade Bone Marrow-Derived Human Mesenchymal Stromal Cells.

    PubMed

    Miller, Renuka P; Hanley, Patrick J

    2016-01-01

    Mesenchymal stromal cells (MSCs) are multipotent cells with both regenerative and immunomodulatory capacities. These unique properties make them appealing as a biologic, with multiple phase 1-3 clinical trials currently testing their safety and efficacy. Although expanding MSCs does not require extensive manipulation, expanding MSCs for use in clinical trials does require the knowledge and safety that are delineated in current good manufacturing practices (GMPs). Here we briefly detail the characteristics of MSCs and considerations for expanding them for clinical use. We then include a step-by-step protocol for expanding MSCs for early phase clinical trials, with important notes to consider during the expansion of these MSCs. PMID:27236680

  8. Mesenchymal Stromal Cells Expressing Heme Oxygenase-1 Reverse Pulmonary Hypertension

    PubMed Central

    Liang, Olin D.; Mitsialis, S. Alex; Chang, Mun Seog; Vergadi, Eleni; Lee, Changjin; Aslam, Muhammad; Fernandez-Gonzalez, Angeles; Liu, Xianlan; Baveja, Rajiv; Kourembanas, Stella

    2012-01-01

    Pulmonary arterial hypertension (PAH) remains a serious disease, and, while current treatments may prolong and improve quality of life, search for novel and effective therapies is warranted. Using genetically-modified mouse lines, we tested the ability of bone marrow-derived stromal cells (MSCs), to treat chronic hypoxia-induced PAH. Recipient mice were exposed for five weeks to normobaric hypoxia (8%–10% O2), MSC preparations were delivered through jugular vein injection and their effect on PAH was assessed after two additional weeks in hypoxia. Donor MSCs derived from wild-type (WT) mice or Heme Oxygenase-1 (HO-1) null mice (Hmox1KO) conferred partial protection from PAH when transplanted into WT or Hmox1KO recipients, whereas treatment with MSCs isolated from transgenic mice harboring a human HO-1 transgene under the control of surfactant protein C promoter (SHO1 line) reversed established disease in WT recipients. SH01-MSC treatment of Hmox1KO animals, which develop right ventricular (RV) infarction under prolonged hypoxia, resulted in normal RV systolic pressure, significant reduction of RV hypertrophy and prevention of RV infarction. Donor MSCs isolated from a bitransgenic mouse line with doxycycline-inducible, lung-specific expression of HO-1 exhibited similar therapeutic efficacy only upon doxycycline treatment of the recipients. In vitro experiments indicate that potential mechanisms of MSC action include modulation of hypoxia-induced lung inflammation and inhibition of smooth muscle cell proliferation. Cumulative, our results demonstrate that MSCs ameliorate chronic hypoxia – induced PAH and their efficacy is highly augmented by lung-specific HO-1 expression in the transplanted cells, suggesting an interplay between HO-1 dependent and HO-1 independent protective pathways. PMID:20957739

  9. Multipotent mesenchymal stromal cells: A promising strategy to manage alcoholic liver disease

    PubMed Central

    Ezquer, Fernando; Bruna, Flavia; Calligaris, Sebastián; Conget, Paulette; Ezquer, Marcelo

    2016-01-01

    Chronic alcohol consumption is a major cause of liver disease. The term alcoholic liver disease (ALD) refers to a spectrum of mild to severe disorders including steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. With limited therapeutic options, stem cell therapy offers significant potential for these patients. In this article, we review the pathophysiologic features of ALD and the therapeutic mechanisms of multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), based on their potential to differentiate into hepatocytes, their immunomodulatory properties, their potential to promote residual hepatocyte regeneration, and their capacity to inhibit hepatic stellate cells. The perfect match between ALD pathogenesis and MSC therapeutic mechanisms, together with encouraging, available preclinical data, allow us to support the notion that MSC transplantation is a promising therapeutic strategy to manage ALD onset and progression. PMID:26755858

  10. [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. PMID:26458460

  11. Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro.

    PubMed

    Mahabal, Shyam; Konala, Vijay Bhaskar Reddy; Mamidi, Murali Krishna; Kanafi, Mohammad Mahboob; Mishra, Suniti; Shankar, Krupa; Pal, Rajarshi; Bhonde, Ramesh

    2016-08-01

    Human skin has continuous self-renewal potential throughout adult life and serves as first line of defence. Its cellular components such as human epidermal keratinocytes (HEKs) and dermal mesenchymal stromal cells (DMSCs) are valuable resources for wound healing applications and cell based therapies. Here we show a simple, scalable and cost-effective method for sequential isolation and propagation of HEKs and DMSCs under defined culture conditions. Human skin biopsy samples obtained surgically were cut into fine pieces and cultured employing explant technique. Plated skin samples attached and showed outgrowth of HEKs. Gross microscopic examination displayed polygonal cells with a granular cytoplasm and H&E staining revealed archetypal HEK morphology. RT-PCR and immunocytochemistry authenticated the presence of key HEK markers including trans-membrane protein epithelial cadherin (E-cadherin), keratins and cytokeratin. After collection of HEKs by trypsin-EDTA treatment, mother explants were left intact and cultured further. Interestingly, we observed the appearance of another cell type with fibroblastic or stromal morphology which were able to grow up to 15 passages in vitro. Growth pattern, expression of cytoskeletal protein vimentin, surface proteins such as CD44, CD73, CD90, CD166 and mesodermal differentiation potential into osteocytes, adipocytes and chondrocytes confirmed their bonafide mesenchymal stem cell like status. These findings albeit preliminary may open up significant opportunities for novel applications in wound healing. PMID:25698160

  12. Data on nitric oxide production by human bone marrow-derived mesenchymal stromal cells.

    PubMed

    Najar, Mehdi; Fayyad-Kazan, Mohammad; Fayyad-Kazan, Hussein; Meuleman, Nathalie; Bron, Dominique; Lagneaux, Laurence

    2016-09-01

    Due to its anti-inflammatory and immunosuppressive potential, Nitric oxide (NO), a gaseous radical, is of special importance during graft-versus-host diseases (GVHD) and feoto-maternal tolerance. NO is a major mediator of murine mesenchymal stromal cells (MSCs)-immunosuppressive capacity. In this data article, we characterized NO production by human bone marrow-derived MSCs (hBMSCs). MSCs, isolated from healthy donors (n=5), were defined according to the International Society for cellular Therapy (ISCT) guidelines. Based on a fluorometric detection system, and upon using Nitrite ([Formula: see text])/Nitrate ( [Formula: see text]) Assay Kit, the amounts of NO metabolites ( [Formula: see text] and [Formula: see text]) produced by hBMSCs, being grown in a culture medium either lacking (constitutive condition) or containing IL-4, IL-10 or a pro-inflammatory cytokine cocktail made of IL-1β, TNF-α, IFN-α and IFN-γ, were assessed. All assays were carried out in triplicates and the mean values are reported. The data from this study supports and corroborates the discussion associated with our previously published work entitled "The Immunomodulatory Potential of Mesenchymal Stromal Cells: A Story of a Regulatory Network" (Najar et al., 2016) [1]. PMID:27536712

  13. Ultrastructural assessment of the differentiation potential of human multipotent mesenchymal stromal cells.

    PubMed

    Pasquinelli, Gianandrea; Valente, Sabrina

    2013-10-01

    Mesenchymal stromal (stem) cells (MSCs) are defined by plastic adherent growth, multiple phenotype expressions, and tripotential mesodermal capability. The authors report examples where electron microscopy (EM) plays a role in stem cell research. MSCs isolated from human arteries are ultrastructurally heterogeneous and become more homogenous after plastic adhesion. EM shows a moderate complement of organelles, mainly mitochondria, rough endoplasmic reticulum, and glycogen aggregates. Clear vacuoles and vesicles are prominent when cells are recovered from plates using an enzymatic method. Since the mesengenic plasticity is the single most important criterion to define a cell as mesenchymal stromal, the authors induced experimentally adipogenic, leiomyogenic, cardiomyogenic, osteo-chondrogenic differentiations. In no case did EM reveal the achievement of complete differentiation. The authors obtained multivacuolated pre-adipocytes and never univacuolated adipocytes typical of mature white fat; myofibroblast and rhabdomyoblast morphotypes, where contractile filaments were not organized to form functional complexes, i.e., dense bodies and sarcomeres. Chondrogenesis and osteogenesis assays resulted in extracellular matrix changes. Collagen and proteoglycan filament/particle deposition was seen when chondrogenesis was promoted. Hydroxyapatite crystals, psammoma bodies, and plaque-like calcified matrix deposits were found in the osteogenic matrix. EM provides detailed structural information on the degree of differentiation induced in stem cells and demonstrates that the methods so far developed are not able to promote complete cell differentiation. These observations contribute to explain why clinical applications with hMSCs have produced results far lower than initial expectations. PMID:24047349

  14. Effect of Initial Seeding Density on Human Umbilical Cord Mesenchymal Stromal Cells for Fibrocartilage Tissue Engineering

    PubMed Central

    Wang, Limin; Seshareddy, Kiran; Weiss, Mark L.

    2009-01-01

    Cells derived from Wharton's jelly from human umbilical cords (called umbilical cord mesenchymal stromal cells herein) are a novel cell source for musculoskeletal tissue engineering. In this study, we examined the effects of different seeding densities on seeding efficiency, cell proliferation, biosynthesis, mechanical integrity, and chondrogenic differentiation. Cells were seeded on non-woven polyglycolic acid (PGA) meshes in an orbital shaker at densities of 5, 25, or 50 million cells/mL and then statically cultured for 4 weeks in chondrogenic medium. At week 0, initial seeding density did not affect seeding efficiency. Throughout the 4-week culture period, absolute cell numbers of the 25 and 50 million-cells/mL (higher density) groups were significantly larger than in the 5 million-cells/mL (lower density) group. The presence of collagen types I and II and aggrecan was confirmed using immunohistochemical staining. Glycosaminoglycan and collagen contents per construct in the higher-density groups were significantly greater than in the lower-density group. Constructs in the high-density groups maintained their mechanical integrity, which was confirmed using unconfined compression testing. In conclusion, human umbilical cord cells demonstrated the potential for chondrogenic differentiation in three-dimensional tissue engineering, and higher seeding densities better promoted biosynthesis and mechanical integrity, and thus a seeding density of at least 25 million cells/mL is recommended for fibrocartilage tissue engineering with umbilical cord mesenchymal stromal cells. PMID:18759671

  15. Isolation, Culture, and Characterization of Human Umbilical Cord Blood-Derived Mesenchymal Stromal Cells.

    PubMed

    Bieback, Karen; Netsch, Philipp

    2016-01-01

    Umbilical cord blood (CB) is considered one of the youngest available sources of adult stem cells. Besides hematopoietic stem cells, CB has been shown to contain endothelial progenitor cells as well as mesenchymal stromal/stem cells (MSC). To isolate MSC from cord blood, CB is collected into a sterile bag containing the anticoagulant citrate-phosphate-dextrose (CPD). The CB is then processed by density-gradient centrifugation to obtain mononuclear cells (MNC). These are cultured until the outgrowth of fibroblastoid cell colonies appears. After reaching a subconfluent stage, cells are harvested, expanded, and characterized as cord blood mesenchymal stromal cells (CB-MSC) according to standard criteria: plastic adherence, fibroblast morphology, CFU-f assay, proliferation potential, immune phenotype, and differentiation potential.Apparently, the frequency of MSC in CB is extremely low. Thus, not every CB unit will provide adequate MSC isolation yields. Different strategies have been proposed aiming to optimize the isolation success by selecting CB units of optimal quality. It is commonly agreed on that a high CB volume, a high cellular content, and a short time frame between birth and MSC isolation are criteria that will enhance the MSC isolation success.The procedures in this chapter are standardized protocols that were established and optimized in the authors' research laboratory; however, various modifications of the protocols are possible. PMID:27236676

  16. Comparison between Stromal Vascular Fraction and Adipose Mesenchymal Stem Cells in Remodeling Hypertrophic Scars

    PubMed Central

    Maumus, Marie; Toupet, Karine; Frouin, Eric; Rigau, Valérie; Vozenin, Marie-Catherine; Magalon, Guy; Jorgensen, Christian; Noël, Danièle

    2016-01-01

    Hypertrophic scars (HTS) are characterized by excessive amount of collagen deposition and principally occur following burn injuries or surgeries. In absence of effective treatments, the use of mesenchymal stem/stromal cells, which have been shown to attenuate fibrosis in various applications, seems of interest. The objectives of the present study were therefore to evaluate the effect of human adipose tissue-derived mesenchymal stem cells (hASC) on a pre-existing HTS in a humanized skin graft model in Nude mice and to compare the efficacy of hASCs versus stromal vascular fraction (SVF). We found that injection of SVF or hASCs resulted in an attenuation of HTS as noticed after clinical evaluation of skin thickness, which was associated with lower total collagen contents in the skins of treated mice and a reduced dermis thickness after histological analysis. Although both SVF and hASCs were able to significantly reduce the clinical and histological parameters of HTS, hASCs appeared to be more efficient than SVF. The therapeutic effect of hASCs was attributed to higher expression of TGFβ3 and HGF, which are important anti-fibrotic mediators, and to higher levels of MMP-2 and MMP-2/TIMP-2 ratio, which reflect the remodelling activity responsible for fibrosis resorption. These results demonstrated the therapeutic potential of hASCs for clinical applications of hypertrophic scarring. PMID:27227960

  17. [The influence of extreme factors on homing multipotent mesenchymal stromal cells].

    PubMed

    Maklakova, I Yu; Grebnev, Y D; Yastrebov, A P

    2015-01-01

    In this study, we studied homing multipotent mesenchymal stromal cells under influence of extreme factors: after radiation exposure, acute blood loss. Absorbed dose ionizing radiation amounted to 4.0 C (causes acute radiation sickness in mice), acute blood loss was caused by bleeding from the tail vein of the mouse in the amount of 2% of the body weight of the animal. Label MMSC used fluorochrome DAPI, ready to use. The experiments were performed on 60 Mature mice (males) age 6-8 months, weighing 20-25 g. Experiments on the culture of multipotent mesenchymal stromal cells from the placenta (chorion) performed on laboratory mice female at the age of 3-4 months in the gestation period of 14 days. Introduction suspensions of MMSC was carried out at a dose of 6 million cells/mouse, suspended in 0.2 ml 0.9% NaCl solution. The control group of laboratory animals MMSC transplantation was carried out also in the amount of 6 million cells/mouse. The assessment was made of tissue chimerism in the peripheral blood, bone marrow, spleen, small intestine, liver, lung, kidney, heart after 1 and 24 hours after transplantation of labeled cells. It was found a significant decrease in the content of labeled MMSC in the peripheral blood at extreme impact, indicating a migration of the transplanted cells in the damaged tissue. Homing transplanted MMSC is realized mainly in those tissues that underwent the most damage. PMID:27116883

  18. Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes

    PubMed Central

    Li, Bojun; Menzel, Ursula; Loebel, Claudia; Schmal, Hagen; Alini, Mauro; Stoddart, Martin J.

    2016-01-01

    Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation. PMID:27198236

  19. Effect of spiperone on mesenchymal multipotent stromal and hemopoietic stem cells under conditions of pulmonary fibrosis.

    PubMed

    Skurikhin, E G; Khmelevskaya, E S; Ermakova, N N; Pershina, O V; Reztsova, A M; Krupin, V A; Stepanova, I E; Reztsova, V M; Reikhart, D V; Dygai, A M

    2014-05-01

    The antifibrotic properties of spiperone and its effect on stem and progenitor cells were studied on the model of reversible bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. Spiperone reduced infiltration of the alveolar interstitium and alveolar ducts with inflammatory cells and prevented the growth of the connective tissue in the parenchyma of bleomycin lungs. Apart from anti-inflammatory effect, spiperone suppressed bone marrow hemopoietic cells (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)-, Sca-1+, c-Kit+, CD34- and progenitor hemopoietic cells (granulocyte-erythroid-macrophage-megakaryocytic and granulocyte CFU). Spiperone-induced disturbances of fi brogenesis were paralleled by restoration of endothelial cells in the lung parenchyma, reduction of the number of circulating bone marrow cells and lung mesenchymopoietic cells (mesenchymal multipotent stromal cells (CD31-, CD34-, CD45-, CD44+, CD73+, CD90+, CD106+) and progenitor fi broblast cells), and suppression of multilineage differentiation of multipotent mesenchymal stromal cells (including fi broblast-lineage cells). PMID:24913578

  20. Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes.

    PubMed

    Li, Bojun; Menzel, Ursula; Loebel, Claudia; Schmal, Hagen; Alini, Mauro; Stoddart, Martin J

    2016-01-01

    Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation. PMID:27198236

  1. Functional differences in mesenchymal stromal cells from human dental pulp and periodontal ligament.

    PubMed

    Vasandan, Anoop Babu; Shankar, Shilpa Rani; Prasad, Priya; Sowmya Jahnavi, Vulugundam; Bhonde, Ramesh Ramachandra; Jyothi Prasanna, Susarla

    2014-02-01

    Clinically reported reparative benefits of mesenchymal stromal cells (MSCs) are majorly attributed to strong immune-modulatory abilities not exactly shared by fibroblasts. However, MSCs remain heterogeneous populations, with unique tissue-specific subsets, and lack of clear-cut assays defining therapeutic stromal subsets adds further ambiguity to the field. In this context, in-depth evaluation of cellular characteristics of MSCs from proximal oro-facial tissues: dental pulp (DPSCs) and periodontal ligament (PDLSCs) from identical donors provides an opportunity to evaluate exclusive niche-specific influences on multipotency and immune-modulation. Exhaustive cell surface profiling of DPSCs and PDLSCs indicated key differences in expression of mesenchymal (CD105) and pluripotent/multipotent stem cell-associated cell surface antigens: SSEA4, CD117, CD123 and CD29. DPSCs and PDLSCs exhibited strong chondrogenic potential, but only DPSCs exhibited adipogenic and osteogenic propensities. PDLSCs expressed immuno-stimulatory/immune-adhesive ligands like HLA-DR and CD50, upon priming with IFNγ, unlike DPSCs, indicating differential response patterns to pro-inflammatory cytokines. Both DPSCs and PDLSCs were hypo-immunogenic and did not elicit robust allogeneic responses despite exposure to IFNγ or TNFα. Interestingly, only DPSCs attenuated mitogen-induced lympho-proliferative responses and priming with either IFNγ or TNFα enhanced immuno-modulation capacity. In contrast, primed or unprimed PDLSCs lacked the ability to suppress polyclonal T cell blast responses. This study indicates that stromal cells from even topographically related tissues do not necessarily share identical MSC properties and emphasizes the need for a thorough functional testing of MSCs from diverse sources with respect to multipotency, immune parameters and response to pro-inflammatory cytokines before translational usage. PMID:24393246

  2. Failure of Intravenous or Intracardiac Delivery of Mesenchymal Stromal Cells to Improve Outcomes after Focal Traumatic Brain Injury in the Female Rat

    PubMed Central

    Turtzo, L. Christine; Budde, Matthew D.; Dean, Dana D.; Gold, Eric M.; Lewis, Bobbi K.; Janes, Lindsay; Lescher, Jacob; Coppola, Tiziana; Yarnell, Angela; Grunberg, Neil E.; Frank, Joseph A.

    2015-01-01

    Mesenchymal stromal cells secrete a variety of anti-inflammatory factors and may provide a regenerative medicine option for the treatment of traumatic brain injury. The present study investigates the efficacy of multiple intravenous or intracardiac administrations of rat mesenchymal stromal cells or human mesenchymal stromal cells in female rats after controlled cortical impact by in vivo MRI, neurobehavior, and histopathology evaluation. Neither intravenous nor intracardiac administration of mesenchymal stromal cells derived from either rats or humans improved MRI measures of lesion volume or neurobehavioral outcome compared to saline treatment. Few mesenchymal stromal cells (<0.0005% of injected dose) were found within 3 days of last dosage at the site of injury after either delivery route, with no mesenchymal stromal cells being detectable in brain at 30 or 56 days post-injury. These findings suggest that non-autologous mesenchymal stromal cells therapy via intravenous or intracardiac administration is not a promising treatment after focal contusion traumatic brain injury in this female rodent model. PMID:25946089

  3. Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy

    PubMed Central

    Sommariva, E.; Brambilla, S.; Carbucicchio, C.; Gambini, E.; Meraviglia, V.; Dello Russo, A.; Farina, F.M.; Casella, M.; Catto, V.; Pontone, G.; Chiesa, M.; Stadiotti, I.; Cogliati, E.; Paolin, A.; Ouali Alami, N.; Preziuso, C.; d'Amati, G.; Colombo, G.I.; Rossini, A.; Capogrossi, M.C.; Tondo, C.; Pompilio, G.

    2016-01-01

    Aim Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder mainly due to mutations in desmosomal genes, characterized by progressive fibro-adipose replacement of the myocardium, arrhythmias, and sudden death. It is still unclear which cell type is responsible for fibro-adipose substitution and which molecular mechanisms lead to this structural change. Cardiac mesenchymal stromal cells (C-MSC) are the most abundant cells in the heart, with propensity to differentiate into several cell types, including adipocytes, and their role in ACM is unknown. The aim of the present study was to investigate whether C-MSC contributed to excess adipocytes in patients with ACM. Methods and results We found that, in ACM patients' explanted heart sections, cells actively differentiating into adipocytes are of mesenchymal origin. Therefore, we isolated C-MSC from endomyocardial biopsies of ACM and from not affected by arrhythmogenic cardiomyopathy (NON-ACM) (control) patients. We found that both ACM and control C-MSC express desmosomal genes, with ACM C-MSC showing lower expression of plakophilin (PKP2) protein vs. controls. Arrhythmogenic cardiomyopathy C-MSC cultured in adipogenic medium accumulated more lipid droplets than controls. Accordingly, the expression of adipogenic genes was higher in ACM vs. NON-ACM C-MSC, while expression of cell cycle and anti-adipogenic genes was lower. Both lipid accumulation and transcription reprogramming were dependent on PKP2 deficiency. Conclusions Cardiac mesenchymal stromal cells contribute to the adipogenic substitution observed in ACM patients' hearts. Moreover, C-MSC from ACM patients recapitulate the features of ACM adipogenesis, representing a novel, scalable, patient-specific in vitro tool for future mechanistic studies. PMID:26590176

  4. Isolation and Characterisation of Mesenchymal Stem/Stromal Cells in the Ovine Endometrium

    PubMed Central

    Deane, James A.; Ulrich, Daniela; Gurung, Shanti; Ong, Y. Rue; Gargett, Caroline E.

    2015-01-01

    Objective Mesenchymal stem/stromal cells (MSC) were recently discovered in the human endometrium. These cells possess key stem cell properties and show promising results in small animal models when used for preclinical tissue engineering studies. A small number of surface markers have been identified that enrich for MSC from bone marrow and human endometrium, including the Sushi Domain-containing 2 (SUSD2; W5C5) and CD271 markers. In preparation for developing a large animal preclinical model for urological and gynecological tissue engineering applications we aimed to identify and characterise MSC in ovine endometrium and determine surface markers to enable their prospective isolation. Materials and Methods Ovine endometrium was obtained from hysterectomised ewes following progesterone synchronisation, dissociated into single cell suspensions and tested for MSC surface markers and key stem cell properties. Purified stromal cells were obtained by flow cytometry sorting with CD49f and CD45 to remove epithelial cells and leukocytes respectively, and MSC properties investigated. Results There was a small population CD271+ stromal cells (4.5 ± 2.3%) in the ovine endometrium. Double labelling with CD271 and CD49f showed that the sorted CD271+CD49f- stromal cell population possessed significantly higher cloning efficiency, serial cloning capacity and a qualitative increased ability to differentiate into 4 mesodermal lineages (adipocytic, smooth muscle, chondrocytic and osteoblastic) than CD271-CD49f- cells. Immunolabelling studies identified an adventitial perivascular location for ovine endometrial CD271+ cells. Conclusion This is the first study to characterise MSC in the ovine endometrium and identify a surface marker profile identifying their location and enabling their prospective isolation. This knowledge will allow future preclinical studies with a large animal model that is well established for pelvic organ prolapse research. PMID:25992577

  5. Multipotent mesenchymal stromal cell therapy in renal disease and kidney transplantation.

    PubMed

    Reinders, Marlies E J; Fibbe, Willem E; Rabelink, Ton J

    2010-01-01

    Cell therapies aim at differentiation of stem cells into the specific cell type required to repair damaged or destroyed cells or tissues. Over recent years, cell therapy has been introduced in a variety of application areas, including cardiovascular repair, diabetes, musculoskeletal disorders and renal repair. Multipotent mesenchymal stromal cells (MSCs), often referred to as mesenchymal stem cells, are of particular interest as a cell therapy model, as this is one of the few cell types that are on the brink of entering the clinical arena in different areas of application. MSCs can be differentiated in vitro and in vivo into various cell types of mesenchymal origin such as bone, fat and cartilage. They have important effects on the innate and adaptive immune system and possess striking anti-inflammatory properties that make them attractive for potential use in diseases characterized by autoimmunity and inflammation. In addition, MSCs have been shown to migrate to sites of tissue injury and to enhance repair by secreting anti-fibrotic and pro-angiogenic factors. In this review, evidence for the renoprotective mechanisms of MSCs as well as their therapeutic possibilities and potential hazards in acute and chronic renal disease and allograft rejection is summarized. PMID:19861311

  6. In Vitro Differentiation Potential of Human Placenta Derived Cells into Skin Cells

    PubMed Central

    Mahmood, Ruhma; Choudhery, Mahmood S.; Mehmood, Azra; Khan, Shaheen N.; Riazuddin, Sheikh

    2015-01-01

    Skin autografting is the most viable and aesthetic technique for treatment of extensive burns; however, this practice has potential limitations. Harvesting cells from neonatal sources (such as placental tissue) is a simple, inexpensive, and noninvasive procedure. In the current study authors sought to evaluate in vitro potential of human placenta derived stem cells to develop into skin-like cells. After extensive washing, amniotic membrane and umbilical cord tissue were separated to harvest amniotic epithelial cells (AECs) and umbilical cord mesenchymal stem cells (UC-MSCs), respectively. Both types of cells were characterized for the expression of embryonic lineage markers and their growth characteristics were determined. AECs and UC-MSCs were induced to differentiate into keratinocytes-like and dermal fibroblasts-like cells, respectively. After induction, morphological changes were detected by microscopy. The differentiation potential was further assessed using immunostaining and RT-PCR analyses. AECs were positive for cytokeratins and E-Cadherin while UC-MSCs were positive for fibroblast specific makers. AECs differentiated into keratinocytes-like cells showed positive expression of keratinocyte specific cytokeratins, involucrin, and loricrin. UC-MSCs differentiated into dermal fibroblast-like cells indicated expression of collagen type 3, desmin, FGF-7, fibroblast activation protein alpha, procollagen-1, and vimentin. In conclusion, placenta is a potential source of cells to develop into skin-like cells. PMID:26229539

  7. 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. PMID:26861667

  8. Mesenchymal Stromal Cells as Cell-Based Therapeutics for Wound Healing

    PubMed Central

    Malhotra, Samir; Hu, Michael S.; Marshall, Clement D.; Leavitt, Tripp; Cheung, Alexander T. M.; Gonzalez, Jennifer G.; Kaur, Harleen; Lorenz, H. Peter; Longaker, Michael T.

    2016-01-01

    Chronic wounds are a source of substantial morbidity for patients and are a major financial burden for the healthcare system. There are no current therapies that reliably improve nonhealing wounds or reverse pathological scarring. Mesenchymal stromal cells (MSCs) are a promising source of novel cell-based therapies due to the ease of their harvest and their integral role in the native wound repair process. Recent work has addressed the problems of loss of plasticity and off-target delivery through use of modern bioengineering techniques. Here we describe the applications of MSCs harvested from different sources to the wound healing process and recent advances in delivery of MSCs to targeted sites of injury. PMID:26966438

  9. Human gingiva-derived mesenchymal stromal cells contribute to periodontal regeneration in beagle dogs.

    PubMed

    Yu, Xinbo; Ge, Shaohua; Chen, Shulan; Xu, Quanchen; Zhang, Jin; Guo, Hongmei; Yang, Pishan

    2013-01-01

    Gingiva-derived mesenchymal stromal cells (GMSCs) have been considered as a promising alternative strategy for periodontal regeneration based on their potential for multilineage differentiation in vitro and the ability to form new bone in vivo. In order to investigate the capacity of GMSCs for periodontal regeneration and the fate of GMSCs during periodontal tissue repair, enhanced green fluorescent protein-labeled GMSCs were transplanted into class III furcation defects created in beagle dogs. The results showed that the transplanted GMSCs significantly enhanced the regeneration of the damaged periodontal tissue, including the alveolar bone, cementum and functional periodontal ligament (PDL). Moreover, GMSCs were able to differentiate into osteoblasts, cementoblasts and PDL fibroblasts in vivo. These findings indicate that GMSCs represent a novel cell source for periodontal tissue reconstruction. PMID:24777155

  10. Characterization and Angiogenic Potential of Human Neonatal and Infant Thymus Mesenchymal Stromal Cells

    PubMed Central

    Wang, Shuyun; Mundada, Lakshmi; Johnson, Sean; Wong, Joshua; Witt, Russell; Ohye, Richard G.

    2015-01-01

    Resident mesenchymal stromal cells (MSCs) are involved in angiogenesis during thymus regeneration. We have previously shown that MSCs can be isolated from enzymatically digested human neonatal and infant thymus tissue that is normally discarded during pediatric cardiac surgical procedures. In this paper, we demonstrate that thymus MSCs can also be isolated by explant culture of discarded thymus tissue and that these cells share many of the characteristics of bone marrow MSCs. Human neonatal thymus MSCs are clonogenic, demonstrate exponential growth in nearly 30 population doublings, have a characteristic surface marker profile, and express pluripotency genes. Furthermore, thymus MSCs have potent proangiogenic behavior in vitro with sprout formation and angiogenic growth factor production. Thymus MSCs promote neoangiogenesis and cooperate with endothelial cells to form functional human blood vessels in vivo. These characteristics make thymus MSCs a potential candidate for use as an angiogenic cell therapeutic agent and for vascularizing engineered tissues in vitro. PMID:25713463

  11. Characteristics of Multipotent Mesenchymal Stromal Cells Isolated from Human Endometrium and Endometriosis Lesions.

    PubMed

    Savilova, A M; Yushina, M N; Rudimova, Yu V; Khabas, G N; Chuprynin, V D; Sukhikh, G T

    2016-08-01

    Cell cultures isolated from endometriosis lesions by enzymatic dissociation consisted of fibroblast-like cells expressing CD90, CD73, and CD105; cell viability in these cultures was >90%, but this parameter decreased by passage 3. Zero passage cultures contained 10-25% epithelial cells expressing cytokeratin-7, but by passage 2, the cultures became more homogeneous and epithelial cells disappeared. The proportion of proliferating cells and population doubling level increased from passage 1 to passage 3. The cultures from the endometrium were induced to adipogenic and osteogenic differentiation in vitro. The cultures derived from ectopic endometrium have properties of multipotent mesenchymal stromal cells that exhibited in vitro similarities and differences from cell cultures from eutopic endometrium, which allows using this cell model for the search and testing of new drugs and technologies aimed at suppression of the growth and spread of endometriosis lesions. PMID:27590769

  12. In vivo therapeutic potential of mesenchymal stromal cells depends on the source and the isolation procedure.

    PubMed

    Bortolotti, Francesca; Ukovich, Laura; Razban, Vahid; Martinelli, Valentina; Ruozi, Giulia; Pelos, Barbara; Dore, Franca; Giacca, Mauro; Zacchigna, Serena

    2015-03-10

    Over the last several years, mesenchymal stromal cells (MSCs) have been isolated from different tissues following a variety of different procedures. Here, we comparatively assess the ex vivo and in vivo properties of MSCs isolated from either adipose tissue or bone marrow by different purification protocols. After MSC transplantation into a mouse model of hindlimb ischemia, clinical and histological analysis revealed that bone marrow MSCs purified on adhesive substrates exerted the best therapeutic activity, preserving tissue viability and promoting formation of new arterioles without directly transdifferentiating into vascular cells. In keeping with these observations, these cells abundantly expressed cytokines involved in vessel maturation and cell retention. These findings indicate that the choice of MSC source and purification protocol is critical in determining the therapeutic potential of these cells and warrant the standardization of an optimal MSC isolation procedure in order to select the best conditions to move forward to more effective clinical experimentation. PMID:25660405

  13. Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

    PubMed Central

    Pelekanos, Rebecca A.; Sardesai, Varda S.; Futrega, Kathryn; Lott, William B.; Kuhn, Michael; Doran, Michael R.

    2016-01-01

    Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use. PMID:27340821

  14. 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

  15. The potential of mesenchymal stromal cells as a novel cellular therapy for multiple sclerosis

    PubMed Central

    Auletta, Jeffery J; Bartholomew, Amelia M; Maziarz, Richard T; Deans, Robert J; Miller, Robert H; Lazarus, Hillard M; Cohen, Jeffrey A

    2012-01-01

    Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the CNS for which only partially effective therapies exist. Intense research defining the underlying immune pathophysiology is advancing both the understanding of MS as well as revealing potential targets for disease intervention. Mesenchymal stromal cell (MSC) therapy has the potential to modulate aberrant immune responses causing demyelination and axonal injury associated with MS, as well as to repair and restore damaged CNS tissue and cells. This article reviews the pathophysiology underlying MS, as well as providing a cutting-edge perspective into the field of MSC therapy based upon the experience of authors intrinsically involved in MS and MSC basic and translational science research. PMID:22642335

  16. Clinical Protocols for the Isolation and Expansion of Mesenchymal Stromal Cells

    PubMed Central

    Bieback, Karen; Schallmoser, Katharina; Klüter, Harald; Strunk, Dirk

    2008-01-01

    Summary Multipotent mesenchymal stromal cells (MSCs) are currently exploited in numerous clinical trials to investigate their potential in immune regulation, hematopoiesis, and tissue regeneration. The low frequency of MSCs necessitates cell expansion to achieve transplantable numbers. The challenge is to assure safe and high-quality cell production. GMP(Good Manufacturing Practice)-graded cell processing such as cell preparation, culture, and manipulation is mandatory for the progress of such advanced cell therapy. This review summarizes protocols to isolate MSCs from bone marrow and adipose tissue and to expand MSCs for clinical use focussing on culture media composition as well as culture devices and assays to ensure and control quality of the final product. PMID:21512644

  17. The therapeutic potential of bone marrow-derived mesenchymal stromal cells on hepatocellular carcinoma.

    PubMed

    Bayo, Juan; Marrodán, Mariano; Aquino, Jorge B; Silva, Marcelo; García, Mariana G; Mazzolini, Guillermo

    2014-03-01

    Mesenchymal stromal cells (MSCs) are more often obtained from adult and extraembryonic tissues, with the latter sources being likely better from a therapeutic perspective. MSCs show tropism towards inflamed or tumourigenic sites. Mechanisms involved in MSC recruitment into tumours are comprehensively analysed, including chemoattractant signalling axes, endothelial adhesion and transmigration. In addition, signals derived from hepatocellular carcinoma (HCC) tumour microenvironment and their influence in MSC tropism and tumour recruitment are dissected, as well as the present controversy regarding their influence on tumour growth and/or metastasis. Finally, evidences available on the use of MSCs and other selected progenitor/stem cells as vehicles of antitumourigenic genes are discussed. A better knowledge of the mechanisms involved in progenitor/stem cell recruitment to HCC tumours is proposed in order to enhance their tumour targeting which may result in improvements in cell-based gene therapy strategies. PMID:24112437

  18. The Control of Mesenchymal Stromal Cell Osteogenic Differentiation through Modified Surfaces

    PubMed Central

    2013-01-01

    Stem cells continue to receive widespread attention due to their potential to revolutionise treatments in the fields of both tissue engineering and regenerative medicine. Adult stem cells, specifically mesenchymal stromal cells (MSCs), play a vital role in the natural events surrounding bone healing and osseointegration through being stimulated to differentiate along their osteogenic lineage and in doing so, they form new cortical and trabecular bone tissue. Understanding how to control, manipulate, and enhance the intrinsic healing events modulated through osteogenic differentiation of MSCs by the use of modified surfaces and biomaterials could potentially advance the fields of both orthopaedics and dentistry. This could be by either using surface modification to generate greater implant stability and more rapid healing following implantation or the stimulation of MSCs ex vivo for reimplantation. This review aims to gather publications targeted at promoting, enhancing, and controlling the osteogenic differentiation of MSCs through biomaterials, nanotopographies, and modified surfaces for use in implant procedures. PMID:23766768

  19. Comparative Ability of Mesenchymal Stromal Cells from Different Tissues to Limit Neutrophil Recruitment to Inflamed Endothelium

    PubMed Central

    Munir, Hafsa; Luu, Nguyet-Thin; Clarke, Lewis S. C.; Nash, Gerard B.; McGettrick, Helen M.

    2016-01-01

    Mesenchymal stromal cells (MSC) are tissue-resident stromal cells capable of modulating immune responses, including leukocyte recruitment by endothelial cells (EC). However, the comparative potency of MSC from different sources in suppressing recruitment, and the necessity for close contact with endothelium remain uncertain, although these factors have implications for use of MSC in therapy. We thus compared the effects of MSC isolated from bone marrow, Wharton’s jelly, and trabecular bone on neutrophil recruitment to cytokine-stimulated EC, using co-culture models with different degrees of proximity between MSC and EC. All types of MSC suppressed neutrophil adhesion to inflamed endothelium but not neutrophil transmigration, whether directly incorporated into endothelial monolayers or separated from them by thin micropore filters. Further increase in the separation of the two cell types tended to reduce efficacy, although this diminution was least for the bone marrow MSC. Immuno-protective effects of MSC were also diminished with repeated passage; with BMMSC, but not WJMSC, completing losing their suppressive effect by passage 7. Conditioned media from all co-cultures suppressed neutrophil recruitment, and IL-6 was identified as a common bioactive mediator. These results suggest endogenous MSC have a homeostatic role in limiting inflammatory leukocyte infiltration in a range of tissues. Since released soluble mediators might have effects locally or remotely, infusion of MSC into blood or direct injection into target organs might be efficacious, but in either case, cross-talk between EC and MSC appears necessary. PMID:27171357

  20. Transcriptome analysis of bone marrow mesenchymal stromal cells from patients with primary myelofibrosis

    PubMed Central

    Martinaud, Christophe; Desterke, Christophe; Konopacki, Johanna; Vannucchi, Alessandro M.; Pieri, Lisa; Guglielmelli, Paola; Dupriez, Brigitte; Ianotto, Jean-Christophe; Boutin, Laetitia; Lataillade, Jean-Jacques; Le Bousse-Kerdilès, Marie-Caroline

    2015-01-01

    Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm whose severity and treatment complexity are attributed to the presence of bone marrow (BM) fibrosis and alterations of stroma impairing the production of normal blood cells. Despite the recently discovered mutations including the JAK2V617F mutation in about half of patients, the primitive event responsible for the clonal proliferation is still unknown. In the highly inflammatory context of PMF, the presence of fibrosis associated with a neoangiogenesis and an osteosclerosis concomitant to the myeloproliferation and to the increase number of circulating hematopoietic progenitors suggests that the crosstalk between hematopoietic and stromal cells is deregulated in the PMF BM microenvironmental niches. Within these niches, mesenchymal stromal cells (BM-MSC) play a hematopoietic supportive role in the production of growth factors and extracellular matrix which regulate the proliferation, differentiation, adhesion and migration of hematopoietic stem/progenitor cells. A transcriptome analysis of BM-MSC in PMF patients will help to characterize their molecular alterations and to understand their involvement in the hematopoietic stem/progenitor cell deregulation that features PMF. PMID:26484208

  1. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

    PubMed

    Bieback, Karen

    2013-10-01

    Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review. PMID:24273486

  2. Non-invasive characterization of the adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells by HS-SPME/GC-MS

    PubMed Central

    Lee, Dong-Kyu; Yi, TacGhee; Park, Kyung-Eun; Lee, Hyun-Joo; Cho, Yun-Kyoung; Lee, Seul Ji; Lee, Jeongmi; Park, Jeong Hill; Lee, Mi-Young; Song, Sun U.; Kwon, Sung Won

    2014-01-01

    A non-invasive method to characterize human mesenchymal stromal cells during adipogenic differentiation was developed for the first time. Seven fatty acid methyl esters (FAMEs), including methyl laurate, methyl myristate, methyl palmitate, methyl linoleate, methyl oleate, methyl elaidate and methyl stearate, were used for characterizing adipogenic differentiation using headspace solid-phase microextraction (HS-SPME) which is a very simple and non-invasive method for the extraction of volatile compounds. Glassware was used for culturing mesenchymal stromal cells rather than the common plasticware to minimize contamination by volatile impurities. The optimal SPME fiber was selected by comparing diverse fibers containing two pure liquid polymers (PDMS and PA) and two porous solids (PDMS/DVB and CAR/PDMS). Using optimized procedures, we discovered that seven FAMEs were only detected in adipogenic differentiated mesenchymal stromal cells and not in the mesenchymal stromal cells before differentiation. These data could support the quality control of clinical mesenchymal stromal cell culture in the pharmaceutical industry in addition to the development of many clinical applications using mesenchymal stromal cells. PMID:25298091

  3. Non-invasive characterization of the adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells by HS-SPME/GC-MS.

    PubMed

    Lee, Dong-Kyu; Yi, TacGhee; Park, Kyung-Eun; Lee, Hyun-Joo; Cho, Yun-Kyoung; Lee, Seul Ji; Lee, Jeongmi; Park, Jeong Hill; Lee, Mi-Young; Song, Sun U; Kwon, Sung Won

    2014-01-01

    A non-invasive method to characterize human mesenchymal stromal cells during adipogenic differentiation was developed for the first time. Seven fatty acid methyl esters (FAMEs), including methyl laurate, methyl myristate, methyl palmitate, methyl linoleate, methyl oleate, methyl elaidate and methyl stearate, were used for characterizing adipogenic differentiation using headspace solid-phase microextraction (HS-SPME) which is a very simple and non-invasive method for the extraction of volatile compounds. Glassware was used for culturing mesenchymal stromal cells rather than the common plasticware to minimize contamination by volatile impurities. The optimal SPME fiber was selected by comparing diverse fibers containing two pure liquid polymers (PDMS and PA) and two porous solids (PDMS/DVB and CAR/PDMS). Using optimized procedures, we discovered that seven FAMEs were only detected in adipogenic differentiated mesenchymal stromal cells and not in the mesenchymal stromal cells before differentiation. These data could support the quality control of clinical mesenchymal stromal cell culture in the pharmaceutical industry in addition to the development of many clinical applications using mesenchymal stromal cells. PMID:25298091

  4. The Murine Bladder Supports a Population of Stromal Sca-1+/CD34+/lin- Mesenchymal Stem Cells.

    PubMed

    Lilly, Meredith A; Kulkulka, Natalie A; Firmiss, Paula R; Ross, Michael J; Flum, Andrew S; Santos, Grace B Delos; Bowen, Diana K; Dettman, Robert W; Gong, Edward M

    2015-01-01

    Bladder fibrosis is an undesired end point of injury of obstruction and often renders the smooth muscle layer noncompliant. In many cases, the long-term effect of bladder fibrosis is renal failure. Despite our understanding of the progression of this disease, little is known about the cellular mechanisms that lead to a remodeled bladder wall. Resident stem (progenitor) cells have been identified in various organs such as the brain, heart and lung. These cells function normally during organ homeostasis, but become dysregulated after organ injury. Here, we aimed to characterize a mesenchymal progenitor cell population as a first step in understanding its role in bladder fibrosis. Using fluorescence activated cell sorting (FACS), we identified a Sca-1+/ CD34+/ lin- (PECAM-: CD45-: Ter119-) population in the adult murine bladder. These cells were localized to the stromal layer of the adult bladder and appeared by postnatal day 1. Cultured Sca-1+/ CD34+/ lin- bladder cells self-renewed, formed colonies and spontaneously differentiated into cells expressing smooth muscle genes. These cells differentiated into other mesenchymal lineages (chondrocytes, adipocytes and osteocytes) upon culture in induction medium. Both acute and partial obstruction of the bladder reduced expression of CD34 and changed localization of Sca-1 to the urothelium. Partial obstruction resulted in upregulation of fibrosis genes within the Sca-1+/CD34+/lin- population. Our data indicate a resident, mesenchymal stem cell population in the bladder that is altered by bladder obstruction. These findings provide new information about the cellular changes in the bladder that may be associated with bladder fibrosis. PMID:26540309

  5. The Murine Bladder Supports a Population of Stromal Sca-1+/CD34+/lin- Mesenchymal Stem Cells

    PubMed Central

    Lilly, Meredith A.; Kulkulka, Natalie A.; Firmiss, Paula R.; Ross, Michael J.; Flum, Andrew S.; Santos, Grace B. Delos; Bowen, Diana K.; Dettman, Robert W.; Gong, Edward M.

    2015-01-01

    Bladder fibrosis is an undesired end point of injury of obstruction and often renders the smooth muscle layer noncompliant. In many cases, the long-term effect of bladder fibrosis is renal failure. Despite our understanding of the progression of this disease, little is known about the cellular mechanisms that lead to a remodeled bladder wall. Resident stem (progenitor) cells have been identified in various organs such as the brain, heart and lung. These cells function normally during organ homeostasis, but become dysregulated after organ injury. Here, we aimed to characterize a mesenchymal progenitor cell population as a first step in understanding its role in bladder fibrosis. Using fluorescence activated cell sorting (FACS), we identified a Sca-1+/ CD34+/ lin- (PECAM-: CD45-: Ter119-) population in the adult murine bladder. These cells were localized to the stromal layer of the adult bladder and appeared by postnatal day 1. Cultured Sca-1+/ CD34+/ lin- bladder cells self-renewed, formed colonies and spontaneously differentiated into cells expressing smooth muscle genes. These cells differentiated into other mesenchymal lineages (chondrocytes, adipocytes and osteocytes) upon culture in induction medium. Both acute and partial obstruction of the bladder reduced expression of CD34 and changed localization of Sca-1 to the urothelium. Partial obstruction resulted in upregulation of fibrosis genes within the Sca-1+/CD34+/lin- population. Our data indicate a resident, mesenchymal stem cell population in the bladder that is altered by bladder obstruction. These findings provide new information about the cellular changes in the bladder that may be associated with bladder fibrosis. PMID:26540309

  6. Intra-Arterial Administration of Multipotent Mesenchymal Stromal Cells Promotes Functional Recovery of the Brain After Traumatic Brain Injury.

    PubMed

    Silachev, D N; Plotnikov, E Yu; Babenko, V A; Danilina, T I; Zorov, L D; Pevzner, I B; Zorov, D B; Sukhikh, G T

    2015-08-01

    We compared the efficiency of delivery of multipotent mesenchymal stem cells into the brain after their intravenous and intra-arterial injection. Analysis of the therapeutic effects of cells after experimental traumatic brain injury revealed improvement of the neurological status and motor functions of the damaged hemisphere, the effect being more pronounced after intraarterial injection of cells. Intra-arterial administration was followed by rapid infiltration of the cells into the brain tissue and their number considerably surpassed that after intravenous infusion. Targeted delivery of multipotent mesenchymal stromal cells into the brain after their injection into the carotid arteries substantially potentiated their neuroprotective effects in traumatic brain injury. PMID:26388566

  7. Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo.

    PubMed

    Chan, Michael C W; Kuok, Denise I T; Leung, Connie Y H; Hui, Kenrie P Y; Valkenburg, Sophie A; Lau, Eric H Y; Nicholls, John M; Fang, Xiaohui; Guan, Yi; Lee, Jae W; Chan, Renee W Y; Webster, Robert G; Matthay, Michael A; Peiris, J S Malik

    2016-03-29

    Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation. PMID:26976597

  8. Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo

    PubMed Central

    Chan, Michael C. W.; Kuok, Denise I. T.; Leung, Connie Y. H.; Hui, Kenrie P. Y.; Valkenburg, Sophie A.; Lau, Eric H. Y.; Nicholls, John M.; Fang, Xiaohui; Guan, Yi; Lee, Jae W.; Chan, Renee W. Y.; Webster, Robert G.; Matthay, Michael A.; Peiris, J. S. Malik

    2016-01-01

    Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium’s protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation. PMID:26976597

  9. Study on viability and chondrogenic differentiation of cryopreserved adipose tissue-derived mesenchymal stromal cells for future use in regenerative medicine.

    PubMed

    González-Fernández, M L; Pérez-Castrillo, S; Ordás-Fernández, P; López-González, M E; Colaço, B; Villar-Suárez, V

    2015-10-01

    Adipose-derived mesenchymal stromal cells are promising as a regenerative therapy tool for defective tissues in mesenchymal lineage, including fat, bone, cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation is an essential fundamental technology. The aim of this study is to define an adequate protocol for the cryopreservation of adipose-derived mesenchymal stromal cells, by comparing various protocols so as to determine the effects of cryopreservation on viability and chondrogenic differentiation potential of adipose-derived stem cells upon freeze-thawing of AT-MSCs colonies cryopreserved with standard and modified protocols, using flow cytometry and confocal microscopy. The study concludes that adipose-derived mesenchymal stromal cells could be long-term cryopreserved without any loss of their proliferative or differentiation potential. PMID:26209137

  10. Dermal Substitutes Support the Growth of Human Skin-Derived Mesenchymal Stromal Cells: Potential Tool for Skin Regeneration

    PubMed Central

    Jeremias, Talita da Silva; Machado, Rafaela Grecco; Visoni, Silvia Beatriz Coutinho; Pereima, Maurício José; Leonardi, Dilmar Francisco; Trentin, Andrea Gonçalves

    2014-01-01

    New strategies for skin regeneration are needed in order to provide effective treatment for cutaneous wounds and disease. Mesenchymal stem cells (MSCs) are an attractive source of cells for tissue engineering because of their prolonged self-renewal capacity, multipotentiality, and ability to release active molecules important for tissue repair. In this paper, we show that human skin-derived mesenchymal stromal cells (SD-MSCs) display similar characteristics to the multipotent MSCs. We also evaluate their growth in a three-dimensional (3D) culture system with dermal substitutes (Integra and Pelnac). When cultured in monolayers, SD-MSCs expressed mesenchymal markers, such as CD105, Fibronectin, and α-SMA; and neural markers, such as Nestin and βIII-Tubulin; at transcriptional and/or protein level. Integra and Pelnac equally supported the adhesion, spread and growth of human SD-MSCs in 3D culture, maintaining the MSC characteristics and the expression of multilineage markers. Therefore, dermal substitutes support the growth of mesenchymal stromal cells from human skin, promising an effective tool for tissue engineering and regenerative technology. PMID:24586857

  11. Bone Marrow Regeneration Promoted by Biophysically Sorted Osteoprogenitors From Mesenchymal Stromal Cells

    PubMed Central

    Poon, Zhiyong; Lee, Wong Cheng; Guan, Guofeng; Nyan, Lin Myint; Lim, Chwee Teck; Han, Jongyoon

    2015-01-01

    Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as “cell factories” that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies. PMID:25411477

  12. Regulation of mesenchymal stromal cells differentiation by a blue laser irradiation

    NASA Astrophysics Data System (ADS)

    Kushibiki, Toshihiro; Awazu, Kunio

    2007-07-01

    Mesenchymal stromal cells (MSCs) are multipotent cells, which are present in adult bone marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, and muscle. Their rapid and selective differentiation should provide the potential of new therapeutic approaches for the restoration of damaged or diseased tissue. However, several fundamental questions must be answered before it will be feasible to usefully predict and control MSCs responses to exogenous cytokines or genes. In particular, a better understanding of how specific factor may alter the fate of differentiation of MSCs is needed. In recent reports, circadian clock protein controls osteogenesis in vitro and in vivo. Here we show that a stimulation of a blue-violet laser irradiation regulates the differentiation of mouse MSCs to osteoblasts by change of the localization of a circadian rhythm protein, mouse Cryptochrome 1 (mCRY1). We found that a blue laser irradiation accelerated osteogenesis of MSCs. After laser irradiation, mCRY1 protein was translocated from cytoplasm to nucleus and mCRY1 mRNA level was downregulated thereafter. These results indicate that mCRY1, a blue-violet-light receptor and a master regulator of circadian rhythm, plays important roles in the regulation of the differentiation of MSCs. Since the differentiation of MSCs was easily regulated only by a laser irradiation, the potential of new therapeutic approaches for the restoration of damaged or diseased tissue is anticipated. Furthermore, our results obtained in this study may prove an excellent opportunity to gain insights into cross-talk between circadian rhythms and bone formation.

  13. Could donor multipotent mesenchymal stromal cells prevent or delay the onset of diabetic retinopathy?

    PubMed

    Ezquer, Fernando; Ezquer, Marcelo; Arango-Rodriguez, Martha; Conget, Paulette

    2014-03-01

    Diabetes mellitus is a complex metabolic disease that has become a global epidemic with more than 285 million cases worldwide. Major medical advances over the past decades have substantially improved its management, extending patients' survival. The latter is accompanied by an increased risk of developing chronic macro- and microvascular complications. Amongst them, diabetic retinopathy (DR) is the most common and frightening. Furthermore, during the past two decades, it has become the leading cause of visual loss. Irrespective of the type of diabetes, DR follows a well-known clinical and temporal course characterized by pericytes and neuronal cell loss, formation of acellular-occluded capillaries, occasional microaneurysms, increased leucostasis and thickening of the vascular basement membrane. These alterations progressively affect the integrity of retinal microvessels, leading to the breakdown of the blood-retinal barrier, widespread haemorrhage and neovascularization. Finally, tractional retinal detachment occurs leading to blindness. Nowadays, there is growing evidence that local inflammation and oxidative stress play pivotal roles in the pathogenesis of DR. Both processes have been associated with pericytes and neuronal degeneration observed early during DR progression. They may also be linked to sustained retinal vasculature damage that results in abnormal neovascularization. Currently, DR therapeutic options depend on highly invasive surgical procedures performed only at advanced stages of the disease, and which have proved to be ineffective to restore visual acuity. Therefore, the availability of less invasive and more effective strategies aimed to prevent or delay the onset of DR is highly desirable. Multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), are promising healing agents as they contribute to tissue regeneration by pleiotropic mechanisms, with no evidence of significant adverse events. Here, we revise the

  14. Human placenta-derived adherent cells improve cardiac performance in mice with chronic heart failure.

    PubMed

    Chen, Hong-Jung; Chen, Chien-Hsi; Chang, Ming-Yao; Tsai, Da-Ching; Baum, Ellen Z; Hariri, Robert; Herzberg, Uri; Hsieh, Patrick C H

    2015-03-01

    Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 10(6) cells per mouse), medium (0.5 × 10(5) cells per mouse), or low (0.5 × 10(4) cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect. PMID:25673767

  15. Bottlenecks in the Efficient Use of Advanced Therapy Medicinal Products Based on Mesenchymal Stromal Cells

    PubMed Central

    Escacena, Natalia; Quesada-Hernández, Elena; Capilla-Gonzalez, Vivian; Soria, Bernat; Hmadcha, Abdelkrim

    2015-01-01

    Mesenchymal stromal cells (MSCs) have been established as promising candidate sources of universal donor cells for cell therapy due to their contributions to tissue and organ homeostasis, repair, and support by self-renewal and multidifferentiation, as well as by their anti-inflammatory, antiproliferative, immunomodulatory, trophic, and proangiogenic properties. Various diseases have been treated by MSCs in animal models. Additionally, hundreds of clinical trials related to the potential benefits of MSCs are in progress. However, although all MSCs are considered suitable to exert these functions, dissimilarities have been found among MSCs derived from different tissues. The same levels of efficacy and desired outcomes have not always been achieved in the diverse studies that have been performed thus far. Moreover, autologous MSCs can be affected by the disease status of patients, compromising their use. Therefore, collecting information regarding the characteristics of MSCs obtained from different sources and the influence of the host (patient) medical conditions on MSCs is important for assuring the safety and efficacy of cell-based therapies. This review provides relevant information regarding factors to consider for the clinical application of MSCs. PMID:26273307

  16. Preclinical Studies with Umbilical Cord Mesenchymal Stromal Cells in Different Animal Models for Muscular Dystrophy

    PubMed Central

    Zucconi, Eder; Vieira, Natassia Moreira; Bueno, Carlos Roberto; Secco, Mariane; Jazedje, Tatiana; Costa Valadares, Marcos; Fussae Suzuki, Miriam; Bartolini, Paolo; Vainzof, Mariz; Zatz, Mayana

    2011-01-01

    Umbilical cord mesenchymal stromal cells (MSC) have been widely investigated for cell-based therapy studies as an alternative source to bone marrow transplantation. Umbilical cord tissue is a rich source of MSCs with potential to derivate at least muscle, cartilage, fat, and bone cells in vitro. The possibility to replace the defective muscle cells using cell therapy is a promising approach for the treatment of progressive muscular dystrophies (PMDs), independently of the specific gene mutation. Therefore, preclinical studies in different models of muscular dystrophies are of utmost importance. The main objective of the present study is to evaluate if umbilical cord MSCs have the potential to reach and differentiate into muscle cells in vivo in two animal models of PMDs. In order to address this question we injected (1) human umbilical cord tissue (hUCT) MSCs into the caudal vein of SJL mice; (2) hUCT and canine umbilical cord vein (cUCV) MSCs intra-arterially in GRMD dogs. Our results here reported support the safety of the procedure and indicate that the injected cells could engraft in the host muscle in both animal models but could not differentiate into muscle cells. These observations may provide important information aiming future therapy for muscular dystrophies. PMID:21785565

  17. Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells.

    PubMed

    González-Vázquez, Arlyng; Planell, Josep A; Engel, Elisabeth

    2014-06-01

    Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8-40mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca(2+)]o) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10mM of Ca(2+). Several experiments were conducted to demonstrate the effect of [Ca(2+)]o on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca(2+)]o induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10mM Ca(2+) stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca(2+). Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca(2+)]o, revealing that the CaSR is a key modulator of these cellular responses. PMID:24525034

  18. Tissue-specific Differentiation Potency of Mesenchymal Stromal Cells from Perinatal Tissues.

    PubMed

    Kwon, Ahlm; Kim, Yonggoo; Kim, Myungshin; Kim, Jiyeon; Choi, Hayoung; Jekarl, Dong Wook; Lee, Seungok; Kim, Jung Min; Shin, Jong-Chul; Park, In Yang

    2016-01-01

    Human perinatal tissue is an abundant source of mesenchymal stromal cells(MSCs) and lacks the ethical concerns. Perinatal MSCs can be obtained from various tissues as like amnion, chorion, and umbilical cord. Still, little is known of the distinct nature of each MSC type. In this study, we successfully isolated and cultured MSCs from amnion(AMSCs), chorion(CMSCs), and umbilical cord(UC-MSCs). Proliferation potential was different among them, that AMSCs revealed the lowest proliferation rate due to increased Annexin V and senescence-associated β-galactosidase positive cells. We demonstrated distinct characteristic gene expression according to the source of the original tissue using microarray. In particular, genes associated with apoptosis and senescence including CDKN2A were up-regulated in AMSCs. In CMSCs, genes associated with heart morphogenesis and blood circulation including HTR2B were up-regulated. Genes associated with neurological system processes including NPY were up-regulated in UC-MSCs. Quantitative RT-PCR confirmed the gene expression data. And in vitro differentiation of MSCs demonstrated that CMSCs and UC-MSCs had a more pronounced ability to differentiate into cardiomyocyte and neural cells, respectively. This study firstly demonstrated the innate tissue-specific differentiation potency of perinatal MSCs which can be helpful in choosing more adequate cell sources for better outcome in a specific disease. PMID:27045658

  19. Octanoate in Human Albumin Preparations Is Detrimental to Mesenchymal Stromal Cell Culture

    PubMed Central

    Wong, Way-Wua; MacKenzie, Andrew D.; Nelson, Vicky J.; Faed, James M.; Turner, Paul R.

    2015-01-01

    Cell therapies hold great promise as the next major advance in medical treatment. To enable safe, effective ex vivo culture whilst maintaining cell phenotype, growth media constituents must be carefully controlled. We have used a chemically defined mesenchymal stromal cell culture medium to investigate the influence of different preparations of human serum albumin. We examined two aspects of cell culture, growth rate as measured by population doubling time and colony forming ability which is a representative measure of the stemness of the cell population. Albumin preparations showed comparative differences in both of these criteria. Analysis of the albumin bound fatty acids also showed differences depending on the manufacturing procedure used. We demonstrated that octanoate, an additive used to stabilize albumin during pasteurization, slows growth and lowers colony forming ability during ex vivo culture. Further to this we also found the level of Na+/K+ ATPase, a membrane bound cation pump inhibited by octanoate, is increased in cells exposed to this compound. We conclude that the inclusion of human serum albumin in ex vivo growth media requires careful consideration of not only the source of albumin, but also the associated molecular cargo, for optimal cell growth and behavior. PMID:26074972

  20. Increased Paracrine Immunomodulatory Potential of Mesenchymal Stromal Cells in Three-Dimensional Culture.

    PubMed

    Follin, Bjarke; Juhl, Morten; Cohen, Smadar; Perdersen, Anders Elm; Kastrup, Jens; Ekblond, Annette

    2016-08-01

    Mesenchymal stromal/stem cells (MSCs) have been investigated extensively through the past years, proving to have great clinical therapeutic potential. In vitro cultivation of MSCs in three-dimensional (3D) culture systems, such as scaffolds, hydrogels, or spheroids, have recently gained attention for tissue engineering applications. Studies on MSC spheroids demonstrated that such cultivation increased the paracrine immunomodulatory potential of the MSCs, accompanied by phenotypic alterations. In this review, we gather results from recent experimental studies on the immunomodulatory abilities of MSCs when cultured as spheroids or in biomaterials like scaffolds or hydrogels compared to regular two-dimensional (2D) culture and show that alterations occurring to MSCs in spheroids also occur in MSCs in biomaterials. We provide a brief description of known mechanisms of MSC immunomodulatory capacity and how they are altered in the two 3D culture systems, together with phenotypic cellular changes. Based on the present knowledge, we highlight vital areas in need of further investigation. The impact of 3D environments on immunomodulation has great potential for tissue engineering and cellular therapy, and this is the first review to gather this knowledge with a comparison across different 3D environments. PMID:26861485

  1. Concise review: optimizing expansion of bone marrow mesenchymal stem/stromal cells for clinical applications.

    PubMed

    Hoch, Allison I; Leach, J Kent

    2014-05-01

    Bone marrow-derived mesenchymal stem/stromal cells (MSCs) have demonstrated success in the clinical treatment of hematopoietic pathologies and cardiovascular disease and are the focus of treating other diseases of the musculoskeletal, digestive, integumentary, and nervous systems. However, during the requisite two-dimensional (2D) expansion to achieve a clinically relevant number of cells, MSCs exhibit profound degeneration in progenitor potency. Proliferation, multilineage potential, and colony-forming efficiency are fundamental progenitor properties that are abrogated by extensive monolayer culture. To harness the robust therapeutic potential of MSCs, a consistent, rapid, and minimally detrimental expansion method is necessary. Alternative expansion efforts have exhibited promise in the ability to preserve MSC progenitor potency better than the 2D paradigm by mimicking features of the native bone marrow niche. MSCs have been successfully expanded when stimulated by growth factors, under reduced oxygen tension, and in three-dimensional bioreactors. MSC therapeutic value can be optimized for clinical applications by combining system inputs to tailor culture parameters for recapitulating the niche with probes that nondestructively monitor progenitor potency. The purpose of this review is to explore how modulations in the 2D paradigm affect MSC progenitor properties and to highlight recent efforts in alternative expansion techniques. PMID:24682286

  2. Ferrociphenol lipid nanocapsule delivery by mesenchymal stromal cells in brain tumor therapy.

    PubMed

    Roger, Mathilde; Clavreul, Anne; Huynh, Ngoc Trinh; Passirani, Catherine; Schiller, Paul; Vessières, Anne; Montero-Menei, Claudia; Menei, Philippe

    2012-02-14

    The prognosis of patients with malignant glioma remains extremely poor despite surgery and improvements in radio- and chemo-therapies. Thus, treatment strategies that specifically target these tumors have the potential to greatly improve therapeutic outcomes. "Marrow-isolated adult multilineage inducible" cells (MIAMI cells) are a subpopulation of mesenchymal stromal cells (MSCs) which possess the ability to migrate to brain tumors. We have previously shown that MIAMI cells were able to efficiently incorporate lipid nanocapsules (LNCs) without altering either their stem cell properties or their migration capacity. In this study, we assessed whether the cytotoxic effects of MIAMI cells loaded with LNCs containing an organometallic complex (ferrociphenol or Fc-diOH) could be used to treat brain tumors. The results showed that MIAMI cells internalized Fc-diOH-LNCs and that this internalization did not induce MIAMI cell death. Furthermore, Fc-diOH-LNC-loaded MIAMI cells produced a cytotoxic effect on U87MG glioma cells in vitro. This cytotoxic effect was validated in vivo after intratumoral injection of Fc-diOH-LNC-loaded MIAMI cells in a heterotopic U87MG glioma model in nude mice. These promising results open up a new field of treatment in which cellular vehicles and nanoparticles can be combined to treat brain tumors. PMID:21554935

  3. Zoledronic acid in vivo increases in vitro proliferation of rat mesenchymal stromal cells.

    PubMed

    Heino, Terhi J; Alm, Jessica J; Halkosaari, Heikki J; Välimäki, Ville-Valtteri

    2016-08-01

    Background and purpose - Bisphosphonates are widely used in the treatment of bone loss, but they might also have positive effects on osteoblastic cells and bone formation. We evaluated the effect of in vivo zoledronic acid (ZA) treatment and possible concomitant effects of ZA and fracture on the ex vivo osteogenic capacity of rat mesenchymal stromal cells (MSCs). Methods - A closed femoral fracture model was used in adult female rats and ZA was administered as a single bolus or as weekly doses up to 8 weeks. Bone marrow MSCs were isolated and cultured for in vitro analyses. Fracture healing was evaluated by radiography, micro-computed tomography (μCT), and histology. Results - Both bolus and weekly ZA increased fracture-site bone mineral content and volume. MSCs from weekly ZA-treated animals showed increased ex vivo proliferative capacity, while no substantial effect on osteoblastic differentiation was observed. Fracture itself did not have any substantial effect on cell proliferation or differentiation at 8 weeks. Serum biochemical markers showed higher levels of bone formation in animals with fracture than in intact animals, while no difference in bone resorption was observed. Interestingly, ex vivo osteoblastic differentiation of MSCs was found to correlate with in vivo serum bone markers. Interpretation - Our data show that in vivo zoledronic acid treatment can influence ex vivo proliferation of MSCs, indicating that bisphosphonates can have sustainable effects on cells of the osteoblastic lineage. Further research is needed to investigate the mechanisms. PMID:27196705

  4. Human mesenchymal stromal cell proteomics: contribution for identification of new markers and targets for medicine intervention.

    PubMed

    Faça, Vitor Marcel

    2012-04-01

    Mesenchymal stem or stromal cells (MSCs) have become of great interest for cell-based therapy owing to their roles in tissue repair and immune suppression. MSCs have the ability to differentiate into specialized tissues, including bone, cartilage and muscle, among several others. Furthermore, it has been found that MSCs can also serve as cellular factories that secrete mediators to stimulate in situ regeneration of injured tissues. Proteomics has contributed significantly to the identification of new proteins to improve cellular characterization of MSCs, to identify new targets for therapeutic intervention and to elucidate important pathways utilized by MSCs to differentiate into distinct tissues. As proteomics technology advances, several studies can be revisited and analyzed in depth, employing state-of-the-art approaches, helping to uncover the cellular mechanisms utilized by MSCs to exert their regenerative functionalities. In this article, we will review the progress made so far and discuss further opportunities for proteomics to contribute to the clinical applications of MSCs. PMID:22462791

  5. Mechanisms of tumor escape from immune system: role of mesenchymal stromal cells.

    PubMed

    Poggi, Alessandro; Musso, Alessandra; Dapino, Irene; Zocchi, Maria Raffaella

    2014-01-01

    Tumor microenvironment represents the site where the tumor tries to survive and escape from immune system-mediated recognition. Indeed, to proliferate tumor cells can divert the immune response inducing the generation of myeloid derived suppressor cells and regulatory T cells which can limit the efficiency of effector antitumor lymphocytes in eliminating neoplastic cells. Many components of the tumor microenvironment can serve as a double sword for the tumor and the host. Several types of fibroblast-like cells, which herein we define mesenchymal stromal cells (MSC), secrete extracellular matrix components and surrounding the tumor mass can limit the expansion of the tumor. On the other hand, MSC can interfere with the immune recognition of tumor cells producing immunoregulatory cytokines as transforming growth factor (TGF)ß, releasing soluble ligands of the activating receptors expressed on cytolytic effector cells as decoy molecules, affecting the correct interaction among lymphocytes and tumor cells. MSC can also serve as target for the same anti-tumor effector lymphocytes or simply impede the interaction between these lymphocytes and neoplastic cells. Thus, several evidences point out the role of MSC, both in epithelial solid tumors and hematological malignancies, in regulating tumor cell growth and immune response. Herein, we review these evidences and suggest that MSC can be a suitable target for a more efficient anti-tumor therapy. PMID:24657523

  6. Evaluation of epithelial chimerism after bone marrow mesenchymal stromal cell infusion in intestinal transplant patients.

    PubMed

    Kilinc, S; Gurkan, U A; Guven, S; Koyuncu, G; Tan, S; Karaca, C; Ozdogan, O; Dogan, M; Tugmen, C; Pala, E E; Bayol, U; Baran, M; Kurtulmus, Y; Pirim, I; Kebapci, E; Demirci, U

    2014-01-01

    Intestinal transplantation is the most effective treatment for patients with short bowel syndrome and small bowel insufficiencies. We evaluated epithelial chimerism after infusion of autologous bone marrow mesenchymal stromal cells (BMSCs) in patients undergoing cadaveric donor isolated intestinal transplantation (I-ITx). BMSCs were isolated from patients' bone marrow via iliac puncture and expanded in vitro prior to infusion. Two out of the 3 patients were infused with autologous BMSCs, and small intestine tissue biopsies collected post-operatively were analyzed for epithelial chimerism using XY fluorescent in situ hybridization and short tandem repeat polymerase chain reaction. We observed epithelial chimeric effect in conditions both with and without BMSC infusion. Although our results suggest a higher epithelial chimerism effect with autologous BMSC infusion in I-ITx, the measurements in multiple biopsies at different time points that demonstrate the reproducibility of this finding and its stability or changes in the level over time would be beneficial. These approaches may have potential implications for improved graft survival, lower immunosuppressant doses, superior engraftment of the transplanted tissue, and higher success rates in I-ITx. PMID:25131122

  7. Intravenous Transplantation of Mesenchymal Stromal Cells to Enhance Peripheral Nerve Regeneration

    PubMed Central

    Matthes, Stella M.; Reimers, Kerstin; Janssen, Insa; Kocsis, Jeffery D.; Vogt, Peter M.; Radtke, Christine

    2013-01-01

    Peripheral nerve injury is a common and devastating complication after trauma and can cause irreversible impairment or even complete functional loss of the affected limb. While peripheral nerve repair results in some axonal regeneration and functional recovery, the clinical outcome is not optimal and research continues to optimize functional recovery after nerve repair. Cell transplantation approaches are being used experimentally to enhance regeneration. Intravenous infusion of mesenchymal stromal cells (MSCs) into spinal cord injury and stroke was shown to improve functional outcome. However, the repair potential of intravenously transplanted MSCs in peripheral nerve injury has not been addressed yet. Here we describe the impact of intravenously infused MSCs on functional outcome in a peripheral nerve injury model. Rat sciatic nerves were transected followed, by intravenous MSCs transplantation. Footprint analysis was carried out and 21 days after transplantation, the nerves were removed for histology. Labelled MSCs were found in the sciatic nerve lesion site after intravenous injection and regeneration was improved. Intravenously infused MSCs after acute peripheral nerve target the lesion site and survive within the nerve and the MSC treated group showed greater functional improvement. The results of study suggest that nerve repair with cell transplantation could lead to greater functional outcome. PMID:24459671

  8. [The influence of low partial oxygen pressure on the biolodical process of mesenchymal stromal cells].

    PubMed

    Berezovskyĭ, V Ia; Plotnikova, L M; Vesel'skyĭ, S P; Litovka, I H

    2014-01-01

    The influence of low partial oxygen pressure (Po2) on the amino acid composition in culture medium of human mesenchymal stromal cell (MSC) lines 4BL has been studied. At 23 mm Hg (3% oxygen), a significant decrease (by 31%) in the concentration of proline and hydroxyproline was registered. Under these conditions, the concentration of serine and aspartic acid decreased by 45% compared to the control. Maximum consumption of free amino acids from the culture medium required for the synthesis of collagen (proline and hydroxyproline by 42%, serine and aspartic acid by 62%) was observed at a gas-phase Po2 of 38 mm Hg (5% O2). At Po2 76 mm Hg (10% O2), a lack of amino acids proline and hydroxyproline was only 21%, while that of glutamine and alanine amounted 12% compared to the control. This intensity ratio of consumption of amino acids may indicate that the maximum of MSC vital functions occurs at Po2 38 mm Hg. PMID:25095672

  9. Heterogeneous functions of perinatal mesenchymal stromal cells require a preselection before their banking for clinical use.

    PubMed

    Peltzer, Juliette; Montespan, Florent; Thepenier, Cédric; Boutin, Laetitia; Uzan, Georges; Rouas-Freiss, Nathalie; Lataillade, Jean-Jacques

    2015-02-01

    Perinatal sources of mesenchymal stromal cells (MSCs) have raised growing interest because they are readily and widely available with minimal ethical/legal issues and can easily be stored for allogeneic settings. In addition, perinatal tissues are known to be important in mediating the fetomaternal tolerance of pregnancy, which confer upon perinatal-MSCs (P-MSCs) a particular interest in immunomodulation. It has been recently shown that it is possible to deeply modify the secreted factor profiles of MSCs with different cytokine stimuli such as interferon gamma or tumor necrosis factor alpha to license MSCs for a better immunosuppresive potential. Therefore, we aimed to compare adult bone marrow-MSCs with MSCs from perinatal tissues (cord blood, umbilical cord, amnion, and chorion) on their in vitro immunological and stromacytic efficiencies under different priming conditions. Our results showed that P-MSCs had a potential to modulate the in vitro immune response and be useful for hematopoietic progenitor cell ex vivo expansion. However, we showed contrasted effects of cytokine priming embedded in an important between-donor variability. In conclusion, our study highlights the importance to elaborate predicitive in vitro tests to screen between-donor variability of perinatal tissues for banking allogeneic standardized MSCs. PMID:25203666

  10. Delivery of improved oncolytic adenoviruses by mesenchymal stromal cells for elimination of tumorigenic pancreatic cancer cells

    PubMed Central

    Kaczorowski, Adam; Hammer, Katharina; Liu, Li; Villhauer, Sabine; Nwaeburu, Clifford; Fan, Pei; Zhao, Zhefu; Gladkich, Jury; Groß, Wolfgang; Nettelbeck, Dirk M.; Herr, Ingrid

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies and has poor therapeutic options. We evaluated improved oncolytic adenoviruses (OAds), in which the adenoviral gene E1B19K was deleted or a TRAIL transgene was inserted. Bone marrow mesenchymal stromal cells (MSCs) served as carriers for protected and tumor-specific virus transfers. The infection competence, tumor migration, and oncolysis were measured in cancer stem cell (CSC) models of primary and established tumor cells and in tumor xenografts. All OAds infected and lysed CSCs and prevented colony formation. MSCs migrated into PDA spheroids without impaired homing capacity. Xenotransplantation of non-infected PDA cells mixed with infected tumor cells strongly reduced the tumor volume and the expression of the proliferation marker Ki67 along with a necrotic morphology. Adenoviral capsid protein was detected in tumor xenograft tissue after intravenous injection of infected MSCs, but not in normal tissue, implying tumor-specific migration. Likewise, direct in vivo treatment correlated with a strongly reduced tumor volume, lower expression of Ki67 and CD24, and enhanced activity of caspase 3. These data demonstrate that the improved OAds induced efficient oncolysis with the OAd-TRAIL as most promising candidate for future clinical application. PMID:26824985

  11. Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?

    PubMed Central

    De Becker, Ann; Riet, Ivan Van

    2016-01-01

    Mesenchymal stromal cells (MSCs) are currently being investigated for use in a wide variety of clinical applications. For most of these applications, systemic delivery of the cells is preferred. However, this requires the homing and migration of MSCs to a target tissue. Although MSC homing has been described, this process does not appear to be highly efficacious because only a few cells reach the target tissue and remain there after systemic administration. This has been ascribed to low expression levels of homing molecules, the loss of expression of such molecules during expansion, and the heterogeneity of MSCs in cultures and MSC culture protocols. To overcome these limitations, different methods to improve the homing capacity of MSCs have been examined. Here, we review the current understanding of MSC homing, with a particular focus on homing to bone marrow. In addition, we summarize the strategies that have been developed to improve this process. A better understanding of MSC biology, MSC migration and homing mechanisms will allow us to prepare MSCs with optimal homing capacities. The efficacy of therapeutic applications is dependent on efficient delivery of the cells and can, therefore, only benefit from better insights into the homing mechanisms. PMID:27022438

  12. The molecular signature of AML mesenchymal stromal cells reveals candidate genes related to the leukemogenic process.

    PubMed

    Binato, Renata; de Almeida Oliveira, Nathalia Correa; Du Rocher, Barbara; Abdelhay, Eliana

    2015-12-01

    Acute myeloid leukemia (AML) is a heterogeneous disease characterized by myeloid precursor proliferation in the bone marrow, apoptosis reduction and differentiation arrest. Although there are several studies in this field, events related to disease initiation and progression remain unknown. The malignant transformation of hematopoietic stem cells (HSC) is thought to generate leukemic stem cells, and this transformation could be related to changes in mesenchymal stromal cell (hMSC) signaling. Thus, the aim of this work was to analyze the gene expression profile of hMSC from AML patients (hMSC-AML) compared to healthy donors hMSCs (hMSC-HD). The results showed a common molecular signature for all hMSC-AML. Other assays were performed with a large number of patients and the results confirmed a molecular signature that is capable of distinguishing hMSC-AML from hMSC-HD. Moreover, CCL2 and BMP4 genes encode secreted proteins that could affect HSCs. To verify whether these proteins are differentially expressed in AML patients, ELISA was performed with plasma samples. CCL2 and BMP4 proteins are differentially expressed in AML patients, indicating changes in hMSC-AML signaling. Altogether, hMSCs-AML signaling alterations could be an important factor in the leukemic transformation process. PMID:26279521

  13. Three dimensional alginate-fucoidan composite hydrogel augments the chondrogenic differentiation of mesenchymal stromal cells.

    PubMed

    Karunanithi, Puvanan; Murali, Malliga Raman; Samuel, Shani; Raghavendran, Hanumanantha Rao Balaji; Abbas, Azlina Amir; Kamarul, Tunku

    2016-08-20

    Presence of sulfated polysaccharides like heparan sulphate has often been implicated in the regulation of chondrogenesis. However, recently there has been a plethora of interest in the use of non-animal extracted analogs of heparan sulphate. Here we remodeled alginate (1.5%) by incorporating fucoidan (0.5%), a natural sulphated polysaccharide extracted from seaweeds to form a composite hydrogel (Al-Fu), capable of enhancing chondrogenesis of human mesenchymal stromal cells (hMSCs). We confirmed the efficiency of fucoidan incorporation by FTIR and EDX analysis. Further, its ability to support hMSC attachment and chondrogenic differentiation was confirmed by SEM, biochemical glycosaminoglycan quantification, real-time quantitative PCR and immunocytochemical analyses of chondrogenic markers Sox-9, Collagen II, Aggrecan and COMP. Effect of Al-Fu hydrogel on hMSC hypertrophy was also confirmed by the downregulation of hypertrophic genes Collagen X and Runx2. This composite scaffold can hence be used as a cartilage biomimetic biomaterial to drive hMSC chondrogenesis and for other cartilage repair based therapies. PMID:27178935

  14. Localization of mesenchymal stromal cells dictates their immune or proinflammatory effects in kidney transplantation.

    PubMed

    Casiraghi, F; Azzollini, N; Todeschini, M; Cavinato, R A; Cassis, P; Solini, S; Rota, C; Morigi, M; Introna, M; Maranta, R; Perico, N; Remuzzi, G; Noris, M

    2012-09-01

    Multipotent mesenchymal stromal cells (MSC) have recently emerged as promising candidates for cell-based immunotherapy in solid-organ transplantation. However, optimal conditions and settings for fully harnessing MSC tolerogenic properties need to be defined. We recently reported that autologous MSC given posttransplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Here, we moved back to a murine kidney transplant model with the aim to define the best timing of MSC infusion capable of promoting immune tolerance without negative effects on early graft function. We also investigated the mechanisms of the immunomodulatory and/or proinflammatory activities of MSC according to whether cells were given before or after transplant. Posttransplant MSC infusion in mice caused premature graft dysfunction and failed to prolong graft survival. In this setting, infused MSC localized mainly into the graft and associated with neutrophils and complement C3 deposition. By contrast, pretransplant MSC infusion induced a significant prolongation of kidney graft survival by a Treg-dependent mechanism. MSC-infused pretransplant localized into lymphoid organs where they promoted early expansion of Tregs. Thus, pretransplant MSC infusion may be a useful approach to fully exploit their immunomodulatory properties in kidney transplantation. PMID:22642544

  15. Adipose Mesenchymal Stromal Cells Isolated From Type 2 Diabetic Patients Display Reduced Fibrinolytic Activity

    PubMed Central

    Acosta, Lourdes; Hmadcha, Abdelkrim; Escacena, Natalia; Pérez-Camacho, Inmaculada; de la Cuesta, Antonio; Ruiz-Salmeron, Rafael; Gauthier, Benoit R.; Soria, Bernat

    2013-01-01

    Stem cells have been successfully used for the treatment of critical limb ischemia (CLI). We conducted a clinical trial to determine the feasibility of using autologous adipose-derived mesenchymal stromal cells (AdMSCs) for the treatment of CLI. Unexpectedly, two diabetic patients developed peripheral microthrombosis. This adverse effect, which contrasts with the reported antithrombotic properties of MSCs, may stem from the diabetic environment that alters the fibrinolytic activity of AdMSCs, thereby increasing the probability of developing thrombosis. Here, we confirm this premise by demonstrating that diabetic AdMSCs cultured in the presence of blood sera expressed and released higher levels of plasminogen activator inhibitor type 1, reduced levels of tissue plasminogen activator, and lower d-dimer formation compared with nondiabetic AdMSCs. Thus, to establish an appropriate cell therapy for diabetic patients, we recommend including new preclinical safety tests, such as the d-dimer and/or the tissue plasminogen activator-to-plasminogen activator inhibitor type 1 ratio tests, to assess fibrinolytic activity of cells before implantation. PMID:24043757

  16. Human mesenchymal stromal cells response to biomimetic octacalcium phosphate containing strontium.

    PubMed

    Birgani, Zeinab Tahmasebi; Malhotra, Angad; van Blitterswijk, Clemens A; Habibovic, Pamela

    2016-08-01

    The incorporation of bioinorganics into synthetic biomaterials is a promising approach to improve the biological performance of bone graft substitutes, while still retaining their synthetic nature. Among these bioinorganics, strontium ions (Sr(2+) ) have reported enhanced bone formation, and a reduced risk of bone fractures. While previous results have been encouraging, more detailed studies are needed to further develop specific applications. This study demonstrates the effects of Sr(2+) on the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) when introduced as either a dissolved salt, or incorporated into biomimetic calcium phosphate (CaP) coatings. Upon attachment, hMSCs seeded in the presence of higher Sr(2+) concentrations presented with a more elongated shape as compared to the controls without Sr(2+) . Both Sr(2+) as a dissolved salt in the medium, or incorporated into CaP coatings, positively influenced hMSC alkaline phosphatase (ALP) activity in a dose-dependent manner. At the mRNA level, the expression of osteogenic markers ALP, bone sialoprotein, bone morphogenetic protein 2, osteopontin, and osteoclacin were increased in the presence of Sr(2+) , independent of the delivery method. Overall, this study demonstrates the positive effects of strontium on the osteogenic differentiation of human MSCs, and supports the use of strontium-incorporated CaPs for bone regeneration applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1946-1960, 2016. PMID:27012665

  17. Mesenchymal Stromal Cells from Neonatal Tracheal Aspirates Demonstrate a Pattern of Lung-Specific Gene Expression

    PubMed Central

    Bozyk, Paul D.; Popova, Antonia P.; Bentley, John Kelley; Goldsmith, Adam M.; Linn, Marisa J.; Weiss, Daniel J.

    2011-01-01

    We have previously isolated mesenchymal stromal cells (MSCs) from the tracheal aspirates of premature neonates with respiratory distress. Although isolation of MSCs correlates with the development of bronchopulmonary dysplasia, the physiologic role of these cells remains unclear. To address this, we further characterized the cells, focusing on the issues of gene expression, origin, and cytokine expression. Microarray comparison of early passage neonatal lung MSC gene expression to cord blood MSCs and human fetal and neonatal lung fibroblast lines demonstrated that the neonatal lung MSCs differentially expressed 971 gene probes compared with cord blood MSCs, including the transcription factors Tbx2, Tbx3, Wnt5a, FoxF1, and Gli2, each of which has been associated with lung development. Compared with lung fibroblasts, 710 gene probe transcripts were differentially expressed by the lung MSCs, including IL-6 and IL-8/CXCL8. Differential chemokine expression was confirmed by protein analysis. Further, neonatal lung MSCs exhibited a pattern of Hox gene expression distinct from cord blood MSCs but similar to human fetal lung fibroblasts, consistent with a lung origin. On the other hand, limiting dilution analysis showed that fetal lung fibroblasts form colonies at a significantly lower rate than MSCs, and fibroblasts failed to undergo differentiation along adipogenic, osteogenic, and chondrogenic lineages. In conclusion, MSCs isolated from neonatal tracheal aspirates demonstrate a pattern of lung-specific gene expression, are distinct from lung fibroblasts, and secrete pro-inflammatory cytokines. PMID:21341990

  18. In vivo biocompatibility of custom-fabricated apatite-wollastonite-mesenchymal stromal cell constructs.

    PubMed

    Lee, Jennifer A; Knight, Charlotte A; Kun, Xiao; Yang, Xuebin B; Wood, David J; Dalgarno, Kenneth W; Genever, Paul G

    2015-10-01

    We have used the additive manufacturing technology of selective laser sintering (SLS), together with post SLS heat treatment, to produce porous three dimensional scaffolds from the glass-ceramic apatite-wollastonite (A-W). The A-W scaffolds were custom-designed to incorporate a cylindrical central channel to increase cell penetration and medium flow to the center of the scaffolds under dynamic culture conditions during in vitro testing and subsequent in vivo implantation. The scaffolds were seeded with human bone marrow mesenchymal stromal cells (MSCs) and cultured in spinner flasks. Using confocal and scanning electron microscopy, we demonstrated that MSCs formed and maintained a confluent layer of viable cells on all surfaces of the A-W scaffolds during dynamic culture. MSC-seeded, with and without osteogenic pre-differentiation, and unseeded A-W scaffolds were implanted subcutaneously in MF1 nude mice where osteoid formation and tissue in-growth were observed following histological assessment. The results demonstrate that the in vivo biocompatibility and osteo-supportive capacity of A-W scaffolds can be enhanced by SLS-custom design, without the requirement for osteogenic pre-induction, to advance their potential as patient-specific bone replacement materials. PMID:25777813

  19. Active control of the nucleation temperature enhances freezing survival of multipotent mesenchymal stromal cells.

    PubMed

    Lauterboeck, L; Hofmann, N; Mueller, T; Glasmacher, B

    2015-12-01

    Cryopreservation is a technique that has been extensively used for storage of multipotent mesenchymal stromal cells (MSCs) in regenerative medicine. Therefore, improving current cryopreservation procedures in terms of increasing cell viability and functionality is important. In this study, we optimized the cryopreservation protocol of MSCs derived from the common marmoset Callithrix jacchus (cj), which can be used as a non-human primate model in various pathological and transplantation studies and have a great potential for regenerative medicine. We have investigated the effect of the active control of the nucleation temperature using induced nucleation at a broad range of temperatures and two different dimethylsulfoxide concentrations (Me2SO, 5% (v/v) and 10%, (v/v)) to evaluate the overall effect on the viability, metabolic activity and recovery of cells after thawing. Survival rate and metabolic activity displayed an optimum when ice formation was induced at -10 °C. Cryomicroscopy studies indicated differences in ice crystal morphologies as well as differences in intracellular ice formation with different nucleation temperatures. High subzero nucleation temperatures resulted in larger extracellular ice crystals and cellular dehydration, whereas low subzero nucleation temperatures resulted in smaller ice crystals and intracellular ice formation. PMID:26499840

  20. Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system.

    PubMed

    Mizukami, Amanda; Orellana, Maristela D; Caruso, Sâmia R; de Lima Prata, Karen; Covas, Dimas T; Swiech, Kamilla

    2013-01-01

    The need for efficient and reliable technologies for clinical-scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood-derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10(7) cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10(8) cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra-Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier-based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical-scale production system. PMID:23420706

  1. Comparative immunophenotyping of equine multipotent mesenchymal stromal cells: an approach toward a standardized definition.

    PubMed

    Paebst, Felicitas; Piehler, Daniel; Brehm, Walter; Heller, Sandra; Schroeck, Carmen; Tárnok, Attila; Burk, Janina

    2014-08-01

    Horses are an approved large animal model for therapies of the musculoskeletal system. Especially for tendon disease where cell-based therapy is commonly used in equine patients, the translation of achieved results to human medicine would be a great accomplishment. Immunophenotyping of equine mesenchymal stromal cells (MSCs) remains the last obstacle to meet the criteria of the International Society for Cellular Therapy (ISCT) definition of human MSCs. Therefore, the surface antigen expression of CD 29, CD 44, CD 73, CD 90, CD 105, CD 14, CD 34, CD 45, CD 79α, and MHC II in equine MSCs from adipose tissue, bone marrow, umbilical cord blood, umbilical cord tissue, and tendon tissue was analyzed using flow cytometry. Isolated cells from the different sources and donors varied in their expression pattern of MSC-defining antigens. In particular, CD 90 and 105 showed most heterogeneity. However, cells from all samples were robustly positive for CD 29 and CD 44, while being mostly negative for CD 73 and the exclusion markers CD 14, CD 34, CD 45, CD 79α and MHC II. Furthermore, it was evident that enzymes used for cell detachment after in vitro-culture affected the detection of antigen expression. These results emphasize the need of standardization of MSC isolation, culturing, and harvesting techniques. As the equine MSCs did not meet all criteria the ISCT defined for human MSCs, further investigations for a better characterization of the cell type should be conducted. PMID:24894974

  2. Bio- chemical and physical characterizations of mesenchymal stromal cells along the time course of directed differentiation

    PubMed Central

    Chen, Yin-Quan; Liu, Yi-Shiuan; Liu, Yu-An; Wu, Yi-Chang; del Álamo, Juan C.; Chiou, Arthur; Lee, Oscar K.

    2016-01-01

    Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds. PMID:27526936

  3. Human turbinate mesenchymal stromal cell sheets with bellows graft for rapid tracheal epithelial regeneration.

    PubMed

    Park, Jeong Hun; Park, Ju Young; Nam, Inn-Chul; Hwang, Se-Hwan; Kim, Choung-Soo; Jung, Jin Woo; Jang, Jinah; Lee, Hyungseok; Choi, Yeongjin; Park, Sun Hwa; Kim, Sung Won; Cho, Dong-Woo

    2015-10-01

    Rapid functional epithelial regeneration on the luminal surface is essential when using artificial tracheal grafts to repair tracheal defects. In this study, we imposed human turbinate mesenchymal stromal cell (hTMSC) sheets for tracheal epithelial regeneration, and then assessed their potential as a new clinical cell source. In vitro, hTMSCs sheets showed high capacity to differentiate into tracheal epithelium. We fabricated a poly(ε-caprolactone) (PCL) tracheal graft by indirect three-dimensional (3D) printing technique and created a composite construct by transplanting the hTMSC sheets to its luminal surface of the tracheal graft, then applied this tissue-engineered tracheal graft to non-circumferential tracheal reconstruction in a rabbit model. 4 weeks after implantation, the luminal surface of tissue-engineered tracheal graft was covered by a mature and highly-ciliated epithelium, whereas tracheal grafts without hTMSC sheets were covered by only a thin, immature epithelium. Therefore, hTMSC sheets on the luminal surface of a tissue-engineered tracheal graft can accelerate the tracheal epithelial regeneration, and the tissue-engineered tracheal graft with hTMSC sheets provides a useful clinical alternative for tracheal epithelial regeneration. PMID:26163763

  4. Mesenchymal stem/stromal cells-a key mediator for regeneration after perinatal morbidity?

    PubMed

    Mueller, Martin; Wolfs, Tim G A; Schoeberlein, Andreina; Gavilanes, Antonio W D; Surbek, Daniel; Kramer, Boris W

    2016-12-01

    Perinatal complications in both term- and preterm-born infants are a leading cause of neonatal morbidities and mortality. Infants face different challenges in the neonatal intensive care unit with long-term morbidities such as perinatal brain injury and bronchopulmonary dysplasia being particularly devastating. While advances in perinatal medicine have improved our understanding of the pathogenesis, effective therapies to prevent and/or reduce the severity of these disorders are still lacking. The potential of mesenchymal stem/stromal cell (MSC) therapy has emerged during the last two decades, and an increasing effort is conducted to address brain- and lung-related morbidities in neonates at risk. Various studies support the notion that MSCs have protective effects. MSCs are an easy source and may be readily available after birth in a clinical setting. MSCs' mechanisms of action are diverse, including migration and homing, release of growth factors and immunomodulation, and the potential to replace injured cells. Here, we review the pathophysiology of perinatally acquired brain and lung injuries and focus on MSCs as potential candidates for therapeutic strategies summarizing preclinical and clinical evidence. PMID:26869264

  5. Forskolin enhances in vivo bone formation by human mesenchymal stromal cells.

    PubMed

    Doorn, Joyce; Siddappa, Ramakrishnaiah; van Blitterswijk, Clemens A; de Boer, Jan

    2012-03-01

    Activation of the protein kinase A (PKA) pathway with dibutyryl cyclic adenosine monophosphate (db-cAMP) was recently shown to enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs) in vitro and bone formation in vivo. The major drawback of this compound is its inhibitory effect on proliferation of hMSCs. Therefore, we investigated whether fine-tuning of the dose and timing of PKA activation could enhance bone formation even further, with minimum effects on proliferation. To test this, we selected two different PKA activators (8-bromo-cAMP (8-br-cAMP) and forskolin) and compared their effects on proliferation and osteogenic differentiation with those of db-cAMP. We found that all three compounds induced alkaline phosphatase levels, bone-specific target genes, and secretion of insulin-like growth factor-1, although 8-br-cAMP induced adipogenic differentiation in long-term cultures and was thus considered unsuitable for further in vivo testing. All three compounds inhibited proliferation of hMSCs in a dose-dependent manner, with forskolin inhibiting proliferation most. The effect of forskolin on in vivo bone formation was tested by pretreating hMSCs before implantation, and we observed greater amounts of bone using forskolin than db-cAMP. Our data show forskolin to be a novel agent that can be used to increase bone formation and also suggests a role for PKA in the delicate balance between adipogenic and osteogenic differentiation. PMID:21942968

  6. Human mesenchymal stromal cells: identifying assays to predict potency for therapeutic selection.

    PubMed

    Deskins, Desirae L; Bastakoty, Dikshya; Saraswati, Sarika; Shinar, Andrew; Holt, Ginger E; Young, Pampee P

    2013-02-01

    Multipotent mesenchymal stromal cells (MSCs) have the potential to repair and regenerate damaged tissues, making them attractive candidates for cell-based therapies. To maximize efficacy of MSCs, prediction of their therapeutic abilities must be made so that only the best cells will be used. Our goal was to identify feasible and reproducible in vitro assays to predict MSC potency. We generated cell lines from 10 normal human bone marrow samples and used the International Society for Cellular Therapy's minimal criteria to define them as MSCs: plastic adherence, appropriate surface marker expression, and trilineage differentiation. Each MSC line was further characterized by its growth, proliferation, and viability as determined by cell count, bromodeoxyuridine incorporation, and cellular ATP levels, respectively. To determine whether these tests reliably predict the therapeutic aptitude of the MSCs, several lines were implanted in vivo to examine their capacity to engraft and form granulation tissue in a well-established murine wound model using polyvinyl alcohol sponges. Long-term engraftment of MSCs in the sponges was quantified through the presence of the human-specific Alu gene in sponge sections. Sections were also stained for proliferating cells, vascularity, and granulation tissue formation to determine successful engraftment and repair. We found that high performance in a combination of the in vitro tests accurately predicted which lines functioned well in vivo. These findings suggest that reliable and reproducible in vitro assays may be used to measure the functional potential of MSCs for therapeutic use. PMID:23362238

  7. β-PIX is critical for transplanted mesenchymal stromal cell migration.

    PubMed

    Koh, Seong-Ho; Huh, Yong-Min; Noh, Min Young; Kim, Hyun Young; Kim, Kyung Suk; Lee, Eun-Sook; Ko, Hyun-Ju; Cho, Goang Won; Yoo, A Rum; Song, Ho-Taek; Hwang, Sejin; Lee, Kwangyeol; Haam, Seungjoo; Frank, Joseph A; Suh, Jin-Suck; Kim, Seung Hyun

    2012-07-20

    Bone marrow-derived mesenchymal stromal cells (MSCs) have been used successfully as a source of stem cells for treating neurodegenerative diseases. However, for reasons that are not clear, autologous MSC transplants have not yielded successful results in human trials. To test one possible reason, we compared the migratory ability of MSCs from amyotrophic lateral sclerosis (ALS) patients with those of healthy controls. We found that MSCs derived from ALS patients (ALS-MSCs) had a reduced ability to migrate, which may explain why autologous transplantation is not successful. We also found that expression of one of the intracellular factors implicated in migration, β-PIX, was significantly reduced in ALS-MSCs compared with healthy stem cells. Restoration of β-PIX expression by genetic manipulation restored the migratory ability of ALS-MSCs, and inhibition of β-PIX expression with shRNA reduced the migration of healthy MSCs. We suggest that transplantation of allogeneic or genetically modified autologous stem cells might be a more promising strategy for ALS patients than transplantation of autologous stem cells. PMID:22087847

  8. Safety and Efficacy Endpoints for Mesenchymal Stromal Cell Therapy in Renal Transplant Recipients

    PubMed Central

    Bank, J. R.; Rabelink, T. J.; de Fijter, J. W.; Reinders, M. E. J.

    2015-01-01

    Despite excellent short-term graft survival after renal transplantation, the long-term graft outcome remains compromised. It has become evident that a combination of sustained alloreactivity and calcineurin-inhibitor- (CNI-) related nephrotoxicity results in fibrosis and consequently dysfunction of the graft. New immunosuppressive regimens that can minimize or eliminate side effects, while maintaining efficacy, are required to improve long-term graft survival. In this perspective mesenchymal stromal cells (MSCs) are an interesting candidate, since MSCs have immunosuppressive and regenerative properties. The first clinical trials with MSCs in renal transplantation showed safety and feasibility and displayed promising results. Recently, the first phase II studies have been started. One of the most difficult and challenging aspects in those early phase trials is to define accurate endpoints that can measure safety and efficacy of MSC treatment. Since both graft losses and acute rejection rates declined, alternative surrogate markers such as renal function, histological findings, and immunological markers are used to measure efficacy and to provide mechanistic insight. In this review, we will discuss the current status of MSCs in renal transplantation with a focus on the endpoints used in the different experimental and clinical studies. PMID:26258149

  9. Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

    PubMed Central

    Muntión, Sandra; Ramos, Teresa L.; Diez-Campelo, María; Rosón, Beatriz; Sánchez-Abarca, Luis Ignacio; Misiewicz-Krzeminska, Irena; Preciado, Silvia; Sarasquete, María-Eugenia; de las Rivas, Javier; González, Marcos; Sánchez-Guijo, Fermín; del Cañizo, María-Consuelo

    2016-01-01

    Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties. PMID:26836120

  10. Concise Review: Mesenchymal Stromal Cells Used for Periodontal Regeneration: A Systematic Review

    PubMed Central

    Monsarrat, Paul; Vergnes, Jean-Noël; Nabet, Cathy; Sixou, Michel; Snead, Malcolm L.; Planat-Bénard, Valérie; Casteilla, Louis

    2014-01-01

    Periodontitis is a chronic infectious disease of the soft and hard tissues supporting the teeth. Recent advances in regenerative medicine and stem cell biology have paved the way for periodontal tissue engineering. Mesenchymal stromal cells (MSCs) delivered in situ to periodontal defects may exert their effects at multiple levels, including neovascularization, immunomodulation, and tissue regeneration. This systematic review had two goals: (a) to objectively quantify key elements for efficacy and safety of MSCs used for periodontal regeneration and (b) to identify patterns in the existing literature to explain differences between studies and suggest recommendations for future research. This systematic review provided good evidence of the capacity of MSCs to regenerate periodontal tissues in animals; however, experimentally generated defects used in animal studies do not sufficiently mimic the pathophysiology of periodontitis in humans. Moreover, the safety of such interventions in humans still needs to be studied. There were marked differences between experimental and control groups that may be influenced by characteristics that are crucial to address before translation to human clinical trials. We suggest that the appropriate combination of cell source, carrier type, and biomolecules, as well as the inclusion of critical path issues for a given clinical case, should be further explored and refined before transitioning to clinical trials. Future studies should investigate periodontal regenerative procedures in animal models, including rodents, in which the defects generated are designed to more accurately reflect the inflammatory status of the host and the shift in their pathogenic microflora. PMID:24744392

  11. Cytomegalovirus infection impairs immunosuppressive and antimicrobial effector functions of human multipotent mesenchymal stromal cells.

    PubMed

    Meisel, Roland; Heseler, Kathrin; Nau, Julia; Schmidt, Silvia Kathrin; Leineweber, Margret; Pudelko, Sabine; Wenning, Johannes; Zimmermann, Albert; Hengel, Hartmut; Sinzger, Christian; Degistirici, Özer; Sorg, Rüdiger Volker; Däubener, Walter

    2014-01-01

    Human mesenchymal stromal cells (MSC) possess immunosuppressive and antimicrobial effects that are partly mediated by the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Therefore MSC represent a promising novel cellular immunosuppressant which has the potential to control steroid-refractory acute graft versus host disease (GvHD). In addition, MSC are capable of reducing the risk of infection in patients after haematopoietic stem cell transplantation (HST). Recent data indicate that signals from the microenvironment including those from microbes may modulate MSC effector functions. As Cytomegalovirus (CMV) represents a prominent pathogen in immunocompromised hosts, especially in patients following HST, we investigated the impact of CMV infection on MSC-mediated effects on the immune system. We demonstrate that CMV-infected MSC lose their cytokine-induced immunosuppressive capacity and are no longer able to restrict microbial growth. IDO expression is substantially impaired following CMV infection of MSC and this interaction critically depends on intact virus and the number of MSC as well as the viral load. Since overt CMV infection may undermine the clinical efficacy of MSC in the treatment of GvHD in transplant patients, we recommend that patients scheduled for MSC therapy should undergo thorough evaluation for an active CMV infection and receive CMV-directed antiviral therapy prior to the administration of MSC. PMID:24782599

  12. Neovascularization Capacity of Mesenchymal Stromal Cells From Critical Limb Ischemia Patients Is Equivalent to Healthy Controls

    PubMed Central

    Gremmels, Hendrik; Teraa, Martin; Quax, Paul HA; den Ouden, Krista; Fledderus, Joost O; Verhaar, Marianne C

    2014-01-01

    Critical limb ischemia (CLI) is often poorly treatable by conventional management and alternatives such as autologous cell therapy are increasingly investigated. Whereas previous studies showed a substantial impairment of neovascularization capacity in primary bone-marrow (BM) isolates from patients, little is known about dysfunction in patient-derived BM mesenchymal stromal cells (MSCs). In this study, we have compared CLI-MSCs to healthy controls using gene expression profiling and functional assays for differentiation, senescence and in vitro and in vivo pro-angiogenic ability. Whereas no differentially expressed genes were found and adipogenic and osteogenic differentiation did not significantly differ between groups, chondrogenic differentiation was impaired in CLI-MSCs, potentially as a consequence of increased senescence. Migration experiments showed no differences in growth factor sensitivity and secretion between CLI- and control MSCs. In a murine hind-limb ischemia model, recovery of perfusion was enhanced in MSC-treated mice compared to vehicle controls (71 ± 24% versus 44 ± 11%; P < 1 × 10−6). CLI-MSC- and control-MSC–treated animals showed nearly identical amounts of reperfusion (ratio CLI:Control = 0.98, 95% CI = 0.82–1.14), meeting our criteria for statistical equivalence. The neovascularization capacity of MSCs derived from CLI-patients is not compromised and equivalent to that of control MSCs, suggesting that autologous MSCs are suitable for cell therapy in CLI patients. PMID:25174586

  13. Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects.

    PubMed

    Marquez-Curtis, Leah A; Janowska-Wieczorek, Anna; McGann, Locksley E; Elliott, Janet A W

    2015-10-01

    Originally isolated from bone marrow, mesenchymal stromal cells (MSCs) have since been obtained from various fetal and post-natal tissues and are the focus of an increasing number of clinical trials. Because of their tremendous potential for cellular therapy, regenerative medicine and tissue engineering, it is desirable to cryopreserve and bank MSCs to increase their access and availability. A remarkable amount of research and resources have been expended towards optimizing the protocols, freezing media composition, cooling devices and storage containers, as well as developing good manufacturing practices in order to ensure that MSCs retain their therapeutic characteristics following cryopreservation and that they are safe for clinical use. Here, we first present an overview of the identification of MSCs, their tissue sources and the properties that render them suitable as a cellular therapeutic. Next, we discuss the responses of cells during freezing and focus on the traditional and novel approaches used to cryopreserve MSCs. We conclude that viable MSCs from diverse tissues can be recovered after cryopreservation using a variety of freezing protocols, cryoprotectants, storage periods and temperatures. However, alterations in certain functions of MSCs following cryopreservation warrant future investigations on the recovery of cells post-thaw followed by expansion of functional cells in order to achieve their full therapeutic potential. PMID:26186998

  14. Biodistribution of mesenchymal stem/stromal cells in a preclinical setting.

    PubMed

    Sensebé, Luc; Fleury-Cappellesso, Sandrine

    2013-01-01

    Due to their multi/pluripotency and immunosuppressive properties, mesenchymal stem/stromal cells (MSCs) are important tools for treatment of immune disorders and tissue repair. The increasing uses of MSCs lead to the development of production processes that need to be in accordance with good manufacturing practices (GMP). In Europe, MSCs are somatic cell-therapy products, referred to as advanced-therapy medicinal products (ATMPs), and in the United States MSCs must comply with current good tissue practice requirements. The safety and efficacy of MSCs must be ensured, whatever the cell source, and studies of dose and biodistribution are important aspects of safety testing. Preclinical data on biodistribution and pharmacodynamics are mandatory for approval. It is important to demonstrate that MSCs do not have unwanted homing that could drive to inappropriate differentiation in some organ or to support cancer development as suggested in some experiments. All these aspects should be addressed in a risk-based approach according to recently published guidelines by EMA. In the present article, we summarize the main approaches for labeling and tracking of infused MSCs, report on current animal models, and give an overview of available results on biodistribution. PMID:24222773

  15. Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review.

    PubMed

    Sensebé, Luc; Gadelorge, Mélanie; Fleury-Cappellesso, Sandrine

    2013-01-01

    Because of their multi/pluripotency and immunosuppressive properties, mesenchymal stem/stromal cells (MSCs) are important tools for treating immune disorders and for tissue repair. The increasing use of MSCs, their definition as advanced-therapy medicinal products in European regulations, and the US Food and Drug Administration requirements for their production and use imply the use of production processes that should be in accordance with Good Manufacturing Practices (GMPs). Complying with GMPs requires precisely defining the production process (es) as well as the multiple criteria required for a quality final product. Such variables include the environment, staff training and qualification, and controls. Developing processes based on well-defined or completely defined media and operating in closed systems or bioreactors is important and will increase safety and reproducibility. One of the most challenging issues remains implementation of relevant and reproducible controls for safety and efficacy. A linking of researchers, research and development teams, producers, and clinicians is mandatory to achieve GMP-compliant processes with relevant controls for producing well-defined, safe, and efficient MSCs. PMID:23751270

  16. Making surrogate β-cells from mesenchymal stromal cells: perspectives and future endeavors.

    PubMed

    Bhonde, Ramesh R; Sheshadri, Preethi; Sharma, Shikha; Kumar, Anujith

    2014-01-01

    Generation of surrogate β-cells is the need of the day to compensate the short supply of islets for transplantation to diabetic patients requiring daily shots of insulin. Over the years several sources of stem cells have been claimed to cater to the need of insulin producing cells. These include human embryonic stem cells, induced pluripotent stem cells, human perinatal tissues such as amnion, placenta, umbilical cord and postnatal tissues involving adipose tissue, bone marrow, blood monocytes, cord blood, dental pulp, endometrium, liver, labia minora dermis-derived fibroblasts and pancreas. Despite the availability of such heterogonous sources, there is no substantial breakthrough in selecting and implementing an ideal source for generating large number of stable insulin producing cells. Although the progress in derivation of β-cell like cells from embryonic stem cells has taken a greater leap, their application is limited due to controversy surrounding the destruction of human embryo and immune rejection. Since multipotent mesenchymal stromal cells are free of ethical and immunological complications, they could provide unprecedented opportunity as starting material to derive insulin secreting cells. The main focus of this review is to discuss the merits and demerits of MSCs obtained from human peri- and post-natal tissue sources to yield abundant glucose responsive insulin producing cells as ideal candidates for prospective stem cell therapy to treat diabetes. PMID:24275096

  17. Rationale for the potential use of mesenchymal stromal cells in liver transplantation.

    PubMed

    Vandermeulen, Morgan; Grégoire, Céline; Briquet, Alexandra; Lechanteur, Chantal; Beguin, Yves; Detry, Olivier

    2014-11-28

    Mesenchymal stromal cells (MSCs) are multipotent and self-renewing cells that reside essentially in the bone marrow as a non-hematopoietic cell population, but may also be isolated from the connective tissues of most organs. MSCs represent a heterogeneous population of adult, fibroblast-like cells characterized by their ability to differentiate into tissues of mesodermal lineages including adipocytes, chondrocytes and osteocytes. For several years now, MSCs have been evaluated for their in vivo and in vitro immunomodulatory and 'tissue reconstruction' properties, which could make them interesting in various clinical settings, and particularly in organ transplantation. This paper aims to review current knowledge on the properties of MSCs and their use in pre-clinical and clinical studies in solid organ transplantation, and particularly in the field of liver transplantation. The first available clinical data seem to show that MSCs are safe to use, at least in the medium-term, but more time is needed to evaluate the potential adverse effects of long-term use. Many issues must be resolved on the correct use of MSCs. Intensive in vitro and pre-clinical research are the keys to a better understanding of the way that MSCs act, and to eventually lead to clinical success. PMID:25469010

  18. Brief report: The potential role of epigenetics on multipotent cell differentiation capacity of mesenchymal stromal cells.

    PubMed

    Yannarelli, Gustavo; Pacienza, Natalia; Cuniberti, Luis; Medin, Jeffrey; Davies, John; Keating, Armand

    2013-01-01

    Human umbilical cord perivascular cells (HUCPVCs) are a readily available source of mesenchymal stromal cells (MSCs) for cell therapy. We were interested in understanding how differences from human bone marrow (BM)-derived MSCs might yield insights into MSC biology. We found that HUCPVCs exhibited increased telomerase activity and longer telomeres compared with BM-MSCs. We also observed enhanced expression of the pluripotency factors OCT4, SOX2, and NANOG in HUCPVCs. The methylation of OCT4 and NANOG promoters was similar in both cell types, indicating that differences in the expression of pluripotency factors between the MSCs were not associated with epigenetic changes. MSC methylation at these loci is greater than reported for embryonic stem cells but less than in dermal fibroblasts, suggesting that multipotentiality of MSCs is epigenetically restricted. These results are consistent with the notion that the MSC population (whether BM- or HUCPV-derived) exhibits higher proliferative capacity and contains more progenitor cells than do dermal fibroblasts. PMID:23097343

  19. Challenges in animal modelling of mesenchymal stromal cell therapy for inflammatory bowel disease

    PubMed Central

    Chinnadurai, Raghavan; Ng, Spencer; Velu, Vijayakumar; Galipeau, Jacques

    2015-01-01

    Utilization of mesenchymal stromal cells (MSCs) for the treatment of Crohn’s disease and ulcerative colitis is of translational interest. Safety of MSC therapy has been well demonstrated in early phase clinical trials but efficacy in randomized clinical trials needs to be demonstrated. Understanding MSC mechanisms of action to reduce gut injury and inflammation is necessary to improve current ongoing and future clinical trials. However, two major hurdles impede the direct translation of data derived from animal experiments to the clinical situation: (1) limitations of the currently available animal models of colitis that reflect human inflammatory bowel diseases (IBD). The etiology and progression of human IBD are multifactorial and hence a challenge to mimic in animal models; and (2) Species specific differences in the functionality of MSCs derived from mice versus humans. MSCs derived from mice and humans are not identical in their mechanisms of action in suppressing inflammation. Thus, preclinical animal studies with murine derived MSCs cannot be considered as an exact replica of human MSC based clinical trials. In the present review, we discuss the therapeutic properties of MSCs in preclinical and clinical studies of IBD. We also discuss the challenges and approaches of using appropriate animal models of colitis, not only to study putative MSC therapeutic efficacy and their mechanisms of action, but also the suitability of translating findings derived from such studies to the clinic. PMID:25944991

  20. Adipose-Derived Mesenchymal Stromal/Stem Cells: Tissue Localization, Characterization, and Heterogeneity

    PubMed Central

    Baer, Patrick C.; Geiger, Helmut

    2012-01-01

    Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stromal/stem cells (ASCs) yields a high amount of stem cells, which is essential for stem-cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in a perivascular niche, whereas the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to plastic. Nevertheless, recent isolation and culture techniques lack standardization. Cultured cells are characterized by their expression of characteristic markers and their capacity to differentiate into cells from meso-, ecto-, and entodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, neural cells, and endothelial and epithelial cells. Nevertheless, recent studies suggest that ASCs are a heterogeneous mixture of cells containing subpopulations of stem and more committed progenitor cells. This paper summarizes and discusses the current knowledge of the tissue localization of ASCs in situ, their characterization and heterogeneity in vitro, and the lack of standardization in isolation and culture methods. PMID:22577397

  1. Chemical nanoroughening of Ti40Nb surfaces and its effect on human mesenchymal stromal cell response.

    PubMed

    Helth, A; Gostin, P F; Oswald, S; Wendrock, H; Wolff, U; Hempel, U; Arnhold, S; Calin, M; Eckert, J; Gebert, A

    2014-01-01

    Samples of low modulus beta-type Ti40Nb and cp2-Ti were chemically treated with 98% H2 SO4 + 30% H2 O2 (vol. ratio 1:1) solution. Surface analytical studies conducted with HR-SEM, AFM, and XPS identified a characteristic nanoroughness of the alloy surface related with a network of nanopits of ∼25 nm diameter. This is very similar to that obtained for cp2-Ti. The treatment enhances the oxide layer growth compared to mechanically ground states and causes a strong enrichment of Nb2 O5 relative to TiO2 on the alloy surface. The in vitro analyses clearly indicated that the chemical treatment accelerates the adhesion and spreading of human mesenchymal stromal cells (hMSC), increases the metabolic activity, and the enzyme activity of tissue non-specific alkaline phosphatase (TNAP). Surface structures which were generated mimic the cytoplasmic projections of the cells on the nanoscale. Those effects are more pronounced for the Ti40Nb alloy than for cp2-Ti. The relation between alloy surface topography and chemistry and cell functions is discussed. PMID:23846980

  2. Recent insights: mesenchymal stromal/stem cell therapy for acute respiratory distress syndrome

    PubMed Central

    Horie, Shahd; Laffey, John G.

    2016-01-01

    Acute respiratory distress syndrome (ARDS) causes respiratory failure, which is associated with severe inflammation and lung damage and has a high mortality and for which there is no therapy. Mesenchymal stromal/stem cells (MSCs) are adult multi-progenitor cells that can modulate the immune response and enhance repair of damaged tissue and thus may provide a therapeutic option for ARDS. MSCs demonstrate efficacy in diverse in vivo models of ARDS, decreasing bacterial pneumonia and ischemia-reperfusion-induced injury while enhancing repair following ventilator-induced lung injury. MSCs reduce the pro-inflammatory response to injury while augmenting the host response to bacterial infection. MSCs appear to exert their effects via multiple mechanisms—some are cell interaction dependent whereas others are paracrine dependent resulting from both soluble secreted products and microvesicles/exosomes derived from the cells. Strategies to further enhance the efficacy of MSCs, such as by overexpressing anti-inflammatory or pro-repair molecules, are also being investigated. Encouragingly, early phase clinical trials of MSCs in patients with ARDS are under way, and experience with these cells in trials for other diseases suggests that the cells are well tolerated. Although considerable translational challenges, such as concerns regarding cell manufacture scale-up and issues regarding cell potency and batch variability, must be overcome, MSCs constitute a highly promising potential therapy for ARDS. PMID:27408702

  3. 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. Stem Cells 2016;34:781-790. PMID:26731338

  4. Mesenchymal Stromal Cell-Based Therapies for Chronic Lung Disease of Prematurity.

    PubMed

    Strueby, Lannae; Thébaud, Bernard

    2016-09-01

    Advances in perinatal care allow the survival of ever more premature infants. By approaching the biological limit of viability, survival free of injury becomes more challenging. As a consequence, bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, remains one of the main complications in infants born before 28 weeks' gestation. Currently, there is no treatment for BPD. Recent progress in understanding the biology of stem cells has opened unprecedented therapeutic options to mitigate lung injury and promote lung growth. Perinatal tissue, such as the umbilical cord and the placenta, represents a rich source of potent repair cells. Thus far, mesenchymal stromal cell (MSC)-based therapies demonstrate the most potential for protecting the developing lung from injury. Preclinical evidence supporting this potential therapeutic role has provided the basis for the initiation of phase I and II clinical trials in preterm neonates. This brief review summarizes the current knowledge accumulated over the past 10 years about MSCs and their repair potential in BPD. PMID:27603532

  5. Transient proteolytic modification of mesenchymal stromal cells increases lung clearance rate and targeting to injured tissue.

    PubMed

    Kerkelä, Erja; Hakkarainen, Tanja; Mäkelä, Tuomas; Raki, Mari; Kambur, Oleg; Kilpinen, Lotta; Nikkilä, Janne; Lehtonen, Siri; Ritamo, Ilja; Pernu, Roni; Pietilä, Mika; Takalo, Reijo; Juvonen, Tatu; Bergström, Kim; Kalso, Eija; Valmu, Leena; Laitinen, Saara; Lehenkari, Petri; Nystedt, Johanna

    2013-07-01

    Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes. PMID:23734061

  6. Transient Proteolytic Modification of Mesenchymal Stromal Cells Increases Lung Clearance Rate and Targeting to Injured Tissue

    PubMed Central

    Hakkarainen, Tanja; Mäkelä, Tuomas; Raki, Mari; Kambur, Oleg; Kilpinen, Lotta; Nikkilä, Janne; Lehtonen, Siri; Ritamo, Ilja; Pernu, Roni; Pietilä, Mika; Takalo, Reijo; Juvonen, Tatu; Bergström, Kim; Kalso, Eija; Valmu, Leena; Laitinen, Saara; Lehenkari, Petri; Nystedt, Johanna

    2013-01-01

    Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes. PMID:23734061

  7. Tracking mesenchymal stromal cells using an ultra-bright TAT-functionalized plasmonic-active nanoplatform.

    PubMed

    Yuan, Hsiangkuo; Gomez, Jose A; Chien, Jennifer S; Zhang, Lunan; Wilson, Christy M; Li, Shuqin; Fales, Andrew M; Liu, Yang; Grant, Gerald A; Mirotsou, Maria; Dzau, Victor J; Vo-Dinh, Tuan

    2016-04-01

    High-resolution tracking of stem cells remains a challenging task. An ultra-bright contrast agent with extended intracellular retention is suitable for in vivo high-resolution tracking of stem cells following the implantation. Here, a plasmonic-active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide-functionalized gold nanostars (TAT-GNS) that emit ultra-bright two-photon photoluminescence capable of tracking MSCs under high-resolution optical imaging. In vitro experiment showed TAT-GNS-labeled MSCs retained a similar differentiability to that of non-labeled MSCs controls. Due to their star shape, TAT-GNS exhibited greater intracellular retention than that of commercial Q-Tracker. In vivo imaging of TAT-GNS-labeled MSCs five days following intra-arterial injections in mice kidneys showed possible MSCs implantation in juxta-glomerular (JG) regions, but non-specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic-active nanoplatforms may be a useful intracellular tracking tool for stem cell research. An ultra-bright intracellular contrast agent is developed using TAT peptide-functionalized gold nanostars (TAT-GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two-photon photoluminescence and superior labeling efficiency than commercial Q-Tracker. Following renal implantation, some TAT-GNS-labeled MSCs permeate blood vessels and migrate to the juxta-glomerular region. PMID:27095616

  8. 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.

  9. Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells

    PubMed Central

    Zhou, Y; Guan, XX; Zhu, ZL; Guo, J; Huang, YC; Hou, WW; Yu, HY

    2010-01-01

    BACKGROUND AND PURPOSE Caffeine is consumed extensively in Europe and North America. As a risk factor for osteoporosis, epidemiological studies have observed that caffeine can decrease bone mineral density, adversely affect calcium absorption and increase the risk of bone fracture. However, the exact mechanisms have not been fully investigated. Here, we examined the effects of caffeine on the viability and osteogenesis of rat bone marrow-derived mesenchymal stromal cells (rBMSCs). EXPERIMENTAL APPROACH Cell viability, apoptosis and necrosis were quantified using thymidine incorporation and flow cytometry. Sequential gene expressions in osteogenic process were measured by real-time PCR. cAMP, alkaline phosphatase and osteocalcin were assessed by immunoassay, spectrophotometry and radioimmunoassay, respectively. Mineralization was determined by calcium deposition. KEY RESULTS After treating BMSCs with high caffeine concentrations (0.1–1 mM), their viability decreased in a concentration-dependent manner. This cell death was primarily due to necrosis and, to a small extent, apoptosis. Genes and protein sequentially expressed in osteogenesis, including Cbfa1/Runx2, collagen I, alkaline phosphatase and its protein, were significantly downregulated except for osteocalcin and its protein. Moreover, caffeine inhibited calcium deposition in a concentration- and time-dependent manner, but increased intracellular cAMP in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS By suppressing the commitment of BMSCs to the osteogenic lineage and selectively inhibiting gene expression, caffeine downregulated some important events in osteogenesis and ultimately affected bone mass. PMID:20726981

  10. Three-dimensional spherical spatial boundary conditions differentially regulate osteogenic differentiation of mesenchymal stromal cells

    PubMed Central

    Lo, Yin-Ping; Liu, Yi-Shiuan; Rimando, Marilyn G.; Ho, Jennifer Hui-Chun; Lin, Keng-hui; Lee, Oscar K.

    2016-01-01

    The spatial boundary condition (SBC) arising from the surrounding microenvironment imposes specific geometry and spatial constraints that affect organogenesis and tissue homeostasis. Mesenchymal stromal cells (MSCs) sensitively respond to alterations of mechanical cues generated from the SBC. However, mechanical cues provided by a three-dimensional (3D) environment are deprived in a reductionist 2D culture system. This study investigates how SBC affects osteogenic differentiation of MSCs using 3D scaffolds with monodispersed pores and homogenous spherical geometries. MSCs cultured under SBCs with diameters of 100 and 150 μm possessed the greatest capability of osteogenic differentiation. This phenomenon was strongly correlated with MSC morphology, organization of actin cytoskeleton, and distribution of focal adhesion involving α2 and α5 integrins. Further silencing either α2 or α5 integrin significantly reduced the above mentioned mechanosensitivity, indicating that the α2 and α5 integrins as mechano-sensitive molecules mediate MSCs’ ability to provide enhanced osteogenic differentiation in response to different spherical SBCs. Taken together, the findings provide new insights regarding how MSCs respond to mechanical cues from the surrounding microenvironment in a spherical SBC, and such biophysical stimuli should be taken into consideration in tissue engineering and regenerative medicine in conjunction with biochemical cues. PMID:26884253

  11. Induced Pluripotent Mesenchymal Stromal Cell Clones Retain Donor-derived Differences in DNA Methylation Profiles

    PubMed Central

    Shao, Kaifeng; Koch, Carmen; Gupta, Manoj K; Lin, Qiong; Lenz, Michael; Laufs, Stephanie; Denecke, Bernd; Schmidt, Manfred; Linke, Matthias; Hennies, Hans C; Hescheler, Jürgen; Zenke, Martin; Zechner, Ulrich; Šarić, Tomo; Wagner, Wolfgang

    2013-01-01

    Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is an epigenetic phenomenon. It has been suggested that iPSC retain some tissue-specific memory whereas little is known about interindividual epigenetic variation. We have reprogrammed mesenchymal stromal cells from human bone marrow (iP-MSC) and compared their DNA methylation profiles with initial MSC and embryonic stem cells (ESCs) using high-density DNA methylation arrays covering more than 450,000 CpG sites. Overall, DNA methylation patterns of iP-MSC and ESC were similar whereas some CpG sites revealed highly significant differences, which were not related to parental MSC. Furthermore, hypermethylation in iP-MSC versus ESC occurred preferentially outside of CpG islands and was enriched in genes involved in epidermal differentiation indicating that these differences are not due to random de novo methylation. Subsequently, we searched for CpG sites with donor-specific variation. These “epigenetic fingerprints” were highly enriched in non-promoter regions and outside of CpG islands–and they were maintained upon reprogramming. In conclusion, iP-MSC clones revealed relatively little intraindividual variation but they maintained donor-derived epigenetic differences. In the absence of isogenic controls, it would therefore be more appropriate to compare iPSC from different donors rather than a high number of different clones from the same patient. PMID:23032973

  12. Tissue-specific Differentiation Potency of Mesenchymal Stromal Cells from Perinatal Tissues

    PubMed Central

    Kwon, Ahlm; Kim, Yonggoo; Kim, Myungshin; Kim, Jiyeon; Choi, Hayoung; Jekarl, Dong Wook; Lee, Seungok; Kim, Jung Min; Shin, Jong-Chul; Park, In Yang

    2016-01-01

    Human perinatal tissue is an abundant source of mesenchymal stromal cells(MSCs) and lacks the ethical concerns. Perinatal MSCs can be obtained from various tissues as like amnion, chorion, and umbilical cord. Still, little is known of the distinct nature of each MSC type. In this study, we successfully isolated and cultured MSCs from amnion(AMSCs), chorion(CMSCs), and umbilical cord(UC-MSCs). Proliferation potential was different among them, that AMSCs revealed the lowest proliferation rate due to increased Annexin V and senescence-associated β-galactosidase positive cells. We demonstrated distinct characteristic gene expression according to the source of the original tissue using microarray. In particular, genes associated with apoptosis and senescence including CDKN2A were up-regulated in AMSCs. In CMSCs, genes associated with heart morphogenesis and blood circulation including HTR2B were up-regulated. Genes associated with neurological system processes including NPY were up-regulated in UC-MSCs. Quantitative RT-PCR confirmed the gene expression data. And in vitro differentiation of MSCs demonstrated that CMSCs and UC-MSCs had a more pronounced ability to differentiate into cardiomyocyte and neural cells, respectively. This study firstly demonstrated the innate tissue-specific differentiation potency of perinatal MSCs which can be helpful in choosing more adequate cell sources for better outcome in a specific disease. PMID:27045658

  13. [Intracellular immunoglobulins in Namalwa and U266 cells co-cultivated with mesenchymal stromal cells].

    PubMed

    Aĭzenshtadt, A A; Ivanova, N A; Bagaeva, V V; Smolianinov, A B; Pinevich, A A; Samoĭlovich, M P; Klimovich, V B

    2014-01-01

    There are contradictory data concerning the influence of mesenchymal stromal cells (MSC) on immunoglobulin (Ig) production. Most of them were obtained using MSC from bone marrow. Properties of MSC from other tissues are elusive. In the present work MSC cultures were derived from umbilical cord, adipose tissue, and bone marrow of healthy donors, as well as from bone marrow of patients with autoimmune diseases. MSC from all these sources had similar surface markers phenotype. The influence of co-cultivation with MSC at exponential or stationary phase on IgM and IgE content in Namalva and U266 cells was evaluated. MSC from bone marrow of healthy donors had no effect on IgM and IgE production. Proliferating MSC obtained from patients with Crohn's disease and multiple sclerosis stimulated Ig production. Exponentially growing MSC derived from umbilical cord and adipose tissue also stimulated Ig synthesis. MSC at stationary cultures amplified IgM production in Namalva cells and suppressed IgE synthesis in U266. Thus, MSC with similar phenotype but derived from different sources differ in their capacity to modulate Ig production in B-lymphoid cells. The effect of MSC depends on their growth stage and may differ for lymphoblastoid and myeloma cells. PMID:25509151

  14. Bio- chemical and physical characterizations of mesenchymal stromal cells along the time course of directed differentiation.

    PubMed

    Chen, Yin-Quan; Liu, Yi-Shiuan; Liu, Yu-An; Wu, Yi-Chang; Del Álamo, Juan C; Chiou, Arthur; Lee, Oscar K

    2016-01-01

    Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds. PMID:27526936

  15. Mesenchymal Stem/Stromal Cells from Discarded Neonatal Sternal Tissue: In Vitro Characterization and Angiogenic Properties

    PubMed Central

    Wang, Shuyun; Mundada, Lakshmi; Colomb, Eric; Ohye, Richard G.; Si, Ming-Sing

    2016-01-01

    Autologous and nonautologous bone marrow mesenchymal stem/stromal cells (MSCs) are being evaluated as proangiogenic agents for ischemic and vascular disease in adults but not in children. A significant number of newborns and infants with critical congenital heart disease who undergo cardiac surgery already have or are at risk of developing conditions related to inadequate tissue perfusion. During neonatal cardiac surgery, a small amount of sternal tissue is usually discarded. Here we demonstrate that MSCs can be isolated from human neonatal sternal tissue using a nonenzymatic explant culture method. Neonatal sternal bone MSCs (sbMSCs) were clonogenic, had a surface marker expression profile that was characteristic of bone marrow MSCs, were multipotent, and expressed pluripotency-related genes at low levels. Neonatal sbMSCs also demonstrated in vitro proangiogenic properties. Sternal bone MSCs cooperated with human umbilical vein endothelial cells (HUVECs) to form 3D networks and tubes in vitro. Conditioned media from sbMSCs cultured in hypoxia also promoted HUVEC survival and migration. Given the neonatal source, ease of isolation, and proangiogenic properties, sbMSCs may have relevance to therapeutic applications. PMID:26770206

  16. Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy

    PubMed Central

    Bieback, Karen; Brinkmann, Irena

    2010-01-01

    Cell-based regenerative medicine is of growing interest in biomedical research. The role of stem cells in this context is under intense scrutiny and may help to define principles of organ regeneration and develop innovative therapeutics for organ failure. Utilizing stem and progenitor cells for organ replacement has been conducted for many years when performing hematopoietic stem cell transplantation. Since the first successful transplantation of umbilical cord blood to treat hematological malignancies, non-hematopoietic stem and progenitor cell populations have recently been identified within umbilical cord blood and other perinatal and fetal tissues. A cell population entitled mesenchymal stromal cells (MSCs) emerged as one of the most intensely studied as it subsumes a variety of capacities: MSCs can differentiate into various subtypes of the mesodermal lineage, they secrete a large array of trophic factors suitable of recruiting endogenous repair processes and they are immunomodulatory. Focusing on perinatal tissues to isolate MSCs, we will discuss some of the challenges associated with these cell types concentrating on concepts of isolation and expansion, the comparison with cells derived from other tissue sources, regarding phenotype and differentiation capacity and finally their therapeutic potential. PMID:21607124

  17. Concise review: mesenchymal stromal cells used for periodontal regeneration: a systematic review.

    PubMed

    Monsarrat, Paul; Vergnes, Jean-Noël; Nabet, Cathy; Sixou, Michel; Snead, Malcolm L; Planat-Bénard, Valérie; Casteilla, Louis; Kémoun, Philippe

    2014-06-01

    Periodontitis is a chronic infectious disease of the soft and hard tissues supporting the teeth. Recent advances in regenerative medicine and stem cell biology have paved the way for periodontal tissue engineering. Mesenchymal stromal cells (MSCs) delivered in situ to periodontal defects may exert their effects at multiple levels, including neovascularization, immunomodulation, and tissue regeneration. This systematic review had two goals: (a) to objectively quantify key elements for efficacy and safety of MSCs used for periodontal regeneration and (b) to identify patterns in the existing literature to explain differences between studies and suggest recommendations for future research. This systematic review provided good evidence of the capacity of MSCs to regenerate periodontal tissues in animals; however, experimentally generated defects used in animal studies do not sufficiently mimic the pathophysiology of periodontitis in humans. Moreover, the safety of such interventions in humans still needs to be studied. There were marked differences between experimental and control groups that may be influenced by characteristics that are crucial to address before translation to human clinical trials. We suggest that the appropriate combination of cell source, carrier type, and biomolecules, as well as the inclusion of critical path issues for a given clinical case, should be further explored and refined before transitioning to clinical trials. Future studies should investigate periodontal regenerative procedures in animal models, including rodents, in which the defects generated are designed to more accurately reflect the inflammatory status of the host and the shift in their pathogenic microflora. PMID:24744392

  18. Characterization of Gaucher disease bone marrow mesenchymal stromal cells reveals an altered inflammatory secretome

    PubMed Central

    Campeau, Philippe M.; Rafei, Moutih; Boivin, Marie-Noëlle; Sun, Ying; Grabowski, Gregory A.

    2009-01-01

    Gaucher disease causes pathologic skeletal changes that are not fully explained. Considering the important role of mesenchymal stromal cells (MSCs) in bone structural development and maintenance, we analyzed the cellular biochemistry of MSCs from an adult patient with Gaucher disease type 1 (N370S/L444P mutations). Gaucher MSCs possessed a low glucocerebrosidase activity and consequently had a 3-fold increase in cellular glucosylceramide. Gaucher MSCs have a typical MSC marker phenotype, normal osteocytic and adipocytic differentiation, growth, exogenous lactosylceramide trafficking, cholesterol content, lysosomal morphology, and total lysosomal content, and a marked increase in COX-2, prostaglandin E2, interleukin-8, and CCL2 production compared with normal controls. Transcriptome analysis on normal MSCs treated with the glucocerebrosidase inhibitor conduritol B epoxide showed an up-regulation of an array of inflammatory mediators, including CCL2, and other differentially regulated pathways. These cells also showed a decrease in sphingosine-1-phosphate. In conclusion, Gaucher disease MSCs display an altered secretome that could contribute to skeletal disease and immune disease manifestations in a manner distinct and additive to Gaucher macrophages themselves. PMID:19587377

  19. Effect of calcium phosphate ceramic substrate geometry on mesenchymal stromal cell organization and osteogenic differentiation.

    PubMed

    Urquia Edreira, Eva R; Hayrapetyan, Astghik; Wolke, Joop G C; Croes, Huib J E; Klymov, Alexey; Jansen, John A; van den Beucken, Jeroen J J P

    2016-01-01

    The composition of calcium phosphate (CaP) ceramics in combination with surface features have been shown to influence biological performance, and micro- and nano-scale topography is known to stimulate osteogenic differentiation of mesenchymal stromal cells (MSCs). In view of this, adipose tissue derived MSCs were cultured on CaP disks featuring hemispherical concavities of various sizes (440, 800 or 1800 μm diameter). It was hypothesized that (i) surface concavities would promote cell proliferation, cellular organization within the concavities, and osteogenic differentiation, as a result of a more pronounced 3D micro-environment and CaP nucleation in concavities, and (ii) MSC proliferation and osteogenic differentiation would increase with smaller concavity size due to more rapidly occurring 3D cell-cell interactions. We found that concavities indeed affect cell proliferation, with 440 μm concavities increasing cell proliferation to a larger extent compared to 800 and 1800 μm concavities as well as planar surfaces. Additionally, concavity size influenced 3D cellular organization within the concavity volume. Interestingly, concavity size promoted osteogenic differentiation of cells, as evidenced by increased osteocalcin gene expression in 440 μm concavities, and osteocalcin staining predominantly for 440 and 800 μm concavities, but not for 1800 μm concavities and only slightly for planar surface controls. PMID:27150445

  20. Current Perspectives in Mesenchymal Stromal Cell Therapies for Airway Tissue Defects

    PubMed Central

    Petrella, Francesco; Rizzo, Stefania; Borri, Alessandro; Casiraghi, Monica; Spaggiari, Lorenzo

    2015-01-01

    Lung cancer is the leading cause of cancer death and respiratory diseases are the third cause of death in industrialized countries; for this reason the airways and cardiopulmonary system have been the focus of extensive investigation, in particular of the new emerging branch of regenerative medicine. Mesenchymal stromal cells (MSCs) are a population of undifferentiated multipotent adult cells that naturally reside within the human body, which can differentiate into osteogenic, chondrogenic, and adipogenic lineages when cultured in specific inducing media. MSCs have the ability to migrate and engraft at sites of inflammation and injury in response to cytokines, chemokines, and growth factors at a wound site and they can exert local reparative effects through transdifferentiation and differentiation into specific cell types or via the paracrine secretion of soluble factors with anti-inflammatory and wound-healing activities. Experimental and clinical evidence exists regarding MSCs efficacy in airway defects restoration; although clinical MSCs use, in the daily practice, is not yet completely reached for airway diseases, we can argue that MSCs do not represent any more merely an experimental approach to airway tissue defects restoration but they can be considered as a “salvage” therapeutic tool in very selected patients and diseases. PMID:26167186

  1. Conditioned Medium From Human Amniotic Mesenchymal Stromal Cells Limits Infarct Size and Enhances Angiogenesis

    PubMed Central

    Danieli, Patrizia; Malpasso, Giuseppe; Ciuffreda, Maria Chiara; Cervio, Elisabetta; Calvillo, Laura; Copes, Francesco; Pisano, Federica; Mura, Manuela; Kleijn, Lennaert; de Boer, Rudolf A.; Viarengo, Gianluca; Rosti, Vittorio; Spinillo, Arsenio; Roccio, Marianna

    2015-01-01

    The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC-CM by cytokine array. Our results strongly support the concept that the administration of hAMC-CM favors the repair process after acute myocardial infarction. PMID:25824141

  2. Conditioned medium from human amniotic mesenchymal stromal cells limits infarct size and enhances angiogenesis.

    PubMed

    Danieli, Patrizia; Malpasso, Giuseppe; Ciuffreda, Maria Chiara; Cervio, Elisabetta; Calvillo, Laura; Copes, Francesco; Pisano, Federica; Mura, Manuela; Kleijn, Lennaert; de Boer, Rudolf A; Viarengo, Gianluca; Rosti, Vittorio; Spinillo, Arsenio; Roccio, Marianna; Gnecchi, Massimiliano

    2015-05-01

    The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC-CM by cytokine array. Our results strongly support the concept that the administration of hAMC-CM favors the repair process after acute myocardial infarction. PMID:25824141

  3. Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

    PubMed Central

    Osiecki, Michael J.; Michl, Thomas D.; Kul Babur, Betul; Kabiri, Mahboubeh; Atkinson, Kerry; Lott, William B.; Griesser, Hans J.; Doran, Michael R.

    2015-01-01

    Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs. PMID:26660475

  4. Interferon-{gamma} and NF-{kappa}B mediate nitric oxide production by mesenchymal stromal cells

    SciTech Connect

    Oh, I.; Ozaki, K. . E-mail: ozakikat@jichi.ac.jp; Sato, K.; Meguro, A.; Tatara, R.; Hatanaka, K.; Nagai, T.; Muroi, K.; Ozawa, K. . E-mail: kozawa@ms2.jichi.ac.jp

    2007-04-20

    Mesenchymal stromal cells (MSCs) have been shown to have an immunosuppressive effect. Previously, we demonstrated that nitric oxide (NO) is one of the immunomodulatory mediators of MSCs. We herein show that primary mouse bone marrow MSCs and three cell lines that mimic MSCs suppress both differentiation and proliferation in Th1 condition, whereas the suppression in Th2 condition is mild. NO production is inversely correlated with T cell proliferation in Th1 and Th2 conditions. NO is highly induced in Th1 and minimally induced in Th2. Moreover, an inhibitor of NO synthase restores both proliferation and interferon-{gamma} (IFN-{gamma}) production in Th1 condition. Furthermore, an anti-IFN-{gamma} antibody strongly inhibits NO production and an inhibitor of NF-{kappa}B reduces the level of induction of inducible NO synthase (iNOS) in MSCs. Taken together, our results suggest that NO plays a significant role in the modification of Th1 and Th2 differentiation by MSCs, and that both IFN-{gamma} and NF-{kappa}B are critical for NO production by MSCs.

  5. Toward Brain Tumor Gene Therapy Using Multipotent Mesenchymal Stromal Cell Vectors

    PubMed Central

    Bexell, Daniel; Scheding, Stefan; Bengzon, Johan

    2010-01-01

    Gene therapy of solid cancers has been severely restricted by the limited distribution of vectors within tumors. However, cellular vectors have emerged as an effective migratory system for gene delivery to invasive cancers. Implanted and injected multipotent mesenchymal stromal cells (MSCs) have shown tropism for several types of primary tumors and metastases. This capacity of MSCs forms the basis for their use as a gene vector system in neoplasms. Here, we review the tumor-directed migratory potential of MSCs, mechanisms of the migration, and the choice of therapeutic transgenes, with a focus on malignant gliomas as a model system for invasive and highly vascularized tumors. We examine recent findings demonstrating that MSCs share many characteristics with pericytes and that implanted MSCs localize primarily to perivascular niches within tumors, which might have therapeutic implications. The use of MSC vectors in cancer gene therapy raises concerns, however, including a possible MSC contribution to tumor stroma and vasculature, MSC-mediated antitumor immune suppression, and the potential malignant transformation of cultured MSCs. Nonetheless, we highlight the novel prospects of MSC-based tumor therapy, which appears to be a promising approach. PMID:20407426

  6. Mesenchymal Stem Cells with Increased Stromal Cell-Derived Factor 1 Expression Enhanced Fracture Healing

    PubMed Central

    Ho, Chih-Yuan; Hua, Jia; Coathup, Melanie; Kalia, Priya; Blunn, Gordon

    2015-01-01

    Treatment of critical size bone defects pose a challenge in orthopedics. Stem cell therapy together with cytokines has the potential to improve bone repair as they cause the migration and homing of stem cells to the defect site. However, the engraftment, participation, and recruitment of other cells within the regenerating tissue are important. To enhance stem cell involvement, this study investigated overexpression of stem cells with stromal cell-derived factor 1 (SDF-1) using an adenovirus. We hypothesized that these engineered cells would effectively increase the migration of native cells to the site of fracture, enhancing bone repair. Before implantation, we showed that SDF-1 secreted by transfected cells increased the migration of nontransfected cells. In a rat defect bone model, bone marrow mesenchymal stem cells overexpressing SDF-1 showed significantly (p=0.003) more new bone formation within the gap and less bone mineral loss at the area adjacent to the defect site during the early bone healing stage. In conclusion, SDF-1 was shown to play an important role in accelerating fracture repair and contributing to bone repair in rat models, by recruiting more host stem cells to the defect site and encouraging osteogenic differentiation and production of bone. PMID:25251779

  7. Alveolar macrophages are critical for the inhibition of allergic asthma by mesenchymal stromal cells.

    PubMed

    Mathias, Louisa J; Khong, Sacha M L; Spyroglou, Lisa; Payne, Natalie L; Siatskas, Christopher; Thorburn, Alison N; Boyd, Richard L; Heng, Tracy S P

    2013-12-15

    Multipotent mesenchymal stromal cells (MSCs) possess reparative and immunoregulatory properties, making them attractive candidates for cellular therapy. However, the majority of MSCs administered i.v. encounter a pulmonary impasse and soon disappear from the lungs, raising the question of how they induce such durable immunosuppressive effects. Using a mouse model of allergic asthma, we show that administration of MSCs isolated from human bone marrow, umbilical cord, or adipose tissue provoked a pronounced increase in alveolar macrophages and inhibited hallmark features of asthma, including airway hyperresponsiveness, eosinophilic accumulation, and Th2 cytokine production. Importantly, selective depletion of this macrophage compartment reversed the therapeutic benefit of MSC treatment on airway hyperresponsiveness. Our data demonstrate that human MSCs exert cross-species immunosuppressive activity, which is mediated by alveolar macrophages in allergic asthma. As alveolar macrophages are the predominant immune effector cells at the air-tissue interface in the lungs, this study provides a compelling mechanism for durable MSC effects in the absence of sustained engraftment. PMID:24249728

  8. Extracellular vesicles derived from mesenchymal stromal cells: a therapeutic option in respiratory diseases?

    PubMed

    Abreu, Soraia C; Weiss, Daniel J; Rocco, Patricia R M

    2016-01-01

    Extracellular vesicles (EVs) are plasma membrane-bound fragments released from several cell types, including mesenchymal stromal cells (MSCs), constitutively or under stimulation. EVs derived from MSCs and other cell types transfer molecules (such as DNA, proteins/peptides, mRNA, microRNA, and lipids) and/or organelles with reparative and anti-inflammatory properties to recipient cells. The paracrine anti-inflammatory effects promoted by MSC-derived EVs have attracted significant interest in the regenerative medicine field, including for potential use in lung injuries. In the present review, we describe the characteristics, biological activities, and mechanisms of action of MSC-derived EVs. We also review the therapeutic potential of EVs as reported in relevant preclinical models of acute and chronic respiratory diseases, such as pneumonia, acute respiratory distress syndrome, asthma, and pulmonary arterial hypertension. Finally, we discuss possible approaches for potentiating the therapeutic effects of MSC-derived EVs so as to enable use of this therapy in clinical practice. PMID:27075363

  9. Mesenchymal stromal cells loading curcumin-INVITE-micelles: a drug delivery system for neurodegenerative diseases.

    PubMed

    Tripodo, Giuseppe; Chlapanidas, Theodora; Perteghella, Sara; Vigani, Barbara; Mandracchia, Delia; Trapani, Adriana; Galuzzi, Marta; Tosca, Marta Cecilia; Antonioli, Barbara; Gaetani, Paolo; Marazzi, Mario; Torre, Maria Luisa

    2015-01-01

    This work reports on the formation of a carrier-in-carrier device for the systemic delivery and targeting of hydrophobic drugs mediated by micelle-loaded mesenchymal stromal cells (MSCs) (carrier-in-carrier) to be administered by intravenous injection. The innate ability of MSCs to reach injured tissues such as the central nervous system or other damaged tissues, is the key for the second order delivery and first order targeting. Inulin-D-alfa-tocopherol succinate micelles (INVITE M) are able to incorporate highly hydrophobic drugs and, due to their dimensions (≈7 nm diameter), to penetrate the cell membrane easily and quickly. This study demonstrates that the curcumin loaded micelles (INVITE MC), sterilized by filtration, reached the maximum loading in MSCs in few minutes and that the loading was concentration-dependent. When "naked" curcumin was used, an evident cytotoxicity on MSCs was detected, while INVITE micelles protected them from this effect. Moreover, MSCs loaded with INVITE MC are able to release the entrapped drug. This study strongly supports the feasibility of the carrier-in-carrier approach for the therapy of selected diseases, i.e., this innovative drug delivery system will be proposed for the treatment of the amyotrophic lateral sclerosis (ALS). PMID:25524221

  10. Human mesenchymal stromal cell-enhanced osteogenic differentiation by contact interaction with polyethylene terephthalate nanogratings.

    PubMed

    Antonini, Sara; Meucci, Sandro; Parchi, Paolo; Pacini, Simone; Montali, Marina; Poggetti, Andrea; Lisanti, Michele; Cecchini, Marco

    2016-01-01

    Among the very large number of polymeric materials that have been proposed in the field of orthopedics, polyethylene terephthalate (PET) is one of the most attractive thanks to its flexibility, thermal resistance, mechanical strength and durability. Several studies have been proposed that interface nano- or micro-structured surfaces with mesenchymal stromal cells (MSCs), demonstrating the potential of this technology for promoting osteogenesis. All these studies were carried out on biomaterials other than PET, which remains almost uninvestigated in terms of cell shaping, alignment and differentiation. Here, we study the effect of PET 350-depth nanogratings (NGs) with a ridge and lateral groove size of 500 nm (T1) or 1 μm (T2), on bone marrow-derived human MSC (hMSC) differentiation in relation to the osteogenic fate. We demonstrate that these substrates, especially T2, can promote the osteogenic phenotype more efficiently than standard flat surfaces and that this effect is more marked if cells are cultured in osteogenic medium than in basal medium. Finally, we show that the shape and disposition of calcium hydroxyapatite granules on the different substrates was influenced by the substrate symmetry, being more elongated and spatially organized on NGs than on flat surfaces. PMID:27388559

  11. Mesenchymal Stromal Cells but Not Cardiac Fibroblasts Exert Beneficial Systemic Immunomodulatory Effects in Experimental Myocarditis

    PubMed Central

    Miteva, Kapka; Pappritz, Kathleen; Westermann, Dirk; Schefold, Joerg C.; Fusch, Gerhard; Weithäuser, Alice; Rauch, Ursula; Becher, Peter-Moritz; Klingel, Karin; Ringe, Jochen; Kurtz, Andreas; Schultheiss, Heinz-Peter; Tschöpe, Carsten

    2012-01-01

    Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1β and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs. PMID:22815907

  12. Pre-differentiation of mesenchymal stromal cells in combination with a microstructured nerve guide supports peripheral nerve regeneration in the rat sciatic nerve model.

    PubMed

    Boecker, Arne Hendrik; van Neerven, Sabien Geraldine Antonia; Scheffel, Juliane; Tank, Julian; Altinova, Haktan; Seidensticker, Katrin; Deumens, Ronald; Tolba, Rene; Weis, Joachim; Brook, Gary Anthony; Pallua, Norbert; Bozkurt, Ahmet

    2016-02-01

    Many bioartificial nerve guides have been investigated pre-clinically for their nerve regeneration-supporting function, often in comparison to autologous nerve transplantation, which is still regarded as the current clinical gold standard. Enrichment of these scaffolds with cells intended to support axonal regeneration has been explored as a strategy to boost axonal regeneration across these nerve guides Ansselin et al. (1998). In the present study, 20 mm rat sciatic nerve defects were implanted with a cell-seeded microstructured collagen nerve guide (Perimaix) or an autologous nerve graft. Under the influence of seeded, pre-differentiated mesenchymal stromal cells, axons regenerated well into the Perimaix nerve guide. Myelination-related parameters, like myelin sheath thickness, benefitted from an additional seeding with pre-differentiated mesenchymal stromal cells. Furthermore, both the number of retrogradely labelled sensory neurons and the axon density within the implant were elevated in the cell-seeded scaffold group with pre-differentiated mesenchymal stromal cells. However, a pre-differentiation had no influence on functional recovery. An additional cell seeding of the Perimaix nerve guide with mesenchymal stromal cells led to an extent of functional recovery, independent of the differentiation status, similar to autologous nerve transplantation. These findings encourage further investigations on pre-differentiated mesenchymal stromal cells as a cellular support for peripheral nerve regeneration. PMID:26296589

  13. Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β.

    PubMed

    Mele, Valentina; Muraro, Manuele G; Calabrese, Diego; Pfaff, Dennis; Amatruda, Nunzia; Amicarella, Francesca; Kvinlaug, Brynn; Bocelli-Tyndall, Chiara; Martin, Ivan; Resink, Therese J; Heberer, Michael; Oertli, Daniel; Terracciano, Luigi; Spagnoli, Giulio C; Iezzi, Giandomenica

    2014-06-01

    Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells. PMID:24214914

  14. Intracoronary Delivery of Human Mesenchymal/Stromal Stem Cells: Insights from Coronary Microcirculation Invasive Assessment in a Swine Model

    PubMed Central

    Fiarresga, António; Mata, Márcia F.; Cavaco-Gonçalves, Sandra; Selas, Mafalda; Simões, Irina N.; Oliveira, Eunice; Carrapiço, Belmira; Cardim, Nuno; Cabral, Joaquim M. S.; Ferreira, Rui Cruz; da Silva, Cláudia L.

    2015-01-01

    Background Mesenchymal stem/stromal cells have unique properties favorable to their use in clinical practice and have been studied for cardiac repair. However, these cells are larger than coronary microvessels and there is controversy about the risk of embolization and microinfarctions, which could jeopardize the safety and efficacy of intracoronary route for their delivery. The index of microcirculatory resistance (IMR) is an invasive method for quantitatively assessing the coronary microcirculation status. Objectives To examine heart microcirculation after intracoronary injection of mesenchymal stem/stromal cells with the index of microcirculatory resistance. Methods Healthy swine were randomized to receive by intracoronary route either 30x106 MSC or the same solution with no cells (1% human albumin/PBS) (placebo). Blinded operators took coronary pressure and flow measurements, prior to intracoronary infusion and at 5 and 30 minutes post-delivery. Coronary flow reserve (CFR) and the IMR were compared between groups. Results CFR and IMR were done with a variance within the 3 transit time measurements of 6% at rest and 11% at maximal hyperemia. After intracoronary infusion there were no significant differences in CFR. The IMR was significantly higher in MSC-injected animals (at 30 minutes, 14.2U vs. 8.8U, p = 0.02) and intragroup analysis showed a significant increase of 112% from baseline to 30 minutes after cell infusion, although no electrocardiographic changes or clinical deterioration were noted. Conclusion Overall, this study provides definitive evidence of microcirculatory disruption upon intracoronary administration of mesenchymal stem/stromal cells, in a large animal model closely resembling human cardiac physiology, function and anatomy. PMID:26479722

  15. Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells

    PubMed Central

    2013-01-01

    Background Mesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3. Methods The tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed. Results The AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil. Conclusions Our work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses. PMID:24209831

  16. Effect of TGF-β1 Stimulation on the Secretome of Human Adipose-Derived Mesenchymal Stromal Cells

    PubMed Central

    Rodríguez, Tania M.; Saldías, Alejandro; Irigo, Marcelo; Zamora, Jorge Velasco; Perone, Marcelo J.

    2015-01-01

    Adipose tissue is an attractive source of mesenchymal stromal cells (MSCs) owing to the relative ease of obtaining large volumes with more MSC abundance compared with other sources. Increasing evidence supports the fact that trophic factors secreted by MSCs play a pivotal therapeutic role. Several strategies in regenerative medicine use MSCs, mainly exploiting their immunosuppressive effect and homing capacity to sites of damage. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that, depending on the cell niche, can display either anti-inflammatory or proinflammatory effects. TGF-β1 expression increases in various tissues with damage, especially when accompanied by inflammation. Thus, we analyzed the effect of TGF-β1 on the secretion by adipose-derived mesenchymal stromal cells (ASCs) of a panel of 80 cytokines/chemokines using an antibody array. To avoid a possible effect of fetal bovine serum (FBS) on ASCs secretion, we performed our analysis by culturing cells in FBS-free conditions, only supplemented with 0.1% of bovine serum albumin. We report the cytokine profile secreted by ASCs. We also found that TGF-β1 exposure modulates 8 chemokines and 18 cytokines, including TGF-β1 and -β2, and other important cytokines involved in immunosuppression, allergic responses, and bone resorption. Significance Mesenchymal stromal cells (MSCs) secrete a broad spectrum of bioactive macromolecules that are both immunoregulatory and serve to structure regenerative microenvironments in fields of tissue injury. Increases or decreases in the production of TGF-β1 have been linked to numerous disease states, including autoimmune diseases and cancer. The secretome of MSCs stimulated with TGF-β1 is largely unknown. Thus, the present study makes an important contribution toward a better understanding of how MSCs could be affected by a cytokine normally upregulated in various diseases. PMID:26025982

  17. Mesenchymal Stromal Cells Implantation in Combination with Platelet Lysate Product Is Safe for Reconstruction of Human Long Bone Nonunion

    PubMed Central

    Fazeli, Roghayeh; Mohseni, Fatemeh; Hosseini, Seyedeh Esmat; Moghadasali, Reza; Mardpour, Soura; Azimian, Vajiheh; Ghorbani Liastani, Maede; Mirazimi Bafghi, Ali; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser

    2016-01-01

    Objective Nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell (MSC) implantation in combination with platelet lysate (PL) product for treating human long bone nonunion. Materials and Methods In this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells (20 million cells implanted once into the nonunion site using a fluoroscopic guide) in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects (local or systemic, serious or non-serious, related or unrelated) were observed during this time period. Results From a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control Xray evidence, bony union had occurred. Conclusion Results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179). PMID:27602311

  18. Effects of Freeze-Thawing and Intravenous Infusion on Mesenchymal Stromal Cell Gene Expression.

    PubMed

    Hoogduijn, Martin J; de Witte, Samantha F H; Luk, Franka; van den Hout-van Vroonhoven, Mirjam C G N; Ignatowicz, Lech; Catar, Rusan; Strini, Tanja; Korevaar, Sander S; van IJcken, Wilfred F J; Betjes, Michiel G H; Franquesa, Marcella; Moll, Guido; Baan, Carla C

    2016-04-15

    Mesenchymal stromal cells (MSC) are increasingly used as an investigative therapeutic product for immune disorders and degenerative disease. Typically, MSC are isolated from human tissue, expanded in culture, and cryopreserved until usage. The safety and efficacy of MSC therapy will depend on the phenotypical and functional characteristics of MSC. The freeze-thawing procedure may change these characteristics. Furthermore, the cells encounter a microenvironment after administration that may impact their properties. It has been demonstrated that the majority of MSC localize to the lungs after intravenous infusion, making this the site to study the effects of the in vivo milieu on administered MSC. In this study, we investigated the effect of freeze-thawing and the mouse lung microenvironment on human adipose tissue-derived MSC. There were effects of freeze-thawing on the whole genome expression profile of MSC, although the effects did not exceed interdonor differences. There were no major changes in the expression of hemostatic regulators on transcriptional level, but significantly increased expression of procoagulant tissue factor on the surface of thawed adipose MSC, correlating with increased procoagulant activity of thawed cells. Exposure for 2 h to the lung microenvironment had a major effect on MSC gene expression and affected several immunological pathways. This indicates that MSC undergo functional changes shortly after infusion and this may influence the efficacy of MSC to modulate inflammatory responses. The results of this study demonstrate that MSC rapidly alter in response to the local milieu and disease-specific conditions may shape MSC after administration. PMID:26914168

  19. Assessment of human herpesvirus-6 infection in mesenchymal stromal cells ex vivo expanded for clinical use.

    PubMed

    Pessina, A; Bonomi, A; Coccè, V; Bernardo, M E; Cometa, A M; Ferrari, M; Sisto, F; Cavicchini, L; Locatelli, F

    2009-12-01

    Infection or reactivation of human herpesvirus (HHV)-6 represents a potentially serious complication (often involving the central nervous system) in patients receiving either solid organ or hematopoietic stem cell transplantation. The objective of this study was to assess the risk of HHV-6 infection/reactivation in mesenchymal stromal cells (MSCs). MSCs are multipotent cells displaying immunomodulatory properties that have been already successfully used in the clinical setting to enhance hematopoietic stem cell engraftment and to treat steroid-refractory acute graft-versus-host disease. We analyzed 20 samples of ex vivo expanded MSCs, at different passages of culture, isolated both from bone marrow and from umbilical cord blood. Through Western blotting and immunocytochemistry techniques, we investigated the presence of the HHV-6 receptor (CD46) on cell surface, whereas the presence of HHV-6 DNA was evaluated by nested polymerase chain reaction assay. All of the MSC samples tested were positive for the virus receptor (CD46), suggesting their potential susceptibility to HHV-6. However, none of the MSC samples derived from cultures, performed in the perspective of clinical use, was found to harbor HHV-6. This preliminary observation on a consistent number of MSC samples, some of them tested at late in vitro passages, indicates a good safety profile of the product in terms of HHV-6 contamination. Nevertheless, it remains important to set up in vitro experimental models to study MSCs' susceptibility to HHV-6 (and HHV-7) infection, to verify their capacity to integrate the virus into cellular DNA, and to investigate which experimental conditions are able to induce virus reactivation. PMID:19664021

  20. T cell subsets differently regulate osteogenic differentiation of human mesenchymal stromal cells in vitro.

    PubMed

    Grassi, Francesco; Cattini, Luca; Gambari, Laura; Manferdini, Cristina; Piacentini, Anna; Gabusi, Elena; Facchini, Andrea; Lisignoli, Gina

    2016-04-01

    T lymphocytes play a key role in the regulation of bone homeostasis and bone healing. The inflammatory response at the site of bone injury is essential to the initiation of the bone repair program; however, an uncontrolled exposure to inflammatory environment has a negative effect on tissue regeneration - indeed, activated T cells were shown to inhibit osteogenic differentiation on human mesenchymal stromal cells (MSCs). Whether resting T cells can induce osteogenic differentiation of MSCs and what role specific T cells subset play in this process is still elusive. In this study, we sought to analyse the osteogenic gene expression profile of whole T cells, CD4 and CD8 T cells isolated from healthy donors and investigated whether secreted factors from each group modulate osteogenic differentiation of human MSCs. Gene expression profiling identified a pool of 51 genes involved at various stages in bone growth which are expressed above detectable levels in CD4 and CD8 T cells. Most genes of this pool were expressed at higher levels in the CD4 subset. In vitro mineralization assays revealed that conditioned medium from CD4 T cells, but not from CD8 cells, significantly increased mineralization in osteogenic cultures of human MSCs; furthermore, mRNA expression of Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) in MSCs was significantly upregulated in the presence of CD4-conditioned medium but not with that obtained from CD8. The results show a differential role for CD4 and CD8 T cells in supporting bone formation and identify an osteogenic gene signature of each subset. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23653421

  1. Isolation and Characterization of Human Mesenchymal Stromal Cell Subpopulations: Comparison of Bone Marrow and Adipose Tissue.

    PubMed

    Busser, Hélène; Najar, Mehdi; Raicevic, Gordana; Pieters, Karlien; Velez Pombo, Rafael; Philippart, Pierre; Meuleman, Nathalie; Bron, Dominique; Lagneaux, Laurence

    2015-09-15

    Preparations of mesenchymal stromal cells (MSCs) are generally obtained from unfractionated tissue cells, resulting in heterogeneous cell mixtures. Several markers were proposed to enrich these cells, but the majority of these markers are defined for bone marrow (BM). Moreover, the surface markers of freshly isolated MSCs also differ from those of cultured MSCs in addition to a phenotypic variation depending on the MSC source. For tissue engineering applications, it is crucial to start with a well-defined cell population. In this study, we performed immunomagnetic selections with five single surface markers to isolate MSC subpopulations from BM and adipose tissue (AT): CD271, SUSD2, MSCA-1, CD44, and CD34. We determined the phenotype, the clonogenicity, the proliferation, the differentiation capacity, and the immunoregulatory profile of the subpopulations obtained in comparison with unselected cells. We showed that native BM-MSCs can be enriched from the positive fractions of MSCA-1, SUSD2, and CD271 selections. In contrast, we observed that SUSD2 and MSCA-1 were unable to identify MSCs from AT, meaning they are not expressed in situ. Only the CD34(+) selection successfully isolated MSCs from AT. Interestingly, we observed that CD271 selection can define AT cell subsets with particular abilities, but only in lipoaspiration samples and not in abdominoplasty samples. Importantly, we found a population of clear CD34(+) fresh BM-MSCs displaying different properties. A single marker-based selection for MSC enrichment should be more advantageous for cell therapy and would enable the standardization of efficient and safe therapeutic intervention through the use of a well-identified and homogeneous cell population. PMID:26086188

  2. Deterministic and stochastic approaches in the clinical application of mesenchymal stromal cells (MSCs)

    PubMed Central

    Pacini, Simone

    2014-01-01

    Mesenchymal stromal cells (MSCs) have enormous intrinsic clinical value due to their multi-lineage differentiation capacity, support of hemopoiesis, immunoregulation and growth factors/cytokines secretion. MSCs have thus been the object of extensive research for decades. After completion of many pre-clinical and clinical trials, MSC-based therapy is now facing a challenging phase. Several clinical trials have reported moderate, non-durable benefits, which caused initial enthusiasm to wane, and indicated an urgent need to optimize the efficacy of therapeutic, platform-enhancing MSC-based treatment. Recent investigations suggest the presence of multiple in vivo MSC ancestors in a wide range of tissues, which contribute to the heterogeneity of the starting material for the expansion of MSCs. This variability in the MSC culture-initiating cell population, together with the different types of enrichment/isolation and cultivation protocols applied, are hampering progress in the definition of MSC-based therapies. International regulatory statements require a precise risk/benefit analysis, ensuring the safety and efficacy of treatments. GMP validation allows for quality certification, but the prediction of a clinical outcome after MSC-based therapy is correlated not only to the possible morbidity derived by cell production process, but also to the biology of the MSCs themselves, which is highly sensible to unpredictable fluctuation of isolating and culture conditions. Risk exposure and efficacy of MSC-based therapies should be evaluated by pre-clinical studies, but the batch-to-batch variability of the final medicinal product could significantly limit the predictability of these studies. The future success of MSC-based therapies could lie not only in rational optimization of therapeutic strategies, but also in a stochastic approach during the assessment of benefit and risk factors. PMID:25364757

  3. The effect of local anesthetic on pro-inflammatory macrophage modulation by mesenchymal stromal cells.

    PubMed

    Gray, Andrea; Marrero-Berrios, Ileana; Weinberg, Jonathan; Manchikalapati, Devasena; SchianodiCola, Joseph; Schloss, Rene S; Yarmush, Joel

    2016-04-01

    Administering local anesthetics (LAs) peri- and post-operatively aims to prevent or mitigate pain in surgical procedures and after tissue injury in cases of osteoarthritis (OA) and other degenerative diseases. Innovative tissue protective and reparative therapeutic interventions such as mesenchymal stromal cells (MSCs) are likely to be exposed to co-administered drugs such as LAs. Therefore, it is important to determine how this exposure affects the therapeutic functions of MSCs and other cells in their target microenvironment. In these studies, we measured the effect of LAs, lidocaine and bupivacaine, on macrophage viability and pro-inflammatory secretion. We also examined their effect on modulation of the macrophage pro-inflammatory phenotype in an in vitro co-culture system with MSCs, by quantifying macrophage tumor necrosis factor (TNF)-α secretion and MSC prostaglandin E2 (PGE2) production. Our studies indicate that both LAs directly attenuated macrophage TNF-α secretion, without significantly affecting viability, in a concentration- and potency-dependent manner. LA-mediated attenuation of macrophage TNF-α was sustained in co-culture with MSCs, but MSCs did not further enhance this anti-inflammatory effect. Concentration- and potency-dependent reductions in macrophage TNF-α were concurrent with decreased PGE2 levels in the co-cultures further indicating MSC-independent macrophage attenuation. MSC functional recovery from LA exposure was assessed by pre-treating MSCs with LAs prior to co-culture with macrophages. Both MSC attenuation of TNF-α and PGE2 secretion were impaired by pre-exposure to the more potent bupivacaine and high dose of lidocaine in a concentration-dependent manner. Therefore, LAs can affect anti-inflammatory function by both directly attenuating macrophage inflammation and MSC secretion and possibly by altering the local microenvironment which can secondarily reduce MSC function. Furthermore, the LA effect on MSC function may persist

  4. Human Olfactory Mucosa Multipotent Mesenchymal Stromal Cells Promote Survival, Proliferation, and Differentiation of Human Hematopoietic Cells

    PubMed Central

    Diaz-Solano, Dylana; Wittig, Olga; Ayala-Grosso, Carlos; Pieruzzini, Rosalinda

    2012-01-01

    Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit–granulocyte/macrophage (CFU-GM) progenitors and CD34+ cells were found, at 43 days of co-culture. Reverse transcriptase–polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines. PMID:22471939

  5. Mesenchymal Stromal Cells Differentiating to Adipocytes Accumulate Autophagic Vesicles Instead of Functional Lipid Droplets.

    PubMed

    Gruia, Alexandra T; Suciu, Maria; Barbu-Tudoran, Lucian; Azghadi, Seyed Mohammad Reza; Cristea, Mirabela I; Nica, Dragos V; Vaduva, Adrian; Muntean, Danina; Mic, Ani Aurora; Mic, Felix A

    2016-04-01

    Adult bone marrow mesenchymal stromal cells (BMSCs) can easily be differentiated into a variety of cells. In vivo transplantation of BMSCs-differentiated cells has had limited success, suggesting that these cells may not be fully compatible with the cells they are intended to replace in vivo. We investigated the structural and functional features of BMSCs-derived adipocytes as compared with adipocytes from adipose tissue, and the structure and functionality of lipid vesicles formed during BMSCs differentiation to adipocytes. Gas chromatography-mass spectrometry showed fatty acid composition of BMSCs-derived adipocytes and adipocytes from the adipose tissue to be very different, as is the lipid rafts composition, caveolin-1 expression, caveolae distribution in their membranes, and the pattern of expression of fatty acid elongases. Confocal microscopy confirmed the absence from BMSCs-derived adipocytes of markers of lipid droplets. BMSCs-derived adipocytes cannot convert deuterated glucose into deuterated species of fatty acids and cannot uptake the deuterated fatty acid-bovine serum albumin complexes from the culture medium, suggesting that intra-cellular accumulation of lipids does not occur by lipogenesis. We noted that BMSCs differentiation to adipocytes is accompanied by an increase in autophagy. Autophagic vesicles accumulate in the cytoplasm of BMSCs-derived adipocytes and their size and distribution resembles that of Nile Red-stained lipid vesicles. Stimulation of autophagy in BMSCs triggers the intra-cellular accumulation of lipids, while inhibition of autophagy prevents this accumulation. In conclusion, differentiation of BMSCs-derived adipocytes leads to intra-cellular accumulation of autophagic vesicles rather than functional lipid droplets, suggesting that these cells are not authentic adipocytes. J. Cell. Physiol. 231: 863-875, 2016. © 2015 Wiley Periodicals, Inc. PMID:26332160

  6. Mesenchymal Stromal Cells Protect Endothelial Cells from Cytotoxic T Lymphocyte-Induced Lysis.

    PubMed

    Cahill, E F; Sax, T; Hartmann, I; Haffner, S; Holler, E; Holler, B; Huss, R; Günther, C; Parolini, O; Kolch, W; Eissner, G

    2016-09-01

    The integrity of the vasculature plays an important role in the success of allogeneic organ and haematopoietic stem cell transplantation. Endothelial cells (EC) have previously been shown to be the target of activated cytotoxic T lymphocytes (CTL) resulting in extensive cell lysis. Mesenchymal stromal cells (MSC) are multipotent cells which can be isolated from multiple sites, each demonstrating immunomodulatory capabilities. They are explored herein for their potential to protect EC from CTL-targeted lysis. CD8(+) T cells isolated from human PBMC were stimulated with mitotically inactive cells of a human microvascular endothelial cell line (CDC/EU.HMEC-1, further referred to as HMEC) for 7 days. Target HMEC were cultured in the presence or absence of MSC for 24 h before exposure to activated allogeneic CTL for 4 h. EC were then analysed for cytotoxic lysis by flow cytometry. Culture of HMEC with MSC in the efferent immune phase (24 h before the assay) led to a decrease in HMEC lysis. This lysis was determined to be MHC Class I restricted linked and further analysis suggested that MSC contact is important in abrogation of lysis, as protection is reduced where MSC are separated in transwell experiments. The efficacy of multiple sources of MSC was also confirmed, and the collaborative effect of MSC and the endothelium protective drug defibrotide were determined, with defibrotide enhancing the protection provided by MSC. These results support the use of MSC as an adjuvant cellular therapeutic in transplant medicine, alone or in conjunction with EC protective agents such as defibrotide. PMID:27384426

  7. Brief report: importance of SOX8 for in vitro chondrogenic differentiation of human mesenchymal stromal cells.

    PubMed

    Herlofsen, Sarah R; Høiby, Torill; Cacchiarelli, Davide; Zhang, Xiaolan; Mikkelsen, Tarjei S; Brinchmann, Jan E

    2014-06-01

    The transcription factor SOX9 is believed to be the master regulator of chondrogenesis. SOX8 is another SOX group E transcription factor with a high degree of homology to SOX9. Here, we demonstrate that SOX8 mRNA levels decrease during in vitro dedifferentiation of human articular chondrocytes and increase during chondrogenic differentiation of mesenchymal stromal cells. Knockdown of SOX9 reduced the expression of SOX8, COL2A1, and a range of other chondrogenic molecules. SOX8 knockdown reduced the expression of a large number of overlapping chondrogenic molecules, but not SOX9. Neither siSOX9 nor siSOX8 altered expression of the hypertrophic marker gene COL10A1. siSOX9, but not siSOX8 led to upregulation of hypertrophy associated genes MMP13 and ALPL. Transfection of synthetic SOX5, 6, and 9 mRNA trio upregulated SOX8, COL2A1, and ACAN, but not COL10A1 mRNA. Replacement of synthetic SOX9 by SOX8 in the SOX trio showed similar but lower chondrogenic effect. We conclude that SOX8 expression is regulated by SOX9, and that both together with SOX5 and SOX6 are required as a SOX quartet for transcription of COL2A1 and a large number of other chondrogenic molecules. Neither SOX8 nor SOX9 affect COL10A1 expression, but SOX9 inhibits chondrocyte hypertrophy through inhibition of MMP13 and ALPL expression. PMID:24449344

  8. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

    PubMed Central

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

  9. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation.

    PubMed

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

  10. Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation.

    PubMed

    Perico, Norberto; Casiraghi, Federica; Gotti, Eliana; Introna, Martino; Todeschini, Marta; Cavinato, Regiane Aparecida; Capelli, Chiara; Rambaldi, Alessandro; Cassis, Paola; Rizzo, Paola; Cortinovis, Monica; Noris, Marina; Remuzzi, Giuseppe

    2013-09-01

    Bone marrow-derived mesenchymal stromal cells (MSC) have emerged as useful cell population for immunomodulation therapy in transplantation. Moving this concept towards clinical application, however, should be critically assessed by a tailor-made step-wise approach. Here, we report results of the second step of the multistep MSC-based clinical protocol in kidney transplantation. We examined in two living-related kidney transplant recipients whether: (i) pre-transplant (DAY-1) infusion of autologous MSC protected from the development of acute graft dysfunction previously reported in patients given MSC post-transplant, (ii) avoiding basiliximab in the induction regimen improved the MSC-induced Treg expansion previously reported with therapy including this anti-CD25-antibody. In patient 3, MSC treatment was uneventful and graft function remained normal during 1 year follow-up. In patient 4, acute cellular rejection occurred 2 weeks post-transplant. Both patients had excellent graft function at the last observation. Circulating memory CD8(+) T cells and donor-specific CD8(+) T-cell cytolytic response were reduced in MSC-treated patients, not in transplant controls not given MSC. CD4(+) FoxP3(+) Treg expansion was comparable in MSC-treated patients with or without basiliximab induction. Thus, pre-transplant MSC no longer negatively affect kidney graft at least to the point of impairing graft function, and maintained MSC-immunomodulatory properties. Induction therapy without basiliximab does not offer any advantage on CD4(+) FoxP3(+) Treg expansion (ClinicalTrials.gov number: NCT 00752479). PMID:23738760

  11. Different Balance of Wnt Signaling in Adult and Fetal Bone Marrow-Derived Mesenchymal Stromal Cells.

    PubMed

    Paciejewska, Maja M; Maijenburg, Marijke W; Gilissen, Christian; Kleijer, Marion; Vermeul, Kim; Weijer, Kees; Veltman, Joris A; von Lindern, Marieke; van der Schoot, C Ellen; Voermans, Carlijn

    2016-06-15

    Mesenchymal stromal cells (MSCs) are applied as novel therapeutics for their regenerative and immune-suppressive capacities. Clinical applications, however, require extensive expansion of MSCs. Fetal bone marrow-derived MSCs (FBMSCs) proliferate faster than adult bone marrow-derived MSC (ABMSCs). To optimize expansion and function of MSC in general, we explored the differences between ABMSC and FBMSC. Gene expression profiling implicated differential expression of genes encoding proteins in the Wnt signaling pathway, including excreted inhibitors of Wnt signaling, particularly by ABMSC. Both MSC types had a similar basal level of canonical Wnt signaling. Abrogation of autocrine Wnt production by inhibitor of Wnt production-2 (IWP2) reduced canonical Wnt signaling and cell proliferation of FBMSCs, but hardly affected ABMSC. Addition of exogenous Wnt3a, however, induced expression of the target genes lymphocyte enhancer-binding factor (LEF) and T-cell factor (TCF) faster and at lower Wnt3a levels in ABMSC compared to FBMSC. Medium replacement experiments indicated that ABMSC produce an inhibitor of Wnt signaling that is effective on ABMSC itself but not on FBMSC, whereas FBMSC excrete (Wnt) factors that stimulate proliferation of ABMSC. In contrast, FBMSC were not able to support hematopoiesis, whereas ABMSC displayed hematopoietic support sensitive to IWP2, the inhibitor of Wnt factor excretion. In conclusion, ABMSC and FBMSC differ in their Wnt signature. While FBMSC produced factors, including Wnt signals, that enhanced MSC proliferation, ABMSC produced Wnt factors in a setting that enhanced hematopoietic support. Thus, further unraveling the molecular basis of this phenomenon may lead to improvement of clinical expansion protocols of ABMSCs. PMID:27154244

  12. Mesenchymal stromal cells derived from acute myeloid leukemia bone marrow exhibit aberrant cytogenetics and cytokine elaboration

    PubMed Central

    Huang, J C; Basu, S K; Zhao, X; Chien, S; Fang, M; Oehler, V G; Appelbaum, F R; Becker, P S

    2015-01-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play a fundamental role in the BM microenvironment (BME) and abnormalities of these cells may contribute to acute myeloid leukemia (AML) pathogenesis. The aim of the study was to characterize the cytokine and gene expression profile, immunophenotype and cytogenetics of BM-MSCs from AML patients compared to normal BM-MSCs from healthy donors. AML BM-MSCs showed decreased monocyte chemoattractant protein-1 levels compared to normal BM-MSCs. AML BM-MSCs expressed similar β1 integrin, CD44, CD73, CD90 and E-cadherin compared to normal BM-MSCs. Cytogenetic analysis revealed chromosomal aberrations in AML BM-MSCs, some overlapping with and others distinct from their corresponding AML blasts. No significant difference in gene expression was detected between AML BM-MSCs compared to normal BM-MSCs; however, comparing the differences between AML and MSCs from AML patients with the differences between normal hematopoietic cells and normal MSCs by Ingenuity pathway analysis showed key distinctions of the AML setting: (1) upstream gene regulation by transforming growth factor beta 1, tumor necrosis factor, tissue transglutaminase 2, CCAAT/enhancer binding protein alpha and SWItch/Sucrose NonFermentable related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4; (2) integrin and interleukin 8 signaling as overrepresented canonical pathways; and (3) upregulation of transcription factors FBJ murine osteosarcoma viral oncogene homolog and v-myb avian myeloblastosis viral oncogene homolog. Thus, phenotypic abnormalities of AML BM-MSCs highlight a dysfunctional BME that may impact AML survival and proliferation. PMID:25860293

  13. Immunomodulation of endothelial differentiated mesenchymal stromal cells: impact on T and NK cells.

    PubMed

    El Omar, Reine; Xiong, Yu; Dostert, Gabriel; Louis, Huguette; Gentils, Monique; Menu, Patrick; Stoltz, Jean-François; Velot, Émilie; Decot, Véronique

    2016-04-01

    Wharton's jelly mesenchymal stromal cells (WJ-MSCs) are promising candidates for tissue engineering, as their immunomodulatory activity allows them to escape immune recognition and to suppress several immune cell functions. To date, however, few studies have investigated the effect of differentiation of the MSCs on this immunomodulation. To address this question, we sought to determine the impact of differentiation toward endothelial cells on immunoregulation by WJ-MSCs. Following differentiation, the endothelial-like cells (ELCs) were positive for CD31, vascular endothelial cadherin and vascular endothelial growth factor receptor 2, and able to take up acetylated low-density lipoproteins. The expression of HLA-DR and CD86, which contribute to MSCs immunoprivilege, was still weak after differentiation. We then co-cultured un- and differentiated MSCs with immune cells, under conditions of both direct and indirect contact. The proliferation and phenotype of the immune cells were analyzed and the mediators secreted by both ELCs and WJ-MSCs quantified. Interleukin (IL)-6, IL-1β, prostaglandin E2 and in particular indoleamine-2,3-dioxygenase expression were upregulated in ELCs on stimulation by T and NK cells, suggesting the possible involvement of these factors in allosuppression. ELCs co-cultured with T cells were able to generate CD25(+) T cells, which were shown to be of the CD4(+)CD25(+)FoxP3(+) regulatory subset. Direct contact between NK cells and ELCs or WJ-MSCs decreased the level of NK-activating receptor natural-killer group 2, member D. Moreover, direct co-culturing with ELCs stimulates CD73 acquisition on NK cells, a mechanism which may induce adenosine secretion by the cells and lead to an immunosuppressive function. Taken together, our results show that ELCs obtained following differentiation of WJ-MSCs remain largely immunosuppressive. PMID:26510892

  14. miR-92a regulates angiogenic activity of adipose-derived mesenchymal stromal cells.

    PubMed

    Kalinina, Natalia; Klink, Galina; Glukhanyuk, Eugeniy; Lopatina, Tatiana; Efimenko, Anastassia; Akopyan, Zhanna; Tkachuk, Vsevolod

    2015-11-15

    Mesenchymal stromal cells including those from adipose tissue (MSCs) regulate angiogenesis in adult tissues. MicroRNAs (miRs), small noncoding RNAs that control gene expression by binding to target mRNAs, reducing their stability and/or inhibiting translation, appear to be important regulators of blood vessel growth. In this study, we examined the impact of angio-miRs on paracrine activities of MSCs. Using Illumina microarrays we found that miR-92a is one of the most abundant angio-miRs in human MSCs. We transfected MSC with pre-miR-92a or anti-miR-92a which led to the coordinated changes of known miR-92a target mRNA levels. Then we tested the ability of conditioned medium from transfected cells to stimulate tube formation by HUVECs. MSC overexpressing miR-92a completely lost the ability to stimulate tubes formation by endothelial cells. However, knocking-out miR-92a by transfection with anti-miR-92a did not increase the ability of MSC to stimulate tube formation. Secretion of hepatocyte growth factor (HGF) and angiopoetin-1 was significantly lower in the medium of miR-92a overexpressing MSC, whereas VEGF secretion did not change significantly. The replenishment of HGF but not angiopoietin-1 has restored the ability of conditioned medium from miR-92a overexpressing MSC to stimulate the tube formation. We conclude that overexpression of miR-92a in MSC suppresses angiogenic properties of these cells by down-regulation of HGF secretion. PMID:26477824

  15. Multiple myeloma mesenchymal stromal cells: Contribution to myeloma bone disease and therapeutics

    PubMed Central

    Garcia-Gomez, Antonio; Sanchez-Guijo, Fermin; del Cañizo, M Consuelo; San Miguel, Jesus F; Garayoa, Mercedes

    2014-01-01

    Multiple myeloma is a hematological malignancy in which clonal plasma cells proliferate and accumulate within the bone marrow. The presence of osteolytic lesions due to increased osteoclast (OC) activity and suppressed osteoblast (OB) function is characteristic of the disease. The bone marrow mesenchymal stromal cells (MSCs) play a critical role in multiple myeloma pathophysiology, greatly promoting the growth, survival, drug resistance and migration of myeloma cells. Here, we specifically discuss on the relative contribution of MSCs to the pathophysiology of osteolytic lesions in light of the current knowledge of the biology of myeloma bone disease (MBD), together with the reported genomic, functional and gene expression differences between MSCs derived from myeloma patients (pMSCs) and their healthy counterparts (dMSCs). Being MSCs the progenitors of OBs, pMSCs primarily contribute to the pathogenesis of MBD because of their reduced osteogenic potential consequence of multiple OB inhibitory factors and direct interactions with myeloma cells in the bone marrow. Importantly, pMSCs also readily contribute to MBD by promoting OC formation and activity at various levels (i.e., increasing RANKL to OPG expression, augmenting secretion of activin A, uncoupling ephrinB2-EphB4 signaling, and through augmented production of Wnt5a), thus further contributing to OB/OC uncoupling in osteolytic lesions. In this review, we also look over main signaling pathways involved in the osteogenic differentiation of MSCs and/or OB activity, highlighting amenable therapeutic targets; in parallel, the reported activity of bone-anabolic agents (at preclinical or clinical stage) targeting those signaling pathways is commented. PMID:25126382

  16. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Methods for ATMP Release.

    PubMed

    Radrizzani, Marina; Soncin, Sabrina; Lo Cicero, Viviana; Andriolo, Gabriella; Bolis, Sara; Turchetto, Lucia

    2016-01-01

    Mesenchymal stromal/stem cells (MSC) are promising candidates for the development of cell-based therapies for various diseases and are currently being evaluated in a number of clinical trials (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014). MSC for therapeutic applications are classified as advanced therapy medicinal products (ATMP) (Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004) and must be prepared according to good manufacturing practices ( http://ec.europa.eu/health/documents/eudralex/vol-4 ). They may be derived from different starting materials (mainly bone marrow (BM), adipose tissue, or cord blood) and applied as fresh or cryopreserved products, in the autologous as well as an allogeneic context (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014; Sensebé and Bourin, Transplantation 87(9 Suppl):S49-S53, 2009). In any case, they require an approved and well-defined panel of assays in order to be released for clinical use.This chapter describes analytical methods implemented and performed in our cell factory as part of the release strategy for an ATMP consisting of frozen autologous BM-derived MSC. Such methods are designed to assess the safety (sterility, endotoxin, and mycoplasma assays) and identity/potency (cell count and viability, immunophenotype and clonogenic assay) of the final product. Some assays are also applied to the biological starting material (sterility) or carried out as in-process controls (sterility, cell count and viability, immunophenotype, clonogenic assay).The validation strategy for each analytical method is described in the accompanying Chapter 20 . PMID:27236681

  17. Effect of Nano-HA/Collagen Composite Hydrogels on Osteogenic Behavior of Mesenchymal Stromal Cells.

    PubMed

    Hayrapetyan, Astghik; Bongio, Matilde; Leeuwenburgh, Sander C G; Jansen, John A; van den Beucken, Jeroen J J P

    2016-06-01

    This study aimed to comparatively evaluate the in vitro effect of nanosized hydroxyapatite and collagen (nHA/COL) based composite hydrogels (with different ratios of nHA and COL) on the behavior of human mesenchymal stromal cells (MSCs), isolated from either adipose tissue (AT-MSCs) or bone marrow (BM-MSCs). We hypothesized that (i) nHA/COL composite hydrogels would promote the osteogenic differentiation of MSCs in an nHA concentration dependent manner, and that (ii) AT-MSCs would show higher osteogenic potential compared to BM-MSCs, due to their earlier observed higher proliferation and osteogenic differentiation potential in 2D in vitro cultures [1]. The obtained results indicated that AT-MSCs show indeed high proliferation, differentiation and mineralization capacities in nHA/COL constructs compared to BM-MSCs, but this effect was irrespective of nHA concentration. Based on the results of alkaline phosphatase (ALP) activity and osteocalcin (OCN) protein level, the osteogenic differentiation of BM-MSCs started in the beginning of the culture period and for AT-MSCs at the end of the culture period. At a molecular level, both cell types showed high expression of osteogenic markers (bone morphogenic protein 2 [BMP2], runt-related transcription factor 2 [RUNX2], OCN or COL1) in both an nHA concentration and time dependent manner. In conclusion, AT-MSCs demonstrated higher osteogenic potential in nHA/COL based 3D micro-environments compared to BM-MSCs, in which proliferation and osteogenic differentiation were highly promoted in a time dependent manner, irrespective of nHA amount in the constructs. The fact that AT-MSCs showed high proliferation and mineralization potential is appealing for their application in future pre-clinical research as an alternative cell source for BM-MSCs. PMID:26803618

  18. Different wound healing properties of dermis, adipose, and gingiva mesenchymal stromal cells.

    PubMed

    Boink, Mireille A; van den Broek, Lenie J; Roffel, Sanne; Nazmi, Kamran; Bolscher, Jan G M; Gefen, Amit; Veerman, Enno C I; Gibbs, Susan

    2016-01-01

    Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis-, adipose-, and gingiva-derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin-derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation. PMID:26542883

  19. Mesenchymal stromal cells derived from acute myeloid leukemia bone marrow exhibit aberrant cytogenetics and cytokine elaboration.

    PubMed

    Huang, J C; Basu, S K; Zhao, X; Chien, S; Fang, M; Oehler, V G; Appelbaum, F R; Becker, P S

    2015-01-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play a fundamental role in the BM microenvironment (BME) and abnormalities of these cells may contribute to acute myeloid leukemia (AML) pathogenesis. The aim of the study was to characterize the cytokine and gene expression profile, immunophenotype and cytogenetics of BM-MSCs from AML patients compared to normal BM-MSCs from healthy donors. AML BM-MSCs showed decreased monocyte chemoattractant protein-1 levels compared to normal BM-MSCs. AML BM-MSCs expressed similar β1 integrin, CD44, CD73, CD90 and E-cadherin compared to normal BM-MSCs. Cytogenetic analysis revealed chromosomal aberrations in AML BM-MSCs, some overlapping with and others distinct from their corresponding AML blasts. No significant difference in gene expression was detected between AML BM-MSCs compared to normal BM-MSCs; however, comparing the differences between AML and MSCs from AML patients with the differences between normal hematopoietic cells and normal MSCs by Ingenuity pathway analysis showed key distinctions of the AML setting: (1) upstream gene regulation by transforming growth factor beta 1, tumor necrosis factor, tissue transglutaminase 2, CCAAT/enhancer binding protein alpha and SWItch/Sucrose NonFermentable related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4; (2) integrin and interleukin 8 signaling as overrepresented canonical pathways; and (3) upregulation of transcription factors FBJ murine osteosarcoma viral oncogene homolog and v-myb avian myeloblastosis viral oncogene homolog. Thus, phenotypic abnormalities of AML BM-MSCs highlight a dysfunctional BME that may impact AML survival and proliferation. PMID:25860293

  20. Cell surface structures influence lung clearance rate of systemically infused mesenchymal stromal cells.

    PubMed

    Nystedt, Johanna; Anderson, Heidi; Tikkanen, Jonne; Pietilä, Mika; Hirvonen, Tia; Takalo, Reijo; Heiskanen, Annamari; Satomaa, Tero; Natunen, Suvi; Lehtonen, Siri; Hakkarainen, Tanja; Korhonen, Matti; Laitinen, Saara; Valmu, Leena; Lehenkari, Petri

    2013-02-01

    The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells. PMID:23132820

  1. Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

    PubMed

    Neves, Sara C; Mota, Carlos; Longoni, Alessia; Barrias, Cristina C; Granja, Pedro L; Moroni, Lorenzo

    2016-06-01

    Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 μm to 1.950 ± 0.553 μm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 μm and 396.1 ± 32.3 μm, in the z-axis between 36.5 ± 5.3 μm and 70.7 ± 8.8 μm, average fiber diameters between 69.4 ± 6.1 μm and 99.0 ± 9.4 μm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 μm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 μm). PMID:27219645

  2. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    SciTech Connect

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  3. Phenotypic and immunomodulatory properties of equine cord blood-derived mesenchymal stromal cells.

    PubMed

    Tessier, Laurence; Bienzle, Dorothee; Williams, Lynn B; Koch, Thomas G

    2015-01-01

    Multipotent mesenchymal stromal cells (MSC) have attracted interest for their cytotherapeutic potential, partly due to their immunomodulatory abilities. The aim of this study was to test the robustness of our equine cord blood (CB) MSC isolation protocol, to characterize the CB-MSC before and after cryopreservation, and to evaluate their immunosuppressive phenotype. We hypothesized that MSC can be consistently isolated from equine CB, have unique and reproducible marker expression and in vitro suppress lymphoproliferation. Preliminary investigation of constitutive cytoplasmic Toll-like receptor (TLR) 3 and 4 expression was also preformed due to their possible association with anti- or pro-inflammatory MSC phenotypes, respectively. Surface markers were assessed for antigen and mRNA expression by flow cytometry and quantitative polymerase chain reaction (qPCR). Immunomodulatory properties were evaluated in mixed lymphocyte reaction assays, and TLR3 and TLR4 expression were measured by qPCR and immunocytochemistry (ICC). CB-MSC were isolated from each off nine cord blood samples. CB-MSC highly expressed CD29, CD44, CD90, and lacked or had low expression of major histocompatibility complex (MHC) class I, MHC-II, CD4, CD8, CD11a/18 and CD73 before and after cryopreservation. CB-MSC suppressed in vitro lymphoproliferation and constitutively expressed TLR4. Our findings confirmed CB as a reliable MSC source, provides an association of surface marker phenotype and mRNA expression and suggest anti-inflammatory properties of CB-MSC. The relationship between TLRs and lymphocyte function warrants further investigation. PMID:25902064

  4. Enzymatic Detachment of Therapeutic Mesenchymal Stromal Cells Grown on Glass Carriers in a Bioreactor

    PubMed Central

    Salzig, Denise; Schmiermund, Alexandra; P. Grace, Pablo; Elseberg, Christiane; Weber, Christian; Czermak, Peter

    2013-01-01

    Cell therapies require the in vitro expansion of adherent cells such as mesenchymal stromal cells (hMSCs) in bioreactor systems or other culture environments, followed by cell harvest. As hMSCs are strictly adherent cells, cell harvest requires cell detachment. The use of hMSCs for cell therapy requires GMP production in accordance with the guidelines for advanced therapeutic medical products. Therefore, several GMP-conform available proteolytic enzymes were investigated for their ability to promote hMSC detachment. An allogeneic hMSC cell line (hMSC-TERT) that is used in clinical trials in the form of alginate cell capsules was chosen as a model. This study investigated the influence of several factors on the outcome of proteolytic hMSC-TERT detachment. Therefore, hMSC-TERT detachment was analyzed in different cultivation systems (static, dynamic) and in combination with further cell processing including encapsulation. Only two of the commercially available enzymes (AccutaseTM, TrypZeanTM) that fulfill all process requirements (commercial availability, cost, GMP conditions during manufacturing and non-animal origin) are found to be generally suitable for detaching hMSC-TERT. Combining cell detachment with encapsulation demonstrated a high impact of the experimental set up on cell damage. It was preferable to reduce the temperature during detachment and limit the detachment time to a maximum of 20 minutes. Cell detachment in static systems was not comparable with detachment in dynamic systems. Detachment yields in dynamic systems were lower and cell damage was higher for the same experimental conditions. Finally, only TrypZeanTM seemed to be suitable for the detachment of hMSC-TERT from dynamic reactor systems. PMID:24478807

  5. Umbilical Cord Mesenchymal Stromal Cells Affected by Gestational Diabetes Mellitus Display Premature Aging and Mitochondrial Dysfunction

    PubMed Central

    Kim, Jooyeon; Piao, Ying; Pak, Youngmi Kim; Chung, Dalhee; Han, Yu Mi; Hong, Joon Seok; Jun, Eun Jeong; Shim, Jae-Yoon

    2015-01-01

    Human umbilical cord mesenchymal stromal cells (hUC-MSCs) of Wharton's jelly origin undergo adipogenic, osteogenic, and chondrogenic differentiation in vitro. Recent studies have consistently shown their therapeutic potential in various human disease models. However, the biological effects of major pregnancy complications on the cellular properties of hUC-MSCs remain to be studied. In this study, we compared the basic properties of hUC-MSCs obtained from gestational diabetes mellitus (GDM) patients (GDM-UC-MSCs) and normal pregnant women (N-UC-MSCs). Assessments of cumulative cell growth, MSC marker expression, cellular senescence, and mitochondrial function-related gene expression were performed using a cell count assay, senescence-associated β-galactosidase staining, quantitative real-time reverse transcription–polymerase chain reaction, immunoblotting, and cell-based mitochondrial functional assay system. When compared with N-UC-MSCs, GDM-UC-MSCs showed decreased cell growth and earlier cellular senescence with accumulation of p16 and p53, even though they expressed similar levels of CD105, CD90, and CD73 MSC marker proteins. GDM-UC-MSCs also displayed significantly lower osteogenic and adipogenic differentiation potentials than N-UC-MSCs. Furthermore, GDM-UC-MSCs exhibited a low mitochondrial activity and significantly reduced expression of the mitochondrial function regulatory genes ND2, ND9, COX1, PGC-1α, and TFAM. Here, we report intriguing and novel evidence that maternal metabolic derangement during gestation affects the biological properties of fetal cells, which may be a component of fetal programming. Our findings also underscore the importance of the critical assessment of the biological impact of maternal–fetal conditions in biological studies and clinical applications of hUC-MSCs. PMID:25437179

  6. Genetic stability of bone marrow-derived human mesenchymal stromal cells in the Quantum System

    PubMed Central

    Jones, Mark; Varella-Garcia, Marileila; Skokan, Margaret; Bryce, Steven; Schowinsky, Jeffrey; Peters, Rebecca; Vang, Boah; Brecheisen, Michelle; Startz, Thomas; Frank, Nathan; Nankervis, Brian

    2014-01-01

    Background aims The Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality. Methods A rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum. Results BM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation. Conclusions Quantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates. PMID:23992670

  7. [Therapeutic potential of human mesenchymal stromal cells secreted components: a problem with standartization].

    PubMed

    Sagaradze, G D; Grigorieva, O A; Efimenko, A Yu; Chaplenko, A A; Suslina, S N; Sysoeva, V Yu; Kalinina, N I; Akopyan, Zh A; Tkachuk, V A

    2015-01-01

    Regenerative medicine approaches, such as replacement of damaged tissue by ex vivo manufactured constructions or stimulation of endogenous reparative and regenerative processes to treat different diseases, are actively developing. One of the major tools for regenerative medicine are stem and progenitor cells, including multipotent mesenchymal stem/stromal cells (MSC). Because the paracrine action of bioactive factors secreted by MSC is considered as a main mechanism underlying MSC regenerative effects, application of MSC extracellular secreted products could be a promising approach to stimulate tissue regeneration; it also has some advantages compared to the injection of the cells themselves. However, because of the complexity of composition and multiplicity of mechanisms of action distinguished the medicinal products based on bioactive factors secreted by human MSC from the most of pharmaceuticals, it is important to develop the approaches to their standardization and quality control. In the current study, based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for the complex of extracellular products secreted by human adipose-derived MSC on key indicators, such as "Identification", "Specific activity" and "Biological safety". Developed approaches were tested on the samples of conditioned media contained products secreted by MSC isolated from subcutaneous adipose tissue of 30 donors. This strategy for the standardization of innovative medicinal products and biomaterials based on the bioactive extracellular factors secreted by human MSC could be applicable for a wide range of bioactive complex products, produced using the different types of stem and progenitor cells. PMID:26716748

  8. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    PubMed

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. PMID:24438898

  9. Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.

    PubMed

    Bieback, Karen; Hecker, Andrea; Kocaömer, Asli; Lannert, Heinrich; Schallmoser, Katharina; Strunk, Dirk; Klüter, Harald

    2009-09-01

    Mesenchymal stromal cells (MSCs) are promising candidates for novel cell therapeutic applications. For clinical scale manufacturing, human factors from serum or platelets have been suggested as alternatives to fetal bovine serum (FBS). We have previously shown that pooled human serum (HS) and thrombin-activated platelet releasate in plasma (tPRP) support the expansion of adipose tissue-derived MSCs. Contradictory results with bone marrow (BM)-derived MSCs have initiated a comprehensive comparison of HS, tPRP, and pooled human platelet lysate (pHPL) and FBS in terms of their impact on MSC isolation, expansion, differentiation, and immunomodulatory activity. In addition to conventional Ficoll density gradient centrifugation, depletion of lineage marker expressing cells (RosetteSep) and CD271+ sorting were used for BM-MSC enrichment. Cells were cultured in medium containing either 10% FBS, HS, tPRP, or pHPL. Colony-forming units and cumulative population doublings were determined, and MSCs were maximally expanded. Although both HS and tPRP comparable to FBS supported isolation and expansion, pHPL significantly accelerated BM-MSC proliferation to yield clinically relevant numbers within the first two passages. MSC quality and functionality including cell surface marker expression, adipogenic and osteogenic differentiation, and immunosuppressive action were similar in MSCs from all culture conditions. Importantly, spontaneous cell transformation was not observed in any of the culture conditions. Telomerase activity was not detected in any of the cultures at any passage. In contrast to previous data from adipose tissue-derived MSCs, pHPL was found to be the most suitable FBS substitute in clinical scale BM-MSC expansion. PMID:19544413

  10. Mesenchymal Stromal (Stem) Cell Therapy Fails to Improve Outcomes in Experimental Severe Influenza

    PubMed Central

    Darwish, Ilyse; Banner, David; Mubareka, Samira; Kim, Hani; Besla, Rickvinder; Kelvin, David J.; Kain, Kevin C.; Liles, W. Conrad

    2013-01-01

    Rationale Severe influenza remains a major public health threat and is responsible for thousands of deaths annually. Increasing antiviral resistance and limited effectiveness of current therapies highlight the need for new approaches to influenza treatment. Extensive pre-clinical data have shown that mesenchymal stromal (stem) cell (MSC) therapy can induce anti-inflammatory effects and enhance repair of the injured lung. We hypothesized that MSC therapy would improve survival, dampen lung inflammation and decrease acute lung injury (ALI) in a murine model of severe influenza. Methods C57Bl/6 mice were infected with influenza A/PuertoRico/8/34 (mouse-adapted H1N1) or influenza A/Mexico/4108/2009 (swine-origin pandemic H1N1) and administered human or mouse MSCs via the tail vein, either pre- or post- infection. MSC efficacy was evaluated as both an independent and adjunctive treatment strategy in combination with the antiviral agent, oseltamivir. Weight loss and survival were monitored. Inflammatory cells, cytokine/chemokines (IFN-γ, CXCL10, CCL2 and CCL5) and markers of ALI (total protein and IgM), were measured in bronchoalveolar lavage fluid and lung parenchyma. Results Administration of murine MSCs or human MSCs in a prophylactic or therapeutic regimen failed to improve survival, decrease pulmonary inflammation/inflammatory cell counts or prevent ALI in influenza virus-infected mice. MSCs administered in combination with oseltamivir also failed to improve outcomes. Conclusions Despite similarities in the clinical presentation and pathobiology of ALI and severe influenza, our findings suggest that MSC therapy may not be effective for prevention and/or treatment of acute severe influenza. PMID:23967240