Medium Of Mesenchymal Stem Cells Research Articles

Retention of the stemness of mouse adipose-derived stem cells by their expansion on human bone marrow stromal cell-derived extracellular matrix.

Mesenchymal stem cells (MSCs) usually lose their stemness during in vitro expansion as they are deprived of their niche environment. Cell-extracellular matrix (ECM) interaction is known to play important roles in preserving the stemness of the cells in their stem cell niche environment. Previously, coating with bone marrow MSC (BMSC)-derived ECM was found able to maintain the differentiation potential of in vitro cultured MSCs. This study aimed to determine if this ECM coating could also maintain the stemness of cultured murine adipose-derived stem cells (ASCs) using a regular culture flask as a control. Cells were expanded in ECM-coated and ECM-noncoated flasks for two and four passages and then harvested for various analyses. The results showed that ASCs exhibited fibroblast-like spindle morphology in ECM-coated flasks, whereas ASCs gradually spread and enlarged in the ECM-noncoated flasks. After three and five passages, both groups of cells exhibited similar cytokinetics in the MSC culture medium (MesenPRO RS™ Medium). However, when cultured in Dulbecco's modified Eagles medium (DMEM) plus 10% fetal bovine serum, coating group cells exhibited more potent proliferation than control group cells with a significant difference in both passages 3 and 5 (p<0.01). When seeded at low density (500 cells/10-cm dish), coating group cells formed significantly more and larger sized cell colonies than control group cells with significant difference in cell colony numbers between two groups (p<0.05). In addition, coated colony cells were much smaller and more compactly arranged compared to control colony cells. Furthermore, ASCs expanded in coated flasks exhibited greater potentials for adipogenic, osteogenic, and chondrogenic differentiations than the cells expanded in regular flasks. Quantitatively, the Oil Red O staining area, Alizarin staining area, and Toluidine Blue staining area were all significantly larger than the respective staining areas of control cells (p<0.05). Real-time polymerase chain reaction also revealed significantly higher gene expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein (C/EBP), Runx2, osteocalcin, Sox9, collagen II, and aggrecan in ECM-coated group cells than in control group cells (p<0.05). Collectively, these results suggest that human BMSC decellular ECM coating helps to preserve the stemness of cultured murine ASCs.

Abstract P4-04-09: Mesenchymal stem cells and macrophage interactions promote inflammatory breast cancer cell invasion and self renewal

Abstract Background: Inflammatory breast cancer (IBC) is responsible for 10% of breast cancer deaths. The hallmarks of IBC are skin involvement and a high propensity to metastasize. Our lab has shown previously, "normal" breast tissue from women with an IBC diagnosis had significantly greater macrophage infiltration and increased cells with stem cell markers compared to non IBC "normal" breast tissue. These changes were present prior to diagnosis in two patients where pre-IBC biopsies were available. Therefore, we hypothesized changes in the normal breast microenvironment prior to tumor formation contributes to the IBC phenotype. Methods: To study our hypothesis we used a co-culture system to measure the interactions between normal macrophages (Raw 264.7 cell line), bone-marrow derived mesenchymal stem cells (MSCs), and IBC cells (SUM 149, IBC3, and KPL4). Conditioned media (CM) from MSC culture was added to macrophages overnight. The macrophages were subsequently analyzed for their surface markers and cytokine production. Reciprocally, MSCs were "educated" by macrophages by adding CM from polarized M2 macrophages to the MSC culture media at a 1:1 ratio. MSCs and cancer cells were co-cultured in trans-wells (Boyden chambers) for 24 hours. Migration and invasion in vitro was determined by adding MSCs or IBC cells to the insert of the trans-well and cultured in combination with either polarized macrophages or macrophage educated MSCs for 24 hours. After co-culture, IBC cells were analyzed for their ability to form mammospheres. Results: Mouse macrophages polarized (using IL-4) into a M2 phenotype (Tumor associated macrophage) secreted 3 fold more IL-6 compared to unstimulated or M1 polarized macrophages. The addition of MSC CM to the macrophage culture for 24 hours polarized the macrophages into a similar M2 phenotype described above with 4-fold increase in IL-6 secretion compared to unstimulated macrophages (P&amp;lt;0.01). When M2 macrophages at the bottom of the co-culture system were co-cultured with MSC, the number of MSCs migrating increased 2 fold with the addition of M2 macrophages compared to media alone, or unstimulated macrophages (P&amp;lt;0.05). IBC cells showed a 2 fold enhancement of invasion towards M2 educated MSCs compared to either uneducated MSCs, or media alone (P&amp;lt;0.05). IBC cells co cultured with M2 educated MSCs grew 3 fold more mammospheres compared to IBC cells grown with uneducated MSCs (P&amp;lt;0.01). IBC cells added to the bottom of the co-culture system also enhanced migration of MSCs and did so to a greater extent than non-IBC cells (P&amp;lt;0.01). Lastly, the addition of IL-6 neutralizing antibody inhibited IBC induced MSC migration. Conclusions: Herein we demonstrate reciprocal tumor interactions between normal cells in the IBC microenvironment. MSC and macrophages can influence each other to increase the tumor promoting influence of each on IBC cells. Our results suggest IL-6 a mediator of these tumor promoting influences and is important for the IBC induced migration of MSCs. Currently we are investigating the in vivo interactions between macrophages and MSCs in an orthotopic IBC mouse model. Citation Format: Adam R Wolfe, Rachel Atkinson, Bisrat G Debeb, Yan Zhang, Brian Ruffel, James M Reuben, Naoto T Ueno, Wendy A Woodward. Mesenchymal stem cells and macrophage interactions promote inflammatory breast cancer cell invasion and self renewal [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-09.

Mesenchymal Stem Cells & Endothelial Function

Mesenchymal Stem Cells & Endothelial Function

Mesenchymal stem cells-regulated Treg cells suppress colitis-associated colorectal cancer.

IntroductionPrevious studies have produced controversial results regarding whether mesenchymal stem cells (MSCs) promote or inhibit tumor development. Given the dual role of MSCs in inflammation and cancer, in this study the colitis-associated colorectal cancer (CAC) model was used to examine whether umbilical cord tissue-derived MSCs could prevent neoplasm by inhibiting chronic inflammation.MethodsMSCs were obtained and identified using flow cytometry. Colitis-associated colorectal cancer model was induced using azoxymethane (AOM) and dextran sulfate sodium (DSS) and MSCs were injected intravenously twice. Levels of immune cells in mesenteric lymph node including regulatory T (Treg) cells were detected using flow cytometry. Naïve T cells and Jurkat cells were co-cultured with MSCs and the effect of MSCs on Treg cells differentiation was evaluated.ResultsAfter injection through tail vein, MSCs could migrate to colon and suppress colitis-related neoplasm. This tumor suppressive effect was characterized by longer colon length, decreased tumor numbers and decreased expression of Ki-67. Moreover, MSCs alleviated the pathology of inflammation in the colitis stage of CAC model and inhibited inflammation cytokines both in colon and serum. Furthermore, Treg cells were accumulated in mesenteric lymph node of MSCs-treated mice while the percentage of T helper cells 2 (Th2) and Th17 were not changed. Of note, MSCs secreted transforming growth factor-β (TGF-β) enhanced the induction of Treg cells from naïve T cells. The conditioned medium of MSCs also activated Smad2 signaling, which has been reported to regulate Treg cells.ConclusionsThese results proved that MSCs could migrate to colon tissues and induce the differentiation of Treg cells via Smad2 as so to inhibit the colitis and suppress the development of CAC.

Odyssey of human dental pulp stem cells and their remarkable ability to survive in extremely adverse conditions.

Adult tissues contain stem cells, which proliferate to compensate for tissue loss throughout the life of the organism (Li and Clevers, 2010; Jimenez-Rojo et al., 2012). Therefore, adult stem cells are important for tissue repair and regeneration after injury. In seriously injured or carious teeth, stem cells residing in the dental pulp are responsible for the repair and regeneration of the damaged dental tissues (Caton et al., 2011; Mitsiadis et al., 2011). Dental pulp stem cells (DPSCs) can be isolated after labeling of the pulp cells with fluorescent mesenchymal stem cell (MSC) markers such as STRO-1, CD146, and CD44, followed by fluorescence-activated cell sorting (FACS) (Mitsiadis et al., 2012). Additional identification procedures include morphological criteria, adherence properties, proliferation, and differentiation potential, as well as tissue repair abilities of the sorted stem cell populations. The potential of DPSCs for the regeneration of bone tissue in patients has been demonstrated with a successful trial assay that has been realized in Napoli (Italy) several years ago (Caton et al., 2011; Mitsiadis et al., 2012). We have established a close scientific collaboration with the Napoli stem cell team headed by Prof. Gianpaolo Papaccio for the exchange of knowledge and materials. Several months ago, we have requested for a bunch of freshly isolated DPSCs in order to complete a number of experiments that we are currently realizing in our laboratories in Zurich (Switzerland). DPSCs were shipped with an express delivery company on the 26/11/2014 from Napoli with destination to Zurich. The shipped DPSCs were grown as a monolayer in a 25 cm2 culture flask containing the standard MSC culture medium. The flask that was sealed with Parafilm and left in ambient temperature should normally arrive to the destination the next day, but for unknown reasons the journey of DPSCs was longer and more exciting than expected. The DPSCs have followed an astonishing itinerary from Italy, to France (Paris), Great Britain, South Africa (Johannesburg), back to France, thereafter to Spain, France again. From France the cells finally arrived to Zurich Switzerland through Basel on the 4/12/2014. This itinerary can be considered as an Odyssey of DPSCs, which had to develop sophisticated mechanisms for their survival under these extremely adverse conditions. Sometimes discoveries are arising by chance, following a mistake or having an unexpected behavior. It was expected that the flask, upon its late arrival, would contain only dead cells. However, surprisingly, DPSCs (or a big part of them) were still alive after their journey (Figure ​(Figure1A).1A). DPSCs were extremely proliferative and became confluent within 3 days, after changing the medium (Figures 1B,C). How this could be explained? Recent findings indicate that quiescent (reserve) and proliferating (active) stem cell pools may coexist in separate but adjacent compartments of many tissues (Li and Clevers, 2010). It has been proposed that stem cells can adopt a reversible quiescence state characterized by reduced metabolic activity in conditions such as lack of oxygen and/or nutrients (Cheung and Rando, 2013). However, the mechanisms that allow quiescent stem cells to survive metabolic or environmental stress, to preserve their cellular and genomic integrity and to assure long-term survival are not yet elucidated. It has been suggested that quiescence is an actively maintained state regulated by intrinsic mechanisms to sustain metabolic function during persistent environmental stress, and thus ensuring stem cell survival (Cheung and Rando, 2013). Viable stem cells were found in post-mortem tissues (Latil et al., 2012). These cells were able to maintain their functional properties after prolonged storage in anoxia in vitro and after transplantation (Latil et al., 2012). Similarly, it has been shown that several stem cell populations reside in poorly oxygenated niches (Simsek et al., 2010). Quiescent stem cells have the ability to sense environmental changes and respond by re-entering the cell cycle for proliferation (Cheung and Rando, 2013). Severe hypoxia could be critical for maintaining the viability of DPSCs. It is possible that mechanisms compatible with the low metabolic state of quiescence have allowed rapid responses for DMSCs re-activation. Figure 1 Visualization of human dental pulp stem cells (hDPSCs): (A) upon arrival to Zurich (first passage), (B) after 6 h of culture (second passage), and (C) after 3 days of culture (second passage). Scale bars indicate the magnifications. This Odyssey revealed that DPSCs are able to survive for prolonged periods of time in conditions of extreme stress.

Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes

PurposeCorneal stromal cells transform to precursor cells in spheroid culture. We determined whether keratocytes derived from spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes.MethodsSpheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes.ResultsStromal cells formed spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in spheroids as compared to adherent cells whereas alpha-smooth muscle actin (α-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and α-SMA in spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the spheroids.ConclusionStemness transcription factor genes are upregulated in spheroids. Keratocytes derived from spheroids resemble tissue resident keratocytes, thus increasing manifolds the quantity of these cells for in-vitro experiments.

Embryonic stem cells conditioned medium enhances Wharton’s jelly-derived mesenchymal stem cells expansion under hypoxic condition

Mesenchymal stem cells (MSCs) are accepted as a promising tool for therapeutic purposes. However, low proliferation and early senescence are still main obstacles of MSCs expansion for using as cell-based therapy. Thus, clinical scale of cell expansion is needed to obtain a large number of cells serving for further applications. In this study, we investigated the value of embryonic stem cells conditioned medium (ESCM) for in vitro expansion of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) as compared to typical culture medium for MSCs, Dulbecco's modified Eagle's medium with 1.0g/l glucose (DMEM-LG) supplemented with 10% FBS, under hypoxic condition. The expanded cells from ESCM (ESCM-MSCs) and DMEM-LG (DMEM-MSCs) were characterized for both phenotype and biological activities including proliferation rate, population doubling time, cell cycle distribution and MSCs characteristics. ESCM and DMEM-LG could enhance WJ-MSCs proliferation as 204.66±10.39 and 113.77±7.89 fold increase at day 12, respectively. ESCM-MSCs could express pluripotency genes including Oct-4, Oct-3/4, Nanog, Klf-4, C-Myc and Sox-2 both in early and late passages whereas the downregulations of Oct-4 and Nanog were detected in late passage cells of DMEM-MSCs. The 2 cell populations also showed common MSCs characteristics including normal cell cycle, fibroblastic morphology, cell surface markers expressions (CD29(+), CD44(+), CD90(+), CD34(-), CD45(-)) and differentiation capacities into adipogenic, chondrogenic and osteogenic lineages. Moreover, our results revealed that ESCM exhibited as a rich source of several factors which are required for supportive WJ-MSCs proliferation. In conclusion, ESCM under hypoxic condition could accelerate WJ-MSCs expansion while maintaining their pluripotency properties. Our knowledge provide short term and cost-saving in WJ-MSCs expansion which has benefit to overcome insufficient cell numbers for clinical applications by reusing the discarded cell culture supernates from human ES culture system. Moreover, these findings can also apply for stem cell banking, regenerative medicine and pharmacological applications.

Abstract 4802: Human breast carcinoma-associated mesenchymal stem cells from primary breast cancer metastasis promote proliferation, invasion and mammosphere formation in breast cancer

Abstract Background: Accumulating evidence suggests that mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment; however controversy exists regarding their role in solid tumors. In this study, we identified the presence of carcinoma-associated MSCs (CA-MSCs) isolated from breast cancer metastasis to a human lymph node (LNM) and liver (LM). Methods: Tissue harvesting and culture: CA-MSCs were isolated from fresh samples of lymph nodes and liver breast cancer metastasis obtained from the operating room. A portion of each specimen was mechanically dissected and digested with collagenase. Red cells were lysed with ACK buffer and cell suspensions were cultured in Human Epithelial Medium and isolated adherent CA-MSCs were subculture in MSC medium for up to 12 passages for each experiment. Another portion of the tumor samples were subjected to IHC for H&amp;E staining. Differentiation assays: For bone differentiation cells were plated in StemPro Osteogenesis differentiation or control medium and allowed to grow for 14 days, and then washed and stained with Alizarin Red S. For cartilage differentiation cells were pelleted and grown in 15 ml tubes for 21 days, immobilized in histogel and stained with Alizarin Blue. For adipose differentiation cells were plated in StemCell differentiation and control medium, after 21 days the cells were stained with 0.3% Oil Red O. Proliferation, invasion, mammosphere assays, cytokine array and RT-PCR for EMT signature were performed seeding the same number of cells and comparing the single culture of CA-MSCs with co-culture of CA-MSCs and a panel of breast cancer cells lines Results: CA-MSCs had spindle morphology, normal karyotype, were nontumorigenic, and possessed tri-lineage differentiation ability (osteoblast, adipocyte, and chondrocyte). When co-cultured with breast cancer cells, CA-MSCs promoted proliferation, invasion, and mammosphere formation, increased IL6 and IL8 secretion into the media, and induced a gene expression profile of EMT when compared to the single cultures. Conclusion: We successfully isolated and characterized CA-MSCs, confirming their presence in human breast metastasis. We found that they provide a favorable microenvironment for tumor cell growth, enhance mammosphere formation, and promote an EMT signature and invasion in breast cancer cells. Citation Format: Maria E. Gonzalez, Kathy A. Toy, Celina G. Kleer. Human breast carcinoma-associated mesenchymal stem cells from primary breast cancer metastasis promote proliferation, invasion and mammosphere formation in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4802. doi:10.1158/1538-7445.AM2014-4802

Mesenchymal stem cells contribute to the chemoresistance of hepatocellular carcinoma cells in inflammatory environment by inducing autophagy.

BackgroundMesenchymal stem cells (MSCs) have been reported to play an important role in tumor growth. Inflammation is an important feature of hepatocellular carcinoma (HCC). Certain inflammatory cytokines produced in tumor microenvironment modulate functional activities of MSCs. At the present time, however, the role of MSCs in the development of HCC cell resistance to chemotherapy in the inflammatory microenvironment during tumor growth has not yet been identified.MethodsMTT and PI/Annexin V-FITC assay were employed to examine the proliferation and apoptosis of HCC cell lines. The expression of TGF-β are detected by Realtime PCR and Western blot. GFP tagged LC3 expression vector and electron microscopy are utilized to demonstrate the occurrence of autophagy.ResultsWe observed that MSCs pretreated with the combination of IFN-γ and TNF-α induced resistance to chemotherapy in HCC cell lines in both the in vitro and in vivo circumstances. Following exposure to conditioned medium of MSCs that were pre-treated with IFN-γ plus TNF-α, HCC cell line cells underwent autophagy which serves as a protective mechanism for HCC cells to resist the cell toxicity of chemotherapeutic agents. Treatment of HCC cell line cells with autophagy inhibitor effectively reversed the MSCs-induced resistance to chemotherapy in these cells. Stimulation with the combination of IFN-γ and TNF-α provoked expression of TGF-β by MSCs. MSCs-induced chemoresistance in HCC cell lines was correlated with the up-regulation of TGF-β expression by MSCs. Knockdown of TGF-β expression by MSCs with siRNA attenuated MSCs-induced chemoresistance in HCC cells.ConclusionsThese results suggest that increase in TGF-β expression by MSCs in the inflammatory microenvironment of HCC promotes the development of chemoresistance in HCC cells.

Mesenchymal stem cells infected with Mycoplasma arginini secrete complement C3 to regulate immunoglobulin production in B lymphocytes.

Mesenchymal stem cells (MSCs) have immunomodulatory functions such as the suppression of T and B cells. MSCs suppress immunoglobulin (Ig) production by B cells via cell–cell contact as well as via secretion of soluble factors. Our study showed that the conditioned medium (CM) of MSCs infected with a mycoplasma strain, Mycoplasma arginini, has marked inhibitory effects on Ig production by lipopolysaccharide/interleukin-4-induced B cells compared with mycoplasma-free MSC-CM. We analyzed mycoplasma-infected MSC-CM by fast protein liquid chromatography and liquid chromatography to screen the molecules responsible for Ig inhibition. Complement C3 (C3) was the most critical molecule among the candidates identified. C3 was shown to be involved in the suppression of the Ig production of B cells. C3 was secreted by mycoplasma-infected MSCs, but not by mycoplasma-free MSCs or B cells. It was able to directly inhibit Ig production by B cells. In the presence of a C3 inhibitor, Ig inhibition by MSC-CM was abrogated. This inhibitory effect was concomitant with the downregulation of B-cell-induced maturation protein-1, which is a regulator of the differentiation of antibody-secreting plasma cells. These results suggest that C3 secreted from mycoplasma-infected MSCs has an important role in the immunomodulatory functions of MSCs. However, its role in vivo needs to be explored.

Cultivation and cryopreservation of cord tissue MSCs with Cord Blood AB Plasma

Neonatal tissues, cord and placenta, are explored as alternate sources of mesenchymal stem cells (MSCs) for their therapeutic applications. Conventionally, MSCs isolated from cord tissues are maintained and propagated in FBS containing medium for promotion of growth and survival of cells. However, for therapeutic use, FBS use is not encouraged as it is of animal origin. Thus, there is a need for replacement of FBS by equally potent and clinically acceptable cost effective sources. The current study is designed to compare the effect of cord blood plasma (CBP) with MSC qualified FBS (M FBS) during culture and cryopreservation of MSCs. MSCs were isolated from cord and placenta and propagated in either M FBS or CBP. The efficiency of the cultures was analyzed by growth curve, morphology, phenotype and functionality. The cryo-protective role of the CBP was evaluated by using it in freezing medium of MSCs. Our data showed that CBP is equivalent to M FBS for culturing placental MSCs with respect to the phenotype, proliferation rate and differentiation to various lineages. However, cord MSCs displayed slow growth rate and reduction in surface expression of CD105 marker in CBP, whereas, the other parameters were comparable. Freezing of MSCs with CBP resulted in reduction of the late apoptotic and necrotic population. Thus, CBP imparts superior protection against cryogenic insults, and appears to be a valuable substitute to M FBS for cultivation and freezing of MSCs.

Progenitor cells suppress t-cell proliferation via a paracrine mechanism

Purpose: Limited treatment options are available for heart failure patients. Stem cell therapy has recently become a potential new way of achieving repair of injured cardiac tissue. Several different progenitor cell sources have been investigated, but most promising for cardiac therapy are mesenchymal stem cells (MSC) and cardiomyocyte progenitor cells (CMPC). Cardiac stem cell therapy using MSC or CMPC improved cardiac function, despite low engraftment of the cells. It is likely that paracrine factors, produced by the injected cells are causing these improvements. Many studies are performed on the paracrine effects, however, modulation of the immune response in cardiac stem cell therapy, especially the strong influence of T-lymphocytes on adverse remodeling, has not been explored extensively. Material and methods: Human fetal MSC and CMPC were characterized using flow cytometry and tested for multipotency by determining differentiation into all mesenchymal or cardiac lineages. The immunosuppressive properties of both cell types were tested in co-culture with freshly isolated, allogeneic peripheral blood mononuclear cells (PBMC) or T-lymphocytes stimulated with IL-2 and PMA. Proliferation was measured by CFSE-analysis using flow cytometry. Results: Proliferation of PBMC and T-lymphocytes was significantly reduced in the presence of MSC (65±8%) or CMPC (97±0.6%). In addition, the inflammatory cytokine panel of the cells in culture changed, with strong downregulation of IFN-gamma and TNF-alpha. This effect was observed in both direct cell contact as well as in transwell co-culture systems (MSC: 58±10%; CMPC: 62±9%). Transfer of conditioned medium from these co-cultures to unrelated, activated PBMC or T-lymphocytes abrogated proliferation in these cells to a similar extent as the original co-culture (MSC: 51±8%; CMPC: 97±0.7%). Interestingly, exosomes isolated from the conditioned medium of MSC and CMPC prevented T-lymphocyte proliferation in a dose-dependent fashion. At a concentration of 1.5μg, T-lymphocyte proliferation was significantly suppressed (MSC-exosomes: 73±12%; CMPC-exosomes: 77±10%). Conclusion: Both MSC and CMPC have a strong capacity for in vitro immunosuppression, which is mediated by paracrine factors. One potent immunosuppressive factor secreted by both MSC and CMPC are exosomes, which prevented T-lymphocyte proliferation in a dose-dependent fashion.

Abstract 003: Progenitor Cells Suppress T-Lymphocyte Proliferation Via a Paracrine Mechanism

PURPOSE: Limited treatment options are available for heart failure patients. Stem cell therapy has recently become a potential new way of repairing injured cardiac tissue. Different progenitor cell sources have been investigated, but most promising for cardiac therapy are mesenchymal stem cells (MSC) and cardiomyocyte progenitor cells (CMPC). Cardiac stem cell therapy using MSC or CMPC improved cardiac function, despite low engraftment of the cells. Paracrine factors, produced by the injected cells, presumably cause these improvements. Many studies are performed on the paracrine effects, yet modulation of the immune response in cardiac stem cell therapy, especially the strong influence of T-lymphocytes on adverse remodeling, has not been explored extensively. Methods: Human fetal MSC and CMPC were characterized and tested for multipotency. The immunosuppressive properties of both cell types were tested in co-culture with allogeneic peripheral blood mononuclear cells (PBMC) or T-lymphocytes stimulated with IL-2 and PMA. Proliferation was measured by CFSE-analysis using flow cytometry. Results: Proliferation of PBMC and T-lymphocytes was significantly reduced in the presence of MSC (65 ± 8%) or CMPC (97 ± 0.6%). In addition, production of inflammatory cytokines IFN-gamma and TNF-alpha was strongly downregulated. This effect was observed in both direct cell contact as well as in transwell co-culture systems (MSC: 58 ± 10%; CMPC: 62 ± 9%). Transfer of conditioned medium from these co-cultures to unrelated, activated PBMC or T-lymphocytes abrogated proliferation in these cells to a similar extent as the original co-culture (MSC: 51 ± 8%; CMPC: 97 ± 0.7%). Interestingly, exosomes isolated from the conditioned medium of MSC and CMPC prevented T-lymphocyte proliferation in a dose-dependent fashion. At a concentration of 1.5μg, T-lymphocyte proliferation was significantly suppressed (MSC-exosomes: 73 ± 12%; CMPC-exosomes: 77 ± 10%). Conclusion: Both MSC and CMPC have a strong capacity for in vitro immunosuppression, which is mediated by paracrine factors. One potent immunosuppressive factor secreted by both MSC and CMPC are exosomes, which prevented T-lymphocyte proliferation in a dose-dependent fashion.

Induce and differenctiation of marrow mesenchymal stem cell into photoreceptor-like cell in vitro and microenvironment

Background Marrow mesenchymal stem cells （MSCs） has been successful induced to differentiate into corneal cells,retinal ganglion cells （RGCs） and retinal neuron-like cells in recent years, but there are few studies about MSCs induced into photoreceptor cells and their microenvironment. Objective The aim of this study was to explore the induce and differentiation of bone marrow mesenehymal stem cells （BMSCs） into photoreeeptor-like cells in vitro and mieroenvironment. Methods The second generation of human BMSCs strain and RPE cells strain were cultured and passaged, respectively, and the fourth generation of BMSCs and the third generation of RPE cells were used in the experiment. BMSCs were eoeuhured using the mesenchymal stem cells medium （MSCM） contained 20 μg/L basic fibroblast growth factor （bFGF） ,20 μg/L epithelial growth factor （EGF） and 20μg/L brain derived neurotrophie factor （BDNF） with RPE cells to induce the differentiation of BMSCs in the induced group, and BMSCs were cultured in MSCM only in the control group. The morphology of induced and differentiated cells were observed under the inverted microscope. Inmmunocytoehemistry was used in induced for 3-, 5-,7-day cells to detect the expression rate of rhodopsin protein for the identification of phenotype of the differentiated cells. RT-PCR was used in induced for 5-,7-day cells to detect the expressions of rhodopsin mRNA and recoverin mRNA. Results Cultured BMSCs grew well with the spindle shape,and passaged RPE cells showed the uniform size and polygon shape with the abundant pigment in the cells. Some induced cells appeared to be the neuron-like cells with round shape and long prominence and the secondary reticular branches. The expression rates of rhodopsinin the cells were （5.83±0.29）%, （20. 36±0. 32）% and （29.80±2. 30）% in the third,fifth and seventh day after induce, which were significantly higher than （0.71 ±0. 35 ） % , （ 2. 56±0.24 ） % and （ 2. 32±0. 42 ） % of control cells （ t3 d = 41. 510, t5 d = 107. 290, t7 d = 30. 036, P〈0. 01 ）. The grey scales of rhodopsin mRNA and recoverin mRNA were significantly elevated in the induced and differentiated cells compared with control cells in the fifth and seventh day （ rhodopsin mRNA ：t5 d = 103. 506, t7 d = 122. 584, P〈0. 01 ;reeoverin mRNA ：t5 d = 106. 674, t7 d = 189. 992 ,P〈0. 01 ）. Conclusions BMSCs can be successfully indueed to differentiate into photoreeeptor cells after cocultured by conditioned medium with RPE cells. Key words: Mesenehymal stem cell; Bone marrow; Indueed and differentiation; Photoreceptor-like cell; Mieroenvironment; Conditioned medium; Rhodopsin; Reeoverin

Effective induction of cells expressing GABAergic neuronal markers from mouse embryonic stem cell

Successful derivations of specific neuronal and glial cells from embryonic stem cells have enormous potential for cell therapies and regenerative medicine. However, the low efficiency, the complexity of induction method, and the need for purification represent obstacles that make their application impractical. In this study, we found that PDGFRα(+) cells derived from mouse embryonic stem cells (mESC) can serve as a useful source from which to induce cells that express γ-aminobutyric-acid (GABA)-releasing (GABAergic) neuronal markers. PDGFRα(+) cells were induced from mESC on collagen IV-coated plates in mesenchymal stem cell (MSC) culture medium with limited exposure to retinoic acid, sorted by fluorescence-activated cell sorter and maintained in MSC culture medium containing Y-27632, a Rho-associated kinase inhibitor. We found that supplementation of vascular endothelial growth factor, fibroblast growth factor-basic, and sodium azide (NaN3) to MSC culture medium effectively differentiated PDGFRα(+) cells into cells that express GABAergic neuronal markers, such as Pax2, Dlx2, GAD67 NCAM, and tubulin-βIII, while markers for oligodendrocyte (Sox2) and astrocyte (Glast) were suppressed. Immunostaining for GABA showed the majority (86 ± 5%) of the induced cells were GABA-positive. We also found that the PDGFRα(+) cells retained such differentiation potential even after more than ten passages and cryopreservation. In summary, this study presents a simple and highly efficient method of inducing cells that express GABAergic neuronal markers from mESC. Together with its ease of maintenance in vitro, PDGFRα(+) cells derived from mESC may serve as a useful source for such purpose.

SAT0171 High oxidation status induced the rearrangement of F-ACTIN cytoskeleton of bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus via downregulation of RHOA signaling pathway

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease which has been described as a hematopoietic stem cell (HSC) disorder. Besides HSCs, bone marrow contains mesenchymal stem cells (MSCs) which is...

Human mesenchymal stem cells derived from adipose tissue reduce functional and tissue damage in a rat model of chronic renal failure

Therapeutic approaches for CKD (chronic kidney disease) have been able to reduce proteinuria, but not diminish the disease progression. We have demonstrated beneficial effects by injection of BM (bone marrow)-derived MSCs (mesenchymal stem cells) from healthy donors in a rat model with CKD. However, it has recently been reported that BM-MSCs derived from uraemic patients failed to confer functional protection in a similar model. This suggests that autologous BM-MSCs are not suitable for the treatment of CKD. In the present study, we have explored the potential of MSCs derived from adipose tissue (AD-MSCs) as an alternative source of MSCs for the treatment of CKD. We have isolated AD-MSCs and evaluated their effect on the progression of CKD. Adult male SD (Sprague-Dawley) rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×10(6) AD-MSCs or MSC culture medium alone. The therapeutic effect was evaluated by plasma creatinine measurement, structural analysis and angiogenic/epitheliogenic protein expression. AD-MSCs were detected in kidney tissues from NPX animals. This group had a significant reduction in plasma creatinine levels and a lower expression of damage markers ED-1 and α-SMA (α-smooth muscle actin) (P<0.05). In addition, treated rats exhibited a higher level of epitheliogenic [Pax-2 and BMP-7 (bone morphogenetic protein 7)] and angiogenic [VEGF (vascular endothelial growth factor)] proteins. The expression of these biomarkers of regeneration was significantly related to the improvement in renal function. Although many aspects of the cell therapy for CKD remain to be investigated, we provide evidence that AD-MSCs, a less invasive and highly available source of MSCs, exert an important therapeutic effect in this pathology.

High glucose mediates endothelial-to-chondrocyte transition in human aortic endothelial cells

BackgroundVascular calcification is one of the common complications in diabetes mellitus. Many studies have shown that high glucose (HG) caused cardiovascular calcification, but its underlying mechanism is not fully understood. Recently, medial calcification has been most commonly described in the vessels of patients with diabetes. Chondrocytes were involved in the medial calcification. Recent studies have shown that the conversion into mesenchymal stem cells (MSCs) via the endothelial-to-mesenchymal transition (EndMT) could be triggered in chondrocytes. Our previous research has indicated that HG induced EndMT in human aortic endothelial cells (HAECs). Therefore, we addressed the question of whether HG-induced EndMT could be transitioned into MSCs and differentiated into chondrocytes.MethodsHAECs were divided into three groups: a normal glucose (NG) group, HG group (30 mmol/L), and mannitol (5.5 mmol/L NG + 24.5 mmol/L) group. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of endothelial markers, such as CD31, and fibroblast markers, such as fibroblast-specific protein 1 (FSP-1). The expression of FSP-1 was detected by real time-PCR and western blots. Endothelial-derived MSCs were grown in MSC medium for one week. The expression of the MSCs markers STRO-1, CD44, CD10 and the chondrocyte marker SOX9 was detected by immunofluorescence staining and western blots. Chondrocyte expression was detected by alcian blue staining. Calcium deposits were analyzed by alizarin red staining.ResultsThe incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype. Double staining of the HAECs indicated a co-localization of CD31 and FSP-1. The expression of FSP-1 was significantly increased in the HG group, and the cells undergoing EndMT also expressed STRO-1, CD44 and SOX9 compared with the controls (P < 0.05). Additionally, alcian blue staining in the HG group was positive compared to the NG group. Consistent with the evaluation of SOX9 expression, calcium deposits analyzed by alizarin red staining were also enhanced by the HG treatment. Specifically, we showed that HG-induced EndMT is accompanied by the activation of the canonical Snail pathway.ConclusionsOur study demonstrated that HG could induce endothelial cells transdifferentiation into chondrocyte-like cells via the EndMT, which is mediated in part by the activation of the Snail signaling pathway.

Mesenchymal stem cells and cardiac regeneration: a sophisticated approach depends on trophic effects—what's left over? Focus on “Activation of Toll-like receptor 3 amplifies mesenchymal stem cell trophic factors and enhances therapeutic potency”

application of stem cells for tissue, in particular cardiac, regeneration represents a promising field of investigation but has not achieved clinical utility ([Fig. 1][1]). From the latter point of view, Strauer and colleagues ([7][2]) in 2001 achieved an important milestone by their administration

OC-158 Endogenous production of antibiotics by mesenchymal stem cells and the potential value in crohn's fistula healing

IntroductionPotent effects of human mesenchymal stem cells (MSCs) on immune responses have been demonstrated in vitro studies and animal models, encouraging further investigations into their clinical value. Studies of MSCs...