Background: The Notch signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. This study was designed to determine the role of Notch signaling in adipogenic differentiation of human bone marrow derived MSCs (BM-MSCs). Methods: The Notch signaling was inhibited by the γ-secretase inhibitor N-[N-(3,5-difluor- ophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT). The markers involving adipogenic differentiation of MSCs, the relative pathway PTEN-PI3K/Akt/mTOR and autophagy activation were then analyzed. Furthermore, the autophagy inhibitor chloroquine (CQ) and 3-methyladenine (3-MA) were used to study the role of autophagy in the DAPT-induced the adipogenic differentiation of MSCs. Results: We first confirmed the down -regulation of Notch gene expression during MSCs adipocyte differentiation, and showed that the inhibition of Notch signaling significantly enhanced adipogenic differentiation of MSCs. Furthermore, Notch inhibitor DAPT induced early autophagy by acting on PTEN-PI3K/Akt/mTOR pathway. The autophagy inhibitor CQ and 3-MA dramatically abolished the effects of DAPT-induced autophagy and adipogenic differentiation of MSCs. Conclusion: Our results indicate that inhibition of Notch signaling could promote MSCs adipogenesis mediated by autophagy involving PTEN-PI3K/Akt/mTOR pathway. Notch signaling could be a novel target for regulating the adipogenic differentiation of MSCs.
IntroductionMesenchymal stem cells (MSCs) represent a heterogeneous cell population that is promising for regenerative medicine. The present study was designed to assess whether VCAM-1 can be used as a marker of MSC subpopulation with superior angiogenic potential.MethodsMSCs were isolated from placenta chorionic villi (CV). The VCAM-1+/− CV-MSCs population were separated by Flow Cytometry and subjected to a comparative analysis for their angiogenic properties including angiogenic genes expression, vasculo-angiogenic abilities on Matrigel in vitro and in vivo, angiogenic paracrine activities, cytokine array, and therapeutic angiogenesis in vascular ischemic diseases.ResultsAngiogenic genes, including HGF, ANG, IL8, IL6, VEGF-A, TGFβ, MMP2 and bFGF, were up-regulated in VCAM-1+CV-MSCs. Consistently, angiogenic cytokines especially HGF, IL8, angiogenin, angiopoitin-2, μPAR, CXCL1, IL-1β, IL-1α, CSF2, CSF3, MCP-3, CTACK, and OPG were found to be significantly increased in VCAM-1+ CV-MSCs. Moreover, VCAM-1+CV-MSCs showed remarkable vasculo-angiogenic abilities by angiogenesis analysis with Matrigel in vitro and in vivo and the conditioned medium of VCAM-1+ CV-MSCs exerted markedly pro-proliferative and pro-migratory effects on endothelial cells compared to VCAM-1−CV-MSCs. Finally, transplantation of VCAM-1+CV-MSCs into the ischemic hind limb of BALB/c nude mice resulted in a significantly functional improvement in comparison with VCAM-1−CV-MSCs transplantation.ConclusionsVCAM-1+CV-MSCs possessed a favorable angiogenic paracrine activity and displayed therapeutic efficacy on hindlimb ischemia. Our results suggested that VCAM-1+CV-MSCs may represent an important subpopulation of MSC for efficient therapeutic angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0297-0) contains supplementary material, which is available to authorized users.
Mesenchymal stem cells (MSCs), which are poorly immunogenic and have potent immunosuppressive activities, have emerged as a promising candidate for cellular therapeutics for the treatment of disorders caused by abnormal immune responses. In this study we investigated whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) could ameliorate colitis in a trinitrobenzene sulfonic acid (TNBS)-induced colitis model. TNBS-treated colitic mice were infused with hUC-MSCs or vehicle control. The mice were sacrificed on day 1, 3, and 5 after infusion, and their clinical and pathological conditions were evaluated by body weight, colon length, and histological analysis. The expression levels of proinflammatory cytokine proteins in colon were examined by ELISA. The homing of hUC-MSCs was studied by live in vivo imaging and immunofluorescent microscopy. hUC-MSCs were found to migrate to the inflamed colon and effectively treated the colitic mice with improved clinical and pathological signs. The levels of IL-17 and IL-23 as well as IFN-γ and IL-6 were significantly lower in the colon tissues of the hUC-MSC-treated mice in comparison with the vehicle-treated mice. Coculture experiments showed that hUC-MSCs not only could inhibit IFN-γ expression but also significantly inhibit IL-17 production by lamina propria mononuclear cells (LPMCs) or splenocytes of the colitic mice or by those isolated from normal animals and stimulated with IL-23. Systemically infused hUC-MSCs could home to the inflamed colon and effectively ameliorate colitis. In addition to the known suppressive effects on Th1-type immune responses, hUC-MSC-mediated modulation of IL-23/IL-17 regulated inflammatory reactions also plays an important role in the amelioration of colitis.
Th type 17 (Th17) cells have been identified as a proinflammatory T-cell subset. Here, we investigated the regulation of human Th17 cells by fetal BM-derived mesenchymal stem cells (FBM-MSC). We cocultured FBM-MSC with human PBMC or CD4 1 T cells from healthy donors. FBM-MSC significantly suppressed the proliferation of CD4 1 T cells stimulated by PHA and recombinant IL-2. Significantly higher levels of IL-17 were observed in FBM-MSC cocultured with either PBMC or CD4 1 T cells than that in PBMC cultured alone or CD4 1 T cells cultured alone. Flow cytometry analysis showed that the percentage of Th17 cells in coculture of FBM-MSC and CD4 1 T cells was significantly higher than that in CD4 1 T-cell cultured alone. FBM-MSC did not express IL-17 protein. Consistent with the augmentation of Th17 cells, significantly higher levels of IL-6 and IL-1 were observed in coculture of FBM-MSC and CD4 1 T cells than that in CD4 1 T-cell culture, while the levels of IL-23 were similar between FBM-MSC 1 PBMC coculture and PBMC alone, or FBM-MSC 1 CD4 1 T-cell and CD4 1 T-cell alone. The presence of FBM-MSC decreased the percentage of Th1 cells, but minimally affected the expansion of CD4 1 CD25 1 T cells. In conclusion, our data demonstrate for the first time that FBM-MSC promote the expansion of Th17 cells and decrease IFN-c-producing Th1 cells. These data suggest that IL-6 and IL-1, instead of IL-23, may be partly involved in the expansion of Th17 cells.Key words: Fetal BM-derived mesenchymal stem cells . IL-1 . IL-6 . Th1 . Th17 IntroductionMesenchymal stem cells (MSC) are multipotent stem cells found in many tissues in the body, such as BM, muscle, adipose, synovial membrane, amniotic fluid [1][2][3][4], umbilical cord, cord blood [5] and fetal tissues [6]. Accumulating evidence has shown that MSC perform an immune regulatory function partly by suppressing the proliferation of T cells in vitro [7][8][9]. MSC-induced immunotolerance has been used therapeutically for reducing GVH disease and for modulation of autoimmune disorders [10,11].Classically, effector CD4 1 Th cells have been categorized into two subsets: Th1 and Th2. Th1 cells produce IFN-g, which is à These authors contributed equally to this work. 2840required for the clearance of certain intracellular pathogens; while Th2 cells produce IL-4, which is required for the clearance of parasites [12]. Recently, a new effector CD4 1 T-cell lineage, Th17, has been identified in an EAE model. Th17 cells are characterized by their production of a distinct profile of cytokines, including IL-17, IL-17F and IL-6, and are thought to be involved in inflammatory and autoimmune diseases [13,14]. There have been many investigations in mouse Th17 cells, but the knowledge of human Th17 cells remains limited.So far little is known about the interaction of fetal BM-derived MSC (FBM-MSC) with immune cells, especially human Th17 cells. Therefore, in this study, we cocultured FBM-MSC with human PBMC or CD4 1 T cells and demonstrated for the first time that FBM-MSC had the capability to p...
In contrast to hematopoietic stem cells, there is still a lack of definitive cell markers for specific isolation and identification of mesenchymal stem cells (MSCs). Thus a homogenous population of MSCs is only obtained after several passages, when multilineage potential or other distinctive features of very early progenitors may be already somewhat compromised. Recently a novel surface marker the neural ganglioside GD2 has been reported to distinguish MSCs from all other cells within marrow. Here, we found that MSCs derived from umbilical cord (UC-MSCs) also expressed this marker at early-passages. More importantly, UC-MSCs were the only cells within umbilical cord expressing this marker. Compared to unsorted cells, GD2+-sorted cells not only possessed much higher clonogenicity and proliferation capacity but also had significantly stronger multi-differentiation potentials. Flow cytometric analysis revealed that GD2+-sorted cells showed increased expression of SSEA-4, Oct-4, Sox-2 and Nanog, the typical markers expressed in embryonic stem cells, in comparison to unsorted or GD2-negative MSCs. Take together, our data demonstrate that the cells selected by GD2 are a subpopulation of MSCs with feature of primitive precursor cells and provide evidence that GD2 can be a cell surface marker suitable for the isolation and purification of UC-MSCs in early-passage culture.
Neovasculogenesis induced by stem cell therapy is an innovative approach to improve critical limb ischemia (CLI) in diabetes. Mesenchymal stem cells (MSCs) are ideal candidates due to their angiogenic and immunomodulatory features. The aim of this study is to determine the therapeutic effects of human placenta-derived MSCs (P-MSCs) on diabetic CLI, with or without exogenous insulin administration, and the underlying mechanism of any effect. A series of in vitro experiments were performed to assess the stemness and vasculogenic activity of P-MSCs. P-MSCs were intramuscularly injected at two different doses with and without the administration of insulin. The efficacy of P-MSC transplantation was evaluated by ischemia damage score, ambulatory score, laser Doppler perfusion image (LDPI), capillary, and vascular density. In vivo imaging was applied to track the implanted P-MSCs. In vivo differentiation and in situ secretion of angiogenic cytokines were determined. In vitro experimental outcomes showed the differentiation potential and potent paracrine effect of P-MSCs. P-MSCs survived in vivo for at least 3 weeks and led to the acceleration of ischemia recovery, due to newly formed capillaries, increased arterioles, and secretion of various proangiogenic factors. P-MSCs participate in angiogenesis and vascularization directly through differentiation and cytokine expression.
Mesenchymal stem cells (MSC) have emerged as alternative sources of stem cells for regenerative medicine because of their multipotency and strong immune-regulatory properties. Also, human leukocyte antigen G (HLA-G) is an important mediator of MSC-mediated immunomodulation. However, it is unclear whether MSC retain their immune-privileged potential after differentiation. As promising candidates for cartilage tissue engineering, the immunogenic and immunomodulatory properties of chondro-differentiated MSC (chondro-MSC) require in-depth exploration. In the present study, we used the alginate/hyaluronic acid (Alg/HA) hydrogel scaffold and induced both bone marrow- and adipose tissue-derived MSC into chondrocytes in three-dimensional condition. Then, MSC before and after chondrocyte differentiation were treated or not with interferon γ and tumor necrosis factor α mimicking inflammatory conditions and were compared side by side using flow cytometry, mixed lymphocyte reaction, and immunostaining assays. Results showed that chondro-MSC were hypoimmunogenic and could exert immunosuppression on HLA-mismatched peripheral blood mononuclear cells as well as undifferentiated MSC did. This alloproliferation inhibition mediated by MSC or chondro-MSC was dose dependent. Meanwhile, chondro-MSC exerted inhibition on natural killer cell-mediated cytolysis. Also, we showed that HLA-G expression was upregulated in chondro-MSC under hypoxia context and could be boosted in allogenic settings. Besides, the Alg/HA hydrogel scaffold was hypoimmunogenic and its addition for supporting MSC chondrocyte differentiation did not modify the immune properties of MSC. Finally, considering their chondro-regenerative potential and their retained immunosuppressive capacity, MSC constitute promising allogenic sources of stem cells for cartilage repair.
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