| Background:Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have a strong and more stable proliferation and differentiation, convenient source of donor without harm, low pathogen infection. hUC-MSCs have a high immunodulatory ability, which are allogeneic stem cells with clinical effects and safty. Currently, the traditional hUC-MSCs culture system containing a certain percentage of animal serum component (Serum Containing Medium) with potential safty problems in clinical therapy.The study compared the serum and serum-free culture system expanded hUC-MSCs’ proliferation, aging, the cell surface and totipotent stem cell markers,and multipotency, focusing on the difference between the two culture systems in immunodulatory,and explore the standardized culture methods to achieve clinical application standards.Objective:To verify that different individuals of umbilical mesenchymal stem cells are cultured in serum-free culture system security and immunomodulatory effects. Serum-free culture system can be safe used in clinical treatment.Methods:(1) comparison of hUC-MSCs in serum and serum-free culture system in vitro expansion and aging; detect the growth curve of hUC-MSCs by CCK-8 Kit; long-term culture to the senecence phase, using Cellular Sencence Assay Kit to detect age-related β-galactosidase activity; (2) detect the immunophenotype and the pluripotent marker expression of hUC-MSCs in serum and serum-free culture system in vitro immunophenotype and the pluripotent marker expression by flow cytometry; (3) compare the multipotency of hUC-MSCs in serum and serum-free culture system in vitro for a long-term culture; induce hUC-MSCs in serum and serum-free culture system differentiation in vitro:osteogenic, adipogenic, chondrogenic; (4) the activation and quiescent NK cells of healthy adults peripheral blood co-cultured were different individual hUC-MSCs which cultured in serum and serum-free culture system in vitro, detecting the%IFN-y-positive NK cells by flow cytometry;(5) Compare PBMC of of healthy adults peripheral blood were co-cultured with different individual hUC-MSCs which cultured in serum and serum-free culture system in vitro,detecting the inhibiton of PBMC proliferation by BrdU ELISA Kit; (6) Comparing the immune suppression of hUC-MSCs in serum and serum-free culture system in vitro, hUC-MSCs were co-cultured with healthy adult peripheral blood mononuclear cells,detected the secretion of IFN-γ,PGE2, IDO, IL-6 in culture media supernate using ELISA Kit; detecting the mRNA expression level of of immunodulatory factor TNF-betal, IL-10, IL-27, Galectin-1, Galectin-3, IDO1, COX2, HGF and IL-6 by Real-time PCR.Results:(1)serum-free culture of hUC-MSCs were typically long shuttle-like adherent cells,compared with hUC-MSCs cultured Serum-containing meida culture system, the growth curve is flat, logarithmic appeared later, and β-galactosidase activity occurd eralier; (2) serum-free culture of hUC-MSCs have a high expression of CD29, CD44, CD73 between CD90, CD 105 and CD 106 and other mesenchymal stem cell surface markers, do not express CD34, CD45, CD11b, CD19, HLA-DR, as well as hUC-MSCs cultured serum-containing meida culture system, but the totipotent markers of hUC-MSCs cultured in serum-free culture system have a higher positive rate; (3) hUC-MSCs cultured in serum-free culture and serum containing culture system have a similar multipotency (4) hUC-MSCs were cultured respectively with the activation and stationary phase NK cells, the expression of IFN-y-positive NK cells were significantly reduced by flow cytometry, have a stronger suppression to the stationary phase NK cells than the activation phase NK cells, both culture systems have no significant difference; (5) hUC-MSCs inhibited the proliferation of peripheral blood mononuclear cells, significantly inhibited IFN-y secretion of PBMC, the secretion of PGE2, IDO, IL-6 have no significantly difference between hUC-MSCs in serum and serum-free culture system, the secretion of HGF is higher in serum-free culture system; (6) compared with hUC-MSCs cultured Serum-containing meida culture system, hUC-MSCs cultured in serum-free culture system express higher HGF, TGF-beta1,IL-6 and lower Galectin-lin mRNA level.Conclusion:(1) hUC-MSCs cultured in serum-free culture system have a slower proliferation and aging earlier, compared with hUC-MSCs cultured in the traditional serum culture system in vitro; (2) the biological characteristics and the cell surface of serum-free culture hUC-MSCs are more similar to serum-containing culture hUC-MSCs.while serum-free culture hUC-MSCs have a higher totipotent markers; (3) serum-free culture and serum-containing culture hUC-MSCs have similar immunodulatory in adapt and innate immune.Background:Mesenchymal stem cells (MSCs) are pluripotent stem cells with highly self-renewal and multipotency, MSCs differentiate into nerve cells, osteoblast cells and other tissue systems under certain conditions.In addition, MSCs play a regulatory role in hematopoietic, immune, inflammatory response, angiogenesis and other important functions in the body.Now we put more concentration on the low immunogenicity and immunosuppression. Human umbilical cord mesenchymal stem cells (hUC-MSC) isolated from umbilical cord with higher accessibility and fewer ethical issues, have a potential to be scaled up as an alternative.The best example of allogeneic hUC-MSC immunosuppressive effect is effective in the treatment of severe graft-versus-host disease. In vitro studies have shown that hUC-MSC can inhibit T cell immune response. Current immunomodulatory mechanism of hUC-MSC is not vague, while more and more studies have shown that direct contact, mainly through such PD1-PD-L1, and soluble factors such as prostaglandin E2 (prostaglandin E2, PGE2),indole-2,3-Dioxide (indoleamine2,3-dioxygenase, IDO), Galectin-1 (Galectin-1, Gal-1), Galectin-3(Galectin-3, Gal-3), transforming growth factor-β (transforming growth factor-beta, TGF-P), HGF (hepatocyte growth factor, HGF) and ect exert immunosuppressive function. hUC-MSC therapy of autoimmune diseases, graft-versus-host disease has entered the stage of clinical trials and experimental studies with vast perspectives, but the differences of immunosuppressive among individuals immunomodulatory mechanism are large. hUC-MSC have been used in the clinical treatment for autoimmune diseases, but lack of a unified quantitative method for evaluating the ability of immunosuppression.Objective:Verify that the immunosuppressive of different individuals hUC-MSC immunosuppressive have a stable individual difference.Take human peripheral blood mononuclear co-culture system as a method to detect the effectiveness of hUC-MSC immunosuppressive have many uncertain factors, so we need to explore the the quantitative detection method,laying the foundation for clinical treatment.Methods:(1) different individuals hUC-MSC co-culture with healthy adult peripheral blood mononuclear cells after activation, detecting the secretion of IFN-y in culture media supernate by ELISA Kit,and the inhibiton ratio of IFN-y secretion represent the immunosuppression of hUC-MSC;(2) under the stimulation of inflammatory factors, hUC-MSCs are cultured for 24 hours in vitro, collecting the supernate.The secretion of PGE2, IDO, Galectin-1, Galectin-3, TGF-betal, HGF can be quantitative detection and study the correlation betweeen these secretion with the immunosuppression of hUC-MSC; (3) a specific inhibitor of indole amine 2,3-dioxygenase (IDO),1-M-Trp, prostaglandin E2 (PGE2) synthesis inhibitor NS-398,healthy adult peripheral blood mononuclear cells co-cultured with hUC-MSC, respectively blocked with inhibitor 1-M-Trp or NS-398, after culturing 72h,and detect the secretion of IFN-y in culture media supernate using ELISA Kit; (4)under the stimulation of inflammatory factors, different individuals hUC-MSC express immune-related factor by RT-PCR.Results:(1) inhibition level of different individuals hUC-MSC to the IFN-y secretion were different, and that significant differences exist stably. (2) a variety of cytokines expression are related to immunosuppression, while PGE2 has the highest positive correlation with immunosupression, IDO followed. (3) blocking PGE2, IDO secretion don’t completely reverse hUC-MSC immunosuppression. (4)under the stimulus of inflammatory cytokine,IDO1, COX2, TGF-betal, Galectin-1, HGF, IL-6, Galectin-3, and other immune-related factors significantly increased in mRNA level, IL-10,IL-27 don’t have clearly change.Conclusions:different individuals hUC-MSC have different immunosuppression, we seed 1.5×105/well hUC-MSCs in 24-well plates for 6 hours.Then supernatant was discarded, continuous culturing for 24 hours under the inflammatory cytokines IFN-y stimulation (5μg/ml)+TNF-α(1μg/ml) of, if PGE2 secretion of hUC-MSC>409.89ng /ml, designated as a stronger immunosuppression, which can be used in clinical treatment. |
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