In Vitro Study Of The Biologic Character Of Adipose Stromal Cell And Roles Of Its Immunomodulative Effect

PARTⅠComparison Study of Biologic Character of Human Adipose Stromal Cells and Bone Marrow Mesenchymal Stem Cells【Objective】This study aimed to compare the biologic character of adipose stromal cells (ASC) and bone marrow mesenchymal stem cells (MSC), construct a series of methods of isolation, purification, proliferation and identification of ASC, and make a foundation for cell therapy based on ASC.【Methods】Human adipose tissues were isolated, and digested with typeⅠcollagenase solution. Then the cells were cultured in plastic culture flasks. The numbers of cells were counted and cell growth curve were drawn. Flow cytometry and immunofluorescence were used to identify their phenotype. At the same time, mononuclear cells were isolated from human bone marrow and MSC were derived by cultivation. The numbers of ASC and MSC were counted before and after the cultivation. The surface phenotype of the two populations of cells was examined by flow cytometry. Effects of ASC or MSC on mixed lymphcyte response and PHA-induced lymphocyte transformation were investigated.【Results】The cells isolated from human adipose tissue displayed a fibroblast-like morphology adhering to the culturing plate 3 days later, and double counts time was 48 hours. The phenotype of ASC was CD31- CD34- CD45- HLA-DR- CD29+ CD105+ CD13+ CD44+. The numbers of nucleated cells isolated from bone marrow was significantly higher than those from adipose tissue (P<0.001), while no significant differences were observed for the yield of adherent stromal cells (P>0.05). Both cells expressed CD13,CD29,CD44,CD105. Differences in expression were noted for CD49d and CD106. The same number of ASC and MSC showed comparatively negative immunomodulative functions by inhibiting the mixed lymphocyte response and induction of transformation (P>0.05).【Conclusion】Adipose stromal cells were successfully cultured. ASC and MSC showed both similarities and differences in the features of cell surface markers and immunosuppressive properties. So ASC was some kind of cells quite different from MSC.PARTⅡMultiple Differentiative Potent of Adipose Stromal Cells【Objective】This study aimed to observe the capacity of human adipose stromal cells (ASC) to differentiate into cardiomyocyte-like cells, neuron-like cells, osteoblasts and adipocytes in vitro.【Methods】ASC was isolated and cultured, then induced by 5-Azacytidine (5-aza), all-trans retinoic acid (ATRA), various concentrations of dexamethasone combined with vitamine C andβ-glycerophosphate, respectively. Microscope was used to observe their morphology. Reverse-transcription polymerase chain reaction was used to detect the expression of ANP, cTnT, cTnI,αMHC and nestin. Immunofluorescence and western blot were used to detect the expression of cTnT, desmin and connexin43, respectively. Immunohistochemistry and Western blot were used to detect the expression of neurofilament (NF) and neuron specific enolase (NSE). Von Kossa staining was used to detect the deposit of calcium salts and Oil O staining to detect the leipo-drops.【Results】ASC expressed ANP, cTnT, cTnI,αMHC, desmin and connexin43 after adding 5-Aza in the culture system. Various passages of ASC all have the capacity of neuron differentiation. They expressed nestin, NF and NSE 10 days after adding ATRA in the culture system. ASC can also differentiate into osteoblasts and adipocytes under suitable conditions.【Conclusion】ASC has the character of proliferation and low-differentiation. Under suitable conditions, they can differentiate into cardiomyocyte-like cells, neuron-like cells, osteoblasts and adipocytes in vitro. ASC can be used as an alternative source of cells for tissue engineering.PARTⅢEffects of Adipose Stromal Cell on Allo-genetic T lymphocytes and Dendritic Cells and Its Possible Role【Objective】This study aimed to observe the effect of adipose stromal cells (ASC) on the phenotype and secreting cytokines of allogenetic T lymphocytes and dendritic cells (DC), and tried to elucidate the possible role of the immunomodulation of adipose stromal cell.【Methods】(1) The supernatant of ASC and ASC itself were co-cultured with allogenetic T lymphocytes, respectively. MTT assays were used to detect the proliferative rates of T cells. Annexin-Ⅴ/PI staining was used to detect the apoptotic rates, and flow cytometry to detect the proportion of CD4+CD25+ cells. Enzyme-linked immunoabsorbent assay was used to detect the secretion level of IL-10 and TGF-β1. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression level of FOXP3 gene.(2) The supernatant of ASC and ASC itself were co-cultured with allogenetic dendritic cells. Flow cytometry was used to detect the expression of CD80, CD83 and CD86. Enzyme-linked immunosorbent assay was used to detect the secretion level of IL-12, RT-PCR to detect the expression level of indoleamine 2, 3-dioxyenase (IDO).【Results】(1) The supernatant of ASC had no deep impact on the proliferation and apoptosis of T lymphocytes. ASC had the direct inhibitory effect on the growth of T lymphocytes, while did not affect the apoptosis. Both the supernatant of ASC and ASC itself can increase the proportion of CD4+CD25+ cells in T lymphocytes, and increase the level of IL-10 and TGF-β1. They can also up-regulate the expression level of FOXP3 gene.(2) The expression level of CD80, CD83 and CD86 on the surface of dendritic cells was down-regulated after the supernatant of ASC were added on the culture system of DC. The secretion of IL-12 was reduced, while the expression of IDO was up-regulated in the presence of ASC supernatant and ASC.【Conclusion】ASC can affect the function of T lymphocytes and DC via both cell-to-cell contact and secretion cytokines. Through these ways ASC showed a kind of negative immunomodulative effect and played an important role on inducing immune tolerance.