| Mesenchymal stem cells (MSCs) are multipotent cells, which origin from the mesoderm and reside in most tissue. They can self-renew and differentiate into multiple lineages. As has been reported, MSC was first isolated from bone marrow and contented rarely. Bone marrow derived MSC has been shown to contamined with virus and age decreasing. Their proliferation and differentiation capacity have obviously decreased dependent the age. Recently, most studies determined that human umbilical cord had MSC, and MSC has been identified from umbilical cord of matrix, umbilical vein endothelial separation, and the placental membrane. The umbilical cord tissue constitute very simple, including superficial skin (amniotic epithelial cells), Wharton 's Jelly and vessels, which was neither capillary nor lymphatic, but the cell characteristics in amniotic blood vessels , vessels and vessels related area determined significantly difference by structural, immunohistochemical, and in vitro functional assay. Hence, our project aims to explore the effects of MSC derived from various human umbilical cord and compare their immunologic and hematopoietic function. It will be provided an important instruction for MSC in clinic therapy.First, we isolated the adherent cells from Amnion, Wharto 's Jelly and vessels by mechanical separation and collagenase digestion method. After adherent treatment, we got AM - MSC, WJ - MSC, VS– MSC, and UC-MSC. Using the density gradient centrifugation, the bone marrow MSC was purified. Morever, various derived MSC was compared and characterized by morphology, growth curve, cell cycle, pluripotentiality and immunophenotype. The result showed that five groups of MSC expressed the similar biological characteristics. 1) the cells displayed fibroblastoid morphology; 2) the rates of expansion potential was remarkably increased in vitro; 3) more than 80% of the cells were G0 / G1, indicating the typically stem cell proliferation characteristic and a few cells in active period. Otherwise, the proliferation potential of BM-MSC should be added the low concentration of bFGF, and the growth ability was reduced after 5 passages. For the UC-MSC, they could be proliferated under the low nutrition culture system; 4) Differentiation potential of UC-MSC indicated that it could be induced into adipocytes, osteocytes, and chondrocytes. The osteogenesis of BM-MSC was slightly lower comparing with UC-MSC; 5) Immunophenotypic analysis by FACS demonstrated that CD29, CD44, CD73, CD90, CD105, CD106, CD166, HLA-ABC and pluripotent stem cells antigen TRA-1-60 were positive, but was low or negative expression of CD14, CD31, CD34, HLA-DR, CD80, CD86, CD40 and CD40L. The expression of CD166 was significantly differences, and CD31, CD106 and HLA-DR was also expressed dramatic significantly differences.Secondly, the immunologic regulation effects was tested by the lymphocytic transformation and mixed lymphocyte reaction. The results indicated that UC-MSC could suppress T- lymphocyte proliferation in vitro when stimulated by nonspecific mitogens or allogeneic lymphocytess. This could be suppressed in a dose-dependent way by the addition of increasing numbers of UC-MSC to the culture system, resulting in a significantly lower response. Real-time PCR assay showed that the UC-MSC expressed a set of immunologic cytokines including IL-10, IL-11 and TGF-β1 and there was no significantly difference. For the IL-6, there was a highly significant difference between UC-MSC and the set of WJ,VS,AM,BM(P=0.0000). The same results was found in WJ-MSC and AM-MSC(p=0.008).Finally, to determine whether various UC-MSC was capable of supporting hematopoiesis. The various UC-MSC was as a feed layer and cocultured with CD34+ cells in free-serum culture system. The results demonstrated that UC-MSC could expand hematopoietic cells in vitro, there was displayed a significantly difference comparing with control groups. Furthermore, the total number of mononuclear cells, CD34+ cells, and CFC was evalued after 7 days of inoculation. Consistant with previous reports, the expansion magnitude of total nucleated cells, CD34+ cells and CFC by UC-MSC was far higher than WJ,VS and AM derived MSC. However, there was no significant difference in CFU-Mix. Comparing the other 3 groups, BFU-E and CFU-GM generated from WJ-MSC and VS-MSC were increased than that in AM-MSC. Real-time PCR assay showed that UC-MSC expressed hematopoietic cytokines IL-3, IL-11, TGF-β1, HGF, TPO, EPO, VEGF, M-CSF and G-CSF. Comparing the cytokines expression in various groups, IL-6, SCF, FLT-3 and LIF in UC-MSC displayed more high quality than the other groups.These data suggest that the biological capacity and immunologic effects of umbilical cord derived MSC were strikingly similar to those in human bone marrow. However, hematopoiesis supporting function was different. Further MSC data are required to confirm its function and clinical application. As the seed cells for tissue engineering, the mechanism underlying various tissue derived MSC requires further investigation using appropriate method. |
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