The isolation culture and identification of HUCB MSCs in low serum condition

Objective To investigate the isolation, cultivation, differentiation and identification of human umbilical cord blood (UCB) mesenchymal stem cells(MSCs) in in low serum (2%) condition.and to observe the feasibility of using MSCs as seed cells in experimental furthermore.Methods UCB was collected on normal full term delivery of infants.Mononuclear cells were isolated from UCB by gravity centrifugation and cultured with low serum DMEM/F12medium to produce adherent layer.The morphology, proliferation curve, cell cycle, immunophenotype, osteogenic, adipogenic differentiation and the mRNA expression of some cytokines were investigated.Results The UCB-derived mononuclear cells, when set in culture, gave rise to adherent cells, which exhibited a fibroblast-like homogenous morphology. Cell cycle analysis revealed that more than 97% of MSC cells were in G0/G1 phase. Flowcytometry revealed that MSCs were negative for hematopoietic marker CD34, CD45, CD14, CD3、HLA-DR、endothelial cells marker CD31 and integrin CD11a,UCB-derived MSCs were found to be positive for CD29,CD44,CD90,CD73 and CD105,weak positive CD144、vWF and CD49d.Exposure of these cells to agents for inducing differentiation resulted in a formation of osteoblasts and adipocytes cells. RT-PCR analysis showed that the mRNA expression of cytokines such as IL-6, SCF, FL and G-CSF in UCB derived MSCs.Conclusion MSCs in UCB can be isolated and cultivated in low serum condition.which could be regarded as an alternative source of MSCs for further experimental and clinical applications.