Basic biological characteristics, separation and purification of human mesenchymal stem cell derived from adult bone marrow

Objective To isolate human mesenchymal stem cell (MSC) from adult bone marrow and investigate its basic biological characteristics.Methods Human MSC was separated from adult human marrow by specific gravity 1.073 Ficoll reagent and expanded in culture medium.The proliferation and growth characteristics were observed in primary and passage culture.The surface antigens and cell cycle of human MSC were analysed by flow cytometer. The karyotype of human MSC was detected.Results The isolated human MSC comprised a single phenotypic population and displayed a fibroblast like morphology.Human MSC had a strong self renewal capacity.Three days later,MSC began to adhere to culture bottle and displayed spindle shape in culture.After ten days,it began to fuse as fibroblast like.After primary culture,approximately(4～6)×10 5 cells were obtained.After passage 10,(1～5)×10 10 cells were got.The proliferative ability decreased gradually after five or six passages.These expanded attached human MSC was positive for CD13?CD29?CD44?CD71 but did not express CD14?CD33?CD34?CD38?CD45?HLA DR.Karyotype of human MSC was normal.Analysis for cell cycle included G 0/G 1 phase:79.48%,G 2/M phase:6.10%,S phase:14.42%.Conclusion Human MSC has strong self renewal capacity and can be used as the seed cells for tissue engineering during passages3～6 in vitro.