Isolation and culture of bone marrow mesenchymal stem cells from human fetus and their biological properties

Abstract

AbstractThe population doubling number (70–80 times) of human fetal bone marrow mesenchymal stem cells (BMMSCs) is about two times higher than that (30–40 times) of adult BMMSCs, and their differentiation capacity is superior to that of their adult counterparts. In this study, BMMSCs were isolated from long bones of 2- to 3-month-old human abortuses by rinsing and selectively culturing whole marrow cells. Basic medium and serum concentration were optimized and growth curves plotted, both by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] reduction assay. Isolated cells were identified by flow cytometry and immunocytochemistry for their antigen markers. The biosafety of isolated cells was evaluated by karyotype analysis and a tumour-forming experiment. The results indicated that lengthwise scissoring of fetal long bones and rinsing of their marrow cells was practical and useful for recovery of BMMSCs from the investigated human abortuses. In this experiment, α-MEM (minimum essential medium, alpha medium)+20% FCS (fetal calf serum) was the best for in vitro culture of BMMSCs. The third-passage BMMSCs expressed Oct4, SSEA3 and SSEA4 besides the surface markers of their adult counterparts. The population doubling time of the BMMSCs of passage 6, 12 and 24 were 34, 36 and 40 h, respectively. Cells in all passages showed a diploid karyotype and formed no tumours in nude mice. The BMMSCs used in this study proved to be biologically safe and ideal seed cells for research on human tissue engineering and regeneration medicine.