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

The population doubling number(70～80 times) of human fetal bone marrow mesenchymal stem cells(BMMSCs) is about two times more than that (30～40 times) of the adult BMMSCs, and their differentiation capacity is superior to that of their adult counterparts. In this study, BMMSCs were isolated from 2- to 3-month-old human abortuses by scissoring their long bones lengthwise, followed by rinsing and culturing whole marrow cells. Basic medium and serum concentration for BMMSCs culture were optimized and growth curves made, both with MTT(3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) reduction assay. Isolated cells were identified with flow-cytometry and immunocytochemistry for their antigen markers. The biosafety of isolated cells was evaluated by karyotype analysis and tumor forming experiment. The results indicated that lengthwise scissoring of fetal long bones and rinsing of their marrow cells was practical and useful to obtain the BMMSCs from human abortuses at the age of 2～3 months. In this experiment, α-MEM(minimum essential medium alpha medium) +20% FCS(fetal cattle serum) was the best system for the BMMSCs in vitro. The third passage BMMSCs expressed Oct4, SSEA3 and SSEA4 beside 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. The cells in all the passages showed diploid karyotype and formed no tumor in the nude mice. The BMMSCs of human abortuses at the age of 2～3 months were proved to be biologically safe and ideal seed cells for researches on human tissue engineering and regeneration medicine.