Abstract：ABSTRACT Aim 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. Especially, human BMMSCs from first-trimester fetus are closer to embryonic stem cells, show greater telomerase activity, more plasticity and faster propagation than the mid- and late-trimester fetal BMMSCs, thus being the ideal seed cells for human tissue engineering and regeneration medicine. The purposes of this study are to establish a method for isolating human BMMSCs from 2- to 3-month-old abortuses, select culture system for the cells, identify their biological properties and biosafety and provide seed cells for their tissue engineering and induced differentiation in vitro.
Methods This experiment was conducted in Shaanxi Branch of National Stem Cell Engineering and Technology Centre of China from April to December 2005. 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 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.
Results Lengthwise scissoring of fetal long bones and rinsing their marrow cells was practical and useful to recover the BMMSCs from human abortuses at the age of 2-3 months. In the present experiment, α-MEM+20%FBS was the best system for culturing such 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 h、36 h and 40h respectively. The cells in all the passages showed diploid karyotype and formed no tumor in the nude mice.
Conclusion The BMMSCs of human abortuses at the age of 2-3 months could be isolated and cultured in vitro and expressed some of the antigen markers of embryonic stem cells. Compared with their adult counterparts, fetal BMMSCs grew faster and their population doubling level was higher. They were proved to be biologically safe and ideal seed cells for researches on human tissue engineering and regeneration medicine.
