Isolation And In Vitro Culture Of Human Embryonic Germ (EG) Cells

Human pluripotent stem cells were mainly derived form inner cell mass (ICM) of pre-implantation embryo and primordial germ cells (PGCs) of early post-implantation embryo through prolonged differentiation inhibited culture, and were named human embryonic stem (ES) cells and human embryonic germ (EG) cells respectively. These cells were able to propagate indefinitely and differentiated in to all cell type of three embryonic germ layers.This article focuses mainly on optimization of human EG cell isolation and in vitro culture system. Although 4 labs have reported the derivation of human EG cell lines after Shamblott et al first did in 1998, these culture systems still have some defects such as low derivation efficiency, highly spontaneous differentiation rate and xeno-material contamination, they were need to be further optimized.To raise the stability, derivation efficiency and safety for the future clinical application use of human EG cell culture system, we first establish an human feeder system, then analyzed some vital factors (included isolation method, cell factors, feeders, serum and embryo age) which affect the derivation of human EG cell lines, the proliferation of human EG cells in an xeno-free system was examined.1. Establishment of human feeder systemThe method for isolation and in vitro culture, cryopreservation-recovery and feeder preparation of human skin fibroblast (hSF) cells was established. 3 hSF cell lines from the skins of 3 human fetuses at 5-8 month age were established. Cells have no obvious change in vigor and morphology after cryopreservation -recovery and passaged for 15 times.2. Effect of isolation method, cell factors, serum, feeders and embryo age on the proliferation of human EG cellsEffect of different disaggregation method on the proliferation of human EG cells in primary culture was examined, the results show that gentle pipetting after digestion provided higher colony forming efficiency.Effect of LIF and/or bFGF on the proliferation of human EG cells was examined, the results show that LIF was essential for human EG cells proliferation in vitro, LIF and bFGF act through cooperation.