Separation And Culture Of Human Primordial Germ Cell(PGC)

Human pluripotent stem cells, which include embryonic stem (ES) cells which can be derived from the preimplanted blastocyst and embryonic germ (EG) cells which can be derived from the early embryo, are undifferentiated pluripotential stem cells that have unlimited growth and differentiation potential. These cells have broad application in basic research and transplantation due to their characteristics. Especially, the potential to produce any types of replacement cells, tissues, and organs for clinical treatment would revolutionize medicine.We separated human PGC from genital ridge in 5-9 weeks human embryo, and studied the cultivation and the differentiation in vitro.1. The separation of primary mouse embryonic fibroblast (PMEF) and human embryonic fibroblast (HEF).PMEF were obtained from 13-14 days fetal mouse; HEF were obtained from 5-9 weeks human embryos of termination of pregnancy. Both the two cells, which were treated by 0.25% trypsin and 0.04% EDTA for 3-5 min, were cultured in DMEM containing 10% serum. PMEF and HEF were inactivated by treatment with 10 u g -ml"1 mitomycin C for 2-3 hours and cultured in DMEM as feeder layer to maintain PGC. PMEF and HEF were purified according to the difference of fibroblast sticking time. We also find PMEF and HEF have no significant ('ifference as feeder layer to PGC.2. Making conditioned medium(CM) by rat myocardial cellsThe hearts of newborn (2 days) rat were treated by 0.1% trypsin for 3-5 times, 1-2 min every times. After 10 hours myocardial cells begin to stick to thewall and beat; after 24 hours, about 60% beat, and some clumps are synchronous beat (frequency 80-100 beats ?min"1). Because trypsin can destroy protein of myocardial cell membrane and EDTA can chelate Ca2+ and Mg2+ in the cell, we used low concentration of trypsin and reduced the time of treatment that can effectually decrease the lesion to cells. We also used differential attachment technique to purify myocardial cells and obtained more than 90% pure myocardial cells. Cultured myocardial cells generally can live 10 days. We collected cultured medium of 3-6 days myocardial cells to make conditioned medium to culture PGC because its beats frequency is most stable and its form is best. 3. Separation and culture of human primordial germ cellsPrimordial germ cells (PGC) collected from the genital ridge of 5-9 weeks (postfertilization) human embryos were cultured on PMEF and HEF feeder layer cells with or without conditioned medium (rat myocardial cells). Initially in culture, there were three types of PGC clone expressed alkaline phosphatase (AP) activity: the larger included somatic cells and PGC which didn't separate by trypsin treating had fast proliferation and didn't apt to differentiation; single PGC can proliferation and form clone, but very easily differentiate; the clone which formed by PGC moving together is also larger. Feeder layer and CM can provide leukemia inhibitory factor (LIF), stem cells factor (SCF) and growth factor that can support PGC in vitro survival and proliferation. These cultures can be maintained on feeder layers for 4 passages, and under appropriate conditions give rise to embryo bodies (EB) and to multiple differentiated cells phenotypes in monolayer culture and in tumor (teratoma) in nude mouse.