Isolation And Culture Of Mouse Embryonic Stem Cells And Human Primordial Germ Cells

Inner cell mass (ICM) of the blastocyst stage embryo and primordial germ cells (PGCs) of fetus are the mostly used materials for derivation of pluripotent stem cells. Pluripotent stem cells may be employed to understand fundamental events in embryonic development, such as the mechanism of differential gene expression and so on. In addition, it may also be used as an ideal source of tissue engineering and model of pharmaceutical research and clinical medicine.This study mainly focused on isolating and culturing ES cells from mouse embryo and PGCs from human counterpart. We also investigated the factors including feeder layer cells, growth factors and complements, which may affect the cultivation and differentiation of these cells in vitro.The main contents as follows:(1) Isolation and culture of mouse embryonic fibroblast (PMEF) and preparation the feeder layers.Mouse Embryonic Fibroblast were obtained from mouse embryo 13.5 dpc or 14~16dpc. After treatment with 0.05%trypsin and 0.04%EDTA for 3~5min, it was seeded in flask using Dulbecco's Modified Essential Medium containing 10% serum. MEF were inactivated by treatment with 10ug/ml mitomycin C for 2.5-3 hours or y irradiation 50Gy/75Gy dose and grew in DMEM as feeder layer to help mouse ES cells and human PGCs. It was found that MEF by treatment with mitomycin C has no significant difference as feeder layer as with y irradiation.(2) Isolation and culture of mouse embryonic stem (ES) cellsThree days after ovulation, 166 blastocysts were recoverd by a surgical uterine flush technique form several strains of mice and plated onto MEF feeder layer mitotically inactivated by mitomycin C or r irradiation. After 4 days of growth, the intact inner cell mass (ICM) were separated with 0.05% trypsin-EDTA and replated on feeder layer in DMEM containing 15% serum, 0.1mmol/L nonessential amino acids, 0.1mol/L 2-mercaptoethanol, 2mmol/L glutamine, 100 units/ml ofstreptomycin and 100 units/ml of penicillin. After 4-6 days 5 ES cell colonies were selected and expanded in which alkaline alkaline phosphatase was detected. If MES medium was withdrawn they tend to differentiate into several types of cells such as heart muscle-like cells, including fat-like cells. It was suggested that the genenic background of the mouse is very important in the cultivation of mouse ES cells. (3) Isolation and culture of human primordial germ cells (PGCs). Human PGCs collected from gonadal ridges and mesenteries were grown on mouse feeder layers in the presence of human recombinant leukemia inhibitory factor (LIF), human recombinant basic fibroblast growth factor, and forskolin as described previously. Initially, PGCs were visualized by alkaline phosphatase activity staining. Over a period of 4-7days, PGCs gave rise to large multicellular colonies resembling those of mouse embryonic stem cells. Throughout the culture period, most cells within the colonies continued to be alkaline phosphatase positive and Specific Stage Embryonic Antigen (SSEA-1) positive which has been used routinely to characterize embryonic stem and PGCs cells. Among all PGC-derived cultures, it was found that one derivation under went 12 passages. Taken together, these results suggested that human PGC-derived cultures might be developed into long-term proliferated pluripotent stem cells.