Derivation And Directed Differentiation Of Embryonic Stem Cells

Since the first embryonic stem cell derivation from mouse blastocyst by British scientists in 1981, groups of scientists were devoted into the research field of stem cells. In 1998, two scientific groups of America obtained human embryonic stem cells and human embryonic germ cells respectively from human blastocysts and gonadal ridge of early embryos. The cultivation of embryonic stem cell provides vast space for the research on cell differentiation and tissue development. It also provides experiment models for the research of gene functioning. Our research work is based on the derivation and cultivation of human embryonic germ cell and mouse embryonic stem cell, and the directed differentiation of mouse embryonic stem cells.The first experiment is the derivation and cultivation of human embryonic germ cell. The primary primordial germ cells is obtain from the human embryos after 4-8 weeks of pregnancy. After mechanical desection and enzymic digestion, the cells were cultured on mouse embryonic flbroblast or human embryonic flbroblast feeder layer inactivated by mitomycin. The medium contains several cytokines:LIF(leukemia inhibitory factor), bFGF and Foskolin. There was about 7 days interval between every two passages. EG cell clones can be found in each primary culture from those embryos of appropriate age. In culture of one embryonic cell line, EG cell clones maintained after 9 passages. The alkaline phosphatase activity test showed these cells maintained the character of primordial germ cells.The second experiment is derivation and cultivation of mouse embryonic stem cells. The blastocyst was obtain from the mouse after 3.5 days of pregnancy, and was cultivated on the mouse embryonic flbroblast feeder layer. The blastocyst usually attach to the feeder layer after 48 hours, then the inner cell mass began to grow and form a big cell mass of embryonic stem cells. These cells can form cell clones with the conformation of embryonic stem cell. There was about 5-7 days of interval between every two passages. The AKP activity test showed these cells had maintained the character of stem cells. The ES cell clones decreased and disappeared gradually after 9 passages.In the third experiment, we first constructed a test model of differentiation frommouse ES cells to cardiomyocytes. Then series of dilutions of three chemicals were screened on this platform. The combination of all trans retinoic acid and DMSO is employed as positive control. One of the three chemicals, HG(code), showed potent inducement effect (43.3%) in a certain concentration in the course of differentiation.