Isolation, Cultivation And Initial Identification Of Human Embryonic Stem Cells

Objective: In order to explore a new method to culture primary EG cell in vitro, humanembryonic stem cell were cultured without any cytokines in vitro, then were observedand identified incipiently.Methods: Human embryonic genital ridges, dorsal mesenteries and mesomephric ridges(5~10 weeks postfertilization) were cultured without any cytokines .The fibroblastsderived from their original embryonic tissue were used as the feeder layer.Histochemistry and cytomorphology were employed to identify the cultures.Results: After 12h culture, fibroblasts appeared around the tissue. After 24h, a few EGcells were observed sporadically. 4~20d later, embryonic germ cells grew in clusters onthe fibroblast layers. The EG cells stably maintained normal karyotype and highlyexpressed alkaline phosphatase. 42 human embryos (5-10 weeks postfertilization) werestudied in our experiments, 29 of which generated colonies.Conclusions: Tissue from genital ridges, dorsal mesenteries and mesomephroic ridgescan be cultured in vitro without any cytokines. The embryonic stem cell colonies can beobserved on the feeder layers which was constituted of the fibroblasts from their originalembryonic tissue. This method is highly prone to succeed.