Isolation And Culture Of Embryonic Stem Cells And Neuronal Differentiation Of Cultured Human Embryonic Germ Cells

ObjectiveEmbryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of preimplantation blastocyst or from the primordial germ cells (PGCs). ESCs have the essential characteristics of prolonged undifferentiated proliferation and stable developmental potential to form derivatives of all three embryonic layers after prolonged culture in vitro. Since 1998, because of its pluripotentency, manipulation and plastics, the embryonic stem (ES) cells have been the focus of researches in the fields of medical and life science. ESCs will not only be an important researching model in the biomedical field, but also provide us with a plentiful resource of donor for cell transplantation therapy, and e-ven make it possible for us to manufacture any kinds of tissues and organs in lab. 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. The extensive application with embryo - derived stem cell can largely promote the research fields about establishing the animal modle of human disease, the medical protein production and drug discovery . So, the utilization of hESCs has an unimaginable prospect in the field of life science. Up to now, there are some reports of the establishment of hESC lines from the ICM in the world, but only Gearhart group had established hEGC lines and still no other report in our country or abroad.In our experiments, we want to get some experiences in isolation and culture both of mESCs and hPGCs on the HEF and MEF feeder layer, then put them in differentiation - inhibited culture in vitro and identify them. Our pur-pose is to search for a practical method for the derivation, culture and neuronal differentiation of hEGCs and lay the foundation for the establishment of hEGC lines and further research.Methods1. The mouse ICM and hPGCs were isolated and cultured on feeder layer, then passaged regularly, cryopreserved and thawed repeatedly in vitro. At the same time, the morphology and biological characters of them were observed. Then analysis of effect on the separation of mouse ICM and hEGCs about trypsin and mitomycine C were gived.2. The Immunostaining method and enzyme cytochemical method were used to detect the expression of SSEA - 1,SSEA - 4 and alkaline phosphatase of the cells cultured in vitro. Karyotype analysis and differentiation in vivo of hEGCs were undertaked.3. The effects of mouse embryonic fibroblast (MEF) and human embryonic fibroblast (HEF) were observed.4. Retinoic acid(1 × 10^-6M/L) was made an revulsant(inducer) to the neuronal differentiation of hEGCs and the derived cells were detected with the expression of Nestin, NSE, NF - 200 and GFAP, which are the characteristic markers of neural precursor cell and neuron.Results1. We successfully isolated and cultured mESCs and hEGCs, Obtaining typical colonies. one derivation of mESCs underwent 5 passages and could formed multicellular colonies. The active mobility of the stem cell was observed, the hEGCs remain the growth behavior of colony forming and the undifferentiated morphological characteristics after passaged regularly (the maxium passage number has reached to 9 passages) or cryopreserved and thawed repeatedly. The cultured cells can naturally differentiate into epithelial tike cells, cardiac cells, neuron - like cells and embryonic bodies. It would be more effective to isolation...