Experimental Study On Isolation, Cultivation Of Human Embryonic Germ Cells In Vitro And Committed Differentiation Of Hegcs To Hematopoietic Cells

Human pluripotent stem cell lines are mainly derived from inner cell mass of blastocysts and primordial germ cells of embryos, which are the cell lines cultivated by the way of inhibited differentiation in vitro and characterized by infinite proliferation potentiality and development totipotency. Research of human pluripotent stem cell is becoming the hottest, most active and front-line subject in field of life science. Doing further research on human pluripotent stem cells is of important significance, which mainly shows: firstly, as the ideal model of developmental biology, it can understand precisely incipient development mechanism and related controlling factors. Secondly, human pluripotent stem cell is characterized by infinite proliferation potentiality and development multipotency, and therefore it provides infinite seed cell working as the basic model for cell, tissue and organ transplantation research. Thirdly, the genetic manipulation feasibility of human pluripotent stem cell makes it the carrier model of genetic and cellular therAKPy. In reality, scientists across the world are fastening the research of human pluripotent stem cell by aid of enormous investment and other channels, for it is obvious who can gain research achievement first will get the leader possition of the life science development of 21th century, and do great contribution to improving the social economy and people' health. It is of great urgency for all researchers around the globe to set up human pluripotent stem cell line or human pluripotent stem cell bank. By far, research of human pluripotent stem cell is mainly on human ES cell lines which originated from inner cell mass of blastocysts, and the usual feeder layer for cultivating human pluripotent stem cell is murine embryonic fibroblasts. There is only one successful research case of human EG cell lines set up across the world. However, there is no report about successful setting-up of hEGCs in China. So,it is still spacious to research the human embryonic germ cells originated from PGCs. In order to recognize further human EG cells, we have researches as followed: (1) further research the germination, migration and surface marker of human embryonic germ cells which are the origin of human EG cells. (2) using cryobiological technology to freeze, thaw 6-8week primordial germ cells, and prove feasibility of reserving human embryonic primordial germ cells under low temperature. (3) using 3-5m human embryonic lung as material to isolate, purify, cultivate and identify human embryonic lung fibroblasts, choose the best time and density of mitomycin C to inactivate the feeder layer. (4) using human embryonic lung fibroblasts divided and cultivated by ourselves as the feeder layer to isolate and cultivate human embryonic germ cells and identify. (5) comparing influence on growth of human EG cells by STO and human embryonic lung fibroblasts respectively as feeder layers. (6) Finally, using soluble cytokines as the inducer to make human EG cells differentiate committed to hematopoietic cells. 1. The origin, migration and phenotype of human embryonic primordial germ cells:The aim of this research is to lay the basis for recognization of human PGCs biological characteristics and gaining of human embryonic germ cells derived from human primordial germ cells. Collect 4.5 to 12 week postfertilazation human embryos, slice the gonadal ridges, stain with HE and observe with light microscope. Then the slices of gonadal ridges are analyzed for the expression of alkaline phosphatase and immunologic markers (SSEA-1,SSEA-4) that have been used to routinely to characterize embryonic germ cells. Lastly , detect the expression of transcriptional factor Oct-4 by RT-PCR. Experiment results show that there is no PGC migrated to gonad ridgres before 5 w, up to 5-11 weeks, PGCs gradually migrate to gonad ridges and proliferate. During the period, genital ridges consists of AKP positive cells which express SSEA-1 and SSEA-4 and express transcription factor Oct-4. 12 weeks later, there are still AKP positiv...