Study of primate embryonic stem (ES) cells is not only useful for basic research on biomidecal science, but also very helpful for therapeutic cloning. Currently, many questions in the field of primate ES cells are also required for further elucidations, such as the improvement of culture system, highly enriched specific subtypes derived from ES cells and the possible mechanisms of cell differentiation. In order to improve the culture system of rhesus money ES (rES) cells and promote the neural progenitors (NPs) and oligodendrocytes differentiation derived from rES cells, homologous cells as feeders and new neural differentiation system were established in the study. Furthermore, the possibilities of NPs as transplantable donor cells are further evaluated. The main results were as follows. The four homologous feeder cell lines (MESF, MOF, MFG and CMESF) can be used to support the undifferentiated growth and maintenance of pluripotency in rES cells. Comparative studies showed there are different among abilities of the four feeders to support the growth of rES cells, and MOF was preferred homologous feeders, then MESF, CMESF, MFQ respectively. Further studies showed that the abilities variation of the feeders might be related with some genes expression difference. It was found that hepatocyte growth factor (HGF) and G5 as inducers promoted NPs differentiation from rES cells. Alone HGF or G5 only induced rES cells differentiation into 65±8.3% or 69±14% of NPs, respectively. However, the use of HGF and G5 combined markedly increased the NPs differentiation rate (88.3±8.1%). Additional purification resulted in NPs preparations that were 98% nestin positive. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes and oUgodendcocytes, The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, ^25% of transplanted NPs survived and 65-80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. SuMoning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro, and in vivo in rat brains. 3) 75 ±6.8% of oligodendrocyte precursors and 81 ±8.6% of mature oligodendrocytes from rES cells were obtained in five steps using HGF and G5 as our culture system. HGF significantly increased the NPs proliferation speed by both decreasing cell apoptosis rate and shortening cell cycle time in presence of G5. Our studies also revealed that HGF promoted oligodendrocyte maturation by increasing both the length and number of branches and the expression of myelin basic protein. The results from different temporal treatments and combinations of HGF and G5 demonstrated that the ability of HGF responsiveness to initiate oligodendrocyte differentiation was regulated by G5 and alone HGF without G5 induced rES cells differentiation into neurons. Further studies showed that the crucial time point of G5 action was from EBs to NPs.These works are significant for study of the primate stem cells, great helpful for future neural replacement therapies, and provide a platform for probing the molecular mechanisms that control neural induction and oligodendrocyte differentiation.
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