| hESCs derived from inner cell mass (ICM) of blastula before implantation possesses pluri-potential and unlimited self-renew capacity, providing a good model for human embryo development research and ideal materials for clinic cell replacement therapy. At present, it is the cell culture system that is an obstacle of hESCs researches, the production of hESCs in feeder culture system can not meet the requirement of clinic and basal research, owing to labor intension and hard scale, at the same time, the mechanism of hESCs is not explored yet, which is critical for cell-lineage induction. So it's very important to establish a new simple, stable and suitable for large scale non-feeder hESCs culture system. This feeder free system is helpful to elucidate the mechanism of pluri-potency, and avoid the animal viral containments. The main aim of our study is to establish a feeder-free hESCs culture system and make a primary study of it.In chapter one, the feeder-free hESCs culture system (DMEM/F12 plus N2 and B27, namely NB culture system) was established, hESCs in different FGF2 concentrations (4ng/ml,40ng/ml, and 100ng/ml) show undifferentiated morphologies (high nucleus-plasma ratio) and positive for Oct4, SSEA4 and Tra-1-60, express multi-potent genes, such as Oct4, Sox2, Nanog and Rex1, differentiate to three germ layer cell types. At the some time, the effects of different FGF2 doses are compared. At 4ng/ml FGF2 condition in NB culture system (NB4 system), the percent of undifferentiated hESCs clones (PUC) decreases and reaches the deepest point (the PUC is about 30%), and then it increases nearly to 100%at passage 11. The PUC of hESCs in different FGF2 concentrations (40ng/ml,NB40;1 00ng/ml,NB 100)are higher and nearly reaching 100% after 8 passages in NB culture system.All hESCs clones at different FGF2 concentrations in NB culture system are undifferentiated hESCs clones.High concentrations of FGF2 have important supports to maintain hESCs pluri-potent,when they transferred from HEF to NB system.In general,NB culture system is a stable and high effcient feeder-free culture system.In chapter two,the characteristics of hESCs in NB were compared with those in HEF in six aspects including cell double time,cell cycle, cell apoptosis, FACS analysis of SSEA4 and Tra-1-60, EBs differentiation inclination and Ranks of carcinomas.hESCs in NB have shorter cell double time,the double times in HEF,NB4,NB40 and NB100 are 40.07±2.01 hrs,37.33±0.79 hrs,30.94±1.63 hrs和29.22±2.29 hrs respectively.The cell cycles are not changed in the two different systems,the percents of S phase are 41.75%±1.05%,42.90%±3.02%,41.48%±2.04%和41.34%±5.72%,and the apoptosis are 10.8%±1.51%,8.44%±1.11%,5.96%±1.27%和3.61±%1.05% respectively,so the more important factor is apoptosis not cell cycle that influent hESCs amplification.hESCs in NB system are more purer than those in HEF system in term of FACS of SSEA4 and Tra-1-60.the percents of Tra-1-60 are 71.6%±1.80%,93.0%±2.8%,87.8%±1.8%,83.1%±7.8%,and the percents of SSEA4 are 81.5±5.46%,97.3%±0.8%,95.5%±2.5%,95.9%±2.4%respectively.When hESCs in NB system were transferred back to HEF system,the percents of Tra-1-60 and SSEA4 decrease,the percents of Tra-1-60 are 86.1%±4.6%,84.8 %±2.0%,87.6%±1.0%and the percents of SSEA4 are 89.2%±1.9 %,85.8%±1.2%,87.9%±0.7%respectively.In EBs differentiation inclinations, hESCs in NB system show anti-differentiation capacity, highly expressing multi-potent genes (Oct4, Nanog and Rexl) and lowery or not expressing differentiation genes (ectoderm genes:Sox3, Pax6 and Soxl; mesoderm:Brachyury; endoderm:Sox17, AFP, Gata6), and the differentiation inclinations are recovered in some degree, when hESCs are transferred back to HEF system for 13 passage. There may be co-relationship between percents of SSEA4 and Tra-1-60 and differentiation inclination, the more purer of hESCs, the more difficult to differentiate. In general, hESCs in NB system exhibit more efficient amplification, lower apoptosis, more purer, and their differentiation inclination can be recovered when transferred back to HEF system, there may be some relationship between the percents of SSEA4 and Tra-1-60 and differentiation inclination.In chapter three, the autocrines of hESCs are discussed. We find that the percents of undifferentiated hESCs clones are influent by the hESCs clones density, providing a new clue that the autocrines of hESCs may have positive roles in maintaining hESCs undifferentiated states. In our results, hESCs can maintain their undifferentiated state in high density of culture clones(>34 clones/cm2) in NB culture system, at the same time, the BMP-like induction of culture medium can be modulated by the density of hESCs clones; So we detect expression of ligands of three pathway (Wnt, FGF and TGF signaling) thought important to hESCs pluri-potential. hESCs in NB culture system do not express ligands of Wnt signaling, highly express FGF2 and Nodal. Owing to the expression of FGF2, hESCs maintain their undifferentiating states even if FGF2 is withdraw. hESCs also maintain their undifferentiating states without addition of FGF2 when they are transferred from HEF to NB system, expressing Oct4, Nanog and Rexl and markers (Oct4, SSEA4 and Tra-1-60). However, there exists some unknown factors that support hESCs culture in vitro. Some researchers have proved that the factors secreted by hESCs can are helpful for establishment and maintainace their undifferentiating states. The autocrines may provide a new way to elucidate the mechanism of hESCs pluri-potential maintenance.
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