| There were two aspects involved in induced pluripotent stem cell (iPS) technology. Primarily, activation the expression of endogenous gene in target cells by the transduction of specific transcription factors, and transform the epigenetic markers, so that the cells could be maintained in a intermediate activated state. Subsequently, under specific cultural conditions, the intermediate state cells were induced to specific destinations, and selected using provided pluripotent stem cell cultural conditions. Finally, ES-like iPSCs were obtained.In the current pig iPS studies, there were great differences among the established iPS cell lines, and most of the iPSCs had exogenous gene expression. Although pig iPSCS were used to generate chimera in one report, but they could not be repeated in the following studies. It was surprising that the embryo developing rate of pig iPSCs was extremely lower than normal stomic cell in nucler transfer experiments, let alone the in vivo embryo development rate, pluripotency level of pig iPSCs could not accord to mouse iPSCs, that were the main obstacles in transgenetic pig production and disease model establishment using pig iPSCs, moreover, there was little new information obtained with the study of pig ES cells, no admitted pig iPSCs and cultural conditions could be used as references. It was difficult to maintain the pluripotency of pig iPSCs in vitro as the insufficient study of pig developmental biology.Based on the characteristics of iPS technology and problems existed in the current pig iPS study, the generation, selsction, passage and pluripotency maintence were studied in this study, to facilitate these process, a DOX-mediated gene expression system was used to generate two sourse of piPSCs, effects on the generation rate, profication ability were studied, and two cultural systems were adopted to generate two different state of piPSCs, their biology characteristics and molecular mechanism of pluripotency maintence were compared, the main results were as follows:1. Effects on the generation rate and profication ability of piPSCs derived from fibroblasts and BMSCs(1) Pig fibroblasts and BMSCs were reprogramed by3factors,4factors and6factos using Tet-on mediated gene expression system, respectively. Morphology, alkaline phosphatase activity, immunofluorescence, gene expression, karyotype, in vitro and in vivo differentiation ability of the two kinds of piPSCs were identified. The results showed that the generation rate of the two kinds of piPSCs were same, they all had ES-like characteristics. In the3generation methods, clones in3factors group were AP negative, there were no significant differences in4and6factors groups, generation rate in6factors group was highest, but all the clones had exogenous gene expression.(2) Growth characteristics of fibroblasts and BMSCs derived piPSCs in different feeder layer were studied. The two kind of piPSCs in CF1feeder layer growed well, they all could be passaged for more than20generations, and their cloning formation ability was most powerful, the mean doubling time was shortest, there were no significant differences of culture effects in MEF and pig fibroblasts (P>0.05), while these two kinds of piPSCs could not be well expanded in STO feeder layer.(3) Effects of single cell passage, small and medium clone passage on the profication ability were studied. The results showed that fibroblasts and BMSCs derived piPSCs could not accommodate with single cell passage for the cloning formation rate was extremely low, there was no significant differences of cloning formation rate and mean doubling time in small and medium clone passage. After passage, piPSCs clones were tight, and no differentiation was found, they could be passaged for more than20generations.(4) Effects of ROCK inhibitor Y-27632on frozen/thowling rate of fibroblasts and BMSCs derived piPSCs were studied. The results showed that Y-27632could significantly increase the survival rate of piPSCs after thowing, and accelerate their adhesion and cloning formation ability, but higher concentration of Y-27632would affect the morphology of piPSCs.(5) Cell cycle related protein CDK2and CDC25b were purified, their effects on piPSCs cloning formation and mean doubling time were studied. The results showed that recombinate CDK2anf CDC25b proteins could increase the cloning formation rate of fibroblasts and BMSCs derived piPSCs, and decrese their mean doubling time, among the two proteins, CDC25b had the greatest effect.2. Biological characteristics and molecular mechanisms of two state piPSCs(1) BMSCs derived piPSCs (piPB4-6) were passaged according to normal human ES culture method, the clones were incompact, formed flat colonies, cell boundary in the colonies could be obsearved, after changing culture system, mouse ES-like clones (piPB4-6_m) were obtained, these clones could be passaged for more than20generations. Morphology of piPB4-6_m was different from piPB4-6_h, it’s size was relatively small, boundaries of colonies were obvious, cells in the conlonies were tightly accumulated, and there was no obvious boundary between cells.(2) piPB4-6_m could be passaged without DOX, there was rarely differentiation, it was indicated that DOX was not indispensable in the culturation. After conversion, colony number in the unit area increased significantly, cell vitality was also increased compared with piPB4-6_h.(3) After identification, piPB4-6_m could reach the criterion of pluripotent stem cell, while there were some differences, piPB4-6__m expressed the embryonic stem cell surface marker SSEA1, that was different from piPB4-6_h. Exogenous Oct4and sox2were not silenced both in piPB4-6_m and piPB4-6_h, but the expression level of them was different, piPB4-6_m had a higher expression of exogenous Sox2than piPB4-6_h. In terms of endogenous gene expression, although expression of endogenous Oct4, Sox2and Nanog were all started, but their expression level were relatively lower than exogenous genes, piPB4-6_h had a higher Sox2and Nanog expression than piPB4-6_m.(4) X chromosome state of piPB4-6_h and piPB4-6_m was analyzed by the detection of Xist34and Xist45, and the expression of SSEA4was also detected. The results showed that Xist and SSEA4expression in piPB4-6_m were decreased, indicating that the converted piPB4-6_m could approach to mouse ES-like state.(5) The expression of signal pathway related genes was analyzed. The results showed that the pluripotent maintenance of piPB4-6_m depended on LIF and BMP4pathway, but they also had a high expression of FGF2, while piPB4-6_h depended on bFGF pathway. Expression of imprinted genes DLK1, GTL2of piPB4-6_m were higher than piPB4-6_h, it could be supposed that piPB4-6_m had a more pluripotent level. |
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