Establishment Of High Quality Porcine IPS Cells

Induced pluripotent stem cells (iPS cells) can be artificially reprogrammed from somatic cells by overexpression of exogenous transcription factors. IPS cells from somatic cells hold great potential for regenerative medicine. The efficiency in generation of iPS cells has been greatly improved in recent years. However, generation of high quality of iPS cells remains great interest. The pig has increasingly become an important large animal model for preclinical tests and studies of human diseases. Thus, the generation of porcine iPS cells will facilitate research into the efficacy and safety of stem cell therapy.The purpose of this study is to establish high quality porcine iPS cells using pig as a human disease model. We mainly designed four aspects of experiments:(1) Generation of high quality mouse iPS cells using optimized methods as a base of porcine iPS cells study.(2) Generation of high quality pig iPS cells using variety of optimized methods.(3) In vitro and in vivo developmental capacity of pig iPS cells by differentiation and chimera tests.(4) Molecular mechanisms of reprogramming and maintenance of iPS cells.We have accomplished following studies over the last six years.(1) Knockout serum replacement (KSR) accelerates iPS induction and improves the quality of iPS cells, as evidenced by generation of chimeras and all iPS offspring with germline transmission competency. Furthermore, inhibition of MAPK/ERK by a specific inhibitor PD0325901in KSR rather than in FBS significantly increases Nanog-GFP+cells.(2) Optimization of pig iPS induction methods and improvement in the efficiency of iPS programming, using variety of methods, different precursor cell types, factor combinations, and induction and culture conditions. We suggest that high expression of exogenes and good precursor donor cells benefit the generation of high quality porcine iPS cells.(3) Establishment16porcine iPS cell lines from2059picked colnes. And our porcine iPS cells showed pluripotency, as evidenced by expressing pluripotent markers, by differentiation into three germ layers in vitro following embryoid body formation, as well as by efficiently forming teratomas containing three germ layers in immunodeficient mice. Significantly, our porcine iPS cells could contribute to chimeras.(4) Furthermore, we generated porcine iPS cells by using only two porcine transcription factors in combination with small molecules.(5) Systematic analysis and comparison of transcriptome of porcine iPS lines and donor cells. We compareed difference between donor cells and iPS cells and found that Rexl indexed high quality porcine iPS cells and iPS cells had open and quick epigenetic state.Although our chimeric pig don’t have coat color mosaic, these attempts represent the first step toward generating truly pluripotent porcine iPS cells. Further dissection of mechanism in reprogramming and maintenance of porcine pluripotent cells is still need.