The Study On The Improvement Of Chimeric Ability Of Porcine Induced Pluripotent Stem Cells

Induced pluripotent stem cells(iPSCs)produced by reprogramming somatic cells.In general,the pluripotent phenotype of iPSCs is resembling as Embryonic Stem Cells(ESCs)which have the ability to self-renew and multi-directional differentiation.The successful establishment of iPSCs avoids the technology challenge and ethical concerns surrounding the use of human embryonic stem cells.Pigs are closer to humans than mouse in terms of metabolism,anatomical structure,and organ size.Pigs are ideal animal models for preclinical testing in human medicine,such as disease models and xenotransplantation.Therefore,the establishment of porcine iPSCs with “na?ve” pluripotency,and the detection and analysis of iPSCs’ safety and effectiveness has become a hot spot in scientific research.Porcine doxycycline-iPSCs(Dox-iPSCs)were obtained in the laboratory early.On this basis,this study introduced new transcription factors to explore the effects of different transcription factors on the interspecies chimeric ability of porcine iPSCs.Isolated ESCs or reprogrammed iPSCs are all necessary to assess the expression level of pluripotency factors and differentiation ability by gene expression profiling,embryoid induction and teratoma experiments.Ultimately,chimeric experiments are the gold standard for identifying pluripotent states in terms of spanning multiple cell lineages in vivo,and participating in embryonic development,producing chimeric offspring with germline transmission ability.This study used mouse diploid embryos as the recipient embryos and explore the chimeric ability of porcine iPSCs which have different phenotypes and the Extraembryonic endoderm(XEN)cells which are an intermediate state cells during somatic reprogramming.So that we can evaluate their pluripotent state and developmental potential profoundly.1.Firstly,we explored the chimeric ability of porcine Dox-iPSCs with overexpressing BCL2.We microinjected BCL2-iPSCs into 8-16-cell stage mouse embryos.The results showed that the chimeric ability of BCL2-iPSCs was significantly increased compared to the control-iPSCs.But,BCL2-iPSCs only exhibited clone-like growth in mouse embryos and almost no migration phenomenon occurred.Immunofluorescence staining results showed that BCL2-iPSCs were positive for SOX2 and negative for OCT4.2.Secondly,the chimeric ability of porcine Dox-iPSCs with overexpressing TBX3 was explored.TBX3-iPSCs were mainly localized to inner cell mass(ICM)in chimeric blastocyst,and a few cells distributed in trophectoderm(TE).After culturing to the post-implantation stage in vitro,TBX3-iPSCs can migrate to the Epiblast region.Immunofluorescence results showed that TBX3-iPSCs were localized to the OCT4 positive region.The control-iPSCs have the ability to migrate to the TE under a state where the density of the feeder cells is reduced.3.The interspecies chimeric ability of XEN cells was explored.We microinjected XEN cells into mouse embryos.They were localized in the ICM region in early embryonic development.The embryos were cultured in vitro for E10.5 d.Immunofluorescence showed that XEN cells were positive for the XEN marker GATA4.This study mainly explored the interspecies chimeric ability of porcine BCL2-iPSCs,TBX3-iPSCs and XEN cells in mouse early embryos.The results indicated that the overexpression of BCL2 didn’t solve the chimeric problem of porcine Dox-iPSCs fundamentally.While TBX3 can significantly increase the chimeric contribution of porcine Dox-iPSCs.At the same time,porcine XEN cells did not have a tendency to switch to a pluripotent state in the pluripotent environment of recipient embryos.We established the operating parameters in vitro of porcine-mouse interspecies chimeric embryos.The experiments set the foundation for the establishment of naive iPSCs.