The Pluripotent Stem Cells Derived From Porcine Spermatogonial Stem Cells

Induced pluripotent stem cells(iPSCs)possess pluripotent differentiation abilities which will have extensive use in the human disease treatment,drug screening and animal gene editing.The pigs are both valuable livestock and important biomedical model organism.Therefore,porcine iPSCs establishment became the third most important stem cell research after the mice and human iPSCs were established.In the past twenty years,there have been some reports about established porcine iPSCs,but they were not considered as the real porcine iPSCs by the whole field.The cells obtained from present induction methods are of low quality and consistency,so it is difficult to unify the porcine iPSCs' biological characteristics.Meanwhile,the embryonic stem cells(ESCs)research on the pigs makes slow progress as the consequence of lacking established porcine ESCs and suitable culture system.Above-mentioned problems impede the porcine iPSCs establishment and the molecular mechanism behind porcine iPSCs reprogramming.During the induced process of fibroblast cells into the iPSCs undergoes a transformation to epithelioid cells.Recent studies have showed that the transformation process to epithelioid can be skipped and improve the induction efficiency.Spermatogonial Stem Cells(SSCs),located at the seminiferous epithelium in testes,are epithelioid adult stem cells which have high self-renewal capacity and pluripotent differentiation potential.Previous studies introduced the so-called "Yamanaka-Factors"(Oct4,Sox2,Klf4 and c-Myc)system to achieve the conversion of mice spermatogonial stem cells(SSCs)into iPSCs.The induction period from SSCs to iPSCs could shorten half comparing with the fibroblast induced reprogramming period.The studies also found that the iPSCs induced from SSCs showed lower tumorigenicity than the iPSCs induced from other kinds of cells.This study employs four kinds of transcription factors and related small molecular compounds to induce the Guanzhong black pig SSCs into iPSCs.The main content of this study is as follows:Isolation culture and identification of Porcine SSCs:The mice SSCs isolated culture and identification is relatively mature,however the porcine isolation and culture methods have been rarely investigated.We use classical SSCs isolation methods to obtain the porcine SSCs and identify them.Porcine SSCs were inducted into pluripotent stem cells:We obtained the porcine SSCs by primary isolation.After that,we transducted the mice Oct4/Sox2/Klf4/c-Myc four factors into the porcine SSCs by using lentivirus-mediated gene transfer system.The porcine SSCs weres induced into the iPSCs in certain culture conditions,the reprogramming cells' identification was carried out on both molecular biological and cell biological levels.The porcine iPSCs expressed pluripotent gene and had the capacity to differentiate into cellular derivations of all three primary germ layers.