Cultural Condition Of Porcine Indueced Pluripotent Stem Cells

As a method of somatic cell nuclear reprogramming, induced pluripotent stem cells(iPSCs) have obtained success in many species. iPSCs technology is stable and easy tooperate relatively. Meanwhile, it avoids the ethics and immune rejection problems ofapplication of embryonic stem cells. Thus iPSCs have potential values in basic biologyresearch and clinical applications. So far, several porcine iPSC lines had been established bydifferent research groups, these cells did not have the uniform and authentic characteristics ofpluripotency. At the same time, culture conditions of mostly porcine iPSCs were referring tothe culture conditions of human or mouse iPSCs, and the molecular mechanism duringreprogramming remained unclear. In this study, through reprogramming porcine somatic cellsby transfected four different combinations of retroviral plasminds carrying human five factors(OCT4/SOX2/KLF4/C-MYC/TERT) and cultured in two different media, we optimized theinduction and culture condition of porcine iPSCs and revealed the expression changes ofpluripotent genes at early stage of cell reprogramming. Then we reprogrammed PEF and POG cellsunder the optimized condition into pluripotency. The results were obtained as follow:1. Optimized induction and cultrue condition for porcine iPSCsPEF and POG cells were isolated. PEF were infected with four different combinationsof retrovirus containing human OCT4(O)/SOX2(S)/KLF4(K)/C-MYC(M)/TERT(T),including OSKM(4F), OSKMT(4F+T), SKM(3F), SKMT(3F+T). Then the infected cellswere cultured in two specific culture media: M1(KO-DMEM supplemented with10%KSR,10%FBS,0.1mM NEAA,1mM L-Glu,0.1mM β-Me,1000u/ml LIF and10ng/ml bFGF)and M2(KO-DMEM supplemented with10%KSR,10%FBS,0.1mM NEAA,1mML-Glu,0.1mM β-Me,100ng/ml Insulin,1uM PD0325901and3uM CHIR99021(2i) forreprogramming. We monitored molecular and cell biology change at the early stage of PEFreprogramming. The results showed that cell proliferation rate, clone formation efficiency,alkaline phosphatase (AP) positive rate, efficiency of endogenous pluripotent gene activationwere all obviously higher in M1medium than that in M2medium; compared inducedefficiency on the same culture conditions, we found that4F and4F+T can more effectivelyinduced PEF reprogramming. Under the optimized conditions, that is,4F/4F+T as inducing factors and M1training condition, we induced PEF and POG cells reprogramming, andacquired two iPS cell lines from PEF.2. Gene expression and regulation at the early stage of reprogrammingIn our study, real-time PCR analysis the expression of porcine pluripotent OCT4, TERTand NANOG presented the similar expression profile that the endogenous genes weregradually upregulated along with the induction time, and the The sequence of activated geneis OCT4, TERT and NANOG orderly. Porcine endogenous OCT4, SOX2，KLF4, TERT andNANOG genes were all activated at induction10day. Immunofluorescence analysis of10-dayinducted PEF showed SSEA4and NANOG were positive, yet Tra-1-60and Tra-1-81as thelater stage of reprogramming markers were undetectable, suggesting10-day inducted PEFwere at the early reprogramming stage. Optimization of induced factors implied OCT4playan important role during reprogrammed PEF, TERT may provide new clues for promotingporcine somatic cell reprogramming. Optimization of culture condition indicated thatLIF/JAK-STAT3signaling pathway is important in the establishment and maintainance ofporcine pluripotent cell lines.