The Preliminary Study On Na(?)ve State-Like Induced Pluripotent Stem Cells Of Guangxi Bama Mini-Pigs

Porcine fetal fibroblasts (PFF), porcine bone marrow menenchymal stem cells (PBMSCs) and porcine adipose-derived stem cells (PASCs), which derived from Guangxi bama mini-pig, were used for induced pluripotent stem cells (iPS) induction in this study. Inducible lentiviral vectors encoded oct4, sox2, c-myc, klf4, lin28, nanog were introduced into the different porcine donor cells, the efficiencies of induced pluripotent stem cells(iPSCs) formation were compared in different cells and different culture conditions, and the biological characteristics of porcine induced pluripotent stem cells (piPSCs) were identified. And two inhibitors SB203580 and SP600125 which affect p38 and JNK signal pathway respectively were added, in order to obtain na(?)ve state porcine induced pluripotent stem cells. The primary results were as followed:(1) The efficiencies of iPS induction derived from different cells and different culture conditions were compared. After lentivirus infection, the iPS clonies derived from different cells were generated at day 10-day 12 induction under mTeSR conditions which is the most widely published feeder-free cell culture medium for human embryonic stem cells and induced pluripotent stem cells; After combined with clonies morphology and AP staining assay, we found the reprogramming efficiencies of iPSCs derived from PBMSCs, PASCs, PFF were 0.28%,0.25%,0.23% respectively. The iPSCs formation efficiencies derived from PBMSCs group was significantly higher than PFF group, but there was no significant difference between PASCs and PBMSCs, PFF cell lines. But until now we didn’t establish the stable iPSCs which could unlimited passage.In order to improve the culture condition, we further chose PBMSCs as the donor cells, compared the iPS induction efficiencies derived from PBMSCs after treated by three induction condition which was bFGF,2i+Lif, 2i+Lif+bFGF respectively. We found that the efficiencies of iPS induction derived from PBMSCs in bFGF,2i+Lif,2i+Lif+bFGF was 1.12%,0.84% and 1.12% respectively. The efficiencies of iPS clonies formation in bFGF, 2i+Lif+bFGF group were significantly higher than 2i+Lif, but there was no significant difference between bFGF and 2i+Lif+bFGF group; In order to confirm the best condition for iPS subculture, we picked the piPSC clonies from different conditions and assay the expression of endogenous pluripotent related genes. As results we found the expression levels of endogenous oct4, klf4 from bFGF group was lowest, and the clonies couldn’t passage and proliferate after P10; Clonies derived from 2i+Lif culture condition was more round state, but the cells proliferate was slowly and stopped expand after P6. The expression levels of endogenous oct4, klf4 were highest in clonies derived from 2i+Lif+bFGF group, the morphology of clonies could keep the round state and proliferate more than P15. Considering the reprogramming efficiency, clonies passage and expression levels of endogenous pluripotent related genes, which suggested that the 2i+Lif+bFGF was fit to use for piPSCs induction and passage.(2) The induction and biology identification of piPSCs from different cells. We successfully obtained iPSC clonies derived from PBMSCs, PASCs, PFF under the 2i+Lif+bFGF culture condition. The biology characteristic identification results showed that:piPSCs were positive for alkaline phosphatase activity; these clonies expressed the endogenous pluripotent related genes like Oct4, Sox2, E-cadherin, Dnmt3b, GDF3, at the same time, they expressed the marker genes FGF5 and Klf4, Klf5 that attribute to primed and na(?)ve state respectively. After culture these iPSCs withdrawing the DOX, piPSCs can still subculture and exogenous pluripotent related genes were silent gradually; The speeds of piPSCs outgrow was faster than the original cells, the time for iPSCs double proliferated derived from PBMSCs, PASCs, PFF were 0.95d,0.93d and 0.97d respectively; piPSCs has 38 chromosomes, the normal karyotype rate were more than 70%; Culturing piPSCs in low adhesion dish, they could form embryoid bodies (EBs) on day 7 and express three germ layers marker genes, has the potential of differentiation in vitro; Considering the identify results above, we suggested hold that the piPSCs have the characteristics of primed and na(?)ve state.(3) The preliminary study of SB203580 and SP600125 on promote piPSCs shift to na(?)ve state. In order to obtain the na(?)ve state iPSCs, SB203580 and SP600125, which was the inhibitor of p38 and JNK signal pathway respectively, were used on the basis culture of 2i+Lif+bFGF. Firstly, we used cck-8 method to assay different concentrations of SB203580 and SP600125 on the effect of PBMSCs proliferation, eventually we used 5 μM SB203580 and SP600125 for subsequent experiments; PBMSCs were chose as donor cells for iPS induction and successfully obtain clonies and identified biological characteristics. Results indicated that the piPSCs clonies were AP staining positive; these clonies expressed na(?)ve state candidate marker genes like Oct4, Sox2, K1f4, Klf5, Rex1, etc., but the primed state marker gene FGF5 was not expressed in these clonies. RT-PCR results showed that the expression level of K1f4 derived from SB203580 and SP600125 treated condition was significantly higher than 2i+Lif+bFGF; In addition, piPSCs clonies could tolerate trypsin digestion and formed monoclone; These datas suggested that SB203580 and SP600125 can promote piPSCs shift to na(?)ve state,2s-piPSCs have some characteristics of na(?)ve state pluripotent stem cells.The above results showed that PBMSCs has higher efficiency on reprogramme to piPSCs, the conditions of 2i+Lif+bFGF more suitable for piPSCs subcultue, the inhibitors SB203580 and SP600125 which targeting p38 and JNK signalling pathways respectively can promote piPSCs shift to na(?)ve state,2s-piPSCs have some characteristics of na(?)ve state pluripotent stem cells.