Establishment Of Sheep Induced Pluripotent Stem Cells Using Sheep Reprogramming Factors And Exploration Of Pluripotency Maintenance Mechanism

Induced pluripotent stem cells(i PSCs)have similar biological functions as embryonic stem cells(ESCs).It has overcome the problems related to the source of acquisition,moral ethics,and immune rejection after allotransplantation,which has shown irreplaceable advantages in the fields of cell therapy,pharmacology,and toxicology.In recent years,human and mouse i PSCs technology have been relatively mature,and there have been some achievements in the study of large livestock i PSCs such as pigs,bovine,and sheep.As a major domestic animal,sheep has significant value in both biomedical and agricultural fields.Although the sheep i PSCs(si PSCs)have been established by researchers in several laboratories around the world,they were basically induced by heterologous reprogramming factors such as from human and mouse.Moreover,the present si PSCs have problems such as low reprogramming efficiency,low safety,long positive cloning formation cycle,low pluripotent gene expression,and need to rely on the continuous expression of exogenous reprogramming factors.The efficient preparation of si PSCs is still challenging.Therefore,the purpose of this study was to reprogram sheep fetal fibroblasts(SFFs)into si PSCs using sheep derived reprogramming factors to establish an induction method with higher reprogramming efficiency,higher safety,and better differentiation potential,and to provide material for preparation of sheep modified with special functional genes and construction of disease model.At the same time,the mechanism of si PSCs pluripotency maintenance was further explored to lay the foundation for exploring the mechanism of somatic cell reprogramming and stem cell directed differentiation in sheep.Firstly,multiple expression vectors of sheep derived reprogramming transcription factors were constructed based on piggy Bac transposon system,SFFs were isolated and cultured,and the electrotransfection conditions were optimized.On this basis,SFFs were reprogrammed into si PSCs using different amounts of reprogramming factor induction systems from different species.The obtained si PSCs were identified by clone morphology,karyotype identification,expression detection of pluripotent genes and proteins,embryoid body formation,and differentiation potential,so as to compare the reprogramming effects of different systems.In addition,combined with transcriptome sequencing(RNA-seq)and bioinformatics analysis techniques,the pluripotent gene regulatory network of si PSCs was deeply explored,and the roles and mechanisms of some pluripotent genes in reprogramming were explored by transfecting single factor expression vectors.The results are as follows:1 The electrotransfection conditions of piggy Bac transposon system in SFFs were systematically optimized.It was found that DMEM/F12 basal medium as electroperforation buffer had good transfection effect.The optimized conditions of voltage,pulse number and duration were also important for transfection results.The optimized electroporation system(DMEM/F12 buffer,200 V,2 pulses,20 ms length,and 20 μg DNA(3 μg/μL)in 4 mm cuvette)could significantly improve the transfection efficiency and cell viability of SFFs,which provided a basis for the stable expression of transgene.2 Eight sheep derived reprogramming factor vectors were successfully constructed.By comparing different reprogramming induction systems,it was found that S10 F induction system(s OSKM+s NL+h RL+h TERT+SV40 LT)containing sheep derived reprogramming factors had the best reprogramming effect.The si PSCs cell line established on this basis could be stably passaged to more than60 generations with stable karyotype,expressing a variety of pluripotency marker genes and proteins,and it could form embryoid bodies in vitro with the potential of spontaneous differentiation into three germ layers and directional osteogenic differentiation.3 Five groups of si PSCs and SFFs were analyzed and compared by RNA-seq analysis.The stem cell related signaling pathways in five groups of si PSCs,including MAPK,PI3K-AKT,TGF-β,WNT,and "regulating pluripotency of stem cells" signaling pathways,were screened by KEGG enrichment and the expression of related genes were analyzed.Among the five groups of si PSCs,si PSC180 and si PSC185 showed more obvious activation or inhibition of various pathways,indicating their pluripotency were better.In the process of sheep somatic reprogramming,it might be difficult to activate the PI3K-AKT signaling pathway and inhibit the WNT signaling pathway,which might be the key to the difficulty of reprogramming and the low quality of i PSCs in sheep compared with other species.Based on this,the existing si PSCs reprogramming and culture systems are expected to be improved.Compared with SFFs,3657 differentially expressed genes(DEGs)were shared by the five groups of si PSCs,and 1253 DEGs were shared between si PSC180 and si PSC185.Through protein-protein interaction(PPI)analysis,the Top1 subnetworks and its hub genes of 1253 DEGs were further selected,and KEGG pathway enrichment was performed on the Top1 subnetworks.It was found that the inhibition of oxidative phosphorylation(OXPHOS)played an important role in the maintenance of si PSCs pluripotency,indicating that whether OXPHOS was inhibited could be used as an indicator of si PSCs pluripotency.Moreover,inhibition of OXPHOS or improvement of glycolysis may be beneficial to sheep reprogramming.4 Through analysis,16 potential reprogramming factors was identified in sheep,including PRDM14,LOC106991721,DLX5,ZIC3,OTX2,TFAP2 C,LOC101106129,DPPA4,and ZIC2,which have been studied for the role of pluripotency in other species,as well as HHLA2,CKB,MME,RFX4,ZYG11 A,MIP,and GREB1,which have not been reported in pluripotent studies and may be new pluripotent functional genes.5 Overexpression of OCT4,SOX2,KLF4 or MYC single reprogramming factor could promote the proliferation of SFFs to varying degrees,among which overexpression of MYC had the most obvious effect and could lead to the formation of clone-like cells capable of passage,which may be related to the promotion of transition of SFFs cell cycle from G1 to S phase.The overexpression of the four factors could also affect the original epigenetic modification status of SFFs to a certain extent and promote the formation of SFFs tight junction at the initial stage,but sustained tight junction required the combination of multiple reprogramming factors.In conclusion,in this study,we optimized the electrotransfection conditions of piggy Bac transposon system,established the optimal induction system of S10 F based on the sheep derived reprogramming factor,and obtained si PSCs with high pluripotency gene expression and good differentiation potential.Based on RNA-seq analysis,MAPK,PI3K-AKT,TGF-β,WNT and "regulating pluripotency of stem cells" signaling pathways which are related to the pluripotency maintenance of i PSCs in sheep were found.It was found that whether OXPHOS was inhibited could be used as an indicator of si PSCs pluripotency.16 potential sheep reprogramming factors were identified,including 7 new functional genes that may be associated with pluripotency.In addition,it was found that OCT4,SOX2,KLF4 and MYC had different roles and mechanisms in reprogramming,which were related to promoting cell proliferation,and affecting cell cycle,cell junction and epigenetic modification,respectively or jointly.Overexpression of MYC could make the SFFs form clone-like cells capable of passage.