Study Of The Aberrant Epigenetic Reprogramming In PiPSCs And PiPSC Nuclear Transfer Embryos

Embryonic stem cells (ESCs) have proven to be powerful tools for generating genetically modified animals. Successful derivations of ESCs have been reported from mouse, rat, rhesus monkey and human.Most genetically modified pigs have been produced using genetically modified somatic cells nuclear transfer(SCNT). Due to limited proliferative capacity of somatic cells, it is difficult to establish genetically modified somatic cell lines via gene targeting. As a substitute of ESCs, induced pluripotent stem cells (iPSCs) generated from mouse somatic cells by overexpressing defined cocktails of transcription factors can produce germline-competent chimeras and tetraploid complementation mice.Porcine induced pluripotent stem cells (iPSCs) have been established by several groups which potentially serve as substitutes of somatic cells for producing genetically modified pigs via nuclear transfer. A lot of efforts have been made to generate cloned pigs from piPSCs, but the clone efficiency of piPSCs is such extremely low that only one group reported generation of live piPSC-NT piglets, all of which died in less than two months. Mechanism that leads to the low efficiency of piPSC cloning has not been studied so far. Compromised pluripotency of mouse iPSCs have been attributed to epigenetic errors originating from reprogramming process by several groups. It has also been proposed that aberrant reprogramming of somatic epigenetic modification patterns in SCNT embryos may contribute to developmental failure. In order to investigate causes of the low efficient piPSC cloning, we identified possible aberrant epigenetic changes during reprogramming in piPSCs and piPSC-NT embryos,including DNA hyper/hypo-methylation, miscoded genomic imprinting and deregulated X chromosome inactivation (XCI).In this study, we generated iPSCs from pig fetal fibroblasts (PFFs) by using two types of vectors. One is pSTEM-h100p which included eight reprogramming factors of human OCT4, SOX2, KLF4, C-MYC,NANOG, LIN28, NR5A2 and miR302.The other is a DOX-inducible piggyBac vector bearing four factors of human OCT4, SOX2, KLF4 and C-MYC. These piPSCs were positive for alkaline phosphatase(AP), OCT4, NANOG, SSEA-1 and SSEA-3 by immunofluorescence analysis and could differentiate into derivatives of all three germ layers in teratomas. Quantitative-PCR (qPCR) analysis showed that piPSCs expressed endogenous OCT4 gene at very low or even undetectable levels, which were consistent with the fact of hyper-methylation of endogenous OCT4 promoter region in all piPSC lines.To examine how genomic imprinting may be perturbed in piPSCs, we derived multiple piPSC clones from hybrid PFFs of Duroc × Rongchang pig carrying single nucleotide polymorphisms (SNPs)at some imprinted gene locus. First, we assayed four imprinted genes MAGEL2, MIR296, NECDIN and GTL2 which were mono-allelic expressed in PFFs by allele-specific pyrosequencing analysis. In addition, we performed allele-specific pyrosequencing analysis to determine if mono-allelic expression profile of the four imprinted genes was retained in piPSCs. The results showed that the imprint of MAGEI and NECDIN were variably lost among these piPSC clones, whereas GTL2 was silenced in all piPSCs.SNPs of an X-linked genes, G6PD, were identified in hybrid PFFs of Duroc × Rongchang pig. We performed allele-specific RT-PCR analysis to determine the XCI status in female piPSCs. Sequencing results of the RT-PCR products showed that mono-allelic expression of G6PD in all heterozygous piPS cell lines, including five mouse ESC-like piPSCs which had no dense sign of H3K27 methylation enrichment on X chromosome. The results indicated that no piPSCs in this study have two active X chromosomes as naive mESCs.Hyper-methylation of endogenous OCT4 promoter was also found in piPSC NT embryos, with 15 percent of DNA methylation level higher than that of endogenous OCT4 promoter in PFFs-NT embryos.Hyper-methylation of endogenous OCT4 promoter correlated with lower OCT4 expression in piPSC NT embryos. When piPSCs and PFFs transfected with pOCT4-GFP reporters were used as donor cells for NT, most of piPSC NT embryos were GFP negative, revealing that silenced OCT4 in piPSCs were intractable to reprogramming in piPSC NT embryos.XCI patterns in piPSCs embryos were analyzed by immunostaining for H3K27me3. Less proportion of blastmeres which contained one inactive X chromosome was found in undifferentiated piPSC NT blastocysts, compared with in vitro fertilizaton (IVF), parthenogenesis (PA) and SCNT blastocysts. All differentiated piPSC NT blastocysts had two inactive X chromosomes in some blastomeres, indicating aberrant XCI patterns in both undifferentiated and differentiated piPSC NT enbryos.In order to improve development of piPSC NT embryos, we tried to complement epigenetic disturbances in piPSC NT embryos by aggregating two 4-cell differentiated piPSC NT embryos with two 4-cell PFF NT embryos. A total of 824 aggregation blastocysts of differentiated piPSC NT embryos and PFF NT embryos were transplanted into surrogate pigs, resulting in 2 pigs pregnant and 6 cloned piglets born. All piglets were only GFP positive for complete origin of PFFs, indicating no chimeric piglets from both piPSC NT embryos and PFF NT embryos were obtained.