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Generation and Characterization of Stable Pig Pre-gastrulation Epiblast Stem Cell Lines [single-cell RNA-seq]

ABSTRACT: Despite ongoing attempts since the 1990s, no stable embryonic stem cell line has been established in pigs. Here, guided by insights from large-scale single-cell RNA-sequencing profiling of pig embryos from embryonic day (E) 0 to E14, we developed an in vitro culture system for establishing and maintaining stable pluripotent stem cell lines from pig E10 pre-gastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in culture, the pgEpiSCs maintain their pluripotency and normal karyotypes after more than 200 passages. Strikingly, ultra-deep in situ Hi-C analysis revealed functional impacts of 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in the pgEpiSCs. Practically, we confirmed that the pgEpiSCs can readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of large model animal stem cells and open new avenues for biological research, animal husbandry, and biomedicine.

ORGANISM(S): Sus scrofa

PROVIDER: GSE168106 | GEO | 2023/12/13

REPOSITORIES: GEO

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Project description:The wide application of pig disease model has caused a surge of interest in the study of derivation of pig induced pluripotent cells (iPSCs). Here we performed genome-wide analysis of gene expression profiling by RNA-seq and small RNA-seq and DNA methylation profile by MeDIP-seq in pig iPSCs through comparison with somatic cells. We identified mRNA and microRNA transcripts that were specifically expressed in pig iPSCs. Our analysis identifies the genes up-regulated in pig iPS compared with somatic cells and also the differentially expressed genes between pig iPSCs under different culture medium. We then pursued comprehensive bioinformatics analyses, including functional annotation of the generated data within the context of biological pathways, to uncover novel biological functions associated with maintenance of pluripotency in pig. This result supports that pig iPS have transcript profiles linked to “ribosome”, “chromatin remodeling”, and genes involved in “cell cycle “that may be critical to maintain their pluripotency, plasticity, and stem cell function. Our analysis demonstrates the key role of RNA splicing in regulating the pluripotency phenotype of pig cells. Specifically, the data indicate distinctive expression patterns for SALL4 spliced variants in different pig cell types and highlight the necessity of defining the type of SALL4 when addressing the expression of this gene in pig cells. MeDIP-seq data revealed that the distribution patterns of methylation signals in pig iPS and somatic cells along the genome. We identify 25 novel porcine miRNA, including pluripotency-related miR-302/367cluster up-regulated in pig iPSCs. At last, we profile the dynamic gene expression signature of pluripotent genes in the preimplantation development embryo of pig. The resulting comprehensive data allowed us to compare various different subsets of pig pluripotent cell. This information provided by our analysis will ultimately advance the efforts at generating stable naïve pluripotency in pig cells.

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Generation and Characterization of Stable Pig Pre-gastrulation Epiblast Stem Cell Lines [single-cell RNA-seq]

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