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

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. We generated H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) data for pEpiSCs and pEFs with 2 replicates, respectively.