Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Abstract

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs 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 pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Dynamic 3D genome reorganization during development and metabolic stress of the porcine liver

“…We next measured the spatial proximity between promoters, which represents an additional active transcriptional program responding to signaling and environmental cues 36 . Consistent with the previous observations in mouse 68 , human 69 , and pig 70 , genes with relatively high expression exhibited an elevated extent of spatial associations, which were most likely to be occupied by common transcription factors (Supplementary Fig. S14a ).…”

Deeply conserved super-enhancers maintain stem cell pluripotency in placental mammals

“…4A). To understand the functions of identified enhancers, we combined transcriptome data, H3K4me3 (marks active promoters) ChIP-seq data, and Hi-C data (51, 52), to link enhancers to their potential target genes. We identified 3067 shared TE-linked genes (about 27.7 % of porcine TE linked genes) and 58 shared (about 11.2 % of total porcine SE linked genes) SE-linked genes in all three species (Fig.…”

“…In this study, we assigned the ChIP-defined SEs to their neighboring genes, allowing a maximal distance of 100 kb between SE and the target TSS, in combined with transcriptome data, H3K4me3 (marks active promoters) ChIP-seq data, and avaiblale Hi-C data from pigs, mice, and humans (51, 52). The chromosome conformation was analyzed by HiCExplorer (75).…”

Allele-specific Effects of Three-dimensional Genome Architecture in Hybrid Pigs

“…To elucidate how this observed extensive rewiring of PEIs may contribute to the transcriptomic divergence across haploid genomes, we calculated the regulatory potential score (RPS, a spatial proximity-based index of the combined regulatory effects of multiple enhancers for a given gene) for each promoter using a uni ed set of PEIs from all haplotypes of a given tissue 27,42 ; see Methods). As expected, genes with larger RPS had higher expression within haplotypes (Supplementary Fig.…”

“…Sus scrofa (i.e., pig or swine) is a primary source of calories for humans; the world's pig population reached almost 1 billion, with China alone accounting for 49 percent 12 Moreover, pig is rapidly emerging as a versatile and informative biomedical model for human developmental processes 13,14 and complex diseases 15,16 , in addition to its utility in vaccine 17 and drug design 18 due to the relatively close anatomical 19,20 , physiological 21,22 , immunological 23 and genetic [24][25][26] similarities with humans. The ability to generate genome-editing mutations in pig combined with somatic nuclear cloning procedures has resulted in a number of new models for various human diseases 27 and suggests the potential use of pigs as a source for xenotransplantation 28 . Extensive genomic divergence is known to have occurred between European and Asian pigs, attributable to the relative isolation of European and Asian lineages (at least one million years ago and their independent domestication in multiple locations across Eurasia in the past ∼10,000 years 25 .…”