Extraembryonic Endoderm (XEN) Cells Capable of Contributing to Embryonic Chimeras Established from Pig Embryos

Abstract

Summary Most of our current knowledge regarding early lineage specification and embryo-derived stem cells comes from studies in rodent models. However, key gaps remain in our understanding of these developmental processes from nonrodent species. Here, we report the detailed characterization of pig extraembryonic endoderm (pXEN) cells, which can be reliably and reproducibly generated from primitive endoderm (PrE) of blastocyst. Highly expandable pXEN cells express canonical PrE markers and transcriptionally resemble rodent XENs. The pXEN cells contribute both to extraembryonic tissues including visceral yolk sac as well as embryonic gut when injected into host blastocysts, and generate live offspring when used as a nuclear donor in somatic cell nuclear transfer (SCNT). The pXEN cell lines provide a novel model for studying lineage segregation, as well as a source for genome editing in livestock.

Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States

“…We also compared our RNA sequencing data for pXEN cells with publicly available datasets from mouse XEN cells, porcine XEN-like cells, and porcine yolk sac (day 28), as well as porcine hypoblast cells of early embryos ( Figure 7 G). Hierarchical cluster analysis demonstrated that the pXEN cells derived in our lab clustered closely with the pXENpg cell lines ( Figure 7 H) derived by Park et al [ 27 ]. We chose the signaling pathway-related genes as well ( Supplementary Table S1 ) and analyzed the expression of them in these cell lines.…”

“…The epiblast fraction of the primary blastocyst outgrowths from pigs usually fails to proliferate under the routine ESC derivation conditions used for rodents and primates. Instead, the cells are rapidly replaced by expanding extra-embryonic cells, which are often mistaken for epiblast-derived primed ESCs, as found in previous studies [27,36]. Therefore, the discovery of ways to inhibit the growth of the PrE and to promote the proliferation of EPI may be significant for obtaining authentic porcine ESCs.…”

“…The heterogeneity of pluripotency gene expression of pXEN cells (Figure 7E) may be due to the embryonic origin, since parthenogenetic embryos themselves are not normal naturally acquired embryos. RNA sequencing data analysis was performed on our XEN cells and published data from mouse XEN cells (the data for mXEN were from GSE106158 on NCBI), porcine XEN-like cells (data for pXENL were from GSE140414 [26]; data for pXENpg and pXENvv were from GSE128149 [27]), and porcine yolk sac (d 28, data for PYS were from GSE128149 [27]), as well as on porcine hypoblast cells from early embryos (data for pHYPO were retrieved from a previous study [35]). PCA plot (G) and heatmap (H) of global gene expression of these cells.…”

“…In mice, XEN cells can be obtained in three ways: by directly derivation from blastocysts [ 19 ]; by conversion of ESCs through the expression of transcription factors [ 20 , 21 , 22 ], or through chemical modification of the culture medium [ 23 , 24 ]; and by parallel generation of induced XEN during induced pluripotent stem (iPS) cell production [ 25 ]. Isolation of porcine XEN cells has rarely been reported; only two groups have reported deriving XEN cells from porcine blastocysts [ 26 , 27 ], and one group has derived XEN cells from porcine pluripotent stem cells [ 28 ]. These researchers obtained XEN cells by adding Fibroblast Growth Factor (FGF) and Leukemia Inhibitory Factor (LIF) to the culture medium, but made little mention of the specific signaling pathways that XEN cells rely on for self-renewal or the differences between XEN cells and different states of porcine ESCs.…”

Fumarylacetoacetate hydrolase gene as a knockout target for hepatic chimerism and donor liver production

“…Another strategy to target donor cells to the endoderm uses extraembryonic endoderm stem (XEN) cells as donor cells instead of iPSCs. XEN cells are stem cells derived from the primitive endoderm in pigs and rodents, which contribute to both extraembryonic and gut endoderm in chimeras ( Niakan et al., 2013 ; Park et al., 2021 ). Intraspecies pig chimeras generated using pig XEN donor cells resulted in donor cells restricted exclusively to the endoderm and extraembryonic structures.…”