Efficient Derivation and Transcriptional Characterization of Mouse Extra-Embryonic Endoderm Stem Cell Lines Generated...

Somatic cell nuclear transfer (SCNT) holds great promise for regenerative medicine and agriculture, but its application is severely hampered by low efficiency, primarily attributable to aberrant epigenetic reprogramming. Although embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have been successfully derived from cloned embryos, an in vitro counterpart of the primitive endoderm (PrE) lineage has remained unavailable. To address this gap, this study reports the first successful establishment of extra-embryonic endoderm stem cell lines (XENs) from mouse SCNT-derived blastocysts (NT-XENs). Under conventional culture conditions, NT-XENs were generated from hybrid B6D2F1 blastocysts at a high efficiency of 55%, statistically comparable to that of fertilization-derived XEN lines (FD-XENs, 50%), whereas derivation from inbred C57BL/6J SCNT-derived blastocysts was markedly lower (12.5%). Immunofluorescence and NanoString multiplex gene expression profiling confirmed that NT-XENs robustly expressed specific marker genes for PrE/XENs (e.g., Gata4, Gata6, and Sox17), while exhibiting negligible or absent expression of pluripotency and trophoblast markers. Based on NanoString assay data, NT-XENs and FD-XENs shared highly similar gene expression patterns, yet also exhibited some nonnegligible differences, exemplified by the differentially expressed genes (DEGs) Pecam1, Gtl2, Thbd, and Xlr3b. These differences raise a preliminary hypothesis that the NT-XENs might exhibit a slight transcriptional propensity toward a more differentiated state, and potentially reflect lingering traces of SCNT-associated epigenetic errors, such as localized dysregulation of imprinted genes and X-linked transcripts. In summary, this study successfully establishes NT-XEN cell lines, providing a valuable in vitro model for investigating the reprogramming scenarios of PrE lineage in SCNT and the mechanisms underlying developmental failure of cloned embryos.