We investigated whether sequential reprogramming via porcine induced pluripotent stem cells (piPSCs) or exposure to oocyte cytoplasm following nuclear transfer could generate nuclear transfer-derived ESCs (piPSCs-ntESCs). Nuclear transfer embryos were reconstructed with piPSCs possessing a ZsGreen fluorescent marker for expression of exogenous Nanog and Lin28. Reconstructed oocytes developed to morphologically normal 8-cell/morulae (35/93, 37.6%) and blastocysts (12/93, 12.9%). Although most green fluorescent protein-positive blastocysts showed efficient outgrowth (8/10, 80%), none formed primary colonies and all cultures degenerated. Conversely, 15% of fluorescent positive 8-cell/morula stage embryos showed outgrowth (6/40), with three forming primary colonies (7.5%). All three were expanded and maintained as piPSC-ntESC lines. These cell lines expressed stem cell marker genes and proteins. Despite inactivation of one X chromosome, all piPSC-ntESC lines formed teratomas comprising derivatives from all three embryonic germ layers. Strong SSEA1, 3, and 4 expression was detected at the 8-cell/morula stage in embryos reconstructed from both piPSCs and porcine embryonic fibroblasts (PEFs). SSEA3 was notably absent from IVF controls at pre-implantation embryo stages. Finally, we attempted to establish ntESCs from 8-cell/morulae reconstructed with PEFs using the same culture conditions as those for piPSC-ntESC derivation. Although eight primary colonies arose from 107 embryos (7.5%), they all degenerated after the first passage culture. Early and sustained expression of key reprogramming regulatory factors may be critical for pluripotent stem cell derivation to derive piPSC-ntESCs from 8-cell/morula stages, while the expression of SSEAs may be involved in the initial stem cell colony formation phases.

中文翻译：

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以诱导多能干细胞为供体核的猪核移植胚胎干细胞的建立

我们研究了通过猪诱导的多能干细胞 (piPSCs) 或在核移植后暴露于卵母细胞质的顺序重编程是否可以产生核移植衍生的 ESCs (piPSCs-ntESCs)。使用具有 ZsGreen 荧光标记的 piPSC 重建核移植胚胎，用于表达外源 Nanog 和 Lin28。重建的卵母细胞发育为形态正常的 8 细胞/桑椹胚 (35/93, 37.6%) 和囊胚 (12/93, 12.9%)。尽管大多数绿色荧光蛋白阳性囊胚显示出有效的生长 (8/10, 80%)，但没有形成原代集落并且所有培养物都退化。相反，15% 的荧光阳性 8 细胞/桑椹胚阶段胚胎显示出生长 (6/40)，形成三个原代集落 (7.5%)。所有三个都被扩展并维持为 piPSC-ntESC 系。这些细胞系表达干细胞标记基因和蛋白质。尽管一条 X 染色体失活，但所有 piPSC-ntESC 系都形成了包含来自所有三个胚胎胚层的衍生物的畸胎瘤。在从 piPSC 和猪胚胎成纤维细胞 (PEF) 重建的胚胎中，在 8 细胞/桑椹胚阶段检测到强 SSEA1、3 和 4 表达。在植入前胚胎阶段，IVF 对照中明显不存在 SSEA3。最后，我们尝试使用与 piPSC-ntESC 衍生相同的培养条件，从用 PEF 重建的 8 细胞/桑葚中建立 ntESC。尽管 107 个胚胎（7.5%）产生了 8 个原代集落，但在第一次传代培养后它们都退化了。