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Chimeric pigs harboring organs derived from human stem cells are promising for patient-specific regenerative therapies. Induced pluripotent stem cells (iPSCs) can contribute to all cell types of the fetus, including germline after injection into embryos. However, ethical concerns prohibit testing human iPSCs in chimera assays. Here, we evaluated porcine embryos as hosts for an interspecies chimera assay using iPSCs from either cynomolgus monkeys (cyiPSCs) or mouse (miPSCs). To establish an in vitro culture system compatible for cyiPSCs and porcine embryos, we determined blastocyst development in eight different stem cell media. The highest developmental rates of blastocysts were achieved in Knockout Dulbecco's modified Eagle's medium with 20% knockout serum replacement. We found that cyiPSCs injected into porcine embryos survived in vitro and were mostly located in the trophectoderm (TE). Instead, when miPSCs were injected into porcine embryos, the cells rapidly proliferated. The behavior of chimeras developed in vitro was recapitulated in vivo; cyiPSCs were observed in the TE, but not in the porcine epiblast. However, when miPSCs were injected into in vivo derived porcine embryos, mouse cells were found in both, the epiblast and TE. These results demonstrate that porcine embryos could be useful for evaluating the interspecies chimera-forming ability of iPSCs from different species. [ABSTRACT FROM AUTHOR]

In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin-nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity.