Blastocyst complementation generates exogenous donor-derived liver in ahepatic pigs

Liver diseases are one of the leading causes of morbidity and mortality worldwide. Globally, liver diseases are responsible for approximately 2 million deaths annually (1 of every 25 deaths). Many of the patients with chronic liver diseases can benefit from organ transplantation. However, stringent criteria for placement on organ transplantation waitlist and chronic shortage of organs preclude access to patients. To bridge the shortfall, generation of chimeric human organs in pigs has long been considered as an alternative. Here, we report feasibility of the approach by generating chimeric livers in pigs using a conditional blastocyst complementation approach that creates a vacant niche in chimeric hosts, enabling the initiation of organogenesis through donor-derived pluripotent cells. Porcine fetal fibroblasts were sequentially targeted for knockin of CRE into the endogenous FOXA3 locus (FOXA3^CRE) followed by floxing of exon 1 of HHEX (FOXA3^CREHHEX^loxP/loxP) locus. The conditional HHEX knockout and constitutive GFP donor (COL1A^{CAG:LACZ 2A EGFP}) were used as nuclear donors to generate host embryos by somatic cell nuclear transfer, and complemented and transferred into estrus synchronized surrogates. In the resulting fetuses, donor EGFP blastomeres reconstituted hepatocytes as confirmed by immunohistochemistry. These results potentially pave the way for exogenous donor-derived hepatogenesis in large animal models.