26 IN VIVO EXOGENIC ORGAN GENERATION WITH ORGANOGENESIS-DISABLED CLONED PIGS AS A PLATFORM

Abstract

The generation of organs from pluripotent stem cells (PSC) is one of the ultimate goals of regenerative medicine. We have demonstrated that functional organs can be generated in vivo from xenogenic PSC in the body of organogenesis-disabled mice using blastocyst complementation. To apply this principle in generating human organs, a technical platform using large non-rodent mammals is essential. The aim of the present study was to establish a blastocyst complementation system using cloned pig embryos. We generated transgenic-cloned pigs with an apancreatic phenotype via the overexpression of Hes1 (hairy and enhancer of split-1) under the Pdx1 promoter (pancreatic and duodenal homeobox-1). Cloned embryos of apancreatic pigs (host embryos, male) were complemented (i.e. chimerized) by blastomeres of cloned embryos (donor cells, female) with normal developmental competence. Chimeric embryos were cultured for 1 or 2 days before being transferred into the uteri of oestrus-synchronized gilts. The complementation of 292 Pdx1-Hes1 cloned embryos gave rise to 260 (89.0%) blastocysts. The transfer of these blastocysts resulted in 5 male chimeric pigs. Chimerism was confirmed by the detection of host embryo-derived Pdx1-Hes1 and marker transgenes of the donor cells, such as humanized Kusabira-Orange (huKO) or Pdx1-Venus. Chimeric pigs possessed normally formed pancreata entirely derived from the exogenous donor cells. We thus established a blastocyst complementation system in the pig using cloned embryos that would otherwise give rise to apancreatic animals. Chimeric pigs obtained developed normally, maintaining normal serum glucose concentrations up to maturity, and became fertile boars. Mating the chimeric boars with 7 wild-type sows gave rise to 72 fetuses/piglets of which 37 (51.4%) exhibited the apancreatic phenotype. These results indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize with a cloned dysorganogenetic embryo. Blastocyst complementation using cloned porcine embryos may permit the use of a large animal for the generation of functional organs from xenogenic PSC, including human iPSC. The chimeric boar produced by blastocyst complementation sired fetuses/offspring with the apancreatic phenotype in a Mendelian fashion. Porcine fetuses with an organogenesis-disabled phenotype may provide a useful platform for organ regeneration research. Table 1.Production of chimeric pigs by complementation and of Pdx1-Hes1 cloned embryos