The present study was performed to investigate the effect of Hh-Ag1.5, a small-molecule chemical agonist of SMOothened receptor, on the in vitro maturation and development of in vitro fertilized (IVF) embryos in pigs. Oocytes or fertilized embryos were cultured in a maturation or embryo culture medium supplemented with 0 (control), 25, 50 or 100 nM of Hh-Ag1.5, respectively. Although the maturation rate were not different among treatment groups, the blastocyst formation rate in the group treated with 25 nM Hh-Ag1.5 was significantly increased compared to other groups (P<0.05). While the highest dose of Hh-Ag1.5 (100 nM) did negatively affect to the embryo development and cell number in blastocysts compared to other groups (P<0.05), the apoptotic cell index in blastocysts was significantly lower in 25 and 50 nM groups than in control and 100 nM groups (P<0.05). The mRNA expression of the proapoptotic gene Bax and the ratio of Bax/Bcl-XL decreased in among treatment groups compared to control (P<0.05). The embryo quality related genes, Tert and Zfp42, were significantly decreased in 50 and 100 nM groups compared with control and 25 nM groups (P<0.05). In conclusion, the addition of 25 nM Hh-Ag1.5 to in vitro maturation and culture medium can enhance the developmental potential as well as quality of IVF embryos in pig.

The generation and application of porcine iPSCs (piPSCs) as a large animal model may enable the test for the efficacy and safety of the therapy in the field of human regenerative medicine. Here, we report the generation of piPSC from wild (a 10-day-old Massachusetts General Hospital miniature pig; MGH minipig) and genetically modified pig, alpha1,3-Galactosyltransferase knock-out (—/—) (GalT KO homo) and human CD46 (membrane cofactor protein) knock-in (hCD46 KI) MGH minipig (a 10-day-old). Fibroblasts were isolated from the ear skin of wild and MGH minipigs, respectively. After 2 passages, each of fibroblasts was transduced with cocktail of 6 human factors (POU5F1, NANOG, SOX2, C-MYC, KLF4, and LIN28) and cultured on a mitotically inactive mouse embryonic fibroblast (MEF) monolayer. Both of reprogrammed somatic cells expressed the classical pluripotency markers (POU5F1, NANOG, and SOX2) and surface marker (SSEA1). Similar to mouse ESCs, both piPSCs from wild and transgenic minipigs were negative for SSEA3, Tra-1-60, and Tra-1-81. Further these cells could form embryoid body (EB) and differentiate into 3 germ layers in vitro (ectoderm: FOXJ3 and PAX6, mesoderm: HAND2, and endoderm: SOX17 and GATA6). Our piPSCs may provide useful source as a large animal model for studying approaches that can reduce an immune- rejection of cell or organ transplantation.