183 Generation of porcine embryonic stem cell lines derived from nuclear transfer embryos reconstructed with induced pluripotent stem cells

To establish a porcine embryonic stem (ES) cell line that not only maintains self-renewing capacity but also exhibits pluripotency [Haraguchi et al. 2012 J. Reprod. Dev. 58, 707-716], 6 synthetic porcine RNAs (Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28) were chemically transfected into outgrowth cultured cells derived from the inner cell mass of in vitro-produced porcine embryos. Subsequently, cells grew as compact, dome-shaped colonies displaying alkaline phosphatase activity and were cultured for more than 20 passages. Although 13 candidate cell lines were generated (13/43, 30%), none formed teratomas after injection of the cells into SCID (sever combined immunodeficiency) mice. We also observed that when transfection of the exogeneous RNAs was discontinued, the cells no longer maintained a stem cell morphology and began to differentiate (13/13, 100%). This suggests that continuous expression of exogenous reprogramming factors is necessary to maintain induced pluripotency in the pig. Next, we used cloned embryos reconstructed with porcine induced pluripotent stem cells (piPSC), which were created using a recombinant lentivirus expression vector carrying 6 mouse reprogramming factor genes (the same as above) and green fluorescent protein (GFP) (Fukuda et al. 2017 J. Cell Biochem. 118, 537-553]. The piPSC were dispersed to a single cell suspension and electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes. A second cytoplast was then fused to the first reconstruct (double cytoplast nuclear transfer). Reconstructs were electrically activated and cultured in microwells with porcine zygote medium-3 (PZM3). After 5 days, reconstructed embryos developed to GFP-positive blastocysts (10/93, 11%) and 4- to 8-cell stages (25/93, 27%). The blastocysts (10) and 4- to 8-cell-stage embryos (25) were transferred onto mouse embryonic fibroblast feeder cells for outgrowth culture in FCS-based ES cell medium supplemented with 2% polyvinylpyrrolidone. After 24h, the medium was changed to piPSC medium containing CHIR99021, PD0325901, thiazovivin, and GF-109203x. Embryos attached to the feeder cells began to outgrow (8/10 of blastocysts and 6/25 of 4- to 8-cell-stage embryos). To date, 3 ES-like cell lines have been established from blastomeres of embryos (3/25, 12%) but not from blastocysts (0/10, 0%). They show GFP fluorescence and have been maintained continuously in culture for more than 20 passages without any overt changes in morphology. These results suggest that the constant expression of reprogramming factors and the use of combinations of specific small molecule inhibitors largely contribute to the establishment of pluripotent cells in the pig. Further characterisation of the cells is ongoing, including methylation status of the X chromosome and the capacity for in vivo differentiation.