Efficient derivation of transgene-free porcine induced pluripotent stem cells enables in vitro modeling of species-specific developmental timing

Sus domesticus (pig) are an excellent large mammalian model for comparative studiesdue to their relatively comparable physiology and organ size to humans. The derivation of transgene-free porcine pluripotent stem cells (PiPSCs) will therefore benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. Established PiPSCs can robustly differentiate into derivates representing the primary germ layers in vitro and in vivo. Furthermore, the transgene- free PiPSCs preserve intrinsic species-specific developmental timing in culture. This capacity is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at ~ 3.7 hours, a time scale recapitulating in vivo porcine somitogenesis. We therefore propose that these transgene-free PiPSCs can serve as a powerful tool for modeling development and disease, informing both conserved and unique features of mammalian pluripotency and developmental timing mechanisms. Overall design: A total of 36 bulk RNA-seq samples: (1) compare undifferentiated clonal PiPSC lines to fibroblasts. (2) PiPSC differentiation to various lineage-specific progenitors. (3) PiPSC differentiation to primitive streak, PSM and somite cell fates. To construct the RNA-seq library, total RNA from each sample (an input of ~100 ng total (RNA) was used following the LM-seq protocol