Generation of somatic chimeras using genetically-reprogrammed rabbits induced pluripotent stem cells [KEPi]

Little is known about the molecular underpinnings of pluripotent stem cells (PSCs)’ ability to colonize preimplantation embryo epiblast to generate chimeras. Using rabbit PSCs as a model system, we conducted unbiased screening of a cDNA library encoding a panel of 36 pluripotency factors. From this, we identified KLF2, ERAS and PRMT6 whose overexpression endows the ability to self-renew in a KOSR/FGF2-free culture medium supplemented with LIF, activin A, PKC and WNT inhibitors. The reprogrammed cells acquired transcriptomic and epigenetic features of naïve pluripotency, including the reactivation of the 2nd X-chromosome. Leveraging on these PSC lines, we determined the transcriptomic signature of embryonic colonization-competence, demonstrating transcriptional repression of genes involved in MAPK, WNT, HIPPO, and EPH signaling pathways, and activation of genes involved in amino-acid metabolism, NF-kB signaling, and p53 pathway. Remarkably, these reprogrammed cells acquired the ability to produce chimeric embryos and fetuses with high contribution from PSCs in all major organs. Transcriptomic profiling of chimeric competent KEP rabbit iPS cells.