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GENERATION OF SYSTEMIC CHIMERAS WITH RABBIT INDUCED PLURIPOTENT STEM CELLS REPROGRAMMED WITH KLF2, ERAS AND PRMT6
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International audience. Embryo-derived and induced pluripotent stem cells (ESCs and iPSCs, respectively) exist in two different states, designated naive and primed. Naïve and primed states differ by the signalling pathways, transcription factors and epigenetic regulators that hold the cells in one of either state. In rabbits, ESCs and iPSCs only exist in the primed state of pluripotency.We developed a strategy that led to identify factors capable of reprogramming rabbit iPSCs to a naïve-like, embryo-colonization competent state. We conducted an unbiased screening of a cDNA library encoding a panel of 36 factors, including transcription factors, epigenetic regulators and signalling molecules associated with naïve-state pluripotency in rodents and primates. All the factors were randomly introduced into rabbit primed iPSCs by means of lentivector infection, followed by clonogenic growth in a FGF2/KOSR-deprived culture media containing LIF, activin A, PKC and WNT inhibitors. The three transgenes most frequently detected were PRMT6 that encodes an arginine methyl transferase, ERAS that encodes a GTPase, and KLF2 that encodes Krüppel-like factor 2. To investigate the transgene action, KLF2, ERAS and PRMT6 were overexpressed, either separately, in pairwise combinations, or all three together, in rabbit iPSC cells, either constitutively or with induction. KLF2 was necessary and sufficient to sustain self-renewal in a FGF2/KOSR-deprived culture regimen. The addition of both ERAS and PRMT6 led to the appearance of a subset of cells expressing the naïve state-specific marker CD75 at a high level. These CD75high cell population underwent epigenetic reconfiguration typical of naïve-state pluripotency including reactivation of the 2nd X-chromosome. Remarkably, the CD75high cells gained the ability to produce chimeric embryos and fetuses, showing a high contribution in all major organs. Our results describe for the first systemic chimeras in the rabbit species, similar to the chimeras produced in mice with ES cells.
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