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A novel combination of transcription factors and chromatin modifiers supporting naïve pluripotency in rabbit stem cells
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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 state. Using rabbit iPSCs as a model system, 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 new culture media designated VALGöX.The three transgenes most frequently detected and associated with naive-state pluripotency include an arginine methyl transferase, a GTPase, and a transcription factor of the KLF family (designated KEP transgene combination). In order to confirm their action, these three genes were overexpressed in rabbit iPSC cells. Remarkably, the reprogrammed cells underwent epigenetic reconfiguration typical of naïve-state pluripotency including reactivation of the 2nd X-chromosome, and produced chimeric embryos with a high efficiency. Overall, our findings underscore a synergy between the VALGöX culture regimen and the KEP transgenes, enabling rabbit iPSCs to acquire the molecular and functional characteristics of naïve pluripotency.
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