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Epigenetic reprogramming of rabbit pluripotent stem cells
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International audience. During reprogramming, induced pluripotent stem cells (iPSCs) may retain residual epigenetic “memory” from their tissue of origin, resulting in cellular heterogeneity and constraining their differentiation potential. Pluripotent stem cells (PSCs) exist in two distinct states of pluripotency called ‘naïve’ and ‘primed’. These two states differ in signaling pathways, growth factor dependence, cell cycle duration, epigenetic regulations and developmental potentials. Specifically, only naïve PSCs possess the remarkable ability to colonize host embryos and generate germline chimaeras. However, the attainment of a naïve-like state in iPSC is a considerable challenge in non-rodent mammals. In this context, rabbits represent a valuable model for elucidating novel mechanisms and markers of pluripotency, given their physiological and developmental similarities with primates and their ease of use. Like human and monkey PSCs, rabbit PSCs exhibit characteristics of primed state pluripotency and are unable to form chimeras. Our project aims to overcome epigenetic barriers hindering the reprogramming and maintenance of naïve-state pluripotency in rabbit stem cells. To achieve this, we are conducting CRISPR perturbation screening to identify chromatin modifiers (CMs) which promote reprogramming to naïve state. The optimal combinations of CMs will subsequently be activated and/or repressed using CRISPRai-based systems to generate rabbit naïve stem cells. Additionally, we have developed a novel culture media (called ALGöX) composed of Activin A, LIF, Gö6983 (PKC inhibitor) and XAV939 (tankyrase inhibitor) aimed at inducing naïve-like characteristics in rabbit iPSCs. Epigenetic inhibitory compounds will then be included to modulate relevant chromatin modification pathways without any genetic modification. Ultimately, through epigenetic reprogramming, we anticipate the generation of transgene-free rabbit iPSCs in the naïve-state of pluripotency. These cells could be used for various applications, including the generation of organoids for disease modeling and advancing our understanding of pluripotency dynamics.
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