Pluripotency is the term used to describe the ability of a stem cell to give rise to all cell types in mature organisms. Pluripotent stem cells (PSCs) in mice comprise the following two main types: (i) embryonic stem cells (mESCs), derived from the early epiblast of the blastocyst, which epitomise the naïve (or ground) state of pluripotency, and (ii) epiblast stem cells (EpiSCs), derived from the late epiblast of the egg cylinder–stage embryo, which epitomise the primed state of pluripotency. Only the naïve mESCs can colonise the epiblast of the blastocyst, contribute to the development of all tissue types and generate chimeras; PSCs in the primed state cannot. Thus, chimeric competency is a hallmark of naïve pluripotency. PSC lines established in other mammals, such as primates and rabbits, display nearly all the characteristic features of primed pluripotency although they are generated from the early epiblast of the blastocyst, similar to rodent ESCs. In non-rodent species, including humans, it is challenging to capture the original state of pluripotency of early epiblast cells in PSCs. In particular, the scarcity of primate embryos makes it difficult to address this issue. As a surrogate model, the rabbit is perfectly suited to explore the nature and mechanisms of acquisition and maintenance of pluripotency in the epiblast cells and ESCs for a wide range of non-rodent mammals, including primates. The aim of our proposal is to explore naïve pluripotency and chimeric competency in the rabbit. The project’s main objectives are to (1) characterise the transcriptome of the rabbit epiblast throughout pre-implantation development and identify rabbit-specific markers of naïve pluripotency using single-embryo and single-cell RNA sequencing (RNA-seq), (2) identify new genes and small molecules for reprogramming conventional rabbit PSCs to naïve-state pluripotency and (3) capture naïve-state pluripotency from pre-implantation rabbit embryos using the identified markers and molecules. To achieve these objectives, the following two complementary approaches will be implemented: (i) unbiased screening of a library of lentiviral vectors that express transcription factors, histone-modifying enzymes and chromatin-remodelling factors, and (ii) high-throughput screening of a small-molecule library. From this study using the rabbit model, we will glean new information about the naive state of pluripotency in primates, which would be applicable to the generation of somatic chimeras in monkeys. The project comprises four partners with complementary expertise. Pierre Savatier (Stem Cell and Brain Research Institute, INSERM; Partner 1) has extensive expertise in the study of PSCs in the mouse, macaque, human and rabbit. P. Savatier recently developed the tools and methods for testing chimeric competency of PSCs using pre-implantation rabbit embryos. Veronique Duranthon (INRA; Partner 2) has extensive expertise in the molecular analysis of pre-implantation mammalian embryos at both the genomic and epigenetic levels and was first to publish a characterisation of the rabbit epiblast transcriptome. V. Duranthon specialises in bioinformatics and statistical analyses of high-throughput Omics data in domestic species, including the rabbit. Dr Romeo Ricci (IGBMC; Partner 3) has gained extensive expertise in cell biology and cellular signalling by successfully using cellular-screening approaches. R. Ricci conducts genetic and chemical cellular screening in the context of stem-cell maintenance and differentiation. Dr Fredrik Lanner, assistant Professor at the Karolinska Institutet (Partner 4), has a strong background in mouse and human pluripotency and embryo development. Through single-cell RNA-seq, Dr Lanner’s laboratory has described lineage specifications in the mouse and has established a transcriptional roadmap of the human embryo that also identified bi-allelic dosage compensation as an in vivo hallmark of the naïve pluripotent state.