THE DEVELOPMENTAL POTENCY OF NAÏVE HIPSCS IN A THREE-DIMENSIONAL MORPHOGENETIC CONTINUUM

To properly recapitulate human morphogenesis, models that mimic spatial organization and cell complexity of in vivo developing tissues are required. 3D models, by combining the differentiation potential of human pluripotent stem cells (hPSCs) with intrinsic self-organization ability of cells, can recapitulate key features of morphogenesis. Blastoids and organoids are new models of in vivo epiblast development and organogenesis, respectively. Technical and ethical limitations restrict the use of blastoids up to gastrulation-like stages. Organoids, instead, which are based on the use of primed hPSCs, are limited by differentiation biases related to the epigenetic landscape of cells, that lead to batch-to-batch heterogeneity in the differentiation outcomes. Naïve hPSCs represent a possible alternative to overcome the limitations of these models. Indeed, naïve pluripotency has been defined as a developmental “ground state” due to its unique epigenetic and transcriptional properties. The earlier developmental stage of naïve hPSCs represent a “blank page” from which multiple differentiation trajectories can be explored, allowing the characterization of underlying transcriptional, epigenetic and metabolic events. Moreover, with naïve hPSCs it is possible to model physiological epigenetic processes, including X-chromosome inactivation and epigenetic-associated diseases. In this context, this work aims to exploit the developmental potential of naïve induced PSCs to generate a new 3D model of human epiblast development and neurogenesis relevant for the investigation of inaccessible in vivo processes in an in vitro developmental continuum. To achieve this, a new 3D culture system for naïve hPSCs, based on the use of a hydrogel rich in extracellular matrix (ECM) components, has been developed. This permits to avoid the use of feeders, commonly used in naïve cultures and a source of variability during differentiation. Moreover, this allows the intrinsic self-organization of cells in space during differentiation, thus supporting the possibility to properly model morphogenetic processes in vitro. This work contributes to extend the knowledge in the hPSCs field, by providing, for the first time, a description of ECM organization in the naïve microenvironment and shows how the 3D organization of laminin is linked to the maintenance of naïve clonogenicity. Moreover, the 3D-ECM rich hydrogel maintained both naïve identity after prolonged passaging and the differentiation potency towards extraembryonic lineages. Single naïve cells, cultured in the 3D-ECM rich hydrogel, were tested for their capacity to recapitulate 3D human morphogenesis in vitro. New protocols for the direct differentiation of naïve cells into 3D post-implantation epiblast-like cysts or into 3D neural organoids have been established. By characterizing the differentiation trajectories, this work shows that naïve cells can acquire a neuroepithelial-like identity only after transiently adopting a post-implantation epiblast-like state, which was never observed before. Moreover, for the first time naïve have been guided to acquire precise neuroepithelial progenitor identities, along the in vivo antero-posterior axis, including posterior neuromesodermal progenitor identity. Finally, as reported for primed hPSCs-derived organoids, this work demonstrates that naïve-derived cortical organoids can mimic in vivo-like features of the maturing human cortex, like cell type complexity, partial cortical layering and functional neuronal activity. In conclusion, this work describes the establishment of a new in vitro model that opens the realistic possibility of recapitulating key stages of human development in a 3D context, similar to that observed in vivo, starting from single naïve hiPSCs which give access to earlier state transitions relevant for human embryogenesis