Inzicht in Menselijke Naïeve Pluripotentie: Extra-embryonaal Mesoderm Modellering en de Rol van DNMT3L

In this PhD thesis, I aimed to investigate the developmental potential of naive human pluripotent stem cells (hPSCs) toward postimplantation extraembryonic lineages. I established a new model enabling the study and manipulation of this important cell type in vitro. This study impacts our ability to model and study the molecular mechanisms of early human postimplantation development and related diseases. In addition, my goal was to determine the function of DNMT3L, the most abundant chromatin protein in naive hPSCs, on human naive pluripotency and the developmental potential of naive hPSCs. To achieve this, we knocked out DNMT3L using CRISPR/Cas9 combined with comprehensive downstream multi-omics analyses. Our study provides insights into the roles of DNMT3L in naive hPSCs. In article 1, I found that naive hPSCs are able to give rise to extraembryonic mesoderm cells (EXMCs) by inhibiting Nodal signaling and GSK3B (Pham et al. 2022). These EXMCs express human and non-human primate extraembryonic genes. EXMCs arise following an epithelial-to-mesenchymal transition and are capable of self-renewal. They also recapitulate other features of human and monkey EXMCs such as chromatin accessibility of regulatory elements, gene regulatory network activity, signaling, and extracellular matrix gene expression. We also found that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. In article 2, I determined the function of DNMT3L on human naive pluripotency and the developmental potential of naive hPSCs by perturbing DNMT3L and conducting comprehensive downstream molecular profiling. Our results suggested that DNMT3L is dispensable to maintain naive human pluripotency. Interestingly, our transposable element analyses revealed a decreased expression of naive hominoid-specific transposons upon DNMT3L knockout. In contrast, expression of primed-specific transposons increased in DNMT3L knockout naive hPSCs. Our data revealed the reduction in global DNA methylation in the DNMT3L knockout naive hPSCs compared to their wild-type counterparts. This finding provides insights into the role of DNMT3L in humans. Furthermore, we also showed that DNMT3L knockout facilitates naive hPSCs to trophectoderm differentiation, suggesting that DNMT3L potentially prevents differentiation of naive hPSCs into trophectoderm. In addition, blastoids generated from DNMT3L knockout cells exhibited smaller sizes and improper segregation of epiblast and trophectoderm. Our study provides insights into the roles of DNMT3L in naive hPSCs and early human embryo development.