Establishment of Human Formative Pluripotent Stem Cell Like Cells Exhibiting Amniotic Differentiation Potentials [WGBS]

Human embryos undergo pivotal morphogenetic remodelling shortly after implantation. The understanding of this crucial stage is severely impeded by the scarcity of embryonic samples and ethical constraints. Pluripotent stem cells with the competence for somatic and germline differentiation serve as in vitro models of epiblast. In this study, we established human formative pluripotent stem cells-like cell (hfPSC-LCs) from naive human embryonic stem cells (hESCs), conventional hESCs, human induced pluripotent stem cells (hiPSCs), as well as human blastocysts using the three dimensional (3D) Matrigel culture system. Similar to pre gastrula stage epiblast hfPSC-LCs self-organize into self-renewing colonies with an apical lumen and exhibit several hallmarks of formative pluripotency, consistent with the properties observed in mouse fPSCs. Notably, single cells of hfPSC-LCs could differentiate into amnion-like precursor cells (hALPCs) which are transcriptionally and morphologically similar to the bona fide amnion. Meanwhile, hfPSC-LCs directly respond to primordial germ cell (PGC) induction signals, generating PGC-like cells (PGCLCs) either as single - cell aggregates or intact colonies, with an efficiency of approximately 50%. Chromatin accessibility analysis revealed that the differentiation capacity of hfPSC-LCs for gametes and amnion lineages might correlate with the accessible chromatin architecture of PGC and amnion associated genes. Loss of 3D-Matrigel niche disrupts formative pluripotency in both mouse and human, manifesting as downregulated formative markers and compromised differentiation capacity. Collectively, our findings establish hfPSC-LCs as a 3D model for investigating formative pluripotency of humans, thereby probably addressing a critical gap in the understanding of human pluripotency transitions.