Three Dimensional Matrigel Endows Human Formative Pluripotent Stem Cells with Pre-gastrula Stage Epiblast Features [ATAC-seq]

Human embryos undergo pivotal morphogenetic remodeling shortly after implantation into the uterus. However, our 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 simulants of epiblast pluripotency continuum. Here, we establish human formative pluripotent stem cells (hfPSCs) from naïve human embryonic stem cells (hESCs), conventional hESCs of different backgrounds, human induced pluripotent stem cells (hiPSCs) as well as human blastocysts in three-dimensional (3D) Matrigel. Similar to pre-gastrula stage epiblast, hfPSCs self-organize into renewal clones with apical lumen and exhibit unique features of formative pluripotency. Functionally, hfPSCs correspond to pre-amnion epiblast cells and exhibit the enhanced tri-lineage differentiation capacity and superior germline competence. Mechanistically, Matrigel-based three-dimensional culture conditions ensure the formative pluripotency through dual ways: biomechanical support and biochemical priming. Thus, hfPSCs open new avenues for creating faithful stem cell-based embryo models and developing the relevant differentiation protocols for regenerative medicine. Overall design: To decipher the multi-omics landscape of hfPSCs cultured in three-dimensional Matrigel, we conducted bulk RNA-seq, single-cell RNA-seq (scRNA-seq), ATAC-seq, and whole-genome bisulfite sequencing (WGBS).