Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq (scRNA-seq) analyses have revealed differences between humans and mice, necessitating the study of human embryos 1-8 . Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation stage studies. Thus, recent efforts have focused on developing in vitro self-organising models using human stem cells 9-17 . Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (nHyC)-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naïve human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naïve hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naïve hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pre-gastrula stage, whose emergence can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have thus successfully modelled and revealed the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.