Human non-neural ectoderm trajectory separates into surface ectoderm and amniotic ectoderm in accordance with cellular density

Mechanisms specifying amniotic ectoderm and surface ectoderm are still equivocal in human due to their resemblances in expression patterns and signal requirements. This lack of knowledge hinders legitimate strategies to recapitulate embryogenesis. Here, we developed a human pluripotent stem cell model to investigate the divergence between these primordial tissues. We established a culture condition that upregulates amnioblast genes and non-neural ectodermal genes in the function of cell density. Single-cell RNA sequencing analysis on the in vitro amnioblast differentiation showed that the developmental trajectory of amnioblast goes through a TFAP2A-abundant state which may be permissive for surface ectoderm commitment. Interestingly, our culture system also generated extraembryonic mesoderm-like cells from primed pluripotent state, some of which transiently expressed embryonic mesodermal markers. Taken together, our study brings an integrative understanding about the development of human amniotic and surface ectoderms, and provides a new model to examine the generation of human extraembryonic mesoderm.