TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [RNAseq_CRISPRi_ZNF398]

Pluripotency is the ability to give rise to all cell types of the body and is first observed in a mass of disorganised cells of the embryo. Upon implantation, pluripotent cells form a columnar epithelium and undergo lumenogenesis. At gastrulation, a portion of the pluripotent epiblast will undergo epithelial to mesenchymal transition (EMT), forming the primitive streak (PS). It still remains unclear what molecular mechanism supports the epithelial identity of the pluripotent epiblast before gastrulation. Here we developed an optimised, chemically defined 3D model of human pluripotent epiblast formation in which conventional pluripotent stem cells (PSCs) self-organise into a columnar epithelium with a lumen in 48 hours. From 72 hours we observed spontaneous symmetry breaking and specification of PS-like cells, as confirmed by single-cell RNA sequencing. We found that Insulin and FGF signalling are both required for the proliferation and survival of the pluripotent epiblast model. Conversely, TGF-beta signalling maintains epithelial identity. Epithelial identity appears uncoupled from the expression of canonical pluripotency markers OCT4, NANOG and PRDM14, but under the control of ZNF398. Once the pluripotent epithelium is established, TGF-beta inhibition is inconsequential, and stimulation with Activin A leads to highly efficient PS induction. We conclude that TGF-beta dynamically orchestrates epithelial identity of human pluripotent cells. Overall design: Bulk RNA-seq profiling of gRNA-ZNF398 PSCs in 2D and 3D-hE-gastruloids, with (ZNF398 Knock-Down) or without (Control) Dox+TMP induction. 8 samples, 2 replicates per condition.