A temporally resolved BMP4 response in the peri-implantation human embryonic disc is critical for the induction of multipotent TFAP2A + cells and EMT

The unilaminar embryonic disc, a sheet of epithelized SOX2+ epiblast cells, undergoes complex morphogenesis to create a multi-dimensional human gastrula. This process endows early lineage specification and spatial identities that begin at the posterior end of the disc to create a coordinate system for embryogenesis. Here, we find that simply adding basement membrane extracts (BMEs) to the stem cell media is sufficient to model the early peri-gastrulating embryonic disc. We demonstrate that the emergence of amnion, primordial germ cells (PGCs), and mesoderm arise from BMP4-responsive disc cells that induce transient TFAP2A expression and SOX2 repression. We track the order of embryonic events that take place during disc morphogenesis and show that amnion-like cells (AMLCs) and PGC-like cells (PGCLCs) are specified first from epithelized TFAP2A+ progenitors. Shortly after, gastrulating mesoderm-like cells (MeLCs) arise from transiently expressed TFAP2A+ disc cells that then undergo EMT. We find that BMP4-responsiveness is critical to SOX2 repression, TFAP2A expression, lineage induction and EMT, while the process of EMT itself is not. Thus, we show that the extracellular matrix is necessary to promote disc morphogenesis and EMT. GiMO culture of UCLA hPSCs at d1,2 and 3 were first washed with cold D-PBS 3X in 30sec increments to dissolve Geltrex overlay. Next, samples were treated with 0.25% Trypsin/EDTA for 10 mins at 37 °C and 5% CO2. After incubation, cells were quenched with cold E8 medium and centrifuged at 1.2 rpm for 3.5 min. Cells were then resuspended with FACS buffer, D-PBS containing 0.5% Bovine Serum Albumin (BSA, Sigma-Aldrich, # A3311), as a wash step and centrifuged at 0.7 rpm for 4 min. Cells were resuspended again with FACS buffer and dissociated to single cells using a Falcon 100 mm Cell Strainer (Corning, # 352360) and manually counted. hPSCs were then further dissociated using EASYstrainer Cell Sieves 40 mm filter and centrifuged at 0.3 rpm for 3 min and resuspended in FACS buffer at a total volume of 1000 cells mL-2. Within 1hr after cell dissociation, cells were loaded into the 10x Genomics Chromium. Alignment was performed by Cellranger v5.0.1 to hg38 2020A.