Loss of E-cadherin promotes extraembryonic fates accompanying multilineage differentiation

Embryonic development involves a range of complex morphogenic events as the embryo generates various cell types that go onto form functional tissues. This process is heavily reliant on symmetry breaking events that control the emergence of separate lineages that then spatial organize and physically arranging themselves within the embryo often via regulation of cell-cell adhesions. This work examines how changing adhesions in 3D aggregates of human pluripotent stem cells (hPSCs) encapsulated in hydrogel mixed with extracellular matrix allows for emergent population and morphological behaviors reminiscent of early embryonic behaviors and morphologies. In this context, knockdown of the cell adhesion molecule CDH1 generated aggregates with protrusion morphologies that did not arise in aggregates cultured without encapsulation. Genomic analysis revealed that the combination of CDH1 knockdown and encapsulation promoted emergence of extra-embryonic lineages. Further the transcriptomes of encapsulated CDH1(-) hPSCs were comparable to previously published single cell RNA-sequencing data of human embryos. Overall, these results establish a hPSC culture system that resembles early embryonic fate decisions and morphologies. Additionally, these studies highlight a potential relationship between changing adhesions, microenvironment, and acquisition of specific lineage fates, progressing our understanding of early human embryonic biology. WTC hiPSC cells with DOX inducible CRISPRi targeting CDH1 were treated for 5 days, then mixed with WT cells at defined ratios and force aggregated. Aggregates were then either encapsulated in agarose or unencapsulated and cultured in suspension for 6 days. At days 1, 3, and 6, WT, CDH1-KD and mixed aggregates were dissociated and prepared for single cell RNA-seq.