Mapping Gene Circuits Essential for Germ Layer Differentiation via Loss-of-Function Screens in Haploid Human Embryonic Stem Cells

Pluripotent stem cells can differentiate into all embryonic germ layers, yet the genes essential for these cell fate transitions in human remain elusive. Here, we mapped the essential genes for the differentiation of human pluripotent stem cells (hPSCs) into the three germ layers by using a genome-wide loss-of-function library established in haploid hPSCs. Strikingly, we observed a high fraction of essential genes associated with plasma membrane, highlighting signaling pathways needed for each lineage differentiation. Interestingly, analysis of all hereditary neurological disorders uncovered high essentiality among microcephaly-causing genes. Furthermore, we demonstrated lineage-specific hierarchies among essential transcription factors and a set of Golgi- and endoplasmic reticulum-related genes needed for the differentiation into all germ layers. Our work sheds light on the gene networks regulating early gastrulation events in human by defining essential drivers of specific embryonic germ layer fates and essential genes for the exit from pluripotency.