摘要：SummaryUnderstanding the biological processes that determine the entry of three germ layers of human pluripotent stem cells (hPSCs) is a central question in developmental and stem cell biology. Here, we genetically engineered hPSCs with the germ layer reporter and inducible CRISPR/Cas9 knockout system, and a genome-scale screening was performed to define pathways restricting germ layer specification. Genes clustered in the key biological processes, including embryonic development, mRNA processing, metabolism, and epigenetic regulation, were centered in the governance of pluripotency and lineage development. Other than typical pluripotent transcription factors and signaling molecules, loss of function of mesendodermal specifiers resulted in advanced neuroectodermal differentiation, given their inter-germ layer antagonizing effect. Regarding the epigenetic superfamily, microRNAs enriched in hPSCs showed clear germ layer-targeting specificity. The cholesterol synthesis pathway maintained hPSCs via retardation of neuroectoderm specification. Thus, in this study, we identified a full landscape of genetic wiring and biological processes that control hPSC self-renewal and trilineage specification.Graphical abstractDisplay OmittedHighlights•Lineage reporter and CRISPR screening are powerful tools for studying cell fates•Lineage-specification preventing genes (LPGs) are identified in hPSCs•LPGs maintain pluripotency via targeting one or multiple germ layers•LPGs are clustered into distinct functional modulesBiological Sciences; Cell Biology; Stem Cells Research; Developmental Biology
