Abstract

The shift from collective migration to differentiation is a crucial process in epithelial biology but recreating this intricate transition has thus far proved elusive. We provide experimental, mechanistic, in vivo, and bioinformatic data supporting an undoubtable link between human pluripotent stem cell (hPSC)- derived collectively migrating hepatoblasts (MHB), and transcriptionally mature, functional hPSC- hepatocytes (HEPs), which incorporates two unrecognized steps. The protocol induces FOXA-dependent induction of HBs, leading to TBX3-positive, YAP-TEAD active MHBs which provide a transcriptional match with murine liver E9.5 MHBs. Simple cultivation changes trigger MHBs to rapidly form functional day 18 HEPs, predicted by a deep-learning designed gene circuit, resulting in a [~]236% fold- increase in maturation (PACNet), on par with the highest score, but with enhanced global transcriptional shaping. Overall, incorporating the MHB to HEP transition establishes a new, unrecognized, and highly efficient mechanism for differentiation that can be cumulatively integrated with existing methods to overcome barriers to maturation.