Accelerated human liver progenitor generation from pluripotent stem cells by inhibiting formation of unwanted lineages

Abstract Despite decisive progress in differentiating pluripotent stem cells (PSCs) into diverse cell-types, the often-lengthy differentiation and functional immaturity of such cell-types remain pertinent issues. Here we address the first challenge of prolonged differentiation in the generation of hepatocyte-like cells from PSCs. We delineate a roadmap describing the extracellular signals controlling six sequential branching lineage choices leading from pluripotency to endoderm, foregut, and finally, liver progenitors. By blocking formation of unwanted cell-types at each lineage juncture and manipulating temporally-dynamic signals, we accelerated generation of 89.0±3.1% AFP + human liver bud progenitors and 87.3±9.4% ALBUMIN + hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. 81.5±3.2% of hepatocyte-like cells expressed metabolic enzyme FAH (as assayed by a new knock-in reporter line) and improved short-term survival in the Fah -/- Rag2 -/- Il2rg -/- mouse model of liver failure. Collectively the timed signaling interventions indicated by this developmental roadmap enable accelerated production of human liver progenitors from PSCs.