FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA‐seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)‐hepatic differentiation, siRNA knockdown demonstrated de‐differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.

Organoids bearing human stem cell-derived progenitors enable basic and applied investigation of organogenesis in a wide range of epithelial tissues. During liver organogenesis (LO), E9.5 collectively migrating hepatoblasts (MHs) arise from the E9.0 liver diverticulum (LD) and directly penetrate the surrounding mesoderm (MES) tissue, forming cell strands that link migration, differentiation, and growth. Currently, human pluripotent stem cell (hPSC) organoid protocols model the E10.5 liver bud and forward differentiation, but not the LD or the LD-derived MHs, in spite of their significance. In fact, the transcriptome underlying MHs, the niche that drives their migration, and methods to induce them from hPSC remain key questions. We performed bioinformatics analysis of single cell RNA-seq data, in vivo transplantation, and in vitro hPSC differentiation with organoid formation, microscopy, gene and protein expression, small molecule inhibitor screening of growth, and organoid culture in bioengineered devices to assess tissue tension. Our in depth bioinformatic analysis of early murine LO demonstrates pathway up-regulation of an unexpected wide array of soluble signaling factors, as well as cell cycle, chromatin modification, and metabolic reprogramming, in addition to a widespread cell stress-response. These findings led us hypothesize that the LD and MES tissue form a tissue complex (LD-MESC) that drives MH induction. Using this LD-MESC concept, we designed an in vivo transplant system, as well as a three-step in vitro protocol for inducing hPSC-derived MHs, both of which recapitulate liver growth, morphogenesis, differentiation. We show that Hippo signaling pathway, in agreement with murine MH data, mediates migration and growth of hPSC-MH in vitro. These data substantiate the LD-MESC model developed here, and directly address key challenges facing liver regenerative medicine. Our bioinformatics, in vitro, and in vivo data all support the concept that the LD-MESC initiates LO. This concept can be used to change protocols to emphasize linking of migration, growth, with differentiation. Modeling epithelial collective migration for LO bolsters not only organogenesis studies of alternate endodermal organs, but also in vivo transplantation efforts, and facilitates employing migrating organoids to therapeutically target human tumor migration/metastasis.