Background: The process of liver organogenesis has served as a paradigm for organ formation. However, there remains a lack of understanding regarding early mouse and human liver bud morphogenesis and early liver volumetric growth. Elucidating dynamic changes in liver volumes is critical for understanding organ development, implementing toxicological studies, and for modeling hPSC-derived liver organoid growth. New visualization, analysis, and experimental techniques are desperately needed.Results: Here, we combine observational data with digital resources, new 3D imaging approaches, retrospective analysis of liver volume data, mathematical modeling, and experiments with hPSC-derived liver organoids. Mouse and human liver organogenesis, characterized by exponential growth, demonstrate distinct spatial features and growth curves over time, which we mathematically modeled using Gompertz models. Visualization of liver-epithelial and septum transversum mesenchyme (STM) interactions suggests extended interactions, which together with new spatial features may be responsible for extensive exponential growth. These STM interactions are modeled with a novel in vitro human pluripotent stem cell (hPSC)-derived hepatic organoid system that exhibits cell migration. Conclusions: Our methods enhance our understanding of liver organogenesis, with new 3D visualization, analysis, mathematical modeling, and in vitro

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.

Foxa2 has garnered considerable interest in its pioneer functions and its role endoderm gut, and liver differentiation, and development, and initiator of gene regulatory networks (GRN) in these tissues.Although Foxa2 has been investigated in these systems, Foxa1 also compensates for its function, and thus may also have compensatory effects in studies of Foxa2 regulation. In this study, we focus on the role for both Foxa1 and Foxa2 in controlling endoderm GRN, liver activation in gut tube, and liver GRN. We first compare endoderm induction protocols, and develop a novel model of liver induction under hypoxic conditions that relies on minimal growth factors and enhanced morphology. We employ an RNAi (siRNA and shRNA) approach to demonstrate the effects of Foxa1/2 on endoderm induction, gut tube activation of the albumin gene. Our data demonstrates widespread regulation of the endoderm and mesendoderm GRN, and albumin, which is significant for activation of the liver differentiation program. We then analyze the Foxa1/2 phenotype in stable liver cell lines, and engineer stable cell lines that demonstrate potential reversibility of cell state. Finally, we perform RNA-seq and bioinformatics analysis that demonstrates global GRN changes and transcription changes due to Foxa1/2 perturbation, including expansive changes in cellular differentiation and metabolism. These data suggest that Foxa1/2 phenotype has time-dependent effects on GRN and widespread effects on GRN, the liver transcriptome, and liver metabolism.