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.

Catalytic oxidation of CH4 over nonprecious Ni/CeO2 catalysts has attracted wide attention. Controlling the morphology of a CeO2 support can enhance the CH4 oxidation activity without changing the catalyst composition. Herein, a series of 2 wt % Ni/CeO2 nanocatalysts with different CeO2 support morphologies (nanoparticles (P), rods (R), cubes (C)) and synthetic procedures (precipitation, sol-gel (SG)) were evaluated for their CH4 oxidation performance. The redox properties of CeO2 supports and corresponding Ni loaded catalysts were characterized by H2-temperature-programmed reduction and oxygen storage capacity (OSC) measurements. The relationship among the CeO2 morphologies, surface areas, redox properties, and CH4 oxidation activity for both CeO2 supports and Ni/CeO2 catalysts was established. The findings suggest that CeO2-R has a greater amount of surface oxygen vacancies as well as an improved OSC and CH4 oxidation activity compared to CeO2-P and CeO2-C supports. The same CH4 oxidation activity pattern was observed for the Ni containing catalysts (Ni/CeO2-R > Ni/CeO2-P > Ni/CeO2-C). Increasing the CeO2 surface area by using a sol-gel synthesis method (CeO2-SG) improved the amount of surface oxygen vacancies and CH4 oxidation performance of CeO2-SG and Ni/CeO2-SG compared to CeO2-R and Ni/CeO2-R, respectively. Finally, all studied Ni/CeO2 nanocatalysts showed improved hydrothermal stability compared to conventional Pd/Al2O3.

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Liver organogenesis has thus far served as a paradigm for solid organ formation, and has recently attracted interest due to challenges faced in liver regenerative medicine. Murine genetic studies indicate that early steps in morphogenesis are required, suggesting that three-dimensional imaging of early liver morphogenesis at high resolution can improve our understanding. Unfortunately, existing approaches to image early liver morphogenesis have been unable to achieve high spatial resolution (1-5 um) required. In this study, we focused on imaging, visualization, and analysis of early liver development. We utilized available online databases for both mouse (EMAP) and human (3D Atlas of Human Embryology) liver development. To visualize liver bud morphogenesis at high spatial resolution, we performed 3D reconstructions of stacked, digital tissue sections. We show dynamic 3D hepatic cord formation in the mouse in humans. Interestingly, when we quantified fetal liver growth, we showed that 3D fetal liver growth appears to occur in spurts rather continuously, and 3D images suggest that there could be considerable remodeling during these stages. Further, our analysis of the STM, in both mouse and humans, demonstrates that it increases in size during early fetal liver growth, that is highly interconnecting with liver epithelium, and that it can have strong local effects on growth. Finally, we identify and visualize and identify human hepatic cord formation followed by rapid sheet-like growth, which we propose could be an under-appreciated morphological feature that enables rapid growth of early human fetal liver. These studies will motivate future approaches to employ in vitro culture and organoid technology to improve human PSC differentiation, and improve disease modeling, and therapeutic opportunities for liver diseases. In conclusion, compared to 2D sectioning, high spatial resolution imaging of the mouse and human 3D liver bud morphogenesis enables greatly improved visualization of the hepatic cords, 3D sheet-like liver cell growth, STM-epithelial cell interactions, and quantitative comparisons between mouse and human liver bud morphogenesis.