(490d) Growth to Differentiation Switch in Early Murine and Human Pluripotent Stem Cell-Derived Liver Organogenesis

The liver establishes its mass, architecture, and functions during early murine liver organogenesis (E8.5-11.5) (LION), switching between early migration/growth and differentiation. However, it is currently not well understood how early hepatoblasts (HBs) within the E9.0 liver diverticulum (LD) undergo a growth to differentiation switch (GDS), as part of the epithelial (gut) to hepatic transition (EHT), and whether a GDS is relevant for the design of human pluripotent stem cell (hPSC)-derived hepatocyte (HEP) directed-differentiation protocols, organoids, or methods for liver tissue replacement.

We first performed bioinformatic analysis of the GDS during murine LION, identified a list of unique signaling and cell stress pathways at E9.5, and metabolic changes at E10.5. We then used this information to design a novel human hepatic stem cell differentiation protocol to model early LION and the GDS. Under complete hypoxic conditions, low serum-derived conditions, day 4 definite endoderm cells were continuously exposed to a unique combination of liver regeneration mediators (VEGF, EGF, IGF-1, FGF, Heparin) (H + M medium) for HB induction, with no maturating agents, resulting in albumin-positive HBs by day 14. To validate hepatic commitment in this 14-day protocol, we performed triple knockdown of FOXA1/2/3 to establish that FOXA factors initiate liver differentiation, as they do in murine liver. We formed day 15 hepatic organoids to model the LD and provide insight towards the GDS. We imposed growth conditions to the LD organoids by continued exposure to H + M growth medium, with or without matrigel droplets to emulate the surrounding mesoderm tissue complex (MESC). We performed whole transcriptome analysis (RNA-seq), microscopy, and molecular analysis (qRT-PCR, Western Blot, tissue sectioning) to determine levels of GLD. CellNet Liver Classification Score analysis was used to assess extent of liver differentiation. We further developed new methods for finding and quantifying enriched gene pathways between conditions.

With continuous exposure to H + M medium, LD organoids in suspension either entered a pre-migration/growth phase, or a differentiation phase, spontaneously, distinguished by liver gene regulatory network expression, AMP-activated protein kinase (AMPK) expression, Hippo pathway, Hypoxia Inducible Factor (HIF1A), and TCA cycle. In the LD-MESC model, H + M medium in the LD-MESC model resulted in overt migration. Further, rapid removal of H + M and replacement with control medium from days 16-18 days, induced rapid hepatic maturation. This GDS was distinguished by p53, Hippo, VEGF, AMPK, TCA, and oxidative phosphorylation. We note that the Liver Classification Scores of the LD and the LD-MESC, although they express disparate liver GRN, are competitive with the highest Liver Scores based upon 26 published studies despite not using any maturation agents. However, our cells undergoing growth/migration had much lower Liver Classification Scores and were additionally found to have higher scores for alternative lineages. Using both conventional and novel methods, comparison of GDS in the LD-MESC model demonstrated nearly identical changes in transcription observed in the transition between E9.5-10.5 in mouse. To functionally assess pathways that induce LD-MESC migration, we performed a 24-condition chemical screen, and identified Hippo signaling pathway as an inhibitor. Finally, we applied LION principles to create a simple in vivo transplantation model, demonstrating evidence of GDS.

In summary, we developed new human models of the GDS in suspension (LD) and matrigel droplets (LD-MESC) that accurately correlate with mouse GDS between E9.5-10.5, demonstrate multiple paths to high levels of maturity, showed how the hepatic GRN, metabolism, and cell signaling can be used to identify GDS, identified new markers and mechanisms of cell migration/growth, identified functional pathways of growth, and proposed key roles for signaling and metabolism in the process.