Poster BB6 - Linking collective migration to differentiation boosts global shaping of the transcriptome and maturation

Introduction: Procuring functionally mature epithelial cells remains a formidable challenge for the clinical translation of human pluripotent stem cells (hPSCs). Epithelial cells, including hepatoblasts in the liver, exhibit migratory behavior prior to differentiation, a phenomenon often overlooked in studies of cell maturation. Drawing from our comprehensive analysis of mouse and human liver organogenesis, we hypothesized that faithfully progressing the transcription factor (TF) gene circuit governing migrating hepatoblasts (MHBs) in an in vitro model could drive enhanced hepatocyte maturation. Surprisingly, this novel approach demonstrates remarkable efficacy in promoting epithelial maturation, offering a promising avenue to surmount barriers hindering the maturation of hepatocytes and advancing regenerative medicine.

Materials and

Methods: We initially analyzed single-cell RNA sequencing (scRNA-seq) data from mouse and human to explore the transcriptional landscape of MHBs. Leveraging a deep learning model, we uncovered a progressive TF gene circuit, previously overlooked, underlying transcriptional maturation. This information guided the development of a human hepatic stem cell differentiation protocol to mimic MHB development. Operating under hypoxic conditions and low serum-derived environments, definite endoderm cells were exposed to liver regeneration mediators (VEGF, EGF, IGF-1, FGF, Heparin) (H + M medium) from day 4 onwards, inducing hepatoblast (HB) formation without maturation agents, leading to albumin-positive HBs by day 14. To confirm hepatic commitment, we conducted a triple knockdown of FOXA1/2/3, revealing FOXA factors' pivotal role in liver differentiation. Hepatic organoids formed on day 15 allowed insight into the growth to differentiation switch (GDS), with growth conditions introduced using H + M growth medium with or without matrigel droplets. This facilitated scrutiny of gene expression patterns through whole transcriptome analysis (RNA-seq) and molecular techniques such as microscopy, qRT-PCR, Western Blotting, and tissue sectioning to assess liver differentiation. Additionally, CellNet Classification evaluated liver differentiation extent. We also developed novel methodologies for analyzing liver-specific transcription factors' activation, maturation markers, and enriched gene pathways across conditions.

Results, Conclusions, and Discussions: Continuous exposure to H + M medium led to distinct developmental pathways in liver bud (LD) organoids, with cells either entering a pre-migration/growth phase or committing to differentiation spontaneously. This was marked by changes in transcription factor expression (FOXA, TBX3, CEBPA), AMPK activity, Hippo pathway signaling, HIF1A levels, and the TCA cycle. A chemical screen of 24 conditions further identified the Hippo signaling pathway as a potent inhibitor of LD-MESC migration. In the LD-MESC model, H + M medium induced overt migration, while subsequent replacement with control medium led to rapid hepatic maturation, characterized by changes in signaling pathways and metabolic activity. LD and LD-MESC organoids exhibited competitive CellNet classification scores with the highest liver scores reported in 26 studies, despite the absence of maturation agents. LD-MESC organoids also showed superior activation of liver-specific genes compared to all other hPSC-derived hepatocyte populations analyzed. Conversely, cells undergoing growth/migration displayed lower liver CellNet Classification Scores and higher scores for alternative lineages, highlighting the rapid transcriptomic changes between conditions. In summary, our models replicate transcriptional profiles observed in murine and human fetal liver development, offering insights into hepatic migration, maturation, and the roles of gene regulatory networks, metabolism, and cell signaling in a migration to differentiation switch.

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