Engineered microtissues to model the effects of dynamic heterotypic cell signaling on iPSC-derived human hepatocyte maturation.

In vitro human liver models are indispensable for compound metabolism/toxicity screening, disease modeling, and regenerative medicine. While induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) mitigate the sourcing limitations with primary human hepatocytes (PHHs), their functional maturity is rate-limiting for application use. During development, immature hepatoblasts interact with different non-parenchymal cell (NPC) types, such as mesenchyme and endothelia, in a spatiotemporal manner to progress through functional maturation. Modeling such interactions in vitro is critical to elucidate the key regulators of iHep maturation. Here, we utilized high-throughput droplet microfluidics to encapsulate iHeps within monodisperse collagen I microgels (Ø ∼ 250 µm), which were coated with NPCs to generate 'microtissues' placed within microwells in multiwell plates. Embryonic fibroblasts and liver sinusoidal endothelial cells (LSECs) induced the highest level of iHep maturation over 4+ weeks of culture compared to adult hepatic stellate cells (myofibroblastic), liver portal fibroblasts, dermal fibroblasts, and human umbilical vein endothelial cells. Combining iHep microtissues in plates with Transwell inserts containing different NPC types enabled the modeling of dynamic heterotypic signaling on iHep maturation; introducing embryonic fibroblast signaling first, followed by LSECs, led to the highest iHep maturation. Unique cytokine secretion profiles were detected across the top-performing microtissue configurations; stromal-derived factor-1 alpha was validated as one factor that enhanced iHep maturation. Lastly, gene expression patterns and regulatory networks showed adult PHH-like maturation in LSEC/iHep microtissues compared to iHep-only microtissues. Overall, microtissues are useful for elucidating the microenvironmental determinants of iHep maturation and for future use in downstream applications. STATEMENT OF SIGNIFICANCE: Induced pluripotent stem cell-derived hepatocyte-like cells (iHeps) hold great promise for drug screening, disease modeling, and regenerative medicine but often exhibit immature phenotypes. We utilized high-throughput droplet microfluidics to generate 3D microtissues containing iHeps and non-parenchymal cell (NPC) types to elucidate the effects of dynamic NPC signaling on iHep maturation. We observed that iHep maturation is significantly enhanced with embryonic fibroblasts and liver sinusoidal endothelial cells (LSEC) compared to adult liver fibroblasts and non-liver endothelia; the LSEC/iHep microtissues showed adult liver-like gene expression signatures. The highest iHep maturation in microtissues was achieved when mesenchymal stimulation was introduced first, followed by LSEC stimulation. Our platform provides a robust framework to elucidate cellular and molecular mediators of iHep maturation and biomedical applications.