Effect of 2D and 3D ECM and Biomechanical Cues on Human iPSC‐Derived Liver Progenitor Cell Differentiation

The differentiation of human liver tissue from induced pluripotent stem cells presents a powerful platform for drug screening and disease modeling. While 3D organoid systems effectively recapitulate tissue development, systematic investigation of microenvironmental parameters remains challenging. Here, complementary approaches are developed utilizing 2D cellular microarrays and 3D polyethylene glycol(PEG)-based microwells to examine human stem cell-derived hepatoblast differentiation. High-throughput microarray platform enables systematic evaluation of 60 distinct microenvironmental conditions, combining 15 ECM compositions with 4 growth factor treatments. Position-dependent expression of hepatocytic and cholangiocytic markers is observed including HNF4a, SOX9, and CK19, with differentiation patterns varying substantially across ECM and growth factor conditions. Based on these findings, specific ECM combinations are selected - Collagen 1, Fibronectin, and their combination - for integration into a modular 3D PEG hydrogel system. This 3D platform provides independent control over microtissue geometry and matrix composition, enabling investigation of spatial organization in hepatic differentiation. Through this integrated approach combining high-throughput screening and defined 3D culture, a framework is established for dissecting the microenvironmental regulation of human liver development.