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

## TL;DR

This study explores how 2D and 3D environments affect the differentiation of liver cells from stem cells, offering insights into liver development and potential applications in drug screening.

## Contribution

The paper introduces a novel integrated approach combining high-throughput screening and 3D culture to study microenvironmental regulation of liver cell differentiation.

## Key findings

- Different ECM compositions and growth factors significantly influence hepatocytic and cholangiocytic marker expression.
- Collagen 1, Fibronectin, and their combination were identified as effective in 3D culture for liver progenitor differentiation.
- The 3D PEG hydrogel system allows independent control over microtissue geometry and matrix composition.

## Abstract

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.

An engineered microtissue model is developed and optimized to investigate human liver development. Extracellular matrix composition and mechanical forces are tuned using high‐throughput assays. Human induced pluripotent stem cells serve as the cellular source, enabling the observation of de novo patterned differentiation.