Tunable Synthetic Hydrogel Modulates Hepatic Lineage Specification of Human Liver Organoid

Abstract Hydrogel has been widely applied in the 3D cultivation of stem cells and organoids as supporting scaffolds. However, precise engineering of cell differentiation state in 3D remains a critical challenge and demands proper design of a functionally tunable micro‐niche. In this study, a tunable synthetic “click” hydrogel is presented based on dendritic polyglycerol‐bicyclononyne (dPG‐BCN) and poly(N‐isopropylacrylamide)‐ co ‐polyethylene glycol azide (pNIPAAm‐ co ‐PEG‐N 3 ), tunable in mechanical and biochemical properties. The hydrogel is capable of hosting long‐term 3D proliferation of human induced pluripotent stem cells (hiPSC) with sustained pluripotency, and induction of human liver organoid (HLO) with controlled hepatic lineage specification. Apart from mechanical regulation in HLO growth, RGD conjugation to hydrogel promotes cholangiocyte differentiation via enhanced TGF‐β activation, whereas RGD‐free hydrogel results in low TGF‐β activation and major hepatocyte differentiation of HLO cells. In agreement with this, RGD‐free hydrogel also supports the development of hepatoblast organoids (HBOs) into hepatocyte lineage. Taken together, the reported hydrogel system provides a synthetic alternative to animal‐derived matrices for 3D tissue culture, offering a customizable platform for hepatic lineage specification with application potential in regenerative medicine and disease modeling.