The extracellular matrix (ECM) plays a central role in regulating cell behavior, tissue morphogenesis, and differentiation through a combination of biochemical and mechanical cues. However, most conventional cell-culture substrate materials are chemically poorly defined and exhibit batch-to-batch variability, hindering mechanistic investigations into how individual matrix parameters influence cellular decision-making. To overcome these limitations, this thesis presents the design and characterization of a fully synthetic, tunable hydrogel platform that enables systematic exploration of cell–matrix interactions under defined and reproducible conditions. In the first part of this work, hydrogels were synthesized via strain-promoted alkyne–azide cycloaddition (SPAAC) between dendritic polyglycerol-bicyclononyne (dPG-BCN) and azide-functionalized pNIPAM-co-PEG copolymers. This bioorthogonal click chemistry provided independent control over matrix stiffness, viscoelasticity, and biochemical functionality. The platform supported long-term three dimensional culture of human induced pluripotent stem cells (hiPSCs) and guided their differentiation into hepatic organoids. Functionalization with cyclic RGD peptides revealed that matrix adhesiveness alone was sufficient to direct lineage specification via integrin-mediated activation of the TGF-β signaling cascade, identifying a mechanosensitive integrin–MMP–TGF-β axis as a key regulator of hepatic fate decisions. In the second part, the hydrogel chemistry was expanded using a thiol–maleimide coupling approach, and sulfate groups were introduced as a biomimetic, charge-tunable component. These hydrogels exhibited enhanced mechanical stability, slower enzymatic degradation, and selective molecular diffusion profiles as demonstrated by fluorescence recovery after photobleaching (FRAP) compared to the unsulfated hydrogel. Sulfation significantly improved cell viability and adhesion in both two- and three-dimensional cultures, highlighting the importance of electrostatic interactions in modulating cell matrix communication. The combined findings demonstrate that synthetic hydrogels can be rationally designed to decouple and quantify the effects of stiffness, adhesiveness, charge density, and viscoelasticity on stem-cell function. Beyond their fundamental value for understanding ECM-guided morphogenesis, these chemically defined and scalable materials provide a promising foundation for translational applications in regenerative medicine, drug testing, and tissue engineering. By replacing the ECM using rational designed hydrogels, this work contributes to the next generation of biomaterials capable of guiding cellular identity and function by design.
