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Xeno‐free, chemically defined poly(ethylene glycol) (PEG)‐based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene‐click chemistries are integrated to form synthetic, dynamically tunable PEG–peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol–norbornene hydrogels crosslinked by multiarm PEG–norbornene (PEG–NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine–norbornene (Tz–NB) click reaction is then employed to dynamically stiffen the cell‐laden hydrogels. Fast reactive Tz and its stable derivative methyltetrazine (mTz) are tethered to multiarm PEG, yielding mono‐functionalized PEG‐Tz, PEG‐mTz, and dualfunctionalized PEG‐Tz/mTz that react with PEG–NB to form additional crosslinks in the cell‐laden hydrogels. The versatility of Tz‐NB stiffening is demonstrated with different Tz‐modified macromers or by intermittent incubation of PEG‐Tz for temporal stiffening. Finally, the Tz–NB‐mediated dynamic stiffening is explored for 4D culture and definitive endoderm differentiation of hiPSCs. Overall, this dynamic hydrogel platform affords exquisite controls of hydrogel crosslinking for serving as a xeno‐free and dynamic stem cell niche.

Photoresponsive hydrogels have become invaluable 3D culture matrices for mimicking aspects of the extracellular matrix. Recent efforts have focused on using ultraviolet (UV) light exposure and multifunctional macromers to induce secondary hydrogel crosslinking and dynamic matrix stiffening in the presence of cells. This contribution reports the design of a novel yet simple dynamic poly(ethylene glycol)–peptide hydrogel system through flavin mononucleotide (FMN) induced di‐tyrosine crosslinking. These di‐tyrosine linkages effectively increase hydrogel crosslinking density and elastic modulus. In addition, the degree of stiffening in hydrogels at a fixed PEG macromer content can be readily tuned by controlling FMN concentration or the number of tyrosine residues built‐in to the peptide linker. Furthermore, tyrosine‐bearing pendant biochemical motifs can be spatial‐temporally patterned in the hydrogel network via controlling light exposure through a photomask. The visible light and FMN‐induced tyrosine dimerization process produces a cytocompatible and physiologically relevant degree of stiffening, as shown by changes of cell morphology and gene expression in pancreatic cancer and stromal cells. This new dynamic hydrogel scheme should be highly desirable for researchers seeking a photoresponsive hydrogel system without complicated chemical synthesis and secondary UV light irradiation.