Tuning Embryoid Germ Layer Specification in Tryptophan Zipper Hydrogels Through Pendant Matrix‐Derived Motifs

The ability of stem cells to sense and respond to nanoscale features in their microenvironment underscores the need for tunable hydrogel nanostructures in matrices for biofabrication. Here, a modular hydrogel platform is presented that uses tryptophan zipper (Trpzip) based peptides functionalized with extracellular matrix-derived adhesive motifs to create shear thinning bioactive synthetic hydrogels with tunable mechanical and biochemical properties. Rheological and small-angle neutron scattering analyses reveal that while the addition of GRGDS, GFOGER, YIGSR, or IKVAV motifs has minimal impact on nanofiber diameter and bulk hydrogel stiffness, they significantly alter nanofiber flexibility and microscale hierarchical organization of Trpzip networks. In concert with adhesive cues, these subtle differences in overall hydrogel architecture influence lineage commitment of 3D encapsulated human induced pluripotent stem cell embryoids. Trpzip matrices presenting GRGDS best support mesodermal fates, IKVAV promotes ectodermal commitment, and unmodified Trpzip matrices with no adhesive cues enhance endodermal differentiation. The results suggest that ligand-driven modulation of nanofiber rigidity and entanglement can contribute to pluripotent stem cell lineage specification, supporting the potential of Trpzip hydrogels as customizable reagents for biofabrication, stem cell manufacture, tissue engineering, and regenerative medicine.