Orchestrating Chemical and Physical Cross-Linking in Protein Hydrogels to Regulate Embryonic Stem Cell Growth

Protein hydrogels are ideal candidates for next-generation biomaterials due to their genetically programmable properties. Herein, we report an entirely protein-based hydrogel as an artificial extracellular matrix (ECM) for regulating the embryonic stem cell growth. A synergy between chemical and physical cross-linking was achieved in one step by SpyTag/SpyCatcher reaction and P zipper association at 37 °C. The hydrogels' stress relaxation behaviors can be tuned across a broad spectrum by single-point mutation on a P zipper. It has been found that faster relaxation can promote the growth of HeLa tumor spheroids and embryonic stem cells, and mechanical regulation of embryonic stem cells occurs via retention of the cells at the G1 phase. The results highlight the promise of genetically encoded protein materials as a platform of artificial ECM for understanding and controlling the complex cell–matrix interactions in a 3D cell culture.