Significance
Stem-cell microenvironment has been identified as an important modulator of plasticity, self-renewal, and differentiation. This work details the development of a hydrogel system tailored to promote human pluripotent stem cell (HPSC) self-renewal with a simple chemical microenvironmental switch to direct differentiation. Furthermore, the timing of switching post hydrogel fabrication can promote specific lineage differentiation as in vivo. This system highlights the role of microenvironment on fate choices of pluripotent cells and demonstrates that it may be tailored to control differentiation in vitro. Importantly, this approach may improve the generation of fully differentiated tissues, as demonstrated for cardiogenic differentiation. Our combination of hydrogels allows dense tissue structures to be produced from HPSCs by using a single-step process inaccessible to any current methodology.