Templated\nPluripotent Stem Cell Differentiation via\nSubstratum-Guided Artificial Signaling

The emerging field\nof synthetic morphogenesis implements\nsynthetic\nbiology tools to investigate the minimal cellular processes sufficient\nfor orchestrating key developmental events. As the field continues\nto grow, there is a need for new tools that enable scientists to uncover\nnuances in the molecular mechanisms driving cell fate patterning that\nemerge during morphogenesis. Here, we present a platform that combines\ncell engineering with biomaterial design to potentiate artificial\nsignaling in pluripotent stem cells (PSCs). This platform, referred\nto as PSC-MATRIX, extends the use of programmable biomaterials to\nPSCs competent to activate morphogen production through orthogonal\nsignaling, giving rise to the opportunity to probe developmental events\nby initiating morphogenetic programs in a spatially constrained manner\nthrough non-native signaling channels. We show that the PSC-MATRIX\nplatform enables temporal and spatial control of transgene expression\nin response to bulk, soluble inputs in synthetic Notch (synNotch)-engineered\nhuman PSCs for an extended culture of up to 11 days. Furthermore,\nwe used PSC-MATRIX to regulate multiple differentiation events via\nmaterial-mediated artificial signaling in engineered PSCs using the\northogonal ligand green fluorescent protein, highlighting the potential\nof this platform for probing and guiding fate acquisition. Overall,\nthis platform offers a synthetic approach to interrogate the molecular\nmechanisms driving PSC differentiation that could be applied to a\nvariety of differentiation protocols.