Templated Pluripotent Stem Cell Differentiation via Substratum-Guided Artificial Signaling.

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