Induced Neural Progenitor Specification from Human Pluripotent Stem Cells by a Refined Synthetic Notch Platform

Historically, studying the development of brain and central nervous system (CNS) tissues has been challenging. Human pluripotent stem cell (hPSC) technology has allowed for the in vitro reconstitution of relevant, early cell trajectories by using small molecules and recombinant proteins to guide differentiation of cells toward relevant brain and CNS phenotypes. However, many of these protocols fail to recapitulate the cell-guided differentiation programs intrinsic to embryonic development, particularly the signaling centers that emerge within the neural tube during brain formation. Located on the ventral end of the neural tube, the floor plate acts as one such signaling center to pattern the dorsal/ventral axis by secreting the morphogen Sonic Hedgehog (SHH). Here, we present a method for cell-guided differentiation using the synthetic Notch (synNotch) receptor platform to regulate SHH production and subsequent cell fate specification. We show that the widely used configuration of the orthogonal synNotch ligand green fluorescent protein (GFP) mounted on a platelet-derived growth factor receptor-β transmembrane chassis does not allow for robust artificial signaling in synNotch-hPSCs ("receivers") cocultured with ligand-presenting hPSCs ("senders"). We discovered that refined designs of membrane-bound GFP-ligand allow for efficient receptor activation in hPSC receivers. A variant of this enhanced synNotch system drives the production of SHH in hPSC sender:hPSC receiver cocultures and gives rise to floor plate-like cell types seen during neural tube development. This revised synNotch platform has the potential to pattern hPSC differentiation programs in synthetic morphogenesis studies designed to uncover key paradigms of human CNS development.