Synthetic Morphogenesis to Instruct Early Brain and Central Nervous System Development

Abstract

Studying brain and central nervous system (CNS) formation remains challenging due to the complexity of morphogenetic signaling during early embryonic development. During embryogenesis, Sonic Hedgehog (SHH) from the notochord induces floor plate formation, which subsequently patterns the neural tube along the dorsal-ventral axis. Human pluripotent stem cells (hPSCs) offer a platform to model these processes in vitro, typically using small molecules and recombinant proteins. However, conventional two-dimensional (2D) and three-dimensional (3D) differentiation protocols fail to recapitulate intrinsic, cell-driven signaling centers that regulate development in vivo. Synthetic morphogenesis aims to address these limitations by engineering multicellular systems to drive tissue formation and differentiation. While many synthetic morphogenetic strategies rely on externally controlled drug- or light-inducible systems, they still lack endogenous cell-cell signaling cues. Here, we investigate synthetic Notch (synNotch) as an alternative platform for cell-guided differentiation. However, synNotch has not yet been applied in hPSC:hPSC juxtacrine signaling. We first demonstrate that the widely used synNotch ligand, green fluorescent protein (GFP), presented on a platelet-derived growth factor receptor-β transmembrane domain, fails to induce robust synNotch activation in hPSC co-cultures. We identify an optimized ligand-presenting chassis, Epithelial Cadherin (E-Cadherin), which enables efficient receptor activation, driving mCherry reporter expression in sender-receiver co-cultures, as confirmed by flow cytometry. Using this optimized system, we establish a 2D synNotch-driven differentiation strategy in which receiver hPSCs express SHH upon ligand recognition, resulting in the emergence of floor plate-like cells. Single-cell RNA sequencing confirms the presence of floor plate markers in these cultures, and these cells exhibit comparable marker expression to a conventional 2D differentiation strategy using bulk recombinant SHH protein. To extend this approach into a 3D model, we integrate synNotch into brain organoids, polarizing SHH-expressing receiver hPSCs and surrounding them with ligand-presenting senders. Immunofluorescence analysis reveals markers of floor plate, ventral, and dorsal neural tube identity, indicating spatially organized patterning. This work represents the first demonstration of synNotch-mediated hPSC:hPSC juxtacrine signaling in both 2D and 3D, expanding synthetic morphogenesis strategies for engineering cell-cell communication, directing hPSC differentiation, and modeling developmental processes.