Human neuron subtype programming via single-cell transcriptome-coupled patterning screens

Human neurons programmed through transcription factor (TF) overexpression model neuronal differentiation and disease. However, the diversity of neuronal subtypes programmable in vitro remains unresolved. We modulated developmental signaling pathways combined with TF overexpression to explore the spectrum of neuron subtypes generated from pluripotent stem cells. We screened 480 morphogen signaling modulations coupled with TF induction using a multiplexed single-cell transcriptomic readout. Analysis of 700,000 cells identified diverse excitatory and inhibitory neurons patterned along the developmental axes of the neural tube. Patterning neural progenitors prior to TF overexpression expanded neuronal diversity by enabling access to regulons active in primary tissue counterparts. Our approach provides a strategy for programming diverse human cell subtypes as well as investigating how cooperative signaling drives neuronal fate.