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. Editor's summary: The use of human induced pluripotent stem cells to generate induced neurons is used in a broad range of neuroscience studies, from disease modeling to drug screening. However, replicating the large heterogeneity of human neurons is challenging. Lin et al. performed a systematic screening of 480 morphogen combinations coupled with overexpression of one of two different transcription factors to generate a library of induced neurons successively analyzed using single-cell RNA sequencing. The authors identified a broad range of neuronal subtypes and mapped them along the anterior-posterior and dorsal-ventral axes of neural tube development. The results provide a valuable resource for engineering human neurons. —Mattia Maroso INTRODUCTION: Human excitatory and inhibitory neurons can be induced from pluripotent stem cells (PSCs) in vitro through forced expression of pioneer transcription factors. These induced neurons (iNs) are widely used for studying neural development, differentiation, and neurological diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In addition, programmed neurons show promise for cell replacement therapies to restore function after neurological damage. Systematic strategies to expand the diversity of neuron types is essential for driving future breakthroughs. RATIONALE: To create diverse neuron types in vitro, we propose that broadly expressed pro-neural transcription factors (TFs), such as NGN2 and ASCL1 combined with morphogens, can guide regional patterning across the central and peripheral nervous system. RESULTS: We designed a systematic morphogen screening approach to explore how different morphogens work together with pro-neural TFs to generate different types of neurons from PSCs. Using high-throughput single-cell RNA-sequencing (scRNA-seq), we analyzed nearly 700,000 cells across 480 unique combinations of morphogen conditions. We identified diverse iN subtypes resembling those found in the human body, including neurons from the forebrain, midbrain, hindbrain, spinal cord, and peripheral nervous system. These iNs also share characteristics with human neurons that produce neurotransmitters such as glutamate, GABA, dopamine, and acetylcholine. In addition, the diversity of iN subtypes is evident in their electrical activity patterns, suggesting that they are functionally distinct. To understand how morphogens influence the formation of specific iN subtypes, we use scRNA-seq data to infer gene regulatory networks. We identified key TFs and their downstream target genes, collectively called regulons, that are activated by combinations of morphogens to guide neurons into specific subtypes. To verify our findings, we used genetic methods such as overexpression and CRISPR-Cas9 knockouts to perturb key TFs. When key TFs are absent, morphogens can no longer direct iNs into specific subtypes. Conversely, overexpressing key TFs is sufficient to drive the formation of specific iN subtypes without morphogens. We also found that exposing PSCs to morphogens before inducing pro-neural TFs can activate regulons found in human neurons, generating iN subtypes that are more uniform and more closely resemble primary human neurons. CONCLUSION: We expanded the diversity of human iNs generated in vitro and uncovered how cooperative signaling drives cell fate acquisition. We identified regulons that direct the emergence of iN subtypes, which will enable the targeted generation of pure cultures of specific neuron types in the future. Our dataset linking morphogens to cell fate outcomes is especially suited for predictive modeling to infer cell fates under novel conditions. Our approach is generalizable to diverse cell types beyond neurons and holds great potential to advance understanding of human biology, disease mechanisms, and therapeutic innovation. We programmed human neuron subtypes from PSCs combining pioneer TFs with morphogens involved in nervous system development.: Using highly multiplexed scRNA-seq readouts, we profiled 700,000 cells in 480 conditions, identifying a wide range of neuron subtypes with expanded diversity compared to control. Patterned neuron subtypes are morphologically, functionally, and transcriptionally distinct. Regulatory network inference revealed cooperative signaling and key regulons in neuron fate specification. [ABSTRACT FROM AUTHOR]