Modular derivation of diverse, regionally discrete human posterior CNS neurons enables discovery of transcriptomic patterns

Our inability to derive the neuronal diversity that comprises the posterior central nervous system (pCNS) using human pluripotent stem cells (hPSCs) poses an impediment to understanding human neurodevelopment and disease in the hindbrain and spinal cord. Here, we establish a modular, monolayer differentiation paradigm that recapitulates both rostrocaudal (R/C) and dorsoventral (D/V) patterning, enabling derivation of diverse pCNS neurons with discrete regional specificity. First, neuromesodermal progenitors (NMPs) with discrete HOX profiles are converted to pCNS progenitors (pCNSPs). Then, by tuning D/V signaling, pCNSPs are directed to locomotor or somatosensory neurons. Expansive single-cell RNA-sequencing (scRNA-seq) analysis coupled with a novel computational pipeline allowed us to detect hundreds of transcriptional markers within region-specific phenotypes, enabling discovery of gene expression patterns across R/C and D/V developmental axes. These findings highlight the potential of these resources to advance a mechanistic understanding of pCNS development, enhance in vitro models, and inform therapeutic strategies.