<i>In vivo</i> human embryonic spinal cord atlas validates stem cell–derived human dorsal interneurons and reveals ASD spinal signatures

Spinal cord injuries (SCI) result in the loss of motor and sensory function. We are working towards restoring sensation by developing directed differentiation protocols to generate dorsal spinal interneurons (dIs; dI1-dI6) from human embryonic stem cells (hESCs). Here, we present an improved method that produces human dIs via a neuromesodermal progenitor state, the physiological intermediate for spinal cord development. We show that retinoic acid (RA), bone morphogenetic protein 4 (BMP4) and growth differentiation factor (GDF) 11 direct dI identity, while GDF11 and extended time in culture promotes posterior spinal identities. Together, these protocols generate the full complement of dorsal subtypes along the entire anterior-posterior axis of the spinal cord. To benchmark <i>in vitro</i>-derived dIs, we constructed a single-cell RNA-Seq atlas of the human embryonic spinal cord and used it to show that hESC-derived dIs closely match their endogenous counterparts. The atlas also reveals that the dI4/dI5 populations dramatically expand in comparison with the other spinal lineages. Moreover, they have mechanosensory circuit signatures linked to autism spectrum disorder, implicating spinal circuits in autistic phenotypes.