Integration of central and peripheral neurons in human elongating multilineage organized (EMLO) gastruloids

Human stem cell technologies including self-assembling 3D tissue models provide unprecedented access to early neurodevelopment and are enabling fundamental insights into neuropathologies. Gastruloid models have yet to be used to investigate developing neuronal systems. Here we generate elongating multi-lineage-organized (EMLO) gastruloids with trunk identity that co-develop central and peripheral nervous system (CNS, PNS) correlates. We identify neural crest cells that differentiate to form peripheral neurons integrated with an upstream spinal cord region. This follows initial EMLO polarization events and is coordinated with primitive gut tube elongation and multicellular spatial reorganization. We evaluate EMLOs over a forty-day period, applying immunofluorescence of multi-lineage and functional biomarkers, including day 16 single-cell RNA-Seq, and use them to investigate the impact of mu opioid receptor modulation on neuronal activity. This comprehensive study demonstrates the first combined human CNS-PNS model of early organogenesis in the trunk to benefit biomedical research.