Summary: V2a interneurons derived from human stem cells show promise for repairing spinal cord injuries by restoring long-range connections that coordinate movement.
Source: Gladstone Institute.
Gladstone researchers report the first successful production of human stem cell–derived V2a interneurons, a cell type that could help restore motor function after spinal cord injury.
Scientists at the Gladstone Institutes have developed a reliable method to generate V2a interneurons from human pluripotent stem cells. V2a interneurons are excitatory spinal cord neurons that relay signals from the brain to motor circuits, coordinating locomotion and respiration by projecting long distances along the spinal cord. Damage to these interneurons is a key contributor to the loss of communication between the brain and limbs after spinal cord injury, and replacing them could provide a route to re-establishing neural circuitry and improving movement.
The Gladstone team identified a precise combination and timing of signaling molecules that guides stem cells through spinal cord progenitor stages into CHX10+ V2a interneurons. By calibrating the concentrations and scheduling of retinoic acid, sonic hedgehog, and Notch pathway modulators, the researchers optimized differentiation to produce a high yield of cells exhibiting the molecular and functional properties of V2a interneurons rather than alternative neural fates such as motor neurons.
In collaboration with researchers at the University of California, San Francisco, the team transplanted the stem cell–derived V2a interneurons into the spinal cords of healthy mice. After transplantation the cells survived, matured in situ, extended axons several millimeters in both directions, and formed putative synaptic contacts with host neurons. Importantly, mice retained normal motor behavior following transplantation, indicating that the grafts did not produce overt dysfunction in the intact spinal cord.
Lead researchers emphasize that the defining properties of V2a interneurons are their long-range projections and role in central pattern generators that coordinate rhythmic activities such as walking and breathing. The ability of transplanted human V2a interneurons to extend processes and begin to connect with host circuitry is a critical proof of principle that supports further testing in injury models.
Next steps described by the authors include transplanting V2a interneurons into animal models of spinal cord injury to determine whether the cells can restore disrupted pathways and improve motor recovery. The team also plans to explore how these neurons might be applied to neurodegenerative movement disorders where interneuron dysfunction contributes to disease progression.
Study details and significance
The study, published in the Proceedings of the National Academy of Sciences, reports directed differentiation of CHX10+ V2a interneurons from multiple human pluripotent stem cell lines. Differentiated cultures showed elevated expression of V2a marker genes (including CHX10 and SOX14), neuronal markers such as neurofilament and NeuN, and glutamatergic identity indicated by VGlut2 expression. Electrophysiological maturation was observed as increasing action potential frequency over time. Single-cell RNA sequencing confirmed the presence of CHX10+ neurons among the differentiated population, which also contained some glial and progenitor cells.
Two weeks after intraspinal transplantation in mice, grafted V2a interneurons survived at the injection site, coexpressed neuronal markers, extended neurites beyond 5 mm, and formed presumptive synapses with host neurons. These outcomes indicate both molecular fidelity to the V2a interneuron identity and the capacity to integrate anatomically with spinal circuits—key attributes for modeling central nervous system development and testing cell-based therapies for spinal cord injury.
Contributors and funding
Authors on the study include Jessica C. Butts, Dylan A. McCreedy, Jorge A. Martinez-Vargas, Frederico N. Mendoza-Camacho, Tracy A. Hookway, Casey A. Gifford, Praveen Taneja, Linda Noble-Haeusslein, and Todd C. McDevitt, with contributions from colleagues at UC Berkeley and UCSF. Funding was provided by the California Institute for Regenerative Medicine, the National Science Foundation, the UCSF Alvera Kan Endowed Chair, the Howard Hughes Medical Institute, and the Damon Runyon Cancer Research Foundation.
Funding: California Institute for Regenerative Medicine; National Science Foundation; UCSF Alvera Kan Endowed Chair; Howard Hughes Medical Institute; Damon Runyon Cancer Research Foundation.
Source: Julie Langelier – Gladstone Institute
Original research: Butts JC, McCreedy DA, Martinez-Vargas JA, Mendoza-Camacho FN, Hookway TA, Gifford CA, Taneja P, Noble-Haeusslein L, McDevitt TC. “Differentiation of V2a interneurons from human pluripotent stem cells.” Proceedings of the National Academy of Sciences. Published online April 24, 2017. doi:10.1073/pnas.1608254114
Abstract (summary): The spinal cord contains diverse neuronal types that control motor functions and arise from distinct developmental domains. Excitatory V2a interneurons are integral to central pattern generators governing respiration and locomotion, but a robust human source has been lacking. This study reports an optimized small-molecule protocol to produce CHX10+ V2a interneurons from human pluripotent stem cells, yielding approximately 25% CHX10+ cells across four cell lines. Differentiated cells express V2a markers and neuronal proteins, show functional maturation, and after transplantation into mouse spinal cord survive, extend long neurites, and form putative synapses with host neurons. These findings support the use of hPSC-derived V2a interneurons for modeling spinal cord development and for investigating cell replacement strategies for spinal cord injury.