Differentiation of V2a interneurons from human pluripotent stem cells

Abstract

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however, the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here, we report the directed differentiation of CHX10 V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid, sonic hedgehog, and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10 cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time, CHX10 cells expressed neuronal markers [neurofilament, NeuN, and vesicular glutamate transporter 2 (VGlut2)], and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10 cells within the differentiated population, which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice, hPSC-derived V2a cultures survived at the site of injection, coexpressed NeuN and VGlut2, extended neurites >5 mm, and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI.

Transsynaptic tracing and its emerging use to assess graft-reconstructed neural circuits

“…The initiation of monosynaptic tracing from specific therapeutically relevant populations of neurons, relying on the use of transgenic Cre driver lines and/or intersectional tracing methods, is a clear next step. Good candidates for such an approach in PD and SCI models would be graft tissue prepared from dopaminergic and V2a interneuronal Cre driver lines, respectively. Rabies tracing could also be initiated from host motor or premotor neurons caudal to the injury site following SCI, in order to identify graft neurons that contribute to motor output, for example.…”

Derivation of Specific Neural Populations From Pluripotent Cells for Understanding and Treatment of Spinal Cord Injury

“…Although the initial methodology has undergone revision and review (Okada et al, 2004;Dasen et al, 2008;Peljto et al, 2010), it still paved the way for later extrapolation of these protocols to drive human embryonic and induced pluripotent stem cells to MN fates. Protocols outlining the induction of hESC/hiPSC derived-MNs frequently use SMAD inhibitors in the initial stages of differentiation to ensure efficient neurogenesis (Chambers et al, 2009;Reinhardt et al, 2013;Ben-Shushan et al, 2014;Butts et al, 2017). Following neuroepithelialization, RA treatment caudalizes the cells, and SAG is used for ventral specification.…”

“…The initial step forward to producing V2a INs from mESCs, as a tool for potential therapy, was recently extrapolated to hESC/ hIPSCs by Butts et al (2017). Briefly, hESCs and hiPSCs were differentiated using methods analogous to hESC derived MNs, with SMAD inhibitors and basal medium for the first segment of neuro-epithelialization (Peljto et al, 2010;Ben-Shushan et al, 2014).…”

Microfluidic Chip for Label-Free Removal of Teratoma-forming Cells from Therapeutic Human Stem Cells

“…hiPSC-derived NPCs (hiPSC-NPCs) are poised to overcome these obstacles and have demonstrated cell integration into host tissue [19]. In addition to chronic neurodegenerative diseases, acute neurodegeneration may result from traumatic brain injury (TBI) 20or spinal cord injury [20][21][22]. Human NPC transplantation has shown promising results to treat TBI and has demonstrated neuroprotection and improved cognitive outcomes following TBI [23].…”