Significance Spinal cord injury (SCI) significantly disrupts normal neural circuitry, leading to severe degradation of motor and sensory function. Excitatory interneurons that relay signals from the brain to neural networks throughout the spinal cord, including glutamatergic V2a interneurons that coordinate respiration and locomotion, are lost after SCI. Thus, transplantation of V2a interneurons after SCI could provide a novel therapy to restore functional connections between the brain and spared downstream neurons. This study describes the generation of V2a interneurons from human pluripotent stem cells, using developmentally relevant morphogenic signaling pathways. This work provides initial insight into the development of excitatory human interneurons and enables the examination of their therapeutic efficacy for SCI repair.

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.

Cell replacement is a promising therapeutic strategy to restore motor function and sensation after traumatic injury to the central nervous system (CNS). Several clinical trials are currently investigating the therapeutic efficacy of various transplanted neural cells to treat spinal cord injury (SCI), including oligodendrocyte progenitors derived from human embryonic stem cells (hESCs) (1), autologous Schwann cells (2), and fetal-derived neural stem cells (3) and precursor cells (4, 5). Despite such efforts, however, the ability of specific neuronal cell types to functionally restore damaged neural networks needs further investigation.

Pluripotent stem cells (PSCs) provide a renewable source of cells that can differentiate into a variety of neurons, provided that the necessary signaling cues are presented in an appropriate spatiotemporal manner. Different types of neurons, including forebrain and midbrain neural cell types, such as cortical neurons (6), dopaminergic neurons (7), and inhibitory interneurons from the medial ganglionic eminence (8, 9), have been differentiated from hPSCs, but motor neurons are the only caudal neuronal population to be generated from hPSCs to date (10, 11).

V2a spinal interneurons in particular are crucial to the transmission and coordination of motor and sensory functions (12, 13). Glutamatergic V2a interneurons, identified by expression of the CHX10 transcription factor (14), are distributed throughout the hindbrain and spinal cord in mammals and relay excitatory stimuli to central pattern generators that regulate motor function for breathing and locomotion (15⇓⇓⇓–19). Ablation of V2a interneurons in mice results in disruption of normal breathing patterns (18), impaired forelimb reaching tasks (15), and loss of left-right hindlimb coordination (16, 17).

Combinations of various morphogenic signals are responsible for patterning of the neural tube during development. The rostral-caudal location of spinal cell types is patterned by retinoic acid (RA) released from adjacent somites (20). In parallel, an orthogonal ventral-dorsal gradient of sonic hedgehog (Shh), secreted from the floor plate and notochord, specifies independent progenitor domains (21) that yield mature cell types with distinct roles in motor function. In the ventral neural tube, excitatory V2a and inhibitory V2b interneurons arise coincidentally from the p2 domain (22, 23), which is located immediately dorsal to the pMN domain. Notch signaling dictates the balance between V2a and V2b interneurons, with Notch signaling necessary for the specification of V2b interneurons and Notch inhibition increasing the proportion of V2a interneurons (Fig. 1A) (24).

Fig. 1. Morphogen concentrations modulate V2a interneuron population. (A) Schematic of the developing neural tube. RA, released from the somites, and Shh, released from the floorplate and notochord, pattern the different progenitor domains of the neural tube. Notch signaling is necessary for generating V2b interneurons, whereas Notch inhibition promotes V2a interneuron differentiation. (B) Timeline of the V2a interneuron protocol. (C) Flow cytometry analysis of CHX10 expression as RA concentration was varied, and Shh agonist pur and DAPT concentrations were held constant. CHX10 expression using 100 nM RA was greater (P < 0.05, one-way ANOVA and Tukey’s post hoc comparison) than that in the vehicle control (V.C., DMSO), 10 nM, 30 nM, and 1 μM groups. (D) Flow cytometry analysis of CHX10 expression as pur concentration was varied and RA and DAPT concentrations were held constant. CHX10 expression using 100 nM pur was greater (P < 0.05, one-way ANOVA and Tukey’s post hoc comparison) than that of all groups. (E) Flow cytometry of CHX10 expression as DAPT concentration was varied, and RA and pur concentrations were held constant. CHX10 expression using 1 μM and 5 μM DAPT was greater (P < 0.05, one-way ANOVA and Tukey’s post hoc comparison) than that in the V.C, 100 nM, and 500 nM groups. (F–H) Immunostaining for CHX10 (green) and nuclei labeling (blue) of differentiations with 100 nM RA (F), 100 nM pur (G), or 1 μM DAPT (H). n = 3. Data represent mean ± SD. (Scale bar: 100 μm.)

This study provides a description of human V2a interneurons differentiated from PSCs with small molecule agonists and antagonists of developmental signaling pathways. We investigated the specificity of the defined protocol and the ability of the differentiated cells to mature into excitatory neurons in vitro and in vivo. These results provide a robust source of human V2a interneurons that can be used to further define their molecular profile and in vitro electrophysiological properties, as well as to examine their therapeutic potential for repair of spinal cord injury.

Methods All work with human ESC and iPSC lines was approved by the University of California – San Francisco Human Gamete, Embryo and Stem Cell Research (GESCR) Committee. All human PSC lines were grown to 70% confluence and passaged as single cells every 3 d. Dissociated cells were replated on Matrigel-coated cultureware at a density of 10,000 cells per cm2 with 10 μM ROCK inhibitor in mTeSR. The experimental procedures, including V2a interneuron differentiation, neuronal maturation, endpoint analysis, imaging analysis and quantification, electrophysiology testing, single-cell RNAseq, spinal transplantation, and statistical analysis, are described in detail in SI Appendix, Methods.

Acknowledgments We thank the following individuals for helpful discussions and assistance with manuscript preparation: Dr. Shelly Sakiyama-Elbert, Dr. Biljana Djukic, Alex Williams and staff the Gladstone Bioinformatics Core, Meredith Calvert and staff at the Gladstone Histology and Light Microscopy Core, staff at the Gladstone Stem Cell Core, Oriane Matthys, Dr. Nathaniel Huebsch, Giovanni Maki, and David Joy. We also thank Dr. Bruce Conklin for generously providing the WTB and WTC hiPSC cell lines and Dr. Thomas Fandel for guidance and assistance in cell transplantation. This work was supported by California Institute of Regenerative Medicine Grant LA1-08015 (to T.C.M.) and the Alvera Kan Endowed Chair (L.N.-H.). J.C.B. was supported by a National Science Foundation (NSF) Graduate Research Fellowship and previously by an NSF Stem Cell Biomanufacturing Integrative Graduate Education and Research Traineeship (DGE 0965945). C.A.G. is a Howard Hughes Medical Institute Fellow of the Damon Runyon Cancer Research Foundation (DRG-2206-14).