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Edited by Tom Maniatis, Columbia University Medical Center, New York, NY, and approved March 29, 2017 (received for review June 13, 2016)
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.
Footnotes
- ↵1To whom correspondence should be addressed. Email: todd.mcdevitt{at}gladstone.ucsf.edu.
Author contributions: J.C.B., D.A.M., J.A.M.-V., T.A.H., C.A.G., P.T., L.N.-H., and T.C.M. designed research; J.C.B., D.A.M., J.A.M.-V., F.N.M.-C., C.A.G., and P.T. performed research; C.A.G. contributed new reagents/analytic tools; J.C.B., D.A.M., T.A.H., C.A.G., P.T., L.N.-H., and T.C.M. analyzed data; and J.C.B., D.A.M., J.A.M.-V., T.A.H., C.A.G., P.T., L.N.-H., and T.C.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequencing data have been deposited in the National Center for Biotechnology Information’s Sequence Read Archive (accession no. GSE97564).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1608254114/-/DCSupplemental.
- Jessica C. Buttsa,b,
- Dylan A. McCreedya,c,d,
- Jorge Alexis Martinez-Vargase,
- Frederico N. Mendoza-Camachoa,
- Tracy A. Hookwaya,
- Casey A. Gifforda,
- Praveen Tanejaf,
- Linda Noble-Haeussleinc,d, and
- Todd C. McDevitta,g,1
- aGladstone Institute of Cardiovascular Disease, San Francisco, CA 94158;
- bGraduate Program in Bioengineering, University of California, San Francisco, CA 94158;
- cDepartment of Neurosurgery, University of California, San Francisco, CA 94143;
- dDepartment of Physical Therapy and Rehabilitation Science, University of California, San Francisco, CA 94143;
- eDepartment of Bioengineering, University of California, Berkeley, CA 94709;
- fGladstone Institute of Neurological Disease, San Francisco, CA 94158;
- gDepartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94158
Edited by Tom Maniatis, Columbia University Medical Center, New York, NY, and approved March 29, 2017 (received for review June 13, 2016)
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.
- V2a interneurons
- human pluripotent stem cells
- differentiation
- single-cell RNAseq
Footnotes
- ↵1To whom correspondence should be addressed. Email: todd.mcdevitt{at}gladstone.ucsf.edu.
Author contributions: J.C.B., D.A.M., J.A.M.-V., T.A.H., C.A.G., P.T., L.N.-H., and T.C.M. designed research; J.C.B., D.A.M., J.A.M.-V., F.N.M.-C., C.A.G., and P.T. performed research; C.A.G. contributed new reagents/analytic tools; J.C.B., D.A.M., T.A.H., C.A.G., P.T., L.N.-H., and T.C.M. analyzed data; and J.C.B., D.A.M., J.A.M.-V., T.A.H., C.A.G., P.T., L.N.-H., and T.C.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequencing data have been deposited in the National Center for Biotechnology Information’s Sequence Read Archive (accession no. GSE97564).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1608254114/-/DCSupplemental.
V2a interneurons from human pluripotent stem cells
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