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V2a interneuron differentiation from mouse and human pluripotent stem cells

An Author Correction to this article was published on 08 November 2019

This article has been updated


V2a interneurons are located in the hindbrain and spinal cord, where they provide rhythmic input to major motor control centers. Many of the phenotypic properties and functions of excitatory V2a interneurons have yet to be fully defined. Definition of these properties could lead to novel regenerative therapies for traumatic injuries and drug targets for chronic degenerative diseases. Here we describe how to produce V2a interneurons from mouse and human pluripotent stem cells (PSCs), as well as strategies to characterize and mature the cells for further analysis. The described protocols are based on a sequence of small-molecule treatments that induce differentiation of PSCs into V2a interneurons. We also include a detailed description of how to phenotypically characterize, mature, and freeze the cells. The mouse and human protocols are similar in regard to the sequence of small molecules used but differ slightly in the concentrations and durations necessary for induction. With the protocols described, scientists can expect to obtain V2a interneurons with purities of ~75% (mouse) in 7 d and ~50% (human) in 20 d.

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Fig. 1: Schematic of developing neural tube.
Fig. 2: Schematic of mouse and human V2a interneuron differentiation protocol.
Fig. 3: Phase and fluorescence imaging throughout mouse and human differentiations.
Fig. 4: End point analyses of mouse and human differentiations.
Fig. 5: Flow cytometry gating strategy.
Fig. 6: Enrichment of V2a interneuron populations.
Fig. 7: Maturation of V2a interneuron cultures.
Fig. 8: Cryopreservation of human V2a interneuron cultures.
Fig. 9: Seeding density is critical to human V2a interneuron differentiation.
Fig. 10: Mouse V2a interneuron aggregate formation.

Data availability

Data are available from the authors upon request.

Change history

  • 08 November 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.


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We acknowledge C. Brown for her dedication and hard work in co-developing the original mouse V2a interneuron protocol. In addition, we acknowledge the Gladstone Stem Cell Core for providing cell culture facilities and the Gladstone Communications Department for graphics creation. This work was made possible through funding by NIH NINDS F31 NS090760 (N.I.), CIRM LA1 C14-08015 (T.C.M.), The Roddenberry Foundation L02593 (T.C.M.), and NIH NINDS R01 NS090617 (S.S.-E.). We thank B. Conklin (Gladstone Institutes) for the kind gifts of WTB iPSCs, WTC10 iPSCs, and WTC11 AAVS1::GCaMP6f iPSCs.

Author information

Authors and Affiliations



J.C.B. designed, performed, and analyzed the human and mouse differentiation experiments. N.I., N.W., and R.T. designed, performed, and analyzed the mouse differentiation experiments. S.S.-E. designed the mouse differentiation experiments. T.C.M. designed the human differentiation experiments. J.C.B., N.I., S.S.-E., and T.C.M. prepared the manuscript.

Corresponding authors

Correspondence to Shelly Sakiyama-Elbert or Todd C. McDevitt.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Chian-Yu Peng and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Butts, J. C. et al. Proc. Natl. Acad. Sci. USA 114, 4969–4974 (2017):

Iyer, N. R., Huettner, J. E., Butts, J. C., Brown, C. R., & Sakiyama-Elbert, S. E. Exp. Neurol. 277, 305–316 (2016):

Brown, C. R., Butts, J. C., McCreedy, D. A. & Sakiyama-Elbert, S. E. Stem Cells Dev. 23, 1765–1776 (2014):

Integrated supplementary information

Supplementary Figure 1 Cell detachment during hPSC V2a differentiation.

The differentiation appears to be normal on D3 and D5 as nice confluent cell layers are forming. On D7, areas where the cells have peeled off are visible (*). By D13, neurites are visible (inset (i), arrowhead) indicating neurons are present but dense monolayers are not visible. Scale bar in D3 = 100 μm, scale bar in inset = 25 μm.

Supplementary information

Supplementary Figure 1

Cell detachment during hPSC V2a differentiation.

Reporting Summary

Supplementary Video 1

Calcium flux in mouse V2a-Olig2 aggregate cultures. Video played back at 2× speed. Scale bar, 250 μm.

Supplementary Video 2

Calcium flux in human V2a interneuron cultures. The differentiation was performed with the WTC hiPSC cell line harboring the genetically-encoded calcium sensor GCaMP6f. Video played back at 2× speed. Scale bar, 100 μm.

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Butts, J.C., Iyer, N., White, N. et al. V2a interneuron differentiation from mouse and human pluripotent stem cells. Nat Protoc 14, 3033–3058 (2019).

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