Differentiation and single cell RNA sequencing of V2a interneurons from human pluripotent stem cells

Authors
*J. BUTTS1, D. A. MCCREEDY2, J. A. MARTINEZ-VARGAS3, C. A. GIFFORD4, L. J. NOBLE-HAEUSSLEIN5, T. C. MCDEVITT4;
1Bioengineering, Gladstone Inst. of Cardiovasc. Dis., San Francisco, CA; 2Neurolog. Surgery, UCSF, San Francisco, CA; 3Univ. of California – Berkeley, Berkeley, CA; 4Gladstone Inst., San Francisco, CA; 5Dept. of Neurosurg and Physical Therapy and Rehabil. Sci., Univ. California, San Francisco, CA
Disclosures
J. Butts: None. D.A. McCreedy: None. J.A. Martinez-Vargas: None. C.A. Gifford: None. L.J. Noble-Haeusslein: None. T.C. McDevitt: None.

LINK: Society for Neuroscience

V2a interneurons (INs) are a critical population of neurons found in the hindbrain and spinal cord that are necessary for coordinated motor function, including control of breathing and locomotion. V2a INs span several spinal cord segments and relay excitatory information to adjacent INs and downstream motor neurons. While studies of murine V2a INs have provided critical early characterization of this population, there is no robust source of human V2a INs to phenotypically characterize this cell population in vitro as a potential therapy for human motor dysfunction. Therefore, we recently described a protocol to differentiate V2a INs, marked by the CHX10 transcription factor, from human pluripotent stem cells (hPSCs). Drawing inspiration from neural tube development, the critical signaling molecules retinoic acid (RA), Purmorphamine (Pur, a Shh agonist), and DAPT (a Notch inhibitor) were varied systematically until a CHX10+ population of approximately 30% was obtained with 100nM RA, 100nM Pur, and 1μM DAPT. After starting with human ESCs (H7), the protocol was reproduced in three additional hPSC lines (H1 ESCs, WTC and WTB induced PSCs), to yield CHX10 percentages between 25-50%. Expression of V2a IN lineage markers (CHX10, SOX14) was highly upregulated (~100-fold) when compared to a published motor neuron protocol. Single cell RNA sequencing was performed to identify the cell populations in the V2a IN differentiation. K-means clustering of twelve principle components revealed seven distinct cell populations including mature neurons, progenitor neurons, and glial cells. The cluster containing the majority of CHX10-expressing cells also expressed other markers of V2a interneurons (SOX21 and SHOX2), as well as genes associated with a glutamatergic phenotype (PCP4 and OAT). V2a cultures expressed a mature glutamatergic neuronal phenotypic (NeuN+ and VGlut2+) and demonstrated increased action potential frequency when cultured for up to 60 days. To determine if V2a interneuron cultures could integrate with the endogenous spinal cord, dissociated cultures were transplanted into T9 vertebrae of uninjured mice and resulted in survival and neurite extension greater than 5mm rostral and caudal to the transplantation site. Transplanted cells expressed the presynaptic maker synaptophysin on neurite terminals adjacent to host NeuN+ neurons, indicating integration with host circuitry. In summary, we have developed the first protocol to differentiate V2a INs from hPSCs that will allow for further characterization of novel phenotypic and electrophysiological properties of these cells.

Gladstone Article

Grant Support
CIRM Grant LA1-08015

This entry was posted in Chronic Spinal Cord Injury Research, Neuroscience Abstracts, Regenerative Medicine, Spinal Research, Stem Cell Research and tagged , . Bookmark the permalink.

One Response to Differentiation and single cell RNA sequencing of V2a interneurons from human pluripotent stem cells

  1. Alma sierra says:

    Estoy muy interested en participate en esto.yo. soy quadriplegic????? Or paraplegic…….que necesito hacer????

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