A pinboard by
Jessica Butts

Graduate Student, Gladstone Institutes


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

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 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 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 12 principal components revealed 7 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). To determine if V2a interneuron cultures 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 permit further characterization of novel phenotypic and electrophysiological properties of these cells.


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.

Pub.: 26 Apr '17, Pinned: 21 Jun '17

Pax3- and Pax7-mediated Dbx1 regulation orchestrates the patterning of intermediate spinal interneurons.

Abstract: Transcription factors are key orchestrators of the emergence of neuronal diversity within the developing spinal cord. As such, the two paralogous proteins Pax3 and Pax7 regulate the specification of progenitor cells within the intermediate neural tube, by defining a neat segregation between those fated to form motor circuits and those involved in the integration of sensory inputs. To attain insights into the molecular means by which they control this process, we have performed detailed phenotypic analyses of the intermediate spinal interneurons (IN), namely the dI6, V0D, V0VCG and V1 populations in compound null mutants for Pax3 and Pax7. This has revealed that the levels of Pax3/7 proteins determine both the dorso-ventral extent and the number of cells produced in each subpopulation; with increasing levels leading to the dorsalisation of their fate. Furthermore, thanks to the examination of mutants in which Pax3 transcriptional activity is skewed either towards repression or activation, we demonstrate that this cell diversification process is mainly dictated by Pax3/7 ability to repress gene expression. Consistently, we show that Pax3 and Pax7 inhibit the expression of Dbx1 and of its repressor Prdm12, fate determinants of the V0 and V1 interneurons, respectively. Notably, we provide evidence for the activity of several cis-regulatory modules of Dbx1 to be sensitive to Pax3 and Pax7 transcriptional activity levels. Altogether, our study provides insights into how the redundancy within a TF family, together with discrete dynamics of expression profiles of each member, are exploited to generate cellular diversity. Furthermore, our data supports the model whereby cell fate choices in the neural tube do not rely on binary decisions but rather on inhibition of multiple alternative fates.

Pub.: 20 Jun '17, Pinned: 21 Jun '17