V2a interneuron differentiation from mouse and human pluripotent stem cells

Abstract

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, which 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 to induce differentiation 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 the sequence of small molecules used, but

Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice

“…Human induced pluripotent stem cell-derived V2a interneurons were generated as previously described ( Butts et al, 2019 ). For transplantation into the intact spinal cord, adult female NSG mice were anesthetized with 2% isoflurane and a laminectomy was performed at T9.…”

Passive clearing and 3D lightsheet imaging of the intact and injured spinal cord in mice

“…Human induced pluripotent stem cell (hiPSC)-derived V2a interneurons were generated as previously described (Butts et al 2019). For transplantation into the intact spinal cord, adult female NSG mice were anesthetized with 2% isoflurane and a laminectomy was performed at T9.…”

Stem Cell Neurodevelopmental Solutions for Restorative Treatments of the Human Trunk and Spine

“…Notch signaling promotes the inhibitory V2b subtype, but directly inhibits the generation of excitatory V2a neurons. In a recent protocol (Butts et al, 2019 ), the γ-secretase inhibitor DAPT was added early in neural induction to inhibit Notch, and along with dual SMAD inhibition and optimized RA/Shh concentrations was sufficient to bias towards V2a excitatory interneuron fate. These advancements begin to open up studies and therapeutic applications of interneurons.…”

“…However, by applying NMPs, the ability to begin to model circuit formation event in vitro is expanding (Nedelec and Martinez-Arias, 2021). Recent advancements out of the Sakiyama-Elbert lab have enabled new protocols to produce V3 excitatory commissural interneurons using mESCs (Xu and Sakiyama-Elbert, 2015), and V2a excitatory interneurons both from mESCs (Brown et al, 2014) and human PSCs (Butts et al, 2019). V3 interneuron (NKX-2.2 + /SIM1+) differentiation was similar to SMN protocols but modified to decrease RA concentration (10 nM) and lengthen the exposure to the potent Shh agonist, SAG (∼18% efficiency NKX-2.2 cells).…”