Protocol for inducing dorsal spinal sensory interneurons from mouse embryonic stem cell-derived neuromesodermal progenitors

Here, we present a protocol comprising two differentiation techniques to derive dorsal sensory spinal interneurons (dIs) from mouse embryonic stem cells (mESCs) through a neuromesodermal progenitor (NMP) precursor. First, we describe the steps for using retinoic acid (RA) to induce dI4s, dI5s, and dI6s, which mediate pain, itch, touch, and sensorimotor integration. Second, we detail the procedures for generating dI1s, dI2s, and dI3s, which mediate proprioception and mechanosensation, using RA together with bone morphogenetic protein (BMP) 4. For complete details on the use and execution of this protocol, please refer to Gupta et al. 1 • Instructions to expand mESCs on MEF feeders before starting the differentiation • Steps to induce NMPs by the timed addition of bFGF and CHIR • Procedures to pattern NMPs with RA or RA+BMP4 to produce the dI1–dI6 populations • Guidance on two procedures to assess the identity of dorsal interneuron subtypes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol comprising two differentiation techniques to derive dorsal sensory spinal interneurons (dIs) from mouse embryonic stem cells (mESCs) through a neuromesodermal progenitor (NMP) precursor. First, we describe the steps for using retinoic acid (RA) to induce dI4s, dI5s, and dI6s, which mediate pain, itch, touch, and sensorimotor integration. Second, we detail the procedures for generating dI1s, dI2s, and dI3s, which mediate proprioception and mechanosensation, using RA together with bone morphogenetic protein (BMP) 4.