Accelerated Production and Maturation of Human Induced Pluripotent Stem Cell Derived Nociceptors

Abstract

The scarce availability and prohibitive costs of primary human DRG tissues limits the scalability of in vitro models for pain drug discovery and disease modeling. Human induced pluripotent stem cell (hiPSC)-derived sensory neurons provide a near-limitless amount of tissue for these applications. Using an accelerated, step-wise, directed differentiation protocol that proceeds through a novel ‘primal ectoderm’ population, we are able to manufacture nociceptors rapidly and at scale by relying on a simplified chemically-defined basal medium, recombinant growth factors and small molecules. Following production and only one week of maturation, these nociceptors have a consistent molecular phenotype and express key therapeutic pain targets such as human voltage-gated sodium channels Nav1.7, 1.8, and 1.9, which are primarily responsible for eliciting nociceptor action potentials, as well as the human capsaicin receptor TRPV1. Using whole-cell patch clamp electrophysiology, we demonstrate that these neurons fire action potentials and possess burst-firing ability by day 9 in vitro as assessed by performing stepwise depolarizing current injections in current clamp mode. We also show these neurons exhibit inward and outward currents in response to voltage steps in voltage clamp mode. We have also demonstrated that these neurons are responsive to ligands such as capsaicin using calcium imaging region-of-interest analysis. Greater than 30% of neuronal cell bodies respond to 100 nM capsaicin following an additional 2 to 3 weeks of maturation. Importantly, this response was ablated by capsazepine, thus confirming response specificity. These data demonstrate that hiPSC nociceptors have the potential to screen and identify novel analgesic or anti-nociceptive compounds for use in humans.