Small molecule approach to direct differentiation of human induced pluripotent stem cells to sensory neurons

Abstract

BACKGROUND: Strategies that exploit induced pluripotent stem cells (iPSCs) to derive neurons have relied on cocktails of cytokines and/or growth factors to bias cell signaling events in the course of fate choice. These are often costly and inefficient, involving multiple steps. OBJECTIVE: In this study, we took an alternative approach and selected five small molecule compounds (SMCs) of key signaling pathways to test out a new protocol for the derivation of sensory neurons from human iPSCs. RESULTS: Within 8 days of the differentiation protocol, iPSCderived sensory neurons were achieved at > 80% efficiency. The derived cells were positive for cytoskeletal markers common to neurons, Tuj1 and neurofilament, and specific markers for sensory neurons, Islet, peripherin and Brn3a. Whole-cell patch-clamp recordings of the neurons showed firing in response to membrane depolarization, capable of generating action potentials sensitive to tetrodotoxin (TTX). In co-culture with rat Schwann cells in myelinating medium, axon bundles of the derived sensory neurons underwent myelination, showing internodal segments that were positive for myelin associated proteins. The phenotype of the iPSCderived sensory neurons was sustainable in Neurobasal medium supplemented with maintenance growth factors but without SMCs. CONCLUSION: Our rapid and efficient induction protocol promises controlled production of sensory neurons from human iPSCs as a pool for developmental studies and disease modeling.