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

Abstract

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. In this study, we took an alternative approach and selected five small-molecule inhibitors of key signaling pathways to improve efficiency in derivation of sensory neurons from human iPSCs. Within 8 days of the differentiation protocol, iPSC-derived sensory neurons were achieved as denoted by marker expression, >80% being immuno-positive for Tuj1, NeuN and NF200, Islet1, peripherin and Brn3a but immuno-negative for neural progenitor markers (Pax6 and nestin) as well as neural crest cell markers (AP2, HNK1 and p75). Patch-clamp recordings on the derived neurons revealed healthy resting membrane potentials averaging -60 mV. Single action potentials were elicited in response to depolarizing step currents whereas multiple action potentials could be evoked with increasing intensity of the depolarizing current. Spiking was blocked by bath administration of tetrodotoxin. The derived cells therefore demonstrated electrophysiological properties characteristic of functional neurons. Neurite bundles that extended from the derived neurons were amenable to myelination in co-culture with rat Schwann cells, showing internodal segments that were immuno-positive for myelin-related proteins, P0, MBP and GALC. The phenotype of the iPSC-derived neurons was sustainable in Neurobasal medium supplemented with maintenance growth factors but without the small-molecule inhibitors. With this rapid and efficient induction protocol, we expect production of sensory neurons from human iPSCs on demand for developmental studies and disease modeling.