Nociceptors differentiation from human pluripotent stem cell follows ontogenesis of embryonic sensory neuronal development

Vijayaragavan, K. 1 , Iceta, R. 1 , Devesa Giner , I. 3 , Mathivanan, S. 3 , Torrecilla Sesma, M. 2 , Bruzos-Cidon , C. 2 , Ugedo, L. 2 , Ferrer-Montiel, A. 3 & Vijayaragavan, K. 1

1 Neurodegenerative Disease Program, Inbiomed Foundation
2 Dept. Pharmacology, University of the Basque Country
3 Inst. Molecular Biology y Cellular, Universidad Miguel Hernández

Human embryonic stem cells (hESCs) and the induced pluripotent stem cells (hiPSCs), collectively known as human pluripotent stem cells (hPSCs), represent an invaluable opportunity to dissect complex cellular and molecular events that occur during early nociceptor development. Importantly the differentiation process from hPSCs allow the unique means to track and isolate intermediate cells (precursors) that only transiently exist in vivo like the neural crest cells and progenitor cells for the peripheral nervous system. While specification of peripheral sensory neurons has been studied in animal models, investigation into the embryonic determinants for the emergence of human nociceptive sensory neurons remains elusive. Here we present a study cataloguing the hierarchical differentiation and lineage relationship of sensory neurons, nociceptors and smooth muscles as they are generated from neural crest cells (NCCs) of the hPSCs. Molecular programs leading to the emergence are delineated by the temporal appearance of dorsal neuroepithelial and truck NCCs identities and early embryonic dorsal root ganglion markers. Early during differentiation (day 0), neuroepithelial cells derived from hPSCs stained positive for p75, HNK1, A2B5 and MAP5. The neuroepithelial cells were also positive for BRN3a, however were localized in the perinuclear region as opposed to discreet punctuated pattern later during the differentiation (days 25-30). We also observed two waves of SOX2 expression peaking at days 0-4 and days 18-30. Interestingly at day18, we observed a mutually exclusive localisation of high SOX2+ and high BRN3a+ neurons. By day 30, the neurons were positive for PERIPHERIN, TRKA, Nav1.7 and Nav1.8 channels. TRKA+ neurons were positive for Nav1.7 and Nav1.8, however Nav1.7 staining in the soma was co-localised with TRKA, while this arrangement was not observed along the axon. As for Nav1.8 positive neurons, TRKA did not co-localise with the TTX-R channel neither in the soma nor the axon. Whole cell recording revealed two types of Na currents, a fast gating and voltage dependence component showing TTX sensitivity or typical slow gating Na currents with persistent properties.