582 Differentiation of human skin-derived precursors (SKPs) into functional sensory neurons

Abstract

Skin-derived precursor cells (SKPs) have been shown as a population of stem cells that express some markers common to the neural crest stem cells (NCSCs) as progenitor neural nestin, the low affinity neurotrophin receptor p75 and transcription factors as Sox9 and 10 and Pax3, Slug, Snail. SKPs are NCSCs that persist in certain adult tissues, particularly in the skin, which can generate a large part of the peripheral nervous system, particularly sensory neurons (SN). In this study, we reported a protocol to obtain SNs from SKPs. For this, we rely on a protocol already established by Reinhardt et al. in 2013 from Embryonic stem cells (ESCs) and induced Pluripotent stem cells (iPSCs). The SKPs were induced to generate SNs by treating cells with trophic factors and / or chemicals that had previously been shown to be important for the generation of SNs: Purmorphamine, CHIR99021, BMP4 and GDNF, BDNF, NGF. We show that the differentiation of SKPs into SNs was regulated by neurogenins 1, 2 and 3. At the end of the differentiation, the protocol allowed gene transcription of BRN3A and PRDM12 (marker of pain-sensing nerve cells). Expression was from 1000 to 2500 times higher for PRDM12 and BRN3A respectively versus undifferentiated SKP. Using immunostaining we showed that 65% and 80% of cells expressed BRN3A and peripherin, marker for peripheral neurons. Furthermore, cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, H1, B3-tubulin. Using calcium imaging, a proportion of cells was responding for histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKP are NCSC, that able to differentiate directly into functional sensory neuron.