Generation of iPSC-Derived Human Peripheral Sensory Neurons Releasing Substance P Elicited by TRPV1 Agonists.

Marília Z P Guimarães,Jaroslaw K Sochacki,Rodrigo De Vecchi,Newton G Castro,Igor Lima,Felipe Rodrigues Da Silva,Lionel Breton,Bruna S Paulsen,Stevens K Rehen,Rodrigo F M Da Costa,Gabriela Vitória

doi:10.3389/fnmol.2018.00277

Abstract

Neural crest stem cells (NCPCs) have been shown to differentiate into various cell types and tissues during embryonic development, including sensory neurons. The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases in vitro.

Highlights

Human induced pluripotent stem cells are used to generate different neuronal types to study biology, pharmacology and to screen for potential new drugs
Chambers et al (2012) showed TRPV1 expression via quantitative PCR and correlated with only 1–2% of the cells responding to capsaicin
Others showed electrophysiological activity of human sensory neurons directly reprogrammed from fibroblasts, but not capsaicin-elicited signals (Wainger et al, 2015)

Summary

Introduction

Human induced pluripotent stem cells (hiPSCs) are used to generate different neuronal types to study biology, pharmacology and to screen for potential new drugs. In the field of skin irritation, Human SP Releasing Sensory Neurons the activity of transient receptor potential (TRP) channels are of valuable importance, since these channels detect a range of topical nociceptive irritants, such as capsaicin and mustard oil (Nilius and Szallasi, 2014). Transient receptor potential channels comprise a diverse family of ligand-gated, mostly non-selective, cation channels that are robustly expressed in sensory systems throughout species (Nilius and Szallasi, 2014). TRPV1 is the most well-studied and is considered to be the prototypical TRP channel present in somatosensory neurons (Basbaum et al, 2009). One of the receptors that seem to inhibit TRPV1 activation is the cannabinoid 1 receptor (CB1), present in somatosensory neurons (Julius and Basbaum, 2001). An endogenous agonist of CB1, anandamide, is a TRPV1 agonist, albeit with an EC50 one order of magnitude higher in the latter (Zygmunt et al, 1999)

Methods

Results

Conclusion