Robust induction of neural crest cells to derive peripheral sensory neurons from human induced pluripotent stem cells

Yoshie Umehara,Mitsutoshi Tominaga,Yayoi Kamata,Nobuaki Takahashi,François Niyonsaba,Hironori Matsuda,Sumika Toyama,Hideoki Ogawa,Kenji Takamori

doi:10.1038/s41598-020-60036-z

Yoshie Umehara, Mitsutoshi Tominaga + Show 7 more

Open Access

https://doi.org/10.1038/s41598-020-60036-z

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Abstract

Because intractable itch reduces quality of life, understanding the fundamental mechanisms of itch is required to develop antipruritic treatments. Itch is mediated by peripheral sensory neurons, which originate from the neural crest (NC) during development. Itch-associated signaling molecules have been detected in genetically engineered animals and in cultures of peripheral neurons from dorsal root ganglia (DRG). Ethical difficulties collecting peripheral neurons from human DRG have limited analysis of itch in humans. This study describes a method of differentiating peripheral neurons from human induced pluripotent stem cells (hiPSCs) for physiological study of itch. This method resulted in the robust induction of p75 and HNK1 double-positive NC cells from hiPSCs. The expression of NC markers TFAP2A, SOX10 and SNAI1 increased during NC induction. The induction efficiency was nearly 90%, and human peripheral neurons expressing peripherin were efficiently differentiated from hiPSC-derived NC cells. Moreover, induced peripheral neurons expressed the sensory neuronal marker BRN3A and the itch-related receptors HRH1, MRGPRX1, IL31R and IL-4R. Calcium imaging analyses indicated that these peripheral neurons included sensory neurons responsive to itch-related stimuli such as histamine, BAM8-22, IL-31 and IL-4. These findings may enable detailed analyses of human DRG neurons and may result in new therapies for intractable itch.

Highlights

Because intractable itch reduces quality of life, understanding the fundamental mechanisms of itch is required to develop antipruritic treatments
SB431542 inhibits the activation of activin receptor-like kinase (ALK) 5, which is induced by transforming growth factor (TGF)-β or activin, resulting in the suppression of SMAD2 signaling
The efficiency of neural crest (NC) induction was evaluated by fluorescence activated cell sorting (FACS) analyses of the levels of expression on cells of the NC markers p75 and HNK1, using the same clones of antibodies as used in the NSB culture system[10]

Summary

Introduction

Because intractable itch reduces quality of life, understanding the fundamental mechanisms of itch is required to develop antipruritic treatments. This study describes a method of differentiating peripheral neurons from human induced pluripotent stem cells (hiPSCs) for physiological study of itch. Somatic sensations, including itch and pain are mediated by primary sensory neurons, which have cell bodies in dorsal root ganglia (DRG) or trigeminal ganglia. These neurons differ in somal size, expression of ion channels and receptors, innervation territories, and electrophysiological properties[1]. Mas-related G protein-coupled receptors (Mrgprs), especially MrgprAs, MrgprB4, MrgprB5, MrgprC11 and MrgprD, have been reported involved in histamine-independent itch pathways in mice, with the expression of these Mrgprs restricted to small-diameter DRG neurons[4]. This study reports that these hiPSC-derived NC cells have the potential to differentiate into human peripheral sensory neurons that respond to itch-related stimuli

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