iPSC-derived human sensory neurons reveal a subset of TRPV1 antagonists as anti-pruritic compounds

Given the inaccessibility of human sensory neurons (SNs), it is yet to be established whether the signalling pathway between histamine 1 receptor (H1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) is conserved between humans and mammalian models. Accessible human SNs are vital for identifying TRPV1 antagonists with higher potential for success in clinical trials targeting histaminergic itch, especially given TRPV1's species specificity concerns. Hence to build a humanized histamine-dependent itch model, we derived peripheral sensory neurons from human pluripotent stem cells (hiPSC-SNs) and validated channel functionality using immunostaining, calcium imaging and multielectrode array (MEA) recordings. Here, we demonstrated that a subset of hiPSC-SNs exhibits co-expression of H1R and TRPV1, responding to both histamine and capsaicin agonists. We found that inhibiting TRPV1 prevented histamine activation. Moreover, we show that silencing histamine-sensitive neurons reduces capsaicin response, and silencing capsaicin-sensitive neurons diminishes histamine response. To assess the ability of hiPSC-SNs in TRPV1 antagonist drug screening, we evaluated two well-established hyperthermic and three thermal-neutral TRPV1 antagonists. Our finding identifies SB366791, a thermal-neutral antagonist, as a potent inhibitor of H1R activation. The use of hiPSC-SNs may therefore provide a physiologically relevant means to perform large-scale screening to discover anti-pruritic agents predictive of actual efficacy in human clinical trials. 46-day old hiPSC-SNs were dissociated using papain and analyzed using scRNA-seq.

鉴于人类感觉神经元（human sensory neurons, SNs）难以获取，目前尚无法确定组胺H1受体（histamine 1 receptor, H1R）与瞬时受体电位阳离子通道V亚家族成员1（transient receptor potential cation channel subfamily V member 1, TRPV1）之间的信号通路在人类与哺乳动物模型中是否保守。可获取的人类SNs对于筛选在靶向组胺能瘙痒的临床试验中更具成功潜力的TRPV1拮抗剂至关重要，尤其是考虑到TRPV1存在物种特异性相关问题。因此，为构建人源化组胺依赖性瘙痒模型，我们从人类多能干细胞中诱导得到外周感觉神经元（hiPSC-SNs），并通过免疫染色、钙成像及多电极阵列（multielectrode array, MEA）记录验证了通道功能。本研究证实，一部分hiPSC-SNs可共表达H1R与TRPV1，能够对组胺和辣椒素激动剂产生应答。我们发现抑制TRPV1可阻断组胺介导的激活。此外，我们还证实，沉默组胺敏感神经元会减弱辣椒素应答，而沉默辣椒素敏感神经元则会削弱组胺应答。为评估hiPSC-SNs用于TRPV1拮抗剂药物筛选的能力，我们评测了2种经典的热敏型与3种热非热敏型TRPV1拮抗剂。本研究发现SB366791——一种热非热敏型拮抗剂——可强效抑制H1R介导的激活。因此，利用hiPSC-SNs或许可提供一种生理相关的实验手段，开展大规模筛选以发现可预测人类临床试验实际疗效的抗瘙痒药物。我们使用木瓜蛋白酶解离培养46天的hiPSC-SNs，并通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）完成了分析。