Abstract：
In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9000 cells were used to establish the \"fraction of reactive cells\" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.
摘要：
周围神经系统的体外模型将受益于进一步的改进，以更好地支持神经病变的研究。特别是,在人类细胞培养物中,疼痛相关信号的评估仍然很困难.这里,我们利用诱导多能干细胞(iPSCs)产生富含伤害感受器的外周感觉神经元.目的是产生具有适用于药理学和毒理学研究的信号传导端点的培养系统。通过常规分化方案产生的神经元表达中等水平的P2X3嘌呤能受体和只有低水平的TRPV1辣椒素受体，当成熟时间保持在6周的实际有用上限时。作为替代方法，我们用诱导型NGN1转基因产生细胞。在确定的分化时间窗口中，该转录因子的异位表达导致高度富集的伤害感受器培养物。如通过功能（P2X3和TRPV1受体）和免疫细胞化学表型确定，辅以广泛的转录组分析。来自>9000个细胞的Ca2+指示剂荧光的单细胞记录用于建立刺激群体中的“反应性细胞分数”作为实验终点，看起来很健壮，透明和可量化。为了提供一个应用于生物医学研究的例子，在非细胞毒性浓度下检查了长期暴露于化疗药物奥沙利铂的功能后果。我们发现（i）神经元（异常性疼痛样）对其他非激活性机械刺激的超敏反应，可被电压门控钠通道的调节剂阻断；（ii）对TRPV1受体刺激的高反应性。这些发现和一些其他测量的功能改变表明该模型适用于与周围神经病变相关的药理学和毒理学研究。
