Transcriptomic analysis and high throughput functional characterization of human induced pluripotent stem cell derived sensory neurons (bulk RNA-Seq). Transcriptomic analysis and high throughput functional characterization of human induced pluripotent stem cell derived sensory neurons (bulk RNA-Seq)

Peripheral sensory neurons are a primary effector in pain neurotransmission, and have become a useful cellular model for the study of pain. While rodent tissue has historically served as a source of these neurons, it has become increasingly clear that pain mechanisms in rodents and humans are substantially divergent. Sensory neurons harvested from cadaveric human tissue serve as a superior translational model for studying pain mechanisms, however their relative paucity limits their widespread utility. Theoretically, sensory neurons manufactured from human pluripotent stem cells (hPSCs) could help bridge this translational gap given their relative abundance and potential similarity to primary human tissue. However, hPSC-derived sensory neurons manufactured with the most common methodologies correlate poorly to human tissue both transcriptionally and functionally. In the present work, we compare a novel population of hPSC-derived sensory neurons to previously published datasets and find this novel population to more closely resemble human primary dorsal root ganglia transcriptionally. Furthermore, we evaluate the heterogeneity of this novel population via single nucleus RNA sequencing and find it resembles specific nociceptor and mechanoreceptor subsets found in vivo. Finally, we assay the functionality of this population with high throughput automated patch clamp electrophysiology with respect to voltage-gated sodium (Nav) and potassium channels (Kv), and ligand-gated ionotropic GABA and P2X receptors. Overall, we find this population of hPSC-derived sensory neurons to be of relatively high fidelity, and suitable for interrogating numerous potential pain targets on a fully humanized platform. Overall design: Three lots of RealDRG manufactured from the same hiPSC line were thawed in Senso-MM (Anatomic Incorporated, #1030) at 100,000 neurons/cm2 in T25 format. Total RNA was collected at weekly timepoints for each line through four weeks.

外周感觉神经元（peripheral sensory neurons）是疼痛神经传递（pain neurotransmission）的核心效应器，现已成为疼痛研究的重要细胞模型（cellular model）。长期以来，啮齿类动物组织（rodent tissue）一直是这类神经元的主要来源，但越来越多的研究表明，啮齿类与人类的疼痛机制存在显著差异。从尸体人类组织（cadaveric human tissue）中获取的感觉神经元是研究疼痛机制的更优质转化模型（translational model），但其相对稀缺性极大限制了其广泛应用。理论上，由人多能干细胞（human pluripotent stem cells, hPSCs）诱导生成的感觉神经元，凭借其相对充足的供给量以及与原代人类组织的潜在相似性，有望填补这一转化鸿沟（translational gap）。然而，当前主流技术体系诱导得到的hPSC来源感觉神经元，在转录组学与功能层面均与人类原代组织相关性较差。

在本研究中，我们将一种新型hPSC来源感觉神经元群体与已发表的数据集进行比对，发现该群体在转录组学层面更贴近人类原代背根神经节（dorsal root ganglia）。此外，我们通过单细胞核RNA测序（single nucleus RNA sequencing）解析了该新型群体的异质性，发现其与体内存在的特定伤害性感受器（nociceptor）和机械感受器（mechanoreceptor）亚型高度相似。最后，我们采用高通量自动化膜片钳电生理技术（high throughput automated patch clamp electrophysiology），针对电压门控钠（Nav）通道、钾（Kv）通道，以及配体门控离子型GABA受体与P2X受体，对该群体的功能进行了检测。

总体而言，本研究证实该hPSC来源感觉神经元群体具有较高的保真度，适用于在完全人源化平台上探究各类潜在疼痛靶点。

总体实验设计：将三批由同一人类诱导多能干细胞（human induced pluripotent stem cells, hiPSC）系诱导生成的RealDRG细胞，以100,000个神经元/cm²的密度接种于T25培养瓶中，使用Senso-MM（Anatomic Incorporated，货号#1030）进行复苏培养。每株细胞系在复苏后的四周内，于每周时间点收集总RNA。