Hulme Amy J, McArthur Jeffrey R, Maksour Simon, Miellet Sara, Ooi Lezanne, Adams David J, Finol-Urdaneta Rocio K, Dottori Mirella
Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia.
School of Medicine, University of Wollongong, Wollongong, NSW, Australia.
Front Cell Neurosci. 2020 Dec 4;14:600895. doi: 10.3389/fncel.2020.600895. eCollection 2020.
Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons (iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.
感觉知觉是日常生活的基础,但由于组织可用性的限制,在分子水平上对人类感觉生理学的理解受到阻碍。研究和表征周围神经病变的新兴策略涉及使用分化为背根神经节(DRG)感觉神经元的人类多能干细胞(hPSC)。然而,神经元的功能和成熟度有限且未得到充分探索。一种将hPSC分化为功能成熟神经元的最新且有前景的方法涉及Neurogenin-2(NGN2)的外源表达。这里描述的优化方案通过病毒诱导的NGN2表达从hPSC来源的神经嵴(NC)祖细胞生成感觉神经元。NC细胞通过小分子抑制剂方法从hPSC中获得,并富集迁移的NC细胞(66% SOX10+细胞)。在蛋白质和转录水平上,由此产生的NGN2诱导感觉神经元(iSNs)表达感觉神经元标记物,如BRN3A(82% BRN3A+细胞)、ISLET1(91% ISLET1+细胞)、TRKA、TRKB和TRKC。重要的是,iSNs通过电压门控钠、钾和钙电导重复激发动作电位(APs)。对iSN兴奋性分子基础的深入分析揭示了与初级传入神经元兴奋性相关的离子通道的功能性表达,如Nav1.7、Nav1.8、Kv1.2、Kv2.1、BK、Cav2.1、Cav2.2、Cav3.2、ASICs和HCN等其他离子通道,我们为此提供了功能和转录证据。我们对干细胞来源的感觉神经元的表征揭示了人类感觉生理学的分子基础,并突出了使用hPSC来源的感觉神经元来模拟人类DRG发育的适用性及其在人类周围神经病变和药物治疗研究中的潜力。