Abstract
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 in vitro 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 via 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 (NGN2iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2iSN 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.
Highlights
Basic sensory experiences such as pain, temperature, pressure, and spatial positioning require specialized sensory neurons within the peripheral nervous system to detect and transmit stimuli to the central nervous system for processing
Ion channels have a critical role in the development and progression of peripheral neuropathies strides have been made towards the development of cancer therapies aimed at curtailing excitability remodeling and neurotoxicity (Keenan et al, 2020)
A significant proportion of NGN2 derived sensory neuron (NGN2iSN) derived from the described protocol was positive for the pan-sensory neuron markers BRN3A and ISLET1 (∼80–90% positive), which is notably higher than previous protocols not implementing NGN2 expression in neural crest (NC) cells (∼5% BRN3A+/ISLET1+ cells; Alshawaf et al, 2018)
Summary
Basic sensory experiences such as pain, temperature, pressure, and spatial positioning require specialized sensory neurons within the peripheral nervous system to detect and transmit stimuli to the central nervous system for processing. Dysfunction, and damage to sensory neurons can alter the expression, distribution, density, and conductance of membrane ion channels These changes can dramatically alter action potential (AP) transduction producing changes in the excitability of specific sensory neurons resulting in the symptoms of peripheral neuropathies (Rasband et al, 2001; Chaplan et al, 2003; Chen et al, 2009; Zhang and Dougherty, 2014; Pitake et al, 2019). Ion channels have a critical role in the development and progression of peripheral neuropathies strides have been made towards the development of cancer therapies aimed at curtailing excitability remodeling and neurotoxicity (Keenan et al, 2020)
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