Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons

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.

Keywords: NGN2; dorsal root ganglia; electrophysiology; excitability; human sensory neurons; ion channels; pluripotent stem cells.

Figure 1

Generation of hPSC-derived Neural crest (NC) cells and _NGN2iSN. (A) Schematic of the protocol to derive _NGN2iSNs. Briefly, the protocol involves the formation of caudal neural progenitors (days 1–5) and neurospheres (days 5–12). Neurospheres are plated down onto a monolayer (day 12) and then removed after 48 h (day 14), to enrich for NC cells. NC cells are transduced with the NGN2 and reverse tetracycline transactivator lentiviruses (day 14) and NGN2 expression is induced for 96 h (days 15–19) and successfully transduced cells are obtained via puromycin selection (days 17–19). Finally, proliferating cells are removed using an antimitotic agent (AraC; days 24–26) and the _NGN2iSN are sampled for staining and RT-qPCR (day 34) or patch clamping (day 34–44). (B–C) Plating neurospheres for 48 h using the above protocol enriches for SOX10+ and P75^NTR+ NC cells. Neurospheres were either disaggregated into single cells or plated as whole spheres in NM supplemented with 10 μM Y-27632 for 24 h or 48 h. (B) Representative immunocytochemistry images of the NC markers P75^NTR (green) and SOX10 (pink). (C) The percentage of SOX10 positive cells, n = 3 biological replicates, >300 cells counted per biological replicate, error bars presented as SEM, **p < 0.01, ***p < 0.001.

Figure 2

Protein and mRNA expression of sensory neuron markers in _NGN2iSNs. (A) _NGN2iSN was positive for the protein expression of the neuronal marker ß-III-TUBULIN (green) and the sensory neuron markers BRN3A (green), ISLET1 (red), TRKA (red), TRKB (red), and TRKC (red), nuclei shown in blue. (B) The percentage of neurons expressing either BRN3A or ISLET1, n = 4 biological replicates, >200 cells counted per biological replicate. (C) Fold change in mRNA expression of POU4F1, ISL1, and PRPH normalized the GFP control. (D) Relative mRNA expression of NTRK1, NTRK2, NTRK3 (normalized to the house-keeping genes B2M, PPIA, and GAPDH), error bars presented as SEM, n = 5 biological replicates.

Figure 3

Excitability profile of _NGN2iSN. (A) Current clamp recording of a 17 pF _NGN2iSN. Representative membrane potential voltage responses recorded under current-clamp conditions elicited by progressive current injections (from −60 to +180 pA, Δ 10 pA, 500 ms). Traces corresponding to rheobase and 2× rheobase are highlighted in red and orange, respectively. Inset: first derivative of the trace at rheobase. (B–F) Summary of excitability properties: resting membrane potential (RPM, mV, B), rheobase (pA, C), number of fired action potential (APs at 2× rheobase, D), AP width (at 0 mV, ms, E), and AP firing rate (Hz, F). (G) Potassium-channel mediated membrane hyperpolarization upon current injections (0 to −55 pA, Δ 5 pA, 500 ms). The membrane potential traces in response to −55 pA current injection is highlighted in red. (H) Bar graph showing mV change in peak vs. steady-state current amplitude after a 55 pA hyperpolarizing pulse. (I) Relative mRNA expression of HCN1-4 transcripts detected in _NGN2iSN (normalized to the house-keeping genes B2M, PPIA, and GAPDH), error bars presented as SEM, n = 3 biological replicates.

Figure 4

Voltage-gated sodium currents in _NGN2iSN. Sodium currents (I_Na) from _NGN2iSN are predominantly mediated by TTX-S Nav channels. (A,B) Representative I_Na current traces in control (A) and the presence of 300 nM TTX (B) elicited by the voltage protocol shown in the inset. (C) _NGN2iSN sodium current (I_Na) density upon depolarization to −10 mV (highlighted in red in A and B) calculated from peak total and TTX-R I_Na current in nA/pF. (D) Voltage- dependence of activation (filled symbols) and steady-state inactivation (SSI; empty symbols) of the total (●, ○) and TTX-R (300 nM TTX, ▪, □) components of I_Na. (E) Relative mRNA expression of SCN9A and SCN10A transcripts in _NGN2iSN (normalized to the house-keeping genes B2M, PPIA, and GAPDH), error bars presented as SEM, n = 3–4 biological replicates. (F) Immunoreactivity against human Nav1.7 and Nav1.8 antibodies.

Figure 5

Voltage-gated potassium currents in _NGN2iSN. (A,B) Representative delayed rectifier potassium currents (I_K) recorded in a 14.56 pF cell upon standard activation (inset in A) and SSI (inset in B) protocols. (C) I_K current density (pA/pF) quantification. (D) Voltage dependence of activation (▪) and SSI (□) of I_K. (E,F) The molecular identity of _NGN2iSN I_K. (E) Stacked bar plots of 14 different _NGN2iSNs showing the fraction of total outward I_K current inhibited by inhibitors of Kv1 ( 300 nM RIIIJ and 10 nM Urotoxin), Kv2 ( 300 nM GxTx), Kv3 ( 100 μM 4-AP), and Kv4 ( 1 μM AmmTx3) channels. The fraction of I_K remaining in the presence of all inhibitors is shown in gray ( Res). (F) Relative mRNA expression of the transcripts encoding the major Kv channels mediating I_K in sensory neurons (KCNA2, KCNB1, and KCNMA1). The inset displays cell-attached single-channel recordings of a large conductance potassium channel consistent with the functional expression of BK (~500 nM [Ca²⁺]_i, pulse to +80 mV, scale bars: 4 pA, 500 ms, o, open; c, closed).

Figure 6

Voltage-gated calcium currents (I_Ca) present in _NGN2iSN. _NGN2iSN I_Ca evidence contribution from high and low voltage-activated calcium channels (HVA and LVA, respectively). (A) Representative HVA at 0 mV (black) and LVA at −40 mV (gray) I_Ca traces. Stimulation protocol is shown in the inset (100 ms Vh −90 mV, 0.2 Hz). (B) Bar graph summarizing I_Ca current density (pA/pF) of the HVA and LVA mediated components. (C) Relative mRNA expression of transcripts encoding the major calcium channels mediating HVA (CACNA1A, CACNA1B) and LVA (CACNA1H) in _NGN2iSN (normalized to the house-keeping genes B2M, PPIA, and GAPDH), error bars presented as SEM, n = 3 biological replicates.

Figure 7

_NGN2iSN proton- activated currents. _NGN2iSN I_pH6.0 evidences contribution from ASIC channels. (A) Current trace at pH 7.4 (gray bar), representative inward currents upon rapid change to pH 6.0 drop (red bar) and pH 6.0 drop in the presence of 10 μM amiloride (dashed bar; V_h = −80 mV). (B) Bar graph summarizing I_pH6.0 current density (pA/pF) of the total (black, n = 13) and the 10 μM amiloride-sensitive current (red, n = 6).