iPSC-derived human sensory neurons reveal a subset of TRPV1 antagonists as anti-pruritic compounds

Abstract

Signaling interplay between the histamine 1 receptor (H1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in mediating histaminergic itch has been well-established in mammalian models, but whether this is conserved in humans remains to be confirmed due to the difficulties in obtaining human sensory neurons (SNs) for experimentation. Additionally, previously reported species-specific differences in TRPV1 function indicate that use of human SNs is vital for drug candidate screening to have a higher chance of identifying clinically effective TRPV1 antagonists. In this study, we built a histamine-dependent itch model using peripheral SNs derived from human induced pluripotent stem cells (hiPSC-SNs), which provides an accessible source of human SNs for pre-clinical drug screening. We validated channel functionality using immunostaining, calcium imaging, and multielectrode array (MEA) recordings, and confirmed the interdependence of H1R and TRPV1 signalling in human SNs. We further tested the amenability of our model for pre-clinical studies by screening multiple TRPV1 antagonists in parallel, identifying SB366791 as a potent inhibitor of H1R activation and potential candidate for alleviating histaminergic itch. Notably, some of the results using our model corroborated with efficacy and side effect findings from human clinical trials, underscoring the importance of this species-specific platform. Taken together, our results present a robust in vitro human model for histaminergic itch, which can be used to further interrogate the molecular basis of human SN function as well as screen for TRPV1 activity-modifying compounds for a number of clinical indications.

Keywords: Histamine receptor; Human pluripotent stem cells; Itch; MEA; Sensory neurons; TRPV1; TRPV1 antagonist.

Fig. 1

Derivation of hiPSC-SNs and characterization of H1R and TRPV1 signalling elements. (A) Directed differentiation protocol to obtain Sensory neurons from human induced pluripotent stem cells (hiPSC-SNs) as early as within 28 days. (B,C) Identification of cell types present in 45-day old hiPSC-SN culture using single cell RNA-seq. UMAP plot showing the 8 clusters identified (B). UMAP plots of the expression of gene markers that were used to label neuronal clusters (STMN2 and NEFL) and Sensory neuron clusters (PRPH and ISL1) (C). (D) qPCR analysis in hiPSC-SNs showing upregulation of SN canonical markers, i.e. TRKA, PRPH, BRN3A and ISL1 relative to undifferentiated BJ-iPSC controls at day 15 post differentiation. Gene expression was normalized to ACTINB and GAPDH. N = 3 independent biological differentiations. (E) Immunostaining of hiPSC-SNs at various developmental stages exhibiting SN canonical markers, i.e. PRPH, BRN3A and ISL1. (F) qPCR analysis in hiPSC-SNs showing upregulation of H1R/TRPV1 signalling pathway elements, i.e. H1R, TRPV1, SCN9A and SCN11A relative to undifferentiated BJ-iPSC controls at day 28 post differentiation, respectively. Gene expression was normalized to ACTINB and GAPDH. N = 3 independent biological differentiations. (G) Immunostaining of hiPSC-SNs at day 50 demonstrating co-localization of H1R and TRPV1 elements.

Fig. 2

Functional characterization of H1R and TRPV1 in hiPSC-SNs. (A) Time trace of fluorescence change in calcium influxes within five representative hiPSC-SNs in response to either H1R agonist, i.e. histamine (His) or TRPV1 agonist, i.e. capsaicin (Cap). (B, C) Quantitation of the percentages of neurons responding to histamine or capsaicin in the presence of H1R antagonist, i.e. mepyramine (B) or TRPV1 antagonist, i.e. AMG9810 (C), respectively. N = 2–3 independent biological differentiations (colour coded as blue, red and orange spots). For each independent differentiation, 3–4 technical repeats (spots indicated in same colour) were performed with 60–100 neurons analyzed in each repeat. (D, E) Multielectrode array (MEA) measurements demonstrating an increase in hiPSC-SNs’ burst spike events in response to stimulation with histamine (D) or capsaicin (E). Hyperexcitabilities are reversibly suppressed in the presence of H1R antagonist, i.e. mepyramine (D) or TRPV1 antagonist, i.e. AMG9810 when tracked in the same group of neurons. N = 3 independent biological differentiations (colour coded as blue, red and orange spots). For each independent differentiation, all active electrode channels recording at multiple locations of a single hiPSC culture well are indicated as spots of the same colour. (F) Increase in hiPSC-SNs’ excitabilities/burst spike count correspond to noxious heat exposure from 37 to 42 °C (1–9 min) and gradually decreases as temperature cools back to 37 °C (9–20 min). Heat response is reversibly ameliorated in the presence of AMG9810. N = 3 independent biological differentiations but only one representative time course result is shown here. Data is shown as means ± SD. ^nsP > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired Student T-test (B, C) or Friedman test with Dunn’s multiple comparisons test (D, E).

Fig. 3

H1R signals through TRPV1 in a proportion of hiPSC-SNs. (A,B) Fluorescence images of calcium response at baseline and after applications of histamine and capsaicin (A) and pie chart showing the proportion of SNs that responded to either or both agonists (B). N = 6 independent biological differentiations. For each independent differentiation, 2–4 technical repeats were performed with 40–140 neurons analyzed in each repeat. (C–E) Histamine-mediated activation of H1R is inhibited in the presence of a TRPV1 antagonist, i.e. AMG9810 as shown in a MEA measurement (C), calcium imaging (D) and schematic illustration I. (F–H) Co-administration of histamine and sodium channel blocker, i.e. QX-314 followed by a subsequent stimulation with capsaicin reveals a suppression in capsaicin response in both MEA analysis (F), calcium imaging (G) and schematic illustration (H). (I–K) Co-administration of capsaicin and sodium channel blocker, i.e. QX-314 followed by a subsequent stimulation with histamine reveals a suppression in histamine response in both MEA analysis (I), calcium imaging (J) and schematic illustration (K). Data is shown as means ± SD. ^nsP > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, Friedman test with Dunn’s multiple comparisons test (C,F,I), Mann-Whitney test (D) or unpaired Student T-test (G, J). N = 1–3 independent biological differentiations (colour coded as blue, red and orange spots) in all experimental set-ups described in (C–K). For each independent differentiation described in MEA experiments (C,F,J), active electrode channels recording at multiple locations of a single hiPSC culture well are indicated as spots of the same colour. For each independent differentiation described in calcium experiments (D,G,J), 3–4 technical repeats (spots indicated in same colour) were performed with 35–150 neurons analyzed in each repeat.

Fig. 4

Efficacies of various TRPV1 antagonists to block capsaicin and histamine activations in hiPSC-SNs. (A–E) MEA based assessments of hyperthermia-inducing TRPV1 antagonists, i.e. AMG517 (A) and ABT102 (B) and hyperthermia-free TRPV1 antagonists, i.e. SB366791 (C), SB705498 (D) and PAC-14,028 (E) on TRPV1 blockage in capsaicin activation mode. (F–J) Effect of hyperthermia-inducing TRPV1 antagonists, i.e. AMG517 (F) and ABT102 (G) and hyperthermia-free TRPV1 antagonists, i.e. SB366791 (H), SB705498 (I) and PAC-14,028 (J) in blocking acute effects of histamine-mediated H1R activation. Data is shown as means ± SD. ^nsP > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, Friedman test with Dunn’s multiple comparisons test, N = 3 independent biological differentiations in all experimental set-ups (colour coded as blue, red and orange spots). For each independent differentiation, all active electrode channels recording at multiple locations of a single hiPSC culture well are indicated as spots of the same colour.