Profiling human iPSC-derived sensory neurons for analgesic drug screening using a multi-electrode array

Abstract

Chronic pain is a global health issue, yet effective treatments remain limited due to poor preclinical-to-human translation. To address this, we developed a high-content screening (HCS) platform using hiPSC-derived nociceptors to identify analgesics targeting the peripheral nervous system. These cells, cultured on multi-well microelectrode arrays, achieved nearly 100% active electrodes by week 2, maintaining stable activity for at least 2 weeks. After 28 days, we assessed drug effects on neuronal activity, achieving strong assay performance (robust Z' > 0.5). Pharmacological tests confirmed responses to key analgesic targets, including ion channels (Nav, Cav, Kv, and TRPV1), neurotransmitter receptors (AMPAR and GABA-R), and kinase inhibitors (tyrosine and JAK1/2). Transcriptomic analysis validated target expression, though levels differed from primary human DRG cells. The platform was used to screen over 700 natural compounds, demonstrating its potential for analgesic discovery. This HCS platform facilitates the rapid discovery of uncharacterized analgesics, reducing preclinical-to-human translation failure.

Keywords: CP: Stem cell; DRG; analgesic discovery; chronic pain; hiPSC; high-content screening; human induced pluripotent stem cell; nociceptor.

Graphical abstract

Figure 1

hiPSC nociceptor growth, neural activity, Z′, and robust Z′ factors on a 48-well MEA plate hiPSC nociceptors were cultured on a 48-well microelectrode array (MEA) plate for 4 weeks. (A) Representative micrographs of hiPSC nociceptors spot seeded at different cell densities (2,000, 35,000, and 65,000 cells/well) and two time points (day 2 and day 28). These micrographs were imaged at 100× magnification. (B–E) Across the 4 weeks of culture, cells were evaluated for (B) total spike count per well; (C) mean firing rate per well; (D) impedance, a measure of cell viability of hiPSC nociceptors; and (E) active electrode yield (AEY), indicating the percentage of electrodes successfully recording neural activity. At the 28 day time point, cells were challenged with inhibitors, and the Z′ and robust Z′ factors were calculated to measure the quality of the assay. (F and G) Z′ (F) and robust Z′ (G) factors were calculated at different densities, showing high quality with the robust Z′ factor at several seeding densities. (H) Scatterplots comparing well total spike count data before (blue) and 2 h post treatment with 500 μM lidocaine (pink) for a representative density of 35,000 cells/well. (I) Normal quantile-quantile (QQ) plot assessing the normality of data distribution before and after lidocaine treatment (five wells for each density). Next, two independent experiments were conducted to evaluate the Z′ and robust Z′ of the assay to three neuronal inhibitors (lidocaine, tetrodotoxin [TTX], and huwentoxin [HWTX]) for cells at 28 days in culture at 35,000 cells/well. (J and K) Z′ factor (J) and the robust Z′ values (K) for different inhibitors in experiment 1 (12 wells for each inhibitor). (L and M) Z′ factor (L) and the robust Z′ values (M) for different blockers in experiment 2 using 35,000/well (12 wells for each inhibitor). 0.5 dotted lines represent the Z′ threshold for a “good” quality assay. Data from these experiments represent three distinct sets of experiments, including two confirmatory experiments as shown. Data from (B)–(E) are presented as mean ± SEM (standard error of the mean).

Figure 2

Inhibitory effects of sodium channel blockers on hiPSC nociceptor activity The inhibitory effects of various sodium channel blockers on the activity of hiPSC nociceptors were assessed after 28 days of culture. (A)–(C), (D)–(F), (G)–(I), and (J)–(L) represent data obtained using tetrodotoxin (TTX, a Nav1.7 blocker), huwentoxin-IV (HWTX, a Nav1.7 blocker), GX201 (a Nav1.7 blocker), and A887826 (a Nav1.8 blocker), respectively. For each blocker, the percentage of inhibition of neuronal activity at 37°C and 47°C is shown (A), (B), (D), (E), (G), (H), (J), and (K) and was calculated relative to 37°C or 42°C baseline, respectively. Additionally, the spike rate of hiPSC nociceptors was monitored for 10 min before and after treatment with the respective blocker (C), (F), (I), and (L) at 37°C to evaluate acute effects (6 wells for each concentration). Pilot studies were conducted to determine the effective concentration range. Data are presented as mean ± SEM (standard error of the mean). See also Table S1 for statistical comparisons and Figure S6 for relevant gene expression for these targets.

Figure 3

Effects of calcium channel blockers and potassium channel openers on hiPSC nociceptor activity The inhibitory effects of various drugs on the activity of hiPSC nociceptors were assessed after 28 days of culture. (A)–(C) and (D)–(F) represent data obtained using mibefradil (a T-type Ca²⁺ blocker) and nifedipine (an L-type Ca²⁺ blocker), respectively. (G)–(I) and (J)–(L) represent data obtained using retigabine (a Kv7 opener) and diazoxide (a K-ATP channel opener), respectively. For each blocker or activator, the percentage of inhibition of neuronal activity at 37°C and 47°C is shown (A), (B), (D), (E), (G), (H), (J), and (K) and was calculated relative to 37°C or 42°C baseline, respectively. Additionally, the spike rate of hiPSC nociceptors was monitored for 10 min before and after treatment with the respective blocker (C), (F), (I), and (L) at 37°C to evaluate acute effects (6 wells for each concentration). Pilot studies were conducted to determine the effective concentration range. Data are presented as mean ± SEM (standard error of the mean). See also Table S1 for statistical comparisons and Figure S6 for relevant gene expression for these targets.

Figure 4

Effects of AMPA/kainite receptor antagonism, TRPV1 channel inhibition, and GABA receptor activation on hiPSC nociceptor activity The inhibition of glutamate AMPA/kainite receptors and TRPV1 channels and the activation of GABA receptors on the activity of hiPSC nociceptors were assessed after 28 days of culture. (A)–(C), (D)–(F), and (G)–(I) represent data obtained using DNQX (an AMPA/kainate antagonist), capsazepine (a TRPV1 antagonist), and GABA (a pan-GABA receptor agonist), respectively. For each drug, the percentage of inhibition of neuronal activity at 37°C and 47°C is shown (A), (B), (D), (E), (G), and (H) and was calculated relative to 37°C or 42°C baseline, respectively. Additionally, the spike rate of hiPSC nociceptors was monitored for 10 min before and after treatment with the respective blocker (C), (F), and (I) at 37°C to evaluate acute effects (6 wells for each concentration). Pilot studies were conducted to determine the effective concentration range. Data are presented as mean ± SEM (standard error of the mean). See also Table S1 for statistical comparisons and Figures S6 and S7 for relevant gene expression for these targets.

Figure 5

Inhibitory effects of kinase inhibitors and atypical analgesics on hiPSC nociceptors The inhibitory impact of various kinase inhibitors as well as atypical serotonin-norepinephrine reuptake inhibitor (SNRI) and tricyclic antidepressant (TCA) analgesics on the activity of hiPSC nociceptors following a 28-day culture period is evaluated. (A)–(C) and (D)–(F) represent data obtained using dasatinib (a multiple tyrosine kinase inhibitor) and baricitinib (a JAK1/2 inhibitor), respectively. (G)–(I) and (J)–(L) represent data obtained using duloxetine (SNRI) and amitriptyline (TCA), respectively. For each inhibitor, the percentage of inhibition of neuronal activity at 37°C and 47°C is shown (A), (B), (D), (E), (G), (H), (J), and (K) and was calculated relative to 37°C or 42°C baseline, respectively. Additionally, the spike rate of hiPSC nociceptors was monitored for 10 min before and after treatment with the respective blocker (C), (F), (I), and (L) at 37°C to evaluate acute effects (6 wells for each concentration). Pilot studies were conducted to determine the effective concentration range. Data are presented as mean ± SEM (standard error of the mean). See also Table S1 for statistical comparisons and Figure S7 for relevant gene expression for these targets.

Figure 6

Functional channels, receptors, and kinases in hiPSC nociceptors at 37°C and during 42°C heat ramps Each time point indicates peak drug efficacy at (A) 37°C or (B) during 42°C heat ramps. ns indicates the maximum point for non-significant treatments. Negative changes from baseline show inhibition of firing.