Sickle cell disease iPSC-derived sensory neurons exhibit increased excitability and sensitization to patient plasma Free

Sickle cell disease (SCD), the most common inherited hemoglobinopathy, affects >100 000 Black and Hispanic Americans,^1-3 and >3 million individuals globally.^1-3 SCD pathophysiology includes red blood cell sickling, chronic hemolysis, vaso-occlusion with resultant ischemia-reperfusion injury, and chronic vascular inflammation, leading to multiorgan damage and premature death. Individuals with SCD often suffer from both acute^4-8 and chronic (steady state) pain^9-13 that cannot be explained by known SCD pathology.^13-16 Opioids are the main analgesic for acute and chronic SCD pain; however, they are fraught with serious aversive side effects^16-19 and often do not provide effective analgesia. SCD disproportionately affects Black individuals who also experience multifactorial health care disparities including reduced likelihood for pain treatment.^20-22 These disparities, the current opioid crisis, and ineffective analgesia of opioids, create a critical need for effective nonopioid SCD pain therapies. To accomplish this goal, improved understanding of the neurobiological mechanisms underlying human SCD pain is needed.

Humanized SCD mouse models exhibit pronounced acute and chronic pain behaviors.^23-27 However, growing evidence indicates striking differences in gene expression, transcriptional regulators, and functional responsiveness between human and mouse dorsal root ganglia (DRG) sensory neurons.^28-36 Human DRGs may constitute a translational bridge between preclinical rodent models and clinical testing of targets.^33,36-40 However, obtaining DRGs from individuals with SCD is highly impractical because of rare clinical indications for DRG resection.

Human induced pluripotent stem cell (iPSC)-derived sensory neurons (iSNs) have recently been used to model chronic pain disorders.⁴¹ iSNs express canonical markers of human nociceptors^42-51 and exhibit functional responses to noxious stimuli.^47,49,51 Therefore, we differentiated iSNs from individuals with SCD and age- and race-matched healthy controls (HCs) and observed sensitization to electrical simulation and SCD plasma and plasma-derived factors in SCD iSNs compared with HC iSNs. Importantly, this study establishes a human-specific model of SCD and proposes the novel finding that both intrinsic and extrinsic factors may underly SCD pain.

We used 3 SCD lines and 3 age- and race-matched HC iPSC lines (WiCell, Madison, WI; supplemental Table 1, available on Blood website). SCD donors were physician diagnosed with hemoglobin-β (Hb-β [HBB]) HBB(E6V) sickle cell anemia (HbSS genotype). Karyotypically normal and mycoplasma-negative iPSCs were maintained on Matrigel (Corning) or Geltrex (Gibco) in Essential 8 medium (Gibco) and passaged every 4 to 6 days. iPSCs were differentiated into iSNs based on previous reports.^41,49 After dual SMAD inhibition (100 nM LDN 193189, 10 μM SB 431542, Selleck Chemical), SNs were patterned using 3 μM CHIR99021, 10 μM SU5402, and 10 μM DAPT (Selleck Chemical) starting on day 2 with a stepwise addition of N2 medium (25% Dulbecco modified Eagle medium, 25% F12, 50% neurobasal medium, 1× N2 supplement, 1× B27 supplement; ThermoFisher) every other day starting on day 4. On day 12, cells were dissociated using Accutase (ThermoFisher) and replated onto triple-coated (15 μg/mL poly-L-ornithine hydrobromide [Sigma], 2 μg/mL laminin [Fisher Scientific], and 2 μg/mL fibronectin [Fisher Scientific]) 96-well plates (20 000 cells per well), 6-well plates (480 000 cells per well), or glass coverslips (52 500 cells per coverslip) in 24-well plates and maintained in N2 media supplemented with neuronal growth factors (10 ng/mL human nerve growth factor β, neurotrophin-3, brain-derived neurotrophic factor, and Glial cell line–derived neurotrophic factor; PeproTech) throughout the duration of the experiments. iSNs were considered mature at day 35 and used experimentally between days 35 to 45 (supplemental Figure 1).

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and rinsed with phosphate-buffered saline (PBS). After blocking and permeabilization, cells were incubated with primary antibodies overnight at 4°C, then labeled with appropriate secondary antibodies (supplemental Table 2). Nuclei were labeled with DAPI (4′,6-diamidino-2-phenylindole). Representative images were taken on a Zeiss confocal microscope using a 20× air objective and are displayed as a maximum intensity projection of z-stack image series for Figure 1 and supplemental Figures 1 and 2.

RNA was isolated from HC and SCD iSNs using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions, quantified using a Nanodrop spectrophotometer, treated with RQ1 RNase-free DNase (Promega), and converted to complementary DNA using the Promega Reverse Transcription system (Promega). SYBR green quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in triplicate using complementary DNA and run on the Bio-Rad CFX384 thermocycler. Primer sequences are shown in supplemental Table 3. Cq values for each target were normalized to glyceraldehyde-3-phosphate dehydrogenase and calculated using the ΔΔCq method. A minimum of 3 differentiations for each line were collected and run in technical triplicates.

On day 12 of differentiation, cells were dissociated and plated onto PLL (poly-L-lysine)-laminin-fibronectin–coated 48-well CytoView microelectrode array plates (Axion Biosystems) at a density of 30 000 cells per well. Spontaneous and electrically evoked motor neuron activity was recorded every other day for 2 weeks starting from day 16 using the first-generation Maestro system (Axion Biosystems). Cells were maintained on a preheated (37°C) multielectrode array (MEA) stage for 10 minutes before starting the 4-minute recordings. Version 2.4.2.13 of the AxIS acquisition software (Axion Biosystems) was used to record activity across 16 electrodes per well with a sampling frequency of 12.5 kHz and a digital high-pass filter of 5 Hz IIR (infinite impulse response). The spike-detecting threshold of 5 spikes per minute was defined as voltage exceeding 6 standard deviations away from the mean background noise. Butterworth digital filter settings with high (200 Hz) and low (3 kHz) pass cutoff frequencies were applied during recordings. For electrically evoked recordings, the neural stimulation with artifact elimination (0.5 V for 400 μs [36 400 μs total operation time]) was applied to the cells by stimulating 1 electrode per well once every 10 seconds. Statistic Compiler files and waveform images were extracted from raw AxIS (spontaneous recordings) and raw AxIS Artifact Eliminator files (electrically evoked recordings) for weighted mean firing rates. Values <0.15 Hz were considered background noise, as previously determined⁵² and were removed from data sets. Analyses were performed using PENN022i-89-1 and CREM032i-SS48.

Coverslips were incubated in 2.5 μg/mL of the ratiometric dye Fura-2-AM in 2% bovine serum albumin for 45 minutes, followed by a 30-minute wash in extracellular normal HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffer (ENH) before imaging. Coverslips were mounted on a Nikon Eclipse TE200 inverted microscope and were superfused with ENH (pH 7.4 ± 0.03; 320 ± 3 mOsm; 150 mM NaCl, 10 mM HEPES, 8 mM glucose, 5.6 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂; Sigma Aldrich). Fluorescence images were obtained at 340 and 380 nm using Nikon Elements software (Nikon Instruments). Following a 1-minute baseline incubation in ENH, neurons were exposed to 100 μM glutamate (abcam) or 50 μM α,β-methyleneadenosine 5’-triphosphate trisodium salt (ab-meATP; Tocris) for 2 minutes; or 0.1 μM, 1 μM, 10 μM, or 50 μM capsaicin (Sigma Aldrich) for 30 seconds, ENH for 3 minutes, and 50 mM KCl (Sigma Aldrich) for 1 minute. All buffers were superfused at a rate of 6 mL/min. Neurons displaying a ≥20% increase in the 340:380 nm ratio relative to baseline in response to agonist treatment were included in the quantification.

Calcium flux was measured in iSNs seeded in a 96-well plate using the Fluo-4 NW Calcium Assay Kit (ThermoFisher) per the manufacturer’s instructions. Growth medium was removed, and 100 μL of dye loading solution was added to each well and incubated for 30 minutes at 37°C followed by 30 minutes at room temperature. Just before measuring fluorescence, 25 μL of the appropriate agonist or PBS were spiked into each well. Fluorescence (excitation, 494 nm; emission, 516 nm) was immediately measured using a GloMax microplate reader. No differences in fluorescence were found between preagonist background fluorescence and PBS-stimulated wells (supplemental Figure 3); relative fluorescence (% above PBS baseline) was calculated by subtracting the fluorescence of PBS-stimulated wells from each test well, then dividing by PBS-stimulated well value, and multiplying by 100. Agonist solutions were made fresh daily and included 250 mM KCl (final concentration, 50 mM); 500 μM L-glutamate (final concentration, 100 μM); 250 μM ab-meATP (final concentration, 50 μM); and 250 μM, 50 μM, 5 μM, or 0.5 μM capsaicin (final concentration, 50 μM, 10 μM, 1 μM, or 0.1 μM).

Human samples were collected under an institutional human research review board approved protocol. Written informed consent was obtained from the study participant and/or legal guardian, and written informed assent was obtained from the participant when age appropriate.

Human plasma samples, collected via routine venipuncture, were obtained from individuals with SCD during baseline health (SS BL) and during hospitalization for an acute pain event (SS IP) as paired samples from the same individual, and from healthy Black individuals without SCD (HCs; supplemental Table 4). Plasma was processed and immediately stored at −80°C. Plasma was thawed overnight at 4°C on ice before usage; freeze/thaw cycles were limited to 2. iSNs were treated with 10% plasma diluted in iSN maturation media. After a 30-minute treatment, all media were removed, and 100 μL of dye loading solution was immediately added for the Fluo-4 NW calcium flux assay.

SS BL with paired SS IP samples and race-matched and relatively age-matched HC plasma samples (supplemental Table 5; n = 25 per group) were analyzed by Metabolon, Inc, using its in-house methods for sample processing, ultrahigh performance liquid chromatography–tandem mass spectroscopy, quantification and data normalization, and statistical analyses (Welch 2-sample t tests and matched-pairs t tests).

iSNs were treated with 100 ng/mL recombinant human endothelin 1 (ET1; R&D) or C-C motif chemokine ligand 2 (CCL2; PeproTech) diluted in iSN maturation media. After a 30-minute treatment, cells were immediately processed for the Fluo-4 NW calcium flux assay. Recombinant human protein treatments (ET1 and CCL2) were performed on only PENN022i-89-1 and CREM032i-SS48-1 lines in biological triplicates by incubating iSNs with 100 μM endothelin receptor type A (ETRa) antagonist BQ123 (Tocris) or 100 μM ETRb antagonist BQ788 (Tocris) diluted in iSN maturation media for 24 hours at 37°C before 30-minute ET1 or plasma treatment. Finally, 30-minute spermine treatments were performed by treating iSNs with 100 nM, 100 μM, or 500 μM spermine (Sigma) diluted in iSN maturation media for 30 minutes at 37°C before the Fluo-4 NW calcium flux assay.

Whole-cell patch clamp recordings in current-clamp mode were performed on the PENN022i-89-1 and CREM032i-SS48 iSNs. iSNs were continuously superfused with extracellular solution: 140 mM NaCl, 2.8 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, and 8.8 mM sucrose (pH 7.4 and 310 mOsm). Patch electrodes were made from borosilicate glass capillaries with 4-8MΩ resistance when filled with internal solution: 135 mM KCl, 4.1 mM MgCl2, 2 mM EGTA, 0.2 mM NaGTP, 2.5 mM ATPNa2, and 10 mM HEPES (pH 7.2 and 290 mOsm). Recordings we performed at room temperature using either EPC10 amplifier with Patchmaster software (HEKA Electronics) or Axopatch 200B amplifier with pCLAMP software (Molecular Devices). Cells were held at −60 mV, and neuronal excitability was examined using a series of 500-ms depolarizing current pulses (range, 0-190 pA; 10-pA increments).

Experiments were performed in technical triplicates for a minimum of 3 independent experiments unless otherwise noted. Data were analyzed using GraphPad Prism software, and the appropriate statistical tests were performed including regression and outlier removal (ROUT) analysis to remove statistical outliers, Student t test, Welch t test, paired 2-sample t test, 1-way analysis of variance (ANOVA), and 2-way ANOVA followed by Tukey post hoc analysis of significance. Changes were considered statistically significant when P < .05.

Three SCD iPSC lines and 3 age- and race-matched HC iPSC lines were differentiated into iSNs following published protocols^41,49 (Figure 1A; supplemental Figures 1 and 2). HC and SCD iSNs showed similar transcript expression levels for the mature neuron marker neurofilament heavy chain (NF200) and the sensory neuron marker calcitonin gene-related peptide as measured by qRT-PCR (Figure 1B). Immunocytochemistry confirmed appropriate neuronal morphology and expression of calcitonin gene-related peptide (CGRP), type III intermediate filament protein (peripherin), and transient receptor potential cation channel subfamily V member 1 (TRPV1; Figure 1C-D) with virtually no expression of the glial marker glial acidic fibrillary protein or proliferative marker Ki67 (supplemental Figures 1 and 2). MEA recordings detected spontaneous and electrically evoked action potentials with no differences in weighted mean firing rate between HC and SCD iSNs (Figure 1E-F) indicating iSN maturation.

Next, we used qRT-PCR to assess expression of sensory neuron and pain-relevant ligands and receptors (Table 1) in healthy and disease iSN (Table 2). We found no significant differences in cytokine receptor transcripts, including CCL2, despite their involvement in peripheral immune response and inflammatory pain,^53-57 or in receptors specific for peripheral immune response and vasoconstriction including ETRa and ETRb (Table 2). Similarly, we found no difference in expression levels between HC and SCD iSNs for nociceptor-relevant ion channels (Table 2) and ion channels potentially involved in sensitization of sensory neurons during injury (Table 2). These findings indicate that SCD iPSCs and the age- and race-matched HCs can be consistently differentiated into iSNs expressing canonical and functional sensory neuron markers.

Next, we tested calcium responses in HC and SCD iSNs to 50 mM KCl, 100 μM glutamate, and 50 μM ab-meATP using ratiometric calcium imaging analysis (Figure 2A-B). No differences in percentage of iSNs responding or magnitude of response were found between the HC and SCD iSN (χ², 1-way ANOVA, P > .05). Previous studies had challenges recording calcium responses to capsaicin of differentiated iSNs, with 1 μM concentrations resulting in 1% to 2% of cells responding,⁴⁹ and 10 μM concentrations resulting in 2% to 10% of cells responding.⁴¹ Likewise, we found very few calcium responders to 0.1 μM capsaicin (Figure 2C; 0.67%-5.26%) and 1 μM capsaicin (0.90%-6.45%), and more responses to 10 μM capsaicin (12.99%-14.55%). We did note lower peaks in Fura-2 ratios above baseline when comparing 1 μM responses (Figure 2D; 64% increase above baseline) with responses to 10 μM (396% above baseline) and 50 μM capsaicin (522% above baseline).

To better capture capsaicin responders, we used a calcium flux assay (Fluo4-NW) to measure total intracellular calcium in response to agonist treatment (Figure 2E). By measuring changes in the entire population of cells in each well, we captured calcium flux increases in response to 0.1 and 1 μM capsaicin in 40% to 50% of stimulated wells (Figure 2F, left), and consistently high response rates to 10 μM (88.9%) and 50 μM (90%) capsaicin. The magnitude of these responses, calculated as the percent increase of Fluo-4 fluorescence after agonist stimulation compared with background fluorescence measured before agonist (Figure 2F, right; supplemental Figure 4), increased in 10 μM (8.87%) and 50 μM (16.25%) capsaicin conditions compared with 0.1 μM (3.35%) and 1 μM (2.41%) capsaicin conditions. Using these data, we labeled 1 μM capsaicin as subthreshold, 10 μM capsaicin as low-concentration, and 50 μM capsaicin as high-concentration, for future experiments.

Using the Fluo4-NW assay, we again found no differences in HC and SCD iSN population-based response to 50 mM KCl (Figure 2G; t test, P > .05), 100 μM glutamate (Figure 2H; P > .05), or 50μ M ab-meATP (Figure 2I; P > .05). When comparing HC and SCD iSNs responses to 10 and 50 μM capsaicin, we found no differences in response based on disease state (Figure 2J-K; P > .05). These data confirm that the iSNs respond to stimulation with an increase in intracellular calcium associated with neuronal firing.

In SCD, a minimal blood nerve barrier at the level of DRGs becomes compromised and leaky,⁵⁸ thereby allowing circulating ligands access to receptors and ion channels on peripheral neuronal processes. Therefore, we treated HC and SCD iSNs with age- and race-matched plasma taken from healthy individuals without SCD (HC plasma, n = 4), individuals with SCD during baseline health (SS BL plasma, n = 4), and those same individuals with SCD during hospitalization for acute pain (SS IP, n = 4). Initially, we found no differences between the plasma-treated HC and SCD iSNs in response to 50 mM KCl (Figure 3A; supplemental Figure 3), 100 μM glutamate (Figure 3B), and 50 μM ab-meATP (Figure 3C; 2-way ANOVA, P > .05). However, SCD iSNs did show a significant increase in response to 10 μM capsaicin after SS IP plasma treatment compared with HC iSNs (Figure 3D; P = .0240). This difference was not present in the 50 μM capsaicin condition (Figure 3E; P > .05), suggesting we had reached a maximal threshold response. We also found that SS IP plasma increased SCD iSN response to 100 μM glutamate (Figure 3F, left; 2-way ANOVA, P = .0435) and 10 μM capsaicin (Figure 3F, right; P = .0266) compared with untreated SCD iSNs. HC iSNs did not show these same effects but did have increased response to 50 μM ab-meATP after SS IP plasma treatment compared with untreated SCD iSNs (P = .0405) and HC plasma–treated conditions (P = .0089). These data reveal possible intrinsic differences within SCD iSNs that may contribute to acute pain associated with vaso-occlusive events.

To further define intrinsic differences between SCD iSNs and HC iSNs, we used patch clamp electrophysiology and found stimulus-induced sensitization in SCD iSNs compared with HC iSNs. Current-evoked intrinsic excitability was substantially increased at 190 pA current injection for SCD iSNs relative to HC iSNs (Figure 3G-H), but there was no significant change in resting membrane potential (average ± standard error of the mean, HC iSNs: −61.1 ± 2.0 mV; SCD iSNs: −57.5 ± 3.0 mV). Together, these data show that SCD iSNs have altered intrinsic excitability compared with HC iSNs that can be further affected by extrinsic exposure to plasma-derived factors and may contribute to neuronal sensitivity and pain in SCD.

Because SCD is characterized by a mutation in the Hb gene and recent studies have discovered unexpected expression of Hb in neuronal cultures,^59-62 we measured transcript expression of the Hb-α (HBA) and Hb-β (HBB) chains. Both HBA and HBB transcripts were expressed at similar levels in HC and SCD iSNs (Figure 4A; ANOVA, P > .05). These transcript levels were also not differentially expressed after treatment with HC, SS BL, or SS IP plasma (Figure 4B; 2-way ANOVA, P > .05). Although there is evidence linking Hb expression in neurons to expression of genes associated with oxidative phosphorylation and mitochondrial function,⁵⁹ targets involved in protection against oxidative stress⁶³ were not significantly different between HC and SCD iSNs with or without plasma treatments (Figure 4C; 2-way ANOVA, P > .05).

Next, we hypothesized that other intrinsic differences between HC and SCD iSNs may contribute to altered calcium responses in SCD iSNs (Figure 4D). Previous studies have shown that plasma from individuals with SCD contains increased levels ET1.^64-69 Signaling of ET1 through increased ETRa receptor expression in SCD iSNs can contribute to decreased nitric oxide release^70,71 contributing to vasoconstriction,^72,73 increased intracellular calcium,^73,74 and increased expression of ion channels.^64,75-77 Moreover, increased CCL2 production by SCD iSNs can result in increased neuronal excitability,^78,79 ion channel expression and function,⁸⁰ release of glutamate,⁸¹ and recruitment of immune cells resulting in inflammation.^54,82 Therefore, we added 100 ng/mL of recombinant human CCL2 (Figure 4E) and ET1 (Figure 4F) to HC and SCD iSN cultures. ET1 significantly increased calcium flux of the SCD iSNs in response to 50 mM KCl (P = .0333), 100 μM glutamate (P = .0186), and 10 μM capsaicin (P = .0333) compared with HC iSNs. CCL2 did not induce these direct increases in calcium flux (P > .05), although it did increase the range of calcium responses of SCD iSN to glutamate and 10 μM capsaicin (Figure 4E).

To determine whether the effect of SS IP plasma on SCD iSNs associated with signaling through ETRa, we first pretreated iSNs with 100 μM of ETRa or ETRb antagonist for 24 hours before addition of ET1. As expected, both ETRa and ETRb inhibition significantly reduced the effects of ET1 on HC (Figure 4G; 2-way ANOVA, P < .0001) and SCD iSNs (Figure 4H; 2-way ANOVA, P < .0001) when stimulated with 10 μM capsaicin. We then applied HC, SS BL, and SS IP plasma onto HC (Figure 4I) and SCD iSNs (Figure 4J) pretreated with ETRa or ETRb antagonists. Neither ETRa nor ETRb inhibition significantly altered baseline HC iSN response to 10 μM capsaicin (Figure 4I; 2-way ANOVA, P > .05, P > .05), although ETRb inhibition did decrease HC calcium response to both SS BL (P = .0264) and SS IP plasma (P = .0220) compared with untreated HC iSNs. ETRa inhibition also decreased HC iSN response to SS IP plasma compared with untreated HC iSNs (P = .0425) but, surprisingly, did not significantly decrease HC iSN response to SS BL plasma (P > .05). Similarly, both ETRa and ETRb inhibition significantly reduced SCD iSN response to 10 μM capsaicin when treated with SS IP plasma (Figure 4J; 2-way ANOVA, P < .0001, P < .0001) compared with untreated iSNs. Unlike HC iSNs, inhibition of ETRa (P = .0405) and ETRb (P = .0265) did significantly reduce the effect of SS BL plasma on SCD iSN response to 10 μM capsaicin, although the range of responses was again greater when inhibiting ETRa compared with ETRb. Furthermore, ETRa inhibition significantly reduced responses of SCD iSNs to 10 μM capsaicin at baseline (P = .0386) and when treated with HC plasma (P = .0026), suggesting a role for this receptor in baseline sensitization of SCD iSNs.

Post-hoc analyses revealed variable calcium response levels and a significant number of SCD iSNs with abnormally high baseline calcium levels compared with HC iSNs (Figure 4K; P = .0024) supporting the idea that some SCD iSNs are residing in an abnormal state, thereby potentially contributing to increased sensitivity to circulating plasma factors during acute SCD pain.

Although ETR inhibition did suppress the influence of ET1 on iSN calcium flux, the effect of ET1 treatment was neither capsaicin specific nor SCD iSN specific (Figure 4I-J) and do not explain why SCD iSNs are particularly sensitized to 10 μM capsaicin by SS IP plasma (Figure 3D). Therefore, we compared the metabolomic profiles of HC, SS BL, and SS IP plasma samples (n = 25 per group, supplemental Table 5). We chose to focus on arginine metabolism pathways, because arginine is known to be altered in SCD and decreased levels are associated with increased pain in patients with SCD.^83-85 Spermidine is a polyamine produced as a metabolite of arginine metabolism through ornithine consumption⁸⁶ (Figure 5A). Both ornithine and urea were decreased in SS IP plasma (Figure 5B), which may be explained by increased consumption of ornithine for polyamine synthesis. In support of this, the polyamines spermidine, spermine, N(‘1)-acetylspermidine, N-acetylputrescine, and acisoga had significantly higher expression in SS BL and SS IP plasma than in HC plasma (Figure 5B-C; Welch t test, P < .05).

Spermine has been shown to directly activate TRPV1 and potently sensitize TRPV1 to capsaicin activation.⁸⁷ Therefore, we examined whether spermine pretreatment would sensitize iSNs to 10 μM or 50 μM capsaicin (Figure 5D). Treatment with 100 μM spermine significantly increased the HC iSN calcium response (2-way ANOVA, P = .0038) and induced a trending increase in the SCD iSNs (P = .0949) to 10 μM capsaicin. No increase occurred in the 50 μM capsaicin condition in HC (P > .05) or SCD iSNs (P > .05), suggesting a possible ceiling effect at this high capsaicin concentration. Strikingly, we found that, compared with HC iSNs, spermine treatment significantly increased the calcium response of SCD iSNs to the subthreshold concentration of 1 μM capsaicin (Figure 5E, 2-way ANOVA, P < .0001). Together, these data show that plasma-derived factors can sensitize SCD iSNs to capsaicin and may contribute to the neuronal sensitivity and pain in SCD.

SCD iPSCs have been used to study erythrocyte biology,^88-90 but here we used SCD iPSCs to study intrinsic and dynamic mechanisms of nociception in SCD. Our data confirm that iSNs are morphologically and functionally consistent with in vivo sensory neurons regarding channel and receptor expression⁹¹ and electrophysiological function.^37,39 Although we found no differences in marker expression or select transcripts between HC and SCD iSNs, a more unbiased RNA sequencing approach would be needed to fully assess potential differences between SCD and HC iSNs. Nevertheless, we did find that SCD iSNs had altered baseline calcium levels, increased excitability compared with HC iSNs, and were sensitized to nociceptive stimuli by patient-derived SCD plasma suggesting a novel finding that SCD iSNs have intrinsic alterations in excitability that may contribute to pain phenotypes.

Although ET-1 induces oxidative stress through both ETRa and ETRb signaling in other cell types,^92-94 we did not identify any changes in oxidative stress markers after exposure of HC and SCD iSNs to patient-derived SCD plasma. Additionally, although increased CCL2 is associated with glutamate signaling in sensory neurons,⁸¹ application of CCL2 increased response variability but did not lead to significant sensitization of SCD iSNs response compared with HC iSN responses. These pathways may be more driven by extrinsic factors in vivo or require interactions of sensory neurons with other cell types.

Beyond ET1 and CCL2, we hypothesized other factors in patient-derived SCD plasma sensitize iSNs. Our metabolomics data revealed several dysregulated factors involved in arginine and polyamine metabolism in patient-derived SCD plasma compared to plasma from HCs but also identified differences between baseline and acute pain state of patients with SCD. Further investigation into these metabolites could inform ongoing clinical trials seeking to reduce SCD pain through arginine supplementation.⁸⁵ We found increased SCD iSN response after spermine treatment to 1 μM capsaicin, a concentration that in untreated iSNs does not elicit increased intracellular calcium. This finding indicates an intrinsic mechanism by which SCD iSNs exhibit increased sensitivity to TRPV1 activation and support our previous findings that nociceptor TRPV1 is sensitized in mice with SCD.²⁴ Our metabolomic analysis focused on polyamines, but future experiments could assess both the plasma composition and transcriptional profiles of iSNs to determine additional mechanisms contributing to iSN sensitization.

The iSN platform has limitations, and in vivo studies may still be more appropriate to examine sensory neuron phenotypes or disease development across the lifespan. Although iSNs recapitulate phenotypes identified in mouse and primary human cultures, the concentration of capsaicin required to induce a robust calcium response in iSNs is higher than that required for primary DRG cultures in either mouse or human.³⁸ The purified nature of iSN cultures is an advantage to studying intrinsic cell-specific mechanisms of sensitization, but interactions with other cell types likely contribute to maturation and sensitization for native sensory neurons in vivo. Our iSN cultures do not appear to contain peripheral glia, but other non-capsaicin responsive neurons in the culture may contribute to the SCD iSN sensitivity. Future experiments could better investigate individual neuronal cell types or use established coculture techniques to grow iSNs with other relevant interacting cell types within the in vivo milieu. Because a limited number of SCD iPSC lines are available, as well as age- and race-matched controls for these lines, we cannot fully address patient heterogeneity or differences based on sex nor can we fully generalize these findings to all individuals with SCD. Nevertheless, these data are a foundation to assess molecular mechanisms in iSNs that may be relevant to pain neurobiology. Although SCD affects males and females equally, recent analyses show that females have significantly worse pain frequency and severity,⁹⁵ indicating that future in-depth analyses are necessary to provide additional mechanistic insight.

In summary, our data indicate that SCD iSNs exhibit intrinsic excitability and sensitization to external blood-derived stimuli compared with HC iSNs, which suggests a combination of central and peripheral mechanisms are involved in SCD pain.

The authors thank and acknowledge individuals with and without SCD who contributed to this study, and also thank Bhavya Dharanikota and Benjamin O’Brien for technical assistance with calcium imaging. Figures 2E and 4D, supplemental Figure 1, and the visual abstract were created with BioRender.com.

This work was supported by funding from the National Institutes of Health (NIH), National Institute of Neurological Disorders and Stroke (R01NS070711 [C.S.], R37NS108278 [C.S.]), NIH, National Heart, Lung, and Blood Institute (1K23HL114636-01A1 [A.M.B.], 1R01HL142657-01 [A.M.B.]), and Advancing a Healthier Wisconsin Endowment (A.D.E., C.S., and A.M.B.).

Contribution: R.L.A., E.W., V.E., A.B., and O.I. performed and analyzed experiments; J.H. coordinated plasma sample preparation and delivery; all authors designed experiments and interpreted data; A.M.B., C.S., and A.D.E. supervised the study and provided funding; R.L.A. wrote the manuscript; R.L.A., E.W., V.E., O.I., A.B., and D.N.T. created figures; and all authors edited and approved the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Allison D. Ebert, Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226; email: aebert@mcw.edu.