Investigating the roles of Hb, ETRa, and CCL2 in SCD iSN response to painful stimuli. (A) Unexpected transcript production for both Hbα (HBA) and Hbβ (HBB) subunits in iSNs show no significant differences in expression between HC and SCD iSNs. (B) Hb transcript levels do not change when iSNs are treated with HC, SS BL, or SS IP plasma. (C) No significant differences in transcripts to indicate oxidative stress (catalase, HMOX1, and Nqo1) at baseline or with plasma treatments in HC and SCD iSNs. (D) Schematic of proposed roles of ETRa and CCL2 in SCD pathogenesis using a human iPSC model and connections to previously published mechanisms. Findings established using this iSN model are shown in red; purple arrows are suggested connections to previously published data (gray boxes). Treatment of iSNs with 100 ng/mL recombinant human CCL2 did not induce sensitivity of iSNs to agonists but did increase variability of SCD iSN responses compared with HC iSNs (t tests, ns) (E), whereas treatment of iSNs with 100 ng/mL recombinant human ET1 induced significantly higher responses to 50 mM KCl, 100 μM glutamate, and 10 μM capsaicin in SCD iSNs than in HC iSNs (t tests, ∗P < .05 [F]). Pretreatment with 100 μM BQ123 (ETRa inhibitor) and 100 μM BQ788 (ETRb inhibitor) reduced the effect of ET1 when paired with 10 μM capsaicin agonism in both HC (G) and SCD iSNs (ANOVA, ∗∗∗∗P < .0001) (H). Pretreatment with both ETRa and ETRb inhibitors significantly reduce responses to 10 μM capsaicin in HC (I) and SCD iSNs (J) even with the addition of HC, SS BL, and SS IP plasma treatment (ANOVA, ∗P < .05, ∗∗P < .005, and ∗∗∗∗P < .0001). Specifically, the increased sensitivity of SCD iSNs to 10 μM capsaicin after SS IP plasma was significantly reduced by ETRa and ETRb inhibition (ANOVA, ∗∗∗∗P < .0001). (K) Post-hoc analyses reveal high degree of variability and abnormal baseline intracellular calcium levels in SCD iSNs compared with HCs (t test, ∗∗P < .01).