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3677 Sickle Cell Disease Patient Plasma Sensitizes iPSC-Derived Sensory Neurons from Sickle Cell Disease Patients

Program: Oral and Poster Abstracts
Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Sickle Cell Disease, Hemoglobinopathies, Diseases, Technology and Procedures, molecular testing
Monday, December 12, 2022, 6:00 PM-8:00 PM

Reilly L. Allison1*, Anthony Burand, PhD2*, Amanda M. Brandow, DO, MS3, Cheryl Stucky, PhD2* and Allison D. Ebert, PhD2*

1Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Wauwatosa, WI
2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI
3Pediatric Hematology/Oncology, Medical College of Wisconsin and Children’s Research Institute of the Children’s Hospital of Wisconsin, Milwaukee, WI

Individuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. The underlying neurobiology of the complex pain of SCD is not well understood. Limited and often ineffective treatment options and multifactorial healthcare disparities create an immediate need to develop non-opioid based SCD pain therapies. However, in order to develop novel therapies, we must better understand the neurobiological mechanisms underlying SCD pain. Although humanized mouse models of SCD do exist, growing evidence indicates there are striking differences in gene expression and functional responsiveness between human and mouse dorsal root ganglia (DRG) sensory neurons. Human DRG tissues are extremely limited in availability, postmortem tissues only provide static, endpoint molecular signatures, and obtaining DRGs from patients with SCD is untenable. These are all barriers to gaining mechanistic insight into pathogenic pain processes.

Human induced pluripotent stem cell (iPSC)-derived sensory neurons (iSNs) have recently been used to model the altered function and expression of molecules in native DRG neurons in chronic pain disorders. These iPSC-derived SNs express canonical markers of human nociceptors and exhibit functional responses to noxious stimuli similar to those properties of native mammalian nociceptors. The objective of this study was to utilize iSNs differentiated from SCD patient iPSCs as well as age- and race-matched healthy controls (HCs) to characterize baseline receptor expression and function of SCD iSNs compared to HC iSNs. Our goal is to assess the intrinsic properties of SCD nociceptors that may contribute to the persistent sensitization of nociceptors in SCD patients with pain. To this end, both HC and SCD iSNs expressed transcripts for canonical markers of mature sensory neurons (NF200, CGRP), immune receptors (CCR1/2/3/5, CD4, CRLF2, CSF2Ra/b), and functional ion channels (HCN1, Nav1.7, P2X3/4, CACNA2D1, CHRNA9) (Table 1). Further, they expressed transcripts for ion channels relevant to SCD pain (Piezo1, Piezo2, TRPA1, TRPC5, TRPM3, TRPM8, TRPV1, TRPV4). We confirmed the expression of Peripherin, CGRP, TRPV1 at the protein level using immunocytochemistry. There were no significant differences in expression for any of these targets in iSNs derived from SCD patients compared to those from HCs. However, we did identify trends for higher endothelin 1 receptor type A (ETRa) and CC motif chemokine ligand 2 (CCL2) transcript production by SCD iSNs compared to HC iSNs (Table 1).

Additionally, we observed robust responses with no significant differences in baseline function between HC and SCD iSNs when stimulated with known agonists of human SNs (KCl, Glutamate, ab-meATP, capsaicin) in traditional or high-throughput calcium imaging studies. To begin defining extrinsic mechanisms that may sensitize human SCD SNs, we tested the effects of SCD patient plasma (n=4 from SCD baseline [SS BL] and acute pain [SS IP]- paired samples, n=4 Black controls [HC]) on iSN hypersensitivity by exposing iSNs in vitro to human plasma diluted 1:10 in maintenance media for 30 minutes before analyzing calcium flux. Plasma samples from SCD patients experiencing acute pain (SS IP) were found to significantly increase SCD iSN response to capsaicin (p<0.05) compared to HC iSNs treated with SS IP plasma, while race-matched HC plasma or SCD patient plasma at baseline pain states (SS BL) did not induce this effect (Figure 1).

Excitingly, these findings reproduce results identified in native DRG neurons from SCD mice and suggest that iSNs derived from patient iPSCs can be used in parallel with native SCD neurons from mouse models to investigate mechanisms underlying the chronic sensory neuron sensitization in SCD. They also identify potential intrinsic mechanisms of SCD pain which may extend beyond a blood-based disease pathology.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH