Characterizing functional and molecular signatures of inflammatory sensitization in human pluripotent stem cell-derived sensory neurons

Authors

• D Castellano
• VM Jovanovic
• R Tharakan
• Y Qu
• K Gerrish
• P Ormanoglu
• D Tao
• CA LeClair
• CA Tristan

Abstract

Specialized nociceptive sensory neurons are critical for the detection and transmission of noxious stimuli. During inflammation, sensory neurons exhibit increased excitability, known as sensitization, which plays a role in the development of chronic pain. Understanding the molecular and cellular mechanisms regulating inflammatory sensitization is crucial for elucidating the pathophysiology of chronic pain and identifying novel therapeutic targets. Although inflammatory sensitization has been extensively studied using rodent models, transcriptomic and physiological differences across species indicate that human nociceptors exhibit distinct gene expression signatures during sensitization, which are not well understood. Characterization of human sensory neurons is often constrained by the limited availability of donor dorsal root ganglia (DRGs) and challenges associated with obtaining postmortem samples. Recently, our group published a novel and scalable differentiation protocol that reproducibly generates human pluripotent stem cell derived-sensory neurons (hPSC-SNs) that functionally resemble peripheral sensory neurons found in the native human DRG. Using multi-electrode arrays, we demonstrate that our hPSC-SNs can be sensitized after a 24 h treatment with cocktail of inflammatory mediators (1 μM prostaglandin E2, 10 μM bradykinin, 1 μM histamine, and 0.5 μM serotonin; hereby referred to as inflammatory soup; IS). This sensitization can be blocked by the protein synthesis inhibitor cycloheximide (CHX; 50 μg/mL). Data-independent acquisition mass spectrometry revealed that IS or CHX treatment resulted in differentially expressed proteins present within cytosolic and membrane fractions. These findings demonstrate the utility of our hPSC-SNs as a cellular sensitization model to identify proteins that play a role in inflammation and chronic pain.

Scientific Focus Area: Stem Cell Biology

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