(I-355) Phenotypic screening of FDA-approved compounds as potential analgesics using iPSC-based DRG tissue mimic

Introduction:: Chronic pain affects 1 in 5 adults and is the number one cause of long-term disability in the world. Opioids are prescribed to manage chronic pain but are associated with deleterious side effects, tolerance, and abuse. The Pain Research Lab at UMass Lowell has developed an innovative DRG tissue model of acute and chronic nociception using human induced pluripotent stem cell (hiPSC)-derived sensory neurons and astrocytes co-cultured on microelectrode arrays (MEAs). This novel approach enables non-invasive, moderate-throughput, high-content phenotypic screening of prospective therapeutic compounds. Our overarching goal is to identify novel compounds and classes of compounds that may serve as analgesics or therapeutics for inflammatory pain conditions. Here, we screen 100 FDA-approved compounds, selected based on their off-label overlap with pathways associated with inflammatory nociception, and report their phenotypic profiles generated by our iPSC sensory co-culture assay. In total, our data suggests that iPSC-based sensory co-cultures on MEAs represent an excellent assay and that distinct phenotypic profiles may be generated depending on the compound’s mechanism of action.


Materials and Methods:: hiPSC sensory neurons and glia are co-cultured on 48-well MEA plates and recorded every other day for 30 min using Axion Biosystem’s Maestro Pro. Upon reaching a stable baseline – defined as three consecutive recordings with statistically similar electrode yields and mean firing rates (MFR) – sensory co-cultures are incubated with TNF-α (50 ng/mL) for 24 h to induce an inflammatory nociceptive state. FDA-approved compounds are added at 10 µM initial concentration to ≥ 4 wells and recorded continuously for 3 h. Following spontaneous recordings, astrocyte reactivity and sensory neuron temperature responsiveness are measured during application of electric stimulation and temperature ramp to 42 °C, respectively. Assay quality metric (Z’) is calculated in the cases of spontaneous and evoked activity and assay hits are defined as mean log spike count/well greater or less the 3x MAD of TNF-α treated baseline.


Results, Conclusions, and Discussions:: Sensory co-cultures were consistently found to reach stable baselines between 27 and 42 days in vitro (DIV), with active electrode yield percentage ≥ 60%. TNF-α induced statistically significant increases in MFR and temperature evoked responsiveness rates. Z’-based assay quality based on changes in MFR in the presence of positive (TNF-α) and negative (100 µM) controls was determined to be ‘excellent’ (0.56). Likewise, Z’ values based on electrically evoked glial activity were determined to be ‘excellent’ (0.62). In total, these data suggest that we have developed an effective assay for evaluating compound response in terms of spontaneous and evoked activity. 37% of FDA-approved compounds exhibited statistically significant changes in spontaneous activity (≤ 3x MAD of TNF-α treated baseline) following bolus addition (10 mM, 1:1000). Further assessment of spontaneous activity (burst rate, interspike interval, area under the normalized cross-correlogram) as well evoked activity (total power over baseline) enabled generation of distinct phenotypic profiles of assay ‘hits’ as well as ‘hit’ classes.

Phenotypic screening using iPSC sensory co-cultures on MEAs represents a promising model to find alternative therapeutics for inflammatory nociception. ‘Hits’ from our assay that are deemed non-cytotoxic are now being forwarded to preclinical animal testing in behavioral models of inflammatory arthritis. Correlation of behavioral outcomes will inform further optimization of our model and/or its continued use in screening analgesic effects of off-label compounds. Additionally, we plan to extend and validate our model in other types of chronic nociception (e.g., diabetic and chemotherapy induced peripheral neuropathies).


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