(L-448) Electrically stimulated oscillating calcium transients in healthy and pathological iPSC-derived glia

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row L - Poster # 448

Presenting Author(s)

Mauricio F. De Almeida, Undergraduate (he/him/his)

Undergraduate Research Assistant
University of Massachusetts Lowell
Leominster, Massachusetts, United States

Co-Author(s)

JL

Jennifer Lawson

Graduate Research Assistant
University of Massachusetts Lowell, United States
EL

Elijah LaVancher

Undergraduate Research Assistant
University of Massachusetts Lowell, United States

Primary Investigator(s)

BB

Bryan Black

Assistant Faculty
University of Massachusetts Lowell
Lowell, Massachusetts, United States

Introduction:: Support glia in the peripheral and central nervous systems regulate neuronal function through ion buffering and bidirectional molecular (i.e., paracrine) signaling. Many recent 2D and 3D phenotypic assays proposed for screening neuropharmaceuticals make use of heterogeneous co-cultures of neurons and support glia. However, the assay’s measured outcomes often fail to evaluate a drug’s potential impact on glia, potentially leading to false negatives. Assays based on multi-well microelectrode arrays (MEAs) have confirmed the ability to stimulate and measure oscillating calcium transients (OCa²⁺Ts) or ‘high-frequency oscillations’. However, until recently, none have been developed or tested using induced pluripotent stem cell (iPSC)-derived glia. We recently published, and here expand upon, electrically stimulated OCa²⁺Ts in mono-cultures of iPSC spinal astrocytes as well as co-cultures of iPSC glia and iPSC sensory neurons. In both cases, we observed dose-dependent changes in OCa²⁺T magnitude (defined as total power over pseudo-trial baselines) in response to balanced biphasic electrical stimuli. Futhermore, we demonstrated the role of gap junctions in OCa²⁺T activity through the use of AP5/CNQX, carbenoxolone, nicardipine, and everolimus. Lastly, we assess differences in OCa²⁺T magnitude and duration in glia derived from patients suffering from amyotrophic lateral sclerosis (ALS). In total, these findings suggest that OCa2+T stimulation and measurement may be, and perhaps should be, incorporated into future moderate-to-high throughput phenotypic assays aimed at identifying novel analgesics. Additionally, these results suggest a broader applicability to pharmacological assays targeting glia or co-cultures of neuronal and non-neuronal support cells.

Materials and Methods:: Co-cultures and mono-cultures of hiPSCs SNs and glia were cultured on a 48-well MEA plate and stimulated/recorded using an Axion Biosystems Maestro Pro. Mono- and co-cultures were electrically stimulated with a biphasic (600 μs/ph), cathodic leading waveforms, with a pulse frequency of 0.025 Hz and amplitude of 800 mV in the presence of inflammatory cytokines and/or the compounds listed above. All electrical stimulation spectrogram data were analyzed in NeuroExplorer (Nex Technologies) and a boutique MATLAB script. OCa²⁺Ts were calculated and reported as the total power over baseline (TPoB); comparing stimulated trials to baseline pseudo-trials.

Results, Conclusions, and Discussions:: OCa²⁺Ts could be readily observed in real time, persisting for up to 20 s. OCa²⁺T amplitudes are reported as total power over baseline (TPoB) as compared with each electrode’s pseudo-trial average. TPoBs increased in a dose-dependent manner in mono-cultures of spinal astrocytes as well as sensory co-cultures and were significantly attenuated in the presence of gap junction inhibitor carbenoxolone, but not glutamate receptor antagonists AP5/CNQX. Using carbenoxolone as a positive control, we then calculated an assay quality metric (Z’) which suggested this approach serves as an ‘excellent’ stand-alone assay. Screening cherry-picked FDA-approved compounds in sensory co-cultures from different classes of analgesics, we measured significant attenuation (classified here as ‘hits’) due to nicardipine and everolimus. Lastly, we observed significant differences in OCa²⁺T profiles in mono-cultures of pathological (ALS patient-derived) astrocytes versus ‘healthy’ controls. In brief, these data suggest that electrically stimulated OCa²⁺Ts, in either mono- or co-cultures of glia and associated neurons, may serve as a necessary supplement to phenotypic screening assays for neuropharmaceutical hit and lead detection.

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