Human IPSC‐Derived PreBötC‐Like Neurons and Development of an Opiate Overdose and Recovery Model

Abstract

Opioid overdose is the leading cause of drug overdose lethality, posing an urgent need for investigation. The key brain region for inspiratory rhythm regulation and opioid‐induced respiratory depression (OIRD) is the preBötzinger Complex (preBötC) and current knowledge has mainly been obtained from animal systems. This study aims to establish a protocol to generate human preBötC neurons from induced pluripotent cells (iPSCs) and develop an opioid overdose and recovery model utilizing these iPSC‐preBötC neurons. A de novo protocol to differentiate preBötC‐like neurons from human iPSCs is established. These neurons express essential preBötC markers analyzed by immunocytochemistry and demonstrate expected electrophysiological responses to preBötC modulators analyzed by patch clamp electrophysiology. The correlation of the specific biomarkers and function analysis strongly suggests a preBötC‐like phenotype. Moreover, the dose‐dependent inhibition of these neurons’ activity is demonstrated for four different opioids with identified IC50's comparable to the literature. Inhibition is rescued by naloxone in a concentration‐dependent manner. This iPSC‐preBötC mimic is crucial for investigating OIRD and combating the overdose crisis and a first step for the integration of a functional overdose model into microphysiological systems.

Investigating the neurobiology of maternal opioid use disorder and prenatal opioid exposure using brain organoid technology

“…In future, the increased inclusion of midbrain, hindbrain, and brainstem organoid cultures may help provide greater insight into the mechanisms underlying OUD and POE ( Tieng et al, 2014 ; Jo et al, 2016 ; Qian et al, 2016 ; Eura et al, 2020 ; Nickels et al, 2020 ; Valiulahi et al, 2021 ). The value of this approach is underscored not only by the involvement of these brain regions in the opioid addiction cycle, but also by prior efforts to use iPSC-derived midbrain dopaminergic or brainstem pre-Bötzinger Complex neurons to investigate the etiology or impact of OUD ( Sheng et al, 2016b ; Halikere et al, 2020 ; Guo et al, 2023 ).…”

“…The circulating miRNAs served as biomarkers for OUD progression and also influenced transcriptional programs associated with neurotransmitter dynamics, neurite outgrowth, and neural growth at a cellular level ( Chen et al, 2022 ). The following year, Guo et al (2023) , developed a model of opioid overdose by generating iPSC-derived neurons representing the preBötzinger Complex (preBötC), a brainstem structure necessary for inspiratory rhythm generation, which is suppressed by opioids. These neurons exhibited dose-dependent cessations in activity due to four μ-opioids (fentanyl, codeine, DAMGO, and methadone) and recovery upon naloxone administration ( Guo et al, 2023 ).…”

“…The following year, Guo et al (2023) , developed a model of opioid overdose by generating iPSC-derived neurons representing the preBötzinger Complex (preBötC), a brainstem structure necessary for inspiratory rhythm generation, which is suppressed by opioids. These neurons exhibited dose-dependent cessations in activity due to four μ-opioids (fentanyl, codeine, DAMGO, and methadone) and recovery upon naloxone administration ( Guo et al, 2023 ). Although the cells in both studies were not derived from OUD patients, they helped to demonstrate the utility of 2D neuronal cultures for parsing the cellular and molecular changes associated with specific phases of the addiction cycle, as well as identifying valuable biomarkers and therapeutic targets for its sequelae.…”