A human stem cell-derived neuronal model of morphine exposure reflects brain dysregulation in opioid use disorder: Transcriptomic and epigenetic characterization of postmortem-derived iPSC neurons

Abstract

IntroductionHuman-derived induced pluripotent stem cell (iPSC) models of brain promise to advance our understanding of neurotoxic consequences of drug use. However, how well these models recapitulate the actual genomic landscape and cell function, as well as the drug-induced alterations, remains to be established. New in vitro models of drug exposure are needed to advance our understanding of how to protect or reverse molecular changes related to substance use disorders.MethodsWe engineered a novel induced pluripotent stem cell-derived model of neural progenitor cells and neurons from cultured postmortem human skin fibroblasts, and directly compared these to isogenic brain tissue from the donor source. We assessed the maturity of the cell models across differentiation from stem cells to neurons using RNA cell type and maturity deconvolution analyses as well as DNA methylation epigenetic clocks trained on adult and fetal human tissue. As proof-of-concept of this model’s utility for substance use disorder studies, we compared morphine- and cocaine-treated neurons to gene expression signatures in postmortem Opioid Use Disorder (OUD) and Cocaine Use Disorder (CUD) brains, respectively.ResultsWithin each human subject (N = 2, 2 clones each), brain frontal cortex epigenetic age parallels that of skin fibroblasts and closely approximates the donor’s chronological age; stem cell induction from fibroblast cells effectively sets the epigenetic clock to an embryonic age; and differentiation of stem cells to neural progenitor cells and then to neurons progressively matures the cells via DNA methylation and RNA gene expression readouts. In neurons derived from an individual who died of opioid overdose, morphine treatment induced alterations in gene expression similar to those previously observed in OUD ex-vivo brain tissue, including differential expression of the immediate early gene EGR1, which is known to be dysregulated by opioid use.DiscussionIn summary, we introduce an iPSC model generated from human postmortem fibroblasts that can be directly compared to corresponding isogenic brain tissue and can be used to model perturbagen exposure such as that seen in opioid use disorder. Future studies with this and other postmortem-derived brain cellular models, including cerebral organoids, can be an invaluable tool for understanding mechanisms of drug-induced brain alterations.

Single nuclei transcriptomics in human and non-human primate striatum implicates neuronal DNA damage and proinflammatory signaling in opioid use disorder

“…DNA damage markers were identified in both the striatum of humans and monkeys, suggesting chronic opioid use associated with OUD leads to augmented DNA modifications ( e.g., double-stranded breaks, chromatin accessibility). Elevated markers for neuroinflammation, the involvement of microglia-dependent signaling in striatum, and alterations in synaptic signaling in OUD are consistent with previous bulk transcriptomics findings from postmortem brains of subjects with OUD 2, 34, 108, 126, 127 .…”

Human pluripotent stem cells as a translational toolkit in psychedelic research in vitro

“…At least partially motivated also by regulatory pressures to reduce animal experimentation by exploring in vitro alternatives, the scope of diseases modeled with PSCs has expanded to include psychedelic-targeted disorders such as post-traumatic stress disorder (PTSD), 76 SUD, 77 and depression 78 ( Table 2 ). Despite the increasing popularity of these studies, which have predominantly emerged in the past decade, clinical trials of compounds identified or repurposed via PSC-based models remain constrained and in their nascent stages.…”

“… 91 In a study assessing the effects of morphine treatment as a proxy for chronic opioid exposure, the expression patterns of neuron-related genes such as egr-1 were found to be similar between PSC-derived neurons and the dorsolateral prefrontal cortex of individuals deceased with opioid use disorder. 77 Also, direct neuronal transdifferentiation has emerged as a means to bypass intermediate embryonic-like pluripotent stages, which may erase or modify disease-associated epigenetic traits.…”

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

“…Altogether, such rapid developments in the generation of iPSC-derived neuronal models of OUD provoked questions regarding their fidelity to in vivo signatures of the disorder. To address this, Mendez et al (2023) , engineered novel iPSC-derived cortical neurons from skin fibroblasts of individuals who had died of an opioid overdose. Following chronic treatment with morphine, these neurons remarkably showed transcriptional alterations paralleling those observed in the postmortem, ex vivo frontal cortex tissue of individuals with OUD ( Mendez et al, 2023 ).…”

“…To address this, Mendez et al (2023) , engineered novel iPSC-derived cortical neurons from skin fibroblasts of individuals who had died of an opioid overdose. Following chronic treatment with morphine, these neurons remarkably showed transcriptional alterations paralleling those observed in the postmortem, ex vivo frontal cortex tissue of individuals with OUD ( Mendez et al, 2023 ). These included developmental and synaptic genes associated with substance use disorders ( Gallo et al, 2018 ; Seney et al, 2021 ), as well G-protein-coupled receptor (GPCR) pathways, of interest given that opioid receptors are GPCRs themselves.…”