Table_8_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.XLSX

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.

引言 源自人类的诱导多能干细胞（induced pluripotent stem cell, iPSC）脑模型，有望推动我们对药物使用所引发神经毒性后果的认知。然而，此类模型能否精准重现真实的基因组特征与细胞功能，以及药物诱导的分子改变，仍有待验证。亟需构建新型药物暴露体外模型，以深化我们对如何保护或逆转物质使用障碍相关分子改变的认知。
方法 本研究从培养的死后人类皮肤成纤维细胞中，构建了一种新型诱导多能干细胞来源的神经前体细胞（neural progenitor cells）与神经元模型，并将其与供体来源的同基因脑组织直接进行比对。本研究利用RNA细胞类型与成熟度反卷积分析，以及基于成人和胎儿人类组织训练的DNA甲基化表观遗传时钟，评估了细胞模型从干细胞向神经元分化过程中的成熟状态。作为该模型用于物质使用障碍研究的概念验证，我们分别将吗啡和可卡因处理的神经元，与死后阿片类物质使用障碍（Opioid Use Disorder, OUD）和可卡因使用障碍（Cocaine Use Disorder, CUD）患者脑组织的基因表达特征进行了比对。
结果 在每名人类受试者（N=2，每名受试者对应2个克隆）中，大脑额叶皮层的表观遗传年龄与皮肤成纤维细胞的表观遗传年龄一致，且与供体的实际年龄高度吻合；从成纤维细胞诱导获得干细胞的过程，可将表观遗传时钟有效重置至胚胎期年龄；干细胞向神经前体细胞再向神经元的分化过程，可通过DNA甲基化与RNA基因表达检测结果，逐步推动细胞走向成熟。在一名因阿片类药物过量死亡的供体来源的神经元中，吗啡处理诱导的基因表达改变与此前在OUD离体脑组织中观察到的改变相似，包括即刻早期基因EGR1的差异表达——该基因已知会因阿片类药物使用而失调。
讨论 综上，本研究构建了一种源自人类死后成纤维细胞的iPSC模型，该模型可与对应的同基因脑组织直接比对，还可用于模拟阿片类物质使用障碍等情境下的扰动因子暴露。未来，利用该模型及其他死后来源的脑细胞模型（包括大脑类器官）开展的研究，将成为解析药物诱导脑组织改变机制的宝贵工具。