Single cell transcriptomics reveals distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with opioid use disorder. Single cell transcriptomics reveals distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with opioid use disorder

The opioid epidemic represents a national crisis. Oxycodone is one of the most prescribed opioid medications in the United States, whereas buprenorphine is currently the most prescribed medication for opioid use disorder (OUD) pharmacotherapy. Given the extensive use of prescription opioids and the global opioid epidemic, it is essential to understand how opioids modulate brain cell type function at the single-cell level. We performed single nucleus RNA-seq (snRNA-seq) using iPSC-derived forebrain organoids from three male OUD subjects in response to oxycodone, buprenorphine, or vehicle for seven days. We utilized the snRNA-seq data to identify differentially expressed genes following drug treatment using the Seurat integrative analysis pipeline. We utilized iPSC-derived forebrain organoids and single-cell sequencing technology as an unbiased tool to study cell-type-specific and drug-specific transcriptional responses. After quality control filtering, we analyzed 25787 cells and identified sixteen clusters using unsupervised clustering analysis. Our results reveal distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with OUD. Specifically, buprenorphine displayed a significant influence on transcription regulation in glial cells. However, oxycodone induced type I interferon signaling in many cell types, including neural cells in brain organoids. Finally, we demonstrate that oxycodone, but not buprenorphine activated STAT1 and induced the type I interferon signaling in patients with OUD. These data suggest that elevation of STAT1 expression associated with OUD might play a role in transcriptional regulation in response to oxycodone. In summary, our results provide novel mechanistic insight into drug action at single-cell resolution. Overall design: This study was designed to identify gene expression profiles associated with drug treatment with oxycodone or buprenorphine drug treatment in iPSC-derived brain organoids from OUD patients. Drug treatment was conducted at 83-90 days of forebrain organoid differentiation and at 28-34 days of forebrain neuron differentiation. The concentrations of oxycodone (50 µg/L) and buprenorphine (2 ng/mL) used to perform these experiments were selected to fall within the range of blood drug concentrations in patients taking standard clinical doses of these two drugs. 3-D iPSC-derived brain organoids were cultured in the bioreactors with the drugs for seven days, and the medium was changed daily.

阿片类药物泛滥是一场国家级公共卫生危机。羟考酮（oxycodone）是美国处方量最高的阿片类药物之一，而丁丙诺啡（buprenorphine）是目前阿片类使用障碍（opioid use disorder, OUD）药物治疗的首选处方药物。鉴于处方阿片类药物的广泛使用以及全球范围内的阿片类药物流行，解析阿片类药物在单细胞水平如何调控脑细胞功能至关重要。本研究针对3名男性OUD患者的诱导多能干细胞（induced pluripotent stem cell, iPSC）来源前脑类器官，分别以羟考酮、丁丙诺啡或溶剂对照处理7天，随后开展单细胞核RNA测序（single nucleus RNA-seq, snRNA-seq）。我们采用Seurat整合分析流程，基于snRNA-seq数据鉴定药物处理后的差异表达基因。本研究借助iPSC来源前脑类器官与单细胞测序技术，作为无偏倚工具探究细胞类型特异性与药物特异性的转录响应。经过质控过滤后，我们共分析了25787个细胞，并通过无监督聚类分析鉴定出16个细胞簇。研究结果显示，OUD患者的iPSC来源脑类器官对羟考酮与丁丙诺啡呈现截然不同的转录响应。具体而言，丁丙诺啡对胶质细胞的转录调控具有显著影响；而羟考酮则在脑类器官的多种细胞类型（包括神经细胞）中诱导I型干扰素信号通路。此外，我们证实仅羟考酮可激活OUD患者细胞中的STAT1并诱导I型干扰素信号通路，而非丁丙诺啡。上述数据表明，与OUD相关的STAT1表达上调可能在响应羟考酮的转录调控中发挥作用。综上，本研究以单细胞分辨率为药物作用机制提供了全新的见解。整体实验设计：本研究旨在鉴定OUD患者iPSC来源脑类器官经羟考酮或丁丙诺啡处理后的基因表达谱。药物处理于前脑类器官分化第83-90天、前脑神经元分化第28-34天进行。本实验选用的羟考酮（50 µg/L）与丁丙诺啡（2 ng/mL）浓度，处于临床标准剂量给药患者的血液药物浓度范围内。三维iPSC来源脑类器官与药物共同置于生物反应器中培养7天，每日更换培养基。