A new brain organoid model for NeuroHIV and the impact of opioids

摘要 HIV-1感染中枢神经系统(CNS)的常驻细胞，导致神经发病。艾滋病毒-- 神经发病机制可能是由直接和间接的病毒和宿主因素引起的。然而，确切的潜在原因是 机制仍不清楚。尽管抗逆转录病毒疗法(ART)在抑制艾滋病毒复制方面取得了成功， 近一半的艾滋病毒携带者(PLWH)仍有不同程度的艾滋病毒相关神经疾病 (手)。也有证据表明，中枢神经系统是艾滋病毒的宿主和避难所，可能会使低血压病 严重的病毒血症，导致持续的神经炎症。人类免疫缺陷病毒的分子进化 神经发病机制很难描述，部分原因是缺乏现实的艾滋病毒动物模型，以及 人脑组织很少能用于研究，直到病人死亡，通常是由于晚期疾病。 人脑皮质有机体(BCO)是研究神经病理学的一种新兴的前沿技术 疾病；由于它们起源于人类，它们更符合发育中的基因组和结构特征 将人脑与动物模型进行比较。该模型由自组装的动态3-D结构组成， 提供不同细胞类型的相互作用，这在传统的单层培养中是有限的。我们优化了 协议，以产生长期可行和有效的BCO。我们的BCO模型有一种前所未有的细胞类型 多样性，通过从祖细胞到神经细胞的动态发展，变得夹杂着 随着时间的推移，星形胶质细胞处于静止状态；这是一种难以在体外获得的表型。利用蜂窝组件 功能神经网络的生成到位，我们的BCO模型显示了强大的细胞外电活动 在早期阶段，并逐渐发展为一个有组织的振荡网络。此外，我们还有 先前建立的用于将IPSC衍生的小胶质细胞整合到BCO中以形成组合物的方法， 这对这项研究至关重要。包含相关免疫细胞类型的BCO集合体模型将使 人类免疫缺陷病毒易感性及不同细胞类型对人类免疫缺陷病毒神经系统影响的研究 感染。利用这个功能强大的BCO组合体，我们建议开发一种新的人体模型来研究 HIV神经发病的细胞和分子机制，以及潜在的相互作用， 抗逆转录病毒治疗(ART)和阿片类药物暴露的相加或协同效应。这种注入了小胶质细胞的BCO 内源性星形胶质细胞将允许艾滋病毒感染及其相关的病理生理事件，并有助于 理清相关免疫细胞在神经发病机制中的作用和相互作用。

Abstract HIV-1 infects resident cells of the central nervous system (CNS) leading to neuropathogenesis. HIV- neuropathogenesis is likely caused by direct and indirect viral and host factors. However, the exact underlying mechanisms remain unclear. Despite the success of antiretroviral therapy (ART) in suppressing HIV replication, near half of people living with HIV (PLWH) still have varying degrees of HIV-associated neurological disorders (HAND). There is also evidence that the CNS serves as an HIV reservoir and sanctuary site that may allow low level viremia, contributing to persistent neuroinflammation. The evolving molecular events underlying HIV neuropathogenesis are difficult to delineate, partially due to the lack of realistic HIV animal models and because human brain tissues rarely become available for studies until patients die, often due to advanced diseases. Human brain cortical organoids (BCO) are an emerging, cutting-edge technology for studying neuropathological disorders; because of their human origin, they better match the genomic and structural features of the developing human brain compared to animal models. This model consists of a self-assembled dynamic 3-D structure that provides an interplay of different cell types, which is limited in traditional monolayer cultures. We optimized protocols to generate long-term viable and functional BCO. Our BCO model has an unprecedented cell type diversity, via a dynamic development from progenitor cells to neuronal cells, that become interspersed with quiescent astrocytes over time; a difficult phenotype to obtain ex vivo. With the cellular components for generation of a functional neural network in place, our BCO model shows a robust extracellular electrical activity at early stages and progressively develops into an organized oscillatory network. Additionally, we have previously established methods for integration of iPSC-derived microglia into the BCO forming an assembloid, which is crucial tor this study. A BCO assembloid model containing relevant immune cell types will enable susceptibility to HIV and the study of the contributions of different cell types to the neurological consequences of infection. Using this robust and functional BCO assembloid, we propose to develop a new human model to study the cellular and molecular mechanisms underlying HIV neuropathogenesis, and the potential interactive, additive, or synergistic effects of antiretroviral treatment (ART) and opioid exposure. This microglia-infused BCO with endogenous astrocytes will allow HIV infection and its related pathophysiological events and help to disentangle the contribution and interplay of relevant immune cells to neuropathogenesis.