Brain barrier and support in a dish Deep within the brain, the choroid plexus filters blood and secretes cerebrospinal fluid (CSF), a nutrient-rich liquid that bathes and supports the brain and protects it from entry of toxic compounds. Current understanding of this vital tissue in humans is limited. Pellegrini et al. developed choroid plexus organoids that quantitatively predict human brain permeability of small molecules and secrete an isolated CSF-like fluid (see the Perspective by Silva-Vargas and Doetsch). This CSF model reveals secretion of developmental factors and disease-related biomarkers by key cell types and provides a testing ground for drug entry into the brain. Science, this issue p. eaaz5626; see also p. 143 Choroid plexus organoids secrete cerebrospinal fluid and model the tight, selectively permeable human blood-brain barrier. INTRODUCTION The choroid plexus is a secretory epithelial tissue of the central nervous system (CNS) responsible for cerebrospinal fluid (CSF) production and functions as a barrier that regulates entry of compounds and nutrients into the brain. The CSF plays key roles in the delivery of nutrients to the brain, circulation of instructive signaling molecules, and clearance of toxic by-products such as protein aggregates. RATIONALE Current understanding of the choroid plexus and CSF has primarily come from animal models or CSF collected from human volunteers. These have yielded insight into general CSF composition, but the specific cellular and tissue sources of various secreted proteins have remained elusive. There is also limited understanding of the development of the choroid plexus in humans and of the relative changes in CSF composition over time. All of these deficiencies in our understanding come from a lack of experimental access to the human choroid plexus. Although several previous studies have successfully generated cells with a choroid plexus identity from human pluripotent stem cells, none have been able to recapitulate the morphology, maturation, and function of the choroid plexus, and currently, no in vitro model exists for authentic human CSF. Knowledge of the processes that regulate choroid plexus development and CSF composition could provide better strategies to manipulate and therapeutically target this vital brain tissue. RESULTS To study the development and function of the human choroid plexus, we developed a pluripotent stem cell–derived organoid model. Choroid plexus organoids recapitulate key morphological and functional features of human choroid plexus. First, organoids form a tight barrier that selectively regulates the entry of small molecules such as dopamine. We demonstrate that organoids can qualitatively and quantitatively predict the permeability of new drugs, and we take advantage of this system to reveal a potential toxic accumulation of BIA 10-2474, a drug that caused severe neurotoxicity only in humans and not in animal models tested. Second, choroid plexus organoids secrete a CSF-like fluid containing proteins and known biomarkers within self-contained compartments. We examine changes in secretion of CSF proteins over time and identify distinct cell types within the epithelium that contribute to dynamic changes in CSF composition. We find that these cell types can be traced to rather obscure descriptions in the literature of “dark” and “light” cells, and we demonstrate that these cells exhibit opposing features related to mitochondria and cilia. We also uncover a previously unidentified cell type in the choroid plexus: myoepithelial cells. These interacting subpopulations exhibit distinct secretory roles in CSF production and reveal previously uncharacterized human-specific secreted proteins that may play important roles in human brain development. CONCLUSION Human choroid plexus organoids provide an easily tractable system to study the key functions of this organ: CSF secretion and selective transport into the CNS. As such, they can predict CNS permeability of new compounds to aid in the development of neurologically relevant therapeutics. They also provide a source of more authentic CSF and can be used to understand development of this key organ in brain development and homeostasis. CSF-producing choroid plexus organoids predict CNS permeability of drugs. Choroid plexus organoids develop highly intricate folded tissue morphology (section stained for choroid plexus markers shown at top right) similar to choroid plexus tissue in vivo (top left) and, later, self-contained fluid-filled compartments containing a CSF-like fluid (top middle) that is separate from media. (Bottom left) Choroid plexus organoids accurately predict the permeability of small molecules such as dopamine and levodopa (l-dopa) and quantitatively predict the permeability of a range of therapeutic molecules. The graph shows the correlation between permeability in vivo and in vitro for the drugs tested. R2, coefficient of determination. (Bottom right) Single-cell RNA sequencing reveals newly identified epithelial subtypes (colored dark and light) that participate in filtration and specialized secretion of CSF proteins. The Venn diagram shows overlap between proteins detected in CSF in vivo and in the organoid. Cerebrospinal fluid (CSF) is a vital liquid, providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP), a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here, we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally, the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes.

中文翻译：

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具有脑脊液产生的人类中枢神经系统屏障形成类器官

脑屏障和培养皿中的支撑 在大脑深处，脉络丛过滤血液并分泌脑脊液 (CSF)，脑脊液是一种营养丰富的液体，可以沐浴和支撑大脑，并保护大脑免受有毒化合物的进入。目前对人类这一重要组织的了解还有限。佩莱格​​里尼等人。开发了脉络丛类器官，可以定量预测人脑对小分子的渗透性并分泌一种分离的脑脊液样液体（参见 Silva-Vargas 和 Doetsch 的观点）。该脑脊液模型揭示了关键细胞类型分泌发育因子和疾病相关生物标志物，并为药物进入大脑提供了试验场。科学，本期第 14 页。eaaz5626; 另见 p. 143 脉络丛类器官分泌脑脊液并模拟紧密的、选择性渗透的人类血脑屏障。简介 脉络丛是中枢神经系统 (CNS) 的分泌性上皮组织，负责脑脊液 (CSF) 的产生，并作为调节化合物和营养物质进入大脑的屏障。脑脊液在向大脑输送营养物质、指导性信号分子循环以及清除蛋白质聚集物等有毒副产物方面发挥着关键作用。基本原理 目前对脉络丛和脑脊液的了解主要来自动物模型或从人类志愿者收集的脑脊液。这些研究使我们对脑脊液的一般组成有了深入的了解，但各种分泌蛋白的特定细胞和组织来源仍然难以捉摸。对于人类脉络丛的发育以及脑脊液成分随时间的相对变化的了解也很有限。我们理解中的所有这些缺陷都源于缺乏对人类脉络丛的实验访问。尽管之前的几项研究已经成功地从人多能干细胞中产生了具有脉络丛特性的细胞，但没有一个能够重现脉络丛的形态、成熟和功能，而且目前还没有真正的人脑脊液的体外模型。了解调节脉络丛发育和脑脊液成分的过程可以提供更好的策略来操纵和治疗靶向这一重要的脑组织。结果 为了研究人类脉络丛的发育和功能，我们开发了一种多能干细胞衍生的类器官模型。脉络丛类器官概括了人类脉络丛的关键形态和功能特征。首先，类器官形成紧密的屏障，选择性地调节多巴胺等小分子的进入。我们证明类器官可以定性和定量预测新药的渗透性，并且我们利用该系统揭示了 BIA 10-2474 的潜在毒性积累，这种药物仅在人类中引起严重的神经毒性，而在测试的动物模型中则没有。第二，脉络丛类器官在独立的隔室中分泌一种类似脑脊液的液体，其中含有蛋白质和已知的生物标志物。我们检查脑脊液蛋白分泌随时间的变化，并识别上皮内导致脑脊液成分动态变化的不同细胞类型。我们发现这些细胞类型可以追溯到文献中对“暗”和“光”细胞相当模糊的描述，并且我们证明这些细胞表现出与线粒体和纤毛相关的相反特征。我们还发现了脉络丛中以前未识别的细胞类型：肌上皮细胞。这些相互作用的亚群在脑脊液产生中表现出独特的分泌作用，并揭示了以前未表征的人类特异性分泌蛋白，这些蛋白可能在人类大脑发育中发挥重要作用。结论 人脉络丛类器官提供了一个易于处理的系统来研究该器官的关键功能：脑脊液分泌和选择性转运到中枢神经系统。因此，他们可以预测新化合物的中枢神经系统渗透性，以帮助开发神经相关疗法。它们还提供了更真实的脑脊液来源，可用于了解大脑发育和稳态中这一关键器官的发育。产生脑脊液的脉络丛类器官可预测药物的中枢神经系统渗透性。脉络丛类器官发育出高度复杂的折叠组织形态（右上显示脉络丛标记染色部分），类似于体内脉络丛组织（左上），并且后来形成包含脑脊液样液体的独立的充满液体的隔室（顶部中间）与媒体分开。（左下）脉络丛类器官可准确预测多巴胺和左旋多巴 (l-dopa) 等小分子的渗透性，并定量预测一系列治疗分子的渗透性。该图显示了所测试药物的体内和体外渗透性之间的相关性。R2，决定系数。（右下）单细胞 RNA 测序揭示了新发现的参与脑脊液蛋白过滤和专门分泌的上皮亚型（深色和浅色）。维恩图显示了体内脑脊液和类器官中检测到的蛋白质之间的重叠。脑脊液 (CSF) 是一种重要的液体，可提供营养和信号分子并清除大脑中的有毒副产物。脑脊液由脉络丛 (ChP) 产生，脉络丛是一种保护性上皮屏障，还可防止有毒分子或药物从血液中自由进入。在这里，我们建立了人类 ChP 类器官，在独立的隔室中具有选择性屏障和类似脑脊液的液体分泌功能。我们表明，这种体外屏障对小分子表现出与体内 ChP 相同的选择性，并且 ChP-CSF 类器官可以预测新化合物的中枢神经系统 (CNS) 渗透性。ChP-CSF 类器官的转录组和蛋白质组特征显示与体内 ChP 高度相似。最后，单细胞转录组学和蛋白质组学分析的交叉揭示了先前未识别的特殊上皮亚型产生的关键人类脑脊液成分。