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Neurosciences

体外血脑屏障重建，模拟和治疗神经系统疾病

Published: October 20, 2023
doi:
10.3791/65921
, , , , , , ,
1Icahn School of Medicine at Mount Sinai, 2Black Family Stem Cell Institute at Mount Sinai, 3Ronald M. Loeb Center for Alzheimer’s Disease at Mount Sinai, 4Friedman Brain Institute at Mount Sinai, 5Nash Family Department of Neuroscience at Mount Sinai

Summary

血脑屏障（BBB）在维持稳定和健康的大脑环境方面起着至关重要的作用。血脑屏障功能障碍与许多神经系统疾病有关。我们开发了一种 3D 干细胞衍生的 BBB 模型，用于研究脑血管病理学、BBB 完整性以及 BBB 如何被遗传和疾病改变。

Abstract

血脑屏障 （BBB） 是中枢神经系统 （CNS） 的关键生理组成部分，可维持营养、清除废物并保护大脑免受病原体的侵害。血脑屏障固有的屏障特性对治疗药物递送至中枢神经系统以治疗神经系统疾病提出了挑战。血脑屏障功能受损与神经系统疾病有关。脑淀粉样血管病 （CAA） 是淀粉样蛋白在脑血管系统中的沉积导致 BBB 受损，是大多数阿尔茨海默病 （AD） 病例的合并症，表明 BBB 功能障碍或分解可能与神经退行性变有关。由于对人体血脑屏障组织的访问有限，导致正常血脑屏障功能和血脑屏障变性的机制仍然未知。为了解决这些局限性，我们通过将内皮细胞、周细胞和星形胶质细胞掺入 3D 基质中，开发了一种人类多能干细胞衍生的 BBB （iBBB）。iBBB 自组装以概括 BBB 中存在的解剖结构和细胞相互作用。用淀粉样蛋白接种 iBBB 可捕获 CAA 的关键方面。此外，iBBB提供了一个灵活的平台来调节与脑血管疾病和神经退行性变有关的遗传和环境因素，以研究遗传和生活方式如何影响疾病风险。最后，iBBB可用于药物筛选和药物化学研究，以优化向中枢神经系统的治疗输送。在该方案中，我们描述了由人类多能干细胞产生的三种细胞（内皮细胞，周细胞和星形胶质细胞）的分化，如何将分化的细胞组装到iBBB中，以及如何使用外源性淀粉样蛋白 在体外 模拟CAA。该模型克服了使用具有生物学保真度和实验灵活性的系统研究活体人脑组织的挑战，并能够研究人类血脑屏障及其在神经退行性变中的作用。

Introduction

血脑屏障 （BBB） 是将中枢神经系统 （CNS） 与外周分开的关键微血管网络，以维持正常神经元功能的理想环境。它通过维持代谢稳态 1,2,3,4、清除废物 4,5,6 以及保护大脑免受病原体和毒素的侵害，在调节物质流入和流入中枢神经系统方面起着关键作用 7,8。

血脑屏障的主要细胞类型是内皮细胞（EC）。源自中胚层谱系的内皮细胞形成脉管系统的壁 1,9。微血管 EC 彼此形成紧密连接，大大降低其膜的通透性10、11、12、13、14，同时表达转运蛋白以促进营养物质进出中枢神经系统1、4、12、14.微血管 EC 被周细胞 （PC） 壁细胞包围，这些细胞调节微血管功能和稳态，对于调节 BBB 对分子和免疫细胞的通透性至关重要 15,16,17。星形胶质细胞是一种主要的神经胶质细胞类型，是构成血脑屏障的最终细胞类型。星形胶质细胞端脚缠绕在 EC-PC 血管管周围，而细胞体延伸到脑实质，在神经元和脉管系统之间形成连接1。不同的溶质和底物转运蛋白位于星形胶质细胞端脚（例如水通道蛋白 4 [AQP-4]）上，这些转运蛋白在 BBB 功能中起关键作用18、19、20、21。

血脑屏障对于维持正常的大脑健康功能至关重要，许多神经系统疾病都报告了血脑屏障的功能障碍，包括阿尔茨海默病 （AD）22、23、24、25、多发性硬化症7、26、27、28、癫痫29、30 和中风31,32.人们越来越认识到，脑血管异常在神经退行性变中起着核心作用，导致对缺血和出血事件的易感性增加。例如，超过 90% 的 AD 患者患有脑淀粉样血管病 （CAA），其特征是淀粉样蛋白 β （Aβ） 沿脑血管系统沉积。CAA 增加 BBB 通透性并降低 BBB 功能，使中枢神经系统容易出现缺血、出血事件和加速认知能力下降33。

我们最近开发了一种人类血脑屏障的 体外 模型，该模型源自患者诱导的多能干细胞，其中包含封装在 3D 基质中的 EC、PC 和星形胶质细胞（图 1A）。iBBB 概括了生理相关的相互作用，包括血管管的形成和星形胶质细胞端足与脉管系统的定位 24。我们应用 iBBB 来模拟 APOE4 介导的 CAA 的易感性（图 1B）。这使我们能够确定 APOE4 促进 CAA 的因果细胞和分子机制，并利用这些见解开发治疗策略，以减少 CAA 病理学并改善 APOE4 小鼠 体内 的学习和记忆24。在这里，我们提供了一个详细的实验方案和视频教程，用于从人iPSCs重建BBB并在 体外对CAA进行建模。

Protocol

1. 将 iPSC 分化为 iBBB 细胞 注：这些分化方案先前已在Mesentier-Louro等人34中描述过。 包被细胞培养板在4°C下解冻还原生长因子（GF）膜基质过夜。 在 49.5 mL DMEM 中稀释 500 μL 基底膜基质。保持该溶液低温，以防止涂层溶液过早聚合。 每孔加入 1-2 mL 的 6 孔组织培养处理板。在用适当的加热培养基替换之前，将包衣溶液在37°C?…

Representative Results

正确形成的iBBB凝固成单个半透明圆盘（图3A）。几天后，iBBB从首次移液的表面脱落是正常的。这是无法避免的，但如果在更换介质时小心不要意外吸出 iBBB，则这不是 iBBB 正确形成的主要问题。24小时后，均匀分布的单个细胞可以在明场显微镜下鉴定（图3B）。2周后，可以看到更明显的结构，尽管很难用定义来辨认（图3C）。 <…

Discussion

血脑屏障功能障碍是许多神经系统疾病的合并症，并且可能是原因或加重因素7,40,41。然而，几乎不可能研究人类神经血管疾病患者血脑屏障功能障碍和分解的分子和细胞生物学。本方案中介绍的诱导型BBB（iBBB）提供了一个体外系统，该系统概括了BBB的重要细胞相互作用，包括血管管的形成和星形胶质细胞端足与脉管系?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作得到了 NIH 3-UG3-NS115064-01、R01NS14239、治愈阿尔茨海默氏症基金会、NASA 80ARCO22CA004、Chan-Zuckerberg Initiative、MJFF/ASAP 基金会和美国脑损伤协会的支持。CG由NIH F31NS130909支持。 图 1A 是用 BioRender.com 创建的。

Materials

6e10 amyloid-β antibody Biolegend SIG-39320 Used at 1:1000
Accutase Innovative Cell Technologies AT104
Activin A Peprotech 20-14E
Alexa Fluor 488, 555, 647 secondary antibodies Invitrogen Various Used at 1:1000
Amyloid-beta 40 fibril AnaSpec AS-24235
Amyloid-beta 42 fibril AnaSpec AS-20276
Aquaporin-4 antibody Invitrogen PA5-53234 Used at 1:300
Astrocyte basal media and supplements ScienCell 1801
B-27 serum-free supplement Gibco 17504044
BMP4 Peprotech 120-05ET
CHIR99021 Cyamn Chemical 13112
DMEM/F12 with GlutaMAX medium Gibco 10565018
Doxycycline Millipore-Sigma D3072-1ML
FGF-basic Peprotech 100-18B
Fluoromount-G slide mounting medium VWR 100502-406
Forskolin R&D Systems 1099/10
GeltrexTM LDEV-Free hESC-qualified Reduced Growth Factor Basement  Gibco A1413302
Glass Bottom 48-well Culture Dishes Mattek Corporation P48G-1.5-6-F
GlutaMAX supplement Gibco 35050061
Hoechst 33342  Invitrogen H3570
Human Endothelial Serum-free medium Gibco 11111044
LDN193189 Tocris 6053
Minimum Essential Medium Non-essential Amino Acid Solution (MEM-NEAA)  Gibco 11140050
N-2 supplement Gibco 17502048
Neurobasal medium Gibco 21103049
Normal Donkey Serum Millipore-Sigma S30-100mL Use serum to match secondary antibody host
Paraformaldehyde (PFA)  ThermoFisher 28908
PDGF-BB Peprotech 100-14B
PDGFRB (Platelet-derived growth factor receptor beta) antibody R&D Systems AF385 Used at 1:500
Phosphate Buffered Saline (PBS), pH 7.4 Gibco 10010031
Pecam1 (Platelet endothelial cell adhesion molecule 1) antibody R&D Systems AF806 Used at 1:500
Penicillin-Streptomycin Gibco 15140122
PiggyBac plasmid (PB_iETV2_P2A_GFP_Puro) AddGene  Catalog #168805
S100B antibody Sigma-Aldrich S2532-100uL Used at 1:500
SB43152 Reprocell 04-0010
Thioflavin T Chem Impex 22870 Used at 25uM
Triton X-100  Sigma-Aldrich T8787-250mL
VE-cadherin (CD144) antibody R&D systems AF938 Used at 1:500
VEGF-A Peprotech 100-20
Y27632 R&D Systems 1254/10
ZO-1 Invitrogen MA3-39100-A488 Dilution = 1:500
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Tags

Keywords: Blood-brain BarrierIn Vitro ModelAlzheimer’s DiseaseNeurodegenerationCerebrovascular PathologyAPOE4AmyloidInduced Pluripotent Stem CellsPersonalized TherapyDrug Screening3D ModelBBB FunctionCerebral Amyloid Angiopathy
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Goldman, C., Suhy, N., Schwarz, J. E., Sartori, E. R., Rooklin, R. B., Schuldt, B. R., Mesentier-Louro, L. A., Blanchard, J. W. Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease. J. Vis. Exp. (200), e65921, doi:10.3791/65921 (2023).
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