用于线粒体疾病建模的人脑类器官的生成
1Department of General Pediatrics, Neonatology and Pediatric Cardiology, Duesseldorf University Hospital, Medical Faculty,Heinrich Heine University, 2Institute of Neurobiology,Heinrich Heine University, 3Berlin Institute for Medical Systems Biology (BIMSB),Max Delbrueck Center for Molecular Medicine (MDC), 4Seaver Autism Center for Research and Treatment,Icahn School of Medicine at Mount Sinai, 5Max Delbrueck Center for Molecular Medicine (MDC)
Summary
我们描述了产生人类诱导多能干细胞衍生脑类器官及其在线粒体疾病建模的详细方案。
Abstract
线粒体疾病是最大的一类先天性代谢缺陷，目前无法治愈。这些疾病导致神经发育缺陷，其潜在机制仍有待阐明。一个主要障碍是缺乏有效的模型来概括患者中早发性神经元损伤。诱导多能干细胞（iPSCs）技术的进步使三维（3D）脑类器官的产生成为可能，可用于研究疾病对神经系统发育和组织的影响。包括这些作者在内的研究人员最近引入了人脑类器官来模拟线粒体疾病。本文报告了人类iPSC衍生的脑类器官的强大生成及其在线粒体生物能量分析和成像分析中的应用的详细方案。这些实验将允许使用脑类器官来研究代谢和发育功能障碍，并可能为剖析线粒体疾病的神经元病理学提供关键信息。
Introduction
线粒体疾病是最大的一类先天性代谢缺陷1。它们是由破坏不同线粒体过程的基因突变引起的，包括氧化磷酸化（OXPHOS）2，呼吸链组装，线粒体动力学和线粒体DNA转录或复制3。有能量需求的组织尤其受线粒体功能障碍的影响4。因此，患有线粒体疾病的患者通常会出现早发性神经系统表现。
目前尚无针对线粒体疾病患儿的治疗方法5。线粒体疾病药物开发的一个主要障碍是缺乏概括人类疾病过程的有效模型6。目前研究的几种动物模型没有表现出患者中存在的神经缺陷7。因此，线粒体疾病神经元病理学的潜在机制仍未完全清楚。
最近的研究从受线粒体疾病影响的患者中产生了iPSCs，并使用这些细胞来获得患者特异性神经元细胞。例如，已发现与线粒体疾病Leigh综合征相关的遗传缺陷可导致细胞生物能量学8，9，蛋白质合成10和钙稳态9，11的畸变。这些报告为线粒体疾病中发生的神经元损伤提供了重要的机制线索，为这些不治之症的药物发现铺平了道路12。
然而，二维（2D）文化无法调查3D器官的建筑复杂性和区域组织13。为此，使用源自患者特异性iPSCs14 的3D脑类器官可能使研究人员能够获得额外的重要信息，从而有助于剖析线粒体疾病如何影响神经系统的发育和功能15。使用iPSC衍生的脑类器官来研究线粒体疾病的研究开始揭示线粒体疾病的神经发育成分。
脊髓类器官携带与线粒体疾病、线粒体脑病、乳酸性酸中毒和卒中样发作综合征（MELAS）相关的突变，显示神经发生缺陷和运动神经元分化延迟16。来自线粒体疾病Leigh综合征患者的皮质类器官显示尺寸减小，神经上皮芽生成缺陷和皮质结构丧失17。来自Leigh综合征患者的脑类器官表明，疾病缺陷始于神经祖细胞的水平，其不能致力于线粒体代谢，导致异常神经元分支和形态发生18。因此，神经祖细胞可能代表线粒体疾病的细胞治疗靶点，促进其线粒体功能的策略可能支持神经系统的功能发育。
脑类器官的使用可能有助于揭示线粒体疾病的神经发育成分。线粒体疾病主要被认为是早发性神经变性5。然而，受线粒体疾病影响的患者也存在神经发育缺陷，包括发育迟缓和认知障碍19。患者特异性脑类器官可能有助于解决这些方面，并阐明线粒体疾病如何影响人脑发育。线粒体功能障碍也可能在其他更常见的神经系统疾病中起致病作用，例如阿尔茨海默病，帕金森病和亨廷顿病4。因此，使用脑类器官阐明线粒体缺陷对神经发育的影响也可能有助于研究这些疾病。本文描述了一种用于生成可重复脑类器官的详细方案，可用于进行线粒体疾病的疾病建模。
Protocol
注意：使用人类 iPSC 可能需要获得伦理批准。本研究中使用的iPSCs来自当地伦理批准后的健康对照个体（#2019-681）。所有细胞培养程序必须在无菌细胞培养罩下进行，在转移到罩下之前仔细消毒所有试剂和耗材。用于分化的人类iPSC的传代数应低于50，以避免在广泛培养时可能发生的潜在基因组畸变。细胞的多能状态应在类器官生成之前进行验证，例如，通过监测多能性相关标志物（如NANOG或OCT4）?…
Representative Results
这里描述的协议有助于圆类器官的稳健生成（图1A）。生成的类器官包含成熟的神经元，可以使用轴突（SMI312）和树突（微管相关蛋白2（MAP2））特异性的蛋白质标记物进行可视化（图1B） ）。成熟的类器官不仅含有神经元细胞（MAP2阳性），还含有神经胶质细胞（例如，星形胶质细胞标志物S100钙结合蛋白B（S100ß）…
Discussion
本文描述了人类iPSC衍生的脑类器官的可重复生成及其在线粒体疾病建模中的应用。此处描述的协议是根据以前发表的 work20 进行修改的。本方案的一个主要优点是它不需要手动将每个类器官嵌入到支架基质中。实际上，基质溶液只是简单地溶解到细胞培养基中。此外，无需使用昂贵的生物反应器，因为类器官可以在标准组织培养物中培养6孔板，放置在培养箱内的轨道振荡器上。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Miriam Bünning的技术支持。我们感谢德国联邦医疗协会（DFG）（PR1527/5-1至AP）、Spark和柏林卫生研究院（BIH）（BIH验证基金至AP）、联合线粒体疾病基金会（UMDF）（利综合征国际财团资助AP）、杜塞尔多夫大学医院（Forschungskommission UKD至AP）和德国联邦教育与研究部（BMBF）（e：生物年轻研究员向AP授予AZ 031L0211）。C.R.R.实验室的工作得到了DFG的支持（FOR 2795″压力下的突触”，Ro 2327/13-1）。
Materials
| 2-mercaptoethanol | Gibco | 31350-010 | |
| Affinity Designer | Serif (Europe) Ltd | Layout software; Vector graphics editor | |
| Alexa Fluor 488 donkey anti-guinea pig | Sigma Aldrich | SAB4600033-250UL | 1:300 |
| Alexa Fluor 488 donkey anti-mouse | Thermo Fisher Scientific | A-31571 | 1:300 |
| Antimycin A | Sigma Aldrich | 1397-94-0 | |
| Anti-β-Tubulin III (TUJ-1) | Sigma Aldrich | T8578 | 1:2000 |
| Argon Laser | Melles Griot | Any other Laser, e.g., diode lasers emitting 488 is fine, too | |
| Ascorbic acid | Sigma | A92902 | |
| B-27 with Vitamin A | Gibco | 17504044 | |
| Bacto Agar | Becton Dickinson | 3% in PBS, store solution at -20 °C | |
| BDNF | Miltenyi Biotec | 130-093-0811 | |
| cAMP | Sigma | D0627 | |
| Cell Star cell culture 6 well plate | Greiner-Bio-One | 657160 | |
| Chemically Defined Lipid Concentrate | Gibco | 11905031 | |
| Confocal laser scanning microscope C1 | Nikon Microscope Solutions | Modular confocal microscope system | |
| Corning Matrigel Growth Factor Reduced (GFR) Basement membrane matrix, Phenol Red-free, LDEV-free | Corning | 356231 | Matrix component |
| CyQUANT Cell Proliferation Assay Kit | Thermo Fisher | C7026 | |
| DMEM/F12 | ThermoFisher | 31330038 | |
| DMSO | Sigma | D2660-100ML | |
| Donkey anti-goat Cy3 | Merck Millipore | AP180C | 1:300 |
| Donkey anti-mouse Cy3 | Merck Millipore | AP192C | 1:300 |
| Donkey anti-rabbit Cy3 | Merck Millipore | AP182C | 1:300 |
| DPBS | Gibco | 14190250 | |
| DS-Q1Mc camera | Nikon Microscope Solutions | ||
| Eclipse 90i upright widefield microscope | Nikon Microscope Solutions | ||
| Eclipse E 600FN upright microscope | Nikon Microscope Solutions | ||
| Eclipse Ts2 Inverted Microscope | Nikon Microsope Solutions | ||
| EZ-C1 Silver Version 3.91 | Nikon Microscope Solutions | Imaging software for confocal microscope | |
| FCCP | Sigma Aldrich | 370-86-5 | |
| Fetal Bovine Serum | Gibco | 10270-106 | |
| GDNF | Miltenyi Biotec | 130-096-291 | |
| Glasgow MEM | Gibco | 11710-035 | |
| Glass Pasteur pipette | Brand | 747715 | Inverted |
| Glutamax | Gibco | 35050-061 | |
| Helium-Neon Laser | Melles Griot | Every other Laser, e.g., diode lasers emitting 594 is fine, too | |
| Heparin | Merck | H3149-25KU | |
| HERACell 240i CO2 Incubator | Thermo Scientific | 51026331 | |
| Hoechst 33342 | Invitrogen | H3570 | 1:2500 |
| Image J 1.53c | Wayne Rasband National Institute of Health | Image processing Software | |
| Injekt Solo 10 mL/ Luer | Braun | 4606108V | |
| Knockout Serum Replacement | Gibco | 10828010 | |
| Laser (407 nm) | Coherent | Any other Laser, e.g., diode lasers emitting 407 is fine, too | |
| Map2 | Synaptic Systems | No. 188004 | 1:1000 |
| Maxisafe 2030i | |||
| MEM NEAA | Gibco | 11140-050 | |
| mTeSR Plus | Stemcell Technology | 85850 | iPSC medium |
| Multifuge X3R Centrifuge | Thermo Scientific | 10325804 | |
| MycoAlert Mycoplasma Detection Kit | Lonza | # LT07-218 | |
| N2 Supplement | Gibco | 17502-048 | |
| Needle for single usage (23G x 1” TW) | Neoject | 10016 | |
| NIS-Elements Aadvanced Research 3.2 | Nikon | Imaging software | |
| Oligomycin A | Sigma Aldrich | 75351 | |
| Orbital Shaker Heidolph Unimax 1010 | Heidolph | 543-12310-00 | |
| PAP Pen | Sigma | Z377821-1EA | To draw hydrophobic barrier on slides. |
| Papain Dissociation System kit | Worthington | LK003150 | |
| Paraformaldehyde | Merck | 818715 | 4% in PBS, store solution at -20 °C |
| Pasteur pipette 7mL | VWR | 612-1681 | Graduated up to 3 mL |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| Plan Apo VC 20x / 0.75 air DIC N2 ∞/0.17 WD 1.0 | Nikon Microscope Solutions | Dry Microscope Objective | |
| Plan Apo VC 60x / 1.40 oil DIC N2 ∞/0.17 WD 0.13 | Nikon Microscope Solutions | Oil Immersion Microscope Objective | |
| Polystyrene Petri dish (100 mm) | Greiner Bio-One | 664161 | |
| Polystyrene round-bottom tube with cell-strainer cap (5 mL) | Falcon | 352235 | |
| Potassium chloride | Roth | 6781.1 | |
| ProLong Glass Antifade Moutant | Invitrogen | P36980 | |
| Qualitative filter paper | VWR | 516-0813 | |
| Rock Inhibitior | Merck | SCM075 | |
| Rotenone | Sigma | 83-794 | |
| S100β | Abcam | Ab11178 | 1:600 |
| SB-431542 | Cayman Chemical Company | 13031 | |
| Scalpel blades | Heinz Herenz Hamburq | 1110918 | |
| SMI312 | Biolegend | 837904 | 1:500 |
| Sodium bicarbonate | Merck/Sigma | 31437-1kg-M | |
| Sodium chloride | Roth | 3957 | |
| Sodium dihydrogen phosphate | Applichem | 131965 | |
| Sodium Pyruvate | Gibco | 11360070 | |
| SOX2 | Santa Cruz Biotechnology | Sc-17320 | 1:100 |
| StemPro Accutase Cell Dissociation Reagent | Gibco/StemPro | A1110501 | Reagent A |
| Super Glue Gel | UHU | 63261 | adhesive gel |
| SuperFrost Plus | VWR | 631-0108 | |
| Syringe for single usage (1 mL) | BD Plastipak | 300015 | |
| TB2 Thermoblock | Biometra | ||
| TC Plate 24 Well | Sarstedt | 83.3922 | |
| TC Plate 6 Well | Sarstedt | 83.392 | |
| TGFbeta3 | Miltenyi Biotec | 130-094-007 | |
| Tissue Culture Hood | ThermoFisher | 51032711 | |
| TOM20 | Santa Cruz Biotechnology | SC-11415 | 1:200 |
| Triton-X | Merck | X100-5ML | |
| UltraPure 0.5M EDTA | Invitrogen | 15575020 | |
| Vibratome Microm HM 650 V | Thermo Scientific | Production terminated, any other adjustable microtome is fine, too. | |
| Vibratome Wilkinson Classic Razor Blade | Wilkinson Sword | 70517470 | |
| Whatman Benchkote | Merck/Sigma | 28418852 | |
| Wnt Antagonist I | EMD Millipore Corp | 3378738 | |
| XF 96 extracellular flux analyser | Seahorse Bioscience | 100737-101 | |
| XF Assay DMEM Medium | Seahorse Bioscience | 103680-100 | |
| XF Calibrant Solution | Seahorse Bioscience | 100840-000 | |
| XFe96 FluxPak (96-well microplate) | Seahorse Bioscience | 102416-100 |
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Cite This Article
Le, S., Petersilie, L., Inak, G., Menacho-Pando, C., Kafitz, K. W., Rybak-Wolf, A., Rajewsky, N., Rose, C. R., Prigione, A. Generation of Human Brain Organoids for Mitochondrial Disease Modeling. J. Vis. Exp. (172), e62756, doi:10.3791/62756 (2021).