人诱导多能干细胞及其神经和神经胶质衍生物中多种线粒体参数的流式细胞术分析
1Department of Clinical Medicine (K1),University of Bergen, 2Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases,Haukeland University Hospital, 3Department of Neurosurgery, Qilu Hospital and Institute of Brain and Brain-Inspired Science, Cheeloo College of Medicine,Shandong University, 4Shandong Key Laboratory of Brain Function Remodeling
Summary
本研究报道了一种基于流式细胞术和两种荧光报告基因或抗体双重染色测量多种线粒体功能参数的新方法，以检测线粒体体积、线粒体膜电位、活性氧水平、线粒体呼吸链组成和线粒体 DNA 的变化。
Abstract
线粒体在许多神经退行性疾病的病理生理学中很重要。线粒体体积、线粒体膜电位 （MMP）、线粒体活性氧产生 （ROS） 和线粒体 DNA （mtDNA） 拷贝数的变化通常是这些过程的特征。本报告详细介绍了一种基于流式细胞术的新型方法，用于测量不同细胞类型中的多种线粒体参数，包括人诱导多能干细胞（iPSC）和iPSC来源的神经和神经胶质细胞。这种基于流动的策略使用活细胞来测量线粒体体积、MMP 和 ROS 水平，并使用固定细胞来估计线粒体呼吸链 （MRC） 和 mtDNA 相关蛋白质（如线粒体转录因子 A （TFAM））的成分。
通过与荧光报告基因（包括 MitoTracker Green （MTG）、四甲基罗丹明乙酯 （TMRE） 和 MitoSox Red 共同染色，可以量化线粒体体积、MMP 和线粒体 ROS 的变化，并与线粒体含量相关。用针对MRC复合物亚基和线粒体外膜20转位酶（TOMM20）的抗体进行双重染色，可以评估MRC亚基的表达。由于TFAM的量与mtDNA拷贝数成正比，因此每个TOMM20的TFAM测量可以间接测量每个线粒体体积的mtDNA。整个协议可以在2-3小时内进行。重要的是，这些协议允许使用流式细胞术测量线粒体参数，包括总水平和每线粒体体积的特定水平。
Introduction
线粒体是存在于几乎所有真核细胞中的必需细胞器。线粒体通过氧化磷酸化产生三磷酸腺苷（ATP）来负责能量供应，并作为生物合成和代谢的代谢中介。线粒体深入参与许多其他重要的细胞过程，如ROS生成、细胞死亡和细胞内Ca2+ 调节。线粒体功能障碍与各种神经退行性疾病有关，包括帕金森病 （PD）、阿尔茨海默病 （AD）、亨廷顿病 （HD）、弗里德赖希共济失调 （FRDA） 和肌萎缩侧索硬化症 （ALS）1。线粒体功能障碍和mtDNA异常的增加也被认为有助于人类衰老2，3。
神经退行性疾病中会出现各种类型的线粒体功能障碍，线粒体体积的变化、MMP去极化、ROS的产生和mtDNA拷贝数的改变是常见的4，5，6，7。因此，在研究疾病机制和测试潜在治疗剂时，测量这些和其他线粒体功能的能力非常重要。此外，鉴于缺乏忠实复制人类神经退行性疾病的动物模型，建立合适的体外模型系统来概括脑细胞中的人类疾病是朝着更好地了解这些疾病和开发新疗法迈出的重要一步2，3，8，9。
人iPSCs可用于产生各种脑细胞，包括神经元和非神经元细胞（即神经胶质细胞），并且在两种细胞类型中都发现了与神经退行性疾病相关的线粒体损伤3，10，11，12，13。iPSC分化为神经和神经胶质谱系的适当方法可用14，15，16。这些细胞为体外疾病建模和药物筛选提供了独特的人/患者平台。此外，由于这些来自患者，iPSC衍生的神经元和神经胶质细胞提供了更准确地反映人类正在发生的事情的疾病模型。
迄今为止，很少有方便可靠的方法来测量iPSCs中的多个线粒体功能参数，特别是活神经元和神经胶质细胞。流式细胞术的使用为科学家提供了一种强大的工具来测量单细胞中的生物学参数，包括线粒体功能。该协议提供了生成不同类型的脑细胞的详细信息，包括来自 iPSC 的神经干细胞 （NSC）、神经元和神经胶质星形胶质细胞，以及基于流式细胞术的新型方法，用于测量不同细胞类型中的多个线粒体参数，包括 iPSC 和 iPSC 衍生的神经和神经胶质细胞。该协议还提供了使用流式细胞术测量线粒体体积、MMP、线粒体 ROS 水平、MRC 复合物和 TFAM 的共染色策略。通过结合线粒体体积或质量的测量，这些协议还允许测量每个线粒体单位的总水平和特定水平。
Protocol
注意：请参阅 材料 表和 补充表S1 ，了解本协议中使用的所有培养基和溶液的配方。 1. 人 iPSC 分化为 NCS、多巴胺能 （DA） 神经元和星形胶质细胞 准备基质包被的板。将一瓶 5 mL 基质在冰上解冻过夜。用 99 mL 冷的高级杜尔贝克改良鹰培养基/火腿的 F-12（高级 DMEM/F12）（1% 终浓度）稀释 1 mL 基质。制作1mL等分试样并将其储存?…
Representative Results
分化方法和流式细胞术策略的示意图如图 3所示。将人iPSC分化为神经花环，然后提升到悬浮培养物中分化为神经球体。神经球进一步分化并成熟为DA神经元。神经球被解离成单细胞以产生神经胶质星形胶质细胞，作为NSC重新铺在单层中，然后分化成星形胶质细胞。该协议提供了通过流式细胞术获取和分析样品所需的策略，以测量MMP，线粒体体积，线粒体ROS水平，MRC复合物亚?…
Discussion
以下是用于生成iPSC衍生的神经元和星形胶质细胞以及使用流式细胞术评估线粒体功能的多个方面的协议。这些方案允许将人iPSCs有效地转化为神经元和神经胶质星形胶质细胞，并详细表征线粒体功能，主要是在活细胞中。该协议还提供了基于共染色流式细胞术的策略，用于获取和分析多种线粒体功能，包括活细胞中的体积、MMP 和线粒体 ROS 水平以及固定细胞中的 MRC 复合物和 TFAM。具体来说，这些…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们衷心感谢挪威卑尔根大学的分子成像中心和流式细胞术核心设施。这项工作得到了挪威研究委员会（资助号：229652），Rakel og Otto Kr.Bruuns legat和中国国家留学基金委（项目编号：201906220275）的资助。
Materials
| anti-Oct4 | Abcam | ab19857, RRID:AB_445175 | Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody. |
| anti-SSEA4 | Abcam | ab16287, RRID:AB_778073 | Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody. |
| anti-Sox2 | Abcam | ab97959, RRID:AB_2341193 | Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody. |
| anti-Pax6 | Abcam | ab5790, RRID:AB_305110 | Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody. |
| anti-Nestin | Santa Cruz Biotechnology | sc-23927, RRID:AB_627994 | Primary Antibody; use as 1:50, 20 μL in 1000 μL staining solution; use Alexa Fluor ® 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody. |
| anti-GFAP | Abcam | ab4674, RRID:AB_304558 | Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 594 goat anti-chicken IgG (1:800, Thermo Fisher Scientific, Catalog # A-11042) as secondary antibody. |
| anti-S100β conjugated with Alexa Fluor 488 | Abcam | ab196442, RRID:AB_2722596 | Primary Antibody; use as 1:400, 2.5 μL in 1000 μL staining solution; |
| anti-TH | Abcam | ab75875, RRID:AB_1310786 | Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody. |
| anti-Tuj 1 | Abcam | ab78078, RRID:AB_2256751 | Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody. |
| anti-Synaptophysin | Abcam | ab32127, RRID:AB_2286949 | Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody. |
| anti-PSD-95 | Abcam | ab2723, RRID:AB_303248 | Primary Antibody; use as 1:100, 10 μL in 1000 μL staining solution; use Alexa Fluor ® 594 goat anti-chicken IgG (1:800, Thermo Fisher Scientific, Catalog # A-11042) as secondary antibody. |
| anti-TFAM conjugated with Alexa Fluor 488 | Abcam | ab198308 | Primary Antibody; use as 1:400, 2.5 μL in 1000 μL staining solution; use mouse monoclonal IgG2b Alexa Fluor® 488 as an isotype control. |
| anti-TOMM20 conjugated with Alexa Fluor 488 | Santa Cruz Biotechnology | Cat# sc-17764 RRID:AB_628381 | Primary Antibody; use as 1:400, 2.5 μL in 1000 μL staining solution; use mouse monoclonal IgG2a Alexa Fluor® 488 as an isotype control. |
| anti-NDUFB10 | Abcam | ab196019 | Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control. |
| anti-SDHA | Abcam | ab137040 | Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control. |
| anti-COX IV | Abcam | ab14744, RRID:AB_301443 | Primary Antibody; use as 1:1000, 1 μL in 1000 μL staining solution; use Alexa Fluor ® 488 goat anti-mouse IgG (1:400, Thermo Fisher Scientific, Catalog # A-11001) as secondary antibody; use mouse monoclonal IgG as an isotype control. |
| Activin A | PeproTech | 120-14E | Astrocyte differentiation medium ingredient |
| ABM Basal Medium | Lonza | CC-3187 | Basal medium for astrocyte culture |
| AGM SingleQuots Supplement Pack | Lonza | CC-4123 | Supplement for astrocyte culture |
| Antibiotic-Antimycotic | Thermo Fisher Scientific | 15240062 | CDM ingredient |
| Advanced DMEM/F-12 | Thermo Fisher Scientific | 12634010 | Basal medium for dilute Geltrex |
| Bovine Serum Albumin | Europa Bioproducts | EQBAH62-1000 | Blocking agent to prevent non-specific binding of antibodies in immunostaining assays and CDM ingredient |
| BDNF | PeproTech | 450-02 | DA neurons medium ingredient |
| B-27 Supplement | Thermo Fisher Scientific | 17504044 | Astrocyte differentiation medium ingredient |
| BD Accuri C6 Plus Flow Cytometer | BD Biosciences, USA | ||
| Chemically Defined Lipid Concentrate | Thermo Fisher Scientific | 11905031 | CDM ingredient |
| Collagenase IV | Thermo Fisher Scientific | 17104019 | Reagent for gentle dissociation of human iPSCs |
| CCD Microscope Camera Leica DFC3000 G | Leica Microsystems, Germany | ||
| Corning non-treated culture dishes | Sigma-Aldrich | CLS430589 | Suspension culture |
| DPBS | Thermo Fisher Scientific | 14190250 | Used for a variety of cell culture wash |
| DMEM/F-12, GlutaMAX supplement | Thermo Fisher Scientific | 10565018 | Astrocyte differentiation basal Medium |
| EDTA | Thermo Fisher Scientific | 15575020 | Reagent for gentle dissociation of human iPSCs |
| Essential 8 Basal Medium | Thermo Fisher Scientific | A1516901 | Basal medium for iPSC culture |
| Essential 8 Supplement (50X) | Thermo Fisher Scientific | A1517101 | Supplement for iPSC culture |
| EGF Recombinant Human Protein | Thermo Fisher Scientific | PHG0314 | Supplement for NSC culture |
| FGF-basic (AA 10–155) Recombinant Human Protein | Thermo Fisher Scientific | PHG0024 | Supplement for NSC culture |
| Fetal Bovine Serum | Sigma-Aldrich | 12103C | Medium ingredient |
| FGF-basic | PeproTech | 100-18B | Astrocyte differentiation medium ingredient |
| FCCP | Abcam | ab120081 | Eliminates mitochondrial membrane potential and TMRE staining |
| Fluid aspiration system BVC control | Vacuubrand, Germany | ||
| Formaldehyde (PFA) 16% | Thermo Fisher Scientific | 28908 | Cell fixation |
| Geltrex | Thermo Fisher Scientific | A1413302 | Used for attachment and maintenance of human iPSCs |
| GlutaMAX Supplement | Thermo Fisher Scientific | 35050061 | Supplement for NSC culture |
| GDNF | Peprotech | 450-10 | DA neurons medium ingredient |
| Glycine | Sigma-Aldrich | G8898 | Used for blocking buffer |
| Ham's F-12 Nutrient Mix | Thermo Fisher Scientific | 31765027 | Basal medium for CDM |
| Heregulin beta-1 human | Sigma-Aldrich | SRP3055 | Astrocyte differentiation medium ingredient |
| Hoechst 33342 | Thermo Fisher Scientific | H1399 | Stain the nuclei for confocal image |
| Heracell 150i CO2 Incubators | Fisher Scientific, USA | ||
| IMDM | Thermo Fisher Scientific | 21980032 | Basal medium for CDM |
| Insulin | Roche | 1376497 | CDM ingredient |
| InSolution AMPK Inhibitor | Sigma-Aldrich | 171261 | Neural induction medium ingredient |
| Insulin-like Growth Factor-I human | Sigma-Aldrich | I3769 | Astrocyte differentiation medium ingredient |
| KnockOut DMEM/F-12 medium | Thermo Fisher Scientific | 12660012 | Basal medium for NSC culture |
| Laminin | Sigma-Aldrich | L2020 | Promotes attachment and growth of neural cells in vitro |
| Leica TCS SP8 STED confocal microscope | Leica Microsystems, Germany | ||
| Monothioglycerol | Sigma-Aldrich | M6145 | CDM ingredient |
| MitoTracker Green FM | Thermo Fisher Scientific | M7514 | Used for mitochondrial volume indicator |
| MitoSox Red | Thermo Fisher Scientific | M36008 | Used for mitochondrial ROS indicator |
| N-Acetyl-L-cysteine | Sigma-Aldrich | A7250 | Neural induction medium ingredient |
| N-2 Supplement | Thermo Fisher Scientific | 17502048 | Astrocyte differentiation medium ingredient |
| Normal goat serum | Thermo Fisher Scientific | PCN5000 | Used for blocking buffer |
| Orbital shakers – SSM1 | Stuart Equipment, UK | ||
| Poly-L-ornithine solution | Sigma-Aldrich | P4957 | Promotes attachment and growth of neural cells in vitro |
| Poly-D-lysine hydrobromide | Sigma-Aldrich | P7405 | Promotes attachment and growth of neural cells in vitro |
| Purmorphamine | STEMCELL Technologies | 72204 | Promotes DA neuron differentiation |
| ProLong Gold Antifade Mountant | Thermo Fisher Scientific | P36930 | Mounting the coverslip for confocal image |
| PBS 1x | Thermo Fisher Scientific | 18912014 | Used for a variety of wash |
| Recombinant Human/Mouse FGF-8b Protein | R&D Systems | 423-F8-025/CF | Promotes DA neuron differentiation |
| SB 431542 | Tocris Bioscience | TB1614-GMP | Neural Induction Medium ingredient |
| StemPro Neural Supplement | Thermo Fisher Scientific | A10508-01 | Supplement for NSCs culture |
| TrypLE Express Enzyme | Thermo Fisher Scientific | 12604013 | Cell dissociation reagent |
| Transferrin | Roche | 652202 | CDM ingredient |
| TRITON X-100 | VWR International | 9002-93-1 | Used for cells permeabilization in immunostaining assays |
| TMRE | Abcam | ab113852 | Used for mitochondrial membrane potential staining |
| Water Bath Jb Academy Basic Jba5 JBA5 Grant Instruments | Grant Instruments, USA |
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Cite This Article
Liang, K. X., Chen, A., Kristiansen, C. K., Bindoff, L. A. Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives. J. Vis. Exp. (177), e63116, doi:10.3791/63116 (2021).