Flow cytometrisk analyse af flere mitokondrieparametre i humaninducerede pluripotente stamceller og deres neurale og gliale derivater
Summary
Denne undersøgelse rapporterer en ny tilgang til måling af flere mitokondrielle funktionelle parametre baseret på flowcytometri og dobbeltfarvning med to fluorescerende reportere eller antistoffer til påvisning af ændringer i mitokondrievolumen, mitokondriemembranpotentiale, reaktivt iltartsniveau, mitokondrie respiratorisk kædesammensætning og mitokondrie-DNA.
Abstract
Mitokondrier er vigtige i patofysiologien af mange neurodegenerative sygdomme. Ændringer i mitokondrievolumen, mitokondriemembranpotentiale (MMP), mitokondrieproduktion af reaktive iltarter (ROS) og mitokondrie-DNA (mtDNA) kopinummer er ofte træk ved disse processer. Denne rapport beskriver en ny flowcytometribaseret tilgang til måling af flere mitokondrieparametre i forskellige celletyper, herunder humaninducerede pluripotente stamceller (iPSC’er) og iPSC-afledte neurale og gliaceller. Denne flowbaserede strategi bruger levende celler til at måle mitokondrievolumen, MMP og ROS-niveauer samt faste celler til at estimere komponenter i mitokondrie-respiratorisk kæde (MRC) og mtDNA-associerede proteiner såsom mitokondrietranskriptionsfaktor A (TFAM).
Ved co-farvning med fluorescerende journalister, herunder MitoTracker Green (MTG), tetramethylrhodamin ethylester (TMRE) og MitoSox Red, kan ændringer i mitokondrievolumen, MMP og mitokondrie ROS kvantificeres og relateres til mitokondrieindhold. Dobbeltfarvning med antistoffer mod MRC-komplekse underenheder og translocase af ydre mitokondriemembran 20 (TOMM20) tillader vurdering af MRC-underenhedsekspression. Da mængden af TFAM er proportional med mtDNA-kopinummeret, giver målingen af TFAM pr. TOMM20 en indirekte måling af mtDNA pr. mitokondrievolumen. Hele protokollen kan udføres inden for 2-3 timer. Det er vigtigt, at disse protokoller tillader måling af mitokondrieparametre, både på det samlede niveau og det specifikke niveau pr. Mitokondrievolumen, ved hjælp af flowcytometri.
Introduction
Mitokondrier er essentielle organeller, der findes i næsten alle eukaryote celler. Mitokondrier er ansvarlige for energiforsyning ved at producere adenosintrifosfat (ATP) via oxidativ phosphorylering og fungere som metaboliske formidlere for biosyntese og metabolisme. Mitokondrier er dybt involveret i mange andre vigtige cellulære processer, såsom ROS-generering, celledød og intracellulær Ca2+ regulering. Mitokondrie dysfunktion har været forbundet med forskellige neurodegenerative sygdomme, herunder Parkinsons sygdom (PD), Alzheimers sygdom (AD), Huntingtons sygdom (HD), Friedreichs ataksi (FRDA) og amyotrofisk lateral sklerose (ALS)1. Øget mitokondrie dysfunktion og mtDNA abnormitet menes også at bidrage til menneskets aldring 2,3.
Forskellige typer mitokondriedysfunktion forekommer i neurodegenerative sygdomme, og ændringer i mitokondrievolumen, MMP-depolarisering, produktion af ROS og ændringer i mtDNA-kopinummer er almindelige 4,5,6,7. Derfor er evnen til at måle disse og andre mitokondriefunktioner af stor betydning, når man studerer sygdomsmekanismer og tester potentielle terapeutiske midler. I betragtning af manglen på dyremodeller, der trofast replikerer humane neurodegenerative sygdomme, er etablering af egnede in vitro-modelsystemer, der rekapitulerer den menneskelige sygdom i hjerneceller, et vigtigt skridt i retning af en større forståelse af disse sygdomme og udvikling af nye terapier 2,3,8,9.
Humane iPSC’er kan bruges til at generere forskellige hjerneceller, herunder neuronale og ikke-neuronale celler (dvs. gliaceller), og mitokondrieskader forbundet med neurodegenerativ sygdom er fundet i begge celletyper 3,10,11,12,13. Passende metoder til iPSC-differentiering i neurale og gliale afstamninger er tilgængelige14,15,16. Disse celler giver en unik menneskelig / patientplatform til in vitro sygdomsmodellering og lægemiddelscreening. Da disse stammer fra patienter, giver iPSC-afledte neuroner og gliaceller sygdomsmodeller, der mere præcist afspejler, hvad der sker hos mennesker.
Til dato er der kun få praktiske og pålidelige metoder til måling af flere mitokondrielle funktionelle parametre i iPSC’er, især levende neuroner og gliaceller, tilgængelige. Anvendelsen af flowcytometri giver forskeren et kraftfuldt værktøj til måling af biologiske parametre, herunder mitokondriefunktion, i enkeltceller. Denne protokol indeholder detaljer om generering af forskellige typer hjerneceller, herunder neurale stamceller (NSC’er), neuroner og glialastrocytter fra iPSC’er samt nye flowcytometribaserede tilgange til måling af flere mitokondrieparametre i forskellige celletyper, herunder iPSC’er og iPSC-afledte neurale og glialceller. Protokollen giver også en co-farvningsstrategi til brug af flowcytometri til måling af mitokondrievolumen, MMP, mitokondrie-ROS-niveau, MRC-komplekser og TFAM. Ved at inkorporere målinger af mitokondrievolumen eller masse tillader disse protokoller også måling af både det samlede niveau og det specifikke niveau pr. Mitokondrieenhed.
Protocol
Representative Results
Discussion
Heri er protokoller til generering af iPSC -afledte neuroner og astrocytter og evaluering af flere aspekter af mitokondriefunktion ved hjælp af flowcytometri. Disse protokoller muliggør effektiv omdannelse af humane iPSC’er til både neuroner og gliablissytter og den detaljerede karakterisering af mitokondriefunktionen, hovedsagelig i levende celler. Protokollerne giver også en co-farvning flow cytometri-baseret strategi til erhvervelse og analyse af flere mitokondriefunktioner, herunder volumen, MMP og mitokondrie RO…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Vi takker venligst Molecular Imaging Centre og Flow Cytometry Core Facility ved Universitetet i Bergen i Norge. Dette arbejde blev støttet af midler fra Det Norske Forskningsråd (Bevillingsnummer: 229652), Rakel og Otto Kr.Bruuns legat og China Scholarship Council (projektnummer: 201906220275).
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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