Summary

Flow cytometrisk analyse af flere mitokondrieparametre i humaninducerede pluripotente stamceller og deres neurale og gliale derivater

Published: November 08, 2021
doi:

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

BEMÆRK: Se materialetabellen og den supplerende tabel S1 for opskrifter af alle medier og løsninger, der anvendes i denne protokol. 1. Differentiering af humane iPSC’er i NCS’er, dopaminerge (DA) neuroner og astrocytter Forbered matrixbelagte plader.Tø et hætteglas med 5 ml matrix på is natten over. Fortynd 1 ml matrix med 99 ml kold Advanced Dulbecco’s Modified Eagle Medium/Ham’s F-12 (Advanced DMEM/F12) (1% endelig kon…

Representative Results

En skematisk beskrivelse af differentieringsmetoden og flowcytometriske strategier er vist i figur 3. Humane iPSC’er differentieres til neurale rosetter og løftes derefter ind i suspensionskultur til differentiering i neurale sfærer. Neurale sfærer differentieres yderligere og modnes til DA-neuroner. Neurale kugler dissocieres i enkeltceller for at generere glial astrocytter, omlagt i monolag som NSC’er og derefter differentieret til astrocytter. Denne protokol giver de strategier, der er…

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…

Disclosures

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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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).

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