Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Summary

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Abstract

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM).

By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

Introduction

Mitochondria are essential organelles present in almost all eukaryotic cells. Mitochondria are responsible for energy supply by producing adenosine triphosphate (ATP) via oxidative phosphorylation and act as metabolic intermediaries for biosynthesis and metabolism. Mitochondria are deeply involved in many other important cellular processes, such as ROS generation, cell death, and intracellular Ca²⁺ regulation. Mitochondrial dysfunction has been associated with various neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Friedreich's ataxia (FRDA), and amyotrophic lateral scleros....

Protocol

NOTE: See the Table of Materials and the Supplemental Table S1 for recipes of all media and solutions used in this protocol.

1. Differentiation of human iPSCs into NCSs, dopaminergic (DA) neurons, and astrocytes

1. Prepare matrix-coated plates.
   1. Thaw a vial of 5 mL of matrix on ice overnight. Dilute 1 mL of matrix with 99 mL of cold Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 (Advanced DMEM/F12) (1% final c.......

Discussion

Herein are protocols for generating iPSC−derived neurons and astrocytes and evaluating multiple aspects of mitochondrial function using flow cytometry. These protocols allow efficient conversion of human iPSCs into both neurons and glial astrocytes and the detailed characterization of mitochondrial function, mostly in living cells. The protocols also provide a co-staining flow cytometry-based strategy for acquiring and analyzing multiple mitochondrial functions, including volume, MMP, and mitochondrial ROS lev.......

Materials

Name	Company	Catalog Number	Comments
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