Name: flow cytometric analysis of multiple mitochondrial parameters in human induced pluripotent stem cells and their neural and glial derivatives
Uploaded: 2021-11-08T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives at JoVE.com

We kindly thank the Molecular Imaging Centre and the Flow Cytometry Core Facility at the University of Bergen in Norway. This work was supported by funding from the Norwegian Research Council (Grant number: 229652), Rakel og Otto Kr.Bruuns legat and the China Scholarship Council (project number: 201906220275).

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
<ol>
	<li>Prepare matrix-coated plates.
	<ol>
		<li>Thaw a vial of 5 mL of matrix on ice overnight. Dilute 1 mL of matrix with 99 mL of cold Advanced Dulbecco&#39;s Modified Eagle Medium/Ham&#39;s F-12 (Advanced DMEM/F12) (1% final c...

<ol>
	<li>Wang, Y., Xu, E., Musich, P. R., Lin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+neurodegenerative+diseases+and+the+potential+countermeasure.">Mitochondrial dysfunction in neurodegenerative diseases and the potential countermeasure.</a> CNS Neuroscience &amp; Therapeutics. 25 (7), 816-824 (2019).</li><li>Chen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinamide+riboside+and+metformin+ameliorate+mitophagy+defect+in+induced+pluripotent+stem+cell-derived+astrocytes+with+POLG+mutations.">Nicotinamide riboside and metformin ameliorate mitophagy defect in induced pluripotent stem cell-derived astrocytes with POLG mutations.</a> Frontiers in Cell and Developmental Biology. 9, 737304 (2021).</li><li>Liang, K. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-specific+phenotypes+in+iPSC-derived+neural+stem+cells+with+POLG+mutations.">Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations.</a> EMBO Molecular Medicine. 12 (10), 12146 (2020).</li><li>Chen, H., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics--fusion,+fission,+movement,+and+mitophagy--in+neurodegenerative+diseases.">Mitochondrial dynamics--fusion, fission, movement, and mitophagy--in neurodegenerative diseases.</a> Human Molecular Genetics. 18, 169-176 (2009).</li><li>Lin, M. T., Beal, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+oxidative+stress+in+neurodegenerative+diseases.">Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases.</a> Nature. 443 (7113), 787-795 (2006).</li><li>Singh, A., Kukreti, R., Saso, L., Kukreti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress:+A+key+modulator+in+neurodegenerative+diseases.">Oxidative stress: A key modulator in neurodegenerative diseases.</a> Molecules. 24 (8), 1583 (2019).</li><li>Kondadi, A. K., Anand, R., Reichert, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interplay+between+cristae+biogenesis,+mitochondrial+dynamics+and+mitochondrial+DNA+integrity.">Functional interplay between cristae biogenesis, mitochondrial dynamics and mitochondrial DNA integrity.</a> International Journal of Molecular Sciences. 20 (17), 4311 (2019).</li><li>Sterneckert, J. L., Reinhardt, P., Sch&#246;ler, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+human+disease+using+stem+cell+models.">Investigating human disease using stem cell models.</a> Nature Reviews. Genetics. 15 (9), 625-639 (2014).</li><li>Patani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell+models+of+disease+and+the+prognosis+of+academic+medicine.">Human stem cell models of disease and the prognosis of academic medicine.</a> Nature Medicine. 26 (4), 449 (2020).</li><li>Liang, K. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetylcysteine+amide+ameliorates+mitochondrial+dysfunction+and+reduces+oxidative+stress+in+hiPSC-derived+dopaminergic+neurons+with+POLG+mutation.">N-acetylcysteine amide ameliorates mitochondrial dysfunction and reduces oxidative stress in hiPSC-derived dopaminergic neurons with POLG mutation.</a> Experimental Neurology. , 337 (2021).</li><li>Kikuchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPS+cell-derived+dopaminergic+neurons+function+in+a+primate+Parkinson's+disease+model.">Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model.</a> Nature. 548 (7669), 592-596 (2017).</li><li>Juopperi, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+generated+from+patient+induced+pluripotent+stem+cells+recapitulate+features+of+Huntington's+disease+patient+cells.">Astrocytes generated from patient induced pluripotent stem cells recapitulate features of Huntington's disease patient cells.</a> Molecular Brain. 5, 17 (2012).</li><li>Liang, K. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+derived+astrocytes+with+POLG+mutations+and+mitochondrial+dysfunction+including+abnormal+NAD++metabolism+is+toxic+for+neurons.">Stem cell derived astrocytes with POLG mutations and mitochondrial dysfunction including abnormal NAD+ metabolism is toxic for neurons.</a> bioRxiv. , (2020).</li><li>Liu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+crest+stem+cells+derived+from+human+ESCs+and+induced+pluripotent+stem+cells:+induction,+maintenance,+and+differentiation+into+functional+schwann+cells.">Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells: induction, maintenance, and differentiation into functional schwann cells.</a> Stem Cells Translational Medicine. 1 (4), 266-278 (2012).</li><li>Hong, Y. J., Do, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+lineage+differentiation+from+pluripotent+stem+cells+to+mimic+human+brain+tissues.">Neural lineage differentiation from pluripotent stem cells to mimic human brain tissues.</a> Frontiers in Bioengineering and Biotechnology. 7, 400 (2019).</li><li>Lundin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPS-derived+astroglia+from+a+stable+neural+precursor+state+show+improved+functionality+compared+with+conventional+astrocytic+models.">Human iPS-derived astroglia from a stable neural precursor state show improved functionality compared with conventional astrocytic models.</a> Stem Cell Reports. 10 (3), 1030-1045 (2018).</li><li>Liang, K. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetylcysteine+amide+ameliorates+mitochondrial+dysfunction+and+reduces+oxidative+stress+in+hiPSC-derived+dopaminergic+neurons+with+POLG+mutation.">N-acetylcysteine amide ameliorates mitochondrial dysfunction and reduces oxidative stress in hiPSC-derived dopaminergic neurons with POLG mutation.</a> Experimental Neurology. 337, 113536 (2021).</li><li>Pendergrass, W., Wolf, N., Poot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+MitoTracker+Green&#8482;+and+CMXrosamine+to+measure+changes+in+mitochondrial+membrane+potentials+in+living+cells+and+tissues.">Efficacy of MitoTracker Green&#8482; and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues.</a> Cytometry. Part A. 61 (2), 162-169 (2004).</li><li>Keij, J. F., Bell-Prince, C., Steinkamp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+of+mitochondrial+membranes+with+10-nonyl+acridine+orange,+MitoFluor+Green,+and+MitoTracker+Green+is+affected+by+mitochondrial+membrane+potential+altering+drugs.">Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs.</a> Cytometry. 39 (3), 203-210 (2000).</li><li>Buckman, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MitoTracker+labeling+in+primary+neuronal+and+astrocytic+cultures:+influence+of+mitochondrial+membrane+potential+and+oxidants.">MitoTracker labeling in primary neuronal and astrocytic cultures: influence of mitochondrial membrane potential and oxidants.</a> Journal of Neuroscience Methods. 104 (2), 165-176 (2001).</li><li>Zanchetta, L. M., Kirk, D., Lyng, F., Walsh, J., Murphy, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-density-dependent+changes+in+mitochondrial+membrane+potential+and+reactive+oxygen+species+production+in+human+skin+cells+post+sunlight+exposure.">Cell-density-dependent changes in mitochondrial membrane potential and reactive oxygen species production in human skin cells post sunlight exposure.</a> Photodermatology, Photoimmunology &amp; Photomedicine. 26 (6), 311-317 (2010).</li></ol>

Herein are protocols for generating iPSC&#8722;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&#160;lev...

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&#39;s disease (PD), Alzheimer&#39;s disease (AD), Huntington&#39;s disease (HD), Friedreich&#39;s ataxia (FRDA), and amyotrophic lateral sclerosis (ALS)1. Increased mitochondrial dysfunction and mtDNA abnormality are also thought to contribute to human aging2,3.
Various types of mitochondrial dysfunction occur in neurodegenerative diseases, and changes in mitochondrial volume, MMP depolarization, production of ROS, and alterations in mtDNA copy number are common4,5,6,7. Therefore, the ability to measure these and other mitochondrial functions is of great importance when studying disease mechanisms and testing potential therapeutic agents. Moreover, in view of the lack of animal models that faithfully replicate human neurodegenerative diseases, establishing suitable in vitro model systems that recapitulate the human disease in brain cells is an important step towards a greater understanding of these diseases and the development of new therapies2,3,8,9.
Human iPSCs can be used to generate various brain cells, including neuronal and non-neuronal cells (i.e., glial cells), and mitochondrial damage associated with neurodegenerative disease has been found in both cell types3,10,11,12,13. Appropriate methods for iPSC differentiation into neural and glial lineages are available14,15,16. These cells provide a unique human/patient platform for in vitro disease modeling and drug screening. Further, as these are derived from patients, iPSC-derived neurons and glial cells provide disease models that reflect what is happening in humans more accurately.
To date, few convenient and reliable methods for measuring multiple mitochondrial functional parameters in iPSCs, particularly living neurons and glial cells, are available. The use of flow cytometry provides the scientist with a powerful tool for measuring biological parameters, including mitochondrial function, in single cells. This protocol provides details for the generation of different types of brain cells, including neural stem cells (NSCs), neurons, and glial astrocytes from iPSCs, as well as novel flow cytometry-based approaches to measure multiple mitochondrial parameters in different cell types, including iPSCs and iPSC-derived neural and glial cells. The protocol also provides a co-staining strategy for using flow cytometry to measure mitochondrial volume, MMP, mitochondrial ROS level, MRC complexes, and TFAM. By incorporating measures of mitochondrial volume or mass, these protocols also allow the measurement of both total level and specific level per mitochondrial unit.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-Oct4</td>
			<td>Abcam</td>
			<td>ab19857, RRID:AB_445175</td>
			<td>Primary Antibody; use as 1:100, 10 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-rabbit IgG&#160; (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-SSEA4</td>
			<td>Abcam</td>
			<td>ab16287, RRID:AB_778073</td>
			<td>Primary Antibody; use as 1:100, 10 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-Sox2</td>
			<td>Abcam</td>
			<td>ab97959, RRID:AB_2341193</td>
			<td>Primary Antibody; use as 1:100, 10 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-rabbit IgG&#160; (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-Pax6</td>
			<td>Abcam</td>
			<td>ab5790, RRID:AB_305110</td>
			<td>Primary Antibody; use as 1:100, 10 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-rabbit IgG&#160; (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-Nestin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-23927, RRID:AB_627994</td>
			<td>Primary Antibody; use as 1:50, 20 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-GFAP</td>
			<td>Abcam</td>
			<td>ab4674, RRID:AB_304558</td>
			<td>Primary Antibody; use as 1:100, 10 &#956;L in 1000 &#956;L staining solution;&#160; use Alexa Fluor &#174; 594 goat anti-chicken IgG (1:800, Thermo Fisher Scientific, Catalog # A-11042) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-S100&#946;&#160; conjugated with Alexa Fluor 488</td>
			<td>Abcam</td>
			<td>ab196442, RRID:AB_2722596</td>
			<td>Primary Antibody; use as 1:400, 2.5 &#956;L in 1000 &#956;L staining solution;</td>
		</tr>
		<tr>
			<td>anti-TH</td>
			<td>Abcam</td>
			<td>ab75875, RRID:AB_1310786</td>
			<td>Primary Antibody; use as 1:100, 10 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-rabbit IgG&#160; (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-Tuj 1</td>
			<td>Abcam</td>
			<td>ab78078, RRID:AB_2256751</td>
			<td>Primary Antibody; use as 1:1000, 1 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 594 goat anti-mouse IgG (1:800, Thermo Fisher Scientific, Catalog # A-11005) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-Synaptophysin</td>
			<td>Abcam</td>
			<td>ab32127, RRID:AB_2286949</td>
			<td>Primary Antibody; use as 1:100, 10 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-rabbit IgG&#160; (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-PSD-95</td>
			<td>Abcam</td>
			<td>ab2723, RRID:AB_303248</td>
			<td>Primary Antibody; use as 1:100, 10 &#956;L in 1000 &#956;L staining solution;&#160; use Alexa Fluor &#174; 594 goat anti-chicken IgG (1:800, Thermo Fisher Scientific, Catalog # A-11042) as secondary antibody.</td>
		</tr>
		<tr>
			<td>anti-TFAM conjugated with Alexa Fluor 488</td>
			<td>Abcam</td>
			<td>ab198308</td>
			<td>Primary Antibody; use as 1:400, 2.5 &#956;L in 1000 &#956;L staining solution; use mouse monoclonal IgG2b&#160; Alexa Fluor&#174; 488 as an isotype control.</td>
		</tr>
		<tr>
			<td>anti-TOMM20 conjugated with Alexa Fluor 488</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Cat# sc-17764 RRID:AB_628381</td>
			<td>Primary Antibody; use as 1:400, 2.5 &#956;L in 1000 &#956;L staining solution; use mouse monoclonal IgG2a&#160; Alexa Fluor&#174; 488 as an isotype control.</td>
		</tr>
		<tr>
			<td>anti-NDUFB10</td>
			<td>Abcam</td>
			<td>ab196019</td>
			<td>Primary Antibody; use as 1:1000, 1 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-rabbit IgG&#160; (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control.</td>
		</tr>
		<tr>
			<td>anti-SDHA</td>
			<td>Abcam</td>
			<td>ab137040</td>
			<td>Primary Antibody; use as 1:1000, 1 &#956;L in 1000 &#956;L staining solution;&#160; use Alexa Fluor &#174; 488 goat anti-rabbit IgG&#160; (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control.</td>
		</tr>
		<tr>
			<td>anti-COX IV</td>
			<td>Abcam</td>
			<td>ab14744, RRID:AB_301443</td>
			<td>Primary Antibody; use as 1:1000, 1 &#956;L in 1000 &#956;L staining solution; use&#160; Alexa Fluor &#174; 488 goat anti-mouse IgG&#160; (1:400, Thermo Fisher Scientific, Catalog # A-11001) as secondary antibody; use mouse monoclonal IgG as an isotype control.</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>PeproTech</td>
			<td>120-14E</td>
			<td>Astrocyte differentiation medium ingredient</td>
		</tr>
		<tr>
			<td>ABM Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3187</td>
			<td>Basal medium for astrocyte culture</td>
		</tr>
		<tr>
			<td>AGM SingleQuots Supplement Pack</td>
			<td>Lonza</td>
			<td>CC-4123</td>
			<td>Supplement for astrocyte culture</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td>CDM ingredient</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634010</td>
			<td>Basal medium for dilute Geltrex</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Europa Bioproducts</td>
			<td>EQBAH62-1000</td>
			<td>Blocking agent to prevent non-specific binding of antibodies in immunostaining assays and CDM ingredient</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>PeproTech</td>
			<td>450-02</td>
			<td>DA neurons medium ingredient</td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td>Astrocyte differentiation medium ingredient</td>
		</tr>
		<tr>
			<td>BD Accuri C6 Plus Flow Cytometer</td>
			<td>BD Biosciences, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11905031</td>
			<td>CDM ingredient</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Thermo Fisher Scientific</td>
			<td>17104019</td>
			<td>Reagent for gentle dissociation of human iPSCs</td>
		</tr>
		<tr>
			<td>CCD Microscope Camera Leica DFC3000 G</td>
			<td>Leica Microsystems, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning non-treated culture dishes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430589</td>
			<td>Suspension culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190250</td>
			<td>Used for a variety of cell culture wash</td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10565018</td>
			<td>Astrocyte differentiation basal Medium</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td>Reagent for gentle dissociation of human iPSCs</td>
		</tr>
		<tr>
			<td>Essential 8 Basal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1516901</td>
			<td>Basal medium for iPSC culture</td>
		</tr>
		<tr>
			<td>Essential 8 Supplement (50X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1517101</td>
			<td>Supplement for iPSC culture</td>
		</tr>
		<tr>
			<td>EGF Recombinant Human Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0314</td>
			<td>Supplement for NSC culture</td>
		</tr>
		<tr>
			<td>FGF-basic (AA 10&#8211;155) Recombinant Human Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0024</td>
			<td>Supplement for NSC culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12103C</td>
			<td>Medium ingredient</td>
		</tr>
		<tr>
			<td>FGF-basic</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>Astrocyte differentiation medium ingredient</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Abcam</td>
			<td>ab120081</td>
			<td>Eliminates mitochondrial membrane potential and TMRE staining</td>
		</tr>
		<tr>
			<td>Fluid aspiration system BVC control</td>
			<td>Vacuubrand, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde (PFA) 16%</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>Cell fixation</td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1413302</td>
			<td>Used for attachment and maintenance of human iPSCs</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Supplement for NSC culture</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td>DA neurons medium ingredient</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td>Used for blocking buffer</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>31765027</td>
			<td>Basal medium for CDM</td>
		</tr>
		<tr>
			<td>Heregulin beta-1 human</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3055</td>
			<td>Astrocyte differentiation medium ingredient</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H1399</td>
			<td>Stain the nuclei for confocal image</td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubators</td>
			<td>Fisher Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Thermo Fisher Scientific</td>
			<td>21980032</td>
			<td>Basal medium for CDM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Roche</td>
			<td>1376497</td>
			<td>CDM ingredient</td>
		</tr>
		<tr>
			<td>InSolution AMPK Inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>171261</td>
			<td>Neural induction medium ingredient</td>
		</tr>
		<tr>
			<td>Insulin-like Growth Factor-I human</td>
			<td>Sigma-Aldrich</td>
			<td>I3769</td>
			<td>Astrocyte differentiation medium ingredient</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>12660012</td>
			<td>Basal medium for NSC culture</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Promotes attachment and growth of neural cells in vitro</td>
		</tr>
		<tr>
			<td>Leica TCS SP8 STED confocal microscope</td>
			<td>Leica Microsystems, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monothioglycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M6145</td>
			<td>CDM ingredient</td>
		</tr>
		<tr>
			<td>MitoTracker Green FM</td>
			<td>Thermo Fisher Scientific</td>
			<td>M7514</td>
			<td>Used for mitochondrial volume indicator</td>
		</tr>
		<tr>
			<td>MitoSox Red</td>
			<td>Thermo Fisher Scientific</td>
			<td>M36008</td>
			<td>Used for mitochondrial ROS indicator</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A7250</td>
			<td>Neural induction medium ingredient</td>
		</tr>
		<tr>
			<td>N-2 Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td>Astrocyte differentiation medium ingredient</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>PCN5000</td>
			<td>Used for blocking buffer</td>
		</tr>
		<tr>
			<td>Orbital shakers - SSM1</td>
			<td>Stuart Equipment, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4957</td>
			<td>Promotes attachment and growth of neural cells in vitro</td>
		</tr>
		<tr>
			<td>Poly-D-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P7405</td>
			<td>Promotes attachment and growth of neural cells in vitro</td>
		</tr>
		<tr>
			<td>Purmorphamine</td>
			<td>STEMCELL Technologies</td>
			<td>72204</td>
			<td>Promotes DA neuron differentiation</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36930</td>
			<td>Mounting the coverslip for confocal image</td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Thermo Fisher Scientific</td>
			<td>18912014</td>
			<td>Used for a variety of wash</td>
		</tr>
		<tr>
			<td>Recombinant Human/Mouse FGF-8b Protein</td>
			<td>R&#38;D Systems</td>
			<td>423-F8-025/CF</td>
			<td>Promotes DA neuron differentiation</td>
		</tr>
		<tr>
			<td>SB 431542</td>
			<td>Tocris Bioscience</td>
			<td>TB1614-GMP</td>
			<td>Neural Induction Medium ingredient</td>
		</tr>
		<tr>
			<td>StemPro Neural Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10508-01</td>
			<td>Supplement for NSCs culture</td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604013</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Roche</td>
			<td>652202</td>
			<td>CDM ingredient</td>
		</tr>
		<tr>
			<td>TRITON X-100</td>
			<td>VWR International</td>
			<td>9002-93-1</td>
			<td>Used for cells permeabilization in immunostaining assays</td>
		</tr>
		<tr>
			<td>TMRE</td>
			<td>Abcam</td>
			<td>ab113852</td>
			<td>Used for mitochondrial membrane potential staining</td>
		</tr>
		<tr>
			<td>Water Bath Jb Academy Basic Jba5 JBA5 Grant Instruments</td>
			<td>Grant Instruments, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of multiple mitochondrial parameters in human induced pluripotent stem cells and their neural and glial derivatives

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.

A schematic description of the differentiation method and flow cytometric strategies is shown in Figure 3. Human iPSCs are differentiated into neural rosettes and then lifted into suspension culture for differentiation into neural spheres. Neural spheres are further differentiated and matured into DA neurons. Neural spheres are dissociated into single cells to generate glial astrocytes, replated in monolayers as NSCs, and then differentiated into astrocytes. This protocol provides the strate...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives at JoVE.com

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

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.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Our protocol provides the scientist with a powerful tool for marrying multiple micro parameters at single cell level from human IPS and their neuro and clear derivatives. This protocols allow the merriment of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry. This technique can be applied to several cell types including those from other neurodegenerative diseases.So, it can thereby help provide insight into the mechanisms behind these disease types and also potentially aid in therapeutic screening. To begin, seed the cells separately into four wells in a six well plate and incubate the cells until 50 to 60%confluency is achieved. At the end of the culture period, prepare five individual staining solutions containing different combinations of culture medium, FCCP, TMRE, MTG, and mito sock spread.Add them into the respective wells, and incubate the cells as described in the manuscript. Next, aspirate the medium from all the wells and wash with PBS. For cell detachment, incubate the cells with one milliliter of cell dissociation reagent at 37 degree Celsius for five minutes.Neutralize the cell dissociation reagent with one milliliter of DMEM, supplemented with 10%FBS. Then collect the well content in a 15 milliliter chronicle tube. Pellet down the cells by centrification and wash the cell pellet with PBS once or twice.Aspirate all the supernatant, leaving approximately 100 microliters in the tube. Re-suspend the cell pellets in 300 microliters of PBS. After transferring the cell suspension into a 1.5 milliliter micro centrifuge tube, keep the tube in the dark at room temperature.Analyze the cells using the flow cytometer with the band pass filter settings described in the text manuscript. Detach approximately 1 million cells using the cell dissociation reagent and pellet down the cells as demonstrated previously. Neutralize the cell suspension with DMEM plus 10%FBS and collect the suspension in a 15 milliliter tube.Fix the cells with one milliliter of 1.6%paraformaldehyde at room temperature for 10 minutes. Permeabolize the cells with one milliliter of ice cold 90%methanol at minus 20 degrees Celsius for 20 minutes. Then block the samples in one milliliter of blocking buffer, and wash the cells by centrifugation with PBS, as demonstrated.To detect different mitochondrial complexes and subunits, incubate the cells for 30 minutes with the respective primary antibodies described in the text manuscript. After washing the cells once with PBS, incubate the cells with secondary antibody for 30 minutes. At the end of the incubation, wash and re-suspend the cells and PBS as demonstrated.Transfer the cell suspension into a 1.5 milliliter micro centrifuge tube, kept in the dark on ice. Next, analyze the cells on the flow cytometer and detect signals and filter one using a 5 30/30 band pass filter. For each cell subpopulation, select a histogram plot and analyze the median fluorescence intensity or MFI of the different filter channels.Calculate the TMRE levels, specific values for MMP ROS complex subunit and TFAM, as described in the text manuscript. Flow cytometry and live cells was used to investigate MMP mitochondrial volume and ROS levels. In POLG DA neurons, lower specific MMP and higher specific ROS was observed, while no changes in mitochondrial volume, total MMP and ROS.POLG astrocytes had decreased MMP, but no change in mitochondrial volume and ROS. Flow cytometry and fixed cells was used to investigate the level of MRC complex subunit and TFAM. DA neurons showed increased complex one and TFAM versus control, but no change in complex two level.POLG astrocytes showed a reduction of complex one four and specific TFAM. As the cell density can also influence MMP and the relationship between MTG and MMP fluorescence, this is cell specific. Adapting this protocol to other cell types will likely require optimization Following this procedure, Microscopic based asis, for example confocal microcopy can be performed, providing additional details of mitochondrial structures.So, while this strategy detects mitochondrial dysfunction in DA neurons and astrocytes from a known mitochondrial disease, these techniques can also be applied to explore mitochondrial function in any type of cell and disease.

Research

JoVE Journal

Neuroscience

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...