Name: flow cytometric analysis of multiple mitochondrial parameters in human induced pluripotent stem cells and their neural and glial derivatives
Uploaded: 2021-11-08T13:00:00.000+00:00
Duration: 6 min 9 s
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).

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The authors have no conflicts of interest to disclose.

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;levels in live cells and MRC complexes and TFAM in fixed cells. Specifically, these protocols permit the estimation of both total and specific levels per mitochondrial volume. While this strategy detects mitochondrial dysfunction in a known mitochondrial disease (POLG) in DA neurons and astrocytes, these techniques are applicable to any type of cell and disease. Moreover, the protocol is robust and reproducible. Several previous studies have successfully applied this protocol to analyze the mitochondrial changes in fibroblasts, iPSCs, NSCs, DA neurons, and astrocytes2,3,13,17.
There are some critical points to consider while executing this protocol. To ensure consistent and high-efficiency differentiation, it is critical to initiate the conversion with high-quality iPSCs (cells containing &#60;5% differentiated cells). Although other commercially available defined media can be valid alternatives, this study did not address the alternatives. As medium composition and clonal differences of iPSC lines can influence both proliferation of the starting cell population and differentiation efficiency, adapting this protocol to other maintenance media will likely require optimization.
The relationship between MTG and MMP fluorescence has been studied previously3. This is important as MTG fluorescence is reported to be both independent of18 and sensitive to MMP19,20. In previous studies in which iPSCs were titrated with different concentrations of TMRE and co-stained with 150 nM MTG, the MTG level remained the same at lower concentrations of TMRE (5-100 nM), whereas a decreased MTG signal was observed for higher TMRE concentrations (over 100 nM). Therefore, 100 nM TMRE and 150 nM MTG were chosen to measure the specific MMP. As this relationship may be cell-specific, the correlation between MTG and MMP fluorescence must be assessed before using MTG and TMRE dual staining to measure MMP.
Cell density can also influence mitochondrial function and cell metabolism. In this study, cell-density-dependent changes in MMP were observed, which has also been shown in other studies21. Therefore, it is important to choose a similar cell density in all samples-not too high or low-to minimize variation when establishing the co-staining protocol for different cell types. Compared with other microscopy-based assays, flow cytometry has the advantages of speed and reproducibility when analyzing large numbers of cells. In the analysis of microscopic images, the bias of researchers will distort the results to a certain extent, which is not a problem when using flow cytometry. In addition, flow cytometry analysis requires less than one million cells, and analysis of one sample only takes a few minutes, which means that dozens of samples can be analyzed in 1-2 h. This technique can also be applied to a wide variety of cell types, including those from other neurodegenerative diseases, and should therefore be useful for understanding mechanisms and testing potential therapeutics in different neurodegenerative diseases.

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><tbody><tr><td>anti-Oct4</td><td>Abcam</td><td>ab19857, RRID:AB_445175</td><td>Primary Antibody; use as 1:100, 10 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 488 goat anti-rabbit IgG&amp;nbsp; (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 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 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 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 488 goat anti-rabbit IgG&amp;nbsp; (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 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 488 goat anti-rabbit IgG&amp;nbsp; (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 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 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 &amp;mu;L in 1000 &amp;mu;L staining solution;&amp;nbsp; use Alexa Fluor &amp;reg; 594 goat anti-chicken IgG (1:800, Thermo Fisher Scientific, Catalog # A-11042) as secondary antibody.</td></tr><tr><td>anti-S100&amp;beta;&amp;nbsp; conjugated with Alexa Fluor 488</td><td>Abcam</td><td>ab196442, RRID:AB_2722596</td><td>Primary Antibody; use as 1:400, 2.5 &amp;mu;L in 1000 &amp;mu;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 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 488 goat anti-rabbit IgG&amp;nbsp; (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 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 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 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 488 goat anti-rabbit IgG&amp;nbsp; (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 &amp;mu;L in 1000 &amp;mu;L staining solution;&amp;nbsp; use Alexa Fluor &amp;reg; 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 &amp;mu;L in 1000 &amp;mu;L staining solution; use mouse monoclonal IgG2b&amp;nbsp; Alexa Fluor&amp;reg; 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 &amp;mu;L in 1000 &amp;mu;L staining solution; use mouse monoclonal IgG2a&amp;nbsp; Alexa Fluor&amp;reg; 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 &amp;mu;L in 1000 &amp;mu;L staining solution; use Alexa Fluor &amp;reg; 488 goat anti-rabbit IgG&amp;nbsp; (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 &amp;mu;L in 1000 &amp;mu;L staining solution;&amp;nbsp; use Alexa Fluor &amp;reg; 488 goat anti-rabbit IgG&amp;nbsp; (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 &amp;mu;L in 1000 &amp;mu;L staining solution; use&amp;nbsp; Alexa Fluor &amp;reg; 488 goat anti-mouse IgG&amp;nbsp; (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&amp;ndash;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&#039;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&amp;amp;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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

4.0K Views.  University of Bergen. Ons protocol biedt de wetenschapper een krachtig hulpmiddel voor het combineren van meerdere microparameters op het niveau van één cel van menselijke IPS en hun neuro- en heldere derivaten. Dit protocol maakt het mogelijk om mitochondriale parameters te verbeteren, zowel op het totale niveau als op het specifieke niveau per mitochondriaal volume, met behulp van flowcytometrie. Deze techniek kan worden toegepast op verschillende celtypen, waaronder die van andere neurodegeneratieve ziekten.

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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Developmental Biology

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

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 neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

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

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Biology

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain&#39;s three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

This protocol reports a unique method of using a streaming cytometer and multiple antibodies for simultaneous assessment of multiple mitochondrial functional parameters, including changes in mitochondrial volume, amounts of the mitochondrial respiratory chain (MRC) complex subunits, and mitochondrial DNA (mtDNA) replication.

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Measurement of Multiple Mitochondrial Parameters in Neurons using Flow Cytometry

Source: Liang, K. X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63116">Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives.</a> J. Vis. Exp. (2021)
This video demonstrates the labeling of neuronal cells with antibodies for mitochondrial respiratory chain complexes, mitochondrial DNA-associated protein, and mitochondrial outer membrane protein, followed by flow cytometry to quantify their levels.

Source: Liang, K. X., et al. Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and ...

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

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