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 sclerosis (ALS)¹. Increased mitochondrial dysfunction and mtDNA abnormality are also thought to contribute to human aging^2,3.

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 common^4,5,6,7. 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 therapies^2,3,8,9.

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 types^{3,10,11,12,13}. Appropriate methods for iPSC differentiation into neural and glial lineages are available^14,15,16. 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.

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 concentration). Make 1 mL aliquots and store them at -20 °C.
   2. Thaw the matrix solution at 4 °C (keep it cold) and coat 6 wells (1 mL per well in a 6-well plate).
   3. Place the matrix-coated plate in a humidified 5% CO₂/95% air incubator at 37 °C for 1 h. Take the plate out of the incubator and let it equilibrate to room temperature (RT).
      ​NOTE: It is recommended to use the plate within 3 days of coating. However, the coated plate can be stored for up to 2 weeks at 4 °C. Just remember to take it out and let it warm up to RT before use. For longtime storage, add 1 mL of iPSC culture medium to the coated plate to avoid drying of the matrix.
2. Thawing iPSCs
   1. Prewarm the matrix-coated plates at RT or in the incubator at 37 °C for 20-30 min. Prewarm the required amount of iPSC culture medium at RT.
   2. Mix 6 mL of prewarmed iPSC culture medium with 12 µL of Y-27632 ROCK inhibitor to obtain a final concentration of 10 µM.
   3. Partially thaw the frozen vial of iPSCs at 37 °C in a water bath until a small piece of ice remains.
   4. Slowly add 1 mL of prewarmed iPSC culture medium with 12 µL of ROCK inhibitor dropwise to the cells. Transfer the liquid content of the vial with iPSCs dropwise into one well of a 6-well precoated plate using a 5 mL pipette.
   5. Move the plate in perpendicular directions to mix the well contents and return the plate to the incubator. Change the iPSC culture medium after 24 h after washing with Dulbecco's phosphate-buffered saline (DPBS) (1x) (Ca²⁺/Mg²⁺-free) (4 mL per well in a 6-well plate).
      NOTE: Do not add ROCK inhibitor to subsequent feedings. Change the iPSC culture medium daily.
3. Subculturing of iPSCs
   1. Prewarm the matrix-coated plates at RT or in the incubator at 37 °C for 20-30 min. Prewarm the required amount of iPSC culture medium at RT.
   2. Aspirate the culture medium from the plates containing the cells. Rinse the iPSCs with DPBS (1x) (Ca²⁺/Mg²⁺-free) (4 mL per well in a 6-well plate).
   3. Add EDTA (0.5 mM) (1 mL per well in a 6-well plate). Incubate the plates at 37 °C until the edges of the colonies start to lift from the well (usually 3-5 min). Aspirate the EDTA.
   4. Add prewarmed iPSC culture medium (4 mL per well in a 6-well plate) and forcefully detach the iPSC colonies using a 10 mL sterile pipette once. Do not pipette up and down as this may break cell clumps into single cells.
   5. Transfer the contents of each well into two individual wells in a matrix-coated 6-well plate (2 mL per well in a 6-well plate) and incubate at 37 °C. Do not generate bubbles in the suspension while pipetting.
      NOTE: Shake the plate gently before keeping it in the incubator. The split ratio can be 1:2 (one well into 2 new wells) to 1:4 (one well into 4 new wells).
   6. Replace the medium daily until the colonies reach 60% confluence with good size and connections.
4. Neural induction and neural progenitor generation
   1. Prepare 500 mL of Chemically Defined Medium (CDM), 500 mL of Neural Induction Medium (NIM), and 500 mL of Neural Stem Cell Serum-free (NSC SF) medium.
   2. Rinse the cells with DPBS (1x) (Ca²⁺/Mg²⁺-free) (4 mL per well in a 6-well plate) and add NIM (3 mL per well in a 6-well plate). Set up as Day 0.
   3. Replace the NIM (3 mL per well in a 6-well plate) on Day 1, Day 3, and Day 4 and observe under the microscopy daily.
   4. On Day 5, detach the neural rosettes into suspension culture as described below.
      1. Wash once gently with DPBS (1x) (Ca²⁺/Mg²⁺-free) (4 mL per well in a 6-well plate). Add collagenase IV (1 mL per well in a 6-well plate) and keep in an incubator for 1 min. Aspirate the collagenase IV and wash once with DPBS (1x) (4 mL per well in a 6-well plate) gently.
      2. Add 2 mL of NSC SF Medium per well to a 6-well plate. Detach the cells by scraping the bottoms of the wells by drawing grids using a 200 µL pipette tip.
      3. Collect the cell suspension from the 6-well plate into a 10 cm non-adherent dish. Make up the volume to 12 mL with NSC SF Medium.
      4. Shake the non-adherent dish at 65-85 rpm on an orbital shaker in an incubator to prevent aggregation.
5. DA neuron differentiation
   1. On Day 7, add 12 mL of CDM supplemented with 100 ng/mL fibroblast growth factor-8b (FGF-8b) and place the dish on the orbital shaker in the incubator.
   2. On Days 8-13, change the medium every 2 days and observe under the microscopy daily.
   3. On Day 14, add 12 mL of CDM supplemented with 100 ng/mL FGF-8b and 1 µM purmorphamine (PM). Place the dish on the orbital shaker in the incubator.
   4. On Days 15-20, change the medium every 2 days and observe the cells under the microscopy daily.
   5. Mechanically passage the spheres by using 1000 µL tips to break up the larger spheres.
      NOTE: The ratio can be 1:2 (one dish into 2 new dishes).
6. Termination of differentiation
   1. Coat a 6-well plate or coverslips with Poly-L-Ornithine (PLO) and laminin as described below.
      1. Coat a 6-well plate with 1 mL of PLO per well, and incubate the plate at 37 °C for 20 min. Aspirate the PLO solution.
      2. Sterilize the plate under UV for 20 min. Rinse the wells twice with DPBS (1x) (4 mL per well in a 6-well plate).
      3. Add 5 µg/mL laminin solution (1 mL per well in a 6-well plate) to the well and incubate at 37 °C for 2 h. Aspirate the laminin and wash the wells briefly with DPBS (1x) (4 mL per well in a 6-well plate) once before plating.
   2. Collect all spheres (from step 1.5.5) in 50 mL tubes and top up with DPBS (1x). Spin at 300 × g for 5 min. Aspirate the supernatant.
   3. Incubate with 2 mL of cell dissociation reagent (see the Table of Materials) for 10 min at 37 °C in a water bath followed by gentle trituration with a 200 µL pipette (20-50 times depending on the size of the spheres, avoiding bubble formation).
   4. Neutralize the cell dissociation reagent with 2 mL of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and centrifuge the 50 mL conical tube containing the cells at 300 × g for 5 min at RT. Aspirate the supernatant.
   5. Add 1 mL of CDM supplemented with 10 ng/mL Brain-Derived Neurotrophic Factor (BDNF) and 10 ng/mL Glial cell line-derived neurotrophic factor (GDNF) to resuspend the cell pellets by gently pipetting up and down to obtain single-cell suspensions.
   6. Aspirate the laminin solution from the plate (step 1.6.1.3), wash briefly with DPBS (1x) (4 mL per well in a 6-well plate), and seed the cells (from step 1.6.5) in the precoated plates or coverslips in 3 mL of CDM supplemented with 10 ng/mL BDNF and 10 ng/mL GDNF. Feed the cells every 4 days.
      NOTE: Differentiating cultures can be maintained for many weeks up to 3 months. The neural morphology usually appears after 2 weeks of termination and can be used for downstream analyses from that point onwards. BDNF and GDNF are not necessary for culturing for longer maintenance (up to 2 months).
7. NSC generation
   1. Coat matrix plates.
   2. Collect all neural spheres (generated from step 1.4) in 50 mL tubes and top up with DPBS (1x) (Ca²⁺/Mg²⁺-free). Spin at 300 × g for 5 min. Aspirate the supernatants.
   3. Incubate the pellets with 2 mL of cell dissociation reagent for 10 min at 37 °C in a water bath, followed by gentle trituration with a 200 µL pipette (20-50 times depending on the size of the spheres, avoiding bubble formation).
   4. Neutralize with 2 mL of DMEM with 10% FBS and centrifuge the 50 mL conical tubes containing the cells at 300 × g for 5 min at RT. Aspirate the supernatants. Resuspend the cell pellets by gently pipetting up and down to obtain single-cell suspensions.
   5. Aspirate the matrix solution from a precoated plate and seed the cells in the precoated plates in NSC SF Medium (3 mL per well in a 6-well plate). Feed the cells every 2-3 days and split the cells when confluent.
      NOTE: From this stage onwards, NSCs can be expanded and frozen down.
8. Glia astrocyte differentiation
   1. Astrocyte differentiation from NSCs
      1. Prepare 500 mL of Astrocyte Differentiation Medium.
      2. Seed NSCs on Poly-D-Lysine (PDL)-coated plates/coverslips with NSC SF Medium. 
      3. The following day, rinse the cells with DPBS (1x) (Ca²⁺/Mg²⁺-free) (4 mL per well in a 6-well plate) and add Astrocyte Differentiation Medium (3 mL per well in a 6-well plate). Set up as Day 0.
      4. Observe the NSCs under the microscope daily and replace the Astrocyte Differentiation Medium (3 mL per well in a 6-well plate) every 2 days from Days 1 to 27.
   2. Astrocyte maturation
      1. Prepare Astrocyte Maturation Medium.
      2. On Day 28, rinse the cells with DPBS (1x) (4 mL per well in a 6 well plate) and add Astrocyte Maturation Medium (3 mL per well in a 6-well plate).
      3. On day 29 and onwards, observe the cells under the microscope daily and replace the Astrocyte Maturation Medium (3 mL per well in a 6-well plate) every 2 days.
      4. After one month of maturation, expand the cells and cryopreserve them from this stage onwards.
         ​NOTE: During this phase, the number of cells will increase. When splitting the cells, PDL-coated coverslips are not necessary for culturing.

2. Cell characterization by immunocytochemistry and immunofluorescence staining

1. At the end of the culture period, transfer the coverslips with the cells to a 12-well plate.
   Rinse the cells with phosphate-buffered saline (PBS) (1x) two times and incubate for 10 min in 4% Paraformaldehyde (PFA) (0.5 mL per well in a 12-well plate) at RT.
   NOTE: The fixed sample can be covered with 2 mL of PBS (1x) and stored at 4 °C until required for immunostaining. PFA is toxic and is suspected of causing cancer. Prevent skin and eye exposure, and work under a chemical fume hood.
2. Block and permeabilize the cells and incubate with blocking buffer containing PBS (1x), 0.3% Triton X-100, and 10% normal goat serum for 1 h at RT.
3. Incubate with primary antibodies in blocking buffer overnight at 4 °C: stain iPSCs with anti-octamer-binding transcription factor 4 (Oct4) and anti-stage-specific embryonic antigen-4 (SSEA4), NSCs with anti-sex-determining region Y box-2 (Sox2) and anti-Nestin, neural spheres with anti-paired box-6 (Pax6) and anti-Nestin, astrocytes with anti-glial fibrillary acidic protein (GFAP) and anti-S100 calcium-binding protein β (S100β), and DA neurons with anti-tyrosine hydroxylase (TH), anti-β III Tubulin (Tuj 1), anti-Synaptophysin, and anti-PSD-95 (0.5 mL of primary antibody solution per well in a 12-well plate; see the Table of Materials for details).
4. Wash the samples with PBS (1x) three times for 10 min each with gentle rocking.
5. Incubate with secondary antibody solution (1:800 in blocking buffer, 0.5 mL of Alexa Fluor secondary antibody solution per well in a 12-well plate) for 1 h at RT with gentle rocking.
6. Incubate the cells with Hoechst 33342 (1:5,000, 0.5 mL per well in a 12-well plate) in PBS (1x) for 15 min at RT to label the nuclei.
7. Mount the cells with mounting medium and dry overnight at RT for imaging under a fluorescence microscope in the dark. See Supplemental Figure S1 for the microscope settings and parameters.

3. Flow cytometry measurement of mitochondrial volume, MMP, and mitochondrial ROS in live cells

1. Seed the cells separately into 4 wells in a 6-well plate until the cells reach 50%-60% confluency. Label these four wells as #1, #2, #3, and #4.
2. At the end of the culture period, prepare 5 individual staining solutions (500 µL per well in a 6-well plate) as follows: #1 only culture medium (to well #1 containing only cells for control); #2-1 containing FCCP (100 µM); #2-2 containing FCCP (100 µM) + TMRE (100 nM) + MTG (150 nM) in culture medium; #3 containing TMRE (100 nM) + MTG (150 nM) in culture medium; #4 containing MitoSox Red (10 µM) + MTG (150 nM) in PBS (1x) with 10% FBS. See Figure 1A, Supplemental Table S2, and the Table of Materials for details about these compounds and flow cytometry setup.
   NOTE: Use the culture medium to prepare the staining solution. Warm up the medium and PBS (1x) at RT before using. FCCP is toxic; prevent skin and eye exposure and work under a chemical fume hood.
3. Aspirate the medium from the #2 well and add #2-1 solution (FCCP only). Incubate the cells at 37 °C for 10 min.
4. Aspirate the medium from #2 and #3 wells and add #2-2 solution (FCCP + TMRE + MTG) in the #2 well and #3 solution (TMRE + MTG) in #3. Incubate the cells at 37 °C for 45 min.
5. Aspirate the medium from the #4 well and add #4 solution (MitoSox Red + MTG). Incubate the cells at 37 °C for 15 min.
6. Aspirate the medium from all wells. Wash with PBS (1x) (4 mL per well in a 6-well plate). Detach the cells using 1 mL of cell dissociation reagent (1 mL per well in a 6-well plate) at 37 °C for 5 min. Neutralize the cell dissociation reagent in 1 mL of DMEM with 10% FBS (2 mL per well in a 6-well plate).
7. Collect the contents of all the wells in 15 mL conical tubes. Centrifuge the tubes at 300 × g for 5 min. Wash the pellets with PBS (1x) once or twice.
8. Aspirate the supernatants but leave approximately 100 µL in the tubes. Resuspend the cell pellets in 300 µL of PBS (1x). Transfer the cells to 1.5 mL microcentrifuge tubes. Keep the tubes in the dark at RT.
9. Analyze the cells using a flow cytometer (with a 3 blue and 1 red laser configuration). Detect MTG in filter 1 (FL1) using a 530/30 bandpass filter, TMRE in filter 2 (FL2) using the bandpass filter 585/40, and MitoSox Red in filter 3 (FL3) using a 510/580 bandpass filter.

4. Flow cytometry measurement of MRC complex subunits and TFAM in fixed cells

1. At the end of the culture period, detach the cells (~10⁶) by adding the cell dissociation reagent; then, pellet and collect the cells in a 15 mL tube. Wash the cells by centrifugation with PBS (1x) twice by centrifuging at 300 × g for 5 min.
2. Fix the cells in 1.6% PFA (1 mL of 1.6% PFA in a 15 mL tube) at RT for 10 min. Wash the cells by centrifugation with PBS (1x) twice by centrifuging at 300 × g for 5 min.
3. Permeabilize the cells with ice-cold 90% methanol (1 mL of 90% methanol in a 15 mL tube) at -20 °C for 20 min.
4. Block the samples in blocking buffer containing 0.3 M glycine, 5% goat serum, and 1% bovine serum albumin (BSA) - Fraction V in PBS (1x) (1 mL of blocking buffer in a 15 mL tube). Wash the cells by centrifugation with PBS (1x) twice (as in step 3.7).
5. Incubate the cells with the following primary antibodies for 30 min: anti-NDUFB10 (1:1,000) for measurement of complex I subunit, anti-succinate dehydrogenase complex flavoprotein subunit A (SDHA, 1:1,000) for measurement of complex II subunit and anti-COX IV (1:1,000) for measurement of complex IV subunit, and anti-TFAM antibody conjugated with Alexa Fluor 488 (1:400). Stain the same number of cells separately with anti-TOMM20 antibody conjugated with Alexa Fluor 488 (1:400) for 30 min (1 mL of primary antibody solution in a 15 mL tube; see the Table of Materials for details about the antibodies).
6. Wash the cells with PBS (1x) once with centrifugation at 300 × g for 5 min. Add secondary antibody (1:400) into tubes of NDUFB10, SDHA, and COX IV and incubate the cells with these solutions for 30 min.
7. Wash the cells with PBS (1x) once by centrifuging at 300 × g for 5 min. Aspirate the supernatants, leaving approximately 100 µL in the tubes. Resuspend the cell pellets in 300 µL of PBS (1x). Transfer the cells to 1.5 mL microcentrifuge tubes kept in the dark on ice.
8. Analyze the cells on the flow cytometer (with a 3 blue and 1 red laser configuration). Detect signals in filter 1 (FL1) using a 530/30 bandpass filter. See Supplemental Figure S2 for the microscope settings and parameters.

5. Flow cytometry acquisition and analysis

1. Use the non-stained control tube to set the forward scatter area (FSC-A) and side scatter area (SSC-A) scatter plots based on the size and complexity of the cell population analyzed. See Supplemental Figure S2 for the microscope settings and parameters.
   NOTE: Set up non-stained controls for individual cell types.
2. Use the non-stain control tubes to select the positive gates, and use single-color control tubes to compensate for the fluorescence spectral overlap between MTG (fluorophore-1 [FL-1]) and TMRE (FL-2) in multicolor flow cytometry. Use isotype control for negative control to monitor background staining in MRC and TFAM samples. Use the FCCP tube as a depolarization control for TMRE staining.
3. Gate out extraneous debris to select live cells and the main gating from the forward and side scatter plot (Figure 2A). Gate out doublets using a forward scatter height (FSC-H) versus (vs.) FSC-A density plot to exclude doublets and also construct a side scatter height (SSC-H) vs. SSC-A plot (Figure 2A).
4. Data acquisition (flow cytometer)
   1. Using the unstained or isotype samples as a negative control, create a gate above the main population of the single-cell events while viewing SSC-A and the various filters (FL1, FL2, FL3, FL4) (Figure 1B and Figure 2B).
5. Data analysis (CFlow software)
   1. Copy the position of the gates onto the stained cell samples, and record the amount of positively stained cells for the positive staining.
   2. For each cell subpopulation, select a histogram plot and analyze the median fluorescence intensity (MFI) of the different filter channels (FL1, FL2, FL3, FL4) (x-axis).
      1. Calculate the TMRE levels by subtracting the MFI of FL2 of #2 FCCP-treated cells from the MFI of FL2 from #3 TMRE-stained samples in a histogram, as in Eq (1) below.
         ​TMRE levels = MFI of FL2 from #3 TMRE-stained samples - MFI of FL2 of #2 FCCP-treated cells (1)
      2. Calculate the specific values for MMP and mitochondrial ROS by MFI in TMRE or MitoSox Red, dividing the mitochondrial volume indicator MTG.
      3. Calculate the specific value for complex subunit and TFAM by using MFI in complex expression or TFAM, dividing the mitochondrial volume indicator TOMM20.

Representative Results

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 strategies needed for acquiring and analyzing the samples by flow cytometry for the measurement of MMP, mitochondrial volume, mitochondrial ROS levels, expression levels of MRC complex subunits and TFAM (an indirect measurement of relative mtDNA copy number). Specifically, co-staining with fluorescent reporters, TMRE and MTG, was used to detect and quantify changes in MMP and mitochondrial volume. Co-staining with MitoSox Red and MTG permits measurement of mitochondrial ROS production in live cells. Staining with antibodies against MRC complex subunits together with TOMM20 permits the assessment of the MRC and staining of TFAM and TOMM20 for indirect assessment of mtDNA copy number. Importantly, MTG and TOMM20 allow the measurement per mitochondrial volume, counteracting the influence of mitochondrial volume on these parameters.

DA neurons are generated from iPSCs through dual SMAD inhibition and exposed to FGF-8b and the Sonic hedgehog (SHH) agonist PM, as shown in Figure 4A. Human iPSCs are seeded in iPSC culture medium on matrix-coated plates. When the cells reach 50%-80% confluency, the medium is changed to NIM using a CDM supplemented with SB431542, AMPK inhibitor, Compound C, and N-acetylcysteine for 5 days. After 5 days, the iPSCs (Figure 4B, a) progress to a neural epithelial stage exhibiting clear neural rosette structures (Figure 4B, b). On day 5, neural spheres are generated by lifting the neural epithelium into suspension culture and culturing them in NSC SF medium on an orbital shaker inside the incubator. Round, well-defined spheres are shown in Figure 4B, c. On day 7, the medium is changed into CDM supplemented with 100 ng/mL FGF-8b. On day 14, the medium is changed into the CDM supplemented with 100 ng/mL FGF-8b and 1 µM PM. On day 21, the spheres are dissociated into DA neurons in a monolayer by dissociating them into single cells and culturing them in CDM supplemented with 10 ng/mL BDNF and 10 ng/mL GDNF in PLO and laminin-coated plates/coverslips. Neurons (Figure 4B, e) matured for 15-30 days are further used for mitochondrial functional measurements.

NSCs are produced by dissociating neural spheres into single cells and then replating them in monolayers to generate astrocytes. These NSCs show a classic neural progenitor appearance (Figure 4B, d). NSCs at this stage can readily be expanded and banked for further use. To initiate the astrocyte differentiation, NSCs are plated on PDL-coated coverslips in NSC SF medium. The following day, the medium is changed into astrocyte differentiation medium for 28 days. After 28 days, the differentiated astrocytes are further matured in astrocyte maturation medium. At this stage, astrocytes should display star-shaped morphology (Figure 4B, f), and these cells can be expanded and banked for further use, including mitochondrial functional assessment.

During differentiation, cell identity is confirmed using immunofluorescence staining. In Figure 5A, immunostaining shows that the iPSCs express the specific pluripotent markers, SSEA4 and Oct4. Figure 5B shows that neural spheres exhibit positive staining of Nestin and Pax6, while Figure 5C shows that iPSC-derived NSCs in monolayers exhibit positive staining of Nestin and Sox2. To identify DA neurons, cells are stained with the neural marker β III Tubulin (Tuj 1) and the DA neuronal marker tyrosine hydroxylase (TH) (Figure 6B). In addition, DA neurons show staining for the synaptic markers, synaptophysin and PSD-95, confirming their functional synaptic connections (Figure 5B). Immunostaining of iPSC-derived astrocytes shows the expression of the astrocyte markers, glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein β (S100β).

The investigation of mitochondrial function in differentiated neurons and astrocytes using flow cytometry is performed as described above in protocol sections 3 and 4. A flow cytometer was used for data acquisition and CFlow Sampler for data analysis, as shown in Figure 1B.

Figure 2 demonstrates the method for gating live single cells. Dead cells and cell debris are excluded using an FSC vs. SSC plot (Figure 2A, a). Cell doublets are excluded using an FSC-H vs. FSC-A plot (Figure 2A, b) followed by an SSC-H vs. SSC-A plot (Figure 2A, c). Background fluorescence is properly assessed if the negative population of a particular cell type is compared with the positive population within that same cell type (Figure 2B, a). For MMP samples, treating the cells with FCCP eliminates interference from mitochondrial membrane potential and TMRE staining (Figure 2B, b).

These flow cytometric approaches have been used to study DA neurons generated from the human iPSCs carrying mutation(s) in the catalytic subunit of mitochondrial DNA polymerase, POLG (W748S), and compare them with disease-free samples generated from Detroit 551 fibroblasts. As reported previously⁴, this study also demonstrated decreased MMP and increased specific mitochondrial ROS levels in POLG DA neurons (Figure 7). However, the mitochondrial volume, total MMP, and total mitochondrial ROS level were unchanged. In Figure 8, the results show a decrease in the specific complex I levels, lower total and specific TFAM levels, but similar specific complex II levels in mutant DA neurons compared to controls.

This approach was also used to study astrocytes generated from the same iPSC lines. As reported previously⁷ and shown in Figure 9, the results show that POLG-mutated astrocytes had lower total and specific MMP but similar mitochondrial volume and mitochondrial ROS compared with controls, as well as decreased levels of specific complexes I and IV (Figure 10). However, there were no changes in the total levels of complexes I and IV and no change in the total and specific levels of complex II in POLG astrocytes. Overall, these data suggest that flow cytometric analysis of multiple mitochondrial parameters provides a first-step approximation that is valuable in evaluating mitochondrial function in cells such as iPSCs and their neural and glial derivatives.

Figure 1: Setup for flow cytometry. (A) MMP, mitochondrial volume, and mitochondrial ROS staining; (B) an example of data acquisition in a C6 flow cytometer. Abbreviations: MMP = mitochondrial membrane potential; ROS = reactive oxygen species; FCCP = carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TMRE = tetramethylrhodamine ethyl ester; MTG = MitoTracker Green. Also, see Supplemental Table S2. Please click here to view a larger version of this figure.

Figure 2: Gating strategies. (A) Data acquisition; (B) the histograms of the fluorescence with MTG and TMRE staining in live cells. Abbreviations: SSC-A = side scatter area; FSC-A = forward scatter area; SSC-H = side scatter height; FSC-H = forward scatter height; FL#-A = fluorophore # area; MTG = MitoTracker Green; FCCP = carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TMRE = tetramethylrhodamine ethyl ester. Please click here to view a larger version of this figure.

Figure 3: Schematic representation of the protocol workflow. Abbreviations: DA = dopaminergic; MMP = mitochondrial membrane potential; ROS = reactive oxygen species; NDUFB 10 = NADH: Ubiquinone oxidoreductase subunit 10; SDHA = succinate dehydrogenase complex flavoprotein subunit A; COX IV = cytochrome c oxidase complex IV; mtDNA = mitochondrial DNA; TFAM = mitochondrial transcription factor A. Please click here to view a larger version of this figure.

Figure 4: iPSC differentiation. (A) Flow chart and (B) representative images for the cells from different stages during the differentiation, including iPSCs (a), neural rosette (b), neural spheres (c), NSCs (d), DA neurons (e), and astrocytes (f). Scale bars = 25 µm. Abbreviations: iPSC = induced pluripotent stem cell; NSC = neural stem cell; DA = dopaminergic; SFM = serum-free medium; CDM = chemically defined medium; FGF-8b = fibroblast growth factor-8b; PM = purmorphamine; BDNF = brain-derived neurotrophic factor; GDNF = glial cell line-derived neurotrophic factor; PLO = poly-L-ornithine. Please click here to view a larger version of this figure.

Figure 5: Representative confocal images of iPSCs and their neural derivatives. (A) Immunostaining of SSEA4 (red) and Oct4 (green) in iPSCs. (B) Immunostaining of Nestin (red) and Pax6 (green) in iPSC-derived neuron spheres. (C) Immunostaining of Nestin (red) and Sox2 (green) in iPSC-derived NSCs. Nuclei are stained with DAPI (blue). Scale bars = 50 µm. Abbreviations: iPSCs = induced pluripotent stem cells; NSCs = neural stem cells; SSEA4 = stage-specific embryonic antigen-4; Oct4 = octamer-binding transcription factor 4; Pax6 = paired box-6; Sox2 = sex-determining region Y box-2; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Figure 6: Representative confocal images of the iPSC-derived astrocytes and DA neurons. (A) Immunostaining of GFAP (red) and S100β (green) in iPSC-derived astrocytes. (B) Immunostaining of neural lineage marker TH (green), Tuj 1 (red), and neural functional marker Synaptophysin (green) and PSD-95 (red) in iPSC-derived DA neurons. Nuclei are stained with DAPI (blue). Scale bars = 25 µm. Abbreviations: iPSCs = induced pluripotent stem cells; GFAP = glial fibrillary acidic protein; S100β = S100 calcium-binding protein β; Tuj 1 = β III Tubulin; PSD-95 = postsynaptic density protein 95; DA = dopaminergic; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Figure 7: Flow cytometric analysis for DA neurons derived from one clone of POLG patient iPSCs and one clone of Detroit 551 control iPSCs. (A) Mitochondrial volume measured by MTG, (B) total MMP measured by TMRE, (C) specific MMP level calculated by total TMRE/MTG, (D) total mitochondrial ROS measured by MitoSox Red, and (E) specific mitochondrial ROS level calculated by total MitoSox Red/MTG. Data presented as mean ± standard error of the mean (SEM) for the number of samples (n ≥ 3 per clone). Data analyzed and produced using GraphPad Prism software. Mann-Whitney U test was used to assess statistical significance for variables. Significance is denoted for P < 0.05. *P < 0.05; ns, not significant. Abbreviations: DA = dopaminergic; POLG = DNA polymerase subunit gamma; iPSCs = induced pluripotent stem cells; MMP = mitochondrial membrane potential; ROS = reactive oxygen species; MTG = MitoTracker Green; TMRE = tetramethylrhodamine ethyl ester. Please click here to view a larger version of this figure.

Figure 8: Flow cytometric analysis for astrocytes derived from one clone of POLG patient iPSCs and one clone of Detroit 551 control iPSCs. (A) Mitochondrial volume measured by MTG, (B) total MMP measured by TMRE, (C) specific MMP level calculated by total TMRE/MTG, (D) total Rmitochondrial ROS S measured by MitoSox Red, and (E) specific mitochondrial ROS level calculated by total MitoSox Red/MTG. Data presented as mean ± standard error of the mean (SEM) for the number of samples (n ≥ 3 per clone). Data analyzed and produced using GraphPad Prism software. Mann-Whitney U test was used to assess statistical significance for variables. Significance is denoted for P < 0.05. *P < 0.05; ns, not significant. Abbreviations: POLG = DNA polymerase subunit gamma; iPSC = induced pluripotent stem cell; MMP = mitochondrial membrane potential; ROS = reactive oxygen species; MTG = MitoTracker Green; TMRE = tetramethylrhodamine ethyl ester. Please click here to view a larger version of this figure.

Figure 9: Flow cytometric analysis for DA neurons derived from one clone of POLG patient iPSCs and one clone of Detroit 551 control iPSCs. (A) Total complex I measured by NDUFB10, (B) specific complex I level calculated by total NDUFB10/TOMM20, (C) total complex II measured by SDHA, (D) specific complex II level calculated by total SDHA/TOMM20, (E) total TFAM measured by TFAM, and (F) specific TFAM level calculated by total TFAM/TOMM20. Data presented as mean ± standard error of the mean (SEM) for the number of samples (n ≥ 3 per clone). Data analyzed and produced using GraphPad Prism software. Mann-Whitney U test used to assess statistical significance for variables. Significance is denoted for P < 0.05. *P < 0.05; ns, not significant. Abbreviations: DA = dopaminergic; POLG = DNA polymerase subunit gamma; iPSC = induced pluripotent stem cell; NDUFB10 = NADH: Ubiquinone oxidoreductase subunit 10; TOMM20 = translocase of outer mitochondrial membrane 20; SDHA = succinate dehydrogenase complex flavoprotein subunit A; TFAM = mitochondrial transcription factor A. Please click here to view a larger version of this figure.

Figure 10: Flow cytometric analysis for astrocytes derived from one clone of POLG patient iPSCs and one clone of Detroit 551 control iPSCs. (A) Total complex I measured by NDUFB10, (B) specific complex I level calculated by total NDUFB10/TOMM20, (C) total complex II measured by SDHA, (D) specific complex II level calculated by total SDHA/TOMM20, (E) total complex IV measured by COX IV, (F) specific complex IV level calculated by COX IV/TOMM20, (G) total TFAM measured by TFAM, and (H) specific TFAM level calculated by total TFAM/TOMM20. Data presented as mean ± standard error of the mean (SEM) for the number of samples (n ≥ 3 per clone). Data analyzed and produced using GraphPad Prism software. Mann-Whitney U test was used to assess statistical significance for variables. Significance is denoted for P < 0.05. *P < 0.05; ** P < 0.01; ns, not significant. Abbreviations: POLG = DNA polymerase subunit gamma; iPSC = induced pluripotent stem cell; NDUFB10 = NADH: Ubiquinone oxidoreductase subunit 10; TOMM20 = translocase of outer mitochondrial membrane 20; SDHA = succinate dehydrogenase complex flavoprotein subunit A; TFAM = mitochondrial transcription factor A; COX IV = cytochrome c oxidase complex IV. Please click here to view a larger version of this figure.

Supplemental Figure S1: Settings of the confocal laser scanning fluorescence microscope and steps for taking images. (A) Choose the Configuration tool and select the correct laser type from Current available laser and set its power. (B) Choose Acquire-Acquisition Mode-SEQ and select the corresponding fluorescence wavelength photo mode from the database. (C) Choose Sequential Scan-Load and import corresponding mode. (D) Choose 40x objective lens and add dropwise. (E) Specific setting parameters for taking photos at different wavelengths. (F) Set photo parameters, preview, and save the photo. Please click here to download this File.

Supplemental Figure S2: Steps and settings for flow cytometry. (A) Open Cflow software, choose the File tool, and select Open CFlow file or template. (B) Set up 40000 events and select the gate containing only the live single cells in the Run Limits panel. Choose Medium speed in the Fluidics panel. (C) Choose FSC-A vs. FSC-A plot (a) for setting up the main gating. Choose FSC-A vs. FSC-H plot (b) and SSC-A vs. SSC-H plot (c) to exclude doublets. Choose the corresponding filters, such as FL1 or FL2, and use FL1-A vs. FSC-A plot (d) or FL2-A vs. FSC-A plot to draw the positive events when running the unstained cells. (D) Set up the same parameters, preview, run the stained samples and save the photo. Also, see Supplemental Table S2. Please click here to download this File.

Supplemental Table S1: Media and solution recipes. Please click here to download this Table.

Supplemental Table S2: Setup for flow cytometric staining. Please click here to download this Table.

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 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 astrocytes^2,3,13,17.

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 <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 MMP^19,20. 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.

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