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Assessment of Energy Metabolic Changes in Adipose Tissue-Derived Stem Cells

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1553)

Abstract

Adipose tissue-derived stem cells (ADSC) are promising candidates for therapeutic applications in cardiovascular regenerative medicine. By definition, the phenotype ADSCs, e.g., the ubiquitous secretion of growth factors, cytokines, and extracellular matrix components is not met in vivo, which renders ADSC a culture “artefact.” The medium constituents therefore impact the efficacy of ADSC. Little attention has been paid to the energy source in medium, i.e., glucose, which feeds the cell’s power plants: mitochondria. The role of mitochondria in stem cell biology goes beyond their function in ATP synthesis, because it includes cell signaling, reactive oxygen species (ROS) production, regulation of apoptosis, and aging. Appropriate application of ADSC for stem cells therapy of cardiovascular disease warrants knowledge of their mitochondrial phenotype and function. We discuss several methodologies for assessing ADSC mitochondrial function and structural changes under environmental cues, in particular, increased ROS caused by hyperglycemia.

Key words

ADSC Mitochondria Hyperglycemia ROS Energy metabolism 
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1 Introduction

Adipose tissue-derived stem cells (ADSC) are a promising source of white adipose tissue stromal cells for use in cardiovascular regenerative medicine; for their differentiation potential, their ease of isolation and their secretion of therapeutically relevant trophic factors [1, 2]. The therapeutic potential of ASCS has been assessed in various animal models with specific disorders such as Parkinson’s disease [3] and Alzheimer’s disease [4, 5], bone and cartilage defects [6, 7], skin wound healing [8], myocardial infarction [9, 10], and diabetic retinopathy [11, 12].

There is new evidence that, in addition to growth factors and extracellular matrix cues, the (energy) metabolism of stem cell directs self-renewal and differentiation [13, 14, 15].

Recent studies have revealed limitations in the therapeutic efficacy of ADSC derived from patients who were compromised by diabetes or aging or obesity. It has been demonstrated that ADSC from these patients have impaired differentiation and migration [16, 17, 18, 19]. Recently, we also showed in vitro that ADSC respond to chronic hyperglycemic exposure by increased apoptosis caused by amplified ROS. In addition, hyperglycemically cultured ADSC showed an altered mitochondrial membrane potential and changes in mitochondrial network morphology. Interestingly, we found an altered glycolysis and glucose uptake potential in ADSC upon culture under hyperglycemic conditions (30 mM d-glucose) compared with normaglycemically (5 mM d-glucose) cultured ADSC. These data confirm the well-established fact that mitochondrial disorders have a key role in apoptosis [20] and it contributes to a wide number of diseases, including mitochondrial myopathies [21], mitochondrial neuropathies [22], and diabetes [23]. Mitochondria are a main source of reactive oxygen species (ROS) in the cell [24]. In healthy cells, the inner membrane of mitochondria is impermeable to ions [25] which allows the electrons transport chain (ETC) to build up the proton gradient required to generate energy. The mitochondrial membrane potential (ΔΨm) results from the difference in electrical potential generated by the electrochemical gradient across the inner membrane [26]. Mitochondria are the source for ROS, but also the major target of their damaging effects, demonstrating the trigger for several mitochondrial dysfunctions. Chronic increases in ROS production cause the accumulation of ROS-associated damage in DNA, proteins, and lipids, and are headed by severe perturbations in mitochondrial function detected as a decrease in ΔΨm. This reduction in ΔΨm is accompanied by the production of ROS contributing to cell apoptosis [27].

Alterations of the glucose metabolism may cause mitochondrial dysfunction, i.e., affect the energy metabolism, and may be responsible for further cellular damage and disease pathogenesis. The failure to manage cellular energy pathways either the aerobic respiration or glycolysis via mitochondria may result in serious complications in diseases such as diabetes [28]. Detecting mitochondrial dysfunction in therapeutic used ADSC is a prerequisite in the development of novel stem cell therapies for diseases such as diabetes.

2 Materials

2.1 Isolation of ADSC

  1. 1.

    Human subcutaneous fat tissue or liposuction-derived fat.

     
  2. 2.

    Phosphate-buffered saline (PBS).

     
  3. 3.

    PBS/1 % Bovine serum albumin (BSA).

     
  4. 4.

    0.1 % Collagenase dissolved in PBS/1%BSA, freshly prepared prior to use (Dissociation medium).

     
  5. 5.

    Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 100 U/ml Penicillin, 100 μg/ml Streptomycin, 2 mM l-glutamine, and 1 (gr/L) d-glucose (control medium). Store at 4 °C.

     
  6. 6.

    50-ml Centrifuge tube.

     
  7. 7.

    40-μm Nylon mesh.

     
  8. 8.

    Lymphoprep.

     
  9. 9.

    Lysisbuffer.

     
  10. 10.

    Trypan blue.

     

2.2 ADSC Culture

  1. 1.

    DMEM supplemented with 10 % fetal bovine serum (FBS) and 100 U/ml Penicillin, 100 μg/ml Streptomycin, 2 mM l-glutamine, and 1 (gr/L) d-glucose (control medium) or 4.5 (gr/L) d-glucose (hyperglycemic medium). Store at 4 °C.

     
  2. 2.

    Trypsin (0.25 %) and ethylenediaminetetraacetic acid (EDTA, 1 mM).

     

2.3 Apoptosis Detection of ADSC (Using FACS Calibur Flow-Cytometer)

  1. 1.

    Annexin V-Ethidium Homodimer lll (EthD-III): Apoptotic/Necrotic Cells Detection Kit (#PK-CA707-30018-Promokine) .

     
  2. 2.

    Trypsin (0.25 %) and ethylenediaminetetraacetic acid (EDTA, 1 mM).

     
  3. 3.

    FACS tubes.

     

2.4 Intracellular ROS and Mitochondrial ROS Measurement in ADSC (Using FACS Calibur Flow-Cytometer)

  1. 1.

    2′,7′-Dichlorofluorescin diacetate: H2DCFDA (Thermo Fisher).

     
  2. 2.

    MitoSOX™ Red: Mitochondrial superoxide indicator (Thermo Fisher).

     
  3. 3.

    Trypsin (0.25 %) and ethylenediaminetetraacetic acid (EDTA, 1 mM).

     
  4. 4.

    Control medium/PBS.

     

2.5 Monitoring Mitochondrial Health

2.5.1 Assessment of Mitochondrial Membrane Potential

  1. 1.

    MitoProb ™ JC1 (5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide(Thermo Fisher)).

     
  2. 2.

    CCCP (carbonyl cyanide 3-chlorophenylhydrazone).

     
  3. 3.

    Trypsin (0.25 %) and ethylenediaminetetraacetic acid (EDTA, 1 mM).

     
  4. 4.

    Control medium/PBS.

     

2.5.2 Mitochondrial Morphology Analysis

  1. 1.

    Mito-Tracker Green [MTG] (Thermo Fisher).

     
  2. 2.

    Trypsin (0.25 %) and ethylenediaminetetraacetic acid (EDTA, 1 mM).

     
  3. 3.

    Control medium/PBS.

     

2.6 ADSC Bioenergetics Profiling

  1. 1.

    V7-PS XF24 cell culture microplates (Seahorse Bioscience), XF24 extracellular flux assay kits (Seahorse Bioscience).

     
  2. 2.

    DMEM-XF containing, 1 mM glutamine, 1 % FBS, 1 (gr/L) d-glucose (control medium), or 4.5 (gr/L) d-glucose (hyperglycemic medium) and pyruvate-free (Unbuffered medium).

     
  3. 3.

    Oligomycin (Seahorse Bioscience) .

     
  4. 4.

    FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone).

     
  5. 5.

    Rotenone and Antimycin A.

     
  6. 6.

    2-deoxy-d-glucose.

     
  7. 7.

    Pierce™ BCA Protein Assay Kit (Thermo Fisher).

     

2.7 Assessment of Glucose Uptake

  1. 1.

    2-Deoxy d-glucose.

     
  2. 2.

    PBS.

     
  3. 3.

    DMEM supplemented with 10 % fetal bovine serum (FBS) and 100 U/ml Penicillin, 100 μg/ml Streptomycin, 2 mM l-glutamine, and 1(gr/L) d-glucose (control medium) or 4.5 (gr/L) d-glucose (hyperglycemic medium). Store at 4 °C (serum-free medium).

     
  4. 4.

    Insulin.

     
  5. 5.

    2-Deoxy-d-[14C]glucose (14C-2-DOG).

     
  6. 6.

    NaOH.

     
  7. 7.

    β-Scintillation cocktail and β-scintillation counter.

     
  8. 8.

    Pierce™ BCA Protein Assay Kit (Thermo Fisher).

     

3 Methods

3.1 Isolation of ADSC

  1. 1.

    Mince the fat tissue with fine scissors in culture dishes, and transfer the material into a 50 ml centrifuge tube. Alternatively, lipoaspirated fat can be transferred to centrifuge tubes directly.

     
  2. 2.

    Wash the fat three times with PBS, centrifuge at 300 × g for 3 min each time.

     
  3. 3.

    Add an equal volume of dissociation medium with the fat, stir for 90 min in 37 °C water bath.

     
  4. 4.

    Filter the digested fat through 40-μm Nylon mesh; collect the flow-through in 50 ml tubes.

     
  5. 5.

    Centrifuge the cell suspension at 600 × g for 10 min to obtain a high-density ADSC pellet.

     
  6. 6.

    Aspirate the supernatant , being careful not to disturb the cell pellets.

     
  7. 7.

    Resuspend the cell pellets in 30 ml PBS/1%BSA and add the cell-suspension gently on the top of 15 ml Lymphoprep.

     
  8. 8.

    Centrifuge at 4 °C, 1000 × g for 20 min.

     
  9. 9.

    Carefully aspirate the cells from the interphase.

     
  10. 10.

    Resuspend cells in lysis buffer and place on ice for 5 min, centrifuge at 600 × g for 10 min.

     
  11. 11.

    Count the cells using Trypan blue and seed at a concentration of 1.25 × 105 cells/cm2 in culture flasks.

     

3.2 ADSC Culture

  1. 1.

    Maintain the primary ADSC in control medium at 37 °C in 5 % carbon dioxide. Change the culture medium every 3 days.

     
  2. 2.

    Once adherent cells become confluent, aspirate the culture medium and wash the cells with 5 ml of PBS. Add 1–3 ml of trypsin–EDTA at 37 °C for 5 min to detach the cells.

     
  3. 3.

    Resuspend the ADSC with an equal volume of control medium.

     
  4. 4.

    Centrifuge the cell suspension at 300 × g for 10 min at 4 °C and split the cells 1:3 in control medium.

     
  5. 5.

    Use the cells from passage 2–7 for the experiments (e.g., to expose the cells to an apoptotic condition such as hyperglycemia).

     

3.3 Apoptosis Detection of ADSC (Using FACS Calibur Flow Cytometer)

  1. 1.

    Harvest ADSC from control/treated group: aspirate the culture medium and wash the cells with 5 ml of PBS. Add 1–3 ml of trypsin–EDTA at 37 °C for 5 min to detach the cells.

     
  2. 2.

    Wash the cells, centrifuge at 300 × g for 10 min at 4 °C, and resuspend the cells in 500 μl binding buffer (100,000 cells/500 μl) in FACS tubes.

     
  3. 3.

    Stain the cells with 2.5 μl FITC-Annexin V (marker for apoptosis) and 2.5 μl of Ethidium Homodimer lll (marker for necrosis) in the dark at room temperature for 15 min.

     
  4. 4.

    Analyze the samples (FITC-AnnexinV-Ex/Em= ~492/514 nm and EthD-III-Ex/Em= ~528/617 nm) using a FACS Calibur flow cytometer within 1 h after staining .

     

3.4 Intracellular ROS and Mitochondrial ROS Measurement in ADSC (Using FACS Calibur Flow-Cytometer)

  1. 1.

    Harvest ADSC from control/treated groups: aspirate the culture medium and wash the cells with 5 ml of PBS. Add 1–3 ml of trypsin–EDTA at 37 °C for 5 min to detach the cells.

     
  2. 2.

    Wash the cells, centrifuge at 300 × g for 10 min at 4 °C, and resuspend the cells in FACS tubes with 1 ml of warm control medium followed by incubation with 20 μM H2DCFDA (see Note 1 ) or 5 μM MitoSOX™ Red (see Note 2 ) in the dark at 37 °C for 15 min.

     
  3. 3.

    Analyze the samples (DCF: Ex/Em= ~492/527 nm and oxidized MitoSOX: Ex/Em= ~510/580 nm) directly without washing, using a FACS Calibur flow-cytometer within 30 min after the staining.

     

3.5 Monitoring Mitochondrial Health

3.5.1 Assessment of Mitochondrial Membrane Potential

  1. 1.

    Harvest ADSC from control/treated groups: aspirate the culture medium and wash the cells with 5 ml of PBS. Add 1–3 ml of trypsin–EDTA at 37 °C for 5 min to detach the cells.

     
  2. 2.

    Wash the cells , centrifuge at 300 × g for 10 min at 4 °C and resuspend the cells in 1 mL warm control medium at 1 × 106 cells/ml, followed by adding 2 μM MitoProbe JC-1 and 50 μM CCCP as control.

     
  3. 3.

    Incubate the samples at 37 °C for 15 min.

     
  4. 4.

    Determine the mitochondrial accumulation of the probe by fluorescence emission shift from green (~529 nm) to red (~590 nm) by FACS Calibur flow-cytometer within 15 min after staining.

     

3.5.2 Mitochondrial Morphology Analysis

  1. 1.

    Once ADSC cultures from control/treated group become confluent, aspirate the culture medium and wash the cells with PBS twice.

     
  2. 2.

    Incubate the ADSC at 37 °C in a 5 % CO2 humidified chamber with 120 nM membrane potential–independent dye: Mito-Tracker Green [MTG] in culture medium for 45 min.

     
  3. 3.

    Wash the cells three times with PBS and refresh the medium.

     
  4. 4.

    Keep the cells at 37 °C in a 5 % CO2 humidified microscope stage chamber and image the cells live by confocal microscope with a 60× oil immersion objective (MTG: Ex/Em= ~488/550 nm).

     
  5. 5.

    Acquire in series of six slices per cell ranging in thickness from 0.5 to 0.8 μm per slice to visualize individual mitochondria as well as their interconnective network or disturbances.

     
  6. 6.

    Analyze the mitochondrial length and circularity (see Note 3 ) of ADSC with ImageJ software.

     

3.6 ADSC Bioenergetics Profiling

  1. 1.

    Plate the ADSC on V7-PS microplate under control or treatment condition to reach a confluent monolayer.

     
  2. 2.

    Wash the cells three times with PBS.

     
  3. 3.

    Replace the unbuffered medium and incubate cell at 37 °C in a CO2-free incubator for 60 min.

     
  4. 4.

    For oxygen consumption rate (OCR (see Note 4 )): pipet 2 μM oligomycin in port A, 5 μM FCCP in port B and a mixture containing 2 μM rotenone and 2 μM antimycin A in port C.

    For extracellular acidification rate (ECAR (see Note 5 )): pipet the saturating concentration of glucose (10 mM) in port A, 2 μM oligomycin in port B, and 100 μM 2-deoxy-d-glucose in port C.

     
  5. 5.
    Place the microplate in XF24 Extracellular Flux Analyzer; Seahorse Bioscience to measure extracellular flux changes (Follow the protocol in Fig. 1c).
    Fig. 1

    Mitochondrial respiration graph of ADSC : Basal respiration, ATP production and proton leak after injecting oligomycin, maximal respiration after exposure the ADSC to FCCP and spare respiratory capacity of the cells were measured by using a Seahorse XF-analyzer and plotted (a). The glycolytic function of ADSC: glycolysis, glycolytic capacity after injecting oligomycin and glycolytic reserve are shown (b). Protocol commands for ADSC extracellular flux analyzing by Seahorse XF-analyzer (c)

     
  6. 6.

    Normalize the results from the measurement to total cellular proteins in each well.

     

3.7 Assessment of Glucose Uptake

  1. 1.

    Once ADSC cultures from control/treated group reach confluency, aspirate the culture medium and wash the cells twice with PBS.

     
  2. 2.

    Incubate the cells in serum-free medium at 37 °C for 4 h.

     
  3. 3.

    Stimulate the ADSC with 100 nM insulin for 20 min at 37 °C or leave untreated.

     
  4. 4.

    Remove the medium; wash the cells twice with warm PBS.

     
  5. 5.

    Add 1 ml of PBS containing 0.1 μCi 2-deoxy-d-[14C]glucose (14C-2-DOG) and unlabeled 100 μM 2-deoxy-d-glucose to each well.

     
  6. 6.

    Incubate the cells at 37 °C for 45 min.

     
  7. 7.

    Terminate the glucose transport by washing twice with ice-cold PBS.

     
  8. 8.

    Lyse the cells in 500 μl 0.05 M NaOH.

     
  9. 9.

    Use 400 μl of the aliquot for β-scintillation determination.

     
  10. 10.

    Use the remained 100 μl for the determination of protein concentration with the Pierce™ BCA Protein Assay Kit.

     

4 Notes

  1. 1.

    DCF formation is reflected to H2O2 production but it cannot be used to measure H2O2 production exclusively inside mitochondria. For imaging mitochondrial H2O2 in living cells we recommend peroxy-yellow-1 (MitoPY1), a new type of fluorophore [31], although advanced studies have to be performed to stablish its efficacy.

     
  2. 2.

    MitoSOX is considered a superoxide-specific probe to visualize superoxide ions inside mitochondria [29]. The MitoSox specify for hydrogen peroxide or reactive nitrogen species is quite low [30].

     
  3. 3.

    Mitochondrial circularity is a measure of the “roundness” of mitochondria with 0 referring to a straight line and 1 as a perfect circle. Cells containing a majority of long interconnected mitochondrial networks were classified as cells with tubular mitochondria. Cells with a majority of short mitochondria were classified as fragmented and cells with mostly sparse small round mitochondria were classified as very fragmented [32].

    As an example we show a mainly long and tubular mitochondrial network morphology in the healthy ADSC cultured in 5 mM d-glucose medium, which changed to a very fragmented morphology when cultured in medium with a non-physiologically high (50 mM) concentration of d-glucose (Fig. 2). The tubular mitochondria networks are desired for a normal function of the mitochondria by regulation of fusion and fission events that involve the formation or breaking of the mitochondria network, respectively. A decrease in the rate of fusion and a simultaneous increase in the rate of fission cause fragmentation of the mitochondrial network which results in shorter and rounder mitochondria [33, 34].
    Fig. 2

    Confocal immunofluorescent analysis of ADSC using staining with MitoTracker Green FM. (Blue pseudocolor = DAPI, nucleus staining). Long and tubular mitochondrial network morphology in healthy ADSC cultured in 5 mM d-glucose medium (a). Very fragmented mitochondrial morphology of ADSC after culturing in non-physiologically high (50 mM) d-glucose concentration medium (b)

     
  4. 4.
    OCR measurement steps:
    1. (a)

      Measuring the basal OCR.

       
    2. (b)

      Inhibitory analysis using injections of oligomycin (Olig) at 2 μM which inhibits ATP synthase [ATP-linked respiration = OCRpre-Olig−OCRpost-Olig], [proton leak = OCRpost-Olig−OCRpost-AntA/R].

       
    3. (c)

      Applying proton ionophore FCCP at 5 μM, which uncouples mitochondria to obtain the maximum oxygen consumption rates [maximal respiration = OCRpost-FCCP−OCRpost-AntA/R], [respiratory capacity = OCRpost-FCCP−OCRpre-Olig].

       
    4. (d)

      Adding a mixture of an electron transport blocker , antimycin A (AntA) at 2 μM and rotenone at 2 μM as an inhibitor of mitochondrial complex to confirm that respiration changes were due mainly to mitochondrial respiration.

       
     
  5. 5.
    ECAR measurement steps:
    1. (a)

      Measuring the basal ECAR in a medium without glucose or pyruvate.

       
    2. (b)

      Measuring glycolysis rate of cells in saturating concentration of glucose [basic glycolysis = ECARpre-Olig].

       
    3. (c)

      Inhibitory analysis using injections of oligomycin (Olig) at 2 μM which inhibits ATP synthase and shifts the energy production pathway to glycolysis to reach to the cellular maximum glycolytic capacity[glycolytic capacity = ECARpost-Olig], [ glycolytic reserve =ECARpost-Olig−ECARpre-Olig].

       
    4. (d)

      Inhibiting the glycolysis by using 100 μM2-deoxy-glucose, a glucose analog.

      After normalization and analyzing the data, the mentioned mitochondrial respiration and glycolytic indexes can be calculated (Fig. 1a, b).

       
     

Notes

Acknowledgments

This project has received funding from the Marie Curie International Research Staff Exchange Scheme with the 7th European Community Framework Program under grant agreement No. 295185 - EULAMDIMA.

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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Cardiovascular Regenerative Medicine Research Group of the Department of Pathology and Medical BiologyUniversity of Groningen, University Medical Center GroningenGroningenThe Netherlands

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