Method Article

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

DOI:

10.3791/55245

⸱

February 22nd, 2017

 ,  ,  ,  ,  ,  , 
1Institute for Regenerative Medicine and Biotherapy (IRMB), INSERM U1183, Montpellier University, 2Institute of Neurosciences of Montpellier (INM), INSERM U1051, Montpellier University

In This Article

Summary

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Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

Abstract

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Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.

Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Introduction

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Mitochondria are organelles found in eukaryotic cells where they play a central role in nutrient uptake as well as in energy and metabolite production. These organelles contain circular mitochondrial DNA (mtDNA), 16.6 kb long, that encodes proteins of the electron transport chain complexes, tRNAs and rRNAs 1. The functionality of these organelles is critical for cell homeostasis and several pathologies have been associated with mitochondria dysfunction 1,2,3. The mitochondria status has for instance been linked to inflammation, infectious diseases and cancer, in this latter case with consequences for metastasis and resistance to therapy 4,5,6,7.

Mitochondria display the remarkable capacity of getting transferred between "donor" and "target" cells. This leads to changes in the energetic metabolism of the target cells as well as in other functional modifications such as tissue repair and resistance to chemotherapeutic agents, as recently shown by different laboratories 8,9,10,11,12,13,14,15,16. The human mesenchymal stem cells (MSCs) display this ability to transfer mitochondria to a wide variety of target cells, including cardiomyocytes, endothelial cells, pulmonary alveolar epithelial cells, renal tubular cells and cancer cells, leading to modifications of the functional properties of these cells 8,9,10,12,17,18.

Mitochondria exchange now appears as a widely used mechanism that allows a number of different cell types to communicate with one another and modify their biological properties. This mitochondria exchange can occur through tunneling nanotubes (TNT) formation, involving connexin 43-containing gap junctions 8 or M-Sec/TNFaip2 and the exocyst complex 19. Alternatively, the mitochondria transfer was also shown to be mediated by arrestin domain-containing protein 1-mediated microvesicles (ARMMs) 20. Interestingly, the efficacy of the mitochondria transfer was linked to the expression rate of the Rho GTPase 1 MIRO1 21, a key factor for explaining the differences in mitochondria transfer efficacies between iPSC-MSCs and adult BM-MSCs 22.

In spite of this wealth of data concerning cell-to-cell mitochondria exchange, relatively little is known about the metabolic and biological outcome of this mitochondria transfer. Therefore, it fully warrants setting up the appropriate tools to fully assess the biological effects of this transfer. Over the years, several technical approaches to transfer mitochondria from donor to acceptor cells have been proposed. This includes direct injection of mitochondria into oocytes 23,24,25, cell fusion to generate transmitochondrial cybrids 26,27 and, more recently, transfer of isolated mitochondria using photothermal nanoblades 28.

We and others previously demonstrated the capacity of isolated mitochondria to be internalized by living cells, as observed both in vitro and in vivo 29,30,31, through mechanisms proposed to involve macropinocytosis 32. We further developed a method, called MitoCeption, to quantitatively transfer isolated mitochondria (from MSCs) to target cells, as exemplified with the (adherent) MDA-MB-231 breast cancer cell line 31. This protocol was adapted here for the transfer of isolated human MSC mitochondria to glioblastoma stem cells (GSCs).

Glioblastoma are aggressive malignant tumors of the brain that rapidly become resistant to treatment, mainly due to glioblastoma stem cells (GSC) present within the tumor 33. These GSCs grow as neurospheres in vitro and generate tumors in xenograft models. Cancer cells within glioblastoma have the capacity to make cell-to-cell connections, as shown recently for astrocytic brain tumor cells that interconnect via extended microtubes, through which mitochondria (as well as calcium and cell nuclei) can migrate, resulting in radiotherapy-resistant astrocytoma networks 34. Glioblastoma can recruit many different cells within the tumor microenvironment, including MSCs 35,36. We showed that MSCs can make cell-cell connections with GSCs in coculture and transfer their mitochondria (data not shown), which is expected to modify GSC functional properties. The present protocol describes how the MitoCeption technique can be used to transfer mitochondria, isolated beforehand from human MSCs, to human GSCs with the purpose of determining their functional biological outcome. The multipotent and highly tumorigenic Gb4 GSC line 37 was used in this study.

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Protocol

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Day 1

1. Labeling of the Mesenchymal Stem Cell (MSC) Mitochondria (Optional)

  1. Two days before the mitochondria preparation, seed human MSCs in a 100 mm culture dish, in 10 ml αMEM/FBS 10%, so as to have 4 x 105 MSCs in culture on Day 1.
  2. Rinse MSCs with PBS (4 ml) and add 4 ml αMEM/FBS 1% (prewarmed to 37 °C).
  3. Add the required amount of mitochondria vital dye and incubate cells for 30 min in the 37 °C incubator.
  4. Remove the mitochondria dye solution, rinse cells twice with 4 ml prewarmed (37 °C) αMEM/FBS 1% and add back 4 ml αMEM/FBS 10%. Incubate cells at 37 °C.
  5. Change the culture medium (10 ml αMEM/FBS 10%) after 30 min and, another time, 2 hr later.

Day 1

2. Labeling of the Glioblastoma Stem Cells (GSC) (Optional, See Discussion Section)

Cell culture mediumComposition
GSC basal medium DMEM/F-12 supplemented with 
Insulin 20 mg/ml
N2 supplement 1x
Glucose 3 g/L
L-glutamine 2 mM
GSC proliferation mediumAdd to the basal medium: 
B27 supplement 1x
EGF 10 ng/ml
bFGF 10 ng/ml
Fungine 10 mg/ml
Fungizone 0.25 mg/ml
Heparin 2 mg /ml
Ciprofloxacin 2 μg / ml
Gentamicin 2 μg/ml
MSC proliferation medium αMEM supplemented with
L-glutamine 2 mM
10% FBS
bFGF 2 ng/ml

Table 1: Culture Media.

  1. Dissociate GSCs (Gb4 cell line 32 (10 x 106 cells) grown as neurospheres on poly-HEMA coated cell culture flasks (see steps 3.1 to 3.12).
  2. Seed GSCs in a 48-well plate at 105 cells/well in GSC proliferation medium (500 µl) (see Table 1).
  3. Centrifuge the plate at 270 x g for 5 min at 20 °C.
  4. Add the required amount of cell vital dye and incubate for 30 min at 37 °C.
  5. Add 500 µl of GSC basal medium (Table 1) per well of the 48-well plate.
  6. Centrifuge the plate at 270 x g at 20 °C for 5 min. Aspirate the supernatant.
  7. Repeat steps 2.5 to 2.6.
  8. Add 500 µl of GSC basal medium and incubate at 37 °C for 30 min.
  9. Centrifuge the plate at 270 x g at 20 °C for 5 min. Aspirate the supernatant.
  10. Add 500 µl of GSC proliferation medium per well of the 48-well plate and incubate in the 37 °C incubator.
    Note: The amount of GSCs indicated (10 x 106 cells) allows performing the different dose-response experiments and FACS controls. Once the experimental conditions are more precisely defined this amount can be scaled down.

Day 2

3. Seeding of Glioblastoma Stem Cells

  1. Collect the GSC neurospheres (10 x 106 cells) by centrifugation at 270 x g for 5 min, at 20 °C in a 50 ml tube.
  2. Wash cells with 5 ml of HBSS, and centrifuge at 270 x g for 5 min at 20 °C.
  3. Aspirate the supernatant.
  4. Gently resuspend the GSC pellet in 100 µl trypsin-EDTA (0.25%) (per 10 x 106 cells)
  5. Incubate at 37 °C for 3 min.
  6. Add 10 µl CaCl2 (20 mM) and 2 µl DNase I (10 mg/ml).
  7. Dissociate the neurospheres by gently pipetting up and down (30-50x) with a P200 pipette. Avoid bubbles. Check under the microscope that all GSCs are dissociated.
  8. Add 10 µl trypsin inhibitor (5%) and 10 ml HBSS.
  9. Centrifuge GSCs at 270 x g for 7 min at 20 °C.
  10. Discard the supernatant and add 10 ml GSC basal medium (Table 1).
  11. Count GSCs with a Thoma counting chamber and then centrifuge the cells at 270 x g for 7 min at 20 °C.
  12. Add the appropriate volume of GSC proliferation medium (Table 1) to reach the cellular concentration of 106 GSCs/ml.
  13. Seed 105 GSCs (100 µl of the cell suspension) per well of a 96-well plate.
  14. Centrifuge the plates at 270 x g for 7 min at 20 °C to get GSCs at the bottom of wells.
  15. Incubate the 96-well plate at 37 °C until Section 5.

Day 2

4. MSC Mitochondria Isolation

  1. Adjust the microtube centrifuge temperature to 4 °C.
  2. Prepare two 1.5 ml tubes "A" and "C" containing the reagents for the mitochondria extraction (200 µl reagent A and 400 µl reagent C, both containing the EDTA-free protease inhibitors). Prepare 2 other tubes, labeled "MSC" and "Mito". Keep all tubes on ice. Also prepare tubes for the mitochondria serial dilutions.
  3. Wash MSCs with 10 ml prewarmed (37 °C) PBS.
  4. Wash MSCs with 2 ml trypsin (no EDTA) for 10 sec and add 1 ml trypsin (no EDTA). Incubate cells for 5-10 min at 37 °C.
  5. Recover the MSCs by adding 10 ml αMEM/FBS 10%, transfer to a 50 ml tube.
  6. Centrifuge cells at 270 x g for 5 min at 20 °C.
  7. Discard the supernatant and add 10 ml αMEM/FBS 10% to the cell pellet.
  8. Count the MSCs with a Malassez counting chamber.
  9. Centrifuge MSCs (4-5 x 105) at 270 x g for 5 min at 20 °C.
  10. Discard the supernatant, add 1 m ice-cold αMEM/FBS 10% to the cell pellet and transfer the cells to the "MSC"-labeled tube (prepared at step 4.2). Keep the tube on ice.
  11. Centrifuge the tube containing the MSCs at 900 x g for 5 min, at 4 °C.
  12. Remove all residual medium from the tube.
  13. Add 200 µl of Mitochondria Isolation Reagent A (containing the EDTA-free protease inhibitors). Vortex at medium speed for 5 sec and leave tubes on ice for exactly 2 min.
  14. Add 2.5 µl of Mitochondria Isolation Reagent B. Vortex at maximum speed for 10 sec, and then leave tubes on ice. Repeat every 30 sec for 5 min.
  15. Add 200 µl of Mitochondria Isolation Reagent C (containing the EDTA-free protease inhibitors). Mix by tilting the tube (roughly 30 times, do not vortex). Centrifuge the tube at 700 x g for 10 min at 4 °C.
  16. Transfer the supernatant (containing the MSC mitochondria) to the "Mito" tube (prepared at step 4.2).
  17. Centrifuge at 3,000 x g, 15 min at 4 °C, to get the mitochondria pellet. Discard the supernatant. The pellet contains the isolated mitochondria.
  18. Rinse the mitochondria pellet with 200 µl of the Reagent C. Then, centrifuge at 12,000 x g, 5 min at 4 °C, to get the mitochondria pellet.

5. Transfer of Isolated MSC Mitochondria to GSCs (MitoCeption)

  1. Add 200 µl of the pre-cooled (0 °C) GSC proliferation medium to the mitochondria pellet isolated from MSCs (4 x 105).
  2. Dilute the mitochondria preparation (in GSC proliferation medium) to consistently add 20 µl of mitochondrial suspension to the GSCs.
  3. Add the volume of isolated mitochondria into the wells of the 96-well plate containing the GSCs (from step 3.15), at the desired concentration (0.1 to 10 µg). Add the mitochondria slowly, close to the bottom of the well, covering the entire surface at least once.
  4. For controlling MSC mitochondria vital dye leakage, add the same amounts of the mitochondria preparation (0.1 to 10 µg) to wells of a 96-well plate containing GSC proliferation medium only (no GSCs) (see part 6.2 for vital dye leakage detection).
  5. Centrifuge the 96-well plate containing the mitochondria recipient GSCs with the MSC mitochondria (step 5.3) and the control plate (step 5.4) at 1,500 x g for 15 min at 4 °C.
  6. Place the culture plates in the 37 °C cell incubator immediately after centrifugation.
    Note: The mitochondria transfer protocol relies on the centrifugation of the mitochondria suspension on the cultured cells at the adequate centrifugation force, with a number of centrifugations that can be adjusted depending on the system of mitochondria donor/recipient cells.

Day 3

6. Analysis of the Mitochondria Transfer by FACS and Confocal Imaging

  1. Preparation of GSC samples for FACS analysis
    Note: If MSC mitochondria were labeled with a vital dye beforehand (Section 1), the efficiency can be monitored by FACS, 24 hr after the mitochondria transfer.
    1. Centrifuge the 96-well plate with the MitoCepted GSCs at 270 x g for 5 min at 20 °C.
    2. Discard the supernatant.
    3. Add 100 µl trypsin (no EDTA) in each well. Incubate at 37 °C for 3 min. Pipet up and down to dissociate the neurospheres formed in the 24 hr time period.
    4. Add 100 µl of GSC basal medium.
    5. Centrifuge the plate at 270 x g for 5 min at 20 °C and discard the supernatant.
    6. Resuspend GSCs in 300 µl GSC basal medium and transfer to FACS adapted tubes.
    7. Perform the FACS analysis.
  2. Control for mitochondria vital dye leakage from the isolated mitochondria (FACS)
    Note: The purpose of this step is to determine the background FACS signal that would not reflect a genuine MSC mitochondria transfer but, rather, the mere leakage of vital dye from the labeled MSC mitochondria used for MitoCeption (step 5.3).
    1. Seed GSC cells as described in steps 3.1 to 3.15.
    2. Incubate the cells at 37 °C for 1 hr.
    3. Centrifuge the 96-well plate containing mitochondria only (from step 5.4 to 5.6) at 1,500 x g for 5 min at 20 °C.
    4. Aspirate the culture medium from the GSC 96-well plate (step 6.2.2) and replace it by the medium incubated with mitochondria only (supernatant from step 6.2.3).
    5. Incubate the 96-well plate containing the GSCs at 37 °C for 2 hr.
    6. Proceed as in steps 6.1.1 to 6.1.7 for FACS analysis.
  3. Preparation of GSC samples for confocal imaging
    Note: If the transfer of MSC mitochondria to GSCs is to be analyzed by fluorescence imaging, both MSCs and GSCs need to be labeled beforehand, respectively with mitochondria (step 1) and cell (step 2) vital dyes.
    1. Prepare GSCs as in steps 6.1.1 to 6.1.5.
    2. Resuspend GSCs in 2 ml GSC proliferation medium containing 2% FBS and seed in 35 mm culture dishes (glass bottom).
    3. Perform confocal imaging on the GSCs with the transferred fluorescent MSC mitochondria 24 hr later.

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Results

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The procedural steps outlining the isolation of mitochondria from mesenchymal stem cells (MSC) and their transfer to the targeted glioblastoma stem cells (GSC) by MitoCeption are shown in Figure 1. GSCs are cancer stem cells grown as neurospheres to preserve their stem cell properties. For the protocol, GSCs are seeded as single cells a couple of hours before the transfer of mitochondria (step 3) to allow higher mitochondria transfer efficiency (see FACS data Figu...

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Discussion

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An increasing number of studies show that cells can exchange mitochondria and that these mitochondria have profound effects on the target cell metabolism and functions. Therefore, it is essential to master the tools to quantitatively transfer mitochondria from the donor cells to these target cells to enable an accurate study of their biological effects.

The protocol described here was originally worked out to transfer mitochondria isolated from human mesenchymal stem cells to the adherent canc...

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Disclosures

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INSERM (Institut National de la Santé et de la Recherche Médicale), with which C.J. and M-L.V. are affiliated, has filed a patent application on the MitoCeption technique (EP14306154.7).

Acknowledgements

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We thank Andrea Parmeggiani (L2C and DIMNP, Montpellier), Benoit Charlot (IES, Montpellier) as well as members of the laboratory for helpful discussions, Christophe Duperray for help with the FACS analysis, the Montpellier RIO imaging facility (MRI) for providing the adequate environment for FACS and confocal microscopy. B.N.M was supported by a graduate fellowship from the LabEx Numev (convention ANR-10-LABX-20). A.B. was supported by an undergraduate fellowship from the University of Warsaw and European Union (n° POKL.04.01.02-00-221/12). M.L.V is a staff scientist from the National Center for scientific research (CNRS).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Mitochondria Isolation Kit for Tissue Fisher Scientific 10579663
N-2 Supplement (100x)Fisher Scientific11520536
B-27 Supplement W/O VIT A (50x)Fisher Scientific 11500446
HBSS w/o Ca2+ w/o Mg2+ SigmaH4385
poly Heme Sigma P3932
aMEM w/o glutamineOzymeBE12-169F
DMEM/F-12 without glutamineFisher Scientific 11540566
L-Glutamine Invitrogen 25030-024 
Glucose Sigma G7021
Insuline Sigma I 1882 
Human bFGF R&D Systems233-FB-025
Human EGF Peprotech AF-100-15 
HeparinSigmaH3149 
CaCl2MERCK2382
Trypsine InhibitorSigma T9003
DNase ISIGMA 10104159001
Trypsine 0.25% / EDTA 1 mMInvitrogen 25200056
TrypsinGibco 15090-046
Protease inhibitors EDTA freeSigma4693159001
Ciprofloxacine Sigma17850-5G-F
Fungine Invivogenant-fn-1
Fungizone Thermofisher15290018
GentamycinEuromedexEU0410
MitoTracker Green FMMolecular ProbesM7514
MitoTracker Red CMXRosMolecular Probes M7512
MitoTracker Deep Red FMMolecular Probes M22426 
CellTracker Green CMFDAMolecular ProbesC7025
CellTracker Blue CMF2HCMolecular ProbesC12881
RIPASanta Cruzsc-24948
FluoroDish Sterile Culture DishWorld Precision InstrumentsFD35-100
HemacytometerFisher Scientific267110
FACS tubesBeckman Coulter2,523,749
FACS apparatusGallios  3L 10C
LC FAST START DNA MASTER PLUS Roche3515885001

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Tags

Mitochondria TransferMesenchymal Stem CellsMitochondria IsolationFluorescence Activated Cell SortingConfocal ImagingMitochondrial DNA AnalysisCell Metabolism AssessmentNeurosphere CultureTunneling Nanotubes

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