Name: mitoception: transferring isolated human msc mitochondria to glioblastoma stem cells
Uploaded: 2017-02-22T15:00:00
Description: Watch this Scientific Journal Video about MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells at JoVE.com

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&#176; POKL.04.01.02-00-221/12). M.L.V is a staff scientist from the National Center for scientific research (CNRS).

Day 1
1. Labeling of the Mesenchymal Stem Cell (MSC) Mitochondria (Optional)
<ol>
	<li>Two days before the mitochondria preparation, seed human MSCs in a 100 mm culture dish, in 10 ml &#945;MEM/FBS 10%, so as to have 4 x 105 MSCs in culture on Day 1.</li>
	<li>Rinse MSCs with PBS (4 ml) and add 4 ml &#945;MEM/FBS 1% (prewarmed to 37 &#176;C).</li>
	<li>Add the required amount of mitochondria vital dye and incubate cells for 30 min in the 37 &#176;C incubator.</li>
	<li>Remove the mit...

<ol>
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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...

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 &#34;donor&#34; and &#34;target&#34; 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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	<tbody>
		<tr>
			<td>Mitochondria Isolation Kit for Tissue&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>10579663</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11520536</td>
		</tr>
		<tr>
			<td>B-27 Supplement W/O VIT A (50X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11500446</td>
		</tr>
		<tr>
			<td>HBSS w/o Ca2+ w/o Mg2+</td>
			<td>&#160;Sigma</td>
			<td>H4385</td>
		</tr>
		<tr>
			<td>poly Heme&#160;</td>
			<td>Sigma&#160;</td>
			<td>P3932</td>
		</tr>
		<tr>
			<td>aMEM w/o glutamine</td>
			<td>Ozyme</td>
			<td>BE12-169F</td>
		</tr>
		<tr>
			<td>DMEM/F-12 without glutamine,&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>11540566</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>25030-024&#160;</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma&#160;</td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>Insuline&#160;</td>
			<td>Sigma&#160;</td>
			<td>I 1882&#160;</td>
		</tr>
		<tr>
			<td>&#160;Human&#160;bFGF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
		</tr>
		<tr>
			<td>Human EGF&#160;</td>
			<td>Peprotech&#160;</td>
			<td>AF-100-15&#160;</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>MERCK</td>
			<td>2382</td>
		</tr>
		<tr>
			<td>Trypsine Inhibitor&#160;</td>
			<td>Sigma&#160;</td>
			<td>T9003</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>SIGMA&#160;</td>
			<td>10104159001</td>
		</tr>
		<tr>
			<td>Trypsine 0.25% /EDTA 1 mM</td>
			<td>Invitrogen&#160;</td>
			<td>25200056</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco&#160;</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>Protease inhibitors EDTA free</td>
			<td>Sigma</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>Ciprofloxacine&#160;</td>
			<td>Sigma</td>
			<td>17850-5G-F</td>
		</tr>
		<tr>
			<td>Fungine&#160;</td>
			<td>Invivogen</td>
			<td>ant-fn-1</td>
		</tr>
		<tr>
			<td>Fungizone&#160;</td>
			<td>Thermofisher</td>
			<td>15290018</td>
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			<td>Gentamycin</td>
			<td>Euromedex</td>
			<td>EU0410</td>
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			<td>M7514</td>
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			<td>&#160;MitoTracker Red CMXRos</td>
			<td>Molecular Probes</td>
			<td>&#160;M7512</td>
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			<td>MitoTracker Deep Red FM</td>
			<td>Molecular Probes</td>
			<td>&#160;M22426&#160;</td>
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			<td>&#160;CellTracker Green CMFDA</td>
			<td>Molecular Probes</td>
			<td>C7025</td>
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			<td>&#160;CellTracker Blue CMF2HC</td>
			<td>Molecular Probes</td>
			<td>C12881</td>
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			<td>RIPA</td>
			<td>Santa Cruz</td>
			<td>sc-24948</td>
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		<tr>
			<td>FluoroDish Sterile Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
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			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
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			<td>FACS tubes</td>
			<td>Beckman Coulter</td>
			<td>2,523,749</td>
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			<td>FACS apparatus</td>
			<td>Gallios&#160;</td>
			<td>&#160;3L 10C</td>
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			<td>LC FAST START DNA MASTER PLUS&#160;</td>
			<td>Roche</td>
			<td>3515885001</td>
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mitoception: transferring isolated human msc mitochondria to glioblastoma stem cells

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.

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...

Watch this Scientific Journal Video about MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells at JoVE.com

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

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.

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

The overall goal of this procedure is to transfer previously isolated mitochondria from donor cells such as mesenchymal stem cells to target cells such as glioblastoma stem cells. This procedure can help answer key questions in the metabolism field such as the metabolic and biological effects of the mitochondria that are transferred spontaneously between cells by processes such as tunneling nanotubes or microvesicles. The main advantage of this technique is that the effects of the mitochondria can be assessed independently of other factors present in the micro environment such as cytokines or metabolites and also of other compounds that can also be trafficking in the tunneling nanotubes.For imaging purposes, MSC mitochondria and GSC can be labeled with vital dyes before the mitochondria transfer by MitoCeption. To begin, after pre-labeling GSCs with vital dyes according to the text protocol, collect GSC neurospheres in a 50 milliliter tube by centrifugation at 270 times g at 20 degrees Celsius for five minutes. Use five milliliters of HBSS to wash the cells and spin the tube again.Aspirate the supernatant and with 100 microliters of 0.25%Trypsin-EDTA gently re-suspend the GSC pellet. Then incubate the cells at 37 degrees Celsius for three minutes. Next, add 10 microliters of 20 millimolar calcium chloride in two microliters at 10 milligram per milliliter of DNase I to the tube.Dissociate the neurospheres by using a P200 pipette to gently pipette up and down 30 to 50 times avoiding the formation of bubbles. Add 10 microliters of 5%Trypsin inhibitor in 10 milliliters of HBSS to the cells and centrifuge the GSCs for seven minutes. Then discard the supernatant and 10 milliliters of GSC Basal Medium to the sample.Now, using a Thoma counting chamber, count the cells before spinning again. Add the appropriate volume of GSC proliferation medium to reach a cellular concentrate of 10 to the sixth GSCs per milliliter. Then seed 10 to the fifth GSCs per well of a 96 well plate.Centrifuge the plate so that the GSCs settle at the bottom of the wells. Check the cells under the microscope. Then incubate the plate at 37 degrees Celsius.To carry out MSC mitochondria isolation, begin by adjusting the microcentrifuge temperature to four degrees Celsius. Prepare two 1.5 milliliter tubes labeled A and C containing reagents for the mitochondria extraction. Prepare two additional tubes labeled MSC and MiTo.Keep the tubes on ice. Also prepare tubes for the mitochondria serial dilutions that will be set up with the mitochondria preparation. Use 10 milliliters of PBS pre-warmed to 37 degrees Celsius to wash the MSCs.Then with two milliliters of EDTA-free Trypsin, wash the cells for 10 seconds and add an additional one milliliter of Trypsin before incubating the cells at 37 degrees Celsius for five to 10 minutes. The use of EDTA should be avoided as per protocol, in particular for MSC Trypsinization and for protease inhibitor used in the mitochondria preparation. Recover the MSCs by adding 10 milliliters of Alpha MEM 10%FBS and transfer the suspension to a 50 milliliter tube.Then centrifuge the cells. Discard the supernatant and add 10 milliliters of Alpha MEM 10%FBS to the cell pellet. Then use a Malassez counting chamber to count the MSCs before spinning down four to five times 10 to the fifth cells.Discard the supernatant, add one milliliter of ice cold Alpha MEM 10%FBS to the cell pellet and transfer the cells to the previously prepared MSC-labeled tube while continuing to keep it on ice. Then centrifuge the MSC tube at 900 times g at four degrees Celsius for five minutes and remove all residual medium from the tube. Next, add 200 microliters of mitochondria isolation reagent A with EDTA-free protease inhibitors to the MSCs and vortex at medium speed for five seconds.Then leave the tubes on ice for exactly two minutes. Add 2.5 microliters of mitochondria isolation reagent B and vortex the cells at maximum speed for 10 seconds before placing the tubes back on ice. Repeat the vortex every 30 seconds for five minutes.Now, add 200 microliters of mitochondria isolation reagent C with EDTA-free protease inhibitors to the tube and mix the contents by tilting the tube approximately 30 times. Centrifuge the tubes at 700 times g at four degrees Celsius for 10 minutes. Transfer the supernatant containing the MSC mitochondria to the previously prepared MiTo tube.Centrifuge the sample at 3, 000 times g at four degrees Celsius for 15 minutes. After discarding the supernatant, use 200 microliters of reagent C to rinse the mitochondrial pellet. Then spin the suspension at 12, 000 times g at four degrees Celsius for five minutes to pellet the organelles.To transfer isolated MSC mitochondria to GSCs, add 200 microliters of pre-cooled GSC proliferation medium to the mitochondrial pellet. Dilute the mitochondrial preparation in GSC proliferation medium to consistently allow the addition of 20 microliters of mitochondrial suspension to the GSCs. Next, slowly add the mitochondria close to the bottom of the well.To control MSC mitochondria vital dye leakage, add the same amount of mitochondria preparation to the wells of a 96 well plate containing GSC proliferation medium only. Centrifuge the plates at 1, 500 times g at four degrees Celsius for 15 minutes. Then immediately place the cultures at 37 degrees Celsius.The following day, to analyze the control wells for vital dye leakage, seed GSC cells as described earlier, centrifuge the plate, and then incubate the cultures at 37 degrees Celsius for one hour. Centrifuge the 96 well plate containing only mitochondria. Then aspirate the medium from the GSC 96 well plate and replace it with the mitochondria supernatant.Incubate the GSCs with the mitochondria supernatant at 37 degrees Celsius for two hours. Finally, carry out analysis of mitochondria transfer, FACS analysis and confocal imaging according to the text protocol. GSCs are cancer stem cells grown as neurospheres to preserve their stem cell properties.Just before mitochondrial transfer, they are seeded as single cells to allow a high mitochondria transfer efficiency and the following day as shown here, the cells are observed again forming neurospheres. In this figure, fluorescently-labeled MSC mitochondria can be seen localized inside Gb4 GSCs. The MSC mitochondria appear throughout the GSCs similar to the GSC mitochondria.The transfer of MSC mitochondria could be observed for most GSCs in 3D reconstruction from GSC confocal images confirm the intercellular localization of the transferred MSC mitochondria. Pre-labeling GSCs with both blue cellular and red mitochondrial vital dyes allowed visualizing the localization of the transferred MSC mitochondria seen in green relative to the endogenous GSC mitochondria. As shown here, MitoCeption with increasing amounts of fluorescent MSC mitochondria led to increased FACS signals and with several orders of magnitude higher than the control signal.The mitochondria transfer protocol presented here performed on single cell GSCs showed a dose-dependent response and relative mean fluorescence intensity. Finally, although MSC mitochondria transfer was also achieved on GSC neurospheres, the efficacy of the transfer to GSCs under these conditions was lower as compared to mitochondrial transferred to GSCs first seeded as single cells. Once mastered, this technique can be done in three hours if it's performed properly.While attempting this procedure, it is important to remember to keep the mitochondria preparation on ice at all times. Following these procedures, other methods like FACS, confocal microscopy, QPCR on mitochondrial DNA can be performed in order to visualize and quantify the MSC mitochondrial transfer to the target cells. Functional studies can also be performed to determine the biological effects of the transferred mitochondria.Keep in mind that these studies will generally be performed with lower amounts of mitochondria than those used to visualize the mitochondria transfer. This technique will allow to explore the outcome and biological effects of mitochondria that are spontaneously transferred between cells either in normal physiological or pathological conditions and will thus contribute to develop novel therapeutic treatments.

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