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>
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			<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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			<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 ...

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