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 mitochondria dye solution, rinse cells twice with 4 ml prewarmed (37 &#176;C) &#945;MEM/FBS 1% and add back 4 ml &#945;MEM/FBS 10%. Incubate cells at 37 &#176;C.</li>
	<li>Change the culture medium (10 ml &#945;MEM/FBS 10%) after 30 min and, another time, 2 hr later.</li>
</ol>
Day 1
2. Labeling of the Glioblastoma Stem Cells (GSC) (Optional, See Discussion Section)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture medium</td>
			<td>Composition</td>
		</tr>
		<tr>
			<td rowspan="5">GSC basal medium&#160;</td>
			<td>DMEM/F-12 supplemented with&#160;</td>
		</tr>
		<tr>
			<td>Insulin 20 mg/ml</td>
		</tr>
		<tr>
			<td>N2 supplement 1x</td>
		</tr>
		<tr>
			<td>Glucose 3 g/L</td>
		</tr>
		<tr>
			<td>L-glutamine 2 mM</td>
		</tr>
		<tr>
			<td rowspan="9">GSC proliferation medium</td>
			<td>Add to the basal medium:&#160;</td>
		</tr>
		<tr>
			<td>B27 supplement 1x</td>
		</tr>
		<tr>
			<td>EGF 10 ng/ml</td>
		</tr>
		<tr>
			<td>bFGF 10 ng/ml</td>
		</tr>
		<tr>
			<td>Fungine 10 mg/ml</td>
		</tr>
		<tr>
			<td>Fungizone 0.25 mg/ml</td>
		</tr>
		<tr>
			<td>Heparin 2 mg /ml</td>
		</tr>
		<tr>
			<td>Ciprofloxacin 2 &#956;g / ml</td>
		</tr>
		<tr>
			<td>Gentamicin 2 &#956;g/ml</td>
		</tr>
		<tr>
			<td rowspan="4">MSC proliferation medium&#160;</td>
			<td>&#945;MEM supplemented with</td>
		</tr>
		<tr>
			<td>L-glutamine 2 mM</td>
		</tr>
		<tr>
			<td>10% FBS</td>
		</tr>
		<tr>
			<td>bFGF 2 ng/ml</td>
		</tr>
	</tbody>
</table>
Table 1: Culture Media.
<ol>
	<li>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).</li>
	<li>Seed GSCs in a 48-well plate at 105 cells/well in GSC proliferation medium (500 &#181;l) (see Table 1).</li>
	<li>Centrifuge the plate at 270 x g for 5 min at 20 &#176;C.</li>
	<li>Add the required amount of cell vital dye and incubate for 30 min at 37 &#176;C.</li>
	<li>Add 500 &#181;l of GSC basal medium (Table 1) per well of the 48-well plate.</li>
	<li>Centrifuge the plate at 270 x g at 20 &#176;C for 5 min. Aspirate the supernatant.</li>
	<li>Repeat steps 2.5 to 2.6.</li>
	<li>Add 500 &#181;l of GSC basal medium and incubate at 37 &#176;C for 30 min.</li>
	<li>Centrifuge the plate at 270 x g at 20 &#176;C for 5 min. Aspirate the supernatant.</li>
	<li>Add 500 &#181;l of GSC proliferation medium per well of the 48-well plate and incubate in the 37 &#176;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.</li>
</ol>
Day 2
3. Seeding of Glioblastoma Stem Cells
<ol>
	<li>Collect the GSC neurospheres (10 x 106 cells) by centrifugation at 270 x g for 5 min, at 20 &#176;C in a 50 ml tube.</li>
	<li>Wash cells with 5 ml of HBSS, and centrifuge at 270 x g for 5 min at 20 &#176;C.</li>
	<li>Aspirate the supernatant.</li>
	<li>Gently resuspend the GSC pellet in 100 &#181;l trypsin-EDTA (0.25%) (per 10 x 106 cells)</li>
	<li>Incubate at 37 &#176;C for 3 min.</li>
	<li>Add 10 &#181;l CaCl2 (20 mM) and 2 &#181;l DNase I (10 mg/ml).</li>
	<li>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.</li>
	<li>Add 10 &#181;l trypsin inhibitor (5%) and 10 ml HBSS.</li>
	<li>Centrifuge GSCs at 270 x g for 7 min at 20 &#176;C.</li>
	<li>Discard the supernatant and add 10 ml GSC basal medium (Table 1).</li>
	<li>Count GSCs with a Thoma counting chamber and then centrifuge the cells at 270 x g for 7 min at 20 &#176;C.</li>
	<li>Add the appropriate volume of GSC proliferation medium (Table 1) to reach the cellular concentration of 106 GSCs/ml.</li>
	<li>Seed 105 GSCs (100 &#181;l of the cell suspension) per well of a 96-well plate.</li>
	<li>Centrifuge the plates at 270 x g for 7 min at 20 &#176;C to get GSCs at the bottom of wells.</li>
	<li>Incubate the 96-well plate at 37 &#176;C until Section 5.</li>
</ol>
Day 2
4. MSC Mitochondria Isolation
<ol>
	<li>Adjust the microtube centrifuge temperature to 4 &#176;C.</li>
	<li>Prepare two 1.5 ml tubes &#34;A&#34; and &#34;C&#34; containing the reagents for the mitochondria extraction (200 &#181;l reagent A and 400 &#181;l reagent C, both containing the EDTA-free protease inhibitors). Prepare 2 other tubes, labeled &#34;MSC&#34; and &#34;Mito&#34;. Keep all tubes on ice. Also prepare tubes for the mitochondria serial dilutions.</li>
	<li>Wash MSCs with 10 ml prewarmed (37 &#176;C) PBS.</li>
	<li>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 &#176;C.</li>
	<li>Recover the MSCs by adding 10 ml &#945;MEM/FBS 10%, transfer to a 50 ml tube.</li>
	<li>Centrifuge cells at 270 x g for 5 min at 20 &#176;C.</li>
	<li>Discard the supernatant and add 10 ml &#945;MEM/FBS 10% to the cell pellet.</li>
	<li>Count the MSCs with a Malassez counting chamber.</li>
	<li>Centrifuge MSCs (4-5 x 105) at 270 x g for 5 min at 20 &#176;C.</li>
	<li>Discard the supernatant, add 1 m ice-cold &#945;MEM/FBS 10% to the cell pellet and transfer the cells to the &#34;MSC&#34;-labeled tube (prepared at step 4.2). Keep the tube on ice.</li>
	<li>Centrifuge the tube containing the MSCs at 900 x g for 5 min, at 4 &#176;C.</li>
	<li>Remove all residual medium from the tube.</li>
	<li>Add 200 &#181;l 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.</li>
	<li>Add 2.5 &#181;l 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.</li>
	<li>Add 200 &#181;l 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 &#176;C.</li>
	<li>Transfer the supernatant (containing the MSC mitochondria) to the &#34;Mito&#34; tube (prepared at step 4.2).</li>
	<li>Centrifuge at 3,000 x g, 15 min at 4 &#176;C, to get the mitochondria pellet. Discard the supernatant. The pellet contains the isolated mitochondria.</li>
	<li>Rinse the mitochondria pellet with 200 &#181;l of the Reagent C. Then, centrifuge at 12,000 x g, 5 min at 4 &#176;C, to get the mitochondria pellet.</li>
</ol>
5. Transfer of Isolated MSC Mitochondria to GSCs (MitoCeption)
<ol>
	<li>Add 200 &#181;l of the pre-cooled (0 &#176;C) GSC proliferation medium to the mitochondria pellet isolated from MSCs (4 x 105).</li>
	<li>Dilute the mitochondria preparation (in GSC proliferation medium) to consistently add 20 &#181;l of mitochondrial suspension to the GSCs.</li>
	<li>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 &#181;g). Add the mitochondria slowly, close to the bottom of the well, covering the entire surface at least once.</li>
	<li>For controlling MSC mitochondria vital dye leakage, add the same amounts of the mitochondria preparation (0.1 to 10 &#181;g) to wells of a 96-well plate containing GSC proliferation medium only (no GSCs) (see part 6.2 for vital dye leakage detection).</li>
	<li>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 &#176;C.</li>
	<li>Place the culture plates in the 37 &#176;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.</li>
</ol>
Day 3
6. Analysis of the Mitochondria Transfer by FACS and Confocal Imaging
<ol>
	<li>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.
	<ol>
		<li>Centrifuge the 96-well plate with the MitoCepted GSCs at 270 x g for 5 min at 20 &#176;C.</li>
		<li>Discard the supernatant.</li>
		<li>Add 100 &#181;l trypsin (no EDTA) in each well. Incubate at 37 &#176;C for 3 min. Pipet up and down to dissociate the neurospheres formed in the 24 hr time period.</li>
		<li>Add 100 &#181;l of GSC basal medium.</li>
		<li>Centrifuge the plate at 270 x g for 5 min at 20 &#176;C and discard the supernatant.</li>
		<li>Resuspend GSCs in 300 &#181;l GSC basal medium and transfer to FACS adapted tubes.</li>
		<li>Perform the FACS analysis.</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>Seed GSC cells as described in steps 3.1 to 3.15.</li>
		<li>Incubate the cells at 37 &#176;C for 1 hr.</li>
		<li>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 &#176;C.</li>
		<li>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).</li>
		<li>Incubate the 96-well plate containing the GSCs at 37 &#176;C for 2 hr.</li>
		<li>Proceed as in steps 6.1.1 to 6.1.7 for FACS analysis.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Prepare GSCs as in steps 6.1.1 to 6.1.5.</li>
		<li>Resuspend GSCs in 2 ml GSC proliferation medium containing 2% FBS and seed in 35 mm culture dishes (glass bottom).</li>
		<li>Perform confocal imaging on the GSCs with the transferred fluorescent MSC mitochondria 24 hr later.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Vafai, S. B., Mootha, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+disorders+as+windows+into+an+ancient+organelle.">Mitochondrial disorders as windows into an ancient organelle.</a> Nature. 491 (7424), 374-383 (2012).</li><li>Nunnari, J., Suomalainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+in+sickness+and+in+health.">Mitochondria: in sickness and in health.</a> Cell. 148 (6), 1145-1159 (2012).</li><li>Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+as+signaling+organelles.">Mitochondria as signaling organelles.</a> BMC Biol. 12 (1), 34 (2014).</li><li>Schulze, A., Harris, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+cancer+metabolism+is+tuned+for+proliferation+and+vulnerable+to+disruption.">How cancer metabolism is tuned for proliferation and vulnerable to disruption.</a> Nature. 491 (7424), 364-373 (2012).</li><li>Peiris-Pages, M., Martinez-Outschoorn, U. E., Pestell, R. G., Sotgia, F., Lisanti, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+metabolism.">Cancer stem cell metabolism.</a> Breast Cancer Res. 18 (1), 55 (2016).</li><li>LeBleu, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGC-1alpha+mediates+mitochondrial+biogenesis+and+oxidative+phosphorylation+in+cancer+cells+to+promote+metastasis.">PGC-1alpha mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis.</a> Nat Cell Biol. 16 (10), 992-1003 (2014).</li><li>Liu, S., Feng, M., Guan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+sensing+by+STING+signaling+participates+in+inflammation,+cancer+and+beyond.">Mitochondrial DNA sensing by STING signaling participates in inflammation, cancer and beyond.</a> Int J Cancer. 139 (4), 736-741 (2016).</li><li>Islam, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transfer+from+bone-marrow-derived+stromal+cells+to+pulmonary+alveoli+protects+against+acute+lung+injury.">Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury.</a> Nat Med. 18 (5), 759-765 (2012).</li><li>Pasquier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+transfer+of+mitochondria+from+endothelial+to+cancer+cells+through+tunneling+nanotubes+modulates+chemoresistance.">Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance.</a> J Transl Med. 11, 94 (2013).</li><li>Plotnikov, E. Y., Khryapenkova, T. G., Galkina, S. I., Sukhikh, G. T., Zorov, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasm+and+organelle+transfer+between+mesenchymal+multipotent+stromal+cells+and+renal+tubular+cells+in+co-culture.">Cytoplasm and organelle transfer between mesenchymal multipotent stromal cells and renal tubular cells in co-culture.</a> Exp Cell Res. 316 (15), 2447-2455 (2010).</li><li>Spees, J. L., Olson, S. D., Whitney, M. J., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transfer+between+cells+can+rescue+aerobic+respiration.">Mitochondrial transfer between cells can rescue aerobic respiration.</a> Proc Natl Acad Sci U S A. 103 (5), 1283-1288 (2006).</li><li>Liu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+rescue+injured+endothelial+cells+in+an+in+vitro+ischemia-reperfusion+model+via+tunneling+nanotube+like+structure-mediated+mitochondrial+transfer.">Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer.</a> Microvasc Res. 92, 10-18 (2014).</li><li>Bukoreshtliev, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+block+of+tunneling+nanotube+(TNT)+formation+inhibits+intercellular+organelle+transfer+between+PC12+cells.">Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells.</a> FEBS Lett. 583 (9), 1481-1488 (2009).</li><li>Gurke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunneling+nanotube+(TNT)-like+structures+facilitate+a+constitutive,+actomyosin-dependent+exchange+of+endocytic+organelles+between+normal+rat+kidney+cells.">Tunneling nanotube (TNT)-like structures facilitate a constitutive, actomyosin-dependent exchange of endocytic organelles between normal rat kidney cells.</a> Exp Cell Res. 314 (20), 3669-3683 (2008).</li><li>Vallabhaneni, K. C., Haller, H., Dumler, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+cells+initiate+proliferation+of+mesenchymal+stem+cells+by+mitochondrial+transfer+via+tunneling+nanotubes.">Vascular smooth muscle cells initiate proliferation of mesenchymal stem cells by mitochondrial transfer via tunneling nanotubes.</a> Stem Cells Dev. 21 (17), 3104-3113 (2012).</li><li>Wang, X., Gerdes, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+mitochondria+via+tunneling+nanotubes+rescues+apoptotic+PC12+cells.">Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells.</a> Cell Death Differ. 22 (7), 1181-1191 (2015).</li><li>Plotnikov, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+cross-talk+between+mesenchymal+stem+cells+and+cardiomyocytes+in+co-culture.">Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.</a> J Cell Mol Med. 12 (5A), 1622-1631 (2008).</li><li>Acquistapace, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+reprogram+adult+cardiomyocytes+toward+a+progenitor-like+state+through+partial+cell+fusion+and+mitochondria+transfer.">Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer.</a> Stem Cells. 29 (5), 812-824 (2011).</li><li>Hase, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-Sec+promotes+membrane+nanotube+formation+by+interacting+with+Ral+and+the+exocyst+complex.">M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex.</a> Nat Cell Biol. 11 (12), 1427-1432 (2009).</li><li>Phinney, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+use+extracellular+vesicles+to+outsource+mitophagy+and+shuttle+microRNAs.">Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs.</a> Nat Commun. 6, 8472 (2015).</li><li>Ahmad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miro1+regulates+intercellular+mitochondrial+transport+&amp;+enhances+mesenchymal+stem+cell+rescue+efficacy.">Miro1 regulates intercellular mitochondrial transport &amp; enhances mesenchymal stem cell rescue efficacy.</a> Embo J. 33 (9), 994-1010 (2014).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPSC-MSCs+with+High+Intrinsic+MIRO1+and+Sensitivity+to+TNF-a+Yield+Efficacious+Mitochondrial+Transfer+to+Rescue+Anthracycline-Induced+Cardiomyopathy.">iPSC-MSCs with High Intrinsic MIRO1 and Sensitivity to TNF-a Yield Efficacious Mitochondrial Transfer to Rescue Anthracycline-Induced Cardiomyopathy.</a> Stem Cell Reports. 7 (4), 749-763 (2016).</li><li>Takeda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+intergeneric/interspecies+mitochondrial+injection;+parthenogenetic+development+of+bovine+oocytes+after+injection+of+mitochondria+derived+from+somatic+cells.">Influence of intergeneric/interspecies mitochondrial injection; parthenogenetic development of bovine oocytes after injection of mitochondria derived from somatic cells.</a> J Reprod Dev. 58 (3), 323-329 (2012).</li><li>Takeda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+cytoplasm+or+mitochondria+derived+from+somatic+cells+affects+parthenogenetic+development+of+murine+oocytes.">Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes.</a> Biol Reprod. 72 (6), 1397-1404 (2005).</li><li>Van Blerkom, J., Sinclair, J., Davis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transfer+between+oocytes:+potential+applications+of+mitochondrial+donation+and+the+issue+of+heteroplasmy.">Mitochondrial transfer between oocytes: potential applications of mitochondrial donation and the issue of heteroplasmy.</a> Hum Reprod. 13 (10), 2857-2868 (1998).</li><li>Ishikawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS-generating+mitochondrial+DNA+mutations+can+regulate+tumor+cell+metastasis.">ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis.</a> Science. 320 (5876), 661-664 (2008).</li><li>Kaipparettu, B. A., Ma, Y., Wong, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+effects+of+cancer+mitochondria+on+energy+metabolism+and+tumorigenesis:+utility+of+transmitochondrial+cybrids.">Functional effects of cancer mitochondria on energy metabolism and tumorigenesis: utility of transmitochondrial cybrids.</a> Ann N Y Acad Sci. 1201, 137-146 (2010).</li><li>Wu, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Transfer+by+Photothermal+Nanoblade+Restores+Metabolite+Profile+in+Mammalian+Cells.">Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells.</a> Cell Metab. 23 (5), 921-929 (2016).</li><li>Masuzawa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+autologously+derived+mitochondria+protects+the+heart+from+ischemia-reperfusion+injury.">Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury.</a> Am J Physiol Heart Circ Physiol. 304 (7), H966-H982 (2013).</li><li>Kitani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+human+mitochondrial+transfer:+a+novel+concept+based+on+the+endosymbiotic+theory.">Direct human mitochondrial transfer: a novel concept based on the endosymbiotic theory.</a> Transplant Proc. 46 (4), 1233-1236 (2014).</li><li>Caicedo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MitoCeption+as+a+new+tool+to+assess+the+effects+of+mesenchymal+stem/stromal+cell+mitochondria+on+cancer+cell+metabolism+and+function.">MitoCeption as a new tool to assess the effects of mesenchymal stem/stromal cell mitochondria on cancer cell metabolism and function.</a> Sci Rep. 5, 9073 (2015).</li><li>Kesner, E. E., Saada-Reich, A., Lorberboum-Galski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Mitochondrial+Transformation+into+Human+Cells.">Characteristics of Mitochondrial Transformation into Human Cells.</a> Sci Rep. 6, 26057 (2016).</li><li>Lathia, J. D., Mack, S. C., Mulkearns-Hubert, E. E., Valentim, C. L., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+glioblastoma.">Cancer stem cells in glioblastoma.</a> Genes Dev. 29 (12), 1203-1217 (2015).</li><li>Osswald, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumour+cells+interconnect+to+a+functional+and+resistant+network.">Brain tumour cells interconnect to a functional and resistant network.</a> Nature. 528 (7580), 93-98 (2015).</li><li>Shinojima, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+mediates+homing+of+bone+marrow-derived+human+mesenchymal+stem+cells+to+glioma+stem+cells.">TGF-beta mediates homing of bone marrow-derived human mesenchymal stem cells to glioma stem cells.</a> Cancer Res. 73 (7), 2333-2344 (2013).</li><li>Velpula, K. K., Dasari, V. R., Rao, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homing+of+human+cord+blood+stem+cells+to+sites+of+inflammation:+unfolding+mysteries+of+a+novel+therapeutic+paradigm+for+glioblastoma+multiforme.">The homing of human cord blood stem cells to sites of inflammation: unfolding mysteries of a novel therapeutic paradigm for glioblastoma multiforme.</a> Cell Cycle. 11 (12), 2303-2313 (2012).</li><li>Guichet, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+neuronal+differentiation+of+glioblastoma+stem-like+cells+induced+by+neurogenic+transcription+factors.">Cell death and neuronal differentiation of glioblastoma stem-like cells induced by neurogenic transcription factors.</a> Glia. 61 (2), 225-239 (2013).</li><li>Lyons, E. A., Scheible, M. K., Sturk-Andreaggi, K., Irwin, J. A., Just, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+Sanger+strategy+for+human+mitochondrial+genome+sequencing.">A high-throughput Sanger strategy for human mitochondrial genome sequencing.</a> BMC Genomics. 14, 881 (2013).</li></ol>

INSERM (Institut National de la Sant&#233; et de la Recherche M&#233;dicale), with which C.J. and M-L.V. are affiliated, has filed a patent application on the MitoCeption technique (EP14306154.7).

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 cancer cell line MDA-MB-231 33. As shown here, it can be adapted for cancer stem cells that grow as neurospheres in vitro. Although the mitochondria transfer does occur when MSC mitochondria are added to the GSC neurospheres, it was found to be more efficient on single cell GSCs, as described in the present protocol. This protocol could also be extrapolated to other non-adherent cells, like T cells, with the need to adapt the seeded cell concentration for the MitoCeption step.
The mitochondria preparation should be performed on ice to maintain their integrity. To obtain mitochondria preparations with reduced contamination from other cytosol compounds, centrifugation for the recovery of mitochondria is performed at 3,000 x g for 15 min. To monitor the amount of MSC mitochondria used for the transfer to the GSCs, the mitochondria protein content is determined. For this purpose, mitochondria protein extracts are prepared using RIPA buffer supplemented with protease inhibitors. The protein concentration was determined using the bicinchoninic acid assay, according to the manufacturer&#39;s instructions. Bovine serum albumin was used as a standard. On average 10 &#181;g of proteins were obtained from 100,000 MSCs.
Labeling MSC mitochondria with a vital dye allows quantifying the efficiency of the mitochondria transfer, by fluorescence-activated cell sorting (FACS), and determining the localization of the transferred mitochondria by confocal imaging. The GSCs can be labelled with the same mitochondria vital dye as the MSCs. These cells will be used as a positive control for the FACS analysis. Alternatively, GSCs can be labeled with a mitochondria vital dye different from that of the MSCs, allowing the localization of the transferred MSC mitochondria relative to the endogenous GSC mitochondria. The deep red mitochondria vital dye is well suited for FACS analysis while the green and red mitochondria dyes are preferred for confocal imaging. Of great importance, MSC exposure to EDTA should be avoided at all steps of the protocol. Therefore, cell trypsinization is recommended with trypsin and no EDTA; the cocktail of protease inhibitors used for the mitochondria preparation should, as well, be devoid of EDTA. In the presence of EDTA, a leakage of the mitochondria vital dye from MSC mitochondria was observed. If the mitochondria transfer to GSCs is to be analyzed by microscopy, the recipient glioblastoma cells should also be labeled with a vital dye. The green and blue cell dyes (see Materials and Reagents Table) were preferred as they give a more homogeneous cell labeling, enabling 3D reconstruction from confocal images.
Once the protocol has been validated by the user with his specific cells, mitochondria labeling will be left out for functional studies, to preclude possible biological biases. Dose-response analyses over a wide range of MSC mitochondria concentrations, with serial dilutions of the mitochondria preparation, is advisable. In fact, it should be noted that biological effects for the transferred mitochondria could be achieved for low mitochondria concentrations, in the lower range for detection by FACS or imaging. These functional studies, including investigations on GSC metabolism, proliferation and response to therapy, are carried out the day following the mitochondria transfer.
If MSCs and GSCs are isolated from different donors (with different mtDNA haplotypes), the transferred MSC mitochondria can be monitored by measuring the concentrations of MSC mtDNA in the targeted GSCs. The discrimination between the MSC and endogenous GSC mtDNAs is based on the presence of SNPs, specific for each cell type, in the mtDNA hypervariable D-loop region. Therefore, among the various applications of the MitoCeption technique, the possibility of transferring mitochondria harboring mitochondrial DNA with different haplotypes not only allows detection of the transferred mitochondria but also provides the tools to study the biology of mtDNA maintenance, including the analysis of haplotype exclusion.

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.

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

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 Figure 3B). Following Section 5 (day 3), these cells are observed again as forming neurospheres (Figure 1).
Imaging of the Gb4 GSCs, 72 hr after MitoCeption with fluorescently-labeled MSC mitochondria, shows that the MSC mitochondria are localized inside the Gb4 GSCs, as demonstrated by the orthogonal views obtained from confocal images (Figure 2B). The transferred MSC mitochondria appear localized throughout the GSCs, as this is also the case for the endogenous GSC mitochondria (Figure 2A). Confocal imaging of the MitoCepted GSCs was also performed with MSC mitochondria prelabeled with a deep red vital dye so that the same GSC samples were analyzed both by FACS (see Figure 3A) and imaging (Figure 2C). The transfer of MSC mitochondria could be observed for most GSCs (panel a) and 3D reconstruction from GSC confocal images confirmed the intracellular localization of the transferred MSC mitochondria (panels b, c). Prelabeling GSCs (on day 1) with both blue cellular and red mitochondrial vital dyes allowed visualizing the localization of the transferred MSC mitochondria (colored in green) relative to the endogenous GSC mitochondria (in red), following the MSC mitochondria transfer (Figure 2D).
While confocal imaging allows checking the intracellular localization of the transferred MSC mitochondria following MitoCeption, fluorescence-activated cell sorting (FACS) is the tool of choice to quantify the mitochondria transfer, provided MSC mitochondria were prelabeled with a vital dye. As shown in Figure 3A, MitoCeption with increasing amounts of fluorescent MSC mitochondria led to increased FACS signals. Noteworthy, this signal (in red) was several orders of magnitude higher than the signal obtained in the control conditions (in blue, see step 6.2). This further provided evidence that the FACS signal is representative of MSC mitochondria transferred inside GSCs and not merely the result of the fluorescent vital dye leakage from the labeled MSC mitochondria. The mitochondria transfer protocol presented here, performed on single cell GSCs, showed a dose-dependent response (Figure 3B). The efficiency of the MSC mitochondria transfer was also evaluated on neurospheres. Although this also led to MSC mitochondria transfer to the GSCs, the efficacy of the transfer was lower, as shown in a representative experiment (Figure 3B).
The transfer of MSC mitochondria and their maintenance inside the GSCs can be followed by quantifying MSC mitochondrial DNA (mtDNA) in the GSCs. This requires MSCs and GSCs to be obtained from different donors, with distinct haplotypes. Figure 3C shows mtDNA sequences found within the D-loop of two donors with single nucleotide polymorphisms (SNPs) indicated at the 3&#39; end position. For the design of PCR primers, to specifically amplify mtDNA from either donor 1 or donor 2, an additional mutation was introduced at position n-2 relative to the 3&#39; end of the PCR primers. The mtDNA from donor 1 could be amplified with the set of primers: 5&#39;-TAACAGTACATAGCACATACAA-3&#39; and 5&#39;-GAGGATGGTGGTCAAGGGA-3&#39; (universal sequencing primer 38), while the set of primers: 5&#39;-TTAACTCCACCATTAGCACC-3&#39; (universal sequencing primer from 38) and 5&#39;-AGTATTTATGGTACCGTCCG-3&#39; was used to amplify the mtDNA from donor 2.
<img alt="Figure 1" src="/files/ftp_upload/55245/55245fig1.jpg" /> 
Figure 1: Workflow for the transfer of isolated MSC mitochondria to GSCs by MitoCeption. The 7 steps for the MitoCeption of MSC mitochondria to GSCs and the subsequent GSC analyses are shown. The step numbers are indicated as in the protocol section. If MSC and GSC labeling is not performed, as for functional analyses, steps 1 and 2 can be omitted and the protocol can be followed from day 2 on. Phase contrast images represent Gb4 GSCs as neurospheres (day 1), as unicellular culture ready for the MSC mitochondria transfer (day 2) and 24 hr after the mitochondria transfer (day 3). Scale bar = 100 &#181;m. In step 4, representative photomicrograph of isolated MSC mitochondria, prelabeled with MitoTracker Red CMXRos before isolation from MSCs. Scale bar = 5 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55245/55245fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55245/55245fig2.jpg" /> 
Figure 2: Imaging of Gb4 endogenous mitochondria and of MSC mitochondria after transfer to the Gb4 GSCs by MitoCeption. (A, B, C) Gb4 GSCs were labeled with a green vital dye. (B, C, D) Gb4 GSCs were MitoCepted with 2.5 &#181;g MSC mitochondria preparation (for 105 GSCs). (A) GSCs were labeled with MitoTracker Red CMXRos and cultured for 48 hr (GSC proliferation medium w/o EGF/bFGF). (B) GSC confocal section and orthogonal views, 72 hr after MitoCeption with MitoTracker Red CMXRos-labeled MSC mitochondria (culture medium: GSC proliferation medium/FBS 2%). (C, D) MSCs were prelabeled with a deep red mitochondria vital dye (same conditions as for FACS analysis) before the transfer of MSC mitochondria to GSCs. After the MSC mitochondria transfer, GSCs were seeded in GSC proliferation medium containing FBS 10%. (C) Isosurface views (b, c) with xy plane section (c) of a GSC 3D reconstruction from confocal images (48 hr after MitoCeption). (D) Gb4 GSCs were labeled with a blue cell vital dye and a red mitochondria vital dye before the transfer of MSC mitochondria prelabeled with a deep red mitochondria vital dye (imaged in green). Isosurface views with xz (b) and xy (c) plane sections of a GSC 3D reconstruction from confocal images (72 hr after the MSC mitochondria transfer). All cells were seeded on culture dishes with glass bottom and images were taken on live cells. Scale bars = 5 &#181;m, except for image C panel (a) where Scale bar = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55245/55245fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55245/55245fig3.jpg" /> 
Figure 3: Quantification of the mitochondria transfer by fluorescence-activated cell sorting (FACS) and detection of MSC mitochondrial DNA (mtDNA) in the GSCs. (A, B) GSCs were MitoCepted with MSC mitochondria prelabeled with a deep red mitochondria vital dye and analyzed by FACS. (A) Representative FACS results obtained with MitoCepted GSCs (in red, from top to bottom: 0.6; 1.2; 2.5; 5; 10 &#181;g MSC mitochondria preparation (for 105 GSCs)). In blue, for each panel, control FACS profile of GSCs incubated with the culture medium conditioned with the same amount of MSC mitochondria as used for MitoCeption (see step 5.4). Control conditions with unlabeled GSCs in black (top). (B) Relative mean fluorescence intensity (MFI) obtained after the mitochondria transfer on single cell GSCs (- &#8226;) (average of 3 experiments, with SEM), on one-day GSC neurospheres (- o) and with control mitochondria supernatants on GSC single cells (--). (C) DNA sequences within the mtDNA D-loop and SNP differences between donors 1 and 2. SNPs are indicated in blue at the 3&#39; end of the sequences. Design of primers for specific PCR amplification of mtDNA from either donor (primers spe 1 and 2). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55245/55245fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

mitoception-transferring-isolated-human-msc-mitochondria-to

Nanyang Technological University

Research

JoVE Journal

Biology

12.8K Views.  Montpellier University.  Hier, wordt een protocol (MitoCeption) voorgelegd aan mitochondriën geïsoleerd uit menselijke mesenchymale stamcellen (MSC) over te dragen, glioblastoma stamcellen (GSC), met als doel het bestuderen van hun biologische effecten op het metabolisme en de functies SGR. Een soortgelijk protocol kan worden aangepast aan mitochondriën dragen tussen andere celtypen. 

Video: MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Neuroscience

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

Cancer Research

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular ...

Developmental Biology

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Immunology and Infection

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Medicine

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Investigating Mitochondrial Transfer in MSC and ARPE19 Co-Cultures

Mitochondrial Transfer Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods.
We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.

Author Spotlight: Bidirectional Mitochondrial Transfer between MSCs and Retinal Pigment Epithelium Cells &#8212; Pathways and In Vivo Challenges

Here, we present a protocol including mitochondrial tracing, direct co-culture procedures of mesenchymal stem cells (MSCs) and retinal pigment epithelial cells (ARPE19), as well as the methods for observing and statistically analyzing tunneling nanotubes (TNT) formation and mitochondrial transfer to characterize mitochondrial exchange via TNTs between MSCs and ARPE19 cells.

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important ...

Centrifugation-Assisted Mitochondrial Transfer to Glioblastoma Stem Cells

Source: Mombo, B. N., et al.&#160;<a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55245">MitoCeption: transferring isolated human MSC mitochondria to glioblastoma stem cells.</a> J. Vis. Exp. (2017)
In this video, a method is demonstrated to enable the transfer of fluorescently labeled mitochondria into glioblastoma stem cells (GSCs) through centrifugation. The successful uptake and functional integration of the donor mitochondria are confirmed by detecting the fluoresce using confocal microscopy.

Source: Mombo, B. N., et al.&#160;MitoCeption: transferring isolated human MSC mitochondria to glioblastoma stem cells. J. Vis. Exp. (2017)
In this video, ...

JoVE Encyclopedia of Experiments

Neuropathology

Tumors

A Lentiviral Transcriptional Reporter System to Generate a Glioblastoma Stem Cell Differentiation Reporter Line

To begin seed 1 million glioblastoma stem cells or GSCs in 2 milliliters of complete neural stem cell growth medium in a 6-well plate. Next, transduce the cells with the lentivirus reporter at a multiplicity of infection equal to five.
To the cell and virus mixture, add 2 microliters of polybrene such that the final concentration is 8 micrograms per milliliter, and incubate the cells at 37 degrees Celsius and 5% CO2 overnight. After incubation, replace the growth medium to remove unbound virus, and centrifuge cells at 360 times g for 5 minutes. Then, carefully aspirate medium and plate cells back in a 6-well plate, and continue to incubate the cells for 24 hours.
Harvest 0.5 milliliters of the total culture volume. Pellet cells by centrifugation at 360 times g for 5 minutes at room temperature. Resuspend cells in 0.5 milliliters fresh neural stem cell growth medium.
Place the plate back in the incubator. To allow for expansion for at least 72 hours. Add 200 microliters of dissociation reagent, and incubate for five minutes in a water bath set to 37 degrees Celsius. Dissociate cells by pipetting up and down gently. To the dissociated cells, add 800 microliters of HBSS to ensure minimal cell attachment or aggregation.
Next, transfer 200 microliters of each cell suspension into wells of a 96 multi-well plate. Determine the percentage of cells expressing the green fluorescent protein by flow cytometry. Record at least 10,000 viable cells in each acquisition.

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Samenvatting

Meer video's verkennen