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>

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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 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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			<td>Mitochondria Isolation Kit for Tissue&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>10579663</td>
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			<td>N-2 Supplement (100X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11520536</td>
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			<td>B-27 Supplement W/O VIT A (50X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11500446</td>
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			<td>HBSS w/o Ca2+ w/o Mg2+</td>
			<td>&#160;Sigma</td>
			<td>H4385</td>
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			<td>poly Heme&#160;</td>
			<td>Sigma&#160;</td>
			<td>P3932</td>
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			<td>aMEM w/o glutamine</td>
			<td>Ozyme</td>
			<td>BE12-169F</td>
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			<td>DMEM/F-12 without glutamine,&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>11540566</td>
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			<td>L-Glutamine&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>25030-024&#160;</td>
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			<td>Sigma&#160;</td>
			<td>I 1882&#160;</td>
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			<td>&#160;Human&#160;bFGF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
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			<td>Human EGF&#160;</td>
			<td>Peprotech&#160;</td>
			<td>AF-100-15&#160;</td>
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			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149&#160;</td>
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			<td>CaCl2</td>
			<td>MERCK</td>
			<td>2382</td>
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			<td>Trypsine Inhibitor&#160;</td>
			<td>Sigma&#160;</td>
			<td>T9003</td>
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			<td>DNase I</td>
			<td>SIGMA&#160;</td>
			<td>10104159001</td>
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			<td>Trypsine 0.25% /EDTA 1 mM</td>
			<td>Invitrogen&#160;</td>
			<td>25200056</td>
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			<td>Trypsin</td>
			<td>Gibco&#160;</td>
			<td>15090-046</td>
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			<td>Protease inhibitors EDTA free</td>
			<td>Sigma</td>
			<td>4693159001</td>
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			<td>Ciprofloxacine&#160;</td>
			<td>Sigma</td>
			<td>17850-5G-F</td>
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			<td>Fungine&#160;</td>
			<td>Invivogen</td>
			<td>ant-fn-1</td>
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			<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>&#160;MitoTracker Green FM</td>
			<td>Molecular Probes</td>
			<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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		<tr>
			<td>&#160;CellTracker Green CMFDA</td>
			<td>Molecular Probes</td>
			<td>C7025</td>
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		<tr>
			<td>&#160;CellTracker Blue CMF2HC</td>
			<td>Molecular Probes</td>
			<td>C12881</td>
		</tr>
		<tr>
			<td>RIPA</td>
			<td>Santa Cruz</td>
			<td>sc-24948</td>
		</tr>
		<tr>
			<td>FluoroDish Sterile Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Beckman Coulter</td>
			<td>2,523,749</td>
		</tr>
		<tr>
			<td>FACS apparatus</td>
			<td>Gallios&#160;</td>
			<td>&#160;3L 10C</td>
		</tr>
		<tr>
			<td>LC FAST START DNA MASTER PLUS&#160;</td>
			<td>Roche</td>
			<td>3515885001</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 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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12.7K Views.  Montpellier University. 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.

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...