<meta charset="utf8"></head><body>Investigando a transferência mitocondrial em coculturas de MSC e ARPE19

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotubular+highways+for+intercellular+organelle+transport.">Nanotubular highways for intercellular organelle transport.</a> Science. 303 (5660), 1007-1010 (2004).</li><li>Cordero Cervantes, D., Zurzolo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peering+into+tunneling+nanotubes-the+path+forward.">Peering into tunneling nanotubes-the path forward.</a> Embo j. 40 (8), e105789 (2021).</li><li>Qin, 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=The+functions,+methods,+and+mobility+of+mitochondrial+transfer+between+cells.">The functions, methods, and mobility of mitochondrial transfer between cells.</a> Front Oncol. 11, 672781 (2021).</li><li>Jiang, D., 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=Bioenergetic+crosstalk+between+mesenchymal+stem+cells+and+various+ocular+cells+through+the+intercellular+trafficking+of+mitochondria.">Bioenergetic crosstalk between mesenchymal stem cells and various ocular cells through the intercellular trafficking of mitochondria.</a> Theranostics. 10 (16), 7260-7272 (2020).</li><li>Domhan, 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=Intercellular+communication+by+exchange+of+cytoplasmic+material+via+tunneling+nano-tube+like+structures+in+primary+human+renal+epithelial+cells.">Intercellular communication by exchange of cytoplasmic material via tunneling nano-tube like structures in primary human renal epithelial cells.</a> PLoS One. 6 (6), e21283 (2011).</li><li>Zhang, J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+nanotubes:+Novel+communication+between+distant+cells.">Membrane nanotubes: Novel communication between distant cells.</a> Sci China Life Sci. 56 (11), 994-999 (2013).</li><li>Cheng, X. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>NEST</td>
			<td>703001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>BD Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>NEST</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>ARPE19 cells</td>
			<td>ATCC</td>
			<td>CRL-2302</td>
			<td>Cell lines</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Beyotime</td>
			<td>ST025</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace violet</td>
			<td>Invitrogen</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>C141905005BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12-GlutaMAX</td>
			<td>Gibco</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VivaCell</td>
			<td>C04002-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FluorSave Reagent</td>
			<td>Millipore</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr>
			<td>MSCs</td>
			<td>Nuwacell</td>
			<td>RC02003</td>
			<td>Cell lines</td>
		</tr>
		<tr>
			<td>ncMission</td>
			<td>Shownin</td>
			<td>RP02010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>pCT-Mito-GFP</td>
			<td>SBI</td>
			<td>CYTO102-PA-1</td>
			<td>Plasmid; From&#160; https://www.systembio.com/mitochondria-cyto-tracer-pct-mito-gfp-cmv</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>MCE</td>
			<td>HY-B1743A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Axygen</td>
			<td>TF-1000-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Invitrogen</td>
			<td>A22287</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell plate</td>
			<td>Corning</td>
			<td>3470</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial transfer via tunneling nanotubes between mesenchymal stem cells and retinal pigment epithelium in vitro

Mitochondria serve as the primary energy source for most cell types, with mitochondrial dysfunction particularly impacting high-energy-demanding tissues like the retina1. Metabolic alterations in the retina can trigger a bioenergetic crisis, ultimately resulting in the death of photoreceptors and/or RPE cells2. Mesenchymal stem cell (MSC)-based therapies have demonstrated efficacy in treating ocular degeneration, and one of the precise mechanisms underlying the beneficial effects of MSCs on retinal tissues may be attributed to functional mitochondrial transfer3,4,5,6. In 2004, Rustom et al. first reported the phenomenon of mitochondrial transfer through a novel cell-to-cell interaction facilitated by tunneling nanotubes (TNTs)7.
In 2D culture, tunneling nanotubes (TNTs) are identified by their thin (20-700 nm) membrane protrusions ranging from tens to hundreds of nanometers in length, which are suspended over the substrate and can directly establish connections between two or more homotypic and heterotypic cells. These structures are notably enriched in F-actin and facilitate the transport of cargo, such as mitochondria, between cells. Additionally, TNTs possess openings at both ends, enabling the continuity of cytoplasmic content between interconnected cells8.
It is difficult to detect TNT-mediated mitochondrial transfer in vivo due to the dense cellular arrangement and challenges in tracking mitochondria. In vitro experimentation, utilizing cell co-culture and mitochondrial tracing techniques, allows for the observation of TNT formation and mitochondrial transfer8,9. We also observed the phenomenon of TNT-mediated mitochondrial transfer by co-culturing MSCs and retinal pigment epithelial cells in vitro10.
Many previous studies have only observed unidirectional mitochondrial transfer from MSCs to other cells3,4,5,6. Previously, we also tried to analyze the bidirectional mitochondrial transfer using two kinds of cells labeled with mito-tracker green and mito-tracker red, respectively, but the crosstalk of the dyes interfered with the experimental results. To study mitochondrial bidirectional transfer more precisely, here, we constructed two cell lines with different mitochondrial fluorescence using the lentiviral transfection technique, and subsequently, observed and analyzed the phenomena of TNT formation and mitochondrial bidirectional transfer by direct co-culture in vitro.
In brief, a step-by-step and actionable protocol is described here as to how to trace mitochondria, co-culture MSCs with ARPE19 cells, and analyze TNT formation and mitochondrial transfer. The results of this experiment demonstrated TNT-mediated bidirectional mitochondrial transfer, which not only proved that mitochondrial transport is a common physiological phenomenon but also showed the potential therapeutic ability of MSCs on retinal cells.

<meta charset="utf8"></head><body>Autor em destaque: Transferência mitocondrial bidirecional entre MSCs e células do epitélio pigmentar da retina - vias e desafios in vivo

Transferência mitocondrial via nanotubos de tunelamento entre células-tronco mesenquimais e epitélio pigmentar da retina in vitro

Our study focused on bi-direction mitochondrial transfer between MSC and the retinal pigment epithelial cells. We are investigating the transfer of mitochondria between cells and determining the ratio of mitochondrial transfer in two different directions. It is difficult to detect TNT-mediated mitochondrial transfer in vivo due to the dense senior arrangement and challenges to in and tracking mitochondria.It is also difficult to label the mitochondria of two different cells with different colors. Our previous study observed the phenomena of TNT-mediated mitochondrial transfer by co-capturing MSC in retinal cells, including corneal endothelial cells, photoreceptors, and retinal pigment signal cells in vitro. Functional mitochondrial transfer from MSC can rescue damaged and retinal cells.

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

mitochondrial-transfer-via-tunneling-nanotubes-between-mesenchymal

Research

JoVE Journal

Biology

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

443 visualizações.  Capital Medical University. Nosso estudo se concentrou na transferência mitocondrial bidirecional entre as CTM e as células epiteliais pigmentares da retina. Estamos investigando a transferência de mitocôndrias entre células e determinando a proporção de transferência mitocondrial em duas direções diferentes. É difícil detectar a transferência mitocondrial mediada por TNT in vivo devido ao denso arranjo sênior e aos desafios para entrar e rastrear mitocôndrias.Tamb...