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

Vi takker Andrea Parmeggiani (L2C og DIMNP, Montpellier), Benoit Charlot (IES, Montpellier) samt medlemmer af laboratoriet for nyttige diskussioner, Christophe Duperray efter hjælp til FACS-analyse, at Montpellier RIO imaging facilitet (MRI) for at give tilstrækkelig miljø for FACS og konfokal mikroskopi. BNM blev understøttet af en kandidat fællesskab fra LABEX Numev (konvention ANR-10-LabX-20). AB blev støttet af en bachelor stipendium fra universitetet i Warszawa og Den Europæiske Union (n ° POKL.04.01.02-00-221 / 12). MLV er et personale forsker fra National Center for videnskabelig forskning (CNRS).

Dag 1 1. Mærkning af Mesenchymale Stem Cell (MSC) Mitokondrier (valgfri) <ol><li> To dage før mitokondrier forberedelse, frø humane MSC&#39;er i en 100 mm dyrkningsskål i 10 ml aMEM / FBS 10%, for således at have 4 x 10 5 MSC&#39;er i kulturen på dag 1. </li><li> Skyl MSC&#39;er med PBS (4 ml) og tilsæt 4 ml aMEM / FBS 1% (forvarmet til 37 ° C). </li><li> Tilsæt nødvendige mængde mitokondrier vitale farvestof og inkuber cellerne i 30 minutter i 37 ° C inkubator. </li><l...

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Et stigende antal undersøgelser viser, at cellerne kan udveksle mitokondrier, og at disse mitokondrier har dybtgående virkninger på målcellens stofskifte og funktioner. Derfor er det vigtigt at beherske de værktøjer, der kan hældes mitokondrier fra donorcellerne til disse målceller for at muliggøre en nøjagtig undersøgelse af deres biologiske virkninger. Protokollen beskrevet her var oprindeligt arbejdede til overføre mitokondrier isoleret fra humane mesenkymale stamceller til de...

Mitokondrier er organeller, der findes i eukaryote celler, hvor de spiller en central rolle i optagelsen af ​​næringsstoffer samt i energi- og metabolit-produktion. Disse organeller indeholder cirkulære mitokondrie-DNA (mtDNA), 16,6 kb lang, der koder for proteiner af elektrontransportkæden komplekser, tRNA&#39;er og rRNA&#39;er 1. Funktionaliteten af disse organeller er kritisk for cellehomeostase og adskillige patologier er blevet forbundet med mitokondrier dysfunktion 1, 2, 3. Den mitokondrier status for eksempel været forbundet med inflammation, infektionssygdomme og cancer, i sidstnævnte tilfælde med konsekvenser for metastase og modstandsdygtighed over for terapi 4, 5, 6, 7. Mitokondrier vise den bemærkelsesværdige evne<html> blive overført mellem &quot;donor&quot; og &quot;mål&quot; celler. Dette fører til ændringer i den energiske metabolisme af målcellerne såvel som i andre funktionelle modifikationer, såsom vævsreparation og modstandsdygtighed over for kemoterapeutiske midler, som for nyligt vist ved forskellige laboratorier 8, 9, 10, 11, 12, 13, 14, 15 , 16. De humane mesenkymale stamceller (MSC&#39;er) viser denne evne til at overføre mitokondrier til en bred vifte af målceller, herunder cardiomyocytter, endotelceller pulmonale alveolære epitelceller, renale tubulære celler og cancerceller, hvilket fører til ændringer af de funktionelle egenskaber af disse celler 8,&gt; 9, 10, 12, 17, 18. Mitokondrier udveksling fremstår nu som en meget anvendt mekanisme, der tillader et antal forskellige celletyper til at kommunikere med hinanden og ændre deres biologiske egenskaber. Denne mitokondrier udveksling kan ske gennem tunneling nanorør (TNT) dannelse, der involverer connexin 43-holdige gap junctions 8 eller M-Sec / TNFaip2 og exocyst komplekse 19. Alternativt blev mitokondrier overførsel også vist at være medieret af arrestin-domæne-holdige protein 1-medierede mikrovesikler (ARMMs) 20. Interessant nok blev effekten af mitokondrierne overførsel knyttet til udtrykket satsen for Rho GTPase en MIRO1 21, en afgørende faktor for at forklare forskellene i mitokondrier overføre effektiviteter mellem iPSC-MSC og voksen BM-MSC 22. På trods af dette væld af data om celle-til-celle mitokondrier udveksling, er relativt lidt kendt om metabolisk og biologisk resultat af denne mitokondrier overførsel. Derfor er det fuldt ud berettiger oprette de nødvendige værktøjer til fuldt ud at vurdere de biologiske virkninger af denne overførsel. I årenes løb til adskillige tekniske metoder overføre mitokondrier fra donor til acceptor-celler er blevet foreslået. Dette omfatter direkte injektion af mitokondrier i oocytter 23, 24, 25, cellefusion at generere transmitochondrial cybrids 26, 27 og for nylig, overførsel af isolerede mitokondrier hjælp fototermisk nanoblades 28. Vi og andre tidligere påvist kapacitet isolerede mitochondria skal internaliseres af levende celler, som observeret både in vitro og in vivo 29, 30, 31, gennem mekanismer foreslået at inddrage macropinocytosis 32. Vi udviklede endvidere en fremgangsmåde, kaldet MitoCeption, for kvantitativt at overføre isolerede mitokondrier (fra MSC&#39;er) til målceller, som eksemplificeret med (klæbende) MDA-MB-231 brystcancercellelinje 31. Denne protokol blev tilpasset her til overførsel af isolerede humane MSC mitokondrier til glioblastom stamceller (GSCS). Glioblastoma er aggressive maligne tumorer i hjernen, der hurtigt bliver resistente over for behandling, primært som følge af glioblastom stamceller (GSC) til stede i tumoren 33. Disse GSCS vokse som neurosfærer in vitro og generere tumorer i xenograftmodeller. Cancerceller inden glioblastom harkapacitet til at gøre celle-til-celle-forbindelser, som vist for nylig for astrocytiske hjerne tumorceller, interconnect via udvidede mikrorør, hvorigennem mitochondrier (samt calcium- og cellekerner) kan migrere, hvilket resulterer i radioterapi-resistent astrocytom net 34. Glioblastom kan rekruttere mange forskellige celler i tumoren mikromiljø, herunder MSC&#39;er 35, 36. Vi viste, at MSC kan gøre celle-celle-forbindelser med GSCS i cokultur og overføre deres mitokondrier (data ikke vist), hvilket forventes at modificere GSC funktionelle egenskaber. Den nuværende protokol beskriver, hvordan MitoCeption teknik kan bruges til at overføre mitokondrier, isoleret på forhånd fra humane MSC&#39;er, menneskelige GSCS med henblik på fastlæggelsen af ​​deres funktionelle biologiske resultat. Den multipotente og yderst tumorigene GB4 GSC linie 37 blev anvendt i denne undersøgelse.

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	<tbody>
		<tr>
			<td>Mitochondria Isolation Kit for Tissue&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>10579663</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11520536</td>
		</tr>
		<tr>
			<td>B-27 Supplement W/O VIT A (50X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11500446</td>
		</tr>
		<tr>
			<td>HBSS w/o Ca2+ w/o Mg2+</td>
			<td>&#160;Sigma</td>
			<td>H4385</td>
		</tr>
		<tr>
			<td>poly Heme&#160;</td>
			<td>Sigma&#160;</td>
			<td>P3932</td>
		</tr>
		<tr>
			<td>aMEM w/o glutamine</td>
			<td>Ozyme</td>
			<td>BE12-169F</td>
		</tr>
		<tr>
			<td>DMEM/F-12 without glutamine,&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>11540566</td>
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mitoception: transferring isolated human msc mitochondria to glioblastoma stem cells

Mitokondrier spiller en central rolle for cellens stofskifte, energiproduktion og kontrol af apoptose. Utilstrækkelig mitokondriefunktionen har vist sig ansvarlig for meget forskellige sygdomme, der spænder fra neurologiske patologier til kræft. Interessant nok har mitokondrier nylig vist at vise evnen til at blive overført mellem celletyper, navnlig fra humane mesenchymstamceller (MSC) til cancerceller i cokultur betingelser, med metaboliske og funktionelle konsekvenser for mitokondrierne recipientceller, yderligere forøgelse af det aktuelle interesse for de biologiske egenskaber af disse organeller. Evaluering af virkningerne af den overførte MSC mitokondrier i målcellerne er af største betydning for at forstå den biologiske resultatet af disse celle-celle-interaktioner. Den her beskrevne MitoCeption protokollen gør det muligt at overføre mitokondrierne isolerede forhånd fra donor celler til målcellerne, hjælp MSC mitokondrierog glioblastoma stamceller (GSR) som et modelsystem. Denne protokol er tidligere blevet anvendt til at overføre mitokondrier isoleret fra MSC&#39;er, at adhærente MDA-MB-231 cancerceller. Denne mitokondrier transfer protocol er tilpasset her GSCS der udgør den specifikke særlige ved vokser som neurosfærer in vitro. Overførsel af de isolerede mitokondrier kan følges ved fluorescens-aktiveret cellesortering (FACS) og konfokal billeddannelse under anvendelse mitokondrier vitale farvestoffer. Brugen af ​​mitochondrier donor- og target celler med forskellige haplotyper (SNP&#39;er) tillader også påvisning af de overførte mitokondrier baseret på koncentrationen af ​​deres cirkulære mitokondrie-DNA (mtDNA) i målcellerne. Når protokollen er blevet valideret med disse kriterier, kan de celler, der huser de overførte mitokondrier yderligere analyseret for at bestemme virkningerne af de eksogene mitokondrier på biologiske egenskaber såsom cellens stofskifte, plasticitet, spredning og respons på behandling.

De proceduremæssige skridt skitserer isolering af mitokondrier fra mesenkymale stamceller (MSC) og deres overførsel til de målrettede glioblastom stamceller (GSC) ved MitoCeption er vist i figur 1. GSCS er cancer stamceller dyrkes som neurosfærer at bevare deres stamcelle egenskaber. For protokollen, er GSCS seedet som enkelte celler et par timer før overførsel af mitokondrier (trin 3) for at tillade højere mitokondrier transfer effektivitet (se FACS data ...

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

Her er en protokol (MitoCeption) præsenterede at overføre mitokondrier, isoleret fra humane mesenkymale stamceller (MSC), til glioblastom stamceller (GSC), med det mål at studere deres biologiske virkninger på GSC metabolisme og funktioner. En lignende protokol kan tilpasses til at overføre mitokondrier mellem andre celletyper.

MitoCeption: Overførsel isoleret humant MSC Mitokondrier til Glioblastoma Stamceller

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

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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 ...

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 ...

Immunocytochemistry: Human Neural Stem Cells

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 ...

Purification of Mitochondria from Yeast Cells

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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 ...

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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