Name: multimodal imaging of stem cell implantation in the central nervous system of mice
Uploaded: 2012-06-13T13:00:00
Description: Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

<p class="jove_content"&gt; Il lavoro è stato sostenuto da autori di assegno di ricerca ID-BOF 2006 dell&#39;Università di Anversa (concessa a PPO e AVDL), con assegno di ricerca G.0136.11 e G.0130.11 (concessa a AVDL, ZB e PPO) e 1.5.021.09. N.00 (concessa a PPO) del Fondo per la ricerca scientifica-Flanders (FWO-Vlaanderen, Belgio), da SBO assegno di ricerca IWT-60838: BRAINSTIM dell&#39;Istituto fiammingo per la Scienza e la Tecnologia (concessi a ZB e AVDL), in parte da una ricerca Methusalem borsa di studio del governo fiammingo (concessa a ZB), in parte da EC-FP6-NoE DIMI (LSHB-CT-2005-512146), EC-FP6-NoE EMIL (LSHC-CT-2004-503569) , e dalla Università Inter poli di attrazione IUAP-NIMI-P6/38 (concessa a AVDL). Nathalie De Vocht ha conseguito un PhD-borsa di studio dalla FWO-Vlaanderen. Peter Ponsaerts è un post-doctoral fellow del FWO-Vlaanderen.</p

1. Preparazione cellulare <ol><li> Gli esperimenti deve essere iniziato con ex vivo in coltura popolazioni di cellule staminali geneticamente modificate per esprimere le proteine ​​reporter della luciferasi e eGFP. Qui usiamo Luciferase / eGFP-murine esprimono osseo cellule stromali derivate dal midollo (BMSC-Luc/eGFP) come precedentemente descritti da Bergwerf et al. 2, 5. </li><li> Due giorni prima marcatura delle cellule, le cellule BMSC-Luc/eGFP piastra ad una densità di 8 x 10 5</sup...

<ol>
	<li>Rodriguez-Porcel, M., Wu, J. C., Gambhir, S. 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> Molecular imaging of stem cells. , (2008).</li><li>Bergwerf, I., De Vocht, N., Tambuyzer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+gene-expressing+bone+marrow-derived+stromal+cells+are+immune-tolerated+following+implantation+in+the+central+nervous+system+of+syngeneic+immunocompetent+mice.">Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice.</a> BMC Biotechnol. 9, 1 (2009).</li><li>Bergwerf, I., Tambuyzer, B., De Vocht, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+cellular+implants+by+the+brain's+innate+immune+system.">Recognition of cellular implants by the brain's innate immune system.</a> Immunol. Cell Biol. 89, 511-516 (2011).</li><li>Bradbury, M. S., Panagiotakos, G., Chan, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+bioluminescence+imaging+of+human+ES+cell+progeny+in+the+rodent+CNS.">Optical bioluminescence imaging of human ES cell progeny in the rodent CNS.</a> J. Neurochem. 102, 2029-2039 (2007).</li><li>De Vocht, N., Bergwerf, I., Vanhoutte, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+Luciferase/eGFP-Expressing+Bone+Marrow-Derived+Stromal+Cells+with+Fluorescent+Micron-Sized+Iron+Oxide+Particles+Improves+Quantitative+and+Qualitative+Multimodal+Imaging+of+Cellular+Grafts+In+Vivo.">Labeling of Luciferase/eGFP-Expressing Bone Marrow-Derived Stromal Cells with Fluorescent Micron-Sized Iron Oxide Particles Improves Quantitative and Qualitative Multimodal Imaging of Cellular Grafts In Vivo.</a> Mol Imaging Biol. , (2011).</li><li>Reekmans, K., Praet, J., Daans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Challenges+for+the+Advancement+of+Neural+Stem+Cell+Biology+and+Transplantation+Research.">Current Challenges for the Advancement of Neural Stem Cell Biology and Transplantation Research.</a> Stem Cell Rev. , (2011).</li><li>Reekmans, K. P., Praet, J., De Vocht, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+potential+of+intravenous+neural+stem+cell+delivery+for+treatment+of+neuroinflammatory+disease+in+mice.">Clinical potential of intravenous neural stem cell delivery for treatment of neuroinflammatory disease in mice.</a> Cell Transplant. 20, 851-869 (2011).</li><li>Contag, C. H., Bachmann, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+in+vivo+bioluminescence+imaging+of+gene+expression.">Advances in in vivo bioluminescence imaging of gene expression.</a> Annu. Rev. Biomed. Eng. 4, 235-260 (2002).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging.">Bioluminescence imaging.</a> Proc. Am. Thorac. Soc. 2, 511-540 (2005).</li><li>Sykova, E., Jendelova, P., Herynek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+tracking+of+stem+cells+in+living+recipients.">MR tracking of stem cells in living recipients.</a> Methods Mol. Biol. 549, 197-215 (2009).</li><li>Daadi, M. M., Li, Z., Arac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+magnetic+resonance+imaging+of+human+embryonic+stem+cell-derived+neural+stem+cell+grafts+in+ischemic+rat.">Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat.</a> 17, 1282-1291 (2009).</li><li>Heyn, C., Ronald, J. A., Ramadan, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MRI+of+cancer+cell+fate+at+the+single-cell+level+in+a+mouse+model+of+breast+cancer+metastasis+to+the+brain.">In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain.</a> Magn. Reson. Med. 56, 1001-1010 (2006).</li><li>Hoehn, M., Kustermann, E., Blunk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+implanted+stem+cell+migration+in+vivo:+a+highly+resolved+in+vivo+magnetic+resonance+imaging+investigation+of+experimental+stroke+in+rat.">Monitoring of implanted stem cell migration in vivo: a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 16267-16272 (2002).</li><li>Jendelova, P., Herynek, V., DeCroos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+fate+of+implanted+bone+marrow+stromal+cells+labeled+with+superparamagnetic+nanoparticles.">Imaging the fate of implanted bone marrow stromal cells labeled with superparamagnetic nanoparticles.</a> Magn. Reson. Med. 50, 767-776 (2003).</li><li>Modo, M., Mellodew, K., Cash, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+transplanted+stem+cell+migration+after+a+stroke:+a+serial,+in+vivo+magnetic+resonance+imaging+study.">Mapping transplanted stem cell migration after a stroke: a serial, in vivo magnetic resonance imaging study.</a> Neuroimage. 21, 311-317 (2004).</li><li>Boutry, S., Brunin, S., Mahieu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+labeling+of+non-phagocytic+adherent+cells+with+iron+oxide+nanoparticles:+a+comprehensive+study.">Magnetic labeling of non-phagocytic adherent cells with iron oxide nanoparticles: a comprehensive study.</a> Contrast Media Mol. Imaging. 3, 223-232 (2008).</li><li>Mailander, V., Lorenz, M. R., Holzapfel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxylated+superparamagnetic+iron+oxide+particles+label+cells+intracellularly+without+transfection+agents.">Carboxylated superparamagnetic iron oxide particles label cells intracellularly without transfection agents.</a> Mol Imaging Biol. 10, 138-146 (2008).</li><li>Modo, M., Hoehn, M., Bulte, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+MR+imaging.">Cellular MR imaging.</a> Mol. Imaging. 4, 143-164 (2005).</li><li>Hinds, K. A., Hill, J. M., Shapiro, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+endosomal+labeling+of+progenitor+and+stem+cells+with+large+magnetic+particles+allows+magnetic+resonance+imaging+of+single+cells.">Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells.</a> Blood. 102, 867-872 (2003).</li><li>Shapiro, E. M., Sharer, K., Skrtic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+single+cells+by+MRI.">In vivo detection of single cells by MRI.</a> Magn. Reson. Med. 55, 242-249 (2006).</li><li>Shapiro, E. M., Skrtic, S., Sharer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+detection+of+single+particles+for+cellular+imaging.">MRI detection of single particles for cellular imaging.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 10901-10906 (2004).</li><li>Crabbe, A., Vandeputte, C., Dresselaers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+MRI+contrast+agents+on+the+stem+cell+phenotype.">Effects of MRI contrast agents on the stem cell phenotype.</a> Cell Transplant. 19, 919-936 (2010).</li><li>Szarecka, A., Xu, Y., Tang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+firefly+luciferase+inhibition+by+general+anesthetics:+Gaussian+and+anisotropic+network+analyses.">Dynamics of firefly luciferase inhibition by general anesthetics: Gaussian and anisotropic network analyses.</a> Biophys. J. 93, 1895-1905 (2007).</li><li>Keyaerts, M., Heneweer, C., Gainkam, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+protein+binding+of+luciferase+substrates+influences+sensitivity+and+accuracy+of+bioluminescence+imaging.">Plasma protein binding of luciferase substrates influences sensitivity and accuracy of bioluminescence imaging.</a> Mol. Imaging. Biol. 13, 59-66 (2011).</li><li>Keyaerts, M., Verschueren, J., Bos, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+bioluminescence+imaging+for+quantitative+tumour+burden+assessment+using+IV+or+IP+administration+of+D:+-luciferin:+effect+on+intensity,+time+kinetics+and+repeatability+of+photon+emission.">Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission.</a> Eur. J. Nucl. Med. Mol. Imaging. 35, 999-1007 (2008).</li><li>Zhang, Y., Bressler, J. 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In questo rapporto, descriviamo un protocollo ottimizzato per la combinazione di tre modalità di imaging complementari (BLI, MRI e istologia) per la caratterizzazione dettagliata degli impianti cellulari nel SNC di topi immuni competenti. Una combinazione di etichettatura gene reporter di cellule, basata sulla modificazione genetica con i geni reporter lucciola, luciferasi e eGFP, e una cella di etichettatura diretta con GB MPIO, porta ad una valutazione accurata degli innesti di cellule staminali in vivo. </p...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> IMDM </td><td> Lonza </td><td> BE12-722F </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> Siero fetale bovino </td><td> Gibco </td><td> 10270-106 </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> Siero di cavallo </td><td> Gibco </td><td> 1605-122 </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> Penicillina-streptomicina </td><td> Gibco </td><td> 15140 </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> Fungizone </td><td> Gibco </td><td> 15290-018 </td><td> Componente del CEM mezzo di crescita delle cellule </td></tr><tr><td> PBS </td><td> Gibco </td><td> 14190 </td><td></td></tr><tr><td> Puromycine </td><td> Invivogen </td><td> ant-pr-1 </td><td></td></tr><tr><td> tripsina </td&gt; <td> Gibco </td><td> 25300 </td><td></td></tr><tr><td> GB MPIO </td><td> Bangs Laboratories </td><td> ME04F/7833 </td><td></td></tr><tr><td> D-luciferina </td><td> Promega </td><td> E1601 </td><td></td></tr><tr><td> Ketamina (Ketalar) </td><td> Pfizer </td><td></td><td></td></tr><tr><td> Xilazina (Rompun) </td><td> Bayer Sanità </td><td></td><td></td></tr><tr><td> Isoflurano </td><td> Isoflo </td><td> 05260-05 </td><td></td></tr><tr><td> 0,9% NaCl </td><td> Baxter </td><td></td><td></td></tr><tr><td> paraformaldeide </td><td> Merck </td><td> 1.04005.1000 </td><td></td></tr><tr><td> saccarosio </td><td> Applichem </td><td> A1125 </td><td></td></tr><tr><td> Micro-pompa di iniezione </td><td> KD scientifica </td><td> KDS100 </td><td></td></tr><tr><td> Photon imager </td><td> Biospace Lab </td><td></td><td></td></tr><tr&gt; <td> 9.4T MR scanner </td><td> Bruker Biospin </td><td> Biospec 94/20 USR </td><td></td></tr><tr><td> BX51 microscopio </td><td> Olimpo </td><td> BX51 </td><td></td></tr><tr><td> Mycrom HM criostato </td><td> Prosan </td><td> HM525 </td><td></td></tr><tr><td> siringa </td><td> Hamilton </td><td> 7635-01 </td><td></td></tr><tr><td> 30 gauge </td><td> Hamilton </td><td> 7762-03 </td><td></td></tr><tr><td> Foto Vision software </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> M3vision software </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> ParaVision 5,1 software </td><td> Bruker Biospin </td><td></td><td></td></tr><tr><td> Amira 4,0 software </td><td> Visage Imaging </td><td></td><td></td></tr></tbody></table>

multimodal imaging of stem cell implantation in the central nervous system of mice

Nel corso dell&#39;ultimo decennio, il trapianto di cellule staminali ha acquisito un crescente interesse come primario o secondario modalità terapeutica per una varietà di malattie, sia negli studi preclinici e clinici. Tuttavia, ad oggi i risultati per quanto riguarda i risultati funzionali e / o la rigenerazione dei tessuti a seguito di trapianto di cellule staminali sono molto diverse. In generale, un beneficio clinico si osserva senza profonda comprensione del meccanismo sottostante (s) 1. Pertanto, gli sforzi multipli hanno portato allo sviluppo di diverse modalità di imaging molecolare per monitorare cellule staminali innesto con l&#39;obiettivo finale di valutare con precisione la sopravvivenza, destino e fisiologia delle cellule staminali innestate e / o loro micro-ambiente. I cambiamenti osservati in uno o più parametri determinati dalla imaging molecolare potrebbe essere correlato all&#39;effetto clinico osservato. In questo contesto, i nostri studi si concentrano sull&#39;uso combinato di imaging bioluminescenza (BLI), risonanza magnetica (MRI) e l&#39;analisi dell istologicas per valutare cellule staminali innesto. BLI viene comunemente utilizzato per non invasivo eseguire il monitoraggio delle cellule e la sopravvivenza cellulare monitorare in tempo successivo trapianto 2-7, basato su una reazione biochimica in cui le cellule che esprimono il gene reporter luciferasi sono in grado di emettere luce seguente interazione con il suo substrato (per esempio D- luciferina) 8, 9. MRI invece è una tecnica non invasiva che è clinicamente applicabile 10 e può essere utilizzata per localizzare con precisione innesti cellulari ad altissima risoluzione 11-15, sebbene la sua sensibilità dipende fortemente dal contrasto generato dopo marcatura delle cellule con un agente di contrasto MRI . Infine, post-mortem analisi istologica è il metodo di scelta per convalidare i risultati della ricerca ottenuti con tecniche non invasive con la più alta risoluzione e sensibilità. Inoltre end-point analisi istologica ci permette di eseguire una dettagliata analisi fenotipica delle cellule innestate e / o il tessuto circostante, based sull&#39;uso di proteine ​​reporter fluorescenti e / o etichettatura diretto di cellule con anticorpi specifici. In sintesi, siamo qui visivamente dimostrare la complementarietà di BLI, MRI e istologia di svelare cellule staminali e diversi / o l&#39;ambiente associate le seguenti caratteristiche innesto di cellule staminali nel sistema nervoso centrale di topi. Come esempio, midollo osseo, le cellule stromali geneticamente ingegnerizzati per esprimere la proteina fluorescente verde maggiore (EGFP) e luciferasi di lucciola (Fluc), e marcato con blu di ferro particelle fluorescenti dimensioni micron di ossido (MPIOs), verrà innestata nella SNC di topi immuno-competenti ei risultati saranno monitorati da BLI, MRI e istologia (Figura 1).

Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Questo articolo descrive una sequenza ottimizzata di eventi per l&#39;imaging multimodale di innesti cellulari nel cervello di roditori utilizzando: (i) in bioluminescenza vivo e la risonanza magnetica, e (ii) post mortem l&#39;analisi istologica. La combinazione di queste modalità di imaging su un singolo animale consente la valutazione del trapianto cellulare ad alta risoluzione, sensibilità e specificità.

Imaging multimodale impianto di cellule staminali nel sistema nervoso centrale di topi

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

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Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

Registrazione elettrofisiologica dell'attività del sistema nervoso centrale di terza-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...