The authors work was supported by research grant ID-BOF 2006 of the University of Antwerp (granted to PPo and AVdL), by research grant G.0136.11 and G.0130.11 (granted to AVdL, ZB and PPo) and 1.5.021.09.N.00 (granted to PPo) of the Fund for Scientific Research-Flanders (FWO-Vlaanderen, Belgium), by SBO research grant IWT-60838: BRAINSTIM of the Flemish Institute for Science and Technology (granted to ZB and AVDL), in part by a Methusalem research grant from the Flemish government (granted to ZB), in part by EC-FP6-NoE DiMI (LSHB-CT-2005-512146), EC-FP6-NoE EMIL (LSHC-CT-2004-503569), and by the Inter University Attraction Poles IUAP-NIMI-P6/38 (granted to AVDL). Nathalie De Vocht holds a PhD-studentship from the FWO-Vlaanderen. Peter Ponsaerts is a post-doctoral fellow of the FWO-Vlaanderen.

1. Cell Preparation
<ol>
	<li>Experiments should be initiated using ex vivo cultured stem cell populations genetically engineered to express the Luciferase and eGFP reporter proteins. Here we use Luciferase/eGFP-expressing murine bone marrow-derived stromal cells (BMSC-Luc/eGFP) as previously described by Bergwerf et al.2, 5.</li>
	<li>Two days before cell labeling, plate BMSC-Luc/eGFP cells at a density of 8 x 105 cells per T75 culture flask in 15 ml complete expansion medium (CEM) suppl...

<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. 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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. 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In this report, we describe an optimized protocol for the combination of three complementary imaging modalities (BLI, MRI and histology) for detailed characterization of cellular implants in the CNS of immune competent mice. A combination of reporter gene labeling of cells, based on genetic modification with the reporter genes firefly Luciferase and eGFP, and a direct cell labeling with GB MPIO, leads to an accurate assessment of stem cell grafts in vivo.
For particle labeling of BMSC...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Lonza</td>
 <td>BE12-722F</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Gibco</td>
 <td>10270-106</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>Gibco</td>
 <td>1605-122</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Gibco</td>
 <td>15140</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>Fungizone</td>
 <td>Gibco</td>
 <td>15290-018</td>
 <td>Component of the cell growth medium CEM</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Gibco</td>
 <td>14190</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Puromycine</td>
 <td>Invivogen</td>
 <td>ant-pr-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>trypsin</td>
 <td>Gibco</td>
 <td>25300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GB MPIO</td>
 <td>Bangs Laboratories</td>
 <td>ME04F/7833</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-luciferin</td>
 <td>Promega</td>
 <td>E1601</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ketamine (Ketalar)</td>
 <td>Pfizer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Xylazine (Rompun)</td>
 <td>Bayer Health care</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Isoflo </td>
 <td>05260-05</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.9% NaCl solution</td>
 <td>Baxter</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>paraformaldehyde</td>
 <td>Merck</td>
 <td>1.04005.1000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>sucrose</td>
 <td>Applichem</td>
 <td>A1125</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro-injection pump</td>
 <td>KD scientific</td>
 <td>KDS100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Photon imager</td>
 <td>Biospace Lab</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>9.4T MR scanner</td>
 <td>Bruker Biospin</td>
 <td>Biospec 94/20 USR </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BX51 microscope </td>
 <td>Olympus</td>
 <td>BX51</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mycrom HM cryostat</td>
 <td>Prosan</td>
 <td>HM525</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>syringe</td>
 <td>Hamilton</td>
 <td>7635-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>30 gauge needle</td>
 <td>Hamilton</td>
 <td>7762-03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Photo Vision software</td>
 <td>Biospace Lab</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>M3vision software</td>
 <td>Biospace Lab</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paravision 5.1 software</td>
 <td>Bruker Biospin</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Amira 4.0 software</td>
 <td>Visage Imaging</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 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

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

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

multimodal-imaging-stem-cell-implantation-central-nervous-system

Research

JoVE Journal

Neuroscience

11.0K vistas.  University of Antwerp. En este artículo se describe una secuencia optimizada de eventos para obtener imágenes multimodal de los injertos celulares en el cerebro de los roedores por medio de: (i) en la bioluminiscencia in vivo y la resonancia magnética, y (ii) el análisis post mortem histológico. La combinación de estas modalidades de imágenes en un solo animal permite la evaluación del injerto celular con alta resolución, sensibilidad y especificidad.