在中央小鼠中枢神经系统的干细胞移植的多模态成像
Özet
本文介绍了优化多式联运成像啮齿动物脑细胞的使用移植的事件序列：（i）在活体动物和磁共振成像，及（ii）验尸的病理分析。结合在一个单一的动物允许这些成像分辨率高，灵敏度和特异性细胞移植评估。
Abstract
在过去十年中，造血干细胞移植已越来越为原发性或继发多种疾病，在临床前和临床研究的治疗方法的兴趣。然而，迄今为止，有关功能的结果和/或组织再生以下干细胞移植的结果是多种多样的。一般来说，临床受益观察没有深刻理解底层机制（S）1。因此，多的努力已导致不同的分子成像的发展，以监察其最终目的的干细胞移植，以准确评估移植的干细胞和/或他们的微环境的生存，命运和生理。分子成像确定一个或多个参数的变化可能与观察到的临床效果。在此背景下，我们的研究侧重于生物发光成像的结合使用。（BLI），磁共振成像（MRI）和病理analysis到评价干细胞移植。
，劳保局常用非侵入性细胞跟踪和监视细胞的生存时间，移植后2-7细胞表达荧光素酶报告基因的生化反应的基础上能够发出光以下的相互作用，其基板（例如D-荧光素），8，9。另一方面，MRI是一种非侵入性的技术，这是临床上适用10，可用于精确定位，具有很高的分辨率11-15细胞移植，但其灵敏度高，取决于细胞标记后生成的磁共振造影剂的对比。最后，验尸的病理分析的首选方法，以验证获得最高的分辨率和灵敏度的非侵入性技术的研究成果。此外终点的病理分析，让我们进行详细的表型分析移植的细胞和/或周围组织，BASED上使用荧光记者蛋白质和/或直接与特异性抗体细胞标记。
总之，我们在这里直观地展现劳保局，MRI和组织学的互补性，解开不同的干细胞和/或环境相关的特征后，干细胞移植在小鼠中枢神经系统。作为一个例子，骨髓基质细胞，基因工程表达增强型绿色荧光蛋白（EGFP）和萤火虫荧光素酶（fLuc），和蓝色荧光微米大小的氧化铁粒子（MPIOs）标记，将嫁接中枢神经系统免疫能力的小鼠和成果将监测劳保局，MRI和组织学（ 图1）。
Protocol
1。细胞的制备应开始实验使用体外培养干细胞基因工程表达的荧光素酶和绿色荧光蛋白记者蛋白质的人群。在这里，我们使用荧光素酶/ EGFP表达小鼠骨髓来源的基质细胞（BMSC-Luc/eGFP）以前Bergwerf 等。2，5。 细胞标记，在8×10 5％的T75培养瓶中的细胞密度在15毫升完整的扩展介质（CEM），辅以1微克/毫升Puromycine板BMSC-Luc/eGFP细胞前两天。 让细胞成长为48小?…
Discussion
在这份报告中，我们描述了一个在免疫小鼠中枢神经系统的细胞植入的详细描述。（BLI，MRI和组织学）三个互补的成像方式的组合优化的协议。记者细胞的基因标记，记者萤火虫荧光素酶和绿色荧光蛋白基因，并直接与GB的MPIO细胞标记基因改造的基础上，组合会导致体内的干细胞移植在准确的评估。
粒子标记的骨髓间充质干细胞，GB的的MPIO粒径组合与阴离子表?…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
<p class="jove_content">作者的工作得到了转炉研究资助ID-2006年的安特卫普大学（PPO和AVDL授予），研究资助G.0136.11和G.0130.11（授予AVDL，ZB和PPO）和1.5.021.09。 N.00（授予PPO）的科学研究，法兰德斯（FWO Vlaanderen，比利时），服务营办商研究资助内陆水运-60838：BRAINSTIM佛兰德科技研究所（ZB和AVDL授予），在基金1玛土撒拉的研究经费从弗拉芒政府（授予ZB型），部分由EC-FP6的NOE迪米（LSHB-CT-2005-512146），EC-FP6的NOE EMIL（LSHC-CT-2004-503569的部分）间大学景点波兰人IUAP-NIMI-P6/38（授予AVDL）。纳塔莉德Vocht FWO-Vlaanderen持有博士学位的奖学金。彼得Ponsaerts是的FWO-Vlaanderen的博士后研究员。</p>
Materials
| Name of the reagent | Company | Catalogue number | Yorumlar |
| IMDM | Lonza | BE12-722F | Component of the cell growth medium CEM |
| Fetal bovine serum | Gibco | 10270-106 | Component of the cell growth medium CEM |
| Horse serum | Gibco | 1605-122 | Component of the cell growth medium CEM |
| Penicillin-streptomycin | Gibco | 15140 | Component of the cell growth medium CEM |
| Fungizone | Gibco | 15290-018 | Component of the cell growth medium CEM |
| PBS | Gibco | 14190 | |
| Puromycine | Invivogen | ant-pr-1 | |
| trypsin | Gibco | 25300 | |
| GB MPIO | Bangs Laboratories | ME04F/7833 | |
| D-luciferin | Promega | E1601 | |
| Ketamine (Ketalar) | Pfizer | ||
| Xylazine (Rompun) | Bayer Health care | ||
| Isoflurane | Isoflo | 05260-05 | |
| 0.9% NaCl solution | Baxter | ||
| paraformaldehyde | Merck | 1.04005.1000 | |
| sucrose | Applichem | A1125 | |
| Micro-injection pump | KD scientific | KDS100 | |
| Photon imager | Biospace Lab | ||
| 9.4T MR scanner | Bruker Biospin | Biospec 94/20 USR | |
| BX51 microscope | Olympus | BX51 | |
| Mycrom HM cryostat | Prosan | HM525 | |
| syringe | Hamilton | 7635-01 | |
| 30 gauge needle | Hamilton | 7762-03 | |
| Photo Vision software | Biospace Lab | ||
| M3vision software | Biospace Lab | ||
| Paravision 5.1 software | Bruker Biospin | ||
| Amira 4.0 software | Visage Imaging |
Referanslar
- Rodriguez-Porcel, M., Wu, J. C., Gambhir, S. S. . Molecular imaging of stem cells. , (2008).
- Bergwerf, I., De Vocht, N., Tambuyzer, B. Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice. BMC Biotechnol. 9, 1 (2009).
- Bergwerf, I., Tambuyzer, B., De Vocht, N. Recognition of cellular implants by the brain’s innate immune system. Immunol. Cell Biol. 89, 511-516 (2011).
- Bradbury, M. S., Panagiotakos, G., Chan, B. K. Optical bioluminescence imaging of human ES cell progeny in the rodent CNS. J. Neurochem. 102, 2029-2039 (2007).
- De Vocht, N., Bergwerf, I., Vanhoutte, G. 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. Mol Imaging Biol. , (2011).
- Reekmans, K., Praet, J., Daans, J. Current Challenges for the Advancement of Neural Stem Cell Biology and Transplantation Research. Stem Cell Rev. , (2011).
- Reekmans, K. P., Praet, J., De Vocht, N. Clinical potential of intravenous neural stem cell delivery for treatment of neuroinflammatory disease in mice. Cell Transplant. 20, 851-869 (2011).
- Contag, C. H., Bachmann, M. H. Advances in in vivo bioluminescence imaging of gene expression. Annu. Rev. Biomed. Eng. 4, 235-260 (2002).
- Sadikot, R. T., Blackwell, T. S. Bioluminescence imaging. Proc. Am. Thorac. Soc. 2, 511-540 (2005).
- Sykova, E., Jendelova, P., Herynek, V. MR tracking of stem cells in living recipients. Methods Mol. Biol. 549, 197-215 (2009).
- Daadi, M. M., Li, Z., Arac, A. Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat. 17, 1282-1291 (2009).
- Heyn, C., Ronald, J. A., Ramadan, S. S. In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain. Magn. Reson. Med. 56, 1001-1010 (2006).
- Hoehn, M., Kustermann, E., Blunk, J. Monitoring of implanted stem cell migration in vivo: a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat. Proc. Natl. Acad. Sci. U.S.A. 99, 16267-16272 (2002).
- Jendelova, P., Herynek, V., DeCroos, J. Imaging the fate of implanted bone marrow stromal cells labeled with superparamagnetic nanoparticles. Magn. Reson. Med. 50, 767-776 (2003).
- Modo, M., Mellodew, K., Cash, D. Mapping transplanted stem cell migration after a stroke: a serial, in vivo magnetic resonance imaging study. Neuroimage. 21, 311-317 (2004).
- Boutry, S., Brunin, S., Mahieu, I. Magnetic labeling of non-phagocytic adherent cells with iron oxide nanoparticles: a comprehensive study. Contrast Media Mol. Imaging. 3, 223-232 (2008).
- Mailander, V., Lorenz, M. R., Holzapfel, V. Carboxylated superparamagnetic iron oxide particles label cells intracellularly without transfection agents. Mol Imaging Biol. 10, 138-146 (2008).
- Modo, M., Hoehn, M., Bulte, J. W. Cellular MR imaging. Mol. Imaging. 4, 143-164 (2005).
- Hinds, K. A., Hill, J. M., Shapiro, E. M. Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells. Blood. 102, 867-872 (2003).
- Shapiro, E. M., Sharer, K., Skrtic, S. In vivo detection of single cells by MRI. Magn. Reson. Med. 55, 242-249 (2006).
- Shapiro, E. M., Skrtic, S., Sharer, K. MRI detection of single particles for cellular imaging. Proc. Natl. Acad. Sci. U.S.A. 101, 10901-10906 (2004).
- Crabbe, A., Vandeputte, C., Dresselaers, T. Effects of MRI contrast agents on the stem cell phenotype. Cell Transplant. 19, 919-936 (2010).
- Szarecka, A., Xu, Y., Tang, P. Dynamics of firefly luciferase inhibition by general anesthetics: Gaussian and anisotropic network analyses. Biophys. J. 93, 1895-1905 (2007).
- Keyaerts, M., Heneweer, C., Gainkam, L. O. Plasma protein binding of luciferase substrates influences sensitivity and accuracy of bioluminescence imaging. Mol. Imaging. Biol. 13, 59-66 (2011).
- Keyaerts, M., Verschueren, J., Bos, T. J. 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. Eur. J. Nucl. Med. Mol. Imaging. 35, 999-1007 (2008).
- Zhang, Y., Bressler, J. P., Neal, J. ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging. Cancer Res. 67, 9389-9397 (2007).
- Brightwell, G., Poirier, V., Cole, E. Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector. Gene. 194, 115-123 (1997).
- Grassi, G., Maccaroni, P., Meyer, R. Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87. Carcinogenesis. 24, 1625-1635 (2003).
- Krishnan, M., Park, J. M., Cao, F. Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging. FASEB J. 20, 106-108 (2006).
- Svensson, R. U., Barnes, J. M., Rokhlin, O. W. Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy. Cancer Res. 67, 10445-10454 (2007).
Bu Makaleden Alıntı Yapın
De Vocht, N., Reekmans, K., Bergwerf, I., Praet, J., Hoornaert, C., Le Blon, D., Daans, J., Berneman, Z., Van der Linden, A., Ponsaerts, P. Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice. J. Vis. Exp. (64), e3906, doi:10.3791/3906 (2012).