Bergwerf Irene, De Vocht Nathalie, Tambuyzer Bart, Verschueren Jacob, Reekmans Kristien, Daans Jasmijn, Ibrahimi Abdelilah, Van Tendeloo Viggo, Chatterjee Shyama, Goossens Herman, Jorens Philippe G, Baekelandt Veerle, Ysebaert Dirk, Van Marck Eric, Berneman Zwi N, Linden Annemie Van Der, Ponsaerts Peter
Laboratory of Experimental Hematology, University of Antwerp, Antwerp, Belgium.
BMC Biotechnol. 2009 Jan 7;9:1. doi: 10.1186/1472-6750-9-1.
BACKGROUND: Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics. RESULTS: In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-gamma-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation. CONCLUSION: We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.
背景：细胞移植可能会成为治疗中枢神经系统（CNS）各种创伤性和缺血性损伤的重要治疗工具。然而，在许多临床前细胞治疗研究中，对CNS中细胞植入物进行报告基因辅助成像以及潜在的报告基因和/或基于细胞的免疫原性，仍然是具有挑战性的研究课题。 结果：在本研究中，我们使用从ROSA26-L-S-L-Luciferase转基因小鼠培养的自体（同基因）表达荧光素酶的骨髓间充质干细胞（BMSC-Luc），以及用编码增强型绿色荧光蛋白（eGFP）和嘌呤霉素抗性基因（Pac）的慢病毒进行基因改造的BMSC-Luc（BMSC-Luc/eGFP/Pac），在免疫活性小鼠的CNS中进行细胞植入实验。尽管引入的报告蛋白具有潜在免疫原性，但通过实时生物发光成像（BLI）和植入后不同时间点的组织学分析表明，两种经报告基因修饰的BMSC群体在免疫活性小鼠的CNS中均表现出高植入能力。相比之下，如植入后不同时间点的实时BLI所示，BMSC-Luc和BMSC-Luc/eGFP/Pac在肌肉内细胞植入后均无法存活。此外，ELISPOT分析表明，肌肉内细胞植入后可诱导产生IFN-γ的CD8+T细胞，但脑内细胞植入后则不会，这表明BMSC-Luc和BMSC-Luc/eGFP/Pac在CNS中具有免疫耐受性。然而，在我们的实验移植模型中，结果还表明，报告基因特异性免疫反应性T细胞应答并非肌肉内细胞植入后BMSC-Luc或BMSC-Luc/eGFP/Pac免疫排斥的主要因素。 结论：我们在此证明，源自ROSA26-L-S-L-Luciferase转基因小鼠的经报告基因修饰的BMSC在同基因免疫活性小鼠的CNS中植入后具有免疫耐受性，为在无免疫抑制治疗的情况下通过实时BLI和/或组织学分析研究自体BMSC植入物在CNS中的存活和定位提供了一个研究模型。
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