MONITORING STEM CELL GRAFTS USING A NOVEL MRI REPORTER

项目摘要 我们建议开发一种新型的MRI报告基因系统，该系统使用MRI来监测基因的表达和 细胞移植在体内的研究现状。目前的磁共振成像技术能够在10米范围内获得高空间分辨率，并且 在各种疾病的研究和临床诊断中得到了广泛的应用1-7。活体内 对细胞移植物的监测对于未来细胞替代疗法的发展是至关重要的。 尽管核磁共振在细胞跟踪和监测方面的应用显示出巨大的前景，但技术挑战 也得到了认可。由于细胞移植物的水分含量预计不会与周围组织不同， 它们本身并不能产生核磁共振检测到的信号。已经探索了几种方法来克服 这种敏感性的限制包括使用外源性金属螯合造影剂2，3，6，8，9。 在磁共振成像中使用造影剂的最新想法是利用基于金属的对比剂的转基因表达 材料的内生性。这种方法有可能对细胞进行非侵入性、长期的活体监测 移植，特别是在细胞分裂期间，最近受到了相当大的关注2，10。 报告基因是铁蛋白的编码基因，铁蛋白是一种铁螯合蛋白，用于在生命系统中储存铁。首字母 对铁蛋白的研究已经产生了令人振奋的结果，表明核磁共振报告基因2的潜力。 另一个可能的候选者是MAGA，它调节铁的运输和磁铁矿的形成 (Fe3O4)晶体存在于某些类型的细菌中11.磁铁矿是一种超磁性粒子，可以诱导大量的 水松弛时间的变化，因此被认为是一种很好的磁共振造影剂8，9。 MAGA没有像铁蛋白那样受到关注，因为它的表达仅限于细菌。最近， 我们的实验室成功地在哺乳动物细胞系中表达了MAGA，并首次证实了 在哺乳动物细胞中形成磁小体，这在磁共振成像12中可以很容易地检测到。我们假设 MAGA可以在小鼠胚胎干细胞(MESCs)中表达而不会对 干细胞的特性使得mESC移植物可以通过核磁共振无创监测。我们向他求婚 扩大这项研究，并对MAGA作为MRI报告基因的潜力进行批判性评估。我们也是 有兴趣探索它在体内细胞移植监测中的应用，这是细胞前进的主要障碍之一 替代研究。我们的三个具体目标是：(1)确定表达MAGA基因的效果， 鉴定哺乳动物细胞中的磁小体，并在体外研究MAGa磁共振报告的敏感性， (2)通过动物模型确定MAGA是否可以在体内用作MRI报告，以及(3) 表征由MAGa产生的磁小体的成像特性，并开发成像方法 表达MAGA的移植干细胞移植的体内成像和示踪。

Project Summary We propose to develop a novel MRI reporter gene system that uses MRI to monitor gene expression and the status of cell grafts in vivo. Current MRI technology is capable of high spatial resolution at 10-¿m range and has found wide applications in research as well as in the clinical diagnosis of various diseases 1-7. In vivo monitoring of cell grafts is a critical function for the future development of cell replacement based therapy. Although applications of MRI in cell tracking and monitoring have shown great promise, technical challenges have also been recognized. As cell grafts are not expected to differ in water content from surrounding tissues, they alone do not generate signals detected by MRI. Several approaches have been explored to overcome the limitation of this sensitivity including the use of exogenous metal-chelate contrast agents 2, 3, 6, 8, 9. One of the most novel ideas for using contrast agents in MRI is to utilize transgene expression of metal based contrast materials endogenously. This approach has the potential for non-invasive, long-term in vivo monitoring of cell grafts, especially during cell division, and has recently received considerable attention 2, 10. An example of a reporter gene is the one coding for ferritin, an iron chelating protein for iron storage in living systems. Initial studies with ferritin have generated promising results that suggest the potential of MRI reporter genes 2. Another possible candidate is MagA, which regulates the transport of iron and the formation of magnetite (Fe3O4) crystal in certain types of bacteria 11. Magnetite is a supermagnetic particle that can induce substantial changes in water relaxation times, and is therefore considered an excellent MRI contrast agent 8, 9. Previously MagA hasn't received as much attention as ferritin since its expression has been limited to bacteria. Recently, our lab has successfully expressed MagA in mammalian cell lines and, for the first time, confirmed the formation of magnetosomes in mammalian cells, which can be readily detected in MRI 12. We hypothesize that MagA could be expressed in mouse embryonic stem cells (mESCs) without an adverse effect on the stem cell properties that allow an mESC graft to be monitored noninvasively by MRI. We proposed to expand this research and critically evaluate the potential of MagA as an MRI reporter gene. We are also interested in exploring its applications in cell graft monitoring in vivo, one of the major barriers in advancing cell replacement research. Our three specific aims are: (1) Determine the effects of expressing MagA genes, characterize magnetosomes in mammalian cells and investigate the sensitivity of MagA MRI reporter in vitro, (2) Determine whether MagA could be used as an MRI reporter in vivo using an animal model, and (3) Characterize the imaging properties of magnetosome produced from MagA and develop imaging methods for in vivo imaging and tracking of transplanted stem cell grafts expressing MagA.