MONITORING STEM CELL GRAFTS USING A NOVEL MRI REPORTER

描述(申请人提供)：我们建议开发一种新的MRI报告基因系统，该系统使用MRI来监测体内细胞移植物的基因表达和状态。目前的磁共振成像技术能够在10&lt；m范围内具有高空间分辨率，并在各种疾病的研究和临床诊断中得到了广泛的应用1-7。对细胞移植物的体内监测对于未来基于细胞替代的治疗的发展至关重要。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，但也认识到了技术挑战。由于细胞移植物的水分含量预计不会与周围组织不同，因此它们本身不会产生核磁共振检测到的信号。为了克服这种敏感性的局限性，已经探索了几种方法，包括使用外源性金属螯合对比剂2、3、6、8、9。在MRI中使用对比剂的最新想法之一是利用内源性金属基对比剂的转基因表达。这种方法有可能对细胞移植进行非侵入性的、长期的体内监测，特别是在细胞分裂期间，最近受到了相当大的关注2，10。报告基因的一个例子是编码铁蛋白的基因，铁蛋白是生命系统中储存铁的一种铁络合蛋白。对铁蛋白的初步研究已经产生了令人振奋的结果，表明了磁共振报告基因2的潜力。另一个可能的候选基因是MAGA，它调控着某些类型细菌11中铁的运输和磁铁矿(Fe3O4)晶体的形成。磁铁矿是一种超磁性粒子，可以引起水松弛时间的实质性变化，因此被认为是一种优秀的磁共振造影剂8，9。以前，MAGA没有像铁蛋白那样受到那么多的关注，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，并首次证实了哺乳动物细胞中磁小体的形成，这在MRI 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产生不利影响，从而使MRI能够非侵入性地监测mESC移植物。我们建议扩大这项研究，并对MagA作为MRI报告基因的潜力进行批判性评估。我们也有兴趣探索它在体内细胞移植监测中的应用，这是推进细胞替代研究的主要障碍之一。我们的三个具体目标是：(1)确定MAGA基因的表达效果，鉴定哺乳动物细胞中的磁小体，并研究MAGA磁共振报告基因的体外敏感性，(2)通过动物模型确定MAGA是否可以作为MRI报告基因，以及(3)表征MAGA产生的磁小体的成像特性，并建立体内成像和示踪表达MAGA的移植干细胞的成像方法。 公共卫生相关性：拟议的项目旨在评估新型转基因MRI报告及其作为胚胎干细胞移植的非侵入性监测的应用。因此，可以监测和追踪ES细胞的发育。

DESCRIPTION (provided by applicant): 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 graft expressing MagA. PUBLIC HEALTH RELEVANCE: The proposed project seeks to evaluate the novel transgenic MRI reporter and its application as a marker for noninvasive monitoring of embryonic stem (ES) cell grafts. Thus, the development of ES cells can be monitored and traced.