MONITORING STEM CELL GRAFTS USING A NOVEL MRI REPORTER

描述（由申请人提供）：我们建议开发一种新的MRI报告基因系统，该系统利用MRI监测基因表达和细胞移植物的体内状态。目前的MRI技术具有10-<m范围的高空间分辨率，在各种疾病的研究和临床诊断中得到了广泛的应用1-7。细胞移植的体内监测是未来细胞替代疗法发展的关键功能。虽然核磁共振成像在细胞跟踪和监测方面的应用显示出巨大的前景，但技术挑战也已被认识到。由于细胞移植物的含水量与周围组织没有差异，因此它们本身不会产生MRI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MRI中使用造影剂的最新颖的想法之一是利用金属基造影剂的内源性转基因表达。这种方法具有对移植物进行无创、长期体内监测的潜力，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，铁蛋白是一种铁螯合蛋白，用于在生命系统中储存铁。对铁蛋白的初步研究已经产生了令人鼓舞的结果，表明MRI报告基因的潜力。另一个可能的候选物质是MagA，它调节铁的运输和某些类型细菌中磁铁矿（Fe3O4）晶体的形成。磁铁矿是一种超磁性颗粒，可以引起水弛豫时间的实质性变化，因此被认为是一种优秀的MRI造影剂8,9。此前，MagA并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，并首次证实了哺乳动物细胞中磁小体的形成，这在MRI 12中很容易检测到。我们假设MagA可以在小鼠胚胎干细胞（mESCs）中表达，而不会对干细胞特性产生不利影响，从而使mESC移植物能够通过MRI无创监测。我们建议扩大这项研究，并批判性地评估MagA作为MRI报告基因的潜力。我们也有兴趣探索其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)确定表达MagA基因的影响，表征哺乳动物细胞中的磁小体，并研究MagA MRI报告基因的体外敏感性；(2)通过动物模型确定MagA是否可以在体内用作MRI报告基因；(3)表征由MagA产生的磁小体的成像特性，并开发表达MagA的移植干细胞移植物的体内成像和跟踪成像方法。

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.