一种新的超顺磁性氧化铁阳离子微囊可控标记干细胞的MR成像

目的研究高效、可控标记干细胞进行MR成像的可行性。方法人工合成一类新的阳离子、超顺磁氧化铁纳米颗粒微囊标记小鼠骨髓的间充质干细胞,不使用二次转染剂。评价最优标记环境与可控性,标记对细胞活性、增殖性和多向分化能力的影响。在18只大鼠中诱导产生局部缺血性脑损伤,随机注入标记0、8和20mV微囊的1×106个细胞(每组n=6)。活体MR扫描随访对侧纹状体内的移植细胞情况,结果与组织学进行相关分析。结果最优细胞标记条件包括浓度为3.15μgFe/mL的微囊、20mV的正电和1h孵育时间。标记效率随着微囊电势升高而呈线性变化。标记不能影响细胞的活性、增殖性和多向分化能力。MR成像能够显示标记细胞的分布与迁移。组织学证实移植细胞仍然保持标记和活性。结论阳离子、超顺磁氧化铁纳米颗粒微囊能在可控方式下安全、有效地标记干细胞进行分子MR成像。     

干细胞移植治疗心肌梗死的MR示踪观察

各种原因引起的心肌缺血梗死是临床上重要的死亡原因.以前认为心肌坏死只能通过形成疤痕组织进行修复,近年来,许多动物实验证实,用间质干细胞(mesenchymal stem cells,MSCs)移植治疗心肌梗死,可增加有功能的心肌细胞的数量和现存心肌细胞的质量,改善心脏功能.MRI的分辨率达25～50 μm,接近单一细胞水平,因此可用来对移植的间质干细胞进行活体示踪.应用MR对比剂超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)等对MSCs进行标记,可以对心肌梗死区MSCs移植后的迁徙、增殖情况进行动态观察.     

超顺磁性氧化铁标记鼠骨髓间充质干细胞的体外MR成像研究

目的: 探讨不同浓度超顺磁性氧化铁(SPIO)颗粒标记鼠骨髓间充质干细胞(MSCs)的标记率和对细胞活力的影响,以及MR成像显示磁标记干细胞的可行性.材料和方法: 将不同浓度的SPIO-PLL复合物与培养基混合,行普鲁士蓝染色观察细胞内铁和检测细胞活力.应用1.5T MR仪,以T1WI和T2WI行磁标记干细胞成像.结果: SPIO可有效标记MSCs,标记后的铁颗粒位于细胞质内.SPIO标记的MSCs可引起T2WI信号降低,50、100、150较25μg Fe /ml的信号强度降低明显.结论: SPIO可以简便标记MSCs,并在适当浓度下对细胞活力没有影响,此技术为干细胞移植的MR活体内示踪奠定基础.     

铁羧葡胺标记猪间充质干细胞的体内外磁共振成像研究

目的 探讨铁羧葡胺-多聚赖氨酸复合物标记猪骨髓间充质干细胞(MSC)的体外和活体心脏内MR成像的特点.方法 分离培养猪MSC,用含铁羧葡胺-多聚赖氨酸复合物标记细胞24 h.分别于标记后0、4、8、12、16、20 d行普鲁士蓝染色观察细胞内铁,原子吸收分光光度仪测定细胞内含铁量,锥虫蓝排除试验检测细胞活力.对不同时间点、不同细胞数的磁标记干细胞进行1.5 T MR仪体外成像,并对植入猪心肌内的标记细胞进行体内MR成像.结果 ①MSC标记后见胞质中大量普鲁士蓝着色颗粒,标记率达100%,铁离子含量平均为(13.13±2.30)pg/细胞;随细胞的分裂增殖,细胞内铁离子含量逐渐减少,16 d时铁离子含量下降到(0.68±0.20)pg/细胞,接近标记前水平[(0.21±0.06)pg/细胞,P＞0.05].干细胞磁标记后各时间点的锥虫蓝拒染率与未标记细胞无统计学差异(P＞0.05).②3种成像序列中GRE T2*WI信号改变最为明显,成像细胞数量越多,信号强度变化越明显.1×106个细胞进行MR成像,发现随着标记后体外培养时间延长,T2*WI磁标低信号逐渐消失,12 d以后信号强度和标记前无差异(P＞0.05).体外MR成像最少能检测到5×104～1×105个标记的猪MSC.③标记细胞心肌内移植后1周MR成像显示低信号区.结论 应用铁羧葡胺-多聚赖氨酸复合物标记猪MSC安全、高效;体外MR信号强度能一定程度上反映磁标记细胞的数量及增殖情况;1.5 T临床MR成像仪可对植入心脏的磁标记细胞进行活体显像示踪.     

骨髓间充质干细胞修复关节软骨损伤的MRI示踪研究进展

骨髓间充质干细胞能促进骨关节损伤的修复,使用磁共振成像(MRI)对移植入人体的细胞分布状况进行活体示踪具有重要的临床意义,也是目前分子影像学研究的热点之一.MRI示踪需要对干细胞进行标记,目前方法较多,标记物主要有钆、超顺磁性氧化铁以及报告基因成像.本文就骨髓间充质干细胞BMSCs对修复软骨缺损的作用及BMSCs在磁共振分子成像中的研究进展综述如下.     

干细胞移植的磁性标记及磁共振成像活体示踪

干细胞移植的临床应用需要解决植入活体内干细胞在体内存活、迁移及分化的监测问题。通过对干细胞进行顺磁性标记,磁共振成像(MRI)能够在活体上显示标记的干细胞,并进行特异性地追踪及定位,是目前干细胞活体示踪极具前景的方法。干细胞进行磁性标记主要利用铁类或钆类对比剂,两者各有优缺点。利用铁类或钆类对比剂标记干细胞并进行MRI活体监测取得了成功。并在心脑缺血损伤的疾病模型中得到应用,但在干细胞磁性标记的载体选用及其标记率、标记的持久性、标记对细胞活力及遗传性状方面尚存在一定的问题。     

脑梗死大鼠脑内移植超顺磁性氧化铁颗粒标记神经干细胞后的MR示踪研究

目的探讨超顺磁性氧化铁颗粒（SP10）标记的胎鼠神经干细胞（NSCs）在脑梗死模型大鼠脑内移植后，MR示踪观察的可行性。方法大鼠脑梗死模型24只，按随机数字表法分为3组：第1组大鼠同侧尾状核移植SP10和5-溴脱氧尿核苷（BrdU）双标记的NSCs；第2组对侧尾状核移植双标记的NSCs；第3组对侧尾状核移植未标记的NSCs。移植后1、3、5、7周后进行MR示踪观察，选择T2WI和梯度回波（GRE）序列，成像后相应时间点每组处死2只大鼠，取脑组织冰冻切片后进行普鲁士蓝染色及BrdU染色。结果移植后1周MRI显示：移植标记细胞组在注射点处可见类圆形低信号影，未标记细胞组注射点未见异常信号影；3周后，第1组梗死皮层下可见线状低信号影；移植5周后，第2组沿胼胝体走行可见扇形低信号影，尖端指向病灶。GRE序列显示标记细胞较清晰，而T2WI显示梗死病灶和大鼠脑正常结构较清晰。相应时间点相应部位普鲁士蓝染色及BrdU染色可见阳性细胞，与MRI结果相符。结论超顺磁性氧化铁颗粒和BrdU双标记的神经干细胞移植至大鼠脑内后可迁移到病灶区；MR成像能够在活体内连续示踪观察神经干细胞的迁移及分布情况。     

心肌细胞移植的多模态分子成像:第二部分.骨髓基质细胞在猪体内的PET/CT和MR成像

正摘要目的运用临床PET报告基因成像和MR预标记细胞成像来定量确定猪心肌细胞治疗(CCT)后骨髓基质细胞(MSC)可检测的极限值。方法动物研究由动物实验制度     

心脏磁共振在体示踪移植细胞的可行性研究

目的：寻找一种能够对移植细胞进行在体示踪的标记方法,为移植细胞存留、迁移提供重要观察手段。方法：从中华小型猪髂骨处抽取骨髓,体外培养扩增骨髓间充质干细胞（MSCs）。将SPIO和MSCs共同孵育培养36h。普鲁士蓝染色评价细胞的标记效率;通过MTT比色实验评价SPIO对细胞生长能力的影响;台盼蓝染色检验标记后细胞的活性;使用Costar Transwell方法评价铁离子对细胞迁移能力的影响;用细胞分化诱导液培养标记后的细胞评价其向成脂肪细胞和成骨细胞的分化能力。在体内实验中将SPIO标记或未标记的自体MSCs注射到心肌内,通过心脏磁共振检查对移植细胞进行在体示踪观察,取材动物心脏行病理检查观察移植细胞的存活、存留。结果：MSCs经铁离子标记后普鲁士蓝染色阳性率在98%以上,可见蓝色颗粒位于细胞浆内,标记细胞电镜切片可见高密度铁颗粒位于细胞浆内。随着培养液中SPIO浓度的增加细胞增殖能力没有明显改变;标记后98%的细胞保持活性;SPIO标记后的细胞保持原有的形态,可继续培养、传代;SDF-1和VEGF诱导的迁移实验发现标记细胞迁移能力没有降低;铁离子标记后细胞仍可向成脂肪细胞和成骨细胞分化。注射到心肌内的SPIO标记的MSCs可通过心脏磁共振检查进行在体示踪,动态观察显示SPIO标记细胞在磁共振图像上表现为低信号,并且在移植后4周仍可成像。病理学检查可以看到移植细胞呈普鲁士蓝染色阳性,并和影像学有很好的一致性。结论：临床使用的SPIO磁共振对比剂可以安全、有效地标记MSCs,心脏磁共振检查可以实现SPIO标记的移植细胞的在体示踪。     

超顺磁性氧化铁纳米粒子在细胞标记及细胞成像中的应用

近10年来,细胞成像技术是一个快速发展的领域,其目的在于可视化活体内靶细胞,获得细胞在活体内的生物学分布及迁徙情况。细胞成像作为一种活体内细胞监测的技术具有非侵人性及可重复性的特点。MRI由于其高分辨率的优点,可以达到细胞水平,所以成为细胞成像中的研究热点。靶细胞必须经MR对比剂标记后才能区别于背景组织而被MR监测到,目前常用的MR对比剂为钆类及氧化铁类。钆类对比剂具有低弛豫率、细胞清除快、非生物相容性等缺点,且去螯合化后对细胞的毒性作用目前知道的很少。氧化铁类中的超顺磁性氧化铁纳米（SPIO）材料是继钆之后一种新的用于MR对比剂的材料。     

Serial in vivo positive contrast MRI of iron oxide-labeled embryonic stem cell-derived cardiac precursor cells in a mouse model of myocardial infarction

Myocardial regeneration with stem-cell transplantation is a possible treatment option to reverse deleterious effects that occur after myocardial infarction. Since little is known about stem cell survival after transplantation, developing techniques for "tracking" cells would be desirable. Iron-oxide-labeled stem cells have been used for in vivo tracking using MRI but produce negative contrast images that are difficult to interpret. The aim of the current study was to test a positive contrast MR technique using reduced z-gradient rephasing (GRASP) to aid in dynamically tracking stem cells in an in vivo model of mouse myocardial infraction. Ferumoxides and protamine sulfate were complexed and used to magnetically label embryonic stem cell-derived cardiac-precursor-cells (ES-CPCs). A total of 500,000 ES-CPCs were injected in the border zone of infarcted mice and MR imaging was performed on a 9.4T scanner using T(2)*-GRE sequences (negative contrast) and positive contrast GRASP technique before, 24 hours, and 1 week after ES-CPC implantation. Following imaging, mice were sacrificed for histology and Perl's staining was used to confirm iron within myocardium. Good correlation was observed between signal loss seen on conventional T(2)* images, bright areas on GRASP, and the presence of iron on histology. This demonstrated the feasibility of in vivo stem cell imaging with positive contrast MRI.     

Magnetic resonance imaging of the heart: perfusion, function, and structure

The use of interventional therapy in acute myocardial infarction has intensified the desire to obtain accurate information on regional myocardial perfusion. MR imaging using ultrafast techniques and contrast agents may be useful to estimate myocardial perfusion in patients with coronary artery disease. In addition, MR imaging with contrast agents is capable of defining the infarcted region and the area at risk after coronary artery occlusion. Quantitative evaluation of regional myocardial contractile function with and without pharmacologic stress testing further improves the utility of MR imaging in defining the effects of reperfusion therapy on dysfunctional myocardium and in detecting myocardial ischemia. Furthermore, cine MR techniques are now used extensively to assess cardiac function and volumes and to obtain flow velocity maps. Cardiac MR applications are evolving rapidly and the clinical significance is expanding.     

Long‐term MR cell tracking of neural stem cells grafted in immunocompetent versus immunodeficient mice reveals distinct differences in contrast between live and dead cells

Neural stem cell (NSC)‐based therapy is actively being pursued in preclinical and clinical disease models. Magnetic resonance imaging (MRI) cell tracking promises to optimize current cell transplantation paradigms, however, it is limited by dilution of contrast agent during cellular proliferation, transfer of label from dying cells to surrounding endogenous host cells, and/or biodegradation of the label. Here, we evaluated the applicability of magnetic resonance imaging for long‐term tracking of transplanted neural stem cells labeled with superparamagnetic iron oxide and transfected with the bioluminescence reporter gene luciferase. Mouse neural stem cells were transplanted into immunodeficient, graft‐accepting Rag2 mice or immunocompetent, graft‐rejecting Balb/c mice. Hypointense voxel signals and bioluminescence were monitored over a period of 93 days. Unexpectedly, in mice that rejected the cells, the hypointense MR signal persisted throughout the entire time‐course, whereas in the nonrejecting mice, the contrast cleared at a faster rate. In immunocompetent, graft‐rejecting Balb/c mice, infiltrating leukocytes, and microglia were found surrounding dead cells and internalizing superparamagnetic iron oxide clusters. The present results indicate that live cell proliferation and associated label dilution may dominate contrast clearance as compared with cell death and subsequent transfer and retention of superparamagnetic iron oxide within phagocytes and brain interstitium. Thus, interpretation of signal changes during long‐term MR cell tracking is complex and requires caution. Magn Reson Med, 2011. © 2010 Wiley‐Liss, Inc.     

Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents

PURPOSE: To label mammalian and stem cells by combining commercially available transfection agents (TAs) with superparamagnetic iron oxide (SPIO) magnetic resonance (MR) imaging contrast agents. MATERIALS AND METHODS: Three TAs were incubated with ferumoxides and MION-46L in cell culture medium at various concentrations. Human mesenchymal stem cells, mouse lymphocytes, rat oligodendrocyte progenitor CG-4 cells, and human cervical carcinoma cells were incubated 2-48 hours with 25 microg of iron per milliliter of combined TAs and SPIO. Cellular labeling was evaluated with T2 relaxometry, MR imaging of labeled cell suspensions, and Prussian blue staining for iron assessment. Proliferation and viability of mesenchymal stem cells and human cervical carcinoma cells labeled with a combination of TAs and ferumoxides were evaluated. RESULTS: When ferumoxides-TA or MION-46L-TA was used, intracytoplasmic particles stained with Prussian blue stain were detected for all cell lines with a labeling efficiency of nearly 100%. Limited or no uptake was observed for cells incubated with ferumoxides or MION-46L alone. For TA-SPIO-labeled cells, MR images and relaxometry findings showed a 50%-90% decrease in signal intensity and a more than 40-fold increase in T2s. Cell viability varied from 103.7% +/- 9 to 123.0% +/- 9 compared with control cell viability at 9 days, and cell proliferation was not affected by endosomal incorporation of SPIO nanoparticles. Iron concentrations varied with ferumoxides-TA combinations and cells with a maximum of 30.1 pg +/- 3.7 of iron per cell for labeled mesenchymal stem cells. CONCLUSION: Magnetic labeling of mammalian cells with use of ferumoxides and TAs is possible and may enable cellular MR imaging and tracking in experimental and clinical settings.     

Contrast-enhanced MR imaging of the heart: overview of the literature

The use of magnetic resonance (MR) imaging for cardiac diagnosis is expanding, aided by the administration of paramagnetic contrast agents for a growing number of clinical applications. This overview of the literature considers the principles and applications of cardiac MR imaging with an emphasis on the use of contrast media. Clinical applications of contrast material-enhanced MR imaging include the detection and characterization of intracardiac masses, thrombi, myocarditis, and sarcoidosis. Suspected myocardial ischemia and infarction, respectively, are diagnosed by using dynamic first-pass and delayed contrast enhancement. Promising new developments include blood pool contrast media, labeling of myocardial precursor cells, and contrast-enhanced imaging at very high fields.     

Monitoring bone marrow‐originated mesenchymal stem cell traffic to myocardial infarction sites using magnetic resonance imaging

How stem cells promote myocardial repair in myocardial infarction (MI) is not well understood. The purpose of this study was to noninvasively monitor and quantify mesenchymal stem cells (MSC) from bone marrow to MI sites using magnetic resonance imaging (MRI). MSC were dual‐labeled with an enhanced green fluorescent protein and micrometer‐sized iron oxide particles prior to intra‐bone marrow transplantation into the tibial medullary space of C57Bl/6 mice. Micrometer‐sized iron oxide particles labeling caused signal attenuation in T₂*‐weighted MRI and thus allowed noninvasive cell tracking. Longitudinal MRI demonstrated MSC infiltration into MI sites over time. Fluorescence from both micrometer‐sized iron oxide particles and enhanced green fluorescent protein in histology validated the presence of dual‐labeled cells at MI sites. This study demonstrated that MSC traffic to MI sites can be noninvasively monitored in MRI by labeling cells with micrometer‐sized iron oxide particles. The dual‐labeled MSC at MI sites maintained their capability of proliferation and differentiation. The dual‐labeling, intra‐bone marrow transplantation, and MRI cell tracking provided a unique approach for investigating stem cells' roles in the post‐MI healing process. This technique can potentially be applied to monitor possible effects on stem cell mobilization caused by given treatment strategies. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

Contrast media for MR imaging of the heart

The role of contrast media for quantitative characterization of ischemic myocardial events with magnetic resonance (MR) imaging has advanced considerably in the past few years. Contrast material-enhanced MR imaging is useful for identifying and sizing myocardial infarcts and for distinguishing between occlusive and reperfused myocardial infarcts. Recent results suggest that contrast-enhanced MR imaging can also be used to identify areas of cell death in regions of reperfused myocardial infarction. With the aid of MR contrast media, fast MR imaging techniques may be useful in estimating regional myocardial perfusion. Although no simple relationship between signal intensity and concentration exists, contrast-enhanced MR perfusion imaging can demonstrate the presence and relative severity of hypoperfused myocardium. Combining myocardial perfusion imaging with the anatomic and functional information provided by other MR imaging techniques could make MR imaging a comprehensive noninvasive means of evaluating ischemic cardiac disease.     

Gene expression and gene therapy imaging

The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters.     

MRI in guiding and assessing intramyocardial therapy

Cardiovascular intervention, using MRI guidance, is challenging for clinical applications. Real-time imaging sequences with high spatial resolution are needed for monitoring intramyocardial delivery of drug, gene, or stem cell therapies. New generation MR scanners make local intramyocardial and vascular wall therapies feasible. Contrast-enhanced MRI is used for assessing myocardial ischemia, infarction, and scar tissue. Active (microcoils) and passive (T1 and T2* mechanisms) tracking methods have been used for visualization of endovascular catheters. Safety issues related to potential heating of endovascular devices is still a major obstacle for MRI-guided interventions. Fabrication of MRI-compatible interventional devices is limited. Noninvasive imaging strategies will be critical in defining spatial and temporal characteristics of angiogenesis and myocardial repair as well as in assessing the efficacy of new therapies in ischemic heart disease. MRI contrast media improve the capability of MRI by delineating the target and vascular tree. Labeling stem cells enables MRI to trace distribution, differentiation, and survival in myocardium and vascular wall. In the long term, MRI in guiding and assessing intramyocardial therapy may circumvent the limitations of peripherally administered cell therapy, X-ray angiography, and nuclear imaging. MRI represents a highly attractive discipline whose systematic development will foster the implementation of new cardiac and vascular therapies.This revised version was published online in March 2005 with a correction to the last authors name, C. Higgins.     

Cell motility of neural stem cells is reduced after SPIO‐labeling,which is mitigated after exocytosis

MRI is used for tracking of superparamagnetic iron oxide (SPIO)‐labeled neural stem cells. Studies have shown that long‐term MR tracking of rapidly dividing cells underestimates their migration distance. Time‐lapse microscopy of random cellular motility and cell division was performed to evaluate the effects of SPIO‐labeling on neural stem cell migration. Labeled cells divided symmetrically and exhibited no changes in cell viability, proliferation, or apoptosis. However, SPIO‐labeling resulted in decreased motility of neural stem cells as compared with unlabeled controls. When SPIO‐labeled neural stem cells and human induced pluripotent stem cells were transplanted into mouse brain, rapid exocytosis of SPIO by live cells was observed as early as 48 h postengraftment, with SPIO‐depleted cells showing the farthest migration distance. As label dilution is negligible at this early time point, we conclude that MRI underestimation of cell migration can also occur as a result of reduced cell motility, which appears to be mitigated following SPIO exocytosis. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.