胎鼠神经干细胞超顺磁性氧化铁颗粒标记移植后MRI研究

目的:探讨超顺磁性氧化铁颗粒(SPIO)标记神经干细胞的方法,以及标记细胞正常大鼠脑内移植后MR成像的方法学研究。方法:多聚左旋赖氨酸介导的SPIO标记胎鼠神经干细胞,进行台盼兰染色和普鲁士兰染色分别检测标记细胞的存活率和标记率。选取SD大鼠15只,简单随机法分为3组:第1组于大鼠右侧尾状核移植未标记的NSCs,第2组于大鼠右侧尾状核移植标记的NSCs,第3组右侧尾状核移植游离的SPIO颗粒,移植后第1、4、8周进行MRI。8周后处死大鼠,行组织切片普鲁士兰染色。结果:体外标记的神经干细胞普鲁士兰染色发现铁颗粒聚集于细胞浆内,标记率为100%;标记细胞与未标记细胞的台盼兰染色结果无显著差异。移植后MRI,第1组注射点未见低信号影;第2组注射点T2WI及GRE序列均可见类圆形低信号影;第3组大鼠注射后1周注射点可见低信号影,4周后低信号影变淡且边缘变模糊,8周后低信号影T2WI已不明显。与T2WI序列比较,GRE序列显示标记细胞更清晰,但显示范围较扩散。脑组织切片的普鲁士兰染色显示,第1组大鼠脑组织切片未见异常蓝染细胞,第2组注射点可见蓝染细胞,第3组注射点可见稍许散在蓝色颗粒状物质。结论:多聚左旋赖氨酸介导下SPIO可用于标记神经干细胞,标记细胞移植后MRI可以无创性观察移植神经干细胞的位置及分布情况。     

PET molecular imaging in stem cell therapy for neurological diseases

Human neurological diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, spinal cord injury and multiple sclerosis are caused by loss of different types of neurons and glial cells in the brain and spinal cord. At present, there are no effective therapies against these disorders. Discovery of the therapeutic potential of stem cells offers new strategies for the treatment of neurological diseases. Direct assessment of stem cells’ survival, interaction with the host and impact on neuronal functions after transplantation requires advanced in vivo imaging techniques. Positron emission tomography (PET) is a potential molecular imaging modality to evaluate the viability and function of transplanted tissue or stem cells in the nervous system. This review focuses on PET molecular imaging in stem cell therapy for neurological diseases.     

In Vivo Cell Tracking with Bioluminescence Imaging

Molecular imaging is a fast growing biomedical research that allows the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In vivo tracking of cells is an indispensable technology for development and optimization of cell therapy for replacement or renewal of damaged or diseased tissue using transplanted cells, often autologous cells. With outstanding advantages of bioluminescence imaging, the imaging approach is most commonly applied for in vivo monitoring of transplanted stem cells or immune cells in order to assess viability of administered cells with therapeutic efficacy in preclinical small animal models. In this review, a general overview of bioluminescence is provided and recent updates of in vivo cell tracking using the bioluminescence signal are discussed.

     

MR示踪技术在干细胞移植治疗软骨缺损中的研究进展

关节软骨缺损临床十分常见, 但目前的治疗方法均存在修复不完全的缺陷。间充质干细胞移植治疗的发展为再生修复关节软骨缺损提供了新的治疗策略, 但是作为组织修复执行者的干细胞移植后的在体迁徙分布、增殖及转归过程, 目前尚无安全无创、实时动态的监测手段, 因此难以明确外源性干细胞在关节软骨缺损再生修复中所扮演的角色。而MR在体示踪细胞技术为解决上述问题提供了新思路。MRI具有无创、无电离辐射、时间空间分辨率高、对比度好等优点, 协同MRI对比剂, 既可无创提供关节软骨的详细解剖结构信息, 还可动态评估移植干细胞的归宿。笔者就MR示踪技术在干细胞移植治疗软骨缺损中的最新研究进展进行综述, 探讨其优势、局限性及未来前景。     

In vivo MR tracking of mesenchymal stem cells in rat liver after intrasplenic transplantation

PURPOSE: To prospectively track in vivo in rats intrasplenically transplanted stem cells labeled with superparamagnetic particles by using magnetic resonance (MR) imaging. MATERIALS AND METHODS: The study was approved by the institutional Committee on Animal Research. Liver damage in 12 rats was induced with subcutaneous injection of carbon tetrachloride (CCl4). Intrasplenic transplantation of 6x10(6) rodent bone mesenchymal stem cells (BMSCs) with (n=6) and without (n=6) superparamagnetic particle Fe2O3-poly-L-lysine (PLL) labeling was performed via direct puncture. Cell labeling efficiency was assessed in vitro by using Prussian blue stain and an atomic absorption spectrometer. MR examinations were performed immediately before and 3 hours and 3, 7, and 14 days after transplantation. Liver-to-muscle contrast-to-noise ratios (CNRs) on T2*-weighted MR images obtained before and after injection were measured and correlated with histomorphologic studies. Statistical analyses were performed by using repeated-measures analysis of variance. RESULTS: Rat BMSCs could be effectively labeled with approximately 100% efficiency. Migration of transplanted labeled cells to the liver was successfully documented with in vivo MR imaging. CNRs on T2*-weighted images decreased significantly in the liver 3 hours after injection of BMSCs (P<.05) and returned gradually to the level achieved without labeled cell injection in 14 days. Histologic analyses confirmed the presence of BMSCs in the liver. The labeled cells primarily localized in the sinusoids of periportal areas and the foci of CCl4-induced liver damage. Quantitative analysis of Prussian blue-stained cells indicated gradual decrease of dye pigments from 3 hours to 3, 7, and 14 days after injection. No free iron particles were found in the interstitium or within hepatic microvessels. CONCLUSION: The rat BMSCs could be efficiently labeled with Fe2O3-PLL and the relocation of the labeled cells to rat livers after intrasplenic transplantation could be depicted at in vivo MR imaging.     

Serial in vivo MR tracking of magnetically labeled neural spheres transplanted in chronic EAE mice.

Neural stem cell (NSC) transplantation has been shown to attenuate the severity of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Central to the future success of NSC transplantation in MS is the ability of transplanted cells to migrate from the site of transplantation to relevant foci of disease. Using magnetically labeled mouse neurospheres and human embryonic stem cell (hESC)-derived neurospheres, we applied serial magnetic resonance imaging (MRI) to assess the biodynamics of transplanted cell migration in a chronic mouse EAE model. Magnetic labeling did not affect the in vitro and in vivo characteristics of cells as multipotential precursors. Cell migration occurred along white matter (WM) tracts (especially the corpus callosum (CC), fimbria, and internal capsule), predominantly early in the acute phase of disease, and in an asymmetric manner. The distance of cell migration correlated well with clinical severity of disease and the number of microglia in the WM tracts, supporting the notion that inflammatory signals promote transplanted cell migration. This study shows for the first time that hESC-derived neural precursors also respond to tissue signals in an MS model, similarly to rodent cells. The results are directly relevant for designing and optimizing cell therapies for MS, and achieving a better understanding of in vivo cell dynamics and cell-tissue interactions.     

Cell Tracking

Cell based therapies such as stem cell therapies or adoptive immunotherapies are currently being explored as a potential treatment for a variety of diseases such as Parkinson's disease, diabetes or cancer. However, quantitative and qualitative evaluation of adoptively transferred cells is indispensable for monitoring the efficiency of the treatment. Current approaches mostly analyze transferred cells from peripheral blood, which cannot assess whether transferred cells actually home to and stay in the targeted tissue. Using cell-labeling methods such as direct labeling or transfection with a marker gene in conjunction with various imaging modalities (MRI, optical or nuclear imaging), labeled cells can be followed in vivo in real-time, and their accumulation as well as function in vivo can be monitored and quantified accurately. This method is usually referred to as "cell tracking" or "cell trafficking" and is also being applied in basic biological sciences, exemplified in the evaluation of genes contributing to metastasis. This review focuses on principles of this promising methodology and explains various approaches by highlighting recent examples.     

报告基因显像监测干细胞治疗的研究进展

干细胞治疗的研究展示了广阔的应用前景,但干细胞治疗作为一种新的疾病治疗手段,仍存在着很多亟待解决的问题.报告基因显像是近年来发展迅速的一种无创、灵敏的监测干细胞的方法,特别是放射性核素报告基因显像具有灵敏度高和特异性强的优点,并可进行深部组织显像和重复显像,是在活体条件下示踪干细胞最具潜力的技术,展示了良好的应用前景.     

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。     

In vivo serial evaluation of superparamagnetic iron‐oxide labeled stem cells by off‐resonance positive contrast

MRI is emerging as a diagnostic modality to track iron‐oxide‐labeled stem cells. This study investigates whether an off‐resonance (OR) pulse sequence designed to generate positive contrast at 1.5T can assess the location, quantity, and viability of delivered stem cells in vivo. Using mouse embryonic stem cell transfected with luciferase reporter gene (luc‐mESC), multimodality validation of OR signal was conducted to determine whether engraftment parameters of superparamagnetic iron‐oxide labeled luc‐mESC (SPIO‐luc‐mESC) could be determined after cell transplantation into the mouse hindlimb. A significant increase in signal‐ and contrast‐to‐noise of the SPIO‐luc‐mESC was achieved with the OR technique when compared to a gradient recalled echo (GRE) sequence. A significant correlation between the quantity of SPIO‐luc‐mESC and OR signal was observed immediately after transplantation (R² = 0.74, P < 0.05). The assessment of transplanted cell viability by bioluminescence imaging (BLI) showed a significant increase of luciferase activities by day 16, while the MRI signal showed no difference. No significant correlation between BLI and MRI signals of cell viability was observed. In conclusion, using an OR sequence the precise localization and quantitation of SPIO‐labeled stem cells in both space and time were possible. However, the OR sequence did not allow evaluation of cell viability. Magn Reson Med 60:1269–1275, 2008. © 2008 Wiley‐Liss, Inc.     

间充质干细胞移植治疗作用机制

间充质干细胞治疗的疾病谱比造血干细胞更广。迄今为止,国外已有十多个间充质干细胞产品被批准临床应用,国内也有数个产品被批准临床试验研究。间充质干细胞通过直接和间接两种方式发挥治疗作用。直接方式包括归巢、促进外源干细胞植入患处,以及定向分化与多向分化等;间接方式包括双向调节免疫反应和炎症反应、抗氧化应激、抗纤维化、抗凋亡、造血支持作用及参与或促进血管再生、激活内源干细胞增殖分化及患病组织器官已分化的正常细胞去分化,以及抗衰老、抗瘢痕等。间充质干细胞移植治疗的作用机制十分复杂,迄今仍没有完全弄清,还需要进行深入研究。研究间充质干细胞的移植治疗作用机制,对于间充质干细胞临床应用具有重要意义。     

MR示踪技术在干细胞治疗心肌梗死中的研究进展

活体细胞MR示踪成像技术能有效在体内实时、准确评价干细胞治疗心肌梗死的效果，可分为MR对比剂标记细胞成像和MR报告基因成像。自体移植干细胞治疗心肌梗死的疗法中，能标记干细胞的MR对比剂主要有以钆剂为主的顺磁性对比剂、化学交换饱和转移成像（CEST）相关对比剂以及氧化铁类对比剂；MR 报告基因导入后能使细胞表达产生MR信号改变的蛋白质，以膜表面蛋白为主，包括铁蛋白受体、膜表面抗原、酶等。目前的MR细胞示踪技术对自体移植干细胞治疗的疗效评价、机制研究有一定的指导作用。     

核素分子显像监测胚胎干细胞及诱导性多潜能干细胞移植的研究进展

胚胎干细胞（ESCs）具有分化成多种细胞的能力,而由转录因子转染体细胞获得的诱导性多潜能干细胞（iPSCs）具有与ESCs相似的生物学特性,且不涉及伦理学相关问题,在干细胞治疗领域得到广泛的应用.近年来,干细胞移植治疗的分子影像学监测得到了快速发展,并取得了显著的成果.该文仅就核素分子显像监测ESCs及iPSCs移植的研究进展进行综述.     

MR-based imaging of neural stem cells

The efficacy of therapies based on neural stem cells (NSC) has been demonstrated in preclinical models of several central nervous system (CNS) diseases. Before any potential human application of such promising therapies can be envisaged, there are some important issues that need to be solved. The most relevant one is the requirement for a noninvasive technique capable of monitoring NSC delivery, homing to target sites and trafficking. Knowledge of the location and temporospatial migration of either transplanted or genetically modified NSC is of the utmost importance in analyzing mechanisms of correction and cell distribution. Further, such a technique may represent a crucial step toward clinical application of NSC-based approaches in humans, for both designing successful protocols and monitoring their outcome. Among the diverse imaging approaches available for noninvasive cell tracking, such as nuclear medicine techniques, fluorescence and bioluminescence, magnetic resonance imaging (MRI) has unique advantages. Its high temporospatial resolution, high sensitivity and specificity render MRI one of the most promising imaging modalities available, since it allows dynamic visualization of migration of transplanted cells in animal models and patients during clinically useful time periods. Different cellular and molecular labeling approaches for MRI depiction of NSC are described and discussed in this review, as well as the most relevant issues to be considered in optimizing molecular imaging techniques for clinical application. Dr. Letterio Politi was awarded the Lucien Appel Prize in 2006 by the European Society of Neuroradiology for his research into MR imaging of neural stem cells.     

Cell tracking with optical imaging

Adaptability, sensitivity, resolution and non-invasiveness are the attributes that have contributed to the longstanding use of light as an investigational tool and form the basis of optical imaging (OI). OI, which encompasses numerous techniques and methods, is rapid (<5 min), inexpensive, noninvasive, nontoxic (no radiation) and has molecular (single-cell) sensitivity, which is equal to that of conventional nuclear imaging and several orders of magnitude greater than MRI. This article provides a comprehensive overview of emerging applications of OI-based techniques for in vivo monitoring of new stem cell-based therapies. Different fluorochromes for cell labeling, labeling methods and OI-based cell-tracking techniques will be reviewed with respect to their technical principles, current applications and aims for clinical translation. Advantages and limitations of these new OI-based cell-tracking techniques will be discussed. Non-invasive mapping of cells labeled with fluorochromes or OI marker genes has the potential to evolve further within the clinical realm.     

In vivo tracking of implanted stem cells using radio-labeled transferrin scintigraphy

The possibility of monitoring stem cells in vivo with radionuclide imaging after transplantation was investigated. Based on the results of a radioligand receptors assay that human mesenchymal stem cells (hMSCs) express a high level of transferrin receptors, iodinated transferrin (131I-Tf(Fe)2) was chosen as the radiotracer for imaging the cells implanted into the spinal cords of rabbits. Accumulation of radioactivity at the cell transplanted sites was assessed 16 and 24 hours post-intrathecal injection of 131I-Tf(Fe)2. Transferrin receptors expression and Tf binding of the implanted cells were verified by immunofluorescence and ex vivo phosphor imaging. The specificity of Tf uptake of hMSCs was proved through control experiments, i.e., replacing 131I-Tf(Fe)2 with 131I labeled human serum albumin as the tracer or substituting hMSCs with phosphate buffered saline as the grafts. Despite some defects, such as the invasive administration of the tracer and the non-specificity of transferrin receptors as a marker of stem cells in this preliminary study, the technique of nuclear medicine imaging is considered to have great potential in tracking implanted cells in vivo.     

In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium

OBJECTIVE: The conventional method for the analysis of myocardial cell transplantation depends on postmortem histology. Here, we have sought to demonstrate the feasibility of a longitudinal monitoring of transplanted cell survival in living animals, accomplished with optical imaging techniques and pharmacological interventions. METHODS: Human cord blood (50 ml) was donated with parental consent. After getting cord blood derived mesenchymal stem cells (CBMSCs), cells were transfected (MOI = 100) overnight with adenovirus encoding firefly luciferase gene (Ad-CMV-Fluc). Our experimental Sprague-Dawley rats (n = 12) were given intramyocardial injections containing 1 x 10(6) CBMSCs, which had been made to express the firefly luciferase (Fluc) reporter gene. Optical bioluminescence imaging was then conducted using a cooled charged-coupled device (CCD) camera (Xenogen), beginning on the day after the transplantation (day 1). Groups of mice were intraperitoneally injected with cyclosporine (5 mg/kg) or tacrolimus (1 mg/kg), in an attempt to determine the degree to which cell survival had been prolonged, and these values were then compared with the cell survival values of the negative control group. The presence of transplanted CBMSCs on in vivo images confirmed by in situ hybridization for human specific Alu in the myocardium. RESULTS: Cardiac bioluminescence signals were determined to be present for 6 days after transplantation: day 1 (97000 +/- 9100 x 10(5) p/s/cm2/sr), day 3 (9600 +/- 1110 p/s/cm2/sr), and day 5 (3200 +/- 550 p/s/cm2/sr). The six mice that received either cyclosporine or tacrolimus displayed cardiac bioluminescence signals for a period of 8 days after transplantation. We observed significant differences between the treated group and the non-treated group, beginning on day 3 (tacrolimus; 26500 +/- 4340 p/s/cm2/sr, cyclosporine; 27200 +/- 3340 p/s/cm2/sr, non-treated; 9630 +/- 1180 p/s/cm2/sr, p < 0.01), and persisting until day 7 (tacrolimus; 12500 +/- 2946 p/s/cm2/sr, cyclosporine; 7310 +/- 1258 p/s/cm2/sr, non-treated; 2460 +/- 160 p/s/cm2/sr, p < 0.01). The human-derived CBMSCs were detected in the myocardium 3 days after transplantation by in situ hybridization. CONCLUSIONS: The locations, magnitude, and survival duration of the CBMSCs were noninvasively monitored with a bioluminescence optical imaging system. We determined that optical molecular imaging expedites the fast throughput screening of pharmaceutical agents, allowing for the noninvasive tracking of cell survival within animals. In rat cardiac CBMSC transplant models, transient immunosuppressive treatment with tacrolimus or cyclosporine was shown to improve donor cell survival.     

创伤性脑损伤亚低温治疗期间实施干细胞移植的实验研究

目的 研究在亚低温环境中实施干细胞移植的可行性,为创伤性脑损伤(traumatic brain injuries,TBI)患者接受亚低温治疗期间实施干细胞移植提供实验依据.方法 利用温度敏感型猿猴病毒40大T抗原(tsSV40LT)建立温度敏感型人源性脐带间充质干细胞系(tsUCMSOs),观察其在亚低温(33℃)和正常体温(37℃)下细胞形态、核增殖指数(PIx)及端粒酶活性的变化;建立小鼠TBI亚低温治疗模型,在创伤灶周围半损伤带移植tsUCMSCs,检测其存活率、增殖和凋亡指数,并进行神经功能缺陷评分.结果 tsUCMSCs在33℃培养时,胞体呈长梭形,折光性强,增殖指数和端粒酶活性均较高,但在37℃时胞体扁平,折光性差,细胞老化明显,增值指数和端粒酶活性均明显降低.移植于创伤灶周围半损伤带的tsUCMSCs较对照组(非温度敏感型UCMSCs移植组)细胞存活率明显增高(P<0.05),细胞核增殖抗原高表达,凋亡细胞少见,小鼠神经功能明显好转(P<0.05).结论 tsUCMSCs的建立使TBI亚低温治疗期间实施干细胞移植成为可能,为未来脑创伤的救治提供了新的方法.     

磁标记大鼠骨髓间充质干细胞活体内移植后向肝癌细胞趋向性迁移的研究

目的 探讨磁标记大鼠骨髓间充质干细胞(BMSCs)活体内移植后对大鼠肝细胞癌的趋向性迁移及其机制.方法 培养大鼠BMSCs,超顺磁性氧化铁粒子标记.制备大鼠肝癌模型24只,数字表法随机分为3组:实验组(n=12)经脾植入磁标记的BMSCs;对照组A(n=6)移植未标记的BMSCs;对照组B(n=6)不作任何处理.分别于移植前及移植后1、3、7和14 d行MR扫描,选用T_2*WI序列进行移植细胞的示踪并测量肿瘤组织与正常肝组织的信号强度的比值(SI/SI*),结果行单因素方差分析;取肿瘤组织、瘤旁正常肝组织行普鲁士蓝染色,分析BMSCs在体内的分布并与MR对照.结果 BMSCs的磁标记率为90%以上.移植后实验组T_2*WI显示肿瘤信号强度值明显减低,移植前及移植后1、3、7和14 d的SI/SI*值分别为3.18±0.21、1.98±0.20、2.38±0.28、2.70±0.25及3.16±0.24,差异有统计学意义(F=56.65,P<0.05);与移植前相比,1、3、7 d肿瘤信号强度的减低有统计学意义(t值分别为1.20、0.79、0.48,P值均<0.05).对照组移植前后各SI/SI*值差异无统计学意义(P>0.05).免疫组织化学显示实验组肿瘤边缘及内部有大量监染的普鲁士蓝阳性细胞分布,标记细胞在肿瘤内的分布与MR信号改变基本一致.对照组肿瘤组织普鲁士蓝染色均为阴性结果.结论 BMSCs在活体内对肝癌细胞有明显的趋向迁移特性,有望成为基因治疗肝细胞癌的载体.     

Positron emission tomography based in-vivo imaging of early phase stem cell retention after intramyocardial delivery in the mouse model

Purpose

To establish PET as a tool for in-vivo quantification and monitoring of intramyocardially transplanted stem cells after labelling with FDG in mice with induced myocardial infarction.

Methods

After inducing myocardial infarction in C57BL/6 mice, murine embryonic stem cells were labelled with FDG and transplanted into the border zone of the infarction. Dynamic PET scans were acquired from 25 to 120 min after transplantation, followed by a scan with 20 MBq FDG administered intravenously for anatomical landmarking. All images were reconstructed using the OSEM 3D and MAP reconstruction algorithms. FDG data were corrected for cellular tracer efflux and used as marker for cellular retention. FACS analysis of transplanted cells expressing enhanced green fluorescent protein was performed to validate the PET data.

Results

We observed a rapid loss of cells from the site of transplantation, followed by stable retention over 120 min. Amounts of retention were 5.3?±?1.1 % at 25 min, 5.0?±?0.9 % at 60 min and 5.7?±?1.2 % at 120 min. FACS analysis showed a high correlation without significant differences between the groups (P?>?0.05). FDG labelling did not have any adverse effects on cell proliferation or differentiation.

Conclusion

Up-to-date imaging is a powerful method for tracking and quantifying intramyocardially transplanted stem cells in vivo in the mouse model. This revealed a massive cell loss within minutes, and thereafter a relatively stable amount of about 5 % remaining cells was observed. Our method may become crucial for further optimization of cardiac cell therapy in the widely used mouse model of infarction.