Contrast Agents and Cell Labeling Strategies for in Vivo Imaging

Regenerative medicine has become a new therapeutic approach in which stem cells or genetically reprogrammed cells are delivered to diseased areas in the body with the intention that such multipotent cells will differentiate into healthy tissue and exchange damaged tissue. The success of such cell-based therapeutic approaches depends on precise dosing and delivery of the cells to the desired site in the human body. To determine the accuracy and efficacy of the therapy, tracking of the engrafted cells in an intact living organism is crucial. There is a great need for sensitive, noninvasive imaging methods, which would allow clinicians to monitor viability, migration dynamics, differentiation towards specific cell type, regeneration potential and integration of transplanted cells with host tissues for an optimal time period. Various in vivo tracking methods are currently used including: MRI (Magnetic Resonance Imaging), PET (Positron Emission Tomography), SPECT (Single Photon Emission Computer Tomography), optical imaging (OI), photoacoustic imaging (PAI) and ultrasound (US). In order to carry out the detection with each of the aforementioned techniques, the cells must be labeled either exogenously (ex vivo) or endogenously (in vivo). For tracking the administrated cells, scientists usually manipulate cells outside the living organism by incorporating imaging contrast agents (CAs) or reporter genes. Strategies for stem cell labeling using CAs will be reviewed in the light of various imaging techniques.

Water-soluble Cu30 nanoclusters as a click chemistry catalyst for living cell labeling via azide-alkyne cycloaddition

Cu(I)-catalyzed azide-alkyne cycloadditions(CuAAC)have gained increasing interest in the selective labeling of living cells and organisms with biomolecules.However,their application is constrained either by the high cytotoxicity of Cu(I)ions or the low activity of CuAAC in the internal space of living cells.This paper reports the design of a novel Cu-based nanocatalyst,watersoluble thiolated Cu30 nanoclusters(NCs),for living cell labeling via CuAAC.The Cu30 NCs offer good biocompatibility,excellent stability,and scalable synthesis(e.g.,gram scale),which would facilitate potential commercial applications.By combining the highly localized Cu(I)active species on the NC surface and good structural stability,the Cu30 NCs exhibit superior catalytic activities for a series of Huisgen cycloaddition reactions with good recyclability.More importantly,the biocompatibility of the Cu30 NCs enables them to be a good catalyst for CuAAC,whereby the challenging labeling of living cells can be achieved via CuAAC on the cell membrane.This study sheds light on the facile synthesis of atomically precise Cu NCs,as well as the design of novel Cu NCs-based nanocatalysts for CuAAC in intracellular bioorthogonal applications.

Superparamagnetic Iron Oxide Labeling of Neural Stem Cells and 4.7T MRI Tracking in vivo and in vitro

Neural stem cells were labeled with superparamagnetic iron oxide （SPIO） and tracked by MRI in vitro and in vivo after implantation, Rat neural stem cells were labeled with SPIO combined with PLL by the means of receptor-mediated endocytosis. Prussian blue staining and electron microscopy were conducted to identify the iron particles in these neural stem cells. SPIO-labeled cells were tracked by 4.7T MRI in vivo and in vitro after implantation, The subjects were divided into 5 groups, including 5× 10^5 labeled cells cultured for one day after labeling, 5 × 10^5 same phase unlabeled cells, cell culture medium with 25μg Fe/mL SPIO, cell culture medium without SPIO and distilled water. MR/scanning sequences included TIWI, T2WI and T2＊WI. R2 and R2＊ of labeled cells were calculated. The results showed： （1） Neural stem cells could be labeled with SPIO and labeling efficiency was 100%. Prussian blue staining showed numerous blue-stained iron particles in the cytoplasm; （2） The average percentage change of signal intensity of labeled cells on TIWI in 4.7T MRI was 24.06%, T2WI 50.66% and T2＊WI 53.70% respectively; （3） T2 of labeled cells and unlabeled cells in 4.7T MRI was 516 ms and 77 ms respectively, R2 was 1.94 s^-1 and 12.98 s^-1 respectively, and T2＊ was 109 ms and 22.9 ms, R2＊ was 9.17 s^-1 and 43.67 s^-1 respectively; （4） Remarkable low signal area on T2WI and T2＊WI could exist for nearly 7 weeks and then disappeared gradually in the left brain transplanted with labeled cells, however no signal change in the right brain implanted with unlabeled cells. It was concluded that neural stem cells could be labeled effectively with SPIO. R2 and R2＊ of labeled cells were increased obviously. MRI can be used to track labeled cells in vitro and in vivo.

Tracking of Labelled Stem Cells Using Molecular MR Imaging in a Mouse Burn Model <i>in Vivo</i>as an Approach to Regenerative Medicine

Therapies based on stem cell transplants offer significant potential in the field of regenerative medicine. Monitoring the fate of the transplanted stem cells in a timely manner is considered one of the main limitations for long-standing success of stem cell transplants. Imaging methods that visualize and track stem cells in vivo non-invasively in real time are helpful towards the development of successful cell transplantation techniques. Novel molecular imaging methods which are non-invasive particularly such as MRI have been of great recent interest. Hence, mouse models which are of clinical relevance have been studied by injecting contrast agents used for labelling cells such as super-paramagnetic iron-oxide (SPIO) nanoparticles for cellular imaging. The MR techniques which can be used to generate positive contrast images have been of much relevance recently for tracking of the labelled cells. Particularly when the off-resonance region in the vicinity of the labeled cells is selectively excited while suppressing the signals from the non-labeled regions by the method of spectral dephasing. Thus, tracking of magnetically labelled cells employing positive contrast in vivo MR imaging methods in a burn mouse model in a non-invasive way has been the scope of this study. The consequences have direct implications for monitoring labeled stem cells at some stage in wound healing. We suggest that our approach can be used in clinical trials in molecular and regenerative medicine.

Artificial light-harvesting systems and their applications in photocatalysis and cell labeling

Photosynthesis is the basis for the survival of organisms in nature;consequently,the fabrication of artificial light-harvesting systems(LHSs)that simulate natural photosynthesis is of significant interest.Recently,a variety of artificial LHSs have been successfully constructed using fluorescence resonance energy transfer(FRET).However,it is crucial to fabricate artificial LHSs with a sequential energy transfer process when considering that the natural photosynthetic process involves a multistep sequential energy transfer process rather than a simple one-step energy transfer.Moreover,many previously reported LHSs have been used as imaging agents for cell labeling and bioimaging or as catalysts in photocatalytic reactions,showing promise for applications simulating natural photosynthesis.In this review,we have summarized recently published representative work on artificial LHSs.In addition,the application of LHSs in photocatalysis and cell labeling has been described in detail.

Experimental Study of Cell Migration and Functional Differentiation of Transplanted Neural Stem Cells Co-labeled with Superparamagnetic Iron Oxide and Brdu in an Ischemic Rat Model

Objective To explore the migration of transplanted neural stem cells co-labeled with superparamagnetic iron oxide （SPIO） and bromodeoxyuridine （Brdu） using the 4.7T MR system and to study the cell differentiation with immuno-histochemical method in ischemic rats. Methods Rat neural stem cells （NSCs） co-labelled with SPIO mediated by poly-L-lysine and bromodeoxyuridine （BrdU） were transplanted into the unaffected side of rat brain with middle cerebral artery occlusion （MCAO）. At weeks 1, 2, 3, 4, 5, and 6 after MCAO, migration of the labelled cells was monitored by MRI. At week 6 the rats were killed and their brain tissue was cut according to the migration site of transplanted ceils indicated by MRI and subjected to Prussian blue staining and immunohistochemical staining to observe the migration and differentiation of the transplanted NSCs. Results Three weeks after transplantation, the linear hypointensity area derived from the migration of labelled NSCs was observed by MRI in the corpus callosum adjacent to the injection site. Six weeks after the transplantation, the linear hypointensity area was moved toward the midline along the corpus callosum. MRI findings were confirmed by Prussian blue staining and immunohistochemical staining of the specimen at week 6 after the transplantation. Flourescence co-labelled immunohistochemical methods demonstrated that the transplanted NSCs could differentiate into astrocytes and neurons. Conclusion MRI can monitor the migration of SPIO-labelled NSCs after transplantation in a dynamical and non-invasive manner. NSCs transplanted into ischemic rats can differentiate into astrocytes and neurons during the process of migration.

FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer

Fluorescein isothiocyanate-labeled insulin(FITC-insulin)has been widely used for bioanalytical applications.Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature,there is still a need to develop a cost effective,reliable and quick labeling method for insulin.The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer.FITC labeling of insulin gives mono-,di-or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin.Mono-conjugate with unlabeled insulin,mixture of di-and tri-conjugate,and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h.Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer.Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient.However,tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.

Growth and differentiation of neural stem cells with superparamagnetic iron oxides labeling in vitro

Objective: To study the growth and differentiation of superparamagnetic iron oxides(SPIOs) labeled neural stem cells(NSCs).Methods: After NSCs were cultured and subcultured from newborn rat brain,they were magnetically labeled with ferumoxides(a kind of SPIOs).Growth,differentiation and other biology properties of the cells were investigated with immunocytochemistry,transmission electron microscopy(TEM) and Prussian blue staining.Results: Nestin positive cells were found in the culture and offspring clones.NSCs could be differentiated into positive GFAP and NF200 cells in serum culture.When NSCs incubated with ferumoxides,the iron particles were seen in intracellular as well as in offspring clones.With the increase in concentration of ferumoxides(5.6-11.2 μg/ml),ferumoxides showed no significant difference effects on the growth and differentiation of NSCs.When the concentration of ferumoxides exceeded 22.4 μg/ml,there was significant difference(P<0.05). Conclusion: We successfully label NSCs with ferumoxides,it is useful for tracking of magnetic labeled NSCs in vivo with MRI.

A RAPID ESTIMATION OF CELL CYCLE PARAMETERS：ONE HOUR COUNTINUOUS BROMODEOXYURIDINE（BrdUrd）LABELING METHOD

ＡＲＡＰＩＤＥＳＴＩＭＡＴＩＯＮＯＦＣＥＬＬＣＹＣＬＥＰＡＲＡＭＥＴＥＲＳ：ＯＮＥＨＯＵＲＣＯＵＮＴＩＮＵＯＵＳＢＲＯＭＯＤＥＯＸＹＵＲＩＤＩＮＥ（ＢｒｄＵｒｄ）ＬＡＢＥＬＩＮＧ ＭＥＴＨＯＤＳｈｉＸｕｅｇｅｎｇ（石学耕）（ＤｅｐａｒｔｍｅｎｔｏｆＢ...

Preparation of liposome-coated oligonucleotide labeled with ^(99m)Tc and its uptake in vascular smooth muscle cells

To explore the preparation method of liposome-coated 99mTc-labeled antisense oligonucleotide (ASON), targeteing the proliferating cell nuclear antigen (PCNA), and to explore the biological characteristics and the uptake kinetics of a radiolabeled probe in vascular smooth muscle cells, an 18-base single-stranded antisense oligonucleotide targeting PCNA mRNA and the complementary strand (sense oligonucleotide, SON) were synthesized. The ASON (SON) was labeled with 99mTc, by conjugating the bifunctional chelator (hydrazino nicotinamide, HYNIC), and puri- fied through a gel filtration column of Sephadex G-25. The product was then encapsulated in cationic liposome (oli- gofectamineTM). The radiolabeling efficiency, radiochemical purity, stability of the liposome-coated 99mTc-HYNIC- ASON in a phosphate buffered solution (PBS), and fresh human serum and its uptake rate were studied. There was no significant difference between the 99mTc radiolabeling efficiencies of HYNIC-ASON and HYNIC-SON, which were 60.04% ± 1.92% and 59.60% ± 2.53%, respectively (P > 0.05, n = 5). The radiochemical purity of the lipo- some-coated 99mTc-HYNIC-ASON was 94.70% ± 1.90% (n = 5). And after incubation with PBS and fresh human se- rum at a concentration of 1.2 μg·mL-1 for 120 min, the radiochemical purities were 92.27% and 91.55% respectively. At 90 min after transfection, the uptake rate of the liposome-coated 99mTc-HYNIC-ASON reached its peak of 83.8% ± 5.92% in vascular smooth muscle cells (VSMCs) and was much higher than that of the nonliposome-coated 99mTc- HYNIC-ASON, which was 11.16% ± 0.54% (P < 0.01, n = 4). The labeling method of PCNA ASON (SON) conju- gated by HYNIC has been proved successful. The liposome was able to enhance the ASON (SON) uptake in VSMCs, and could be widely used as a safe, convenient, effective gene transfer carrier.

基于Label-free技术的非小细胞肺癌蛋白质差异研究

目的 基于非小细胞肺癌(non-small-cell lung cancer,NSCLC)及与其配对的非癌性邻近组织(non-cancerous adjacent tissues,NATs)的蛋白质组学分析,寻找NSCLC诊断相关蛋白标志物。方法 应用非标记定量蛋白组学(LFP)技术,以NSCLC患者配对的NATs为对照,对5例NSCLC患者进行癌组织蛋白质组学分析。以差异倍数>1.5(上调)或<0.67(下调)且P<0.05为标准筛选差异蛋白。应用String网站和R语言对差异蛋白进行基因本体论(gene ontology,GO)分析、京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析,为进一步研究提供良好基础。结果 本研究发现差异蛋白共644个,其中339个蛋白表达上调,305个蛋白表达下调。PCA结果显示,差异蛋白可明显区分NSCLC与NATs。GO分析结果表明,差异蛋白大多以细胞外泌体的形式存在且主要富集于调节胞外分泌、胞外分泌、骨髓细胞激活参与免疫反应等的生物学过程以及钙粘着蛋白绑定、细胞外基质绑定等的分子功能。KEGG分析结果显示,差异蛋白主要富集在抗坏血酸和醛酸代谢、组氨酸代谢、蛋白质消化吸收、丙酮酸代谢等通路(P<0.05)。结论 NSCLC与NATs存在明显差异,可为发现NSCLC新型标志物提供线索。

THE STUDY OF DIFFERENCE OF PCNA LABELLED CELL INDEXES BETWEEN ENDOMETRIA OF HUMAN AND MOUSE

40 human endometrial tissues of benign diseases were diviided into 8 groups (phases),and 50 BDF1 mousy endometrial tissues were divided into 5 groups (phases).Immunohistochenical staining was performed to show the PCNA (proliferating cell nuclear antigen)positive cell .In human ,the results suggested that the PCNA layer(48%)of mid-proliferative hpase.The PCNA labelled index was low in superficial epithelium.But in mouse, the results suggested that the Pcna labelled index was the highest(22%)in endometrium of estrus and the labelled cells were distributed mainly in superficial epithelium (60%).The results suggested that times that times and positions of cellular proliferation in endometrial tissues of human and mouse differed greatly.

Bone marrow mesenchymal stem cell transplantation for treatment of spinal cord injury An in vivo magnetic resonance imaging tracking study

Non-invasive tracing in vivo can be used to observe the migration and distnbution of grafted stem cells, and can provide experimental evidence for treatment. This study utilized adenovirus-carrying enhanced green fluorescent protein （AD5/F35-eGFP） and superparamagnetic iron oxide （SPIO）-Iabeled bone marrow mesenchymal stem cells （BMSCs）. BMSCs, double-labeled by AD5/F35-eGFP and SPIO, were transplanted into rats with spinal cord injury via the subarachnoid space. MRI tracing results demonstrated that BMSCs migrated to the injured spinal cord over time （T2 hypointensity signals）. This result was verified by immunofluorescence. These results indicate that MRI can be utilized to trace in vivo the SPIO-labeled BMSCs after grafting.

Magnetic resonance evaluation of human mesenchymal stem cells in corpus cavernosa of rats and rabbits

Aim： To investigate whether the biological process of superparamagnetic iron oxide （SPIO）-labeled human mesenchymal stem cells （hMSCs） may be monitored non-invasively by using in vivo magnetic resonance （MR） imaging with conventional 1.5-T system examinations in corpus cavernosa of rats and rabbits. Methods： The labeling efficiency and viability of SP10-labeled hMSCs were examined with Prussian blue and Tripan blue, respectively. After SPIO-labeled hMSCs were transplanted to the corpus cavernosa of rats and rabbits, serial T2-weighted MR images were taken and histological examinations were carried out over a 4-week period. Results： hMSCs loaded with SPIO compared to unlabeled cells had a similar viability. For SPIO-labeled hMSCs more than lx 105 concentration in vitro, MR images showed a decrease in signal intensity. MR signal intensity at the areas of SPIO-labeled hMSCs in the rat and rabbit corpus cavernosa decreased and was confined locally. After injection of SPIO-labeled hMSCs into the corpus cavernosum, MR imaging demonstrated that hMSCs could be seen for at least 12 weeks after injection. The presence of iron was confirmed with Prussian blue staining in histological sections. Conclusion： SP10-labeled hMSCs in corpus cavernosa of rats and rabbits can be evaluated non-invasively by molecular MR imaging. Our findings suggest that MR imaging has the ability to test the long-term therapeutic potential of hMSCs in animals in the setting of erectile dysfunction.

Brainbow示踪小鼠心肌细胞增殖中他莫昔芬最适标记剂量的探究

目的:运用Brainbow 2.1小鼠进行心肌细胞谱系追踪,探讨用于心肌细胞增殖标记的最适他莫昔芬剂量。方法:采用4周龄的Brainbowfl/+;Myh6-MerCreMe+小鼠,通过腹腔注射的方式给予3个不同剂量(3、9和27 mg/kg)的他莫昔芬诱导Cre重组酶表达,使心肌细胞标上特定的颜色,4周后取材染色,共聚焦显微镜下定量不同剂量的他莫昔芬标记的心肌细胞数量,以及被标记的细胞中单个细胞、临近的2个细胞簇和临近的3个及以上细胞簇的比例。选择合适剂量(9 mg/kg)的他莫昔芬标记Brainbow小鼠,对比假手术组与心肌梗死(MI)模型组心肌细胞增殖情况。结果:(1)注射9 mg/kg的他莫昔芬后标记的单个细胞比例为99.13%±0.03%,临近的2个细胞簇比例为0.82%±0.09%,3个及以上细胞簇比例为0.05%±0.01%。3 mg/kg组标记总数较少;27 mg/kg组出现明显克隆扩增富集。(2)用9 mg/kg的他莫昔芬标记心肌细胞后,MI小鼠2个细胞簇和3个及以上细胞簇标记比例较假手术组均显著升高(P<0.01),MI后心肌细胞增殖比例增加。结论:9 mg/kg他莫昔芬标记的临近心肌细胞簇的比例更接近生理状态下的心肌细胞增殖比例,因此9 mg/kg为示踪心肌细胞增殖的最适他莫昔芬剂量。

Magnet-targeted delivery of bone marrow-derived mesenchymal stem cells improves therapeutic efficacy following hypoxic-ischemic brain injury

hypoxicischemic brain injury;however,the therapeutic efficacy of bone marrow-derived mesenchymal stem cells largely depends on the number of cells that are successfully transferred to the target.Magnet-targeted drug delivery systems can use a specific magnetic field to attract the drug to the target site,increasing the drug concentration.In this study,we found that the double-labeling using superparamagnetic iron oxide nanoparticle and poly-L-lysine(SPIO-PLL)of bone marrow-derived mesenchymal stem cells had no effect on cell survival but decreased cell proliferation 48 hours after labeling.Rat models of hypoxic-ischemic brain injury were established by ligating the left common carotid artery.One day after modeling,intraventricular and caudal vein injections of 1×105 SPIO-PLL-labeled bone marrow-derived mesenchymal stem cells were performed.Twenty-four hours after the intraventricular injection,magnets were fixed to the left side of the rats’heads for 2 hours.Intravoxel incoherent motion magnetic resonance imaging revealed that the perfusion fraction and the diffusion coefficient of rat brain tissue were significantly increased in rats treated with SPIO-PLL-labeled cells through intraventricular injection combined with magnetic guidance,compared with those treated with SPIO-PLL-labeled cells through intraventricular or tail vein injections without magnetic guidance.Hematoxylin-eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining revealed that in rats treated with SPIO-PLL-labeled cells through intraventricular injection under magnetic guidance,cerebral edema was alleviated,and apoptosis was decreased.These findings suggest that targeted magnetic guidance can be used to improve the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for hypoxic-ischemic brain injury.This study was approved by the Animal Care and Use Committee of The Second Hospital of Dalian Medical University,China(approval No.2016-060)on March 2,2016.

邻近标记技术在膜蛋白相互作用中的研究进展

膜蛋白是细胞多种生命活动的主要承担者,膜蛋白之间的相互作用参与调控细胞间的接触与信号传递,并与细胞的分化、成熟息息相关。因此,开发多种深入解析膜蛋白之间相互作用的方法具有重要的理论与现实意义。除了传统的免疫共沉淀方法之外,新开发的邻近标记技术已经逐渐成为了研究膜蛋白相互作用的重要手段。邻近标记技术主要利用生物工具酶标记与诱饵蛋白相互作用的邻近蛋白,并通过流式细胞术、质谱以及共聚焦显微成像技术等方法检测膜蛋白之间的相互作用。本文着重介绍了近些年来新开发的用于研究膜蛋白相互作用的多种邻近标记技术,并展望了该类技术在膜蛋白相互作用领域未来的发展前景。

Magnetic resonance imaging and cell-based neurorestorative therapy after brain injury

Restorative cell-based therapies for experimental brain injury, such as stroke and traumatic brain injury,substantially improve functional outcome. We discuss and review state of the art magnetic resonance imaging methodologies and their applications related to cell-based treatment after brain injury. We focus on the potential of magnetic resonance imaging technique and its associated challenges to obtain useful new information related to cell migration, distribution, and quantitation, as well as vascular and neuronal remodeling in response to cell-based therapy after brain injury. The noninvasive nature of imaging might more readily help with translation of cell-based therapy from the laboratory to the clinic.

酶介导的邻近细胞标记方法探究细胞间相互作用

细胞-细胞相互作用(cell-cell interactions,CCIs)可以通过细胞表面蛋白质、聚糖、脂质等介导的细胞间突触形成而发生,以维持机体稳态和调控生理功能。这些CCIs是复杂的,涉及许多不同的细胞表面和细胞内分子的参与。随着基础研究和转化医学的不断发展,如何准确识别细胞间相互作用并对其进行表征和定量,引起了广泛关注。近年来,研究CCIs的技术手段不断推陈出新,而邻近标记是一种十分有前景的研究细胞间相互作用的化学生物学方法。目前,主要有两种类型的标记策略。一种是依赖基因工程操作,在“诱饵”细胞表面表达外源酶,通过其与相邻细胞上的受体底物的直接结合,以便发生细胞间邻近标记。另一种是使用酶或小分子催化剂(例如光催化剂),通过基因工程重组表达或借助化学(化学酶)方法将它们偶联在“诱饵”细胞表面,在适当的刺激或激活后,靶向递送感兴趣的标记分子,实现邻近标记。其中,酶介导的邻近细胞标记方法在检测和表征CCIs上具有重要的应用价值。该综述将标记过程中涉及有酶参与的方法定义为酶介导的邻近细胞标记方法,这种方法的一个明显优势是,由于酶和受体底物之间直接的物理接触或酶催化产生高反应活性标记分子而实现小的标记半径范围。该综述旨在归纳与总结近年来开发的酶介导的邻近细胞标记方法的原理、优缺点及现有应用。