Effect of transplantation route on stem cell migration to fibrotic liver of rats via cellular magnetic resonance imaging

Background aimsAssessing mesenchymal stromal cells (MSCs) after grafting is essential for understanding their migration and differentiation processes. The present study sought to evaluate via cellular magnetic resonance imaging (MRI) if transplantation route may have an effect on MSCs engrafting to fibrotic liver of rats.MethodsRat MSCs were prepared, labeled with superparamagnetic iron oxide and scanned with MRI. Labeled MSCs were transplanted via the portal vein or vena caudalis to rats with hepatic fibrosis. MRI was performed in vitro before and after transplantation. Histologic examination was performed. MRI scan and imaging parameter optimization in vitro and migration under in vivo conditions were demonstrated.ResultsStrong MRI susceptibility effects could be found on gradient echo-weighted, or T21-weighted, imaging sequences from 24 h after labeling to passage 4 of labeled MSCs in vitro. In vivo, MRI findings of the portal vein group indicated lower signal in liver on single shot fast spin echo-weighted, or T2-weighted, imaging and T21-weighted imaging sequences. The low liver MRI signal increased gradually from 0–3 h and decreased gradually from 3 h to 14 days post-transplantation. The distribution pattern of labeled MSCs in liver histologic sections was identical to that of MRI signal. It was difficult to find MSCs in tissues near the portal area on day 14 after transplantation; labeled MSCs appeared in fibrous tuberculum at the edge of the liver. No MRI signal change and a positive histologic examination were observed in the vena caudalis group.ConclusionsThe portal vein route seemed to be more beneficial than the vena caudalis on MSC migration to fibrotic liver of rats via MRI.     

三种不同转染剂介导钆喷酸葡胺标记人脐带间充质干细胞及体外MR成像的实验研究

人脐带组织富含间充质干细胞（human umbilical cord mesenchymal stem cells,hUCMSCs）,是干细胞研究理想的种子来源,如何从脐带组织中分离间充质干细胞及运用影像技术示踪干细胞生物学行为是当前研究的热点。该实验应用组织块贴壁法从足月孕妇脐带组织中分离纯化间充质干细胞并进行鉴定,结果为hUCMSCs。进一步应用磷酸钙、Effectene、脂质体2000三种转染试剂分别介导Gd-DTPA标记hUCMSCs,通过MRI（magnetic resonance imaging）检测钆喷酸葡胺（Gd-DTPA）标记细胞信号强度变化及细胞内钆离子浓度的测定评价三种转染试剂的转染能力,最终发现在三种转染试剂中,Effectene介导Gd-DTPA标记hUCMSCs效果最佳,为Gd-DTPA标记干细胞体外MR成像奠定了实验基础。     

In vitro labeling of human umbilical cord mesenchymal stem cells with superparamagnetic iron oxide nanoparticles

Human umbilical cord mesenchymal stem cells (hUC‐MSCs) transplantation has been shown to promote regeneration and neuroprotection in central nervous system (CNS) injuries and neurodegenerative diseases. To develop this approach into a clinical setting it is important to be able to follow the fates of transplanted cells by noninvasive imaging. Neural precursor cells and hematopoietic stem cells can be efficiently labeled by superparamagnetic iron oxide (SPIO) nanoparticle. The purpose of our study was to prospectively evaluate the influence of SPIO on hUC‐MSCs and the feasibility of tracking for hUC‐MSCs by noninvasive imaging. In vitro studies demonstrated that magnetic resonance imaging (MRI) can efficiently detect low numbers of SPIO‐labeled hUC‐MSCs and that the intensity of the signal was proportional to the number of labeled cells. After transplantation into focal areas in adult rat spinal cord transplanted SPIO‐labeled hUC‐MSCs produced a hypointense signal using T2‐weighted MRI in rats that persisted for up to 2 weeks. This study demonstrated the feasibility of noninvasive imaging of transplanted hUC‐MSCs. J. Cell. Biochem. 108: 529–535, 2009. © 2009 Wiley‐Liss, Inc.     

Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation

Background aims

Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined “cocktail” of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes.

兔大腿VX2移植瘤模型磁共振扩散成像研究

采用大腿肌肉注射法建立兔VX2肿瘤模型,于造模后第7天、14天及21天进行磁共振成像(MRI)平扫、增强及扩散加权成像(DWI)检查,观察不同时期MRI表现。并于造模后第21天对照大体标本测量结果比较DWI及T2WI图像肿瘤最大径,同时比较肿瘤实体部分与髂窝内转移淋巴结的表观弥散系数(ADC)值。结果显示,造模后第7天,12只模型兔MRI常规及DWI图像均可见成瘤;第14天,2例肿瘤内出现坏死灶;第21天,12例肿瘤内均出现坏死,DWI图像可发现局部淋巴结转移灶,DWI及T2WI图像肿瘤最大径与大体标本测量结果差别无统计学意义。髂窝内转移淋巴结的ADC值与原发肿瘤实体部分ADC值间差别无统计学意义。DWI可以监测兔大腿VX2肿瘤生长,有效区分肿瘤早期坏死成分,并判断相应引流区域淋巴结的性质。     

Encapsulated mesenchymal stromal cells for in vivo transplantation

Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in vitro and in vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 h post-injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post-SCI indicated that as few as 5 × 10(4) encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.     

Application of cell penetrating peptide in magnetic resonance imaging of bone marrow mesenchymal stem cells

Tracking the distribution and differentiation of stem cells by high-resolution imaging techniqueswould have significant clinical and research implications.In this study,a model cell-penetrating peptide wasused to carry gadolinium particles for magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs).MSCs were isolated from rat bone marrow and identified by osteogenic differentiation in vitro.The cell-penetrating peptide labeled with fluorescein-5-isothiocyanate (FITC) and gadolinium was synthesized by asolid-phase peptide synthesis method.Fluorescein imaging analysis confirmed that this new peptide couldinternalize into the cytoplasm and nucleus at room temperature,4℃ and 37℃.Gadolinium were efficientlyinternalized into mesenchymal stem cells by the peptide in a time or concentration-dependent manner,resulting in intercellular shortening of longitudinal relaxation enhancements,which were obviously detectedby 1.5 Tesla Magnetic Resonance Imaging.Cytotoxicity assay and flow cytometric analysis showed thatthe intercellular contrast medium incorporation did not affect cell viability at the tested concentrations.Thein vitro experiment results suggested that the new constructed peptides could be a vector for trackingMSCs.     

Platelet-rich plasma-derived scaffolds increase the benefit of delayed mesenchymal stromal cell therapy after severe traumatic brain injury

Background

Cell therapy using mesenchymal stromal cells (MSCs) offers new perspectives in the treatment of traumatic brain injury (TBI). The aim of the present study was to assess the impact of platelet-rich plasma scaffolds (PRPS) as support of MSCs in a delayed phase after severe TBI in rats.

携带CD基因骨髓间充质干细胞对C6大鼠胶质瘤体内抑制作用研究

为了研究自杀基因——胞嘧啶脱氨酶基因(cytosine deaminase gene,CD基因)对大鼠脑胶质瘤的体内治疗效果,采用基因转导系统将慢病毒包装的CD基因转染骨髓间充质干细胞(MSC)并使其长时间、高效表达,然后移植到使用颅内立体定向接种法制作的40只SD大鼠胶质瘤模型中．按接种细胞类型将实验SD大鼠分为5组, 每组8只：① C6胶质瘤; ②C6+MSCs细胞(1∶1);③ C6+MSCs细胞(1∶2);④ C6+MSC-codA/eGFP细胞(1∶1);⑤ C6+MSC-codA/eGFP细胞(1∶2)．瘤龄7天后腹腔注射500 mg/(kg·d) 5-氟胞嘧啶(5-flucytosine,5-FC),共14天．用磁共振成像(MRI)动态监测肿瘤的体积并进行了大鼠存活期观察、常规病理分析、RT-PCR检测、HE 染色．结果显示,第①组瘤龄14天时病灶呈圆形,中心见坏死区,肿瘤平均246 mm³,均存活期15.3天,第②组平均生存期16.0天,第③组平均生存期16.6天,第④⑤组自然存活期均大于30天,14天时病灶平均体积分别为55 mm³、40 mm³,28天时肿瘤抑制率分别为77.24%、83.28%．MRI扫描可清楚显示肿瘤的大小、形态和内部结构,与病理结果高度相关;MRI动态观察可证实携带CD基因的骨髓间充质干细胞及5-FC治疗系统治疗C6颅内胶质瘤有效;RT-PCR检测结果证实肿瘤组织内有胞嘧啶脱氨酶表达．     

Tolerogenic effect of mesenchymal stromal cells on gliadin-specific T lymphocytes in celiac disease

Background aimsCeliac disease is caused by a dysregulated immune response toward dietary gluten, whose only treatment is a lifelong gluten-free diet. We investigated the effects of mesenchymal stromal cells (MSCs) on gliadin-specific T cells, which are known to induce intestinal lesions, in view of a possible use as new therapy.MethodsBone marrow–derived MSCs and gliadin-specific T-cell lines were obtained from allogeneic donors and mucosal specimens of celiac patients, respectively. The immunosuppressant effect of MSCs was evaluated in terms of proliferative response and interferon (IFN)-γ production upon gliadin stimulation of long-term T-cell lines; the immunomodulant effect was assessed in terms of apoptotic rate, immunophenotype and cytokine profile of short-term T-cell lines generated in the presence of MSCs. Different MSC:T-cell ratios were applied, and statistics were performed as appropriate.ResultsMSCs inhibited both proliferative response and IFN-γ production of long-term T-cell lines in a dose-dependent manner while limiting the expansion of short-term T-cell lines by increasing the apoptotic rate. Moreover, a reduction of the CD4⁺ population and expansion of the regulatory FoxP3⁺ subset were found in T-cell lines cultured with MSCs, in which a significant decrease of interleukin (IL)-21, IFN-γ and IL-10 paralleled by an upregulation of transforming growth factor-β1, IL-6 and IL-8 were observed. Finally, an increase of the indoleamine 2,3-dioxygenase activity was found, possibly playing a key role in mediating these effects.ConclusionsMSCs exert potent immunomodulant effects on gliadin-specific T cells, which may be exploited for future therapeutic application in celiac disease.     

脑网络：从脑结构到脑功能

人脑是自然界中最复杂的系统之一,不同的功能区域相互作用、互相协调,共同构成一个网络来发挥其功能。人脑是一个复杂的网络,具有高效的“小世界”拓扑属性。本文从脑结构到脑功能方面介绍了从不同模态影像学数据构造脑网络的主要进展,并探讨不同的脑疾病患者脑网络拓扑结构是否发生了异常,以及这些异常特征能否用来进行疾病分类,最后对本领域未来的研究做了简单的展望。     

间充质干细胞的细胞成像技术比较与应用

间充质干细胞是一类具有强大增殖、多向分化潜能和免疫调节能力的多功能细胞,研究显示间充质干细胞移植可能治疗多种难治性疾病,例如帕金森病、脊髓损伤以及肿瘤等。但是,人们对移植后的细胞在宿主内的存活、分布、增殖、分化、免疫排斥反应以及成瘤特性等问题尚不清楚,所以许多疾病经过细胞移植治疗后的进展及转归情况仍难以获得确切的科学证据。而细胞成像技术(包括放射性核素成像、超声成像、磁共振成像以及光学成像)可以在体外或者体内实现对间充质干细胞实时、无创的示踪,在以间充质干细胞为研究基础的细胞移植治疗和细胞组织再生的医学领域里有着巨大的应用潜力。该文综述近十年来细胞成像技术应用于示踪间充质干细胞移植疗法的研究进展,旨在比较当下多种热门细胞成像技术的优劣,进而找寻更合适的干细胞示踪策略,为干细胞移植治疗的基础和临床研究提供进一步的理论证据支持和研究思路。     

Physiological studies of cortical spreading depression

Cortical spreading depression (CSD) produces propagating waves of transient neuronal hyperexcitability followed by depression. CSD is initiated by K+ release following neuronal firing or electrical, mechanical or chemical stimuli. A triphasic (30-50 s) cortical potential transient accompanies localized transmembrane redistributions of K+, glutamate, Ca2+, Na+, Cl- and H+. Accumulated K+ in the restricted interstitial space can cause both further neuronal depolarisation and inward movement of K+ into astrocytes that buffers this increased extracellular K+ concentration ([K+])o. However, astrocyte interconnections may then propagate the CSD wave by K+ liberation through an opening of remote K+ channels by volume, Ca2+ or N-methyl-D-aspartate receptor activation. Changes in cerebral blood volume and in apparent water diffusion co-efficient (ADC) accompanying CSD were first studied using magnetic resonance imaging (MRI) in whole lissencephalic brains. Diffusion-weighted echoplanar imaging in gyrencephalic brains went on to demonstrate CSD features that paralleled classical migraine aura. The ADC activity persisted minutes/hours post KCl stimulus. Pixelwise analyses distinguished single primary events and multiple, spatially restricted, slower propagating, secondary events whose detailed features varied with the nature of the originating stimulus. These ADC changes varied reciprocally with T2*-weighted (i.e. referring to spin-spin relaxation times) waveforms reflecting local blood flow. There followed prolonged decreases in cerebral blood flow culminating in late cerebrovascular changes blocked by the antimigraine agent sumatriptan. CSD phenomena have possible translational significance for human migraine aura and other cerebral pathologies such as the peri-infarct depolarisation events that follow ischaemia and brain injury.     

Neurotrophic and neuroprotective potential of human limbus-derived mesenchymal stromal cells

Background aimsThe purpose of this study was to examine neurotrophic and neuroprotective effects of limbus stroma-derived mesenchymal stromal cells (L-MSCs) on cortical neurons in vitro and in vivo.MethodsCultured L-MSCs were characterized by flow cytometry and immunofluorescence through the use of specific MSC marker antibodies. Conditioned media were collected from normoxia- and hypoxia-treated L-MSCs to assess neurotrophic effects. Neuroprotective potentials were evaluated through the use of in vitro hypoxic cortical neuron culture and in vivo rat focal cerebral ischemia models. Neuronal morphology was confirmed by immunofluorescence with the use of anti-MAP2 antibody. Post-ischemic infarct volume and motor behavior were assayed by means of triphenyltetrazolium chloride staining and open-field testing, respectively. Human growth antibody arrays and enzyme-linked immunoassays were used to analyze trophic/growth factors contained in conditioned media.ResultsIsolated human L-MSCs highly expressed CD29, CD90 and CD105 but not CD34 and CD45. Mesenchymal lineage cell surface expression pattern and differentiation capacity were identical to MSCs derived form human bone marrow and adipose tissue. The L-MSC normoxic and hypoxic conditioned media both promoted neurite outgrowth in cultured cortical neurons. Hypoxic conditioned medium showed superior neurotrophic function and neuroprotective potential with reduced ischemic brain injury and improved functional recovery in rat focal cerebral ischemia models. Human growth factor arrays and enzyme-linked immunoassays measurements showed neuroprotective and growth-associated cytokines (vascular endothelial growth factor [VEGF], VEGFR3, brain-derived neurotrophic factor, insulin-like growth factor -2 and hepatocyte growth factor) contained in conditioned media. Hypoxic exposure caused VEGF and brain-derived neurotrophic factor upregulation, possibly contributing to neurotrophic and neuroprotective effects.ConclusionsL-MSCs can secrete various neurotrophic factors stimulating neurite outgrowth and protecting neurons against brain ischemic injury through paracrine mechanism.     

Application of F magnetic resonance to study the efficacy of fluorine labeled drugs in the three-dimensional cultured breast cancer cells

The cellular monitoring of tumor response to treatments is important for drug discovery and drug development in cancer therapy. We studied efficacy of Herceptin, a common breast cancer drug conjugated with a fluorine organic compound, perfluoro-15-crown-5-ether (PFCE) which easily forms biocompatible emulsions. Three new pharmaceutical forms of Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink were synthesized for the ex vivo study of their efficacy in breast cancer treatment. The emulsions were administered to 10⁹ cells mL⁻¹ of HER-2 positive human adenocarcinoma (MCF-7) cells and the same amount of human mammary epithelial cells (HMEC) cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. Following drugs administration ex vivo, fluorine-19 magnetic resonance imaging (¹⁹F MRI) was applied for cells imaging to measure their viability and to study drug efficacy over 72 h. To ensure optimum drug tracking, HydraLink was used to provide stable binding affinity of emulsified Herceptin to receptor while cationic lipid (Lipofectamine) was used to enhance lipophilicity of the emulsions.After 72 h of treatment with Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink the viability of cells was 54 ± 2%, 49 ± 3%, 43 ± 5% and 42 ± 1%, respectively, as compared with control 93 ± 2%. The efficacy (EC₅₀) of Herceptin conjugated with emulsions was found to be 970 ± 13 μg mL⁻¹ for Herceptin/PFCE, 645 ± 11 μg mL⁻¹ for Herceptin/PFCE/Lipoplex, 678 ± 7 μg mL⁻¹ for Herceptin/PFCE/HydraLink and 1000 ± 3 μg mL⁻¹ for Herceptin. The results show that fluorine emulsions improved the efficacy of Herceptin and ¹⁹F signal intensity changes validated drug efficiency. The significant correlations between duration of treatments and MCF-7 cells viability were observed. While we studied breast cancer cells, the fluorine emulsions could be applied for treatment of other cancer cells overexpressing HER-2.     

Adipose-derived mesenchymal stromal cell transplantation at the graft site improves the structure and function of autografted mice ovaries: a stereological and biochemical analysis

Background

Ovarian tissue autografting is a fertility restoration technique that is frequently used in young women with cancer who undergo radio/chemotherapy. A limiting factor in this technique is ischemia-reperfusion (I/R) damage. Because adipose-derived mesenchymal stromal cells (ADMSCs) protect different ischemic tissues against I/R damage, we examined the effect of ADMSC transplantation at the graft site in mice ovary autografting.