MRI活体示踪超顺磁性氧化铁标记兔骨髓间充质干细胞的自体皮下移植

背景：高磁场MRI已被成功应用于示踪移植超顺磁性氧化铁标记骨髓基质干细胞的研究,但目前还有应用低磁场MRI示踪移植干细胞的报道。 目的：探讨用0.2 T MRI活体示踪自体皮下移植磁化标记骨髓基质干细胞的分布和迁移的可行性。 方法：从兔骨髓中分离培养骨髓基质干细胞,采用超顺磁性氧化铁和BrdU双重标记后,与壳聚糖复合植入兔自体大腿皮下。自体皮下移植未标记骨髓基质干细胞和皮下单纯注射超顺磁性氧化铁为对照。 结果与结论：超顺磁性氧化铁标记骨髓基质干细胞经普鲁士蓝染色和电镜检查证实细胞胞浆含致密铁颗粒。超顺磁性氧化铁标记后自体皮下移植的兔骨髓基质干细胞在GRET2*WI序列成像时产生特征性的低信号改变至少维持8周,且信号逐渐从移植部位进入组织深处。但普鲁士蓝染色和BrdU免疫组化显示大部分的移植细胞仍停留在原移植部位。提示体外超顺磁性氧化铁能有效地标记骨髓基质干细胞,利用0.2 T MRI活体示踪自体皮下移植的超顺磁性氧化铁标记兔骨髓基质干细胞分布和迁移是可行的。     

Noninvasive tracking of cardiac embryonic stem cells in vivo using magnetic resonance imaging techniques

Despite rapid advances in the stem cell field, the ability to identify and track transplanted or migrating stem cells in vivo is limited. To overcome this limitation, we used magnetic resonance imaging (MRI) to detect and follow transplanted stem cells over a period of 28 days in mice using an established myocardial infarction model. Pluripotent mouse embryonic stem (mES) cells were expanded and induced to differentiate into beating cardiomyocytes in vitro. The cardiac-differentiated mES cells were then loaded with superparamagnetic fluorescent microspheres (1.63 microm in diameter) and transplanted into ischemic myocardium immediately following ligation and subsequent reperfusion of the left anterior descending coronary artery. To identify the transplanted stem cells in vivo, MRI was performed using a Varian Inova 4.7 Tesla scanner. Our results show that (a) the cardiac-differentiated mES were effectively loaded with superparamagnetic microspheres in vitro, (b) the microsphere-loaded mES cells continued to beat in culture prior to transplantation, (c) the transplanted mES cells were readily detected in the heart in vivo using noninvasive MRI techniques, (d) the transplanted stem cells were detected in ischemic myocardium for the entire 28-day duration of the study as confirmed by MRI and post-mortem histological analyses, and (e) concurrent functional MRI indicated typical loss of cardiac function, although significant amelioration of remodeling was noted after 28 days in hearts that received transplanted stem cells. These results demonstrate that it is feasible to simultaneously track transplanted stem cells and monitor cardiac function in vivo over an extended period using noninvasive MRI techniques.     

Characterization and in vivo testing of mesenchymal stem cells derived from human embryonic stem cells

Mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of tissues through homing to injured areas, especially to hypoxic, apoptotic, or inflamed areas and releasing factors that hasten endogenous repair. In some cases genetic engineering of the MSC is desired, since they are excellent delivery vehicles. We have derived MSCs from the human embryonic stem cell (hESC) line H9 (H9-MSCs). They expressed CD105, CD90, CD73, and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR, the hESC pluripotency markers SSEA-4 and Tra-1-81, and the hESC early differentiation marker SSEA-1. Marrow-derived MSCs showed a similar phenotype. H9-MSCs did not form teratoma in our initial studies, whereas the parent H9 line did so robustly. H9-MSCs differentiated into bone, cartilage, and adipocytes in vitro, and displayed increased migration under hypoxic conditions. Finally, using a hindlimb ischemia model, H9-MSCs were shown to home to the hypoxic muscle, but not the contralateral limb, by 48?h after IV injection. In summary, we have defined methods for differentiation of hESCs into MSCs and have defined their characteristics and in vivo migratory properties.     

In vivo magnetic resonance imaging of cell tropism, trafficking mechanism, and therapeutic impact of human mesenchymal stem cells in a murine glioma model

Stem cells have offered much promise as delivery vehicles for brain tumor therapy, with the development of modalities to track the tumor tropism of stem cells receiving intense focus. Cellular magnetic resonance imaging (MRI) allows serial high-resolution in vivo detection of transplanted stem cells' tropism toward gliomas in the mouse brain once these cells are internally labeled with iron oxide particles, but has been impeded by low labeling efficiencies. In this study, we describe the use of ferucarbotran and protamine (Fer-Pro) complexes for labeling human mesenchymal stem cells (hMSCs) for MRI tracking of glioma tropism in vivo. We found that Fer-Pro was not toxic and was highly efficient for labeling in vitro. Cell labeling with Fer-Pro promoted the migration of hMSCs toward glioma U87MG cells in vitro, which was mediated by stromal-derived factor-1/CXCR4 (SDF-1/CXCR4) signaling. Fer-Pro-labeled hMSCs could migrate specifically toward gliomas in vivo, which was observed with a clinical 1.5-T MRI system. The efficient labeling of Fer-Pro also allowed a tropic mechanism mediated by SDF-1/CXCR4 signaling to be detected by MRI in vivo. Additionally, the potential intrinsic inhibitory effect of hMSCs on glioma progression was estimated simultaneously. This is the first report to have used a clinical MRI modality to simultaneously study the migration, the therapeutic impact on tumors, and above all the trafficking mechanism of bone marrow-derived mesenchymal stem cells from human in a murine glioma xenograft model. The use of Fer-Pro for stem cell labeling may have potential clinical applications in stem cell guided therapy.     

鼠胚神经干细胞移植对帕金森模型大鼠的治疗作用

背景：干细胞移植是治疗帕金森的有潜力的方法之一。 目的：观察神经干细胞纹状体移植对帕金森模型大鼠旋转行为及脑内多巴胺含量的影响。 方法：采用6-羟基多巴胺定点注射毁损黑质纹状体的方法构建帕金森大鼠模型;向造模成功的大鼠纹状体内分别移植1×106(共计20 μL)的第3代胚鼠神经干细胞或等量生理盐水。 结果与结论：神经干细胞移植后,帕金森大鼠的旋转行为明显改善。干细胞移植后3周,免疫组化检测发现移植干细胞的帕金森大鼠脑黑质部位酪氨酸羟化酶阳性细胞数增多,纹状体内可见酪氨酸羟化酶阳性细胞;荧光显微镜下观察发现Hoechst 33324d标记神经干细胞在移植针道附近最为密集,并向远隔部位迁徙。干细胞移植后8周,高效液相色谱检测显示移植干细胞的帕金森大鼠纹状体内多巴胺含量明显增高(P < 0.01)。说明神经干细胞脑内移植能够减轻6-羟基多巴胺引起的大鼠中脑黑质多巴胺能神经元的损伤,改善大鼠的旋转行为。     

大鼠骨髓间充质干细胞的PKH26标记和示踪☆

背景：骨髓间充质干细胞标记是体内迁移分化研究的重要环节。 目的：对大鼠骨髓间充质干细胞进行PKH26标记,探讨PKH26标记对骨髓间充质干细胞的生长特征、分化的影响及体内示踪情况。 方法：培养扩增大鼠骨髓间充质干细胞,第2代细胞按PKH26标记程序进行细胞标记,观察标记组和未标记组细胞的增殖、周期和凋亡情况,对标记组细胞行成骨成脂体外诱导分化。尾静脉移植PKH26标记细胞,6周后荧光显微镜观察骨髓间充质干细胞在子宫内膜的分布情况。 结果与结论：PKH26标记对细胞增殖、凋亡和周期无明显影响,不影响成骨成脂诱导分化。子宫内膜组织中PKH26标记阳性的细胞分布于腺上皮和间质细胞。PKH26标记技术可用于示踪骨髓间充质迁移转归和干细胞移植方面的实验研究。      

人骨髓胎儿间充质干细胞在小鼠体内的分化

目的：探讨人骨髓胎儿间充质干细胞（ hfMSC）的特性及其在X染色体- 连锁肌萎缩（ mdx）小鼠体内分 化的可能性。 方法：从胎儿骨髓中分离扩增hfMSC,检测hfMSC Oct-4 和Nanog-3 的表达。经DIR 标记后注射 hfMSC 到mdx小鼠腹股沟三角皮下,采用活体成像法观察小鼠离体后肢肌肉植入细胞的存活状态,观察是否出 现畸胎瘤,并采用免疫荧光染色法检测dystrophin 的表达。结果：从胎儿骨髓中分离到表达Oct-4 和Nanog-3 的 hfMSC。在注射hfMSC 的mdx小鼠离体后肢肌肉中检测到明显的荧光信号,并在肌肉组织中检测到人dystrophin 的 表达,而没有发现畸胎瘤的出现。结论： hfMSC 表达多潜能抗原标志Oct-4 和Nanog-3,hfMSC 在mdx小鼠体内可 以产生dystrophin,且不会形成畸胎瘤。     

大鼠骨髓间充质干细胞体内分化为心肌细胞的相关基因

目的研究大鼠骨髓间充质干细胞(MSCs)在体内诱导分化为心肌细胞的过程中相关调控基因的时序表达,揭示MSCs体内分化为心肌细胞的分子调控机制。方法从大鼠骨髓中分离MSCs并传至第8代,用DAPI标记后注射到正常大鼠心肌组织内,分别于细胞移植后1 d、1、2、3、4、6和8周处死大鼠,在注射点获取心肌标本;采用HE染色观察MSCs植入后的形态变化,荧光免疫组化检测心肌特异性蛋白MHC和connexin43的表达;半定量RT-PCR检测TGF-β、Nkx-2.5、GATA-4、MEF-2C、TEF-1和RARα等相关调控基因的动态时序表达。结果MSCs注入正常心肌组织后可逐渐向心肌细胞转化,从小圆形未分化细胞逐渐转变为与周围宿主组织连接的心肌样细胞;移植1周后出现MHC的表达,移植4周后宿主心肌细胞与移植细胞间出现connexin43的表达,Nkx-2.5和GATA-4基因诱导后1 d表达开始增强,1周时达高峰,以后维持在高水平,TGF-β、MEF-2C、TEF-1和RARα基因mRNA在诱导前后无明显变化。结论Nkx-2.5和GATA-4基因在MSCs体内转化为心肌细胞的过程中可能发挥重要作用。     

超顺磁性氧化铁纳米颗粒体外标记兔骨髓间充质干细胞的安全性以及MRI成像特征*☆

背景：传统的干细胞标记方法常需要病理组织学等侵袭性手段检测,无法动态观察整个实验过程,也不适合临床研究。 目的：观察超顺磁性氧化铁纳米颗粒体外标记对兔骨髓间充质干细胞生物学特性的影响以及标记兔骨髓间充质干细胞的体外MRI成像特征。 方法：采用红细胞裂解贴壁法培养扩增兔骨髓间充质干细胞。采用不同浓度超顺磁性氧化铁纳米颗粒(100,50,25,12.5 mg/L)联合多聚赖氨酸(0.75 mg/L)标记兔骨髓间充质干细胞。对标记兔骨髓间充质干细胞进行MRI检测,观察其成像特征。 结果与结论：25 mg/L的超顺磁性氧化铁纳米颗粒联合0.75 mg/L多聚赖氨酸标记兔骨髓间充质干细胞具有较好的安全性和有效性。此浓度对细胞的生长活性没有影响,不影响骨髓间充质干细胞的成骨成脂分化能力。MRI扫描在T2*WI序列上能明显有效地显像超顺磁性氧化铁纳米颗粒标记的兔骨髓间充质干细胞。 关键词：超顺磁性氧化铁纳米颗粒;骨髓间充质干细胞;标记;磁共振;安全性 doi:10.3969/j.issn.1673-8225.2012.06.001     

Migration of neurotrophic factors-secreting mesenchymal stem cells toward a quinolinic acid lesion as viewed by magnetic resonance imaging

Stem cell-based treatment is a promising frontier for neurodegenerative diseases. We propose a novel protocol for inducing the differentiation of rat mesenchymal stem cells (MSCs) toward neurotrophic factor (NTF)-secreting cells as a possible neuroprotective agent. One of the major caveats of stem cell transplantation is their fate post-transplantation. To test the viability of the cells, we tracked the transplanted cells in vivo by magnetic resonance imaging (MRI) scans and validated the results by histology. MSCs went through a two-step medium-based differentiation protocol, followed by in vitro characterization using immunocytochemistry and immunoblotting analysis of the cell media. We examined the migratory properties of the cells in the quinolinic acid (QA)-induced striatal lesion model for Huntington's disease. The induced cells were labeled and transplanted posterior to the lesion. Rats underwent serial MRI scans to detect cell migration in vivo. On the 19th day, animals were sacrificed, and their brains were removed for immunostaining. Rat MSCs postinduction exhibited both neuronal and astrocyte markers, as well as production and secretion of NTFs. High-resolution two-dimensional and three-dimensional magnetic resonance images revealed that the cells migrated along a distinct route toward the lesion. The in vivo MRI results were validated by the histological study, which demonstrated that phagocytosis had only partially occurred and that MRI could correctly depict the status of the migrating cells. The results show that these cells migrated toward a QA lesion and therefore survived for 19 days post-transplantation. This gives hope for future research harnessing these cells for treating neurodegenerative diseases. Disclosure of potential conflicts of interest is found at the end of this article.     

The derivation of mesenchymal stem cells from human embryonic stem cells

Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a potentially unlimited source of cells capable of undergoing differentiation into specified cell types. Several preclinical studies and a few clinical studies use human bone marrow stromal cells (hBMSCs) to treat skeletal diseases and repair damaged tissue. However, hBMSCs have limited proliferation and differentiation capacity, suggesting that an alternate cell source is desirable, and hESCs may serve this purpose. Here we describe a protocol for the reproducible derivation of mesenchymal stem cells from hESCs (hES-MSCs). The hES-MSCs have a similar immunophenotype to hBMSCs, specifically they are CD73+, STRO-1+ and CD45-, and are karyotypically stable. The derived hES-MSCs are also capable of differentiating into osteoblasts and adipocytes. When the hES-MSCs were genetically modified with the lineage-specific Col2.3-GFP lentivirus and cultured in osteogenic medium, increased GFP expression was detected over time, indicating the hES-MSCs have the capacity to differentiate down the osteogenic lineage and had progressed toward a mature osteoblast phenotype.     

液压转基因技术应用于大鼠再生肝转基因实验

目的 探讨液压转基因技术(HDT)应用于大鼠再生肝转基因的条件和方法 . 方法 以2ml/s的速度将浓度为30mg/L的含目的 基因的质粒注射入大鼠尾静脉,于注射前/后不同时间进行大鼠2/3肝切除(PH),于PH后不同恢复时间称量大鼠体重(g)和再生肝重(g),计算肝系数(Lc),并从Lc±Lc*0%、*5%、*10%、*15%、*20%、*25%、*30%、*35%等15组中找出最佳组,作为计算不同恢复时间再生肝最适注射质粒溶液量的校正系数(Trc);取大鼠肝右叶中部组织制备冷冻切片,在波长488nm的荧光显微镜下观察、计数1万个细胞中的绿色荧光蛋白阳性细胞百分率. 结果 PH后注射生理盐水和注射空质粒对肝再生的影响与对照(只进行PH)相比无显著差异.PH前液压转基因的合适时间是PH前≥12h;PH后所有时间均可进行液压转基因.PH后对肝再生大鼠进行液压转基因的转基因溶液体积为大鼠体重(g)×9%×1/3×相应的校正系数(Trc).转入基因在体内的表达时间和丰度既受载体影响,又受插入的目的 基因影响. 结论 液压转基因技术亦可有效地应用于大鼠再生肝转基因研究.     

In vivo monitoring of age-related changes in rat brain using quantitative diffusion magnetic resonance imaging and magnetic resonance relaxometry

Ghrelin is a novel peptide that stimulates the release of growth hormone from the pituitary and is involved in hypothalamic feeding regulation. A pre-embedding immunostaining technique was used to study the ultrastructure and synaptic relationships of ghrelin-containing neurons in the rat arcuate nucleus (ARC). Ghrelin-like immunoreactive (ghrelin-LI) neurons were found in the ARC, and were especially abundant in its ventral part. At the electron microscopic level, ghrelin-LI neurons received afferent synapses from many unknown axon terminals. Ghrelin-LI products in the immunoreactive cell bodies, processes, and axon terminals were detected mainly in dense granular vesicles about 110 nm in diameter. Ghrelin-LI presynaptic axon terminals often made synapses with unknown immunonegative neurons. These results suggest that ghrelin acts to regulate food intake through synaptic connections in hypothalamic neuronal networks.     

Fetal programming of hepatic lobular architecture in the rat demonstrated ex vivo with magnetic resonance imaging

We demonstrate that MRI imaging at sub-millimetre resolution can distinguish between periportal and perivenous zones of the rat liver lobule. This made it possible to measure the hepatic lobular radius in ex-vivo perfused fixed livers using MRI. Comparisons of histomorphometric and MRI measurements of lobular radius were in good agreement, although MRI measurements were significantly smaller (P< 0.001). Male rats whose mothers were fed 40% of the protein of controls during gestation and lactation, had a significantly larger hepatic lobular radius than that of controls [449+/-11 microm vs. 373+/-9 microm (mean +/- SEM), respectively, p<0.001, n = 12; histomorphometry data]. The proton T(2) in periportal and perivenous zones was mapped both before and after antegrade or retrograde perfusion of 10 ml of digitonin (4 mg ml(-1)). Only the T(2) of the hypointense regions increased significantly following antegrade perfusion of digitonin and conversely only that of the intense regions following retrograde perfusion. Digitonin causes permeabilization of cells in specific hepatic zones, determined by the direction of perfusion. The intense and hypointense regions of the hepatic MR images thus arise from the perivenous and periportal zones of the hepatic lobule, respectively.     

Longitudinal magnetic resonance imaging quantitation of rat liver regeneration after partial hepatectomy

This report demonstrates the advantages of using a noninvasive soft tissue imaging technique--magnetic resonance imaging (MRI)--to monitor liver regeneration after 70% partial hepatectomy in the rat in a longitudinal manner. Six animals were scanned prior to and on 6 subsequent occasions up to 9 days after surgical removal of the median and left lateral lobes. Within the observed time frame liver volumes were restored to approximately 88% of presurgery values. Final liver volumes correlated well with postmortem liver weights (R = 0.93). Regeneration is well-quantified empirically by a 4 parameter logistic equation: % Regeneration = 84 - (84/(1 + (Days/2.31)(2.34))) The rate of regeneration was maximal at 1.5 days, which coincided with the maximum increase of Mitotic Index--a measure of cell proliferation, determined in a subsequent study. Pre- and postpartial hepatectomy measurements remove two potentially confounding unknowns--the presurgery liver volume, and the amount of liver actually excised. 3D reconstructions of the liver effectively illustrate the morphological changes associated with the procedure, and the regrowth of liver tissue.     

Increased survival and migration of engrafted mesenchymal bone marrow stem cells in 6-hydroxydopamine-lesioned rodents

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra. Attempted replacement of these neurons by stem cells has proved inconclusive. Bone marrow mesenchymal stem cells (MSC) are multipotent, differentiating into a variety of cells, including neuron-like cells. We used the 6-hydroxydopamine (6-OHDA) animal model of Parkinson's disease to assess migration and differentiation of transplanted MSC. We found in rodents that transplanted MSC survive better in the 6-OHDA-induced damaged hemisphere compared to the unlesioned side. Moreover, contralaterally engrafted MSC migrated through the corpus callosum to populate the striatum, thalamic nuclei and substantia nigra of the 6-OHDA-lesioned hemisphere. In conclusion, we demonstrate that 6-OHDA-induced damage increases the viability of transplanted MSC and attracts these cells from the opposite hemisphere.     

IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury

Mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell-based therapy for liver diseases. Recently, the therapeutic potential and immunomodulatory activity of MSCs have been revealed. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), so-called adipose-derived stem cells or adipose stromal cells, because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. The goal of the present study was to evaluate the therapeutic potential of AT-MSCs by their transplantation into nude mice with CCl(4)-caused liver injury. We observed that after transplantation, AT-MSCs can improve liver functions, which we verified by changes in the levels of biochemical parameters. Ammonia, uric acid, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase concentrations returned to a nearly normal level after AT-MSC transplantation. These results raised the question of how AT-MSCs can achieve this. To discover the possible mechanisms involved in this therapeutic ability of AT-MSCs, in vitro production of cytokines and growth factors was analyzed and compared with MSCs from bone marrow (BM-MSCs) and normal human dermal fibroblasts (NHDFs). As a result we observed that AT-MSCs secrete interleukin 1 receptor alpha (IL-1Ralpha), IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 1, nerve growth factor, and hepatocyte growth factor in a volume higher than both BM-MSCs and NHDFs. Thus, our findings suggest that AT-MSCs may account for their broad therapeutic efficacy in animal models of liver diseases and in the clinical settings for liver disease treatment. Disclosure of potential conflicts of interest is found at the end of this article.     

In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models

The potential for human adipose-derived mesenchymal stem cells (AMSC) to traffic into various tissue compartments was examined using three murine xenotransplantation models: nonobese diabetic/severe combined immunodeficient (NOD/SCID), nude/NOD/SCID, and NOD/SCID/MPSVII mice. Enhanced green fluorescent protein was introduced into purified AMSC via retroviral vectors to assist in identification of cells after transplantation. Transduced cells were administered to sublethally irradiated immune-deficient mice through i.v., intraperitoneal, or subcutaneous injection. Up to 75 days after transplantation, tissues were harvested and DNA polymerase chain reaction (PCR) was performed for specific vector sequences as well as for human Alu repeat sequences. Duplex quantitative PCR using human beta-globin and murine rapsyn primers assessed the contribution of human cells to each tissue. The use of the novel NOD/SCID/MPSVII mouse as a recipient allowed rapid identification of human cells in the murine tissues, using an enzyme reaction that was independent of surface protein expression or transduction with an exogenous transgene. For up to 75 days after transplantation, donor-derived cells were observed in multiple tissues, consistently across the various administration routes and independent of transduction parameters. Tissue localization studies showed that the primary MSC did not proliferate extensively at the sites of lodgement. We conclude that human AMSC represent a population of stem cells with a ubiquitous pattern of tissue distribution after administration. AMSC are easily obtained and highly amenable to current transduction protocols for retroviral transduction, making them an excellent avenue for cell-based therapies that involve a wide range of end tissue targets.     

In vivo magnetic resonance imaging explorative study of ectopic bone formation in the rat

In animal studies of tissue engineering of bone, histology remains the standard for assessing bone formation. As longitudinal studies with this method are feasible only at the cost of large numbers of animals, we looked for an alternative. Therefore, demineralized bone matrix (DBM) and inactivated demineralized bone matrix (iDBM) implants were subcutaneously implanted in a rat. At 1, 3, 5, and 7 weeks postimplantation soft X-ray and magnetic resonance imaging (MRI) were done to monitor bone formation in the implants. At 7 weeks, the animal was killed and the implants were retrieved for histology. Our results showed that in vivo MRI is well suited to assess bone formation larger than 0.5 mm in diameter and to monitor the complete three-dimensional shape of the newly formed bone noninvasively and longitudinally. The MRI results matched well with the histology results obtained at 7 weeks. In contrast, X-ray imaging appeared inappropriate to monitor the bone formation process in DBM.