Applicability and limitations of MR tracking of neural stem cells with asymmetric cell division and rapid turnover: the case of the shiverer dysmyelinated mouse brain.

LacZ-transfected C17.2 neural stem cells (NSCs) were labeled with the superparamagnetic iron oxide formulation Feridex prior to ICV injection in shi/shi neonates. Feridex labeling did not alter cell differentiation in vitro and in vivo. Initially, MR images obtained at 11.7T correlated closely to NSC distribution as assessed with anti-dextran and anti-beta-galactosidase double-fluorescent immunostaining. However, at 6 days postgrafting there was already a pronounced mismatch between the hypointense MR signal and the histologically determined cell distribution, with a surprisingly sharp cutoff rather than a gradual decrease of signal. Positive in vivo BrdU labeling of NSCs showed that significant cell replication occurred post-transplantation, causing rapid dilution of Feridex particles between mother and daughter cells toward undetectable levels. Neural differentiation experiments demonstrated asymmetric cell division, explaining the observed sharp cutoff. At later time points (2 weeks), the mismatch further increased by the presence of non-cell-associated Feridex particles resulting from active excretion or cell death. These results are a first demonstration of the inability of MRI to track rapidly dividing and self-renewing, asymmetrically dividing SCs. Therefore, MR cell tracking should only be applied for nonproliferating cells or short-term monitoring of highly-proliferative cells, with mitotic symmetry or asymmetry being important for determining its applicability.     

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.     

Tracking of neural stem cells in rats with intracerebral hemorrhage by the use of 3T MRI.

OBJECTIVE: To access the feasibility of clinically available 3T MRI to detect the migration of labeled neural stem cells (NSCs) in intracerebral hemorrhage (ICH) in a rat model. MATERIALS AND METHODS: The ethics committee of our institution approved this study. ICH was induced by the injection of collagenase type IV into the right striatum of ten Sprague-Dawley rats. Human NSCs conjugated with Feridex (super-paramagnetic iron oxide: SPIO) were transplanted into the left striatum one week after ICH induction. MRI was performed on a 3T scanner during the first, second, third, fourth, and sixth weeks post-transplantation. MRI was obtained using coronal T2- and T2*-weighted sequences. Two rats were sacrificed every week after in vivo MRI in order to analyze the histological findings. RESULTS: ICH in the right striatum was detected by MRI one and two weeks after transplantation without migration of the NSCs. There was no migration of the NSCs as seen on the histological findings one week after transplantation. The histological findings two weeks after transplantation showed a small number of NSCs along the corpus callosum. On MRI three weeks after transplantation, there was a hypointense line along the corpus callosum and decreased signal intensity in the right periventricular region. Histological findings three weeks after transplantation confirmed the presence of the hypointense line representing SPIO-labeled NSCs. MRI four and six weeks after transplantation showed a hypointense spot in the right periventricular region. The histological findings four and six weeks after transplantation showed the presence of prominent NSCs in the right periventricular region. CONCLUSION: 3T MRI can detect the migration of NSCs in rats with ICH along the corpus callosum. Therefore, 3T MRI could be feasible for detecting the migration of NSCs in the clinical setting of stem cell therapy.     

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。     

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。     

In vivo MRI of embryonic stem cells in a mouse model of myocardial infarction.

The therapeutic potential of administering stem cells to promote angiogenesis and myocardial tissue regeneration after infarction has recently been demonstrated. Given the advantages of using embryonic stem cells and mouse models of myocardial infarction for furthering the development of this therapeutic approach, the purpose of this study was to determine if embryonic stem cells could be loaded with superparamagnetic iron oxide (SPIO) particles and imaged in a mouse model of myocardial infarction over time using MRI. Mouse embryonic stem cells were labeled with SPIO particles. When incubated with 11.2, 22.4, and 44.8 microg Fe/ml of SPIO particles, cells took up increasing amounts of iron oxide. Embryonic stem cells loaded with SPIO compared to unlabeled cells had similar viability and proliferation profiles for up to 14 days. Free SPIO injected into infarcted myocardium was not observable within 12 hr after injection. After injection of three 10-microl aliquots of 10(7) SPIO-loaded cells/ml into infarcted myocardium, MRI demonstrated that the mouse embryonic stem cells were observable and could be seen for at least 5 weeks after injection. These findings support the ability of MRI to test the long-term therapeutic potential of embryonic stem cells in small animals in the setting of myocardial infarction.     

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.     

In vivo magnetic resonance imaging of iron oxide-labeled, arterially-injected mesenchymal stem cells in kidneys of rats with acute ischemic kidney injury: detection and monitoring at 3T

PURPOSE: To evaluate MRI for a qualitative and quantitative in vivo tracking of intraaortal injected iron oxide-labeled mesenchymal stem cells (MSC) into rats with acute kidney injury (AKI). MATERIALS AND METHODS: In vitro MRI and R2* measurement of nonlabeled and superparamagnetic iron oxide (SPIO)-labeled MSC (MSC(SPIO)) was performed in correlation to cellular iron content and cytological examination (Prussian blue, electron microscopy). In vivo MRI and R2* evaluation were performed before and after ischemic/reperfusion AKI (N = 14) and intraaortal injection of 1.5 x 10(6) MSC(SPIO) (N = 7), fetal calf serum (FCS) (medium, N = 6), and SPIO alone (N = 1) up to 14 days using a clinical 3T scanner. Signal to noise ratios (SNR), R2* of kidneys, liver, spleen, and bone marrow, renal function (creatinine [CREA], blood urea nitrogen [BUN]), and kidney volume were measured and tested for statistical significance (Student's t-test, P < 0.05) in comparison histology (hematoxylin and eosin [H&E], Prussian blue, periodic acid-Schiff [PAS], CD68). RESULTS: In vitro, MSC(SPIO) showed a reduction of SNR and T2* with R2* approximately number of MSC(SPIO) (R2 = 0.98). In vivo MSC(SPIO) administration resulted in a SNR decrease (35 +/- 15%) and R2* increase (101 +/- 18.3%) in renal cortex caused by MSC(SPIO) accumulation in contrast to control animals (P < 0.01). Liver, spleen, and bone marrow (MSC(SPIO)) showed a delayed SNR decline/R2* increase (P < 0.05) resulting from MSC(SPIO) migration. The increase of kidney volume and the decrease in renal function (P < 0.05) was reduced in MSC-treated animals. CONCLUSION: Qualitative and quantitative in vivo cell-tracking and monitoring of organ distribution of intraaortal injected MSC(SPIO) in AKI is feasible in MRI at 3T.     

Magnetosonoporation: Instant magnetic labeling of stem cells

The purpose of this study was to develop an instant MR cell labeling technique, called magnetosonoporation. First, a magnetosonoporation apparatus was successfully established for MR labeling of stem cells. Then, the safety of this new cell labeling approach was confirmed by evaluation of cell viability, proliferation, and differentiation of magnetosonoporation‐labeled and unlabeled C17.2 neural stem cells. Subsequently, the feasibility of using in vivo MRI to detect magnetosonoporation/Feridex‐labeled stem cells was validated in living animals and confirmed by histologic correlation. The magnetosonoporation technique is expected to be convenient, efficient, and safe for future clinical application of MRI‐guided cell therapies. Magn Reson Med 63:1437–1441, 2010. © 2010 Wiley‐Liss, Inc.     

SWIFT detection of SPIO‐labeled stem cells grafted in the myocardium

We report initial results from studies using sweep imaging with Fourier transformation (SWIFT) to detect superparamagnetic iron oxide (SPIO) particle–labeled stem cells in the rat heart. In experiments performed on phantoms containing titanium balls or SPIO–labeled cells, frequency‐shifted signals surrounding the paramagnetic objects produced a pileup artifact visualized by SWIFT. Total signal intensity was retained to a much greater extent by SWIFT as compared to gradient echo imaging. SWIFT imaging of excised and in vivo hearts showed (a) reduced blooming artifact as compared with gradient echo imaging, which helped reduce ambiguity in the detection of SPIO–labeled cells; (b) enhancement of off‐resonance signals relative to the background in the imaginary component of images; and (c) detailed myocardial anatomy in magnitude images, which provided anatomic reference. These features suggest SWIFT can facilitate the detection of SPIO–laden cells in the cardiovascular system. Magn Reson Med 63:1154–1161, 2010. © 2010 Wiley‐Liss, Inc.     

In vivo “hot spot” MR imaging of neural stem cells using fluorinated nanoparticles

To optimize ¹⁹F MR tracking of stem cells, we compared cellular internalization of cationic and anionic perfluoro‐15‐crown‐5‐ether (PFCE) nanoparticles using cell culture plates with different surface coatings. The viability and proliferation of anionic and cationic PFCE‐labeled neural stem cells (NSCs) did not differ from unlabeled cells. Cationic PFCE nanoparticles (¹⁹F T1/T2 = 580/536 ms at 9.4 Tesla) were superior to anionic particles for intracellular fluorination. Best results were obtained with modified polystyrene culture dishes coated with both carboxylic and amino groups rather than conventional carboxyl‐coated dishes. After injecting PFCE‐labeled NSCs into the striatum of mouse brain, cells were readily identified in vivo by ¹⁹F MRI without changes in signal or viability over a 2‐week period after grafting. These results demonstrate that neural stem cells can be efficiently fluorinated with cationic PFCE nanoparticles without using transfection agents and visualized in vivo over prolonged periods with an MR sensitivity of approximately 140 pmol of PFCE/cell. Magn Reson Med 60:1506–1511, 2008. © 2008 Wiley‐Liss, Inc.     

In vivo molecular MRI of cell survival and teratoma formation following embryonic stem cell transplantation into the injured murine myocardium

Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, nonspecific intracellular accumulation of SPIO limits long‐term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO‐conjugated monoclonal antibody against these antigens (SPIO‐MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO‐MAb. The human and mouse ESCs were transduced with the RG (ESC‐RGs) and transplanted into the peri‐infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO‐MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC‐derived teratoma formation in the injured murine myocardium. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.     

In vivo detection of single cells by MRI.

The use of high-relaxivity, intracellular contrast agents has enabled MRI monitoring of cell migration through and homing to various tissues, such as brain, spinal cord, heart, and muscle. Here it is shown that MRI can detect single cells in vivo, homing to tissue, following cell labeling and transplantation. Primary mouse hepatocytes were double-labeled with green fluorescent 1.63-microm iron oxide particles and red fluorescent endosomal labeling dye, and injected into the spleens of recipient mice. This is a common hepatocyte transplantation paradigm in rodents whereby hepatocytes migrate from the spleen to the liver as single cells. One month later the animals underwent in vivo MRI and punctuated, dark contrast regions were detected scattered through the livers. MRI of perfused, fixed samples and labeled hepatocyte phantoms in combination with histological evaluation confirmed the presence of dispersed single hepatocytes grafted into the livers. Appropriate controls were used to determine whether the observed contrast could have been due to dead cells or free particles, and the results confirmed that the contrast was due to disperse, single cells. Detecting single cells in vivo opens the door to a number of experiments, such as monitoring rare cellular events, assessing the kinetics of stem cell homing, and achieving early detection of metastases.     

In vivo MR imaging tracking of transplanted mesenchymal stem cells in a rabbit model of acute peripheral nerve traction injury

Purpose

To investigate in vivo MRI tracking mesenchymal stem cells (MSCs) in peripheral nerve injures using a clinically available paramagnetic contrast agent (Gd‐DTPA) and commercially available rhodamine‐incorporated transfection reagents (PEI‐FluoR).

Materials and Methods

After bone marrow MSCs were labeled with Gd‐DTPA and PEI‐FluoR complex, the labeling efficacy and longevity of Gd‐DTPA maintenance were measured and cell viability, proliferation, and apoptosis were assessed. Thirty‐six rabbits with acute sciatic nerve traction injury randomly received 1 × 10⁶ labeled (n = 12) or unlabeled MSCs (n = 12) or vehicle alone injection. The distribution and migration of implanted cells was followed by MRI and correlated with histology. The relative signal intensity (RSL) of the grafts was measured.

Results

The labeling efficiency was 76 ± 4.7% and the labeling procedure did not in?uence cell viability, proliferation, and apoptosis. A persistent higher RSL in grafts was found in the labeled group compared with the unlabeled and vehicle groups until 10 days after transplantation (P < 0.05). The distribution and migration of labeled cells could be tracked by MRI until 10 days after transplantation. Transplanted MSCs were not found to transdifferentiate into Schwann‐like cells within 14‐day follow‐up.

Conclusion

Labeling MSCs with the dual agents may enable cellular MRI of the engraftment in the experimental peripheral nerve injury. J. Magn. Reson. Imaging 2010;32:1076–1085. © 2010 Wiley‐Liss, Inc.

     

Transferrin receptor upregulation: in vitro labeling of rat mesenchymal stem cells with superparamagnetic iron oxide

PURPOSE: To prospectively evaluate the influence of superparamagnetic iron oxide (SPIO) or ultrasmall SPIO (USPIO) particles on the surface epitope pattern of adult mesenchymal stem cells (MSCs) by regulating the expression of transferrin receptor and to prospectively evaluate the influence of transfection agents (TAs) on the uptake of SPIO or USPIO particles in MSCs. MATERIALS AND METHODS: The study was approved by the institutional animal care committee of the University of Tübingen. MSCs were isolated from the bone marrow of four rats. To obtain highly homogeneous MSC populations, MSCs from one rat were single-cell cloned. One MSC clone was characterized and selected for the labeling experiments. The MSCs, which were characterized with flow cytometry and in vitro differentiation, were labeled with 200 microg/mL SPIO or USPIO or with 60 microg/mL SPIO or USPIO in combination with TAs. Aggregations of labeled cells were accommodated inside a defined volume in an agar gel matrix. Magnetic resonance (MR) imaging was performed to measure SPIO- or USPIO-induced signal voids. Quantification of cellular total iron load (TIL) (intracellular iron plus iron coating the cellular surface), determination of cellular viability, and electron microscopy were also performed. RESULTS: Labeling of MSCs with SPIO or USPIO was feasible without affecting cell viability (91.1%-94.7%) or differentiation potential. For MR imaging, SPIO plus a TA was most effective, depicting 5000 cells with an average TIL of 76.5 pg per cell. SPIO or USPIO particles in combination with TAs coated the cellular surface but were not incorporated into cells. In nontransfected cells, SPIO or USPIO was taken up. MSCs labeled with SPIO or USPIO but without a TA showed enhanced expression of transferrin receptor, in contrary to both MSCs labeled with SPIO or USPIO and a TA and control cells. CONCLUSION: SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.     

Sizing it up: cellular MRI using micron-sized iron oxide particles.

There is rapidly increasing interest in the use of MRI to track cell migration in intact animals. Currently, cell labeling is usually accomplished by endocytosis of nanometer-sized, dextran-coated iron oxide particles. The limitations of using nanometer-sized particles, however, are that millions of particles are required to achieve sufficient contrast, the label can be diluted beyond observability by cell division, and the label is biodegradable. These problems make it difficult to label cells other than macrophages in vivo, and to conduct long-term engraftment studies. It was recently demonstrated that micron-sized iron oxide particles (MPIOs) can be taken up by a number of cell types. In this study we examined the MRI properties of single MPIOs with sizes of 0.96, 1.63, 2.79, 4.50, and 5.80 microm. Furthermore, the capacity of cells to endocytose these MPIOs was investigated, and the MRI properties of the labeled cells at 7.0 and 11.7 Tesla were measured as a function of image resolution and echo time (TE). Cells labeled with MPIOs generally contained iron levels of approximately 100 pg, which is approximately threefold higher than those obtained with the best strategies to label cells using nanometer-sized particles. On occasion, some cells had levels as high as approximately 400 pg. We demonstrate that these large particles and the cells labeled with them can be detected by spin echo (SE)-based imaging methods. These measurements indicate that MPIOs should be useful for improving cell tracking by MRI.     

Magnetic nanoparticles for imaging dendritic cells

We report the development of superparamagnetic iron oxide (SPIOs) nanoparticles and investigate the migration of SPIO‐labeled dendritic cells (DCs) in a syngeneic mouse model using magnetic resonance (MR) imaging. The size of the dextran‐coated SPIO is roughly 30 nm, and the DCs are capable of independent uptake of these particles, although not at levels comparable to particle uptake in the presence of a transfecting reagent. On average, with the assistance of polylysine, the particles were efficiently delivered inside DCs within one hour of incubation. The SPIO particles occupy approximately 0.35% of cell surface and are equivalent to 34.6 pg of iron per cell. In vivo imaging demonstrated that the labeled cells migrated from the injection site in the footpad to the corresponding popliteal lymph node. The homing of labeled cells in the lymph nodes resulted in a signal drop of up to 79%. Furthermore, labeling DCs with SPIO particles did not compromise cell function, we demonstrated that SPIO‐enhanced MR imaging can be used to track the migration of DCs effectively in vivo. Magn Reson Med 63:1383–1390, 2010. © 2010 Wiley‐Liss, Inc.     

SD大鼠神经干细胞培养鉴定实验研究

为探讨从SD胎鼠室管膜、皮质以及SD成年大鼠脑室下区、皮质分离并鉴定神经干细胞的可行性 ,以从上述部位分离的细胞作为种子细胞来源 ,在本实验室特制“CYTOKINE·神经干细胞培养基”中进行了神经干细胞培养诱导 ,单细胞连续传代培养 ,并分别以Nestin、NSE及GFAP免疫抗体鉴定神经干细胞、神经元和神经胶质细胞。结果发现 ,4种来源的组织细胞在相应培养条件下均有神经干细胞快速增殖 ,并有由多细胞组成的神经球形成 ,经鉴定 ,该细胞球表达特殊的胚胎神经干细胞Nestin抗原 ;进一步将这些细胞球分离成单细胞并重新以克隆密度进行培养 ,单个的细胞又很快形成神经球。对神经干细胞进行连续培养或加血清传代培养可使其进一步分裂增殖 ,有小芽形成并发育成突起、建立神经纤维联系 ,其中有的胞体增大 ,逐渐发育为较成熟的长突起细胞 ,长突起相互连接 ,交织成网 ,经鉴定为神经元及神经胶质细胞。 4种组织来源的细胞中所含有的干细胞数量不同 ,胎鼠较成年鼠更富含神经干细胞 ,室管膜或脑室下区所含神经干细胞较皮质更丰富 ,皮质来源的神经干细胞和室管膜来源的神经干细胞在形态学和分化程度上无特殊区别。该结果提示 ,SD胎鼠室管膜、皮质以及SD成年大鼠脑室下区、皮质 4种组织来源的细胞均含有神经干细胞 ;神经干     

Quantification of superparamagnetic iron oxide (SPIO)-labeled cells using MRI

PURPOSE: To show the feasibility of using magnetic resonance imaging (MRI) to quantify superparamagnetic iron oxide (SPIO)-labeled cells. MATERIALS AND METHODS: Lymphocytes and 9L rat gliosarcoma cells were labeled with ferumoxides-protamine sulfate complex (FE-PRO). The cells were labeled efficiently (more than 95%) and the iron concentration inside each cell was measured by spectrophotometry (4.77-30.21 pg). Phantom tubes containing different numbers of labeled or unlabeled cells, as well as different concentrations of FE-PRO, were made. In addition, labeled and unlabeled cells were injected into fresh and fixed rat brains. RESULTS: Cellular viability and proliferation of labeled and unlabeled cells were shown to be similar. T2-weighted images were acquired using 7T and 3T MRI systems, and R2 maps of the tubes containing cells, free FE-PRO, and brains were made. There was a strong linear correlation between R2 values and labeled cell numbers, but the regression lines were different for the lymphocytes and gliosarcoma cells. Similarly, there was strong correlation between R2 values and free iron. However, free iron had higher R2 values than the labeled cells for the same concentration of iron. CONCLUSION: Our data indicate that in vivo quantification of labeled cells can be done by careful consideration of different factors and specific control groups.     

Labeling of human neural precursor cells using ferromagnetic nanoparticles

Fetal human neural precursor cells (NPCs) are unique with respect to their capacity to proliferate and to preserve their potential to differentiate into neurons and glia. Human mesencephalic neural precursor cells (hmNPCs) provide a source for dopaminergic neurons. Preclinical and clinical research will benefit from reliable in vivo tracking of transplanted cells. Here, we investigate the potency of very small superparamagnetic iron oxide particles (VSOPs) to label hmNPCs, the effect of VSOPs on survival, proliferation, and differentiation of hmNPCs, and the sensitivity of 1.5T magnetic resonance imaging (MRI) to detect labeled cells in living rats following transplantation. When incubated with VSOPs at 1.5 mM, >95% of hmNPCs incorporated VSOPs without detectable impact on cell viability (>90%) or proliferative capacity, as measured by the expression of proliferating cell nuclear antigen (PCNA) and cell cycle distribution. Labeled hmNPCs differentiate into neurons (>30%) and glia with no detectable difference compared to nonlabeled cells. Following transplantation into rat striata, marked paramagnetic signal changes were detected for as long as three months postsurgery using MRI, corresponding to the histologically‐identified graft. Our data indicate that hmNPCs can be labeled with VSOPs without impairment of viability, proliferation, or multipotency. Labeled, transplanted cells are detectable in vivo using 1.5T MRI. Magn Reson Med 60:1321–1328, 2008. © 2008 Wiley‐Liss, Inc.