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Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled
Magnetic Resonance Imaging xxx (2014) xxx–xxx
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Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled☆ Han-Lin Shuai 1, Rui-Ling Yan 1, Hong Song, Dan-Liang Chen, Xin Luo ⁎ Department of Obstetrics and Gynecology, the First Affiliated Hospital of Jinan University, Guangzhou, 510630, China
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Article history: Received 27 November 2013 Revised 27 January 2014 Accepted 3 February 2014 Available online xxxx Keywords: Mesenchymal stem cell Magnetic resonance imaging (MRI) Gd-DTPA Trace
a b s t r a c t Objective: Three different kinds of transfection reagents were used to mediate the transfection of gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) into human umbilical-cord-derived mesenchymal stem cells (hUCMSCs). The efficacy of different transfection reagents and the feasibility of NMR tracer in vitro of magnetized stem cells were estimated. Methods: After purification by tissue explants adherent method, the biological characteristics of hUCMSCs in vitro were identified by subculture and amplification. Calcium phosphate, Effectene and liposome2000 were used to transfect Gd-DTPA-labeled hUCMSCs respectively, and cell counting was used to mediate the transfection of Gd-DTPA into hUCMSCs, which were then induced to lipoblast and osteoblast in vitro. The determination of the transfection activities of the transfection reagents was conducted by measuring the magnetic resonance imaging (MRI) signal intensity of the Gd-DTPA-labeled cells and the concentration of gadolinium ion in the cells. Furthermore, the relationship between the signal intensity of Gd-DTPA-labeled hUCMSCsMRI, cell subculture and generations was studied. Results: Primary cells were obtained by tissue explants adherent for two weeks. The cells displayed a long spindle form and grew in swirl. After two passage generations, the cellular morphology became more homogeneous. The result detected by the flow cytometer showed that CD29C, D44, CD90, and CD105 were highly expressed, while no CD45, CD40, and HLA-DR expression was detected in the third generation cells. Directional induction in vitro caused the differentiation into lipoblast and osteoblast. After transfected by calcium phosphate, Effectene and liposome 2000, the signal intensity of stem cells was 2281.2 ± 118.8, 2031.9 ± 59.7 and 1887.4 ± 40.8 measured by MRI. Differences between these three groups were statistically significant (P b 0.05). The concentrations of gadolinium ion in three groups of stem cells were 0.178 ± 0.009 mg/L, 0.158 ± 0.003 mg/L and 0.120 ± 0.002 mg/L respectively, examined by inductively coupled plasma atomic emission spectrometry. No significant differences were found among these three groups (P b 0.05). The proliferation and differentiation abilities of the Gd-DTPA-labeled stem cells were not affected. A minimum 5 × 104 Gd-DTPA-labeled stem cells could be traced with MRI in vitro and presented in high signal. The trace duration time in vitro was about 12 days. Conclusions: Tissue explants adherent method can be availably applied to purify hUCMSCs. The Effectene method was proved to have the best transfection effect. The proliferation ability and differentiation potency of Gd-DTPAlabeled hUCMSCs were not affected, and the NMR of labeled stem cells in vitro was proved to be feasible. © 2014 Elsevier Inc. All rights reserved.
1. Introduction With the rapid development of the modern medicine, stem cell research and its clinical application have been widely used in ☆ Funding: This paper is funded by China National Natural Science Foundation Project and the project number is 81070459. ⁎ Corresponding author at: Department of Obstetrics and Gynecology, the First Affiliated Hospital of Jinan University, 613 Huangpu Road West, Guangzhou, 510630 China. Tel.: + 86 150 14210277; fax: + 86 150 14210277. E-mail address: [email protected] (X. Luo). 1 These authors contributed equally to this work.
biotechnology in the 21st century [1–3]. However, how to monitor its survival distribution, migration, differentiation and other biological behaviors in vivo after stem cell transplantation is still a problem. Magnetic resonance imaging (MRI) has been used to trace transplanted cells, and cells must be effectively labeled before transplant. Presently, gadolinium chelate and super paramagnetic iron oxide (SPIO) are the most commonly used for MRI contrast agents. Compared with SPIO, gadolinium-diethylene-triamine-pentaacetic acid (Gd-DTPA) is more effective for transfection. According to the pharmacokinetic study of Gd-DTPA, if the tag targeted cells have damages or apoptosis, they will be ruled out of the body within
http://dx.doi.org/10.1016/j.mri.2014.02.008 0730-725X/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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24 hours [4]. In addition, gadolinium chelate is a paramagnetic resonance imaging contrast agent. By means of shortening the T1, the MRI signal displayed a high signal, and prominent contrast enhancement effect, easily distinguishing from the surrounding low signal cells. Shen [5] reported that 30 microliter of Gd-DTPA can effectively mark stem cell, and the marking rate reached up to 90% with no cytotoxicity. In recent years, stem cell transplant treatment effect has been widely used in animal experiments and clinical trials [6–10]. Mesenchymal stem cells (MSCs) are a group of multipotent stem cells existed in the connective tissues and the interstitial substances of organs, and could differentiate into the cells of three embryonic layers under specific conditions [11–13]. Human umbilical cord mesenchymal stem cells (hUCMSCs) were regarded as an ideal cell source for the future of stem cells in clinical treatment and scientific research [14–21]. In comparison with other mesenchymal stem cells, human umbilical cord tissue has rich MSCs, powerful proliferation ability, low immunogenicity and no tumorigenicity. Moreover, hUCMSCs was not involved in social, ethical and legal aspects of debate. The comprehensive judgment identification of HUCMSCs is based primarily on the cell morphology, surface markers and multidirectional differentiation ability. International association of cell therapy [22] maintains that ectomesenchymal stem cells cultured in vitro must meet the following conditions: adherent cell growth; cellular immune phenotypes positive rate of CD105 and CD73, CD90 must be greater than 95%, and positive rates of CD45, CD34, CD14, CD19 and HLA-DR must be less than 2%; owing the ability of differentiating into osteoblast, fat cells and chondrocytes. Hematopoietic cell surface markers of CD45 and surface symbol CD40, HLADR closely related to transplantation immune rejection, are both negative expression, which can be induced into fat cells and osteoblasts in vitro. In this study, three different transfection reagents were used to transfect Gd-DTPA marked umbilical cord mesenchymal stem cells. The changes of the biological characteristics of stem cells after magnetic marker and the use of Gd-DTPA markers human umbilical cord mesenchymal stem cells (hUCMSCs) were investigated to measure the feasibility of magnetic resonance imaging (MRI) in vitro [4,5]. 2. Materials and methods 2.1. Isolation and culture of hUCMSCs in vitro Umbilical cord specimen was obtained from the umbilical cord tissue of a health neonate delivered by full-term cesarean in the first hospital affiliated to Jinan University (the study was approved by the hospital ethics committee and informed consent was obtained from the family members of the donors). The umbilical cord tissue under aseptic conditions was collected. The tissue explants adherent method was used to isolate and purify the MSCs, and then subculture with the ratio 1:2 or 1:3 when the cell fusion rate reached 80%~90%. 2.2. hUCMSCs growth curve assay Cell proliferation ability was detected, and the growth curve was assayed by cell counting. Cell lines of the third generation were seeded in 24-well tissue culture plates (4000 cells per well). The cultures were incubated for 8 days, each day at the same time, cells from 3 random wells were counted after distribution by trypsin, using Blood Cell Counter and inverted microscope (Olympus, Japan). 2.3. hUCMSCs phenotype and the cell cycle Single-cell suspensions of the third generation cells were obtained by trypsinization (with a concentration of 0.25%). Expres-
sions of surface markers CD29, CD44, CD90, CD105, CD40, CD45 and HLA-DR, and the cell cycle were analyzed by flow cytometry (BD, USA). Atomic force microscope (Thermo Electric, USA) was used to observe hUCMSCs. 2.4. Multipotent differentiation ability of hUCMSCs Cells of the third generation were seeded in six-well plates in complete culture medium. The medium was replaced by differentiation-inducing medium when 80 ~ 90% of cytomixis was achieved. To induce fat cells differentiation: the experimental group was treated with inducing medium (containing 10% FBS, 10 μg/mL insulin, 1 μmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methyl yellow purine, 100U/mL penicillin, 100U/mL streptomycin low glucose Dulbecco's modified Eagle's medium (GIBCO, USA). Culture medium was exchanged every 3 days. Oil red "O" (Sijia biotech, Guangzhou) staining was performed after incubation for 21 days. To induce osteoblast differentiation: the experimental group was treated with osteoblast induction medium (10% FBS, 100 nmol/L dexamethasone, 10 mmol/Lβ- glycerophosphate, 0.2 mmol/L vitamin C, 100U/mL penicillin, 100U/mL streptomycin DMEM/F12 medium); medium was changed every 3 days. Alizarin red staining was performed after incubation for 21 days. 2.5. Gd-DTPA-labeling of hUCMSCs mediated by transfection reagents The third generation of T25 cells were incubated until 70 to 80% cytomixis achieved. Calcium phosphate, Effectene (QIAGEN, China) and liposome 2000 were used to mediate the entry of gadolinium ions (Bayer, Germen) into stem cells. Untransfected Gd-DTPA group was set as a control. (1) Calcium phosphate transfection: culture medium was replaced with fresh complete medium (5 mL, without antibiotics) 1 hour before the transfection. A 30 μL Gd-DTPA and 260 μL calcium chloride were mixed well with 260 μL PBS solution. After incubation at room temperature for 10 ~ 20 min, the mixture was added to T25 culture flasks (Corning, USA). The cells were incubated at 37°C in 5% CO2 for 4 hours, washed three times by PBS, detached by trypsin and then collected in the EP tube. MRI imaging was performed by measuring signal strength of the cells in a plastic container filled with water. (2) Effectene and Lipofectamine 2000 were used in the transfection of gadolinium ions. After MRI imaging analysis, the concentrations of gadolinium ion in stem cells transfected by different reagents were examined by inductively coupled plasma atomic emission spectrometry. 2.6. MR imaging MRI of these tubes was performed with a clinical 1.5-T MR imager (GE, USA), by using an 8-channel wrist coil. Imaging parameters are as follows: SE T1WI sequence: TR were 300 ms, 600 ms, 900 ms, 1200 ms, respectively; images were obtained with a matrix size of 256 × 256, TE = 15 ms, two measurements acquired, layer distance/thickness = 1.0/0 mm and field of view of 12 × 9 cm. SE T1WI sequence: TR/TE = 1500/88 ms, layer distance/thickness = 1.0/0 mm, images were obtained with a matrix size of 384 × 192 with a field of view of 12 × 12 cm, two measurements were acquired. Region of interest for signal intensity (SI) measurement was 1 × 1 mm. 3. Statistical analysis Data were expressed as mean and standard deviation (x± S). The t test or variance analysis was used, and all of the statistics analyses were performed using SPSS17.0 (information of the software), test level α = 0.05, and P b 0.05 was considered to be statistically significant.
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
H-L. Shuai et al. / Magnetic Resonance Imaging xxx (2014) xxx–xxx
4. Results 4.1. hUCMSCs isolation and morphological characteristics One week after planting, cells could be seen with newly formed colonies (Fig. 1A). After first passage, the fibroblast-like MSCs uniformly distributed in the cell culture flasks. Spherical cells suspended in culture medium could be observed using inverted phase contrast microscope after passage. Twenty-four hours later, most of the cells were short rod-like or long fusiform-like and adhered to the flask wall. By changing medium many times and passages, miscellaneous cells were eliminated, and the uniform fibroblast-like cells left were umbilical cord blood MSCs (Fig. 1B). These cells could be further cultured, passed and proliferated rapidly. Most of the cells were long fusiform-like, clear boundaries between cytoplasm and nucleus, and a prominent nucleoli could be seen. The cell volume was large, with a length of more than 100 μm. Numerous projections could be seen around the cell (Fig. 1C).
4.2. Identification of hUCMSCs surface markers Antigenic identification of the 3rd generation of hUCMSCs cells was investigated by flow cytometry analysis. The results demon-
A
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strated high positive signals for hUCMSCs cell markers CD29 (β1 integrin), CD44 (the hyaluronate receptor) and CD105 (endoglin/ SH2). The hUCMSCs cells show a negative signal for cell markers CD40 (TNF-receptor superfamily) and HLA-DR associated with allograft rejection on the surface of hUCMSCs cells (Fig. 2). 4.3. Proliferative capacity of Gd-DTPA labeled hUCMSCs The growth curves of the experiment group and the control group were both S" shaped (Fig. 3A). After inoculation for 1 to 4 days, the cells were in a incubation period and grew slowly; then cells went into the logarithmic growth phase; cell growth rate gradually slowed down from the sixth day. No significant difference between the two groups was observed (P N 0.05), suggesting that gadolinium ions do not affect the proliferation activity of the hUCMSCs. 4.4. Cell cycle of Gd-DTPA labeled hUCMSCs Distributions of cells in different stages were analyzed by flow cytometry. For the labeled hUCMSCs, G1/G0 phase cells accounted for (61.267 ± 4.488)%, S phase cells accounting (23.567 ± 1.401)%, and G2/M phase cells accounted for (15.167 ± 4.631)%. As for the hUCMSCs, G1/G0 phase cells accounted for (65.100 ± 6.756)%, S
B
250µm
C
D
Fig. 1. The morphology of hUCMSCs cells. Structure of primary hUCMSCs (A) the 3rd genetation of hUCMSCs (B) under general microscope; C, D structure of hUCMSCs under atomic force microscope.
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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Fig. 2. Identification of hUCMSCs surface markers. Antigenic identification of the 3rd generation of hUCMSCs cells were investigated by flow cytometry analysis.
phase cells accounted for (15.367 ± 5.014)%, and G2/M phase cells accounted for (19.567 ± 2.013)%. Random block design analysis of variance was used, and the difference between the two groups was not statistically significant (F = 0.00, P = 1.00), (Fig. 3B).
4.5. Gd-DTPA labeled hUCMSCs differentiation in vitro testing It has been reported that hUCMSCs may differentiate along several cell lineages in all three germ layers including adipogenic and osteogenic [11–13]. Adipogenic and osteogenic mediums were used to test the differentiation ability of Gd-DTPA labeled hUCMSCs. As shown in Fig. 4A and B, lipid droplets of adipogenic was stained red by oil red "O," and mineralized nodules of osteogenic was stained red by alizarin red.
4.6. MR imaging After transfection, MSCs were trypsinized, centrifuged in tubes (1 × 10 6 cells each), and scanned by MR. Regions of interest (ROI) were used to measure the signal strength of T1WI sequence cells. Signals of Gd-DTPA labeled hUCMSCs were white high in T1WI (Fig. 5A) and low in T2WI (Fig. 5B); signals of unlabeled hUCMSC were both low in T1WI and T2WI. Signal intensity of the three labeled groups: calcium phosphate, Effectene, Lipofectamine 2000 were 2281.2 ± 118.8, 2031.9 ± 59.7, 1887.4 ± 40.8, respectively. The blank control group was 1306.7 ± 52.0. Variance analysis of completely randomized design was performed. Differences between the four groups were statistically significant (F = 93.1, P b 0.05), suggesting that the three transfection agents successfully transfected Gd-DTPA into stem cells.
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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Fig. 3. Identification of Gd-DTPA labeled hUCMSCs. A. Growth curves of hUCMSCs with and without Gd-DTPA label. B. Cell cycle of hUCMSCs with and without GdDTPA label.
4.7. Gadolinium ion concentration measurement of stem cells by inductively coupled plasma atomic emission spectrometry
4.9. Minimum amount of magnetically labeled hUCMSCs detected by MRI
Gadolinium ion concentrations of the three groups, calcium phosphate, Effectene, Lipofectamine 2000 t were (0.178 ± 0.009) mg/L, (0.158 ± 0.003) mg/L, and (0.120 ± 0.002) mg/L. Completely randomized design analysis of variance was performed, and differences between the four groups were statistically significant (Fig. 6).
The concentrations of magnetic cells were set to be 1 × 10 5/mL, 5 × 10 4/mL, 2.5 × 10 4/ml, 1 × 10 4/mL and 5 × 10 3/mL, respectively. When the concentration of magnetic cells was 2.5 × 10 4/mL, no high T1WI signal intensity could be seen compared with that of the blank control (Fig. 7C). The minimum amount of magnetically labeled hUCMSCs that could be detected was 5 × 10 4/mL.
4.8. Effect of cell proliferation on the signal intensity of gadolinium labeled hUCMSCs
5. Discussion
Gd-DTPA labeled stem cells were scanned by MR. The TIWI signal intensity was 2031.9 ± 59.7. T1WI signal intensity was gradually decreasing with the cells subculture and proliferation. At the 12th day (third generation), the T1WI signal intensity of cells was 1251.1 ± 42.0 (Fig. 7A). No significant difference was found by the independent-sample t test (P N 0.05). The tracing time for the magnetically labeled stem cells was about 12 days (Fig. 7B).
Transplantation of stem cell had an insight into the aspect of biological behavior, such as its survival distribution, migration and differentiation in the body, which was important for evaluating stem cell transplantation effect. Based on the extensive research efforts, a large number of studies have demonstrated that MRI is the best way for traced cells in vivo [23,24]. In this experiment, 1.5 T magnetic resonance imaging (MRI) was applied to mark and measure the different transfection medium of hUCMSCs in in vitro research. Through effective transfection of Gd-DTPA tagged hUCMSCs, testing
Fig. 4. Gd-DTPA labeled hUCMSCs differentiation in vitro testing. Lipid droplets of adipogenic was staining red by oil red "O," and mineralized nodules of osteogenic was staining red by alizarin red. A. Cell differentiation to lipoblast with Gd-DTPA label (400×). B. Cell differentiation to osteoblast with Gd-DTPA label (200×).
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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Fig. 5. MRI of Gd-DTPA labeled hUCMSCs. A. T1 weighted imaging from left to right, calcium phosphate, Effectene and liposome and blank. B. T2 weighted imaging from left to right, calcium phosphate, Effectene and liposome and blank.
its growth curve, cell cycle and multi-directional differentiation capacity, we could draw the conclusion that the biological characteristics and multi-directional differentiation potential of stem cells have not been affected after marked by Gd-DTPA. Gd-DTPA and cell membrane surface are negatively charged, and it is hard to enter cells without the surface modification of Gd-DTPA. In this experiment, we chose calcium phosphate liposome, Effectene and liposomes 2000 as transfection agent for transfection. Through MRI signal intensity testing and inductively coupled plasma atomic emission spectrometry screening, calcium phosphate liposome, Effectene group, liposomes 2000 group, and blank control group were all statistically significant (P b 0.05) among the four groups. The strongest signal is the calcium phosphate group (2281.2 ±
Fig. 7. Effect of cell proliferation on the signal intensity of Gd-DTPA labeled hUCMSCs. A. T1 weighted imaging from left to right: generation P1, P2, P3, P4, blank control. B. T2 weighted imaging from left to right: generation P1, P2, P3, P4, blank control. C. The minimum hUCMSCs traced by MRI. The concentrations of cells from left to right were 105, 5 × 104, 2.5 × 104, 104, 5 × 103.
Fig. 6. Gadolinium ion concentration measurement of stem cells by inductively coupled plasma atomic emission spectrometry.
118.8), followed by Effectene and liposomes 2000 group, which is consistent with the results of inductively coupled plasma atomic emission spectrometry screening. However, we found that calcium phosphate in the experimental process was easy to form calcium phosphate small precipitation after 4 hours, which affects the cell morphology. Based on the comprehensive comparison, Effectene is the better Gd-DTPA transfection reagents. Gadolinium labeled stem cells were set to be five gradients: 1 × 10 5/ml, 5 × 10 4/ml, 2.5 × 10 4/ml, 1 × 10 4/ml and 5 × 10 3/ml, and the results showed that with the increase of cell number, MRI signal intensity increased, indicating that there was a certain relationship between the signal strength and signed cells. The detection
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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limit of magnetic signal cell labeling number is 5 × 10 4/ml using MRI. Given the number of cell transplantation in clinical application is far greater than the minimum cell count, thus the imaging cells threshold is enough to meet the needs of the clinical application. In this experiment, by means of dynamic tracer of 1 × 10 6 gadolinium tag stem cells, we found that MRI signal strength decreases gradually as the cells proliferate, and the reason may be contributed to dilution effect of the cell's proliferation and elimination impact of cell metabolism, gadolinium ions gradually ruling out of the body, indicating that MRI signal intensity has relation to the cell proliferation. When cells reproduce to the fourth generation, there was no obvious difference by MRI between the magnetic marker stem cells signal intensity and untagged signal intensity. Moreover, magnetic markers of stem cells in vitro can trace to the time prolonging to 12 days. In conclusion, Effectene was the best agent for transfection GdDTPA tag hUCMSCs, and the proliferation and multi-directional differentiation potential of marked stem cell has not been affected. References [1] Horibe EK, Sacks J, Unadkat J, Raimondi G, Wang Z, Ikeguchi R. Rapamycinconditioned, alloantigen-pulsed dendritic cells promote indefinite survival of vascularized skin allografts in association with T regulatory cell expansion. Transpl Immunol 2008;18:307–18. [2] Forraz N, Pettengell R, McGuckin CP. Hematopoietic and neuroglial pro-genitors are promoted during cord blood ex vivo expansion. Br J Haematol 2002;119:888. [3] Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–9. [4] Shyu WC, Chen CP, Lin SZ, Lee YJ, Li H. Efficient tracking of non-iron-labeled mesenchymal stem cells with serial MRI in chronic stroke rats. Stroke 2007;38:367–74. [5] Shen J, Cheng LN, Zhong XM, Duan XH, Guo RM, Hong GB. Efficient in vitro labeling rabbit neural stem cell with paramagnetic Gd-DTPA and fluorescent substance. Eur J Radiol 2010;75:397–405. [6] Maijenburg MW, Gilissen C, Melief SM, Kleijer M, Weijer K, Ten Brinke A, et al. Nuclear receptors Nur77 and Nurr1 modulate mesenchymal stromal cell migration. Stem Cells Dev 2012;21(2):228–38. [7] Zhang MJ, Sun JJ, Qian L, Liu Z, Zhang Z, Cao W, et al. Human umbilical mesenchymal stem cells enhance the expression of neurotrophic factors and protect ataxic mice. Brain Res 2011;1402(7):122–31.
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Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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Magnetic Resonance Imaging journal homepage: www.mrijournal.com
Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled☆ Han-Lin Shuai 1, Rui-Ling Yan 1, Hong Song, Dan-Liang Chen, Xin Luo ⁎ Department of Obstetrics and Gynecology, the First Affiliated Hospital of Jinan University, Guangzhou, 510630, China
a r t i c l e
i n f o
Article history: Received 27 November 2013 Revised 27 January 2014 Accepted 3 February 2014 Available online xxxx Keywords: Mesenchymal stem cell Magnetic resonance imaging (MRI) Gd-DTPA Trace
a b s t r a c t Objective: Three different kinds of transfection reagents were used to mediate the transfection of gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) into human umbilical-cord-derived mesenchymal stem cells (hUCMSCs). The efficacy of different transfection reagents and the feasibility of NMR tracer in vitro of magnetized stem cells were estimated. Methods: After purification by tissue explants adherent method, the biological characteristics of hUCMSCs in vitro were identified by subculture and amplification. Calcium phosphate, Effectene and liposome2000 were used to transfect Gd-DTPA-labeled hUCMSCs respectively, and cell counting was used to mediate the transfection of Gd-DTPA into hUCMSCs, which were then induced to lipoblast and osteoblast in vitro. The determination of the transfection activities of the transfection reagents was conducted by measuring the magnetic resonance imaging (MRI) signal intensity of the Gd-DTPA-labeled cells and the concentration of gadolinium ion in the cells. Furthermore, the relationship between the signal intensity of Gd-DTPA-labeled hUCMSCsMRI, cell subculture and generations was studied. Results: Primary cells were obtained by tissue explants adherent for two weeks. The cells displayed a long spindle form and grew in swirl. After two passage generations, the cellular morphology became more homogeneous. The result detected by the flow cytometer showed that CD29C, D44, CD90, and CD105 were highly expressed, while no CD45, CD40, and HLA-DR expression was detected in the third generation cells. Directional induction in vitro caused the differentiation into lipoblast and osteoblast. After transfected by calcium phosphate, Effectene and liposome 2000, the signal intensity of stem cells was 2281.2 ± 118.8, 2031.9 ± 59.7 and 1887.4 ± 40.8 measured by MRI. Differences between these three groups were statistically significant (P b 0.05). The concentrations of gadolinium ion in three groups of stem cells were 0.178 ± 0.009 mg/L, 0.158 ± 0.003 mg/L and 0.120 ± 0.002 mg/L respectively, examined by inductively coupled plasma atomic emission spectrometry. No significant differences were found among these three groups (P b 0.05). The proliferation and differentiation abilities of the Gd-DTPA-labeled stem cells were not affected. A minimum 5 × 104 Gd-DTPA-labeled stem cells could be traced with MRI in vitro and presented in high signal. The trace duration time in vitro was about 12 days. Conclusions: Tissue explants adherent method can be availably applied to purify hUCMSCs. The Effectene method was proved to have the best transfection effect. The proliferation ability and differentiation potency of Gd-DTPAlabeled hUCMSCs were not affected, and the NMR of labeled stem cells in vitro was proved to be feasible. © 2014 Elsevier Inc. All rights reserved.
1. Introduction With the rapid development of the modern medicine, stem cell research and its clinical application have been widely used in ☆ Funding: This paper is funded by China National Natural Science Foundation Project and the project number is 81070459. ⁎ Corresponding author at: Department of Obstetrics and Gynecology, the First Affiliated Hospital of Jinan University, 613 Huangpu Road West, Guangzhou, 510630 China. Tel.: + 86 150 14210277; fax: + 86 150 14210277. E-mail address: [email protected] (X. Luo). 1 These authors contributed equally to this work.
biotechnology in the 21st century [1–3]. However, how to monitor its survival distribution, migration, differentiation and other biological behaviors in vivo after stem cell transplantation is still a problem. Magnetic resonance imaging (MRI) has been used to trace transplanted cells, and cells must be effectively labeled before transplant. Presently, gadolinium chelate and super paramagnetic iron oxide (SPIO) are the most commonly used for MRI contrast agents. Compared with SPIO, gadolinium-diethylene-triamine-pentaacetic acid (Gd-DTPA) is more effective for transfection. According to the pharmacokinetic study of Gd-DTPA, if the tag targeted cells have damages or apoptosis, they will be ruled out of the body within
http://dx.doi.org/10.1016/j.mri.2014.02.008 0730-725X/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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24 hours [4]. In addition, gadolinium chelate is a paramagnetic resonance imaging contrast agent. By means of shortening the T1, the MRI signal displayed a high signal, and prominent contrast enhancement effect, easily distinguishing from the surrounding low signal cells. Shen [5] reported that 30 microliter of Gd-DTPA can effectively mark stem cell, and the marking rate reached up to 90% with no cytotoxicity. In recent years, stem cell transplant treatment effect has been widely used in animal experiments and clinical trials [6–10]. Mesenchymal stem cells (MSCs) are a group of multipotent stem cells existed in the connective tissues and the interstitial substances of organs, and could differentiate into the cells of three embryonic layers under specific conditions [11–13]. Human umbilical cord mesenchymal stem cells (hUCMSCs) were regarded as an ideal cell source for the future of stem cells in clinical treatment and scientific research [14–21]. In comparison with other mesenchymal stem cells, human umbilical cord tissue has rich MSCs, powerful proliferation ability, low immunogenicity and no tumorigenicity. Moreover, hUCMSCs was not involved in social, ethical and legal aspects of debate. The comprehensive judgment identification of HUCMSCs is based primarily on the cell morphology, surface markers and multidirectional differentiation ability. International association of cell therapy [22] maintains that ectomesenchymal stem cells cultured in vitro must meet the following conditions: adherent cell growth; cellular immune phenotypes positive rate of CD105 and CD73, CD90 must be greater than 95%, and positive rates of CD45, CD34, CD14, CD19 and HLA-DR must be less than 2%; owing the ability of differentiating into osteoblast, fat cells and chondrocytes. Hematopoietic cell surface markers of CD45 and surface symbol CD40, HLADR closely related to transplantation immune rejection, are both negative expression, which can be induced into fat cells and osteoblasts in vitro. In this study, three different transfection reagents were used to transfect Gd-DTPA marked umbilical cord mesenchymal stem cells. The changes of the biological characteristics of stem cells after magnetic marker and the use of Gd-DTPA markers human umbilical cord mesenchymal stem cells (hUCMSCs) were investigated to measure the feasibility of magnetic resonance imaging (MRI) in vitro [4,5]. 2. Materials and methods 2.1. Isolation and culture of hUCMSCs in vitro Umbilical cord specimen was obtained from the umbilical cord tissue of a health neonate delivered by full-term cesarean in the first hospital affiliated to Jinan University (the study was approved by the hospital ethics committee and informed consent was obtained from the family members of the donors). The umbilical cord tissue under aseptic conditions was collected. The tissue explants adherent method was used to isolate and purify the MSCs, and then subculture with the ratio 1:2 or 1:3 when the cell fusion rate reached 80%~90%. 2.2. hUCMSCs growth curve assay Cell proliferation ability was detected, and the growth curve was assayed by cell counting. Cell lines of the third generation were seeded in 24-well tissue culture plates (4000 cells per well). The cultures were incubated for 8 days, each day at the same time, cells from 3 random wells were counted after distribution by trypsin, using Blood Cell Counter and inverted microscope (Olympus, Japan). 2.3. hUCMSCs phenotype and the cell cycle Single-cell suspensions of the third generation cells were obtained by trypsinization (with a concentration of 0.25%). Expres-
sions of surface markers CD29, CD44, CD90, CD105, CD40, CD45 and HLA-DR, and the cell cycle were analyzed by flow cytometry (BD, USA). Atomic force microscope (Thermo Electric, USA) was used to observe hUCMSCs. 2.4. Multipotent differentiation ability of hUCMSCs Cells of the third generation were seeded in six-well plates in complete culture medium. The medium was replaced by differentiation-inducing medium when 80 ~ 90% of cytomixis was achieved. To induce fat cells differentiation: the experimental group was treated with inducing medium (containing 10% FBS, 10 μg/mL insulin, 1 μmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methyl yellow purine, 100U/mL penicillin, 100U/mL streptomycin low glucose Dulbecco's modified Eagle's medium (GIBCO, USA). Culture medium was exchanged every 3 days. Oil red "O" (Sijia biotech, Guangzhou) staining was performed after incubation for 21 days. To induce osteoblast differentiation: the experimental group was treated with osteoblast induction medium (10% FBS, 100 nmol/L dexamethasone, 10 mmol/Lβ- glycerophosphate, 0.2 mmol/L vitamin C, 100U/mL penicillin, 100U/mL streptomycin DMEM/F12 medium); medium was changed every 3 days. Alizarin red staining was performed after incubation for 21 days. 2.5. Gd-DTPA-labeling of hUCMSCs mediated by transfection reagents The third generation of T25 cells were incubated until 70 to 80% cytomixis achieved. Calcium phosphate, Effectene (QIAGEN, China) and liposome 2000 were used to mediate the entry of gadolinium ions (Bayer, Germen) into stem cells. Untransfected Gd-DTPA group was set as a control. (1) Calcium phosphate transfection: culture medium was replaced with fresh complete medium (5 mL, without antibiotics) 1 hour before the transfection. A 30 μL Gd-DTPA and 260 μL calcium chloride were mixed well with 260 μL PBS solution. After incubation at room temperature for 10 ~ 20 min, the mixture was added to T25 culture flasks (Corning, USA). The cells were incubated at 37°C in 5% CO2 for 4 hours, washed three times by PBS, detached by trypsin and then collected in the EP tube. MRI imaging was performed by measuring signal strength of the cells in a plastic container filled with water. (2) Effectene and Lipofectamine 2000 were used in the transfection of gadolinium ions. After MRI imaging analysis, the concentrations of gadolinium ion in stem cells transfected by different reagents were examined by inductively coupled plasma atomic emission spectrometry. 2.6. MR imaging MRI of these tubes was performed with a clinical 1.5-T MR imager (GE, USA), by using an 8-channel wrist coil. Imaging parameters are as follows: SE T1WI sequence: TR were 300 ms, 600 ms, 900 ms, 1200 ms, respectively; images were obtained with a matrix size of 256 × 256, TE = 15 ms, two measurements acquired, layer distance/thickness = 1.0/0 mm and field of view of 12 × 9 cm. SE T1WI sequence: TR/TE = 1500/88 ms, layer distance/thickness = 1.0/0 mm, images were obtained with a matrix size of 384 × 192 with a field of view of 12 × 12 cm, two measurements were acquired. Region of interest for signal intensity (SI) measurement was 1 × 1 mm. 3. Statistical analysis Data were expressed as mean and standard deviation (x± S). The t test or variance analysis was used, and all of the statistics analyses were performed using SPSS17.0 (information of the software), test level α = 0.05, and P b 0.05 was considered to be statistically significant.
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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4. Results 4.1. hUCMSCs isolation and morphological characteristics One week after planting, cells could be seen with newly formed colonies (Fig. 1A). After first passage, the fibroblast-like MSCs uniformly distributed in the cell culture flasks. Spherical cells suspended in culture medium could be observed using inverted phase contrast microscope after passage. Twenty-four hours later, most of the cells were short rod-like or long fusiform-like and adhered to the flask wall. By changing medium many times and passages, miscellaneous cells were eliminated, and the uniform fibroblast-like cells left were umbilical cord blood MSCs (Fig. 1B). These cells could be further cultured, passed and proliferated rapidly. Most of the cells were long fusiform-like, clear boundaries between cytoplasm and nucleus, and a prominent nucleoli could be seen. The cell volume was large, with a length of more than 100 μm. Numerous projections could be seen around the cell (Fig. 1C).
4.2. Identification of hUCMSCs surface markers Antigenic identification of the 3rd generation of hUCMSCs cells was investigated by flow cytometry analysis. The results demon-
A
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strated high positive signals for hUCMSCs cell markers CD29 (β1 integrin), CD44 (the hyaluronate receptor) and CD105 (endoglin/ SH2). The hUCMSCs cells show a negative signal for cell markers CD40 (TNF-receptor superfamily) and HLA-DR associated with allograft rejection on the surface of hUCMSCs cells (Fig. 2). 4.3. Proliferative capacity of Gd-DTPA labeled hUCMSCs The growth curves of the experiment group and the control group were both S" shaped (Fig. 3A). After inoculation for 1 to 4 days, the cells were in a incubation period and grew slowly; then cells went into the logarithmic growth phase; cell growth rate gradually slowed down from the sixth day. No significant difference between the two groups was observed (P N 0.05), suggesting that gadolinium ions do not affect the proliferation activity of the hUCMSCs. 4.4. Cell cycle of Gd-DTPA labeled hUCMSCs Distributions of cells in different stages were analyzed by flow cytometry. For the labeled hUCMSCs, G1/G0 phase cells accounted for (61.267 ± 4.488)%, S phase cells accounting (23.567 ± 1.401)%, and G2/M phase cells accounted for (15.167 ± 4.631)%. As for the hUCMSCs, G1/G0 phase cells accounted for (65.100 ± 6.756)%, S
B
250µm
C
D
Fig. 1. The morphology of hUCMSCs cells. Structure of primary hUCMSCs (A) the 3rd genetation of hUCMSCs (B) under general microscope; C, D structure of hUCMSCs under atomic force microscope.
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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Fig. 2. Identification of hUCMSCs surface markers. Antigenic identification of the 3rd generation of hUCMSCs cells were investigated by flow cytometry analysis.
phase cells accounted for (15.367 ± 5.014)%, and G2/M phase cells accounted for (19.567 ± 2.013)%. Random block design analysis of variance was used, and the difference between the two groups was not statistically significant (F = 0.00, P = 1.00), (Fig. 3B).
4.5. Gd-DTPA labeled hUCMSCs differentiation in vitro testing It has been reported that hUCMSCs may differentiate along several cell lineages in all three germ layers including adipogenic and osteogenic [11–13]. Adipogenic and osteogenic mediums were used to test the differentiation ability of Gd-DTPA labeled hUCMSCs. As shown in Fig. 4A and B, lipid droplets of adipogenic was stained red by oil red "O," and mineralized nodules of osteogenic was stained red by alizarin red.
4.6. MR imaging After transfection, MSCs were trypsinized, centrifuged in tubes (1 × 10 6 cells each), and scanned by MR. Regions of interest (ROI) were used to measure the signal strength of T1WI sequence cells. Signals of Gd-DTPA labeled hUCMSCs were white high in T1WI (Fig. 5A) and low in T2WI (Fig. 5B); signals of unlabeled hUCMSC were both low in T1WI and T2WI. Signal intensity of the three labeled groups: calcium phosphate, Effectene, Lipofectamine 2000 were 2281.2 ± 118.8, 2031.9 ± 59.7, 1887.4 ± 40.8, respectively. The blank control group was 1306.7 ± 52.0. Variance analysis of completely randomized design was performed. Differences between the four groups were statistically significant (F = 93.1, P b 0.05), suggesting that the three transfection agents successfully transfected Gd-DTPA into stem cells.
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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Fig. 3. Identification of Gd-DTPA labeled hUCMSCs. A. Growth curves of hUCMSCs with and without Gd-DTPA label. B. Cell cycle of hUCMSCs with and without GdDTPA label.
4.7. Gadolinium ion concentration measurement of stem cells by inductively coupled plasma atomic emission spectrometry
4.9. Minimum amount of magnetically labeled hUCMSCs detected by MRI
Gadolinium ion concentrations of the three groups, calcium phosphate, Effectene, Lipofectamine 2000 t were (0.178 ± 0.009) mg/L, (0.158 ± 0.003) mg/L, and (0.120 ± 0.002) mg/L. Completely randomized design analysis of variance was performed, and differences between the four groups were statistically significant (Fig. 6).
The concentrations of magnetic cells were set to be 1 × 10 5/mL, 5 × 10 4/mL, 2.5 × 10 4/ml, 1 × 10 4/mL and 5 × 10 3/mL, respectively. When the concentration of magnetic cells was 2.5 × 10 4/mL, no high T1WI signal intensity could be seen compared with that of the blank control (Fig. 7C). The minimum amount of magnetically labeled hUCMSCs that could be detected was 5 × 10 4/mL.
4.8. Effect of cell proliferation on the signal intensity of gadolinium labeled hUCMSCs
5. Discussion
Gd-DTPA labeled stem cells were scanned by MR. The TIWI signal intensity was 2031.9 ± 59.7. T1WI signal intensity was gradually decreasing with the cells subculture and proliferation. At the 12th day (third generation), the T1WI signal intensity of cells was 1251.1 ± 42.0 (Fig. 7A). No significant difference was found by the independent-sample t test (P N 0.05). The tracing time for the magnetically labeled stem cells was about 12 days (Fig. 7B).
Transplantation of stem cell had an insight into the aspect of biological behavior, such as its survival distribution, migration and differentiation in the body, which was important for evaluating stem cell transplantation effect. Based on the extensive research efforts, a large number of studies have demonstrated that MRI is the best way for traced cells in vivo [23,24]. In this experiment, 1.5 T magnetic resonance imaging (MRI) was applied to mark and measure the different transfection medium of hUCMSCs in in vitro research. Through effective transfection of Gd-DTPA tagged hUCMSCs, testing
Fig. 4. Gd-DTPA labeled hUCMSCs differentiation in vitro testing. Lipid droplets of adipogenic was staining red by oil red "O," and mineralized nodules of osteogenic was staining red by alizarin red. A. Cell differentiation to lipoblast with Gd-DTPA label (400×). B. Cell differentiation to osteoblast with Gd-DTPA label (200×).
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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Fig. 5. MRI of Gd-DTPA labeled hUCMSCs. A. T1 weighted imaging from left to right, calcium phosphate, Effectene and liposome and blank. B. T2 weighted imaging from left to right, calcium phosphate, Effectene and liposome and blank.
its growth curve, cell cycle and multi-directional differentiation capacity, we could draw the conclusion that the biological characteristics and multi-directional differentiation potential of stem cells have not been affected after marked by Gd-DTPA. Gd-DTPA and cell membrane surface are negatively charged, and it is hard to enter cells without the surface modification of Gd-DTPA. In this experiment, we chose calcium phosphate liposome, Effectene and liposomes 2000 as transfection agent for transfection. Through MRI signal intensity testing and inductively coupled plasma atomic emission spectrometry screening, calcium phosphate liposome, Effectene group, liposomes 2000 group, and blank control group were all statistically significant (P b 0.05) among the four groups. The strongest signal is the calcium phosphate group (2281.2 ±
Fig. 7. Effect of cell proliferation on the signal intensity of Gd-DTPA labeled hUCMSCs. A. T1 weighted imaging from left to right: generation P1, P2, P3, P4, blank control. B. T2 weighted imaging from left to right: generation P1, P2, P3, P4, blank control. C. The minimum hUCMSCs traced by MRI. The concentrations of cells from left to right were 105, 5 × 104, 2.5 × 104, 104, 5 × 103.
Fig. 6. Gadolinium ion concentration measurement of stem cells by inductively coupled plasma atomic emission spectrometry.
118.8), followed by Effectene and liposomes 2000 group, which is consistent with the results of inductively coupled plasma atomic emission spectrometry screening. However, we found that calcium phosphate in the experimental process was easy to form calcium phosphate small precipitation after 4 hours, which affects the cell morphology. Based on the comprehensive comparison, Effectene is the better Gd-DTPA transfection reagents. Gadolinium labeled stem cells were set to be five gradients: 1 × 10 5/ml, 5 × 10 4/ml, 2.5 × 10 4/ml, 1 × 10 4/ml and 5 × 10 3/ml, and the results showed that with the increase of cell number, MRI signal intensity increased, indicating that there was a certain relationship between the signal strength and signed cells. The detection
Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008
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limit of magnetic signal cell labeling number is 5 × 10 4/ml using MRI. Given the number of cell transplantation in clinical application is far greater than the minimum cell count, thus the imaging cells threshold is enough to meet the needs of the clinical application. In this experiment, by means of dynamic tracer of 1 × 10 6 gadolinium tag stem cells, we found that MRI signal strength decreases gradually as the cells proliferate, and the reason may be contributed to dilution effect of the cell's proliferation and elimination impact of cell metabolism, gadolinium ions gradually ruling out of the body, indicating that MRI signal intensity has relation to the cell proliferation. When cells reproduce to the fourth generation, there was no obvious difference by MRI between the magnetic marker stem cells signal intensity and untagged signal intensity. Moreover, magnetic markers of stem cells in vitro can trace to the time prolonging to 12 days. In conclusion, Effectene was the best agent for transfection GdDTPA tag hUCMSCs, and the proliferation and multi-directional differentiation potential of marked stem cell has not been affected. References [1] Horibe EK, Sacks J, Unadkat J, Raimondi G, Wang Z, Ikeguchi R. Rapamycinconditioned, alloantigen-pulsed dendritic cells promote indefinite survival of vascularized skin allografts in association with T regulatory cell expansion. Transpl Immunol 2008;18:307–18. [2] Forraz N, Pettengell R, McGuckin CP. Hematopoietic and neuroglial pro-genitors are promoted during cord blood ex vivo expansion. Br J Haematol 2002;119:888. [3] Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–9. [4] Shyu WC, Chen CP, Lin SZ, Lee YJ, Li H. Efficient tracking of non-iron-labeled mesenchymal stem cells with serial MRI in chronic stroke rats. Stroke 2007;38:367–74. [5] Shen J, Cheng LN, Zhong XM, Duan XH, Guo RM, Hong GB. Efficient in vitro labeling rabbit neural stem cell with paramagnetic Gd-DTPA and fluorescent substance. Eur J Radiol 2010;75:397–405. [6] Maijenburg MW, Gilissen C, Melief SM, Kleijer M, Weijer K, Ten Brinke A, et al. Nuclear receptors Nur77 and Nurr1 modulate mesenchymal stromal cell migration. Stem Cells Dev 2012;21(2):228–38. [7] Zhang MJ, Sun JJ, Qian L, Liu Z, Zhang Z, Cao W, et al. Human umbilical mesenchymal stem cells enhance the expression of neurotrophic factors and protect ataxic mice. Brain Res 2011;1402(7):122–31.
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Please cite this article as: Shuai H-L, et al, Analysis of feasibility of in vitro nuclear magnetic resonance tracking human umbilical cord mesenchymal stem cells by Gd-DTPA labeled, Magn Reson Imaging (2014), http://dx.doi.org/10.1016/j.mri.2014.02.008