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Biologic properties of gadolinium diethylenetriaminepentaacetic acid-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells
Cytotherapy, 2013; 0: 1e10
Biologic properties of gadolinium diethylenetriaminepentaacetic acid-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells
HUA PAN1,*, JIANFA LAN2,*, XIN LUO3, JUN GAO2, XIAODONG XIE1 & HONGYU GUO2 1
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu Province, China, 2Department of Gynaecology, Lanzhou University Second Hospital, Lanzhou, Gansu Province, China, and 3Department of Gynecology and Obstetrics, First Affiliated Hospital Jinan University, Guangzhou, Guangdong Province, China
Abstract Background aims. This study was conducted to characterize gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA)labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells (HuMSCs) and to track them with magnetic resonance imaging (MRI) in vitro and in vivo. Methods. HuMSCs were isolated from umbilical cords and expanded in vitro. Cells were sequentially labeled with Gd-DTPA and PKH26. The labeling efficiency was determined by spectrophotometry measurements, and the longevity of Gd-DTPA maintenance was measured with MRI. The influence of double labeling on cellular biologic properties was assessed by cell proliferation, viability, differentiation, cycle and apoptosis. Transplantation of double-labeled HuMSCs or placebo was performed in 39 female Sprague-Dawley rats. Leak point pressure and maximal bladder capacity were measured in animals 6 weeks after injection. Results. The T1 values and signal intensity on T1-weighted imaging of labeled cells were significantly higher than the control group (P < 0.05). The signal intensity on T1-weighted imaging of labeled cells was retained >14 days in vitro and in vivo. There was no significant difference in the cell cycle, cell apoptosis, cell proliferation and cell viability between labeled and unlabeled HuMSCs (P > 0.05). After double labeling, HuMSCs were still capable of differentiating into osteoblasts and adipocytes. Periurethrally injected HuMSCs in the rats significantly improved leak point pressure and maximal bladder capacity. Conclusions. HuMSCs were successfully labeled with Gd-DTPA and PKH26. This labeling method is reliable and efficient and can be applied for tracking cells in vitro and in vivo without altering cellular biologic properties. Key Words: cell labeling, Gd-DTPA, HuMSCs, MRI, PKH26
Introduction Stem cell-based regenerative medicine has been intensively studied in both pre-clinical and clinical settings in recent decades. It is essential to establish an ideal technique to monitor the cell status in the recipient after transplantation (1,2). Numerous techniques have been developed for tracking mesenchymal stromal cells (MSCs) in vitro or in vivo, such as fluorescence imaging, ultrasound, isotope labels and magnetic resonance imaging (MRI) (3). Fluorescence imaging has the limitation of detection depth for inner organs, and it requires tissue samples for detection (4). Limitations of ultrasound include accuracy in cell quantification and special resolution (3,5). Isotopes for labeling can lead to radiation injury of stem cells (6). However, MRI not only is a
non-invasive technique to monitor the movement of cells in vivo, but it also provides real-time images of the cells in internal organs. It has been widely used as a cell tracking technique in recent years (7e9). An ideal method for in vivo evaluation of cell distribution and its dynamics is extremely desirable for a successful cellular transplantation treatment. The fate of the transplanted cells within the host should be non-invasively, repeatedly and specifically determined. We labeled HuMSCs with standard contrast agents for MRI to track the homing and engraftment of transplanted cells. To obtain better MRI results, the key factor is to select contrast agents. The T2 contrast agents, such as superparamagnetic iron oxide particles (SPIOs),
*These authors contributed equally to this work. Correspondence: Dr Jianfa Lan, Department of Gynaecology, Lanzhou University Second Hospital, Lanzhou, Gansu Province 730030, China. E-mail: [email protected]; Professor Xiaodong Xie, Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu Province 730000, China. E-mail: [email protected] (Received 22 March 2013; accepted 20 May 2013) ISSN 1465-3249 Copyright Ó 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.05.015
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which have high T2 relaxivities, have been widely used for cell labeling during the past 15 years. The high susceptibility of SPIOs is different from other contrast agents, and several studies have demonstrated their high sensitivity for detecting transplanted cells in vivo (10,11). However, because SPIOs could enhance the dark background, SPIOderived images are unable to distinguish cells precisely in tissue, especially under hemorrhagic conditions. In addition, the particulate nature of SPIOs can induce phagocytosis by activating reticulohistiocytic cells, which would cause false-positive images. This phenomenon was recognized and documented by Cahill et al. (11). A study by Baligand et al. (12) suggested that iron-containing particles should be used with extreme caution to evaluate the presence of grafted cells in vivo, and such particles are unsuitable for the long-term tracing the transplanted cells. Gadolinium (Gd) is a contrast agent that has been used extensively in a clinical setting. Use of contrast agents with T1-weighted MRI enhances the ability to produce positive signal intensity in images, facilitating tracking cells in low signal tissues. Among these, Gd is the most effective T1 contrast agent for clinical use. Gd usually forms a complex with the chelating ligand, diethylenetriaminepentaacetic acid (DTPA), which is used for MRI. In biologic tissues, T1 relaxivity is longer than that of T2, and contrast enhancement is better observed with T1-weighted sequences. HuMSCs have many advantageous characteristics as seed cells, such as high proliferative activity, low immunogenicity and non-tumorigenic nature. The potential clinical application of HuMSCs in the fields of tissue engineering and gene therapy has become an area of active stem cell research. Although HuMSCs are widely used as the source of cells for tissue repair or regeneration, their actual function and migration after they are injected into the human body are unknown. To determine the cellular biologic properties and bio-distribution of therapeutic stem cells, it is crucial to develop a safe, reproducible and non-invasive technique to trace them in vivo. In this study, HuMSCs were doublelabeled with Gd-DTPA and PKH26. Parallel studies were performed with labeled and unlabeled cells to identify the possible effects of Gd-DTPA and PKH26 labeling on biologic properties of transplanted HuMSCs, including cell viability, proliferation, differentiation and apoptosis.
Methods Rat model of stress urinary incontinence All experimental protocols were approved by the Chinese Institution of Animal Care Committee at
our institution. We obtained 39 female SpragueDawley rats weighing 180e200 g from the Medical Laboratory Animal Center of Guangdong Province, China. The rats were subjected to development of stress urinary incontinence (SUI) voiding dysfunction by a previously described procedure (13). Briefly, immediately after delivery, the rats underwent vaginal balloon dilation to simulate prolonged labor and dystocia. The rats were anesthetized 2 weeks later, and a midline incision was made in the abdomen and both ovaries were excised. The rats were subjected 1 month later to leak point pressure (LPP) and maximal bladder capacity (MBC) measurement with urodynamic examination. HuMSC isolation and ex vivo expansion Healthy term fetal umbilical cord was collected after cesarean section from informed, consenting mothers and processed as quickly as possible. All umbilical cords were obtained from the Department of Obstetrics at First Affiliated Hospital of Jinan University. Cell isolation, culture and ex vivo expansion were performed as previously described (14). Briefly, immediately after an umbilical cord was collected, the blood was removed, and the cord was cut into 1-mm3 pieces. The chopped tissue was seeded in the culture dish with Dulbecco’s modified Eagle’s medium/Nutrient Mixture F12 (Gibco DMEM/F12; Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 15 mmol/L 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid. The pieces of cord were subsequently incubated at 37 C in humid air with 5% CO2. After 1 week, a few drops of fresh medium were placed in the culture dish every 2e3 days. As soon as enough cellular colonies formed around the chopped tissues, the tissue blocks were removed by changing the culture medium. When cells reached 70e80% confluency, they were trypsinized for subculture. Cell labeling A non-liposomal lipid transfection reagent, Effectene transfection reagent (Qiagen, Hilden, Germany), was used to transfect Gd-DTPA (Magnevist; Schering, Berlin, Germany) into HuMSCs. The encapsulation of Gd-DTPA with cationic lipids, which were formed when negative charged Gd-DTPA particles mixed with Effectene, could be efficiently transferred into cells. To optimize the transfection condition, we performed a pilot test involving various dosages of Gd-DTPA and Effectene and various incubation periods. An incubation time of 4 h and 25 mL GdDTPA and 25 mL Effectene were considered a good
Biologic properties of Gd-DTPA- and PKH26-labeled HuMSCs compromise between labeling effectiveness and cell preservation. This condition of labeling was applied in the present study. In a 25 cm2 tissue culture flask containing 5 mL DMEM/F12 medium, 25 mL GdDTPA (0.5 mol/L) and 25 mL Effectene were added to 1 106 HuMSCs. After 4 h of incubation under standard cell culture conditions, PHK26 (2 108 mol/L) (Sigma-Aldrich, St Louis, MO, USA) was added to the medium for fluorescent labeling following the manufacturer’s instructions. The blank control was 1 106 unlabeled HuMSCs, which were incubated in regular culture medium without addition of any labeling agents. Cell viability, proliferation and differentiation After double labeling, cell viability and proliferation were assessed with trypan blue dye exclusion test and Cell Counting Kit-8 (CCK-8; BiYunTian Biological Technology Research Institute, Chengdu, China) assay. The cell viability was determined by trypan blue exclusion assay at times 0 h (before labeling), 8 h, 24 h and 72 h after initial labeling. There were five cell samples in each group at each time point. For CCK-8 assay, HuMSCs were cultured in flat-bottom 96-well plates at 1 103 cells per well, and cells in 50% of the wells were labeled. Cells in the remaining wells were not labeled and served as controls. After labeling, the cells were washed twice with phosphate-buffered saline (PBS), and 100 mL of DMEM/F12 with 10% FBS was added to each well. The plate was kept in the incubator under standard culture conditions. At each time point of day 1, day 3 and day 6 of incubation, the samples of labeled and unlabeled cells were underwent CCK-8 assay. Each well had 10 mL of CCK-8 added, and cells were incubated for 1 h and measured at a wavelength of 680 nm with 420 nm as the subtracted reference. Adipogenic differentiation was performed with special medium. Double-labeled HuMSCs were inoculated at 1 105 mL1 placed sterile coverslips in six-well plates. When cells grew to 100% fusion, the induced group was exchanged for adipogenic differentiation induced solution (containing 10% FBS, 5 mg/L insulin, 1 mmol/L dexamethasone, 0.5 mmol/L 1-methyl-3-isobutylxanthine and 100 mmol/L indomethacin). The control group was cultured with standard stem cell medium. Both groups were cultured for 21 days with regular medium changes (twice a week). At the end of induction, cells were stained with oil red O and observed under microscopy. For osteogenic differentiation, double-labeled HuMSCs (1 104 mL1 cells) were seeded in sixwell plates until the cells grew to 80% confluence, and the induced group was exchanged for osteogenic
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differentiation-induced solution (medium containing 10% FBS, 100 nmol/L dexamethasone, 100 mmol/L b-glycerophosphate and 0.2 mmol/L ascorbic acid). The negative control group was cultured with standard stem cell medium. Both groups were continuously cultured for 28 days with regular medium changes (twice a week). At the end of induction, cells were stained with alizarin red and observed under microscopy. Flow cytometry detected cell cycle and cell apoptosis The phenotype of HuMSCs was detected with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with BD CellQuest Pro software (BD Biosciences) as previously described (14). Fluorescence-activated cell sorter (FACS) was also used for the detection of cell cycle and apoptosis. Unlabeled or labeled cells (1 106) were centrifuged. After washing with PBS, RNase was added to remove RNA, and cells were stained with propidium iodide dye at 4 C dark for 30 min. The cell cycle was then detected with FACS. Unlabeled or labeled cells were adjusted to a concentration of 1 106/mL. In a 5-mL flow tube, 100 mL of cell suspension was mixed with 5 mL Annexin V-fluorescein isothiocyanate and 10 mL (20 mg/mL) propidium iodide solution. After 15 min incubation at room temperature, 400 mL PBS was added, and cell apoptosis was analyzed by FACS. Spectrophotometry Labeling efficiency was determined by spectrophotometry measurements of the cellular uptake of paramagnetic Gd-DTPA particles (15). Briefly, after labeling, cells were washed three times with PBS and then re-suspended in PBS. The Gd-DTPA concentration within labeled cells was measured with inductively coupled plasma atomic emission spectrometry (IRIS-HR) using a polarized Zeeman atomic absorption spectrometer (Thermo Jarrell Ash, Boston, MA, USA). The labeling efficiency was determined as ratio of the percentage of Gd-DTPA concentration within labeled cells to the concentration of Gd-DTPA that was initially added to the culture medium for labeling. Fluorescent microscopy and electron microscopy After labeling, HuMSCs were washed three times with PBS. One aliquot of PKH26-labeled cells was detected by fluorescent microscopy (Nikon, Tokyo, Japan). Another aliquot of labeled cells was fixed in 3% glutaraldehyde-cacodylate buffer at 3 C for overnight. After being fixed for 1 h in 1% osmium tetroxide, cells were dehydrated in graded dilutions of ethanol, embedded in artificial resin (Merck,
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Figure 1. Labeled and unlabeled cells under different microscopy. (A) Inverted microscopy (40). (B) PKH26-labeled cells under fluorescent microscopy (40). (C, D) Gd-DTPA-labeled cells showed clusters of Gd-DTPA particles existing within cytoplasm. No Gd-DTPA particles were visible within unlabeled cells under transmission electron microscopy (15,500).
Darmstadt, Germany) and processed for electron microscopy. Thin sections of the cell probes were evaluated unstained to prevent false-positive findings. Electron microscopy was performed at 60e80 kV.
Figure 2. Gd-DTPA-labeled HuMSCs were tracked by MRI in vitro. (A1, A2) Cell pellets (1 106) within test tubes were from (left to right) unlabeled cells, Effectene-labeled cells, Gd-labeled cells and Effectene-Gd-labeled cells. (B1, B2) Longevity of maintenance in labeled cells. After labeling, 2 106 cells were cultured and passaged under normal condition. On T1-weighted imaging, passages of labeled cells remained at high signal intensity at 1, 7 and 14 days after the initial labeling procedure; signal intensity disappeared on 15th day. (C1, C2) Minimal detectable number of labeled cells on MRI in vitro. The signal intensity became gradually stronger with the increasing number of Gd-labeled cells on the T1weighted image. The signal intensity of 5 103 Gd-labeled cells was almost the same as the signal intensity of 5 103 unlabeled cells.
In vitro MRI MRI was performed with a clinical 1.5T MRI scanner (Philips Medical Systems, Best, Netherlands), and a circular surface coil with a diameter of 11 cm was used (16). Briefly, after labeling, HuMSCs (1 106) were washed three times with PBS. The cell pellets within the test tubes were imaged. The tubes were placed in a plastic box filled with 37 C water to ensure signal homogeneity and to avoid potential susceptibility artifacts from surrounding air. Labeled and unlabeled HuMSCs were included in each subset of imaging analysis. In addition, the supernatant from labeled HuMSCs was collected and imaged to determine whether there was residual Gd-DTPA in labeled HuMSCs. For MRI pulse sequences, refer to Shen et al. (16). To determine the sensitivity of this application, the decreasing numbers of labeled cells and unlabeled cells (1 106, 5 105, 1 105, 1 104, 5 103) were collected in 1.0-mL eppendorf tubes, re-suspended in 50 mL 4% gelatin solution and imaged by MRI.
Figure 3. Graph shows the minimal detectable number of labeled cells. Data were expressed as mean SD. *P < 0.05 compared with unlabeled cells.
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Figure 4. Graphs show cell viability and proliferation of labeled and unlabeled cells. (A) Cell viability was tested by trypan blue dye exclusion. (B) Cell proliferation was tested by CCK-8.
Transplantation of HuMSCs
In vivo MRI
HuMSCs were transplanted at 8 h after Gd-DTPA and PKH26 labeling. The negative control group was 12 normal rats (group A). We randomly assigned 24 SUI rats that were balloon-injured and ovariectomized into the positive control group (group B, n ¼ 12) and the urethra-HuMSCs group (group C, n ¼ 12). The control animals (groups A and B) received injection of 300 mL PBS in the urethra at 3 o’clock, 6 o’clock and 9 o’clock positions at the bladder urethra junction. Group C received injection of 4.5 106 double-labeled HuMSCs (soluble in 300 mL PBS) at the same positions. In addition, three rats received injection of 4.5 106 unlabeled HuMSCs (in 300 mL of PBS) in the urethra as a control for MRI.
Rats were examined on days 1, 7 and 14 after placebo and labeled or unlabeled HuMSC transplantation. Rats were anesthetized with 10% chloral hydrate and scanned in a rat MRI coil. LPP and MBC test LPP and MBC were quantified before transplantation and at 6 weeks after implantation of HuMSCs. Animals were anesthetized with chloral hydrate as previously described (13). Briefly, an epidural catheter was connected through a three-way pipe to a BL-410 biologic function experiment system instrument (Tai Meng Science and Technology,
Figure 5. Staining identification of adipogenic and osteogenic differentiation. Oil red O staining of adipogenic differentiation at 21 days (A) and control (B). Alizarin red staining of osteogenic differentiation at 28 days (C) and control (D).
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Table I. Determination of apoptosis of labeled and unlabeled human umbilical cord mesenchymal stromal cells with fluorescence-activated cell sorter.
24 h Labeled cells Unlabeled cells P value 72 h Labeled cells Unlabeled cells P value
Normal cells (%)
Early apoptotic cells (%)
Late apoptotic cells (%)
Dead cells (%)
91.581 2.901 92.127 2.896 0.8289
6.049 0.428 5.893 0.369 0.6575
1.398 0.170 1.148 0.164 0.1407
0.967 0.156 0.916 0.190 0.7375
95.200 3.01 94.848 2.98 0.8925
2.200 0.567 2.492 0.345 0.4885
1.511 0.189 1.531 0.200 0.9059
1.101 0.123 1.110 0.167 0.9437
Flow cytometry detected cell apoptosis of labeled and unlabeled cells at 24 h and 72 h.
Chengdu, China), and another interface connected a 1-mL syringe, with sterile saline filling the three-way pipe guaranteeing the pipeline air free. Methylene blue fluid was injected into the bladder using a syringe at a speed of 0.5 mL/min. When the first blue liquid drop was observed in the urethral meatus, the bladder capacity was recorded, which was the MBC of the rat. At the same time, bladder pressure curve recording and regulatory experiment were started. This curve was also the rat LPP diagram. Each rat was tested three times. Immunofluorescence and tracking of PKH26-labeled HuMSCs After HuMSC transplantation, three rats from each experimental group were sacrificed at day 1, day 7 and day 42. The bladder-urethra complex was removed en bloc. Frozen tissue sections were processed to visualize red fluorescence. Statistical analysis Statistical analysis was performed using SPSS 14.0 software for Windows (IBM, Chicago, IL, USA). A P value of <0.05 was considered to indicate a statistically significant difference. Cell apoptosis, cell cycle, trypan blue exclusion assay for toxicity and CCK8-based proliferation data were expressed as percentage of the corresponding unlabeled control cells, and analysis of variance significance was performed to test for statistical significance. Comparisons of signal Table II. Cell cycle determination of human umbilical cord mesenchymal stromal cells with fluorescence-activated cell sorter. Phase G0/G1 (%)
S (%)
G2/M (%)
7 days Labeled cells 66.66 3.073 12.65 1.683 21.09 1.98 Unlabeled cells 67.10 2.103 12.30 1.283 20.78 1.38 P value 0.8478 0.7888 0.8348 Results are shown of cell cycle between the labeled and unlabeled group after cells were labeled on the 7th day.
intensity and T1 values between unlabeled and labeled cells were performed by using the unpaired Student t test. Results Cellular surface markers HuMSC surface marker antigen expressions were positive for CD29, CD44, CD90 and CD105 and negative for CD31, CD34 and CD45, which represent hematopoietic cells. Surface marker antigen expression was extremely low for graft-versus-host disease related factor HLA-DR CD40, explaining its extremely low immunogenicity. Cell labeling After labeling, red fluorescence was observed in most labeled cells under fluorescent microscopy. PKH26 labeling efficiency was detected to be 99%, and red fluorescence was still detectable on cells at passage 10. As measured by atomic emission spectrophotometry, Effectene-Gd-DTPA labeling efficiency was detected to be 85%. Black high-density Gd-DTPA particles were contained and compartmentalized within lysosomes of labeled cells under electron microscopy, whereas no such particles were visible in unlabeled cells (Figure 1). Effectene-Gd-DTPA-labeled cells showed high signal intensity on T1-weighted imaging and low signal intensity on T2-weighted imaging (Figure 2). The signal intensity in Effectene-Gd-DTPA-labeled cells was significantly high compared with unlabeled cells and cells incubated with Gd-DTPA or Effectene alone (Figure 2). The T1 values of Effectene-GdDTPA-labeled cells, cells with Gd-DTPA, cells with Effectene and unlabeled cells were 1125 181, 734 86, 410 97 and 401 37. The signal intensity of labeled cells was significantly higher than the signal intensity of negative control and blank control cells (P < 0.01). Strong signal still could be seen on the 14th day after cells were labeled in vitro (Figure 2). Increasing numbers of labeled cells were suspended in 50 mL gelatin (Figure 2). After diluting in
Biologic properties of Gd-DTPA- and PKH26-labeled HuMSCs
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Figure 6. Double-labeled HuMSCs in the urethra of rats were detected by MRI after transplantation. There was no high signal in unlabeled cells (A). Fat-suppressed T1-weighted images of labeled cells (B1eB3) showed high signal (arrows) that gradually diminished. T2-weighted images showed low signal (arrows) in all specimens treated with HuMSCs (C, D1eD3).
50 mL 4% gelatin solution, the signal intensity on T1weigted imaging of 1 106, 5 105, 1 105, 1 104 and 5 103 HuMSCs was statistically higher than the counterparts of unlabeled cells (P < 0.05), but no significantly higher signal intensity was found between the 5 103 labeled cells and unlabeled cells (P > 0.05) (Figure 3). The minimal detectable number of labeled cells was 1 104 cells. Biologic properties of HuMSCs Trypan blue dye exclusion test showed that there was no reduction in cell viability comparing labeled cells and unlabeled cells (P > 0.05) (Figure 4). CCK-8 assay showed no statistical difference between labeled and unlabeled cells (P > 0.05) (Figure 4). During adipogenic differentiation, with the extension of induction time, the cells began to exhibit vacuolar lipid droplets, which gradually became larger. The cell shape was gradually changed from fibrous to circular or oval. After oil red O staining, oil droplets
were observed in the cytoplasm under the microscope (Figure 5). During osteogenesis induced differentiation, with the extension of induction time, the cells began to exhibit growth of stacked aggregation. The place of cell fusion appeared as a “mesh shape.” After 2 weeks of continuous induction, multiple mineralized nodules were observed. Subsequently, nodules increased gradually and merged with calcified nodules, which were red with Alizarin red staining, until 28 days of induction. This phenomenon was not observed in the cells without special induction (Figure 5). Flow cytometry detected cell apoptosis of labeled and unlabeled cells at 24 h and 72 h. The results are shown in Table I. There were various cell proportions of the labeled group and unlabeled group but no statistical significance (P > 0.05). Table II shows the results of labeled and unlabeled cell cycles. The difference of each period cell proportion between the two groups of cells was not statistically significant (P > 0.05).
Figure 7. Gd-DTPA-labeled and PKH26-labeled HuMSCs in the urethra of rats were tracked in vivo. Frozen sections of intermediate urethra after transplantation are shown under fluorescent microscopy (100) at 1 day (A), 1 week (B) and 6 weeks (C).
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Figure 8. LPP was tested 6 weeks after implantation of HuMSCs or placebo. Group A comprised normal rats, and the average value of LPP was 21.72 mm Hg. Group C comprised 12 SUI rats, and the average value of LPP was 20.18 mm Hg. Group B comprised 12 SUI rats, and the average value of LPP was 13.82 mm Hg.
In vivo MRI and fluorescent microscopy The double-labeled HuMSCs in the rat urethra were distinctly detected by the hyperintense signal on T1weighted imaging after transplantation. The hyperintense signal gradually decreased but remained for 2 weeks. However, no obvious hyperintense signal was found on T1-weighted imaging 1e14 days after transplantation in the unlabeled groups. Low signal intensities were found around the urethra on T2weighted imaging in both experimental and control groups (Figure 6). Frozen tissue sections showed that PKH26-labeled HuMSCs can be tracked for >6 weeks (Figure 7). LPP and MBC test As demonstrated in Figure 8 and Table III, LPP and MBC of HuMSC-transplanted SUI are statistically significantly different from the SUI placebo model (P < 0.01). Discussion Cell tracking plays an important part in stem cell therapy-related studies. A reliable and reproducible labeling method is a prerequisite for monitoring transplanted cells and investigating their biologic properties. MRI, a non-invasive method with high anatomic resolution, has been widely used to track the migration and fate of transplanted stem cells (17,18). At the present time, Gd-based paramagnetic contrast agents or iron oxide-based superparamagnetic
contrast agents are mainly used for cell tracking with MRI. Advantages of Gd have been mentioned previously. In addition, T1-sensitive contrast agents can track the distribution of freshly implanted cells more accurately than T2-wighted ferric oxide nuclear protein-labeled contrast agents (19). There is much more than convenience, safety and feasibility considerations to encourage investigators to opt for Gd chelate loading in many conditions. Gd-DTPA is a clinical standard contrast agent and has been used extensively in various clinical applications. In this study, we selected Gd-DTPA and PKH26 as labeling probes that are commercially Table III. Leak point pressure and maximal bladder capacity quantified before implantation and after implantation.
1 week Group A Group B Group C P value 6th week Group A Group B Group C P value
Average leak point pressure (mm Hg)
Maximal bladder capacity (mL)
23.15 1.21 13.85 1.21 14.05 1.21 0.0001
2.19 0.31 1.42 0.12 1.37 0.12 0.0045
21.05 1.21 13.15 1.21 20.55 1.21 0.0003
2.08 0.31 1.28 0.12 2.10 0.12 0.0041
At the 6th week, average leak point pressure (LPP), P < 0.01 group A versus group B, P < 0.01 group B versus group C, P > 0.05 group A versus group C; maximal bladder capacity, P < 0.01 group A versus group B, P < 0.01 group B versus group C, P > 0.05 group A versus group C.
Biologic properties of Gd-DTPA- and PKH26-labeled HuMSCs available and potentially applicable in humans. Our approach focused on establishing a highly efficient, safe and inexpensive labeling method that offers noninvasive and reliable application in clinical practice. Effectene was used for transfection of the MRI standard contrast agent Gd-DTPA into HuMSCs because it is more efficient and less toxic than calcium, liposome phosphate or viral vectors (20,21). The related transfection protocols are widely used in the field of cellular and molecular biology (22,23). The results of this study showed that HuMSCs can be labeled with Gd-DTPA, and these labeled cells could be visualized >2 weeks using a clinical 1.5T MRI scanner with a minimal detectable quantity of 1 104 cells. Effectene-induced Gd-DTPA transfection efficiency was 85% in this particular cell type. We further traced the labeled HuMSCs in a rat model of SUI using MRI. As demonstrated by fluorescence microscopy, the distribution and migration of transplanted cells could be tracked for 2 weeks, which was reflected by a gradually diminished and obscured T1 hyperintense signal within the injured sphincter. The labeled HuMSCs were not visible 1.5 months after transplantation, which might be due to local contrast agent dilution caused by cell death or continuous division and dispersion of viable cells in the host tissue. Periurethrally injected HuMSCs in an animal model of SUI could significantly improve LPP and MBC. HuMSCs hold the therapeutic potential to treat patients with SUI in the future. PKH26 can be incorporated into the lipid region of the cell membrane. This red fluorescent dye has many advantages, such as fast staining and no transcellular transfection, indicating that this dye cannot be phagocytosed and stain the dead cells (24) and indicating no toxicity to cells. A major obstacle of using fluorescent dyes for stem cell labeling is the fading of the staining. The occurrence of the rapid fading of the staining could limit the long-term tracking of the labeled cells after transplantation. Hass et al. (25) had demonstrated the reliability and stability of PKH26 for neural stem cell transplantation. Their results showed that PKH26-labeled donor cells were detectable 4 months after transplantation in the host brain. In our present study, HuMSCs bi-functionally labeled with Gd-DTPA and PKH26 were very stable and readily detected by MRI and fluorescence microscopy. Our study demonstrated that PKH26 is a reliable fluorescent dye for long-term tracking of labeled cells in vitro and in vivo. In terms of stem cell labeling and tracking, the cell safety should be carefully considered. Our results showed that through a double-labeling method HuMSCs can be precisely tracked with MRI and
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fluorescence microscopy without altering cellular biologic behaviors, such as cell proliferation, differentiation, cycle and apoptosis. Efficient magnetic labeling of cells to be tracked with MRI is important for keeping the detection threshold as low as possible. As determined by spectrometry in this study, the Gd-DTPA labeling efficiency was 85%, which is lower than previously reported Gd-DTPA labeling (15) in neural stromal cells. The high sensitivity was achieved with a minimal number of detectable cells in T1-weighted imaging, which is more sensitive than the results reported by Rudelius et al. (15), who used liposome and calcium acid phosphate to transfer Gd-DTPA into cells. Electron microscopy confirmed the presence of Gd-DTPA particles in the cytoplasm of HuMSCs. In conclusion, periurethrally injected HuMSCs in an animal model of SUI significantly improved LPP and MBC. We demonstrated an inexpensive and safe strategy to label HuMSCs magnetically and fluorescently. This method does not require novel synthesis. It will provide researchers with a simple and straightforward method of magnetically labeling stem cells for tracking in the host body. Acknowledgments We thank Professor Jun Shen and his student Li-Na Cheng for their valuable suggestions and technical advice. This research was supported by the Fundamental Research Funds for the Central Universities (lzujbky—2011-141) in China. Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Vuu K, Xie J, McDonald MA, Bernardo M, Hunter F, Zhang Y, et al. Gadolinium rhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging. Bioconjugate Chem. 2005;16:995e9. 2. Mulder WJ, Strijkers GJ, vanTilborg GA, Griffioen AW, Nicolay K. Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging. NMR Biomed. 2006;19: 142e64. 3. Chemaly ER, Yoneyama R, Frangioni JV, Hajjar RJ. Tracking stem cells in the cardiovascular system. Trends Cardiovasc Med. 2005;15:297e302. 4. Hardy J, Edinger M, Bachmann MH, Negrin RS, Fathman CH, Contag C. Bioluminescence imaging of lymphocyte trafficking in vivo. Exp Hematol. 2001;29:1353e60. 5. Morawski AM, Lanza GA, Wickine SA. Target contrast agents for magnetic resonance imaging and ultrasound. Curr Opin Biotechnol. 2005;16:89e92. 6. Adonai N, Nguyen K, Walsh J, Iyer M, Toyokuni T, Phelps M, et al. Ex vivo cell labeling with 64
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H. Pan et al. Cu-pyruvaldehyde-bis (N4Methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc Natl Acad Sci U S A. 2002;99:3030e5. Hinds K, Hill J, Shapiro E, Laukkanen M, Silva A, Combs C, et al. Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells. Blood. 2003;102:867e72. Lanza GM, Winter PM, Caruthers SD, Morawski AM, Schmieder AH, Crowder KC, et al. Magnetic resonance molecular imaging with nanoparticles. J Nucl Cardiol. 2004; 11:733e43. Leslie LC, Nitin N, Gang B. Magnetic nanoparticle probes. Mat Today. 2005;8:32e8. Bulte JW, Kraitchman DL. Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed. 2004;17:484e99. Cahill KS, Gaidosh G, Huard J, Silver X, Byrne BJ, Walter GA. Noninvasive monitoring and tracking of muscle stem cell transplants. Transplantation. 2004;78:1626e33. Baligand C, Vauchez K, Fiszman M, Vilquin J-T, Carlier PG. Discrepancies between the fate of myoblast xenograft in mouse leg muscle and NMR label persistency after loading with Gd-DTPA or SPIOs. Gene Ther. 2009;16:734e45. Luo X, Lu Y, Yao R, Xie JY, Wang XY, Li YJ, et al. Experimental study on the effect of genitourinary tract structure and function of the model rats established by simulating delivery trauma and menopause. Prog Obstet Gynecol. 2008; 17:767e9. He SQ, Luo ZY, Liu QY, Zhou XR, Deng MQ, Luo X, et al. Isolation of human umbilical cord mesenchymal stem cells and differentiation into adipocytes and osteoblasts. J Clin Rehab Tissue Eng Res. 2010;14:2492e6. Rudelius M, Daldrup-Link HE, Heinzmann U, Piontek G, Settles M, Link TM, et al. Highly efficient paramagnetic labelling of embryonic and neuronal stem cells. Eur J Nucl Med Mol Imaging. 2003;30:1038e44.
16. 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;75:397e405. 17. Walczak P, Bulte JW. The role of noninvasive cellular imaging in developing cell-based therapies for neurodegenerative disorders. Neurodegener Dis. 2007;4:306e13. 18. Kraitchman DL, Gilson WD, Lorenz CH. MRI guidance and monitoring. J Magn Reson Imaging. 2008;27:299e310. 19. Nolte IS, Gungor S, Erber R, Plaxina E, Scharf J, Misselwitz B, et al. In vitro labeling of glioma cells with gadofluorine M enhances T1 visibility without affecting glioma cell growth or motility. Magn Res Med. 2008;59: 1014e20. 20. Daldrup-Link HE, Rudelius M, Oostendorp RA, Settles M, Piontek G, Metz S, et al. Targeting of hematopoietic progenitor cells with MR contrast agents. Radiology. 2003;228: 760e7. 21. van den Bos EJ, Wagner A, Mahrholdt H, Thompson RB, Morimoto Y, Sutton BS, et al. Improved efficacy of stem cell labeling for magnetic resonance imaging studies by the use of cationic liposomes. Cell Transplant. 2003;12:743e56. 22. Malone RW, Felgner PL, Verma IM. Cationic liposomemediated RNA transfection. Proc Natl Acad Sci U S A. 1989; 86:6077e81. 23. Chen CA, Okayama H. Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques. 1988;6: 632e8. 24. Horan PK, Slezak SE. Stable cell membrane labelling. Nature. 1989;340:167e8. 25. Hass SJ, Bauer P, Rolfs A, Wree A. Immunocytochemical characterization of in vitro PKH26-labelled intracerebrally transplanted neonatal cells. Acta Histochem. 2000;102: 273e80.
Biologic properties of gadolinium diethylenetriaminepentaacetic acid-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells
HUA PAN1,*, JIANFA LAN2,*, XIN LUO3, JUN GAO2, XIAODONG XIE1 & HONGYU GUO2 1
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu Province, China, 2Department of Gynaecology, Lanzhou University Second Hospital, Lanzhou, Gansu Province, China, and 3Department of Gynecology and Obstetrics, First Affiliated Hospital Jinan University, Guangzhou, Guangdong Province, China
Abstract Background aims. This study was conducted to characterize gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA)labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells (HuMSCs) and to track them with magnetic resonance imaging (MRI) in vitro and in vivo. Methods. HuMSCs were isolated from umbilical cords and expanded in vitro. Cells were sequentially labeled with Gd-DTPA and PKH26. The labeling efficiency was determined by spectrophotometry measurements, and the longevity of Gd-DTPA maintenance was measured with MRI. The influence of double labeling on cellular biologic properties was assessed by cell proliferation, viability, differentiation, cycle and apoptosis. Transplantation of double-labeled HuMSCs or placebo was performed in 39 female Sprague-Dawley rats. Leak point pressure and maximal bladder capacity were measured in animals 6 weeks after injection. Results. The T1 values and signal intensity on T1-weighted imaging of labeled cells were significantly higher than the control group (P < 0.05). The signal intensity on T1-weighted imaging of labeled cells was retained >14 days in vitro and in vivo. There was no significant difference in the cell cycle, cell apoptosis, cell proliferation and cell viability between labeled and unlabeled HuMSCs (P > 0.05). After double labeling, HuMSCs were still capable of differentiating into osteoblasts and adipocytes. Periurethrally injected HuMSCs in the rats significantly improved leak point pressure and maximal bladder capacity. Conclusions. HuMSCs were successfully labeled with Gd-DTPA and PKH26. This labeling method is reliable and efficient and can be applied for tracking cells in vitro and in vivo without altering cellular biologic properties. Key Words: cell labeling, Gd-DTPA, HuMSCs, MRI, PKH26
Introduction Stem cell-based regenerative medicine has been intensively studied in both pre-clinical and clinical settings in recent decades. It is essential to establish an ideal technique to monitor the cell status in the recipient after transplantation (1,2). Numerous techniques have been developed for tracking mesenchymal stromal cells (MSCs) in vitro or in vivo, such as fluorescence imaging, ultrasound, isotope labels and magnetic resonance imaging (MRI) (3). Fluorescence imaging has the limitation of detection depth for inner organs, and it requires tissue samples for detection (4). Limitations of ultrasound include accuracy in cell quantification and special resolution (3,5). Isotopes for labeling can lead to radiation injury of stem cells (6). However, MRI not only is a
non-invasive technique to monitor the movement of cells in vivo, but it also provides real-time images of the cells in internal organs. It has been widely used as a cell tracking technique in recent years (7e9). An ideal method for in vivo evaluation of cell distribution and its dynamics is extremely desirable for a successful cellular transplantation treatment. The fate of the transplanted cells within the host should be non-invasively, repeatedly and specifically determined. We labeled HuMSCs with standard contrast agents for MRI to track the homing and engraftment of transplanted cells. To obtain better MRI results, the key factor is to select contrast agents. The T2 contrast agents, such as superparamagnetic iron oxide particles (SPIOs),
*These authors contributed equally to this work. Correspondence: Dr Jianfa Lan, Department of Gynaecology, Lanzhou University Second Hospital, Lanzhou, Gansu Province 730030, China. E-mail: [email protected]; Professor Xiaodong Xie, Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu Province 730000, China. E-mail: [email protected] (Received 22 March 2013; accepted 20 May 2013) ISSN 1465-3249 Copyright Ó 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.05.015
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which have high T2 relaxivities, have been widely used for cell labeling during the past 15 years. The high susceptibility of SPIOs is different from other contrast agents, and several studies have demonstrated their high sensitivity for detecting transplanted cells in vivo (10,11). However, because SPIOs could enhance the dark background, SPIOderived images are unable to distinguish cells precisely in tissue, especially under hemorrhagic conditions. In addition, the particulate nature of SPIOs can induce phagocytosis by activating reticulohistiocytic cells, which would cause false-positive images. This phenomenon was recognized and documented by Cahill et al. (11). A study by Baligand et al. (12) suggested that iron-containing particles should be used with extreme caution to evaluate the presence of grafted cells in vivo, and such particles are unsuitable for the long-term tracing the transplanted cells. Gadolinium (Gd) is a contrast agent that has been used extensively in a clinical setting. Use of contrast agents with T1-weighted MRI enhances the ability to produce positive signal intensity in images, facilitating tracking cells in low signal tissues. Among these, Gd is the most effective T1 contrast agent for clinical use. Gd usually forms a complex with the chelating ligand, diethylenetriaminepentaacetic acid (DTPA), which is used for MRI. In biologic tissues, T1 relaxivity is longer than that of T2, and contrast enhancement is better observed with T1-weighted sequences. HuMSCs have many advantageous characteristics as seed cells, such as high proliferative activity, low immunogenicity and non-tumorigenic nature. The potential clinical application of HuMSCs in the fields of tissue engineering and gene therapy has become an area of active stem cell research. Although HuMSCs are widely used as the source of cells for tissue repair or regeneration, their actual function and migration after they are injected into the human body are unknown. To determine the cellular biologic properties and bio-distribution of therapeutic stem cells, it is crucial to develop a safe, reproducible and non-invasive technique to trace them in vivo. In this study, HuMSCs were doublelabeled with Gd-DTPA and PKH26. Parallel studies were performed with labeled and unlabeled cells to identify the possible effects of Gd-DTPA and PKH26 labeling on biologic properties of transplanted HuMSCs, including cell viability, proliferation, differentiation and apoptosis.
Methods Rat model of stress urinary incontinence All experimental protocols were approved by the Chinese Institution of Animal Care Committee at
our institution. We obtained 39 female SpragueDawley rats weighing 180e200 g from the Medical Laboratory Animal Center of Guangdong Province, China. The rats were subjected to development of stress urinary incontinence (SUI) voiding dysfunction by a previously described procedure (13). Briefly, immediately after delivery, the rats underwent vaginal balloon dilation to simulate prolonged labor and dystocia. The rats were anesthetized 2 weeks later, and a midline incision was made in the abdomen and both ovaries were excised. The rats were subjected 1 month later to leak point pressure (LPP) and maximal bladder capacity (MBC) measurement with urodynamic examination. HuMSC isolation and ex vivo expansion Healthy term fetal umbilical cord was collected after cesarean section from informed, consenting mothers and processed as quickly as possible. All umbilical cords were obtained from the Department of Obstetrics at First Affiliated Hospital of Jinan University. Cell isolation, culture and ex vivo expansion were performed as previously described (14). Briefly, immediately after an umbilical cord was collected, the blood was removed, and the cord was cut into 1-mm3 pieces. The chopped tissue was seeded in the culture dish with Dulbecco’s modified Eagle’s medium/Nutrient Mixture F12 (Gibco DMEM/F12; Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 15 mmol/L 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid. The pieces of cord were subsequently incubated at 37 C in humid air with 5% CO2. After 1 week, a few drops of fresh medium were placed in the culture dish every 2e3 days. As soon as enough cellular colonies formed around the chopped tissues, the tissue blocks were removed by changing the culture medium. When cells reached 70e80% confluency, they were trypsinized for subculture. Cell labeling A non-liposomal lipid transfection reagent, Effectene transfection reagent (Qiagen, Hilden, Germany), was used to transfect Gd-DTPA (Magnevist; Schering, Berlin, Germany) into HuMSCs. The encapsulation of Gd-DTPA with cationic lipids, which were formed when negative charged Gd-DTPA particles mixed with Effectene, could be efficiently transferred into cells. To optimize the transfection condition, we performed a pilot test involving various dosages of Gd-DTPA and Effectene and various incubation periods. An incubation time of 4 h and 25 mL GdDTPA and 25 mL Effectene were considered a good
Biologic properties of Gd-DTPA- and PKH26-labeled HuMSCs compromise between labeling effectiveness and cell preservation. This condition of labeling was applied in the present study. In a 25 cm2 tissue culture flask containing 5 mL DMEM/F12 medium, 25 mL GdDTPA (0.5 mol/L) and 25 mL Effectene were added to 1 106 HuMSCs. After 4 h of incubation under standard cell culture conditions, PHK26 (2 108 mol/L) (Sigma-Aldrich, St Louis, MO, USA) was added to the medium for fluorescent labeling following the manufacturer’s instructions. The blank control was 1 106 unlabeled HuMSCs, which were incubated in regular culture medium without addition of any labeling agents. Cell viability, proliferation and differentiation After double labeling, cell viability and proliferation were assessed with trypan blue dye exclusion test and Cell Counting Kit-8 (CCK-8; BiYunTian Biological Technology Research Institute, Chengdu, China) assay. The cell viability was determined by trypan blue exclusion assay at times 0 h (before labeling), 8 h, 24 h and 72 h after initial labeling. There were five cell samples in each group at each time point. For CCK-8 assay, HuMSCs were cultured in flat-bottom 96-well plates at 1 103 cells per well, and cells in 50% of the wells were labeled. Cells in the remaining wells were not labeled and served as controls. After labeling, the cells were washed twice with phosphate-buffered saline (PBS), and 100 mL of DMEM/F12 with 10% FBS was added to each well. The plate was kept in the incubator under standard culture conditions. At each time point of day 1, day 3 and day 6 of incubation, the samples of labeled and unlabeled cells were underwent CCK-8 assay. Each well had 10 mL of CCK-8 added, and cells were incubated for 1 h and measured at a wavelength of 680 nm with 420 nm as the subtracted reference. Adipogenic differentiation was performed with special medium. Double-labeled HuMSCs were inoculated at 1 105 mL1 placed sterile coverslips in six-well plates. When cells grew to 100% fusion, the induced group was exchanged for adipogenic differentiation induced solution (containing 10% FBS, 5 mg/L insulin, 1 mmol/L dexamethasone, 0.5 mmol/L 1-methyl-3-isobutylxanthine and 100 mmol/L indomethacin). The control group was cultured with standard stem cell medium. Both groups were cultured for 21 days with regular medium changes (twice a week). At the end of induction, cells were stained with oil red O and observed under microscopy. For osteogenic differentiation, double-labeled HuMSCs (1 104 mL1 cells) were seeded in sixwell plates until the cells grew to 80% confluence, and the induced group was exchanged for osteogenic
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differentiation-induced solution (medium containing 10% FBS, 100 nmol/L dexamethasone, 100 mmol/L b-glycerophosphate and 0.2 mmol/L ascorbic acid). The negative control group was cultured with standard stem cell medium. Both groups were continuously cultured for 28 days with regular medium changes (twice a week). At the end of induction, cells were stained with alizarin red and observed under microscopy. Flow cytometry detected cell cycle and cell apoptosis The phenotype of HuMSCs was detected with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with BD CellQuest Pro software (BD Biosciences) as previously described (14). Fluorescence-activated cell sorter (FACS) was also used for the detection of cell cycle and apoptosis. Unlabeled or labeled cells (1 106) were centrifuged. After washing with PBS, RNase was added to remove RNA, and cells were stained with propidium iodide dye at 4 C dark for 30 min. The cell cycle was then detected with FACS. Unlabeled or labeled cells were adjusted to a concentration of 1 106/mL. In a 5-mL flow tube, 100 mL of cell suspension was mixed with 5 mL Annexin V-fluorescein isothiocyanate and 10 mL (20 mg/mL) propidium iodide solution. After 15 min incubation at room temperature, 400 mL PBS was added, and cell apoptosis was analyzed by FACS. Spectrophotometry Labeling efficiency was determined by spectrophotometry measurements of the cellular uptake of paramagnetic Gd-DTPA particles (15). Briefly, after labeling, cells were washed three times with PBS and then re-suspended in PBS. The Gd-DTPA concentration within labeled cells was measured with inductively coupled plasma atomic emission spectrometry (IRIS-HR) using a polarized Zeeman atomic absorption spectrometer (Thermo Jarrell Ash, Boston, MA, USA). The labeling efficiency was determined as ratio of the percentage of Gd-DTPA concentration within labeled cells to the concentration of Gd-DTPA that was initially added to the culture medium for labeling. Fluorescent microscopy and electron microscopy After labeling, HuMSCs were washed three times with PBS. One aliquot of PKH26-labeled cells was detected by fluorescent microscopy (Nikon, Tokyo, Japan). Another aliquot of labeled cells was fixed in 3% glutaraldehyde-cacodylate buffer at 3 C for overnight. After being fixed for 1 h in 1% osmium tetroxide, cells were dehydrated in graded dilutions of ethanol, embedded in artificial resin (Merck,
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Figure 1. Labeled and unlabeled cells under different microscopy. (A) Inverted microscopy (40). (B) PKH26-labeled cells under fluorescent microscopy (40). (C, D) Gd-DTPA-labeled cells showed clusters of Gd-DTPA particles existing within cytoplasm. No Gd-DTPA particles were visible within unlabeled cells under transmission electron microscopy (15,500).
Darmstadt, Germany) and processed for electron microscopy. Thin sections of the cell probes were evaluated unstained to prevent false-positive findings. Electron microscopy was performed at 60e80 kV.
Figure 2. Gd-DTPA-labeled HuMSCs were tracked by MRI in vitro. (A1, A2) Cell pellets (1 106) within test tubes were from (left to right) unlabeled cells, Effectene-labeled cells, Gd-labeled cells and Effectene-Gd-labeled cells. (B1, B2) Longevity of maintenance in labeled cells. After labeling, 2 106 cells were cultured and passaged under normal condition. On T1-weighted imaging, passages of labeled cells remained at high signal intensity at 1, 7 and 14 days after the initial labeling procedure; signal intensity disappeared on 15th day. (C1, C2) Minimal detectable number of labeled cells on MRI in vitro. The signal intensity became gradually stronger with the increasing number of Gd-labeled cells on the T1weighted image. The signal intensity of 5 103 Gd-labeled cells was almost the same as the signal intensity of 5 103 unlabeled cells.
In vitro MRI MRI was performed with a clinical 1.5T MRI scanner (Philips Medical Systems, Best, Netherlands), and a circular surface coil with a diameter of 11 cm was used (16). Briefly, after labeling, HuMSCs (1 106) were washed three times with PBS. The cell pellets within the test tubes were imaged. The tubes were placed in a plastic box filled with 37 C water to ensure signal homogeneity and to avoid potential susceptibility artifacts from surrounding air. Labeled and unlabeled HuMSCs were included in each subset of imaging analysis. In addition, the supernatant from labeled HuMSCs was collected and imaged to determine whether there was residual Gd-DTPA in labeled HuMSCs. For MRI pulse sequences, refer to Shen et al. (16). To determine the sensitivity of this application, the decreasing numbers of labeled cells and unlabeled cells (1 106, 5 105, 1 105, 1 104, 5 103) were collected in 1.0-mL eppendorf tubes, re-suspended in 50 mL 4% gelatin solution and imaged by MRI.
Figure 3. Graph shows the minimal detectable number of labeled cells. Data were expressed as mean SD. *P < 0.05 compared with unlabeled cells.
Biologic properties of Gd-DTPA- and PKH26-labeled HuMSCs
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Figure 4. Graphs show cell viability and proliferation of labeled and unlabeled cells. (A) Cell viability was tested by trypan blue dye exclusion. (B) Cell proliferation was tested by CCK-8.
Transplantation of HuMSCs
In vivo MRI
HuMSCs were transplanted at 8 h after Gd-DTPA and PKH26 labeling. The negative control group was 12 normal rats (group A). We randomly assigned 24 SUI rats that were balloon-injured and ovariectomized into the positive control group (group B, n ¼ 12) and the urethra-HuMSCs group (group C, n ¼ 12). The control animals (groups A and B) received injection of 300 mL PBS in the urethra at 3 o’clock, 6 o’clock and 9 o’clock positions at the bladder urethra junction. Group C received injection of 4.5 106 double-labeled HuMSCs (soluble in 300 mL PBS) at the same positions. In addition, three rats received injection of 4.5 106 unlabeled HuMSCs (in 300 mL of PBS) in the urethra as a control for MRI.
Rats were examined on days 1, 7 and 14 after placebo and labeled or unlabeled HuMSC transplantation. Rats were anesthetized with 10% chloral hydrate and scanned in a rat MRI coil. LPP and MBC test LPP and MBC were quantified before transplantation and at 6 weeks after implantation of HuMSCs. Animals were anesthetized with chloral hydrate as previously described (13). Briefly, an epidural catheter was connected through a three-way pipe to a BL-410 biologic function experiment system instrument (Tai Meng Science and Technology,
Figure 5. Staining identification of adipogenic and osteogenic differentiation. Oil red O staining of adipogenic differentiation at 21 days (A) and control (B). Alizarin red staining of osteogenic differentiation at 28 days (C) and control (D).
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Table I. Determination of apoptosis of labeled and unlabeled human umbilical cord mesenchymal stromal cells with fluorescence-activated cell sorter.
24 h Labeled cells Unlabeled cells P value 72 h Labeled cells Unlabeled cells P value
Normal cells (%)
Early apoptotic cells (%)
Late apoptotic cells (%)
Dead cells (%)
91.581 2.901 92.127 2.896 0.8289
6.049 0.428 5.893 0.369 0.6575
1.398 0.170 1.148 0.164 0.1407
0.967 0.156 0.916 0.190 0.7375
95.200 3.01 94.848 2.98 0.8925
2.200 0.567 2.492 0.345 0.4885
1.511 0.189 1.531 0.200 0.9059
1.101 0.123 1.110 0.167 0.9437
Flow cytometry detected cell apoptosis of labeled and unlabeled cells at 24 h and 72 h.
Chengdu, China), and another interface connected a 1-mL syringe, with sterile saline filling the three-way pipe guaranteeing the pipeline air free. Methylene blue fluid was injected into the bladder using a syringe at a speed of 0.5 mL/min. When the first blue liquid drop was observed in the urethral meatus, the bladder capacity was recorded, which was the MBC of the rat. At the same time, bladder pressure curve recording and regulatory experiment were started. This curve was also the rat LPP diagram. Each rat was tested three times. Immunofluorescence and tracking of PKH26-labeled HuMSCs After HuMSC transplantation, three rats from each experimental group were sacrificed at day 1, day 7 and day 42. The bladder-urethra complex was removed en bloc. Frozen tissue sections were processed to visualize red fluorescence. Statistical analysis Statistical analysis was performed using SPSS 14.0 software for Windows (IBM, Chicago, IL, USA). A P value of <0.05 was considered to indicate a statistically significant difference. Cell apoptosis, cell cycle, trypan blue exclusion assay for toxicity and CCK8-based proliferation data were expressed as percentage of the corresponding unlabeled control cells, and analysis of variance significance was performed to test for statistical significance. Comparisons of signal Table II. Cell cycle determination of human umbilical cord mesenchymal stromal cells with fluorescence-activated cell sorter. Phase G0/G1 (%)
S (%)
G2/M (%)
7 days Labeled cells 66.66 3.073 12.65 1.683 21.09 1.98 Unlabeled cells 67.10 2.103 12.30 1.283 20.78 1.38 P value 0.8478 0.7888 0.8348 Results are shown of cell cycle between the labeled and unlabeled group after cells were labeled on the 7th day.
intensity and T1 values between unlabeled and labeled cells were performed by using the unpaired Student t test. Results Cellular surface markers HuMSC surface marker antigen expressions were positive for CD29, CD44, CD90 and CD105 and negative for CD31, CD34 and CD45, which represent hematopoietic cells. Surface marker antigen expression was extremely low for graft-versus-host disease related factor HLA-DR CD40, explaining its extremely low immunogenicity. Cell labeling After labeling, red fluorescence was observed in most labeled cells under fluorescent microscopy. PKH26 labeling efficiency was detected to be 99%, and red fluorescence was still detectable on cells at passage 10. As measured by atomic emission spectrophotometry, Effectene-Gd-DTPA labeling efficiency was detected to be 85%. Black high-density Gd-DTPA particles were contained and compartmentalized within lysosomes of labeled cells under electron microscopy, whereas no such particles were visible in unlabeled cells (Figure 1). Effectene-Gd-DTPA-labeled cells showed high signal intensity on T1-weighted imaging and low signal intensity on T2-weighted imaging (Figure 2). The signal intensity in Effectene-Gd-DTPA-labeled cells was significantly high compared with unlabeled cells and cells incubated with Gd-DTPA or Effectene alone (Figure 2). The T1 values of Effectene-GdDTPA-labeled cells, cells with Gd-DTPA, cells with Effectene and unlabeled cells were 1125 181, 734 86, 410 97 and 401 37. The signal intensity of labeled cells was significantly higher than the signal intensity of negative control and blank control cells (P < 0.01). Strong signal still could be seen on the 14th day after cells were labeled in vitro (Figure 2). Increasing numbers of labeled cells were suspended in 50 mL gelatin (Figure 2). After diluting in
Biologic properties of Gd-DTPA- and PKH26-labeled HuMSCs
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Figure 6. Double-labeled HuMSCs in the urethra of rats were detected by MRI after transplantation. There was no high signal in unlabeled cells (A). Fat-suppressed T1-weighted images of labeled cells (B1eB3) showed high signal (arrows) that gradually diminished. T2-weighted images showed low signal (arrows) in all specimens treated with HuMSCs (C, D1eD3).
50 mL 4% gelatin solution, the signal intensity on T1weigted imaging of 1 106, 5 105, 1 105, 1 104 and 5 103 HuMSCs was statistically higher than the counterparts of unlabeled cells (P < 0.05), but no significantly higher signal intensity was found between the 5 103 labeled cells and unlabeled cells (P > 0.05) (Figure 3). The minimal detectable number of labeled cells was 1 104 cells. Biologic properties of HuMSCs Trypan blue dye exclusion test showed that there was no reduction in cell viability comparing labeled cells and unlabeled cells (P > 0.05) (Figure 4). CCK-8 assay showed no statistical difference between labeled and unlabeled cells (P > 0.05) (Figure 4). During adipogenic differentiation, with the extension of induction time, the cells began to exhibit vacuolar lipid droplets, which gradually became larger. The cell shape was gradually changed from fibrous to circular or oval. After oil red O staining, oil droplets
were observed in the cytoplasm under the microscope (Figure 5). During osteogenesis induced differentiation, with the extension of induction time, the cells began to exhibit growth of stacked aggregation. The place of cell fusion appeared as a “mesh shape.” After 2 weeks of continuous induction, multiple mineralized nodules were observed. Subsequently, nodules increased gradually and merged with calcified nodules, which were red with Alizarin red staining, until 28 days of induction. This phenomenon was not observed in the cells without special induction (Figure 5). Flow cytometry detected cell apoptosis of labeled and unlabeled cells at 24 h and 72 h. The results are shown in Table I. There were various cell proportions of the labeled group and unlabeled group but no statistical significance (P > 0.05). Table II shows the results of labeled and unlabeled cell cycles. The difference of each period cell proportion between the two groups of cells was not statistically significant (P > 0.05).
Figure 7. Gd-DTPA-labeled and PKH26-labeled HuMSCs in the urethra of rats were tracked in vivo. Frozen sections of intermediate urethra after transplantation are shown under fluorescent microscopy (100) at 1 day (A), 1 week (B) and 6 weeks (C).
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Figure 8. LPP was tested 6 weeks after implantation of HuMSCs or placebo. Group A comprised normal rats, and the average value of LPP was 21.72 mm Hg. Group C comprised 12 SUI rats, and the average value of LPP was 20.18 mm Hg. Group B comprised 12 SUI rats, and the average value of LPP was 13.82 mm Hg.
In vivo MRI and fluorescent microscopy The double-labeled HuMSCs in the rat urethra were distinctly detected by the hyperintense signal on T1weighted imaging after transplantation. The hyperintense signal gradually decreased but remained for 2 weeks. However, no obvious hyperintense signal was found on T1-weighted imaging 1e14 days after transplantation in the unlabeled groups. Low signal intensities were found around the urethra on T2weighted imaging in both experimental and control groups (Figure 6). Frozen tissue sections showed that PKH26-labeled HuMSCs can be tracked for >6 weeks (Figure 7). LPP and MBC test As demonstrated in Figure 8 and Table III, LPP and MBC of HuMSC-transplanted SUI are statistically significantly different from the SUI placebo model (P < 0.01). Discussion Cell tracking plays an important part in stem cell therapy-related studies. A reliable and reproducible labeling method is a prerequisite for monitoring transplanted cells and investigating their biologic properties. MRI, a non-invasive method with high anatomic resolution, has been widely used to track the migration and fate of transplanted stem cells (17,18). At the present time, Gd-based paramagnetic contrast agents or iron oxide-based superparamagnetic
contrast agents are mainly used for cell tracking with MRI. Advantages of Gd have been mentioned previously. In addition, T1-sensitive contrast agents can track the distribution of freshly implanted cells more accurately than T2-wighted ferric oxide nuclear protein-labeled contrast agents (19). There is much more than convenience, safety and feasibility considerations to encourage investigators to opt for Gd chelate loading in many conditions. Gd-DTPA is a clinical standard contrast agent and has been used extensively in various clinical applications. In this study, we selected Gd-DTPA and PKH26 as labeling probes that are commercially Table III. Leak point pressure and maximal bladder capacity quantified before implantation and after implantation.
1 week Group A Group B Group C P value 6th week Group A Group B Group C P value
Average leak point pressure (mm Hg)
Maximal bladder capacity (mL)
23.15 1.21 13.85 1.21 14.05 1.21 0.0001
2.19 0.31 1.42 0.12 1.37 0.12 0.0045
21.05 1.21 13.15 1.21 20.55 1.21 0.0003
2.08 0.31 1.28 0.12 2.10 0.12 0.0041
At the 6th week, average leak point pressure (LPP), P < 0.01 group A versus group B, P < 0.01 group B versus group C, P > 0.05 group A versus group C; maximal bladder capacity, P < 0.01 group A versus group B, P < 0.01 group B versus group C, P > 0.05 group A versus group C.
Biologic properties of Gd-DTPA- and PKH26-labeled HuMSCs available and potentially applicable in humans. Our approach focused on establishing a highly efficient, safe and inexpensive labeling method that offers noninvasive and reliable application in clinical practice. Effectene was used for transfection of the MRI standard contrast agent Gd-DTPA into HuMSCs because it is more efficient and less toxic than calcium, liposome phosphate or viral vectors (20,21). The related transfection protocols are widely used in the field of cellular and molecular biology (22,23). The results of this study showed that HuMSCs can be labeled with Gd-DTPA, and these labeled cells could be visualized >2 weeks using a clinical 1.5T MRI scanner with a minimal detectable quantity of 1 104 cells. Effectene-induced Gd-DTPA transfection efficiency was 85% in this particular cell type. We further traced the labeled HuMSCs in a rat model of SUI using MRI. As demonstrated by fluorescence microscopy, the distribution and migration of transplanted cells could be tracked for 2 weeks, which was reflected by a gradually diminished and obscured T1 hyperintense signal within the injured sphincter. The labeled HuMSCs were not visible 1.5 months after transplantation, which might be due to local contrast agent dilution caused by cell death or continuous division and dispersion of viable cells in the host tissue. Periurethrally injected HuMSCs in an animal model of SUI could significantly improve LPP and MBC. HuMSCs hold the therapeutic potential to treat patients with SUI in the future. PKH26 can be incorporated into the lipid region of the cell membrane. This red fluorescent dye has many advantages, such as fast staining and no transcellular transfection, indicating that this dye cannot be phagocytosed and stain the dead cells (24) and indicating no toxicity to cells. A major obstacle of using fluorescent dyes for stem cell labeling is the fading of the staining. The occurrence of the rapid fading of the staining could limit the long-term tracking of the labeled cells after transplantation. Hass et al. (25) had demonstrated the reliability and stability of PKH26 for neural stem cell transplantation. Their results showed that PKH26-labeled donor cells were detectable 4 months after transplantation in the host brain. In our present study, HuMSCs bi-functionally labeled with Gd-DTPA and PKH26 were very stable and readily detected by MRI and fluorescence microscopy. Our study demonstrated that PKH26 is a reliable fluorescent dye for long-term tracking of labeled cells in vitro and in vivo. In terms of stem cell labeling and tracking, the cell safety should be carefully considered. Our results showed that through a double-labeling method HuMSCs can be precisely tracked with MRI and
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fluorescence microscopy without altering cellular biologic behaviors, such as cell proliferation, differentiation, cycle and apoptosis. Efficient magnetic labeling of cells to be tracked with MRI is important for keeping the detection threshold as low as possible. As determined by spectrometry in this study, the Gd-DTPA labeling efficiency was 85%, which is lower than previously reported Gd-DTPA labeling (15) in neural stromal cells. The high sensitivity was achieved with a minimal number of detectable cells in T1-weighted imaging, which is more sensitive than the results reported by Rudelius et al. (15), who used liposome and calcium acid phosphate to transfer Gd-DTPA into cells. Electron microscopy confirmed the presence of Gd-DTPA particles in the cytoplasm of HuMSCs. In conclusion, periurethrally injected HuMSCs in an animal model of SUI significantly improved LPP and MBC. We demonstrated an inexpensive and safe strategy to label HuMSCs magnetically and fluorescently. This method does not require novel synthesis. It will provide researchers with a simple and straightforward method of magnetically labeling stem cells for tracking in the host body. Acknowledgments We thank Professor Jun Shen and his student Li-Na Cheng for their valuable suggestions and technical advice. This research was supported by the Fundamental Research Funds for the Central Universities (lzujbky—2011-141) in China. Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Vuu K, Xie J, McDonald MA, Bernardo M, Hunter F, Zhang Y, et al. Gadolinium rhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging. Bioconjugate Chem. 2005;16:995e9. 2. Mulder WJ, Strijkers GJ, vanTilborg GA, Griffioen AW, Nicolay K. Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging. NMR Biomed. 2006;19: 142e64. 3. Chemaly ER, Yoneyama R, Frangioni JV, Hajjar RJ. Tracking stem cells in the cardiovascular system. Trends Cardiovasc Med. 2005;15:297e302. 4. Hardy J, Edinger M, Bachmann MH, Negrin RS, Fathman CH, Contag C. Bioluminescence imaging of lymphocyte trafficking in vivo. Exp Hematol. 2001;29:1353e60. 5. Morawski AM, Lanza GA, Wickine SA. Target contrast agents for magnetic resonance imaging and ultrasound. Curr Opin Biotechnol. 2005;16:89e92. 6. Adonai N, Nguyen K, Walsh J, Iyer M, Toyokuni T, Phelps M, et al. Ex vivo cell labeling with 64
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