original article tracing mesenchymal stem cells in treated ... · magnetic resonance imaging of...

Int J Clin Exp Med 2016;9(2):773-783www.ijcem.com /ISSN:1940-5901/IJCEM0016330

Original Article

Tracing mesenchymal stem cells in treated uterine horns trauma of rats with magnetic resonance imaging

Bai Zhou1*, Xin-An Li1*, Li-Jun Ding2, Jing-Mei Wang3, Feng-Chao Zang4, Yun Cao1, Qiang-Bin Wang5, Zong-Wu Deng5, Jian-Wu Dai6, Ya-Li Hu1

1Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Affiliated to Nanjing Medical University, Nanjing 210008, China; 2Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China; 3Department of Pathology, Nanjing Drum Tower Hospital, Affiliated to Nanjing University Medical School, Nanjing 210008, China; 4China Jiangsu Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School of Southeast University, Nanjing 210009, China; 5Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215125, China; 6Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100190, China. *Equal contributors.

Received September 17, 2015; Accepted November 30, 2015; Epub February 15, 2016; Published February 29, 2016

Abstract: Stem cells have shown a great promise to treat uterine injury. However, long-term and noninvasive track-ing of stem cells in uterine horns is still challenging. In the present study, high-field magnetic resonance imaging (MRI) was applied in the tracking mesenchymal stem cells derived from human umbilical cord blood (UCB-MSCs) in reparation of uterine horns trauma of rats. Nano-sized MR contrast agent SPIO@SiO2 (silica nanoparticle coated super-paramagnetic iron oxide nanoparticle) labeled UCB-MSCs. We found that SPIO@SiO2 labeled UCB-MSCs could be detected in the surgical region in vivo 7 days postoperatively using 7.0 T MRI. By 30 days, the labeled cells only could be detected using 11.7 T MRI in vivo. SPIO@SiO2 labeled UCB-MSCs were mainly in the myometrium and serosal junction area. MRI findings were highly correlated to Prussian blue staining results. Our findings indicate that high-field MRI monitoring of the early phase of this model may provide unique opportunities to study stem cells implantation.

Keywords: Umbilical cord blood-derived mesenchymal stem cells, super-paramagnetic iron oxide nanoparticle, silica nanoparticle, magnetic resonance imaging, uterine reparation

Introduction

Severe damages of the endometrium caused by curettage or endometritis lead to endome-trium scar formation which is a major issue in infertility clinics [1, 2]. Series of exploration have been carried out in the treatment of severe endometrial scars. However, few meth-ods are available to solve these problems [3-6]. Stem cell therapy, because of the differentia-tion potentialities and paracrine abilities of stem cells, holds the promise of tissue regen-eration for endometrium damages. Previous reports have shown that MSCs contributed to the regeneration of endometrium [7-10]. Our previous study has revealed that MSCs com-bined with collagen scaffold could restore endometrial morphology and pregnant function in the model of rat uterine severe injury [11].

With the promising results from these studies, the roles of stem cells are under intensive investigation. Several issues remain to be clari-fied prior to clinical application of MSCs trans-plantation for severe endometrial scars. One of the crucial points is to monitor the migration, distribution and differentiation of transplanted cells, as well as verify the efficiency and func-tional capability. Currently, several biomedical and histological methods are used for tracking stem cells in vitro, which can be accomplished only by obtain tissues via autopsy invasively. Therefore, for both basic and preclinical trials, the development of real-time, long-term and noninvasive techniques to track transplanted stem cells in the host organ in vivo is impera-tive. MRI with different pulse sequences which offers great advantages of high temporal and

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Magnetic resonance imaging of tracing mesenchymal stem cells in rats

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spatial resolution and good soft-tissue contrast to reveal multiple physiologic parameters is promising for dynamically tracking cell after transplantation in vivo [12]. It has been used to perform serially tracking of a number of implanted cell lines in vivo including MSCs [13-16].

However, few studies have identified three-dimensional anatomical structures of rat uter-us in MRI processing [17]. It is an important step in supporting tasks such as visualization, tracking and more. One reason is that it is diffi-cult to identify each plane of the crimped horns and differentiate them from the intestines and ureter. Even more, no techniques have been reported to track transplanted stem cell in the host uterus for long periods of time. For MR detection, to visualize the transplanted cells from the surrounding tissues, magnetic tags are used to label the cells to increase their intrinsic sensitivity. Super-paramagnetic iron oxide nanoparticle (SPIO) which can induce strong field in homogeneities in proximal water molecules during MRI has been used as ideal probe to investigate the fate of labeled cells in model animals and a few clinical settings [18-21].

Herein, we designed the non-cytotoxic and bio-compatible SiO2-coated core shell SPIO (SPIO@SiO2) as magnetic tags for labeling UCB-MSCs. MSCs combined with collagen scaffold was implanted to severe uterine damage rat model. High-field MRI was applied to identify three-dimensional anatomical structures of rat uter-us and evaluate the migration, distribution and differentiation abilities of UCB-MSCs in the treatment of uterine horns trauma in rats.

Materials and methods

UCB-MSCs culture and identification

UCB-MSCs were donated by the stem cell bank of Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences. Cells were cultured (37°C in 5% CO2) at a density of 2 × 103 cells/cm2 in 100-mm cell culture dish with the following growth medium: low glucose Dulbecco’s modified Eagle medium (LG-DMEM) supplemented with 15% fetal bovine serum, 1 × Insulin-Transferrin-Selenium, 100 U/mL peni-cillin and 100 mg/mL streptomycin (all pur-chased from Gibco).

Cells of 1 × 107 were incubated with 0.4% try-pan blue dye for 15 min at room temperature before microscopic examination. Images of four randomly selected fields were captured. The percentage of viable cells was calculated: Cell survival rate (%) = (total number of cells-num-ber of dyed cells)/total number of cells × 100%.

Flow cytometric analysis of UCB-MSCs was per-formed at the fifth passage. Cells were detached from the culture dish using trypsin-EDTA (0.25%: 0.02%) (Gibco) and re-suspended in PBS at a density of 106 cells/mL. Then cells were incubated with 5 µL of fluorescein isothio-cyanate-labeled or phycoerythrin (PE)-conju- gated anti-human CD73, CD29, CD34, CD45, CD90 in the dark at 4°C for 5 min. After being washed twice with PBS, cytometric analysis was performed using a flow cytometer (BD FACSCalibur). UCB-MSCs were cultured in osteogenic, adipogenic, and neurogenic media (all purchased from Gibco) to evaluate the mul-tipotent potential of the cells.

SPIO@SiO2 preparation

Fe3O4 nanoparticles were first synthesized though a facile polyol reduction method by reducing Fe(acac)3 with 1,2-dodecanediol which were protected by oleic acid and oleyl-amine. The average diameters of these mag-netic cores were about 8 nm. Then the surfaces of SPIO nanoparticles were modified with silica through alkaline hydrolysis of tetraethyl ortho-silicate (TEOS). The nanoparticles were charac-terized with the transmission electron micros-copy (TEM) which demonstrated that the syn-thesized nanocomposites had a core-shell structure with light contrast silica shells and dark contrast of SPIO cores.

After sterilization in liquid nitrogen, SPIO@SiO2 was prepared for cell labeling by adding serum-free LG-DMEM to dilute the concentration to 100 µg Fe/ml.

UCB-MSCs labeling with SPIO@SiO2

UCB-MSCs suspension of passages 5 were used for cell labeling. SPIO@SiO2 of 100 µg Fe/ml was added with Poly-L-lysine (PLL) (Fe:PLL = 1:0.03) and incubated at 37°C for 1 h. Cell labeling was initiated by sucking out original serum-containing culture medium, equal amounts with growth medium containing


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SPIO@SiO2-PLLmixture added into 107 cells of UCB-MSCs in culture dish and incubated at 37°C in a humid atmosphere consisting of 5% CO2. After 12 h incubation, growth medium con-taining SPIO@SiO2 was removed and cells were rinsed with PBS. Then cells were detached from the culture dish using trypsin-EDTA (0.25%: 0.02%) and re-suspended with LG-DMEM for the following experiments.

Prussian blue staining of SPIO@SiO2-labeled UCB-MSCs in vitro

Prussian blue staining was performed accord-ing to the manufacturer’s instructions. Briefly, samples of labeled cells were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, incubated with working solu-tion for 30 min, washed with PBS again and counterstained with Eosin. The samples were visualized on an Olympus microscope (Olympus America Inc.) with an Optronics camera attach-ment. The percentage of positive cells = the total number of cells showing positive blue stain/the total number of cells from five inde-pendent fields of view. The cells without SPIO@SiO2 labeling were used as the control group.

MR detection of SPIO@SiO2-labeled UCB-MSCs in vitro

The labeled UCB-MSCs were suspended in 1% gelatin at the cell concentrations of 104 cells/mL, 105 cells/mL and 106 cells/mL. Unlabeled cells were used as the control group. All sam-ples were placed in 1.5 ml Eppendorf tube. MRI was performed on an 11.7-T magnet with a 15 mm birdcage coil. The axial T2*-weighted image (T2*WI) was acquired by means of a fast low angle shot sequence: repetition time (TR) = 30 ms, echo time (TE) = 2 ms, slice thickness = 1 mm, slice space = 0 mm, flip-angle = 15°, view = 10 × 10 mm, data matrix = 128 × 128, and number of excitations = 16. The signal intensity

(SI) was calculated as mean ± standard devia-tion (SD).

SPIO@SiO2-labeled UCB-MSCs culture on col-lagen scaffolds

Culture of SPIO@SiO2-labeled UCB-MSCs on collagen scaffolds was performed according to the procedure in our previous report [11]. Collagen scaffolds (1.5 cm × 0.5 cm) were ob- tained from Zhenghai Biotechnology (Yantai, Shandong Province, China). 20 µL of SPIO@SiO2-labeled UCB-MSCs suspension containing 104, 105 and 106 cells, respectively was dripped onto each scaffold (Figure 1). The cell-seeded scaffolds were incubated in humid air consist-ing of 5% CO2 at 37°C for 1 h. The scaffolds were then added with growth medium for the following experiments.

Rat uterine horns full-thickness trauma and SPIO@SiO2-labeled UCB-MSCs transplantation

All animals were treated in accordance with the guidelines of the Experimental Animals Management Committee (Jiangsu Province, China). As description in our previous report [11], female Sprague-Dawley rats (224-330 g) were randomly assigned to collagen/PBS group and collagen/SPIO@SiO2-labeled UCB-MSCs group (104, 105 and 106 cells, respectively). After the rats were anesthetized, a low abdomi-nal midline incision was made to expose the uterine horns (Figure 2A). A 1.5 cm long and 0.5 cm wide tissue was excised from each uter-ine horn leaving the mesometrium intact (Figure 2B). Different kinds of collagen scaf-folds were sutured to replace the excised seg-ment (Figure 2C) and then the rectus fascia and skin were sutured. Intramuscular injection of penicillin twice a day was given to all animals for three days.

MR detection of SPIO@SiO2-labeled UCB-MSCs in uterine horns in vivo

MR was performed 2 days, 7 days and 14 days postoperatively. Rats were anesthetized with 4% isoflurane and a gas mixture of 2% isoflu-rane-nitrogen/oxygen (70:30) was used to maintain the anesthetic. Plastic rods were placed inside the vagina as an indicator. MRI was performed on a 7.0-T magnet with a 16 cm diameter of horizontal scan frame and body RF coil. The MRI scan positions of sagittal, coronal and axial planes of rat abdominal were taken

Figure 1. SPIO@SiO2-labeled labeled with different number of UCB-MSCs culture on collagen scaffolds.


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separately with T2-weighted image (T2WI) and proton density-weighted image (PDWI). T2WI was acquired using turbo RARE sequence: TR = 1642 ms, TE = 32 ms, slice thickness = 2 mm, slice space = 0.5 mm, flip-angle = 180°, view = 60 × 30 mm, data matrix = 256 × 256, and number of excitations = 3. PDWI was acquired using fat suppression sequence: TR = 4000 ms, TE = 40 ms, slice thickness = 2 mm, slice space = 0.5 mm, flip-angle = 120°, view = 60 × 30 mm, data matrix = 256 × 256, and number of excitations = 3.

MR detection of SPIO@SiO2-labeled UCB-MSCs in uterine horns in vitro

After rat was euthanasia at 30 days postopera-tively, entire uterus was dissected. The vagina was sutured with silk to the Eppendorf tube cap with sterile distilled water. MRI was performed on an 11.7-T magnet with a 38 mm coil. The coronal and oblique coronal T2*WI was acquired using fast low angle shot sequence: TR = 3000 ms, TE = 20 ms, slice thickness = 1 mm, slice space = 0 mm, flip-angle = 15°, view = 60 × 10 mm, data matrix =128 × 128, and number of excitations = 3. The axial T2*WI was acquired using fast inversion recovery sequence: TR = 6000 ms, TE = 100 ms, slice thickness = 2 mm, slice space = 0 mm, flip-angle = 15°, view = 60 × 10 mm, data matrix = 128 × 128, and number of excitations = 3.

Histological analysis

30 days postoperatively, after MRI detection, the uterine horns were fixed with 4% parafor-maldehyde, dehydrated in graded alcohols and embedded in paraffin. Sections (5 μm) of the uterus were sliced transversally. Hematoxylin-eosin staining was applied to observe the tis-sue structure and Prussian blue staining was

used to reveal the SPIO@SiO2-labeled UCB-MSCs under light microscopy. For immunohis-tochemistry, tissue sections were labeled with antibodies against α-smooth muscle actin (1:200, ab5694, Abcam, Cambridge).

Statistical analysis

Two independent observers performed the his-tological measurement with the average was taken for subsequent analysis. Data were pre-sented as mean ± SD. Multiple comparisons were determined by one-way analysis of vari-ance using Statistics Package for Social Science (SPSS 17.0). Pairwise comparisons using the SNK-q test. P < 0.05 was considered statistically significant (*P < 0.05).

Results

UCB-MSCs identification

The viability of the UCB-MSCs was 95% which was examined using the trypan blue exclusion assay. The cells became adherent 3 h after seeding in the culture dish, appearing round or polygonal in shape. Spindle or star like cells with pseudopodia appeared 12 h later. At day 2 post seeding, radially arranged colonies were observed. At 3 days, the cells were swirl shaped and between 80 and 90% confluent. UCB-MSCs were negative for hematopoietic stem cell markers CD34 and CD45. And the surface markers of CD29, CD73, and CD90 were posi-tive (Figure 3A-G). These cells displayed the capacity of differentiation into chondrocytes (Figure 3H, 3I), adipocytes (Figure 3J, 3K) and neuron (Figure 3L, 3M).

UCB-MSCs labeled with SPIO@SiO2

Prussian blue staining was used to detect the iron particles in UCB-MSCs. Phase contrast microscopy revealed that the presence of blue iron particles in 95.3% of the cell cytoplasm in the SPIO@SiO2 labeled UCB-MSCs.

Compared with the unlabeled UCB-MSCs, the SPIO@SiO2 labeled cells exhibited a markedly decreased signal on the T2*WI sequence and the signal intensity were related to the number of labeled cells (Figure 4).

In vivo MR detection of SPIO@SiO2-labeled UCB-MSCs in uterine horns

A plastic rod which showed decreased signal on the T2WI sequence was placed inside the vagi-

Figure 2. Surgical procedure of rat uterine horn damage/regeneration model. Uterine horns were exposed via abdominal midline incision (A). (A) seg-ment of approximately 1.5 cm in length and 1/2-2/3 of the total area was excised and removed (B). Col-lagen scaffold with UCB-MSCs was grafted to the re-maining uterine horns (C).


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na as an indicator (Figure 5A, 5B) for the loca-tion of uterus, because the uterine horns’ loca-tion was affected by the uterine spontaneous motility and intestinal peristalsis.

For 7.0-T MR system detection, SPIO@SiO2-labeled UCB-MSCs suspension containing 104, 105 and 106 cells were loaded on the collagen scaffold for transplantation. Two days after

transplantation, only in 106 cells group of axial view of the uterus low signals could be detect-ed. Large reductions of MRI signal intensity were found at surgical region on the T2WI sequence. With the change of scanning angle, areas with low intensity MRI signals of flaky, cordal and linear shapes could be visualized. In the corresponding regions of the PDWI sequence similar signal decrease were also

Figure 3. Phenotype and multi-differentiation of UCB-MSCs. A. UCB-MSCs showed homogenous, fibroblast-like mor-phology at the 3rd passage. B-G. Flow cytometry analysis of immune-markers in UCB-MSCs. B, isotope controls for FITC. Results confirmed that UCB-MSCs were positive for CD29 (E), CD73 (F) and CD90 (G), but negative for CD34 (C) and CD45 (G). H-J. UCB-MSCs had the potential to differentiate to multiple lineages. Phase contrast microscopic appearance of calcified nodule (H) and stained with Alizarin red S (I) indicated that UCB-MSCs differentiated to os-teogenic lineage. Adipocytes with intracellular lipid droplet induced from UCB-MSCs were detected by microscope at the day 14 (J), and determined by Oil red O staining (K). Differentiation of UCB-MSCs to neuronal lineage after 21 days induction displayed the multipolar neuron under microscope (L) and these cells were positive for NSE by immunofluorescence staining (M).


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detected (Figure 6A). By seven days, the low intensity signal decreased over time. In the T2WI and PDWI sequences of the same uterus, the signal from the entire tissue decreased col-lectively. In the same area, the low-signal regions decreased in size (Figure 6B). Subsequently, the signal intensity decreased and became indistinct at 14 days. The MRI scan positions of sagittal and coronal were unable to make uterine horns visible in the imaging plane.

In vitro MR detection of SPIO@SiO2-labeled UCB-MSCs in uterine horns

The coronal, oblique coronal and axial T2*WI sequences under the 11.7-T MR system were used for in vitro detection. The endometrial,

muscular and serosal layers were clearly visible at all three planes. Low intensity MRI signals were detected at surgical region (Figure 7). To validate that the low intensity MRI signals were caused by SPIO@SiO2 labeling, adjacent tissue slides were treated with Prussian blue staining, a large number of viable cells with blue-positive particles were seen at the surgical region (Figure 7).

30 days postoperatively, Prussian blue staining revealed that labeled UCB-MSCs were found to distribute in the surgical region. Cells stained blue-positive were mainly in the myometrium and serosal junction area (Figure 8A, 8B), how-ever, some also distributed in the endometrium and adipose tissue (Figure 8C, 8D). The per-

Figure 4. MR detection sensitivity of SPIO@SiO2-labeled UCB-MSCs in vitro. T2*-weighted MR imaging of SPIO@SiO2-labeled MSCs at the cell concentration of 104 cells/mL, 105 cells/mL and 106 cells/mL. Unlabeled cells were used as the control group. The cells are suspended in 1% gelatin.

Figure 5. The Method for the uterine horns’ location. A. Plastic materials come from the Digene Hybrid Capture 2 High-Risk HPV DNA Test (HC2); B. T2WI sequence MR images of plastic material as an indicator for the location of uterus.


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horns are slender organs, with one end fixes to the vagina and the other free. In MR images, adjacent tissues including the intestines and ureters are shown as curved tubular objects that are difficult to distinguish from the gracile uterus. Thus, very few MR imaging studies have been conducted on the uterine horns. We used plastic materials which showed decreased sig-nal on the T2WI sequence to display the top of the vagina in MR images. By altering the scan position, the sinuous tissues with free and slightly swollen terminus could be discerned as the uterine horns. Even so, it was difficult to make bilateral uterine horns simultaneously visible in the same imaging plane. Because rats’ uterine horns show spontaneous rhythmic contractions and meanwhile intestinal motility may also induce uterine movement. Since high-

centage of Prussian blue staining positive area was positively correlated with the number of transplanted cells. The percentage of Prussian blue staining positive area in 106 cells transplantation group (1.53% ± 0.17%) was significantly higher than that of 105 cells group (0.71% ± 0.07%) and 104 cells group (0.13% ± 0.01%) (P < 0.01) (Figure 8E).

Discussion

Stem cells therapy is a novel area of investigation in tissue repair and regeneration. Cells migration and homing are momentous steps for cell transplantation. Noninvasive- ly tracing of the location, dis-tribution, and viability of the transplanted cells is impera-tive. MRI is a powerful imag-ing method that capable of demonstrating cells labeled with an MRI contrast agent in vivo.

In vivo MRI tracking studies in rat models focused mainly on large organs with fixed posi-tions such as the brain, heart, and liver [16, 22, 23]. The internal reproductive organs of rats are located in the lower abdomen. The uterine

Figure 6. In vivo PD-weighted and T2-weighted MR images of labeled MSCs (106) loading on collagen scaffolds implanted onto injured uterus 2 day (A), 7 d (B).

Figure 7. In vitro the coronal, oblique coronal and axial T2*-weighted MR images of uterine horns.


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field imaging scans take a long time, the posi-tion of the uterus can be highly variable.

In vitro MR detection of SPIO@SiO2-labeled UCB-MSCs showed that the low signal in Flash

T2*WI sequence images decreased as the num-ber of labeled cells increased, suggesting that as the number of SPIO@SiO2-labeled cells increased the negative enhancement of MR images became more noticeable. This facilitat-

Figure 8. Distribution of cells stained blue-positive. A-D. Cells stained blue-positive were mainly in the myometrium and serosal junc-tion area, some also distributed in the endometrium and adipose tis-sue. E. The percentage of Prussian blue staining positive area was posi-tively correlated with the number of transplanted cells.


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ed imaging of SPIO@SiO2-labeled cells after in vivo transplantation. In the present study, to localize stem cells we employed a local MSCs delivery method. Collagen scaffolds with pore diameters in the range of 120-200 mm were used for 3D culture and deliver of MSCs. SPIO @SiO2-labeled UCB-MSCs of 104, 105 and 106 cells were respectively loaded on collagen scaffold as a complex for treatment of rats’ uterine horns trauma. In vivo MR images were conducted at 7.0 T magnets. Two days after delivering of labeled UCB-MSCs, only in 106 cells group low signals were expressed in the T2WI and PDWI sequence in the axial view. Seven days after transplantation, only small area of decreased signal could be detected. Fourteen days after the procedure, the low sig-nal could no longer be detected, indicating that the low signals of 106 labeled cells in vivo can only be detected for 1 week after surgery using 7.0 T MRI tracking. A decrease in the area of the low-signal region may be caused by the fol-lowing reasons: partial apoptosis of transplant-ed cells and subsequent phagocytosis of iron particles by macrophages, migration of a frac-tion of transplanted cells to other parts of the body, and transfer of the iron particles to prog-eny cells via cell proliferation and division.

To achieve high sensitivity, on measures is using higher magnetic field scanner, better RF coil and gradient echo sequences with longer TE. In conventional clinical scanners, 105 is the minimum number of cells that can be visualized [24], but at a high field strength of > 11 T, the sensitivity can be increased to a small amount of, or even single-cell level when contrast agents were used [25, 26]. In the present study, 105 SPIO@SiO2-labeled UCB-MSCs were loaded on collagen scaffold to conduct in vitro imag-ing at 11.7 T, using a small animal micro-imag-ing system 30 days postoperatively. In the coro-nal and the oblique coronal views, the in vitro MRI displayed the uterine cavity, uterine wall and surgical area clearly. In the axial position, the images displayed a blurry view of the endo-metrium, the myometrium, and the uterine serosal. In the surgical region, low intensity MRI signals were detected which was validated to be cells with iron particles by Prussian blue staining.

Previous studies suggested that the low-signal area could be detected for more than 4 weeks

when SPIO-labeled MSCs were injected into the myocardial infarction border zone or into mouse brain [22, 23]. However, in the present case, the much shorter in vivo tracing period may have been associated with the slenderness of the uterus and difficulty of imaging.

In the cross-sections of in vitro genital tissues 30 days after the procedure, Prussian blue staining showed that the positive area was cor-related with the number of transplanted cells. Blue-positive particles were mainly located in the repaired area, scattered across the junc-tion between the endometrium and the myome-trium and the endometrial stroma. In our earlier studies [11], we found that 4 weeks after trans-planting MSCs labeled with a membrane fluo-rescent dye, most MSCs were found in the repaired area, while a small number of cells had differentiated into endometrial stromal cells. Results from both studies were highly consistent, which suggested that SPIO@SiO2 labeling does not affect the survival of MSCs.

Conclusion

Our present study described a simple protocol for efficient SPIO labeling of UCB-MSCs by coat-ing of SPIO with SiO2. An in vivo 7.0 T MRI detected the labeled UCB-MSCs on collagen scaffold in surgical region of rats’ uterine horns trauma 7 days after implantation. In vitro 11.7 T MRI detected the labeled UCB-MSCs in surgi-cal area 30 days postoperatively, which were also further confirmed by Prussian blue stain-ing. The results presented a valuable step for monitoring of SPIO@SiO2 labeled stem cells in uterine tissue reparation by noninvasive MRI, which could greatly improve the efficiency of stem cells tracking in both animal and clinical trials. These results also enhance our under-standing of stem cell based therapy for uterine reparation.

Acknowledgements

The authors thank Dr Qiang Zhou for his techni-cal assistance in immunohistochemistry. This work is supported by grants from the “Strategic Priority Research Program” of the Chinese Academy of Sciences (XDA01030401); the National Natural Sciences Foundation of China (81401166); the Fundamental Research Funds for the Central Universities (20620140672); Maternal-Fetal Medicine from Jiangsu Province


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Health Department of China; Key Laboratory of Obstetric & Gynecologic Diseases, National Ministry of Health and Nanjing Health De- partment.

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Ya-Li Hu, Depart- ment of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, 321 Zhongshan Road, Nanjing 210008, China. Tel: 0086-10-82614426; Fax: 0086-10-82614426; E-mail: [email protected]; Dr. Jian-Wu Dai, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 3 Nanyitiao, Zhongguancun, Beijing 100190, China. Tel: 0086- 25-83317016; Fax: 0086-25-83317016; E-mail: [email protected]; Dr Zong-Wu Deng, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215125, China. Tel: 0086-512-62872559; Fax: 0086-512-62872559; E-mail: [email protected]

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