Abstract
The aim of this study was to determine whether double labelling of human umbilical cord mesenchymal stem cells (hUCMSCs) with gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA) and PKH26 influences their biological characteristics. A tissue adherence technique was used to separate and purify the hUCMSCs and flow cytometry was performed to detect the surface markers expressed on them. Gd-DTPA and PKH26 were used to label the stem cells and MRI and fluorescence microscopy were used to detect the double-labelled hUCMSCs. A MTT assay was used to delineate the growth curve. Transmission electron microscopy (TEM) and atomic force microscopy were used to demonstrate the ultrastructural features of the hUCMSCs. Flow cytometry showed that hUCMSCs highly expressed CD29, CD90, CD44 and CD105. No expression of CD31, CD34 and CD45 was detected. Very low expression of HLA-DR and CD40 was detected. Atomic force microscopy showed these cells were long, spindle shaped, and the cytoplasm and nucleus had clear boundaries. After double labelling, TEM showed Gd particles aggregated in the cytoplasm in a cluster pattern. The proliferation activity, cell cycle, apoptosis and differentiation of the stem cells were not influenced by double labelling. Thus a tissue adherence technique is helpful to separate and purify hUCMSCs effectively; and Gd-DTPA and PKH26 are promising tracers in the investigation of migration and distribution of hUCMSCs in vivo.
Keywords: Gd-DTPA, magnetic resonance imaging, mesenchymal stem cells, PKH26, Tracing
Human umbilical cord mesenchymal stem cells (hUCMSCs) are a group of cells with self-renewal and multipotent differentiation potentials. These stem cells have been ideal seed cells in stem cell studies and in clinical application (Sanberg et al. 2005; Jin et al. 2013). There are many advantages of hUCMSCs; and the disadvantages of the stem cells in repairing tissues are not well understood. Evaluation of the behaviour of these cells in tissue repair, their migratory dynamics, and the degree of cell survival, integration into the newly formed tissue and fate in vivo after transplantation has not been investigated previously. This may be because how to monitor the biological behaviours in both animal models and in clinical trials after stem cell transplantation remains a problem. Bioluminescence, radioactive substrates, near-infrared fluorescence, post-mortem histological analysis and magnetic resonance imaging (MRI) contrast agents have been employed to detect migration and homing of the transplanted cells (Michalet et al. 2005; Thorne 2008). MRI has been said to be a promising way for clinical use and for in vivo studies. It has been used to trace transplanted cells. However, a prerequisite is that these cells must be labelled efficiently and effectively before transplant. Compared with other reagents for MRI, gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA) can mark stem cells effectively, and the marking rate reached up to 90% without cytotoxicity (Shyu et al. 2007; Cheng et al. 2011; Mahmoudi et al. 2011; Shen et al. 2010a,b; Guenoun et al. 2012). The red fluorescence dye, PKH26, is a lipophilic membrane binding dye that can irreversibly bind to the lipid bilayer of cell membranes. PKH26 is also less toxic, has no influence on cell proliferation, and can be used to stain a variety of cells (Modo et al. 2009). Thus, PKH26 is also one of the ideal tools used to label cells. Therefore the aim of this study was to observe and trace hUCMSCs when the hUCMSCs had been labelled with Gd-DTPA and PKH26 together and to determine the effect of double labelling on the biological characteristics of the hUCMSCs.
Materials and methods
Materials
Umbilical cords were collected from full-term, healthy neonates during caesarean sections. Informed consent was obtained before enrolment in the study. The following equipment was used: an inverted phase contrast microscope (Olympus, Tokyo, Japan), atomic force microscope (Thermomicroscopes, Sunnyvale, CA, USA), FACSCalibur flow cytometer (BDBiosciences, San Jose, CA, USA) and PhiliPCM-10 transmission electron microscope (Philips, Amsterdam, Holland). The following reagents were used: DMEM/F12 powder (GIBCO BRL, Gaithersburg, MD, USA), alizarin red and oil red O staining solutions (Guangzhou Sijia Biotech Co., Ltd., Guangzhou, China), Gd-DTPA injection (Bayer, Leverkusen, North Rhine-Westphalia, Germany), Effectene (Qiagen, Hilden, North Rhine-Westphalia, Germany), trypan blue staining kit (Beyotime Institute of Biotechnology), PKH26 (Sigma, St. Louis, MO, USA), and 1.5T HDXT MR (GE Healthcare, Milwaukee, WI, USA).
Ethical Approval statement
This study was approved by the Medical Ethics Committee of Jinan University First Affiliated Hospital.
Methods
Separation and culture of hUCMSCs and detection of the hUCMSC phenotypes by flow cytometry
Under aseptic conditions, umbilical cords were harvested and MSCs were separated and purified using a tissue adherence technique. The cord was washed in D-Hank's balanced salt solution to remove blood cells. The vessels were removed. The cord was cut into cubic millimetre small pieces and filtered through 150 μm strainers. The small pieces of tissues were seeded in six-well plates, covered with coverslip and cultured in DMEMF/F12 with 15% FBS (culture medium). After one week, cells had grown around the tissues and the medium was refreshed. When cell confluence reached 80–90%, the adherent cells could be detached with trypsin/EDTA. Passaging was performed at a ratio of 1:3. Passage3 cells were digested routinely and then washed three times with phosphate buffered saline (PBS). Single cell suspensions were prepared at a cell density of 1 × 106 cells/ml. Flow cytometry was employed to detect the following surface markers: CD29; CD44; CD90; CD105; CD40; CD45; and HLA-DR.
Double labelling of hUCMSCs with Gd-DTPA and PKH26
Effective transfection: Passage3 T25 cells were cultured. When cell confluence reached 70–80%, the culture medium (4 ml) was refreshed. One hour later, 25 μl of Gd-DTPA and 120 μl of buffer EC were added to a 1.5 ml tube, followed by the addition of 8 μl of Enhance. The cell culture was incubated at room temperature for 5 min. Then, 25 μl of Enhance was added, followed by incubation at room temperature for 10 min. Subsequently, 1 ml of culture medium was added to the tube, and the mixture was added to the cell-containing dish, followed by incubation at 37°C for 4 h in 5% CO2. The cells were harvested and labelled with PKH26 according to the manufacturer's instructions. The double-labelled stem cells were maintained in an incubator.
Observation of hUCMSCs by confocal microscopy, transmission electron microscopy and atomic force electron microscopy
The double-labelled hUCMSCs (Passage3) were seeded into a dish at a density of 105/ml (100 μl) and incubated overnight for confocal microscopy (the cells were in logarithmic growth phase). The cells were washed twice with PBS (5 min each), then fixed in 4% paraformaldehyde at room temperature for 15 min and examined by electron microscopy. Samples were routinely prepared, and transmission electron microscopy (TEM) and atomic force electron microscopy were performed to observe the ultrastructural features of hUCMSCs with and without double labelling.
Detection of cell viability by trypan blue staining
At 1, 24, and 72 h after double labelling, cell viability was detected using trypan blue. Stem cells of the same generation served as controls. After labelling, cells were counted on a haemocytometer. At least 500 cells were counted. The cell viability was calculated as follows: cell viability = (total cells–blue cells)/total cells × 100%.
Growth curve of hUCMSCs with and without double labelling
Cells of the same generation with and without double labelling were harvested after digestion and seeded into 96-well plates (103 cells/well). Eight days later detection was carried out using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay in five wells. The results were read out on a microplate reader and the growth curve of cells with and without double labelling was delineated.
Analysis of cell cycle activity and levels of apoptosis
Human umbilical cord mesenchymal stem cells at passage3 with and without double labelling were routinely digested and harvested to prepare a single cell suspension. The cells were counted, and the cell density was adjusted to 106 cells/ml. Then, the cell suspension was centrifuged at 400 g for 5 min. The supernatant was removed, and 75% ethanol at −20°C was added. Before detection, the cell suspension was centrifuged at 400 g for 5 min, and the supernatant was removed. Rnase was added to remove RNA. After the addition of propidium iodide (PI), the cells were incubated at 4°C in the dark for 30 min. The cell cycle distribution profile was recorded by flow cytometry.
Human umbilical cord mesenchymal stem cells at passage3 with and without double labelling were routinely digested and harvested to prepare single cell suspensions. Cells at 4°C were twice washed with PBS and re-suspended in PBS. The cell density was adjusted to 106/ml. Then, 100 μl of cell suspension was added to a 5-ml tube, followed by addition of 5 μl of Annexin V/FITC and 10 μl of PI (20 μg/ml). The resultant cell suspension was incubated at room temperature for 15 min in the dark. After addition of 400 μl of PBS, flow cytometry was used to detect apoptotic cells.
Detection of multipotent differentiation potentials of hUCMSCs with and without double labelling
Human umbilical cord mesenchymal stem cells at passage3 with and without double labelling were seeded into six-well plates and maintained in complete medium. When cell confluence reached 80–90%, induction differentiation was performed. Adipogenic induction medium was added (10% foetal bovine serum, 10 μg/ml insulin, 1 μmol/l dexamethasone, 0.5 mmol/l 3-isobutyl-1-methylxanthine, 100 U/ml penicillin and 100 U/ml streptomycin in LG-DMEM). The medium was refreshed once every 3 days. On day 21, oil red ‘O’ staining was performed. Osteogenic induction medium was added (10% foetal bovine serum, 100 nmol/l dexamethasone, 10 mmol/l β-glycerophosphate, 0.2 mmol/l vitamin C, 100 U/ml penicillin, and 100 U/ml streptomycin in DMEM/F12). The medium was refreshed once every 3 days. On day 21, alizarin red staining was performed.
Detection of Gd-DTPA-labelled hUCMSCs by MRI
Four hours after Gd-DTPA labelling, a fraction of cells were re-suspended in PBS and added to 1-ml tubes. Then, cells with and without labelling were subjected to MRI. The remaining labelled cells were incubated at 37°C in a humidified environment with 5% CO2 and passaged. When cell confluence approached 100%, cells were routinely digested and passaged at a ratio of 1:3. Cells at passage3 with and without Gd-DTPA staining were addedto a 1-ml tube and subjected to MRI until hyperintensity was not observed. The Gd-DTPA-stained and Gd-DTPA-unstained hUCMSCs were added to a 1.0-ml tube and centrifuged. Then, these cells were re-suspended in 4% gelatine solution for MRI. The number of cells detected was gradually reduced (106, 5 × 105, 105, 104, and 5 × 103) until the hyperintensity was not observed.
Magnetic resonance imaging was performed with a 1.5T MRI system and an 8-channel wrist coil. The detection conditions were as follows: SE T1WI sequence, TR = 300 ms, 600 ms, 900 ms and 1200 ms; TE = 15 ms; Slice distance/thickness = 1.0/0 mm, matrix = 256 × 256 pixels; slice distance/thickness = 1.0/0 mm, matrix = 384 × 192; and the view = 12 cm × 12 cm. Signal collection was performed twice. The area of interest was used to evaluate the intensity of cells. The area of interest was 1mm2.
Statistical analysis
Statistical analysis was performed with spss 13.0. Data are expressed as 
, and comparisons were done with t-test. P < 0.05 was considered statistically significant.
Results
Separation, culture and observation of hUCMSCs morphology
A tissue adherence technique was used to separate hUCMSCs. After 24 h the cells were adherent to the wall of culture dishes. These cells were spindle shaped or polygonal (Figure1a). Refreshing the medium and passaging, hUMSCs were purified. The passaged cells had uniform morphology. Passaging was performed once every 3 days and more than 25 times.
Figure 1.
Morphology of hUCMSCs. (a) Inverted phase contrast microscopy (40×) 24 h after culture of third generation hUCMSCs; (b) Ultrastructure of third generation hUCMSCs under a transmission electron microscope (4800×); (c) Ultrastructure of third generation hUCMSCs under an atomic force microscope.
Under an atomic force microscope, hUCMSCs were spindle shaped and more than 100 μm in length. The cytoplasm and nucleus had a clear boundary. Cells extended several pseudopodia-like protrusions with an axon-like skeleton, which play important roles in the maintenance of cell morphology, cell movement and substance transportation. Microfilaments and microtubules formed observable net-like intercellular connections, suggesting the possibility of intercellular communication and material exchange. Observation of the focal structure of stem cells showed some pseudopodia at the edge of cells and a branch- or fishtail-like cytoskeleton, which was complex in morphology (Figure1c).
Transmission electron microscopy showed that hUCMSCs were oval and had a single nucleus. There was a large amount of nucleoplasm. The nucleolus was large and clear. The cells were rich in cytoplasm with a large number of microfilaments, ribosomes and mitochondria. However, there are few other organelles. The cells had a large amount of heterochromatin, but a small amount of euchromatin. There were some microvilli on the cells, which is a characteristic of undifferentiated cells. Indeed, the cells had the hallmark characteristics of MSCs (Figure1b).
Flow cytometry of hUCMSCs after double staining
Flow cytometry demonstrated that hUCMSCs did not express CD31, CD34 or CD45, but had high expression of CD29, CD105, CD44 and CD90. In addition, the expression of HLA-DR and CD40 (factors related to graft-versus-host disease) was very low (Figure2).
Figure 2.
Expression of hUCMSC surface markers. hUCMSCs did not express CD31, CD34 or CD45, but had high expression of CD29, CD105, CD44 and CD90. In addition, low expression of HLA-DR and CD40 is detected.
In vitro MRI of Gd-DTPA-labelled hUCMSCs
1.5T MRI showed that Gd-DTPA-labelled hUCMSCs had hyperintensity on T1WI and T2WI (Figures3 and 4). MRI showed that the intensity on T1 was reduced with the reduction of cell number. When 5 × 103 cells were subjected to MRI, hyperintensity was not observed on T1. The minimal number of cells that were detectable was 104, which was significantly different from the number of cells labelled with Gd-DTPA after transfection (5 × 103) on T1 (P < 0.01). However, the number of Gd-DTPA-labelled and unlabelled cells was comparable on T1 (the number of cells was approximately 5 × 103). After effective transfection, Gd was detectable by MRI for 14 days in vitro and disappeared approximately 15 days. Thus, the maximal time to trace stem cells stained by Gd-DTPA was approximately 14 days.
Figure 3.
MRI of different numbers of Gd-DTPA-labelled hUCMSCs. 1.5T MRI showed that Gd-DTPA-labelled hUCMSCs had hyperintensity on T1WI and T2WI. MRI showed that the intensity on T1 was reduced with the reduction in cell number. When 5 × 103 cells were subjected to MRI, hyperintensity was not observed on T1.
Figure 4.
MRI of Gd-DTPA-labelled hUCMSCs. (a) and (b) show that the intensity changed with the increase in cell passage in T1WI and T2WI::passage1 (P1), passage2 (P2), passage3 (P3), passage4 (P4), and negative control (NC).
Laser scanning confocal microscope
Cells stained with PKH26 were fibroblast-like with similar morphologies. Cell viability remained unchanged, and trypan blue staining showed the cell viability to be approximately 99%. Cell growth was good. Under a laser scanning confocal microscope, cells were red, the dye was evenly distributed within the cell membrane, the cell outline was clear, and the staining efficiency was as high as 100%. With adherence growth and passaging, the intensity of red fluorescence on the cell membrane was gradually reduced, and granule-like spots were observed (Figure5a: images from laser scanning confocal microscopy).
Figure 5.
hUCMSCs labelled with PKH26 under a laser scanning confocal microscope. (a) Shows that the intensity of red fluorescence on the cell membrane was gradually reduced and granule-like spots were observed with the growth of hUCMSCs. (b) Shows that the intensity of red fluorescence on the cell membrane was gradually reduced with the prolongation of time to PKH26 staining.
With the prolongation of time to PKH26 staining, the intensity of red fluorescence on the cell membrane was gradually reduced. hUCMSCs at passage6 had minimal red fluorescence (Figure5b: images from fluorescence microscopy).
TEM of hUCMSCs after double staining
After Gd-DTPA staining, TEM showed that the Gd granules located in the cytoplasm were black and aggregated in a cluster pattern, which were not observed in cells not stained with Gd-DTPA (Figure6).
Figure 6.
hUCMSCs under a transmission electron microscope. (a) and (b) show that hUCMSCs without labelling or with Gd-DTPA respectively. Arrow: Gd granules (14800).
Viability, growth curve and proliferation of hUCMSCs with and without staining
Trypan blue staining showed that the cell viability was approximately 99%. The viability was comparable between unlabelled cells and labelled cells (P > 0.05). After double labelling, the morphology of hUCMSCs remained unchanged; the hUCMSCs were still long, spindle-shaped and fibroblast-like, and had a parallel arrangement. After passaging, cells with double labelling had rapid proliferation and were stably passaged. Passaging was performed once every 2–3 days. The doubling time was similar between cells that were and were not double stained.
Cell cycle and apoptosis of cells that were and were not double stained
Flow cytometry was performed to detect cells in different phases (Table1). The proportion of cells in S and G2/M phases (G2/M+S%) was more than 30%, indicating active proliferation in both groups. A t-test showed the proportion of cells in different phases was similar between unlabelled cells and double-labelled cells (P > 0.05, Figure7), suggesting that double labelling had no effect on the cell cycle of hUCMSCs. Flow cytometry was performed to detect the apoptotic cells (Table2). The proportion of apoptotic cells that were similar between unlabelled cells and double-labelled cells (P > 0.05), suggesting that double labelling had no effect on the apoptosis of hUCMSCs (Figure8).
Table 1.
Detection of cells in different phases at 7 days after double labelling by flow cytometry
| Hour | Phase | ||
|---|---|---|---|
| G0/G1 (%) | S (%) | G2/M (%) | |
| 7 days | |||
| Labelled cells | 66.66 ± 3.073 | 12.65 ± 1.683 | 21.09 ± 1.98 |
| Unlabelled cells | 67.10 ± 2.103 | 12.30 ± 1.283 | 20.78 ± 1.38 |
| P value | 0.8478 | 0.7888 | 0.8348 |
Figure 7.
Analysis of cell cycle activity by flow cytometry. The proportion of cells in S and G2/M phases (G2/M+S%) was more than 30%. The percentage of cells in different phases was similar between unlabelled cells and double-labelled cells.
Table 2.
Detection of apoptotic cells that were and were not double labelled at different time points by flow cytometry
| Hour | Cells | |||
|---|---|---|---|---|
| Normal cells (%) | Early apoptotic cells (%) | Late apoptotic cells (%) | Dead cells (%) | |
| 24 h | ||||
| Labelled cells | 91.581 ± 2.901 | 6.049 ± 0.428 | 1.398 ± 0.170 | 0.967 ± 0.156 |
| Unlabelled cells | 92.127 ± 2.896 | 5.893 ± 0.369 | 1.148 ± 0.164 | 0.916 ± 0.190 |
| P value | 0.8289 | 0.6575 | 0.1407 | 0.7375 |
| 72 h | ||||
| Labelled cells | 95.200 ± 3.01 | 2.200 ± 0.567 | 1.511 ± 0.189 | 1.101 ± 0.123 |
| Unlabelled cells | 94.848 ± 2.98 | 2.492 ± 0.345 | 1.531 ± 0.200 | 1.110 ± 0.167 |
| P value | 0.8925 | 0.4885 | 0.9059 | 0.9437 |
Figure 8.
Analysis of cell apoptosis by flow cytometry. The proportion of apoptotic cells was the same in both unlabelled cells and double-labelled cells.
Differentiation potential of cells after double staining
Human umbilical cord mesenchymal stem cells were independently maintained in adipogenic and osteogenic induction medium for 21 days and followed by oil red O staining and alizarin red staining. Lipid droplets and mineralized nodules were independently observed in cells after differentiation induction (Figure9). There is no obvious difference in the ability of labelled and unlabelled hUCMSCs to undergo osteogenic and chondrogenic differentiation.
Figure 9.
(a) and (b) Oil red O staining for unlabelled cell and labelled cells after adipogenic induction for 21 days (200×). (c) and (d) alizarin red staining for unlabelled cell and labelled cells after osteogenic induction for 28 days (200×).
Discussion
In recent years, the therapeutic efficacy of stem cell transplantation has been confirmed in animal studies and some clinical trials (Neuss et al. 2010; Talavera-Adame et al. 2011; Wang et al. 2011; Yang et al. 2011; Zhang et al. 2011; Maijenburg et al. 2012); especially, human umbilical cord blood (UCB) has been regarded as an alternative source for cell transplantation and cell therapy because of its haematopoietic and non-haematopoietic (mesenchymal) potential. Besides the advantages in cell therapy and their potential applications, the disadvantages of using stem cells in repairing tissues is not well understood. The tracing of stem cells after transplantation to monitor the biological behaviour of stem cells in vivo is still a challenge in practice. Some reagents have been employed to detect migration and homing of the transplanted cells. However, the cytotoxicity and effectiveness limit their use. In the present study, we labelled primary hUCMSCs with Gd-DTPA and PKH26 together and observe the influence on biological characteristics of hUCMSCs. We found that Gd-DTPA and PKH26 had no influence on the intrinsic biological characteristics of hUCMSCs.
A tissue adherence technique was used to separate and purify the hUCMSCs. In this study, explants culture and enzymatic digestion are two basic methods to isolate hUMSCs from the human umbilical cord. Enzymatic digestion is often used by other researchers but overdigestion of tissue may result in diminished cellular viability and altered cellular function. In our laboratory the viability of cells after enzymatic digestion is 54%. However, the viability of cells using the adherence technique is about 89% (data not shown). These cells were long, spindle-shaped and had swirl-like growth (Li & Cai 2012; Han et al. 2013).
After passaging, the cells grew rapidly and show uniform morphology. The growth curves showed these cells entered the logarithmic growth phase by day 4 of culture. hUCMSCs at passage3 had high expression of CD29, CD44, CD90 and CD105, but no expression of CD45 (a surface marker of hematopoietic cells), CD40 and HLA-DR (markers related to allograft rejection).
In vitro labelling for hUCMSCs is the basis of tracing of stem cells after transplantation. Different methods have been tried to optimize it. Each has its own advantages and disadvantages (Tao et al. 2014).
The red fluorescence dye, PKH26, is a lipophilic membrane binding dye that can irreversibly bind to the lipid bilayer of cell membranes. Several minutes after PKH26 staining, the cell membrane is evenly stained with PKH26 when viewed under a fluorescence microscope. With cell division, PKH26 was evenly divided into two daughter cells, and the fluorescence intensity was reduced by one half. However, when the PKH26 stained cells died, the PKH26 may not be phagocytized by other cells. Thus, there are no false-positive phenomena. PKH26 is less toxic, has no influence on cell proliferation, can be used to stain a variety of cells. It can not be transmitted into unstained cells and can be retained in vivo for as long as 1 year (Modo et al. 2009). Thus, PKH26 has been one of the ideal tools used to label cells. In the present study, red fluorescence PKH26 was used to label hUCMSCs, which were then passaged in vitro. With the increase in cell passage, the fluorescence intensity was gradually reduced. hUCMSCs at passage6 had minimal fluorescence intensity, which was different from a previous report. This finding might be attributed to the long-term exposure to light during passaging, which resulted in a rapid decay of the fluorescence.
Related studies have shown that MRI is non-invasive, has a lengthy effective imaging time, high spatial and temporal resolution and good contrast. Thus, MRI is regarded as an optimal method for in vivo tracing (Bos et al. 2004; Kim et al. 2007; Shen et al. 2010a,b) It is reported that Gd-DTPA (as little as 30 μl) could be used to effectively stain stem cells, and more than 90% of cells were labelled. In addition, MRI is not cytotoxic. In the present study, 1.5T MRI was used to detect the labelled hUCMSCs in vitro. Gd-DTPA-labelled cells showed hyperintensity in T1-weighted images of MRI, and the Gd-DTPA was retained in these cells for approximately 14 days. Thus, Gd-DTPA can be used to non-invasively and repeatedly trace cells. The migration of these stem cells may be determined by signal intensity, and the signal intensity may be used to determine the aggregation of stained cells, which is beneficial for the repeated detection of stained cells and the collection of more objective information.
PKH26 can be safely used to label cells and retained in the labelled cells for a long time. However, tracing of PKH26-stained cells requires the killing of animals. Thus, this is an invasive method, and only applicable in animal studies, and has limited application in clinical practice. Conversely Gd-DTPA staining for tracing cells is non-invasive and can be performed repeatedly. Of note, Gd-DTPA is retained in cells for a short time, which significantly limits its wide application. In the present study, Gd-DTPA and PKH26 were successfully used to label cells. The advantages of both markers were used to trace these stem cells, which may be effective in tracing the migration, distribution and proliferation of stem cells in vivo. Further investigation showed, with respect to the biological characteristics of cells, the morphology, viability and proliferation of double-labelled cells were similar to unlabelled cells. In addition, the proportion of cells in different phases and that of apoptotic cells were comparable between cells that were and were not double labelled. These findings suggest that Gd-DTPA and PKH26 can be used to effectively stain stem cells and have no influence on the biological characteristics of UC MSCs. The intracellular ultrastructure of cells undergoing double staining was similar to that of cells that were not labelled, indicating that double labelling has no impact on the cell structure (such as some microvilli, evident cytoskeleton, abundant organelles and active metabolism). These cells still presented with characteristics of undifferentiated cells. The above findings suggest that the separated cells have the characteristics of MSCs. TEM showed that Gd granules were black and aggregated in the cytoplasm, which was not observed in unstained cells. Moreover, after double labelling, hUCMSCs exhibited adipogenic and osteogenic differentiation after adipogenic and osteogenic induction, respectively, suggesting that double labelling had no impact on the multipotent differentiation potentials of stem cells.
Taken together, our results reveal that a tissue adherence technique can be used to separate and purify MSCs from umbilical cords. Double labelling with Gd-DTPA and PKH26 had no effect on the intrinsic biological characteristics of UCMSCs. Thus, Gd-DTPA and PKH26 can be used to trace UCMSCs. Our findings provide a theoretical basis for further in vivo tracing of stem cells. However, in vivo experiments still should be conducted to evaluate the influence of Gd-DTPA and PKH26.
Conflict of interest
The authors declare that they have no conflict of interest.
Funding source
This work is supported by China National Natural Science Foundation Project (No. 81070459).
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