Stem cell therapy with potential to regenerate damaged myocardium is an emerging treatment modality for ischemic heart disease [
Thus far, tracking of cardiovascular delivered stem cells in a clinical setting has been limited to direct cell labeling with radioisotopes and tracking with gamma-cameras, single-photon emission computed tomography, or positron emission tomography [
Tracking of cells labeled with superparamagnetic iron-oxide (SPIO) or ultrasmall superparamagnetic iron-oxide (USPIO) nanoparticles using magnetic resonance imaging (MRI) offers high spatial resolution in combination with high soft tissue detail, without exposing the patient to ionizing radiation. Furthermore, the cells can be tracked for months. Cellular labeling methods with SPIO or USPIO are relatively simple, fast and inexpensive.
Iron-oxide is nontoxic, since iron is a naturally occurring metal in the human body, and the iron oxide core is coated with biocompatible shell, allowing its eventual assimilation via endogenous metabolic iron cycles. The use of SPIO and USPIO labeling is clinically safe and does not influence cell function [
MRI tracking of SPIO and USPIO labeled cells has been utilized
There has been some concern that MRI signals from SPIO and USPIO labeled cells may originate from macrophages that have engulfed the labeled cells. This was seen in a few rat studies [
For tracking of nonphagocytic cells, USPIO particles are probably more suitable than SPIO particles, due to higher cellular uptake [
The aim of the present study was to determine
Bone marrow was obtained from the iliac crest by needle aspiration from healthy donors. The studies were conducted under local ethical approval. Mononuclear cells were then isolated by gradient centrifugation and cultured in complete medium consisting of Dulbecco’s modified Eagle medium supplemented with HEPES and L-glutamine, (PAA Laboratories, Austria), 10% fetal bovine serum (PAA Laboratories, Austria), and 1% penicillin/streptomycin (Invitrogen, Austria). Cells were incubated at 37°C in humid air with 5% CO2. Medium was changed twice a week. The cells were grown to confluence before each passage. After two passages, the cells were washed with PBS (Invitrogen, Austria) and harvested with TrypLE Select (Invitrogen, Austria). Cells from each donor were characterized by flow cytometry for CD90, CD73, CD105, CD13, CD45, and CD34, in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells [
Tat-peptide derivatized USPIO nanoparticles coated with dextran (IODEX-TAT-FITC; 15–20 nm) were prepared in our laboratory using the method described by Josephson et al. [
Dose titrating evaluation of iron concentrations added to cells and resulting amounts of iron bound to cells by Josephson et al. [
MSCs were labeled by incubation with USPIO nanoparticles at a concentration of either 5
MSCs in a volume corresponding to 1 × 105 cells were transferred to microfuge tubes and centrifuged for 5 min at 500 g. Cell pellet was frozen and stored at –20°C until date of quantification. Then, the cells were resuspended in 50
Labeled and unlabeled MSCs were transferred to microfuge tubes with 2.5 × 105, 5 × 105, or 1 × 106 MSCs per tube. Tubes were centrifuged at 500 g for 5 minutes. The cells were then suspended in 500
Number of MRI phantoms.
Number of MSCs | USPIO dose | USPIO incubation time | ||
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2 hours | 6 hours | 21 hours | ||
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full | 13 | 10 | 13 |
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full | 11 | 7 | 9 |
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full | 12 | 7 | 8 |
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half | 14 | 5 | 9 |
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half | 11 | 5 | 8 |
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half | 11 | 4 | 8 |
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0 | 14 | 5 | 12 |
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0 | 10 | 6 | 11 |
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0 | 12 | 5 | 8 |
MSC: mesenchymal stromal cell; USPIO: ultrasmall super-paramagnetic iron-oxide. USPIO dose—full = 10
MRI phantoms. (a) Two phantoms containing USPIO labeled cells. (b) MRI image of 2 phantoms with an ellipsoid region of interest placed in the upper phantom. USPIO: ultrasmall superparamagnetic iron-oxide.
Phantoms were scanned using a 1.5T GE Signa Excite HD MRI scanner with a 4-channel receive-transmit brain coil (GE Healthcare). Two phantoms and one reference phantom with no cells were scanned concurrently. Phantoms were placed in an Eppendorf tube rack, with the reference phantom in the center and a randomly selected MSC phantom on each side with 4 cm distance to the reference phantom. The rack was placed and fixated with tape on top of 4 other racks inside the coil to achieve a central position within the coil. Images were acquired using a brain-hemorrhage T2*-weighted gradient-echo (GRE) sequence with repetition-time (TR) = 620 ms, echo-time (TE) = 15.7 ms, flip-angle = 35°, matrix = 192 × 256, field of view (FOV) = 140 × 140 mm, and slice thickness = 7 mm.
Image analysis was performed using an Advantage Workstation AW4.3-05 (GE Healthcare). An ellipsoid region of interest (ROI) of 20 mm2 was placed on the images in the center of each phantom, avoiding the edges. The postprocessing tool produces mean intensity values for each ROI. Each pixel in the ROI is given an intensity value between 0 and 4095. The mean intensity value is the mean of these values for all the pixels in the ROI (Operators manual, GE Healthcare). For comparative analysis, the difference in mean intensity values between reference and cell phantom was used. Figure
Two hearts from freshly slaughtered pigs were placed and fixated with small wooden sticks in a polystyrene box. The hearts were MRI scanned before and after injection of MSCs. One heart was injected with 4 injections of USPIO labeled MSCs (full dose—21 hours incubation), each injection with approximately 2 × 106 MSCs in 0.4 mL. The other heart received 4 injections with unlabeled cells. Care was taken that the hearts remained in the exact same position before and after injections.
Hearts were scanned using a 1.5T Siemens Magnetom Avanto MRI scanner and a body matrix coil (Siemens AG, Germany). The scanning protocol was a thalassemia T2* weighted GRE sequence with TR = 200 ms, flip angle = 20°, matrix = 96 × 256, FOV = 135 × 180 mm, and slice thickness of 5 mm. The entire left ventricle was scanned with concurrent slice thickness of 5 mm with no gaps. The protocol produces 8 images for each slice, with different TE times (3.05, 5.89, 8.73, 11.57, 14.41, 17.25, 20.09, and 22.93 ms).
Statistical analysis was carried out using SPSS 20 (SPSS Inc., USA). One-way ANOVA tests were used for comparing cellular iron content and MRI intensity differences between groups. A
Normality was determined for each group with Kolmogorov-Smirnov and Shapiro-Wilk tests. Equal variances were determined with Levene’s test for homogeneity of variances.
Determination of the cellular iron load showed a positive correlation between iron content per cell and the length of the USPIO incubation period. The results are illustrated in Figure
Cellular iron content.
Group | Iron content per cell |
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Multiple comparisons (Bonferroni corrected) | ||||||
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Group A | Group B | Group C | Group D | Group E | Group F | Group G | |||
(A) Unlabeled | 0.48 ± 0.17 pg | 44 |
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ns |
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(B) Half dose, 2 hours incubation | 1.22 ± 0.52 pg | 17 |
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ns | ns |
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(C) Full dose, 2 hours incubation | 1.54 ± 0.83 pg | 17 |
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ns |
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(D) Half dose, 6 hours incubation | 2.36 ± 0.65 pg | 17 |
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ns |
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(E) Full dose, 6 hours incubation | 2.71 ± 0.86 pg | 21 |
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(F) Half dose, 21 hours incubation | 4.26 ± 1.59 pg | 37 |
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(G) Full dose, 21 hours incubation | 5.24 ± 1.50 pg | 44 |
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Values are shown ± SD.
USPIO: ultrasmall super-paramagnetic iron-oxide. USPIO dose—full = 10
Cellular iron content. The iron content per cell was determined in unlabeled MSC and MSC incubated with half or full dose USPIO for 2, 6, and 21 hours. MSC: mesenchymal stromal cells. USPIO: ultrasmall superparamagnetic iron-oxide. USPIO dose—full: 10
After 2 hours USPIO incubation time, the cellular iron content was only slightly higher than that of the unlabeled cells. This increase was only significant for the full USPIO dose compared to the unlabeled cells. After 6 hours USPIO incubation time, there was a highly significant increase in cellular iron content compared to unlabeled cells. When comparing to 2-hour incubation times, only the full USPIO dose was significantly higher after 6 hours. After 21 hours, the increase in cellular iron content was highly significant compared to both unlabeled and labeled cells for 2 and 6 hours at both USPIO doses. The cells labeled for 21 hours with the full USPIO dose also had significantly higher iron content than the cells labeled for 21 hours with only half USPIO dose.
Overall MRI intensity diminished with increasing cell numbers and USPIO dosage. A graphical illustration of the
MRI intensity differences after 21-hour USPIO incubation.
USPIO dose |
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Full |
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Half |
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Unlabeled |
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Multiple comparisons |
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Full versus unlabeled |
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Full versus half |
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ns | ns |
Half versus unlabeled | ns | ns | ns |
The MRI intensities are mean pixel intensities (values between 0 and 4095) of a 20 mm2 region of interest in the center area of each phantom, supplied by the imaging software. Values are shown ± SD.
USPIO: ultrasmall super-paramagnetic iron-oxide. USPIO dose—full = 10
Absolute phantom MRI intensities after 21 hour USPIO incubation. MRI intensities are absolute mean values. USPIO: ultrasmall superparamagnetic iron-oxide. USPIO dose: full = 10
Phantoms intensity differences after 21-hour USPIO incubation. MRI intensity differences are the mean numeric difference between absolute MRI intensities of phantoms and reference gels. USPIO = ultrasmall superparamagnetic iron-oxide. USPIO dose: full = 10
USPIO labeled MSCs in amounts of 250.000, 500.000, and 1.000.000 could all be significantly separated on MRI from the same number of unlabeled cells, when using USPIO incubation time of 21 hours and full USPIO dosage.
MSCs labeled with half USPIO dosage could not be separated from unlabeled MSCs at any concentration on MRI. With 2 and 6 hours of incubation time, it was not possible to differentiate between labeled and unlabeled cells at any dose or concentration on MRI (see Tables
MRI intensity differences after 6-hour USPIO incubation.
USPIO dose |
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Full |
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Half |
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Unlabeled |
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ns | ns | ns |
The MRI intensities are mean pixel intensities (values between 0 and 4095) of a 20 mm2 region of interest in the center area of each phantom, supplied by the imaging software. Values are shown ± SD.
USPIO: ultrasmall super-paramagnetic iron-oxide. USPIO dose—full = 10
MRI intensity differences after 2-hour USPIO incubation.
USPIO dose |
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Full |
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Half |
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Unlabeled |
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ns | ns | ns |
The MRI intensities are mean pixel intensities (values between 0 and 4095) of a 20 mm2 region of interest in the center area of each phantom, supplied by the imaging software. Values are shown ± SD.
USPIO: ultrasmall super-paramagnetic iron-oxide. USPIO dose—full = 10
Therefore, the MRI detection limits are as low as 250.000 cells when using full USPIO dose and 21 hours of incubation time. For cells labeled with lower USPIO dose and lower incubation times, no significant difference was detected on MRI compared to unlabeled cells, and the detection limits for cells labeled using these conditions will therefore be at least several million cells.
There are distinct differences in the before and after images when looking at MRI images from porcine hearts receiving USPIO labeled MSCs (Figure
MRI images of porcine myocardium before and after USPIO labeled MSC injection. T2*-images of porcine myocardium before injection (a1–a5) and after injection (b1–b5) of USPIO-labeled MSCs. USPIO labeled MSCs are identified as hypointense areas (arrows). MSCs: mesenchymal stromal cells. USPIO: ultrasmall superparamagnetic iron-oxide.
MRI images from the heart receiving unlabeled cells were without visual differences; thus, unlabeled MSCs are undetectable on MRI (Figure
MRI images of porcine myocardium before and after unlabeled MSC injection. T2*-images of porcine myocardium before injection (a1–a5) and after injection (b1–b5) of unlabeled MSCs. Unlabeled cells cannot be identified. MSCs: mesenchymal stromal cells. USPIO: ultrasmall superparamagnetic iron-oxide.
In the present study, we have examined MRI detection limits of USPIO labeled MSCs
MRI tracking of cells labeled with iron-oxide based nanoparticles in cardiovascular disease has been utilized in a variety of animal studies. In one study, rats were subjected to MI and intramyocardial injection of SPIO labeled allogeneic MSCs [
Interesting results were found in a study using a rat MI model, where SPIO labeled MSCs were injected intramyocardially directly into the infarct lesion [
Moreover, five other studies have also histologically evaluated intramyocardial injection [
A major concern with MRI tracking of SPIO and USPIO labeled cells has been that the obtained MRI signals could originate from macrophages that consumed the SPIO particles after cell death of the original labeled cells. The vast majority of animal studies have found the labeled cells to live at the injection sites with no signs of macrophages or other phagocytic cells. Therefore, concern for phagocytic engulfment of injected cells seems overrated and should not hinder future studies in this area.
Another concern has been that the number of cells that remain in the heart for a prolonged period of time may be limited. A number of animal studies have attempted to assess the number of cells that remain in the heart at different time points after intramyocardial injection. The study by Tran et al. [
MRI tracking of iron-oxide-based nanoparticles labeled cells has yet to be carried out in a clinical cardiovascular setting, but both SPIO and USPIO have been used successfully in different clinical studies. In one study, 10 patients with spinal cord injury received spinal injections of autologous CD34+ cells labeled with magnetic beads [
For clinical use, commercial SPIO and USPIO products were available a few years ago, but these products have been taken of the market. The USPIO particles used in the present study (IODEX) were developed for clinical use and can be used as such, when produced under Good Manufacturing Practice (GMP) conditions. The IODEX particles are designed to remain stable for months without releasing the iron core. This is achieved with additional cross-linking of the dextran coating with epichlorohydrin [
As an intracellular contrast agent for nonphagocytic cells, we find that USPIO particles might be more suitable than SPIO particles for clinical use. In a comparison study, USPIO particles exhibited significantly higher uptake in nonphagocytic cells compared to SPIO particles [
The present study demonstrated that to label MSCs for MRI tracking, the preferable USPIO incubation time and dosage were 21 hours and 10
The authors declare that they have no conflict of interests.
The authors would like to thank chief physician Niels Fogh-Andersen, Department of Clinical Biochemistry, Herlev Hospital, Denmark for assistance with quantification of cellular iron content and lending of equipment.