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Published online before print June 11, 2007, 10.1148/radiol.2442060599
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(Radiology 2007;244:514-523.)
© RSNA, 2007

Molecular Imaging

Transferrin Receptor Upregulation: In Vitro Labeling of Rat Mesenchymal Stem Cells with Superparamagnetic Iron Oxide1

Richard Schäfer, MD, Rainer Kehlbach, PhD, Jakub Wiskirchen, MD, Rüdiger Bantleon, PhD, Jörg Pintaske, PhD, Bernhard R. Brehm, MD, Annika Gerber, Hartwig Wolburg, PhD, Claus D. Claussen, MD, and Hinnak Northoff, MD

1 From the Institute of Clinical and Experimental Transfusion Medicine (R.S., A.G., H.N.) and Departments of Diagnostic Radiology (R.K., J.W., R.B., J.P., C.D.C.), Pathology (H.W.), and Cardiology (B.R.B.), University Medical Center Tübingen, Hoppe-Seyler-Str 3, D-72076 Tübingen, Germany. Received April 4, 2006; revision requested June 2; revision received July 12; accepted August 23; final version accepted November 13. Address correspondence to R.K. (e-mail: rainer.kehlbach{at}med.uni-tuebingen.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To prospectively evaluate the influence of superparamagnetic iron oxide (SPIO) or ultrasmall SPIO (USPIO) particles on the surface epitope pattern of adult mesenchymal stem cells (MSCs) by regulating the expression of transferrin receptor and to prospectively evaluate the influence of transfection agents (TAs) on the uptake of SPIO or USPIO particles in MSCs.

Materials and Methods: The study was approved by the institutional animal care committee of the University of Tübingen. MSCs were isolated from the bone marrow of four rats. To obtain highly homogeneous MSC populations, MSCs from one rat were single-cell cloned. One MSC clone was characterized and selected for the labeling experiments. The MSCs, which were characterized with flow cytometry and in vitro differentiation, were labeled with 200 µg/mL SPIO or USPIO or with 60 µg/mL SPIO or USPIO in combination with TAs. Aggregations of labeled cells were accommodated inside a defined volume in an agar gel matrix. Magnetic resonance (MR) imaging was performed to measure SPIO- or USPIO-induced signal voids. Quantification of cellular total iron load (TIL) (intracellular iron plus iron coating the cellular surface), determination of cellular viability, and electron microscopy were also performed.

Results: Labeling of MSCs with SPIO or USPIO was feasible without affecting cell viability (91.1%–94.7%) or differentiation potential. For MR imaging, SPIO plus a TA was most effective, depicting 5000 cells with an average TIL of 76.5 pg per cell. SPIO or USPIO particles in combination with TAs coated the cellular surface but were not incorporated into cells. In nontransfected cells, SPIO or USPIO was taken up. MSCs labeled with SPIO or USPIO but without a TA showed enhanced expression of transferrin receptor, in contrary to both MSCs labeled with SPIO or USPIO and a TA and control cells.

Conclusion: SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Adult mesenchymal stem cells (MSCs) are in the focus of research in the emerging field of regenerative medicine (1–3). The expression of many surface epitopes (ie, CD29, CD44, CD59, CD90, CD117), including the transferrin receptor (CD71), varies among the hetereogeneous populations of adherent cells, even when such cells have MSC-like properties (4–6). Transferrin receptor expression is known to be modulated by intracellular iron concentration, cell proliferation, cell differentiation, and antigen and mitogen stimulation (7–9). Depending on the cell type, the intracellular iron concentration is directly or inversely associated with transferrin receptor expression (7,8,10).

For clinical applications of MSCs, labeling and tracking are crucial to evaluate cell distribution and homing. Principally, two ways of cell labeling suitable for further magnetic resonance (MR) imaging have already been described: (a) cellular labeling with paramagnetic agents like gadolinium (11,12) and (b) cellular labeling with superparamagnetic agents like small (mean particle size, 60 nm) or ultrasmall (mean particle size, 20 nm) particles of iron oxide coated with carboxydextran—that is, superparamagnetic iron oxide (SPIO) or ultrasmall SPIO (USPIO). Cell labeling with iron oxide particles seems to be superior to labeling techniques involving gadolinium in that the imaging artifact caused by iron oxide particles is several times larger than the labeled cell.

The labeling of MSCs and hematopoietic stem cells with SPIO and USPIO has been established (13–16) and is thought to have no influence on the biology of the target cells. SPIO labeling of nonclonal MSCs, for example, has not been noted to affect cell viability or differentiation potential (17). Currently, several SPIO-based contrast agents like ferumoxides (Endorem; Guerbet, Paris, France) or ferucarbotran (Resovist; Schering, Berlin, Germany) are already approved in some countries and are in clinical use, while other agents like the USPIO Sinerem (Guerbet) or Resovist C (Schering) are under clinical observation. A major aim of any kind of cell labeling is to emphasize the target cell with high contrast in minimal time without affecting cell biology.

Thus, the purpose of our study was to prospectively evaluate the influence of SPIO or USPIO particles on the surface epitope pattern of adult MSCs by regulating the expression of transferrin receptor and to prospectively evaluate the influence of transfection agents (TAs) on the uptake of SPIO or USPIO in MSCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Bone Marrow Preparation and Animals
Our study was approved by the institutional animal care committee of the University of Tübingen. Four male Sprague-Dawley rats (age, 12–20 weeks) weighing 420–530 g were anesthetized with intraperitoneal injection of ketamine (100 mg per kilogram of body weight) and xylazine (5 mg/kg) (B.R.B.). Whole bone marrow was obtained in sterile conditions (R.S.), as described previously (5): After the ends of the femurs and tibiae were clipped off, the whole bone marrow was washed out with phosphate-buffered saline (PBS) (Cambrex Bio Science, Verviers, Belgium). To obtain highly homogenous MSC populations, MSCs from one rat were single-cell cloned. One MSC clone was characterized and selected for the labeling experiments.

MSC Isolation, Cloning, and Cell Culture
To isolate MSCs from whole bone marrow, we used the density gradient technique (R.S.) described previously (4). Briefly, 10 mL bone marrow was resuspended in 10 mL PBS (Cambrex Bio Science) and laid over 20 mL sodium diatrizoate 9.1% wt/vol, ficoll 5.7% wt/vol (Lymphoflot; Biotest, Dreieich, Germany). After centrifugation (20 minutes at 1000g without braking), the mononuclear cells were harvested, washed twice with PBS, transferred to a 75-cm2 culture flask (Corning, Schiphol-Rijk, the Netherlands) and incubated (37°C, 5% humidified CO2) with normal medium containing desoxyribonucleotides, ribonucleotides, ultraglutamine 1 ({alpha}-MEM; Cambrex Bio Science), 100 IU/mL penicillin (Cambrex Bio Science), 100 µg/mL streptomycin (Cambrex Bio Science), and 10% heat-inactivated fetal calf serum (Cambrex Bio Science). After 24 hours, the nonadherent cells were removed, and the adherent cells were cultured (R.S., A.G.) (passage 0). The medium was changed every 3 or 4 days. At subconfluence (70%), the cells were detached with Accutase (PAA Laboratories, Cölbe, Germany) to start the cloning procedure. Briefly, normal medium was added to the cell suspension to achieve a dilution of one cell per 200 µL. Two hundred microliters of this diluted cell suspension was pipetted into each well of a 96-well plate (BD Biosciences, Heidelberg, Germany) and cultured (passage 1). After 4 weeks of culture, single-cell derived clones could be identified and were transferred to six-well plates (BD Biosciences, Heidelberg, Germany) (passage 2) and 75-cm2 culture flasks (Corning) (passage 3 and higher).

In Vitro Differentiation and Cell Staining
MSCs are functionally characterized with in vitro differentiation assays (2). We evaluated the differentiation potential into three mesenchymal lineages: adipogenic, osteogenic, and chondrogenic. The differentiation and staining procedures were performed by two authors (R.S. and A.G., with 1 year of experience in the technique), and the interpretation of the staining results was performed in consensus by two authors (R.S. and A.G., with 1 year of experience in the technique).

Three samples of clonal MSCs (one unlabeled, one labeled with Resovist, and one labeled with Resovist C) were each treated for 21 days with adipogenic medium, and three samples of clonal MSCs (one unlabeled, one labeled with Resovist, and one labeled with Resovist C) were each treated for 21 days with osteogenic medium, as described previously (18,19), with the modification that there was no addition of amphotericin B. The adipogenic medium contained Dulbecco's modified Eagle's medium plus 20% fetal calf serum with 1.0 µmol/L dexamethasone (Sigma, Deisenhofen, Germany), 0.5 mmol/L isobutylmethylxanthine (Sigma), 0.2 mmol/L indomethacin (Sigma), and 0.01 mg/mL insulin (Sigma). Osteogenic medium contained normal medium with 10–8 mol/L dexamethasone (Sigma), 0.2 mmol/L ascorbic acid (Sigma), and 10 mmol/L ß-glycerolphosphate (Sigma).

Six samples of control cells from the same clone (two unlabeled, two labeled with Resovist, and two labeled with Resovist C) were cultured with normal medium. After 21 days, the cell culture was stained with oil red O for in vitro adipogenesis. Briefly, after the medium was removed and washed twice with PBS, 2 mL of 10% formalin was added, followed by incubation for 30 minutes. After the formalin was removed and the cell layer was washed with sterile water, 2 mL isopropanol 60% (Bio Whittaker, Verviers, Belgium) was added for 2 minutes. The isopropanol was removed, and 2 mL of a filtered working solution of oil red O (three parts oil red O stock solution [300 mg oil red O powder {Sigma} plus 100 mL 99% isopropanol {Sigma}] plus two parts deionized water) was pipetted onto the cells and left there for 5 minutes. Thereafter, the plate was rinsed with tap water and the cells were counterstained with 2 mL hematoxylin (Sigma) for 1 minute. At the same passage, the osteogenic differentiated cells, as well as the control cells, were cytochemically stained for alkaline phosphatase by using a commercial staining kit according to the manufacturer`s (Cambrex Bio Science) recommendations: After the medium was removed and washed twice with PBS, 2 mL citrate fixative (12.5 mL citrate solution plus 32.5 mL acetone plus 4 mL 37% formaldehyde) was added for 1 minute, followed by staining with 2 mL alkaline dye (0.5 mL sodium nitrite plus 0.5 mL fast red violet alkaline solution plus 22.5 mL deionized water plus 0.5 mL naphthol AS-BI alkaline solution) for 30 minutes. The cell layer was washed twice and counterstained with 2 mL hematoxylin (Sigma) for 1 minute.

Chondrogenic differentiation was performed by using a commercially available mesenchymal functional differentiation kit (R&D Systems, Wiesbaden, Germany). Three samples of clonal MSCs (one unlabeled, one labeled with Resovist, and one labeled with Resovist C) were treated with the chondrogenic differentiation procedure: 250 x 103 cells were transferred into a 15 mL tube. After centrifugation (200g), 1.0 mL basal medium (D-MEM/F-12; Bio Whittaker) and 0.5 mL chondrogenic differentiation medium were added and replaced every 2–3 days. The cell suspension was cultured in the tube, forming a solid pellet. After 14 days, the chondrocyte pellet was removed, squeezed onto a glass slide, and stained with 1% Alcian blue 8GX (Serva, Heidelberg, Germany) in 3% acetic acid (pH 2.5).

Flow Cytometry
Fluorescent-activated cell scanning (FACS) analysis was performed with a flow cytometer (FACScan; BD Biosciences, San Jose, Calif) and software (CellQuest Pro; BD Biosciences, San Jose, Calif) (R.S. and A.G., with 1 year of experience in the technique). At subconfluency (1 x 106 cells), the cells were detached with Accutase (PAA Laboratories) and washed (PBS plus AccuMax [PAA Laboratories]). Each probe contained a cell suspension with 5 x 105 cells in FACS buffer (PBS plus 1% bovine serum albumin [Sigma] plus 0.1% fetal calf serum [Cambrex Bio Science]). The fluorescein isothiocyanate– or phycoerythrin-conjugated antibody (anti-rat CD4, CD11b, CD29, CD31, CD45, CD59, CD71, CD90, CD106, or CD117) was added. After an incubation time of 20 minutes and two washing steps, the probe was ready for analysis. All antibodies were from BD Biosciences.

Contrast Media and Cell Labeling
Resovist, or SHU 555A (Schering), an MR imaging contrast agent already approved for clinical use in Europe, Japan, and Australia, is organ-specific and is used for liver imaging (20–22). It consists of SPIO nanoparticles (4–6 nm) (23) coated with carboxydextran (mean hydrodynamic diameter, 60 nm), which is accumulated by phagocytosis in Kupffer cells. Resovist C (SHU 555C or Supravist; Schering) is a small-molecular-size subfraction of Resovist. Resovist C has a mean hydrodynamic diameter of about 20 nm (24).

For enhanced loading of the cells, the liposomal transfection reagent DOSPER (Roche Diagnostics, Mannheim, Germany) and a linear polyethylenimine, jetPEI (PolyPlus Transfection, Illkirch, France), were used.

Cell labeling was performed for 4 and 15 hours with 200 µg/mL iron alone or for 4 hours with 60 µg/mL iron in combination with the TAs in six-well plates 2 days after seeding of 7 x 105 cells (R.K., with 2 years of experience in the technique). This protocol was the result of previous experiments to evaluate the optimal iron load for each labeling procedure (data not shown). First, 10 µL of each TA in a total volume of 50 µL PBS was carefully added to 50 µL of the iron solution, mixed, and preincubated for 30 minutes. The following transfection was performed in the incubator at standard conditions (37°C, 5% humidified CO2). After the labeling, cells were washed three times with PBS, harvested, and processed for viability, total iron load (TIL) analysis, and MR imaging. The SPIO or USPIO load of the cells in culture was estimated with light microscopy during several passages (R.K. and R.B., both of whom had 2 years of experience in the technique, in consensus). The clonal MSCs were labeled with SPIO or USPIO with or without transfection reagent at different passages (passages 7, 10, 13, and 16). After two more passages (passages 9, 12, 15, and 18), the transferrin receptor (CD71) expression of the cells was evaluated with FACS. For FACS analysis, the cells were seeded at 1.0 x 105 cells per well in six-well plates and harvested after one more passage at subconfluency (10 x 105 cells per well).

Quantification of TIL of Cells with Photometry
After incubation with Resovist for 4 hours (n = 4), Resovist for 15 hours (n = 5), Resovist C for 4 hours (n = 4), or Resovist C for 15 hours (n = 5), as well as incubation with the same agents and a TA (n = 4 for each condition), labeled cells and control cells (n = 7) were washed three times with PBS, harvested, and counted; the cell pellet was then dried for 2 hours at 80°C. Then, samples were incubated overnight at room temperature and for another 2 hours at 60°C in perchloric and nitric acid at a 3:1 ratio to completely digest the cells and expose iron oxide from the dextran-coated nanoparticles. For photometric determination of the TIL, a ferrozine-based spectrophotometric assay (Eisen Ferene S Plus; Rolf Greiner Biochemica, Flacht, Germany) was used. Fe2+ forms a blue complex with ferene that can be measured at 595 nm. The extinction of the sample relates directly to the iron concentration, which was calculated with the help of a defined standard curve (R.K. and R.B., with 2 years of experience in the technique).

Determination of Viability
Cellular viability after the different incubation conditions and a time course of two passages was examined by one author (R.K., with 5 years experience in the technique) with the help of a cell analysis system (CASY2 Cell Counter and Analyser System, model TT; Schärfe System, Reutlingen, Germany) according to the electric current exclusion method described by Lindl et al (25).

MR Imaging
To assess the imaging detection limits of the labeling procedures, in vitro MR imaging was performed. The cell preparation for MR imaging was performed by one author (R.B., with 2 years of experience in the technique), while the imaging was performed by another author (J.P., with 2 years of experience in the technique) who specializes in experimental radiology. The interpretation of the imaging results was performed by two authors (J.P. and J.W., with 7 years of experience in the technique) in consensus. An agar matrix was used as a suitable environment for measuring SPIO-labeled MSCs. The agar solution (1%) was boiled and embedded in nonferromagnetic boxes before it became stable. By using a special stamp, a series of identical cone-shaped cavities was created in the agar block (Fig 1). For MR measurement, cell numbers from 1000 to 100 000 MSCs were used. Cells were centrifuged at 200g for 5 minutes, dissolved in 8% gelatin (20 µL), and implanted into the cone-shaped cavities within the agar matrix. After the gelatin solidified, the hollows were closed with agar. Thus, it was possible to achieve a homogeneous distribution of the target cells in a defined volume of 20 µL within a homogeneous agar block.


Figure 1A
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  		Figure 1a: (a) Agar matrix with embedded iron contrast medium–containing cells. (b) Wrist coil for measurement with 3.0-T MR imaging unit.

 

Figure 1B
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  		Figure 1b: (a) Agar matrix with embedded iron contrast medium–containing cells. (b) Wrist coil for measurement with 3.0-T MR imaging unit.

 
Cell preparations were scanned systematically by using a clinical MR imaging unit operating at 3.0 T (Magnetom Trio; Siemens Medical Solutions, Erlangen, Germany), a wrist coil, and a spoiled gradient-echo sequence (fast low-angle shot) with an echo time of 20 msec, a repetition time of 30 msec, and a flip angle of 20°. Further parameters were as follows: section thickness, 0.30 mm; field of view, 128 x 128 mm2; base resolution matrix, 384 x 384; isotropic voxel size, 0.3 x 0.3 x 0.3 mm; number of signals acquired, one; and acquisition time, 11.5 seconds per section.

Aggregations of labeled cells were reflected as either low-signal-intensity spots or signal voids, particularly with gradient-echo sequences. In the present study, the labeling efficiency of various concentrations of labeled cells was detected with qualitative interpretation of the SPIO- or USPIO-induced signal voids at MR imaging.

Electron Microscopy
To assess the uptake and localization of the SPIO or USPIO particles and their possible influence on the cellular ultrastructure, electron microscopy was performed. Cells grown as described above and treated as indicated were fixed in 2.5% glutaraldehyde (Paesel-Lorei, Frankfurt, Germany) buffered with 0.1 M cacodylate buffer (pH 7.4), postfixed in 1% OsO4 in 0.1-mol/L cacodylate buffer, and scraped off the plastic (R.K., with 1 year of experience in the technique). The pellet was then dehydrated in an ethanol series (50%, 70%, 96%, 100%). The 70% ethanol was saturated with uranyl acetate for contrast enhancement. Dehydration was completed in propylene oxide. The specimens were embedded in Araldite (Serva). Ultrathin slices were cut with a microtome (FCR Reichert Ultracut; Leica, Bensheim, Germany), mounted on pioloform-coated copper grids, contrasted with lead citrate, and analyzed and documented with an electron microscope (EM 10A; Zeiss, Oberkochen, Germany). The electron microscopy procedure and revision of the images were performed by one author (H.W.) with decades of expertise in this field.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Characterization of Clonal MSCs
After treatment for 21 days with adipogenic medium, staining with oil red O showed adipogenic differentiation (red droplets that represented lipid vacuoles) (Fig 2). After treatment for 21 days with osteogenic medium, staining for alkaline phosphatase showed osteogenic differentiation (in pink) (Fig 2). A 14-day treatment with chondrogenic medium resulted in chondrocyte pellets. Alcian blue stained the mucopolysaccharides blue to bluish green (Fig 2). No differences in differentiation potential could be detected between labeled and nonlabeled MSCs.


Figure 2
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  		Figure 2: Results of light microscopy of adipogenic, osteogenic, and chondrogenic differentiated labeled (Resovist [R] or Resovist C [RC]) and nonlabeled MSCs (passage 14). SPIO or USPIO could be identified as brown particles. a, Adipogenic differentiation after 21 days of treatment with adipogenic medium. (Oil red O stain, hematoxylin counterstain; original magnification, x400.) Red droplets represent lipid vacuoles. Arrows indicate adipocytes. o, Osteogenic differentiation after 21 days of treatment with osteogenic medium. (Alkaline phosphatase stain, hematoxylin counterstain; original magnification, x400.) Positive cells are stained pink to violet. c, Chondrogenic differentiation after 14 days of treatment with chondrogenic medium. Mucopolysaccharides are stained blue to bluish green. (Alcian blue stain; original magnification, x200.)

 
The clonal MSCs before SPIO or USPIO labeling showed the following surface epitope pattern (at FACS analysis): negative for CD4, CD11b, CD31, CD45, CD71, CD106, and CD117 and positive for CD29, CD59, and CD90.

Epitope Change after Cell Labeling with SPIO and USPIO
The MSCs that were labeled with SPIO and USPIO without transfection reagent showed an enhanced expression of transferrin receptor (CD71). SPIO- or USPIO-labeled cells that were also labeled with transfection reagent and control cells showed no or only slightly increased expression of transferrin receptor (CD71), respectively. The maximum of this reproducible effect could be observed at passage 12 during a susceptible phase between passage 9 and passage 18 (Fig 3).


Figure 3
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  		Figure 3: CD71 expression (at passage [P] 9, 12, 15, and 18) of MSCs labeled with Resovist (R) or Resovist C (RC) without TA at passages 7, 12, 13, and 16. The maximum of this effect was observed at passage 12 during a susceptible phase between passage 9 and passage 18. Red indicates isotype control. Green indicates probes. X-axis indicates fluorescence intensity (FL1-H) (100–104). Y-axis indicates counts (0–120).

 
TIL of Cells after Labeling with SPIO and USPIO
The mean TIL of nonlabeled control cells was 0.73 pg per cell ± 0.65 (standard deviation), while a 4-hour incubation period led to a mean TIL of 5.20 pg per cell ± 1.70 with Resovist alone and a mean TIL of 1.67 pg per cell ± 1.38 with Resovist C. Incubation with Resovist for 15 hours increased TIL to 24.7 pg per cell ± 10.5, while incubation with Resovist C resulted in a TIL of 3.8 pg per cell ± 2.5. The values for DOSPER and Resovist and DOSPER and Resovist C (at 4 hours) were 27.5 pg per cell ± 11.5 and 9.9 pg per cell ± 6.2, respectively. The highest amounts of iron were reached with the combinations of jetPEI and Resovist C (86.1 pg per cell ± 44.8), followed by jetPEI and Resovist (76.5 pg per cell ± 31.7) (Fig 4). In particular, jetPEI was very effective in augmenting the TIL, while the use of DOSPER did not lead to substantial differences compared with the nontransfected cells incubated for 15 hours. Observations at light microscopy during several passages after cell divisions showed no evidence of a decrease in the total cellular load of SPIO or USPIO.


Figure 4
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  		Figure 4: Bar graph shows TIL (intracellular iron plus iron coating cellular surface) of cells after labeling with SPIO or USPIO alone or in combination with a TA. Error bars indicate standard deviations. DR = DOSPER plus Resovist (4 hours incubation), DRC = DOSPER plus Resovist C (4 hours incubation), jPR = jetPEI plus Resovist (4 hours incubation), jPRC = jetPEI plus Resovist C (4 hours incubation), R = Resovist, RC = Resovist C.

 
Cell Viability after Labeling with SPIO and USPIO
The viability differed slightly between the different attempts. The results were between 94.7% ± 1.2 (with DOSPER and Resovist) and 91.1% ± 1.9 (with jetPEI and Resovist), with 93.8% ± 1.1 for the untreated control immediately after the labeling. One week after the treatment, the values changed, and 2 weeks after treatment, no differences in viability could be detected (Table).


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  		Viability of MSCs after Different Labeling Conditions

 
MR Imaging
The qualitative interpretation of the SPIO- or USPIO-induced signal voids was related to their diameter and homogeneity, which were dependent on the cell count and the contrast agent used, respectively. In our experimental setup, the combination of jetPEI and Resovist was the most effective: 5000 cells in a total volume of 20 µL were detected reliably in our in vitro model (Fig 5). Without TA, the detection limit was 50 000 cells for Resovist and 100 000 cells for Resovist C.


Figure 5
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  		Figure 5: MR imaging of cell counts from 1000 to 100 000 MSCs in agar solution (1%) at 3.0 T. Experimental parameters were as follows: 10 µL jetPEI per milliliter and 60 µg Resovist (jPR) or Resovist C (jPRC) per milliliter and 4 hours incubation time. Arrow indicates lowest cell count (5 x 103 cells) at which a signal was detectable at sagittal, coronal, and transverse imaging. R = Resovist, RC = Resovist C.

 
Electron Microscopy
Cells were treated with Resovist or Resovist C for 4 and 15 hours and with a TA for 4 hours. In the TA group, the SPIO- or USPIO-based contrast agent was observed to be in direct contact with the membrane of the cultured cells; however, it was obviously not incorporated into the cells but instead coated the cellular surface (Fig 6). However, in nontransfected cells, Resovist was taken up by the cells. After 4 hours, only a few cells incorporated Resovist (Fig 6), but after 15 hours, Resovist was incorporated in nearly all cells. The membrane-surrounded vacuoles were in the magnitude of 0.5–1.0 µm (Fig 6).


Figure 6A
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  		Figure 6a: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

 

Figure 6B
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  		Figure 6b: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

 

Figure 6C
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  		Figure 6c: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

 

Figure 6D
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  		Figure 6d: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

 

Figure 6E
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  		Figure 6e: Electron microscopic investigation of (a, b) transfected and (c–e) nontransfected cells treated with Resovist (R). (a, b) Cells transfected (with jetPEI) were not able to ingest the iron-containing Resovist at 4 hours incubation. Resovist remains on the cellular surface. (c–e) Nontransfected cells showed a consistent and time-dependent uptake of Resovist. In c (15 hours incubation), * shows phagocytic vacuole; this detail is shown at greater magnification in insert. In d (15 hours incubation), the Resovist-containing vacuole lies directly beneath both an extracellular deposit of Resovist and a cistern of rough endoplasmic reticulum (rER), indicating a high rate of protein synthesis in this cell. The dynamic process of Resovist incubation is demonstrated in e (4 hours incubation), which shows extracellular Resovist and some uptaken Resovist already. Black arrows show Resovist, which was taken up by the cells. White arrows show heterochromatin. N = nucleus. Bars in a, b, c, and e = 1 µm; bar in d = 0.5 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
For tracking MSCs after transplantation, the MR imaging technique offers several advantages like excellent spatial resolution, high tissue contrast, and the lack of ionizing radiation. Furthermore, in contrast to the short-lived tracers used in nuclear medicine (single-photon emission computed tomography, positron emission tomography), MR imaging–based cell trafficking is possible even several days after the initial labeling procedure. By using cardiac MR imaging, for example, the delivery procedure of MSCs could be monitored and cardiac function parameters could be evaluated (26). Therefore, iron-based MSC labeling procedures have been established without affecting cell viability, proliferation, and differentiation potential in vitro (27,28). The previously reported inhibition of the chondrogenic differentiation potential of human MSCs after labeling with SPIO (29,30) could not be detected in the rat model used in our study.

At comparison of the TILs of labeled cells without TAs, Resovist-labeled MSCs showed higher TILs than Resovist C–labeled MSCs. This corresponds to published results of other investigators that suggest a better intracellular uptake of SPIO than USPIO (31,32). Regarding the combination with a TA, the highest TIL was achieved with jetPEI. Interestingly, the most effective labeling combination for MR imaging was jetPEI and Resovist, although the TIL was not as high as that with jetPEI and Resovist C. The observed differences in the extension of the image signal void might be attributed to differences in the cellular distribution of the iron oxide nanoparticles on the microscopic scale. It is likely that a different spatial distribution causes changes in the microscopic field inhomogeneity and, due to the size of the particles, a different sensitivity of the MR imaging signal decay to spin diffusion (33,34). The complete understanding of the processes involved is complex, and it is reasonable to assume that the signal dephasing effects are more pronounced for jetPEI and Resovist–labeled cells than for jetPEI and Resovist C–labeled cells, yielding a more intense dephasing of magnetic moments in gradient-echo MR imaging, although the average iron load is smaller.

The uptake mechanisms of SPIO and USPIO include phagocytosis, pinocytosis, and receptor-mediated endocytosis (31). Owing to the biodegradeablility of dextran-coated iron oxide nanoparticles, the uptaken iron could be metabolized and introduced into the normal plasma iron pool (28). In our study, we investigated the influence of iron-based MR imaging labeling reagents on MSCs, focusing on transferrin receptor expression. Intracellular iron concentration modulates the expression of transferrin receptor (CD71). In various nonerythroid cells, transferrin receptor expression is inversely associated with the intracellular iron concentration (8), whereas in monocytes, iron upregulates transferrin receptor expression (10). By monitoring the transferrin receptor expression of labeled and unlabeled MSCs during several passages, we were able to identify a distinct period in the cell culture time when transferrin receptor expression was upregulated.

This observation raises two major questions: (a) Which mechanism(s) is responsible for this unexpected effect? and (b) Does the upregulation of transferrin receptor have an influence on the desired and/or undesired clinical effects of MSC transplantation? Regarding a, enhanced transferrin receptor expression could be a result of the change in the intracellular iron content (high or low) (7,8). Transferrin receptor synthesis is mediated by regulation of the stability of transferrin receptor messenger RNA through iron-regulatory protein 1 (IRP-1) (35). The presentation of transferrin receptor on the cellular surface is part of a dynamic process recycling apoprotein–transferrin receptor complex from the cytoplasm to the surface and back (9). In our experiments, the cells that upregulated transferrin receptor did not show a higher TIL than other labeled cells (ie, they showed a lower one) but did show a higher one than the control cells. Only the cells of this group incorporated the iron particles. The uptake mechanism was activated and time dependent. The electron microscopic examination of the iron particles showed no structural differences. Therefore, we exclude an influence of the molecular structure of SPIO or USPIO on transferrin receptor expression, but we hypothesize that the upregulation of transferrin receptor is a result of the enhanced iron uptake. Testa et al (10) described a positive association between the intracellular iron concentration and transferrin receptor expression during monocyte-macrophage maturation. Cell differentiation is known to be related to transferrin receptor expression (8). In our study, labeled MSCs upregulating the transferrin receptor did not show an altered differentiation capacity. Further known parameters that could have had an effect on transferrin receptor expression—like cell proliferation (8) or culture density (36)—could be excluded because of the identical culture conditions and cell counts. In summary, we hypothesize that the incorporation of iron results in enhanced transferrin receptor expression. Whether this effect is mediated by cytokines like interleukin-2 (9) or interferon-{gamma} (10) is unclear. Revealing these mechanisms will be the focus of further investigations like microarray-based screening on cytokines and iron-regulatory protein. Additionally, experiments blocking the transferrin cycle at distinct stages should be performed.

Regarding b, to our knowledge, transferrin receptor is not involved in homing of MSCs like CXCR4 (37,38). However, other interactions of MSC-expressed transferrin receptor with various cell types and tissue are unknown. Furthermore, it is not clear what impact transferrin receptor has on MSC differentiation potential and/or immunomodulation in vivo. This requires further investigation. In other cell types, the transferrin receptor is involved in mechanisms that are different from iron metabolism. In T lymphocytes, for example, transferrin receptor mediates uptake and presentation of hepatitis B envelope antigen (39). The incubation of MSCs with SPIO or USPIO without transfection reagent was not as effective as with jetPEI. Interestingly, the higher TIL was not a result of an efficient transfection process, because we had no evidence of iron uptake into the cells of this group. Rather, the complexes of SPIO or USPIO and TA coated the cellular surface. Although MSC labeling with SPIO or USPIO and transfection reagent does result in satisfactory MR imaging without enhancement of transferrin receptor expression, a possible impact on MSC homing could not be excluded and should be evaluated in further experiments. Homing of MSCs in vivo is mediated by chemokine receptors of the CC and CXC family (38). It is not unlikely that coating of the cellular surface could compromise cell homing by blocking these receptors and inhibiting the specific interactions with their ligands in the prospective homing area. In our observations with light microscopy during several passages after cell division, we had no evidence that the total load of SPIO or USPIO decreased. We therefore do not expect an important loss of surface-coating SPIO or USPIO in vivo, as long as the cells survive after transplantation. A possible impact of SPIO or USPIO on MSC homing could be evaluated by the detection of homing receptors before and after labeling, by in vivo MR imaging of transplanted labeled MSCs, and by ex vivo investigations of transplanted labeled MSCs with electron microscopy and FACS. For clinical application, preferably labeling agents that have been shown to influence neither cell biology nor homing should be used. These investigations should proceed to improve the clinical safety of these products.

There were some limitations to our study. Our findings were based exclusively on results of in vitro experiments. To evaluate the in vivo situation, clinical experiments have to be performed (eg, detection of transferrin receptor expression of labeled MSCs before and after transplantation). Moreover, the results generated in the animal model (rat) have to be verified in the human system with human MSCs. Further investigations with other iron-based contrast agents with or without TAs (eg, poly-l-lysine) that were not tested in this study will show whether the reported effects are more general or are restricted to distinct substances.

Practical applications: We were able to show that MSC labeling with clinically established SPIO or USPIO particles is feasible without affecting cell viability. Contrary to results reported in the current literature, our results reveal that SPIO or USPIO labeling without a transfection reagent does have a biologic impact on MSCs by upregulating transferrin receptor and suggest caution with use of this labeling procedure for clinical application. Furthermore, SPIO or USPIO labeling with transfection reagent will coat the cellular surface; therefore, an influence on MSC homing after transplantation could not be excluded. Further experiments are crucial to elucidate the mechanisms and consequences of these effects.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   ACKNOWLEDGMENTS
 
The technical assistance in cell culture by Ursula Hermanutz-Klein and Brigitte Maurer and in electron microscopy by Gabriele Frommer-Kästle is gratefully acknowledged.


   FOOTNOTES
 

Abbreviations: FACS = fluorescent-activated cell scanning • MSC = mesenchymal stem cell • PBS = phosphate-buffered saline • SPIO = superparamagnetic iron oxide • TA = transfection agent • TIL = total iron load • USPIO = ultrasmall SPIO

Authors stated no financial relationship to disclose.

Author contributions: Guarantor of integrity of entire study, R.S.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, R.S., R.K., J.W.; experimental studies, R.S., R.K., R.B., J.P., A.G., H.W.; statistical analysis, R.S., R.K., R.B.; and manuscript editing, R.S., R.K., J.W., B.R.B., H.W., C.D.C., H.N.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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