Rüdiger Bantleon

Labeling of human mesenchymal stromal cells with superparamagnetic iron oxide leads to a decrease in migration capacity and colony formation ability

BACKGROUND AIMS Labeling of stem cells is crucial to allow tracking of stem cell homing and engraftment after transplantation. In this study we evaluated the influence of cell labeling procedures using clinically approved small particles of iron oxide (SPIO) with or without transfection reagents (TA) on functional parameters of human mesenchymal stem cells (MSC). METHODS The study was approved by the institutional review board of the University of Tubingen, Germany. Seven populations of bone marrow (BM)-derived human mesenchymal stem cells (MSC) were labeled with SPIO alone or in combination with various TA. Directly after labeling and two passages after labeling migration assays, quantification of colony-forming units and quantitative evaluation of the differentiation potential were performed. Quantification of the cellular total iron load (TIL), determination of the cellular viability and electron microscopy were also performed. RESULTS Labeling of mesenchymal stem cells with SPIO with or without TA did not affect cell viability and differentiation potential significantly. SPIO in combination with TA coated the cellular surface directly after labeling but was incorporated into the cells after two passages. Labeling of mesenchymal stem cells with TA led to a significant decrease of migration capacity. This effect was abolished after two passages. Labeling with and without TA led to a significant decrease in colony formation ability. This effect could also be observed after two passages. CONCLUSIONS The observed decrease of migration capacity and colony-formation ability was not associated with either TIL or localization of particles of iron oxide. SPIO labeling with and without TA had functional effects on human mesenchymal stem cells by decreasing the migration capacity and colony-formation ability of the stem cells.

Functional investigations on human mesenchymal stem cells exposed to magnetic fields and labeled with clinically approved iron nanoparticles.

BackgroundFor clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSCs biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed.ResultsHuman MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs.ConclusionsIn our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.

Detection of DNA double-strand breaks using γh2AX after MRI exposure at 3 Tesla: An in vitro study

To evaluate the effects of the static magnetic field and typical imaging sequences of a high‐field magnetic resonance scanner (3 Tesla) on the induction of double‐strand breaks (DSBs) in two different human cell lines.

Positive contrast imaging of iron oxide nanoparticles with susceptibility-weighted imaging

Superparamagnetic iron oxide particles can be utilized to label cells for immune cell and stem cell therapy. The labeled cells cause significant field distortions induced in their vicinity, which can be detected with magnetic resonance imaging (MRI). In conventional imaging, the signal voids arising from the field distortions lead to negative contrast, which is not desirable, as detection of the cells can be masked by native low signal tissue. In this work, a new method for visualizing magnetically labeled cells with positive contrast is proposed and described. The technique presented is based on the susceptibility‐weighted imaging (SWI) post‐processing algorithm. Phase images from gradient‐echo sequences are evaluated pixel by pixel, and a mask is created with values ranging from 0 to 1, depending on the phase value of the pixel. The magnitude image is then multiplied by the mask. With an appropriate mask function, positive contrast in the vicinity of the labeled cells is created. The feasibility of this technique is proved using an agar phantom containing superparamagnetic iron oxide particles–labeled cells and an ex vivo bovine liver. The results show high potential for detecting even small labeled cell concentrations in structurally inhomogeneous tissue types. Magn Reson Med, 2010.

In Vivo Tracking of Th1 Cells by PET Reveals Quantitative and Temporal Distribution and Specific Homing in Lymphatic Tissue

Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. Methods: We developed protocols that minimize the inhibitory effects of 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-γ–producing CD4+ T (Th1) cells with 0.7–2.2 MBq of 64Cu-PTSM and analyzed cell viability, IFN-γ production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular 64Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 107 64Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of 64Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 107 64Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. Results: PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, 64Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered 64Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of 64Cu-OVA-Th1 cells in the pulmonary LNs (6.8 ± 1.1 percentage injected dose per cubic centimeter [%ID/cm3]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity–diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 ± 0.4 %ID/cm3). As expected, 64Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 ± 0.5 %ID/cm3) when compared with phosphate-buffered saline–challenged animals (4.6 ± 0.5 %ID/cm3). Conclusion: Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for 64Cu-PTSM–labeled T cells in clinical trials and novel therapy concepts focusing on T-cell–based immunotherapies of autoimmune diseases or cancer.

A preparation technique for quantitative investigation of SPIO-containing solutions and SPIO-labelled cells by MRI

Abstract Ziel: Entwicklung einer Präparationstechnik zum Nachweis von SPIO (superparamagnetic iron oxide) -haltigen Lösungen und SPIO-markierten Zellen mittels MRT ex-vivo. Detektion und Quantifizierung der magnetischen Markierung über den Durchmesser der Signalauslöschung in MR-Gradientenecho-Aufnahmen. Funktionale Beschreibung des Zusammenhangs zwischen lokaler Eisenkonzentration und Durchmesser der messbaren Signalauslöschung für die gewählten Versuchsbedingungen. Untersuchung des Einflusses der Echozeit und der räumlichen Auflösung auf die Signalverteilung im MR-Tomogramm. Material und Methoden: Verwendung von Resovist, (SHU 555A) als superparamagnetisches Kontrastmittel. Einbringen von SPIO-haltigen Lösungen (0,75 – 15 mg Fe/10 ml) und SPIO-markierten SK-Mel28 Zellen (25.000 – 1.000.000 Zellen/10ml) in ein definiertes Volumen innerhalb einer Agar-Matrix. Messung des Durchmessers der Signalauslöschung in Abhängigkeit von der lokalen Eisenkonzentration, der Echozeit (5-25 ms) und der räumlichen Auflösung (isotrop mit 0,25-0,60 mm Kantenlänge der Bildelemente) bei Verwendung einer 3D Gradientenechosequenz (FLASH) an einem klinischen 3 Tesla Ganzkörpertomographen. Ergebnisse: Die Ausdehnung der Signalauslöschung im MR-Bild nahm mit geringerer Auflösung und längerer Echozeit zu. Die Sensitivität zum Nachweis der magnetischen Markierung war am größten bei TE 25 ms. Eine möglichst ortstreue Abbildung magnetisch markierter Bezirke konnte bei TE 5 ms und einer Auflösung von 0,25 mm erreicht werden. Der Durchmesser der Auslöschung war eine logarithmische Funktion der lokalen Eisenkonzentration. Unter den gegebenen Messbedingungen konnten Konzentrationen ab 0,75 mg Fe/10ml (SPIO-haltige Lösung) bzw. 1,25 mg Fe/10ml (SPIO-markierte SK-Mel28 Zellen) sicher erfasst werden. Schlussfolgerungen: Die vorgestellte Präparationstechnik mit definierter Verteilung magnetischen Materials in einer Agar-Matrix zeigt sehr gut reproduzierbare und quantitativ verwertbare Ergebnisse und erscheint üblichen Präparationen mit zufälliger Materialverteilung in Glasröhrchen überlegen. Purpose. This work aims to present a preparation technique for ex-vivo MR examination of SPIO (superparamagnetic iron oxide) containing solutions or SPIO labeled cells. Accumulations of SPIO particles and labeled cells were prepared in different concentrations using agar gel phantoms. Signal extinction around accumulations of magnetic material was examined systematically by gradient echo sequences with variable echo times and spatial resolution. The correlation between local iron concentration and diameter of signal extinction in MR gradient echo images was investigated. Methods. Resovist, (SHU 555A) was used as superparamagnetic contrast medium. Different concentrations of SPIO-containing solutions (0.75 – 15 mg Fe/10 ml) and magnetically labeled SK-Mel28 cells (25.000 – 1.000.000 cells/10 ml) were accommodated inside a defined volume in an agar matrix. Diameters of signal void were assessed in dependence on local iron concentration, echo time (5 – 25 ms) and isotropic spatial resolution (length of voxel 0.25 – 0.60 mm). Measurements were performed on a clinical MR whole body scanner (3 Tesla) using a spoiled gradient echo sequence (FLASH). Results. For the present experimental conditions sensitivity to detect the magnetic label was maximized using TE 25 ms. In contrast, the area of signal cancellation was minimized using TE 5 ms and isotropic resolution of 0.25 mm. In the latter case the image indicated the area of magnetic material most precisely. Diameter of signal cancellation was a logarithmic function on local iron concentration. In the presented set-up detection of concentrations as low as 0.75 mg Fe/10 ml in SPIO-containing solution or 1.25 mg Fe/10 ml in SPIO-labeled SK-Mel28 cells was certainly possible. Conclusion. The proposed preparation strategy with a well defined spatial distribution of the magnetic material in an agar gel phantom produced reliable results and appears clearly superior compared to set-ups with randomly distributed material in glass tubes. The diameter of the signal extinction in gradient echo images was significantly affected by the choice of echo time and spatial resolution. The calibration of signal cancellation versus iron concentrations may be valuable to assess SPIO concentrations and possibly numbers of labeled cells under specific conditions in vitro or even in vivo.

In vitro evaluation of magnetic resonance imaging at 3.0 tesla on clonogenic ability, proliferation, and cell cycle in human embryonic lung fibroblasts.

Objectives:We investigated the influence of magnetic resonance (MR) at 3.0 T on clonogenic ability, proliferation, and cell cycle in an embryonic human cell line. Materials and Methods:Cells (human lung fibroblasts Hel 299) were exposed to the static magnetic field (3.0 T) of a magnetic resonance imager (MRI) and to a turbo spin echo sequence at 3.0 T within clinical limitations (specific absorption rate 0.92 W/kg). A special MR-compatible incubation system was used. A control group (sham-exposed) and a MRI group (exposed) were set up. We investigated 3 biologic endpoints: colony forming, cell cycle, and proliferation ability. The exposure time was 2 hours in each experiment. Results:In the statistical analysis, none of these tests showed relevant differences between the exposed and sham-exposed group. Conclusions:No influences of the static field alone as well as a turbo spin echo sequence at 3.0 T on clonogenic ability, proliferation, or cell cycle in eugenic human lung fibroblasts were found.

Effects of MRI contrast agents on human embryonic lung fibroblasts.

Rationale and Objectives:The objective of this investigation was to evaluate 6 magnetic resonance contrast media (CM) with regard to their different effects on human embryonic lung fibroblasts (HEL-299). Methods:Human embryonic fibroblasts (HEL-299) were incubated with 1×, 5×, 10×, and 20× of the normal molar blood concentration (1×, 5×, 10×, 20× conc.) reached through routine contrast media applications for MRI examinations. Four gadolinium-based CM, ie, Gadovist, Magnevist, Multihance, Omniscan, Teslascan (Manganese-based), and Resovist (Iron-based), with incubation periods over 4 hours and 24 hours were investigated. Proliferation kinetics, colony formation, and viability assays were performed after 4 and 24 hours of treatment. Apoptotic cells were quantified after tetramethylrhodamine ethyl ester staining following 24 hours of CM media incubation (20× conc.) by fluorescence activated cell sorting cytometry. Furthermore, immunofluorescence images with vimentin staining were obtained (20× conc., 24 hours treatment). Cell cycle analysis was performed after 24 hours of incubation and 20× conc. directly after incubation and 24 hours later (fluorescence activated cell sorting cytometry). Results:The proliferation kinetics performed with 20× conc. revealed a persistent increase in cell numbers until day 11 for all CM without significant differences after 4 hours of incubation. A significant reduction in initial cell numbers was recorded in the 24-hours-group after 4 days of CM incubation with Magnevist, Multihance, Omniscan, and Teslascan. Solely cells incubated with Resovist and Gadovist failed to show decreased cell numbers when compared with the control group. However, a considerable cell regain occurred afterward reaching control-group levels on day 21. Colony numbers were significantly reduced (about 20%, respectively) with Magnevist at 10× and 20× conc., as well as Omniscan and Multihance at 20× conc. when compared with all other groups, P < 0.05. Cell-cycle distribution showed a reduction of S-phase cells for Magnevist, Omniscan, and Multihance (2.9%–10.5%) when compared with Gadovist, Resovist and Teslascan (16.7%–21.0%). Twenty-four hours after incubation, the percentiles of cells in S-phase were significantly increased for Magnevist, Omniscan, and Multihance (31.4%–38.5%) when compared with Gadovist, Resovist, and Teslascan (18.6%–26.8%), P < 0.05. Viability was not impaired by administration of any CM and no apoptosis was seen after tetramethylrhodamine ethyl ester staining at 24 hours of incubation. Cell morphology remained unchanged in vimentin-staining for all CM and conditioning regimens. Conclusions:No toxic effects on embryonic fetal lung fibroblasts were detectable after 4 and 24 hours of incubation in 6 MRI CM and 10× to 20× conc. in our setting. Antiproliferative effects, initially detected with Magnevist, Omniscan and Multihance, were rapidly compensated for.

Effects of magnetic resonance imaging contrast agents on human umbilical vein endothelial cells and evaluation of magnetic resonance imaging contrast media-triggered transforming growth factor-beta induction in dermal fibroblasts (HSF) as a model for nephrogenic systemic fibrosis.

Rationale and Objectives:The objective of this study was to evaluate effects of 6 commercially available magnetic resonance contrast media (CM) on human umbilical vein endothelial cells (HUVEC) and the induction of transforming growth factor-beta (TGF-&bgr;) in dermal fibroblasts (HSF) as a possible model for the pathogenesis of nephrogenic systemic fibrosis. Methods:HUVECs were incubated with 10× and 20× of the molar standard blood concentration achieved with CM applications for magnetic resonance imaging examinations (10× and 20× concentration) for 24 hours using gadolinium-based CM Gadovist, Magnevist, Multihance, and Omniscan, as well as Teslascan (Manganese-based), and Resovist (Iron-based). Proliferation kinetics (PK), colony formation, and viability assays were performed. Additionally, human dermal fibroblasts (HSF) were incubated for 24 hours with 1× and 20× concentration in all 6 CM, and TGF-&bgr; levels were assessed directly after the incubation period as well as on days 3 and 8 postincubation. Results:HUVEC PK data show similar gains in cell numbers for all 6 CM in both concentration groups over the 17-day assessment period. Only cells incubated with Omniscan and Teslascan differed from the other groups on days 3 and 7 postincubation (P < 0.05). After day 7, a cell regain occurred in the Omniscan and Teslascan groups reaching the numbers of the other groups in sequel. Differences in colony formation were consistent with PK results with a statistically significant reduction in clonogenic activity for Teslascan and Omniscan in HUVEC cells, P < 0.05. No reduction in viability was seen for all groups and conditions. TGF-&bgr; expression of HSF cells incubated with 1× concentration and all CM did not differ significantly from control cells for any point in time investigated. At 20× concentration directly after incubation, TGF-&bgr; was significantly reduced for the Teslascan and Resovist group as 3 compared with control and all other CM groups, P < 0.05. On day 3 postincubation, only Resovist-incubated HSF cells showed a significant reduction of TGF-&bgr; (1.614, standard deviations: 89) as compared with the control group (2.883, standard deviations: 30) and the other CM. TGF-&bgr; was slightly reduced for all CM groups 8 days after incubation (not statistically significant, P > 0.05). Conclusions:After 24 hours of incubation with Omniscan and Teslascan (10× and 20× concentration), considerable short-term antiproliferative effects in HUVECs were observed. HSF cells (20× concentration) showed a reduction of TGF-&bgr; for Resovist and Teslascan directly after incubation, whereas TGF-&bgr; levels in HSF cells were slightly reduced for all CM 8 days after incubation. Therefore, TGF-&bgr;-mediated proliferative effects on fibroblasts or on collagen synthesis potentially leading to nephrogenic systemic fibrosis may mainly be triggered by tissue monocytes and macrophages in the peripheral blood instead of dermal fibroblasts.

Do static or time‐varying magnetic fields in magnetic resonance imaging (3.0 T) alter protein–gene expression?—A study on human embryonic lung fibroblasts

To evaluate the influence of magnetic resonance imaging (MRI) on gene expression in embryonic human lung fibroblasts (Hel 299).