The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells

Abstract

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with ferucarbotran using various protocols: cells were labeled with 100 microg Fe/ml for 4 and 18 h and additional samples were cultured for 6 or 12 days after the 18 h labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP-spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared with unlabeled controls by staining with Alcian blue and Hematoxylin and Eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3 T was investigated on days 1 and 14 of chondrogenic differentiation by measuring signal-to-noise ratios on T(2)-SE and T(2)*-GE sequences. Iron uptake was significant for all labeling protocols (p < 0.05). The uptake was highest after transfection with protamine sulfate (25.65 +/- 3.96 pg/cell) and lowest at an incubation time of 4 h without transfection (3.21 +/- 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4 h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose-dependent. MR imaging showed markedly lower SNR values of all labeled cells compared with the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose-dependent manner when using simple incubation techniques. When decreasing the incubation time to 4 h, inhibition of chondrogenesis was not significant.

Figure 1

The different samples are arranged along the x-axis according to their iron content. Graph A shows the mean uptake of iron per cell immediately after labeling of undifferentiated hMSC ( n = 3 ). Graph B displays the average amount of glucosaminoglycans synthesized after 14 days of chondrogenic differentiation by the different pellets (n = 3). Except for the cells incubated with protamine sulfate, the relation between chondrogenic differentiation and cellular iron uptake is inversely proportional. Photographs (C) show chondrogenic pellets (black arrows). Note the brown coloring of the labeled pellets due to the iron oxides.

Figure 2

MR imaging at 3T of chondrogenic pellets at 3T at day 1 and day 14 of differentiation (n = 1). The MR images demonstrate the contrast agent effect of all labeled chondrogenic pellets as acquired with axial T2 SE- (TR 2000/TE 15) and T2*GE-sequences (TR 500/TE 14). The bar graph shows the signal-to-noise ratio for all samples as acquired with these sequences. Of note: The black bars under the centrifuge tubes correspond to the rack used for positioning the tubes in the water bath.

Figure 3

Comparison of different labeling protocols versus unlabeled control: Anti-dextran staining of undifferentiated hMSC (green) demonstrates intracellular localization of contrast agent within the labeled cells (first column). Electron microscopy images of all undifferentiated labeled hMSC samples depict iron oxide particles within secondary lysosomes (second column, black arrows in B, C, D). Note the membrane-adsorbed iron oxide particles after labeling with 100 μg Fe for 18 h (gray arrowhead in B). H&E and alcian blue staining (third and fourth column) of differentiated cell pellets show different grades of chondrogenesis. Note the differentiated spindled cells in the magnified window (lower right corner, third column, A-C) versus the undifferentiated round cells (upper right corner, third column, A). The brown deposits on H&E and Alcian blue stains indicate iron oxide particles. Note: The high density of these contrast agent particles compared to the electron microscopy images are due to the thin slice thickness (80 nm vs 5 μm) and the high magnification of electron microscopy images.