Our study demonstrates that hMSCs can be effectively labeled by simple incubation with DiD and DiD/ferucarbotran for depiction with OI and MR, without impairing the cells viability or proliferation. However, while histopathological evaluation of both unlabeled and labeled cells demonstrated differentiation into chondrocytes of similar morphology, labeled cells exhibited significantly less GAG production. Thus, our data suggest that the applied fluorescent and iron oxide contrast agent label do not interfere with morphological cell characteristics but may impair cell function.
Our data are in accordance with studies by Bulte and colleagues and Henning and colleagues, who recently described a dose-dependent inhibition of chondrogenesis with the SPIO based iron oxide labels ferumoxides and ferucarbotran [3
Other authors reported a decline in cell proliferation after labeling neurons with Gd-based contrast agents [7
], a diminished self-repair of mesenchymal stem cells after ferumoxides labeling [8
] and a decreased function of natural killer cells after labeling with FeCl3 [9
On the other hand, these same cells need iron for their metabolism and normal development [10
]. Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport, and many essential redox reactions involving hemoproteins [10
]. Iron oxide nanoparticles are in fact appealing as contrast agents for cell labeling since two preparations are already FDA-approved (ferumoxides and ferumoxytol) and since their cellular uptake and intracellular metabolism have been extensively investigated in patients [12
]. Likewise, labeling with fluorochromes has been reported to interfere with cell functions. For example, the fluorochrome calcein acetomethylester impaired the migration capacity of monocytes [15
]. Conversely, other authors reported an unimpaired function of DiD-labeled natural killer cells [17
A major challenge of cellular imaging is to develop optimized cell-labeling techniques, which provide a compromise between the minimal possible concentration for sensitive cell detection, and maximal possible concentration that does not impair cell function. Our data showed significantly less impairment in GAG production for chondrocytes labeled with DiD/ferucarbotran as opposed to cells labeled with DiD alone. A possible explanation for this observation may be a decreased uptake of DiD after prelabeling with ferucarbotran. These data support the theory of Bulte et al.
that inhibition of chondrogenesis by contrast agent labeling is dose dependent [3
]. In addition, DiD integrates into the cell membrane and may interfere with the cell–cell contact needed for chondrogenesis [18
We recognize several limitations to our study: Firstly, we examined stem cell differentiation in vitro; differentiation processes are more complex in vivo and have to be re-evaluated in this context. Secondly, we focused our investigations on the fluorescent dye DiD and the iron oxide contrast agent ferucarbotran. Further studies have to investigate the effect of other fluorescent dyes and other MR contrast agents on stem cell differentiation. Lastly, we examined human mesenchymal stem cells; other stem cell types may be more or less sensitive to contrast agent labeling.