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 100μg Fe/ml for 4h and 18h and additional samples were cultured for 6 or 12 days after the 18-hour 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 to unlabeled controls by staining with alcian blue and hematoxylin & eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3T was investigated on day 1 and day 14 of chondrogenic differentiation by measuring signal-to-noise ratios on T2-SE and T2*-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 4h 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 4h 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 to 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 4h, inhibition of chondrogenesis was not significant.

Direct Labeling of hMSC with SPIO: the Long-Term Influence on Toxicity, Chondrogenic Differentiation Capacity, and Intracellular Distribution

“…A recent report also demonstrated successful chondrogenesis following direct SPIO labeling [23]. A dose-dependent inhibition of chondrogenesis was observed, but there was no significant decrease in GAG synthesis after labeling for 4 h without transfection agent, while strong T2 and T2* contrast was preserved in MR images [23].…”

“…Using other transfection agents in molecular imaging might cause potential hazards. Once the transfection agent was used, there was a mechanism other than SPIO dosedependency observed in the inhibition of chondrogenesis, and a possible explanation is that the transfection agent may change surface-linked cell−cell interaction or the interaction with differentiation-linked intracellular structures or substrates [23]. There were several studies suggesting the possible clinical use of direct SPIO labeling on cells without transfection [20,21].…”

“…Using this agent, it was shown that the detection of directly labeled stem cells, without the use of transfection agents, is possible even at the single cell level [20][21][22]. The amount of intracellular iron uptake following Ferucarbotran incubation without transfection agents was adequate to produce strong signals on MR studies, and no significant adverse effects were found in short-term studies of cell viability, proliferation, or differentiation capacities [20,21,23]. Since different intracellular environments might alter how and at what speed the iron is released, it is important to know in which pathway the cells metabolize iron.…”

The use of dopamine-hyaluronate associate-coated maghemite nanoparticles to label cells

“…Although iron oxide nanoparticles are generally considered as safe, there are several reports in the literature that differently coated superparamagnetic iron oxide particles negatively affect the chondrogenic differentiation of MSCs. 31,32 Some superparamagnetic iron oxides are suspected to produce reactive oxygen species 33 or affect the cytoskeleton and cell membrane. 34 Since HA is present in cartilage, the chondrogenic differentiation of rat MSCs labeled with HA-γ- 5 Da, when added to the culture medium of an explant culture of articular cartilage, inhibited both aggrecan and HA synthesis in a concentrationdependent manner.…”

Magnetization Transfer Imaging Provides a Quantitative Measure of Chondrogenic Differentiation and Tissue Development

“…[26][27][28][29] For each sample, the entire construct was digested in 1 mL of protease (2.5 mg/mL; Type XIV Bacterial from Streptomyces griseus; Sigma) in trisbuffered saline in a 558C water bath overnight. A 100 mL aliquot of the digest was assayed for total GAG content by addition of 2 mL of 1,9-dimethyl methylene blue dye solution (Polyscience, Northampton, United Kingdom).…”