Labeling human mesenchymal stem cells with fluorescent contrast agents: the biological impact

Abstract

Purpose: This study aims to determine the effect of human mesenchymal stem cell (hMSC) labeling with the fluorescent dye DiD and the iron oxide nanoparticle ferucarbotran on chondrogenesis.

Procedures: hMSCs were labeled with DiD alone or with DiD and ferucarbotran (DiD/ferucarbotran). hMSCs underwent confocal microscopy, optical imaging (OI), and magnetic resonance (MR) imaging. Chondrogenesis was induced by transforming growth factor-b and confirmed by histopathology and glycosaminoglycan (GAG) production. Data of labeled and unlabeled hMSCs were compared with a t test.

Results: Cellular uptake of DiD and ferucarbotran was confirmed with confocal microscopy. DiD labeling caused a significant fluorescence on OI, and ferucarbotran labeling caused a significant T2* effect on MR images. Compared to nonlabeled controls, progenies of labeled MSCs exhibited similar chondrocyte morphology after chondrogenic differentiation, but the labeled cells demonstrated significantly reduced GAG production (p < 0.05).

Conclusion: DiD and DiD/ferucarbotran labeling of hMSC does not interfere with cell viability or morphologic differentiation into chondrocytes, but labeled cells exhibit significantly less GAG production compared to unlabeled cells.

Fig. 1

High-power (×40) confocal microscopy of a unlabeled control, b DiD-labeled hMSCs, and c DiD/ferucarbotran-labeled hMSCs stained with antidextran FITC stain. The nuclei of all hMSC have been counterstained with DAPI (blue channel). There was significant uptake of both contrast agents within the hMSC (b, c).

Fig. 2

A serial dilution was prepared of DiD/ferucarbotran-labeled hMSCs and unlabeled controls with decreasing cell numbers. a OI signal of DiD/ferucarbotran-labeled hMSCs (units in efficiency) and nonlabeled controls showed increased fluorescent signal with increased cell numbers. b MR images of DiD/ferucarbotran-labeled hMSCs demonstrated a significantly decreased T2*-signal with increasing cell number.

Fig. 3

Optical images (upper row) and MR images (lower row) of a, nonlabeled control pellet, b DiD-labeled pellet, and c DiD/ferucarbotran-labeled pellet. A representative color scale is provided which displays the fluorescence intensity above a certain threshold. All labeled pellets demonstrate a significant fluorescent signal on OI for the given threshold (b, c). The fluorescence signal is not homogenously distributed on the optical images due to the fact that the cells were centrifuged prior to imaging which caused the pellet to stick to one side. T2*-weighted MR images of DiD/ferucarbotran-labeled pellets demonstrate a significant decrease in signal (c). The MR signal seen in the lower row of c shows a blooming effect compared to the corresponding OI, which is caused by an extended range susceptibility effect of the magnetic particles within the cells.

Fig. 4

Alcian blue stains of chondrogenic pellets: 1a unlabeled control, 1b DiD labeled, and 1c DiD/ferucarbotran labeled. 1a–c Stained positive (blue stain of the intracellular and extracellular matrix). Safranin-O stains of chondrogenic pellets: 2a unlabeled control, 2b DiD labeled, and 2c DiD/ferucarbotran labeled. Chondrocytes are visible in all samples with the morphology of 2b and 2c similar to control. Iron can be visualized in 1c and 2c.

Fig. 5

Average total and relative GAG content of exogenously labeled chondrogenic pellets and controls: Data are displayed as means of triplicate samples with standard deviation.