Superparamagnetic Iron Oxide Particles (VSOPs) Show Genotoxic Effects but No Functional Impact on Human Adipose Tissue-Derived Stromal Cells (ASCs)

Abstract

Adipose tissue-derived stromal cells (ASCs) represent a capable source for cell-based therapeutic approaches. For monitoring a cell-based application in vivo, magnetic resonance imaging (MRI) of cells labeled with iron oxide particles is a common method. It is the aim of the present study to analyze potential DNA damage, cytotoxicity and impairment of functional properties of human (h)ASCs after labeling with citrate-coated very small superparamagnetic iron oxide particles (VSOPs). Cytotoxic as well as genotoxic effects of the labeling procedure were measured in labeled and unlabeled hASCs using the MTT assay, comet assay and chromosomal aberration test. Trilineage differentiation was performed to evaluate an impairment of the differentiation potential due to the particles. Proliferation as well as migration capability were analyzed after the labeling procedure. Furthermore, the labeling of the hASCs was confirmed by Prussian blue staining, transmission electron microscopy (TEM) and high-resolution MRI. Below the concentration of 0.6 mM, which was used for the procedure, no evidence of genotoxic effects was found. At 0.6 mM, 1 mM as well as 1.5 mM, an increase in the number of chromosomal aberrations was determined. Cytotoxic effects were not observed at any concentration. Proliferation, migration capability and differentiation potential were also not affected by the procedure. Labeling with VSOPs is a useful labeling method for hASCs that does not affect their proliferation, migration and differentiation potential. Despite the absence of cytotoxicity, however, indications of genotoxic effects have been demonstrated.

Keywords: ASCs; MRI; VSOP; adipose tissue-derived stromal cells; cell labeling; iron oxide nanoparticles; toxicity.

Figure 1

Detection of VSOP-labeled hASCs: Prussian Blue staining of human adipose tissue-derived stromal cells (hASCs) labeled with 1.5 mM citrate-coated very small superparamagnetic iron oxide particles (VSOPs): (A) Intracellular blue spots indicate the uptake of iron oxide particles into the hASCs. (B) No blue staining was detected in unlabeled controls. Magnification ×200; scale bars represent 100 µm. Transmission electron microscopy (TEM) image analysis: (C) Intracellular distribution of vesicles containing VSOPs in hASCs after labeling with 1.5 mM VSOPs. The insert shows individual vesicles at higher magnification with intravesicular VSOPs highlighted by black arrows. (D) Within the unlabeled hASCs, no intracellular deposition of VSOPs was detected. Scale bars represent 2.5 µm.

Figure 2

High-resolution MR imaging: (A) Magnetic resonance (MR) images of VSOPs-labeled hASCs seeded in agarose gel show the typical hypointense dark spots due to the signal decrease by iron oxide particles. The signal intensity is directly proportional to the concentration of VSOPs used for the labeling procedure. (B) The single hypointense spots in the gels with the unlabeled hASCs correspond to microscopic air bubbles located within or at the surfaces of the agarose gels.

Figure 3

Comet Assay: No significant increase in olive tail moment (OTM) values and thus in DNA fragmentation was observed after labeling of hASCs. A significant increase in OTM values after exposure to 200 µM MMS, which served as a positive control, was determined compared to unlabeled hASCs. Significance is indicated by asterisks (* p < 0.0001).

Figure 4

Chromosomal aberration test: (A) There was a significant increase in chromosomal aberrations from a concentration of 0.6 mM onwards compared to the unlabeled control (* p < 0.001: 0.6 mM, 1 mM; * p < 0.05: 1.5 mM). Furthermore, there was a significant increase in chromosomal aberrations after exposure of the hASCs to 200 µM MMS (positive control, * p < 0.0001). Significance is indicated by asterisks. (B) Chromatid and chromosomal breaks were observed: the example shows a chromatid break of chromosome 4.

Figure 5

Trypan Blue exclusion test: There was no difference in the viability of the VSOPs-labeled hASCs compared to the unlabeled control.

Figure 6

MTT and proliferation assay: (A) There was no difference between the viability of hASCs, which were labeled with 0.015 mM (light grey columns) and 1.5 mM VSOPs (dark grey columns), and unlabeled cells (white columns) immediately after the labeling procedure and after 24, 48 and 72 h. The values of the unlabeled cells were normalized to a viability of 100% for each single patient. Mean extinction values for each patient and concentration were normalized to the respective values of unlabeled hASCs from the same patient. Human ASCs treated with tert-butylhydroperoxide (t-BHP) (black columns) showed a significant decrease in cell viability compared to VSOPs-labeled and unlabeled hASCs. Significance is indicated by asterisks (* p < 0.0001). (B) VSOPs-labeled hASCs showed no difference in proliferation capacity compared to the unlabeled controls.

Figure 7

Scratch assay: To evaluate the influence of VSOPs-labeling on the migration ability of hASCs, the scratch assay was used. After 24 h, there was no remarkable difference between labeled and unlabeled cells.

Figure 8

Histological analysis after adipogenic, osteogenic and chondrogenic differentiation: Red-stained, intracellular lipid droplets are apparent in both VSOPs-labeled and unlabeled hASCs after adipogenic differentiation (Row (R) 1 and 2, column (C) 1). The deposition of extracellular calcium was confirmed by the von Kossa stain (C2) and Alizarin Red stain (C3) in both groups. Chondrogenic differentiation was verified by the blue-turquoise staining of acid glycosaminglykans of the extracellular matrix (C4). VSOPs-labeled and unlabeled hASCs, maintained in expansion medium, did not show lipid vacuoles or any extracellular matrix deposition (R3, 4/ C1–4). Magnification ×200; scale bars represent 100 µm in all figures.

Figure 9

Real time-PCR analyses after adipogenic, osteogenic and chondrogenic differentiation: The presented values (∆CT values) are normalized to the gene expression values of GAPDH. No differences in the expression of specific marker genes after adipogenic, osteogenic and chondrogenic induction between labeled and unlabeled hASCs were detected. Native tissues such as adipose tissue, bone and cartilage were used as positive controls.