DiI Labeling of Human Adipose-Derived Stem Cells: Evaluation of DNA Damage, Toxicity and Functional Impairment

Froelich, Katrin; Steussloff, Gudrun; Schmidt, Karsten; Tirado, Mario Ramos; Technau, Antje; Scherzed, Agmal; Hackenberg, Stephan; Radeloff, Andreas; Hagen, Rudolf; Kleinsasser, Norbert

doi:10.1159/000346714

Cited by 12 publications

(15 citation statements)

References 35 publications

(59 reference statements)

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“…This finding is supported by the enhanced levels of formazan formation in the scaffolds loaded with PDGF as determined by the MTT assay. Along with other studies, our model did not directly discriminate between PDGF induced proliferation and migration, as fibroblasts that undergo cell division remain DiI-positive [23] , [24] , [29] . We considered investigating the impact of proliferation on our model.…”

Section: Discussionmentioning

confidence: 71%

Cell Population Kinetics of Collagen Scaffolds in Ex Vivo Oral Wound Repair

et al. 2014

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Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

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Section: Discussionmentioning

confidence: 71%

Cell Population Kinetics of Collagen Scaffolds in Ex Vivo Oral Wound Repair

et al. 2014

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“…However, it is known that the intracellular concentration of the labelling substance decreases during proliferation. This is partly due to the uptake of the particles by the daughter cells, and partly due to the degradation of the particles via iron metabolic pathways [3,14,38]. This may lead to a short detectability of the labelled cells in the MRI, which could limit long-term observations over months [3].…”

Section: Discussionmentioning

confidence: 99%

“…Four weeks after the labelling procedure (day 28), hASCs from passage 6 of all 6 patients were used to evaluate the multilineage differentiation potential after VSOPs-labelling. The adipogenic, chondrogenic and osteogenic differentiation assays were performed as described previously [38]. According to the protocol of Pittenger [39] and modified by Nöth et al [40], EM-DMEM supplemented with 1 µg/mL insulin and 10 µM dexamethasone, 100 µM indomethacin, as well as 500 µM 1-methyl-3-isobutylxanthine, was used for adipogenic differentiation.…”

Section: Multidifferentiation Capacitymentioning

confidence: 99%

Toxicity and Functional Impairment in Human Adipose Tissue-Derived Stromal Cells (hASCs) Following Long-Term Exposure to Very Small Iron Oxide Particles (VSOPs)

Radeloff

Tirado

et al. 2020

Nanomaterials

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Magnetic nanoparticles (NPs), such as very small iron oxide NPs (VSOPs) can be used for targeted drug delivery, cancer treatment or tissue engineering. Another important field of application is the labelling of mesenchymal stem cells to allow in vivo tracking and visualization of transplanted cells using magnetic resonance imaging (MRI). For these NPs, however, various toxic effects, as well as functional impairment of the exposed cells, are described. The present study evaluates the influence of VSOPs on the multilineage differentiation ability and cytokine secretion of human adipose tissue derived stromal cells (hASCs) after long-term exposure. Human ASCs were labelled with VSOPs, and the efficacy of the labelling was documented over 4 weeks in vitro cultivation of the labelled cells. Unlabelled hASCs served as negative controls. Four weeks after labelling, adipogenic and osteogenic differentiation was histologically evaluated and quantified by polymerase chain reaction (PCR). Changes in gene expression of IL-6, IL-8, VEGF and caspase 3 were determined over 4 weeks. Four weeks after the labelling procedure, labelled and unlabelled hASCs did not differ in the gene expression of IL-6, IL-8, VEGF and caspase 3. Furthermore, the labelling procedure had no influence on the multidifferentiation ability of hASC. The percentage of labelled cells decreased during in vitro expansion over 4 weeks. Labelling with VSOPs and long-term intracellular disposition probably have no influence on the physiological functions of hASCs. This could be important for the future in vivo use of iron oxide NPs.

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“…The multilineage differentiation potential was evaluated 24 hours and 2 weeks after cell labeling ( n = 3). To measure this, adipogenic, osteogenic, and chondrogenic differentiation potential were induced as previously described [ 3 ].…”

Section: Methodsmentioning

confidence: 99%

“…Ideally, the labeling process meets the following criteria: (1) it should not interfere with the proliferation and vitality of labeled cells, (2) it should not impair the differentiation potential, (3) it should be persistent long-term, and (4) it should not contaminate adjacent cells or structures. Fluorescent dyes are commonly used for cell labeling, and, among others, fluorochromes like PKH26, CSFE [ 2 ], or Dil [ 3 ] were investigated for labeling ASCs. Another material that is often used as a cell tracker is Hoechst 33342 (H33342), which has been widely used in lymphocyte migration assays [ 4 , 5 ].…”

Section: Introductionmentioning

confidence: 99%

Labeling Adipose‐Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

Schendzielorz

Froelich

Rak

et al. 2016

Stem Cells International

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Adipose-derived stem cells (ASCs) have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342) represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 μg/mL and 5 μg/mL), with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 μg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 μg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique.

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DiI Labeling of Human Adipose-Derived Stem Cells: Evaluation of DNA Damage, Toxicity and Functional Impairment

Cited by 12 publications

References 35 publications

Cell Population Kinetics of Collagen Scaffolds in Ex Vivo Oral Wound Repair

Cell Population Kinetics of Collagen Scaffolds in Ex Vivo Oral Wound Repair

Toxicity and Functional Impairment in Human Adipose Tissue-Derived Stromal Cells (hASCs) Following Long-Term Exposure to Very Small Iron Oxide Particles (VSOPs)

Labeling Adipose‐Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

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