Antje Technau

SILVER NANOPARTICLES: EVALUATION OF DNA DAMAGE, TOXICITY AND FUNCTIONAL IMPAIRMENT IN HUMAN MESENCHYMAL STEM CELLS

Silver nanoparticles (Ag-NPs) are the most frequent commercialized nanomaterial currently. Due to a distinct lack of information on hazardous properties of Ag-NPs in human cells, a study was conducted to evaluate Ag-NP induced DNA damage, cell death and functional impairment in human mesenchymal stem cells (hMSCs). Initially, Ag-NPs and their cellular distribution were characterized by transmission electron microscopy (TEM). Ag-NPs were exposed to hMSCs for 1, 3 and 24h. Cytotoxicity was measured by the trypan blue exclusion test and the fluorescein-diacetate test, DNA damage was evaluated by the comet assay and chromosomal aberration test. Cytokine release of IL-6, IL-8 and VEGF was observed using the ELISA technique. Additionally, hMSC migration capability was tested in a transwell system. TEM revealed a Ag-NP distribution to cytoplasm and nucleus. Cytotoxic effects were seen at concentrations of 10 μg/ml for all test exposure periods. Both, comet assay and chromosomal aberration test showed DNA damage after 1, 3, and 24h at 0.1 μg/ml. A significant increase of IL-6, IL-8 and VEGF release indicates hMSC activation. Migration ability was not impaired at subtoxic concentrations. In conclusion, we demonstrated cyto- and genotoxic potential of Ag-NPs in hMSCs at significantly higher concentrations as compared to antimicrobial effective levels.

Cytotoxic, genotoxic and pro-inflammatory effects of zinc oxide nanoparticles in human nasal mucosa cells in vitro

Despite increasing application of zinc oxide nanoparticles (ZnO-NPs) for industrial purposes, data about potential toxic properties is contradictory. The current study focused on the cyto- and genotoxicity of ZnO-NPs in comparison to ZnO powder in primary human nasal mucosa cells cultured in the air-liquid interface. Additionally, IL-8 secretion as a marker for pro-inflammatory effects was measured. Particle morphology and intracellular distribution were evaluated by transmission electron microscopy (TEM). ZnO-NPs were transferred into the cytoplasm in 10% of the cells, whereas an intranuclear distribution could only be observed in 1.5%. While no cyto- or genotoxicity could be seen for ZnO powder in the dimethylthiazolyl-diphenyl-tetrazolium-bromide (MTT) test, the trypan blue exclusion test, and the single-cell microgel electrophoresis (comet) assay, cytotoxic effects were shown at a ZnO-NP concentration of 50 μg/ml (P<0.01). A significant enhancement in DNA damage was observed starting from ZnO-NP concentrations of 10 μg/ml (P<0.05) in comparison to the control. IL-8 secretion into the basolateral culture medium was increased at ZnO-NP concentrations of 5 μg/ml (P<0.05), as shown by ELISA. Our data indicates cyto- and genotoxic properties as well as a pro-inflammatory potential of ZnO-NPs in nasal mucosa cells. Thus, caution should be taken concerning their industrial and dermatological application. Additionally, further investigation on repetitive NP exposure is needed to estimate the impact of repair mechanisms.

Adipose tissue-derived stem cells show both immunogenic and immunosuppressive properties after chondrogenic differentiation

BACKGROUND AIMS The chondrogenic differentiation potential of mesenchymal stromal cells (MSC), as well as their immunosuppressive properties, have been studied extensively. So far, only a few studies have addressed the question of whether MSC still retain their immunosuppressive qualities after transdifferentiation. In particular, the expression of immunogenic markers, such as human leukocyte antigen (HLA)-DR, after differentiation has never been investigated. METHODS Chondrogenic transdifferentiation was induced in human adipose tissue-derived stem cell (ADSC) pellet cultures derived from 10 different patients, using 10 ng/mL transforming growth factor (TGF)-β3. Samples were harvested over a time-course of 28 days and analyzed by immunohistochemistry and reverse transcription (RT)-polymerase chain reaction (PCR). The cytokine levels in the supernatants of the samples were measured semi-quantitatively by dot-blots and quantitatively by enzyme-linked immunosorbant assays (ELISA). RESULTS Undifferentiated ADSC were negative for chondrogenic markers, as well as HLA-ABC and HLA-DR epitopes in immunofluorescence. In contrast, TGF-β3-induced pellet cultures showed both expression of chondrogenic differentiation markers, such as transcription factor 9 (Sox 9), collagen type IIa and aggrecan, and an up-regulation of HLA-DR, beginning at day 7 after induction. Interferon-γ (INF-γ) is known to up-regulate HLA-DR. Therefore we measured INF-γ levels in the supernatants of TGF-β3-induced pellets and, indeed, INF-γ was up-regulated during chondrogenesis in ADSC pellet cultures. However, both undifferentiated and TGF-β3-induced ADSC also showed expression of immunosuppressive HLA-G and interleukin (IL)-10 up-regulation. CONCLUSIONS These results suggest that the immunogenicity of adult stem cell-derived tissue should be tested in animal models before clinical trials for allogeneic engineered tissue are considered.

Effects of salinomycin on human bone marrow-derived mesenchymal stem cells in vitro

Various hypotheses on the origin of cancer stem cells (CSCs) exist, including that CSCs develop from transformed human bone marrow mesenchymal stem cells (hBMSC). Since the polyether antibiotic salinomycin selectively kills CSCs, the present study aims to elucidate the effects of salinomycin on normal hBMSC. The immunophenotype of hBMSC after salinomycin exposure was observed by flow cytometry. The multi-differentiation capacity of hBMSC was evaluated by Oil Red O and van Kossa staining. Cytotoxic effects of salinomycin were monitored by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. Furthermore, spheroid formation and migration capacity were assessed. There were no differences in the immunophenotype and multi-differentiation capacity of hBMSC induced by salinomycin treatment. Cytotoxic effects were observed at concentrations of 30 μM and above. Neither the migration capability nor the ability to form spheroids was affected. Essential functional properties of hBMSC were unaffected by salinomycin. However, dose-dependent cytotoxicity effects could be observed. Overall, low dose salinomycin showed no negative effects on hBMSC. Since mesenchymal stem cells from various sources respond differently, further in vitro studies are needed to clarify the effect of salinomycin on tissue-specific stem cells.

Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro

BACKGROUND AIMS Adipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. METHODS After isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. RESULTS During in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. CONCLUSIONS The study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell-based therapy.

Multipotent Stromal Cells for Autologous Cell Therapy Approaches in the Guinea Pig Model

Multipotent stromal cells have become of increasing interest due to their potential to provide therapeutic approaches for autologous tissue repair. However, these cells are not well defined in the guinea pig, which represents an important model in hearing research. Adipose-tissue-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) were isolated from different donor sites, and growth curves were generated to judge the proliferation potential. Adipogenic, chondrogenic and osteogenic differentiation was induced and confirmed histologically. Finally, the capability of guinea pig ADSC to differentiate into neuron-like cells was investigated. With regard to the expansion potential, total cell number and doubling time, ADSC from the neck were the most suitable cells of the tested donor sites. Both ADSC and BMSC showed nearly identical behaviour and ability to undergo multilineage differentiation. Thus, we identified ADSC from the neck as a promising cell source for autologous cell-based approaches in hearing research using the guinea pig model.

Influence of different growth factors on chondrogenic differentiation of adipose-derived stem cells in polyurethane-fibrin composites

INTRODUCTION Chondrogenic differentiation of adipose-derived stem cells (ASCs) has proven to be feasible. To compensate for laryngeal palsy or cartilage defects after surgery or trauma using tissue engineering, a formable and stable scaffold material is mandatory. METHODS ASCs were seeded in fibrin-polyurethane scaffolds and cultured in chondrogenic differentiation medium adding the growth factors TGF-b1, TGF-b3, and BMP-2 for up to 35 days. RESULTS Histological examination showed acid glycosaminoglycans in the extracellular matrix in all groups. Immunofluorescence presented positive staining for collagen II, aggrecan, and SOX-9 in the TGF-b1-, TGF-b3-, and BMP-2-group. With Real-time PCR analyses, chondrogenic differentiation became apparent by the expression of the specific genes COL2A1 (collagen II), AGC 1 (aggrecan), and SOX-9, whereas collagen II expression was low in all groups compared to bone marrow-derived stem cells (BMSC) due to reduced chondrogenic ability. CONCLUSIONS These findings demonstrate the general ability of ASCs to differentiate into matrix-producing chondrocytes in fibrin-polyurethane scaffolds. However, further experiments are necessary to enhance this chondrogenic potential of ASCs seeded in fibrin-polyurethane scaffolds in order to produce a suitable regeneration method for treating cartilage defects or an implantable medialization material for vocal cord palsy.

Tumor antigen-specific T cells for immune monitoring of dendritic cell-treated glioblastoma patients.

BACKGROUND AIMS CD8(+) T cells are part of the adaptive immune system and, as such, are responsible for the elimination of tumor cells. Dendritic cells (DC) are professional antigen-presenting cells (APC) that activate CD8(+) T cells. Effector CD8(+) T cells in turn mediate the active immunotherapeutic response of DC vaccination against the aggressive glioblastoma (GBM). The lack of tumor response assays complicates the assessment of treatment success in GBM patients. METHODS A novel assay to identify specific cytotoxicity of activated T cells by APC was evaluated. Tumor antigen-pulsed DCs from HLA-A*02-positive GBM patients were cultivated to stimulate autologous cytotoxic T lymphocytes (CTL) over a 12-day culture period. To directly correlate antigen specificity and cytotoxic capacity, intracellular interferon (IFN)-γ fluorescence flow cytometry-based measurements were combined with anti-GBM tumor peptide dextramer staining. IFN-γ response was quantified by real-time polymerase chain reaction (PCR), and selected GBM genes were compared with healthy human brain cDNA by single specific primer PCR characterization. RESULTS Using CTL of GBM patients stimulated with GBM lysate-pulsed DCs increased IFN-γ messenger RNA levels, and intracellular IFN-γ protein expression was positively correlated with specificity against GBM antigens. Moreover, the GBM peptide-specific CD8(+) T-cell response correlated with specific GBM gene expression. Following DC vaccination, GBM patients showed 10-fold higher tumor-specific signals compared with unvaccinated GBM patients. DISCUSSION These data indicate that GBM tumor peptide-dextramer staining of CTL in combination with intracellular IFN-γ staining may be a useful tool to acquire information on whether a specific tumor antigen has the potential to induce an immune response in vivo.

Immunotherapy in atypical teratoid-rhabdoid tumors: Data from a survey of the HGG-Immuno Group

BACKGROUND AIMS Atypical rhabdoid/teratoid tumors (AT/RT) are the most common brain tumors in infants and associated with a dismal prognosis. Although intensification of first-line therapy has resulted in improvement of overall survival, novel treatment strategies are needed. Because immunotherapy has resulted in remarkable results in several adult tumor entities, incorporation of immunotherapy into AT/RT treatment offers a novel alternative. METHODS We retrospectively analyzed data from 7 AT/RT patients from five countries treated within the HGG-Immuno Consortium. Two patients were ≤1 year and 4 patients were ≤2 years of age at diagnosis. All received immunotherapy with autologous, tumor-lysate-loaded dendritic cells (DCs) on a compassionate use basis using a schedule of three to four weekly DC vaccinations with up to 2 × 10(7) DCs per vaccine, followed by three lysate boosts each 1 month apart. RESULTS Monocyte collections (median age at apheresis 31.5, range 20-143 months) and vaccinations were uneventful without any severe adverse event related to the vaccine, demonstrating feasibility and safety in this very young age group. Two children received immunotherapy during their primary and the remaining five during second- or third-line therapy. Three of seven patients survived long term with a follow-up of 143, 138 and 46 months, with at least two of them harboring somatic mutations. One long-term survivor was vaccinated during primary treatment and the other two after first or second relapse/progression. Two analyzed patients showed positive CD8(+) T-cell responses after vaccination. DISCUSSION Our data demonstrate that anti-tumor immunotherapy with autologous DCs is feasible and safe in young children with ATRTs and that this approach warrants further investigation in controlled clinical trials.

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

Introduction: Adipose tissue-derived stem cells (ASCs) have become the primary focus of tissue engineering research. To understand their functions and behavior in in vitro and in vivo models, it is mandatory to track the implanted cells and distinguish them from the resident or host cells. A common labeling method is the use of fluorescent dyes, e.g. the lipophilic carbocyanine dye, DiI. This study aimed to analyze potential DNA damage, toxicity and impairment of the functional properties of human ASCs after labeling with DiI. Methods: Cytotoxicity was measured using the MTT assay and DNA damage was determined by means of the comet assay. Potential apoptotic effects were determined using the annexin V-propidium iodide test. Differentiation potential was evaluated by trilineage differentiation procedures in labeled and unlabeled ASCs. Proliferation as well as migration capability was analyzed, and the duration and stability of DiI labeling in ASCs during in vitro expansion was observed over a period of 35 days. Results: DiI labeling did not cause genotoxic effects 15, or 30 min or 24 h after the labeling procedure, and there were no cytotoxic effects until 72 h afterwards. No impairment of proliferation or migration capability or differentiation potential could be determined. However, after 35 days, only 37% of labeled cells could be detected using the fluorescence microscope, which indicates a decrease in staining stability during in vitro expansion. Conclusion: DiI is a convenient method for ASCs labeling which causes no toxic effects and does not impair the proliferation, migration or differentiation potential of ASCs after the labeling procedure.