Center for Regenerative Therapies Dresden Cluster of Excellence

Dresden, Germany

Center for Regenerative Therapies Dresden Cluster of Excellence

Dresden, Germany
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Ozkucur N.,TU Dresden | Ozkucur N.,University of California at Davis | Ozkucur N.,Tufts University | Song B.,University of California at Davis | And 9 more authors.
Cellular and Molecular Life Sciences | Year: 2014

Endogenous electric fields (EF) may provide an overriding cue for directional cell migration during wound closure. Perceiving a constant direction requires active sodium-hydrogen exchanger (pNHE3) at the leading edge of HEK 293 cells but its activation mechanism is not yet fully understood. Because protein kinase C (PKC) is required in electrotaxis, we asked whether NHE3 is activated by PKC during wound healing. Using pharmacological (pseudosubstrate and edelfosine) inhibition, we showed that inhibition of PKCη isoform impairs directional cell migration in HEK 293 cells in the presence of a persistent directional cue (0.25-0.3 V/mm of EF for 2 h). Further, we found that pNHE3 forms complexes with both PKCη and γ-tubulin, suggesting that these molecules may regulate the microtubule-organizing center. In addition, cellular pNHE3 content was reduced significantly when PKCη was inhibited during directional cell migration. Taken together, these data suggest that PKCη-dependent phosphorylation of NHE3 and the formation of pNHE3/PKCη/γ-tubulin complexes at the leading edge of the cell are required for directional cell migration in an EF. © 2014 Springer Basel.

Tolar J.,University of Minnesota | Adair J.E.,Fred Hutchinson Cancer Research Center | Antoniou M.,King's College London | Bartholomae C.C.,German Cancer Research Center | And 29 more authors.
Molecular Therapy | Year: 2011

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. © The American Society of Gene & Cell Therapy.

Uckermann O.,Carl Gustav Carus University Hospital | Galli R.,TU Dresden | Anger M.,Carl Gustav Carus University Hospital | Anger M.,Center for Regenerative Therapies Dresden Cluster of Excellence | And 7 more authors.
International Journal of Radiation Biology | Year: 2014

Purpose: Vibrational spectroscopy enables the label-free characterization of cells and tissue by probing the biochemical composition. Here, we evaluated these techniques to identify glioblastoma stem cells. Materials and methods: The biochemical fingerprints of glioblastoma cells were established in human cell lines with high and low content of CD133 (cluster of differentiation 133)-positive cells using attenuated total reflection Fourier-transform infrared (ATR FT-IR) on vital cells and FT-IR mapping, which delivers spatially resolved spectroscopic datasets. After data preprocessing, unsupervised cluster analysis was applied. CD133 was addressed with flow cytometry and immunohistochemistry and used as a stemness marker. Results: In all preparations, the algorithm was able to correctly classify the spectra, differentiating CD133-rich and -poor populations. The main spectral differences were found in the region of 1000 cm- 1 to 1150 cm- 1 that can be assigned to vibrations of chemical bonds of DNA, RNA, carbohydrates and phospholipids. Interestingly, this spectral region is a key feature to discern glioblastoma from normal brain parenchyma, as FT-IR spectroscopic mapping of experimental brain tumors demonstrated. Conclusions: We were able to show biochemical differences between glioblastoma cell populations with high and low content of cancer stem cells that are presumably related to changes in the RNA/DNA content. © 2014 Informa UK, Ltd.

Mergler S.,Charité - Medical University of Berlin | Valtink M.,TU Dresden | Coulson-Thomas V.J.,Federal University of São Paulo | Lindemann D.,Center for Regenerative Therapies Dresden Cluster of Excellence | And 5 more authors.
Experimental Eye Research | Year: 2010

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1-3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1-3 functional activity was evident based on capsaicin-induced Ca2+ transients and induction of these responses through rises in ambient temperature from 25 °C to over 40 °C. The currents underlying Ca2+ transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10-20 μM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 °C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La3+) (100 μM) and ruthenium-red (RuR) (10 μM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 μM) led to a reversible increase in intracellular Ca2+ indicating putative TRPV1-3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions. © 2010 Elsevier Ltd.

Wittig D.,TU Dresden | Wang X.,University of Bergen | Walter C.,TU Dresden | Gerdes H.-H.,University of Bergen | And 3 more authors.
PLoS ONE | Year: 2012

Background: Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. Methodology and Principal Findings: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 μm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca 2+ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. Conclusions and Significance: Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic. © 2012 Wittig et al.

Perike S.,TU Dresden | Ozkucur N.,TU Dresden | Ozkucur N.,Tufts University | Sharma P.,TU Dresden | And 5 more authors.
Experimental Cell Research | Year: 2014

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02μM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (Vmem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only Vmem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and Vmem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis. © 2014 Elsevier Inc.

Pflueger M.,Helmholtz Center Munich | Seppanen-Laakso T.,VTT Technical Research Center of Finland | Suortti T.,VTT Technical Research Center of Finland | Hyotylainen T.,VTT Technical Research Center of Finland | And 4 more authors.
Diabetes | Year: 2011

OBJECTIVE - Islet autoimmunity precedes type 1 diabetes and often initiates in childhood. Phenotypic variation in islet autoimmunity relative to the age of its development suggests heterogeneous mechanisms of autoimmune activation. To support this notion, we examined whether serum metabolite profiles differ between children with respect to islet autoantibody status and the age of islet autoantibody development. RESEARCH DESIGN AND METHODS - The study analyzed 29 metabolites of amino acid metabolism and 511 lipids assigned to 12 lipid clusters in children, with a type 1 diabetic parent, who first developed autoantibodies at age 2 years or younger (n = 13), at age 8 years or older (n = 22), or remained autoantibodynegative, and were matched for age, date of birth, and HLA genotypes (n = 35). Ultraperformance liquid chromatography and mass spectroscopy were used to measure metabolites and lipids quantitatively in the first autoantibody-positive and matched autoantibody-negative serum samples and in a second sample after 1 year of follow-up. RESULTS - Differences in the metabolite profiles were observed relative to age and islet autoantibody status. Independent of age-related differences, autoantibody-positive children had higher levels of odd-chain triglycerides and polyunsaturated fatty acid-containing phospholipids than autoantibody-negative children and independent of age at first autoantibody appearance (P < 0.0001). Consistent with our hypothesis, children who developed autoantibodies by age 2 years had twofold lower concentration of methionine compared with those who developed autoantibodies in late childhood or remained autoantibody-negative (P < 0.0001). CONCLUSIONS - Distinct metabolic profiles are associated with age and islet autoimmunity. Pathways that use methionine are potentially relevant for developing islet autoantibodies in early infancy. © 2011 by the American Diabetes Association.

Teichmann J.,TU Dresden | Teichmann J.,Leibniz Institute of Polymer Research | Valtink M.,TU Dresden | Gramm S.,Leibniz Institute of Polymer Research | And 6 more authors.
Acta Biomaterialia | Year: 2013

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options. © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

PubMed | TU Dresden, Center for Regenerative Therapies Dresden Cluster of Excellence and Leibniz Institute of Polymer Research
Type: Journal Article | Journal: Journal of functional biomaterials | Year: 2014

Functional impairment of the human corneal endothelium can lead to corneal blindness. In order to meet the high demand for transplants with an appropriate human corneal endothelial cell density as a prerequisite for corneal function, several tissue engineering techniques have been developed to generate transplantable endothelial cell sheets. These approaches range from the use of natural membranes, biological polymers and biosynthetic material compositions, to completely synthetic materials as matrices for corneal endothelial cell sheet generation. This review gives an overview about currently used materials for the generation of transplantable corneal endothelial cell sheets with a special focus on thermo-responsive polymer coatings.

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