TY - THES A1 - Gotthard, Hannes T1 - Targeting Colorectal Cancer Stem Cells with Hemibodies T1 - Eliminierung von Krebsstammzellen des kolorektalen Karzinoms mithilfe von Hemibodies N2 - The cancer stem cell hypothesis is a cancer development model which elicited great interest in the last decades stating that cancer heterogeneity arises from a stem cell through asymmetrical division. The Cancer Stem Cell subset is described as the only population to be tumorigenic and having the potential to renew. Conventional therapy often fails to eradicate CSC resulting in tumor relapse. Consequently, it is of great inter-est to eliminate this subset of cells to provide the best patient outcome. In the last years several approaches to target CSC were developed, one of them being immunotherapeu-tic targeting with antibodies. Since markers associated with CSC are also expressed on normal stem cells or healthy adjacent tissue in colorectal cancer, dual targeting strate-gies are preferred over targeting only a single antigen. Subsequently, the idea of dual targeting two CSC markers in parallel by a newly developed split T cell-engaging anti-body format termed as Hemibodies emerged. In a preliminary single cell RNA sequenc-ing analysis of colorectal cancer cells CD133, CD24, CD166 and CEA were identified as suitable targets for the combinatorial targeting strategy. Therefore, this study focused on trispecific and trivalent Hemibodies comprising a split binding moiety against CD3 and a binding moiety against either CD133, CD24, CD166 or CEA to overcome the occurrence of resistance and to efficiently eradicate all tumor cells including the CSC compartment. The study showed that the Hemibody combinations CD133xCD24, CD133xCD166 and CD133xCEA are able to eliminate double positive CHO cells with high efficacy while having a high specificity indicated by no killing of single antigen positive cells. A thera-peutic window ranging between one to two log levels could be achieved for all combina-tions mentioned above. The combinations CD133xCD24 and CD133xCD166 further-more proved its efficacy and specificity on established colorectal cancer cell lines. Be-sides the evaluation of specificity and efficacy the already introduced 1st generation of Hemibodies could be improved into a 2nd generation Hemibody format with increased half-life, stability and production yield. In future experiments the applicability of above-mentioned Hemibodies will be proven on patient-derived micro tumors to also include variables like tumor microenvironment and infiltration. N2 - In den letzten Jahrzenten wurde neben der klonalen Evolution ein weiteres Modell zur Krebsentstehung und dessen Heterogenität entwickelt: die Krebsstammzellhypothe-se. Diese Hypothese besagt, dass die Heterogenität eines Tumors durch asymmetri-sche Teilung von sogenannten Krebsstammzellen entsteht. Nur diese sind tumorigen und in der Lage Metastasen zu bilden. Außerdem werden Krebsstammzellen als re-sistent gegen konventionelle Therapien beschrieben, weshalb es nach einer anfängli-chen Tumorregression oft zu einem Rezidiv durch erneutes Auswachsen von zurück-bleibenden Krebsstammzellen kommt. Deshalb ist es von großem Interesse genau diese Population abzutöten, um eine erfolgreiche Therapie zu gewährleisten. In den letzten Jahren wurden zahlreiche Medikationen entwickelt, um Krebsstammzellen ge-zielt anzugreifen. Ein vielversprechender Ansatz ist hierbei die immuntherapeutische Adressierung mittels Antikörpern gegen Krebsstammzellmarkern. Einzelne Marker sind allerdings auch auf normalen Stammzellen und gesundem Gewebe exprimiert, weshalb Therapien, die auf mindestens zwei verschiedene Oberflächenproteine ab-zielen, erfolgsversprechender sind. In dieser Arbeit wurde ein neues T-Zell rekrutie-rendes Antikörperformat entwickelt, sogenannte Hemibodies. Hierbei handelt es sich um ein trispezifisches und trivalentes Format, bestehend aus jeweils zwei Fragmen-ten. Jedes Fragment besteht aus einer Bindedomäne gegen ein Krebsstammzellmar-ker und einer geteilten Bindedomäne gegen CD3. Durch Bindung beider Fragmente an einen Stammzellmarker kommt es zur Komplementierung der geteilten anti-CD3 Domäne und zur T-Zellrekrutierung. Der erste Teil der Arbeit befasst sich mit der bioin-formatischen Analyse von Einzelzell-RNA-Daten des kolorektalen Karzinoms (KRK) zur Identifizierung von potentiellen Krebsstammzellmarkern. Dabei konnten die Ober-flächenproteine CD24, CD133, CD166 und CEA und besonders deren Kombination als geeignete Zielstrukturen identifiziert werden. Die gegen oben genannte Antigene gerichteten Hemibodies zeigten in den Kombinationen CD133xCD24, CD133xCD166 und CD133xCEA auf doppelt positiven CHO-Zellen eine hohe Effektivität. Außerdem konnte die Spezifität durch ein Ausbleiben von Zelltod auf einzel-positiven CHO Zellen bewiesen werden. Die Kombinationen CD133xCD24 und CD133xCD166 konnten Effektivität und Spezifität auch auf etablierten Krebszellen zeigen. Die oben genann-ten Kombinationen waren in einem therapeutischen Fenster von ein bis zwei Logstu-fen funktional. Neben der Testung verschiedener Hemibody-Kombinationen konnten die bereits publizierten Hemibodies der ersten Generation in ein neues Format der zweiten Generation weiterentwickelt werden. Das neue Format zeigte eine verbesser-te Halbwertszeit, Stabilität und Produzierbarkeit. In zukünftigen Experimenten werden die in der Thesis benutzten Hemibodies auf Mikrotumoren getestet, um weitere Vari-ablen, die die Effektivität und Spezifität beeinflussen zu ermitteln. KW - Monoklonaler bispezifischer Antikörper KW - Antikörper KW - T-Lymphozyt KW - Immunreaktion KW - Dickdarmkrebs KW - Hemibody KW - Hemibodies KW - Colorectal Cancer KW - trispecific KW - T-cell engager KW - dual targeting KW - Bispecific T-cell engager KW - stem cells KW - Kolorektales Karzinom Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-303090 ER - TY - JOUR A1 - Krstic, Jelena A1 - Herrmann, Marietta A1 - Gadjanski, Ivana A1 - Mojsilovic, Slavko T1 - Editorial: Microenvironment-derived stem cell plasticity JF - Frontiers in Cell and Developmental Biology N2 - No abstract available. KW - plasticity KW - stem cells KW - microenvironment KW - imaging KW - extracellular vesicles (EVs) KW - oxygen tension KW - tissue regeneration KW - immunomodulation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197424 SN - 2296-634X VL - 5 ER - TY - JOUR A1 - Radeloff, Katrin A1 - Radeloff, Andreas A1 - Tirado, Mario Ramos A1 - Scherzad, Agmal A1 - Hagen, Rudolf A1 - Kleinsasser, Norbert H. A1 - Hackenberg, Stephan T1 - Long-Term Impact of Zinc Oxide Nanoparticles on Differentiation and Cytokine Secretion of Human Adipose-Derived Stromal Cells JF - Materials N2 - Zinc oxide nanoparticles (ZnO-NPs) are widely utilized, for example in manufacturing paints and in the cosmetic industry. In addition, there is raising interest in the application of NPs in stem cell research. However, cytotoxic, genotoxic and pro-inflammatory effects were shown for NPs. The aim of this study was to evaluate the impact of ZnO-NPs on cytokine secretion and differentiation properties of human adipose tissue-derived stromal cells (ASCs). Human ASCs were exposed to the subtoxic concentration of 0.2 mu g/mL ZnO-NPs for 24 h. After four weeks of cultivation, adipogenic and osteogenic differentiation procedures were performed. The multi-differentiation potential was confirmed histologically and using polymerase chain reaction (PCR). In addition, the gene expression of IL-6, IL-8, vascular endothelial growth factor (VEGF) and caspase 3 was analyzed. Over the course of four weeks after ZnO-NPs exposure, no significant differences were detected in the gene expression of IL-6, IL-8, VEGF and caspase 3 compared to non-exposed cells. The differentiation was also not affected by the ZnO-NPs. These findings underline the fact, that functionality of ASCs is likely to be unaffected by ZnO-NPs, despite a long-term disposition of NPs in the cells, supposing that the starting concentration was safely in the non-toxic range. This might provide important information for single-use nanomedical applications of ZnO-NPs. KW - zinc oxide KW - nanoparticles KW - toxicity KW - differentiation potential KW - human adipose-derived stromal cells KW - stem cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224779 VL - 12 IS - 1823 ER - TY - JOUR A1 - Radeloff, Katrin A1 - Radeloff, Andreas A1 - Ramos Tirado, Mario A1 - Scherzad, Agmal A1 - Hagen, Rudolf A1 - Kleinsasser, Norbert H. A1 - Hackenberg, Stephan T1 - Toxicity and functional impairment in human adipose tissue-derived stromal cells (hASCs) following long-term exposure to very small iron oxide particles (VSOPs) JF - Nanomaterials N2 - 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. KW - iron oxide nanoparticles KW - VSOP KW - nanoparticles KW - toxicity KW - differentiation potential KW - human adipose-derived stromal cells KW - stem cells KW - long-term exposure Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203676 SN - 2079-4991 VL - 10 IS - 4 ER - TY - JOUR A1 - Nose, Naoko A1 - Nogami, Suguru A1 - Koshino, Kazuhiro A1 - Chen, Xinyu A1 - Werner, Rudolf A. A1 - Kashima, Soki A1 - Rowe, Steven P. A1 - Lapa, Constantin A1 - Fukuchi, Kazuki A1 - Higuchi, Takahiro T1 - [18F]FDG-labelled stem cell PET imaging in different route of administrations and multiple animal species JF - Scientific Reports N2 - Stem cell therapy holds great promise for tissue regeneration and cancer treatment, although its efficacy is still inconclusive and requires further understanding and optimization of the procedures. Non-invasive cell tracking can provide an important opportunity to monitor in vivo cell distribution in living subjects. Here, using a combination of positron emission tomography (PET) and in vitro 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) direct cell labelling, the feasibility of engrafted stem cell monitoring was tested in multiple animal species. Human mesenchymal stem cells (MSCs) were incubated with phosphate-buffered saline containing [18F]FDG for in vitro cell radiolabelling. The pre-labelled MSCs were administrated via peripheral vein in a mouse (n=1), rats (n=4), rabbits (n=4) and non-human primates (n=3), via carotid artery in rats (n=4) and non-human primates (n=3), and via intra-myocardial injection in rats (n=5). PET imaging was started 10 min after cell administration using a dedicated small animal PET system for a mouse and rats. A clinical PET system was used for the imaging of rabbits and non-human primates. After MSC administration via peripheral vein, PET imaging revealed intense radiotracer signal from the lung in all tested animal species including mouse, rat, rabbit, and non-human primate, suggesting administrated MSCs were trapped in the lung tissue. Furthermore, the distribution of the PET signal significantly differed based on the route of cell administration. Administration via carotid artery showed the highest activity in the head, and intra-myocardial injection increased signal from the heart. In vitro [18F]FDG MSC pre-labelling for PET imaging is feasible and allows non-invasive visualization of initial cell distribution after different routes of cell administration in multiple animal models. Those results highlight the potential use of that imaging approach for the understanding and optimization of stem cell therapy in translational research. KW - biomarkers KW - molecular medicine KW - stem-cell research KW - stem cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260590 VL - 11 IS - 1 ER - TY - JOUR A1 - McNeill, Rhiannon V. A1 - Ziegler, Georg C. A1 - Radtke, Franziska A1 - Nieberler, Matthias A1 - Lesch, Klaus‑Peter A1 - Kittel‑Schneider, Sarah T1 - Mental health dished up — the use of iPSC models in neuropsychiatric research JF - Journal of Neural Transmission N2 - Genetic and molecular mechanisms that play a causal role in mental illnesses are challenging to elucidate, particularly as there is a lack of relevant in vitro and in vivo models. However, the advent of induced pluripotent stem cell (iPSC) technology has provided researchers with a novel toolbox. We conducted a systematic review using the PRISMA statement. A PubMed and Web of Science online search was performed (studies published between 2006–2020) using the following search strategy: hiPSC OR iPSC OR iPS OR stem cells AND schizophrenia disorder OR personality disorder OR antisocial personality disorder OR psychopathy OR bipolar disorder OR major depressive disorder OR obsessive compulsive disorder OR anxiety disorder OR substance use disorder OR alcohol use disorder OR nicotine use disorder OR opioid use disorder OR eating disorder OR anorexia nervosa OR attention-deficit/hyperactivity disorder OR gaming disorder. Using the above search criteria, a total of 3515 studies were found. After screening, a final total of 56 studies were deemed eligible for inclusion in our study. Using iPSC technology, psychiatric disease can be studied in the context of a patient’s own unique genetic background. This has allowed great strides to be made into uncovering the etiology of psychiatric disease, as well as providing a unique paradigm for drug testing. However, there is a lack of data for certain psychiatric disorders and several limitations to present iPSC-based studies, leading us to discuss how this field may progress in the next years to increase its utility in the battle to understand psychiatric disease. KW - hiPSC KW - iPSC KW - stem cells KW - mental disorders KW - affective disorders KW - ADHD Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235666 SN - 0300-9564 VL - 127 ER - TY - JOUR A1 - Gensler, Marius A1 - Leikeim, Anna A1 - Möllmann, Marc A1 - Komma, Miriam A1 - Heid, Susanne A1 - Müller, Claudia A1 - Boccaccini, Aldo R. A1 - Salehi, Sahar A1 - Groeber-Becker, Florian A1 - Hansmann, Jan T1 - 3D printing of bioreactors in tissue engineering: A generalised approach JF - PLoS One N2 - 3D printing is a rapidly evolving field for biological (bioprinting) and non-biological applications. Due to a high degree of freedom for geometrical parameters in 3D printing, prototype printing of bioreactors is a promising approach in the field of Tissue Engineering. The variety of printers, materials, printing parameters and device settings is difficult to overview both for beginners as well as for most professionals. In order to address this problem, we designed a guidance including test bodies to elucidate the real printing performance for a given printer system. Therefore, performance parameters such as accuracy or mechanical stability of the test bodies are systematically analysed. Moreover, post processing steps such as sterilisation or cleaning are considered in the test procedure. The guidance presented here is also applicable to optimise the printer settings for a given printer device. As proof of concept, we compared fused filament fabrication, stereolithography and selective laser sintering as the three most used printing methods. We determined fused filament fabrication printing as the most economical solution, while stereolithography is most accurate and features the highest surface quality. Finally, we tested the applicability of our guidance by identifying a printer solution to manufacture a complex bioreactor for a perfused tissue construct. Due to its design, the manufacture via subtractive mechanical methods would be 21-fold more expensive than additive manufacturing and therefore, would result in three times the number of parts to be assembled subsequently. Using this bioreactor we showed a successful 14-day-culture of a biofabricated collagen-based tissue construct containing human dermal fibroblasts as the stromal part and a perfusable central channel with human microvascular endothelial cells. Our study indicates how the full potential of biofabrication can be exploited, as most printed tissues exhibit individual shapes and require storage under physiological conditions, after the bioprinting process. KW - stem cells KW - technology Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231368 VL - 15 IS - 11 ER - TY - JOUR A1 - Weissenberger, Manuel A1 - Weissenberger, Manuela H. A1 - Wagenbrenner, Mike A1 - Heinz, Tizian A1 - Reboredo, Jenny A1 - Holzapfel, Boris M. A1 - Rudert, Maximilian A1 - Groll, Jürgen A1 - Evans, Christopher H. A1 - Steinert, Andre F. T1 - Different types of cartilage neotissue fabricated from collagen hydrogels and mesenchymal stromal cells via SOX9, TGFB1 or BMP2 gene transfer JF - PLoS One N2 - Objective As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors. Design Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX)9,transforming growth factor beta (TGFB) 1or bone morphogenetic protein (BMP) 2cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy. Results Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenesSOX9,TGFB1andBMP2as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9). Conclusions Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factorsSOX9,TGFB1andBMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage. KW - stem cells KW - in vitro KW - chondrogenic differentiation KW - repair KW - chondrocytes KW - transplantation KW - stimulation KW - scaffolds KW - defects KW - therapy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230494 VL - 15 IS - 8 ER - TY - JOUR A1 - Alzheimer, Mona A1 - Svensson, Sarah L. A1 - König, Fabian A1 - Schweinlin, Matthias A1 - Metzger, Marco A1 - Walles, Heike A1 - Sharma, Cynthia M. T1 - A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni JF - PLoS Pathogens N2 - The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens. KW - in vitro KW - stem cells KW - invasion KW - host KW - adhesion KW - epithelial cells KW - translocation KW - virulence KW - responses KW - microenvironment Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229454 VL - 16 IS - 2 ER - TY - JOUR A1 - Simsekyilmaz, Sakine A1 - Liehn, Elisa A. A1 - Weinandy, Stefan A1 - Schreiber, Fabian A1 - Megens, Remco T. A. A1 - Theelen, Wendy A1 - Smeets, Ralf A1 - Jockenhövel, Stefan A1 - Gries, Thomas A1 - Möller, Martin A1 - Klee, Doris A1 - Weber, Christian A1 - Zernecke, Alma T1 - Targeting In-Stent-Stenosis with RGD- and CXCL1-Coated Mini-Stents in Mice JF - PLoS ONE N2 - Atherosclerotic lesions that critically narrow the artery can necessitate an angioplasty and stent implantation. Long-term therapeutic effects, however, are limited by excessive arterial remodeling. We here employed a miniaturized nitinol-stent coated with star-shaped polyethylenglycole (star-PEG), and evaluated its bio-functionalization with RGD and CXCL1 for improving in-stent stenosis after implantation into carotid arteries of mice. Nitinol foils or stents (bare metal) were coated with star-PEG, and bio-functionalized with RGD, or RGD/CXCL1. Cell adhesion to star-PEG-coated nitinol foils was unaltered or reduced, whereas bio-functionalization with RGD but foremost RGD/CXCL1 increased adhesion of early angiogenic outgrowth cells (EOCs) and endothelial cells but not smooth muscle cells when compared with bare metal foils. Stimulation of cells with RGD/CXCL1 furthermore increased the proliferation of EOCs. In vivo, bio-functionalization with RGD/CXCL1 significantly reduced neointima formation and thrombus formation, and increased re-endothelialization in apoE\(^{-/-}\) carotid arteries compared with bare-metal nitinol stents, star-PEG-coated stents, and stents bio-functionalized with RGD only. Bio-functionalization of star-PEG-coated nitinol-stents with RGD/CXCL1 reduced in-stent neointima formation. By supporting the adhesion and proliferation of endothelial progenitor cells, RGD/CXCL1 coating of stents may help to accelerate endothelial repair after stent implantation, and thus may harbor the potential to limit the complication of in-stent restenosis in clinical approaches. KW - carotid arteries KW - polymers KW - stent implantation KW - coatings KW - endothelial cells KW - mice KW - fluorescence microscopy KW - stem cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179745 VL - 11 IS - 5 ER -