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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.
Oligodendrocytes are the myelinating glia of the central nervous system and ensure rapid saltatory conduction. Shortage or loss of these cells leads to severe malfunctions as observed in human leukodystrophies and multiple sclerosis, and their replenishment by reprogramming or cell conversion strategies is an important research aim. Using a transgenic approach we increased levels of the transcription factor Sox10 throughout the mouse embryo and thereby prompted Fabp7-positive glial cells in dorsal root ganglia of the peripheral nervous system to convert into cells with oligodendrocyte characteristics including myelin gene expression. These rarely studied and poorly characterized satellite glia did not go through a classic oligodendrocyte precursor cell stage. Instead, Sox10 directly induced key elements of the regulatory network of differentiating oligodendrocytes, including Olig2, Olig1, Nkx2.2 and Myrf. An upstream enhancer mediated the direct induction of the Olig2 gene. Unlike Sox10, Olig2 was not capable of generating oligodendrocyte-like cells in dorsal root ganglia. Our findings provide proof-of-concept that Sox10 can convert conducive cells into oligodendrocyte-like cells in vivo and delineates options for future therapeutic strategies.
Zinkoxid-Nanopartikel (ZnO-NP) finden in vielen Produkten des täglichen Verbrauchs Verwendung. Daten über die toxikologischen Eigenschaften von ZnO-NP werden kontrovers diskutiert. Die menschliche Haut ist in Bezug auf die ZnO-NP Exposition das wichtigste Kontakt-Organ. Intakte Haut stellt eine suffiziente Barriere gegenüber NP dar. Bei defekter Haut ist ein Kontakt zu den proliferierenden Stammzellen möglich, sodass diese als wichtiges toxikologische Ziel für NP darstellen. Das Ziel dieser Dissertation war die Bewertung der genotoxischen und zytotoxischen Effekte an humanen mesenchymalen Stammzellen (hMSC) durch niedrig dosierte ZnO-NP nach 24 stündiger Exposition, repetitiven Expositionen und im Langzeitversuch bis zu 6 Wochen. Zytotoxische Wirkungen von ZnO-NP wurden mit 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid-Test (MTT) gemessen. Darüber hinaus wurde die Genotoxizität durch den Comet-Assay bewertet. Zur Langzeitbeobachtung bis zu 6 Wochen wurde die Transmissionselektronenmikroskopie (TEM) verwendet. Zytotoxizität nach 24-stündiger ZnO-NP-Exposition war ab einer Konzentration von 50 µg/ml nachweisbar. Genotoxizität konnten bereits bei Konzentrationen von 1 und 10 µg/ml ZnO-NP beschrieben werden. Wiederholte Exposition verstärkte die Zyto-, aber nicht die Genotoxizität. Eine intrazelluläre NP-Akkumulation mit Penetration der Zellorganelle wurde bei einer Exposition bis zu 6 Wochen beobachtet. Die Ergebnisse deuten auf zytotoxische und genotoxisches Effekte von ZnO-NP hin. Bereits geringe Dosen von ZnO-NP können bei wiederholter Exposition toxische Wirkungen hervorrufen sowie eine langfristige Zellakkumulation. Diese Daten sollten bei der Verwendung von ZnO-NP an geschädigter Haut berücksichtigt werden.
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.
Primäre Nestin-positive adulte Stamm-/Vorläuferzellen aus menschlichen Langerhans'schen Inseln besitzen einen mesenchymalen Charakter und das prinzipielle Potenzial zur in vitro-Differenzierung in Insulin produzierende Phänotypen. Allerdings ist die Entwicklung effektiver Differenzierungsstrategien bisher noch nicht gelungen. Dies ist unter anderem durch das limitierte Wachstumsverhalten dieser Primärzellen in Kultur begründet, das in der vorliegenden Arbeit ausführlich charakterisiert wurde. So besitzt die Gesamtpopulation aus pankreatischen humanen Langerhansschen Inseln auswachsender Zellen (hIZ) ein begrenztes Wachstumspotenzial von im Mittel 19 Passagen. Diese Tatsache limitiert zum einen die Entwicklung von Protokollen zur Differenzierung dieser Zellen und führt zum anderen zu einer Limitierung der Vision in vitro vermehrbaren und differenzierbaren Vorläuferzellmaterials, das nach Differenzierung transplantiert werden und in vivo die beta-Zellfunktion ersetzen könnte. Vor diesem Hintergrund zeigt die vorliegende Arbeit anhand des Nestin-positiven und mesenchymalen Zellmodells der menschlichen Knochenmarksstammzelllinie hMSC-TERT weiterhin, dass sich eine gentechnisch induzierte transiente und stabile Überex-pression des wachstums- und proliferationsassoziierten Proteins p8 fördernd auf das Wachstumsverhalten dieser Zelllinie auswirkt. Dieser Effekt beruht, wie an stabil generierten p8-überexprimierenden Zelllinien gezeigt werden konnte, zum einen auf der Steigerung der Proliferationsrate. Zum anderen ist das verbesserte Wachstumsverhalten jedoch auch auf eine bis dato unbekannte Verminderung der basalen Apoptoserate von hMSC-TERT zurückzuführen. Das Protein p8 konnte erstmals als molekularer Mediator des Wachstums und Überlebens mesenchymaler Nestin-positiver und zu beta-Zellähnlichen Phänotypen differenzierbarer Vorläuferzellen charakterisiert werden. Es kann somit einen entscheidenden Beitrag zur Lösung des Problems begrenzten differenzierbaren Stammzellmaterials auf der Suche nach einer zellbasierten kurativen, breit und risikoarm einsetzbaren Therapiestrategie für den Diabetes mellitus leisten.
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.
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.
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.
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.
Maintenance of hematopoietic stem cells and their potential to give rise to progenitors of differentiated lymphoid and myeloid cells are accomplished by a network of regulatory processes. As a part of this network, the heteromeric transcription factor GA-binding protein (GABP) plays a crucial role in self-renewal of murine hematopoietic and leukemic stem cells. Here, we report the consequences of functional impairment of GABP in human hematopoietic and in leukemic stem/progenitor cells. Ectopic overexpression of a dominant-negative acting GABP mutant led to impaired myeloid differentiation of CD34\(^{+}\) hematopoietic stem/progenitor cells obtained from healthy donors. Moreover, drastically reduced clonogenic capacity of leukemic stem/progenitor cells isolated from bone marrow aspirates of chronic myeloid leukemia (CML) patients underlines the importance of GABP on stem/progenitor cell maintenance and confirms the relevance of GABP for human myelopoiesis in healthy and diseased states.