Research

Osteoarthritis (OA) represents a considerable societal and economic burden in today’s society. Despite the tremendous developments in the field of articular cartilage tissue engineering (AC TE) in the recent decade, none of the TE-based approaches has been able to regenerate the cartilage to levels of native tissue. The established paradigm of AC TE involves employment of undifferentiated MSCs in combination with 3D scaffolds/hydrogels and appropriate growth factors to induce chondrogenic differentiation of cells and deposition of ECM components like collagen and glycosaminoglycans. Once successful tissue has been formed in vitro, engineered cartilage grafts can be studied in vivo in large animal models to assess safety and efficacy of such grafts. Unfortunately, a considerable amount of grafts fails in vivo, which indicates the overall unsuitability and immaturity of the engineered tissues to function in the mechanically demanding environment of the joint in vivo. More importantly, there is no incentive to publish or submit for publication unsuccessful studies, which indicates that the number of failed studies employing large animal models could be considerably higher. Therefore, there is a need for novel strategies to screen and identify in vitro engineered cartilage grafts that have higher chances of success in vivo. In addition to increasing the success rate of such studies, this approach would have a great potential to reduce the number of animals utilized in such studies. In this context, there is evidence suggesting that chondrogenically differentiating MSCs respond anabolically to mechanical stress at later stages of differentiation by producing ECM components like glycosaminoglycans. Interestingly, the differentiation of MSCs is also associated with metabolic changes, where glycolysis is reduced and oxidative phosphorylation is enhanced as maturation progresses. The goal of this project is to develop a platform that could be used to assess such metabolic changes by sampling metabolites in- and outside the developing cartilage grafts to make statements concerning the maturity. By establishing such platform an additional readout would be available, in addition to commonly used biochemical and histological techniques within AC TE, that would facilitate a more informed decision making prior to an in vivo transition of a potential cartilage graft.

Ein Haupthindernis für die Entwicklung von Arzneimitteln für pädiatrische Krebserkrankungen ist der Mangel an präklinischen Modellen, die menschliche Krankheiten rekapitulieren, aufgrund unvollständiger Kenntnis der Gewebeherkunft. Dies gilt insbesondere für das Ewing-Sarkom (EwS), das durch EWSR1/ETS-Gen-Rearrangements verursacht wird. Mesenchymale Stammzellen (MSC) wurden als Kandidatenzelltypen vorgeschlagen, jedoch konnte die gezielte Übertragung von EWS-FLI1 auf die mesenchymale Knochenzelllinie während der Embryogenese der Maus bisher nicht zu einer Tumorentstehung führen. Über die genauen Entwicklungswege der verschiedenen MSC-Zelltypen während der normalen und gestörten Differenzierung ist wenig bekannt. In diesem Projekt verfolgen wir drei Ansätze zur Entschlüsselung des Gewebe- und Differenzierungsursprungs für EwS: i) Basierend auf Einzelzell-Transkriptomanalysen werden wir den ersten zeit- und linienaufgelösten Einzelzell-Referenzatlas der menschlichen MSC-Entwicklung, naiv und entlang der induzierten Differenzierung, erstellen. Wir werden dann die Veränderung der normalen Differenzierungsverläufe nach Induktion der ektopischen EWS-FLI1-Expression aufzeigen, um den Zelltyp, das Differenzierungsstadium und die Chromatinarchitektur zu definieren, die der EwS am nächsten kommen, und die Entwicklung von EwS-Tumorzellen aus Einzelzell- und Bulk-Analysen von EwS-Tumorproben zurückverfolgen, indem wir sie mit dem MSC-Differenzierungs-Referenzatlas abgleichen. ii) Basierend auf der Beobachtung, dass das Krebs-Epigenom bei der Verwendung von Enhancern das Gedächtnis des Ursprungsgewebes bewahrt, werden wir nach Konvergenz in der artenübergreifenden Aktivität dieser Enhancer auf bestimmte Zelltypen und Entwicklungsstadien während der Entwicklung des Zebrafisches suchen. Unter Verwendung dieser Enhancer, um die fluoreszierende Reporteraktivität durch Cre-vermittelte Rekombination irreversibel einzuschalten, werden wir das Entwicklungsschicksal dieser Zellen bis ins Erwachsenenalter verfolgen. iii) Schließlich werden wir Experimente zur Abstammungsverfolgung von seltenen EwS-ähnlichen Tumoren, die sich im Zebrafisch entwickeln, mit mosaikförmiger EWS-FLI1-Expression durchführen, um Zelltypen zu identifizieren, die eine EWS-FLI1-vermittelte Transformation erlauben.

Mesenchymal stem cells (MSCs) are being investigated as potential cell therapeutics because of their immunomodulatory properties. In this context, therapy with MSCs appears to show promise in the treatment of diseases with autoimmune and inflammatory components. However, the role of MSCs in the context of inflammatory diseases has not been adequately characterized. The tasks of toll-like receptors (TLR) on and in MSCs are varied and include the control of the proliferation and migration of the MSCs, the repair of damaged tissue, the promotion of angiogenesis and the regulation of the immune system. In this project we plan to build new cell lines from mesenchymal stem cells in which the specific signaling pathways of TLR 3 and 4 can be switched on and off with the help of optogenetics through light induction. The introduction of reporter genes also enables real-time detection of the signal pathways. The light-activatable cell lines obtained in this way are cultivated under physiological conditions (hypoxia and 3D culture) and both for their multipotency (stem cell character) and for the potential for pro-inflammatory (MSC1; TLR4 activation) or anti-inflammatory (MSC2; TLR3 activation) phenotypes train, tested. These two biological phenotypes will be precisely characterized with the help of multiplex ELISA, gene expression and quantitative proteome analyzes and compared with the phenotype of non-stimulated cells or primary cells. The competencies acquired within the scope of this project will strengthen the collaboration between the University of Natural Resources and Life Sciences, the IMC FH Krems and the LifeTaq-Analytics company, lead to further collaborations with biotech companies and contribute to a long-term expansion of the respective fields of competence in the field of regenerative medicine.