Final Activity Report Summary - ADSC-CARDIAC (Differentiation of adipose-derived stem cells info cardiomyocytes: understanding and application) The projects main objective had been to investigate the potential of adipose tissue-derived stem cells to differentiate into cardiomyocytes and to search for specific inductors. Mesenchymal stem cells have been shown to differentiate into a variety of lineages such as bone, cartilage, fat, neurons and cardiomyocytes. The concept had been to establish immortalised clones that additionally would carry a cardiac-specific reporter construct, to screen in an automated fashion for factors that drive cardiomyocyte differentiation.Several research tools very developed during this project. Adipose derived stem cells were isolated from lipoaspirates from three donors and immortalised using retroviral constructs containing E6/E7 of the human Papiloma Virus. About 500 individual immortalised clones were isolated and tested for their differentiation capacity into cartilage, bone, fat and neuronal cells, following standard differentiation protocols. All clones showed a differentiation capacity albeit at different levels and different lineages. The concept of clonal cell differentiation capacity had been published for mesenchymal stem cells and was thus confirmed in this project for adipose derived stem cells.Mesenchymal stem cells are difficult to stably transfect, and hence a self-inactivating retrovirus was developed that allowed incorporation of cardiac-specific promoters coupled to a reporter gene (copGFP) and a selection cassette (neomycin). Several cardiac specific promoters were cloned and tested in embryonic stem cells for functionality. Embryonic stem cells are known for their high differentiation capability into cardiomyocytes. Upon transfer of these reporter constructs into the immortalised adipose-derived stem cell clones and treatment with the demethylating agent, 5-Azacytidine to induce cardiomyocyte differentiation, no reporter construct expression could be detected. Several groups had published the ability of mesenchymal stem cells to differentiate into cardiomycytes after 5-Azacytidine treatment.The lack of inducibility of the immortalised clones may be due to the immortalisation or lack of cardiomyocyte differentiation. Since all clones retained their differentiation capacity to other lineages, it is more likely that the adipose-derived stem cells are not suited to give rise to cardiomyocytes in a reproducible and possibly therapeutically interesting level.
