Abstract 1028: Molecular Signalling Pathway Determining Cardiac Stem Cell Fate
The regulatory processes that govern cardiac stem cell (CSC) biology are still largely unknown. To exploit the regenerative potential of these unique cells, it is imperative to have a better understanding of the signalling cascade regulating CSC self-renewal and commitment to each of the 3 main cardiac lineages. To this end, c-kitpos CSCs were isolated from the hearts of C57J-B6 adult mice and human myocardial biopsies and their proliferative, clonogenic, self-renewal and differentiation properties quantified. CSCs form cardiospheres (a marker of multipotency). Real time RT-PCR, Western blots and immunocytochemistry showed high expression of Oct-4, Nanog, Notch-1, Bmi-1, and Wnt1-related proteins in the undifferentiated CSCs at the center of the sphere. Immunostaining showed increased expression of Wnt1 and β-catenin in activated, proliferating CSCs at its border. Addition of Wnt3a (one of the Wnt-1 group of secreted proteins) to the culture media increased CSC proliferation and clonal efficiency. This effect was mediated by activation of the canonical Wnt pathway through inhibition of β-catenin phosphorylation and hence its degradation, as shown by its nuclear accumulation and co-localisation with the transcription factor Tcf. Accordingly, lentiviral transfection of Bmi-1 increased CSC BrdU incorporation, clonal efficiency and cardiosphere formation. When expression of β-catenin and Bmi-1 in CSCs were down-regulated through specific si-RNA transfection, CSC proliferation, cardiosphere formation and clonal efficiency were dramatically reduced. Furthermore, β-catenin si-RNA transfection abolished the cellular effects of Wnt3a. When CSC cardiospheres were grown in differentiation medium, containing TGF-β, BMP2, BMP4 and Wnt5, they attached and differentiated with high efficiency into rhythmic spontaneous beating myocytes. These findings document a key role for Bmi-1 and the Wnt and TGF-β gene families in human and murine adult CSC biology, through modulation of clongenesis, self-renewal and commitment to myogenic differentiation. Identifying factors that regulate CSC fate is of great importance to design better protocols and interventions for the regeneration of functional contractile mass following myocardial injury.