Abstract 12611: Economical and Efficient Production of Cardiac Myocytes From ES Cells and iPS Cells Using Small Molecule-Responsive Artificial Receptors For Wnt3a Signal Transduction
Myocardial regeneration therapy using embryonic stem (ES) cells and induced pluripotent stem (iPS) cells has been expected as a promising approach for end-staged heart failure. Recent studies including ours reported that canonical Wnt signal activation by Wnt3a in the early phase of mouse ES/iPS cell differentiation plays a pivotal role in efficient myocardial cell differentiation. Problems about the cost and the quality of recombinant Wnt3a protein, however, compromise the utility of this approach to create sufficient amounts of cardiac myocytes for cell implantation. To address these issues, we attempted to generate small-molecule-responsive artificial receptors which can activate the Wnt3a signal transduction. First, we constructed antibody/receptor chimeras, in which single-chain Fv of anti-fluorescein (FL) antibody was tethered to transmembrane/cytoplasmic domains of Wnt3a receptors, so that they could transduce the Wnt3a signal pathway in response to FL-conjugated BSA (BSA-FL) as a cognate ligand, which costs about 2000 times less than recombinant Wnt3a protein. Signal activations by these chimeric receptors were measured by luciferase reporter assay for TCF/β-catenin dependent transcription, one of the common downstream targets of canonical Wnt signal pathways. ES/iPS cells transfected with the chimeric receptors showed increased luciferase activity by BSA-FL treatment in a dose dependent manner. These activations were similar to the level when the cells are treated with 10 to 100ng/ml of recombinant Wnt3a protein. In addition, these signal activities were not perturbed by Dkk1, an antagonist of Wnt3a, suggesting that the signal transduction via chimeric receptors is independent of the Wnt3a ligand. Finally, BSA-FL treatment in the early phase of differentiation increased the efficiency for cardiac differentiation in ES/iPS cell lines stably expressing these receptors but not in the mock-expressing cell lines, exemplified by the beating ratio (BSA-FL 100% vs Control 53.6%). Application of this technology to ES/iPS cell differentiation could be a powerful tool for efficient and economical creation of cardiac myocytes for heart regeneration therapy.
- © 2011 by American Heart Association, Inc.