Abstract 2114: Generation of Cardiomyocytes From Fabry Mouse-Derived Induced Pluripotent Stem Cells
Background: The reprogramming of mouse and human adult somatic cells to a pluripotent state, which are indistinguishable from embryonic stem (ES) cells, is one of the most exciting breakthroughs in stem cell biology. These induced pluripotent stem (iPS) cells provide the capability of cell therapy, understanding disease pathogenesis, and drug discovery. Especially, disease specific iPS cells are invaluable with regard to understand disease pathogenesis. We have studied on pathogenesis and treatment for Fabry disease including cardiac variant. The purposes of the present study were to generate iPS cells from Fabry mouse and differentiate the cells into cardiomyocytes in order to further investigate cardiac pathogenesis of the disease and to develop new treatment strategy.
Method: Fabry mouse-derived iPS (Fabry iPS) cells were generated from Fabry mouse tail-tip fibroblasts (TTFs) by retroviral transduction of three transcription factors: Oct3/4, Sox2, and Klf4. Cardiomyocytes were generated from Fabry iPS cells by using a standard embryoid body (EB)-based differentiation protocol.
Results: Fabry mouse TTFs were transduced with three factors. Following three weeks of culture, ES-like colonies emerged. These colonies were analyzed by stem cell marker, such as SSEA-1 and alkaline phosphatase (ALP) staining. The pluripotency of SSEA-1 and ALP-double positive cells were examined by teratoma formation. We injected Fabry iPS cells to nude mice. Four weeks later, we obtained tumors containing tissues of all three germ layers. EBs were generated from these Fabry iPS cells. EBs were cultured on OP9 stroma cells for 5–7days and spontaneously contracting EBs could be induced. RT-PCR demonstrated that various cardiac maker genes, such as Nkx2.5, GATA4, cardiac troponin T, MLC2a, and MLC2v, were highly expressed in differentiation cultures of Fabry iPS cells compared to control feeder cells. The expression of α-actinin protein was observed by immunohistochemistry.
Conclusions: We generated Fabry iPS cells and cardiomyocytes could be induced from the iPS cells. This system would contribute to the understanding of pathogenesis of Fabry disease including cardiac variant and the development of new treatment for the disease.