Abstract 11450: Derivation of Cardiac Myocytes from induced Pluripotent Stem Cells of Muscular Dystrophy Models
Duchenne Muscular Dystrophy (DMD) is an x-linked recessive disorder that affects 1:3500 males. The disease is characterized by the absence of the dystrophin protein, leading to severe muscle degeneration. DMD carrier females are heterozygous for the DMD mutation, yet may suffer from cardiac complications due to severe x-chromosome inactivation bias. The mouse model of DMD is the mdx mouse and recapitulates the absence of dystrophin in contractile muscle. We have generated mouse induced pluripotent stem cells (miPSCs) that are heterozygous for the dystrophin gene in order to study dystrophin protein expression in the heterozygote state. Using the original Yamanaka protocol, we used retroviral infection to deliver Oct3/4, Sox2, c-Myc and Klf4 to mouse embryonic fibroblasts that were genomically heterozygous for dystrophin expression. Upon infection, we isolated embryonic stem cell like (ESC) colonies based on morphology. RT-PCR analysis of isolated colonies showed positive expression of the four pluripotency associated markers as well as positivity for endogenously expressed Nanog. These cells also stained positively for alkaline phosphatase activity. To study the cardiac expression of dystrophin in the heterozygous state, we have differentiated these cells as well as mdx (+/−) mESCs into autonomously beating cardiac myocytes in culture. Using the embryoid body technique, we made 30uL droplets of 2,500 cells and allowed them to hang for two days on the lids of culture plates. Following the hanging stage, cells were placed on a shaker platform for three more days to continue differentiation. They were then placed on gelatin-coated plates and allowed to adhere and differentiate completely. The derived myocyte populations beat for up to three weeks in culture and stained positively for troponin T and alpha-actinin. Unexpectedly, we did not detect dystrophin expression in the myocytes via western blot analysis, indicating that x-chromosome inactivation bias may be occurring. In its absence, we detected up-regulation of utrophin. These experiments will be essential for the characterization of these cells for their future transplantation applicability.
- © 2010 by American Heart Association, Inc.