Pluripotent stem cells (ES and iPS cells) have the ability to self-renew and to differentiate into multiple lineages in vitro. This makes these cells powerful tool to study early embryonic developmental pathways and to generate specific cell populations for regenerative medicine and disease investigation. Supported by R01 AR055299, our research group has pioneered methods to derive skeletal myogenic cells from mouse and human pluripotent ES and iPS cells. Until our work, studies in this area were rare since skeletal myogenic differentiation is extremely inefficient during in vitro differentiation. We reasoned that because in vitro systems do not recapitulate neural tube or notochord development, and indeed do not produce structures resembling somites, that the inducing signals that properly pattern paraxial mesoderm were absent. We have overcome this roadblock by activating the myogenic program through transient induction of Pax3 or Pax7 during early mesoderm development. This approach allows for the in vitro generation of large quantities of early embryonic skeletal myogenic progenitors. Transplantation of these cells into dystrophic mice results in myofiber and satellite cell engraftment that is accompanied by improvement in muscle force generation. This renewal application builds on the advances we have brought to the field over the past 5 years and evolves from these into 3 areas: i) understanding the molecular regulation of the embryonic myogenic program by Pax3, ii) investigating the long-term regenerative capacity of human pluripotent donor-derived satellite cells as well as their transcriptional profiling in comparison to in vitro generated myogenic progenitors, and iii) using myotubes from patient-specific pluripotent cells to biochemically and functionally model DMD/BMD pathology and its reversal by therapeutic delivery of dystrophin mini-genes.
This proposal takes advantage of the ability of pluripotent stem cells to uniquely produce differentiated somatic cell types in large quantities. This allows the study of embryonic developmental pathways using biochemical methods, the generation of specific cell populations for regenerative medicine, and the modeling of disease and genetic correction in vitro. Although studying muscle development using pluripotent cell systems has historically been difficult or impossible because of the inefficiency with which pluripotent cells undergo skeletal muscle differentiation, we have developed methods, based on the conditional expression of myogenic master regulators, Pax3 and Pax7, to induce the muscle program in early mesoderm, and thus derive embryonic muscle progenitors with great efficiency. Our investigations will focus on 1) understanding the mechanism by which Pax3 regulates embryonic myogenesis; 2) investigating the regenerative potential of donor-derived satellite cells and their molecular signature; 3) in vitro disease modeling of DMD/BMD through the use of disease-specific iPS cell-derived myotubes.
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