? Human embryonic stem (hES) cells may provide an unlimited source of cells for therapeutic applications in degenerative diseases and cancer. One challenge is to harness the huge differentiation potential of hES cells towards selective derivation of different cell types of interest. We isolated mesenchymal precursors from differentiating hES cells cocultured with mouse OP9 stromal cells, by FACS sorting for the human mesenchymal marker CD73. By applying standard mesenchymal stem cell differentiation protocols, we succeeded in obtaining osteoblasts, adipocytes, chondrocytes, and skeletal myocytes from these precursors. Nevertheless, to ensure that these cells are suitable for future therapeutic applications, we developed a stroma-free differentiation method and demonstrated that hES cells could again be differentiated into CD73+ mesenchymal progenitors. In addition, we set up more specific culture conditions for the efficient generation of mature skeletal myocytes from the CD73+ precursors. Based on our preliminary results, showing that purified hES-derived skeletal myocytes can fuse to form myotubes, this proposal aims to study the function of hES-derived skeletal myocytes in vivo by transplantation into immunodeficient mice and in a mouse model of muscular dystrophy. The advantages associated with hES-derived skeletal muscle cells are their high proliferative capacity, expression of skeletal muscle commitment/stem cell markers Pax3 and Pax7, and the potentially enhanced migratory ability.
The specific aims of this project are: 1) to characterize the in vitro differentiation of hES cells into skeletal myocytes, by optimizing the culture conditions for the selective differentiation of CD73+ precursors into skeletal myocytes, and by identifying putative soluble factor(s) produced by CD73- cells that lead to the enhancement of skeletal muscle differentiation, and 2) to assess the functional capacity of hES-derived skeletal myoblast progeny in vivo after artificial injury in a mouse model. First, we will explore muscle regeneration after cardiotoxin-induced lesions in immunodeficient mice (SCID Beige), and then translate the results into a mouse model of muscular dystrophy (mdx mice). Of note, isolation of factor(s) essential for muscle differentiation from this system could help to obtain better transplantable myocytes from hES, as well as adult stem cells, with consequently important clinical relevance. ? ? ?
