Can myospheres be used to isolate and maintain satellite cells in culture

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DESCRIPTION (provided by applicant): Many strategies to limit progression of muscle disease using cell transplantation to restore some muscle function have been proposed, and while transplantation of freshly isolated donor cells has been successful, the promise of this method for clinical use has been hampered by the inability to expand the donor cells needed without losing their potential to engraft. In an attempt to find a suitable stem cell source that could be used to regenerate muscle, we began to culture muscle derived cells non-adherently as myospheres. The rationale behind this unconventional culturing method was that the 3-dimensional cell-cell interactions would provide a niche-like environment to help maintain cells in a more primitive state. Initial characterization of myospheres indicated that these cells were interstitial cells and not muscle cells because they did not appear to express myogenic markers (MyoD and Pax7), yet they were capable of incorporating into injured muscle and differentiating into cells that express MyoD and Pax7, indicating their origin was myogenic. Our most recent data indicates that in fact, myospheres are composed of two cell populations, one of mesenchymal origin expressing PDGFRα, and a second that is myogenic, expressing MyoD and α7 integrin. The goal of this research is to determine if myosphere cultures can be used to propagate primitive myogenic cells that could be used for transplantation after potential ex-vivo gene therapy treatment. Specific Aim 1 will determine the survival time and expansion potential of myogenic cells cultured within myospheres. Aim 1.1 will use YFP- MyoD and ZsGreen-Pax7 lineage-tracing mice to track the presence of myogenic cells in myospheres over time and Aim 1.2 will determine if the mesenchymal cells within myospheres are needed to maintain the myogenic cells in a primitive state. This will be done by culturing myosphere cells isolated from the MyoD and Pax7 lineage-tracing mice, sorting those cells for the mesenchymal and myogenic fractions, and then monitoring their growth over time. In both aims 1.1 and 1.2 a cell proliferation marker (Violet CellTrace) will be used in conjunction with the lineage-tracing mice to track the proliferation of the mesenchymal and myogenic cells and their relation to one another within myospheres over time. Specific Aim 2 will determine if cells derived from myosphere cultures have adequate potential to regenerate injured muscle. Here we will compare the engraftment potential of fresh satellite cells to myogenic and mixed (both mesenchymal and myogenic) myosphere cell fractions injected into the TA muscle of NOD/RAG null mdx5cv mice. Donor cells will be isolated from ZsGreen-Pax7 mice, labeled with RFP (using a lentiviral vector), and then sorted into myogenic (ZsGreen+, PDGFRα-) and mesenchymal (ZsGreen-, PDGFRα+) fractions. Engraftment will be determined by the expression of RFP alone and RFP in combination Pax7 and dystrophin. The importance of this study is that the concepts behind how myospheres are formed could lead to alternative methods of isolating and maintaining primitive muscle stem cells that are critical for the success of future cell-therapies.