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

描述(申请人提供)：已经提出了许多使用细胞移植来恢复部分肌肉功能来限制肌肉疾病进展的策略，虽然新鲜分离的供体细胞移植已经成功，但这种方法在临床应用的前景受到了阻碍，因为无法在不失去植入潜力的情况下扩大所需的供体细胞。为了寻找合适的可用于肌肉再生的干细胞来源，我们开始非贴壁培养肌球细胞。这种非传统培养方法背后的原理是，三维细胞-细胞相互作用将提供一个类似于利基的环境，以帮助维持细胞处于更原始的状态。肌球的初步鉴定表明，这些细胞是间质细胞，而不是肌肉细胞，因为它们似乎不表达肌源性标记(MyoD和Pax7)，但它们能够整合到受损肌肉中，并分化为表达MyoD和Pax7的细胞，表明它们的起源是肌源性的。我们的最新数据表明，肌球实际上由两个细胞群组成，一个是间充质来源，表达PDGFFRα，另一个是肌源性细胞，表达MyoD和α7整合素。这项研究的目的是确定肌球培养物是否可以用于在潜在的体外基因治疗后用于移植的原始肌源性细胞的繁殖。具体目标1将决定肌球内培养的肌源性细胞的存活时间和扩增潜力。Aim 1.1将使用YFP-MyoD和ZsGreen-Pax7谱系追踪小鼠随着时间的推移追踪肌球中肌源性细胞的存在，Aim 1.2将确定肌球内的间质细胞是否需要维持肌源性细胞在原始状态。这将通过培养从MyoD和Pax7血统追踪小鼠中分离出来的肌球细胞，分离这些细胞中的间质和肌源性成分，然后监测它们随着时间的推移而生长。在两个AIMS 1.1和1.2中，细胞增殖标记(Violet CellTrace)将与血统追踪小鼠一起使用，以跟踪间充质细胞和肌源性细胞的增殖以及它们随着时间的推移在肌球内彼此之间的关系。具体目标2将确定来自肌球培养的细胞是否有足够的潜力再生受损的肌肉。在这里，我们将比较新鲜卫星细胞与注射到NOD/RAG缺失mdx5Cv小鼠TA肌肉中的肌源性和混合性(间质和肌源性)肌球细胞部分的植入潜力。供体细胞将从ZsGreen-Pax7小鼠中分离出来，用绿色荧光蛋白标记(使用慢病毒载体)，然后分成肌源性(ZsGreen+，PDGFRα-)和间质(ZsGreen-，PDGFRα+)组分。植入将由单独的RFP和结合Pax7和dystrophin的RFP的表达决定。这项研究的重要性在于，肌球形成背后的概念可能会导致分离和维护原始肌肉干细胞的替代方法，这对未来细胞疗法的成功至关重要。

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.