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

肌球可以用于分离和维持培养物中的卫星细胞吗

基本信息

批准号：
8820393
负责人：
KAREN A WESTERMAN
金额：
$ 8.54万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-17 至 2015-03-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：已经提出了使用细胞移植来恢复一些肌肉功能以限制肌肉疾病进展的许多策略，并且虽然新鲜分离的供体细胞的移植已经成功，但是这种方法用于临床应用的前景受到了阻碍，因为无法在不失去其移植潜力的情况下扩增所需的供体细胞。为了寻找一种合适的干细胞来源，可以用来再生肌肉，我们开始培养肌肉来源的细胞非贴壁作为肌球。这种非常规培养方法背后的基本原理是，三维细胞-细胞相互作用将提供一个利基样环境，以帮助维持细胞处于更原始的状态。肌球的初步表征表明，这些细胞是间质细胞，而不是肌细胞，因为它们似乎不表达肌源性标记物（MyoD和Pax 7），但它们能够掺入受损肌肉并分化成表达MyoD和Pax 7的细胞，表明它们的起源是肌源性的。我们最近的数据表明，实际上，肌球由两个细胞群组成，一个是表达PDGFRα的间充质来源的细胞群，另一个是表达MyoD和α7整联蛋白的肌源性细胞群。本研究的目的是确定肌球培养物是否可以用于繁殖原始肌源性细胞，这些细胞可以在潜在的离体基因治疗治疗后用于移植。特异性目标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。目标1.1将使用YFP-MyoD和ZsGreen-Pax 7谱系追踪小鼠来追踪肌球中肌源性细胞随时间的存在，并且目标1.2将确定是否需要肌球内的间充质细胞来维持肌源性细胞处于原始状态。这将通过培养从MyoD和Pax 7谱系追踪小鼠中分离的肌球细胞来完成，将这些细胞分选为间充质和肌源性部分，然后监测它们随时间的生长。在目标1.1和1.2中，细胞增殖标记物（Violet CellTrace）将与谱系追踪小鼠联合使用，以追踪间充质细胞和肌源性细胞的增殖及其随时间推移在肌球内的相互关系。特异性目标2将确定来自肌球培养物的细胞是否具有足够的再生受损肌肉的潜力。在这里，我们将比较新鲜的卫星细胞的移植潜力，肌源性和混合（间充质和肌源性）肌球细胞部分注射到TA肌肉的NOD/RAG空mdx 5cv小鼠。将从ZsGreen-Pax 7小鼠中分离供体细胞，用RFP标记（使用慢病毒载体），然后分选为肌源性（ZsGreen+，PDGFRα-）和间充质（ZsGreen-，PDGFRα+）组分。移植将通过单独RFP以及Pax 7和肌营养不良蛋白组合RFP的表达来确定。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持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。