Characterization of a Lung Mesenchymal Progenitor Cell

描述（由申请人提供）：在这个修订的R21资助，我们建议进一步表征一个假定的成人肺间充质祖细胞。我们最初使用高速细胞分选策略分离这样的细胞，该策略利用了来自其他位点的典型间充质祖细胞的特性。这些属性包括：1）Sca-1+/lin阴性。表面特征2）外排Hoechst染料（所谓SP细胞）的能力，3）阴性造血标记物CD 45状态，和4）原始间充质基因的表达。另外的表型表明这些细胞与纤维细胞无关，而是显示经典的多能骨髓间充质干细胞（MSC）的特征。这些共有的特征包括相似标志物的表达，分化成脂肪、平滑肌或软骨的能力，以及在选择的体外条件下以未分化状态增殖。因此，我们假设，成人肺含有一个独特的间充质祖细胞，可以分离的基础上染料流出和表面表型。在目标1中，我们将进一步定义这种细胞的表型，并阐明细胞是单能的还是多能的。我们还提出了一组探索性研究，以确定候选的miRNAs，维持细胞在未分化状态。使用流式细胞术，我们将基于细胞大小和选择的表面抗原的表达来破碎细胞，然后测试确定的组分分化成脂肪、平滑肌和软骨的能力。在我们阐明间充质祖细胞表型后，我们将评估单个细胞的分化库。为此，将克隆扩增单个细胞并测试它们沿沿着不同间充质细胞命运分化的能力。在本研究的最后一部分，我们将利用miRNA表达芯片和实时荧光定量PCR技术来评估miRNA在未分化和分化细胞中的表达模式。目的2是集中于检查这种推定的肺间充质祖细胞在体内肺中的植入潜力。为了调查植入，我们将GFP+细胞注射到2个不同的模型中，所述模型涉及近端肺（CC 10-IL-9转基因）或远端肺（博来霉素）中的组织重塑。在这项工作中，我们将评估体外细胞群体扩增对植入的影响。通过这项工作，我们希望为表征干/祖细胞生物学更发达的器官系统的研究类型奠定基础。

DESCRIPTION (provided by applicant): In this revised R21 grant, we propose to further characterize a putative adult lung mesenchmyal progenitor cell. We initially isolated such cells using a high-speed cell sorting strategy taking advantage of properties that typify mesenchymal progenitors from other sites. These properties include: 1) a Sca-1+/lin neg. surface profile 2) an ability to efflux Hoechst dye (so-called SP cells), 3) negative hematopoietic marker CD45 status, and 4) expression of primitive mesenchymal genes. Additional phenotyping indicate these cells are not related to fibrocytes, but rather display features of classical multipotent marrow mesenchymal stem cells (MSCs). These shared features include expression of similar markers, a capacity to differentiate into fat, smooth muscle, or cartilage, and proliferation in an undifferentiated state under select in vitro conditions. We, thus, hypothesize that the adult lung contains a distinct mesenchymal progenitor cell that can be isolated based on dye efflux and surface phenotype. In Aim 1 we will further define the phenotype of this cell, and we will clarify whether cells are unipotent or multipotent. We also propose a set of exploratory studies to identify candiate miRNAs that maintain cells in an undifferentiate state. Using flow cytometry, we will fractionate cells based on cell size and expression of select surface antigens and then test defined fractions for the ability to differentiate into fat, smooth muscle, and cartilage. After we elucidate the mesenchymal progenitor phenotype, we will evaluate the differentiation repertoire of single cells. To do this, individual cells will be clonally expanded and tested for their ability to differentiate along different mesenchymal cell fates. In the last part of this aim, we will use miRNA expression microarrays and real-time PCR to evaluate miRNA expression patterns in undifferentiated and differentiated cells. Aim 2 is focused on examing the engraftment potential of this putative lung mesenchymal progenitor cell in the lung in vivo. To survey engraftment, we will inject GFP+ cells into 2 distinct models that involve tissue remodeling either in the proximal (CC10-IL-9 transgenic) or distal lung (bleomcyin). In this work, we will evaluate the impact of in vitro cell population expansion on engraftment. Through this work, we hope to establish a foundation for the types of studies that characterize organ systems in which stem/progenitor cell biology is more developed.