Characterization of a Lung Mesenchymal Progenitor Cell

描述(由申请人提供)：在这项修订的R21资助中，我们建议进一步鉴定一种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种细胞，利用了来自其他部位的典型间充质祖细胞的特性。这些性质包括：1)SCA-1+/LIN阴性。表面轮廓2)Hoechst染料外流能力(所谓的SP细胞)，3)造血标记物CD45阴性状态，4)原始间充质基因表达。更多的表型表明这些细胞与纤维细胞无关，而是表现出经典的多能骨髓间充质干细胞(MSCs)的特征。这些共同的特征包括相似的标志物的表达，分化为脂肪、平滑肌肉或软骨的能力，以及在选定的体外条件下未分化状态下的增殖。因此，我们假设成人肺含有一个独特的间充质祖细胞，可以根据染料外流和表面表型进行分离。在目标1中，我们将进一步定义这种细胞的表型，我们将澄清细胞是单能的还是多能的。我们还提出了一系列探索性研究，以确定维持细胞处于未分化状态的候选miRNAs。使用流式细胞术，我们将根据细胞大小和所选表面抗原的表达对细胞进行分离，然后测试确定的部分向脂肪、平滑肌和软骨分化的能力。在我们阐明间充质祖细胞的表型后，我们将评估单细胞的分化能力。为了做到这一点，将对单个细胞进行克隆扩增，并测试它们沿不同间质细胞命运分化的能力。在这个目标的最后部分，我们将使用miRNA表达微阵列和实时荧光定量PCR来评估未分化和已分化细胞中的miRNA表达模式。目的2研究肺间充质前体细胞在体内的植入潜能。为了观察植入情况，我们将GFP+细胞注射到两种不同的模型中，这两种模型涉及近端(CC10-IL-9转基因)或远端肺(博莱姆细胞蛋白)的组织重塑。在这项工作中，我们将评估体外细胞群扩张对植入的影响。通过这项工作，我们希望为描述干细胞/祖细胞生物学更发达的器官系统的类型的研究奠定基础。

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.