Defining a myofibroblast/pericyte as a mesenchymal progenitor cell

描述（由申请人提供）：成体间充质祖细胞在修复医学中具有巨大的应用潜力。骨髓抽吸或吸脂收集的容易获取和分离使这些细胞成为研究分化为各种成体间充质组织用于再生目的的主要目标。如果祖细胞的这种来源要实现广泛的临床应用，祖细胞及其后代的真实身份需要更精确地定义，并且需要了解它们的作用调节。实现这些目标的障碍是无法鉴定和纯化这些细胞，以及缺乏可用于确认祖细胞能够达到终分化表型的状态和功能的体内标记物。我们接受这些挑战，并提出一个假设，将测试周细胞/肌成纤维细胞是否具有间充质祖细胞的特征。我们假设平滑肌a-肌动蛋白（SMA）表达细胞是成人骨组织中骨祖细胞的主要来源。为了确定肌成纤维细胞/周细胞群体，我们将使用先前开发的转基因小鼠，其中周细胞通过SMA启动子- gfp转基因表达（SMAGFP）进行鉴定。这些细胞的分化能力，将使用转基因小鼠进行测试，其中成骨细胞，脂肪细胞或软骨细胞特异性启动子驱动GFP报告基因的表达。这些转基因在谱系的成熟阶段激活，通过组合互补色，我们可以测试分离的SMA+细胞从祖细胞发展到完全成熟状态的能力。此外，我们将利用再生模型和体内新骨形成完成谱系追踪实验。利用SMA- creert2小鼠与Rosa26（cag-tdTomato）报告系杂交，在组织修复模型中包括骨折愈合和BMP2诱导异位骨形成诱导成骨潜能，我们将评估SMA表达细胞的祖细胞能力。在第三个目标中，我们提出确定间充质祖细胞向成熟谱系的分化点。基于谱系定向GFP标记的表达，将分离出特定阶段的间充质祖细胞群体并测试其分化能力。定义分化的控制将允许理解的机制，直接间充质干细胞的骨祖细胞谱系。这些研究的完成将有助于更好地了解间充质祖细胞的特性和表型，并提供它们在损伤和骨折期间骨愈合中的积极作用的信息。

DESCRIPTION (provided by applicant): Adult mesenchymal progenitor cells have enormous potential for use in reparative medicine. The easy access and isolation of bone marrow aspirate or liposuction collection have made these cells a prime target for studies of differentiation into various adult mesenchymal tissues for regenerative purposes. If this source of progenitor cells is to achieve broad clinical utility, the true identity of the progenitors and its progeny need to be more precisely defined and modulation of their commitment needs to be understood. Obstacles to these goals are the inability to identify and purify these cells, and the lack of in vivo markers that can be used to confirm that progenitor cells can attain the state and functionality of terminally differentiated phenotypes. We accept these challenges and propose a hypothesis that will test if pericyte/myofibroblasts have the characteristics of mesenchymal progenitors. We hypothesize that smooth muscle a-actin (SMA) expressing cells are the major source of osteoprogenitors in adult bone tissue. To define a population of myofibroblasts/pericytes we will utilize previously developed transgenic mice in which pericytes are identified by SMA promoter-GFP transgene expression (SMAGFP). The differentiation ability of these cells, will be tested using transgenic mice in which osteoblast, adipocyte or chondrocyte specific promoters drive GFP reporter expression. These transgenes activate at mature stages of the lineage and, by combining complementary colors, we can test for the ability of isolated SMA+ cells to progress from a progenitor to fully mature state. In addition we will complete lineage tracing experiments using regeneration models and new bone formation in vivo. Utilizing an SMA-CreERT2 mouse crossed with a Rosa26(cag-tdTomato) reporter line in models of tissue repair that include fracture healing and induction of osteogenic potential using ectopic bone formation induced by BMP2, we will evaluate the progenitor ability of SMA expressing cells. In the third aim we propose to determine the divergence point of mesenchymal progenitor cell into mature lineages. Based on expression of lineage-directed GFP markers, stage specific populations of mesenchymal progenitors will be isolated and their differentiation ability will be tested. Defining the control of differentiation will allow for the understanding of the mechanisms that direct mesenchymal stem cells down the osteoprogenitor lineage. Completion of the proposed studies will provide a better understanding of the identity and the phenotype of the mesenchymal progenitor cells as well as provide information on their active role in bone healing during injury and bone fractures. PUBLIC HEALTH RELEVANCE: The aims of this application are focused to better identification and characterization of adult mesenchymal progenitor cells. Our study will evaluate their ability to differentiate into mature mesenchymal lineages in vitro and in vivo and determine the diverging point at which mesenchymal progenitor cells commit to mature lineages and factors that can modulate this commitment. The knowledge developed in this grant will provide the foundation for future studies aimed at use of mesenchymal progenitor cells as therapeutic tools for osteoporosis and genetic disorders of bone.