Defining a myofibroblast/pericyte as a mesenchymal progenitor cell

描述(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。易于获取和分离的骨髓抽吸或吸脂收集使这些细胞成为分化为各种成体间充质组织以供再生的研究的主要靶点。如果祖细胞的这一来源要实现广泛的临床应用，需要更准确地定义祖细胞及其后代的真实身份，并需要了解他们的承诺的调节。这些目标的障碍是无法鉴定和纯化这些细胞，以及缺乏体内标记物来证实祖细胞可以达到终末分化表型的状态和功能。我们接受了这些挑战，并提出了一个假设，将检验周细胞/肌成纤维细胞是否具有间充质祖细胞的特征。我们推测，表达平滑肌α-肌动蛋白(SMA)的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/周细胞的群体，我们将利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)来鉴定。这些细胞的分化能力将在转基因小鼠中进行测试，在转基因小鼠中，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，通过结合互补颜色，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，我们还将利用体内再生模型和新骨形成完成谱系追踪实验。利用SMA-CreERT2小鼠与ROSA26(CAG-tdTomato)报告系杂交的组织修复模型，包括骨折愈合和BMP2诱导的异位成骨诱导成骨潜能，我们将评估SMA表达细胞的祖细胞能力。在第三个目标中，我们建议确定间充质祖细胞向成熟谱系的分歧点。基于谱系导向的GFP标志物的表达，将分离出不同阶段的间充质祖细胞群体，并检测其分化能力。确定分化的控制将有助于理解引导间充质干细胞向骨祖细胞方向发展的机制。这些研究的完成将有助于更好地了解间充质前体细胞的特性和表型，并为其在损伤和骨折愈合过程中的积极作用提供信息。 公共卫生相关性：这项应用的目的是为了更好地鉴定和表征成人间充质祖细胞。我们的研究将评估它们在体外和体内分化为成熟间充质系的能力，并确定间充质前体细胞向成熟系分化的分歧点以及调节这一分化的因素。这笔赠款开发的知识将为未来旨在将间充质祖细胞用作骨质疏松症和遗传性疾病的治疗工具的研究奠定基础。

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.