In Vivo and In Vitro Characterization of Bronchio-Alveolar Stem Cells

描述(由申请人提供)： 在全球十大死因中，肺部疾病至少占四个。仅在美国，估计就有1100万人被诊断出患有慢性阻塞性肺疾病，2400万人有肺功能受损的证据。可以想象，肺干细胞功能在表现为细胞受损或耗尽的肺部疾病中受到严重影响。然而，关于内源性肺干细胞的身份和特征的有限信息阻碍了对它们在肺部疾病机制中的作用以及它们潜在的有益用途的了解。我们的长期目标是阐明干细胞在肺内稳态和肺部疾病中的作用。为了实现这一目标，我们鉴定了一组小鼠肺干细胞，命名为细支气管肺泡干细胞(BASCs)。这笔赠款将检验BASCs是在体内维持细支气管和肺泡细胞动态平衡的干细胞的假设。我们证明了BASCs可以在培养中自我更新并分化为细支气管型和肺泡型肺细胞系。我们还发现，在体内，BASCs在细支气管和肺泡细胞损伤后增殖。通过细胞移植和谱系追踪，我们将在体内测试BASCs的分化潜力。首先，我们将开发将基因标记的BASCs移植到受体成年小鼠、胎肺外植体和鸡胚的肺中的方法，以评估它们的效力。其次，我们将在BASCs中产生表达位点特异性重组酶的敲除小鼠。这些小鼠将与重组酶报告等位基因一起用于永久标记内源性BASCs，并跟踪它们在肺损伤后的命运。最后，我们将评估细支气管性肺损伤和肺泡性肺损伤对BASC功能的影响，包括自我更新、分化和基因表达。拟议研究的完成将建立一种肺干细胞的功能分析方法。这项工作还将阐明肺部疾病如何影响肺干细胞的功能。这些进展是基于干细胞的疗法发展的必要前提，可以预防或减轻肺部疾病。BASCs的鉴定已经对肺部生物学和肺癌研究的方式产生了影响。有了这项提案中描述的研究，就有可能创建所需的工具，以努力对肺部疾病患者的临床结果产生积极影响。

DESCRIPTION (provided by applicant): Diseases of the lung contribute to at least four of the top ten causes of death worldwide. In the United States alone, it is estimated that 11 million people are diagnosed with chronic obstructive lung disease and 24 million people have evidence of impaired lung function. It is conceivable that lung stem cell function is critically affected in pulmonary diseases that exhibit damaged or depleted cells. However, limited information about the identity and characteristics of endogenous lung stem cells precludes understanding their role in the mechanisms of lung disease as well as their potential beneficial uses. Our long-term goal is to elucidate the role of stem cells in lung homeostasis and lung disease. Important for this goal, we identified a murine lung stem cell population, named bronchio-alveolar stem cells (BASCs). This grant will test the hypothesis that BASCs are the stem cells that maintain bronchiolar and alveolar cell homeostasis in vivo. We demonstrated that BASCs can self-renew and give rise to bronchiolar and alveolar lung cell lineages in culture. We also showed that BASCs proliferate in response to bronchiolar and alveolar cell damage in vivo. Using cell transplantation and lineage tracing, we will test the differentiation potential of BASCs in vivo. First, we will develop methods to transplant genetically marked BASCs into the lungs of recipient adult mice, fetal lung explants, and chick embryos to assess their potency. Second, we will generate knock-in mice to express site-specific recombinases in BASCs. These mice will be used with recombinase reporter alleles to permanently label endogenous BASCs and track their fate after lung injury. Finally, we will assess the effect of bronchiolar and alveolar lung injury on BASC functions including self-renewal, differentiation, and gene expression using naphthalene and bleomycin treatment. Completion of the proposed studies will establish a functional assay for lung stem cells. This work will also elucidate how lung disease impacts the function of lung stem cells. These advances are a necessary prerequisite to development of stem cell based therapeutics that could prevent or alleviate lung disease. The identification of BASCs has already had an impact on the way lung biology and lung cancer research is approached. With the studies described in this proposal, it will be possible to create the tools needed to work towards a positive impact on the clinical outcome for patients of pulmonary disease.