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

肺部疾病至少是全球十大死亡原因中的四个。在美国 仅据估计，就有1100万人被诊断患有慢性阻塞性肺病，24 100万人有肺功能受损的证据。可以想象，肺干细胞的功能是 严重影响肺部疾病，表现出受损或耗尽的细胞。但受限 关于内源性肺干细胞的身份和特征的信息阻碍了对 它们在肺部疾病机制中的作用以及它们潜在的有益用途。我们的长期目标是 阐明干细胞在肺稳态和肺部疾病中的作用。为了实现这一目标，我们确定了 小鼠肺干细胞群，称为支气管肺泡干细胞（BASC）。这一举措将考验 假设BASC是维持体内细支气管和肺泡细胞稳态的干细胞。 我们证明，BASCs可以自我更新，并产生细支气管和肺泡肺细胞谱系， 文化我们还发现，BASCs增殖响应细支气管和肺泡细胞损伤在体内。 利用细胞移植和谱系追踪，我们将测试BASCs在体内的分化潜力。第一、 我们将开发出将基因标记的BASCs移植到受体成年小鼠、胎儿小鼠和成年小鼠的肺中的方法。 肺外植体和鸡胚以评估其效力。第二，我们将产生基因敲入小鼠， 在BASC中表达位点特异性重组酶。这些小鼠将与重组酶报告基因等位基因一起使用， 永久标记内源性BASCs并追踪其在肺损伤后的命运。最后，我们将评估效果 细支气管和肺泡肺损伤对BASC功能的影响，包括自我更新，分化和基因 表达使用萘和博来霉素处理。完成拟议的研究将建立一个 肺干细胞的功能测定。这项工作还将阐明肺部疾病如何影响肺功能， 肺干细胞这些进展是发展基于干细胞的 可以预防或减轻肺部疾病的治疗方法。BASC的识别已经有了一个 对肺生物学和肺癌研究方法的影响。通过本书中描述的研究， 建议，它将有可能创造所需的工具，努力对临床产生积极的影响， 肺疾病患者的结局。 相关性声明：肺细胞受损或耗竭是许多肺部疾病的标志， 包括囊性纤维化和肺气肿。肺干细胞可能有助于替代受损的肺细胞， 患者这项拟议中的工作将创造出测试动物肺干细胞潜力所需的工具， 使得确定基于肺干细胞的治疗的可行性成为可能。

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. Relevance statement: Injured or depleted lung cells are the hallmark of numerous pulmonary diseases, including cystic fibrosis and emphysema. Lung stem cells might be useful in replacing damaged lung cells in patients. The proposed work will create the tools needed to test the potential of lung stem cells in animals, making it possible to determine the feasibility of lung stem cell based therapy.