Differentiating Nkx2.1-positive lung epithelial progenitors from ES cells

描述(由申请人提供)：人类诱导多能干细胞(IPS)的发现为产生患者特有的分化细胞类型带来了前所未有的机会。尽管目前正在生产来自肺部疾病患者的诱导多能干细胞系，但阻碍使用这些细胞的人类肺部疾病模型发展的主要障碍是我们无法有效地将多能干细胞转化为肺细胞。这项提议旨在开发高效且可重复性的方案，从多能干细胞中产生肺上皮祖细胞。我们从小鼠模型系统开始，以便利用易于操作和培养的小鼠干细胞。小鼠分化平台将允许快速、高通量筛选和机器人细胞成像从多潜能细胞向肺分化。我们选择专注于从MES细胞产生Nkx2.1阳性的肺祖细胞，因为Nkx2.1是已知的最早表达于整个肺原基的转录因子。它标志着早期的肺祖细胞群体，随后产生了成人肺的所有成熟上皮细胞类型。我们将使用两种互补的策略来生产Nkx2.1阳性细胞和更坚定的Nkx2.1/Sox2气道祖细胞。第一种策略依赖于多能细胞首先分化为内胚层，然后分化为前内胚层，然后分化为Nkx2.1阳性的肺祖细胞和Nkx2.1/Sox2气道祖细胞。在第二个策略中，我们将依靠无偏见的高通量化学筛选来确定促进肺祖细胞分化的新途径。最后，Nkx2.1祖细胞 在上述两个平台中产生的干细胞将接受功能分析，测试这些前体细胞是否可以使用新的体内重建试验和众所周知的气液界面培养分化为真正的呼吸道上皮细胞。虽然这些小鼠研究将是肺器官发生的有用模型，但我们也表明，促进MES细胞分化的因素也会导致人类iPS细胞形成肺祖细胞。因此，我们将利用小鼠系统的效率来确定使我们能够 从肺部疾病特异的人iPS细胞中获得呼吸道上皮细胞。这一原则证明将为从多能干细胞制造任何类型的肺细胞奠定基础，并允许在实验室中对患者特定的肺部疾病进行建模的前所未有的能力。

DESCRIPTION (provided by applicant): The discovery of human induced pluripotent stem (iPS) cells has resulted in an unprecedented opportunity to produce patient-specific differentiated cell types. Although induced-pluripotent stem cell lines from patients with lung disease are currently being produced, the major obstacle preventing the development of human lung disease models using these cells is our inability to efficiently convert pluripotent stem cell into lung cells. This proposal aims to develop efficient and reproducible protocols to produce lung epithelial progenitor cells from pluripotent stem cells. We start with the murine model system in order to capitalize on the easy handling and culture of mouse stem cells. The murine platform for differentiation will permit rapid, high throughput screening and robotic cell imaging of pulmonary differentiation from pluripotent cells. We elected to focus on the production of Nkx2.1-positive lung progenitor cells from mES cells because Nkx2.1 is the earliest known transcription factor that is expressed throughout the lung primordium. It marks an early lung progenitor population that subsequently gives rise to all the mature epithelial cell types of the adult lung. We will use two complementary strategies to produce Nkx2.1-positive cells and more committed Nkx2.1+/Sox2+ airway progenitor cells. The first strategy relies on the stepwise differentiation of pluripotent cells first into endoderm, then into anterior endoderm, and subsequently into Nkx2.1-positive lung progenitor cells and Nkx2.1+/Sox2+ airway progenitors. In the second strategy, we will rely on an unbiased high throughput chemical screen to identify novel pathways that promote lung progenitor cell differentiation. Finally, Nkx2.1 progenitors cells produced in both of the above platforms will be subjected to functional analysis by testing whether these progenitors can differentiate into bona fide airway epithelium using a novel in vivo reconstitution assay and well described air-liquid interface culture. Although these murine studies will be useful models of lung organogenesis, we have also shown that factors that promote the differentiation of mES cells also cause human iPS cells to form lung progenitors. Thus, we will exploit the efficiency of the murine system to identify factors that will allow us to make airway epithelium from lung disease specific human iPS cells. This proof of principle will set the stage to make any lung cell type from a pluripotent stem cell and allow the unprecedented ability to model patient specific human lung diseases in the laboratory.