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）的发现为生产患者特异性分化细胞类型带来了前所未有的机会。虽然目前正在生产来自肺病患者的诱导多能干细胞系，但阻止使用这些细胞开发人类肺病模型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。该提案旨在开发有效且可重复的方案，以从多能干细胞产生肺上皮祖细胞。我们从小鼠模型系统开始，以利用小鼠干细胞的易于处理和培养。用于分化的鼠平台将允许从多能细胞进行肺分化的快速、高通量筛选和机器人细胞成像。我们选择专注于从mES细胞产生Nkx2.1阳性肺祖细胞，因为Nkx2.1是已知最早的在整个肺原基中表达的转录因子。它标志着早期肺祖细胞群，随后产生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产生Nkx2.1阳性细胞和更多的定向Nkx2.1+/Sox 2+气道祖细胞。第一种策略依赖于多能细胞首先逐步分化成内胚层，然后分化成前内胚层，随后分化成Nkx2.1阳性肺祖细胞和Nkx2.1+/Sox 2+气道祖细胞。在第二种策略中，我们将依靠无偏的高通量化学筛选来鉴定促进肺祖细胞分化的新途径。最后，Nkx2.1祖细胞 通过使用新的体内重建测定和充分描述的气-液界面培养来测试这些祖细胞是否可以分化成真正的气道上皮，对在上述两个平台中产生的细胞进行功能分析。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iPS细胞形成肺祖细胞。因此，我们将利用小鼠系统的效率来鉴定使我们能够 从肺病特异性人iPS细胞中制备气道上皮。这一原理证明将为从多能干细胞中制造任何肺细胞类型奠定基础，并允许在实验室中建立患者特异性人类肺部疾病模型的前所未有的能力。

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.