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

区分 Nkx2.1 阳性肺上皮祖细胞与 ES 细胞

• 批准号: 8301868
• 负责人: JAYARAJ RAJAGOPAL
• 金额: $ 24.31万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8301868
• 关键词: Activities of Daily Living; Adult; Air; Anterior; Biological Assay; Biological Models; Cell Differentiation process; Cell Line; Cells; Chemicals; Commit; Data; Development; Disease model; Embryo; Endoderm; Endoderm Cell; Epithelial; Epithelial Cells; Event; Goals; Growth; Growth Factor; Human; Human Development; Image Analysis; In Vitro; Knowledge; Laboratories; Learning; Liquid substance; Literature; Lung; Lung diseases; Modeling; Multipotent Stem Cells; Mus; Organogenesis; Pathway interactions; Patients; Pluripotent Stem Cells; Population; Primitive foregut structure; Primordium; Production; Protocols documentation; Robotics; Role; Screening procedure; Signal Pathway; Signal Transduction; Staging; Stem cells; Structure of respiratory epithelium; System; Testing; airway epithelium; base; cell type; cellular imaging; embryonic stem cell; high throughput screening; in vitro Model; in vivo; induced pluripotent stem cell; lung development; novel; prevent; progenitor; reconstitution; regenerative therapy; research study; stem; stem cell differentiation; transcription factor

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. PUBLIC HEALTH RELEVANCE: The discovery of human induced-pluripotent stem (iPS) cells represents an unprecedented opportunity to produce patient-specific cells types that can be used to generate models of human lung disease. The major obstacle preventing the actual development of regenerative therapies using these cells is our inability to efficiently convert them into lung cells. This proposal aims to develop efficient and reproducible protocols to produce lung progenitor cells from stem cells. We use mouse embryonic stem cells to develop our protocols since they are easily cultured, and then show that our findings are readily transferred to human iPS cells.

描述（由申请人提供）：人类诱导多能干（iPS）细胞的发现为生产患者特异性分化细胞类型提供了前所未有的机会。尽管目前正在生产来自肺病患者的诱导多能干细胞系，但阻碍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，以便利用小鼠干细胞的轻松处理和培养。小鼠分化平台将允许对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 细胞中产生 Nkx2.1 阳性肺祖细胞，因为 Nkx2.1 是已知最早的转录因子，在整个肺原基中表达。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 阳性细胞和更定向的 Nkx2.1+/Sox2+ 气道祖细胞。第一种策略依赖于多能细胞首先逐步分化为内胚层，然后分化为前内胚层，然后分化为 Nkx2.1 阳性肺祖细胞和 Nkx2.1+/Sox2+ 气道祖细胞。在第二种策略中，我们将依靠公正的高通量化学筛选来识别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细胞将通过使用新颖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为真正的气道上皮来进行功能分析。尽管这些小鼠研究将成为肺器官发生的有用模型，但我们还表明，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 利用肺部疾病特异性人类 iPS 细胞制造气道上皮。这一原理证明将为利用多能干细胞制造任何类型的肺细胞奠定基础，并赋予在实验室中模拟患者特定人类肺部疾病的前所未有的能力。 公共健康相关性：人类诱导多能干 (iPS) 细胞的发现代表了生产患者特异性细胞类型的前所未有的机会，这些细胞类型可用于生成人类肺部疾病模型。阻碍使用这些细胞进行再生疗法实际开发的主要障碍是我们无法有效地将它们转化为肺细胞。该提案旨在开发有效且可重复的方案，以从干细胞产生肺祖细胞。我们使用小鼠胚胎干细胞来开发我们的方案，因为它们易于培养，然后表明我们的发现很容易转移到人类 iPS 细胞中。