Generation of human alveolar epithelial type I cells from pluripotent stem cells

从多能干细胞产生人肺泡上皮I型细胞

• 批准号: 10528431
• 负责人: Claire Burgess
• 金额: $ 4.67万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10528431
• 关键词: Acute Respiratory Distress Syndrome; Adult; Alveolar; Alveolus; Area; Automobile Driving; Biological Models; Biology; Blood capillaries; CRISPR/Cas technology; Cell Differentiation process; Cell Line; Cell Separation; Cell model; Cells; Chronic Obstructive Pulmonary Disease; Cues; Data; Derivation procedure; Development; Development Plans; Diffusion; Disease; Distal; Epithelial; Epithelial Cells; Exposure to; Fibroblast Growth Factor; Future; Gases; Generations; Genes; Genetic Transcription; Goals; Homeostasis; Human; In Vitro; Injury; Knock-in; Lung; Measures; Methods; Modeling; Molecular; Morphology; Mus; Oxygen; Pathway interactions; Play; Pluripotent Stem Cells; Population; Proteins; Protocols documentation; Publishing; Pulmonary Fibrosis; Recovery; Reporter; Research; Role; Signal Pathway; Signal Transduction; Smoking; Source; Stretching; Structure; Surface; System; Testing; Time; Toxin; Type I Epithelial Receptor Cell; Type II Epithelial Receptor Cell; airway epithelium; alveolar epithelium; base; cell type; design; directed differentiation; drug development; histological studies; human model; improved; in vitro Model; induced pluripotent stem cell; inhibitor; lung development; mechanical signal; molecular phenotype; mouse model; novel; progenitor; protein expression; regenerative therapy; response; smoke inhalation; stem cell model; stem cells; surfactant; tool; transcriptomics

Abstract: The alveolus, where gas exchange takes place in the lung, is composed of two epithelial cell types. Alveolar epithelial type II cells (AEC2s) are cuboidal and produce surfactant while the type I cells (AEC1s) are uniquely flattened in order to allow for the diffusion of oxygen into the capillaries. The fragile structure of AEC1s has made them particularly challenging to study and isolate. As a result, there are no established models for these cells. Little is known about the development of human AEC1s, however lineage tracing studies have shown that in mice, AEC1s originate from an NKX2-1+ progenitor during development and can be derived from adult AEC2 cells during homeostasis and after injury. While many studies have been published on the mechanisms driving AEC1 differentiation, the results have been varied, and in some cases contradictory. Even so, research so far has mostly been performed in mice as there has not been an applicable human model in which to perform these studies. The recent progress in the directed differentiation of induced pluripotent stem cells (iPSC) into different lung epithelial lineages provides an opportunity to study the effects of different signaling pathways on human AEC1 differentiation. We hypothesize that the differentiation of human AEC2s into AEC1s is regulated by a combination of identifiable signaling mechanisms, including activation of the Hippo signaling effector protein YAP, and that this differentiation can be recapitulated in our iPSC in vitro model system, allowing for a source of human AEC1s for future use in basic studies, drug development, or regenerative therapies. In order to test this hypothesis, we will create a novel pluripotent stem cell-based, in vitro fluorescent model to enable the directed differentiation, tracking, and purification of human putative AEC1s. This will be a bi-fluorescent reporter iPSC line with NKX2-1GFP to enable the tracking and purification of lung progenitor cells, as well as AGERtdTomato as a marker of AEC1s. Using this reporter cell line, we will be able to interrogate different signaling mechanisms to determine their role in human AEC1 differentiation. We have preliminary data suggesting that activation of the Hippo effector protein, YAP, is likely to play a role in AEC1 differentiation and will interrogate this pathway further. In addition, we will examine other signaling pathways that, based on murine models, we hypothesize may be involved in regulating human AEC1 differentiation, including BMP, TGF, Wnt, and FGF. This iPSC derived model of human AEC1s can be used to further our understanding of AEC1 biology, as well as their involvement in diseases that disrupt the alveolar epithelium, such as pulmonary fibrosis and chronic obstructive pulmonary disease.

摘要： 肺泡是肺中进行气体交换的地方，由两种上皮细胞组成。肺泡 上皮II型细胞（AEC 2）是立方形的，产生表面活性剂，而I型细胞（AEC 1）是独特的 使其变平以允许氧气扩散到毛细血管中。AEC 1的脆弱结构使得 他们特别具有挑战性的研究和隔离。因此，没有建立这些细胞的模型。 关于人类AEC 1的发育知之甚少，然而谱系追踪研究表明， 在小鼠中，AEC 1在发育期间起源于NKX 2 -1+祖细胞，并且可以衍生自成年AEC 2 细胞在体内平衡和损伤后。虽然已经发表了许多关于驱动机制的研究， AEC 1分化，结果各不相同，在某些情况下相互矛盾。即便如此，迄今为止的研究 大多数在小鼠中进行，因为还没有适用的人类模型来进行这些 问题研究本文综述了诱导多能干细胞定向分化为不同分化潜能干细胞的研究进展。 肺上皮谱系提供了一个机会，研究不同的信号通路对人类的影响， AEC 1分化。我们假设人类AEC 2s向AEC 1 s的分化是由 可识别的信号传导机制的组合，包括Hippo信号传导效应物的激活 蛋白雅普，并且这种分化可以在我们的iPSC体外模型系统中重现， 用于基础研究、药物开发或再生的人类AEC 1来源 治疗为了验证这一假设，我们将创建一个新的多能干细胞为基础的，在体外荧光 模型，使定向分化，跟踪和纯化的人推定的AEC 1。这将是一 具有NKX 2 -1GFP的双荧光报告基因iPSC系能够追踪和纯化肺祖细胞， 以及AGERtdTomato作为AEC 1的标记。使用这种报告细胞系，我们将能够询问不同的 信号传导机制，以确定其在人AEC 1分化中的作用。我们有初步数据 提示Hippo效应蛋白雅普的激活可能在AEC 1分化中起作用， 将进一步研究这条路径此外，我们将研究其他信号通路，基于小鼠 模型，我们假设可能参与调节人AEC 1分化，包括BMP，TGF β，Wnt， 和FGF。这种iPSC衍生的人类AEC 1模型可用于进一步理解AEC 1生物学， 以及它们参与破坏肺泡上皮的疾病，如肺纤维化， 慢性阻塞性肺疾病