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

摘要： 肺泡是在肺内进行气体交换的地方，由两种上皮细胞组成。牙槽骨 第二型上皮细胞(AEC2)是立方的，能产生表面活性物质，而I型细胞(AEC1)是独一无二的 为了让氧气扩散到毛细血管内而变得扁平。AEC1的脆弱结构已经制造出来 他们在研究和隔离方面尤其具有挑战性。因此，目前还没有针对这些细胞的既定模型。 人们对人类AEC1的发展知之甚少，但血统追踪研究表明，在 小鼠，AEC1起源于发育期间的NKX2-1+祖细胞，并可源自成年AEC2 动态期和损伤后的细胞。虽然已经发表了许多关于驱动机制的研究 AEC1的分化，结果各不相同，在某些情况下也是矛盾的。即便如此，到目前为止的研究 大多数是在小鼠身上进行的，因为还没有适用的人体模型来执行这些操作 学习。诱导多能干细胞定向分化为不同类型干细胞的研究进展 肺上皮细胞系为研究不同信号通路对人类的影响提供了机会 AEC1分化。我们假设人类AEC2向AEC1的分化受 可识别的信号机制的组合，包括激活河马信号效应器 蛋白质YAP，这种分化可以在我们的IPSC体外模型系统中概括，允许 获取人类AEC1的来源，以供将来用于基础研究、药物开发或再生 治疗。为了验证这一假设，我们将创造一种新的基于多能干细胞的体外荧光 模型，以实现对人类推测的AEC1的定向分化、跟踪和纯化。这将是一个 双荧光报告IPSC与NKX2-1GFP一起实现对肺祖细胞的跟踪和纯化， 以及将AGERtd番茄作为AEC1的标记。使用这个报告细胞系，我们将能够审问不同的 确定它们在人类AEC1分化中的作用的信号机制。我们有初步数据 提示河马效应蛋白YAP的激活可能在AEC1分化和 将进一步审问这条路径。此外，我们将研究其他信号通路，这些信号通路基于小鼠 模型，我们假设可能参与调节人AEC1分化，包括骨形态发生蛋白，转化生长因子，Wnt， 和成纤维细胞生长因子。这种IPSC衍生的人类AEC1模型可以用来加深我们对AEC1生物学的理解， 以及参与破坏肺泡上皮的疾病，如肺纤维化和 慢性阻塞性肺病。

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.