Defining the genetic program of primordial lung progenitors

描述(由申请人提供)：影响肺上皮的疾病不容易治疗，在世界范围内导致显著的发病率和死亡率。特化干细胞具有自我更新或产生分化的功能后代的潜力，已被认为是许多器官组织稳态的关键组成部分，包括肺。使用干细胞治疗肺部疾病的不同可能方法包括促进内源性干细胞分化或体外定向干细胞向肺系分化，然后进行细胞移植。这两种方法都需要了解肺干细胞或祖细胞的身份和分化途径，并对其进行充分的表征。胚胎干细胞(ES)在过去的10-15年中出现，因为它们可以在体外通过几个确定的阶段来概括哺乳动物的发育，因此成为开发基于细胞的治疗的一个很有前途的平台。此外，诱导多能干细胞(IPS)提供了一种诱人的替代人类ES细胞的方法。IPS细胞很容易衍生，不存在伦理问题，并提供了针对患者的特定疗法的可能性。然而，推测处于发育中的肺祖细胞尚未确定，这是限制ES/iPS细胞用于肺部疾病治疗的主要障碍。这个项目的总体目标是确定肺原基的多潜能上皮祖细胞的遗传程序。这代表着朝着我们为影响肺上皮的疾病开发基于细胞的疗法的长期目标迈出了第一步。由于转录因子TITF1是已知最早的肺发育标志，我们创造了一种新的遗传工具，TITF1-GFP敲入小鼠。这只小鼠将被用来鉴定肺和甲状腺的祖细胞，既有TITF1阳性的，也有来自内胚层的。使用这个工具，我们将能够在目标1中基于GFP荧光从肺原基中分离细胞，通过DNA微阵列确定它们的遗传程序，并研究在肺发育中重要的基因的表观遗传学。我们将在目标2中使用TITF1-GFP ES和iPS细胞株在体外研究成纤维细胞生长因子和Wnt信号在肺规范中的作用。已知在体内确定的内胚层中，成纤维细胞生长因子和Wnt配体可以诱导肺的命运，我们将在体外评估它们是否具有类似的功能。在目标3中，我们将使用新的和已建立的系统，如生物人工肺和气液界面培养，分别测试体外来源的肺祖细胞的功能。我们预计，我们研究的结果将是确定从ES/iPS细胞中获得肺祖细胞的最佳方案，以用于新的肺部疾病治疗。

DESCRIPTION (provided by applicant): Diseases affecting the lung's epithelium are not easily treatable and result in significant morbidity and mortality worldwide. Specialized stem cells with the potential to self-renew or give rise to differentiated, functional progeny have been proposed as a critical component of tissue homeostasis for many organs, including the lung. Different potential approaches for the use of stem cells for lung disease treatment include enhancement of endogenous stem cell differentiation or in vitro directed differentiation of stem cells to lung lineages followed by cell transplantation. Both approaches require that the identity and pathways of differentiation of lung stem or progenitor cells be known and well characterized. Embryonic stem (ES) cells have emerged in the last 10-15 years as a promising platform for the development of cell-based therapies, since they can transit through several defined stages in vitro to recapitulate mammalian development. In addition, induced pluripotent stem (iPS) cells offer an attractive alternative to human ES cells. iPS cells are easy to derive, are not fraught with ethical issues and offer the possibility of patient-specific therapies. Nevertheless, the presumptive lung progenitor cells in development have not yet been identified, and this represents a major hurdle limiting the use of ES/iPS cells for lung disease therapies. The overall objective of this project is to define the genetic program of the multipotent epithelial progenitors of the lung primordium. This represents the first step towards our long-term goal of developing cell-based therapies for diseases affecting the lung epithelium. Since the transcription factor Titf1 is the earliest known marker of lung development, we created a new genetic tool, the Titf1-GFP knock-in mouse. This mouse will be used to characterize the progenitor cells of the lungs and the thyroid, both Titf1-positive and of endodermal origin. Using this tool, we will be able in Aim 1 to isolate cells from lung primordium based on GFP fluorescence, define their genetic program by means of DNA microarrays and study the epigenetics of genes important in lung development. We will proceed in Aim 2 to examine the role of FGF and Wnt signaling in lung specification in vitro using the Titf1-GFP ES and iPS cell lines. FGF and Wnt ligands are known to induce lung fate within definitive endoderm in vivo and we will assess whether they have a similar function in vitro. In Aim 3, we will test the functionality of the in vitro derived lung progenitors using novel and established systems, such as a bioartificial lung and air-liquid interface culture respectively. We envision that the outcome of our studies will be to define the optimal protocol for derivation of lung progenitor cells from ES/iPS cells to be used in novel lung disease therapies.