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，并在全球范围内导致显著的发病率和死亡率。具有自我更新或产生分化的功能性后代的潜能的特化干细胞已被提出作为许多器官（包括肺）的组织稳态的关键组分。使用干细胞治疗肺病的不同潜在方法包括增强内源性干细胞分化或干细胞体外定向分化为肺谱系，然后进行细胞移植。这两种方法都需要肺干细胞或祖细胞的分化的身份和途径是已知的，并充分表征。在过去的10-15年中，胚胎干细胞（ES）已经成为开发基于细胞的疗法的有希望的平台，因为它们可以在体外经过几个确定的阶段以重现哺乳动物的发育。此外，诱导多能干细胞（iPS）提供了一个有吸引力的替代人类ES细胞。iPS细胞易于获得，不存在伦理问题，并提供了患者特异性治疗的可能性。然而，尚未鉴定出开发中的推定肺祖细胞，这代表了限制ES/iPS细胞用于肺部疾病治疗的主要障碍。本项目的总体目标是确定肺原基多能上皮祖细胞的遗传程序。这代表了我们朝着长期目标迈出的第一步，即为影响肺上皮的疾病开发基于细胞的疗法。由于转录因子Titf 1是已知的最早的肺发育标志物，我们创造了一种新的遗传工具，Titf 1-GFP基因敲入小鼠。该小鼠将用于表征肺和甲状腺的祖细胞，Titf 1阳性和内胚层来源。使用该工具，我们将能够在目标1中基于GFP荧光从肺原基分离细胞，通过DNA微阵列定义其遗传程序，并研究在肺发育中重要的基因的表观遗传学。我们将在目的2中继续使用Titf 1-GFP ES和iPS细胞系来检查FGF和Wnt信号传导在体外肺特化中的作用。已知FGF和Wnt配体在体内诱导定形内胚层内的肺命运，并且我们将评估它们在体外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，分别测试体外衍生的肺祖细胞的功能。我们设想，我们的研究结果将是确定用于新型肺部疾病治疗的从ES/iPS细胞衍生肺祖细胞的最佳方案。

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.