Generating 3-D lung organoids in vitro from human pluripotent stem cells

描述（由申请人提供）：人类胚胎和诱导多能干细胞研究为产生治疗用组织开辟了令人难以置信的新可能性。此外，多能干细胞衍生组织可用于开展发育和疾病机制的基础科学研究，以及用于药物筛选以确定药物对感兴趣的目标组织的功效。然而，仍有几个重大障碍阻碍了这些细胞的治疗应用。其中一个障碍包括许多PSC衍生细胞类型的胎儿和未成熟性质，这些细胞类型不具有其体内对应物的完整功能。人们普遍认为，更多功能细胞和组织的体外衍生将取决于尽可能地模仿这些组织的体内发育。这包括概括一个驱动正常器官发生的三维、复杂、多谱系的发育。我们最近使用了一种三维方法来生成功能性肠组织，并相信类似的方法将允许生成具有更多功能的其他器官衍生物。在这个建议中，我们将使用三维方法来尝试从胚胎和诱导多能干细胞中产生肺组织。我们的方法将利用我们开发的一种创新方法来生成三维前肠组织，理论上，这种组织可以分化为任何腹侧前肠衍生物（肺、肝、胰腺、胆道系统）。通过模拟胚胎肺的发育，我们打算引导人类多能干细胞首先分化为三维胚胎样肺祖细胞（球体），然后分化为更成熟的肺样器官单位（类器官）。为了成功实现这些目标，将追求两个目标：1)确定从hPSCs诱导三维肺祖类器官的稳健条件。我们假设刺激人类前肠球体和人类前肠单层中的WNT和SHH信号将诱导NKX2.1+肺祖球体。2)确定刺激前肠类器官扩张和肺特异性分化的条件。我们假设刺激WNT和FGF信号将为三维肺祖球体扩张和生长为成熟的肺类器官创造一个支持和允许的环境。基于初步的研究，我们相信本文提出的目标是可以实现的，并且通过产生一种新的工具来研究人类发育和人体组织疾病的机制，将广泛影响肺部生物学领域。三维系统将首次允许对人类肺部发育和疾病进行前所未有的研究，包括基因功能的获得和丧失。此外，肺类器官可用于研究肺内分化为特定细胞类型的分子调控，并可能促进我们对如何产生用于移植的治疗有用的肺特异性细胞类型的理解。

DESCRIPTION (provided by applicant): Human embryonic and induced pluripotent stem cell research has opened up incredible new possibilities for generating therapeutically useful tissues. In addition, pluripotent stem cell derived tissue can be used for performing basic scientific studies into the mechanisms of development and disease, as well as drug screens to determine the efficacy of a drug in a target tissue of interest. However, there are still several significant obstacles that prevent therapeutic use of these cells. One such hurdle includes the fetal and immature nature of numerous PSC derived cell types, which do not have the full functionality of their in vivo counterpart. It is widely believed that derivation of more functiona cells and tissues in vitro will depend on mimicking the in vivo development of these tissues as closely as possible. This includes recapitulating a 3-dimensional, complex, multi-lineage development that drives normal organogenesis. We have recently used a 3-dimensional approach to generate functional intestinal tissue and believe that a similar approach will allow generation of other organ derivatives that have more functionality. In this proposal, we will use a 3-dimensional approach in attempts to generate lung tissue from embryonic and induced pluripotent stem cells. Our approach will utilize an innovative method we have developed to generate 3-dimensional foregut tissue that can, in theory, be directed to differentiate into any ventral foregut derivative (lung, liver, pancreas, Biliary system). By mimicking embryonic lung development, we intend to direct differentiation of human pluripotent stem cells first into 3-dimensional embryonic-like lung progenitors (spheroids) and then in to more mature lung-like organ units (organoids). To successfully achieve these goals, two aims will be pursued: 1) Determine robust conditions to induce 3-dimensional lung progenitor organoids from hPSCs. We hypothesize that stimulating WNT and SHH signaling in human foregut spheroids and in human foregut monolayers will induce NKX2.1+ lung progenitor spheroids. 2) Determine conditions that stimulate expansion and lung-specific differentiation of foregut organoids. We hypothesize that stimulating WNT and FGF signaling will create a supportive and permissive environment for 3-dimensional lung progenitor spheroids to expand and grow into mature lung organoids. Based on preliminary studies, we are confident that the aims proposed herein are achievable and will broadly impact the lung biology field by generating a new tool to investigate the mechanisms of human development and disease in human tissue. For the first time, a 3-dimensional system will allow unprecedented studies of human lung development and disease, including genetic gain- and loss-of-function. Furthermore, lung organoids can be used to study molecular regulation of differentiation into specific cell types within the lung and may advance our understanding of how to generate therapeutically useful, lung-specific cell types for transplantation. PUBLIC HEALTH RELEVANCE: The proposed translational studies will take a fundamentally different approach to generate 3-dimensional lung tissue from human pluripotent stem cells. Development of this novel human lung model provides a stable source of human lung tissue that can be readily manipulated to study specific pathways, developmental events and lung diseases. Such studies would be challenging if not impossible if based solely on human surgical samples. The proposed studies will provide key information to support efforts to generate tissue, cells, and medical therapies for regeneration and repair of the lung, and will provide a novel reproducible method that can be used throughout the scientific community.