Biomechanical determinants of lung cell fate in pluripotent stem cells

描述(由申请人提供)：影响肺上皮的疾病不容易治疗，并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(ESCs)在过去的10-15年里已经成为基于细胞的疗法发展的一个有前途的平台，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。此外，诱导多能干细胞(IPSCs)提供了一种诱人的替代人类ESCs的方法。IPSC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。尽管大多数方案使用可溶性因子来获得所需的细胞类型，但人们逐渐认识到细胞-基质相互作用和细胞培养底物的硬度在ESCs和IPSCs的定向分化中具有重要作用。因此，该项目的总体目标是在肺发育的体外系统中首次系统地研究生物力学线索在肺的具体化和分化中的作用。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。在目标1中，我们将开发一个必要的工具，它是细胞外基质(ECM)蛋白在不同硬度的凝胶上的组合平台。我们将在AIM 2中使用这个平台来确定ECM蛋白和底物硬度的最佳组合，以获得肺祖细胞及其分化。为了监测肺祖细胞及其分化后代的出现，我们将分别使用小鼠Nkx2-1-GFP ESC和SPC-GFP IPSC报告系。Aim 2实验确定的最佳生物力学条件将用于在Aim 3实验中从人IPSCs中获得临床相关的肺祖细胞。然后将这些祖细胞种植在来自正常和纤维化供体的脱细胞肺支架上，以研究肺硬度增加对肺祖细胞分化和分化后代表型的影响。我们预计，我们的研究结果将是确定从ESCs/IPSCs中获得肺祖细胞的最佳方案，用于新的肺部疾病治疗，并了解肺僵硬如何影响移植的体外来源的上皮祖细胞的特性。

DESCRIPTION (provided by applicant): Diseases affecting the lung 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 cells (ESCs) 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 cells (iPSCs) offer an attractive alternative to human ESCs. iPSCs are easy to derive, are not fraught with ethical issues and offer the possibility of patient-specific therapies. Although most protocol use soluble factors to derive the desired cell types it is gradually recognized that cell-matrix interactions and stiffness of the cell culture substratum have important roles in directed differentiation of ESCs and iPSCs. Thus, the overall objective of this project is to undertake the first systematic study of the role of biomechanical cues in lung specification and differentiation n an in vitro system of lung development. This represents the first step towards our long-term goal of developing cell-based therapies for diseases affecting the lung epithelium. In Aim 1 we will develop an essential tool which is a combinatorial platform of extracellular matrix (ECM) proteins on gels of various stiffness. We will use this platform in Aim 2 to identify the optimal combination of ECM proteins and substratum stiffness for the derivation of lung progenitors and their differentiation. To monitor the emergence of lung progenitors and their differentiated progeny we will use the mouse Nkx2-1-GFP ESC and SPC-GFP iPSC reporter lines, respectively. The optimal biomechanical conditions defined from Aim 2 experiments will be used to derive clinically-relevant lung progenitors from human iPSCs in Aim 3 experiments. These progenitors will then be seeded on decellularized lung scaffolds from normal and fibrotic donors to study the effect of increased lung stiffness on both lung progenitor differentiation and phenotype of differentiated progeny. We envision that the outcome of our studies will be to define the optimal protocol for derivation of lung progenitor cells from ESCs/iPSCs to be used in novel lung disease therapies and to understand how lung stiffness affects the properties of engrafted in vitro derived epithelial lung progenitors.