Biomechanical determinants of lung cell fate in pluripotent stem cells

多能干细胞肺细胞命运的生物力学决定因素

• 批准号: 9101843
• 负责人: Laertis Ikonomou
• 金额: $ 40.93万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9101843
• 关键词: Adhesives; Affect; Alveolar; Atomic Force Microscopy; Biological Models; Biomechanics; Biomimetics; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Therapy; Cell Transplantation; Cell model; Cells; Collagen; Complementary DNA; Cues; DNA; Data; Derivation procedure; Development; Developmental Process; Disease; Distal; ES Cell Line; Elasticity; Embryo; Environment; Enzyme-Linked Immunosorbent Assay; Epithelial; Epithelial Cells; Epithelium; Ethical Issues; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Flow Cytometry; Fluorescence; Foundations; Gel; Goals; Growth; Hamman-Rich syndrome; Health; Homeostasis; Human; Imaging Techniques; In Vitro; Knock-in; Knock-in Mouse; Laminin; Lead; Lung; Lung diseases; Mechanics; Microarray Analysis; Monitor; Morbidity - disease rate; Mus; Nidogen; Organ; Outcome; Pathologic; Pathway interactions; Patients; Phenotype; Pluripotent Stem Cells; Population; Property; Protocols documentation; Pulmonary Fibrosis; Regenerative Medicine; Reporter; Respiratory physiology; Robotics; Role; Signal Pathway; Signal Transduction; Somatic Cell; Spottings; Staging; Stem cells; Structure of parenchyma of lung; System; Testing; Tissues; base; cell type; clinically relevant; combinatorial; design; embryonic stem cell; homologous recombination; human embryonic stem cell; immunocytochemistry; in vivo; induced pluripotent stem cell; inhibitor/antagonist; lung development; mortality; neonate; novel; polyacrylamide gels; progenitor; reconstitution; research study; respiratory; scaffold; self-renewal; stem; stem cell differentiation; tool; transcriptome; Adhesives; Affect; Alveolar; Atomic Force Microscopy; Biological Models; Biomechanics; Biomimetics; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Therapy; Cell Transplantation; Cell model; Cells; Collagen; Complementary DNA; Cues; DNA; Data; Derivation procedure; Development; Developmental Process; Disease; Distal; ES Cell Line; Elasticity; Embryo; Environment; Enzyme-Linked Immunosorbent Assay; Epithelial; Epithelial Cells; Epithelium; Ethical Issues; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Flow Cytometry; Fluorescence; Foundations; Gel; Goals; Growth; Hamman-Rich syndrome; Health; Homeostasis; Human; Imaging Techniques; In Vitro; Knock-in; Knock-in Mouse; Laminin; Lead; Lung; Lung diseases; Mechanics; Microarray Analysis; Monitor; Morbidity - disease rate; Mus; Nidogen; Organ; Outcome; Pathologic; Pathway interactions; Patients; Phenotype; Pluripotent Stem Cells; Population; Property; Protocols documentation; Pulmonary Fibrosis; Regenerative Medicine; Reporter; Respiratory physiology; Robotics; Role; Signal Pathway; Signal Transduction; Somatic Cell; Spottings; Staging; Stem cells; Structure of parenchyma of lung; System; Testing; Tissues; base; cell type; clinically relevant; combinatorial; design; embryonic stem cell; homologous recombination; human embryonic stem cell; immunocytochemistry; in vivo; induced pluripotent stem cell; inhibitor/antagonist; lung development; mortality; neonate; novel; polyacrylamide gels; progenitor; reconstitution; research study; respiratory; scaffold; self-renewal; stem; stem cell differentiation; tool; transcriptome

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.

描述（由申请人提供）：影响肺上皮的疾病不容易治疗，并且在全球范围内导致大量发病率和死亡率。专门的干细胞具有自我更新或引起分化的功能性后代的潜力，作为许多器官（包括肺）的组织稳态的关键成分。使用干细胞进行肺部疾病治疗的不同潜在方法包括增强内源性干细胞分化或在体外定向干细胞对肺谱系的分化，然后进行细胞移植。两种方法都要求已知并且表征肺茎或祖细胞分化的身份和途径。胚胎干细胞（ESC）在过去的10至15年中已经成为基于细胞疗法的有前途的平台，因为它们可以通过几个定义的体外阶段过境，以概括哺乳动物的发育。此外，诱导的多能干细胞（IPSC）为人类ESC提供了有吸引力的替代品。 IPSC易于得出，不充满道德问题，并提供特定于患者的疗法的可能性。尽管大多数协议都使用可溶性因子来得出所需的细胞类型，但逐渐认识到细胞 - 矩阵的相互作用和细胞培养基质的刚度在ESC和IPSC的定向分化中具有重要作用。因此，该项目的总体目的是对生物力学提示在肺规范和分化中的作用进行首次系统研究。这是我们朝着我们长期目标开发基于细胞的疗法的疾病的第一步。在AIM 1中，我们将开发一个必需工具，该工具是在各种刚度的凝胶上的细胞外基质（ECM）蛋白的组合平台。我们将在AIM 2中使用此平台来确定ECM蛋白和底层刚度的最佳组合，以促进肺祖细胞及其分化。为了监测肺祖细胞的出现及其分化后代，我们将分别使用小鼠NKX2-1-GFP ESC和SPC-GFP IPSC报告基因线。 AIM 2实验定义的最佳生物力学条件将用于在AIM 3实验中从人IPSC中得出与临床相关的肺祖细胞。然后将这些祖细胞播种在正常和纤维化供体的脱细胞肺支架上，以研究肺僵硬增加对肺祖细胞分化和分化后代的表型的影响。我们设想我们的研究结果将是定义从ESC/IPSC衍生肺祖细胞的最佳方案，这些方案用于新型肺部疾病疗法，并了解肺僵硬如何影响植入体外衍生的上皮上皮肺癌的特性。