Generation of functional lung stem cells from human iPSCs

从人类 iPSC 中生成功能性肺干细胞

基本信息

批准号：
9811053
负责人：
Finn Hawkins
金额：
$ 41.25万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9811053
关键词：
3-Dimensional Address Affect Age Air Alveolar Alveolar Cell Animal Model Antibodies Autologous Transplantation Basal Cell Biological Assay Biology Bleomycin Cell Culture Techniques Cell Line Cell Separation Cell Therapy Cell Transplantation Cell Transplants Cell surface Cells Chronic Obstructive Airway Disease Collaborations Communities Competence Cystic Fibrosis Data Set Derivation procedure Development Disease Disease model Elderly Engineering Engraftment Epigenetic Process Epithelial Epithelial Cells Epithelium Fluorochrome Future Generations Genetic Genetic Transcription Goals Growth Human Immunodeficient Mouse In Vitro Kidney Knock-in Liquid substance Lung Lung diseases Medical Metabolic Methods Modeling Morbidity - disease rate Mus Natural regeneration Newborn Infant Organoids Pathogenesis Patients Pluripotent Stem Cells Population Proteome Proteomics Protocols documentation Publications Publishing Pulmonary Fibrosis Regenerative Medicine Reporter Research Research Personnel Resources Respiratory Tract Diseases Safety Serum Sorting - Cell Movement Source Standardization Stem cells Surface Systems Biology Testing Tissues Translating Transplantation Tumorigenicity United States United States National Institutes of Health Work Xenograft Model base capsule catalyst cell type clinical application clinical translation clinically relevant disease phenotype drug development effective therapy efficacy testing genome integrity genomic data human stem cells improved in vitro Assay in vivo induced pluripotent stem cell innovation lung injury lung regeneration member mortality novel novel therapeutics phosphoproteomics pre-clinical progenitor programs reconstitution regenerative regenerative therapy safety testing self-renewal stem cell population success theories three dimensional cell culture tool transcriptome transcriptomics

项目摘要

From newborns to the elderly, diseases of the respiratory tract are a major cause of morbidity and mortality in the United States. For many of these diseases, there is a desperate need for effective treatments. A regenerative medicine approach using cell-based therapy could, in theory, drastically improve the lives of these patients. The long term goal of this application is to address critical hurdles to the development and application of alveolar and airway stem cells derived from induced pluripotent stem cells (iPSCs) as safe and effective cell-based therapies for lung disease. Significant progress has been made in deriving lung epithelial cells from human iPSCs over the past decade. Increasingly sophisticated directed differentiation protocols have produced more mature and functional airway and alveolar epithelial cells. Despite this progress, current protocols do not generate pure populations of cells. Knock-in fluorescent reporters have been used to purify lung epithelial cells and confirmed their similarity to primary controls but this approach is undesirable for cell-based therapies. There are crucial questions that must be addressed prior to the application of these cells to human patients. What tools are required to generate pure populations of lung stem cells and how can we best determine the safety and efficacy of those cells? In this application we address these key questions. First we develop a panel of new tools that will be required for clinically- relevant directed differentiation protocols through the application of cell-sorting strategies using antibodies against cell-surface makers to purify both alveolar (CKIT/CPM) and airway (ITGA6) stem cells from a panel of human iPSCs. These tools and protocols obviate the need for knock-in reporters, utilize serum-free defined media, and establish standardized manufacturing approaches that will be essential for successful IND applications of these novel cells. Next, and with support from the Regenerative Medicine Innovation Catalyst, we perform the most in-depth characterization to date of iPSC-derived alveolar and airway stem cells. These global proteomic, phosphoproteomic, metabolic/respiromic, transcriptomic, and genomic datasets will provide the research community with an essential resource. Safety of these cells in terms of genetic stability and tumorgenicity remains a major concern when considering clinical applications. We assess the safety profile of iPSC-derived airway and alveolar stem cells over long-term in vitro culture and in vivo. Finally, we assess the efficacy and potency of these cells. We employ established in-vitro assays to address fundamental questions of self-renewal and multi-lineage differentiation potential compared to primary controls and ultimately determine their capacity for engraftment in lung injury models in mice. At the conclusion of this proposal we will have developed new tools to purify alveolar and airway stem cells from iPSCs and will have established detailed datasets to provide a better understanding of the biology, safety and efficacy of these cells to share with the research community and move towards clinical translation.

从新生儿到老年人，呼吸道疾病是发病率和死亡率的主要原因在美国。对于许多这些疾病，迫切需要有效治疗。一个从理论上讲，使用基于细胞的治疗的再生医学方法可以大大改善这些患者。该应用程序的长期目标是解决发展的关键障碍和源自诱导多能干细胞（IPSC）的肺泡和气道干细胞的应用和有效的基于细胞的肺部疾病疗法。得出肺部已经取得了重大进展过去十年来，来自人IPSC的上皮细胞。越来越复杂的定向差异化方案产生了更成熟和功能性气道和牙槽上皮细胞。尽管如此进展，当前方案不会产生纯细胞种群。敲击荧光记者有被用来纯化肺上皮细胞并确认它们与主要对照的相似性，但是这种方法对于基于细胞的疗法是不希望的。在此之前，必须解决一些关键问题这些细胞应用于人类患者。需要什么工具来产生肺的纯种群干细胞，我们如何才能最好地确定这些细胞的安全性和功效？在此应用中，我们解决这些关键问题。首先，我们开发了一组新工具，这些工具在临床上需要 - 相关定向分化方案通过使用抗体应用细胞分类策略针对细胞表面制造商从面板上净化肺泡（CKIT/CPM）和气道（ITGA6）干细胞人类IPSC。这些工具和协议消除了对敲门记者的需求，不使用血清定义的媒体，并建立标准化的制造方法，这对于成功至关重要这些新细胞的IND应用。接下来，在再生医学创新的支持下催化剂，我们在IPSC衍生的肺泡和气道茎上执行最深入的表征细胞。这些全球蛋白质组学，磷酸蛋白质组学，代谢/呼吸组，转录组和基因组学数据集将为研究社区提供基本资源。这些细胞的安全性在考虑临床应用时，遗传稳定性和肿瘤性仍然是主要问题。我们在长期体外培养和体内。最后，我们评估了这些细胞的功效和效力。我们使用建立的体外测定法与相比主要控制并最终确定其在小鼠肺损伤模型中植入的能力。在该提案的结论我们将开发新工具，以净化肺泡和气道干细胞 IPSCS并将建立详细的数据集，以更好地了解生物学，安全性这些细胞与研究界共享并转向临床翻译的功效。