Modeling Polycystic Kidney Disease Using Human Induced Pluripotent Stem Cells

描述(由申请人提供)：来自肾脏疾病患者的诱导多能干细胞(IPSCs)在针对患者的疾病建模和免疫相容组织替代治疗方面具有巨大的潜力。申请者本杰明·弗里德曼博士在导师约瑟夫·邦文特尔博士的实验室中进行了这些领域的开创性研究。这项申请的目标是进一步扩大候选人在这一新颖研究领域的专业知识和发现，成为一个全面的、独立的研究计划。弗里德曼博士最近领导了一项研究，为多囊肾病(PKD)建立了IPSC模型，PKD是导致肾衰竭的主要原因。PKD是由多囊蛋白-1(PC1)、PC2和纤维囊蛋白/多导管蛋白(FPC)突变引起的，这些突变在初级纤毛上相互作用。与来自健康或ARPKD患者的同等培养相比，纤毛PC2减少是ADPKD IPSCs及其后代上皮细胞和肝母细胞的共同特征。野生型PC1的过表达挽救了PC2对纤毛的定位，提示了一种可能的治疗方法。我们实验室最近开发了一种方案，用于将IPSCs定向分化为表达肾脏谱系标志的肾祖细胞(KPC)。利用现有的和创新的PKD iPSC系，弗里德曼博士将在2D培养中测试PKD疾病突变导致去分化和细胞周期表型，在3D培养中导致异常囊变的假设。为了在体内扩展这项工作，IPSC来源的KPC将被植入免疫缺陷小鼠体内，以形成组织生长，这将被仔细检查，以寻找支持肾脏分化和PKD特异性囊变的组织学和免疫组织化学证据。这些实验将促进我们对PKD发病机制的理解，为未来的研究创造创新的细胞系和方法，并扩大弗里德曼博士的技术宝库，包括基因组修改、3D培养和体内分化的新技术。弗里德曼博士将把100%的时间投入到这一奖项下的研究中，布里格姆妇女医院将把他提升到教员职位。Bonventre博士将继续每天指导Freedman博士，为他和他的研究助理提供办公室和长椅空间，并使用Bonventre的所有设施，包括完成这些研究所需的所有设备。弗里德曼博士将由哈佛大学在PKD病理生理学和治疗方面的三位知名专家共同指导：周静博士、弗里德海姆·希尔德布兰特博士和西奥多·斯坦曼博士。导师和共同导师将每六个月会面一次，评估弗里德曼博士的进步。弗里德曼博士将通过1)致力于干细胞、PKD和肾脏生理学的每周会议和研讨会，2)全国干细胞和肾病学会议，以及3)负责任地开展研究课程，来补充他的教育。预计他将在获奖期间每年制作第一作者手稿，并在第三年结束时具有独立研究奖的竞争力。

DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPSCs) from patients with kidney disease have significant potential for patient-specific disease modeling and immunocompatible tissue replacement therapy. The applicant, Dr. Benjamin Freedman, has performed pioneering studies in these areas in the laboratory of the mentor, Dr. Joseph Bonventre. This application's goal is to further expand the candidate's expertise and findings in this novel research area into a well-rounded, independent research program. Dr. Freedman recently led research establishing iPSC models for polycystic kidney disease (PKD), a leading cause of kidney failure. PKD is caused by mutations in polycystin-1 (PC1), PC2, and fibrocystin/polyductin (FPC), which interact at the primary cilium. Reduced ciliary PC2 was found to be a common feature in ADPKD iPSCs and descendant epithelial cells and hepatoblasts, when compared to equivalent cultures from healthy or ARPKD patients. Overexpression of wild-type PC1 rescued PC2 localization to cilia, suggesting a possible therapeutic approach. Protocols have recently been developed in our laboratory for directed differentiation of iPSCs into kidney progenitor-like cells (KPCs) expressing markers of the renal lineage. Utilizing existing and innovative PKD iPSC lines, Dr. Freedman will test the hypothesis that PKD disease mutations result in dedifferentiation and cell cycle phenotypes in 2D culture and aberrant cystogenesis in 3D culture. To extend this work in vivo, iPSC-derived KPCs will be implanted into immunodeficient mice to form tissue growths, which will be carefully examined for histological and immunohistochemical evidence supporting kidney differentiation and PKD-specific cystogenesis. These experiments will advance our understanding of PKD pathogenesis, produce innovative cell lines and methodologies for future research, and expand Dr. Freedman's technical repertoire to include new skills of genome modification, 3D culture, and in vivo differentiation. Dr. Freedman will devote 100 % of his time to research under this award and Brigham and Women's Hospital will promote him to a faculty position. Dr. Bonventre will continue to mentor Dr. Freedman on a daily basis, providing office and bench space to him and his research assistants and access to all of the Bonventre facilities including all the necessary equipment to complete these studies. Dr. Freedman will be co-mentored by three renowned experts in PKD pathophysiology and treatment at Harvard: Dr. Jing Zhou, Dr. Friedhelm Hildebrandt, and Dr. Theodore Steinman. The mentor and co-mentors will meet to evaluate Dr. Freedman's progress every six months. Dr. Freedman will supplement his education with 1) weekly meetings and seminars devoted to stem cells, PKD, and kidney physiology, 2) national stem cell and nephrology conferences, and 3) responsible conduct of research courses. He is expected to produce first author manuscripts on an annual basis during the award period and will be competitive for independent research awards by the end of the third year.