Mini-Livers Derived from Human IPS Cells for Modeling Steatosis and Therapy

源自人 IPS 细胞的微型肝脏用于脂肪变性建模和治疗

• 批准号: 10297425
• 负责人: Deepak Nagrath
• 金额: $ 53.63万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10297425
• 关键词: Adult; Alcoholic Liver Cirrhosis; Animals; Cell Communication; Cell Culture Techniques; Cell model; Cells; Cessation of life; Chronic Disease; Cirrhosis; Clinical; Complex; Cues; Data; Deposition; Detection; Development; Diabetes Mellitus; Disease; Engineering; Environment; Epidemic; Epigenetic Process; Evolution; Excision; Fatty Liver; Fatty acid glycerol esters; Fibroblasts; Fibrosis; Funding; Future; Genetic; Genetic Diseases; Genetic Polymorphism; Goals; Hepatic Stellate Cell; Hepatocyte; Heterogeneity; High Prevalence; Histologic; Human; Human Genetics; In Vitro; Inflammatory; Isotopes; Link; Liver; Liver Dysfunction; Liver Fibrosis; Liver diseases; Mediating; Mesenchymal; Metabolic; Metabolic Diseases; Metabolic Pathway; Metabolism; Modeling; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organ; Organ Culture Techniques; Organ Model; Outcome; Pathogenesis; Pathologic; Patients; Pharmacotherapy; Phenotype; Population; Preventive; Primary carcinoma of the liver cells; Process; Public Health; Rattus; Resources; Role; SIRT1 gene; Single Nucleotide Polymorphism; Source; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Tissue Model; Tissues; Variant; Work; alternative treatment; base; cell type; chronic liver disease; comorbidity; engineered stem cells; fatty acid biosynthesis; genetic variant; high risk; histone modification; human disease; improved; induced pluripotent stem cell; knock-down; lifestyle factors; lipidomics; liver metabolism; macrophage; metabolic profile; metabolomics; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel strategies; prevent; response; small hairpin RNA; stem; stem cell model; stressor; success

ABSTRACT/SUMMARY Our long-term goal remains the same and targets to develop human liver tissue model from stem cells, with multi-cellular cues, metabolic functionality, that incorporates dynamic genetic and epigenetic factors and encompasses the spectrum and evolution of human NAFLD to uncover disease mechanism and therapeutics. The objectives of the proposed study are to continue developing human fatty livers engineered with iPS cells, that incorporates the dynamic expression of the epigenetic-related histone modification SIRT1 in different genetic polymorphisms and elucidate their role as metabolic regulators in the development of human fatty liver disease. The central hypothesis to be tested here is that genetic polymorphisms especially the NAFLD-high- risk PNPLA3 rs738409, combined with epigenetic-related changes (SIRT1) can control hepatic metabolism and contribute to the development of human fatty liver. The rationale is that the ability to generate human diseased liver tissue using genetically modifiable iPS cells from different human populations with single- nucleotide-polymorphisms is a powerful resource, that now make it possible, for the first time, to functionally interrogate their role in disease. Importantly, based on our strong preliminary data of targeted metabolomic analysis in isolated human hepatocytes obtained from either liver resections “Normal Human Hepatocytes” or hepatocytes isolated from explanted NASH livers “NASH Human Hepatocytes” carrying either the NAFLD-high risk PNPLA3 I148M genetic variant or wild type we hypothesize that PNPLA3 I148M hepatocytes have high levels of ferroptosis thereby leading to low metabolic capacity to adapt to stressors and continuous loss of hepatocytes. In the newly proposed aim3, we aim to uncover ferroptosis based therapeutic strategies for patients with NAFLD-high risk PNPLA3 I148M genetic variants. The work described here is expected to i) generate human iPS cells carrying shRNA mediated conditional knockdown of SIRT1 in different genetic backgrounds (especially the NAFLD-high-risk PNPLA3 rs738409 variant), ii) develop a novel approach for modeling an organ-like environment to determine the role of the epigenetic related factor SIRT1 and the genetic variants in the development of fibrotic human mini-livers with fatty liver disease and iii) determine the role of genetic polymorphisms in regulating functional metabolism in mini-human iPS-derived liver tissue with fatty liver disease with especial focus on ferroptosis. Understanding this process alone or in relation to epigenetic changes in a human liver tissue model can result in preventive therapeutic strategies for patients with NAFLD-high risk PNPLA3 I148M genetic variants. The results of this work will also have a positive impact by establishing the basis and platform for future sophisticated organ engineering techniques that incorporates several different cell types from the same iPS cell source, these techniques could be applied to study other liver diseases (e.g. metabolic diseases) and is expected to be a major contribution to the fields of stem cells engineering and liver steatosis.

摘要/总结 我们的长期目标保持不变，目标是利用干细胞开发人类肝脏组织模型， 多细胞线索、代谢功能，融合了动态遗传和表观遗传因素， 涵盖人类 NAFLD 的谱系和演变，以揭示疾病机制和治疗方法。 拟议研究的目标是继续开发用 iPS 细胞改造的人类脂肪肝， 结合了表观遗传相关组蛋白修饰 SIRT1 在不同环境中的动态表达 遗传多态性并阐明其在人类脂肪肝发展中作为代谢调节剂的作用 疾病。这里要检验的中心假设是基因多态性，尤其是 NAFLD 高基因多态性。 风险PNPLA3 rs738409，结合表观遗传相关变化（SIRT1）可以控制肝脏代谢 并有助于人类脂肪肝的发展。其基本原理是，产生人类的能力 使用来自不同人群的基因修饰 iPS 细胞对患病肝组织进行单 核苷酸多态性是一种强大的资源，现在首次使功能性 询问它们在疾病中的作用。重要的是，基于我们针对目标代谢组学的强有力的初步数据 对从肝切除术中获得的分离的人肝细胞进行分析“正常人肝细胞”或 从移植的 NASH 肝脏“NASH 人肝细胞”中分离出的肝细胞携带 NAFLD 高水平 风险 PNPLA3 I148M 遗传变异或野生型 我们假设 PNPLA3 I148M 肝细胞具有高风险 铁死亡水平，从而导致适应应激源的代谢能力低下和持续丧失 肝细胞。在新提出的目标3中，我们的目标是揭示基于铁死亡的治疗策略 患有 NAFLD 高危 PNPLA3 I148M 基因变异的患者。 这里描述的工作预计 i) 生成携带 shRNA 介导的条件条件的人类 iPS 细胞 不同遗传背景下 SIRT1 的敲低（特别是 NAFLD 高危 PNPLA3 rs738409 变体），ii）开发一种新方法来模拟类器官环境以确定 表观遗传相关因子 SIRT1 及其在纤维化人类小肝发育中的遗传变异 脂肪肝疾病和iii)确定遗传多态性在调节功能代谢中的作用 患有脂肪肝疾病的微型人 iPS 衍生的肝组织，特别关注铁死亡。理解 这一过程单独或与人类肝脏组织模型中的表观遗传变化相关，可以产生预防性效果 NAFLD 高危 PNPLA3 I148M 基因变异患者的治疗策略。这样做的结果 工作还将通过为未来的尖端器官建立基础和平台而产生积极影响 工程技术融合了来自同一 iPS 细胞来源的几种不同细胞类型，这些 技术可以应用于研究其他肝脏疾病（例如代谢疾病），并有望成为一种 在干细胞工程和肝脂肪变性领域做出了重大贡献。