Functionally maturing iPSC-derived human hepatocytes in 3D microgels

3D 微凝胶中功能成熟的 iPSC 衍生人肝细胞

• 批准号: 9226831
• 负责人: Salman R Khetani
• 金额: $ 24.06万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9226831
• 关键词: Address; Adult; Albumins; Alpha Cell; Animals; Behavior; Cell Communication; Cell Therapy; Cell physiology; Cells; Chemicals; Chronic; Cirrhosis; Coculture Techniques; Complex; Consensus; Coupled; Cues; Cytochrome P450; Devices; Dimensions; Disadvantaged; Disease; Disease Progression; Drug toxicity; Embryo; Endothelial Cells; Engineering; Evaluation; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Future; Gene Expression; Genetic; Goals; Gold; Hepatic; Hepatic Stellate Cell; Hepatitis B; Hepatocyte; Hour; Human; Hydrogels; In Vitro; Liver; Liver diseases; Malaria; Malignant Neoplasms; Measurement; Measures; Methods; Microfabrication; Microfluidics; Modeling; Molecular; Mus; Non-Insulin-Dependent Diabetes Mellitus; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phenotype; Physiology; Play; Preclinical Drug Evaluation; Proteins; Protocols documentation; Resources; Role; Site; Source; Stimulus; Stromal Cells; Technology; Testing; Time; Tissues; Toxic effect; Urea; Virus; Virus Replication; Wood material; base; cell type; cholangiocyte; combinatorial; cost; drug metabolism; drug sensitivity; drug testing; experimental study; fetal; implantation; in vitro Model; in vivo; induced pluripotent stem cell; inter-individual variation; liver development; liver function; macrophage; man; miniaturize; monolayer; non-alcoholic fatty liver; response; scaffold; scale up; screening; stem cell differentiation; three dimensional cell culture; tool; treatment group; ward

PROJECT SUMMARY Primary human hepatocytes (PHHs) represent a scarce resource to build human liver models due to shortages of donor livers and thus these cells are not suitable for high-throughput drug screening. Induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-hHs) can address the aforementioned limitations of PHHs, allowing for the creation of genetically diverse donor panels to help elucidate inter-individual variations in drug response and disease progression. However, we and others have shown that iPSC-hHs need to be further differentiated towards the adult PHH phenotype. In vivo, a complex extracellular matrix (ECM) and a complex stromal compartment with multiple cell types modulate the hepatic phenotype. However, systematic evaluation of these cues on iPSC-hH functional maturation has not been evaluated to any considerable de- gree. It is well known that 3D microenvironments mimic human physiology better than 2D culture formats for many cell types (i.e. cancer, liver). Our main hypothesis is that a 3D microenvironment, which contains com- plex liver-inspired ECM coupled with key liver stromal cell types, will significantly differentiate iPSC-hHs to- wards the adult PHH phenotype. The challenge to testing this hypothesis using bulk hydrogels is that they are too labor/time intensive and costly to produce for high throughput exploration of optimal culture conditions. Thus, here we will utilize a high-throughput microtissue technology (>45,000 protein-based uniform microtis- sues per hour) to explore the aforementioned cues, which will allow us to have a large numbers of microtissues in each condition (>75) to obtain high statistical power in the results. In aim 1, we will investigate differentiated functions of iPSC-hHs in 3D microtissues of varying ECM compositions, while in aim 2, we will investigate dif- ferentiated functions of iPSC-hHs in co-culture with a complex liver-like stromal compartment in 3D microtis- sues with optimal ECM. Thus, our studies will create the first high-throughput 3D iPSC-hH / stromal co-culture platform with tunable ECM microenvironment, which can be used to investigate the chronic impacts of various stimuli (i.e. differentiation cues, chemicals, viruses, implantation sites for therapies) on liver functions.

项目摘要 原代人肝细胞（PHH）是一种稀缺的资源，建立人类肝脏模型，由于短缺 因此这些细胞不适合用于高通量药物筛选。诱导多能 干细胞衍生的人肝细胞样细胞（iPSC-hH）可以解决上述限制， PHH，允许创建遗传多样性供体面板，以帮助阐明个体间的变异 在药物反应和疾病进展方面。然而，我们和其他人已经表明，iPSC-hH需要 进一步向成人PHH表型分化。在体内，复合细胞外基质（ECM）和 具有多种细胞类型的复杂基质隔室调节肝表型。然而，系统 这些线索对iPSC-hH功能成熟的评估尚未被评估到任何相当大的程度。 同意。众所周知，3D微环境比2D培养形式更好地模拟人类生理学， 许多细胞类型（即癌症，肝脏）。我们的主要假设是，一个3D微环境，其中包含COM- 复杂的肝脏激发的ECM与关键的肝脏基质细胞类型相结合，将使iPSC-hH显著分化为- 成人PHH表型。使用散装水凝胶测试这一假设的挑战在于， 对于最佳培养条件的高通量探索来说，生产起来太劳动/时间密集且成本高。 因此，在这里，我们将利用高通量微组织技术（> 45，000个基于蛋白质的均匀微组织， 每小时100次）来探索上述线索，这将使我们能够获得大量的微组织 在每种条件下（>75），以获得结果的高统计功效。在目标1中，我们将研究差异化 iPSC-hH在不同ECM组成的3D微组织中的功能，而在目标2中，我们将研究iPSC-hH在不同ECM组成的3D微组织中的功能。 iPSC-hH在与3D显微组织中复杂的肝样基质隔室共培养中的生殖功能 最佳ECM的优化。因此，我们的研究将创建第一个高通量3D iPSC-hH /基质共培养物 具有可调ECM微环境的平台，可用于调查各种 刺激（即分化线索、化学品、病毒、治疗植入部位）对肝功能的影响。