Functionally maturing iPSC-derived human hepatocytes in 3D microgels

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。

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.