High-throughput exploration of chemomechanical crosstalk in the maturation of iPSC-derived human hepatocytes

项目总结/摘要 跨物种的药物代谢途径的差异需要使用体外人肝模型用于 筛选药物/工业化学品的代谢和毒性，并发现新的治疗方法 对抗肝脏疾病虽然原代人肝细胞（PHH）是制备人肝细胞的“金标准”，但是， 肝脏模型，供体肝脏的严重短缺限制了它们在高通量筛选中的应用，并阻止了 全面评估疾病的遗传决定因素。相反，诱导多能干细胞- 从遗传多样性供体组产生的衍生的人肝细胞样细胞（iPSC-HH）可以解决 PHH的局限性;然而，产生iPSC-HH的方案不能使这些细胞完全成熟， 成人PHH表型，因此这些方案需要大量的改进。加深了解 肝脏激发的微环境线索如何协同影响iPSC-HH的成熟和维持 因此，迫切需要改进iPSC-HH表型以更有效地利用iPSC-HH用于上述应用。成人肝脏 包含细胞外基质（ECM），其a）呈现生物化学和生物力学信号，和B）可 进一步调节肝细胞与生长因子和细胞因子的相互作用。先前的研究 表明ECM的组成、基质硬度和生长因子可以独立地影响 原代肝细胞和胚胎干细胞衍生的肝细胞样细胞的体外功能。然而，在这方面， 体内发生的这些线索之间的串扰尚不清楚，ECM和 生长因子信号对iPSC-HH功能成熟的影响还有待鉴定。此外，肝脏 有几种非实质细胞（NPC）类型可以影响肝细胞功能，包括肝脏 肝窦内皮细胞（LSEC），在肝硬化过程中为肝细胞提供关键的调节信号 发育，生理学和再生;我们最近的工作也支持LSEC调节的概念， iPSC-HH比其他肝脏NPC类型发挥更大程度的功能。因此，在本提案中，我们将审查 ECM组成/硬度和LSEC细胞间相互作用共同作用的中心假设 显著调节iPSC-HH功能成熟。检验这一假设的部分挑战在于， 以大量培养形式评估大量微环境线索的组合成本太高， 也是劳动和时间密集型的。因此，我们将采用一种细胞微阵列平台， ECM微环境，可溶性因子组合培养，细胞定量评估 表型使用高内容成像读数。在目标1中，我们将系统地研究合作社 ECM组成、底物硬度和生长因子信号传导对iPSC-HH表型的影响。在目标2中， 我们将研究iPSC-HH和LSEC之间的相互作用，并建立一个药物治疗平台。 毒性评价总的来说，这些研究对于进一步开发体外肝脏模型至关重要 包含iPSC-HH用于临床预测药物/化学筛选，并最终用于再生医学。

PROJECT SUMMARY / ABSTRACT Differences in drug metabolism pathways across species necessitate the use of in vitro human liver models for screening the metabolism and toxicity of drugs/industrial chemicals, and for discovering novel therapeutics against liver diseases. While primary human hepatocytes (PHHs) are the ‘gold standard’ for fabricating human liver models, a severe shortage of donor livers limits their utility for high-throughput screening and prevents a comprehensive assessment of genetic determinants of diseases. In contrast, induced pluripotent stem cell- derived human hepatocyte-like cells (iPSC-HHs) created from genetically diverse donor panels can address the limitations of PHHs; however, the protocols to generate iPSC-HHs cannot fully mature these cells towards an adult PHH phenotype and thus such protocols require substantial refinement. An enhanced understanding of how liver-inspired microenvironmental cues synergistically affect iPSC-HH maturation and maintenance of phenotype is urgently needed to utilize iPSC-HHs more effectively for the above applications. The adult liver contains an extracellular matrix (ECM) that a) presents biochemical and biomechanical signals, and b) can further modulate interactions of liver cells with growth factors and cytokines. Previous investigations have demonstrated that the composition of ECM, substrate stiffness, and growth factors can independently influence the in vitro functions of primary hepatocytes and embryonic stem cell-derived hepatocyte-like cells. However, the crosstalk between these cues as occurs in vivo is unclear, and the combinatorial effects of ECM and growth factor signals on the functional maturation of iPSC-HHs have yet to be identified. In addition, the liver has several non-parenchymal cell (NPC) types that can influence hepatocellular functions, including liver sinusoidal endothelial cells (LSECs) that provide critical regulatory signals to hepatocytes during liver development, physiology, and regeneration; our recent work also supports the notion that LSECs regulate iPSC-HH functions to a greater degree than other liver NPC types. Thus, in this proposal we will examine the central hypothesis that ECM composition/stiffness and LSEC intercellular interactions collectively act to significantly modulate iPSC-HH functional maturity. Part of the challenge in testing such a hypothesis is that evaluating a large number of combinations of microenvironmental cues in a bulk culture format is too costly as well as labor and time intensive. Therefore, we will adapt a cellular microarray platform that enables defined ECM microenvironments, combinatorial culture with soluble factors, and quantitative assessment of cell phenotype using high-content imaging readouts. In Aim 1, we will systematically investigate the cooperative effects of ECM composition, substrate stiffness, and growth factor signaling on iPSC-HH phenotype. In Aim 2, we will examine reciprocal interactions between iPSC-HHs and LSECs and establish a platform for drug toxicity assessment. Collectively, these studies are essential for further developing in vitro liver models containing iPSC-HHs for clinically-predictive drug/chemical screening, and ultimately, regenerative medicine.