Recent advances in pluripotent stem cell technology provide an alternative source of human hepatocytes to overcome the limitations of current toxicity tests. However, this approach requires optimization and standardization before it can be used as a fast and reliable toxicity screening system. Here, we designed and tested microwell culture platforms with various diameters. We found that large quantities of uniformly-sized hepatocyte-like cell (HLC) spheroids (3D-uniHLC-Ss) could be efficiently and reproducibly generated in a short period time from a small number of differentiating human pluripotent stem cells (hPSCs). The hPSC-3D-uniHLC-Ss that were produced in 500-μm diameter microwells consistently exhibited high expressions of hepatic marker genes and had no significant signs of cell death. Importantly, a hepatic master gene hepatocyte nuclear factor 4α (HNF4α) was maintained at high levels, and the epithelial-mesenchymal transition was significantly attenuated in hPSC-3D-uniHLC-Ss. Additionally, when compared with 3D-HLC-Ss that were produced in other 3D platforms, hPSC-3D-uniHLC-Ss showed significantly higher hepatic gene expressions and drug-metabolizing activity of the enzyme, CYP3A4. Imaging-based drug toxicity studies demonstrated that hPSC-3D-uniHLC-Ss exhibited enhanced sensitivity to various hepatotoxicants, compared to HLCs, which were differentiated under 2D conditions. Precise prediction of drug-induced hepatotoxicity is a crucial step in the early phases of drug discovery. Thus, the hPSC-3D-uniHLC-Ss produced using our microwell platform could be used as an imaging-based toxicity screening system to predict drug hepatotoxicity.

多能干细胞技术的最新进展为人类肝细胞提供了另一种来源，以克服当前毒性试验的局限性。但是，这种方法需要先进行优化和标准化，然后才能用作快速可靠的毒性筛选系统。在这里，我们设计并测试了各种直径的微孔培养平台。我们发现，可以在短时间内从少量分化的人类多能干细胞（hPSC）中高效且可重复地生成大量均匀大小的肝样细胞（HLC）球体（3D-uniHLC-Ss）。在直径为500μm的微孔中产生的hPSC-3D-uniHLC-Ss始终表现出肝标志物基因的高表达，并且没有明显的细胞死亡迹象。重要的，HNF4α）维持在高水平，和上皮-间充质细胞过渡明显减少了hPSC-3D-uniHLC-Ss。此外，与在其他3D平台中生成的3D-HLC-S相比，hPSC-3D-uniHLC-S显示出明显更高的肝基因表达和该酶CYP3A4的药物代谢活性。基于影像的药物毒性研究表明，与在2D条件下分化的HLC相比，hPSC-3D-uniHLC-S对各种肝毒性表现出更高的敏感性。药物诱发的肝毒性的精确预测是药物发现早期阶段的关键步骤。因此，使用我们的微孔平台生产的hPSC-3D-uniHLC-S可用作基于影像的毒性筛选系统，以预测药物的肝毒性。