Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
Laboratory of Functional Organoid for Drug Discovery, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan.
PLoS One. 2023 May 18;18(5):e0285783. doi: 10.1371/journal.pone.0285783. eCollection 2023.
Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes as a new source of functional hepatocytes in various medical applications. However, the hepatic functions of HLCs are still low and it takes a long time to differentiate them from human iPS cells. Furthermore, HLCs have very low proliferative capacity and are difficult to be passaged due to loss of hepatic functions after reseeding. To overcome these problems, we attempted to develop a technology to dissociate, cryopreserve, and reseed HLCs in this study. By adding epithelial-mesenchymal transition inhibitors and optimizing the cell dissociation time, we have developed a method for passaging HLCs without loss of their functions. After passage, HLCs showed a hepatocyte-like polygonal cell morphology and expressed major hepatocyte marker proteins such as albumin and cytochrome P450 3A4 (CYP3A4). In addition, the HLCs had low-density lipoprotein uptake and glycogen storage capacity. The HLCs also showed higher CYP3A4 activity and increased gene expression levels of major hepatocyte markers after passage compared to before passage. Finally, they maintained their functions even after their cryopreservation and re-culture. By applying this technology, it will be possible to provide ready-to-use availability of cryopreserved HLCs for drug discovery research.
人诱导多能干细胞（iPS）衍生的肝细胞样细胞（HLC）有望替代原代人肝细胞，成为各种医学应用中功能性肝细胞的新来源。然而，HLC 的肝功能仍然较低，并且从人 iPS 细胞分化它们需要很长时间。此外，HLC 的增殖能力非常低，由于在重新接种后肝功能丧失，它们很难传代。为了克服这些问题，我们在本研究中尝试开发一种分离、冷冻保存和重新播种 HLC 的技术。通过添加上皮-间充质转化抑制剂并优化细胞解离时间，我们开发了一种在不损失其功能的情况下传代 HLC 的方法。传代后，HLC 呈现出肝细胞样多角形细胞形态，并表达白蛋白和细胞色素 P450 3A4（CYP3A4）等主要肝细胞标记蛋白。此外，HLC 具有低密度脂蛋白摄取和糖原储存能力。与传代前相比，HLC 还显示出更高的 CYP3A4 活性和主要肝细胞标记物的基因表达水平增加。最后，它们甚至在冷冻保存和再培养后仍保持其功能。通过应用这项技术，将有可能为药物发现研究提供即用型冷冻保存的 HLC。
