Recently, Biomaterials published a paper titled "Two-dimensionally cultured functional hepatocytes generated from human induced pluripotent stem. "cell derived hepatic organoids for pharmaceutical research" by a research team led by Professor Hiroyuki Mizuguchi of the Department of Pharmaceutical Research at Osaka University. Using human induced pluripotent stem cell (iPSC) -derived liver organoids (iHOs), this study successfully obtained powerful iHO-Heps hepatocytes by optimizing culture conditions and developing two-dimensional (2D) culture protocols.

01 Research Background

Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSC) are considered as potential alternatives to primary human hepatocytes (PHHs) for drug development. However, HLCs has some limitations, such as low liver function, long differentiation time, and inability to proliferate after terminal differentiation, making it difficult to prepare in large quantities. To overcome these problems, the research team built iPS cell-derived liver organoids (iHOs) and further developed a two-dimensional culture protocol.

Medium suitable for the establishment and culture of liver organoids

02 Research Results

iHOs is highly proliferative

By optimizing the medium composition and culture conditions, the research team was able to multiply iHOs by about 10⁵ times in 18 days, which is 100 times faster than traditional media. In addition, iHOs can still maintain strong proliferation ability after multiple generations (more than 3 generations), and can even maintain more than 10 generations. This highly efficient proliferative ability provides the possibility for large-scale preparation of functional liver cells, and also lays the foundation for subsequent drug development and toxicity testing.

In addition, iHOs established from terminally differentiated (day 25) hepatocellular like cells (HLCs) were found to be more suitable for generating hepatocytes with higher liver function. These results indicate that iHOs not only have strong proliferative ability, but also maintain good cellular function during passage, providing an ideal in vitro model for drug development.

It showed a significant advantage in the level of gene expression

RNA-seq analysis showed that iHO-Heps had significantly higher expression levels of most major hepatocyte marker genes than traditional hepatocell-like cells (HLCs) and iHOs without two-dimensional culture, and even exceeded the expression of some key genes in primary human hepatocytes (PHHs). For example, iHO-Heps showed higher activity in the expression of genes related to drug metabolism, especially CYP3A4 and CYP2C19, even higher than PHHs. This suggests that iHO-Heps have properties closer to human physiological states in terms of drug metabolism and toxicity testing.

In addition, two-dimensional culture conditions transformed the cells from a proliferative state to a metabolic state, activated related pathways such as drug metabolism, and further enhanced the function of iHO-Heps. This increased level of gene expression makes iHO-Heps potentially more widely used in drug development to more accurately simulate metabolic and toxic reactions in the human liver.

Exhibited a variety of typical hepatocyte functions

iHO-Heps exhibit several key hepatocyte functions, including glycogen storage, uptake and release of indocyanine green (ICG), secretion of albumin and urea, and the ability to form timid ducts. These functions not only reflect the activity of iHO-Heps in metabolism, detoxification, protein synthesis, and bile transport, but also show that it can highly simulate the physiological properties of real liver under two-dimensional culture conditions, making it a promising in vitro model for drug development and toxicity testing.

It showed significant advantages in drug metabolism and toxic reactions

The cytochrome P450 (CYP) enzyme activity of iHO-Heps was significantly higher than that of traditional hepatocellular like cells (HLCs), and the activity of CYP3A4 and CYP2C19 even exceeded that of primary human hepatocytes (PHHs). In response experiments to hepatotoxic drugs, the sensitivity of iHO-Heps to different drugs is similar to that of PHHs, indicating that it can accurately simulate the metabolism and toxic reaction of drugs in the human liver.

Overall, this study successfully overcame the limitations of traditional hepatocell-like cells (HLCs) and established an optimized culture regimen for iPS cell-derived liver organoids (iHOs), with iHO-Heps obtained through 2D culture possessing multiple key liver functions and powerful drug metabolism. This result provides a more reliable hepatocyte model for drug development and helps to more accurately evaluate drug metabolism and toxicity. At the same time, it also provides a new tool for studying the pathogenesis of liver diseases and personalized drug therapy. Although there is still room for further optimization, this study undoubtedly brings an important breakthrough in the field of pharmaceutical research and development, and is expected to promote the rapid development of medical research in the future.