39°C-in vitro culture facilitates hepatic functions of hepatocyte-like cells derived from human embryonic stem cells

Hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) may offer an alternative to primary hepatocytes that are commonly used for drug screenings and toxicity tests. Although tremendous efforts have been made to facilitate hepatic functions of hPSC-HLCs using growth factors and chemicals, these cells have not yet reached hepatic functions that are comparable to primary hepatocytes. Therefore, there exists a critical need to use an alternative trigger to facilitate hepatic functions in hPSC-HLCs. We noted that human liver temperature (around 39°C) is higher than normal human body temperature (around 36.5°C), yet hepatocytes are generally cultured at 37°C. Here, we tested the hypothesis that hepatic functions of hPSC-HLCs would be facilitated under physiologically-relevant hepatic temperatures. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39°C and 42°C. As expected, 42°C caused significantly greater cell death compared to 39°C. Next, we measured the activities of cytochrome P450 3A4 (CYP3A4), which is one of the most important enzymes for hepatic detoxification. The 39°C-treated hESC-HLCs had CYP3A4 activities that were greater than the 37°C-treated hESC-HLCs. Albumin secretion significantly increased in the 39°C-treated hESC-HLCs. In combination with existing hepatic differentiation protocols, the method proposed here may further improve hepatic functions for hPSCs and thereby aid drug discovery efforts and improve drug toxicity tests.