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) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological 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 comparable to hepatocytes in vivo. 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{degrees}C) is higher than normal human body temperature (around 36.5{degrees}C), yet hepatocytes are generally cultured at 37{degrees}C in-vitro. Here we showed that hepatic functions of hPSC-HLCs would be facilitated under physiological liver temperatures. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39{degrees}C and 42{degrees}C. 42{degrees}C-treatment caused significantly greater cell death compared to 39{degrees}C. We also confirmed the increases of hepatic functions, such as secretion of albumin, cytochrome P450 3A4 (CYP3A4) activities, and collagen productions, without severe cell damages. To elucidate the underlying mechanisms of heat-induced hepatic functions, RNA-seq was to identify gene expression signatures due to 39{degrees}C-treated hESC-HLCs. This study also showed the possible mechanisms of heat-induced hepatic function via glucocorticoid receptor pathway and molecular chaperons. In combination with existing hepatic differentiation protocols, the method proposed here may further improve hepatic functions for hPSCs, and lead to the realization of drug discovery efforts and drug toxicological tests. Significance statementHepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. We noted that human liver temperature (around 39{degrees}C) is higher than normal human body temperature (around 36.5{degrees}C), affecting the in-vitro hepatic functions of hPSC-HLCs, such as metabolic activities. Here we showed that hepatic functions of hPSC-HLCs, albumin secretion, CYP3A4 activities, and collagen production would be facilitated under physiological liver temperatures at 39{degrees}C, without severe cell damages. RNA-seq was used to elucidate the underlying mechanisms of heat-induced hepatic functions. This study also showed the possible mechanisms of heat-induced hepatic function via glucocorticoid receptor pathway and molecular chaperons.