In vitro metabolic zonation through oxygen gradient on a chip

Among the multiple metabolic signals involved in the establishment of the hepatic zonation, oxygen could play a key role. Indeed, depending on hepatocyte position in the hepatic lobule, gene expression and metabolism are diferently afected by the oxygen gradient present across the lobule. The aim of this study is to understand whether an oxygen gradient, generated in vitro in our developed device, is sufcient to instruct a functional metabolic zonation during the diferentiation of human embryonic stem cells (hESCs) from endoderm toward terminally diferentiated hepatocytes, thus mimicking the in vivo situation. For this purpose, a microfuidic device was designed for the generation of a stable oxygen gradient. The oxygen gradient was applied to diferentiating hESCs at the pre-hepatoblast stage. The defnitive endoderm and hepatic endoderm cells were characterized by the expression of the transcription factor SOX-17 and alpha-fetoprotein (AFP). Immature and mature hepatocytes were characterized by hepatocyte nuclear factor 4-alpha (HNF-4α) and albumin (ALB) expression and also analyzed for cytochrome P450 (CYP3A4) zonation and glycogen accumulation through PAS staining. Metabolic zonated genes expression was assessed through quantitative real time PCR. Application of the oxygen gradient during diferentiation induced zonated glycogen storage, which was higher in the hepatocytes grown in high pO2 compared to those grown in low pO2. The mRNA levels of glutamine synthetase (GLUL), beta-catenin (CTNNB) and its direct target cyclin D1 (CCND1) showed signifcantly higher expression in the cells grown in low pO2 compared to those grown in high pO2. On the contrary, carbamoyl-phosphate synthetase 1 (CPS1), ALB, the proliferative marker ki67 (MKI67) and cyclin A (CCNA) resulted to be signifcantly higher expressed in cells cultured in high pO2 compared to those cultured in low pO2. These results indicate that the oxygen gradient generated in our device can instruct the establishment of a functional metabolic zonation in diferentiating hESCs. The possibility to obtain diferentiated hepatocytes in vitro may allow in the future to deepen our knowledge about the physiology/pathology of hepatocytes in relation to the oxygen content