Hepatic differentiation of human embryonic stem cells in a 3D bioreactor environment

Abstract

A number of articles addressing the hepatic differentiation of human embryonic stem cells (hESC) in conventional 2D culture systems have been published recently. However, the cell numbers and purity available through this culture method are limited. This study aims to investigate the hepatic differentiation of hESC in a dynamic 3D culture environment that supports cell differentiation and three-dimensional tissue reorganisation in vitro. Based on a perfused 3D-multicompartment bioreactor technology successfully used for primary human liver cell culture, a downscaled system (0.5ml cell volume) was developed to investigate the differentiation, maturation and function of the hESC-derived hepatic cells in vitro. Undifferentiated hESC were inoculated into a miniaturized 3D bioreactor and hepatic differentiation was induced by the sequential application of growth factors, hormones and cytokines. Metabolic parameters indicating metabolic activities (glucose, lactate) of the cells were measured daily. After 30 days the bioreactor was opened and samples for immunofluorescence staining and RNA analysis were taken.The time courses of glucose and lactate indicated stable cell metabolism over 30 days. Immunofluorescence analysis showed the expression of the biliary marker cytokeratin 19 (CK19), and CK18 as hepatocytic / biliary marker indicating hepatic differentiation. Both markers were localized predominantly at the peripheral areas of cell aggregates. These findings are consistent with the detected induction in the expression of albumin, AFP and cytochrome P450 2D6 in samples taken from the bioreactor following hepatic maturation of hESC compared to undifferentiated hESC. In conclusion, the results of the study suggest that the 3D-multicompartment bioreactor supports hepatic differentiation of hESC. Further studies are needed to increase the yield and maturation degree of the cells.