318 DIFFERENTIATION OF ES-DERIVED HEPATIC PROGENITORS AND HepaRG CELL LINE INTO CHOLANGIOCYTE-LIKE CELLS

Abstract

Background and Aims: Primary hepatocytes are difficult to culture ex vivo and their utility is limited by the lack of available human hepatocytes. These problems provide incentive into finding sources of hepatocyte-like cells from stem cell populations. The hepatic differentiation of human embryonic stem cells (hESC) has been extensively described for 2D cultures, however differentiated cells exhibit poor hepatic function and no typical hepatic architecture is observed. Our aims were to investigate whether 3D culture of hESC in bioreactor devices can enhance the hepatic function and whether a more physiological architecture can be promoted. Methods and Results: Human ESC (WA01, WiCell, James A. Thomson, University of Wisconsin, Madison, USA) were initially subjected comparatively to five established protocols [1–5] for promoting hepatic differentiation. In two of these approaches [1,2] we observed successful differentiation of hESC into hepatocyte-like cells, which were obtained for further analysis and culture. The culture and differentiation of hESC was carried out in a miniaturised bioreactor system with a cell compartment volume of 0.4ml and 2D cultures were performed in parallel as controls. The differentiation in the bioreactors was performed by adaptation of the protocols published by Hay et al [1,2]. Medium samples were taken daily and analysed for hepatic differentiation markers. At the end of culture immunohistochemistry for hepatic, biliary and pluripotency markers was performed for both bioreactor and 2D cultures. RT-PCR analysis for hepatocyte-specific genes including albumin and various cytochrome P450 isotypes showed a significant increase in expression in 3D cultures. Furthermore observations of the cellular architecture from bioreactor cultures demonstrated 3D structures that were closer to primary liver cell structures than those observed from 2D cultures. Conclusions: These data show that hESC can be successfully differentiated in 3D bioreactors where they express a number of hepatocyte-specific genes and form 3D structures. Thus, the bioreactor system provides a new instrument for generating functional hepatocyte-like cells from hESC, which has great potential for clinical and pharmacological studies.