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Augmentation of EB‐directed hepatocyte‐specific function via collagen sandwich and SNAP

The development of implantable engineered liver tissue constructs and ex vivo hepatocyte‐based therapeutic devices are limited by an inadequate hepatocyte cell source. In our previous studies, embryoid body (EB)‐mediated stem cell differentiation spontaneously yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB) and cytokeratin‐18 (CK18). However, these cultures neither yielded a homogenous hepatocyte lineage population nor exhibited detoxification function typical of a more mature hepatocyte lineage cell. In this study, secondary culture configurations were used to study the effects of collagen sandwich culture and oncostatin‐M (OSM) or S‐nitroso‐N‐acetylpenicillamine (SNAP) supplementation of EB‐derived hepatocyte‐lineage cell function. Quantitative immunofluorescence and secreted protein analyses were used to provide insights into the long‐term maintenance and augmentation of existing functions. The results of these studies suggest that SNAP, independent of the collagen supplementation, maintained the highest levels of ALB expression, however, mature liver‐specific CK18 was only expressed in the presence of gel sandwich culture supplemented with SNAP. In addition, albumin secretion and cytochrome P450 detoxification studies indicated that this condition was the best for the augmentation of hepatocyte‐like function. Maintenance and augmentation of hepatocyte‐like cells isolated from heterogeneous EB cell populations will be a critical step in generating large numbers of functional differentiated cells for therapeutic use.