Metabolic Engineering of Embryonic Stem Cell Differentiation into Hepatocytes

Embryonic stem (ES) cells serve as a promising technology to obtain hepatocyte-like cells for a number of biomedical applications. Since traditional techniques result in mixed cell populations with compromised hepatic function, we have utilized strategies which (a) generate an enriched population of hepatocyte-like cells, (b) improve function using metabolic engineering principles and (c) quantify the effects of soluble factors on function using a computational model. To generate cell uniformity, we have investigated the use of 2.5 mM sodium butyrate in a monolayer culture configuration to mediate hepatocyte-specific differentiation of murine ES cells. In conjunction with mitochondrial mass and activity measurements, we have shown that ES cell derived hepatocyte-like cells mediate energy metabolism predominantly through glycolysis and thus represent an immature hepatocyte phenotype from a functional and energetic standpoint. In order to mediate further differentiation, we have utilized key regulatory molecules for inducing mitochondrial development in the precursor populations. We have shown that 500 µM S-NitrosoAcetylPenicillamine (SNAP) increased mitochondrial mass and activity, two components implicated in mitochondrial biogenesis in hepatocyte-like cells. In addition, hepatocyte functional characteristics were increased in the treated population. Using, Metabolic Flux Analysis (MFA), we have shown that 500 µM SNAP treated hepatocyte-like cells have higher glycolytic, TCA cycle and urea cycle fluxes as compared to the untreated population during the differentiation process.