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Hepatocytes derived from human embryonic stem cells (hESC) promise to be an inexhaustible source of functional cells for use in biomedical research, drug discovery and treatment of liver diseases. We have developed a unique strategy to efficiently differentiate hESC into functional hepatocyte-like cells (HLC) in vitro. The robustness of our protocol was assessed by duplicating the process of differentiation in two of our in house derived hESC lines, Relicell®hES1 and Relicell®hES2, and in the well studied BG01 cell line. To induce early hepatic commitment, undifferentiated hESC were initially primed with conditioned medium from HepG2, a human hepatoma cell line, which resulted in an enriched population of definitive endoderm. We have also attempted to recapitulate the hepatogenetic events occurring in vivo by sequential application of growth factors involved in liver development, such as aFGF, HGF, oncostatin, dexamethasone and EGF. Our differentiation process yielded a homogenous population of HLC exhibiting the typical polygonal morphology of hepatocytes. This population expressed hepatic lineage markers including HNF4α, AFP and ALBUMIN, and drug metabolizing enzymes such as CYP3A4 (Phase I) and GSTA1 (Phase II). Flow cytometric analysis showed that over 70% of the differentiated cells expressed albumin and CK8/18. The differentiated HLC exhibited hepatic characteristics such as glycogen storage and production of albumin and urea. Our results indicate that functional HLC generated by this method can be utilized in regenerative medicine and as a screening platform in the discovery and development of new drugs

Stage specific differentiation of human embryonic stem cells into hepatocyte-like cells using conditioned medium from a human hepatoma cell line