Efficient derivation of hepatogenic endoderm from human embryonic stem cells using serum-free conditioned medium from l-wnt3a cells

The directed differentiation of human embryonic stem cells (hESCs) into hepatocytes is considered a promising new approach to generate a transplantable and limitless cell source for the treatment of acute and chronic liver diseases. Current protocols for generating hepatocytes from hESCs need to be improved because of the inefficient differentiation procedures which lead to low yields and large cellular heterogeneity. In this study, we describe a simple and efficient three-stage process for differentiating hESCs into functional hepatocyte-like cells. The first step of this optimized protocol was to induce hESCs towards mesendoderm (co-precursor stage of the mesoderm and endoderm) using 80% Wnt3a conditioned medium (Wnt3a-CM) and 20 ng/ml activin A for 5 days. In the second step, the mesoendodermal cells were treated with 40 ng/ml bone morphogenetic protein 4 (BMP4) for 4 days. In the third stage of hepatocyte maturation, cells were cultured in hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone (DEX). During the first stage, the definitive endodermal markers were significantly increased, and under the further differentiation, the expression of hepatocyte precursor markers, especially, albumin and CYP genes, induced sequentially. Furthermore, these cells showed functions associated with hepatocytes, which include ?-glutamyltranspeptidase (GGT), glycogen storage, indocyanine green (ICG) uptake, and albumin secretion. These results may form the basis for further investigation into understanding the fundamental mechanisms of liver development, applying cell therapy and screening cytotoxicity in a new drug to treat liver diseases.