Expression of endoderm and hepatic specific genes after in vitro differentiation of human embryonic stem cells

ABSTRACT

Human embryonic stem cells [hESC], which are derived from the inner cell mass of the blastocysts, have been considered to be pluripotent cells. In this study we examine the differentiating potential of hESC into hepatocytes by characterization of the expression of endoderm and liver-specific genes. hESC were cultivated in suspension to form aggregates, the embryoid bodies. They were allowed to outgrowth on the plated culture with the stepwise addition of growth factors such as acidic fibroblast growth factor [aFGF], hepatocyte growth factor and oncostatin M into the culture medium. The expressions of endodermal and liver specific genes such as hepatocyte nuclear factor 3 beta, alpha-fetoprotein [AFP], albumin [ALB], cytokeratin 8 [CK-8], CK-18, transthyretin, glucose 6-phosphatase and tyrosine aminotransferase were analyzed by reverse transcription-polymerase chain reaction [RT-PCR]. The expressions of ALB and CK-18 in the cytoplasm were analyzed by Immunohistochemistry. The immunoblotting and chemiluminescence of the conditioned media indicated the secretion of ALB and AFP. RT-PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of hESC. Our results showed the expression of endoderm and hepatic specific genes after in vitro differentiation of hESC into hepatocyte-like cells through addition of various growth factors in three dimensional culture systems [collagen type I]. hESC could be a new potential source of hepatocyte for transplantation in patients with liver failure