Embryonic stem (ES) cell s are capabl e of unlimi ted self-renewal and have a diverse dif ferentiation po- tential. The se unique features make ES cells as an attractive source for developmental bi ology stu dies. Having the mature hepatocyte in the lab with func tional activities is valuable in drug disc overy stu dies. Overexp ression of hepatocyte linea ge-speci fi c transcription factors (TFs) be comes a promising approach in plurip otent cell dif ferentiation toward liver cells. Many studies generate t ransgenic ES cell line s to exami ne the effect s of speci fi c TFs overexp ression in cell dif ferentiation. In the present report, we have addressed whether a suspe nsion or adherent model of differentiation is an appropriate way to study the role of Hnf4 a overexpression. We generated ES cells that carrie d a doxycycline (Dox)-in ducible Hn f4a using lentiviral vectors. The transduced cells were subjected to in duced Hnf4a overexp ression through both spontan eous and directed dif ferentiation method s. Gen e expression ana lysis showed substanti ally increased expression of hepati c gene markers, partic ularly Ttrand endogenous Hnf 4a, in t ransduced cells differentiated by the directed approach. These results demon strated t hat forced expression of TFs durin g directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation .
