Differential expression of glutathione S-transferases P1-1 and A1-1 at protein and mRNA levels in hepatocytes derived from human bone marrow mesenchymal stem cells Abdolamir Allameh a, * , Shahnaz Esmaeli a , Somaieh Kazemnejad a , Masoud Soleimani b a Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Aleahmad-Chamran Crossing, Tehran 14115-111, Iran b Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran article info Article history: Received 16 June 2008 Accepted 28 January 2009 Available online 5 February 2009 Keywords: Mesenchymal stem cells Differentiation Hepatocyte Glutathione S-transferase Expression abstract The aim of this study was to find out the profile of cellular glutathione (GSH) and GSH S-transferase (GST) in hepatocytes differentiated from adult mesenchymal stem cells (MSC). For this purpose, we have derived functionally active hepatocyte-like cells from normal human multipotent adult MSC. Then the differentiated cells were characterized by specific hepatic markers. The cellular GSH and GST catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined in hepatocyte-like cells differenti- ated from MSC compared with undifferentiated MSC. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting techniques were used to study GST-P1-1 and GST-A1-1 expression in dif- ferentiated and undifferentiated cells. The results showed that there is more than threefold increase in GST catalytic activity in hepatocytes recovered by day 14 of differentiation. GST-P1-1 mRNA expression was detected in both differentiated hepatocyte-like cells and their undifferentiated progenitors. Under similar conditions, only differentiated hepatocyte-like cells expressed GST-A1-1 mRNA. These results were further confirmed by showing that the undifferentiated cells expressed both GST-A and GST-P pro- teins. Unlike GST, the level of cellular GSH was declined (20%) in hepatocytes derived from MSC as com- pared to that of undifferentiated cells. These data may suggest that hepatogenic differentiation of human bone marrow MSC is accompanied with the regulation of factors participating in GSH conjugation pathway. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Human hepatocytes can potentially be derived from either adult or embryonic stem cells (Suzuki et al., 2002). Adult stem cells are more desirable than embryonic stem cells, since they are more differentiated so they do not cause tumors and these cells are less ethically contentious than embryonic stem cells (O’Donoghue and Nicholas, 2004). Moreover, utilizing patients own bone marrow can avoid or reduce immunological rejection (Shu et al., 2004). Adult stem cells (ASCs), also known as mesenchymal stem cells (MSCs) or multipotent adult progenitor cells (MAPCs) are special- ized cells found within many tissues (Pittenger et al., 1999; Clarke et al., 2000; Toma et al., 2001; Zuk et al., 2001, 2002; Zhao et al., 2003). Previous studies showed that MSCs can be differentiated into wide variety of cell types such as osteoblasts, adipocytes, chondrocytes, endothelial cells, neurons, cardiac myocytes and hepatocytes (Bianco et al., 2001; Chen et al., 2001; Wislet-Gende- bien et al., 2003; Orlic et al., 2001). It has also been reported that MSCs derived hepatocytes-like cells contribute to the regeneration of liver tissue (Lagasse et al., 2000; Wang et al., 2002). Schwartz et al. (2002) showed that human bone marrow MSCs (hBMSCs) could differentiate into functional hepatocytes with capacity of expressing hepatocyte markers such as albumin, alpha-fetoprotein (AFP), cytokeratin-18 (CK-18), cytokeratin-19 (CK-19), cytochrome P450 (CYP1B1 and CYP2B6). Very recently we reported that the hepatocyte-like cells differentiated from human adult MSCs with functional properties can be maintained for 21 days on an engi- neered three-dimensional (3D) scaffold (Kazemnejad et al., 2009). In addition to the liver specific markers normally used for char- acterization of the hepatocytes derived from stem cells, the cells are expected to be functionally active. In this connection the expression of the factors involved in phase-II drug metabolizing system is important with regard to xenobiotic metabolism and drug resistance. Cytochrome P-450 (CYP) enzymes which play a major role in metabolic activation of a wide variety of drugs and chemical carcinogens is believed to be expressed and undergo induction during the differentiation of BMSCs to hepatocyte-like cells (Raucy et al., 2002). Phase I drug metabolizing reactions are always coupled with phase II detoxification reactions and the products of CYP450-med- iated reactions are good substrates for phase-II conjugation 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.01.018 * Corresponding author. Tel.: +98 21 82883585; fax: +98 21 88006544. E-mail address: allameha@modares.ac.ir (A. Allameh). Toxicology in Vitro 23 (2009) 674–679 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit