|A role of Wnt/beta-catenin signals in hepatic fate specification of human umbilical cord blood-derived mesenchymal stem cells. |
Yoshida, Y; Shimomura, T; Sakabe, T; Ishii, K; Gonda, K; Matsuoka, S; Watanabe, Y; Takubo, K; Tsuchiya, H; Hoshikawa, Y; Kurimasa, A; Hisatome, I; Uyama, T; Terai, M; Umezawa, A; Shiota, G
American journal of physiology. Gastrointestinal and liver physiology
Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to be an excellent source of cells for transplantation. In addition, the stem cell plasticity of human UCBMSCs, which can transdifferentiate into hepatocytes, has been reported. However, the mechanisms involved remain to be clarified. To identify the genes and/or signals that are important in specifying the hepatic fate of human UCBMSCs, we analyzed gene expression profiles during the hepatic differentiation of UCBMSCs with human telomerase reverse transcriptase, UCBMSCs immortalized by infection with a retrovirus carrying telomerase reverse transcriptase, but whose differentiation potential remains unchanged. Efficient differentiation was induced by 5-azacytidine (5-aza)/hepatocyte growth factor (HGF)/oncostatin M (OSM)/fibroblast growth factor 2 (FGF2) treatment in terms of function as well as protein expression: 2.5-fold increase in albumin, 4-fold increase in CCAAT enhancer-binding protein alpha, 1.5-fold increase in cytochrome p450 1A1/2, and 8-fold increase in periodic acid-Schiff staining. Consequently, we found that the expression of Wnt/beta-catenin-related genes downregulated, and the translocation of beta-catenin was observed along the cell membrane and in the cytoplasm, although some beta-catenin was still in the nucleus. Downregulation of Wnt/beta-catenin signals in the cells by Fz8-small interference RNA treatment, which was analyzed with a Tcf4 promoter-luciferase assay, resulted in similar hepatic differentiation to that observed with 5-azacytidine/HGF/OSM/FGF2. In addition, the subcellular distribution of beta-catenin was similar to that of cells treated with 5-azacytidine/HGF/OSM/FGF2. In conclusion, the suppression of Wnt/beta-catenin signaling induced the hepatic differentiation of UCBMSCs, suggesting that Wnt/beta-catenin signals play an important role in the hepatic fate specification of human UCBMSCs.
|Synthetic peptide antigens elicit monoclonal and polyclonal antibodies to cytochrome P450 IA2. |
Myers, M J, et al.
Biochem. Biophys. Res. Commun., 169: 171-6 (1990)
Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and polyclonal antibodies. Antisera to both peptides reacted with rat IA2 but not the structurally similar IA1 form as determined by enzyme-linked immunosorbent assay. However, antisera to both peptides detected both rat IA2 and IA1 on immunoblots. In addition immunoblots of human liver microsomes revealed that both antisera recognized human IA2, but not IA1. Monoclonal antibodies generated against one of the peptides recognized rat IA2 and IA1 but did not detect human IA2. These results demonstrate the utility of anti-peptide antisera as a practical approach for the generation of P450 specific antibodies.