Biochemical and Biophysical Research Communications
Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells
Section snippets
Materials and methods
Cell culturing of ES cells and EBs. Undifferentiated ES cells (BL6 cell line) were maintained on 0.1% gelatin-coated dishes in Knockout Dulbecco’s modified Eagles’s medium containing 20% Knockout serum replacement (Gibco-BRL, Grand-Island, NY), 300 μmol/L monothioglycerol (Sigma–Aldrich, St. Louis, MO), 2 mmol/L l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 25 mmol/L Hepes (Gibco-BRL). The medium was supplemented with 1000 U/ml recombinant mouse LIF (Gibco-BRL). To induce
Western blot analysis of ES cells transfected with HA-tagged Wnt4
ES cells were transfected by the electroporation method with 20 μg Wnt4 plasmid containing the neo resistant gene and the HA gene. The cells were cultured in a medium containing G418 to expand the G418-resistant clones. The exogenous Wnt4 expression of each clone at 20 days after the electroporation was examined by Western blot analysis using an anti-HA antibody and an anti-Wnt4 antibody. Fig. 1A demonstrates the Western blot analysis of two typical Wnt4-positive ES clones and control ES cells.
Discussion
During the culturing of EBs derived from Wnt4-ES cells, AQP2 mRNA was expressed within 15–20 days. The expression of AQP2 in Wnt4-EBs was enhanced in the presence of HGF and activin A. Wnt4-EB cells cultured with HGF and activin A in the three-dimensional gels formed tubular-like formations and expressed AQP2 mRNA. These isolated cells from Wnt4-EBs were transplanted into the mouse kidney, resulting in the formation of teratomas that contained tubular-like formations and expressed AQP2.
This
Acknowledgments
This study was supported by Health and Labor Science Research Grants for Research on Specific Diseases from the Ministry of Health, Labor, and Welfare, and Mochida Memorial Foundation.
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