Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells

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Abstract

Embryonic stem (ES) cells have the potential to differentiate into various progenitor cells. Here we investigated the capacity of mouse ES cells to differentiate into renal tubular cells both in vitro and in vivo. After stably transfecting Wnt4 cDNA to mouse ES cells (Wnt4-ES cells), undifferentiated ES cells were incubated by the hanging drop culture method to induce differentiation to embryoid bodies (EBs). During culturing of the EBs derived from the Wnt4-ES cells, aquaporin-2 (AQP2) mRNA and protein were expressed within 15–20 days. The expression of AQP2 in Wnt4-EBs was enhanced in the presence of hepatocyte growth factor (HGF) and activin A. We next performed in vivo experiments by transplanting the Wnt4-EBs into the mouse renal cortex. Four weeks after transplantation, some portions of the EB-derived cells expressing AQP2 in the kidney assembled into tubular-like formations. In conclusion, our in vitro and in vivo experiments revealed two new findings: first, that cultured Wnt4-EBs have an ability to differentiate into renal tubular cells; and second, that Wnt4, HGF, and activin A may promote the differentiation of ES cells to renal tubular cells.

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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.

References (44)

  • A.G. Smith et al.

    Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides

    Nature

    (1988)
  • R.L. Williams et al.

    Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells

    Nature

    (1988)
  • T.C. Doetschman et al.

    The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium

    J. Embryol. Exp. Morphol.

    (1985)
  • A. Leahy et al.

    Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation

    J. Exp. Zool.

    (1999)
  • B. Weinhold et al.

    Srf(−/−) ES cells display non-cell-autonomous impairment in mesodermal differentiation

    EMBO J.

    (2000)
  • M. Schuldiner et al.

    Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells

    Proc. Natl. Acad. Sci. USA

    (2000)
  • A.J. Potocnik et al.

    Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells

    Proc. Natl. Acad. Sci. USA

    (1997)
  • J. Yamashita et al.

    Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors

    Nature

    (2000)
  • J.H. Kim et al.

    Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease

    Nature

    (2002)
  • B. Soria et al.

    Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice

    Diabetes

    (2000)
  • N. Lumelsky et al.

    Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets

    Science

    (2001)
  • J.A. Thomson et al.

    Embryonic stem cell lines derived from human blastocysts

    Science

    (1998)
  • Cited by (0)

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