Repression of Zeb1 and Hypoxia Cause Sequential Mesenchymal-to-Epithelial Transition and Induction of Aid, Oct4, and Dnmt1, Leading to Immortalization and Multipotential Reprogramming of Fibroblasts in Spheres§

Authors

  • Yongqing Liu,

    1. Molecular Targets Program, James Graham Brown Cancer CenterLouisville, Kentucky, USA
    2. Department of OphthalmologyLouisville, Kentucky, USA
    3. Birth Defects Center, University of Louisville Health Sciences Center, Louisville, Kentucky, USA
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  • Partha Mukhopadhyay,

    1. Birth Defects Center, University of Louisville Health Sciences Center, Louisville, Kentucky, USA
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  • M. Michele Pisano,

    1. Birth Defects Center, University of Louisville Health Sciences Center, Louisville, Kentucky, USA
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  • Xiaoqin Lu,

    1. Department of OphthalmologyLouisville, Kentucky, USA
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  • Li Huang,

    1. Department of OphthalmologyLouisville, Kentucky, USA
    2. School of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China
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  • Qingxian Lu,

    1. Molecular Targets Program, James Graham Brown Cancer CenterLouisville, Kentucky, USA
    2. Department of OphthalmologyLouisville, Kentucky, USA
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  • Douglas C. Dean

    Corresponding author
    1. Molecular Targets Program, James Graham Brown Cancer CenterLouisville, Kentucky, USA
    2. Department of OphthalmologyLouisville, Kentucky, USA
    3. Birth Defects Center, University of Louisville Health Sciences Center, Louisville, Kentucky, USA
    4. Department of Biochemistry and Molecular BiologyLouisville, Kentucky, USA
    • University of Louisville Health Sciences Center, 301 E. Muhammad Ali Blvd., Louisville, Kentucky 40202, USA
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    • Telephone: 502-852-4882; Fax: 502-852-0691


  • Author contribution: Y.L.: conception and design; collection/assembly of data; data analysis and interpretation; manuscript writing; final approval of manuscript; P.M., X.L., and L.H.: collection/assembly of data; data analysis and interpretation; final approval of manuscript; M.M.P.: data analysis and interpretation; final approval of manuscript; Q.L.: conception and design; data analysis and interpretation; final approval of manuscript; D.C.D.: conception and design; financial support; data analysis and interpretation; manuscript writing; final approval of manuscript.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    first published online in STEM CELLS EXPRESS April 4, 2013.

Abstract

In this study, we demonstrate that sphere formation triggers immortalization and stable reprogramming of mouse fibroblasts. Cell contact signaling in spheres causes downregulation of the epithelial-to-mesenchymal transition transcription factor Zeb1 leading to rapid mesenchymal-to-epithelial transition. Hypoxia within spheres together with loss of Zeb1 repression synergize to cause superinduction of Hif1a, which in turn leads to induction of the DNA demethylase Aid/Aicda, demethylation of the Oct4 promoter/enhancer and multipotency. Oct4 and Nanog expression diminish when cells are removed from the hypoxic environment of spheres and placed in monolayer culture, but the cells retain multipotential capacity, demonstrating stable reprogramming and a gene expression pattern resembling adult stem cells. Oct4 has been shown to induce Dnmt1 in mesenchymal stem cells, and we link Oct4 and Dnmt1 to silencing of cell cycle inhibitory cyclin dependent kinase inhibitors and Arf, and immortalization of the reprogrammed fibroblasts. Sphere formation then represents a novel and rapid protocol for immortalization and stable reprogramming of fibroblasts to multipotency that does not require exogenous expression of a stem cell factor or a lineage-specifying transcription factor. STEM Cells2013;31:1350–1362

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