Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells

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http://www.sciencedirect.com/science/article/pii/S0092867403003921

Abstract

Embryonic stem (ES) cells undergo extended proliferation while remaining poised for multilineage differentiation. A unique network of transcription factors may characterize self-renewal and simultaneously suppress differentiation. We applied expression cloning in mouse ES cells to isolate a self-renewal determinant. Nanog is a divergent homeodomain protein that directs propagation of undifferentiated ES cells. Nanog mRNA is present in pluripotent mouse and human cell lines, and absent from differentiated cells. In preimplantation embryos, Nanog is restricted to founder cells from which ES cells can be derived. Endogenous Nanog acts in parallel with cytokine stimulation of Stat3 to drive ES cell self-renewal. Elevated Nanog expression from transgene constructs is sufficient for clonal expansion of ES cells, bypassing Stat3 and maintaining Oct4 levels. Cytokine dependence, multilineage differentiation, and embryo colonization capacity are fully restored upon transgene excision. These findings establish a central role for Nanog in the transcription factor hierarchy that defines ES cell identity.

Article Outline

• Introduction
• Results
• • A Functional Screen for ES Cell Self-Renewal Determinants
  • Characterization of the Nanog cDNA
  • Nanog mRNA Appears Confined to Pluripotent Tissues and Cell Lines
  • ES Cell Identity Is Faithfully Maintained by Nanog Expression
  • Relationship of Nanog with Other ES Cell Regulators
• Discussion
• • A Variant Homeodomain Protein Mediates ES Cell Self-Renewal
  • Expression and Function of Nanog
  • The Transcription Factor Network for ES Cell Identity
• Experimental Procedures
• • ES Cell Culture
  • cDNA Library Construction and Characterization
  • cDNA Library Transfection and Screening
  • Preparation of Episomal Constructs
  • Colony Forming Assays
  • RNA Analysis
  • Preparation of Cells Expressing a loxP Flanked Nanog Transgene
  • Immunoblotting