Induced Pluripotent Stem Cell Generation Using a Single Lentiviral Stem Cell Cassette

Stem Cells. 2009 Mar;27(3):543-9. doi: 10.1634/stemcells.2008-1075.

Abstract

Induced pluripotent stem (iPS) cells can be generated using retroviral vectors expressing Oct4, Klf4, Sox2, and cMyc. Most prior studies have required multiple retroviral vectors for reprogramming, resulting in high numbers of genomic integrations in iPS cells and limiting their use for therapeutic applications. Here we describe the use of a single lentiviral vector expressing a "stem cell cassette" composed of the four transcription factors and a combination of 2A peptide and internal ribosome entry site technology, generating iPS cells from postnatal fibroblasts. iPS cells generated in this manner display embryonic stem cell-like morphology, express stem cell markers, and exhibit in vivo pluripotency, as evidenced by their ability to differentiate in teratoma assays and their robust contribution to mouse chimeras. Combining all factors into a single transcript achieves the most efficient reprogramming system to date and allows derivation of iPS cells with a single viral integration. The use of a single lentiviral vector for reprogramming represents a powerful laboratory tool and a significant step toward the application of iPS technology for clinical purposes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Southern
  • Cells, Cultured
  • Genetic Vectors / genetics
  • Kruppel-Like Transcription Factors / genetics
  • Kruppel-Like Transcription Factors / physiology
  • Lentivirus / genetics*
  • Mice
  • Octamer Transcription Factor-3 / genetics
  • Octamer Transcription Factor-3 / physiology
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism*
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / physiology
  • Reverse Transcriptase Polymerase Chain Reaction
  • SOXB1 Transcription Factors / genetics
  • SOXB1 Transcription Factors / physiology

Substances

  • GKLF protein
  • Kruppel-Like Transcription Factors
  • Myc protein, mouse
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • Proto-Oncogene Proteins c-myc
  • SOXB1 Transcription Factors
  • Sox2 protein, mouse