Stable genetic modification of human embryonic stem cells by lentiviral vectors

Mol Ther. 2003 Feb;7(2):281-7. doi: 10.1016/s1525-0016(02)00047-3.

Abstract

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of the early preimplantation embryo. An efficient strategy for stable genetic modification of hES cells may be highly valuable for manipulating the cells in vitro and may promote the study of hES cell biology, human embryogenesis, and the development of cell-based therapies. Here, we demonstrate that vectors derived from self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1) are efficient tools for stable genetic modification of hES cells. Transduction of hES cells by a modified vector derived from SIN HIV-1 and containing the woodchuck hepatitis regulatory element (WPRE) and the central polypurine tract (cPPT) sequence facilitated stable transgene expression during prolonged (38 weeks) undifferentiated proliferation in vitro. Southern blot analysis revealed that the viral vector had integrated into the host cells' DNA. Transgene expression was maintained throughout differentiation into progeny of all three germ layers both in vitro and in vivo in teratomas. Thus, the transduced hES cells retained the capability for self-renewal and their pluripotent potential. Genetic modification of hES cells by lentiviral vectors provides a powerful tool for basic and applied research in the area of human ES cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Southern
  • Cell Differentiation
  • Cell Line
  • Cell Separation
  • Embryo, Mammalian / cytology
  • Flow Cytometry
  • Genetic Vectors*
  • Green Fluorescent Proteins
  • HIV-1 / genetics*
  • Humans
  • Immunohistochemistry
  • Lentivirus / genetics*
  • Luminescent Proteins / metabolism
  • Mice
  • Mice, SCID
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • Stem Cells / metabolism*
  • Teratoma / metabolism
  • Time Factors
  • Transfection
  • Transgenes

Substances

  • Luminescent Proteins
  • Green Fluorescent Proteins