Transduction of CD34(+) and CD34(-)/lin(-) hemopoietic progenitors by lentivirus vectors

Cytotherapy. 1999;1(6):433-8. doi: 10.1080/0032472031000141302.


Background: While hemopoietic stem cells have been thought to reside predominantly in the CD34(+) population, recent data suggests that repopulating cells may, in fact, also reside in the lin(-)/CD34(-) population. Transduction of both these populations by murine retroviral vectors is limited by quiescence of hemopoietic stem cells.

Methods: We therefore sought to transduce these populations using a VSV-G pseudotyped, HIV-based, human lentiviral vector, encoding eGFP. CD34(+) cells and lin(-)/CD34(-) cells were selected from the same BM samples by immunomagnetic beads (StemSep) to deplete lineage-positive cells and by CD34 selection columns (Miltenyi) to separate CD34(+) and CD34(-) populations. We transduced target cells, with or without prestimulation with cytokines, using conventional suspension culture, or fibronectin plates, or flow-through transduction. Transduction efficiency was analyzed by flow cytometry and clonogenic assay.

Results: We found that transduction on fibronectin plates was more efficient than flow-through transduction, or suspension cultures, for both cell populations. Mean transduction rates on fibronectin plates, analyzed by flow cytometry, were 14% and 5% for CD34(+) cells and lin(-)/CD34(-) cells respectively, without prestimulation, and 31% and 20% with prestimulation. By contrast, a murine retroviral vector transduces CD34(+) cells with lower efficiency (mean 16.1% with prestimulation) and does not induce any significant transduction of the CD34(-)/lin(-) population (mean < 2%). When lentiviral transduction was assayed in short- and long-term clonogenic assays there was minimal transduction of CD34 cells without prestimulation, increasing to 20% with prestimulation.

Discussion: Lentiviral eGFP vectors can transduce hematopoietic progenitors effectively and efficiency is improved by cytokine prestimulation and the use of fibronectin. Moreover, the human viral vectors can transduce a candidate stem-cell population that is resistant to murine retroviral transduction.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antigens, CD34 / biosynthesis*
  • Fibronectins / chemistry
  • Fibronectins / metabolism
  • Flow Cytometry / methods
  • Genetic Vectors
  • Green Fluorescent Proteins / chemistry
  • HIV / genetics
  • Hematopoietic Stem Cells / cytology*
  • Humans
  • Immunomagnetic Separation / methods
  • Lentivirus / genetics*
  • Membrane Glycoproteins / metabolism
  • Retroviridae / genetics
  • Stem Cells / cytology
  • Viral Envelope Proteins / metabolism


  • Antigens, CD34
  • Fibronectins
  • G protein, vesicular stomatitis virus
  • Membrane Glycoproteins
  • Viral Envelope Proteins
  • Green Fluorescent Proteins