Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture

F. Berger, D. Soligo, K. Schwarz, P. Bossolasco, H. Schrezenmeier, B. Kubanek, G. Lambertenghi Deliliers, T. Licht

Research output: Contribution to journalArticlepeer-review


Retroviral transduction of hematopoietic cells has resulted in unsatisfactory gene marking in clinical studies. Since cytokine-stimulated stem cells have engrafted poorly in animal models, we investigated phenotypic changes during culture of peripheral blood progenitor cells (PBPC). Human CD34+ HLA-DRlow cells, immunomagnetically separated from PBPC collections, were found to extrude rhodamine-123, which is characteristic for primitive hematopoietic cells. Cells were grown in suspension cultures supplemented with cytokines. While interleukin-3-containing factor combinations promoted cell proliferation they caused loss of rhodamine-123 extrusion and reduced the frequencies of cobblestone area-forming cells (CAFC). Several other cytokines failed to stimulate cell divisions, which are required for retroviral transduction. A combination including Flt-3 ligand (FL), interleukin-6 and stem cell factor (SCF) preserved an immature phenotype for 5 to 6 days and stimulated cell divisions, which was improved upon addition of leukemia inhibitory factor and interleukin-11. Furthermore, the CAFC frequency among cells treated with these cytokines was increased as compared with widely used cocktails containing interleukin-3, interleukin-6 and SCF. Rhodamine-123 appeared to be a particularly sensitive indicator for differentiation of PBPC. For analysis of gene transfer, amphotropic retroviruses conferring an MDR1 cDNA were added repeatedly for 6 days to cytokine-treated PBPC stroma-free cultures. Proviral cDNA was detected by polymerase chain reaction in 68% of cobblestone areas derived from CD34+ HLA-DRlow cells that had been exposed to Flt-3 ligand, interleukin-6 and SCF. In summary, conditions were identified that facilitate efficient transduction of early PBPC with amphotropic retroviruses while preserving a primitive phenotype for extended periods.

Original languageEnglish
Pages (from-to)687-696
Number of pages10
JournalGene Therapy
Issue number9
Publication statusPublished - 2001


  • Gene therapy
  • Growth factors
  • Hematopoietic stem cells
  • Multidrug resistance
  • P-glycoprotein
  • Rhodamine

ASJC Scopus subject areas

  • Genetics


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