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.

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. - In: GENE THERAPY. - ISSN 0969-7128. - 8:9(2001 May), pp. 687-696. [10.1038/sj.gt.3301455]

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

D. Soligo
Secondo
;
P. Bossolasco;G. Lambertenghi Deliliers
Penultimo
;
2001

Abstract

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.
Settore MED/15 - Malattie del Sangue
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/47326
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