Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture.
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-DR(low) 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-DR(low) 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.
Berger
F
F
Fondazione Matarelli, Ospedale Fatebenefratelli e Oftalmico, Milan, Italy.
Soligo
D
D
Schwarz
K
K
Bossolasco
P
P
Schrezenmeier
H
H
Kubanek
B
B
Deliliers
G L
GL
Licht
T
T
D018952:Antigens, CD34 / Q000097:blood; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D019450:Genes, MDR / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006133:Growth Substances / Q000494:pharmacology; D006684:HLA-DR Antigens / Q000097:blood; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D006801:Humans; D012190:Retroviridae / Q000235:genetics*; D020112:Rhodamine 123 / Q000378:metabolism; D014161:Transduction, Genetic
D018952:Antigens, CD34; D016207:Cytokines; D018076:DNA, Complementary; D006133:Growth Substances; D006684:HLA-DR Antigens; D020112:Rhodamine 123