title: Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer.
abstract: Retroviral gene transfer into human myeloid precursor cells allows introduction of marker genes as well as genes conferring resistance to chemotherapeutic drugs. We transduced a human mutant dihydrofolate reductase (DHFR) cDNA into CD34 antigen-positive peripheral blood cells from patients with breast or ovarian cancer obtained after treatment with chemotherapy and granulocyte colony-stimulating factor (G-CSF). This mutant DHFR has been shown to confer resistance to methotrexate (MTX) in murine bone marrow. We established a transduction protocol that permitted ex vivo expansion and selection of transduced early progenitor cells. The number of progenitor cells from transduced CD34-positive cells increased 50-fold after cytokine prestimulation with interleukin-1 (IL-1), c-kit ligand (KL; stem cell factor), and IL-3 and 2 weeks in liquid culture. Transduced colony-forming unit-granulocyte-macrophage (CFU-GM), assayed directly after the transduction procedure, were protected completely against 2 x 10(-8) mol/L MTX, a concentration that significantly reduced the CFU-GM detected in the control population. Gene transfer of the mutant DHFR led to a twofold selective advantage for a pre-CFU population after exposure to MTX in liquid culture (P < .001). Polybrene, in contrast with protamine, significantly inhibited the expansion of progenitors. The presence of proviral DNA was monitored by polymerase chain reaction (PCR) and was detected in greater than 80% of CFU-GM and ex vivo expanded pre-CFU. We have demonstrated that human hematopoietic precursor cells can be expanded extensively after retroviral gene transfer. The same population of early progenitors can be selected ex vivo with low-dose MTX. As long-term expression of transduced genes in human hematopoietic cells remains a problem in vivo, these results may have implications for future clinical trials, especially for the introduction of nonselectable genes.
authors.affiliation: James Ewing Laboratory of Developmental Hematopoiesis, Sloan-Kettering Institute for Cancer Research, New York, NY 10021.
authors.forename: M
authors.initials: M
authors.lastname: Flasshove
authors.forename: D
authors.initials: D
authors.lastname: Banerjee
authors.forename: S
authors.initials: S
authors.lastname: Mineishi
authors.forename: M X
authors.initials: MX
authors.lastname: Li
authors.forename: J R
authors.initials: JR
authors.lastname: Bertino
authors.forename: M A
authors.initials: MA
authors.lastname: Moore
mesh_terms: D015703:Antigens, CD; D018952:Antigens, CD34; D001483:Base Sequence; D015415:Biomarkers; D001943:Breast Neoplasms / Q000097:blood; D002455:Cell Division; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D005822:Genetic Vectors; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006410:Hematopoiesis / Q000187:drug effects; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000097:blood; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D012641:Selection, Genetic; D019089:Stem Cell Factor; D014162:Transfection
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; D018076:DNA, Complementary; D004279:DNA, Viral; D016298:Hematopoietic Cell Growth Factors; D007375:Interleukin-1; D007377:Interleukin-3; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D008727:Methotrexate