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Ex vivo expansion and selection of human CD34+ peripheral blood progenitor
cells after introduction of a mutated dihydrofolate reductase cDNA via
retroviral gene transfer
M Flasshove, D Banerjee, S Mineishi, MX Li, JR Bertino and MA Moore
James Ewing Laboratory of Developmental Hematopoiesis, Sloan-Kettering
Institute for Cancer Research, New York, NY 10021.
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.
Volume 85,
Issue 2,
pp. 566-574,
01/15/1995
Copyright © 1995 by The American Society of Hematology
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