Ex vivo expansion and selection of human CD34(+) peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer Journal Article

  

Authors:	Flasshove, M.; Banerjee, D.; Mineishi, S.; Li, M. X.; Bertino, J. R.; Moore, M. A. S.
Article 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 10-6 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.
Keywords:	gene mutation; human cell; methotrexate; polymerase chain reaction; ovarian neoplasms; biological markers; cells, cultured; cell division; stem cell factor; drug resistance; enzyme activity; transfection; breast neoplasms; gene transfer; genetic transduction; genetic vectors; stem cell; selection (genetics); molecular sequence data; recombinant fusion proteins; hematopoietic stem cells; base sequence; dna, viral; hematopoiesis; bone marrow cell; antigens, cd; dihydrofolate reductase; granulocyte colony-stimulating factor; antigens, cd34; multidrug resistance; marker gene; complementary dna; retrovirus; retroviridae; dna, complementary; virus vector; interleukin-1; virus gene; interleukin-3; virus transformation; human; female; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; proviruses; hematopoietic cell growth factors
Journal Title:	Blood
Volume:	85
Issue:	2
ISSN:	0006-4971
Publisher:	American Society of Hematology
Date Published:	1995-01-15
Start Page:	566
End Page:	574
Language:	English
PUBMED:	7529065
PROVIDER:	scopus
DOI:	10.1182/blood.V85.2.566.566
DOI/URL:	https://www.scopus.com/inward/record.uri?eid=2-s2.0-0028967307&partnerID=40&md5=11f7b752367ff8d232710f57069a2533
Notes:	Article -- Export Date: 28 August 2018 -- Source: Scopus

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