Comparative analysis of retroviral-mediated gene transduction into CD34+ cord blood hematopoietic progenitors in the presence and absence of growth factors.
Hematopoietic stem and progenitor cells present in umbilical cord blood at the birth of a child are efficiently transduced ex vivo by genes using retroviral vectors in combination with exposure of these cells to combinations of growth factors. Because retroviral-mediated gene transduction of adult bone marrow and blood hematopoietic stem and progenitor cells is greatly enhanced by growth factors, we evaluated the possibility that cord blood progenitors, which have extensive proliferative and replating capacity, could be efficiently transduced with a TK neo gene in a retroviral vector in the absence of growth factors, and also determined the influence of exogenously added growth factors on this transduction. Highly purified CD34+ (62% pure) cord blood cells isolated by magnetic bead separation were cultured in suspension for 72 hours with viral supernatant in the absence and presence of interleukin-3 (IL-3), IL-6, and steel factor. Evaluation of progenitor cell-derived colonies and polymerase chain reaction (PCR)/Southern analysis of the TK neo gene in resultant colony cells demonstrated that some gene transduction was apparent in the absence of growth factors (12.8-14.3% by PCR), but that this was greatly enhanced (40.0-44.4%) by addition of growth factors. Reverse transcription PCR analysis of the expression of IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor genes in this population of cells suggested that the transduction, although at a lower efficiency, in the absence of added growth factors might in part be due to "constitutive" and viral supernatant-induced expression of these cytokine genes in the CD34(+)-enriched cell population.(ABSTRACT TRUNCATED AT 250 WORDS)
Shi
Y J
YJ
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Shen
R N
RN
Lu
L
L
Broxmeyer
H E
HE
D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D001483:Base Sequence; D004351:Drug Resistance / Q000235:genetics; D005312:Fetal Blood / Q000166:cytology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018189:Immunomagnetic Separation; D007377:Interleukin-3 / Q000096:biosynthesis / Q000235:genetics; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection
D015703:Antigens, CD; D018952:Antigens, CD34; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D015850:Interleukin-6; D012333:RNA, Messenger; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013937:Thymidine Kinase; D009355:Neomycin