Evaluation of a cytokine combination including thrombopoietin for improved transduction of a retroviral gene into G-CSF-mobilized CD34+ human blood cells.
We examined cell culture conditions with various combinations of cytokines including thrombopoietin (TPO) to obtain the most efficient transduction of recombinant retrovirus vectors into G-CSF-mobilized blood CD34+ cells which were obtained from children and purified with an Isolex 50 system (Baxter; Deerfield, IL). Three different 4-day culture conditions for the stimulation of CD34+ cells were compared in terms of a cell-cycle analysis by fluorometry and gene transduction efficiency as determined by resistance to G418 and NeoR polymerase chain reaction (PCR) for individual colony-forming unit-granulocyte/macrophage (CFU-GM) grown in a methylcellulose culture system. The cytokines tested were: A) interleukin (IL)-6 + stem cell factor (SCF); B) IL-3 + IL-6 + SCF, and C) IL-3 + IL-6 + SCF + TPO. Without a cell culture, the percentage of CD34+ cells in the cell cycle (the percentage of cells in phases S and G2/M) was 4.6%. After a four-day culture (n = 5), this value increased with the addition of IL-3 (22%) or IL-3 + TPO (27%, p < 0.05) as compared to that with the baseline cocktail of IL-6 + SCF (15%). The cell number uniformly increased approximately 10-fold in each culture condition. The average efficiency of gene transfer into incubated CD34+ cells with the corresponding combinations of cytokines was, respectively, 57%, 47%, and 30% for G418-screened CFU-GM and 72%, 68%, and 51% for polymerase chain reaction-positive CFU-GM. A statistically significant difference (p < 0.01) was found for G418/CFU-GM with IL-3 + IL-6 + SCF (57%) versus IL-3 + IL-6 + SCF + TPO (30%). Hence, it is likely that the increased cell proliferation produced by the addition of TPO was not necessarily translated into an increased rate of retroviral-mediated gene transduction, possibly because TPO preferentially induced the differentiation of stem cells into mature progenitors in these culture systems.
Kim
H J
HJ
Department of Pediatrics, University of Tokushima, Japan.
Okamoto
Y
Y
Ito
M
M
Takaue
Y
Y
Kawano
Y
Y
Watanabe
T
T
Yamaue
T
T
Tohda
Y
Y
Ogose
T
T
Shimada
T
T
Shimosaka
A
A
Kuroda
Y
Y
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018952:Antigens, CD34 / Q000097:blood*; D002452:Cell Count / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000494:pharmacology*; D004359:Drug Therapy, Combination; D005069:Evaluation Studies as Topic; D018014:Gene Transfer Techniques; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D005839:Gentamicins / Q000494:pharmacology; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics*; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology*; D014161:Transduction, Genetic / Q000187:drug effects
D000900:Anti-Bacterial Agents; D018952:Antigens, CD34; D016207:Cytokines; D005839:Gentamicins; D007377:Interleukin-3; D015850:Interleukin-6; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D013926:Thrombopoietin; C010680:antibiotic G 418