Cell-cycle kinetics and VSV-G pseudotyped retrovirus-mediated gene transfer in blood-derived CD34+ cells.
As hematopoietic stem and progenitor cells have a low mitotic index, we have quantitated the impact of cytokine combinations on cell cycling of CD34+ cells and, using VSV-G pseudotyped retroviral vectors, correlated our findings with ex vivo gene transfer. We tested nine different combinations of cytokines for induction of human peripheral blood CD34+ cells into cell cycle over 72 hours. Using the 5-bromodeoxyuridine-Hoechst 33258 (BrdU-Hoechst) assay, we measured the cell-cycle kinetics. The combinations of cytokines tested that were most efficient in inducing the CD34+ cells into cycle were stem cell factor (SCF) plus one of the following: interleukin-1 (IL-1), IL-3, granulocyte colony-stimulating factor (G-CSF). The maximum numbers of cells in S+G2M phase were observed after 48 hours of culture. At least 35 +/- 5% of the CD34+ cells remained quiescent in the first G0/G1 phase, however, no matter which cytokine combination was used. Cell-cycle analysis of the CD34+CD38- subset by 7-amino actinomycin D staining did not detect cycling cells during 72 hours of culture with any of the cytokines tested. To investigate whether the cells could be infected by the VSV-G pseudotyped virus containing the neomycin phospho-transferase gene (neo), we exposed CD34+ cells to the virus for 7-8 hours after 0, 36, and 48 hours of cytokine stimulation. Total CD34+ cells and the CD34+CD38- subset were analyzed by polymerase chain reaction (PCR) for reverse-transcribed viral DNA of the neomycin resistance gene (RT-neoDNA). Immediately after exposure to the virus, RT-neoDNA was detectable in CD34+ cells that have been cultured with or without cytokines for 36 to 48 hours. Forty-eight hours postinfection, however, RT-neoDNA could be detected only with cytokine combinations that induced mitosis of the CD34+ cells, consistent with the requirement for mitotic activity for retroviral integration. Similar experiments performed with the 34+CD38- subset showed that RT-reoDNA could not be detected at any time point. Thus, postinfection RT-neoDNA could be immediately detected in noncycling CD34+ cells but not in CD34+CD38- cells. These results suggest during short-term liquid culture, there may be blocks for reverse transcription of retroviral RNA in CD34+CD38-cells in addition to the lack of mitotic activity.
Agrawal
Y P
YP
Department of Medicine, University of California, San Diego School of Medicine, USA.
Agrawal
R S
RS
Sinclair
A M
AM
Young
D
D
Maruyama
M
M
Levine
F
F
Ho
A D
AD
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D017209:Apoptosis; D001483:Base Sequence; D001773:Blood Cells; D002453:Cell Cycle; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016130:Immunophenotyping; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000032:analysis; D011533:Proviruses / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D017931:DNA Primers; D008562:Membrane Glycoproteins; D012194:RNA-Directed DNA Polymerase; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1