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Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates
Masaaki Takatoku, … , Robert E. Donahue, Cynthia E. Dunbar
Masaaki Takatoku, … , Robert E. Donahue, Cynthia E. Dunbar
Published August 1, 2001
Citation Information: J Clin Invest. 2001;108(3):447-455. https://doi.org/10.1172/JCI12593.
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Article

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates

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Abstract

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34+ cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.

Authors

Masaaki Takatoku, Stephanie Sellers, Brian A. Agricola, Mark E. Metzger, Ikunoshin Kato, Robert E. Donahue, Cynthia E. Dunbar

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Figure 1

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(a) In vitro cell growth of PB CD34+ cells. The CD34+ cells were culture...
(a) In vitro cell growth of PB CD34+ cells. The CD34+ cells were cultured in the presence of MGDF/SCF/FLT/FN for 4 days. On day 4 the cells were split into three equal fractions. One was continued in culture in the presence of SCF/MGDF/FLT/FN (MSF/MSF). The second was transferred to SCF/FN (MSF/SCF). The third was maintained without cytokines on FN (MSF/FN). Shown are the mean ± SD of viable cell numbers obtained in independent experiments using cells from three different animals. (b) Cell cycle analysis. PB CD34+ cells were stained with PI and analyzed for DNA content. The percentage of cells in S/G2/M phases of the cell cycle are shown. A significantly lower percentage of cells in active cycle with MSF/SCF treatment than with MSF/MSF at days 6, 8, and 10 (P < 0.05 for each time point). (c) Cell cycle analysis of PB CD34+ cells cultured and transduced for 4 days in the presence of MGDF/SCF/FLT/FN with a retroviral vector expressing the eGFP gene, and then either continued in MGDF/SCF/FLT/FN without further transduction (MSF/MSF) or transferred to SCF/FN (MSF/SCF). The percentage of GFP-positive cells in the S/G2/M phases of the cell cycle are shown. (d) Apoptosis analysis. The percentage of apoptotic cells is shown for cells cultured with SCF/MGDF/FLT/FN for 4 days, and then either continued in the three cytokines (MSF/MSF), transferred to SCF/FN (MSF/SCF), or transferred to FN alone (MSF/FN). Transfer to FN alone (MSF/FN) resulted in a significant increase in apoptosis compared with both MSF/MSF and MSF/SCF (P < 0.005 for both comparisons).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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