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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 3

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Analysis of engraftment with transduced cells in vivo. (a) PCR analysis ...
Analysis of engraftment with transduced cells in vivo. (a) PCR analysis of neo sequences in PBMCs. 96E025 and RC706 received cells transduced with LNL6 for 4 days in MGDF/SCF/FLT/FN (active) and cells transduced with G1Na for 4 days in MGDF/SCF/FLT/FN that were then transferred to SCF/FN for two additional days without further exposure to vector (rested). 96E019 cells received the reverse vector treatment: G1Na for 4 days in MGDF/SCF/FLT/FN (active) and LNL6 for 4 days then transferred to SCF/FN for two additional days without further exposure to vector (rested). DNA from PBMCs at the indicated number of weeks after transplantation (W) was analyzed by PCR. Serial dilutions of G1Na vector–containing DNA with a known number of integrated copies were made using normal rhesus PB DNA at the indicated percentages; known-copy-number LNL6 DNA and mixtures of LNL6 and G1Na controls are shown. Dash (–) indicates concurrently extracted control rhesus PB DNA; H2O: reagent control. (b) Southern blot analysis of genomic marking levels in RC706 and 96E019, using DNA samples from PBMCs at the indicated number of weeks after transplantation, and positive standards made by diluting known-copy-number cell-line DNA into normal rhesus DNA. Samples were digested with KpnI, an enzyme that cuts within the LTRs in both LNL6 and G1Na. Due to residual env sequences in LNL6 that are not present in G1Na, the expected fragment size is 3.0 kb in LNL6 and 2.3 kb in G1Na.

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

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