The impact of ex vivo cytokine stimulation on engraftment of primitive hematopoietic cells in a non-human primate model.
The impairment of engraftment ability after ex vivo or in vivo stimulation of hematopoietic stem cells, potentially related to induction of active cell cycling, has recently been a topic of intense interest. Our group has used the non-human primate autologous transplantation model and genetic marking to investigate a number of questions in hematopoiesis with direct relevance to human clinical applications. The issue of a potential reversible engraftment defect would have many implications for gene therapy and allogeneic or autologous transplantation. Initial in vitro studies with rhesus CD34+ cells indicated that after 4 days of stimulatory culture in stem cell factor (SCF), megakaryocyte growth and development factor (MDGF), and flt3 ligand (FLT), transfer of the cells to SCF alone on retronectin (FN) support resulted in decreased active cycling and a halt to proliferation, without a loss of viability or induction of apoptosis. We then directly compared the engraftment potential of cytokine-stimulated cells versus those transferred to SCF on FN alone before reinfusion, SCF/G-CSF mobilized CD34+ cells from three animals were split into two parts and transduced with either of two retroviral marking vectors for 4 days in the presence of SCF/FLT/MGDF on FN. One aliquot was cryopreserved, and the other was continued in culture without transduction for 2 days in the presence of SCF alone on FN. After total body irradiation, both aliquots were thawed and reinfused into each animal. In all animals, the level of marking from the fraction continued in culture for 2 days with SCF on FN was significantly higher than the level of marking from the aliquot transduced for 4 days without the 2-day period in SCF alone. This approach may allow more efficient engraftment of successfully transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.
Dunbar
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Molecular Hematopoiesis Section, Hematology Branch, NHLBI, NIH, Building 10, Room 7C103, 9000 Rockville Pike, Bethesda, MD 20892, USA. dunbarc@nhlbi.nih.gov
Takatoku
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Donahue
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D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D002465:Cell Movement; D002478:Cells, Cultured / Q000187:drug effects; D005353:Fibronectins; D006085:Graft Survival; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D036341:Intercellular Signaling Peptides and Proteins; D008253:Macaca mulatta; D008565:Membrane Proteins / Q000494:pharmacology*; D011828:Radiation Chimera; D019089:Stem Cell Factor / Q000494:pharmacology*; D014162:Transfection; D014182:Transplantation, Autologous
D005353:Fibronectins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; C053894:mesenchyme-derived growth factor; D016179:Granulocyte Colony-Stimulating Factor