Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability.
The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)-mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with 0.01% in the original IL-3/IL-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells.
Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892-1652, USA. tisdalej@gwgate.nhlbi.nih.gov
J F
JF
Tisdale
Y
Y
Hanazono
S E
SE
Sellers
B A
BA
Agricola
M E
ME
Metzger
R E
RE
Donahue
C E
CE
Dunbar
D000818:Animals; D002478:Cells, Cultured / Q000637:transplantation; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D000069585:Filgrastim; D017930:Genes, Reporter; D005819:Genetic Markers; D005822:Genetic Vectors; D006085:Graft Survival; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000235:genetics; D008253:Macaca mulatta / Q000097:blood*; D011518:Proto-Oncogene Proteins / Q000494:pharmacology; D011828:Radiation Chimera; D020794:Receptor Protein-Tyrosine Kinases / Q000494:pharmacology; D011994:Recombinant Proteins; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology; D040281:Vascular Endothelial Growth Factor Receptor-1; D014916:Whole-Body Irradiation
D005819:Genetic Markers; D007377:Interleukin-3; D015850:Interleukin-6; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D019868:Kanamycin Kinase; D020794:Receptor Protein-Tyrosine Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1; D000069585:Filgrastim