Ex-vivo expansion of CFU-GM and BFU-E in unselected PBMC cultures with Flt3L is enhanced by autologous plasma.
BACKGROUND
Previous ex-vivo expansion studies in our laboratory, comparing unselected and CD34(+)-selected PBMC, have shown no advantage for CD34(+) cell selection, in terms of the expansion achieved. Our goal was to develop procedures for consistent generation of large numbers of hematopoietic progenitor and post-progenitor cells from unselected PBMC.


METHODS
Unselected PBMC, collected from cancer patients undergoing apheresis prior to high-dose chemotherapy and autologous stem cell rescue, were expanded ex vivo in static cultures, without a stromal layer, in the presence of Flt3 ligand (Flt3L), a recombinant GM-CSF/IL-3 fusion protein (PIXY321), G-CSF and GM-CSF for 10 days.


RESULTS
The addition of 2% autologous plasma to this cytokine combination enhanced expansion of total cell numbers (3.2 fold versus 1.9 fold; p < 0.01), colony-forming units granulocyte-macrophage (CFU-GM) (22.0 fold versus 8.1 fold, p < 0.01) and burst-forming units erythroid (BFU-E) (17.6 fold versus 7.0 fold, 0.01 < p < 0.02). The optimal seeding density for a given specimen was inversely related to the frequency of CD34(+) cells in the sample. CFU-GM expansion with the Flt3L-containing cytokine cocktail was equivalent to that obtained with IL-3, IL-6, G-CSF and SCF, whether or not the cultures were supplemented with autologous plasma. In plasma-free cultures, BFU-E expansion was significantly higher with IL-3, IL-6, G-CSF and SCF than with Flt3L, PIXY321, G-CSF and GM-CSF. In the presence of autologous plasma, however BFU-E expansion was higher in the Flt3L-containing media. In comparison studies, autologous plasma suppressed BFU-E expansion in SCF-containing cultures. Consistent with our colony assay results, dual-parameter flow cytometric analysis of the expanded cell population revealed that supplementation with autologous plasma yielded a significant increase in the numbers of myeloid progenitors in Flt3L-containing cultures.


CONCLUSIONS
Unselected PBMC from cancer patients can be effectively expanded ex vivo in Flt3L, PIXY321, G-CSF and GM-CSF, supplemented with autologous plasma, yielding high numbers of myeloid and erythroid progenitors.
Guo
M
M
Robert H Lurie Cancer Center, Department of Medicine, Northwestern University, Evanston and Chicago, USA.
Miller
W M
WM
Papoutsakis
E T
ET
Patel
S
S
James
C
C
Goolsby
C
C
Winter
J N
JN
D018952:Antigens, CD34 / Q000096:biosynthesis*; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D005947:Glucose / Q000378:metabolism; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D055014:Granulocyte-Macrophage Progenitor Cells / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008565:Membrane Proteins / Q000096:biosynthesis*; D009369:Neoplasms / Q000628:therapy; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology*
D018952:Antigens, CD34; D016207:Cytokines; D008565:Membrane Proteins; C084881:flt3 ligand protein; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005947:Glucose