Short-term exposure of umbilical cord blood CD34+ cells to granulocyte-macrophage colony-stimulating factor early in culture improves ex vivo expansion of neutrophils.
OBJECTIVE
Despite the availability of modern antibiotics/antimycotics and cytokine support, neutropenic infection accounts for the majority of chemotherapy-associated deaths. While transfusion support with donor neutrophils is possible, cost and complicated logistics make such an option unrealistic on a routine basis. A manufactured neutrophil product could enable routine prophylactic administration of neutrophils, preventing the onset of neutropenia and substantially reducing the risk of infection. We examined the use of pre-culture strategies and various cytokine/modulator combinations to improve neutrophil expansion from umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HPC).


METHODS
Enriched UCB HPC were cultured using either two-phase pre-culture strategies or a single phase using various cytokine/modulator combinations. Outcome was assessed with respect to numerical expansion, cell morphology, granulation and respiratory burst activity.


RESULTS
Pre-culture in the absence of strong differentiation signals (e.g. granulocyte colony-stimulating factor; G-CSF) failed to provide any improvement to final neutrophil yields. Similarly, removal of differentiating cells during pre-culture failed to improve neutrophil yields to an appreciable extent. Of the cytokine/modulator combinations, the addition of granulocyte-macrophage (GM)-colony-stimulating factor (CSF) alone gave the greatest increase. In order to avoid production of monocytes, it was necessary to remove GM-CSF on day 5. Using this strategy, neutrophil expansion improved 2.7-fold.


CONCLUSIONS
Although all cytokines and culture strategies employed have been reported previously to enhance HPC expansion, we found that the addition of GM-CSF alone was sufficient to improve total cell yields maximally. The need to remove GM-CSF on day 5 to avoid monocyte differentiation highlights the context and time-dependent complexity of exogenous signaling in hematopoietic cell differentiation and growth.
Marturana
Flavia
F
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Queensland, St. Lucia, Australia.
Timmins
Nicholas E
NE
Nielsen
Lars K
LK
D018952:Antigens, CD34 / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004950:Esterases / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D010100:Oxygen / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology; D013997:Time Factors; D014635:Valproic Acid / Q000494:pharmacology
D018952:Antigens, CD34; D016207:Cytokines; D005029:Ethylenediamines; D036341:Intercellular Signaling Peptides and Proteins; D013481:Superoxides; D014635:Valproic Acid; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006493:Heparin; D004950:Esterases; D010100:Oxygen; D016559:Tacrolimus; C034269:tetraethylenepentamine