Ex vivo expansion of neutrophil precursor cells from fresh and cryopreserved cord blood cells.
BACKGROUND
Neutropenia following cord blood (CB) transplantation may be abrogated by infusion of granulopoietic progenitor cells. The purpose of this study was to determine whether myeloid progenitors can be obtained by ex vivo expansion of cryopreserved cord blood aliquots, and whether these progenitors present the morphologic, biologic and functional properties of myeloid progenitors at various stages of differentiation.


METHODS
The cells, plated for 7 days in serum-free medium with SCF, IL-3, G-CSF, Flt3-ligand and thrombopoietin in various combinations were assessed for the expression of CD34, CD38 and CD13. Maturation of cells into the myeloid lineage was evaluated by the expression of CD15, CD11b and CD16 and by the presence of primary (myeloperoxidase) and secondary granules (lactoferrin). The capacity of cells to phagocyte latex beads was evaluated to assess their functionality.


RESULTS
We have shown that a). CD34+ cells isolated from thawed samples were able to produce expansions similar to fresh samples. b). The best combination for the expansion of neutrophil precursor cells was S3FG; c). in these conditions, all stages of myeloid progenitors were represented, but few mature cells were observed. d). However, when the cells were plated on a BM stroma to try to reproduce conditions occurring during transplant, they acquired rapidly the characteristics of mature segmented cells. e). The ex vivo generated granulocytes were able to phagocyte latex beads.


CONCLUSIONS
In conclusion, it seems reasonable to systematically aliquot CB samples before cryopreservation. Some aliquots can then be thawed, enriched in CD34+ cells and ex vivo differentiated into myeloid lineage, while the other aliquots are conserved to be infused without manipulation.
Experimental Hematology, Jules Bordet Institute, Brussels, Belgium.
C
C
De Bruyn
A
A
Delforge
M
M
Bernier
D
D
Bron
D018952:Antigens, CD34 / Q000276:immunology; D001681:Biological Assay; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000502:physiology*; D002455:Cell Division / Q000502:physiology*; D015925:Cryopreservation; D005312:Fetal Blood / Q000166:cytology*; D016179:Granulocyte Colony-Stimulating Factor; D016298:Hematopoietic Cell Growth Factors; D006801:Humans; D007377:Interleukin-3; D023461:Myeloid Progenitor Cells / Q000166:cytology / Q000276:immunology / Q000502:physiology*; D009504:Neutrophils / Q000502:physiology; D010587:Phagocytosis / Q000502:physiology; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
D018952:Antigens, CD34; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C404145:myelopoietin; D016179:Granulocyte Colony-Stimulating Factor