Ex vivo myeloid differentiation of cord blood CD34+ cells: comparison of four serum-free media containing bovine or human albumin.
BACKGROUND
Infusion of ex vivo generated myeloid post-progenitor cells associated with unmanipulated cells appears to be a promising approach to reduce neutropenia following cord blood (CB) transplant. We compared four commercially available serum-free media, two containing BSA and two containing human albumin, on the in vitro differentiation of CB CD34+ cells into post-myeloid progenitor cells.


METHODS
CB CD34+ cells were cultured for 7 days in CTM-H00 (Mabio-International), StemSpanH2000 (StemCell Technologies), RM-B00 (Mabio-International) and Stem(alpha)A (Stem Alpha). The cells were stimulated by SCF, G-SCF, IL3 and Flt3-ligand (FL) added once at Day 0. Expansion was evaluated as the increase of leucocytes, CD34+ cells and CD13+ cells. Maturation of cells into the myeloid lineage was evaluated by expression of CD15, CD11b and CD16 Ags and by the presence of primary (myeloperoxydase, MPO) and secondary granules (lacoferrin, LF). The capacity of cells to phagocyte latex beads was evaluated to assess their functionality.


RESULTS
We observed that: a) the mononuclear cell and CD34+ cell expansions were significantly different according to the medium tested (respectively 61.5 +/- 7.7 and 15.5 +/- 3.4 for RM-B00, 37.3 +/- 5.4 and 10.3 +/- 1.6 for CTM-H00, 23.2 +/- 6.5 and 5.8 +/- 0.9 for StemSpanH2000 and 16.6 +/- 2.4 and 3.9 +/- 0.7 for Stem(alpha)A; b) the expansion of myeloid precursors is higher with RM-B00, similar with CTM-H00 and StemSpanH2000, and lower with Stem(alpha)A. This difference is essentially due to total leucocyte expansion, rather than to a selective expansion of myeloid cells, except for Stem(alpha)A, for which the percentages of neutrophil precursor cells [promyelocytes (CD15+ CD11b+), myelocytes (CD11b+ CD16-) and mature cells (CD11b+ CD16+)] are significantly decreased.


CONCLUSIONS
It appears that during ex vivo differentiation into myeloid lineages, the medium is critical and should be systematically screened before use in preclinical protocols. The use of human rather than bovine albumin, seems to have neither a negative, nor positive impact on the effectiveness of the medium.
Experimental Hematology, Jules Bordet Institute, Brussels, Belgium.
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De Bruyn
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Delforge
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Bron
D000418:Albumins / Q000494:pharmacology*; D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D005260:Female; D018380:Hematopoietic Stem Cell Transplantation / Q000009:adverse effects / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; D023461:Myeloid Progenitor Cells / Q000187:drug effects* / Q000637:transplantation*
D000418:Albumins; D018952:Antigens, CD34; D016895:Culture Media, Serum-Free