Establishment of serum-free pre-colony forming unit assays for differentiation of primitive hematopoietic progenitors: serum induces early macrophage differentiation and inhibits early erythroid differentiation of CD34++CD38- cells.
In this report we show that serum has differentiation-inducing effects on primitive hematopoietic progenitor cells with the CD34++CD38- immunophenotype. Using the pre-colony forming unit (pre-CFU) assay as a model for early myelopoiesis, we compared the effects of serum-containing and serum-free media and evaluated different cytokine cocktails [interleukin (IL)-1, IL-3, IL-6, kit ligand with and without the Flt3/Flk2 ligand (FL)]. In this assay, pre-CFUs are defined as cells unable to form colonies when plated directly in semi-solid assays, but which can differentiate into CFUs when cultured in liquid medium containing early-acting cytokines. In one of the investigated serum-free media, the average myeloid expansion in liquid medium reached up to more than 50% of that obtained in serum-containing medium. In addition, our experiments revealed differences in the clonogenic output between cells cultured in serum-free medium and those cultured in serum-containing medium, demonstrating that serum has a monocyte differentiation-inducing effect on primitive hematopoietic progenitors. Also in serum-free medium, higher proportions of erythroid progenitors were generated. These differentiation-inducing effects of serum further emphasize the need for serum-free culture protocols for hematopoietic graft engineering. Addition of FL to the culture media ameliorated cellular expansion and resulted in a decrease in the proportion of erythroid and granulocyte progenitors and an increase in the proportion of monocyte progenitors. In conclusion, this study shows that good serum-free conditions are available for differentiation assays with primitive hematopoietic progenitors and demonstrates that serum and FL have biasing effects on the initial phase of hematopoietic differentiation, favoring the monocyte lineage.
Laboratory of Experimental Hematology, University of Antwerp, Antwerp University Hospital, Edegem, Belgium.
R
R
Willems
E
E
Henckaerts
M
M
Lenjou
G
G
Nijs
I
I
Rodrigus
A C
AC
Moulijn
H
H
Slegers
Z N
ZN
Berneman
D R
DR
Van Bockstaele
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D000954:Antigens, Surface / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D003114:Colony-Forming Units Assay / Q000379:methods*; D016895:Culture Media, Serum-Free / Q000592:standards; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000276:immunology; D005434:Flow Cytometry; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008024:Ligands; D008264:Macrophages / Q000166:cytology; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000032:analysis; D010641:Phenotype
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D000954:Antigens, Surface; D016895:Culture Media, Serum-Free; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C084881:flt3 ligand protein; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1