Structural Specificity of the Biological Activity of Human and Murine Stem Cell Factor in Stress Erythropoiesis,

Abstract 3395

Stem cell factor (SCF) is a cyokine produced by bone marrow stromal cells that upon engagement of its cognate receptor cKit and in combination with other hematopoietic growth factors initiates downstream phosphorylation events that generate hematopoietic progenitors and precursors of all types. SCF and cKit are encoded by highly conserved genes. The human (hSCF) and murine (mSCF) forms are highly homologous and are 82% identical at the amino acid levels. Both have been reported to sustain differentiation of human mast cells from cord blood (CB) but mast cells generated with mSCF remain immature (Mitzu et al, PNAS 90:735–739). In addition, anecdotic reports indicate that hSCF is 100-fold less active than mSCF on mouse cells while mSCF and hSCF are equally active on human cells.

To clarify if structural differences between the two growth factors have biological significance, we compared cord blood (CB) mononuclear cells and CD34^pos cells as the stem cell source for in vitro production of colony forming cells, CFU-GM and BFU-E, using established techniques, and for ex-vivo expansion of megakaryocytes (MK) (SCF + IL-3 + TPO) and erythroid cells (EB) under standard conditions (IL-3 and EPO) and conditions of stress (IL-3 + EPO + dexamethasone).

In semisolid assay, hSCF and mSCF (20 ng/mL) sustained growth of similar numbers of CFU-GM (51±5 vs 44±5/2×10⁵ MNC and 15.4±0.9 vs 13.3±2.0/500 CD34^pos cells)- and BFU-E (8.5±0.5 vs 8.7±1.2/500 CD34^pos cells)-derived colonies. By day 12 of MK expansion assay, hSCF and mSCF induced similar 2-1.6-fold increases over those observed with IL-3 and TPO alone. In addition, hSCF and mSCF-stimulated cultures contained similar number of MK as identified by morphology and CD41a/CD42b staining (∼40–60% CD41a^posCD42b^pos cells) (Figure 1). By day 13 of EB expansion culture without steroids, hSCF and mSCF generated similar numbers of EB (FI= ∼6–8 with both MNC and CD34^pos cells), ∼60% of which had a mature CD36^posCD235a^high phenotype (Figure 1), but, by day 15 of EB expansion with steroids, hSCF induced significantly greater numbers of EB that mSCF (FI=154±45 vs 54±15 from MNC, and 13,200±740 vs 2,000±550 for CD34^pos cells, respectively, p<0.01 in both cases). In addition, day 8 EB generated with mSCF were more mature than those generated with hSCF (86% vs 31% of CD36^posCD235a^high EB, Figure 1). Similar results were obtained in EB expansion cultures of AB MNC.

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To clarify the difference in biological activity of hSCF and mSCF in cultures containing steroids, expression of glucocorticoid receptor α (GRα) and hSCF and mSCF signaling in adult blood (AB) and CB EB generated with the two growth factors were compared. hSCF-generated EB expressed 2-times more GRα than those generated with mSCF (Figure 2) and the GRα content of hSCF-generated EB was reduced by 50% after 24 h exposure with mSCF. In addition, both transient (within 15 m) and sustained (after 2 h) ERK phosphorylation were observed only in EB stimulated with hSCF while both hSCF and mSCF induced low levels of STAT-5 phosphorylation but neither factor induced AKT phosphorylation.

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In summary, hSCF and mSCF have overlapping biological activities in culture conditions which mimic steady-state hematopoiesis (semisolid cultures, MK and EB expansion without dexamethasone) but hSCF was uniquely capable to sustain GRα expression and was more effective than mSCF in cultures which mimic stress erythropoiesis.