Hematopoietic Stem Cell Defect in c-Kit Deficient Mice.

Abstract 2542 Poster Board II-519 Hematopoietic stem cell (HSC) numbers are traditionally quantitated with competitive transplantation assays. However, the results can be misleading if short-term repopulating cells (STRCs) in the test marrow or their progeny do not optimally expand. For example, when c-mpl −/− and wild-type (WT) bone marrow cells are transplanted into WT recipients at ratios of 4:1 and 8:1, the relative contribution of c-mpl −/− engraftment is low (<13%), but when the cells are transplanted into thrombopoietin-deficient tpo −/− mice, c-mpl −/− and WT marrow cells compete fairly, resulting in 70.2 ± 7.1% (4:1) and 82.1 ± 6.6% (8:1) c-mpl −/− cell engraftment ( Blood 109:5196, 2007). This outcome, with results from parabiotic studies, implies that the stem cell numbers in c-mpl −/− mice are not reduced. Rather their defective differentiation and/or proliferation in a WT thrombopoietin-containing environment accounts for the inferior competitiveness. Marrow cells from c-kit deficient (W, W41 or Wv) also compete poorly with marrow cells form WT mice when transplanted into WT recipients. In competitive transplantation studies in which c-kit deficient marrow is transplanted in 50-fold excess into WT recipients, these cells are still outcompeted by WT HSCs. To determine whether the defect in engraftment reflects a deficient number of HSCs or impaired HSC function as seen in c-mpl −/− mice, we performed studies using a comparable experimental strategy. Stem cell factor (SCF), the ligand for c-kit (CD117), a type III tyrosine kinase receptor, is encoded by the steel (Sl) locus. Homozygous deletion of either Sl or W causes embryonic lethality. However, other less severe mutations exist: for example, Sld, which encodes soluble but not membrane-bound functional SCF and Wv, which contains a point mutation leading to impaired c-kit tyrosine kinase activity. We first performed parabiotic experiments in which W v /W v (CD45.2) mice were surgically joined with Pep3b (WT, CD45.1) mice. After 6 weeks of parabiosis, the mice were separated and the immunophenotype of blood and marrow cells was assayed. In the W v /W v parabiont, a high proportion of marrow granulocytes derived from the Pep3b partner (67.8 ± 3.2%). In contrast, the Pep3b parabiont had very few marrow granulocytes from the W v /W v partner (2.2 ± 0.5%). These results are in accordance with the previous finding that WT marrow can engraft into W v /W v mice without irradiation and suggest that there are many open (unoccupied) marrow niches in W v /W v mice. We then transplanted marrow from each parabiont into irradiated C57BL/6 (WT) mice (traditional experiment) or SCF-deficient Sl d/+ mice (to assure more equal competition between W v /W v and WT (Pep3b) cells). C57BL/6 recipients of W v /W v parabiont marrow had 44.3 ± 20.8% Pep3b HSCs measured by the phenotype of blood and marrow cells three months post transplantation. Importantly, Sl d/+ recipients of W v /W v parabiont marrow had similar numbers of Pep3b HSCs (48.3 ± 22.7%), contrasting the results of our studies of HSCs in c-mpl −/− mice. Therefore, the high level of Pep3b HSC repopulation in the W v /W v parabiont is not exclusively due to a differentiation/proliferation defect in W v /W v HSCs. We next transplanted varying ratios (1:1 to 8:1) of W v /W v and Pep3b marrow cells into lethally-irradiated C57BL/6 or Sl d/+ mice. At all the ratios, engraftment was overwhelmingly dominated by Pep3b cells (>87%) and there was no significant difference of engraftment percentages in C57BL/6 vs. Sl d/+ mice. Even in mice lacking membrane-bound SCF, Pep3b HSCs out-compete c-kit-deficient cells. These data conclusively demonstrate that in distinction from c-mpl −/− mice which have a defect in the commitment of HSCs to STRCs or STRC proliferation, c-kit deficient ( W v /W v ) mice indeed have a severe defect at the level of the hematopoietic stem cell. Disclosures: No relevant conflicts of interest to declare.