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 W^v) 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, Sl^d, which encodes soluble but not membrane-bound functional SCF and W^v, 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.