Fanca-/- hematopoietic stem cells demonstrate a mobilization defect which can be overcome by administration of the Rac inhibitor NSC23766

Abstract

Fanconi anemia is a severe bone marrow failure syndrome resulting from inactivating mutations of Fanconi anemia pathway genes. Gene and cell therapy trials using hematopoietic stem cells and progenitors have been hampered by poor mobilization of HSC to peripheral blood in response to G-CSF. Using a murine model of Fanconi anemia (Fanca(-/-) mice), we found that the Fanca deficiency was associated with a profound defect in hematopoietic stem cells and progenitors mobilization in response to G-CSF in absence of bone marrow failure, which correlates with the findings of clinical trials in Fanconi anemia patients. This mobilization defect was overcome by co-administration of the Rac inhibitor NSC23766, suggesting that Rac signaling is implicated in the retention of Fanca(-/-) hematopoietic stem cells and progenitors in the bone marrow. In view of these data, we propose that targeting Rac signaling may enhance G-CSF-induced HSC mobilization in Fanconi anemia.

Figure 1.

Immunophenotypic characterization of Fanca^−/− versus WT bone marrow (BM) cells. (A) Percentage of LSK cells in the BM. Femora were isolated from either WT or Fanca^−/− mice and were subsequently stained with fluorescent conjugated antibodies directed against Sca-1, c-Kit, and a panel of lineage specific markers, as described in the Methods section. Flow analysis was used to evaluate the percentage of LSK cells in the BM. n=4 per group, in each of two independent experiments. (B) Total number of LSK cells per femur. The absolute number of BM cells per femur was calculated as described in the Methods section. The total femur cellularity was multiplied by the frequency of LSK cells in BM in order to calculate the absolute number of LSK cells per femur. n=4 per group, in each of two independent experiments. (C) Relative expression of cell adhesion molecules in BM LSK population. BM from A was additionally stained with fluorescent antibodies directed against either CD49d, CD49e, CD62L or CXCR4, as described in the Methods section. The relative expression of each of these adhesion molecules within the LSK population was assessed by determining the mean fluorescent intensity of staining by flow analysis. n=4 per group, in each of two independent experiments. □ WTBM; ▪ Fanca^−/− BM.

Figure 2.

Functional characterization of Fanca^−/− versus WT BM cells. (A) CFU homing to the bone marrow (BM). Lethally irradiated mice were injected with 1×10⁷ Fanca^−/− or WT BM, and 16 h later, BM was isolated from the femora and tibiae of recipient mice and plated out in methylcellulose. Homing efficiency is expressed as the percentage of CFU which were recovered from the BM relative to the number of CFU which were injected into the recipient mice. n=5 per group, in each of two independent experiments (B) CFU homing to the spleen. At the time of sacrifice, splenocytes were harvested from the recipient mice described in A. Splenocytes were plated out in methycellulose, and, as for BM, the homing efficiency was calculated as the percentage of CFU recovered from the spleen relative to the number of CFU injected into the recipient mice. n=5 per group, in each of two independent experiments (C) Competitive repopulation activity of Fanca^−/− versus WT BM. BM was harvested from the femora of age matched WT or Fanca^−/−mice (both CD45.2⁺). Lethally irradiated recipient mice (CD45.1⁺) were co-injected with 3×10⁶ competitor BM cells (CD45.1⁺) and either 1/10 femur equivalent of Fanca^−/− BM or 1/10 femur equivalent of WT BM. At both four weeks and five months post-transplant, recipient mice were bled and the percentage of CD45.2⁺ cells in the peripheral blood was determined by flow analysis after staining with fluorescent labeled antibodies. The number of CRU was calculated as described in the Methods section. Data is from one representative experiment of two independent experiments performed. *p<0.05, n=5–6 per group. □ WT BM; ▪ Fanca^−/− BM.

Figure 3.

Relative mobilization of Fanca^−/− versus WT HSC/P. (A) Mobilization of CFU via treatment with G-CSF alone, or G-CSF and NSC23766. Fanca^−/− or WT mice were treated with either G-CSF alone, or G-CSF and NSC23766 as described in the Methods section. Peripheral blood was isolated from treated mice via cardiac puncture. 100 μL of peripheral blood was subject to red cell lysis and was then plated out in methycellulose. CFU were scored seven days later. **p<0.01, n=3 per group, from two independent experiments. (B) Mobilization of CRU via treatment with G-CSF alone, or G-CSF and NSC23766. Fanca^−/− or WT mice were treated with G-CSF alone or G-CSF and NSC23766 as described above. 400 μL of peripheral blood was subject to red cell lysis and was then co-injected into lethally irradiated CD45.1⁺ recipient mice along with 5×10⁵ competitor (CD45.1⁺) bone marrow (BM) cells. At four months post-transplant, recipient mice were bled and the percentage chimerism of CD45.2⁺ cells was determined by staining with fluorescent antibodies prior to flow cytometry as described in the Methods section. The CRU per ml of peripheral blood was determined as described in the Methods section (normalized to the frequency of CRU in the WT group mobilized with G-CSF alone). *p<0.05, **p<0.01, n=6–8 mice in each experimental group from two independent experiments. (C) Secondary transplant of BM from competitive transplant recipients. BM was isolated from the primary competitive transplant recipients described in B, and 1×10⁷ BM cells were injected i.v. into lethally irradiated secondary recipients (CD45.1⁺). At six months post-transplant, peripheral blood was harvested from recipient mice and the percentage of donor chimerism (CD45.2⁺) was determined as above. **p<0.01, n=5–7 mice in each experimental group from two independent experiments. □ WT mobilized peripheral blood; ▪ Fanca^−/− mobilized peripheral blood.