A Critical Role of the Rho Family GTPase Cdc42 in Hematopoietic Stem Cell Mobilization, Homing, Engraftment and Differentiation.

The Rho family GTPase Cdc42 has emerged as a key signaling molecule with unique roles in multiple hematopoietic cell lineages. To investigate the physiologic role of Cdc42 in hematopoietic stem cells/progenitors (HSC/Ps), we bred conditional cdc42flox/floxmice with Mx-Cre mice that allowed interferon-inducible cdc42 deletion in hematopoietic cells in vivo following polyI:C induction. Loss of Cdc42 expression in the bone marrow caused significant leukocytosis with neutrophilia in peripheral blood (PB) (Table). Concurrently, deletion of cdc42 in the bone marrow led to a massive efflux of HSC/Ps from bone marrow to PB, liver and spleen (Table) and increased proliferative activity of HSC/Ps. Consequently, flow analysis further revealed that the number of Lin-Sca-1+c-Kit+ (LSK) HSCs in BM, PB, spleen and liver increased significantly following cdc42 deletion (Table). Transplantation of the cdc42−/− bone marrow cells into NOD/SCID recipient mice or competitive transplantation into BoyJ recipient mice showed that cdc42 deficiency in the bone marrow caused the impaired engraftment (Table) and the failure of hematopoiesis after a transient increase of cell cycle rate of HSCs. The engraftment defect is associated with a homing deficiency (Table). The cdc42−/− (Lin−c-Kit+) HSC/Ps showed defects in cortical F-actin assembly, adhesion to fibronectin and directional migration in response to SDF-1α (Table). These data suggest that defective actin structure and adhesion of the cdc42−/− HSC/Ps may contribute to the loss of retention of the cells in the BM niche and to the mobilization phenotype. Moreover, deletion of cdc42 resulted in significantly reduced thymus cellularity, T cell, B cell population, and decreased Ter119+ cell number associated with markedly decreased CFU-E activity of the bone marrow cells (Table). Two additional complimentary approaches, BM-reconstitution by cdc42−/− HSCs and reciprocal (rescue) transplantation by WT HSCs, further demonstrated that the Cdc42 loss-of-function phenotypes are hematopoietic cell intrinsic. Taken together these results implicate Cdc42 as a critical regulator of HSC mobilization, homing and engraftment, and indicate that Cdc42 is involved in erythropoiesis and in T- and B-cell differentiation and homeostasis. WT Cdc42−/− *p<0.05**p<0.005†p<0.001 White blood cell count, × 109 /L 11.0±2.5 40.8±3.9† Neutrophil count, × 109 /L 4.6±0.8 34.1±4.7† CFU-C per 105 liver cells 0.13±0.12 169±132* CFU-C per 104 spleen cells 1.36±1.05 68.4±55.6** CFU-C per 100μl PB 3.67±2 58.8±47.3** LSK HSCs in BM 0.52%±0.22% 0.94%±0.45%* LSK HSCs in PB 0.13%±0.08% 0.31%±0.15%* LSK HSCs in spleen 0.37%±0.20% 0.63%±0.23%* LSK HSCs in liver 0.23%±0.10% 0.70%±0.18%* Engraftment in NOD/SCID mice 35.0%±4.9% 23.0%±7.1%* Competitive transplantation 61.5%±6.7% 22.2%±2.9%** CFU-C Homing to BM (% injected) 0.85%± 0.11% 0.28%± 0.12%** Adhesion to fibronectin (%plating) 50.8%±2.5% 28.8%±4.0%** Migration to SDF (%input) 26.2%±5.3% 5.4%±0.9%† CD3+ T cells in BM 5.4%±0.22% 2.7%±0.45%** B220+ B cells in BM 8.4%±4.6% 1.5%±2.0%† Ter119+ cells in BM 15.2%±9.3% 6.0%±4.2%* CFU-E per 105 BM cells 87.8±9.7 25.6±4.2†