Bar size=100 m

Bar size=100 m. Notch2 signaling blockade increases HSPC cell cycling and down-regulates CXCR4 expression To understand the mechanism underlying the enhanced sensitization to mobilizing reagents and an expansion of myeloid progenitors from Notch2-blocked donors during transplantation, we assessed the cell-cycling status of the marrow HSPCs by the proliferation marker Ki67 in conjunction with the DNA-specific dye 7-AAD as a measure of quiescence. interaction maintains HSC quiescence and niche retention. Using Notch receptor blocking antibodies, we report that Notch2 blockade, but not Notch1 blockade, sensitizes hematopoietic stem cells F2RL1 and progenitors (HSPCs) to mobilization stimuli and leads to enhanced egress from marrow to the periphery. Notch2 blockade leads to transient myeloid progenitor expansion without affecting HSC homeostasis and self-renewal. We show that transient Notch2 blockade or Notch2-loss in mice lacking Notch2 receptor lead to decreased CXCR4 expression by HSC but increased cell CYN-154806 cycling with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. In addition, we found that Notch2-blocked or Notch2-deficient marrow HSPCs show an increased homing to the marrow, while mobilized Notch2-blocked, but not Notch2-deficient stem cells and progenitors, displayed a competitive repopulating advantage and enhanced hematopoietic reconstitution. These findings suggest that blocking Notch2 combined with the current clinical regimen may further enhance HPC mobilization and improve engraftment during HCT. Introduction Hematopoietic cell transplantation (HCT) is the only curative option for various neoplastic and a few non-neoplastic diseases.1 The vast majority of clinical autologous HCT procedures utilize hematopoietic progenitor cells (HPCs) mobilized into the blood. For a variety of reasons, some patients may not mobilize adequate numbers of HPCs and thus are not candidates for the autologous HCT procedure. In CYN-154806 addition, in some subjects, less than an optimal number of HPCs may be obtained, resulting in slower hematopoietic reconstitution and increased risk of complications during the transplant.2C4 In recent years, the use of CXCR4 antagonizing molecules/peptides (i.e. AMD3100 or plerixafor) has enhanced HPC mobilization and overcome some of these limitations.5 Inadequate mobilization, however, still remains a problem for many patients and the development of more efficacious strategies may enhance patient outcome. 6 The signaling molecule Notch is important for stem cell self-renewal and fate determination in many tissues, including the hematopoietic system. An important feature of Notch is its adhesive nature which was first described by cell aggregation assays in Drosophila studies.7,8 There are 4 Notch receptors (Notch1-4), and 2 families of Notch ligand: Jagged (JAG1-2), and delta-like (DLL1-4) ligand. Notch2 is the major isotype expressed on hematopoietic stem cells (HSC) and non-lymphoid progenitor cells.9C12 However, the precise role and the physiological significance of Notch receptors, either as adhesion and/or signaling molecules, in HSC homeostasis and functional support are still not completely understood. Notch signaling transactivation is consequent to a functional engagement of the Notch receptor with the Notch ligand. We previously reported that hematopoietic stem cell and progenitors (HSPCs) with faulty Notch-ligand interaction due to the loss of CYN-154806 O-fucose modification of Notch display increased cell cycling and decreased adhesion to marrow osteoblastic lineage cells.11 These HSPCs exhibit enhanced egress from the marrow. However, the significance and the mechanism of Notch downstream signaling in the maintenance of HSC quiescence are not clear. Here we report that prior treatment with Notch2 blocking antibody sensitizes HSPC to the mobilizing stimuli of G-CSF and AMD3100 with a 3C4-fold increase in mobilization without affecting the overall bone marrow HSC homeostasis and self-renewal. Moreover, we demonstrate that Notch signaling directly regulates CXCR4 expression, and hence transient Notch2 blockade decreases CXCR4 concentration and increases cell cycling. Consistent with these findings, transient Notch2 blockade leads to greater HSPC homing to the CYN-154806 marrow and a competitive repopulating advantage of the progenitors with enhanced recovery of hematopoietic elements. Methods Mice The Institutional Animal Care and Use Committee of Case Western Reserve University approved all aspects of the animal research described in this study. C57Bl/6 (Ly5.2) and B6.SJL-Ptrca Pep3b/BoyJ (B6.BoyJ:Ly5.1) mice were maintained in the lab. Vav-Cre/Notch2F/F mice were generated by crossing Vav-Cre mice (008610; Jackson Laboratory, Bar Harbor, ME, USA) with Notch2F/F mice (010525; Jackson Laboratory, Bar Harbor, ME, USA). Notch receptor blockade Humanized anti-Notch1 (anti-NRR1, Genentech), anti-Notch2 (anti-NRR2, Genentech) or control antibody (anti-ragweed, Genentech, South San Francisco, CA, USA) have been described previously.11 Antibodies were injected i.p..