Two dimensional gel electrophoresis comparison of bone marrow fluid from G-CSF treated wildtype and em SFK /em ?/? mice revealed a significant decrease in global protein expression in em SFK /em ?/? mice compared to wildtype, suggestive of enhanced proteolysis. WT. (C) Lineage unfavorable Beperidium iodide cells were plated in a methylcellulose assay with varying concentrations of G-CSF. Colonies were scored 7 days later. Bars symbolize the mean colony forming ability from an independent experiment. *?/? vs WT. Hypersensitivity to G-CSF due to SFK deficiency results in enhanced Stat3 activation Previous studies have argued for a critical role for Stat3 in regulating G-CSF responses in HSC/Ps, including G-CSF induced mobilization [24]. To determine whether the increase in G-CSF induced mobilization of stem/progenitors in differentiated. After 48 and 72 hours, cells were harvested and stained with antibodies that identify the cell surface marker Mac-1 and Gr-1. (C) Wildtype and differentiation by monitoring the acquisition of Gr-1 and Mac-1 with time. At the time of initiating the cultures (day 0), the Beperidium iodide expression of Gr-1 and Mac-1 was nearly undetectable in wildtype and experiments to determine if the enhanced G-CSF mobilization in with an inhibitor that completely inhibits the breakdown of VCAM-1 was clearly rescued (Physique 4A). Taken together, these results suggest that although the breakdown of VCAM-1 in G-CSF treated is usually notWildtype and 0.001, em SFK /em ?/? vs WT. (C) Wildtype and em SFK /em ?/? mice were injected with 300 g/kg/day of human G-CSF for 4 days and their bone marrow fluid harvested. SDF-1 concentration was measured by ELISA according to manufactures recommendation. Bars show the mean SDF-1 level from an experiment performed in replicates of three. * em P /em 0.05, WT vs em SFK /em ?/? (control), ** em P /em 0.05 WT vs em SFK /em ?/? (G-CSF treated), *** em P Beperidium iodide /em 0.05 WT (control) vs WT (G-CSF) and **** em P /em 0.05 em SFK /em ?/? (control) vs em SFK /em ?/? (G-CSF). (D) Enhanced AMD3100 induced stem/progenitor cell mobilization and cooperation with G-CSF in em SFK /em ?/? mice. SDF-1 antagonist AMD3100 was injected 1 hr before harvesting peripheral blood (PB) or 1 hr before harvesting PB from mice injected with G-CSF (300 g/kg/day) for three days. Cells were plated in a methylcellulose assay. CFU-Cs were scored 7 days later. Bars show the mean colony forming ability from two impartial experiments performed in replicates of three with 4 mice in each experiment. * em P /em 0.05, G-CSF treated WT vs em SFK /em ?/? mice. ** em P /em 0.05, G-CSF+AMD3100 treated WT vs em SFK /em ?/? mice. *** em P /em 0.05 AMD3100 treated WT vs em SFK /em ?/? mice. Previous studies have suggested a more prominent role for the CXCR4/SDF-1 axis in the retention of HSC/Ps in the bone marrow under basal conditions as well as in response to G-CSF treatment. To determine if the enhanced G-CSF induced mobilization in em SFK /em ?/? mice is usually associated with reduced levels of SDF-1 in the BM of em SFK /em ?/? mice, we examined SDF-1 levels in the bone marrow of em SFK /em ?/? and wildtype mice treated with PBS or with G-CSF. As seen in Physique 4C, a significant reduction in the level of SDF-1 was observed in em SFK /em ?/? bone marrow under basal conditions as well as upon G-CSF treatment in comparison to wildtype controls. Since disruption of the SDF-1/CXCR4 pathway is one of the principal pathways involved in the mobilization of HSC/Ps, we hypothesized that co-administering G-CSF and an Beperidium iodide SDF-1 antagonist AMD3100 would further augment the mobilization of HSC/Ps in em SFK /em ?/? mice compared to wildtype controls. Consistent with this notion, injecting em SFK /em ?/? mice with G-CSF and AMD3100 resulted in a signficant increase in the mobilization of em SFK /em ?/? HSC/Ps compared to wildtype mice treated with the same combination or mice treated with G-CSF alone (Physique 4D). These results strongly implicate the SDF-1/CXCR4 axis in negatively regulating G-CSF induced mobilization of HSC/Ps in em SFK /em ?/? mice. Peripheral blood mobilized HSC/Ps in SFK?/? mice possess long-term and multilineage repopulating ability Even though above studies demonstrate a signficant increase in the mobilization of clonogenic HSC/Ps in em SFK /em ?/? mice treated with G-CSF, they do not provide evidence of stem cell function. To assess whether PB mobilized HSC/Ps in em SFK /em ?/? mice possess stem cell properties, we harvested peripheral blood from G-CSF treated wildtype or em SFK /em ?/? mice and transplanted equivalent numbers of mononuclear cells into wildtype lethally irradiated recipients. As seen in Physique 5A, lethally irradiated recipient mice transplanted with PB derived from G-CSF treated em SFK /em ?/? mice exhibited 100% viability 7 months post transplantation. These mice continue to survive 12 month post transplantation (data not shown). Furthermore, a TMOD3 comparison of the peripheral blood indices between em SFK /em ?/? non-transplanted mice and lethally irradiated recipient mice transplanted with em SFK /em ?/? PB revealed no significant differences (Physique 5B). In addition, G-CSF mobilized em SFK /em ?/? PB cells upon transplantation into a.