Introduction: Recent studies have shown that MSCs can be isolated from the peripheral blood of many different species. Hematopoietic stem cell (HSC) mobilization from the bone marrow to the circulating bloodstream can be induced using granulocyte colony stimulating factors (G-CSF). As it has been shown that HSCs and MSCs have positive interactions with each other, it may be possible that G-CSF also promotes the release of circulating peripheral blood MSCs (PBMSCs). The hypothesis of this study was that G-CSF would increase the mobilization of peripheral blood-derived stromal-like cells.

Materials and Methods: Six sheep with normal hematological profiles were given 5& #956;g/kg Neupogen& #63721; (filgrastim, G-CSF) subcutaneously for five days. Pre- and post-G-CSF treatment, blood was taken 4, 12, 24, and 2 weeks post-treatment. PBMSCs were isolated from the blood and cells plated at a cell density of 4.0 x 10e4 nucleated cells/cm2. Fibroblastic colony forming units (CFU-F) were counted 7 and 14 days after initial culture. The cells were tested for their multipotency by treating them with osteogenic, adipogenic, and chondrogenic supplements, and staining with the Von Kossa, Oil Red ‘O,’ and Alcian Blue stains, respectively, to show differentiation down the different lineages.

Results: No CFU-F formation was observed in all blood samples taken before G-CSF therapy (0 CFU-F) after 7 and 14 days in culture. After G-CSF treatment, CFU-Fs were observed in blood samples taken 4, 12, and 336 hours (2 weeks) post-G-CSF. The CFU-F count was highest after 14 days in culture in the blood samples obtained 2 weeks post-G-CSF administration (1.027 ± 30.1353 CFU-F/cm2), compared to the lowest count, which was at 12 hours post-G-CSF treatment (0.064 ± 0.064 CFU-F/cm2). Hematology showed an increase in white blood cell (WBC), neutrophil, and eosinophil counts 24 hours after G-CSF administration. Two weeks post-G-CSF treatment, WBC, neutrophil, lymphocyte, and monocyte counts dropped back to normal range values. The highest number of CFU-F/cm2 were observed at this time. When WBC numbers were correlated with CFU-F counts using Pearson’s correlation co-efficient, the result was 0.523, a significant value (p=0.023) indicating that 27.4% of the WBC counts were related to CFU-F counts and vice versa. When time was accounted for as a third variable using the test for partial correlation coefficients, the co-efficient was found to be −0.0063, and was not significant (p=0.492). Expanded cells were fibroblastic in morphology, and upon differentiation were positive for the Von Kossa, Oil Red ‘O,’ and Alican Blue stains, indicating differentiation down the osteogenic, adipogenic, and chondrogenic lineages, respectively.

Discussion and Conclusions: We have shown that PBMSCs can be isolated after G-CSF administration in sheep, and that the numbers of CFU-F increase after WBC levels have returned to normal. A previous in vitro study proposed that the increased BMSC growth observed when co-cultured with CD45+ HSCs was due to positive interactions between HSCs and MSCs, indicating a possible steady-state balance. PBMSCs may have important future applications in bone tissue regeneration.