Differential bone marrow stem cell mobilization by G-CSF injection or arterial ligation in baboons
Version of Record online: 8 JUL 2008
© 2008 The Authors Journal compilation © 2009 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Journal of Cellular and Molecular Medicine
Volume 13, Issue 8b, pages 1896–1906, August 2009
How to Cite
Shi, Q., Hodara, V., Butler, S. D., Thomas III, C. A., Hubbard, G. B., VandeBerg, J. L. and Wang, X. L. (2009), Differential bone marrow stem cell mobilization by G-CSF injection or arterial ligation in baboons. Journal of Cellular and Molecular Medicine, 13: 1896–1906. doi: 10.1111/j.1582-4934.2008.00405.x
- Issue online: 26 NOV 2009
- Version of Record online: 8 JUL 2008
- Received: April 7, 2008; Accepted: May 8, 2008
Fig. S1 Immunophenotypic characteristics of PBMNCs 3 days after mobilization by two treatments compared to baseline levels. PBMNCs were freshly isolated from subjects at baseline (white) and after mobilization either by femoral artery ligation (grey) or G-CSF administration (black). Cells were stained with the indicated antibodies and analyzed by FACS. ANOVA was used for among group comparisons and Bonferroni post hoc analysis was used for the between group comparisons. *P < 0.05, comparison to the baseline levels; †P < 0.05, comparison between the two treatments.
Fig. S2 Characteristics of EPCs that formed the colonies. PBMNCs were cultured in endothelial growth medium (EndoCult, StemCell Technologies) on fibronectin-coated 6-well plate at 5 χ 106 per well. As early as day 5, cells adherent to the plate featuring with cobblestone shape morphology appeared (A). The cells continued to proliferate and became a large colony within 14 days (B). Using immunostaining, most of these cells expressed endothelial markers including antigen CD31, vWF, lectin-binding molecules (UEA-1) and moderate CD146 (Fig. 1, C–I). They are negative for hematopoietic cells and monocytes/macrophage (CD45 and CD14) markers or precursor erythroctye cell (CD235a) marker. The immunochemical study is consistent with the cells being endothelial origins. A and B were viewed with 200χ magnification and C–J were viewed under 400χ magnification.
Fig. S3 Characteristics of colonies formed by PBMNCs. PBMNCs from both groups were induced to form EPC colonies. We hand-picked representative colonies from both groups and spun down by Cytofuge (StatSpin) onto slides. Immunostaining with various antibodies were carried out and revealed that the most colonies (11 out of 15 colonies) in G-CSF treated samples expressed a wide range of hematopoietic related proteins (CD45, CD14 and CD235a), as well as endothelial cell markers like CD31 and CD146, but not vWF. However, colonies harvested from arterial ligation group (14 out of 15 colonies) were stained strongly for CD31, CD146, and vWF, moderate CD45 positive cells, and negative for CD14 and CD235a surface antigens.
Fig. S4 Identification of vWF expression using immunohistochemistry. We collected the PBMNCs from the cultures that formed tube-like structures in culture and stained them for the expression of vWF (green fluorescence) – a marker of mature ECs. The percentage of cells showing intense vWF staining was much higher in baboons with artery ligation (1) than those treated with G-CSF (2). Nuclei were stained with DAPI (blue). A magnification of χ600 was used to visualize the cells.
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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.