• 1. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275: 964967.

    • Search Google Scholar
    • Export Citation
  • 2. Urbich C, Dimmeler S. Endothelial progenitor cells functional characterization. Trends Cardiovasc Med 2004;14: 318322.

  • 3. Hristov M, Erl W, Weber PC. Endothelial progenitor cells: mobilization, differentiation, and homing. Arterioscler Thromb Vasc Biol 2003;23: 11851189.

    • Search Google Scholar
    • Export Citation
  • 4. Kovacic JC, Moore J, Herbert A, et al. Endothelial progenitor cells, angioblasts, and angiogenesis—old terms reconsidered from a current perspective. Trends Cardiovasc Med 2008;18: 4551.

    • Search Google Scholar
    • Export Citation
  • 5. Richardson MR, Yoder MC. Endothelial progenitor cells: quo vadis? J Mol Cell Cardiol 2011;50: 266272.

  • 6. Timmermans F, Plum J, Yoder MC, et al. Endothelial progenitor cells: identity defined? J Cell Mol Med 2009;13: 87102.

  • 7. Salter MM, Seeto WJ, DeWitt BB, et al. Characterization of endothelial colony-forming cells from peripheral blood samples of adult horses. Am J Vet Res 2015;76: 174187.

    • Search Google Scholar
    • Export Citation
  • 8. Seeto WJ, Tian Y, Lipke EA. Peptide-grafted poly(ethylene glycol) hydrogels support dynamic adhesion of endothelial progenitor cells. Acta Biomater 2013;9: 82798289.

    • Search Google Scholar
    • Export Citation
  • 9. Hofmann NA, Reinisch A, Strunk D. Isolation and large scale expansion of adult human endothelial colony forming progenitor cells. J Vis Exp 2009;28: 1524.

    • Search Google Scholar
    • Export Citation
  • 10. Reinisch A, Hofmann NA, Obenauf AC, et al. Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo. Blood 2009;113: 67166725.

    • Search Google Scholar
    • Export Citation
  • 11. Hackett CH, Flaminio MJ, Fortier LA. Analysis of CD14 expression levels in putative mesenchymal progenitor cells isolated from equine bone marrow. Stem Cells Dev 2011;20: 721735.

    • Search Google Scholar
    • Export Citation
  • 12. Bakogiannis C, Tousoulis D, Androulakis E, et al. Circulating endothelial progenitor cells as biomarkers for prediction of cardiovascular outcomes. Curr Med Chem 2012;19: 25972604.

    • Search Google Scholar
    • Export Citation
  • 13. Loomans CJ, de Koning EJ, Staal FJ, et al. Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes 2004;53: 195199.

    • Search Google Scholar
    • Export Citation
  • 14. Petrelli A, Di Fenza R, Carvello M, et al. Strategies to reverse endothelial progenitor cell dysfunction in diabetes. Exp Diabetes Res 2012;2012: 471823.

    • Search Google Scholar
    • Export Citation
  • 15. Schmidt-Lucke C, Rossig L, Fichtlscherer S, et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair. Circulation 2005;111: 29812987.

    • Search Google Scholar
    • Export Citation
  • 16. Bai C, Hou L, Zhang M, et al. Characterization of vascular endothelial progenitor cells from chicken bone marrow. BMC Vet Res 2012;8: 54.

    • Search Google Scholar
    • Export Citation
  • 17. Sekiguchi H, Li M, Jujo K, et al. Improved culture-based isolation of differentiating endothelial progenitor cells from mouse bone marrow mononuclear cells. PLoS One 2011; 6: e28639.

    • Search Google Scholar
    • Export Citation
  • 18. Nagano M, Yamashita T, Hamada H, et al. Identification of functional endothelial progenitor cells suitable for the treatment of ischemic tissue using human umbilical cord blood. Blood 2007;110: 151160.

    • Search Google Scholar
    • Export Citation
  • 19. Wu H, Riha GM, Yang H, et al. Differentiation and proliferation of endothelial progenitor cells from canine peripheral blood mononuclear cells. J Surg Res 2005;126: 193198.

    • Search Google Scholar
    • Export Citation

Advertisement

Isolation of endothelial colony-forming cells from blood samples collected from the jugular and cephalic veins of healthy adult horses

View More View Less
  • 1 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 2 Department of Chemical Engineering, Samuel Ginn College of Engineering, Auburn University, Auburn, AL 36849.
  • | 3 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 4 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 5 Department of Chemical Engineering, Samuel Ginn College of Engineering, Auburn University, Auburn, AL 36849.
  • | 6 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

Abstract

OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses.

SAMPLE Jugular and cephalic venous blood samples from 17 adult horses.

PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate–labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor-2, and von Willebrand factor) and hematopoietic (CD14) cell markers by use of indirect immunofluorescence assay (IFA) and flow cytometry.

RESULTS Colonies with cobblestone morphology were isolated from 15 of 17 horses. Blood collected from the cephalic vein yielded colonies significantly more often (14/17 horses) than did blood collected from the jugular vein (8/17 horses). Of 14 cephalic blood samples with colonies, 13 were obtained with DGC and 8 with WBA. Of 8 jugular blood samples with colonies, 8 were obtained with DGC and 4 with WBA. Colony frequency (colonies per milliliter of blood) was significantly higher for cephalic blood samples and samples isolated with DGC. Cells formed vascular tubules, had uptake of DiI-Ac-LDL, and expressed endothelial markers by use of IFA and flow cytometry, which confirmed their identity as ECFCs.

CONCLUSIONS AND CLINICAL RELEVANCE Maximum yield of ECFCs was obtained for blood samples collected from both the jugular and cephalic veins and use of DGC to isolate cells. Consistent yield of ECFCs from peripheral blood of horses will enable studies to evaluate diagnostic and therapeutic uses.

Contributor Notes

The first 2 authors contributed equally to the manuscript.

Address correspondence to Dr. Wooldridge (aaw0002@auburn.edu).