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

Ashley N. Sharpe Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Wen J. Seeto Department of Chemical Engineering, Samuel Ginn College of Engineering, Auburn University, Auburn, AL 36849.

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Randolph L. Winter Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Qiao Zhong Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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Elizabeth A. Lipke Department of Chemical Engineering, Samuel Ginn College of Engineering, Auburn University, Auburn, AL 36849.

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Anne A. Wooldridge Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

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 DVM, PhD
DOI:
https://doi.org/10.2460/ajvr.77.10.1157
Volume/Issue:
Volume 77: Issue 10
Online Publication Date:
01 Oct 2016
Restricted access
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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.

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).
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
Publication Date:
01 Oct 2016

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