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Establishment of outgrowth endothelial cells from peripheral blood

Nature Protocols volume 7, pages 1709–1715 (2012)Cite this article

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Abstract

Blood outgrowth endothelial cells (BOECs) are important tools when investigating diagnostic and therapeutic approaches for vascular disease. In this protocol, mononuclear cells are isolated from peripheral blood and plated on type I collagen at ∼135,000 cells per cm2 in endothelial cell differentiation medium. On average, 0.34 colonies of endothelial cells per milliliter of blood can be obtained. Colonies of endothelial cells become visible after 14–28 d. Upon confluence, these rapidly expanding colonies can be passaged and have been shown to propagate up to 1018-fold. Isolated BOECs are phenotypically similar to vascular endothelial cells, as revealed by their cobblestone morphology, the presence of endothelial cell–specific Weibel-Palade bodies and the expression of endothelial cell markers such as VE-cadherin. The protocol presented here also provides a particularly useful tool for the ex vivo assessment of endothelial cell function from patients with different vascular abnormalities.

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Figure 1: BOEC morphology.
Figure 2: Blood separation.
Figure 3: BOEC characterization by immunostaining.

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Acknowledgements

J.M.-R. and M.H. are supported by a grant from Center for Translational Molecular Medicine, grant no. INCOAG-01C-201-04. R.P.H. is supported by US National Institutes of Health grants HL62931 and HL55552.

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Authors and Affiliations

  1. Department of Plasma Proteins, Landsteiner Laboratory, Academic Medical Centre and Sanquin, University of Amsterdam, Amsterdam, The Netherlands

    Javier Martin-Ramirez, Menno Hofman, Maartje van den Biggelaar & Jan Voorberg

  2. Division of Hematology-Oncology-Transplantation, Department of Medicine, Vascular Biology Center, University of Minnesota Medical School, Minneapolis, Minnesota, USA

    Robert P Hebbel

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  1. Javier Martin-Ramirez
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  3. Maartje van den Biggelaar
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  4. Robert P Hebbel
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  5. Jan Voorberg
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Contributions

J.M.-R., M.H. and J.V. developed the current protocol that is based on the original protocol described by Lin and co-workers. J.M.-R., M.H., M.v.d.B., R.P.H. and J.V. wrote the manuscript. All authors approved the final version of the manuscript.

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Correspondence to Jan Voorberg.

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The authors declare no competing financial interests.

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Martin-Ramirez, J., Hofman, M., van den Biggelaar, M. et al. Establishment of outgrowth endothelial cells from peripheral blood. Nat Protoc 7, 1709–1715 (2012). https://doi.org/10.1038/nprot.2012.093

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