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The isolation and culture of endothelial colony-forming cells from human and rat lungs

Nature Protocols volume 10, pages 1697–1708 (2015)Cite this article

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A Corrigendum to this article was published on 30 December 2015

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Abstract

Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell–driven 'postnatal vasculogenesis' has been suggested as an important mechanism that contributes to new blood vessel formation and organ repair. Among several described progenitor cell types that contribute to blood vessel formation, endothelial colony-forming cells (ECFCs) have received widespread attention as lineage-specific 'true' vascular progenitors. Here we describe a protocol for the isolation of pulmonary microvascular ECFCs from human and rat lung tissue. Our technique takes advantage of an earlier protocol for the isolation of circulating ECFCs from the mononuclear cellular fraction of peripheral blood. We adapted the earlier protocol to isolate resident ECFCs from the distal lung tissue. After enzymatic dispersion of rat or human lung samples into a cellular suspension, CD31-expressing cells are positively selected using magnetic-activated cell sorting and plated in endothelial-specific growth conditions. The colonies arising after 1–2 weeks in culture are carefully separated and expanded to yield pure ECFC cultures after a further 2–3 weeks. The resulting cells demonstrate the defining characteristics of ECFCs such as (i) 'cobblestone' morphology of cultured cell monolayers; (ii) acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding; (iii) tube-like network formation in Matrigel; (iv) expression of endothelial cell–specific surface markers and the absence of hematopoietic or myeloid surface antigens; (v) self-renewal potential displayed by the most proliferative cells; and (vi) contribution to de novo vessel formation in an in vivo mouse implant model. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 4 weeks. Isolation and culture of lung vascular ECFCs will allow assessment of the functional state of these cells in experimental and human lung diseases, providing newer insights into their pathophysiological mechanisms.

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Figure 1: Flow diagram of the steps involved in lung ECFC isolation.
Figure 2: Schematic representation of lung dissection and lung cell suspension preparation (Steps 14–29).
Figure 3: Schematic representation of CD31+ cell selection, plating and ECFC colony isolation (Steps 30–57).
Figure 4: Representative photomicrographs of contaminating cells in ECFC culture.
Figure 5: Representative phenotypic analysis of human lung vascular ECFCs.
Figure 6: Human lung ECFCs contribute to de novo vasculogenesis.
Figure 7: Surface antigen expression and lectin-binding characteristics of rat lung vascular ECFCs.

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  • 02 December 2015

     In the version of this article initially published, the third author's last name was spelled incorrectly. The spelling was initially 'Zong' but should have been 'Zhong'. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by the Canadian Institutes of Health Research (CIHR) and the Canadian Heart and Stroke Foundation. R.S.A. was supported by Alberta Innovates-Health Solutions (AIHS). A.V. was supported by a studentship from the Mazankowski Heart Institute. B.T. was supported by the Canada Foundation for Innovation and the Stollery Children's Hospital Foundation. The work was also supported in part by funds from the Riley Children's Foundation (to M.C.Y.).

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Authors and Affiliations

  1. Department of Pediatrics and Women's and Children's Health Research Institute, University of Alberta, Edmonton, Alberta, Canada

    Rajesh S Alphonse

  2. Department of Pediatrics, Ottawa Hospital Research Institute, Regenerative Medicine Program, Sprott Center for Stem Cell Research, Children's Hospital of Eastern Ontario, University of Ottawa, Ottawa, Ontario, Canada

    Arul Vadivel, Shumei Zhong & Bernard Thébaud

  3. Department of Pediatrics, University of New Mexico, Albuquerque, New Mexico, USA

    Suzanne McConaghy & Robin Ohls

  4. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Mervin C Yoder

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  1. Rajesh S Alphonse
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  2. Arul Vadivel
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  3. Shumei Zhong
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  4. Suzanne McConaghy
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  5. Robin Ohls
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  7. Bernard Thébaud
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Contributions

R.S.A. developed the protocol design, troubleshot and perfected the steps of the procedure, performed experiments, analyzed data and wrote the manuscript. A.V. and S.Z. performed the experiments, helped with experimental design and contributed to manuscript preparation. S.M. and R.O. helped with human tissue acquisition and experimental design. M.C.Y. contributed to protocol design, guided troubleshooting and edited the manuscript. B.T. supervised the project, contributed to experimental design, guided protocol development and edited the manuscript.

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Correspondence to Bernard Thébaud.

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The authors declare no competing financial interests.

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Alphonse, R., Vadivel, A., Zhong, S. et al. The isolation and culture of endothelial colony-forming cells from human and rat lungs. Nat Protoc 10, 1697–1708 (2015). https://doi.org/10.1038/nprot.2015.107

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