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

Developers

Rajesh S. Alphonse, Arul Vadivel, Mervin C. Yoder, Bernard Thébaud, etc.

Description of the technology

Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell-driven «postnatal vasculogenesis» is considered as an important mechanism, contributing to new blood vessel formation and organ repair. Among several described progenitor cell types, taking part in blood vessel formation, endothelial colony-forming cells are of most interest as lineage-specific 'true' vascular progenitors.

New technology provides an isolation of pulmonary microvascular endothelial colony-forming cells from human and rat lung tissue. This method uses the advantages of an earlier protocol for the isolation of circulating endothelial colony-forming cells from the mononuclear cellular fraction of peripheral blood. This protocol has been adapted to isolate resident endothelial colony-forming cells from the distal lung tissue. According to modified protocol, ater 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 endothelial colony-forming cells cultures after a further 2–3 weeks. The resulting cells show the defining characteristics of endothelial colony-forming cells 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.

Practical application

Isolation and culture of lung vascular endothelial colony-forming cells according to this technology will allow assessment of the functional state of these cells in experimental and human lung diseases, provide the understanding of their pathophysiological mechanisms and develop new effective methods of their treatment.

Laboratories

• Department of Pediatrics and Women’s and Children’s Health Research Institute, University of Alberta, Edmonton (Canada)
• Ottawa Hospital Research Institute, Regenerative Medicine Program, Sprott Center for Stem Cell Research, Department of Pediatrics, Children’s Hospital of Eastern Ontario, University of Ottawa, Ottawa (Canada)
• Department of Pediatrics, University of New Mexico, Albuquerque (USA)
• Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis (USA)

Links

Publications

• Alphonse, R.S. et al. «The isolation and culture of endothelial colony-forming cells from human and rat lungs." 10 Nature Protocols (2015): 1697–1708.
• Alphonse, R.S. et al. «Existence, functional impairment, and lung repair potential of endothelial colony-forming cells in oxygen-induced arrested alveolar growth." 129 Circulation (2014): 2144–2157.
• Thebaud, B. & Abman, S.H. «Bronchopulmonary dysplasia: where have all the vessels gone? Roles of angiogenic growth factors in chronic lung disease." 175 Am. J. Respir. Crit. Care Med. (2007): 978–985.