title: The isolation and culture of endothelial colony-forming cells from human and rat lungs.
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.
authors.affiliation: Department of Pediatrics and Women's and Children's Health Research Institute, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Rajesh S
authors.initials: RS
authors.lastname: Alphonse
authors.affiliation: 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, Ontario, Canada.
authors.forename: Arul
authors.initials: A
authors.lastname: Vadivel
authors.forename: Shumei
authors.initials: S
authors.lastname: Zhong
authors.affiliation: 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, Ontario, Canada.
authors.forename: Shumei
authors.initials: S
authors.lastname: Zong
authors.affiliation: Department of Pediatrics, University of New Mexico, Albuquerque, New Mexico, USA.
authors.forename: Suzanne
authors.identifier: http://orcid.org/0000-0001-9409-0067
authors.initials: S
authors.lastname: McConaghy
authors.affiliation: Department of Pediatrics, University of New Mexico, Albuquerque, New Mexico, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Ohls
authors.affiliation: Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Mervin C
authors.initials: MC
authors.lastname: Yoder
authors.affiliation: 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, Ontario, Canada.
authors.forename: Bernard
authors.initials: B
authors.lastname: Thébaud
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000502:physiology*; D006801:Humans; D008168:Lung / Q000166:cytology*; D051381:Rats; D013234:Stem Cells / Q000502:physiology*