Repair of blood vessels is essential to maintain homeostasis and this process is driven endothelial colony-forming cells. These are vascular lineage-specific progenitor cells that have the ability of driving post-natal vasculogenesis. Endothelial progenitor cells are mobilized into circulation by mediators released due to vascular trauma. Initially, it was though that these cells only acted during embryonic development, but now we know that they are equally important to restore the tissues’ vascularization after disease, especially in heart disease and cancer. Therefore, these cells have therapeutical potential in the treatment of conditions affecting the vascular network. The problem is that these are very rare cells in circulation accounting for only 0.01% of all cells.
Hence, the development of new methods for endothelial colony-forming cells isolation and culture is urgent. This has been achieved by taking advantage of an existing protocol for isolation of these cells from mononuclear blood cells and adapting it to the isolation of pulmonary endothelial colony-forming cells. The first step is to obtain a cellular homogenate from distal lungtissue by enzymatic dispersion. Then, the method relies on positive selection by magnetic activated cell sorting, labeling CD31-expressing cells. These cells can be then expanded in vitro by stimulation with cEGM-2. The colonies that form in 1 to 2 weeks in response to the stimuli are then harvested using cloning cylinders and are expanded for 2 to 3 weeks, purified, and plated again in 2 to 4 cycles. After this process, the characterization of purified cells was done by several methods. Isolated endothelial colony-forming cells express endothelial specific cell markers and do not express myeloid or hematopoietic antigens. Furthermore, when transplanted into an in vivo mouse model they exhibit self-renewal and de novoblood vessel formation, characteristic of active endothelial colony-forming cells. The entire procedure takes up to 4 weeks and the cells obtained are pure enough for the study of the pathophysiological methods of lung disease.
The isolation and culture of endothelial colony-forming cells from human and rat lungs. Alphonse, R. S., Vadivel, A., Zong, S., McConaghy, S., Ohls, R., Yoder, M. C., &Thebaud, B. (2015). Nat. Protocols, 10(11), 1697–1708.
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