Endothelial progenitor cells induction by short-term stimulation with phytohaemagglutinin of mononuclear cells
Rossella Di Stefano1, Tatiana Santoni, Chiara Armani
1Cardiovascular Research Laboratory, Cardiac and Thoracic Department, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy. r.distefano@ao-pisa.toscana.it
Background:
The identification of circulating endothelial progenitors cells (EPCs) in the adult has forced to reconsider how new blood vessels grow in physiological and pathological conditions. Neovascularization during adult life has long been attributed to angiogenesis only. However, recent studies have revealed that peripheral blood EPCs may be recruited and incorporated into sites of active neovascularization.
Purpose:
To verify that EPCs are induced from peripheral blood mononuclear cells (PBMCs) and bone marrow derived mononuclear cells (BMMCs) upon short-term stimulation with phytohaemoagglutinin (PHA), a potent T-cell mitogen.
Methods:
PBMCs and BMMCs were isolated from healthy donors. Freshly isolated or depleted of adherent cells (one day and three days of adherence) mononuclear cells (MCs) were cultured in RPMI, 10% FBS, containing PHA (10 microl/10(6) cells) for 24 h. After stimulation with PHA, clusters of adherent cells were further propagated in M199 containing L-glutammine, Hepes, 20% FBS, heparin, antibiotics and bovine retina extract for 1 and 2 weeks. PBMCs and BMMCs cultured without PHA stimulation served as controls. FACS of EPCs was performed on attached cells after 7 and 14 days of culture.
Results And Conclusion:
After stimulation of MCs with PHA for 24 h, many cells clusters were observed and around these clusters some adherent EC-like cells were observed. These cells were ovoid but a very little of these were elongated in morphology, however their number and size gradually increased during culture. However a longer time was needed for obtaining EPCs from MCs harvested after adherence. Thus this indicates that short-term signals provided by PHA must be sufficient for MCs to express the ligands necessary for the induction of EPCs but signals from monocytes/macrophages are important for a more rapid differentiation.
