Different growth conditions for peripheral blood endothelial progenitors.
OBJECTIVE
To compare different growth conditions for endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells (PBMNCs).


METHODS
PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, IGF-I; the same medium with bovine retina-derived extract (RDE); freshly isolated or depleted of adherent cells PBMNCs in HUVEC conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in Ml99 with RDE. PBMNCs were analysed by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. EPCs were seeded alone or with HUVECs on Matrigel to assess in vitro angiogenesis.


RESULTS
With growth factors, numerous cell clusters appeared within 1 week. Spindle-shaped and attached cells sprouted, differentiating in endothelial cell (EC)-like cells within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDE, numerous large cell clusters appeared within 1 week, but the number of cells with an EC morphology decreased during culture. FACS confirmed the endothelial phenotype and attached cells were able to migrate in response to VEGF. When nonadherent cells were cultured in HUVEC conditioned medium, they proliferated readily and EPCs were induced while freshly isolated cells neither proliferated nor induced EPCs. FACS analysis of the cocultures showed the presence of double-labeled PBMNCs expressing endothelial antigens. Capillary-like structures were observed on Matrigel only from cocultures and PBMNCs were able to incorporate in these networks.


CONCLUSIONS
PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions (growth factors, HUVEC conditioned medium, HUVECs).
Di Stefano
Rossella
R
Cardiovascular Research Laboratory, University of Pisa, Pisa, Italy.
Santoni
Tatiana
T
Barsotti
M Chiara
MC
Armani
Chiara
C
Chifenti
Barbara
B
Guida
Chiara
C
Vanacore
Renato
R
Locci
M Teresa
MT
Mariani
Massimo
M
Balbarini
Alberto
A
Mariani
Mario
M
D000925:Anticoagulants / Q000494:pharmacology; D002021:Buffers; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects*; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects*; D005353:Fibronectins / Q000494:pharmacology; D005973:Glutamine / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D006531:HEPES / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects*; D010406:Penicillins / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012016:Reference Values; D013234:Stem Cells / Q000187:drug effects*; D013307:Streptomycin / Q000494:pharmacology
D000925:Anticoagulants; D002021:Buffers; D003470:Culture Media; D005353:Fibronectins; D006133:Growth Substances; D010406:Penicillins; D011500:Protein Synthesis Inhibitors; D005973:Glutamine; D006493:Heparin; D006531:HEPES; D013307:Streptomycin