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Journal of Biochemistry Advance Access published online on February 18, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm060
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© 2007 The Japanese Biochemical Society

Comparison Between Total Endothelial Progenitor Cell Isolation Versus Enriched Cd133+ Culture

Amelia Casamassimi*,{dagger}, Maria Luisa Balestrieri**,{dagger}, Carmela Fiorito*, Concetta Schiano*, Ciro Maione*, Raffaele Rossiello**, Vincenzo Grimaldi*, Vincenzo Del Giudice*, Ciro Balestrieri**, Bartolomeo Farzati*, Vincenzo Sica* and Claudio Napoli*,°

*Department of General Pathology, Division of Clinical Pathology and Excellence Research Center on Cardiovascular Diseases, and **Department of Chemical Biology and Physics, 1st School of Medicine, II University of Naples, Italy.

°Correspondence to: Prof. Claudio Napoli, Department of General Pathology, Division of Clinical Pathology and Excellence Research Center on Cardiovascular Diseases, Complesso S. Andrea delle Dame, 1st School of Medicine, II University of Naples, Naples 80138 Italy- e-mail: claunap{at}tin.it Tel/fax +39-081-293399

Received January 11, 2007; Accepted January 23, 2007


  Abstract

Endothelial progenitor cells (EPCs) play a role in endogenous neovascularization of ischemic tissues. Isolation and characterization of EPCs from circulating mononuclear cells is important for developing targeted cellular therapies and reproducibility of data is the major scientific goals. Here we compared two currently employed isolation methods, i.e. from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133+ cells, by defining the cell morphology and functional activities. We show that EPCs from cultured PBMCs resulted in an adherent population of 23%±4% merged cells positive for Dil-Ac-LDL and lectin, whereas the percentage of double positive cells in cultured CD133+ enriched cells was 50%±7% (P<0.01). These data were obtained through a novel an more complete method of analysis of cell calculations (specifically by dividing each microscope field into 120 sub-fields). When stimulated with TNF-{alpha} and glucose, cell number was reduced in EPCs from total PBMCs and, more consistently, in CD133+ enriched cells. However, both cultured total PBMCs and CD133+ enriched cells respond similarly to TNF-{alpha} or glucose-induced p38-phosphorylation.

EPCs from both procedures show similar results in terms of phenotype and response to modulators of their functional activities. However, when the cell phenotype of CD133+ enrichment-derived cells was compared with that of cells from the total PBMC, a significant increase in CD133+ expression was observed (P<0.01) This may have relevance during intervention studies using cultured EPCs.

Key Words: CD133, Endothelial progenitor cells, glucose, TNF

{dagger}Contributed equally to this study


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