Figure 3

Importance of small-EPCs as large-EPC-CFU-sprouting cells. (a) Flow cytometry analysis of small-EPCs or large-EPCs after culturing for 10 days from freshly isolated bone marrow c-Kit⁺/Sca-1⁺ lineage-negative cells (BM-KSL). (b) Secondary culture assay of small-EPC-CFUs from bone marrow mononuclear cells (BM-MNCs). Representative micrographs of small-EPC-CFUs from BM-MNCs before reseeding are shown on the left, and representative light and fluorescent micrographs of small-EPCs cultured secondarily in methylcellulose-containing medium are shown on the right. Secondary cultured cells were identified as double-positive cells due to 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate-labeled acetylated-low density lipoprotein (acLDL-DiI) uptake (red) and BS-1 lectin reactivity (green). Small-EPCs could differentiate into large-EPCs. (c) The expression of vascular endothelial (VE)-cadherin, Flk-1, and endothelial nitric oxide synthase (eNOS) was measured in small-EPCs, small-EPCs-derived large-EPCs (Large EPCs-1), and large-EPCs (Large EPCs-2) by reverse transcription-polymerase chain reaction analysis. (d) Adhesion assay of small-EPCs, Large EPCs-1, and Large EPCs-2. *P < 0.05, **P < 0.01 versus small-EPCs. (e) Quantification of the number of cells incorporated into tubes in small-EPCs, Large EPCs-1, and Large EPCs-2. *P < 0.05, **P < 0.01 versus small-EPCs.