Figure 3. Identification of optimal culture conditions for the ex-vivo expansion of ACS PB-derived EPC/ECFC.

Primary EPC/ECFC colonies were generated by plating patient PBMC in M⁵¹⁰⁰ medium, as detailed in the Method section. In A, after the colony identification (at day 5 after plating), medium was change (arrow) and replaced either with fresh M⁵¹⁰⁰, or M^EGM or M¹⁹⁹ and the development of the colonies was monitored over the time. The growth kinetics of a representative experiment out of five independent experiments is shown. At each indicated time point, the mean cell number/ECFC was determined by two independent operators; standard deviations were below 10% and are not shown. Asterisk, p<0.05. In B, immunocytochemical analysis of in vitro expanded EPC/ECFC documenting positivity for CD105 antigen (original magnification: 20X) and for the specific endothelial marker Factor VIII (original magnification: 40X). In C, FISH analysis performed on in vitro expanded EPC/ECFC by using the centromeric enumeration probe CEP9 (white arrows) documenting a normal diploid chromosomal pattern (original magnification: 40X).