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In vitro characterization of circulating endothelial progenitor cells isolated from patients with acute coronary syndrome.

Campioni D, Zauli G, Gambetti S, Campo G, Cuneo A, Ferrari R, Secchiero P - PLoS ONE (2013)

Bottom Line: Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133.Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Section of Hematology, Azienda Ospedaliero-Universitaria, Arcispedale Sant'Anna, University of Ferrara, Ferrara, Italy.

ABSTRACT

Background: The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

Methodology/principal findings: By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7-14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

Conclusion/significance: Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

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Related in: MedlinePlus

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 MEGM 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).
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pone-0056377-g003: 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 MEGM 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).

Mentions: For the identification of primary EPC/ECFC, patient PBMC were seeded in three different culture media (as detailed in the Methods). Growth of EPC/ECFC was detected only by using the M5100 medium, while and MEGM and in M199 were ineffective for this purpose. In order to perform further cell characterizations, we searched for the optimal culture conditions for the in vitro expansion of the primary EPC/ECFC, by assessing the change of medium after the initial plating in M5100. Indeed, while M5100 medium was necessary to obtain primary colonies, reaching a mean number of 102±25 cells/colony after 15 days of culture, a switch of the medium to MEGM, which is a medium particularly enriched of angiogenic cytokines, after the colony identification (approximately at day 5 after PBMC plating), significantly (p<0.05) improved the growth kinetics (Figure 3A).


In vitro characterization of circulating endothelial progenitor cells isolated from patients with acute coronary syndrome.

Campioni D, Zauli G, Gambetti S, Campo G, Cuneo A, Ferrari R, Secchiero P - PLoS ONE (2013)

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 MEGM 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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569417&req=5

pone-0056377-g003: 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 MEGM 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).
Mentions: For the identification of primary EPC/ECFC, patient PBMC were seeded in three different culture media (as detailed in the Methods). Growth of EPC/ECFC was detected only by using the M5100 medium, while and MEGM and in M199 were ineffective for this purpose. In order to perform further cell characterizations, we searched for the optimal culture conditions for the in vitro expansion of the primary EPC/ECFC, by assessing the change of medium after the initial plating in M5100. Indeed, while M5100 medium was necessary to obtain primary colonies, reaching a mean number of 102±25 cells/colony after 15 days of culture, a switch of the medium to MEGM, which is a medium particularly enriched of angiogenic cytokines, after the colony identification (approximately at day 5 after PBMC plating), significantly (p<0.05) improved the growth kinetics (Figure 3A).

Bottom Line: Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133.Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Section of Hematology, Azienda Ospedaliero-Universitaria, Arcispedale Sant'Anna, University of Ferrara, Ferrara, Italy.

ABSTRACT

Background: The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

Methodology/principal findings: By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7-14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

Conclusion/significance: Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

Show MeSH
Related in: MedlinePlus