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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

Subcloning potential of EPC/ECFC generated from the PBMC of ACS patients.After ex-vivo expansion, primary EPC/ECFC colonies were trypsinized and assessed for clonogenic potential capacity by single cells replating assay. In A, single cells derived from EPC/ECPF colonies were seeded in collagen I coated wells and monitored day by day (a: day 1; b: day 2; c: day 3; e–f: day 4; a–e: original magnification 25X; f: original magnification 40X). One representative experiment is shown. In B, secondary clones were classified on the basis of their proliferation properties. Data are mean±SD derived from six independent experiments.
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pone-0056377-g005: Subcloning potential of EPC/ECFC generated from the PBMC of ACS patients.After ex-vivo expansion, primary EPC/ECFC colonies were trypsinized and assessed for clonogenic potential capacity by single cells replating assay. In A, single cells derived from EPC/ECPF colonies were seeded in collagen I coated wells and monitored day by day (a: day 1; b: day 2; c: day 3; e–f: day 4; a–e: original magnification 25X; f: original magnification 40X). One representative experiment is shown. In B, secondary clones were classified on the basis of their proliferation properties. Data are mean±SD derived from six independent experiments.

Mentions: After isolation from the ACS PBMC and ex-vivo expansion, primary EPC/ECFC colonies were trypsinized and assessed for: i) their immuno-phenotype, by multi-colors flow cytometry (Figure 4) as well as for ii) clonogenic potential capacity, by single cells sub-culturing (Figure 5). As documented in Figure 4A, EPC/ECFC colonies were characterized by a variable expression of the CD34 antigen, ranging from 20-75% among the different cell samples. Moreover, a 4-colors flow cytometric analysis showed that viable cells from EPC/ECFC colonies were CD45 negative and by gating on cultured CD34+/CD45-/7-AAD- EPC/ECFC, the expression of CD105, CD31 and CD146 resulted uniformly positive (Figure 4B). On the other hand, EPC/ECFC were always negative for CD90, CD117 and CD133, while the expression of CD106 and CD184 was variable (data not shown).


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)

Subcloning potential of EPC/ECFC generated from the PBMC of ACS patients.After ex-vivo expansion, primary EPC/ECFC colonies were trypsinized and assessed for clonogenic potential capacity by single cells replating assay. In A, single cells derived from EPC/ECPF colonies were seeded in collagen I coated wells and monitored day by day (a: day 1; b: day 2; c: day 3; e–f: day 4; a–e: original magnification 25X; f: original magnification 40X). One representative experiment is shown. In B, secondary clones were classified on the basis of their proliferation properties. Data are mean±SD derived from six independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056377-g005: Subcloning potential of EPC/ECFC generated from the PBMC of ACS patients.After ex-vivo expansion, primary EPC/ECFC colonies were trypsinized and assessed for clonogenic potential capacity by single cells replating assay. In A, single cells derived from EPC/ECPF colonies were seeded in collagen I coated wells and monitored day by day (a: day 1; b: day 2; c: day 3; e–f: day 4; a–e: original magnification 25X; f: original magnification 40X). One representative experiment is shown. In B, secondary clones were classified on the basis of their proliferation properties. Data are mean±SD derived from six independent experiments.
Mentions: After isolation from the ACS PBMC and ex-vivo expansion, primary EPC/ECFC colonies were trypsinized and assessed for: i) their immuno-phenotype, by multi-colors flow cytometry (Figure 4) as well as for ii) clonogenic potential capacity, by single cells sub-culturing (Figure 5). As documented in Figure 4A, EPC/ECFC colonies were characterized by a variable expression of the CD34 antigen, ranging from 20-75% among the different cell samples. Moreover, a 4-colors flow cytometric analysis showed that viable cells from EPC/ECFC colonies were CD45 negative and by gating on cultured CD34+/CD45-/7-AAD- EPC/ECFC, the expression of CD105, CD31 and CD146 resulted uniformly positive (Figure 4B). On the other hand, EPC/ECFC were always negative for CD90, CD117 and CD133, while the expression of CD106 and CD184 was variable (data not shown).

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