Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of the clonogenic potential of PBMC derived from ACS patients.PBMC samples obtained from ACS patients (n = 70) were seeded in collagen I coated wells for short-term primary colony assay in liquid culture medium. Cultures were monitored for 15 days for the presence of adherent colonies, scored on the basis of morphological features as: CFU-EC (A, left panel) or EPC/ECFC (B, left panel; arrowheads: hemopoietic mononucleated cells). In A, the right panel shows a monolayer of spindle-shaped endothelial-like monocytes. In B, the right panel shows a representative image of CFU-EC after in vitro expansion. Original magnification: 20X and 25X for the inset. In C, frequency of detection of CFU-EC and EPC/ECFC in PBMC of ACS patients, divided on the basis of the time of blood withdrawal after the hospital admission for the acute cardiovascular events.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3569417&req=5

pone-0056377-g001: Characterization of the clonogenic potential of PBMC derived from ACS patients.PBMC samples obtained from ACS patients (n = 70) were seeded in collagen I coated wells for short-term primary colony assay in liquid culture medium. Cultures were monitored for 15 days for the presence of adherent colonies, scored on the basis of morphological features as: CFU-EC (A, left panel) or EPC/ECFC (B, left panel; arrowheads: hemopoietic mononucleated cells). In A, the right panel shows a monolayer of spindle-shaped endothelial-like monocytes. In B, the right panel shows a representative image of CFU-EC after in vitro expansion. Original magnification: 20X and 25X for the inset. In C, frequency of detection of CFU-EC and EPC/ECFC in PBMC of ACS patients, divided on the basis of the time of blood withdrawal after the hospital admission for the acute cardiovascular events.

Mentions: PBMC samples obtained from the ACS patients were seeded in collagen I coated wells for short-term primary colony assay in liquid culture medium. Cultures were scored up to 15 days of culture for the presence and the morphology of adherent colony forming units of monocytes (CFU-EC; Figure 1A) and endothelial (EPC/ECFC; Figure 1B) origin. CFU-EC colonies, as previously described [6], [24], were characterized by a central cluster of endothelial-like monocytic cells (Figure 1A), sometimes forming also tubular structures. CFU-EC could be frequently (77%) derived from the ACS patients, irrespectively of time of blood withdrawal (Figure 1C). Of note, CFU-EC did not displayed in vitro expansion capacity and their endothelial differentiation resulted defective, in spite of using different endothelial specific media supplemented of pro-angiogenic cytokines.


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)

Characterization of the clonogenic potential of PBMC derived from ACS patients.PBMC samples obtained from ACS patients (n = 70) were seeded in collagen I coated wells for short-term primary colony assay in liquid culture medium. Cultures were monitored for 15 days for the presence of adherent colonies, scored on the basis of morphological features as: CFU-EC (A, left panel) or EPC/ECFC (B, left panel; arrowheads: hemopoietic mononucleated cells). In A, the right panel shows a monolayer of spindle-shaped endothelial-like monocytes. In B, the right panel shows a representative image of CFU-EC after in vitro expansion. Original magnification: 20X and 25X for the inset. In C, frequency of detection of CFU-EC and EPC/ECFC in PBMC of ACS patients, divided on the basis of the time of blood withdrawal after the hospital admission for the acute cardiovascular events.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056377-g001: Characterization of the clonogenic potential of PBMC derived from ACS patients.PBMC samples obtained from ACS patients (n = 70) were seeded in collagen I coated wells for short-term primary colony assay in liquid culture medium. Cultures were monitored for 15 days for the presence of adherent colonies, scored on the basis of morphological features as: CFU-EC (A, left panel) or EPC/ECFC (B, left panel; arrowheads: hemopoietic mononucleated cells). In A, the right panel shows a monolayer of spindle-shaped endothelial-like monocytes. In B, the right panel shows a representative image of CFU-EC after in vitro expansion. Original magnification: 20X and 25X for the inset. In C, frequency of detection of CFU-EC and EPC/ECFC in PBMC of ACS patients, divided on the basis of the time of blood withdrawal after the hospital admission for the acute cardiovascular events.
Mentions: PBMC samples obtained from the ACS patients were seeded in collagen I coated wells for short-term primary colony assay in liquid culture medium. Cultures were scored up to 15 days of culture for the presence and the morphology of adherent colony forming units of monocytes (CFU-EC; Figure 1A) and endothelial (EPC/ECFC; Figure 1B) origin. CFU-EC colonies, as previously described [6], [24], were characterized by a central cluster of endothelial-like monocytic cells (Figure 1A), sometimes forming also tubular structures. CFU-EC could be frequently (77%) derived from the ACS patients, irrespectively of time of blood withdrawal (Figure 1C). Of note, CFU-EC did not displayed in vitro expansion capacity and their endothelial differentiation resulted defective, in spite of using different endothelial specific media supplemented of pro-angiogenic cytokines.

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