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CD34+ cells represent highly functional endothelial progenitor cells in murine bone marrow.

Yang J, Ii M, Kamei N, Alev C, Kwon SM, Kawamoto A, Akimaru H, Masuda H, Sawa Y, Asahara T - PLoS ONE (2011)

Bottom Line: Although CD34(+) cells showed the lowest EPC colony forming activity, CD34(+) cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1.Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34(+) cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34(+) cells.Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34(+) cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others.

View Article: PubMed Central - PubMed

Affiliation: Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation/RIKEN Center for Developmental Biology, Kobe, Japan.

ABSTRACT

Background: Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow.

Methodology/principal findings: CD34(+) cells, c-Kit(+)/Sca-1(+)/Lin(-) (KSL) cells, c-Kit(+)/Lin(-) (KL) cells and Sca-1(+)/Lin(-) (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34(+) cells showed the lowest EPC colony forming activity, CD34(+) cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34(+) cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34(+) cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34(+) cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others.

Conclusion: These findings suggest that mouse CD34(+) cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

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Isolation of KSL, KL, SL, and CD34+ cells by FACS and EPC-CFA.a, Lineage depleted BMMNCs were stained with APC anti-c-kit and PE anti-Sca-1 antibodies followed by FACS sorting. The cell fractions gated with black, red, and blue frames were defined as KSL, KL, and SL cells, respectively. Also BMMNCs were stained with a FITC anti-CD34 antibody followed by FACS sorting for CD34 positive cells. b, The number of colonies was counted in each group. Small EPC colony count in each group and large EPC colony count in each group. c, EPC-CFA was performed with KSL, KL, SL, and CD34+ cells, and the morphologies of small EPC colony and large EPC colony were dispalyed. The framed areas were magnified in lower panels. d, Representative double staining for Fluorescein isolectin B4 (ILB4) and DiI-acLDL in KSL cell-formed colonies. All assays were triplicated and demonstrated similar results.
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pone-0020219-g001: Isolation of KSL, KL, SL, and CD34+ cells by FACS and EPC-CFA.a, Lineage depleted BMMNCs were stained with APC anti-c-kit and PE anti-Sca-1 antibodies followed by FACS sorting. The cell fractions gated with black, red, and blue frames were defined as KSL, KL, and SL cells, respectively. Also BMMNCs were stained with a FITC anti-CD34 antibody followed by FACS sorting for CD34 positive cells. b, The number of colonies was counted in each group. Small EPC colony count in each group and large EPC colony count in each group. c, EPC-CFA was performed with KSL, KL, SL, and CD34+ cells, and the morphologies of small EPC colony and large EPC colony were dispalyed. The framed areas were magnified in lower panels. d, Representative double staining for Fluorescein isolectin B4 (ILB4) and DiI-acLDL in KSL cell-formed colonies. All assays were triplicated and demonstrated similar results.

Mentions: Initially we determined the populations to investigate. For KL, SL and KSL cell isolation, lineage positive cells, counting about 90%, were depleted from total BMMNCs. KL cells and SL cells counted 37.37±0.04% and 13.27±0.01% respectively in lineage negative BMMNCs. KSL cells were included in KL or SL cells, and counted 5.97±0.01% in lineage negative BMMNCs. For CD34+ cell isolation, CD34+ cells were 12.23±0.02% in total BMMNCs (Figure 1a). The levels of expression of CD34 by KSL, KL and SL cells are 89.8%, 72% and 55.9%, respectively (Figure S1).


CD34+ cells represent highly functional endothelial progenitor cells in murine bone marrow.

Yang J, Ii M, Kamei N, Alev C, Kwon SM, Kawamoto A, Akimaru H, Masuda H, Sawa Y, Asahara T - PLoS ONE (2011)

Isolation of KSL, KL, SL, and CD34+ cells by FACS and EPC-CFA.a, Lineage depleted BMMNCs were stained with APC anti-c-kit and PE anti-Sca-1 antibodies followed by FACS sorting. The cell fractions gated with black, red, and blue frames were defined as KSL, KL, and SL cells, respectively. Also BMMNCs were stained with a FITC anti-CD34 antibody followed by FACS sorting for CD34 positive cells. b, The number of colonies was counted in each group. Small EPC colony count in each group and large EPC colony count in each group. c, EPC-CFA was performed with KSL, KL, SL, and CD34+ cells, and the morphologies of small EPC colony and large EPC colony were dispalyed. The framed areas were magnified in lower panels. d, Representative double staining for Fluorescein isolectin B4 (ILB4) and DiI-acLDL in KSL cell-formed colonies. All assays were triplicated and demonstrated similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105013&req=5

pone-0020219-g001: Isolation of KSL, KL, SL, and CD34+ cells by FACS and EPC-CFA.a, Lineage depleted BMMNCs were stained with APC anti-c-kit and PE anti-Sca-1 antibodies followed by FACS sorting. The cell fractions gated with black, red, and blue frames were defined as KSL, KL, and SL cells, respectively. Also BMMNCs were stained with a FITC anti-CD34 antibody followed by FACS sorting for CD34 positive cells. b, The number of colonies was counted in each group. Small EPC colony count in each group and large EPC colony count in each group. c, EPC-CFA was performed with KSL, KL, SL, and CD34+ cells, and the morphologies of small EPC colony and large EPC colony were dispalyed. The framed areas were magnified in lower panels. d, Representative double staining for Fluorescein isolectin B4 (ILB4) and DiI-acLDL in KSL cell-formed colonies. All assays were triplicated and demonstrated similar results.
Mentions: Initially we determined the populations to investigate. For KL, SL and KSL cell isolation, lineage positive cells, counting about 90%, were depleted from total BMMNCs. KL cells and SL cells counted 37.37±0.04% and 13.27±0.01% respectively in lineage negative BMMNCs. KSL cells were included in KL or SL cells, and counted 5.97±0.01% in lineage negative BMMNCs. For CD34+ cell isolation, CD34+ cells were 12.23±0.02% in total BMMNCs (Figure 1a). The levels of expression of CD34 by KSL, KL and SL cells are 89.8%, 72% and 55.9%, respectively (Figure S1).

Bottom Line: Although CD34(+) cells showed the lowest EPC colony forming activity, CD34(+) cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1.Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34(+) cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34(+) cells.Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34(+) cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others.

View Article: PubMed Central - PubMed

Affiliation: Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation/RIKEN Center for Developmental Biology, Kobe, Japan.

ABSTRACT

Background: Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow.

Methodology/principal findings: CD34(+) cells, c-Kit(+)/Sca-1(+)/Lin(-) (KSL) cells, c-Kit(+)/Lin(-) (KL) cells and Sca-1(+)/Lin(-) (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34(+) cells showed the lowest EPC colony forming activity, CD34(+) cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34(+) cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34(+) cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34(+) cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others.

Conclusion: These findings suggest that mouse CD34(+) cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

Show MeSH
Related in: MedlinePlus