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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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Related in: MedlinePlus

Assessment of endothelial markers in cultured KSL, KL, SL, and CD34+ cells by immunocytostaining and real-time RT-PCR.Freshly isolated cells were cultured in 20%FBS/EGM-2MV medium on vitronectin-coated 4 well-chamber slides for 7 days. a, Adherent cells were stained with endothelial markers, VE-cadherin, vWF, and Flk-1. b, mRNA expression levels of the markers in 3-days cultured cells were assessed by quantitative real-time RT-PCR. The mRNA expressions were normalized to GAPDH (n = 3). All assays were triplicated and demonstrated similar results.
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pone-0020219-g003: Assessment of endothelial markers in cultured KSL, KL, SL, and CD34+ cells by immunocytostaining and real-time RT-PCR.Freshly isolated cells were cultured in 20%FBS/EGM-2MV medium on vitronectin-coated 4 well-chamber slides for 7 days. a, Adherent cells were stained with endothelial markers, VE-cadherin, vWF, and Flk-1. b, mRNA expression levels of the markers in 3-days cultured cells were assessed by quantitative real-time RT-PCR. The mRNA expressions were normalized to GAPDH (n = 3). All assays were triplicated and demonstrated similar results.

Mentions: The expression of endothelial markers in cultured KSL, KL, SL, and CD34+ cells was assessed by fluorescent immunocytochemical staining and real-time RT-PCR. Consistent with the previous report that cultured EPCs express endothelial markers such as VE-cadherin, vWF and Flk-1 [23], a significant number of cells expressed VE-cadherin, vWF and Flk-1 protein in each group (Figure 3a). RT-PCR revealed that at day three CD34+ cells had the highest mRNA expression levels of VE-cadherin, Flk-1 and vWF (Figure 3b). While all the cells before culture expressed low and similar levels of these markers (data not shown).


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)

Assessment of endothelial markers in cultured KSL, KL, SL, and CD34+ cells by immunocytostaining and real-time RT-PCR.Freshly isolated cells were cultured in 20%FBS/EGM-2MV medium on vitronectin-coated 4 well-chamber slides for 7 days. a, Adherent cells were stained with endothelial markers, VE-cadherin, vWF, and Flk-1. b, mRNA expression levels of the markers in 3-days cultured cells were assessed by quantitative real-time RT-PCR. The mRNA expressions were normalized to GAPDH (n = 3). 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-g003: Assessment of endothelial markers in cultured KSL, KL, SL, and CD34+ cells by immunocytostaining and real-time RT-PCR.Freshly isolated cells were cultured in 20%FBS/EGM-2MV medium on vitronectin-coated 4 well-chamber slides for 7 days. a, Adherent cells were stained with endothelial markers, VE-cadherin, vWF, and Flk-1. b, mRNA expression levels of the markers in 3-days cultured cells were assessed by quantitative real-time RT-PCR. The mRNA expressions were normalized to GAPDH (n = 3). All assays were triplicated and demonstrated similar results.
Mentions: The expression of endothelial markers in cultured KSL, KL, SL, and CD34+ cells was assessed by fluorescent immunocytochemical staining and real-time RT-PCR. Consistent with the previous report that cultured EPCs express endothelial markers such as VE-cadherin, vWF and Flk-1 [23], a significant number of cells expressed VE-cadherin, vWF and Flk-1 protein in each group (Figure 3a). RT-PCR revealed that at day three CD34+ cells had the highest mRNA expression levels of VE-cadherin, Flk-1 and vWF (Figure 3b). While all the cells before culture expressed low and similar levels of these markers (data not shown).

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