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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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Assessment of cell incorporation into the neovasculature of the ischemic myocardium.KSL, KL, SL, and CD34+ cells were isolated by FACS from GtRosa26 transgenic mice expressing LacZ gene universally, and were systemically injected three days after MI induction. Heart samples were harvested following BS1 lectin systemic perfusion 28 days after cell injection. a, Recruited cells visualized by immunofluorescent staining for β-gal (green) and BS1 lectin perfused capillaries (red) were shown in all groups. Recruited cells co-localizing with capillaries were marked by arrowheads and shown in yellow. b, A representative confocal image from the CD34+ cell-injected group. c, The numbers of recruited cells that co-localized with capillaries were counted under a fluorescence microscope separately and averaged.
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pone-0020219-g006: Assessment of cell incorporation into the neovasculature of the ischemic myocardium.KSL, KL, SL, and CD34+ cells were isolated by FACS from GtRosa26 transgenic mice expressing LacZ gene universally, and were systemically injected three days after MI induction. Heart samples were harvested following BS1 lectin systemic perfusion 28 days after cell injection. a, Recruited cells visualized by immunofluorescent staining for β-gal (green) and BS1 lectin perfused capillaries (red) were shown in all groups. Recruited cells co-localizing with capillaries were marked by arrowheads and shown in yellow. b, A representative confocal image from the CD34+ cell-injected group. c, The numbers of recruited cells that co-localized with capillaries were counted under a fluorescence microscope separately and averaged.

Mentions: To track the injected cells in vivo for 28 days, we employed cells, isolated from ROSA mice, that constitutively express β-gal under the regulation of LacZ gene. Thus, cell differentiation and incorporation into the neovasculature of ischemic myocardium four weeks after surgery can be detected as β–gal+ cells by immunohistochemistry. Double fluorescent immunostaining of ischemic myocardium 28 days after surgery revealed that β-gal-expressing EPCs (green) were frequently observed in ischemic border zone and co-localized with vessels perfused with BS-1 lectin (red) in the CD34+ cell-injected group (Figure 6a). In contrast, a few KSL, KL and SL cells were observed in the ischemic border zone and incorporated into vessels (Figure 6a). Quantitative analysis for cell retention demonstrated that the number of incorporated cells into neovasculature was significantly higher in the CD34+ cell-injected group than those in the other groups (Figure 6c).


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 cell incorporation into the neovasculature of the ischemic myocardium.KSL, KL, SL, and CD34+ cells were isolated by FACS from GtRosa26 transgenic mice expressing LacZ gene universally, and were systemically injected three days after MI induction. Heart samples were harvested following BS1 lectin systemic perfusion 28 days after cell injection. a, Recruited cells visualized by immunofluorescent staining for β-gal (green) and BS1 lectin perfused capillaries (red) were shown in all groups. Recruited cells co-localizing with capillaries were marked by arrowheads and shown in yellow. b, A representative confocal image from the CD34+ cell-injected group. c, The numbers of recruited cells that co-localized with capillaries were counted under a fluorescence microscope separately and averaged.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020219-g006: Assessment of cell incorporation into the neovasculature of the ischemic myocardium.KSL, KL, SL, and CD34+ cells were isolated by FACS from GtRosa26 transgenic mice expressing LacZ gene universally, and were systemically injected three days after MI induction. Heart samples were harvested following BS1 lectin systemic perfusion 28 days after cell injection. a, Recruited cells visualized by immunofluorescent staining for β-gal (green) and BS1 lectin perfused capillaries (red) were shown in all groups. Recruited cells co-localizing with capillaries were marked by arrowheads and shown in yellow. b, A representative confocal image from the CD34+ cell-injected group. c, The numbers of recruited cells that co-localized with capillaries were counted under a fluorescence microscope separately and averaged.
Mentions: To track the injected cells in vivo for 28 days, we employed cells, isolated from ROSA mice, that constitutively express β-gal under the regulation of LacZ gene. Thus, cell differentiation and incorporation into the neovasculature of ischemic myocardium four weeks after surgery can be detected as β–gal+ cells by immunohistochemistry. Double fluorescent immunostaining of ischemic myocardium 28 days after surgery revealed that β-gal-expressing EPCs (green) were frequently observed in ischemic border zone and co-localized with vessels perfused with BS-1 lectin (red) in the CD34+ cell-injected group (Figure 6a). In contrast, a few KSL, KL and SL cells were observed in the ischemic border zone and incorporated into vessels (Figure 6a). Quantitative analysis for cell retention demonstrated that the number of incorporated cells into neovasculature was significantly higher in the CD34+ cell-injected group than those in the other groups (Figure 6c).

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