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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 recruitment and detection of homing molecules in KSL, KL, SL, and CD34+ cells.KSL, KL, SL, and CD34+ cells were freshly isolated by FACS and stained with DiI. DiI-labeled cells were systemically injected into mice three days after MI induction, and heart samples were examined histologically one day after cell injection. a, DiI positive cells (red) detected in ischemic myocardium under a fluorescence microscope. b, Number of DiI positive cells was counted in the ischemic border zone (bilateral sides of peri-infarct area) and averaged (n = 3). c and d, KSL, KL, SL, and CD34+ cells were freshly isolated by FACS, and Integrin β2 and CXCR4 gene expression levels of these cells were examined by quantitative real-time RT-PCR. All assays were triplicated and demonstrated similar results.
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pone-0020219-g005: Assessment of cell recruitment and detection of homing molecules in KSL, KL, SL, and CD34+ cells.KSL, KL, SL, and CD34+ cells were freshly isolated by FACS and stained with DiI. DiI-labeled cells were systemically injected into mice three days after MI induction, and heart samples were examined histologically one day after cell injection. a, DiI positive cells (red) detected in ischemic myocardium under a fluorescence microscope. b, Number of DiI positive cells was counted in the ischemic border zone (bilateral sides of peri-infarct area) and averaged (n = 3). c and d, KSL, KL, SL, and CD34+ cells were freshly isolated by FACS, and Integrin β2 and CXCR4 gene expression levels of these cells were examined by quantitative real-time RT-PCR. All assays were triplicated and demonstrated similar results.

Mentions: Next, we examined the homing capacity of these cells in mouse MI models. For tracking the fate of grafted EPCs in mouse myocardium, transplanted cells were labeled with CM-DiI after isolation by FACS and then delivered systemically three days after MI induction. Heart samples harvested one day after cell injection and examined for the presence of fluorescent-labeled cells. DiI-positive cells were observed in the infarcted myocardium of all examined groups (Figure 5a). However, the most dramatic immediate cell recruitment to ischemic myocardium was detected in the CD34+ cell-injected group (CD34: 112±41/mm2, KSL: 74±34/mm2, KL: 47±21/mm2 and SL: 57±24/mm2; P<0.05, CD34 vs KSL, KL and SL) (Figure 5b). No significant differences among the numbers of recruited cells in the KSL, KL and SL groups could be observed. Integrin β2 has been reported previously to be critical adhesion molecules for the homing of EPCs to ischemic tissue [18], [19], [20], [25]. SDF1/CXCR4 axis has also been shown to be one of the major chemokine/receptor signalings for EPC recruitment [26], [27], [28]. CXCR4 is also crucial for homing of transplanted EPC into ischemic tissues [21]. We thus assessed the mRNA expression levels of these homing-related molecules in the cells that were used for transplantation. We observed a marked up-regulation of integrin β2 and CXCR4 in CD34+ cells (Figure 5c and 5d). KL cells also exhibited higher expression levels of CXCR4 (Figure 5d).


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 recruitment and detection of homing molecules in KSL, KL, SL, and CD34+ cells.KSL, KL, SL, and CD34+ cells were freshly isolated by FACS and stained with DiI. DiI-labeled cells were systemically injected into mice three days after MI induction, and heart samples were examined histologically one day after cell injection. a, DiI positive cells (red) detected in ischemic myocardium under a fluorescence microscope. b, Number of DiI positive cells was counted in the ischemic border zone (bilateral sides of peri-infarct area) and averaged (n = 3). c and d, KSL, KL, SL, and CD34+ cells were freshly isolated by FACS, and Integrin β2 and CXCR4 gene expression levels of these cells were examined by quantitative real-time RT-PCR. 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-g005: Assessment of cell recruitment and detection of homing molecules in KSL, KL, SL, and CD34+ cells.KSL, KL, SL, and CD34+ cells were freshly isolated by FACS and stained with DiI. DiI-labeled cells were systemically injected into mice three days after MI induction, and heart samples were examined histologically one day after cell injection. a, DiI positive cells (red) detected in ischemic myocardium under a fluorescence microscope. b, Number of DiI positive cells was counted in the ischemic border zone (bilateral sides of peri-infarct area) and averaged (n = 3). c and d, KSL, KL, SL, and CD34+ cells were freshly isolated by FACS, and Integrin β2 and CXCR4 gene expression levels of these cells were examined by quantitative real-time RT-PCR. All assays were triplicated and demonstrated similar results.
Mentions: Next, we examined the homing capacity of these cells in mouse MI models. For tracking the fate of grafted EPCs in mouse myocardium, transplanted cells were labeled with CM-DiI after isolation by FACS and then delivered systemically three days after MI induction. Heart samples harvested one day after cell injection and examined for the presence of fluorescent-labeled cells. DiI-positive cells were observed in the infarcted myocardium of all examined groups (Figure 5a). However, the most dramatic immediate cell recruitment to ischemic myocardium was detected in the CD34+ cell-injected group (CD34: 112±41/mm2, KSL: 74±34/mm2, KL: 47±21/mm2 and SL: 57±24/mm2; P<0.05, CD34 vs KSL, KL and SL) (Figure 5b). No significant differences among the numbers of recruited cells in the KSL, KL and SL groups could be observed. Integrin β2 has been reported previously to be critical adhesion molecules for the homing of EPCs to ischemic tissue [18], [19], [20], [25]. SDF1/CXCR4 axis has also been shown to be one of the major chemokine/receptor signalings for EPC recruitment [26], [27], [28]. CXCR4 is also crucial for homing of transplanted EPC into ischemic tissues [21]. We thus assessed the mRNA expression levels of these homing-related molecules in the cells that were used for transplantation. We observed a marked up-regulation of integrin β2 and CXCR4 in CD34+ cells (Figure 5c and 5d). KL cells also exhibited higher expression levels of CXCR4 (Figure 5d).

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