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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

Tube formation assay by HUVECs incorporated with KSL, KL, SL, and CD34+ cells.KSL, KL, SL, and CD34+ cells were isolated via FACS and stained with DiI. DiI-labeled cells and HUVECs were seeded onto Matrigel-coated 96 well plates in 1% FBS/EBM2-MV without growth factors. After 24 hours in culture, incorporation of each cell population into tube-like structures formed with HUVECs was evaluated under fluorescence microscopy. a, Incorporated DiI positive cells were indicated by arrows. b, Number of incorporated DiI positive cells into tube-like structures was counted and averaged. All assays were triplicated and demonstrated similar results.
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pone-0020219-g004: Tube formation assay by HUVECs incorporated with KSL, KL, SL, and CD34+ cells.KSL, KL, SL, and CD34+ cells were isolated via FACS and stained with DiI. DiI-labeled cells and HUVECs were seeded onto Matrigel-coated 96 well plates in 1% FBS/EBM2-MV without growth factors. After 24 hours in culture, incorporation of each cell population into tube-like structures formed with HUVECs was evaluated under fluorescence microscopy. a, Incorporated DiI positive cells were indicated by arrows. b, Number of incorporated DiI positive cells into tube-like structures was counted and averaged. All assays were triplicated and demonstrated similar results.

Mentions: The in vitro incorporation capacity of the cells was assayed via a previously described tube formation assay system [24]. HUVECs were employed to form tube-like structures. KSL, KL, SL and CD34+ cells were seeded together with HUVECs, incorporating into tube-like structures and supporting their growth. One thousand (1,000) DiI-labeled cells and 12,500 HUVECs were seeded onto Matrigel-coated 96 well plates. After 24 hours in culture, incorporation of each cell population into tube-like structures formed by HUVECs was evaluated under fluorescence microscopy. Augmented cell incorporation was found for the CD34+, KSL and KL groups compared to the SL group (CD34: 16±3, KSL: 13±2, KL: 15±2, SL: 8±2; P<0.05, SL vs CD34, KL and KSL) (Figure 4a and 4b).


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)

Tube formation assay by HUVECs incorporated with KSL, KL, SL, and CD34+ cells.KSL, KL, SL, and CD34+ cells were isolated via FACS and stained with DiI. DiI-labeled cells and HUVECs were seeded onto Matrigel-coated 96 well plates in 1% FBS/EBM2-MV without growth factors. After 24 hours in culture, incorporation of each cell population into tube-like structures formed with HUVECs was evaluated under fluorescence microscopy. a, Incorporated DiI positive cells were indicated by arrows. b, Number of incorporated DiI positive cells into tube-like structures was counted and averaged. 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-g004: Tube formation assay by HUVECs incorporated with KSL, KL, SL, and CD34+ cells.KSL, KL, SL, and CD34+ cells were isolated via FACS and stained with DiI. DiI-labeled cells and HUVECs were seeded onto Matrigel-coated 96 well plates in 1% FBS/EBM2-MV without growth factors. After 24 hours in culture, incorporation of each cell population into tube-like structures formed with HUVECs was evaluated under fluorescence microscopy. a, Incorporated DiI positive cells were indicated by arrows. b, Number of incorporated DiI positive cells into tube-like structures was counted and averaged. All assays were triplicated and demonstrated similar results.
Mentions: The in vitro incorporation capacity of the cells was assayed via a previously described tube formation assay system [24]. HUVECs were employed to form tube-like structures. KSL, KL, SL and CD34+ cells were seeded together with HUVECs, incorporating into tube-like structures and supporting their growth. One thousand (1,000) DiI-labeled cells and 12,500 HUVECs were seeded onto Matrigel-coated 96 well plates. After 24 hours in culture, incorporation of each cell population into tube-like structures formed by HUVECs was evaluated under fluorescence microscopy. Augmented cell incorporation was found for the CD34+, KSL and KL groups compared to the SL group (CD34: 16±3, KSL: 13±2, KL: 15±2, SL: 8±2; P<0.05, SL vs CD34, KL and KSL) (Figure 4a and 4b).

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