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A novel early precursor cell population from rat bone marrow promotes angiogenesis in vitro.

Brandl A, Yuan Q, Boos AM, Beier JP, Arkudas A, Kneser U, Horch RE, Bleiziffer O - BMC Cell Biol. (2014)

Bottom Line: Some studies demonstrated therapeutic angiogenesis attributable to the effects of endothelial progenitor cells (EPC), others have reported disappointing results.Based upon these observations in this study we describe a novel and time-saving method for obtaining a pure endothelial precursor cell population as early as 2-3 weeks post isolation that exhibits endothelial abilities in vitro and which still might have retained its early endothelial lineage properties.The rapid isolation and the high angiogenic potential of these syngeneic cells might facilitate and accelerate the pre-vascularization of transplanted tissues and organs also in a human setting in the future.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plastic and Hand Surgery, Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nuremberg, Krankenhausstrasse 12, Erlangen 91054, Germany. oliver.bleiziffer@uk-erlangen.de.

ABSTRACT

Background: Some studies demonstrated therapeutic angiogenesis attributable to the effects of endothelial progenitor cells (EPC), others have reported disappointing results. This may be due to the fact that EPC populations used in these contradictory studies were selected and defined by highly variable and differing experimental protocols. Indeed, the isolation and reliable characterization of ex vivo differentiated EPC raises considerable problems due to the fact there is no biomarker currently available to specifically identify EPC exclusively. On the other hand traditional differentiation of primary immature bone marrow cells towards the endothelial lineage is a time-consuming process of up to 5 weeks. To circumvent these shortcomings, we herein describe a facile method to isolate and enrich a primary cell population from rat bone marrow, combining differential attachment methodology with cell sorting technology.

Results: The combination of these techniques enabled us to obtain a pure population of early endothelial precursor cells that show homogenous upregulation of CD31 and VEGF-R2 and that are positive for CD146. These cells exhibited typical sprouting on Matrigel™. Additionally, this population displayed endothelial tube formation when resuspended in Matrigel™ as well as in fibrin glue, demonstrating its functional angiogenic capacity. Moreover, these cells stained positive for DiI-ac-LDL and FITC-UEA, two markers that are commonly considered to stain differentiating EPCs. Based upon these observations in this study we describe a novel and time-saving method for obtaining a pure endothelial precursor cell population as early as 2-3 weeks post isolation that exhibits endothelial abilities in vitro and which still might have retained its early endothelial lineage properties.

Conclusion: The rapid isolation and the high angiogenic potential of these syngeneic cells might facilitate and accelerate the pre-vascularization of transplanted tissues and organs also in a human setting in the future.

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Flow cytometric analyses 2 weeks after isolation and cultivation of rat bone marrow MNC show heterogeneity of the cell population. (a) Cells were grown in specialized endothelial cell growth medium (EGM2 MV) for 2 weeks after isolation and their morphology was examined using light microscopy. The scale bar depicts 100 μm. The cells were further analyzed by Flow Cytometry for expression of endothelial cell-specific surface markers including CD31 (b), CD146 (c) and VEGF-R2 (d). Grey histograms indicate fluorescence signals of negative controls; white histograms indicate fluorescence signals of specific antigens. Results are representative of 4 separate experiments.
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Figure 1: Flow cytometric analyses 2 weeks after isolation and cultivation of rat bone marrow MNC show heterogeneity of the cell population. (a) Cells were grown in specialized endothelial cell growth medium (EGM2 MV) for 2 weeks after isolation and their morphology was examined using light microscopy. The scale bar depicts 100 μm. The cells were further analyzed by Flow Cytometry for expression of endothelial cell-specific surface markers including CD31 (b), CD146 (c) and VEGF-R2 (d). Grey histograms indicate fluorescence signals of negative controls; white histograms indicate fluorescence signals of specific antigens. Results are representative of 4 separate experiments.

Mentions: Rat MNC isolated from bone marrow by density gradient and cultured on Gelatin-coated (1%) culture plates in EGM MV2 exhibited distinct cell morphology typical of endothelial lineage cells. These cells formed colonies that showed pronounced “cobblestone” morphology (Figure 1a). Analogous observations were made for the rat liver endothelial cell line EC52 that served as a positive control for functional experiments in this study. Furthermore, 2 weeks post-isolation, the population demonstrated to be composed of heterogenous cells in FACS-analyses. A small fraction of the cells expressed CD31 (Platelet endothelial cell adhesion molecule, PECAM-1) (8.3% ± 2.1) (Figure 1b). CD146, also known as the melanoma cell adhesion molecule (MCAM), was already markedly expressed on the cells with different intensity (15.7 ± 5.1%) (Figure 1c). Cells did not express vascular endothelial growth factor receptor 2 (VEGF-R2/KDR) at this time point (Figure 1d).


A novel early precursor cell population from rat bone marrow promotes angiogenesis in vitro.

Brandl A, Yuan Q, Boos AM, Beier JP, Arkudas A, Kneser U, Horch RE, Bleiziffer O - BMC Cell Biol. (2014)

Flow cytometric analyses 2 weeks after isolation and cultivation of rat bone marrow MNC show heterogeneity of the cell population. (a) Cells were grown in specialized endothelial cell growth medium (EGM2 MV) for 2 weeks after isolation and their morphology was examined using light microscopy. The scale bar depicts 100 μm. The cells were further analyzed by Flow Cytometry for expression of endothelial cell-specific surface markers including CD31 (b), CD146 (c) and VEGF-R2 (d). Grey histograms indicate fluorescence signals of negative controls; white histograms indicate fluorescence signals of specific antigens. Results are representative of 4 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3987126&req=5

Figure 1: Flow cytometric analyses 2 weeks after isolation and cultivation of rat bone marrow MNC show heterogeneity of the cell population. (a) Cells were grown in specialized endothelial cell growth medium (EGM2 MV) for 2 weeks after isolation and their morphology was examined using light microscopy. The scale bar depicts 100 μm. The cells were further analyzed by Flow Cytometry for expression of endothelial cell-specific surface markers including CD31 (b), CD146 (c) and VEGF-R2 (d). Grey histograms indicate fluorescence signals of negative controls; white histograms indicate fluorescence signals of specific antigens. Results are representative of 4 separate experiments.
Mentions: Rat MNC isolated from bone marrow by density gradient and cultured on Gelatin-coated (1%) culture plates in EGM MV2 exhibited distinct cell morphology typical of endothelial lineage cells. These cells formed colonies that showed pronounced “cobblestone” morphology (Figure 1a). Analogous observations were made for the rat liver endothelial cell line EC52 that served as a positive control for functional experiments in this study. Furthermore, 2 weeks post-isolation, the population demonstrated to be composed of heterogenous cells in FACS-analyses. A small fraction of the cells expressed CD31 (Platelet endothelial cell adhesion molecule, PECAM-1) (8.3% ± 2.1) (Figure 1b). CD146, also known as the melanoma cell adhesion molecule (MCAM), was already markedly expressed on the cells with different intensity (15.7 ± 5.1%) (Figure 1c). Cells did not express vascular endothelial growth factor receptor 2 (VEGF-R2/KDR) at this time point (Figure 1d).

Bottom Line: Some studies demonstrated therapeutic angiogenesis attributable to the effects of endothelial progenitor cells (EPC), others have reported disappointing results.Based upon these observations in this study we describe a novel and time-saving method for obtaining a pure endothelial precursor cell population as early as 2-3 weeks post isolation that exhibits endothelial abilities in vitro and which still might have retained its early endothelial lineage properties.The rapid isolation and the high angiogenic potential of these syngeneic cells might facilitate and accelerate the pre-vascularization of transplanted tissues and organs also in a human setting in the future.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plastic and Hand Surgery, Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nuremberg, Krankenhausstrasse 12, Erlangen 91054, Germany. oliver.bleiziffer@uk-erlangen.de.

ABSTRACT

Background: Some studies demonstrated therapeutic angiogenesis attributable to the effects of endothelial progenitor cells (EPC), others have reported disappointing results. This may be due to the fact that EPC populations used in these contradictory studies were selected and defined by highly variable and differing experimental protocols. Indeed, the isolation and reliable characterization of ex vivo differentiated EPC raises considerable problems due to the fact there is no biomarker currently available to specifically identify EPC exclusively. On the other hand traditional differentiation of primary immature bone marrow cells towards the endothelial lineage is a time-consuming process of up to 5 weeks. To circumvent these shortcomings, we herein describe a facile method to isolate and enrich a primary cell population from rat bone marrow, combining differential attachment methodology with cell sorting technology.

Results: The combination of these techniques enabled us to obtain a pure population of early endothelial precursor cells that show homogenous upregulation of CD31 and VEGF-R2 and that are positive for CD146. These cells exhibited typical sprouting on Matrigel™. Additionally, this population displayed endothelial tube formation when resuspended in Matrigel™ as well as in fibrin glue, demonstrating its functional angiogenic capacity. Moreover, these cells stained positive for DiI-ac-LDL and FITC-UEA, two markers that are commonly considered to stain differentiating EPCs. Based upon these observations in this study we describe a novel and time-saving method for obtaining a pure endothelial precursor cell population as early as 2-3 weeks post isolation that exhibits endothelial abilities in vitro and which still might have retained its early endothelial lineage properties.

Conclusion: The rapid isolation and the high angiogenic potential of these syngeneic cells might facilitate and accelerate the pre-vascularization of transplanted tissues and organs also in a human setting in the future.

Show MeSH