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Human cord blood-derived AC133+ progenitor cells preserve endothelial progenitor characteristics after long term in vitro expansion.

Janic B, Guo AM, Iskander AS, Varma NR, Scicli AG, Arbab AS - PLoS ONE (2010)

Bottom Line: However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients.During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers.These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, Michigan, United States of America. bjanic@rad.hfh.edu

ABSTRACT

Background: Stem cells/progenitors are central to the development of cell therapy approaches for vascular ischemic diseases. The crucial step in rescuing tissues from ischemia is improvement of vascularization that can be achieved by promoting neovascularization. Endothelial progenitor cells (EPCs) are the best candidates for developing such an approach due to their ability to self-renew, circulate and differentiate into mature endothelial cells (ECs). Studies showed that intravenously administered progenitors isolated from bone marrow, peripheral or cord blood home to ischemic sites. However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients. Hence, the ability to amplify the numbers of autologous EPCs by long term in vitro expansion while preserving their angiogenic potential is critically important for developing EPC based therapies. Therefore, the objective of this study was to evaluate the capacity of cord blood (CB)-derived AC133+ cells to differentiate, in vitro, towards functional, mature endothelial cells (ECs) after long term in vitro expansion.

Methodology: We systematically characterized the properties of CB AC133+ cells over the 30 days of in vitro expansion. During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers. Flow cytometry and immunocytochemistry demonstrated that CB AC133+ cells' expression of endothelial progenitor markers was not affected by long term in vitro culturing. After culturing under EC differentiation conditions, cells exhibited high expression of mature ECs markers, such as CD31, VEGFR-2 and von Willebrand factor, as well as the morphological changes indicative of differentiation towards mature ECs. In addition, throughout the 30 day culture cells preserved their functional capacity that was demonstrated by high uptake of DiI fluorescently conjugated-acetylated-low density lipoprotein (DiI-Ac-LDL), in vitro and in vivo migration towards chemotactic stimuli and in vitro tube formation.

Conclusions: These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage.

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Migration and accumulation of FePro labeled CB AC133+ cells in the matrigel plug detected by MRI and immunohistochemistry.Isotropic 200 µm ex vivo MRI image obtained in coronal plan by FIESTA sequence (A) and the reconstructed 200 µm image of the sagital view (B). Administered FePro labeled CB AC133+ cells generated low signal intensity areas seen on both images (arrows in the circle). Fluorescent DAPI staining of the matrigel section that corresponds the MRI images show multiple cells within the matrigel (C) and the cells are mostly seen within a tube like structures depicted by FITC labeled tomato lectin (D). DAB-enhanced Prussian blue staining of the section very adjacent to the section stained with DAPI and FITC-lectin shows multiple, iron-positive, administered cells within the tube like structure (E). Scale bar  = 100 µm.
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pone-0009173-g008: Migration and accumulation of FePro labeled CB AC133+ cells in the matrigel plug detected by MRI and immunohistochemistry.Isotropic 200 µm ex vivo MRI image obtained in coronal plan by FIESTA sequence (A) and the reconstructed 200 µm image of the sagital view (B). Administered FePro labeled CB AC133+ cells generated low signal intensity areas seen on both images (arrows in the circle). Fluorescent DAPI staining of the matrigel section that corresponds the MRI images show multiple cells within the matrigel (C) and the cells are mostly seen within a tube like structures depicted by FITC labeled tomato lectin (D). DAB-enhanced Prussian blue staining of the section very adjacent to the section stained with DAPI and FITC-lectin shows multiple, iron-positive, administered cells within the tube like structure (E). Scale bar  = 100 µm.

Mentions: One of the important characteristics of the cells of endothelial lineage is the ability to exhibit directional migration, i.e. chemotaxis, along the concentration gradients of biologically active, low molecular weight peptides called chemokines. Therefore we analyzed the effect of long term culturing on CB AC133+ cells' chemotaxis by determining their ability to migrate along the concentration gradients of SDF-1α and Rantes chemokines under the in vitro conditions. In addition to in vitro migration, we tested CB AC133+ cells' migratory capacity in vivo, by employing matrigel plug model in Balb/c nude mice. At days 10–15 and 25–30 of primary culture, CB AC133+ cells were incubated for 4 h in the presence of 50 ng/ml of either SDF-1α or Rantes. Cell migration in response to these two chemokines was significantly higher (p<0.05) than that observed in control conditions (w/o chemoattractant), with no difference between the cells cultured for 10–15 and the cells cultured for 25–30 days (Figure 7). In addition, similar significant increase in migration was observed when cells were incubated in the presence of 10% FBS, that served a positive control for migratory response. These data indicate that during the long term culturing CB AC133+ progenitors maintained functional characteristics with regard to chemotaxis. To assess the capacity of CB AC133+ cells to in vivo migrate and incorporate into the sites of neovascularization we used matrigel plug model in Balb/c nude mice. After in vitro expansion for 10–15 days, CB AC133+ cells were magnetically labeled with FePro and administered intravenously into the matrigel implant bearing Balb/c nude mice. Our extensive previous research on a variety of cell types showed that FePro labeling does not have significant effect on cellular physiology [24], [31]. Seven days post injection MRI and immunohistochemistry revealed that FePro labeled CB AC133+ cells migrated towards neovasculature within the matrigel implants. This migration was detected by ex vivo MRI as hypointensity areas resulting from T2* shortening due to the presence of FePro labeled cells (Figure 8A-B). At the same time lectin (Figure 8C) and DAPI (Figure 8D) fluorescent, and Prussian blue (Figure 8E) staining of sections generated from the areas exhibiting hypointensity MRI signals revealed the co-localization of labeled cells with newly formed tube structures.


Human cord blood-derived AC133+ progenitor cells preserve endothelial progenitor characteristics after long term in vitro expansion.

Janic B, Guo AM, Iskander AS, Varma NR, Scicli AG, Arbab AS - PLoS ONE (2010)

Migration and accumulation of FePro labeled CB AC133+ cells in the matrigel plug detected by MRI and immunohistochemistry.Isotropic 200 µm ex vivo MRI image obtained in coronal plan by FIESTA sequence (A) and the reconstructed 200 µm image of the sagital view (B). Administered FePro labeled CB AC133+ cells generated low signal intensity areas seen on both images (arrows in the circle). Fluorescent DAPI staining of the matrigel section that corresponds the MRI images show multiple cells within the matrigel (C) and the cells are mostly seen within a tube like structures depicted by FITC labeled tomato lectin (D). DAB-enhanced Prussian blue staining of the section very adjacent to the section stained with DAPI and FITC-lectin shows multiple, iron-positive, administered cells within the tube like structure (E). Scale bar  = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2820083&req=5

pone-0009173-g008: Migration and accumulation of FePro labeled CB AC133+ cells in the matrigel plug detected by MRI and immunohistochemistry.Isotropic 200 µm ex vivo MRI image obtained in coronal plan by FIESTA sequence (A) and the reconstructed 200 µm image of the sagital view (B). Administered FePro labeled CB AC133+ cells generated low signal intensity areas seen on both images (arrows in the circle). Fluorescent DAPI staining of the matrigel section that corresponds the MRI images show multiple cells within the matrigel (C) and the cells are mostly seen within a tube like structures depicted by FITC labeled tomato lectin (D). DAB-enhanced Prussian blue staining of the section very adjacent to the section stained with DAPI and FITC-lectin shows multiple, iron-positive, administered cells within the tube like structure (E). Scale bar  = 100 µm.
Mentions: One of the important characteristics of the cells of endothelial lineage is the ability to exhibit directional migration, i.e. chemotaxis, along the concentration gradients of biologically active, low molecular weight peptides called chemokines. Therefore we analyzed the effect of long term culturing on CB AC133+ cells' chemotaxis by determining their ability to migrate along the concentration gradients of SDF-1α and Rantes chemokines under the in vitro conditions. In addition to in vitro migration, we tested CB AC133+ cells' migratory capacity in vivo, by employing matrigel plug model in Balb/c nude mice. At days 10–15 and 25–30 of primary culture, CB AC133+ cells were incubated for 4 h in the presence of 50 ng/ml of either SDF-1α or Rantes. Cell migration in response to these two chemokines was significantly higher (p<0.05) than that observed in control conditions (w/o chemoattractant), with no difference between the cells cultured for 10–15 and the cells cultured for 25–30 days (Figure 7). In addition, similar significant increase in migration was observed when cells were incubated in the presence of 10% FBS, that served a positive control for migratory response. These data indicate that during the long term culturing CB AC133+ progenitors maintained functional characteristics with regard to chemotaxis. To assess the capacity of CB AC133+ cells to in vivo migrate and incorporate into the sites of neovascularization we used matrigel plug model in Balb/c nude mice. After in vitro expansion for 10–15 days, CB AC133+ cells were magnetically labeled with FePro and administered intravenously into the matrigel implant bearing Balb/c nude mice. Our extensive previous research on a variety of cell types showed that FePro labeling does not have significant effect on cellular physiology [24], [31]. Seven days post injection MRI and immunohistochemistry revealed that FePro labeled CB AC133+ cells migrated towards neovasculature within the matrigel implants. This migration was detected by ex vivo MRI as hypointensity areas resulting from T2* shortening due to the presence of FePro labeled cells (Figure 8A-B). At the same time lectin (Figure 8C) and DAPI (Figure 8D) fluorescent, and Prussian blue (Figure 8E) staining of sections generated from the areas exhibiting hypointensity MRI signals revealed the co-localization of labeled cells with newly formed tube structures.

Bottom Line: However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients.During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers.These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, Michigan, United States of America. bjanic@rad.hfh.edu

ABSTRACT

Background: Stem cells/progenitors are central to the development of cell therapy approaches for vascular ischemic diseases. The crucial step in rescuing tissues from ischemia is improvement of vascularization that can be achieved by promoting neovascularization. Endothelial progenitor cells (EPCs) are the best candidates for developing such an approach due to their ability to self-renew, circulate and differentiate into mature endothelial cells (ECs). Studies showed that intravenously administered progenitors isolated from bone marrow, peripheral or cord blood home to ischemic sites. However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients. Hence, the ability to amplify the numbers of autologous EPCs by long term in vitro expansion while preserving their angiogenic potential is critically important for developing EPC based therapies. Therefore, the objective of this study was to evaluate the capacity of cord blood (CB)-derived AC133+ cells to differentiate, in vitro, towards functional, mature endothelial cells (ECs) after long term in vitro expansion.

Methodology: We systematically characterized the properties of CB AC133+ cells over the 30 days of in vitro expansion. During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers. Flow cytometry and immunocytochemistry demonstrated that CB AC133+ cells' expression of endothelial progenitor markers was not affected by long term in vitro culturing. After culturing under EC differentiation conditions, cells exhibited high expression of mature ECs markers, such as CD31, VEGFR-2 and von Willebrand factor, as well as the morphological changes indicative of differentiation towards mature ECs. In addition, throughout the 30 day culture cells preserved their functional capacity that was demonstrated by high uptake of DiI fluorescently conjugated-acetylated-low density lipoprotein (DiI-Ac-LDL), in vitro and in vivo migration towards chemotactic stimuli and in vitro tube formation.

Conclusions: These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage.

Show MeSH
Related in: MedlinePlus