Labeling and Qualification of Endothelial Progenitor Cells for Tracking in Tissue Engineering: An in Vitro Study

Objective: Circulating EPCs (endothelial progenitor cells) play a role in neovascularization and vascular repair. Oxidative stress impairs endothelial progenitor. Flavonoid is a phytochemical compound for antioxidant activity. Flavonoid effects toward oxidative stress, apoptosis, and expression of the cell markers on EPCs are not fully understood. This study was aimed to elucidate the effects of quercetin, kaempferol, and myricetin toward oxidative stress, apoptosis, and cell markers of peripheral blood-derived-EPCs. Methods: EPCs (endothelial progenitor cells) were isolated from peripheral blood mononuclear cells (PBMNCs) using cultivation under EPCs spesific media. Oxidative stress in EPCs was induced by H2O2 and then treated by quercetin, kaempferol, and myricetin. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, while intracellular reactive oxygen species (ROS), apoptosis and characterization of cells, which expressed CD133 and KDR, was measured using flow cytometry. Results: Quercetin, kaempferol, and myricetin at concentration 12.50 µmol/l were not toxic on EPCs as the cells viability were 96.11±4.03%, 95.42±7.75%, and 94.22±9.49%, respectively. Flavonoids decreased intracellular ROS level in EPCs (quercetin: 14.38±1.47%, kaempferol: 20.21±6.25%, and myricetin: 13.88±4.02%) compared to EPCs treated with H2O2 (30.70%±1.04). Percetage of EPCs apoptosis was not significantly different among each treatment. Immunophenotyping showed the increasing of CD133 and KDR expression in EPCs treated with flavonoids. Conclusion: Quercetin, kaempferol, and myricetin were safe for EPCs, decreased ROS levels, and increased CD133 and KDR expression. However, the flavonoids did not significantly affect EPCs apoptosis.

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In vitro cytocompatibility evaluation of collagen based scaffolds using human endothelial progenitor cells for vascular tissue engineering

Biomaterials and Tissue Engineering Bulletin ◽

10.33263/bteb114.010016 ◽

2014 ◽

Vol 1 (1-4) ◽

pp. 10-16 ◽

Cited By ~ 1

Keyword(s):

Tissue Engineering ◽

Blood Vessels ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Vascular Tissue ◽

Vascular Tissue Engineering ◽

Human Umbilical Cord ◽

Collagen Scaffolds ◽

Endothelial Progenitor

Vascular tissue engineering attempts to grow blood vessels through the use of different scaffolds that allows vascular cells such as endothelial cells to form networks and organized in vascular tissue. Various biomaterials are used to produce scaffolds that allow growth and differentiation of stem cells; depending on the cell type and applications some materials are more suitable than other. The aim of this study was to evaluate the cytocompatibility of collagen based scaffolds and to assess the capacity of endothelial progenitor cells (EPC) isolated from human umbilical cord to form vascular networks on these scaffolds. Our results show that after 5 days in culture with collagen scaffolds, the EPC remained viable, a sign of biocompatibility with the 3D scaffolds. Scanning electron microscopy showed that in the collagen scaffolds EPC organize within networks and presents an abundant extracellular matrix that strengthen the links between them. When EPC were cultured on collagenchitosan scaffolds, they are more adherent to the scaffolds compared with collagen, exibiting a good capacity to form networks. This study shows that the collagen and collagen-chitosan scaffolds are not cytotoxic for EPC and they provide the possibility of being used in vascular tissue engineering to help creating blood vessels.

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Gene transfer of endothelial NO-synthase restores migratory capacity of endothelial progenitor cells from patients with coronary artery diseases

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2877 ◽

2007 ◽

Vol 30 (4) ◽

pp. 96

Author(s):

Michael R. Ward ◽

Qiuwang Zhang ◽

Duncan J. Stewart ◽

Michael J.B. Kutryk

Keyword(s):

Risk Factors ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Fold Increase ◽

No Synthase ◽

Endothelial Progenitor ◽

Migratory Capacity ◽

Acute Mi ◽

Cad Risk

Autologous endothelial progenitor cells (EPCs) have been used extensively in the development of cell-based therapy for acute MI. However, EPCs isolated from patients with CAD and/or CAD risk factors have reduced regenerative activity compared to cells from healthy subjects. As in endothelial cells, endothelial NO synthase (eNOS) expression and subsequent NO production are believed to be critical determinants of EPC function. Recently, the ability of EPCs to migrate in vitro in response to chemotactic stimuli has been shown to predict their regenerative capacity in clinical studies. Therefore, we hypothesized that the regenerative function of EPCs from patients with or at high risk for CAD will be enhanced by overexpression of eNOS, as assessed by migratory capacity. Methods: EPCs were isolated from the blood of human subjects with CAD risk factors (>15% Framingham risk score; FRS) (± CAD) by Ficoll gradient separation and differential culture. Following 3 days in culture, cells were transduced using lentivirus vectors containing either eNOS or GFP (sham) at an MOI of 3. The cells were cultured for an additional 5 days before being used in functional assays. Cell migration and chemotaxis in response to VEGF (50 ng/mL) and SDF-1 (100 ng/mL) were assessed using a modified Boyden Chamber assay. Results: Transduction at an MOI of 3 led to a ~90-100-fold increase in eNOS mRNA expression and a 5-6 fold increase in eNOS protein expression, as assessed by qRT-PCR and Western Blotting. Moreover, there was a significant improvement in the migration of EPCs following eNOS transduction compared to sham-transduced EPCs in response to both VEGF (44.3 ± 8.4 vs. 31.1 ± 4.6 cells/high power field; n=10, p < 0.05) and SDF-1 (51.9 ± 11.1 vs. 34.5 ± 3.3 cells/HPF; n=10, p < 0.05). Conclusions: These data show that the reduced migration capacity of EPCs isolated from patients with CAD and/or CAD risk factors can be significantly improved through eNOS overexpression in these cells. Thus, eNOS transduction of autologous EPCs may enhance their ability to restore myocardial perfusion and function following acute MI. We intend to further explore the regenerative potential of eNOS-transduced EPCs using various in vitro and in vivo models.

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