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

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.

Figure 1. Growth kinetics of CB AC133+ cells.

CB AC133+ cells were cultured in the presence of 40 ng/ml of stem cell factor (SCF), 40 ng/ml of FLT3 and 10 ng/ml of thrombopoietin (TPO) for 30 days. Cell numbers were determined by Trypan blue exclusion assay at the time points indicated on the graph. During the ‘lag phase’ (day 0–8) there was no significant increase in numbers with time. Starting at day 8 the living cell population increased rapidly with time at an exponential growth in numbers, and the growth rate increasing with time. Increase in the cell numbers expressed as fold increase (cell numbers at splitting/cell numbers at day 0). Data points, means ± SD. * p<0.05.

Figure 2. Expression of cell surface markers in CB AC133+ progenitor cells during long term in vitro culture.

The data depicts the levels of CD133 and CD34 protein expression in AC133+ cells cultured for 4, 10 and 20 days (A) and the levels of CD31, CD105, CD184, CD29 and CD117 in AC133+ cells cultured for 10 and 20 days (B). Flow cytometric histograms from one representative experiment are shown (n = 3). At least 10,000 live gated cells were analyzed for FITC, PE or PE-Cy5 expression. Isotype controls are shown as solid blue histograms.

Figure 3. Morphological changes in CB AC133+ cells differentiated for 2 weeks.

The similar pattern of morphological changes was observed in cells differentiated at day 15 (A, B) and at day 30 (C, D) of primary culture. Differentiated cells exhibited “cobble stone”-like organizational pattern. Spindle shaped adherent cells sprouted from cell clusters in an attempt to organize into linear, tube-like structures. Phase contrast photomicrographs from 2 representative experiments. Magnifications used: 10x (A and C) and 25x (B and D). Scale bar  = 100 µm.

Figure 4. Expression of cell surface markers in CB AC133+ progenitor cells differentiated for two weeks.

CB AC133+ progenitor cells at day 31 of primary culture were differentiated for two weeks. Flow cytometric analysis showed that after 2 weeks of differentiation cells were positive for CD31, CD54, CD184, KDR, CD62E, CD29, CD150, CD195, CD105 and VE Cadherin. Flow cytometric histograms from one representative experiment are shown (n = 3). At least 10,000 live gated cells were analyzed for FITC, PE or PE-Cy5 expression. Isotype controls are shown as solid blue histograms.

Figure 5. Expression of CD31, vWF and KDR in differentiated CB AC133+ progenitors.

Cells were induced to differentiate at day 5–6 (A), day 10–15 (B) and day 25–30 (C) of primary culture. Positive signals were visualized with FITC conjugated secondary antibody (green) and Rho conjugated secondary antibody (red). Nuclei were visualized with DAPI (blue). CD31 positive (left panels in A and B) and KDR positive (right panel in C) cells also exhibited the uptake of DiI-Ac-LDL (red). Photomicrographs (40x) of differentiated cells representative of 3 other experiments. Scale bar  = 100 µm.

Figure 6. Functional analysis of long term expanded CB AC133+ cells to form tubes in Matrigel.

Tube like structures after 24 h of CB AC133+ cells and HDMVECs co-culture (A-D). Complete tubes in matrigel formed by HDMVECs incubated in the presence of CB AC133+ cells' supernatants (w/o EPCs and VEGF) for 24 h (E). When plated alone, CB AC133+ did not form tube like structures (F). Note in panels A-D, HDMVECs labeled with Calcein (C, green fluorescence) and CB AC133+ cells labeled with DiI (D, red fluorescence) co-localized (yellow; panel A) to form tube like structure. Most of the green fluorescent cells appeared to be structural part of the tubes, while some of the red florescence cells that did not became part of the tube network remained scattered between the tubes. Overlays of bright light microscopy and fluorescent microscopy images (A, C, D). Bright light microscopy only, images shown in panels B, E and F. Magnification 10x.

Figure 7. Effect of long term in vitro expansion on CB AC133+ cell chemotaxis in response to SDF1-α and Rantes.

At days 15 and 30 of primary culture, CB AC133+ cells were incubated for 4 h in the presence of 50 ng/ml of either Rantes or SDF-1α. Cell migration in response to these to chemokines was significantly higher (p<0.05) than that observed in control conditions (w/o chemoatractant; gray bars), with no difference between the cells cultured for 15 and the cells cultured for 30 days. Cells incubated in the presence of 10% FBS (black bars) were used as a positive control. Bars, means ± SD. * p<0.05.

Figure 8. 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.