Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity

<div><p>Introduction</p><p>Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in <i>ex vivo</i> conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.</p><p>Results</p><p>The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.</p><p>Conclusion</p><p>The presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15–20 CPDL is the most adequate maturation stage.</p></div

Expression of genes and soluble antigens involved during sprouting in fibrin matrix in PB-ECFCs at different maturation stages.

<p>Quantitative RT-PCR analysis was performed on total cellular mRNA isolated from PB-ECFCs at different CPDL (open bar 6 CPDL, grey bar 18 CPDL, black bar 31 CPDL). Gene expression levels of uPA (<b>A</b>), uPAR (<b>B</b>), tPA (<b>C</b>), and PAI-1 (<b>D</b>) in PB-ECFCs. Data are expressed as n-fold difference of expression of genes in cells at 6 CPDL. One-way ANOVA with Bonferroni post hoc test (p<0.05).Evaluation of the production of uPA (<b>E</b>) and PAI-1 (<b>F</b>) antigens in CM by PB-ECFCs was performed at 6 CPDL (open bar), 18 CPDL (grey bar), and 31 CPDL (black bar) using ELISA. Results represent the mean ± SEM of uPA or PAI-1 concentration in ng relative to mL of conditioned medium of 3 independent experiments each performed at indicated CPDL. Comparison between different groups was performed one-way ANOVA with Bonferroni post hoc test (p<0.05).</p

Comparison of tube-forming capacity of PB-ECFCs at different time-points of ex vivo expansion upon stimulation with VEGF-A and FGF-2.

<p>PB-ECFCs obtained from 3 different donors were serially expanded in medium supplemented with PL and the sprouting ability of cells in fibrin matrices was assessed at 6, 18, and 31 CPDL. Cells at indicated CPDL (white bar 6 CPDL, grey bar 18 CPDL, black bar 31 CPDL) were either unstimulated (<b>A</b>) or stimulated with FGF-2(<b>B</b>), and VEGF-A(<b>C</b>), FGF-2+VEGF-A (<b>D</b>),TNF-α. Results represent the mean ± SEM of mean tube length of tube-like structures of the 3 donors each performed at indicated CPDL. Comparison between each CPDLs was performed using one-way ANOVA with Bonferroni post hoc test.(*p < 0.05).</p

Comparison of ECFCs colony outgrowth in fetal bovine serum and platelet lysate.

<p>Representative images of ECFC colonies that appeared in FBS-EGM (<b>A</b>) and PL-EGM (<b>B,C</b>) between 10–30 days. (<b>D</b>): The average number of colonies that grew in FBS (open triangle) and PL (black triangle) from 10 isolations at day 10. (<b>E</b>): The number of colonies yields in FBS-EGM-2 (open triangle) or PL-EGM (black triangle) during the isolation period in all donors relative to the number of mL of peripheral blood. Data are expressed as mean±SEM. Statistical analysis was performed with non-parametric Wilcoxon matched pairs test (* p<0.05).</p

Prolonged hypoxia inhibits sprouting of endothelial cells into 3D fibrin matrices.

<p>hMVECs were precultured at 20% (black and dark grey bars) or 1% oxygen (light grey and white bars) for 14 days before seeded on top of 3D fibrin matrices. Subsequently, the hMVECs were stimulated with the combination of VEGF-A/TNFα (4 donors in 7 experiments) or bFGF/TNFα (5 donors in 10 experiments) either at 20% oxygen (black and white bars) or at 1% oxygen (dark grey and light grey bars) (each in quadruple). <b>(A)</b> Representative photos are shown of hMVECs 7 days after seeding and stimulation with VEGF-A/TNFα. Scale bars represent 1 mm. Photos are focused on the sprouts. <b>(B)</b> Tube length was quantified by using Optimas software and expressed as percentage of 20% O₂ with SEM. For statistical analysis repeated measures ANOVA with Bonferroni post-hoc test was used (** p<0.01 *** p<0.001).</p

Phenotypical characterization of PB-ECFCs.

<p>PB-ECFCs monolayers expanded in EGM-2 medium supplemented with 10%PL were assessed for the presence of endothelial cell markers by immunofluorescence cytochemistry (<b>A-C</b>) and flow cytometry as well as for uptake of Dil-Ac-LDL (<b>D</b>). For immunofluorescence cytochemistry staining, cells were seeded on glass cover slips, fixed and stained with antibody against CD31, VE-cadherin or vWF. Cell nuclei were visualized with DAPI staining. Cells were positive for CD31 (<b>A</b>, green), VE-cadherin (<b>B</b>, green), and vWF (<b>C</b>, green). Cell nuclei appear blue. (<b>D</b>): Incorporation of Dil-Ac-LDL by PB-ECFCs (red spots, cell nuclei stained blue with DAPI). Panel <b>E</b>:Flow cytometry characterization of PB-ECFCs for CD31, CD34, CD309, CD144, CD146, CD105, CD14, CD45, and CD133. Plots depict control isotype IgG staining (black histograms) versus specific antibody staining (empty histograms).</p

Addition of recombinant uPA increases endothelial sprouting.

<p>hMVECs were seeded on top of 3D fibrin matrices and stimulated with the combination of VEGF-A/TNFα and the indicated concentration of human recombinant uPA either in normoxia (grey circles) or hypoxia (black squares) (each in quadruple). <b>(A)</b> Representative photos are shown of hMVECs 7 days after seeding and stimulation with VEGF-A/TNFα. Scale bars represent 1 mm. Photos are focused on the sprouts. <b>(B)</b> Tube length of a representative experiment was quantified by using Optimas software and expressed as mm/cm² with SEM. After addition of 20 ng/ml u-PA lysis of fibrin was observed, which interfered with the stability of the tubular structures.</p

Proliferative potential of PB-ECFCs in FBS and PL medium.

<p>For growth kinetics as well as cumulative population doubling determination experiments, cells were serially expanded by seeding 5000 cells/cm² in EGM-2 supplemented with either 10%FBS or 10%PL during period of 40 days. (<b>A</b>): Comparison of proliferative potential of PB-ECFCs maintained in FBS-EGM (open bar) or PL-EGM (closed bar) during 7 days. Results represent the mean ± SEM of counted number of cells relative to cm² of plating surface of 3 independent experiments of three different donors. *p < 0.05 by Student paired t test. (<b>B</b>): Total number of cells yielded during long-term expansion of PB-ECFCs in FBS-EGM (open symbol, n:3) or PL-EGM (closed symbols, n:3). Each symbol indicates total number of cells at each passaging step. (<b>C</b>): Cumulative population doubling levels (CPDL) of PB-ECFCs in PL-EGM (closed bars) or FBS-EGM (open bars) after 10, 20, and 40 days of expansion. Results represent the mean ± SEM of CPDL at three different time points of 3 independent experiments of 3 different donors. Two-way ANOVA with Bonferroni post hoc test.</p

Effect of hypoxia on human microvascular endothelial cell proliferation.

<p>hMVECs were cultured in normoxia (20% O₂, grey circles) or hypoxia (1% O₂, black squares) for 17 days. <b>(A)</b> Photos of confluent monolayers of hMVECs on day 17 are shown. The white scale bar represents 1 mm and the black scale bar represents 0.1 mm. <b>(B)</b> Photos were taken every day and analyzed with ImageJ software, the arrows indicate days that cells were passaged 1:3. Representative experiment out of 3 independent hMVEC donors, showing number of cells/cm² as average of triplicate wells with SD.</p

Effect of prolonged hypoxia on the expression of genes involved in pericellular proteolysis.

<p><b>(A)</b> hMVECs were cultured in normoxia (Un (20%), white bar) or prolonged hypoxia (Un (1%), black bar) for 14 days and transfected with si-HIF-2α (si-H2 (1%), light grey bar) or scrambled (scr (1%), dark grey bar) and stimulated for 24 hours with VEGF/TNFα. mRNA was isolated for analysis by qRT-PCR and the relative mRNA levels were expressed as mean fold change with SEM (4 independent donors). Data was normalized to 1% O₂. <b>(B)</b> uPA and PAI-1 antigens were measured in hMVECs cultured in 20% O₂ (white bar) or 1% O₂ (black bar). Levels were expressed as ng/72h/10⁵ cells with SEM (4 independent donors). <b>(C)</b> uPA antigen was measured in hMVECs cultured in 1% O₂ and either untransfected (Un, black bar), or transfected with scrambled si-RNA (scr, dark grey bar), si-HIF-1α (si-H1, hatched bar), or si-HIF-2α (si-H2, light grey bar). Levels were expressed as ng/72h/10⁵ cells with SEM (3 independent donors). For statistical analysis TWO-way ANOVA with Bonferroni post-hoc test (A), paired student t test (B), or repeated measures ANOVA with Bonferroni post-hoc test was used (C) (** p<0.005, *** p<0.001).</p