Hypoxia Impairs Initial Outgrowth of Endothelial Colony Forming Cells and Reduces Their Proliferative and Sprouting Potential

Vascular homeostasis and regeneration in ischemic tissue relies on intrinsic competence of the tissue to rapidly recruit endothelial cells for vascularization. The mononuclear cell (MNC) fraction of blood contains circulating progenitors committed to endothelial lineage. These progenitors give rise to endothelial colony-forming cells (ECFCs) that actively participate in neovascularization of ischemic tissue. To evaluate if the initial clonal outgrowth of ECFCs from cord (CB) and peripheral blood (PB) was stimulated by hypoxic conditions, MNCs obtained from CB and PB were subjected to 20 and 1% O2 cell culture conditions. Clonal outgrowth was followed during a 30 day incubation period. Hypoxia impaired the initial outgrowth of ECFC colonies from CB and also reduced their number that were developing from PB MNCs. Three days of oxygenation (20% O2) prior to hypoxia could overcome the initial CB-ECFC outgrowth. Once proliferating and subcultured the CB-ECFCs growth was only modestly affected by hypoxia; proliferation of PB-ECFCs was reduced to a similar extent (18–30% reduction). Early passages of subcultured CB- and PB-ECFCs contained only viable cells and few if any senescent cells. Tube formation by subcultured PB-ECFCs was also markedly inhibited by continuous exposure to 1% O2. Gene expression profiles point to regulation of the cell cycle and metabolism as major altered gene clusters. Finally we discuss our counterintuitive observations in the context of the important role that hypoxia has in promoting neovascularization

CD34 expression modulates tube-forming capacity and barrier properties of peripheral blood-derived endothelial colony-forming cells (ECFCs)

Endothelial colony-forming cells (ECFC) are grown from circulating CD34(+) progenitors present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. To evaluate whether the regulation of CD34 characterizes the angiogenic phenotypical features of PB-ECFCs, we investigated the properties of CD34(+) and CD34(−) ECFCs with respect to their ability to form capillary-like tubes in 3D fibrin matrices, tip-cell gene expression, and barrier integrity. Selection of CD34(+) and CD34(−) ECFCs from subcultured ECFCs was accomplished by magnetic sorting (FACS: CD34(+): 95 % pos; CD34(−): 99 % neg). Both fractions proliferated at same rate, while CD34(+) ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3(4−) cells. However, during cell culture CD34(−) cells re-expressed CD34. Cell-seeding density, cell–cell contact formation, and serum supplements modulated CD34 expression. CD34 expression in ECFCs was strongly suppressed by newborn calf serum. Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression. Silencing of CD34 with siRNA resulted in strengthening of cell–cell contacts and increased barrier function of ECFC monolayers as measured by ECIS. Furthermore, CD34 siRNA reduced tube formation by ECFC, but did not affect tip-cell gene expression. These findings demonstrate that CD34(+) and CD34(−) cells are different phenotypes of similar cells and that CD34 (1) can be regulated in ECFC; (2) is positively involved in capillary-like sprout formation; (3) is associated but not causally related to tip-cell gene expression; and (4) can affect endothelial barrier function. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10456-016-9506-9) contains supplementary material, which is available to authorized users

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

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

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

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

Expression of inflammatory activation-related markers.

<p>Quantitative RT-PCR analysis was performed on total cellular mRNA isolated from 3 donors at 6, 18, and 31 CPDL. Gene expression levels of ICAM-1 (<b>A</b>), VCAM-1 (<b>B</b>), and uPA (<b>C</b>) in individual donors PB15 (◆), PB84 (●), and PB224(▲), during long-term expansion. Analysis of uPA expression was performed with the same data set as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129935#pone.0129935.g007" target="_blank">Fig 7A</a>. Data are expressed as n-fold difference of expression of same genes in cells at 6 CPDL.</p