ECFCs possess all biological features to be considered as a cell source of preference for tissue engineering and repair of blood supply. Investigation of regenerative potential of ECFCs in autologous settings in large animal models before clinical application is the next step to clearly outline the most efficient strategy for using ECFCs as treatment.

IntroductionEfficient 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 ex vivo 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.ResultsThe 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.ConclusionThe 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.

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 CD34− 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 materialThe online version of this article (doi:10.1007/s10456-016-9506-9) contains supplementary material, which is available to authorized users.

Processing of pro-interleukin (IL)-1β and IL-18 is regulated by multiprotein complexes, known as inflammasomes. Inflammasome activation results in generation of bioactive IL-1β and IL-18, which can exert potent pro-inflammatory effects. Our aim was to develop a whole blood-based assay to study the inflammasome in vitro and that also can be used as an assay in clinical studies. We show whole blood is a suitable milieu to study inflammasome activation in primary human monocytes. We demonstrated that unprocessed human blood cells can be stimulated to activate the inflammasome by the addition of adenosine 5’-triphosphate (ATP) within a narrow timeframe following lipopolysaccharide (LPS) priming. Stimulation with LPS resulted in IL-1β release; however, addition of ATP is necessary for “full-blown” inflammasome stimulation resulting in high IL-1β and IL-18 release. Intracellular cytokine staining demonstrated monocytes are the major producers of IL-1β in human whole blood cultures, and this was associated with activation of caspase-1/4/5, as detected by a fluorescently labelled caspase-1/4/5 probe. By applying caspase inhibitors, we show that both the canonical inflammasome pathway (via caspase-1) as well as the non-canonical inflammasome pathway (via caspases-4 and 5) can be studied using this whole blood-based model.