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

Abstract

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.

Standardization of methods to quantify and culture endothelial colony‐forming cells derived from peripheral blood

“…Thus, 10% FBS should be preferred to 5% FBS. Plasma and platelet lysate have been proposed as substitutes for serum, and more ECFC colonies have been reported with platelet lysate …”

Gaps in the knowledge of human platelet lysate as a cell culture supplement for cell therapy: a joint publication from the AABB and the International Society for Cell & Gene Therapy

“…Specifically, no immediate or late toxic events against infusions of third-party-expanded MSCs (MSCs from a donor, independent of the patient and the hematopoietic stem cell transplant donor) were observed. Complete and 34 Siegel et al, 35 Tasev et al 36 Hofbauer et al 37 Corneal endothelial cells Improved cell survival and proliferation Thieme et al 38…”

Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal Substitutes in Chronic Phlebostatic Ulcers

“…Because of the large amounts of cytokines and growth factors contained in PL, it presents multiple and significant advantages if applied locally to skin wounds when seeking enhancement of angiogenesis and fibroblast migration, restoration of collagen synthesis and reduction of oxidative stress [46]. In addition, it has been demonstrated that PL is capable of re-establishing skin integrity efficiently [50].…”