The Effect Of Endothelial Colony-forming Cells Conditioned-Medium On Biological Behavior Of Vascular Endothelial Cells And Fibroblasts

Background and objectiveEndothelial colony-forming cells(ECFCs)transplantation exerts beneficial impact on diabetic wound healing.However,the application of ECFCs is encumbered by its low utilization rate,unclear curative effect and the ethical controversy.Taking advantage of the paracrine effect of cells can avoid the above problems effectively.Therefore,we aim to investigate the ECFCs'cytological characteristics and the effect of ECFCs-conditioned medium(ECFCs-CM)on biological behavior of mature endothelial cells and dermal fibroblasts and thereby provide theoretical basis for the use of ECFCs-CM on promoting diabetic wound healing.MethodsDensity gradient centrifugation was used to isolate mononuclear cells(MNCs)from human umbilical cord blood.The MNCs were induced into ECFCs in certain circumstance,after which the primary cells were identified by cell surface markers detection by flow cytometry,immunofluorescence staining and Matrigel assay.The phenotype and function of passage 1 and passage 10 were compared.The antibody microarray and ELISA kits were used to detect the cytokines in serum-free ECFCs-CM.The effects of ECFCs-CM on proliferation,migration and apoptosis of HUVECs and HDFs were tested by CCK-8,transwell culture method,cell scratch experiment and Annexin V-FITC/PI staining flow cytometry,with serum-free EBM-2 used as control.The influence of ECFCs-CM on tube formation ability of HUVECs were examined by Matrigel assay.ResultsThe cells isolated from human cord blood demonstrated typical characteristics of ECFCs,which showed cobblestone appearance when grown and almost oval or spindle shape for a single cell.There was no evidently morphologic change during the serial passages in vitro.Flow cytometry cell surface markers detection showed the passage 1 and passage 10 were both positive for KDR and CD 144(endothelial cell markers),but not CD45(leukocyte marker),there was no statistical difference in these three markers between the two passages(P>0.05).The expression rate of CD34(hematopoietic stem cell marker)of passage 1 was higher than that of passage 10(P<0.05).Passage 1 showed a low expression rate of CD 133(stem cell marker),but passage 10 hardly expressed(P<0.01).Immunofluorescence staining showed both passage 1 and passage 10 were positive for Dil-acLDL uptaking and FITC-UEA-I bonding.Matrigel assay indicated that both passage 1 and passage 10 had the ability to form tube-like structures.The cytokines antibody microarray showed that ECFCs-CM significantly upregulated the expression of 34 kinds of cytokines,such as PDGF-BB,Angiopoietin-2,Angiogenin,MCP-3,ICAM-2,LAP and so on.The quantitative detection by ELISA showed the concentration of PDGF-BB,IL-8 and EGF in ECFCs-CM were 4627.42±457.51pg/ml,4111.36±129.77pg/ml and 129.77±113.35pg/ml(based on 1×106cells/2ml)respectively.Compared with control group,the HUVECs cultured with ECFCs-CM showed significantly improved proliferation ability at 24,48 and 72 hours(P<0.01).Compared with control group,the cells that migrated through the polycarbonate membrane were obviously increased in coculture group(P<0.01).In cell scratch experiment,the migration rate of HUVECs and HDFs in experimental group was significantly higher than that in control group at each time point(P<0.01).Flow cytometry showed that the number of late apoptotic/dead cells of HUVECs was smaller than that of the control group(P<0.01)and both early apoptotic and late apoptotic/dead cells of HDFs decreased compared with control(P<0.05)under the environment of serum starvation.There were more tubular structures and branch points in experimental group than those in control group(P<0.01).ConclusionIn this study,the morphology of ECFCs was stable and showed no remarkable change of phenotype and function during serial passages in vitro.ECFCs-CM contains various kind of cytokines,including some factors that involve in the wound healing process,which indicated that ECFCs were ideal stem/progenitor cell source for researching the function of paracrine effect on wound healing.ECFCs-CM could promote the proliferation and migration ability of HUVECs and HDFs,and inhibit their apoptosis under serum starvation condition,and enhance tube formation abilitiy of HUVECs,which predicts its potential applications in wound healing.