Ex Vivo Derivation Of Endothelial-like Cells From Cord Blood Mononuclear Cells

AIM: To investigate the feasibility of the differentiation of endothelial-like cells induced from cord blood (UCB)mononuclear cells( MNC) ,cultured on extracellular matrix (ECM) with PHA-M,VEGF and bFGF;to determine which ECM protein might modulate or enhance the effects of Endothelial precursors cells on differentiation into mature endothelium.METHODS:MNC isolated by density gradient centrifugation from fresh UCB were cultured in ECM-coated plates in M199 medium containing 10% fetal bovine serum and PHA,VEGF and bFGF. Morphology of adherent cells and Three-dimensional vascular-like structure were observed in vitro ; Endothelial cell markers FactorⅧ,eNOS,vWF and CD31 were found to express by immuocytochemistry,Semiquantinative Reverse Transcription Polymerase Chain Reaction and Flow cytometric analysis; proliferation capacity of adherent cells were also measured at different time.RESULTS:①Outcome of morphological observation:at beginning cells were small,round ,after stimulated by PHA-M for 24 hours ,small clusters of adherent,flat cells were noticeable throughout the plate;at day 5, cells around the clusters became elongated , some cells grew up in line at day 7, and elongated cells became more and more as days on.Moreover ,in functional studies ,these cells can undergo extracellular matrix-dependent morphogenic changes into capillary-like tubular structures.②On the 7th day ,these cells mature to express Endothelial cell markers FactorⅧ,eNOS,vWF and CD31.③eNOS and vWF mRNAlevel of fibronectin-coated group were higher than other groups(P<0.05); its CD31 expression at days 7,14 were notably higher on others(P<0.05),and the value were 48.04±5.34% and 72.90±8.54%. CONCLUSION:①Endothelial precursors cells derived from human umbilical cord blood mononuclear cells can be differentiated into endothelial-like cells by PHA+VEGF+bFGF.②Endothelial-like cells can be induced with PHA from human umbilical cord blood mononuclear cells in short-term culture and PHA can promote synergistic effects with VEGF.③FN significantly promotes the differentiation of endothelial-like cells from UCB mononuclear cells than did collagenⅠ,and is critical for cell proliferation in vitro.④Human umbilical cord blood cells may be an ideal source of stem cells for the treatment of the ischemic disease.