Culture And Differentiation Towards Endothelial Cells Of Human Umbilical Cord Stroma-Derived Stem Cells In Vitro

Objective: Human umbilical cord stroma-derived stem cells, as a new source of stem cells,have been paid more and more attention. The goal of this study was to isolate and culture stem cells derived from human umbilical cord stroma and examine their endothelial potential in vitro.Methods: With the consent of the parents, fresh umbilical cords were collected from normal full-term pregnancies. After removal of blood vessels, the cord was washed extensively with balanced salt solution. Then, the tissue was minced into 1-mm3 pieces with sharp scissors and the tissue pieces were cultured in culture flasks. Tissue pieces were placed in a Dulbecco's modified Eagle's medium (DMEM) with low glucose /Ham's F-12 (DMEM-LG/F12, DF12)supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100μg/ml), 10nmol/L dexamethasone, and 100×Insulin-Tranferrin-Selenium (ITS). The cells were expanded in a humidified incubator at 37℃with 5% CO2. When cells reached 80%～90% confluence around two to three weeks, they were detached with trypsin/ ethylene diamine tetraacetatic acid (EDTA) (0.25%/0.02%) solution and passaged. They were frozen in the freezing medium, which consisted of 10% dimethyl sulfoxide(DMSO) and 90% FBS. The cells growth pattern was showed by cells growth curve. Cells were trypsinized, washed with PBS, and incubated with antibodies against CD34,CD44,CD45, CD54, CD73, CD90, CD105,CD106 and CD133. Analysis was performed with a flow cytometer. Mouse fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated IgG1 were used as controls. Cells were inoculated in new pore plates at a concentration of 5×104/ml and incubated in the differentiation medium which contained 10 ng/ml vascular endothelial growth factor(VEGF), 10 ng/ml basic fibroblast growth factor (b-FGF) and 10%FBS. And then,cells were incubated in a humidified incubator at 37℃with 5% CO2 and observed by inverted phase-contrast microscopy everyday. The endothelial-specific phenotype CD34,von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2) were analyzed by the use of Flow cytometry to observe the difference between undifferentiation and differentiation. Finally, the express of VEGFR-2 and incorporation of DiI-labeled acetylated low-density lipoprotein (DiI-ac-LDL) of differentiated cells were analyzed using a fluorescent microscope.Results: Three to seven days after primary culture, some cells began to come out of fragments , gradually great number of its became adherent to the flasks and grew radiately . They principally displayed a fibroblast-like appearance, while some of them were flat and wide cytoplasmic cells. Two to three weeks after culture, when the cells reached 80%～90% confluence, they were detached with trypsin/EDTA (0.25%/0.02%) solution and were passaged at a ratio of 1:2 to 1:3. Several hours after plating,cells became adherent. They grew faster than before, reached 80%～90% confluence after 3～4 days of passage and gradually formed a compact,whirling-like appearance in low power field. The mophology of cells after freezing and thawing did not change. The cells growth curve showed us that the cells growth pattern was just like"S". Flow cytometry analysis showed that human umbilical cord stroma-derived stem cells expressed putative markers of mesenchymal stem cells,which included CD105,CD73, CD90,CD54 and CD44, but not hematopoietic markers and endothelial cell markers,such as CD34,CD45,CD106 and CD133. Inverted phase-contrast microscopy observation over three days showed that vascular lumina-like structures were formed by human umbilical cord stroma-derived stem cells , after these cells cultured with differentiation medium which contained 10 ng/ml VEGF, 10 ng/ml b-FGF and 10% FBS. Flow cytometry analysis showed the endothelial-specific phenotype CD34, vWF and VEGFR-2 were negative before differentiation and positive partly on the fourteen day of differentiation. The fluorescent microscopy revealed that the differentiated cells could be incorporated by DiI-ac-LDL.Finally, the differentiated cells that could be incorporated by DiI-ac-LDL could also express VEGFR-2.Conclusion: The human umbilical cord stroma-derived stem cells can be isolated,and cultured in vitro .The medium which contains vascular endothelial growth factor and basic fibroblast growth factor can induce human umbilical cord stroma-derived stem cells to differentiated towards endothelial cells in vitro.