| ObjectivesTo isolate and culture human Umbilical Cord-Mesenchymal Stem Cells (UC-MSCs) in vitro; to detect the expression levels of CD309, also called vascular endothelial growth factor receptor2(VEGFR2, Flk-1), hepatocyte growth factor (HGF) and prostaglandin E2(PGE2) by UC-MSCs; to explore the possible relationship of their expression levels with various donor factors, such as maternal age, fetal weight, fetal gender and mode of delivery.Methods:Umbilical cord was donated, and collected from healthy infants. Collection and storage were under sterile condition. UC-MSCs were isolated by tissue adherence method and were expanded after primary culture. During culture, cell morphology was observed. Cell counter was used to count the numbers of harvested UC-MSCs, based on which the growth curve was created. US-MSCs were tested for surface markers by flow cytometry, and identified by osteogenic differentiation and adipogenic differentiation under respective induction conditions.UC-MSCs from82donors were detected for the expression levels of surface marker CD309by flow cytometry.15cell lines were randomly selected and continuously expanded to passage11(P11). UC-MSCs at P1, P3, P5, P7, P10and P11were detected for the secretion levels of HGF and PGE2using enzyme-linked immunosorbent assay (ELISA).28UC-MSCs lines were randomly selected for expansion to P2. Using cell counter, the harvested UC-MSCs were quantified48hours after inoculation. The relationship between the proliferation rate of UC-MSCs and donor factors(such as:maternal age, gestational age, fetal weight, fetal gender and mode of delivery) was analysed.Results:Adherent UC-MSCs appeared in fusiform or spindle, and uniform morphology, which could be maintained more than15passages. The passaged cells were gradually purified, and maintained their biological characteristics after at least15passages in vitro. The growth curves were drawn by quantifying harvested UC-MSCs after different incubation time. The UC-MSCs at P1, P3and P7expressed CD90、CD105and CD166, but not CD14、CD34、CD45、CD79a andHLA-DR. The UC-MSCs at P1could be induced into adipocytes and osteoblasts.The CD309expression levels of UC-MSCs at P1derived from different donors ranged from0%to16.0%(n=82). UC-MSCs line with higher CD309expression had a greater proliferation rate. The levels of HGF and PGE2secreted in the culture media by UC-MSCs at P3were26029±11680(pg/ml) and3046±841(pg/ml)(n=15). The levels of HGF and PGE2in the culture media of UC-MSCs at P5were11660±5137(pg/ml) and2396±569(pg/ml)(n=15), respectively. As UC-MSCs were passaged from P1to P11, the secretion level of HGF decreased gradually, while the secretion level of PGE2had no obvious change.The cell proliferation rates were obtained by28UC-MSCs lines in expansion culture. It was found that the cell proliferation rate in maternal age group24-26is4.39fold, and that in maternal age group31-41is2.99fold; The prolifieration rate of UC-MSCs from the male donors (3.92fold) was higher than that from the female donors (3.03fold); the prolifieration rate of UC-MSCs from the fetal weight<3.5kg (3.89fold) was higher than that from the fetal weight≥3.5kg (2.87fold); however the prolifieration rate was not influenced by gestational age and mode of delivery. Conclusions:UC-MSCs are isolated and expanded successfully in the present study. The expression levels of CD309%HGF and PGE2of the UC-MSCs derived from different donors have significant differences. UC-MSCs line with higher CD309expression had a greater proliferation capacity. As UC-MSCs are passaged, the secretion level of HGF in the’culture medium is decreasing gradually in early passages, and the decrease becomes sharp from P5. However, no obvious change could be seen in the secretion of PGE2. UC-MSCs line derived from the younger mothers, lighter weight or male infants has a higher proliferation capacity. |
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