In vitro study of various factors in isolation, expansion and differentiation of mesenchymal stem cells from human umbilical cord

The Wharton’s Jelly (WJ) of the umbilical cord is a rich source of mesenchymal stem cells (MSCs) with a various potential therapeutic applications. Human umbilical cord mesenchymal stem cells (hUCMCs) can be isolated with different methods, expanded and differentiated into neuron, astrocyte and oligodendrocyte in vitro. In the first study, four methods to isolate hUCMCs were compared. Three of them were based on using an enzyme cocktail containing collagenase, hyaluronidase and trypsin (CHT), collagenase /trypsin (CT) or trypsin alone (TRP) and the other was based on the explant culture (Exp) method. In the second experiment, the competence of DMEM/ F12 and alpha-MEM/Glutamax (α-MEM/GL), also the epidermal growth factor (EGF) and fibroblast growth factor (FGF) role to expand hUCMCs were evaluated. Finally, hUCMCs were examined for their differentiation rate into neural lineage cells by six various cocktails made of a base media (DMEM/LG), 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), EGF and FGF. Comparison of the four isolation methods of the hUCMCs showed that more cells were isolated in the CT, CHT and TRP groups, respectively. Cells were successfully isolated in TRP group but they did not propagate normally. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed on the cell surface in all groups, but there was no expression of hematopoietic lineage markers, CD34 and CD45, in any of the groups. In addition, the expression of C-kit and OCT-4 in enzymatic (CT and CHT) groups was greater than Exp group. One-way ANOVA showed that cell activity rate in the Exp group was significantly higher (P<0.001) than the other groups. A positive alkaline phosphatase activity and differentiation to adipogenic and osteogenic lineage was detected in the hUCMCs of all groups. The ratio of CD44 and CD105 expression cells were similar in DMEM/F12 and α-MEM/GL groups, while in both of the groups expression of the hematopoietic cells surface marker CD34 were negative. Differentiation potential into adipogenic and osteogenic cells were similar in both groups. Student`s t-test for evaluation of cell metabolism with WST-1 showed that the quality of the expanded cells in α-MEM/GL group was significantly higher (P<0.001) than DMEM/F12 group. Population doubling time (PDT) was calculated at 60 hours for DMEM/F12 group, while it was 47 hours in α-MEM/GL group that this difference was statistically significant (P