## 4 Discussion
Secondly, we isolated cells not only from the WJ but also from the SA, which was not performed in the other studies. The SA results that we obtained for initial cell number and growing time were very good as well. The lower number of obtained cells from the SA in comparison with the WJ could be attributed to the fact that the initial amount of cells in the WJ is higher than that in SA, as has already been proven [25, 26]. In addition, in our study, the explants were used again in order to isolate more cells, which eventually led to the faster accumulation of a larger number of MSCs, which was not the case with the rest of the studies. In another paper, [PERSON] et al. (2013) also used the explant method for obtaining MSCs [50]. However, they used the entire umbilical cord tissue, without the distinction of the region of origin, and due to the different approach in the sampling and processing of the cell isolation and transferring of the explants, it was impossible to draw a proper comparison between their method and the one we used. This study also pays heed to the biological characteristics of MSC populations derived from [PERSON]'s jelly and the subam union region by the explant method. The explant method seems to be less stressful for the cells compared to the enzyme digestion method [51]. Moreover, it leads to the isolation of more homogeneous cell populations with higher proliferation rates [33]. The presence of the tissue pieces facilitates the transfer of cells from in vivo to in vitro conditions [35]. Furthermore, the tissue pieces provide a microenvironment important for keeping MSCs in a stem cell-like state during long-term in vitro culturing [50]. In the scientific literature, there are various modifications of the explant method that include long-term explant cultures [42]. In our study, after a culture period of 8-10 days, the cells reached a confluence of around 50%. At this stage, the pieces of the specimens were transferred to new dishes and allowed more cells to migrate. This optimization makes it possible to obtain more cells per low passage. The cells from the same passage were put together and examined. In this way, we received enough cells from the Wharton's jelly and subamnion to perform all experiments on cells between the third and fifth passage. The selection of explant sizes and period of outgrowth from explants was based on the available scientific literature on the topic. The cells isolated from the WJ and SA of the UC by the explant method displayed a similar fibroblast-like morphology, with no significant difference related to their origin. The populations of epithelial-like cells reported in the culture obtained from the subamnion region by [PERSON] et al. were not detected [25]. [PERSON] et al. reported significant differences in the percentages of CD73-, CD105-, and D90-positive cells from the WJ and umbilical cord membrane (contain epithelial and mesenchymal layer) [44]. At early passage (passages 3-5), we found no significant differences between the common MSC signature markers (CD29, CD73, CD90, CD105, and HLA-DR) in the cells from the WJ and SA, which is consistent with the report of [PERSON] et al. [26]. MSCs from the UC and SA demonstrated osteogenic and adipogenic potential, which is in consensus with previous studies on the topic [36, 44, 52]. MSC-based therapies require the production of large cell doses (0.5-12 \(\times\) 10\({}^{6}\) cells/kg body weight) to support the application of MSCs in cell-based therapy and clinical trials [53; 54]. Also, allogeneic MSC doses might go up to 1 \(\times\) 10\({}^{8}\) cells/kg [54]. Hence, the proliferation ability, as an indicator of the MSC potential to be repetitively expanded, should be considered as an essential characteristic for the assessment of MSCs. Our analysis revealed that MSCs derived from the WJ and SA, using the explant method described above, had the same proliferative capacity and displayed a relatively short population doubling time of 29.19 \(\pm\) 7.7 h for WJ-MSCs and 31.96 \(\pm\) 6.4 h for SA-MSCs.