Fig. 1.
Proliferation kinetics of Wj-MSCs and BM-MSCs overextended in vitro propagation. a Number of cumulative population doubling level (PD) as a function of time in culture. b Cell population doubling time (PDT) (hours) after sequential passages. Statistically significant differences using one-way ANOVA, N = 3; *p < 0.05, **p < 0.01, ***p < 0.001. c The capacity of BM- or Wj-MSCs to inhibit the proliferation of stimulated peripheral blood T cells was analyzed. BM- or Wj-MSCs were cultured with 1 × 105 MNCs in different ratios and stimulated with CD3/CD28 beads for 5 days. After, the proliferation of the T cells was measured by thymidine (3H-Thy) incorporation. MSCs from both sources significantly inhibited the proliferation of T cells in a dose-dependent manner (***p < 0.001). The proliferation of T cells in the presence of Wj-MSCs was significantly lower than that obtained using BM-MSCs at the same ratio (##p < 0.01). All data are presented as mean ± SD. N = 3. d Immunophenotypical analysis of Wj-MSCs by flow cytometry. Wj-MSCs were seeded at a density of 5 × 104 cells/cm2 on plastic culture plates (control) (upper panels) or SF patches (bottom panels) for 4 days. After, cells were detached and labeled with specific antibodies for the indicated markers or their control isotypes. Histograms show representative flow cytometry results obtained from N = 3 independent experiments. e To evaluate Wj-MSCs multipotent differentiation properties, cells were cultured in adipogenic, osteogenic, and chondrogenic differentiation media for 14–21 days. After, differentiation was evaluated by staining of lipid droplets with Oil Red O (adipogenic, right), by detection of calcium depositions and alkaline phosphatase activity by Alizarin Red and BCIP-NBT staining (osteogenic, middle left and middle right, respectively), or by detection of glycosaminoglycans by Alcian blue staining (chondrogenic, right). Images shown are representative of N = 3 independent experiments. Scale bar 200 μm. Abbreviations: PD population doubling level, PDT population doubling time, Wj-MSCs Wharton’s jelly MSCs, BM-MSCs bone marrow MSCs