Zinc enhances the cell adhesion, migration, and self-renewal potential of human umbilical cord derived mesenchymal stem cells

Figure 1 Processing and culturing of human umbilical cord tissue. A: Demonstration of the pictographic representation of human umbilical cord processing; B: Show cord tissue comprises one vein and two arteries surrounded by Wharton's jelly; C-F: Shows fibroblast-like morphology of mesenchymal stem cells at 4 × and 10 × magnifications at P₀ (C and D), while P₀ at a later stage shows elongated fibroblast-like morphology, which is interconnected with their extensions and is arranged in colonies (E and F). MSCs: Mesenchymal stem cells.

Figure 2 Characterization of human umbilical cord-derived mesenchymal stem cells. A: Isolated human umbilical cord-derived mesenchymal stem cells appeared as elongated, fibroblast-like cells which were slightly trigonal at P₀ and then gradually became elongated in latter passages (P₁, P₂, P₅); B: These cells positively expressed CD73, CD117, CD29, CD105, Stro1, Vimentin, and Lin28 while exhibiting negative expression of CD45; C: Cell surface analysis by flowcytometry showed that more than 80% of the cell population was positive for CD73, vimentin, and CD105 while less than 4% were positive for hematopoietic marker i.e. CD45; D: The cells were successfully differentiated into adipocytes, osteocytes, and chondrocytes. Oil red O stain, alizarin red S stain, and Alcian blue stain confirmed the presence of lipids vacuoles, calcium deposits, and glycosaminoglycan in adipocytes, osteocytes, and chondrocytes, respectively.

Figure 3 Cytotoxic analysis of ZnCl₂ for human umbilical cord-derived mesenchymal stem cells. A: Concentrations of ZnCl₂ 5 µM, 10 µM, 20 µM, 30 µM, 50 µM, 100 µM, 250 µM, and 500 µM_, were analyzed for their cytotoxicity by MTT assay. Concentrations from 5 µM to 100 µM did not show any significant decrease in viability (Percent) of human umbilical cord (hUC)-derived mesenchymal stem cells (MSCs) compared to control. 250 µM reflected IC₅₀ value of ZnCl_2, and both 250 µM and 500 µM concentrations were found to be significantly (^cP ≤ 0.001) cytotoxic. Results were analyzed by performing one-way ANOVA and Post-Hoc Bonferroni test; B: Alamar blue assay analysis of hUC-MSCs after 24 h after ZnCl₂ did not exhibit any significant difference between control and test groups. But after 48 and 72 h, the proliferation of MSCs was significantly increased (^aP ≤ 0.05, ^bP ≤ 0.01) in all test groups. For further experiments, 20 µM of ZnCl₂ was selected as the test group concentration. C: Test group 20 µM ZnCl₂ exhibited significantly higher (^bP ≤ 0.01) NPD i.e., 2.9 ± 0.14, as compared to the control group, which showed 1.9 ± 0.18. Significantly reduced (^aP ≤ 0.05) D: PDT was observed in the test group i.e., approximately 3 d ± 0.16 in contrast to the control group having population doubling time of 5 ± 0.46 d. Results were analyzed by independent sample t-Test. Data is presented as mean ± SD (n = 3) where the significance level is ^aP ≤ 0.05. NS: Not significant.

Figure 4 Colony forming unit assay and transcriptional analysis of cell cycle and proliferation genes. A: Colonies after crystal violet staining in control and test groups. Denser colonies were observed in ZnCl₂ treated group. Images were captured at 4 × magnification; B: A significantly higher number (^cP ≤ 0.001) of colonies was observed in ZnCl₂ treated group with a mean number of 37 ± 0.57 colonies per T25 flask as compared to control i.e., 15 ± 0.57 colonies per T25 flask. Results were analyzed by SPSS, performing an independent sample t-Test. C: Gene expression of cell cycle genes CDC20, CCNA2, CDCA2 was significantly increased in the test group after 12, 24, 48, and 72 h of treatment with 20 µM ZnCl₂. No significant difference was found in CDK1 gene expression after 12, but it was significantly enhanced after 24, 48, and 72 h. This fold change (2^-ΔΔCt) increase in genes expression was highest in the 24-h group. D: Gene expression of proliferation genes transforming growth factor β1 and GDF5 were significantly increased in all temporal groups i.e. 12, 24, 48, and 72 h, of 20 µM ZnCl₂. HIF1α significantly decreased after 12 h but then significantly increased after 24, 48, and 72 h. Results were analyzed by SPSS, performing an independent sample t-Test. Data was represented as mean ± SD (n = 3) where significance level ^aP ≤ 0.05 (^aP ≤ 0.05, ^bP ≤ 0.01, ^cP ≤ 0.001).

Figure 5 Effect of ZnCl₂ on the migration potential of human umbilical cord-derived mesenchymal stem cells. A: Microscopic examination of human umbilical cord (hUC)-derived mesenchymal stem cells (MSCs) immediately after the scratch was given, i.e., 0 h, and after 2 h, 4 h, 6 h, 8 h, and 24 h. MSCs of the test group (20 µM ZnCl₂) exhibited faster migration as compared to control at all time points up to 8 h, and the scratch was completely healed after 24 h in both groups; B: Percentage of the area of scratch healed at time points, i.e., 0 min, 2 h, 4 h, 6 h, 8 h, and 24 h, analyzed by independent sample t-test. MSCs exhibited a significantly (^bP ≤ 0.01) increased migration (Percent) in the test group (20 µM ZnCl₂). C: Gene expression analysis of migration factors CXCR-4, V-CAM 1, VEGF-A, and MMP2, after 12, 24, 48, and 72 h of 20 µM ZnCl₂ treatment. CRCR-4 exhibited significantly increased expression after 24 h, 48 h, and 72 h of treatment. V-CAM1, and VEGF-A showed significantly higher expression after 48 and 72 h of treatment. MMP2 showed enhanced expression after 24 and 72 h. Results were analyzed by independent sample t-Test. Data was analyzed as mean ± SD (n = 3) where significance level ^aP ≤ 0.05 (^aP ≤ 0.05, ^bP ≤ 0.01, ^cP ≤ 0.001).

Figure 6 Effect ZnCl₂ on adhesion ability of human umbilical cord-derived mesenchymal stem cells. A: Images of human umbilical cord (hUC)-derived mesenchymal stem cells (MSCs) of the control group and 20 µM ZnCl₂ treated groups at 0 min, 15 min, 30 min, 1 h, and 2 h of cell seeding. Bright, rounded, and freely floating cells represent unadhered cells while dark cells represent adhered cells; B: Significant increase was observed in the adhesion ability of hUC-MSCs of the treated group after 15 min (^bP ≤ 0.01), 30 min (^aP ≤ 0.05),1 h (^bP ≤ 0.01), and 2 h (^cP ≤ 0.001). An independent sample t-test was performed to analyze results, and data were represented as mean ± SD (n = 3).

Figure 7 Protein expression of Lin28 and transcriptional analysis of pluripotency genes. A: Positive expression of mesenchymal stem cells (MSCs) specific marker Lin28 was observed after 12 h, 24 h,48 h, and 72 h of 20 µM ZnCl₂ treatment, as visualized by immunocytochemical staining; B: Quantified fluorescent intensities showed that 20 µM ZnCl₂ treated MSCs expressed Lin28 protein (^cP ≤ 0.001); C: Quantitative real-time polymerase chain reaction analysis of pluripotency genes OCT4, SOX2, and NANOG, exhibited significantly increased (^aP ≤ 0.05) expression of OCT4 at 12, 24, 48, and 72 h, while SOX2, and NANOG showed significantly increased expression at 24 and 48 h (^aP ≤ 0.05, ^bp ≤ 0.01). Results were analyzed by SPSS, performing an independent sample t-test. Data were analyzed as mean ± SD (n = 3) where significance level ^aP ≤ 0.05.