Fig 1.
Flow chart of an overview of the experimental procedures that have been done in this paper.
Table 1.
Primer sequences used for the RT-PCR assays.
Table 2.
Sequences of primers used in hTERT gene expression, absolute telomere assay and methylation specific PCR.
Fig 2.
(A) Spindle-shaped morphology of hADSCs 7 days after seeding. Immunocytochemistry staining for the expression of hADSCs cell markers. (B) CD 90, (C) CD 105, (D) CD 45, and (E) CD 56. Nuclei were labeled with propidium iodide (PI) (orange) (bar = 40X).
Fig 3.
The expression of the surface epitopes of hADSCs as analyzed by flow cytometry.
Each antibody was tested individually and the isotopes controls were used as the negative control in this experiment; the ADSCs were positive for CD44 (80.2%) and CD73 (93.1%), and negative for CD31 (0.02%) and CD34 (0.03%). For CD44 and CD73, the isotype control was mouse IgG2b and for CD31 and CD34, the isotype control was mouse IgG1.
Fig 4.
In vitro multilineage differentiation of hADSCs.
(A) Osteogenic differentiation and alizarin red staining of mineralized cell aggregates; (E) Detection of the two bone specific genes (ALP and OCN) by RT-PCR method (B) Generation of lipid vacuoles after adipogenesis; (F) Expression of PPAR-α and PPAR-γ as fat-specific genes; (C) After chondrogenesis of hADSCs, proteoglycan aggregates stain with toluidine blue; (G) RT-PCR analysis of aggrecan and collagen II as chondrocyte-specific genes; (D) The neuronal cells differentiated from hADSCs show extensive somata-associated accumulations of Nissl bodies stained dark black-violet; (H) Expression of nestin and NGF as neural-specific genes (bar = 40X).
Fig 5.
(A and B) Effect of various concentrations of ZnSO4 on the proliferation of hADSCs. Cells were cultured at a density of 2 × 103 cells/wells with various concentrations of ZnSO4 for 24, 48, and 72 hours, and their viability was measured by a MTT assay. The MTT dye solution was added when the hADSCs were cultured for 24, 48, and 72 hours, respectively. After 4 hours of incubation, the culture medium was removed, DMSO was added, and the optical density of each well was measured at a wavelength of 570 nm (*P < 0.05 compared with control group). The results of Fig 5A in the X axis based on the serial dilution. (C) Population doubling time (PDT) of hADSCs after treatment with 1.5×10−8 and 2.99×10−10 M ZnSO4 in comparison to the control. Cells were seeded at a density of 5×104 cells/wells for about 24, 48, and 72 hours in the presence of 1.5×10−8 and 2.99×10−10 M ZnSO4. PDT was calculated according to the following formula: PDT = CT / PDN. The results indicated significantly short PDTs of ZnSO4-treated cells in comparison to that of untreated cells (*P < 0.05 compared with control group). To calculate cell proliferation and PDT for each treatment with ZnSO4, 3 wells of culture plate was considered, this procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. Two-way ANOVA followed by Dunnett’s post hoc test was used in all parts of Fig 5.
Fig 6.
(A) The presence of senescent cells in different passage cultures. Senescent cells were first observed in Passage 5 and their number was increased as the passage number increased. Meanwhile, the treated of hADSCs for 48 hours in the presence of 1.5 × 10−8 and 2.99 × 10−10 M ZnSO4 cause a decrease in the number of stained cells (**P < 0.01 and *** P < 0.001 compared with control group). To count senescent cells for each treatment with ZnSO4, 3 wells of culture plate was considered, this procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. Two-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups. (B) and (C) were included as a positive and negative controls for SA-β-galactosidase staining, respectively.
Fig 7.
Absolute telomere length measurement of hADSCs in the presence of different concentration of ZnSO4 for 48 hours of incubation.
Cells were seeded at a density of 5×104 cells/wells for about 48 hours in the presence of 1.5×10−8 and 2.99×10−10 M ZnSO4. Following, Genomic DNA was isolated, telomere and single copy gene standard curve was created. Real-time PCR technique was used to measure the absolute telomere length. The data were analyzed as kb/reaction and the genome copies/reaction for the telomere and the SCG. As described in results section, 1.5×10−8 M ZnSO4 were significantly increased the telomere length of hADSCs (**P<0.01 compared with control group), this procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. One-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups.
Fig 8.
The schematic figure for showing the 45 CpG sites of the hTERT promoter.
Fig 9.
The results of the original sequence alignment of the hTERT promoter region from 500 bases upstream into the first exon, sequence without treatment, and treated sequences with various concentrations of ZnSO4.
The first sequence from the top is the original sequence (main sequence) that was obtained from the NCBI database; the second, third, and fourth sequence from the top are related to the samples without treatment (control), treated with 1.5×10−8 M, and 2.99×10−10 M ZnSO4, respectively. The CpG sites are shown by the pink arrow.
Fig 10.
The sequence of 500 bases upstream into the first exon of the hTERT promoter and its transcription factor binding sites.
The sequence from bases −223 to −203, and bases 115 to 135 of the hTERT promoter has been shown in the 5′ to 3′ direction, spanning 358 nucleotides, containing 45 CpG sites. The transcription factors whose binding sites have been determined using the online TFBIND INPUT database are presented here in italics.
Table 3.
Methylation of the hTERT promoter region from 500 bases upstream into the first exon before and after treatment with various concentrations of ZnSO4.
The CpG sites are numbered from 1–45, CpG sites are indicated as follows: methylation (M); unmethylation (U); not determined (-).
Fig 11.
Relative hTERT gene expression levels of hADSCs in the presence of ZnSO4 for 48 hours of incubation.
Cells were seeded at a density of 1.5×103 cells/wells for about 48 hours in the presence of 1.5×10−8 and 2.99×10−10 M ZnSO4. Following, total RNA was isolated and was subjected to Real-time PCR Relative mRNA level of hTERT in the presence of 1.5×10−8 and 2.99×10−10 M ZnSO4. As described in results section, 1.5×10−8 M ZnSO4 was significantly increased the hTERT gene expression of hADSCs (*P<0.05 compared with control group). This procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. One-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups.
Fig 12.
Relative telomerase activity measurement of hADSCs in the presence of different concentration of ZnSO4 for 48 hours of incubation as detected by the PCR-ELISA TRAP assay.
Cells were seeded at a density of 5×104 cells/wells for about 48 hours in the presence of 1.5×10−8 and 2.99×10−10 M ZnSO4. Following, cells were lysed and protein were extracted from each sample. Relative telomerase activity was assessed in protein extracts by Telomerase PCR-ELISA kit. Heat inactivated cell extract served as the negative control. As described in results section, 1.5×10−8 M ZnSO4 was significantly increased the relative telomerase activity of hADSCs (*P<0.05 compared with control group). This procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. One-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups.
Fig 13.
ZnSO4 effect on (A) ALP, (B) OCN, (C) PPAR-α and (D) PPAR-γ mRNA expression in hADSCs. Cells were cultured in 6-wells plates at a concentration of 30 × 104 cells /wells. RNA was extracted from cultured hADSCs in the presence and absence of 1.5×10−8 and 2.99×10−10 M ZnSO4 as described in materials and methods section and was subjected to Real-time PCR assay (*P<0.05 compared with control group). This procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. One-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups.