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The fate of adult stem cells is associated with its surrounding microenviroment.Our previous work found that embryonic stem cells (ESCs) micro-environment enhance the stemness of human limbal stem cells (LSCs), but its mechanism has not been elucidated.
This study was to explore the molecular mechanism of ESC micro-environment enhancing the stemness and inhibiting the apoptosis of LSCs.
Human LSCs were cultured by explant culture method with CnT-20 medium and CnT-20+ 20%ES culture supernatant (ESC-CM), respectively.Colony formation assay was used to analyze the proliferation ability of cells.Telomerase reverse transcriptase (TERT) siRNA(19-25nt siRNA) or siRNA(sc-37007) was transfected into the cells of ESC-CM group.Apoptosis and mitochondrial membrane potential were assayed by flow cytometry, and the expressions of telomerase and reactive oxygen species (ROS) in TERT siRNA- or siRNA-F-transfected cells by immunofluorescence and flow cytomery.RT-PCR, immunofluorescence staining and Western blot were employed to determine the expressions of p63, ATP-binding cassette transporer G2 (ABCG2), integrin β1 mRNA and proteins and cytokeratin 3(CK3) in the cells.The levels of focal adhesion kinase (FAK), Akt, glycogen synthase kinase 3β (GSK3β) and p21 protein and phosphorylation proteins in the cells were detected by Western blot.
The LSCs presented an increased proliferative capacity and passaged to the eighth generation with the colony-forming efficiency (CFE) of (7.6±0.6)% in ESC-CM group, but the cells to the sixth generation with the CFE of (5.6±0.6)%, showing a significant difference between them(t=4.454, P=0.011). The apoptotic rates of the cells from 2 through 6 generations were lower in the ESC-CM group than those in the CnT-20 group (all at P<0.05). The apoptotic rate of the cells was (7.67±1.31)% in the siRNA-F transfected group, which was significantly lower than (32.33±3.13)% in the siRNA-TERT transfected group (t=-12.588, P=0.000). No significant differences were seen in the expression levels of p63, ABCG2, integrin β1 mRNA and proteins and TERT protein in the primary cells between the ESC-CM group and the CnT-20 group (all at P>0.05), but significantly declined expressions of CK3 mRNA and protein were found in the ESC-CM group compared with the CnT-20 group (all at P<0.01). However, the expressions of p63, ABCG2, integrin β1 mRNA and proteins and TERT protein in the second generation of the cells were significantly higher in the ESC-CM group compared with the CnT-20 group (all at P<0.01). The telomerase activity was (4.83±0.67) % in the siRNA-TERT transfected group, which was significantly lower than (46.71±1.22) % of the siRNA-F transfected group (t=52.116, P=0.000). The expression of pFAK, pAkt, pGSK3β proteins were weakened, but the expression of p21 was increased in the ESC-CM group after addition of FAK inhibitor, GSK3β inhibitor and TERT-siRNA transfected group.Mitochondrial membrane potential in the second generation of cells was elevated in the ESC-CM group in comparison with the CnT-20 group and the siRNA-TERT transfected group (all at P<0.01), and the rates of ROS positively reaction was lower in the ESC-CM group and the siRNA-F transfected group than those of the CnT-20 group and siRNA-TERT transfected group (all at P<0.01).
ESC-CM culture system can effectively keep the stemness of LSCs and inhibit apoptosis.ESC-CM culture system plays functions probably via telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways.
Key words:
pathway; Apoptosis; Colony-forming units