Enhanced tenogenic differentiation by Scleraxis overexpression in human amniotic mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) are adult stem cells with multipotential differentiation, which can be induced to differentiate into bone, cartilage and other connective tissues. Meanwhile, as a highly specific marker of tenocytes, Scleraxis is involved in aggregation and differentiation of tendon progenitor cells as well as the formation of tendon extracellular matrix. OBJECTIVE: To investigate whether hAMSCs have the ability of differentiation into tenocytes by ectopic expression of Scleraxis. METHODS: Agreed by puerpera, the amniotic membrane from the full-term placenta was separated, and hAMSCs were isolated by a two-step enzyme digestion, observed under inverted phase contrast microscope, and identified by flow cytometry. Passage 3 cells were induced via plasmid-mediated Scleraxis overexpression in overexpression group. Untransfected cells cultured in normal medium served as blank control group, and those with empty plasmid transfection were defined as empty plasmid group. Cell proliferation was tested in each group using cell counting kit-8 within 7 days of culture. Real-time quantitative PCR and western blot were used to assess the tenogenic differentiation of hAMSCs in each group at 3 and 7 days of culture. RESULTS AND CONCLUSION: Findings from the cell counting kit-8 indicated that the cell viability had no significant differences among the groups within 7 days of culture (P > 0.05). Western blot results showed the protein expression of Scleraxis in the treatment group was significantly higher than that in the other two groups (P < 0.05). Real-time PCR results showed, at 3 days of culture, the expression of collagen type I, collagen type III, Fibronectin and Tenascin-C in the overexpression group was significantly higher than that in the empty plasmid group (P < 0.05), but the expression of Tenomodulin had no difference (P > 0.05); at 7 days of culture, the expressions of collagen type I, collagen type III, Fibronectin, Tenascin-C and Tenomodulin in the overexpression group were significantly higher than those in the empty plasmid group (P < 0.05). In summary, hAMSCs can be differentiated into tenocytes by ectopic expression of Scleraxis.