[Differentiation of adipose-derived mesenchymal stem cells after transfection with Pax6 gene].

Objective: To investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene.

Methods: The adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs. The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A), pcDNA3.1 empty vector transfection (group B), and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C), respectively. At 48 hours after transfection, the cells in groups B and C were selected with G418. The cell morphology changes were observed under the inverted microscope. Pax6 protein and level of corneal epithelial cells specific molecular--cytokeratin 12 (CK-12) were measured by Western blot. Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12.

Results: No morphology change was observed in groups A and B. Two different cell clones were found in group C. No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells; No.2 selected clone showed a net-like appearance, with 3-7 cell processes. The Western blot results showed the Pax6 protein expression in 2 clones of group C, but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C, and no expression in the others. The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64 ± 0.73, which was significantly higher than that of No.2 selected clone of group C (0.55 ± 0.42), group B (1.36 ± 0.40), and group A (1.00 ± 0.00) (P < 0.05), and there was no significant difference among groups A, Band No.2 selected clone of group C (P > 0.05).

Conclusion: Pax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels. This result provides a promising strategy of generating corneal epithelium-like cells for construction of tissue engineered cornea.