Effects of Multiple Agents on Epithelial Differentiation of Rabbit Adipose-Derived Stem Cells in 3D Culture

Abstract

Mesenchymal stem cells have been given particular attention in tissue regeneration research due to their multipotency and proliferative activity. In this study, we investigated the possibility of epithelial differentiation of rabbit adipose-derived stem cells (rASCs) in an in vitro 3D culture system. The experimental procedure was performed with different contributing factors including all-trans retinoic acid (ATRA), epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and hydrocortisone in air-liquid interface culture, for modulating proliferation and providing a synergistic effect on epithelial differentiation of rASCs. After induction, immunofluorescence staining, western blot analysis, flow cytometry analysis, and quantitative real-time polymerase chain reaction assay have been performed to detect the expression of epithelial-specific markers and mesenchymal marker alpha-smooth muscle actin (α-SMA). The growth pattern and viability of cells were evaluated by transmission electron microscopy and Hoechst 33258 assay, respectively. After treated with optimized induction medium (including 2.5 μM ATRA, 20 ng/mL EGF, 10 ng/mL KGF, 10 ng/mL HGF, and 0.5 μg/mL hydrocortisone), rASCs were observed to display a stratified epithelial-like morphology, with the expression of cytokeratin 19 and cytokeratin 13 in 63.69%±2.63% and 22.17%±1.51%, respectively, and the relative expression level of cytokeratin 19 increased to 3.152 compared with 0.151 before induction. The expression of α-SMA decreased to 19.40%±1.45% after induction, but almost no expression of involucrin was detected. The results showed that the establishment of an epithelial-specific microenvironment may be a feasible way for epithelial differentiation of ASCs in vitro, and provided an alternative for research on epithelium regeneration.