Department of Urology, Sixth People's Hospital, Jiao Tong University of Shanghai, Shanghai, PR China.
Tissue Eng Part A. 2012 Sep;18(17-18):1760-70. doi: 10.1089/ten.TEA.2011.0424. Epub 2012 Jun 13.
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.
间充质干细胞因其多能性和增殖活性而在组织再生研究中受到特别关注。在这项研究中，我们研究了在体外 3D 培养系统中兔脂肪来源干细胞（rASC）上皮分化的可能性。实验程序是在不同的贡献因素下进行的，包括全反式视黄酸（ATRA）、表皮生长因子（EGF）、角质形成细胞生长因子（KGF）、肝细胞生长因子（HGF）和氢化可的松在气液界面培养中，以调节增殖并对 rASC 的上皮分化产生协同作用。诱导后，通过免疫荧光染色、western blot 分析、流式细胞术分析和实时定量聚合酶链反应检测上皮特异性标志物和间充质标志物α-平滑肌肌动蛋白（α-SMA）的表达。通过透射电子显微镜和 Hoechst 33258 测定分别评估细胞的生长形态和活力。在用优化的诱导培养基（包括 2.5 μM ATRA、20ng/mL EGF、10ng/mL KGF、10ng/mL HGF 和 0.5μg/mL 氢化可的松）处理后，rASC 显示出分层上皮样形态，细胞角蛋白 19 和细胞角蛋白 13 的表达率分别为 63.69%±2.63%和 22.17%±1.51%，并且细胞角蛋白 19 的相对表达水平与诱导前的 0.151 相比增加到 3.152。诱导后α-SMA 的表达降低至 19.40%±1.45%，但几乎没有检测到包裹蛋白的表达。结果表明，建立上皮特异性微环境可能是体外 ASC 上皮分化的一种可行方法，为上皮再生研究提供了一种替代方法。
