Differentiation of bone marrow mesenchymal stem cells into retinal pigment epithelial-like cells by a systematic induction in vitro

Abstract

Objective To investigate the differentiation features of bone marrow mesenchymal stem cells (BMSCs) cultivated with human retinal pigment epithelial cells (HRPECs), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) in vitro. Methods Experimental study. Three experimental groups were established based on the following criteria: HRPECs+EGF, BFGF, BDNF+BMSCs group (group Ⅰ), EGF, BFGF, BDNF+BMSCs group (group Ⅱ) and human BMSCs only (group Ⅲ). For group Ⅰ, HRPECs at passage 3were inoculated at the upper layer of a Transwell 6-well double layer culture plate, and human BMSCs at passage 3 were seeded at the lower layer of the culture plate; a DMEM-LG medium containing 20 ng/ml bFGF, 20 ng/ml EGF, 20 ng/ml BDNF and 10% fetal bovin serum (FBS) was added to each well (for immunocytochemical analysis, a 18 mm×18 mm cover slip was placed under the lower layer to allow cells to grow on it). For group Ⅱ, human BMSCs at passage 3 were seeded at the 6-well culture plate, and a DMEM-LG medium containing 20 ng/ml bFGF, 20 ng/ml EGF, 20 ng/ml BDNF and 10% FBS was added to each well. For group III, human BMSCs at passage 3 were seeded at the 6-well culture plate, and a DMEM-LG medium containing 10% FBS was added to each well. Cell morphology was monitored under an inverted microscope. After 2 weeks, cells were collected for immunocytochemistry and RT-PCR analysis. A Holm-Sidak test was used to analyze data. Results After induction, the cells in group Ⅰ presented a similar appearance to retinal pigment epithelial cells and intracellular pigment particles were visible. However, similar changes did not occur in group Ⅱ and group Ⅲ. Immunocytochemical analysis of RPE65 and keratin-18 showed the optical density value differences between group Ⅰ and group Ⅱ, and between group Ⅰ and group Ⅲwere statistically significant (RPE65: t=37.416, 36.236, P＜0.05; keratin-18: t=38.611,37.532, P＜0.05),while the difference between group Ⅱ and group Ⅲ was not statistically significant (RPE65: t=1.180,P＞0.05; keratin-18: t=1.079, P＞0.05). The expression of RPE65 and keratin-18 was examined by RT-PCR using cell extracted from the 6-well plate, and mRNA expression was calculated after a computer software analysis and a comparison to the internal control β-actin. The inter-group difference showed that the differences between group Ⅰ and group Ⅱ, and between group Ⅰand group Ⅲ were statistically significant (RPE65/β-actin: t=176.110, 174.820, P＜0.05; keratin-18/β-actin:t=243.230, 241.560, P＜0.05), but the difference between group Ⅱ and group Ⅲ was not statistically significant (RPE65/β-actin: t=1.283, P＞0.05; keratin-18/β-actin: t=1.670, P＞0.05). Conclusion BMSCs can be induced into retinal pigment epithelium-like cells when cocultured with HRPECs and BDNF, EGF, bFGF. Key words: Mesenchymal stem cells,bone marrow; Pigment epithelium of eye; Cell differentiation; Cell cultivation techniques