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Background Subretinal transplantation of retinal pigment epithelium (RPE) cells for the treatment of age-related macular degeneration (AMD) have accelerated the drive to develop xeno-free cultivation system that support the rapid differentiation of human embryonic stem cells (hESCs) into ES-RPE cells.Objective This study was to report a modified xeno-free culture system and method for accelerating derivation of hESCs to differentiate into RPE cells.Methods This study was approved by Ethic Committee of Zhongshan Ophthalmic Center.HESC H1 line was cloned and cuhured in Vitronectin XFTM-coated 6-well dish with xenogenetic-free medium.Cells were cultured in 50 ng/ml noggin,10 ng/ml DKK-1 and 10 ng/ml insulin like growth factor-1 (IGF-1) medium for 2 days,and then the concentration of noggin was decreased to 10 ng/ml and 5 ng/ml basic fibroblast growth factor (bFGF) and cultured for the following 2 days.Sequentially,noggin and bFGF were removed and cultured for 2 days.Finally,1 μmol/L CHIR99021 was added in medium for 6 days.Morphological changes in the progress of ESCs differentiation into RPE were observed by Living Cell Imaging System.The expression of Mitf and RPE65,RPE cellsspecific markers,in the cells were detected by immunofluorescence technique,and the relative expression levels of RPE cells-specific marker mRNA were assayed using real time fluorescent quantitation PCR.Results Polygonalshape monolayer cells which contained pigments were initially observed at day 14 after cultured with the cobblestonelike arrangement.Mitf and RPE65 were strongly expressed in the hES-derived RPE cells 35 days after induced,showing red fluorescence,and the cells presented hexagonal shape at cultured day 60 with numerous pigment granules in cytoplasm.Compared with before differentiation,the expression levels of Mitf mRNA in hES-RPE cells increased by (3.43±2.77) folds and (8.91 ± 2.83) folds,and the expression levels of RPE65 mRNA increased by (14.60 ± 3.94) folds and (87.16 ±9.32) folds at day 7 and day 14 after differentiation,respectively (all at P＜0.05).Conclusions A defined xeno-free culture system is successfully established by adding niacinamide,DKK-l,noggin,IGF-1 and CHIR99021 in xeno-free medium,and this system can accelerate the derivation and differentiation of hESCs into RPE-like cells.
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Background Extracellular matrix (ECM) fibrosis leads to corneal scaring during the process of cornea wound healing.Transforming growth factor-β1 (TGF-β1) is known to mediate overproduce of ECM components.Our previous study developed a three-dimensional model for corneal stromal cells culture in vitro.Objective The hypothesis of this study was to apply TGF-β1 in the three-dimensional culture system to establish a corneal stroma ECM fibrosis model.Methods Fresh bovine corneas were extracted for the culture of bovine keratocytes in constructed three-dimension culture system.The Pellets were cultured in the DMEM/F12+ 10％ fetal bovine serum (FBS) medium with 0.5 ng/ml or 1.0 ng/ml TGF-β1 or without TGF-β1,respectively.Calcein AM/(propidium iodide) PI staining was employed to assay the cell viability 2 weeks after culture.The expressions of α-smooth muscle actin (α-SMA),type Ⅰ collagen (Col Ⅰ) and Col Ⅲ mRNA and protein in the cells were detected by real-time PCR and Western blot respectively 48 hours,1 week and 2 weeks after cultured.The results were statistically analyzed.Results Cultured for 48 hours in the Pellet system,corneal stromal cells clustered and was identified alive by Calcein-AM/PI staining in 2 weeks.The relative expression levels of α-SMA,Col Ⅰ and Col m mRNA were elevated in both the 0.5 ng/ml and 1.0 ng/ml TGF-β1 supplement groups in comparison with the only DMEM/F12+10％ FBS group,with marked difference among the three groups (Fgroup =696.745,P＜0.001;Fgroup =35.166,P＜0.001;Fgroup =33.677,P＜0.001),and the expression levels increased with the lapse of culture time (Ftime =5.863,P＜0.05;Ftime =298.614,P＜0.001;Ftime =607.472,P＜0.001).The synthetic rate of Col Ⅲ mRNA was obviously faster than that of Col Ⅰ mRNA.Western blot showed that only a trace of α-SMA,Col Ⅰ and Col Ⅲ were detected 48 hours and 1 week after culture.The expression levels of α-SMA,Col Ⅰ and Col Ⅲ in Pellet system in 0.5 ng/ml TGF-β1 medium were 0.395±0.208,1.060±0.175 and 0.629±0.382,and in 1.0 ng/ml TGF-β1 medium were 0.758±0.228,1.201 ±0.187 and 0.753±0.468,respectively 2 weeks after culture,significant differences were shown among the three groups (α-SMA:F=10.691,P＜0.05;Col Ⅰ:F=14.094,P＜0.05;Col Ⅲ:F=10.995,P＜0.05).Conclusions Addition of TGF-β1 and serum enhance the assembly and fibrosis of ECM,showing the higher expressions of specific fibrotic markers in bovine keratocytes Pellet.This culture systerm can be used as a candidate three-dimensional model for corneal stroma ECM fibrosis.
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Objective To develop a method for the primary culture of retinal Müller cells of adult rabbit in vitro. Methods Retina was isolated from adult rabbit, cut into 1 mm × 1 mm pieces, and placed into Dulbecco modified Eagle medium/F12 containing 20 % fetal bovine serum to culture. Cultured cells were identified by inverted phase contrast microscope, transmissim electron microscope and immunohistochemistry staining method. Results Visible cell processes grew out from the retinal tissues after three days culture, and more cells grew radically around the retina after seven days culture. The cultured cells were often inflated at one side and had one long process at another side, and the nuclei were elliptical and there were two or more than two nucleoli under inverted phase contrast microscope. The cytoplasm was rich and contained abundant microfilaments in eight to ten nanometers under transmission electron microscope. Immunohistochemistry assay showed that 95% of the cells were positive for glial can be cultured by the explant culture method.
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Objective To investigate the feasibility of differentiation of invitro induced rat bone marrow-derived mesenchymal stem cells(rMSCs) into retinal pigment epithelial (RPE) cells.Methods The rMSCs from Brwon-Norway (BN) rats were isolated and cultured by adherent screening method.RPE cells lysate made by repeated freeze-thawing was put into the rMSCs culture system to identify whether the induced cells could express characteristic label cytokeratin(CK)and S-100 simultaneously or not.Results The growth rate of rMSCs induced by RPE cells lysate was slower and protuberant burr surrounded the fusiform cells.The results of immunoblotting and double immunofluorescence showed that partial induced cells expressed CK and S-100 simultaneously.The result of flow cytometry indicated that 14.1% induced cells expressed CK and S-100 simultaneously.Conclusion Induced by RPE cells lysate,rMSCs can differentiate into RPE cells.