Rod with CES to have the functional properties of autologous cells, the tissue-specific therapeutic implications in the clinic can k, Rdern to f. We have shown that stem cell-Ph Phenotype and functional properties of CPB were significantly improved Alvespimycin 17-DMAG by co-culture with CES, especially in a system of direct cell-cell contact. A new strategy was used to the L Isolate of funds-st Bchenf Shaped cells of the mouse by co-culture in the pre-transfection of ESCs with a suicide gene, herpes simplex virus thymidine kinase, which makes them sensitive to the CES ganciclovir. We also showed for the first time that the R Potential of ESCs in the F Promotion of the functional properties of the CEC by, at least in part, was to an integrin PI3K/Akt signaling path-FAK. Second Materials and methods 2.1.
Animals and young cells in New England white E rabbits NVP-BEP800 HSP-90 inhibitor and BALB c / acquired naked from the animal laboratory of Sun Yat-Sen University t. All animal experiments were Sen with permission of the Committee of Ethics in Medicine, Zhongshan Ophthalmic Center, Sun Yat-Universit t, and respect for the Association for Research in Vision and Ophthalmology on the use of animals in ophthalmic research leadership and vision. E14 mouse ES cells were kindly provided by Professor P. provided Xiang Sun Yat-Sen University t, China. E14 mouse ES cells were plated at a density of 400/cm2 in 1% gelatin-coated bo Their tissue culture with a culture medium of mouse ESC. Prim Re CEF were obtained from explants of rabbit peripheral corneal tissue and spread to the middle of the corneal epithelium. 2.2.
Stable transfection of ESCs and ECC All transfections were prepared using Lipofectamine 2000, according to standard Invitrogen protocol. To the CES after coculture with CEC to L Between, ESCs were treated with the HSV-tk-puro vector transfected a suicide gene, herpes simplex virus thymidine kinase, and selected Selects 1 g / ml puromycin. The clones were identified by Western blot. October 4 expression after transfection was analyzed by RT-PCR and immunofluorescence to determine the status of differentiation of ESCs. The cell apoptosis was then induced by treatment of the cultures with 20 M ganciclovir. Cocultured follow the CEC passing a CEC were transfected with EGFP-N1 vector and with 400 g / ml G418. GFP expression was identified by fluorescence microscopy and flow cytometry. 2.3.
Co-culture systems to improve the functional properties of the CES CEC CEC The labeled GFP were grown in the following three conditions: the contr Group: GFP alone increased CEC, in a culture medium ESC transwell coculture: GFP CEC and GFP cells in cell culture medium co-cultured ESC: ESC in CEC with 6 bedrooms and ESC in culture medium of cells in direct contact with cells co-cultured cocultured. ESC in all groups were transfected with the HSV-TK vector puro, and all groups t Resembled changed and subcultured approximately every two days. On day 10 was added 20 mol / L GCV and the cells were harvested from three to 13 days. 2.4. Colony-forming efficiency and growth capacity T to evaluate the proliferative capacity t of the CEC of the three groups, the CFE was studied in cultures in the middle of corneal epithelium with a previous procedure with the modification. CEC of the three groups were plated in triplicate at 500 cells/cm2 in 6-well culture plates