Objective: To develop techniques to culture the human corneal endothelial cells and to identify the cultured human corneal endothelial cells maintain the property of stem cells by ex vivo expansion on amniotic membrane.Materials and Methods: Human donor corneas were selected for studies. To understand the possibility of human corneal endothelium contains stem cells, we set up an organ culture technique for human donor cornea. Human donor cornea was cultured in culture medium with BrdU for labeling 2 weeks then chasing for another 2 weeks. BrdU labeling retention cells will be studied under immunomicroscopy. To further study the human corneal endothelial stem cells can be preserved and cultured on amniotic membrane, human corneal endothelium was obtained from the inner part of human limbal corneal rim, when the central cornea was prepared for PkP. Human corneal endothelium was cut into a small pieces and cultured on the different kind of substrate including amniotic membrane. Series passages were performed, and markers of Na/K ATPase, P63, and ABCG2 were used for immunochemistry studies. Cultured human corneal endothelial cells finally were growth on the human corneal disc.Results: Age of human donor corneas was from 37 y/o to 70 y/o with mean 63±11 y/o. The death to cultured period was from 6 days to 10 days with mean 8.4±1.8 days. The cultured human corneal endothelial cells (HCEC) could be growth on the substrate with basement membrane substance. Serial passages from each HCEC were performed and results with 4.5±2.3 (2 to 9 passages) had been achieved from the aged human corneas. For the study of organ culture of human donor cornea, BrdU labeling retention cells were detected only on the peripheral area of donor corneal endothelial zone, but negative over the central corneal area. To explore the corneal endothelial cells contain stem cells on the cultured system, cultured corneal endothelial cells at different culture period was studied for Na/K ATPase, ABCG2 and P63. The positive staining of ABCG2 and P63 were detected only on the earlier 2 weeks culture period but negative on the longer one month culture period. However, Na/K ATPase detected only on the longer cultured period. When HCEC was cultured on the human corneal disc, TEM demonstrated mosaic pattern of HCEC on the cornea disc.Conclusion: The ex vivo expansion of human corneal endothelial cells systems have been established. The HCEC may maintain the stem cells property on AM. Also, the cultured HCEC could be transplanted on the human corneal disc. These techniques will allow us to further study the engineering of cornea.

Linarin is a flavone glycoside in the plants Flos chrysanthemi indici, Buddleja officinalis, Cirsium setosum, Mentha arvensis and Buddleja davidii, and has been reported to possess analgesic, antipyretic, anti-inflammatory and neuroprotective activities. In this paper, linarin was investigated for its AChE inhibitory potential both in-vitro and ex-vivo. Ellman’ s colorimetric method was used for the determination of AChE inhibitory activity in mouse brain. In-vitro assays revealed that linarin inhibited AChE activity with an IC50 of 3. 801 ± 1. 149 μ M. Ex-vivo study showed that the AChE activity was significantly reduced in both the cortex and hippocampus of mice treated intraperitoneally with various doses of linarin (35, 70 and 140 mg/Kg). The inhibition effects produced by high dose of linarin were the same as that obtained after huperzine A treatment (0. 5 mg/Kg). Molecular docking study revealed that both 4’-methoxyl group and 7-O-sugar moiety of linarin played important roles in ligand-receptor binding and thus they are mainly responsible for AChE inhibitory activity. In view of its potent AChE inhibitory activity, linarin may be a promising therapeutic agent for the treatment of some diseases associated with AChE, such as glaucoma, myasthenia gravis, gastric motility and Alzheimer’ s disease.

The identification and characterization of retinal progenitors with stem cell properties has opened new avenues that may be useful for treating functional impairments caused by the death of specific neural cell populations in the retina. Neuronal degeneration is the cause of debilitating visual impairment associated with prevalent ocular diseases, such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), retinal detachment, and glaucoma. Retinal stem cells/progenitors may help to restore vision in patients who have these diseases by repopulating the damaged retina and/or by rescuing retinal neurons from further degeneration by ex-vivo cell therapy. However there are barriers to this approach, which primarily include sources of cells that can sustain a practical clinical use, free from immunogenic responses, tumor formation, and ethical burden. Significant progress has been made to overcome these barriers.