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Background Corneal transplantation faces a great challenge because of the shortage of corneal donors and difficulty of human corneal endothelial cells (HCECs) regeneration in vitro.So the study on tissue engineering cornea is still a main topic.Previous research showed that mouse embryonic stem cell conditioned medium (ESC-CM) improved the proliferative capacity of HCECs in vitro,and acellular porcine corneal stroma (APCS) was a good saffold material.However,whether HECEs cultured by mouse ESC-CM can form cell sheet in vitro were rarely studied.Objective This study was to investigate the potential that HCECs cultured by mouse ESC-CM form a monolayer cell sheet.Methods The supernatant of ESC-CM was collected after mouse ES-E14 cells were cultured,and the cultured medium was centrifuged and mixed with 75％ human corneal endothelium medium (CEM)at a proportion of 1 ∶ 3 to prepare the 25％ ESC-CM system.Primary cultures of HCECs were established from explants of corneal limbal with Descemet's membrane,and the cells were identified by using reverse-transcription PCR to determine the expressions of collagen Ⅷ (Col Ⅷ) mRNA and neuron-specific enolase (NSE) mRNA in the cells.APCS was prepared by decellularization with phospholipase A2 and bicarbonate solution,and the second generation of HCECs were inoculated on the sterilized APCS at a 800/mm2 density.The morphology of the cells was observed by hematoxylin-eosin staining under the phase-contrast microscope.The expressions of zona occludens protein-1 (ZO-1)and Na+-K+-ATPase in the cell sheet were detected by immunofluorescence staining.Results The second generation of HCECs cultured with 25％ ESC-CM in vitro showed the hexagon in shape with positive expressions for Col Ⅷ mRNA and NSE mRNA.Decellularization APCS was transparent,and no corneal cells were seen,the structures of corneal collagenous fibres were regular.HCECs attached closely to APCS and formed monolayer sheet 7 days after culture on the APCS with the cell density of (2 694±143)/mm2.ZO-1 and Na+-K+-ATPase were positively expressed on the HCECs sheet.Conclusions Twenty-five percent ESC-CM promotes the proliferation and maintains the normal morphology of HCECs.APCS provide a good scoffold and microenvironment for the formation of HCEC sheet.The HCEC sheet Possesses the pump function of HCECs and is a good corneal donor for transplantation.
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OBJECTIVE: Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice and to study cell differentiation and early developmental processes in mice. This study was designated to examine apoptotic processes in ES cells according to culture conditions and to study roles of extracellular matrix on the process. METHODS: Apoptosis was determined by DNA fragmentation and kinase activity during apoptotic process was measured. RESULTS: The apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates. Single cell suspension culture did not affect expression of bcl-2 and bax mRNA. Single cell suspension culture activated stress-activated protein kinase/c-jun-N-terminal kinase (SAPK/JNK), but not p38 kinase. The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from mouse embryonic fibroblasts (MEF), or on the petri dishes coated with fibronectin or laminin, but not with collagen or poly-L-lysine. Culture supernatants from MEF cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and MEF cells is required for the suppression of apoptosis. Activation of SAPK/JNK by single cell suspension was protected by interaction of cells with laminin or fibronectin, but not with collagen or poly-L-lysine. CONCLUSION: The suspension of ES cells as single cells causes serious damage and induces apoptosis, and the apoptotic process is mediated by the activation of SAPK/JNK and is inhibited by the interaction of ES cells with extracellular matrix.