Construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells in vitro

Abstract

Background Corneal transplantation is an effective treatment to severe corneal diseases, but the shortage of cornea donor limits its application.Tissue-engineered cornea is being a new approach to corneal diseases. Objective This study was to investigate the possibility of construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells (hAMSCs) in vitro. Methods Fresh human amniotic membranes were obtained under the approval of Ethic Committee of Affiliated First Hospital of Jinan University and informed consent of maternal women.The 6 cm×6 cm amniotic membrane tissue explant was digested using trypsin+ EDTA, and then the amniotic epithelial cells (AECs) were scraped before putting into collagenase Ⅱ digestion medium to isolate hAMSCs.hAMSCs of passage 3 were cultured to achive 80%-90% confluence, and then the cells were incubated on rabbit deepithelial corneal stroma at a 1×105/ml density.The corneal stroma was co-cultured with hAMSCs at an air-liquid interface till 14 days.Rabbit deepithelial corneal stroma with and without hAMSCs (experimental group and control group) were fixed in 4% para formaldehyde, and sections were prepared for histopathological examination.Immunochemistry and immunofluorescence were empoyed to detect the expressions of cytokeratin3 (CK3) and CK12 in hAMSCs. Results hAMSCs grew well and formed a stratified epidermal structure resembling native corneal epithelium on rabbit corneal stroma in cultured 14 days in the experimental group, with the oval nucleus at basement and fusiform nucleus on the surface of corneal stroma.There was no cell structure in the control group.Immunochemistry revealed brown staining for CK3, CK12 in cytoplasm of hAMSCs on the rabbits corneal stroma, and the green fluorescence for CK3 and CK12 was also seen in the hAMSCs.However, the response for CK3 and CK12 was absent in the control sections either immunochemistry or immunofluorescence test. Conclusions hAMSCs can be induced to differentiate into corneal epithelioid cells at an air-liquid interface on the rabbit corneal stroma. Key words: Amnion; Mesenchymal stem cells; Cell culture; Tissue engineering; Epithelium, corneal; Differentiation; Humans