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Y. Zhu, Y. Hayashida, H. He, S. Chen, Y. Chen, S. C. G. Tseng; Characterization and Comparison of Intercellular Adherent Junctions in Human Cornea Endothelial Cells in vivo and in vitro Sphere Cultures. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3940.
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© ARVO (1962-2015); The Authors (2016-present)
Human corneal endothelial cell (HCEC) proliferation is controlled by their cell-cell junctions, of which the mechanism remains unknown. We recently reported a novel method of cultivating HCEC monolayer by collagenase digestion followed by expansion in the supplemented hormonal epithelial medium (SHEM). Herein we sought to characterize adherent junctional components of in vivo HCECs, compare their gene expression to that of in vitro counterparts, and correlate with their proliferative potential.
Stripped human Descemet’s membranes containing HCECs were digested with 1 mg/mL collagenase A at 37 °C for 16 h. Afterwards, HCEC spheres were cultured in collagen IV-coated dishes for 7, 14, and 21 days in SHEM. BrdU labeling was performed 24 h before termination. Both in vivo HCEC flat mounts and HCEC monolayers cultured at different times were subjected to immunostaining with FITC-phalloidin and antibodies specific to different adherent junctional components and BrdU. Their mRNA expressions were determined by RT-PCR.
In vivo HCECs expressed mRNAs of N- (type I and II), VE-, E- (type I and II), P-cadherins, α-, β-, γ- and p120 catenins. In vitro HCEC counterparts also expressed mRNAs of all these genes except P-cadherin. In vivo HCECs displayed continuous circular N-cadherin, β-catenin, p120, p190 and F-actin as well as discontinuous circular VE-cadherin protein bands at the cell-cell junction, and E-cadherin in the cytoplasm. Immunostaining revealed that gradual maturation of adherent junctions of HCECs cultured for 21days to a pattern similar to that of HCECs in vivo, and such a process was coupled with progressive decline of BrdU labeling.
Both in vivo and in vitro HCECs expressed mRNA and proteins of N-, VE-, E-cadherin, α-, β-, γ- and p120 catenins, and displayed distinct distribution of N-, VE-cadherin, β- and p120 catenins and F-actin at the cell-cell border and E-cadherin in the cytoplasm. Progressive maturation of intercellular adherent junctions was correlated with progressive decline of proliferative potential in HCEC cultures. This baseline information allows us to devise new strategies to engineer HCECs in vitro by targeting these components.
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