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Ray J. Tsai, Ryan Y. Tsai; Cytoplasmic Localization Of P120ctn And N-cadherin In Ex Vivo Expansion Of Human Corneal Endothelial Cells On Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6021.
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Previously, we had developed a unique long term, ex-vivo culture system for human corneal endothelial cells (HCECs) on amniotic membrane (AM), which preserved the stem cell properties, and were serially passaged. To further understand the mechanism and why the HECECs’ stem cell characteristics could be preserved on the amniotic membrane in our define culture system; the following studies have been designed.
HCECs were cultured on amniotic membrane for one month to three months and passaged. HCECs were labeled with BrdU for 2 weeks, chased for another 2 weeks, and then terminated and stained for Anti-BrdU. Stem cell markers, ABCG2, and differentiation marker for HCECs, Na+/K+ ATPase, were studied by immunochemistry. Double staining for ABCG2/p120ctn, ABCG2/N-Cadherin, BrdU/p120ctn, BrdU/N-Cadherin, and p120ctn/N-Cadherin were done and studied. The same markers were also studied using RT-PCR with total RNA extractions from cultured HCEC from different generations.
HCECs were cultured on AM for three generations, BrdU positive cells, ABCG2, Na+/K+ ATPase, p120ctn and N-Cadherin were detected in p0 to p3, with immunochemistry and RT-PCR. Interestingly, p120ctn, N-cadherin, and ABCG2 show co-localization in the cytoplasm.
HCECs could be cultured and serially passaged, while maintaining stem cells properties, in this long-term culture system. The results of this study suggest that the AM provides substrates that can preserve the stem-ness of the HCECs, and modulate the co-localization p120ctn, N-cadherin, and ABCG2.
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