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Hung-Chi Chen, Yi-Jen Hsueh, Tze-Kai Wang, Sung-En Wu, David Hui-Kang Ma, Jan-Kan Chen; Suppression of Hippo pathway in a novel proliferative corneal organ culture system. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5312. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Shortage of available transplants may be attributed to human corneal endothelial cell (HCEC) damage during organ culture or transportation. Previously, we have shown that overexpression of YAP, a core protein in the Hippo pathway, results in proliferation in the HCEC monolayers without change in the normal phenotype. Here, we aimed to investigate the effect of YAP on HCEC proliferation or density in a proliferative corneal organ culture system.
Human or rabbit corneas were excised and cultured for 7 days in standard corneal organ culture medium MEM+2% FBS. Corneas were treated by PBS or adenovirus vector overexpressing YAP (adeno-YAP) from Day 2 on. Cell density and central corneal thickness were measured. Samples were then fixed, paraffin-embedded and examined morphologically by HE-staining and by imunofluorescent staining of YAP-1, ZO-1, Na-K-ATPase, SMA and BrdU labeling. TUNEL assay was performed to detect signs of apoptosis.
Phase contrast microcopy revealed endothelial loss and signs of apoptosis after 24h of organ culture, but no endothelial cell alterations in cultures added with adeno-YAP. From Day 4 to Day 7 the cell density was significantly more while the central corneal thickness was less in cultures added with adeno-YAP. HE-staining failed to demonstrate significant differences between the two groups. Imunofluorescent staining displayed similar patterns of ZO-1, Na-K-ATPase, and SMA, but enhanced staining of YAP and BrdU labeling. TUNEL assay detected signs of apoptosis in the control but not the YAP group.
A stable organ culture system for proliferation can be established, in which overexpression of YAP promoted corneal endothelial proliferation and increased cell density. This novel organ culture protocol may be applied to eye banking, to optimize corneal grafting and to contribute to regenerative medicine.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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