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Y. Zhu, S.-Y. Chen, S. C. G. Tseng; Transient Downregulation of p120 Catenin Promotes Expansion of Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1819.
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© ARVO (1962-2015); The Authors (2016-present)
Human corneal endothelial cells (HCEC) are notorious for their limited proliferative potential, which we hypothesize might be due to contact inhibition controlled by p120 in the adherent junction.
HCEC monolayers were established from Descemet membrane stripped from the peripheral cornea on collagen IV-coated dishes in SHEM. Transfection efficiency of four p120 siRNAs was compared by real-time PCR and western blot in post-confluent ARPE-19 cells. Among them, siRNA3 was most effective and chosen to transfect HCEC monolayers cultured on day 14 at a dose of 40 nM for two weeks before withdrawal for one week. HCEC proliferation was measured by the monolayer size, and double immunostaining to p120 and BrdU. The HCEC phenotype was determined by immunostaining to F-actin, p120, N-cadherin, ZO-1, Na-K-ATPase and the cell density per mm2.
For post-confluent ARPE-19 cells, all four siRNAs resulted in significant downregulation of p120 mRNA and protein without synergistic action. An average number of 14.2 ± 1.9 aggregates per donor or 1.9 ± 1.0 aggregates per 1/8 Descemet membrane could consistently be obtained and each aggregate successfully generated an HCEC monolayer. Control aggregates transfected by scrambled RNA showed a rapid decline of growth after Day 25, leading to cell enlargement in the center of the monolayer and resultant cellular degeneration. In contrast, siRNA3-treated counterparts continued to expand to an average diameter of 2.1 ± 0.4 µm (n=5, p<0.05) without cell enlargement in the center. Furthermore, siRNA3 transfection caused translocation of p120 from intercellular junctions to the nucleus co-localized with BrdU throughout the monolayer. Withdrawal of siRNA3 for one week resulted in restoration of the intercellular distribution of F-actin, p120, N-cadherin, ZO-1 and Na-K-ATPase. The HCEC density was 2241 ± 104 /mm2 in the Descemet membrane, and increased to 2548 ± 93 /mm2 (p<0.05) for HCEC monolayers expanded on Day 14. For the control treated with scrambled RNA, the HCEC density dropped to 2083 ± 86 /mm2 on Day 28 (p>0.05) and 1764 ± 96 /mm2 one week after withdrawal (p<0.05). In contrast, the HCEC density was maintained at 2316 ± 79 /mm2 on Day 28 after siRNA3 treatment (p>0.05), and 2289 ± 113 /mm2 one week after withdrawal (p>0.05) [n=5 for all measurements compared to the in situ density].
Transient downregulation of p120 by siRNA3 followed by its withdrawal can switch proliferation on and off in contact-inhibited HCEC monolayers in vitro. This new strategy can be deployed to expand HCECs from 1/8 Descemet membrane without losing their normal phenotype.
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