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H. He, H.–T. Cho, W. Li, T. Kawakita, L. Jong, S.C. G. Tseng; Signaling Transduction Pathways Required for ex vivo Expansion of Human Limbal Explants on Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2117.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Ex vivo expansion of limbal epithelial progenitor cells by amniotic membrane (AM) is a new surgical approach to treat eyes with limbal stem cell deficiency. Previously, we reported that NGF–TrkA mediated signaling is required. Herein, we sought to further delineate the downstream signaling pathways involved during such expansion. Methods: The human corneolimbal tissue was briefly incubated with 1.2 units/ml Dispase II, cut into explants of approximately 1x1.5x2.5 mm, and cultured on intact AM in SHEM. At day 0 or day 10, different concentrations of small MW inhibitors of PI3K, phospho–AKT, p38, JNK, and p42/p44 MAPK were added, while the control group received the same amount of the vehicle, i.e., DMSO. The epithelial outgrowth was monitored daily for 17 days by digitizing the surface area. The epithelial cells in the outgrowth and the explant were collected for western blotting analysis. Results: In the control, expansion of human limbal epithelial cells was more rapidly from the limbal than the corneal and scleral area during day 5 to 7 and reached ∼80 % confluence at day 17 on a 20 mm diameter AM insert. Compared to the control, LY294002 (PI3K inhibitor) at 50 µM, SR13668 (phosphor–AKT inhibitor) at 50 µM completely, and U0126 at 10 µM significantly suppressed the expansion of limbal epithelial cells on AM (p=0.0006, 0.0005, and 0.0008, respectively). However, the outgrowth was not affected by 10 µM of either SB203580 (MAPK p38 inhibitor) or JNK inhibitor 1 (JNK inhibitor) (p=0.2 and 0.3, respectively). The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible but with a much slower rate. Western blotting analysis showed that SR13668 abolished phosphorylation at Ser473 and Thr308, while LY294002 and U0126 abolished AKT phosphorylation at Thr 308 but down–regulated it at Ser473. All three of them down–regulated FKHRL1 phosphorylation at Thr32. In contrast, U0126 abolished, LY294002 down–regulated, while SR13668, SB203580 and JNK inhibitor 1 did not affect MAPK p42/44 phosphorylation. Conclusions:Both survival signaling pathway mediated by AKT–FKHRL1 and mitogenic pathways mediated by p42/44, but not by p38 and JNK, MAPK are involved in ex vivo expansion of human limbal epithelial progenitor cells on AM.
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