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Signaling-Transduction Pathways Required for Ex Vivo Expansion of Human Limbal Explants on Intact Amniotic Membrane

¹From TissueTech, Inc. and Ocular Surface Center, Miami, Florida; and ²SRI International, Menlo Park, California.

PURPOSE. Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways.

METHODS. The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis.

RESULTS. In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached 80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 µM LY294002 or 50 µM SR13668 and was significantly suppressed by 10 µM U0126, but was not affected by 10 µM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion.

CONCLUSIONS. Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.