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Ursula Schlotzer-Schrehardt, Matthias Zenkel, Angelika Moessner, Johannes Menzel-Severing, Friedrich E. Kruse; Primary Cilia Are Involved In Sonic Hedgehog Signaling In Limbal Epithelial Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1711.
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© ARVO (1962-2015); The Authors (2016-present)
The sonic hedgehog (Shh) pathway has been shown to play a critical role in stem cell maintenance, proliferation, and lineage commitment in various tissues and to be associated with primary cilia. In search for mechanisms regulating corneal epithelial homeostasis, we investigated the expression of primary cilia and Shh signaling components in limbal epithelial progenitor cells in situ and in vitro.
The presence of primary cilia was determined in corneo-limbal specimens obtained from human donor eyes using acetylated alpha-tubulin immunohistochemistry and electron microscopy. Protein expression of Shh pathway components was assessed by light and electron microscopic immunohistochemistry. mRNA expression was analyzed after laser capture microdissection of basal limbal and corneal epithelial cell clusters and pre-amplification of RNA using the Human Hedgehog TaqMan Array (Applied Biosystems) and confirmed by specific real-time PCR assays. Isolated human limbal epithelial stem cells were expanded on a 3T3 feeder layer and cell proliferation was assessed by BrdU assay after stimulation with recombinant Shh.
Primary cilia, projecting from the lateral cell surface into the extracellular space, were detected in basal epithelial cell clusters at the limbus by electron microscopy and immunohistochemistry. Cilia-bearing cells were identified as undifferentiated, transient amplifying, growth-arrested progenitor cells by co-expression of alpha-tubulin with Oct-4, K15, and p63alpha, and lack of Ki67. Essential components of the Shh pathway, i.e. Patched-1 (Ptch1), Smoothened (Smo), and Suppressor of Fused (SuFu) could be localized to primary cilia to a variable extent. PCR-Array analysis revealed increased transcript levels of SuFu, a negative regulator of Shh signaling, in limbal clusters compared to corneal cells in the absence of Shh signaling. Shh pathway activation during limbal stem cell expansion in vitro resulted in decreased levels of SuFu and increased levels of Ptch1, Smo, and Gli transcription factors as well as a significant enhancement of proliferation.
The findings are consistent with a key role of the Shh pathway, mediated through primary cilia, in controlling limbal epithelial progenitor cell proliferation and quiescence, which might be useful for tissue engineering strategies.
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