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M. Notara, A. J. Shortt, A. R. Harris, S. Schrader, J. T. Daniels; IL6 and the Human Limbal Stem Cell Niche: A Mediator of Epithelial-Stromal Interaction. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2045.
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© ARVO (1962-2015); The Authors (2016-present)
The limbal niche, habitat of the limbal epithelial stem cells (LESC), facilitates stromal fibroblast and epithelial stem cell interactions via mediators of cell signaling. In this study, the interactions between LESC and stromal fibroblasts were investigated using imaging of the niche ex vivo and a serum-free (SF) co-culture system in vitro.
Ex vivo imaging of the niche was carried out using confocal microscopy. A SF co-culture where human limbal epithelial (HLE) cells could be propagated in the presence of HLF cells when plated in a ratio 3.1, (3.1SF) was used for in vitro studies. Cytokine dialogues between HLF and HLE were assessed using a protein array, immunocytochemistry, RT-PCR and ELISA.
Imaging of the limbus demonstrated close spatial arrangement between HLE cells located in the crypts and fibroblasts in the stroma. The interactions of the HLE cells and mitotically active human limbal fibroblasts (HLF) were studied for the first time in a serum-free in vitro model that simulated a simplified limbal niche microenvironment.HLE cells cultured using this method expressed high levels of the putative stem cell markers ABCG2 and p63. A protein array revealed that interleukin 6 (IL6) is up-regulated in the 3.1SF system when compared to the HLE culture with growth arrested fibroblasts in the presence of serum (Golden Standard culture, GS). Further assessment showed that IL6 expression by HLE cells has been induced in the 3.1SF conditions whereas it was not expressed by them in GS culture. The in vivo expression of IL6 by HLE and HLF was confirmed by immunostaining of human limbal sections. Neutralization of IL6 in 3.1SF cultures reduced HLE colony forming efficiency suggesting a role for IL6 in preservation of HLE stem cell character.
The 3.1SF system facilitated the investigation of potential paracrine regulators in the human limbal niche by using an in vitro model. This approach identified a role for IL6 in this environment and may allow the elucidation of further signalling mechanisms relevant to stem cell function.
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