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Greg M Hammond, Robert D Young, Bruce Caterson, Andrew J Quantock; Localising potential regulators of the corneal limbal stem cell niche. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3858. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Chondroitin sulphate (CS) glycosaminoglycans are believed to act as regulators of stem cells in developing and adult tissues, but have not yet been investigated in relation to corneal limbal epithelial stem cells. We tested the hypothesis that various CS epitopes will be present in the putative limbal stem cell niche, and will have different distributions in the superior/inferior (S/I) and nasal/temporal (N/T) areas of the limbus, using a porcine experimental model.
S/I and N/T segments of porcine limbal tissue were frozen in optimal cutting temperature compound, cryosectioned and stained with monoclonal antibodies to various CS epitopes associated with stem/progenitor cells in other tissues. Antibodies to putative stem cell and transient amplifying cell markers were also applied. Alexa Fluor® 488- or 594-conjugated secondary antibodies were applied after this. Tissue sections without primary antibodies or with mouse/rabbit immunoglobulins were used as controls. The tissue was then imaged using epifluorescence microscopy.
Invaginations of the limbal epithelium, previously identified as analogous to human limbal crypts, were observed in all sections- though they were less pronounced in N/T sections. Significant staining with antibody 6C3 was observed in the extracellular matrix subjacent to the invaginations in both S/I and N/T tissues sections. 7D4 staining was observed immediately subjacent to the anterior limbal epithelium in S/I sections only. No significant 3B3[-] or 4C3 staining was observed. There was no staining for putative stem cell markers p63 and ABCG2. Vimentin staining was widespread in the stroma and co-localised with 6C3 staining in the subepithelial stroma.
The results showed that the CS epitope recognised by antibody 6C3 is associated with the putative limbal stem cell niche of porcine eyes- the third mammalian species to demonstrate this. There is no significant difference in CS epitope distribution between the horizontal and vertical meridians of the porcine eye, which is suggestive of its widespread distribution around the entire circumference of the porcine limbus. Further immunohistochemical analysis, including of human limbus, is required to determine if CS associates specifically with limbal stem/progenitor cell populations.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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