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Kate Grieve, Djida Ghoubay, Cristina Georgeon, Nacim Bouheraoua, Michel Paques, Vincent M Borderie; Three-dimensional structure of the mammalian limbal stem cell niche. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5827.
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© ARVO (1962-2015); The Authors (2016-present)
The three-dimensional (3D) architecture of the limbal stem cell niche was explored on unfixed whole cadaveric corneoscleral rims from human donors, pigs and mice.
Full-field optical coherence microscopy (FFOCM) was used to image 43 cadaveric human, 10 pig and 14 mouse corneoscleral rims. FFOCM enables non invasive 1µm resolution 3D imaging in unfixed, unsliced tissues. 3D image stacks were analyzed to describe and quantify the limbal crypt network. Following imaging, the limbal epithelium was enzymatically dissociated and cells were cultured at low density with mitomycin-arrested 3T3 feeders to assess colony forming efficiency (CFE). CFE was correlated with crypt-richness, and limbal crypts were imaged with confocal microscopy following the addition of fluorescent markers in order to confirm the presence of stem cells.
In humans, limbal crypts were detected in 94 % of corneoscleral rims. A network of radial crypts was observed at the limbus in 72 % of the human samples, extending between the Vogt palisades into the cornea, and linked in 59% by a non-continuous circumferential crypt ring extending beneath the scleral surface. Rounded or flower-shaped crypts were also detected in 36% of samples. The radial crypts began at the corneal surface in 82%, while light-bulb shaped caverns extended lower into the sclera in 46%, with these caverns being closed off beneath the sclera in 30%. Average crypt depth in humans was 50µm (range 15 µm-100 µm). Crypts were located on two opposing sides in 30%, two neighboring sides in 19%, 3 or 4 sides in 21% and only one side in 25%. In pigs, crypts were located in the sclera rather than extending into the cornea, and were relatively uniformly distributed over 360° as well as having similar depth (30µm) and width (60µm), and all of a radial orientation over a 1.5mm wide limbal zone. Palisades in pig were very fine and crypts large in comparison to human where crypts and palisades were of similar width. In mouse, only the circumferential crypt ring was detected. Colonies grown from dissociated limbal epithelial cells were associated with areas visibly rich in crypts, and fluorescent labeling confirmed presence of stem cells.
While a similar crypt network was detected in all corneas, its visibility in terms of number and depth of crypts was highly variable from one cornea to the next. Limbal crypt architecture differed from species to species.
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