Abstract
Limbal epithelial stem cells (LESCs) are essential to maintain the transparent ocular surface required for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. In the present study we have used in vitro clonal analysis to confirm that limbal crypts provide a niche for the resident LESCs. We have used high-resolution imaging of the basal epithelial layer at the limbus to identify cells with a morphology consistent with stem cells that were only present within the basal layer of the limbal crypts. These cells are proximal to limbal stromal cells suggesting direct cell-to-cell interaction. Serial block-face scanning electron microscopy (SBFSEM) confirmed that the putative LESCs are indeed in direct contact with cells in the underlying stroma, a contact that is facilitated by focal basement membrane interruptions. Limbal mesenchymal cells previously identified in the human limbus collocate in the crypt-rich limbal stromal area in the vicinity of LESCs and may be involved in the cell-to-cell contact revealed by SBFSEM. We also observed a high population of melanocytes within the basal layer of the limbal crypts. From these observations we present a three dimensional reconstruction of the LESC niche in which the stem cell is closely associated and maintained by both dendritic pigmented limbal melanocytes and elongated limbal stromal cells.
Highlights
The cornea, the transparent tissue located at the front of the eye, is a highly specialized tissue that transmits and refracts light onto the retina
Among three human donors and 124 clones analyzed, cells isolated from the limbal crypts’ (LCs) were able to generate the highest proportion of holoclones (17.74%, 11 holoclones among 62 clones) compared to Limbal epithelial cells (LECs) isolated from the non crypt-rich limbus in which only one holoclone was observed among 62 clones analyzed (1.61%)
In the present study we investigated whether limbal mesenchymal cells were involved in the interaction with putative Limbal epithelial stem cells (LESCs) that we revealed by Serial block-face scanning electron microscopy (SBFSEM)
Summary
The cornea, the transparent tissue located at the front of the eye, is a highly specialized tissue that transmits and refracts light onto the retina. LESCs are maintained and concentrated in a stem cell niche where they have the ability to self renew and to preserve their multipotency [3]. Serial histological sectioning revealed ‘distinct anatomical extensions from the peripheral aspect of the limbal palisades’ These were termed ‘limbal epithelial crypts’ and were proposed as a putative LESC niche owing to the presence of cells expressing putative stem cell markers including ABCG2 [17]. In 2007, Shortt et al described epithelial cell filled crypts between the limbal palisades of Vogt corresponding to the ‘interpalisadal epithelial rete ridges’ described by Goldberg and Bron [18]. LCs that were more often observed at the superior and inferior human limbus could not be detected in patients affected by LESC deficiency [19]
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