Three-dimensional structure of the mammalian limbal stem cell niche

Abstract

Although the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its three-dimensional (3D) architecture remains incomplete. The LSC niche was explored on freshly excised and organ-cultured corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full-field optical coherence microscopy (FFOCM). Limbal crypt features were detected in 90% of organ-cultured human corneoscleral rims, extending between the palisades of Vogt as radially oriented rectangular (74% of eyes) and/or rounded (23% of eyes) forms, often branching off to, or becoming interconnected by, sub-scleral radially or circumferentially oriented crypts (in 56% of eyes). Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss-crossing invaginations beneath the scleral surface. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of sampled limbal volume in humans and 9% in pigs. FFOCM combined with fluorescence, and confocal fluorescence microscopy, showed presence of p63-α+ cells and cytokeratin-3+ cells in the limbal crypts. To assess colony forming efficiency (CFE), limbal epithelial cells were cultured at low density with mitomycin-arrested 3T3 feeders. CFE increased with limbal crypt volume and was not significantly decreased in organ-cultured cornea, despite degradation of the epithelial roof, suggesting that stem cells remain protected at the base of crypts during organ culture. CFE in human samples was significantly greater than in pig, and CFE in mouse was zero. Crypt architecture in the three species appears associated with eye exposure to light. LSC density increased with percentage limbal volume occupied by crypts.

The Corneal Epithelial Barrier and Its Developmental Role in Isolating Corneal Epithelial and Conjunctival Cells From One Another

“…The palisades of Vogt have been used by many other studies for determining the location of the junction between the corneal and conjunctival epithelium. 17,18 In sections of DiI-labeled corneas, we observed that the fluorescent label terminated within this transitional area. As shown in Figure 2B, the DiI label terminates within the epithelial layer (arrow), just lateral to the end of Bowman's layer (arrowhead and dashed line).…”

Identification for Differential Localization of Putative Corneal Epithelial Stem Cells in Mouse and Human

“…The thickness of limbal epithelial layer is about 20 µm in both strains, which is account for 50 or 37% of thickness of central corneal epithelium in C57BL/6 or BALB/c mice, respectively 34 . The findings were supported with full-field optical coherence microscopy by Grieve et al ., who detected the palisades of Vogt with limbal crypt features in organ-cultured human corneoscleral rims, but not in fresh mouse cornea, where the limbal region contained only an epithelial trough circumferentially surrounding the cornea 35 .…”

En face coherence microscopy [Invited]

“…Moreover, using high NA objectives enabled us to rely on both the spatial optical sectioning, and the temporal optical sectioning [3]. Using this setup, structures invisible in FF-OCM were revealed [93,94]. With this multimodal setup, we have been able to add specific contrast to the structural FF-OCT image: Fig.…”