Live imaging of limbal niche in Limbal Stem Cell Deficiency patients

Abstract

Purpose: We have shown by in vitro confocal microscopy that unique clusters of CD90 and CD105 positive Mesenchymal Stem Cells (MSCs) in the anterior limbal stroma are a component of limbal niche. The present study aims to identify this limbal niche by live imaging of limbal stroma in healthy subjects and to evaluate whether it is altered in limbal stem cell deficiency (LSCD) patients.

Methods: In vivo confocal microscopic scanning was performed using the HRT3 with Rostock Corneal Module (Heidelberg Engineering, Germany) in partial and total LSCD patients (11 eyes of 6 patients), macular corneal dystrophy patients (3 eyes of 3 patients) and healthy volunteers (30 eyes of 17 individuals) after obtaining their informed consent. A minimum of 5 volume scans were taken in the limbus from epithelium to the deepest stromal level at which structures could be resolved. Mean depth was measured using the IVCM built-in pachymetry from three volume scans.

Results: In concordance with the location and characteristics of MSCs in anterior limbal stroma, clusters of bright hyper-reflective cellular structures subjacent to the basal epithelium were observed at a mean depth of 58.4±1.4µm in normal subjects. They showed a unique morphology compared to neighbouring epithelial cells and keratocytes, and a different branching pattern with respect to neurons and dendritic cells in this region. Similar microstructure was also observed in macular corneal dystrophy patients having corneal stromal opacity but normal corneal and limbal epithelium. However, such hyper reflective structure was not found in LSCD patients indicating the loss of limbal niche in addition to the damage of palisades of Vogt. Other changes were the presence of infiltrating cells, enlarged blood vessels and increase in dendritic cells in the limbal stroma. In partial LSCD, the hyper reflective niche was present in the unaffected limbal region in contrast to the damaged area.

Conclusions: Live imaging of limbal stroma along with epithelium offers a non-invasive approach to the diagnosis and management of limbal stem cell deficiencies. The present study indicates the potential need to provide limbal niche in addition to epithelial stem cell grafting in LSCD patients.

Keywords: 484 cornea: stroma and keratocytes • 551 imaging/image analysis: non-clinical • 596 microscopy: confocal/tunneling