Characterization of the Limbal Epithelial Stem Cell Niche

Limbal epithelial stem cells (LESCs) reside within the LSC niche (LSCN), located at the annular transition zone between the cornea and conjunctiva. LESCs are important for the long-term maintenance of the corneal epithelium and critical for repopulating the corneal epithelium after injury. We have recently identified that a hyaluronan (HA)-rich extracellular matrix (ECM) exists within the LSCN, and that this HA matrix is necessary for maintaining LESCs in the “stem cell” state. Herein we further characterized the composition of the LSCN, identifying key components of the HA-rich matrix. We identified that the hyaladherins IαI, TSG-6 and versican are highly expressed in the limbus when compared to the cornea. For IαI, Heavy chain 5 (HC5) was found to be the most highly expressed HC in the mouse and human cornea, and associates with HA forming HA/HC5 specific matrices. CD44 is the most likely receptor that mediates the interaction between LESCs and the HA-specific matrix. The LSCN is composed of a rich HA matrix that contains HA/HC5. HA/HC5 complexes could be used as an improved substrate for culturing LESCs during ex vivo expansion for limbal stem cell transplantation. To understand the composition of the LSCN we extracted mRNA from dissected corneal buttons and limbal rims of mouse corneas and sequenced them using a NextSeq 500 (Illumina). We then performed gene expression analysis using data obtained from RNA-seq of the two different regions of the cornea. We then performed gene expression analysis of the RNA-seq data of limbus and cornea buttons. The differential expression between cornea and limbus for the identified genes of interest were confirmed by qPCR, Western blotting, and immunohistochemistry.