Porcine limbus as a model for studying the human limbus – results from clonal analysis

Abstract

Purpose : Understanding of the role of the niche in regulating human limbal epithelial stem cell (hLESC) function has significant therapeutic implications. The challenge of obtaining fresh human limbal tissue highlights the need for an alternative animal model of the limbus. To further validate the use of ex vivo porcine tissue as an experimental model of the human limbus, clonal analysis of limbal and central corneal epithelial cells was performed. It was hypothesised that as in humans, epithelial stem cells are enriched in the limbus of the porcine cornea.

Methods : Pig eyes were collected from an abattoir and transported back to the lab on ice to be processed the same day. Single cell suspensions from the limbal and central corneal epithelium of 3 separate eyes were isolated and cultured on a feeder layer of growth-arrested murine fibroblasts for 1 week. Colonies selected at random were isolated and cultured for 12 days, before plates were fixed and stained with 1% Rhodamine. Clones were classified as holo-, mero-, or paraclones according to the proportion of aborted colonies. The expression of putative hLESC markers by the limbal and central corneal epithelium was also explored in frozen sections of porcine cornea.

Results : 60 limbal and 60 central corneal epithelial clones were isolated and characterised. All three colony types were present among both the limbal and central corneal epithelial cell populations. A much higher proportion of limbal epithelial clones were classed as holoclones (38.3% vs. 8.3%). Putative hLESC markers cytokeratin (CK) 15 and p63 were primarily expressed in the porcine basal limbus, while CK3, a marker of epithelial differentiation, was absent in these cells.

Conclusions : The limbus represents the primary source of holoclones in the porcine corneal epithelium, and hLESC markers are enriched in the limbus, suggesting that this is the source of corneal epithelial stem cells in this species. The results of this analysis supports the use of porcine tissue in the study of the human limbal niche.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Holoclones, meroclones, and paraclones were identified among cells isolated from the limbal and central corneal epithelium, with more holoclones present in the limbus. Significant at the 0.01% level, using Fisher’s exact test.

 

Cryosections of limbal tissue were stained with anti-p63 and anti-CK3/15. p63 was expressed in the basal limbus, and staining overlapped with CK15, but not CK3. scalebar=50μm.