Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers.

Abstract

Purpose : To locate and identify novel stem cell markers of the cornea using a transgenic murine approach.

Methods : We used double transgenic K5Tta x TRE-H2BGFP mice to detect slow cycling cells. In these mice the histone H2B-green fluorescence protein (H2B-GFP) is turned off when animals are fed with doxycycline. As the animal ages the fast dividing cells will loose the GFP expression and only those slow cycling cells will retain H2B-GFP labeling. Corneal wholemounts were imaged from K5Tta x TRE-H2BGFP mice after chase periods of 10, 20, 28, 53 and 91 days. Corneal epithelial cells were isolated and FACS used to separate GFP positive or negative for RNA Seq analysis at 28, 42 and 91 chase time points. Immunofluorescence staining for significantly up regulated genes in the GFP+ cells were performed in corneal sections derived from the 35 and 53 days chase period.

Results : After the doxycycline treatment, we observed a gradual decrease of GFP staining in the cornea. After a chase period of 28 days, few cells were visible in the central cornea and a prominent band of GFP+ cells was found around the periphery including the limbus. At 53 days chase onwards fewer GFP+ cells were localized at the limbus and even less after 91 days. High throughput sequencing from the chase periods 28, 53 and 91 days yielded 487, 2341 and 2369 differential genes expression (2 fold or higher) when comparing GFP+ versus GFP- cells. The expression level of some of these genes were determined by immunofluorescence in WT (Sox9 Fzd7, Col6a, Actn1, Anx3a, Kr17) and in the transgenic model (Krt15, P63, Sox9, Actn1) murine corneas.

Conclusions : The K5Tta x TRE-H2BGFP mouse model system is able to localize slow cycling GFP+ cells to the limbal region where stem cells reside thus, enabling an easy way to FACS sort cells from a stem cell rich zone for further analysis by high throughput sequencing to obtain a wide array of differentially expressed genes. Our analysis has provided us with a wide array of novel genes that we are investigating as potential stem cell markers of the cornea.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.