From Embryo to Eye – Human Embryonic Stem Cell Markers OCT–4 and NANOG are Localised to Adult Limbal Epithelium

Abstract

Abstract: : Purpose:Transcription factors OCT–4 and NANOG are expressed by human embryonic stem (ES) cells and are downregulated as these pluripotent cells differentiate. Because the expression of ES cell markers has not been investigated in the limbal epithelium, the niche for the limbal stem cell (LSC), we decided to study the expression of these transcriptions factors in adult human limbal epithelium. Methods:Adult human limbal rings (ages 47–78 years) donated for research were obtained from the UK Transplant Service. Immunohistochemistry for OCT–4 and in situ hybridisation for NANOG were performed on paraffin embedded sections of these limbal rings (which included limbal and peripheral corneal epithelium). In addition, OCT–4 expression in epithelial cells isolated from the limbal rings was quantified using flow cytometry. Epithelial cells isolated from these limbal rings were also cultured on an irradiated 3T3 mouse fibroblast feeder layer for 7 days, prior to confluence of the limbal epithelial colonies. RNA was extracted from these colonies and from the 3T3 fibroblast feeder cells. Reverse transcription – polymerase chain reactions (RT–PCRs) were used to study the expression of OCT–4 and NANOG in these RNA extracts. Results:Immunohistochemistry for OCT–4 on paraffin embedded sections revealed high levels of expression in the limbal epithelium (particularly in the basal layer) and faint expression in the peripheral corneal epithelium. In situ hybridisation for NANOG identified occasional expression by cells in the basal layer of the limbal epithelium and no expression in the peripheral corneal epithelium. Flow cytometric analysis of 1,200 epithelial cells isolated from the limbal rings identified 25.6% of the cells expressing OCT–4. RT–PCRs revealed OCT–4 and NANOG expression in the cultured limbal epithelial cells but not in the 3T3 fibroblasts. Conclusions:OCT–4 and NANOG are markers used to isolate ES cells from their differentiated progeny. Our research has identified the expression of both these markers in the basal layers of the limbal epithelium, the exact site of the LSC. Moreover, we have shown that the expression of these markers of pluripotency is maintained in culture. The possibility of LSC pluripotency and more specific localisation of OCT–4 and NANOG to LSCs isolated by culture methods will require further evaluation.

Keywords: cornea: epithelium • gene/expression • immunohistochemistry