Putative stem cell markers in limbal epithelial cells cultured on intact & denuded human amniotic membrane.

ABSTRACT

BACKGROUND & OBJECTIVES: The ocular surface is an ideal region to study the epithelial stem cell (SC) biology because of the unique spatial arrangement of stem cells and transient amplifying cells. A major challenge in corneal SC biology is the ability to identify SC in vitro and in situ, and one of the major controversies in the field relates to reliable SC markers. This study was carried out to evaluate and compare the expression of the stem cell associated marker ABCG2, keratinocyte stem cell marker p63 and corneal differentiation markers Cnx43 and K3/K12 on limbal explants cultured on human amniotic membrane (HAM) with intact epithelium and HAM denuded of its epithelium. METHODS: Human limbal biopsies obtained from the cadaveric donor eyes were used in this study. The cells were cultured over the HAM with intact and denuded epithelium. Reverse transcriptase PCR, immunohistochemistry, Western blotting for ABCG2, P63, Cnx43 and K3/K12 were done. RESULTS: The limbal epithelial cells cultured over intact HAM expressed the stem cell associated markers (ABCG2, p63) and showed reduced expression of the differentiation markers (Cnx43 and K3/K12) when compared to limbal epithelial cells cultured over denuded HAM, which expressed more differentiation markers at the end of three weeks. BrdU label retaining cells were observed in the limbal epithelial cells cultured over HAM with epihelium only. INTERPRETATION & CONCLUSIONS: Our results showed that the intact HAM supported the growth of limbal epithelial cells expressing stem cell associated markers, and allowing little differentiation of the limbal cells to cornea phenotype. Further studies are needed to understand the properties of the amniotic epithelium that retains the stemness in the cultured limbal stem cells.