Rat limbal niche cells prevent epithelial stem/progenitor cells from differentiation and proliferation by inhibiting notch signaling pathway in vitro

Abstract

PURPOSE. Limbal niche cells (LNCs) play a pivotal role in regulating limbal epithelial stem/ progenitor cells (LESCs). This study aimed to investigate whether Notch signaling is involved in LNCs’ regulation of LESCs. METHODS. Rat limbus was digested by dispase and collagenase, respectively. Limbal niche cells were isolated by serial passage of collagenase-digested cells on coated Matrigel in a modified embryonic stem cell medium (MESCM). Dispase-isolated cells, with or without LNCs, were seeded on three-dimensional (3D) Matrigel. The effects of LNCs, Notch inhibition (by N-(N-[3, 5-difluorophenacetyl]-lalanyl)-S-phenylglycine t-butyl ester [DAPT] or Notch1-siRNA) and activation (by Jagged1) on LESCs were analyzed using quantitative RT-PCR, immunostaining, and Western blot. RESULTS. Dispase isolated pan cytokeratin (PCK)þ limbal epithelial cells (LECs). Collagenase isolated subjacent native LNCs, which were purified and expanded with expression of Oct4, Rex1, Sox2, Nanog, SSEA4, N-cadherin, and CD34. Limbal niche cells reunited with p63aþ LESCs to form clusters and prevented their differentiation on 3D Matrigel. Notch signaling was unactivated in rat corneal and limbal epithelium in vivo, but activated in cultured LECs in vitro. Limbal niche cells inhibited the Notch signaling of LECs in culture. Notch inhibition (by DAPT or Notch1-siRNA) increased p63a expression and decreased CK12 expression in LECs to the level of LNCs’ effects. Notch inhibition by DAPT also decreased Ki67 expression in LECs to the level of LNCs’ effects. CONCLUSIONS. Rat LNCs prevent LESCs from differentiation and proliferation primarily via inhibiting the Notch signaling in vitro. Manipulating the Notch signaling pathway may help to preserve LESCs for corneal epithelial tissue engineering.