Effects of TGF-β1, BMP 2/4 and their antagonists on limbal stem cell properties

Abstract

Stem cells maintenance and function in vivo are controlled by various factors provided by its unique local microenvironment or niche. Although the concept that corneal epithelial stem cells reside mainly in the limbus region has been widely accepted, the molecular mechanism how limbal niche signals govern limbal epithelium stem cells (LESCs) behavior during tissue homeostasis and corneal injury is not well understood. Here we showed that transforming growth factor beta (TGF-β), one of the most important growth factors in corneal wound healing triggered epithelial-to-mesenchymal transition change in LESCs resulted in cells adopted migratory phenotype and lost capability to generate corneal epithelial cells. Inhibition of TGF-β signaling by SB431542, in contrast, promoted stem cell maintenance. TGF-β1 also stimulated expression of BMP antagonists, especially noggin, in limbal stromal fibroblasts and limbal epithelium. To analyze the effect of niche derived BMP signals, we compared the capability of regular 3T3, 3T3 overexpressing BMP4 and noggin feeder systems in maintaining LESCs and studied how each feeder type affected gene expression profile of limbal epithelium. Our data demonstrated that 3T3-BMP4 had superior capability in maintaining epithelial progenitor phenotype, LESCs proliferation and increasing colony forming efficiency (CFE) compared with control 3T3. Although 3T3-Noggin increased clonogenic potential of primary LESCs, it partially promoted EMT change resulted in a marked reduction in their ability to generate epithelial cells upon serial passages. The effect of BMP4 in preventing EMT change and promoting epithelial phenotype maintenance probably be mediated by transcription factors Id-1 and Id-2, inhibitors of differentiation. Our data suggested that modulating niche signals could eventually lead to a way to improve the method for ex vivo expansion of LESCs for therapy.