Retinoic acid promotes conjunctival epithelium differentiation and goblet cell regeneration: evidence from novel 3D conjunctival organoids and whole-mount PAS staining

Abstract

Purpose

To investigate the differentiation effects of retinoic acid on primary conjunctival epithelium using both 2D and 3D models, and to evaluate its in vivo effects on conjunctival epithelium and goblet cells in mice using a novel goblet cell assessment method.

Methods

The differentiation effects of retinoic acid were evaluated in vitro using 2D culture and 3D organoid models. Under 2D conditions, differentiation was assessed using qRT-PCR, light microscopy, immunofluorescence staining, and Western blot analysis for goblet cell markers. In the 3D organoid model, differentiation was confirmed using qRT-PCR, immunofluorescence staining, and AB-PAS staining. In vivo, a novel goblet cell assessment method—whole-mount PAS staining—was introduced, along with H&E staining to evaluate the effects of retinoic acid eye drops.

Results

In the 2D culture model, retinoic acid induced cell fusion, decreased stemness marker expression, and increased goblet cell differentiation markers. In the 3D organoid model, retinoic acid treatment led to elevated expression of goblet cell markers, including Muc5ac, Tff1, and Gcnt3, as confirmed by qRT-PCR, AB-PAS staining, and immunofluorescence. In vivo, retinoic acid eye drops promoted goblet cell generation, as demonstrated by the novel assessment method.

Conclusions

Retinoic acid promotes conjunctival epithelial differentiation and goblet cell regeneration both in vitro and in vivo. A novel method for goblet cell detection is proposed, providing a more accurate and reliable approach for evaluating conjunctival goblet cells in future research.