RSPO3 Promotes Proliferation and Self-Renewal of Limbal Epithelial Stem Cells Through a WNT/β-Catenin-Independent Signaling Pathway

Purpose: R-spondin3 (RSPO3), a mammalian-specific amplifier of Wnt signaling pathway, maintains the homeostasis of various adult stem cells. However, its expression at the limbus and the effect on limbal epithelial stem cells (LESCs) remains unclear. We investigated the impact of RSPO3 on the proliferation and self-renewal of LESCs and explored its molecular mechanisms.

Methods: The expression of four RSPO subtypes at the limbus were detected. Co-cultured with RSPO3 in vitro, the cell outgrowth area and cell density of human LESCs (hLESCs) were measured, along with EdU assay and evaluation of biomarkers of cell proliferation (Ki67) and stemness (△Np63 and ABCG2). The expression of key molecules in Wnt/β-catenin signaling pathway were investigated in RSPO3-co-incubated hLESCs and controls. The effect of RSPO3 on corneal epithelium wound recovery in vivo was investigated in a mouse model of corneal epithelium injury.

Results: Among four subtypes of RSPO protein, only the RSPO3 isoform was stably expressed at the human limbus. RSPO3 promoted the proliferation and stemness maintenance of hLESCs in vitro in a dose-dependent manner when its concentration ≤ 100 ng/mL, and this effect was not impaired when the activation of β-catenin was inhibited by XAV939, indicating that the effect of RSPO3 on hLESCs was not dependent on canonical Wnt/β-catenin signaling pathway. Exogenous RSPO3 accelerated epithelial wound healing by enhancing the proliferation and self-renewal of residual LESCs.

Conclusions: RSPO3 promotes the proliferation and self-renewal of LESCs through a Wnt/β-catenin-independent signaling pathway which might have translational significance in the treatment of corneal epithelium injury and limbal stem cell deficiency.