Amniotic Membrane Stromal Cells Facilitate Corneal Epithelial Tissue Engineering by Promoting Limbal Epithelial Stem Cell Expansion

Purpose: To explore whether amniotic membrane stromal cells (AMSCs) can promote the expansion of limbal stem cells (LSCs) during corneal epithelial tissue engineering. Methods: Rabbit corneal limbal epithelium was cultured on amniotic membrane containing live stromal cells (L-dAM) or amniotic membrane with dead stromal cells (D-dAM). Morphological and microstructural characteristics of in vitro-expanded rabbit limbal epithelial sheets were evaluated using light microscopy and scanning electron microscopy. Stemness properties and differentiation status were assessed through colony forming efficiency (CFE) assays and immunofluorescence staining of ΔNp63α, PAX6, Ki67, and K14. Gene expression in the limbal epithelial sheets was analyzed by quantitative real-time polymerase chain reaction and western blotting. Transcriptomic alterations in LSCs and AMSCs were decoded through RNA sequencing. Intracellular NAD+ dynamics were quantified via liquid chromatography-tandem mass spectrometry/metabolomics and enzymatic NAD+/NADH ratio assays. Results: LSCs cultured on L-dAM exhibited superior tight junction integrity and colony-forming capacity compared with those on D-dAM, with upregulated expression of ΔNp63α, PAX6, Ki67, and K14, alongside downregulated expression of K12. Nicotinamide supplementation in D-dAM cultures restored NAD+-dependent stem cell homeostasis, achieving functional equivalence to L-dAM through complete recovery of CFE and coordinated molecular restitution of stem/progenitor markers with sustained K12 inhibition. Mechanistically, co-cultured AMSCs transferred NAD+ via exosomes to maintain LSC homeostasis, as evidenced by vesicle inhibition causing NAD+ depletion, stemness exhaustion, and accelerated differentiation. Conclusions: AMSCs could promote limbal epithelial stem cell expansion, offering clinical implication for corneal epithelial tissue engineering.