Construction of a Feline Amniotic Membrane Scaffold Loaded with Limbal Stem Cell Grafts

Amniotic membranes are extensively used in feline corneal repair; however, challenges such as rapid degradation and difficulties with preservation across species limit their clinical use. The aim of this study was to develop a feline amniotic membrane-based graft with stem cell activity and provide experimental evidence for its potential use as a corneal epithelial transplant material in cats. FLSCs were first treated with different concentrations of epidermal growth factor (EGF), and 0.5 mg/L EGF was identified as the optimal concentration for promoting cellular activity. Under this condition, FLSCs exhibited enhanced proliferation and wound closure ability, with complete scratch closure observed approximately 24 h earlier than that in the control group. Growth curve analysis showed that FLSCs maintained stable proliferation characteristics, with a calculated population doubling time of approximately 25.2 h. Subsequently, FLSCs were seeded onto the lyophilized feline amniotic membrane scaffold. Light microscopy, scanning electron microscopy, and transmission electron microscopy demonstrated that FLSCs adhered to and proliferated on the scaffold, forming cell-matrix interaction structures, including hemidesmosome-like junctions. Immunofluorescence staining confirmed the expression of stem cell-associated markers ABCG2 and p63 after scaffold culture. These findings demonstrate the successful fabrication of a feline FLSC-loaded amniotic membrane construct in which FLSCs retained the expression of the examined limbal/stem cell-associated markers after scaffold culture. The successfully constructed FLSC-loaded feline amniotic membrane graft demonstrated considerable potential as a novel bioengineered tissue substitute for corneal surface transplantation in clinical applications.