Regenerative Medicine in Ocular Surface Reconstruction: Advancing Cell-Based Therapies for Limbal Stem Cell Deficiency

Limbal stem cell deficiency is a disease caused by the impairment of limbal epithelial stem cells (LESCs), leading to the replacement of the corneal surface with nontransparent conjunctiva, and its treatment is not standardly available worldwide. This work focuses on preparing cells for treating both uni- and bilateral forms of the disease using advanced cell- based therapy. It investigates LESCs in various scaffolds (fibrin, nanofibers) and explores non-limbal cell sources like oral mucosal epithelial cells (OMECs) under standard complex and xenobiotic-free culture conditions. The study uses immunofluorescence and gene expression to detect stem cell markers, proliferation, and differentiation capacity of cultured cells. Moreover, long-term OMECs storage with different media and cryoprotective agents and the healing properties of the amniotic membrane (AM) were also examined. We found that adding interleukin-13 to the culture media enhanced LESCs' stemness. LESCs on fibrin gels showed higher expression of stemness markers, while those on polymers expressed more mesenchymal ones. Using standard and xeno-free media, we successfully prepared OMEC-containing cell sheets on fibrin gel substrates. The cultured cells exhibited high expression of stemness genes (ΔNp63α, NGFR, KLF4) and decreased levels of...