Regeneration of the Corneal Epithelium in the Animal Model of Limbal Stem Cell Deficiency (LSCD) Using the USSC Seeded on Amniotic Membrane

Abstract

The loss of limbal stem cells (LSCs) results in the replacement of corneal epithelial cells with conjunctival epithelial cells, decreasing the usual clarity of the cornea and leading to impaired healing, scarring and vision loss. In this study, we evaluated the beneficial effects of Unrestricted somatic stem cells (USSC) from human cord blood (CB) seeded on the amniotic membrane in rabbits with limbal stem cell deficiency (LSCD) with corneal epithelium wounds. Accordingly, USSCs and LSCs were derived from human CB and limbus tissue. Flowcytometrymetry and karyotype are used to characterize USSCs andchromosomes'chromosomes and numbers. Then, such cells were seeded on the amniotic membrane (AM). The viability of USSCs and LSCs on AM was evaluated by MTT assay within 24, 48 and 72 hours of treatment.Further, the expression of epithelial markers, such as CK3, CK12 and P63, was evaluated using Real-time PCR and immunohistochemistry (IHC) to evaluate the differentiation of cells seeded on AM to corneal epithelium. Further, photographic images in rabbit models of LSCD monitored the healing of corneal epithelium wounds. Based on the analysis, AM could promote cell viability in all periods. Also, increased expression of CK3, CK12 and P63 genes and morphological analysis verified the efficient differentiation of USSCs to corneal epithelium cells. Also, photographic images of rabbit eyes verified the wound healing process in AM and USSCs seeded on AM compared with the control group. In conclusion, we found that transplantation of USSCs seeded on AM could be an effective, innovative therapy to alleviate corneal epithelium wounds in animal models of LSCD.