The 3T3 feeder layer cells are commonly used for stem cell maintenance and expansion. Although feeder free systems are recently being developed for embryonic stem cells culture,feeder base culture system as therapeutic corneal regeneration are still important.In this study, the capability of unrestricted somatic stem cells (USSCs) isolated from umbilical cord blood, is investigated.

Human limbal stem cells were compared between mouse 3T3 fibroblasts, and co-cultured of human USSCs . Cell morphology was monitored by flowcytometry. Furthermore stem cells characteristics were assessed by Immunocytochemistry, real-time PCR for p63, KRT19, KRT3, KRT12 and ABCG2, stem cell markers. Moreover , in order to study isolated cells, colony-forming efficiency (CFE) assay and chromosomal karyotype analysis were performed.

Cord blood somatic stem cells with spindle-shaped morphology can be quickly split up.Real-time PCR analysis and immunostaining showed that more cells at all levels were found to stain positive for ABCG2, p63 and KRT19. Limbal stem cells grown on a USSC feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. Karyotype analysis of USSCs and limbal stem cells represented a normal 46XX karyotype.

USSC provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.