Objectives: In a previous study, we have reported the existence of neural crest-derived stem cell-like cells originating from the corneal limbus of juvenile mice (termed murine corneal cells, MCCs). To yield a sufficient number of MCCs, for example, for cell-therapy approaches, here we have investigated MCCs' ability for extensive proliferation, and we have evaluated their stem cell qualities and genetic stability after large-scale culture.
Materials and methods: MCCs were established from corneal limbal tissue of juvenile mice. To determine their cell proliferation and self-renewing potential, MTT tests and an estimation of colony forming unit efficiency were carried out. Multipotency of cell differentiation was examined by applying adipogenic and osteogenic differentiation protocols. Moreover, karyotyping was performed and expression of stem cell markers and cell cycle-associated genes was analysed.
Results: MCCs, as primary cells, could be cultured for more than 60 passages. We observed increased cell proliferation and high number of colony forming units (CFUs) after extensive culture. Interestingly, there were no changes in expression of MCC markers. Furthermore, cell differentiation potentials remained comparable with MCCs at early passages. However, karyotyping revealed numeric chromosomal aberrations at higher passages. Moreover, tumour suppressor genes such as p16 and p21 were found to be down-regulated after large-scale cell culture.
Conclusions: MCCs immortalize spontaneously after extensive cell culture, but still demonstrate stem cell-like qualities.