The Neural Differentiation Potential of Limbal Stem Cells: A Model for Studying the Multipotency of Limbal Stem Cells.

Abstract

To investigate the multipotency, especially the neural differentiation potential, of limbal stem cells (LSCs) using a coculture system and to provide a relevant literature review. Limbal tissue was harvested from adult New Zealand white rabbits and treated with collagenase A. Small pieces of the resulting limbal epithelial sheets were cocultured with a neuroblastoma cell line (Neuro-2A) in transwells. Morphological observation of the growing epithelial sheets was accomplished by microscopy, and marker expression was detected by immunocytochemistry for ßIII-tubulin and microtubule-associated protein 2. A literature review of associated studies was performed. In the coculture group, directly adherent colonies of neuron-like (DACN) cells were observed among the growing limbal epithelial sheets from day 3. The DACN cells exhibited neuron-like morphology. The control group comprising limbal cell sheets cultured alone showed a very small number of DACN cells at the end of the culture period (day 14). Immunocytochemical staining revealed that the DACN cells were positive for ßIII-tubulin and microtubule-associated protein 2, confirming the neuronal phenotype of the neuron-like cells. By contrast, the DACN cells in the control group produced negative results. In previous reports, LSCs and niches exhibited neural potential, but most differentiated neural cells were observed as floating spheres, in contrast to the DACN cells observed in the present study. We developed a coculture system of LSCs and Neuro-2A neuroblastoma cells and obtained DACN cells with neural differentiation potential. Our findings confirm the neural potential of LSCs, consistent with previous reports, but in a form other than floating spheres.