Trophic factors and neuronal interactions regulate the cell cycle and Pax6 expression in Müller stem cells

The finding that Müller cells have stem cell properties in the retina has led to the hypothesis that they might be a source for replacing neurons lost in neurodegenerative diseases. However, utilization of Müller cells for regenerative purposes in the mammalian eye still requires identifying those factors that regulate their multipotentiality and proliferation. In addition, because Pax6 expression is indispensable for eye development, its regulation would be required during regeneration. In the present study we investigated the regulation of cell-cycle progression and Pax6 expression in pure Müller glial cell cultures and neuroglial cocultures from rat retinas. At early times in vitro, glial cells showed high expression of Pax6 and of nestin, a stem cell marker, and of markers of cell-cycle progression; expression of these markers decreased during development in parallel with increased glial differentiation. The addition of glial-derived neurotrophic factor, basic fibroblast growth factor, and insulin restored proliferation and also Pax6 and nestin expression in glial cells. Noteworthy, in neuroglial cocultures Müller cells retained Pax6 expression for longer periods, and, in turn, neuronal progenitors preserved their proliferative potential for several days in vitro. This suggests that neuroglial interactions mutually regulate their mitogenic capacity. In addition, in glial secondary cultures incubated with insulin, many neuroblast-like cells expressed the neuronal marker NeuN. Our results suggest that the proliferative capacity and the features of eye stem cells of Müller glial cells are regulated by molecular and cellular factors, which might then provide potential tools for manipulating retinal regeneration.