Retinal Glia Differentiation Mediated by LIF in Cooperation with BMP2

Abstract

Abstract: : Purpose:The cell fate of retinal precursor cells (RPCs) is regulated by various mediators during development. This study is conducted in an attempt to clarify roles of cytokines such as LIF and BMP in the determination of the fate of RPCs. Methods:Neural retina from mice on the day of birth (P0) were isolated and dissociated into single cells. Cells were cultured in N2/DMEM/bFGF/EGF medium. The cells proliferated individually and formed colonies (retinal spheres). After 4 days of culture, retinal spheres were dissociated and replaced on Chamber Slides (Nunc) at a density of 2x10⁵ cells per well in N2/DMEM/bFGF medium. The medium supplemented with LIF (80ng/ml), BMP2 (80ng/ml), or both cytokines was used for the analysis of cytokine effect. After 2 days, cells were fixed and analyzed by immunohistochemistry. Expression of LIF, BMP2 and respective receptor molecules in RPCs was examined by RT-PCR. Results:In the control culture without LIF or BMP2, or the culture with either LIF or BMP2 alone, only a small percentage of RPCs expressed GFAP. In contrast, in the culture with both LIF and BMP2, the proportion of GFAP-positive cells was significantly higher than that in the above three culture, suggesting preferential differentiation of RPCs into glial cells. On the other hand, the proportion of ß III-tubulin positive cells remained unchanged. Conclusions: We show in this study that LIF and BMP2 synergistically induce glial differentiation of RPCs in vitro . Because these cytokines and related protein mediators have been shown to be up-regulated in injured retinas, our results may be of importance when considering clinical relevance for retinal regenerative therapy.

Keywords: retinal development • retinal glia • retinal culture