Identification & Characterization of Neural Stem Cells

In vivo lineage studies have clearly demonstrated the presence of multipotent progenitors in the mammalian retina. We have used the neurosphere culture method to isolate and enrich these cells in vitro, which has allowed us to examine their proliferative and differentiation potential in controlled conditions. For example, we observed that retinal stem cells isolated from an early stage of retinal histogenesis (early retinal stem cells) during which retinal ganglion cells (RGCs), cone photoreceptors, horizontal cells and amacrine cells are born, prefer FGF2 to EGF for proliferation. In contrast, those isolated from the late stage of histogenesis (late retinal stem cells), during which rod photoreceptors, bipolar cells and Muller glia are born, are responsive to EGF. The former preferentially generates neurons, while the latter shows proclivity towards glial differentiation. Such distinctions, which are also displayed by the early and neural stem cells/progenitors in other regions of the developing CNS, represent a stage-specific difference in competence, underpinned by altered transcriptional profiles between the two populations. These changes in competence do not mean the acquisition of radical and irreversible differences for generating stage-specific cell types. 

It is rather a sequential acquisition of subtle changes that interact with existing mechanism towards different ends. To further understand the differences and get insights into inherent and acquired potential of these two populations of neural stem cells, we are carrying out transcriptional profiling to identify additional markers and Hoechst dye efflux assay for their direct enrichment. Our search for neural stem cells/progenitors in the adult retina led us to three important findings. First, we identified neural stem cells in the adult ciliary epithelium. Second, we found the positive growth factor responsive progenitors in the periphery of adult retina. Third, we observed that muller glia represent latent stem cells in the adult retina.

Retinal (upper) and Ciliary epithelial (lower) stem cells/progenitors
Retinal (upper) and Ciliary epithelial (lower) stem cells/progenitors