This study identifies and characterizes retinal stem cells (RSCs) in early postnatal to seventh-decade human eyes. Different subregions of human eyes were dissociated and cultured by using a clonal sphere-forming assay. The stem cells were derived only from the pars plicata and pars plana of the retinal ciliary margin, at a frequency of Ϸ1:500. To test for long-term self-renewal, both the sphere assay and monolayer passaging were used. By using the single sphere passaging assay, primary spheres were dissociated and replated, and individual spheres demonstrated 100% selfrenewal, with single spheres giving rise to one or more new spheres in each subsequent passage. The clonal retinal spheres were plated under differentiation conditions to assay the differentiation potential of their progeny. The spheres were produced all of the different retinal cell types, demonstrating multipotentiality. Therefore, the human eye contains a small population of cells (Ϸ10,000 cells per eye) that have retinal stem-cell characteristics (proliferation, self-renewal, and multipotentiality). To test the in vivo potential of the stem cells and their progeny, we transplanted dissociated human retinal sphere cells, containing both stem cells and progenitors, into the eyes of postnatal day 1 NOD͞SCID mice and embryonic chick eyes. The progeny of the RSCs were able to survive, migrate, integrate, and differentiate into the neural retina, especially as photoreceptors. Their facile isolation, integration, and differentiation suggest that human RSCs eventually may be valuable in treating human retinal diseases.

Renewable neurosphere formation in culture is a defining characteristic of certain brain tumor initiating cells. This retrospective study was designed to assess the relationship between neurosphere formation in cultured human glioma, tumorigenic capacity, and patient clinical outcome. Tumor samples were cultured in neurosphere conditions from 32 patients with glioma, including a subpopulation of 15 patients with primary glioblastoma. A subsample of renewable neurosphere cultures was xenografted into mouse brain to determine if they were tumorigenic. Our study shows that both renewable neurosphere formation and tumorigenic capacity are significantly associated with clinical outcome measures. Renewable neurosphere formation in cultured human glioma significantly predicted an increased hazard of patient death and more rapid tumor progression. These results pertained to both the full population of glioma and the subpopulation of primary glioblastoma. Similarly, there was a significant hazard of progression for patients whose glioma had tumorigenic capacity. Multivariate analysis demonstrated that neurosphere formation remained a significant predictor of clinical outcome independent of Ki67 proliferation index. In addition, multivariate analysis of neurosphere formation, tumor grade and patient age, demonstrated that neurosphere formation was a robust, independent predictor of glioma tumor progression. While the lengthy duration of this assay may preclude direct clinical application, these results exemplify how neurosphere culture serves as a clinically relevant model for the study of malignant glioma. Furthermore, this study suggests that the ability to propagate brain tumor stem cells in vitro is associated with clinical outcome.