The mature mammalian retina is thought to lack regenerative capacity. Here, we report the identification of a stem cell in the adult mouse eye, which represents a possible substrate for retinal regeneration. Single pigmented ciliary margin cells clonally proliferate in vitro to form sphere colonies of cells that can differentiate into retinal-specific cell types, including rod photoreceptors, bipolar neurons, and Müller glia. Adult retinal stem cells are localized to the pigmented ciliary margin and not to the central and peripheral retinal pigmented epithelium, indicating that these cells may be homologous to those found in the eye germinal zone of other nonmammalian vertebrates.

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.

SummaryThe utility of stem cells and their progeny in adult transplantation models has been limited by poor survival and integration. We designed an injectable and bioresorbable hydrogel blend of hyaluronan and methylcellulose (HAMC) and tested it with two cell types in two animal models, thereby gaining an understanding of its general applicability for enhanced cell distribution, survival, integration, and functional repair relative to conventional cell delivery in saline. HAMC improves cell survival and integration of retinal stem cell (RSC)-derived rods in the retina. The pro-survival mechanism of HAMC is ascribed to the interaction of the CD44 receptor with HA. Transient disruption of the retinal outer limiting membrane, combined with HAMC delivery, results in significantly improved rod survival and visual function. HAMC also improves the distribution, viability, and functional repair of neural stem and progenitor cells (NSCs). The HAMC delivery system improves cell transplantation efficacy in two CNS models, suggesting broad applicability.

The epithelial layers of the ciliary body (CB) and iris are non-neural structures that differentiate from the anterior region of the eyecup, the ciliary margin (CM). We show here that activation of the canonical Wnt signaling pathway is sufficient and necessary for the normal development of anterior eye structures. Pharmacological activation of beta-catenin signaling with lithium (Li(+)) treatment in retinal explants in vitro induced the ectopic expression of the CM markers Otx1 and Msx1. Cre-mediated stabilization of beta-catenin expression in the peripheral retina in vivo induced a cell autonomous upregulation of CM markers at the expense of neural retina (NR) markers and inhibited neurogenesis. Consistent with a cell autonomous conversion to peripheral eye fates, the proliferation index in the region of the retina that expressed stabilized beta-catenin was identical to the wild-type CM and there was an expansion of CB-like structures at later stages. Conversely, Cre-mediated inactivation of beta-catenin reduced CM marker expression as well as the size of the CM and CB/iris. Aberrant CB development in both mouse models was also associated with a reduction in the number of retinal stem cells in vitro. In summary, activation of canonical Wnt signaling is sufficient to promote the development of peripheral eyecup fates at the expense of the NR and is also required for the normal development of anterior eyecup structures.

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Recently, Notch signaling has been reported to underscore the ability of neural stem cells (NSCs) to self-renew. Utilizing mice deficient in presenilin-1(PS1), we asked whether the function of Notch signaling in NSC maintenance was conserved. At embryonic day 14.5, all NSCs – both similar (cortex-, ganglionic eminence- and hindbrain-derived) and distinct (retinal stem cell) – require Notch signaling in a gene-dosage-sensitive manner to undergo expansionary symmetric divisions, as assessed by the clonal, in vitro neurosphere assay. Within the adult, however, Notch signaling modulates cell cycle time in order to ensure brain-derived NSCs retain their self-renewal property. At face value, the effects in the embryo and adult appear different. We propose potential hypotheses, including the ability of cell cycle to modify the mode of division, in order to resolve this discrepancy. Regardless, these findings demonstrate that PS1, and presumably Notch signaling, is required to maintain all NSCs.

SummarySelf-renewing, multipotential retinal stem cells (RSCs) reside in the pigmented ciliary epithelium of the peripheral retina in adult mammals. RSCs can give rise to rhodopsin positive-cells, which can integrate into early postnatal retina, and represent a potentially useful option for cellular therapy. The ability to purify a stem cell population and direct the differentiation toward a particular cell lineage is a challenge facing the application of stem cells in regenerative medicine. Here we use cell sorting to prospectively enrich mouse RSCs based on size, granularity and low expression of P-cadherin and demonstrate that only rare cells with defined properties proliferate to form colonies. We show that clonally-derived mouse and human RSC progeny are multipotent and can differentiate into mature rhodopsin-positive cells with high efficiency using combinations of exogenous culture additives known to influence neural retinal development, including taurine and retinoic acid. This directed RSC differentiation follows the temporal sequence of photoreceptor differentiation in vivo, and the cells exhibit morphology, protein and gene expression consistent with primary cultures of rods in vitro. These results demonstrate that the RSC, an adult stem cell, can be enriched and directed to produce photoreceptors as a first step toward a targeted cell replacement strategy to treat retinal degenerative disease.