Thomas A. Reh

Efficient generation of retinal progenitor cells from human embryonic stem cells.

The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80% of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cell-derived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptor-specific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations.

Transplantation of Human Embryonic Stem Cell-Derived Photoreceptors Restores Some Visual Function in Crx-Deficient Mice

Some of the most common causes of blindness involve the degeneration of photoreceptors in the neural retina; photoreceptor replacement therapy might restore some vision in these individuals. Embryonic stem cells (ESCs) could, in principle, provide a source of photoreceptors to repair the retina. We have previously shown that retinal progenitors can be efficiently derived from human ESCs. We now show that retinal cells derived from human ESCs will migrate into mouse retinas following intraocular injection, settle into the appropriate layers, and express markers for differentiated cells, including both rod and cone photoreceptor cells. After transplantation of the cells into the subretinal space of adult Crx(-/-) mice (a model of Lebers Congenital Amaurosis), the hESC-derived retinal cells differentiate into functional photoreceptors and restore light responses to the animals. These results demonstrate that hESCs can, in principle, be used for photoreceptor replacement therapies.

Müller glia are a potential source of neural regeneration in the postnatal chicken retina

The retina of warm-blooded vertebrates is believed to be incapable of neural regeneration. Here we provide evidence that the retina of postnatal chickens has the potential to generate new neurons. In response to acute damage, numerous Müller glia re-entered the cell cycle, and shortly thereafter, expressed CASH-1, Pax6 and Chx10, transcription factors expressed by embryonic retinal progenitors. These progenitor-like cells transiently expressed neurofilament. Newly formed cells became distributed throughout the inner and outer nuclear layers of the retina, and remained for at least three weeks after damage. Some of these newly formed cells differentiated into retinal neurons, a few formed Müller glia, and most remained undifferentiated, with continued expression of Pax6 and Chx10. These cells continued to proliferate when grown in culture, with some differentiating into retinal neurons or Müller glia. We propose that, in response to damage, Müller glia in the retina are a potential source of neural regeneration.

EGF and TGF-α stimulate retinal neuroepithelial cell proliferation in vitro

Abstract Peptide growth factors have been shown to have diverse effects on cells of the CNS, such as promoting neuronal survival, neurite outgrowth, and several other aspects of neuronal differentiation. In addition, some of these factors have been shown to be mitogenic for particular classes of glial cells within the brain and optic nerve, and recently two peptide growth factors, fibroblast growth factor and nerve growth factor, have been shown to have mitogenic activity on the CNS neuronal progenitors. We now report that two members of another peptide growth factor, epidermal growth factor and transforming growth factor-α, are mitogenic for retinal neuroepithelial cells in primary cultures and provide evidence for the presence of both of these factors in normal developing rat retina.

Generation, Purification and Transplantation of Photoreceptors Derived from Human Induced Pluripotent Stem Cells

Background Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells. Methodology/Physical Findings In this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers. Conclusions This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.

Stimulation of neural regeneration in the mouse retina.

Müller glia can serve as a source of new neurons after retinal damage in both fish and birds. Investigations of regeneration in the mammalian retina in vitro have provided some evidence that Müller glia can proliferate after retinal damage and generate new rods; however, the evidence that this occurs in vivo is not conclusive. We have investigated whether Müller glia have the potential to generate neurons in the mouse retina in vivo by eliminating ganglion and amacrine cells with intraocular NMDA injections and stimulating Müller glial to re-enter the mitotic cycle by treatment with specific growth factors. The proliferating Müller glia dedifferentiate and a subset of these cells differentiated into amacrine cells, as defined by the expression of amacrine cell-specific markers Calretinin, NeuN, Prox1, and GAD67-GFP. These results show for the first time that the mammalian retina has the potential to regenerate inner retinal neurons in vivo.

Multipotential stem cells and progenitors in the vertebrate retina

The vertebrate retina is derived from paired evaginations from the neural tube in embryonic development and is initially produced by progenitor cells similar to those that generate the neurons and glia of other areas of the central nervous system. In some amphibians and fish, the retina continues to grow along with the eye throughout the life of the animal. The new retinal cells are added at the ciliary margin of the eye from the mitotic activity of neural/glial stem cells in a region known as the germinal zone and are seamlessly incorporated into the existing retinal circuitry. Little is known about the cell or molecular biology of these stem cells; however, studies of retinal progenitor cells in chick and mammalian embryos have led to the identification of several factors that control their proliferation. Moreover, studies of retinal regeneration have shown that retinal stem cells can also be derived from two or perhaps three additional sources after retinal damage: (a) the retinal pigmented epithelium (RPE) in amphibians and embryonic chicks and mammals; (b) a specialized rod progenitor in fish; and (c) the Müller glial cells. While there is currently no evidence for a neural/glial stem cell in the adult mammalian retina, and the retina of the mature mammal does not show regenerative capacity after damage, there is a possibility for the reinitiation of stem cell potential at the peripheral retinal margin, from the RPE or from the Müller glial cells. The application of information derived from the studies of retinal progenitor cells in developing organisms should soon provide a test of these possibilities.

Potential of Müller glia to become neurogenic retinal progenitor cells

The possibility of neural regeneration has gained credence with the identification of neural stem cells seeded within different regions of the adult central nervous system (CNS). Recently, this possibility has received an additional boost from reports that glia, the support cells of the CNS, might provide a source of neural regeneration. We review some of our findings that Müller glia in the chicken retina are a source of proliferating progenitors that can generate neurons. These Müller cells are fully differentiated glial cells that serve functions ascribed to this cell type. In response to damage or exogenous growth factors, Müller glia dedifferentiate, proliferate, express combinations of transcription factors normally expressed by embryonic retinal progenitors, and produce new neurons and glia. In light of these data, the potential of Müller glia as a source of neural regeneration in the retina of nonavian species, namely humans, seems an avenue of investigation that warrants serious consideration. GLIA 43:70–76, 2003.

MIO‐M1 Cells and Similar Müller Glial Cell Lines Derived from Adult Human Retina Exhibit Neural Stem Cell Characteristics

Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor‐like role in developed human retina is unknown. We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO‐M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor‐2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including βIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S‐opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell‐based therapies to restore retinal function.

Regulation of tyrosine hydroxylase-containing amacrine cell number in larval frog retina

To determine whether production of new neurons of a particular type is regulated by the presence of previously differentiated neurons of the same type, we ablated all tyrosine hydroxylase immunoreactive (THIR) cells from larval frog retina with the neurotoxin 6-hydroxydopamine, and examined the retinas in subsequent weeks for newly generated THIR neurons. Three weeks after neurotoxin administration, new THIR cells appeared near the zone of neural proliferation at the ciliary margin at a higher density than that of normal retina, while the densities of other amacrine cell types, serotonin (t-HT) immunoreactive and substance P immunoreactive (SPIR), remained the same as controls. Thus the production of new retinal TRIR cells is selectively up-regulated following ablation of previously differentiated cells of this type.