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.

The Polycomb group (PcG) gene Bmi1 promotes cell proliferation and stem cell self-renewal by repressing the Ink4a/Arf locus. We used a genetic approach to investigate whether Ink4a or Arf is more critical for relaying Bmi1 function in lymphoid cells, neural progenitors, and neural stem cells. We show that Arf is a general target of Bmi1, however particularly in neural stem cells, derepression of Ink4a contributes to Bmi1 −/− phenotypes. Additionally, we demonstrate haploinsufficient effects for the Ink4a/Arf locus downstream of Bmi1 in vivo. This suggests differential, cell type-specific roles for Ink4a versus Arf in PcG-mediated (stem) cell cycle control.Supplemental material is available at http://www.genesdev.org.

Malattia Leventinese (ML), an inherited macular degenerative disease, is closely reminiscent of age-related macular degeneration (AMD), the most common cause of incurable blindness. Both ML and AMD are characterized by extracellular deposits known as drusen between the retinal pigment epithelium (RPE) and Bruch's membrane. The mechanism underlying drusen formation is unknown. An Arg to Trp mutation in a gene of unknown function, EFEMP1, is responsible for ML, indicating EFEMP1 may be important in drusen formation. Here, we show that wild-type EFEMP1 is a secreted protein whereas mutant EFEMP1 is misfolded, secreted inefficiently, and retained within cells. In normal eyes, EFEMP1 is not present at the site of drusen formation. However, in ML eyes, EFEMP1 accumulates within the RPE cells and between the RPE and drusen, but does not appear to be a major component of drusen. Furthermore, in AMD eyes, EFEMP1 is found to accumulate beneath the RPE immediately overlaying drusen, but not in the region where there is no apparent retinal pathology observed. These data present evidence that misfolding and aberrant accumulation of EFEMP1 may cause drusen formation and cellular degeneration and play an important role in the etiology of both ML and AMD.T he macula, a central circular area of the retina 5 to 6 mm in diameter with the fovea at its center, facilitates central vision and high-resolution visual acuity. Various diseases causing macular degeneration result in severe and irreversible loss of vision. Malattia Leventinese (ML), also known as Doyne honeycomb retinal dystrophy, is a rare autosomal dominant macular degenerative disease with high penetrance (1-3). Onset of ML is generally in midlife but can vary from childhood until old age (4). An early characteristic feature of ML is the presence of amorphous sub-retinal pigment epithelium (RPE) deposits known as drusen between the RPE and Bruch's membrane (1, 5). At a later stage of the disease, ML exhibits a variety of clinical and histopathological features, including decreased visual acuity, geographic atrophy, pigmentary changes, and choroidal neovascularization (6). Drusen are also an early hallmark of age-related macular degeneration (AMD), a heterogeneous late onset macular degenerative condition (7). ML exhibits features more consistent with AMD than any other heritable macular disorder. Except for a late age of onset, AMD shares the typical clinical features of ML (8). AMD accounts for approximately 50% of registered blindness in the developed world (9, 10). More than 20% of the population over 65 years of age is affected with AMD. The molecular mechanism responsible for drusen formation and other retinal pathology observed in ML or AMD is currently unknown.A single mutation, Arg-345 to Trp (R345W) in the gene EFEMP1 (for epidermal growth factor-containing fibrillin-like extracellular matrix protein 1), was found to be responsible for ML (11). To date, no mutation in EFEMP1 has been found to be associated with AMD (11). Initially described as S1-5 (12), also kn...

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinson's disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

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Neural stem cells (NSCs), when stimulated with epidermal growth factor (EGF) or fibroblast growth factor-2 (FGF-2), have the capacity to renew, expand, and produce precursors for neurons, astrocytes, and oligodendrocytes. We postulated that the early appearance of insulin-like growth factor (IGF-I) receptors during mouse striatum development implies a role in NSC regulation. Thus, we tested in vitro the action of IGF-I on the proliferation of striatal NSCs. In the absence of IGF-I, neither EGF nor FGF-2 was able to induce the proliferation of E14 mouse striatal cells. However, addition of IGF-I generated large proliferative clusters, termed spheres, in a dose-dependent manner. The newly generated spheres were multipotent, and clonal analysis revealed that EGF or FGF-2, in the presence of IGF-I, acted directly on NSCs. The actions of IGF-I suggest distinct modes of action of EGF or FGF-2 on NSCs. First, continuous versus delayed administration of these neurotrophic factors showed that neither IGF-I nor EGF had an effect on NSC survival, whereas FGF-2 promoted the survival or maintenance of the stem cell state of 50% of NSCs for 6 d. Second, short-term exposure to IGF-I induced the proliferation of NSCs in the presence of EGF, but not of FGF-2, through an autocrine secretion of IGF-I. These findings suggest that IGF-I is a key factor in the regulation of NSC activation and that EGF and FGF-2 control striatal NSC proliferation, in part, through distinct intracellular mechanisms.

Insulin-like growth factor-I (IGF-I) has been reported previously to promote the proliferation, survival, and maturation of sympathetic neuroblasts, the genesis of retinal neurons, and the survival of CNS projection and motor neurons. Here we asked whether IGF-I could promote the in vitro differentiation of postmitotic mammalian CNS neuronal precursors derived from multipotent epidermal growth factor (EGF)-responsive stem cells. In the absence of IGF-I, virtually no neurons were present in cultured stem cell progeny, whereas IGF-I increased neuron number by eight- to 40-fold. Brief exposures (2 hr) to IGF-I were sufficient to allow for neuronal differentiation without affecting proliferation or survival. IGF-I actions could be mimicked by insulin and IGF-II at concentrations that correspond to the pharmacology of the IGF-I receptor, the latter for which the mRNA was detected in undifferentiated stem cell progeny. Although ineffectual alone at low concentrations (10 nM) that would activate its own receptor, insulin was able to potentiate the actions of IGF-I by acting on mitotically active neural precursors. When neuronal precursor differentiation by IGF-I was examined in relation to brain-derived neurotrophic factor (BDNF), two important observations were made: (1) BDNF could potentiate the differentiating actions of IGF-I plus insulin, and (2) BDNF could act on a separate population of precursors that did not require IGF-I plus insulin for differentiation. Taken together, these results suggest that IGF-I and BDNF may act together or sequentially to promote neuronal precursor differentiation.

Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. Our previous observations demonstrate that F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. Here, we establish that NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases. Neural progenitor cells (NPCs)1 are self-renewing multipotent cells that can give rise to all types of neural cells, namely neurons, oligodendrocytes (OLs), and astrocytes. Increasing evidence suggests that this fate commitment of NPCs requires molecular cues provided by extracellular molecules and intrinsic signaling involving various transcription factors (1, 2). Our recent study (3) has demonstrated that the F3/Notch signaling pathway via Deltex1 (DTX1) promotes oligodendrocyte precursor cell (OPC) differentiation into oligodendrocytes (OLs) and up-regulates myelin-associated glycoprotein (MAG) expression in both primary OLs and OLN-93 cells, an OL cell line.