SummaryAge-related macular degeneration (AMD) causes severe visual impairment due in part to age-dependent impairment of retinal pigment epithelium (RPE). It has been suggested that autologous human induced pluripotent stem cells (hiPSCs) may represent a useful cell source for the generation of graft RPE. We generated hiPSC-derived RPE (hiPSC-RPE) cell sheets optimized to meet clinical use requirements, including quality, quantity, consistency, and safety. These cell sheets are generated as a monolayer of cells without any artificial scaffolds, express typical RPE markers, form tight junctions that exhibit polarized secretion of growth factors, and show phagocytotic ability and gene-expression patterns similar to those of native RPE. Additionally, upon transplantation, autologous nonhuman primate iPSC-RPE cell sheets showed no immune rejection or tumor formation. These results suggest that autologous hiPSC-RPE cell sheets may serve as a useful form of graft for use in tissue replacement therapy for AMD.

Ligation of death receptors, such as Fas, triggers apoptosis in many types of cells (28), which is inhibited by cellular FLIP (cFLIP, also known as I-FLICE, FLAME-I, Casper, CASH, MRIT and Usurpin) (8,11,13,16,34,40,41,44). The long form of cFLIP (cFLIP-L) is highly homologous to caspase 8, containing two death effector domains (DED) and a caspaselike domain at the amino and carboxy termini, respectively. cFLIP-L, however, does not have caspase activity due to the lack of a conserved cysteine residue in the caspase-like domain. Upon death receptor ligation, cFLIP-L is recruited to the death receptor complex, together with FADD and caspase 8, and inhibits apoptosis signaling. cFLIP-L is expressed in various cancers (2,16,27,36,43,44), which suggests a role for cFLIP-L in protecting cancer cells from cellular immunity using the Fas system (5, 6, 25).cFLIP, however, does not always inhibit apoptosis signaling but also mediates growth signals in some cases. Under conditions in which the proliferation of CD3-activated human T lymphocytes is increased by recombinant Fas ligand, cFLIP-L interacts with tumor necrosis factor receptor-associated factors 1 and 2, as well as the kinases RIP and Raf-1, resulting in the activation of the NF-B and ERK signaling pathways (17). The ability of cFLIP-L to switch Fas-mediated glucose signaling from a death signal into a proliferation signal in human pancreatic ␤ cells was also reported (24).The Wnt signal transduction pathway plays a critical and evolutionarily conserved role during embryogenesis (3, 10, 46). The Wnt signal is mediated by ␤-catenin, a transcription factor that is normally degraded by the ubiquitin-proteasome system in cytosol. Phosphorylation of ␤-catenin by a large protein complex involving adenomatous polyposis coli (APC) protein, Axin, and glycogen synthase kinase 3␤ (GSK3␤) initiates the ubiquitylation and proteasomal degradation of ␤-catenin (15, 49). Upon Wnt signaling, phosphorylation of ␤-catenin is inhibited, which results in the accumulation and translocation of ␤-catenin into nuclei, thereby inducing the expression of several genes, such as c-myc and the cyclin D gene. Mutations in APC, Axin, and ␤-catenin genes resulting in abolished ␤-catenin ubiquitylation are found in many human cancers (4,9,20,26,30,32,35,37), indicating that inappropriate activation of Wnt signaling plays an important role in human cancers (31,33).In this paper, we report that cFLIP-L inhibits ␤-catenin ubiquitylation and enhances Wnt signaling, which suggests an additional mechanism involved in tumorgenesis, in addition to inhibiting apoptosis signaling. MATERIALS AND METHODSPlasmids. Human cFLIP-L and a short splicing variant of cFLIP (cFLIP-S) were amplified by PCR from a Jurkat cDNA library and subcloned into pcDNAbased mammalian expression vectors (Invitrogen). For deletion mutant constructs, DNA sequences corresponding to different regions of cFLIP-L were

In the nervous system, transcription factor expression in progenitor and/or nascent neurons regulates cell type specification. Although the functions of these transcription factors at early stages are well established, whether or not they are required during late developmental periods remains an open question. To address this issue, we conditionally manipulated gene expression using a recently developed transposon-mediated gene transfer system combined with in ovo electroporation. In chicken retinas, horizontal cells are classified into three subtypes according to their characteristic neuronal morphology. Two LIM family transcription factors, Lim1 and Isl1, begin to be expressed in a distinct subset of nascent retinal neurons, which results in complementary expression of these genes in mature retinas in type I and type II/III horizontal cells, respectively. Overexpression of Isl1 in post-migratory horizontal cells represses endogenous Lim1 expression and increases the number of neurons with a dendritic morphology characteristic of type II horizontal cells, which normally express Isl1. Inhibition of Lim1 function by expression of a dominant negative form Lim1 perturbs axonal morphogenesis of type I horizontal cells. Therefore, we propose that LIM family transcription factors are required for subtype-specific morphogenesis of horizontal cells at later stages of retinal development.

Retinal Müller glia can serve as a source for regeneration of damaged retinal neurons in fish, birds and mammals. However, the proliferation rate of Müller glia has been reported to be low in the mammalian retina. To overcome this problem, growth factors and morphogens have been studied as potent promoters of Müller glial proliferation, but the molecular mechanisms that limit the proliferation of Müller glia in the mammalian retina remain unknown. In the present study, we found that the degree of damage-induced Müller glia proliferation varies across mouse strains. In mouse line 129×1/SvJ (129), there was a significantly larger proliferative response compared with that observed in C57BL/6 (B6) after photoreceptor cell death. Treatment with a Glycogen synthase kinase 3 (GSK3) inhibitor enhanced the proliferation of Müller glia in 129 but not in B6 mouse retinas. We therefore focused on the different gene expression patterns during retinal degeneration between B6 and 129. Expression levels of Cyclin D1 and Nestin correlated with the degree of Müller glial proliferation. A comparison of genome-wide gene expression between B6 and 129 showed that distinct sets of genes were upregulated in the retinas after damage, including immune response genes and chromatin remodeling factors.

The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D 3 was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFPtagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10 ؊8 M 1,25-dihydroxyvitamin D 3 promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus. The vitamin D receptor (VDR)1 is one of the ligand-dependent transcription factors that make up the nuclear hormone receptor superfamily (1-3). To modulate the transcription of target genes in response to its cognate ligand 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), VDR must be localized in nucleus and then bind to an enhancer designated as the vitamin D-responsive element (VDRE), forming a heterodimer with retinoid X receptor (1-6). In contrast to the case for the glucocorticoid receptor (GR), which translocates from the cytoplasm to the nucleus when exposed to its ligand, VDR does not bind to heat shock protein 90, and both immunocytochemical and biochemical fractionation studies suggested the nuclear localization of VDR even in the absence of 1,25(OH) 2 D 3 (7-10).Several reports, however, demonstrated that VDR was located in cytoplasm in the absence of ligand and transported to nucleus in response to 1,25(OH) 2 D 3 (11-13). Although the reason for conflicting results as to the distribution of VDR is not clear, the fixation and cell permeabilization procedures in immunostaining might influence the subcellular distribution of the subject protein. Consistent with this explanation, Barsony et al. (11), by the fixation of cells using a microwave, revealed the cytoplasmic localization of VDR in contrast to the nuclear localization detected by a conventional fixation method utilizing the same antibody against VDR.To avoid the fixation and cell permeabilization steps required in the immunostaining procedure, in the present study we have taken advantage of fusion with green fluorescent protein (GFP), which has been proven to be a useful tag for monitoring the subcellular distribution and trafficking of various proteins in living cell...

The identified C21orf2 mutations decreased protein stability and affected cytoplasmic localization of C21orf2. Since C21orf2 was required for ciliogenesis, our data suggested that reduced levels of functional C21orf2 induced photoreceptor degradation through abnormal cilia formation, leading to arRP or arCRD in the retina.

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.

Inherited optic neuropathies are rare eye diseases of optic nerve dysfunction that present in various genetic forms. Previously, mutation in three genes encoding mitochondrial proteins has been implicated in autosomal recessive forms of optic atrophy that involve progressive degeneration of optic nerve and retinal ganglion cells (RGC). Using whole exome analysis, a novel double homozygous mutation p.L81R and pR212W in malonyl CoA-acyl carrier protein transacylase (MCAT), a mitochondrial protein involved in fatty acid biosynthesis, has now been identified as responsible for an autosomal recessive optic neuropathy from a Chinese consanguineous family. MCAT is expressed in RGC that are rich in mitochondria. The disease variants lead to structurally unstable MCAT protein with significantly reduced intracellular expression. RGC-specific knockdown of Mcat in mice, lead to an attenuated retinal neurofiber layer, that resembles the phenotype of optic neuropathy. These results indicated that MCAT plays an essential role in mitochondrial function and maintenance of RGC axons, while novel MCAT p.L81R and p.R212W mutations can lead to optic neuropathy.