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Retinitis Pigmentosa Stem Cell Treatment

Stem Cell Treatment for Retinitis Pigmentosa

 

Retinitis Pigmentosa treatments using stem cells is now an option here:

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Retinitis pigmentosa is a group of genetic eye conditions that leads to incurable blindness. In the progression of symptoms for Retinitis pigmentosa, night blindness generally precedes tunnel vision by years or even decades. Many people with Retinitis pigmentosa do not become legally blind until their 40s or 50s and retain some sight all their lives. Others go completely blind from Retinitis pigmentosa, in some cases as early as childhood. Progression of Retinitis pigmentosa is different in each case.

Retinitis pigmentosa is a type of progressive retinal dystrophy, a group of inherited disorders in which abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium of the retina lead to progressive visual loss. Affected individuals first experience defective dark adaptation or nyctalopia (night blindness), followed by reduction of the peripheral visual field (known as tunnel vision) and, sometimes, loss of central vision late in the course of the disease.

 

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Stem Cell Treatment for Retinitis Pigmentosa

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Stem Cell Treatment Retinitis Pigmentosa
Related Articles GeneReviews(®) Book. 1993 Authors: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong CT, Smith RJH, Stephens K Abstract DISEASE CHARACTERISTICS: Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. They can be caused by mutation of genes encoded by either nuclear DNA or mitochondrial DNA (mtDNA). While some mitochondrial disorders only affect a single organ (e.g., the eye in Leber hereditary optic neuropathy [LHON]), many involve multiple organ systems and often present with prominent neurologic and myopathic features. Mitochondrial disorders may present at any age. Many individuals with a mutation of mtDNA display a cluster of clinical features that fall into a discrete clinical syndrome, such as the Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with ragged-red fibers (MERRF), neurogenic weakness with ataxia and retinitis pigmentosa (NARP), or Leigh syndrome (LS). However, considerable clinical variability exists and many individuals do not fit neatly into one particular category, which is well-illustrated by the overlapping spectrum of disease phenotypes (including mitochondrial recessive ataxia syndrome (MIRAS) resulting from mutation of the nuclear gene POLG, which has emerged as a major cause of mitochondrial disease. Common clinical features of mitochondrial disease – whether involving a mitochondrial or nuclear gene – include ptosis, external ophthalmoplegia, proximal myopathy and exercise intolerance, cardiomyopathy, sensorineural deafness, optic atrophy, pigmentary retinopathy, and diabetes mellitus. Common central nervous system findings are fluctuating encephalopathy, seizures, dementia, migraine, stroke-like episodes, ataxia, and spasticity. A high incidence of mid- and late pregnancy loss is a common occurrence that often goes unrecognized. DIAGNOSIS/TESTING: In some individuals, the clinical picture is characteristic of a specific mitochondrial disorder (e.g., LHON, NARP, or maternally inherited LS), and the diagnosis can be confirmed by identification of a mtDNA mutation on molecular genetic testing of DNA extracted from a blood sample. In many individuals, such is not the case, and a more structured approach is needed, including family history, blood and/or CSF lactate concentration, neuroimaging, cardiac evaluation, and molecular genetic testing for a mtDNA or nuclear gene mutation. Approaches to molecular genetic testing of a proband to consider are serial testing of single genes, multi-gene panel testing (simultaneous testing of multiple genes), and/or genomic testing (e.g., sequencing of the entire mitochondrial genome exome or exome sequencing to identify mutation of a nuclear gene). In many individuals in whom molecular genetic testing does not yield or confirm a diagnosis, further investigation of suspected mitochondrial disease can involve a range of different clinical tests, including muscle biopsy for respiratory chain function. GENETIC COUNSELING: Mitochondrial disorders may be caused by defects of nuclear DNA or mtDNA. Nuclear gene defects may be inherited in an autosomal recessive or autosomal dominant manner. Mitochondrial DNA defects are transmitted by maternal inheritance. Mitochondrial DNA deletions generally occur de novo and thus cause disease in one family member only, with an approximate recurrence risk of 1 in 24. Mitochondrial DNA single nucleotide variants and duplications may be transmitted down the maternal line. The father of a proband is not at risk of having the mtDNA pathogenic variant, but the mother of a proband (usually) has the mitochondrial pathogenic variant and may or may not have symptoms. A male does not transmit the mtDNA pathogenic variant to his offspring. A female harboring a heteroplasmic mtDNA single nucleotide variant may transmit a variable amount of mutant mtDNA to her offspring, resulting in considerable clinical variability among sibs within the same family. Prenatal genetic testing and interpretation of test results for mtDNA disorders are difficult because of mtDNA heteroplasmy. De novo tissue-specific pathogenic nucleotide variants are rare, but associated with low recurrence risks. MANAGEMENT: Treatment of manifestations: The management of mitochondrial disease is largely supportive and may include early diagnosis and treatment of diabetes mellitus, cardiac pacing, ptosis correction, intraocular lens replacement for cataracts, and cochlear implantation for sensorineural hearing loss. Individuals with complex I and/or complex II deficiency may benefit from oral administration of riboflavin; those with ubiquinone (coenzyme Q10) deficiency may benefit from oral coenzyme Q10 therapy; and those with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) may benefit from hematopoietic stem cell transplantation. PMID: 20301403
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Related Articles Can the injection of the patient's own bone marrow-derived stem cells preserve cone vision in retinitis pigmentosa and other diseases of the eye? Graefes Arch Clin Exp Ophthalmol. 2005 Mar;243(3):187-8 Authors: Kociok N PMID: 15565292 [PubMed - indexed for MEDLINE]
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Related Articles A single, abbreviated RPGR-ORF15 variant reconstitutes RPGR function in vivo. Invest Ophthalmol Vis Sci. 2005 Feb;46(2):435-41 Authors: Hong DH, Pawlyk BS, Adamian M, Sandberg MA, Li T Abstract PURPOSE: The retinitis pigmentosa GTPase regulator (RPGR) is essential for the maintenance of photoreceptor viability. RPGR is expressed as constitutive and ORF15 variants because of alternative splicing. This study was designed to examine whether the retina-specific ORF15 variant alone could substantially substitute for RPGR function. A further objective was to test whether the highly repetitive purine-rich region of ORF15 could be abbreviated without ablating the function, so as to accommodate RPGR replacement genes in adenoassociated virus (AAV) vectors. METHODS: A cDNA representing RPGR-ORF15 but shortened by 654 bp in the repetitive region was placed under the control of a chicken beta-actin (CBA) hybrid promoter. The resultant construct was transfected into mouse embryonic stem cells. Clones expressing the transgene were selected and injected into mouse blastocysts. Transgenic chimeras were crossed with RPGR knockout (KO) mice. Mice expressing the transgene but null for endogenous RPGR (Tg/KO) were studied from 1 month to 18 months of age by light and electron microscopy, immunofluorescence, and electroretinography (ERG). The results were compared with those of wild-type (WT) and RPGR-null control mice. RESULTS: Transgenic RPGR-ORF15 was found in the connecting cilia of rod and cone photoreceptors, at approximately 20% of the WT level. Photoreceptor morphology, cone opsin localization, expression of GFAP (a marker for retinal degeneration) and ERGs were consistent with the transgene exerting substantial rescue of retinal degeneration due to loss of endogenous RPGR. CONCLUSIONS: RPGR-ORF15 is the functionally significant variant in photoreceptors. The length of its repetitive region can be reduced while preserving its function. The current findings should facilitate the design of gene replacement therapy for RPGR-null mutations. PMID: 15671266 [PubMed - indexed for MEDLINE]
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Related Articles Embryonic stem cells: potential source for ocular repair. Semin Ophthalmol. 2005 Jan-Mar;20(1):17-23 Authors: Haruta M Abstract Many ocular diseases, such as retinitis pigmentosa and age-related macular degeneration, reflect damage to specific cells that are not normally repaired or replaced. Preliminary results of animal studies suggest that these degenerative diseases may be treatable by transplantation of healthy fetal cells. However, obtaining a sufficient number of suitable donor cells remains a problem. The isolation of human embryonic stem (ES) cells has drawn much attention because of their potential clinical application for patients with these degenerative diseases. Because ES cells have the potential to generate all adult cell types, ocular diseases resulting from the failure of specific cell types would be potentially treatable through the transplantation of differentiated cells derived from ES cells. In addition, because ES cells can proliferate indefinitely in their undifferentiated state, they are expected to alleviate the problem of the shortage of donor cells for cell-replacement therapy. Recently, reproducible and efficient differentiation methods for the generation of lens cells, retinal neurons, and retinal pigment epithelial (RPE) cells from ES cells have been developed. This review focuses especially on these ocular cells differentiated from ES cells. We will also discuss the potential therapeutic uses of ES cells for the treatment of ocular diseases. PMID: 15804840 [PubMed - indexed for MEDLINE]
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Related Articles Transcriptional factors involved in photoreceptor differentiation. Semin Ophthalmol. 2005 Jan-Mar;20(1):25-30 Authors: Akimoto M Abstract Regenerative medicine constitutes a potentially promising therapy for blind people suffering from retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. For the realization of retinal regeneration, it is necessary to establish 1) a method to produce functional photoreceptor cells in vitro and 2) successful transplantation of the donor cells to connect their axons to the recipient secondary neurons so that they can function properly. The results of experimental transplantation of human retinal photoreceptor cells from cadaveric eyes or of fetal retinal cells into the retina of RP patients have not been satisfactory, but encouraging enough to indicate that the transplantation of developing retinal cells may have beneficial results. Recently, attempts have been made to generate photoreceptor-like cells from stem cells, but it remains to be seen whether they are in fact photoreceptor cells. It is therefore important to fully understand the mechanisms involved in the development of these cells, and to characterize them not only by transcriptome but also by functional analysis. PMID: 15804841 [PubMed - indexed for MEDLINE]
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Related Articles Enhanced neurotrophin synthesis and molecular differentiation in non-transformed human retinal progenitor cells cultured in a rotating bioreactor. Tissue Eng. 2006 Jan;12(1):141-58 Authors: Kumar R, Dutt K Abstract One approach to the treatment of retinal diseases, such as retinitis pigmentosa, is to replace diseased or degenerating cells with healthy cells. Even if all of the problems associated with tissue transplant were to be resolved, the availability of tissue would remain an ongoing problem. We have previously shown that transformed human retinal cells can be grown in a NASA-developed horizontally rotating culture vessel (bioreactor) to form three-dimensional-like structures with the expression of several retinal specific proteins. In this study, we have investigated growth of non-transformed human retinal progenitors (retinal stem cells) in a rotating bioreactor. This rotating culture vessel promotes cell-cell interaction between similar and dissimilar cells. We cultured retinal progenitors (Ret 1-4) alone or as a co-culture with human retinal pigment epithelial cells (RPE, D407) in this system to determine if 3D structures can be generated from non-transformed progenitors. Our second goal was to determine if the formation of 3D structures correlates with the upregulation of neurotrophins, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGFalpha), ciliary neurotrophic factor (CNTF), and brain-delivered neurotrophic factor (BDNF). These factors have been implicated in progenitor cell proliferation, commitment, differentiation, and survival. We also investigated the expression of the following retinal specific proteins in this system: neuron specific enolase (NSE); tyrosine hydroxylase (TH); D(2)D(3), D(4) receptors; protein kinase-C alpha (PKCalpha), and calbindin. The 3D structures generated were characterized by phase and scanning transmission electron microscopy. Retinal progenitors, cultured alone or as a co-culture in the rotating bioreactor, formed 3D structures with some degree of differentiation, accompanied by the upregulation of bFGF, CNTF, and TGFalpha. Brain-derived neurotrophic factor, which is expressed in vivo in RPE (D407), was not expressed in monolayer cultures of RPE but expressed in the rotating bioreactor-cultured RPE and retinal progenitors (Ret 1-4). Upregulation of neurotrophins was noted in all rotating bioreactor-cultured cells. Also, upregulation of D(4) receptor, calbindin, and PKCalpha was noted in the rotating bioreactor-cultured cells. We conclude that non-transformed retinal progenitors can be grown in the rotating bioreactor to form 3D structures with some degree of differentiation. We relied on molecular and biochemical analysis to characterize differentiation in cells grown in the rotating bioreactor. PMID: 16499451 [PubMed - indexed for MEDLINE]
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Related Articles Hematopoietic stem cells provide repair functions after laser-induced Bruch's membrane rupture model of choroidal neovascularization. Am J Pathol. 2006 Mar;168(3):1031-44 Authors: Chan-Ling T, Baxter L, Afzal A, Sengupta N, Caballero S, Rosinova E, Grant MB Abstract Vascular repair by adult hematopoietic stem cells (HSCs) is well-appreciated because these cells are known for their plasticity. We have shown that adult HSCs differentiate into endothelial cells and participate in both retinal and choroidal neovascularization. We asked whether HSCs participated in the wounding response by forming astrocytes, retinal pigment epithelia (RPE), macrophages, and pericytes. Lethally irradiated C57BL6/J mice were reconstituted with HSCs from mice homozygous for green fluorescent protein (GFP) and then subjected to laser-induced rupture of Bruch's membrane. After immunohistochemical examination of ocular tissue, GFP(+) astrocytes were observed concentrated along the edge of the laser wound, where they and mural cells closely ensheathed the neovasculature. GFP(+) vascular endothelial cells and macrophages/microglia were also evident. Large irregularly shaped GFP(+) RPE cells constituted approximately 93% of RPE cells adjacent to the edge of the denuded RPE area. In regions farther away from the wound, GFP(+) RPE cells were integrated among the GFP(-) host RPE. Thus, postnatal HSCs can differentiate into cells expressing markers specific to astrocytes, macrophages/microglia, mural cells, or RPE. These studies suggest that HSCs could serve as a therapeutic source for long-term regeneration of injured retina and choroid in diseases such as age-related macular degeneration and retinitis pigmentosa. PMID: 16507916 [PubMed - indexed for MEDLINE]
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Related Articles Ames Waltzer deaf mice have reduced electroretinogram amplitudes and complex alternative splicing of Pcdh15 transcripts. Invest Ophthalmol Vis Sci. 2006 Jul;47(7):3074-84 Authors: Haywood-Watson RJ, Ahmed ZM, Kjellstrom S, Bush RA, Takada Y, Hampton LL, Battey JF, Sieving PA, Friedman TB Abstract PURPOSE: Mutations of PCDH15, the gene encoding protocadherin 15, cause either nonsyndromic deafness DFNB23 or Usher syndrome type 1F (USH1F) in humans and deafness with balance problems in Ames waltzer (av) mice. Persons with USH1 usually begin to exhibit signs of retinitis pigmentosa (RP) in early adolescence, but av mice are reported to have functional retinas. In this study, the auditory, visual and molecular biological phenotype of Pcdh15av-5J and Pcdh15av-Jfb mice is characterized, and their usefulness as animal models of USH1 is evaluated. METHODS: Hearing thresholds of mice between 6 and 10 weeks of age were measured by auditory brain stem response (ABR). Immunohistochemistry and histology were used to examine the effect of homozygosity of Pcdh15av-5J on stereocilia bundles of inner ear hair cells and on the photoreceptor cells of the retina. Scotopic and photopic Ganzfeld ERGs were recorded from homozygous Pcdh15av-5J and Pcdh15av-Jfb mice at different ages. Heterozygous littermates served as control subjects. Measurements of the width of the outer nuclear layer (ONL) and the length of rod photoreceptor outer segment (ROS) were made. RESULTS: Homozygous Pcdh15av-5J mice have profound hearing loss and disorganized stereocilia bundles of inner ear hair cells. Compared with heterozygous littermates, homozygous Pcdh15av-5J and Pcdh15av-Jfb mutant mice had scotopic ERG amplitudes consistently reduced by approximately 40% at all light intensities. The b-to-a-wave ratio confirmed that the a- and b-waves were reduced proportionally in homozygous mutant mice. Histologic measurements of retinal sections revealed no significant differences in either the ONL width or the ROS length as a function of genotype. The protocadherin 15 labeling pattern with antisera PB303 in the retina of both heterozygous and homozygous Pcdh15av-5J mice was indistinguishable from the wild type. Wild-type Pcdh15 have many alternatively spliced isoforms. A novel isoform was found in the retina of homozygous Pcdh15av-5J mice, which appears to circumvent the effect of the mutant allele (IVS14-2A-->G), which causes skipping of exon 14, a shift in the translation reading frame and a premature stop codon in exon 15. CONCLUSIONS: Pcdh15(av-5J) and Pcdh15(av-Jfb) mice do not faithfully mimic the RP found in USH1 due to mutations of PCDH15, but have significantly attenuated ERG function in the absence of histologic change. The decline in ERG amplitude with a preserved b-to-a-wave ratio suggests a role for Pcdh15 in retinal function and/or generation of the ERG potentials. Understanding the molecular mechanism by which av mice circumvent degeneration of the retina might offer insights into potential therapies for USH1. PMID: 16799054 [PubMed - indexed for MEDLINE]
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Related Articles Differentiation of embryonic stem cells to retinal cells in vitro. Methods Mol Biol. 2006;330:401-16 Authors: Zhao X, Liu J, Ahmad I Abstract Currently, there is no effective treatment for photoreceptor degeneration, the most common cause of blindness caused by diseases like retinitis pigmentosa, age-related macular degeneration, and diabetic retinopathy. Two promising approaches include cell therapy to replace degenerating cells and neuroprotection to rescue affected cells from premature death. Determination of the potential of embryonic stem (ES) cells to differentiate into photoreceptors will provide reagents for both approaches. First, neural progenitors with retinal potential will be available in unlimited supply to test the efficacy of cell therapy; second, the controlled differentiation of ES cells into photoreceptors, in addition to providing cells to replace degenerating photoreceptors, will offer a robust in vitro model of photoreceptor differentiation for better understanding of degenerative processes and screening of neuroprotective drugs/reagents. In addition, it will allow the identification of genes (gene discovery) that play critical roles in photoreceptor differentiation and degeneration. Here, we describe the protocol to promote differentiation of the mouse ES cell-derived neural progenitors into retinal cells, specifically the rod photoreceptors. PMID: 16846039 [PubMed - indexed for MEDLINE]
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Related Articles Transplantation of bone marrow-derived mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse. Graefes Arch Clin Exp Ophthalmol. 2007 Mar;245(3):414-22 Authors: Arnhold S, Absenger Y, Klein H, Addicks K, Schraermeyer U Abstract BACKGROUND: Retinitis pigmentosa belongs to a large group of degenerative diseases of the retina with a hereditary background. It involves loss of retinal photoreceptor cells and consequently peripheral vision. At present there are no satisfactory therapeutic options for this disease. Just recently the use of mesenchymal stem cells has been discussed as one therapeutical option for retinal degeneration, as they have been shown to differentiate into various cell types, including photoreceptor cells. In this article we wanted to investigate the potency of mesenchymal stem cells to induce rescue effects in an animal model for retinitis pigmentosa, the rhodopsin knockout mouse. METHODS: For the experiments, three experimental groups of 10 animals each were formed. The first group consisted of untreated rhodopsin knockout (rho(-/-)) animals used as controls. The second group consisted of rho(-/-) mice that had received an injection of mouse mesenchymal stem cells, which were transduced using an adenoviral vector containing the sequence for the green fluorescent protein (GFP) prior to transplantation. In the third sham group, animals received an injection of medium only. Thirty-five days after transplantation, GFP-expressing cells were detected in whole-mount preparations of the retinas as well as in cryostat sections. For the detection of rescue effects, semi-thin sections of eyes derived from all experimental groups were produced. Furthermore, rescue effects were also analysed ultrastructurally in ultrathin sections. RESULTS: Histological analysis revealed that after transplantation, cells morphologically integrated not only into the retinal pigment epithelium but also into layers of the neuroretina displaying neuronal and glial morphologies. Furthermore, significant rescue effects, as demonstrated by the occurrence of preserved photoreceptor cells, were detected. CONCLUSIONS: Our data indicate that mesenchymal stem cells can prolong photoreceptor survival in the rhodopsin knockout mouse, also providing evidence of a therapeutical benefit in retinitis pigmentosa. PMID: 16896916 [PubMed - indexed for MEDLINE]
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Related Articles Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis. PLoS Med. 2006 Oct;3(10):e347 Authors: Bemelmans AP, Kostic C, Crippa SV, Hauswirth WW, Lem J, Munier FL, Seeliger MW, Wenzel A, Arsenijevic Y Abstract BACKGROUND: RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice. METHODS AND FINDINGS: Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease. CONCLUSIONS: By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65(-/-) mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose. PMID: 17032058 [PubMed - indexed for MEDLINE]
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Related Articles Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease. Stem Cells. 2007 Mar;25(3):602-11 Authors: Lund RD, Wang S, Lu B, Girman S, Holmes T, Sauvé Y, Messina DJ, Harris IR, Kihm AJ, Harmon AM, Chin FY, Gosiewska A, Mistry SK Abstract Progressive photoreceptor degeneration resulting from genetic and other factors is a leading and largely untreatable cause of blindness worldwide. The object of this study was to find a cell type that is effective in slowing the progress of such degeneration in an animal model of human retinal disease, is safe, and could be generated in sufficient numbers for clinical application. We have compared efficacy of four human-derived cell types in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the Royal College of Surgeons rat early in the progress of degeneration. Umbilical tissue-derived cells, placenta-derived cells, and mesenchymal stem cells were studied; dermal fibroblasts served as cell controls. At various ages up to 100 days, electroretinogram responses, spatial acuity, and luminance threshold were measured. Both umbilical-derived and mesenchymal cells significantly reduced the degree of functional deterioration in each test. The effect of placental cells was not much better than controls. Umbilical tissue-derived cells gave large areas of photoreceptor rescue; mesenchymal stem cells gave only localized rescue. Fibroblasts gave sham levels of rescue. Donor cells were confined to the subretinal space. There was no evidence of cell differentiation into neurons, of tumor formation or other untoward pathology. Since the umbilical tissue-derived cells demonstrated the best photoreceptor rescue and, unlike mesenchymal stem cells, were capable of sustained population doublings without karyotypic changes, it is proposed that they may provide utility as a cell source for the treatment of retinal degenerative diseases such as retinitis pigmentosa. PMID: 17053209 [PubMed - indexed for MEDLINE]
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Related Articles Retinal pigment epithelium. Methods Enzymol. 2006;418:169-94 Authors: Klimanskaya I Abstract Retinal pigment epithelium (RPE) arises from neuroectoderm and plays a key role in support of photoreceptor functions. Several degenerative eye diseases, such as macular degeneration or retinitis pigmentosa, are associated with impaired RPE function that may lead to photoreceptor loss and blindness. RPE derived from human embryonic stem (hES) cells can be an important source of this tissue for transplantation to cure such degenerative diseases. This chapter describes differentiation of hES cells to RPE, its subsequent isolation, maintenance in culture, and characterization. PMID: 17141036 [PubMed - indexed for MEDLINE]
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Related Articles Ush1c216A knock-in mouse survives Katrina. Mutat Res. 2007 Mar 1;616(1-2):139-44 Authors: Lentz J, Pan F, Ng SS, Deininger P, Keats B Abstract Usher syndrome is the most common cause of inherited deafness found in combination with blindness. All Usher patients suffer progressive retinitis pigmentosa, with the degree of hearing impairment and the presence or absence of vestibular function differing among subtypes. A cryptic splice site mutation (216G-->A) in exon 3 of the USH1C gene on chromosome 11p, which encodes a PDZ-domain protein, harmonin, was found in Acadian Usher type IC patients in south Louisiana. In vitro analysis using constructs containing the mutant 216A and subsequent analysis of patient cell lines revealed a deletion of 35 bases in the transcript. In order to analyze the impact of this frame-shift mutation, we created a knock-in mouse model containing the human 216G-->A mutation. A targeting construct was made containing 5' and 3' homology arms, each 4kb in length, and a 650 base pair fragment containing exons 3 and 4 of human USH1C cloned from an Acadian patient homozygous for the 216A mutation. W4/129S6 embryonic stem (ES) cells were electroporated with the targeting construct, and after 10 days of neomycin selection, clones were picked and screened by polymerase chain reaction (PCR) and Southern blot analysis for homologous recombination. Two positive clones for targeted insertion were microinjected into C57BL/6 blastocysts which were then transplanted into pseudo-pregnant females. Chimeras were bred with Cre recombinase-expressing mice for simultaneous deletion of the neomycin gene and germline transmission of the 216A allele. Homozygous Ush1c216A (216AA) mice are hyperactive, display circling and head tossing behavior, and do not have a Preyer reflex at 21-25 days old. RT-PCR analysis of the cochlea and retina from 216AA mice shows the same 35 base deletion characteristic of Usher IC patients. PMID: 17174357 [PubMed - indexed for MEDLINE]
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Related Articles Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate. Invest Ophthalmol Vis Sci. 2007 Jan;48(1):446-54 Authors: Canola K, Angénieux B, Tekaya M, Quiambao A, Naash MI, Munier FL, Schorderet DF, Arsenijevic Y Abstract PURPOSE: To characterize the potential of newborn retinal stem cells (RSCs) isolated from the radial glia population to integrate the retina, this study was conducted to investigate the fate of in vitro expanded RSCs transplanted into retinas devoid of photoreceptors (adult rd1 and old VPP mice and rhodopsin-mutated transgenic mice) or partially degenerated retina (adult VPP mice) retinas. METHODS: Populations of RSCs and progenitor cells were isolated either from DBA2J newborn mice and labeled with the red lipophilic fluorescent dye (PKH26) or from GFP (green fluorescent protein) transgenic mice. After expansion in EGF+FGF2 (epidermal growth factor+fibroblast growth factor), cells were transplanted intravitreally or subretinally into the eyes of adult wild-type, transgenic mice undergoing slow (VPP strain) or rapid (rd1 strain) retinal degeneration. RESULTS: Only limited migration and differentiation of the cells were observed in normal mice injected subretinally or in VPP and rd1 mice injected intravitreally. After subretinal injection in old VPP mice, transplanted cells massively migrated into the ganglion cell layer and, at 1 and 4 weeks after injection, harbored neuronal and glial markers expressed locally, such as beta-tubulin-III, NeuN, Brn3b, or glial fibrillary acidic protein (GFAP), with a marked preference for the glial phenotype. In adult VPP retinas, the grafted cells behaved similarly. Few grafted cells stayed in the degenerating outer nuclear layer (ONL). These cells were, in rare cases, positive for rhodopsin or recoverin, markers specific for photoreceptors and some bipolar cells. CONCLUSIONS: These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL. PMID: 17197566 [PubMed - indexed for MEDLINE]
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Related Articles BMI1 loss delays photoreceptor degeneration in Rd1 mice. Bmi1 loss and neuroprotection in Rd1 mice. Adv Exp Med Biol. 2006;572:209-15 Authors: Zencak D, Crippa SV, Tekaya M, Tanger E, Schorderet DE, Munier FL, van Lohuizen M, Arsenijevic Y Abstract Retinitis pigmentosa (RP) is a heterogeneous group of genetic disorders leading to blindness, which remain untreatable at present. Rd1 mice represent a recognized model of RP, and so far only GDNF treatment provided a slight delay in the retinal degeneration in these mice. Bmi1, a transcriptional repressor, has recently been shown to be essential for neural stem cell (NSC) renewal in the brain, with an increased appearance of glial cells in vivo in Bmi1 knockout (Bmi1-/-) mice. One of the roles of glial cells is to sustain neuronal function and survival. In the view of a role of the retinal Miller glia as a source of neural protection in the retina, the increased astrocytic population in the Bmi1-/- brain led us to investigate the effect of Bmi1 loss in Rd1 mice. We observed an increase of Müller glial cells in Rd1-Bmi1-/- retinas compared to Rd1. Moreover, Rd1-Bmi1-/- mice showed 7-8 rows of photoreceptors at 30 days of age (P30), while in Rd1 littermates there was a complete disruption of the outer nuclear layer (ONL). Preliminary ERG results showed a responsiveness of Rd1-Bmi1-/- mice in scotopic vision at P35. In conclusion, Bmi1 loss prevented, or rescued, photoreceptors from degeneration to an unanticipated extent in Rd1 mice. In this chapter, we will first provide a brief review of our work on the cortical NSCs and introduce the Bmi1 oncogene, thus offering a rational to our observations on the retina. PMID: 17249577 [PubMed - indexed for MEDLINE]
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Related Articles Stem cells: potential source for retinal repair and regeneration. Arq Bras Oftalmol. 2007 Mar-Apr;70(2):371-5 Authors: Torquetti L, Castanheira P, de Góes AM, Marcio N Abstract Stem cells have been studied in several fields of Medicine, and their applications are not too far from the clinical practice. Retinal impairment by neuronal death has been considered incurable due to the limited regenerative capacity of the central nervous system. The capacity of stem cells to regenerate tissues, as well as their plasticity makes them a potential source for retinal repair. The stem cells are a great promise for the therapy of inherited retinal disorders and retinal-neuronal degenerative diseases, such as retinitis pigmentosa and allied retinal dystrophies, which can result in blindness. Because of the accessibility, expansibility, and multipotentiality mesenchymal stem cells are expected to be useful for clinical applications, especially in regenerative medicine and tissue engineering. Mesenchymal stem cells are clonogenic, nonhematopoietic stem cells present in the bone marrow. Given the appropriate microenvironment, they could differentiate into cardiomyocytes or even into cells of nonmesodermal derivation including hepatocytes and neurons. So far, the results of a few studies are consistent with the belief that cell-based therapies using mesenchymal stem cells may be effective when it comes to retinal damaged tissue repair. PMID: 17589717 [PubMed - indexed for MEDLINE]
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Related Articles A review of the potential to restore vision with stem cells. Clin Exp Optom. 2008 Jan;91(1):78-84 Authors: Mooney I, LaMotte J Abstract Vision research involving stem cells is a rapidly evolving field. Animal experiments have shown that in response to environmental cues, stem cells can repopulate damaged retinas, regrow neuronal axons, repair higher cortical pathways, and restore pupil reflexes, light responses and basic pattern recognition. Viable corneas have been grown from stem cells and transplanted into humans. Similarly, human trials to repair damaged retinas in retinitis pigmentosa and age-related macular degeneration patients have produced preliminary successes. This review attempts to place the collective contributions toward stem cell/vision research into a broader clinical model of how stem cells might ultimately be used to restore the entire visual pathway. PMID: 18045253 [PubMed - indexed for MEDLINE]
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Related Articles Induction of functional photoreceptor phenotype by exogenous Crx expression in mouse retinal stem cells. Invest Ophthalmol Vis Sci. 2008 Jan;49(1):429-37 Authors: Jomary C, Jones SE Abstract PURPOSE: This study was undertaken to determine whether exogenous expression of the transcription factor Crx can promote the differentiation of mouse retinal stem cells (RSCs) into cells with a functional photoreceptor phenotype exhibiting light-sensitive properties. METHODS: RSCs isolated from mouse ciliary epithelium and maintained in serum-free culture were genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Changes in the expression of stem cell markers (homeodomain transcription factor Pax6; POU transcription factor Oct3/4; proliferating cell nuclear antigen [PCNA]); of neuronal markers (nestin, neuron-specific class III beta-tubulin [beta III Tub] and neurofilament [NF 200]); and of photoreceptor-specific markers (rhodopsin [Rho], cyclic nucleotide-gated cation channel-3 [CNG3], blue-cone opsin, and cGMP phosphodiesterase [PDE]); were evaluated during differentiation by immunocytochemistry and Western blot analysis. Phototransduction cascade activity was assessed by measuring light-induced hydrolysis of cyclic (c)GMP levels with a cGMP enzyme-linked immunoassay. RESULTS: Transient Crx transgene expression was observed in 63% of RSCs. Expression of stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4, was significantly decreased by exogenous Crx expression. Concomitantly, Crx induced expression of the analyzed neuron- and photoreceptor-specific markers. Light-induced cGMP hydrolysis was increased in RSCs expressing exogenous Crx, and inhibition of PDE resulted in elevated cGMP levels. CONCLUSIONS: Crx halted proliferation of RSCs and induced them to differentiate into cells expressing photoreceptor-specific markers and displaying light-induced sensitivity characteristic of an activatable visual phototransduction cascade. This study demonstrates that Crx can successfully induce RSCs to differentiate into cells with functional photoreceptor phenotypes. PMID: 18172122 [PubMed - indexed for MEDLINE]
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Related Articles Derivation of neurons with functional properties from adult limbal epithelium: implications in autologous cell therapy for photoreceptor degeneration. Stem Cells. 2008 Apr;26(4):939-49 Authors: Zhao X, Das AV, Bhattacharya S, Thoreson WB, Sierra JR, Mallya KB, Ahmad I Abstract The limbal epithelium (LE), a circular and narrow epithelium that separates cornea from conjunctiva, harbors stem cells/progenitors in its basal layer that regenerate cornea. We have previously demonstrated that cells in the basal LE, when removed from their niche and cultured in reduced bond morphogenetic protein signaling, acquire properties of neural progenitors. Here, we demonstrate that LE-derived neural progenitors generate neurons with functional properties and can be directly differentiated along rod photoreceptor lineage in vitro and in vivo. These observations posit the LE as a potential source of neural progenitors for autologous cell therapy to treat photoreceptor degeneration in age-related macular degeneration and retinitis pigmentosa. PMID: 18203675 [PubMed - indexed for MEDLINE]
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Related Articles alpha-Aminoadipate induces progenitor cell properties of Müller glia in adult mice. Invest Ophthalmol Vis Sci. 2008 Mar;49(3):1142-50 Authors: Takeda M, Takamiya A, Jiao JW, Cho KS, Trevino SG, Matsuda T, Chen DF Abstract PURPOSE: Retinal Müller glia in higher vertebrates have been reported to possess progenitor cell properties and the ability to generate new neurons after injury. This study was conducted to determine the signals that can activate this dormant capacity of Müller glia in adult mice, by studying their behavior during glutamate stimulation. METHODS: Various concentrations of glutamate and its analogue alpha-aminoadipate, which specifically binds Müller glia, were injected subretinally in adult mice. Proliferating retinal cells were labeled by subretinal injection of 5'-bromo-2'-deoxyuridine (BrdU) followed by immunohistochemistry. Müller cell fates were analyzed in retinal sections by using double immunolabeling with primary antibodies against Müller and other retina-specific cell markers. The effects of glutamate and alpha-aminoadipate were also determined in purified Müller cell cultures. RESULTS: Although high levels of glutamate induce retinal damage, subtoxic levels of glutamate directly stimulate Müller glia to re-enter the cell cycle and induce neurogenesis in vivo and in purified Müller cell cultures. alpha-Aminoadipate, which selectively target glial cells, also induced expression of progenitor cell markers by Müller cells in vitro or stimulated Müller cell migration to the outer nuclear layer (ONL) and to differentiate into photoreceptors in vivo. CONCLUSIONS: Mature Müller glia in adult mice can be induced to dedifferentiate, migrate, and generate new retinal neurons and photoreceptor cells by alpha-aminoadipate or glutamate signaling. The results of this study suggest a novel potential strategy for treating retinal neurodegeneration, including retinitis pigmentosa and age-related macular degeneration, without transplanting exogenous cells. PMID: 18326742 [PubMed - indexed for MEDLINE]
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Related Articles Retinal cells integrate into the outer nuclear layer and differentiate into mature photoreceptors after subretinal transplantation into adult mice. Exp Eye Res. 2008 Apr;86(4):691-700 Authors: Bartsch U, Oriyakhel W, Kenna PF, Linke S, Richard G, Petrowitz B, Humphries P, Farrar GJ, Ader M Abstract Vision impairment caused by degeneration of photoreceptors, termed retinitis pigmentosa, is a debilitating condition with no cure presently available. Cell-based therapeutic approaches represent one treatment option by replacing degenerating or lost photoreceptors. In this study the potential of transplanted primary retinal cells isolated from neonatal mice to integrate into the outer nuclear layer (ONL) of adult mice and to differentiate into mature photoreceptors was evaluated. Retinal cells were isolated from retinas of transgenic mice ubiquitously expressing enhanced green fluorescence protein (EGFP) at either postnatal day (P) 0, P1 or P4 and transplanted into the subretinal space of adult wild-type mice. One week to 11 months post-transplantation experimental retinas were analyzed for integration and differentiation of donor cells. Subsequent to transplantation some postnatal retinal cells integrated into the ONL of the host and differentiated into mature photoreceptors containing inner and outer segments as confirmed by immunohistochemistry and electron microscopy. Notably, the appearance of EGFP-positive photoreceptors was not the result of fusion between donor cells and endogenous photoreceptors. Retinal cells isolated at P4 showed a significant increase in their capacity to integrate into the ONL and to differentiate into mature photoreceptors when compared with cells isolated at P0 or P1. As cell suspensions isolated at P4 are enriched in cells committed towards a rod photoreceptor cell fate it is tempting to speculate that immature photoreceptors may have the highest integration and differentiation potential and thus may present a promising cell type to develop cell replacement strategies for diseases involving rod photoreceptor loss. PMID: 18329018 [PubMed - indexed for MEDLINE]
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Related Articles Activation of bone marrow-derived microglia promotes photoreceptor survival in inherited retinal degeneration. Am J Pathol. 2008 Jun;172(6):1693-703 Authors: Sasahara M, Otani A, Oishi A, Kojima H, Yodoi Y, Kameda T, Nakamura H, Yoshimura N Abstract The role of microglia in neurodegeneration is controversial, although microglial activation in the retina has been shown to provide an early response against infection, injury, ischemia, and degeneration. Here we show that endogenous bone marrow (BM)-derived microglia play a protective role in vascular and neural degeneration in the retinitis pigmentosa model of inherited retinal degeneration. BM-derived cells were recruited to the degenerating retina where they differentiated into microglia and subsequently localized to the degenerating vessels and neurons. Inhibition of stromal-derived factor-1 in the retina reduced the number of BM-derived microglia and accelerated the rate of neurovascular degeneration. Systemic depletion of myeloid progenitors also accelerated the degenerative process. Conversely, activation of BM-derived myeloid progenitors by systemic administration of both granulocyte colony-stimulating factor and erythropoietin resulted in the deceleration of retinal degeneration and the promotion of cone cell survival. These data indicate that BM-derived microglia may play a protective role in retinitis pigmentosa. Functional activation of BM-derived myeloid progenitors by cytokine therapy may provide a novel strategy for the treatment of inherited retinal degeneration and other neurodegenerative diseases, regardless of the underlying genetic defect. PMID: 18483210 [PubMed - indexed for MEDLINE]
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Related Articles Decreased levels of the RNA splicing factor Prpf3 in mice and zebrafish do not cause photoreceptor degeneration. Invest Ophthalmol Vis Sci. 2008 Sep;49(9):3830-8 Authors: Graziotto JJ, Inglehearn CF, Pack MA, Pierce EA Abstract PURPOSE: Pre-mRNA processing factor 3 (PRPF3) is a spliceosomal component essential for pre-mRNA processing. Mutations in PRPF3 have been implicated in retinitis pigmentosa (RP) 18 through an unknown mechanism. The authors created and characterized Prpf3 knockout mice and zebrafish to determine whether RP18 is a result of haploinsufficiency. METHODS: Mice were produced from a Prpf3 gene trap cell line, and parameters of retinal function, structure, and RNA splicing were analyzed. The retinas of prpf3 insertional mutant zebrafish were also analyzed histologically. RESULTS: Homozygous Prpf3 knockout mice do not survive to 14 days postfertilization (dpf), implying that this allele is required for early embryonic development. Homozygous Prpf3 knockout zebrafish die by 4dpf, well beyond the mid-blastula transition at which transcription activates. Zebrafish knockout embryos reveal abnormally high levels of cell death in the developing eye. Heterozygous Prpf3 knockout mice have less than the expected 50% reduction in Prpf3 at the mRNA and protein levels, implying compensatory expression from the wild-type allele. The heterozygous mice develop normally, with no changes in retinal function, no evidence for photoreceptor degeneration at up to 23 months of age, and no decrease in pre-mRNA splicing of transcripts mutated in other forms of RP in the retina. Similarly, heterozygous prpf3 knockout zebrafish develop normally and show no retinal degeneration up to 12 months of age. CONCLUSIONS: These models suggest that RP18 is not a result of haploinsufficiency but instead arises from a toxic gain of function caused by missense mutations in PRPF3. PMID: 18552388 [PubMed - indexed for MEDLINE]
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Related Articles Wnt-signaling in retinal development and disease. Stem Cells Dev. 2009 Jan-Feb;18(1):7-16 Authors: Lad EM, Cheshier SH, Kalani MY Abstract The Wnt-signaling pathway is a known regulator of stem cell maintenance, cellular proliferation and differentiation, and cancer development in various tissues. Wnt proteins play a central role during various stages of retinal development; retinal field establishment, retinal and hyaloid vasculogenesis, cornea and lens development, eye field formation, and maintenance of retinal stem cell and neuronal specification in many species are Wnt-regulated processes. Uncontrolled Wnt signaling may cause retinal diseases such as familial exudative vitroretinopathy, retinitis pigmentosa, and Norrie's disease, further underscoring the importance of the Wnt-signaling pathway in the retina. This review summarizes major developments and discoveries regarding the role of the Wnt-signaling pathway as it pertains to retinal development and disease. PMID: 18690791 [PubMed - indexed for MEDLINE]
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Related Articles [Gene therapy in hereditary hearing loss. Future therapeutic possibility--maybe combined with stem cells]. Lakartidningen. 2008 Sep 3-9;105(36):2406-10 Authors: Palmgren B, Jin Z, Rosenhall U, Duan M PMID: 18831451 [PubMed - indexed for MEDLINE]
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Related Articles Retinal pigment epithelium differentiation of stem cells: current status and challenges. Crit Rev Biomed Eng. 2009;37(4-5):355-75 Authors: Uygun BE, Sharma N, Yarmush M Abstract Degeneration and loss of retinal pigment epithelium (RPE) is the cause of a number of degenerative retinal diseases, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, leading to blindness that affects three million Americans as of now. Transplantation of RPE aims to restore retinal structure and the interaction between the RPE and photoreceptors, which is fundamental to sight. Although a significant amount of progress has been made in the past 20 years in autologous RPE transplantation, sources for RPE cells are limited. Recent advances in stem cell culture and differentiation techniques have allowed the generation of RPE cells from pluripotent stem cells. In this review, we discuss strategies for generating functional RPE cells from human embryonic stem cells and induced pluripotent stem cells, and summarize transplantation studies of these derived RPEs. We conclude with challenges in cell-replacement therapies using human embryonic and induced pluripotent stem cell-derived RPEs. PMID: 20528731 [PubMed - indexed for MEDLINE]
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Related Articles Caspase-8 is involved in neovascularization-promoting progenitor cell functions. Arterioscler Thromb Vasc Biol. 2009 Apr;29(4):571-8 Authors: Scharner D, Rössig L, Carmona G, Chavakis E, Urbich C, Fischer A, Kang TB, Wallach D, Chiang YJ, Deribe YL, Dikic I, Zeiher AM, Dimmeler S Abstract OBJECTIVE: Endothelial progenitor cells (EPCs) comprise a heterogeneous population of cells, which improve therapeutic neovascularization after ischemia. The neovascularization-promoting potential of progenitor cells depends on survival and retention of the infused cells to the tissue. Caspases mediate apoptosis but are also involved in other critical biological processes. Therefore, we aimed to address the role of caspases in proangiogenic cells. METHODS AND RESULTS: The caspase-8 inhibitor zIETD abrogated the ex vivo formation of EPCs, inhibited EPC adhesion and migration, and reduced their capacity to improve neovascularization in vivo. Consistently, cells isolated from caspase-8-deficient mice exhibited a reduced capacity for enhancing neovascularization when transplanted into mice after hindlimb ischemia. Because inhibition of Caspase-8 reduced the adhesion and homing functions of EPCs, we further determined the surface expression of integrins and receptors involved in cell recruitment to ischemic tissues. Pharmacological inhibition of caspase-8 and genetic depletion of caspase-8 reduced the expression of the fibronectin receptor subunits alpha5 and beta1 and the SDF-1 receptor CXCR4. Moreover, we identified the E3 ubiquitin ligase Cbl-b, which negatively regulates integrin and receptor-mediated signaling, as a potential Caspase-8 substrate. CONCLUSIONS: In summary, our data demonstrate a novel apoptosis-unrelated role of caspase-8 in proangiogenic cells. PMID: 19122169 [PubMed - indexed for MEDLINE]
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Related Articles Dynamic expression of epidermal caspase 8 simulates a wound healing response. Nature. 2009 Mar 26;458(7237):519-23 Authors: Lee P, Lee DJ, Chan C, Chen SW, Ch'en I, Jamora C Abstract Tissue homeostasis and regeneration are regulated by an intricate balance of seemingly competing processes-proliferation versus differentiation, and cell death versus survival. Here we demonstrate that the loss of epidermal caspase 8, an important mediator of apoptosis, recapitulates several phases of a wound healing response in the mouse. The epidermal hyperplasia in the caspase 8 null skin is the culmination of signals exchanged between epidermal keratinocytes, dermal fibroblasts and leukocytic cells. This reciprocal interaction is initiated by the paracrine signalling of interleukin 1alpha (IL1alpha), which activates both skin stem cell proliferation and cutaneous inflammation. The non-canonical secretion of IL1alpha is induced by a p38-MAPK-mediated upregulation of NALP3 (also known as NLRP3), leading to inflammasome assembly and caspase 1 activation. Notably, the increased proliferation of basal keratinocytes is counterbalanced by the growth arrest of suprabasal keratinocytes in the stratified epidermis by IL1alpha-dependent NFkappaB signalling. Altogether, our findings illustrate how the loss of caspase 8 can affect more than programmed cell death to alter the local microenvironment and elicit processes common to wound repair and many neoplastic skin disorders. PMID: 19204729 [PubMed - indexed for MEDLINE]
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Related Articles Caspase-8, a double-edged sword for EPC functioning. Arterioscler Thromb Vasc Biol. 2009 Apr;29(4):444-6 Authors: Xiao Q, Xu Q PMID: 19299329 [PubMed - indexed for MEDLINE]
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Related Articles Ex vivo gene therapy using intravitreal injection of GDNF-secreting mouse embryonic stem cells in a rat model of retinal degeneration. Mol Vis. 2009;15:962-73 Authors: Gregory-Evans K, Chang F, Hodges MD, Gregory-Evans CY Abstract PURPOSE: Safe and prolonged drug delivery to the retina is a key obstacle to overcome in the development of new medicines aimed at treating progressive retinal disease. We took advantage of the ability of embryonic stem cells to survive long-term in foreign tissue and used these cells to deliver neuroprotectant molecules to the retina of the rhodopsin TgN S334ter-4 rat model of retinitis pigmentosa (RP). METHODS: Mouse embryonic stem (mES) cells, derived from the pluripotent embryonic stem cell line E14TG2a, were genetically engineered to oversecrete the glial cell-derived neurotrophic factor (GDNF). Cell suspensions, containing approximately 200,000 cells and expressing approximately 35ng/10(6) cells/24 h GDNF, were injected into the vitreous cavity of TgN S334ter rat eyes at postnatal day 21 (P21) without immunosuppression. Histological and immunofluorescence imaging was used to evaluate photoreceptor survival up to P90. Local (vitreous) and systemic (serum) concentrations of GDNF were determined and ocular side effects were monitored. RESULTS: Green fluorescent protein (GFP)-expressing mES cells were observed on the inner limiting membrane of the retina in retinal flatmounts up to P90. In cryostat sections at P45, some GFP-expressing cells had integrated into the inner retina, but did not migrate into the outer nuclear layer. After an initial lag period, the photoreceptor cell counts were significantly higher (p< or =0.05) in animals treated with GDNF-secreting mES cells than in untreated animals, principally in the peripheral retina. Several adverse side effects such as tractional detachments and areas of hyperplasia were seen in a minimal number of treated eyes. Abnormally high levels of GDNF in the peripheral circulation were also observed. CONCLUSIONS: ES cells engineered to secrete GDNF exerted a neuroprotective effect for at least three months on retinal structure in the TgN S334ter rat model of retinal degeneration. Immunosuppression was not required for this. Several adverse effects were identified which require further investigation to make cell-based delivery of neuroprotection a viable clinical strategy. PMID: 19461934 [PubMed - indexed for MEDLINE]
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Related Articles Activation of retinal stem cells in the proliferating marginal region of RCS rats during development of retinitis pigmentosa. Neurosci Lett. 2009 Nov 6;465(1):41-4 Authors: Jian Q, Xu H, Xie H, Tian C, Zhao T, Yin Z Abstract Retinal stem cells (RSCs) have been demonstrated at the proliferating marginal regions from the pars plana of ciliary body to the ciliary marginal zone (CMZ) in adult lower vertebrates and mammals. Investigations in the lower vertebrates have provided some evidence that RSCs can proliferate following retinal damage; however, the evidence that this occurs in mammals is not clear. In this study, we explored RSCs proliferation potential of adult mammalian in proliferating marginal regions of Royal College of Surgeons (RCS) rats, an animal model for retinitis pigmentosa (RP). The proliferation was evaluated using BrdU labeling, and Chx-10 as markers to discern progenitor cell of CMZ in Long-Evan's and RCS rats at different postnatal day (PND) after eye opening. We found that few Chx-10 and BrdU labeled cells in the proliferating marginal regions of Long-Evan's rats, which significantly increased in RCS rats at PND30 and PND60. Consistent with this, Chx-10/Vimentin double staining cells in the center retina of RCS rats increased significantly at PND30 after eye opening. In addition, mRNA expression of Shh, Ptch1 and Smo was up-regulated in RCS rats at PND60 compared to age-matched Long-Evan's rats, which revealed Shh/ptc pathway involving in the activation of RSCs. These results suggest that RSCs in the mammalian retinal proliferating marginal regions has the potential to regenerate following degeneration. PMID: 19651189 [PubMed - indexed for MEDLINE]
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Related Articles Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells. 2009 Oct;27(10):2427-34 Authors: Buchholz DE, Hikita ST, Rowland TJ, Friedrich AM, Hinman CR, Johnson LV, Clegg DO Abstract Human induced pluripotent stem cells (iPSCs) have great promise for cellular therapy, but it is unclear if they have the same potential as human embryonic stem cells (hESCs) to differentiate into specialized cell types. Ocular cells such as the retinal pigmented epithelium (RPE) are of particular interest because they could be used to treat degenerative eye diseases, including age-related macular degeneration and retinitis pigmentosa. We show here that iPSCs generated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which can then be isolated and cultured to form highly differentiated RPE monolayers. RPE derived from iPSCs (iPS-RPE) were analyzed with respect to gene expression, protein expression, and rod outer segment phagocytosis, and compared with cultured fetal human RPE (fRPE) and RPE derived from hESCs (hESC-RPE). iPS-RPE expression of marker mRNAs was quantitatively similar to that of fRPE and hESC-RPE, and marker proteins were appropriately expressed and localized in polarized monolayers. Levels of rod outer segment phagocytosis by iPS-RPE, fRPE, and hESC-RPE were likewise similar and dependent on integrin alpha v beta 5. This work shows that iPSCs can differentiate into functional RPE that are quantitatively similar to fRPE and hESC-RPE and further supports the finding that iPSCs are similar to hESCs in their differentiation potential. PMID: 19658190 [PubMed - indexed for MEDLINE]
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Related Articles Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell. 2009 Oct 2;5(4):396-408 Authors: Idelson M, Alper R, Obolensky A, Ben-Shushan E, Hemo I, Yachimovich-Cohen N, Khaner H, Smith Y, Wiser O, Gropp M, Cohen MA, Even-Ram S, Berman-Zaken Y, Matzrafi L, Rechavi G, Banin E, Reubinoff B Abstract Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases. PMID: 19796620 [PubMed - indexed for MEDLINE]
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Related Articles Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX. PLoS One. 2009;4(10):e7379 Authors: Roduit R, Escher P, Schorderet DF Abstract BACKGROUND: NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD). The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2)). NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients. PMID: 19823680 [PubMed - indexed for MEDLINE]
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Related Articles Cell transplantation to arrest early changes in an ush2a animal model. Invest Ophthalmol Vis Sci. 2010 Apr;51(4):2269-76 Authors: Lu B, Wang S, Francis PJ, Li T, Gamm DM, Capowski EE, Lund RD Abstract Purpose. Usher's syndrome is a combined deafness and blindness disorder caused by mutations in several genes with functions in both the retina and the ear. Here the authors studied morphologic and functional changes in an animal model, the Ush2a mouse, and explored whether transplantation of forebrain-derived progenitor cells might affect the progress of morphologic and functional deterioration. Methods. Ush2a mice were tested at postnatal days (P) 70 to P727 using an optomotor test, which provides a repeatable method of estimating rodent visual acuity and contrast sensitivity. A group of mice that received grafts of forebrain-derived progenitor cells at P80 was tested for up to 10 weeks after grafting. At the end of testing, animals were killed, and eyes were processed for histology. Results. The optomotor test showed that both acuity and contrast sensitivity deteriorated over time; contrast sensitivity showed a deficit even at P70. By contrast, photoreceptor loss was only evident later than 1 year of age, though changes in the intracellular distribution of red/green cone opsin were observed as early as P80. Mice that received transplanted cells performed significantly better than control mice and no longer demonstrated abnormal distribution of red/green opsin where the donor cells were distributed. Conclusions. This study showed that vision impairment was detected well before significant photoreceptor loss and was correlated with abnormal distribution of a cone pigment. Cell transplantation prevented functional deterioration for at least 10 weeks and reversed the mislocalization of cone pigment. PMID: 19959642 [PubMed - indexed for MEDLINE]
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Related Articles Differentiation of mesenchymal stem cell in the microenviroment of retinitis pigmentosa. Int J Ophthalmol. 2010;3(3):216-9 Authors: Huo DM, Dong FT, Yu WH, Gao F Abstract UNLABELLED: CORRESPONDENCE TO: Fang-Tian Dong. Department of Ophthalmology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China. d_fangtian@sina.com AIM: To access the differentiation of rat mesenchymal stem cell (MSC) in the microenvironment of retinal degeneration induced by the administration of sodium iodate. METHODS: In-vitro cultured Lewis rat MSC were injected into the sub-retinal space of NaIO(3) induced retinal degeneration rat eyes (30g/L NaIO(3) 100mg/kg). To observe the trace and differentiation of MSC by immuno-fluorescent method successively in 5 weeks after the surgery. RESULTS: The majority of the transplanted cells stay in retinal pigment epithelium layer and cones & rods layer. From the 2(nd) week after transplantation, the engrafted MSC express PCK and rhodopsin under fluorescent microscope. CONCLUSION: MSC can survive mainly in the outer layer of retina in the microenvironment of retinal degeneration and differentiate forward the RPE cell and photoreceptor. PMID: 22553557 [PubMed]
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Related Articles Targeted disruption of outer limiting membrane junctional proteins (Crb1 and ZO-1) increases integration of transplanted photoreceptor precursors into the adult wild-type and degenerating retina. Cell Transplant. 2010;19(4):487-503 Authors: Pearson RA, Barber AC, West EL, MacLaren RE, Duran Y, Bainbridge JW, Sowden JC, Ali RR Abstract Diseases culminating in photoreceptor loss are a major cause of untreatable blindness. Transplantation of rod photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. Nevertheless, the proportion of cells that integrate into the recipient outer nuclear layer (ONL) is low. The outer limiting membrane (OLM), formed by adherens junctions between Müller glia and photoreceptors, may impede transplanted cells from migrating into the recipient ONL. Adaptor proteins such as Crumbs homologue 1 (Crb1) and zona occludins (ZO-1) are essential for localization of the OLM adherens junctions. We investigated whether targeted disruption of these proteins enhances donor cell integration. Transplantation of rod precursors in wild-type mice achieved 949 +/- 141 integrated cells. By contrast, integration is significantly higher when rod precursors are transplanted into Crb1(rd8/rd8) mice, a model of retinitis pigmentosa and Lebers congenital amaurosis that lacks functional CRB1 protein and displays disruption of the OLM (7,819 +/- 1,297; maximum 15,721 cells). We next used small interfering (si)RNA to transiently reduce the expression of ZO-1 and generate a reversible disruption of the OLM. ZO-1 knockdown resulted in similar, significantly improved, integration of transplanted cells in wild-type mice (7,037 +/- 1,293; maximum 11,965 cells). Finally, as the OLM remains largely intact in many retinal disorders, we tested whether transient ZO-1 knockdown increased integration in a model of retinitis pigmentosa, the rho(-/-) mouse; donor cell integration was significantly increased from 313 +/- 58 cells without treatment to 919 +/- 198 cells after ZO-1 knockdown. This study shows that targeted disruption of OLM junctional proteins enhances integration in the wild-type and degenerating retina and may be a useful approach for developing photoreceptor transplantation strategies. PMID: 20089206 [PubMed - indexed for MEDLINE]
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Related Articles Induced pluripotent stem cells for retinal degenerative diseases: a new perspective on the challenges. J Genet. 2009 Dec;88(4):417-24 Authors: Jin ZB, Okamoto S, Mandai M, Takahashi M Abstract Retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, are the prodominant causes of human blindness in the world; however, these diseases are difficult to treat. Currently, knowledge on the mechanisms of these diseases is still very limited and no radical drugs are available. Induced pluripotent stem (iPS) cells are an innovative technology that turns somatic cells into embryonic stem (ES)-like cells with pluripotent potential via the exogenous expression of several key genes. It can be used as an unlimited source for cell differentiation or tissue engineering, either of which is a promising therapy for human degenerative diseases. Induced pluripotent cells are both an unlimited source for retinal regeneration and an expectant tool for pharmaprojects and developmental or disease modelling. In this review, we try to summarize the advancement of iPS-based technologies and the potential utility for retinal degenerative diseases. We also discuss the challenges of using this technology in the retinology field. PMID: 20090205 [PubMed - indexed for MEDLINE]
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Related Articles Deafness and retinal degeneration in a novel USH1C knock-in mouse model. Dev Neurobiol. 2010 Mar;70(4):253-67 Authors: Lentz JJ, Gordon WC, Farris HE, MacDonald GH, Cunningham DE, Robbins CA, Tempel BL, Bazan NG, Rubel EW, Oesterle EC, Keats BJ Abstract Usher syndrome is the leading cause of combined deaf-blindness, but the molecular mechanisms underlying the auditory and visual impairment are poorly understood. Usher I is characterized by profound congenital hearing loss, vestibular dysfunction, and progressive retinitis pigmentosa beginning in early adolescence. Using the c.216G>A cryptic splice site mutation in Exon 3 of the USH1C gene found in Acadian Usher I patients in Louisiana, we constructed the first mouse model that develops both deafness and retinal degeneration. The same truncated mRNA transcript found in Usher 1C patients is found in the cochleae and retinas of these knock-in mice. Absent auditory-evoked brainstem responses indicated that the mutant mice are deaf at 1 month of age. Cochlear histology showed disorganized hair cell rows, abnormal bundles, and loss of both inner and outer hair cells in the middle turns and at the base. Retinal dysfunction as evident by an abnormal electroretinogram was seen as early as 1 month of age, with progressive loss of rod photoreceptors between 6 and 12 months of age. This knock-in mouse reproduces the dual sensory loss of human Usher I, providing a novel resource to study the disease mechanism and the development of therapies. PMID: 20095043 [PubMed - indexed for MEDLINE]
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Related Articles Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa. Transplantation. 2010 Apr 27;89(8):911-9 Authors: Wang NK, Tosi J, Kasanuki JM, Chou CL, Kong J, Parmalee N, Wert KJ, Allikmets R, Lai CC, Chien CL, Nagasaki T, Lin CS, Tsang SH Abstract BACKGROUND: To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice. METHODS: Yellow fluorescent protein (YFP)-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice. Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation. RESULTS: RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue. CONCLUSIONS: ES cells can differentiate, morphologically, and functionally, into RPE-like cells. Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials. PMID: 20164818 [PubMed - indexed for MEDLINE]
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Related Articles Non-invasive stem cell therapy in a rat model for retinal degeneration and vascular pathology. PLoS One. 2010;5(2):e9200 Authors: Wang S, Lu B, Girman S, Duan J, McFarland T, Zhang QS, Grompe M, Adamus G, Appukuttan B, Lund R Abstract BACKGROUND: Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP. METHODOLOGY/PRINCIPAL FINDINGS: Animals received syngeneic MSCs (1x10(6) cells) by tail vein at an age before major photoreceptor loss. PRINCIPAL RESULTS: both rod and cone photoreceptors were preserved (5-6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs. CONCLUSIONS/SIGNIFICANCE: These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases. PMID: 20169166 [PubMed - indexed for MEDLINE]
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Related Articles Transcriptional regulation of the Alström syndrome gene ALMS1 by members of the RFX family and Sp1. Gene. 2010 Jul 15;460(1-2):20-9 Authors: Purvis TL, Hearn T, Spalluto C, Knorz VJ, Hanley KP, Sanchez-Elsner T, Hanley NA, Wilson DI Abstract Mutations in the human gene ALMS1 cause Alström syndrome, a disorder characterised by neurosensory degeneration, metabolic defects and cardiomyopathy. ALMS1 encodes a centrosomal protein implicated in the assembly and maintenance of primary cilia. Expression of ALMS1 varies between tissues and recent data suggest that its transcription is modulated during adipogenesis and growth arrest. However the ALMS1 promoter has not been defined. This study focused on identifying and characterising the ALMS1 proximal promoter, initially by using 5' RACE to map transcription start sites. Luciferase reporter assay and EMSA data strongly suggest that ALMS1 transcription is regulated by the ubiquitous factor Sp1. In addition, reporter assay, EMSA, chromatin immunoprecipitation and RNA interference data indicate that ALMS1 transcription is regulated by regulatory factor X (RFX) proteins. These transcription factors are cell-type restricted in their expression profile and known to regulate genes of the ciliogenic pathway. We show binding of RFX proteins to an evolutionarily conserved X-box in the ALMS1 proximal promoter and present evidence that these proteins are responsible for ALMS1 transcription during growth arrest induced by low serum conditions. In summary, this work provides the first data on transcription factors regulating general and context-specific transcription of the disease-associated gene ALMS1. PMID: 20381594 [PubMed - indexed for MEDLINE]
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Related Articles Ciliary neurotrophic factor: a survival and differentiation inducer in human retinal progenitors. In Vitro Cell Dev Biol Anim. 2010 Jul;46(7):635-46 Authors: Dutt K, Cao Y, Ezeonu I Abstract Retinitis pigmentosa, age-related macular degeneration, and Parkinson's disease remain major problems in the field of medicine. Some of the strategies being explored for treatment include replacement of damaged tissue by transplantation of healthy tissues or progenitor cells and delivery of neurotrophins to rescue degenerating tissue. One of the neurotrophins with promise is the ciliary neurotrophic factor (CNTF). In this study, we report the role played by CNTF in retinal cell differentiation and survival in retinal progenitors. We found that CNTF is a survival factor for multipotential human retinal cells and increased cell survival by 50%, over a 7-d period, under serum-free conditions, as determined by apoptotic assays (immunohistochemistry and flow cytometry). This effect is dose dependent with a maximum survival at a CNTF concentration of 20 ng/ml. We also report that CNTF might be a cell commitment factor, directing the differentiation mainly toward large multipolar cells with ganglionic and amacrine phenotype. These cells express tyrosine hydroxylase (amacrine cells) as well as, thy 1.1 and neuron-specific enolase (ganglionic cells). Additionally, there was also an increase in protein kinase C alpha, a protein expressed in rod and cone bipolars as well as cone photoreceptors and calbindin, a protein expressed in cone photoreceptors and horizontal cells. In our studies, CNTF doubled the number of cells with ganglionic phenotypes, and basic fibroblast growth factor doubled the number of cells with photoreceptor phenotype. Additionally, CNTF induced a subset of progenitors to undergo multiple rounds of cell division before acquiring the large multipolar ganglionic phenotype. Our conclusion is that CNTF could be an agent that has therapeutic potential and possibly induces differentiation of large multipolar ganglionic phenotype in a subset of progenitors. PMID: 20428961 [PubMed - indexed for MEDLINE]
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Related Articles Centriolar association of ALMS1 and likely centrosomal functions of the ALMS motif-containing proteins C10orf90 and KIAA1731. Mol Biol Cell. 2010 Nov 1;21(21):3617-29 Authors: Knorz VJ, Spalluto C, Lessard M, Purvis TL, Adigun FF, Collin GB, Hanley NA, Wilson DI, Hearn T Abstract Mutations in the human gene ALMS1 cause Alström syndrome, a rare progressive condition characterized by neurosensory degeneration and metabolic defects. ALMS1 protein localizes to the centrosome and has been implicated in the assembly and/or maintenance of primary cilia; however its precise function, distribution within the centrosome, and mechanism of centrosomal recruitment are unknown. The C-terminus of ALMS1 contains a region with similarity to the uncharacterized human protein C10orf90, termed the ALMS motif. Here, we show that a third human protein, the candidate centrosomal protein KIAA1731, contains an ALMS motif and that exogenously expressed KIAA1731 and C10orf90 localize to the centrosome. However, based on deletion analysis of ALMS1, the ALMS motif appears unlikely to be critical for centrosomal targeting. RNAi analyses suggest that C10orf90 and KIAA1731 have roles in primary cilium assembly and centriole formation/stability, respectively. We also show that ALMS1 localizes specifically to the proximal ends of centrioles and basal bodies, where it colocalizes with the centrosome cohesion protein C-Nap1. RNAi analysis reveals markedly diminished centrosomal levels of C-Nap1 and compromised cohesion of parental centrioles in ALMS1-depleted cells. In summary, these data suggest centrosomal functions for C10orf90 and KIAA1731 and new centriole-related functions for ALMS1. PMID: 20844083 [PubMed - indexed for MEDLINE]
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Related Articles Extended extraocular phenotype of PROM1 mutation in kindreds with known autosomal dominant macular dystrophy. Eur J Hum Genet. 2011 Feb;19(2):131-7 Authors: Arrigoni FI, Matarin M, Thompson PJ, Michaelides M, McClements ME, Redmond E, Clarke L, Ellins E, Mohamed S, Pavord I, Klein N, Hunt DM, Moore AT, Halcox J, Sisodiya SM Abstract Mutations in prominin 1 (PROM1) have been shown to result in retinitis pigmentosa, macular degeneration and cone-rod dystrophy. Because of the putative role of PROM1 in hippocampal neurogenesis, we examined two kindreds with the same R373C PROM1 missense mutation using our established paradigm to study brain structure and function. As the protein encoded by PROM1, known as CD133, is used to identify stem/progenitor cells that can be found in peripheral blood and reflect endothelial reparatory mechanisms, other parameters were subsequently examined that included measures of vascular function, endothelial function and angiogenic capacity. We found that aspects of endothelial function assayed ex vivo were abnormal in patients with the R373C PROM1 mutation, with impaired adhesion capacity and higher levels of cellular damage. We also noted renal infections, haematuria and recurrent miscarriages possibly reflecting consequences of abnormal tubular modelling. Further studies are needed to confirm these findings. PMID: 20859302 [PubMed - indexed for MEDLINE]
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Related Articles Stem cell-based therapeutic applications in retinal degenerative diseases. Stem Cell Rev. 2011 Jun;7(2):434-45 Authors: Huang Y, Enzmann V, Ildstad ST Abstract Retinal degenerative diseases that target photoreceptors or the adjacent retinal pigment epithelium (RPE) affect millions of people worldwide. Retinal degeneration (RD) is found in many different forms of retinal diseases including retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy, cataracts, and glaucoma. Effective treatment for retinal degeneration has been widely investigated. Gene-replacement therapy has been shown to improve visual function in inherited retinal disease. However, this treatment was less effective with advanced disease. Stem cell-based therapy is being pursued as a potential alternative approach in the treatment of retinal degenerative diseases. In this review, we will focus on stem cell-based therapies in the pipeline and summarize progress in treatment of retinal degenerative disease. PMID: 20859770 [PubMed - indexed for MEDLINE]
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Related Articles XIAP therapy increases survival of transplanted rod precursors in a degenerating host retina. Invest Ophthalmol Vis Sci. 2011 Mar;52(3):1567-72 Authors: Yao J, Feathers KL, Khanna H, Thompson D, Tsilfidis C, Hauswirth WW, Heckenlively JR, Swaroop A, Zacks DN Abstract PURPOSE: To assess the survival of rod precursor cells transplanted into the Rd9 mouse, a model of X-linked retinal degeneration, and the effect of antiapoptotic therapy with X-linked inhibitor of apoptosis (XIAP) on preventing cell loss. METHODS: Dissociated retinal cells from P4 Nrlp-GFP mice were transplanted into the subretinal space of 2-, 5-, and 8-month-old Rd9 mice. Histology, immunohistochemistry, and quantification of integrated cells were performed every month for up to 3 months after transplantation. XIAP delivery to donor cells was accomplished by transfection with adenoassociated virus (AAV-XIAP). Intraretinal activation of immune modulators was assessed using a quantitative real-time polymerase chain reaction-based immune response array. RESULTS: GFP-positive rod precursors were able to integrate into the outer nuclear layer (ONL) of the Rd9 retina. Transplanted cells underwent morphologic differentiation with the formation of inner and outer segments and synaptic projections to bipolar cells. Integration of donor cells into the ONL increased as a function of host age at the time of transplantation. The number of integrated cells was maximal at 1 month after transplantation and then decreased with time. Survival of integrated cells was significantly increased when donor cells were pretreated with AAV-XIAP. We did not detect any donor cell-specific activation of inflammation within the host retina. CONCLUSIONS: Survival of integrated cells decreases with time after transplantation but can be significantly increased with XIAP antiapoptotic therapy. Preventing programmed cell death through XIAP therapy may be an important component of future therapeutic retinal cell transplantation strategies. PMID: 20926819 [PubMed - indexed for MEDLINE]
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Related Articles Transplantation of photoreceptor and total neural retina preserves cone function in P23H rhodopsin transgenic rat. PLoS One. 2010;5(10):e13469 Authors: Yang Y, Mohand-Said S, Léveillard T, Fontaine V, Simonutti M, Sahel JA Abstract BACKGROUND: Transplantation as a therapeutic strategy for inherited retinal degeneration has been historically viewed to restore vision as a method by replacing the lost retinal cells and attempting to reconstruct the neural circuitry with stem cells, progenitor cells and mature neural retinal cells. METHODS AND FINDINGS: We present evidence for an alternative strategy aimed at preventing the secondary loss of cones, the most crucial photoreceptors for vision, by transplanting normal photoreceptors cells into the eye of the P23H rat, a model of dominant retinitis pigmentosa. We carried out transplantation of photoreceptors or total neural retina in 3-month-old P23H rats and evaluated the function and cell counts 6 months after surgery. In both groups, cone loss was significantly reduced (10%) in the transplanted eyes where the cone outer segments were found to be considerably longer. This morphological effect correlated with maintenance of the visual function of cones as scored by photopic ERG recording, but more precisely with an increase in the photopic b-wave amplitudes by 100% and 78% for photoreceptor transplantation and whole retinal transplantation respectively. CONCLUSIONS: We demonstrate here that the transplanted tissue prevents the loss of cone function, which is further translated into cone survival. PMID: 20976047 [PubMed - indexed for MEDLINE]
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Related Articles Stem cell plasticity, neuroprotection and regeneration in human eye diseases. Curr Stem Cell Res Ther. 2011 Mar;6(1):73-81 Authors: Rodríguez FD, Vecino E Abstract Regeneration and plasticity refer to the ability of certain progenitor cells to produce cell lineages with specific morphological and functional settings. The pathway from a less delineated or immature phenotype to a mature or specialized one follows intricate routes where a monumental array of molecular elements, basically transcription factors and epigenetic regulators that turn off or on a specific phenotypic change, play a fundamental role. Nature itself offers procedures to healing strategies. Therapy approaches to pathologies in the realm of ophthalmology may benefit from the knowledge of the properties and mechanisms of activation of different routes controlling the pathways of cell definition and differentiation. Specification of cell identity, not only in terms of phenotypic traits, but also regarding the mechanisms of gene expression and epigenetic regulation, will provide new tools to manipulating cell fates and status, both forward and backwards. In the human eye, two main locations shelter stem cells: the limbus, which is situated in the limit of the cornea and the conjunctiva, and the ciliary body pars plana. Transplantation of limbal cells is currently used in certain pathologies where corneal epithelium is damaged. Therapeutic applications of retina progenitors are not yet fully developed due to the complexity of the cellular components of the multilayer retinal architecture. Animal models of Retinitis pigmentosa or Glaucoma offer an interesting approach to validate certain techniques, such as the direct injection of progenitors into the vitreal compartment, aimed to restoring retinal function. PMID: 21190534 [PubMed - indexed for MEDLINE]
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Related Articles Possible mechanisms of retinal function recovery with the use of cell therapy with bone marrow-derived stem cells. Arq Bras Oftalmol. 2010 Sep-Oct;73(5):474-9 Authors: Siqueira RC, Voltarelli JC, Messias AM, Jorge R Abstract Bone marrow has been proposed as a potential source of stem cells for regenerative medicine. In the eye, degeneration of neural cells in the retina is a hallmark of such widespread ocular diseases as age-related macular degeneration (AMD) and retinitis pigmentosa. Bone marrow is an ideal tissue for studying stem cells mainly because of its accessibility. Furthermore, there are a number of well-defined mouse models and cell surface markers that allow effective study of hematopoiesis in healthy and injured mice. Because of these characteristics and the experience of bone marrow transplantation in the treatment of hematological disease such as leukemia, bone marrow-derived stem cells have also become a major tool in regenerative medicine. Those cells may be able to restore the retina function through different mechanisms: A) cellular differentiation, B) paracrine effect, and C) retinal pigment epithelium repair. In this review, we described these possible mechanisms of recovery of retinal function with the use of cell therapy with bone marrow-derived stem cells. PMID: 21225138 [PubMed - indexed for MEDLINE]
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Related Articles Modeling retinal degeneration using patient-specific induced pluripotent stem cells. PLoS One. 2011;6(2):e17084 Authors: Jin ZB, Okamoto S, Osakada F, Homma K, Assawachananont J, Hirami Y, Iwata T, Takahashi M Abstract Retinitis pigmentosa (RP) is the most common inherited human eye disease resulting in night blindness and visual defects. It is well known that the disease is caused by rod photoreceptor degeneration; however, it remains incurable, due to the unavailability of disease-specific human photoreceptor cells for use in mechanistic studies and drug screening. We obtained fibroblast cells from five RP patients with distinct mutations in the RP1, RP9, PRPH2 or RHO gene, and generated patient-specific induced pluripotent stem (iPS) cells by ectopic expression of four key reprogramming factors. We differentiated the iPS cells into rod photoreceptor cells, which had been lost in the patients, and found that they exhibited suitable immunocytochemical features and electrophysiological properties. Interestingly, the number of the patient-derived rod cells with distinct mutations decreased in vitro; cells derived from patients with a specific mutation expressed markers for oxidation or endoplasmic reticulum stress, and exhibited different responses to vitamin E than had been observed in clinical trials. Overall, patient-derived rod cells recapitulated the disease phenotype and expressed markers of cellular stresses. Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP. PMID: 21347327 [PubMed - indexed for MEDLINE]
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Related Articles PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa. Hum Mol Genet. 2011 Jun 1;20(11):2116-30 Authors: Tanackovic G, Ransijn A, Thibault P, Abou Elela S, Klinck R, Berson EL, Chabot B, Rivolta C Abstract Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell survival. PMID: 21378395 [PubMed - indexed for MEDLINE]
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Related Articles Pluripotent human stem cells for the treatment of retinal disease. J Cell Physiol. 2012 Feb;227(2):457-66 Authors: Rowland TJ, Buchholz DE, Clegg DO Abstract Despite advancements made in our understanding of ocular biology, therapeutic options for many debilitating retinal diseases remain limited. Stem cell-based therapies are a potential avenue for treatment of retinal disease, and this mini-review will focus on current research in this area. Cellular therapies to replace retinal pigmented epithelium (RPE) and/or photoreceptors to treat age-related macular degeneration (AMD), Stargardt's macular dystrophy, and retinitis pigmentosa are currently being developed. Over the past decade, significant advancements have been made using different types of human stem cells with varying capacities to differentiate into these target retinal cell types. We review and evaluate pluripotent stem cells, both human embryonic stem cells and human induced pluripotent stem cells, as well as protocols for differentiation of ocular cells, and culture and transplant techniques that might be used to deliver cells to patients. PMID: 21520078 [PubMed - indexed for MEDLINE]
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Related Articles Pharmacological enhancement of ex vivo gene therapy neuroprotection in a rodent model of retinal degeneration. Ophthalmic Res. 2012;47(1):32-8 Authors: Gregory-Evans K, Po K, Chang F, Gregory-Evans CY Abstract AIMS: We have previously shown the benefits of cell-based delivery of neuroprotection in a rodent model of retinitis pigmentosa (RP). In order to maximise the effectiveness of this approach, we hypothesised that this could be augmented by combination with an aminoglycoside known to limit the abnormal RNA translation seen in this model. METHODS: A rhodopsin TgN S334ter-4 rat model of RP underwent daily subcutaneous injection of 12.5 μg/g gentamicin from postnatal day 5 (P5). At P21, selected rats also underwent intravitreal injection of cells genetically engineered to oversecrete glial cell-derived neurotrophic factor. Histological imaging was undertaken to evaluate photoreceptor survival at P70 and compared with images from untreated TgN S334ter-4 rats and control Sprague-Dawley rats. RESULTS: Statistically significant (p < 0.05) improvements in outer retinal indices were seen with this combination strategy when compared with results in rats treated with individual therapies alone. This improvement was most apparent in the peripheral retina, where the greatest degeneration was observed. CONCLUSIONS: We have shown that the combination of neuroprotection plus aminoglycoside read-through in an animal model of retinal degeneration improved the histological appearance of the retina such that it was statistically indistinguishable from unaffected controls. Further functional and longitudinal studies of this approach are warranted. PMID: 21691141 [PubMed - indexed for MEDLINE]
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Related Articles Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa. Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):E569-76 Authors: Tucker BA, Scheetz TE, Mullins RF, DeLuca AP, Hoffmann JM, Johnston RM, Jacobson SG, Sheffield VC, Stone EM Abstract Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK-associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies. PMID: 21825139 [PubMed - indexed for MEDLINE]
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Related Articles A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. Stem Cells Dev. 2012 Aug 10;21(12):2262-72 Authors: Zahabi A, Shahbazi E, Ahmadieh H, Hassani SN, Totonchi M, Taei A, Masoudi N, Ebrahimi M, Aghdami N, Seifinejad A, Mehrnejad F, Daftarian N, Salekdeh GH, Baharvand H Abstract We describe a new, efficient protocol that involves the serial addition of noggin, basic fibroblast growth factor (bFGF), retinoic acid, and sonic hedgehog (Shh) for the differentiation of human induced pluripotent stem cells (hiPSC) to retinal pigmented epithelium (RPE) in a serum- and feeder-free adherent condition. hiPSC-RPE cells exhibited RPE morphology and specific molecular markers. Additionally, several hiPSC lines were generated from retinal-specific patients with Leber's congenital amaurosis, Usher syndrome, two patients with retinitis pigmentosa, and a patient with Leber's hereditary optic neuropathy. The RPE cells generated from these disease-specific hiPSCs expressed specific markers by the same RPE lineage-directed differentiation protocol. These findings indicate a new short-term, simple, and efficient protocol for differentiation of hiPSCs to RPE cells. Such specific retinal disease-specific hiPSCs offer an unprecedented opportunity to recapitulate normal and pathologic formation of human retinal cells in vitro, thereby enabling pharmaceutical screening, and potentially autologous cell replacement therapies for retinal diseases. PMID: 22145677 [PubMed - indexed for MEDLINE]
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Related Articles Stanniocalcin-1 rescued photoreceptor degeneration in two rat models of inherited retinal degeneration. Mol Ther. 2012 Apr;20(4):788-97 Authors: Roddy GW, Rosa RH, Oh JY, Ylostalo JH, Bartosh TJ, Choi H, Lee RH, Yasumura D, Ahern K, Nielsen G, Matthes MT, LaVail MM, Prockop DJ Abstract Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD). PMID: 22294148 [PubMed - indexed for MEDLINE]
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Related Articles Norgestrel may be a potential therapy for retinal degenerations. Expert Opin Investig Drugs. 2012 May;21(5):579-81 Authors: Doonan F, Cotter TG Abstract Retinal degenerations cover a broad spectrum of diseases, retinitis pigmentosa being the most common inherited retinal degeneration. It remains an untreatable disorder, partly owing to its genetic complexity and variability. Gene therapies, stem cell transplantation and administration of slow release growth factors are some of the treatments currently under development for the treatment of this disease. More recently, steroid hormones, now known to have functions within the CNS aside from their traditional targets, have been suggested as potential therapeutic agents. Progestogenic hormones are thought to modulate pro-survival pathways in the retina and since these hormones are produced naturally in the body their value as potential therapeutic agents is clear. Current data detailing the pro-survival effects of progestogens in the brain and particularly in the eye will be discussed. PMID: 22375616 [PubMed - indexed for MEDLINE]
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Related Articles Focused magnetic stem cell targeting to the retina using superparamagnetic iron oxide nanoparticles. Cell Transplant. 2012;21(6):1137-48 Authors: Yanai A, Häfeli UO, Metcalfe AL, Soema P, Addo L, Gregory-Evans CY, Po K, Shan X, Moritz OL, Gregory-Evans K Abstract Developing new ways of delivering cells to diseased tissue will be a key factor in translating cell therapeutics research into clinical use. Magnetically targeting cells enables delivery of significant numbers of cells to key areas of specific organs. To demonstrate feasibility in neurological tissue, we targeted cells magnetically to the upper hemisphere of the rodent retina. Rat mesenchymal stem cells (MSCs) were magnetized using superparamagnetic iron oxide nanoparticles (SPIONs). In vitro studies suggested that magnetization with fluidMAG-D was well tolerated, that cells remained viable, and they retained their differentiation capabilities. FluidMAG-D-labeled MSCs were injected intravitreally or via the tail vein of the S334ter-4 transgenic rat model of retinal degeneration with or without placing a gold-plated neodymium disc magnet within the orbit, but outside the eye. Retinal flatmount and cryosection imaging demonstrated that after intravitreal injection cells localized to the inner retina in a tightly confined area corresponding to the position of the orbital magnet. After intravenous injection, similar retinal localization was achieved and remarkably was associated with a tenfold increase in magnetic MSC delivery to the retina. Cryosections demonstrated that cells had migrated into both the inner and outer retina. Magnetic MSC treatment with orbital magnet also resulted in significantly higher retinal concentrations of anti-inflammatory molecules interleukin-10 and hepatocyte growth factor. This suggested that intravenous MSC therapy also resulted in significant therapeutic benefit in the dystrophic retina. With minimal risk of collateral damage, these results suggest that magnetic cell delivery is the best approach for controlled delivery of cells to the outer retina-the focus for disease in age-related macular degeneration and retinitis pigmentosa. PMID: 22405427 [PubMed - indexed for MEDLINE]
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Related Articles Assessment of hereditary retinal degeneration in the English springer spaniel dog and disease relationship to an RPGRIP1 mutation. Stem Cells Int. 2012;2012:685901 Authors: Narfström K, Jeong M, Hyman J, Madsen RW, Bergström TF Abstract Intensive breeding and selection on desired traits have produced high rates of inherited diseases in dogs. Hereditary retinal degeneration, often called progressive retinal atrophy (PRA), is prevalent in dogs with disease entities comparable to human retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA). Recent molecular studies in the English Springer Spaniel (ESS) dog have shown that PRA cases are often homozygous for a mutation in the RPGRIP1 gene, the defect also causing human RP, LCA, and cone rod dystrophies. The present study characterizes the disease in a group of affected ESS in USA, using clinical, functional, and morphological studies. An objective evaluation of retinal function using electroretinography (ERG) is further performed in a masked fashion in a group of American ESS dogs, with the examiner masked to the genetic status of the dogs. Only 4 of 6 homozygous animals showed clinical signs of disease, emphasizing the need and importance for more precise studies on the clinical expression of molecular defects before utilizing animal models for translational research, such as when using stem cells for therapeutic intervention. PMID: 22550515 [PubMed]
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Related Articles Transplanted olfactory ensheathing cells reduce retinal degeneration in Royal College of Surgeons rats. Curr Eye Res. 2012 Aug;37(8):749-58 Authors: Huo SJ, Li YC, Xie J, Li Y, Raisman G, Zeng YX, He JR, Weng CH, Yin ZQ Abstract PURPOSE OF THE STUDY: Retinitis pigmentosa (RP) is a group of genetic disorders and a slow loss of vision that is caused by a cascade of retinal degenerative events. We examined whether these retinal degenerative events were reduced after cultured mixtures of adult olfactory ensheathing cells (OECs) and olfactory nerve fibroblasts (ONFs) were transplanted into the subretinal space of 1-month-old RCS rat, a classic model of RP. MATERIALS AND METHODS: The changes in retinal photoreceptors and Müller cells of RCS rats after cell transplantation were observed by the expression of recoverin and glial fibrillary acidic protein (GFAP), counting peanut agglutinin (PNA)-positive cone outer segments and calculating the relative apoptotic area. The retinal function was also evaluated by Flash electroretinography (ERG). To further investigate the mechanisms, by which OECs/ONFs play important roles in the transplanted retinas, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF) secretion of the cultured cells were analyzed by ELISA. The ability of OECs/ONFs to ingest porcine retinal outer segments and the amount of phagocytosis were compared with retinal pigment epithelium (RPE) cells. RESULTS: Our research showed that the transplantation of OECs/ONFs mixtures restored recoverin expression, protected retinal outer segments, increased PNA-positive cone outer segments, reduced caspase-positive apoptotic figures, downregulated GFAP, and maintained the b-wave of the ERG. Cultured OECs/ONFs expressed and secreted NGF, BDNF, and bFGF which made contributions to assist survival of the photoreceptors. An in vitro phagocytosis assay showed that OECs, but not ONFs, phagocytosed porcine retinal outer segments, and the phagocytic ability of OECs was even superior to that of RPE cells. CONCLUSIONS: These findings demonstrate that transplantation of OECs/ONFs cleaned up the accumulated debris in subretinal space, and provided an intrinsic continuous supply of neurotrophic factors. It suggested that transplantation of OECs/ONFs might be a possible future route for protection of the retina and reducing retinal degeneration in RP. PMID: 22691022 [PubMed - indexed for MEDLINE]
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Related Articles Cell replacement and visual restoration by retinal sheet transplants. Prog Retin Eye Res. 2012 Nov;31(6):661-87 Authors: Seiler MJ, Aramant RB Abstract Retinal diseases such as age-related macular degeneration (ARMD) and retinitis pigmentosa (RP) affect millions of people. Replacing lost cells with new cells that connect with the still functional part of the host retina might repair a degenerating retina and restore eyesight to an unknown extent. A unique model, subretinal transplantation of freshly dissected sheets of fetal-derived retinal progenitor cells, combined with its retinal pigment epithelium (RPE), has demonstrated successful results in both animals and humans. Most other approaches are restricted to rescue endogenous retinal cells of the recipient in earlier disease stages by a 'nursing' role of the implanted cells and are not aimed at neural retinal cell replacement. Sheet transplants restore lost visual responses in several retinal degeneration models in the superior colliculus (SC) corresponding to the location of the transplant in the retina. They do not simply preserve visual performance - they increase visual responsiveness to light. Restoration of visual responses in the SC can be directly traced to neural cells in the transplant, demonstrating that synaptic connections between transplant and host contribute to the visual improvement. Transplant processes invade the inner plexiform layer of the host retina and form synapses with presumable host cells. In a Phase II trial of RP and ARMD patients, transplants of retina together with its RPE improved visual acuity. In summary, retinal progenitor sheet transplantation provides an excellent model to answer questions about how to repair and restore function of a degenerating retina. Supply of fetal donor tissue will always be limited but the model can set a standard and provide an informative base for optimal cell replacement therapies such as embryonic stem cell (ESC)-derived therapy. PMID: 22771454 [PubMed - indexed for MEDLINE]
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Related Articles The mechanosensory structure of the hair cell requires clarin-1, a protein encoded by Usher syndrome III causative gene. J Neurosci. 2012 Jul 11;32(28):9485-98 Authors: Geng R, Melki S, Chen DH, Tian G, Furness DN, Oshima-Takago T, Neef J, Moser T, Askew C, Horwitz G, Holt JR, Imanishi Y, Alagramam KN Abstract Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1(-/-)) show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca(2+) currents and membrane capacitance from inner hair cells that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] loading, and transduction currents pointed to diminished cochlear hair bundle function in Clrn1(-/-) mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip links and staircase arrangement of stereocilia were not primarily affected by Clrn1(-/-) mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant p.N48K failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p.N48K in clarin-1 (Clrn1(N48K)) supports our in vitro and Clrn1(-/-) mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Furthermore, the ear phenotype in the Clrn1(N48K) mouse suggests that it is a valuable model for ear disease in CLRN1(N48K), the most prevalent Usher syndrome III mutation in North America. PMID: 22787034 [PubMed - indexed for MEDLINE]
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Related Articles Long-term safety and efficacy of human-induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa. Mol Med. 2012;18:1312-9 Authors: Li Y, Tsai YT, Hsu CW, Erol D, Yang J, Wu WH, Davis RJ, Egli D, Tsang SH Abstract The U.S. Food and Drug Administration recently approved phase I/II clinical trials for embryonic stem (ES) cell-based retinal pigmented epithelium (RPE) transplantation, but this allograft transplantation requires lifelong immunosuppressive therapy. Autografts from patient-specific induced pluripotent stem (iPS) cells offer an alternative solution to this problem. However, more data are required to establish the safety and efficacy of iPS transplantation in animal models before moving iPS therapy into clinical trials. This study examines the efficacy of iPS transplantation in restoring functional vision in Rpe65(rd12)/Rpe65(rd12) mice, a clinically relevant model of retinitis pigmentosa (RP). Human iPS cells were differentiated into morphologically and functionally RPE-like tissue. Quantitative real-time polymerase chain reaction (RT-PCR) and immunoblots confirmed RPE fate. The iPS-derived RPE cells were injected into the subretinal space of Rpe65(rd12)/Rpe65(rd12) mice at 2 d postnatally. After transplantation, the long-term surviving iPS-derived RPE graft colocalized with the host native RPE cells and assimilated into the host retina without disruption. None of the mice receiving transplants developed tumors over their lifetimes. Furthermore, electroretinogram, a standard method for measuring efficacy in human trials, demonstrated improved visual function in recipients over the lifetime of this RP mouse model. Our study provides the first direct evidence of functional recovery in a clinically relevant model of retinal degeneration using iPS transplantation and supports the feasibility of autologous iPS cell transplantation for retinal and macular degenerations featuring significant RPE loss. PMID: 22895806 [PubMed - indexed for MEDLINE]
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Related Articles Resolution of macular oedema associated with retinitis pigmentosa after intravitreal use of autologous BM-derived hematopoietic stem cell transplantation. Bone Marrow Transplant. 2013 Apr;48(4):612-3 Authors: Siqueira RC, Messias A, Voltarelli JC, Messias K, Arcieri RS, Jorge R PMID: 23000646 [PubMed - indexed for MEDLINE]
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Related Articles Protective effect of carnosic acid, a pro-electrophilic compound, in models of oxidative stress and light-induced retinal degeneration. Invest Ophthalmol Vis Sci. 2012 Nov;53(12):7847-54 Authors: Rezaie T, McKercher SR, Kosaka K, Seki M, Wheeler L, Viswanath V, Chun T, Joshi R, Valencia M, Sasaki S, Tozawa T, Satoh T, Lipton SA Abstract PURPOSE: The herb rosemary has been reported to have antioxidant and anti-inflammatory activity. We have previously shown that carnosic acid (CA), present in rosemary extract, crosses the blood-brain barrier to exert neuroprotective effects by upregulating endogenous antioxidant enzymes via the Nrf2 transcriptional pathway. Here we investigated the antioxidant and neuroprotective activity of CA in retinal cell lines exposed to oxidative stress and in a rat model of light-induced retinal degeneration (LIRD). METHODS: Retina-derived cell lines ARPE-19 and 661W treated with hydrogen peroxide were used as in vitro models for testing the protective activity of CA. For in vivo testing, dark-adapted rats were given intraperitoneal injections of CA prior to exposure to white light to assess protection of the photoreceptor cells. Retinal damage was assessed by measuring outer nuclear layer thickness and by electroretinogram (ERG). RESULTS: In vitro, CA significantly protected retina-derived cell lines (ARPE-19 and 661W) against H(2)O(2)-induced toxicity. CA induced antioxidant phase 2 enzymes and reduced formation of hyperoxidized peroxiredoxin (Prx)2. Similarly, we found that CA protected retinas in vivo from LIRD, producing significant improvement in outer nuclear layer thickness and ERG activity. CONCLUSIONS: These findings suggest that CA may potentially have clinical application to diseases affecting the outer retina, including age-related macular degeneration and retinitis pigmentosa, in which oxidative stress is thought to contribute to disease progression. PMID: 23081978 [PubMed - indexed for MEDLINE]
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Related Articles Brief report: self-organizing neuroepithelium from human pluripotent stem cells facilitates derivation of photoreceptors. Stem Cells. 2013 Feb;31(2):408-14 Authors: Boucherie C, Mukherjee S, Henckaerts E, Thrasher AJ, Sowden JC, Ali RR Abstract Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here, we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx(+) photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells, facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling. PMID: 23132794 [PubMed - indexed for MEDLINE]
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Related Articles Shaping the eye from embryonic stem cells: Biological and medical implications. World J Stem Cells. 2012 Aug 26;4(8):80-6 Authors: Colozza G, Locker M, Perron M Abstract Organogenesis is regulated by a complex network of intrinsic cues, diffusible signals and cell/cell or cell/matrix interactions that drive the cells of a prospective organ to differentiate and collectively organize in three dimensions. Generating organs in vitro from embryonic stem (ES) cells may provide a simplified system to decipher how these processes are orchestrated in time and space within particular and between neighboring tissues. Recently, this field of stem cell research has also gained considerable interest for its potential applications in regenerative medicine. Among human pathologies for which stem cell-based therapy is foreseen as a promising therapeutic strategy are many retinal degenerative diseases, like retinitis pigmentosa and age-related macular degeneration. Over the last decade, progress has been made in producing ES-derived retinal cells in vitro, but engineering entire synthetic retinas was considered beyond reach. Recently however, major breakthroughs have been achieved with pioneer works describing the extraordinary self-organization of murine and human ES cells into a three dimensional structure highly resembling a retina. ES-derived retinal cells indeed assemble to form a cohesive neuroepithelial sheet that is endowed with the intrinsic capacity to recapitulate, outside an embryonic environment, the main steps of retinal morphogenesis as observed in vivo. This represents a tremendous advance that should help resolving fundamental questions related to retinogenesis. Here, we will discuss these studies, and the potential applications of such stem cell-based systems for regenerative medicine. PMID: 23189212 [PubMed]
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Related Articles Integration-free induced pluripotent stem cells derived from retinitis pigmentosa patient for disease modeling. Stem Cells Transl Med. 2012 Jun;1(6):503-9 Authors: Jin ZB, Okamoto S, Xiang P, Takahashi M Abstract We investigated retinitis pigmentosa (RP) caused by a mutation in the gene rhodopsin (RHO) with a patient-specific rod cell model generated from induced pluripotent stem cells (iPSCs) derived from an RP patient. To generate the iPSCs and to avoid the unpredictable side effects associated with retrovirus integration at random loci in the host genome, a nonintegrating Sendai-virus vector was installed with four key reprogramming gene factors (POU5F1, SOX2, KLF4, and c-MYC) in skin cells from an RP patient. Subsequent selection of the iPSC lines was on the basis of karyotype analysis as well as in vitro and in vivo pluripotency tests. Using a serum-free, chemically defined, and stepwise differentiation method, the expressions of specific markers were sequentially induced in a neural retinal progenitor, a retinal pigment epithelial (RPE) progenitor, a photoreceptor precursor, RPE cells, and photoreceptor cells. In the differentiated rod cells, diffused distribution of RHO protein in cytoplasm and expressions of endoplasmic reticulum (ER) stress markers strongly indicated the involvement of ER stress. Furthermore, the rod cell numbers decreased significantly after successive culture, suggesting an in vitro model of rod degeneration. Thus, from integration-free patient-specific iPSCs, RP patient-specific rod cells were generated in vitro that recapitulated the disease feature and revealed evidence of ER stress in this patient, demonstrating its utility for disease modeling in vitro. PMID: 23197854 [PubMed - indexed for MEDLINE]
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Related Articles Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation. Proc Natl Acad Sci U S A. 2013 Jan 15;110(3):1101-6 Authors: Singh MS, Charbel Issa P, Butler R, Martin C, Lipinski DM, Sekaran S, Barnard AR, MacLaren RE Abstract One strategy to restore vision in retinitis pigmentosa and age-related macular degeneration is cell replacement. Typically, patients lose vision when the outer retinal photoreceptor layer is lost, and so the therapeutic goal would be to restore vision at this stage of disease. It is not currently known if a degenerate retina lacking the outer nuclear layer of photoreceptor cells would allow the survival, maturation, and reconnection of replacement photoreceptors, as prior studies used hosts with a preexisting outer nuclear layer at the time of treatment. Here, using a murine model of severe human retinitis pigmentosa at a stage when no host rod cells remain, we show that transplanted rod precursors can reform an anatomically distinct and appropriately polarized outer nuclear layer. A trilaminar organization was returned to rd1 hosts that had only two retinal layers before treatment. The newly introduced precursors were able to resume their developmental program in the degenerate host niche to become mature rods with light-sensitive outer segments, reconnecting with host neurons downstream. Visual function, assayed in the same animals before and after transplantation, was restored in animals with zero rod function at baseline. These observations suggest that a cell therapy approach may reconstitute a light-sensitive cell layer de novo and hence repair a structurally damaged visual circuit. Rather than placing discrete photoreceptors among preexisting host outer retinal cells, total photoreceptor layer reconstruction may provide a clinically relevant model to investigate cell-based strategies for retinal repair. PMID: 23288902 [PubMed - indexed for MEDLINE]
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Related Articles Hypoxia increases the yield of photoreceptors differentiating from mouse embryonic stem cells and improves the modeling of retinogenesis in vitro. Stem Cells. 2013 May;31(5):966-78 Authors: Garita-Hernández M, Diaz-Corrales F, Lukovic D, González-Guede I, Diez-Lloret A, Valdés-Sánchez ML, Massalini S, Erceg S, Bhattacharya SS Abstract Retinitis pigmentosa (RP), a genetically heterogeneous group of diseases together with age-related macular degeneration (AMD), are the leading causes of permanent blindness and are characterized by the progressive dysfunction and death of the light sensing photoreceptors of the retina. Due to the limited regeneration capacity of the mammalian retina, the scientific community has invested significantly in trying to obtain retinal progenitor cells from embryonic stem cells (ESC). These represent an unlimited source of retinal cells, but it has not yet been possible to achieve specific populations, such as photoreceptors, efficiently enough to allow them to be used safely in the future as cell therapy of RP or AMD. In this study, we generated a high yield of photoreceptors from directed differentiation of mouse ESC (mESC) by recapitulating crucial phases of retinal development. We present a new protocol of differentiation, involving hypoxia and taking into account extrinsic and intrinsic cues. These include niche-specific conditions as well as the manipulation of the signaling pathways involved in retinal development. Our results show that hypoxia promotes and improves the differentiation of mESC toward photoreceptors. Different populations of retinal cells are increased in number under the hypoxic conditions applied, such as Crx-positive cells, S-Opsin-positive cells, and double positive cells for Rhodopsin and Recoverin, as shown by immunofluorescence analysis. For the first time, this manuscript reports the high efficiency of differentiation in vivo and the expression of mature rod photoreceptor markers in a large number of differentiated cells, transplanted in the subretinal space of wild-type mice. PMID: 23362204 [PubMed - indexed for MEDLINE]
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Related Articles Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness. Nat Med. 2013 Mar;19(3):345-50 Authors: Lentz JJ, Jodelka FM, Hinrich AJ, McCaffrey KE, Farris HE, Spalitta MJ, Bazan NG, Duelli DM, Rigo F, Hastings ML Abstract Hearing impairment is the most common sensory disorder, with congenital hearing impairment present in approximately 1 in 1,000 newborns. Hereditary deafness is often mediated by the improper development or degeneration of cochlear hair cells. Until now, it was not known whether such congenital failures could be mitigated by therapeutic intervention. Here we show that hearing and vestibular function can be rescued in a mouse model of human hereditary deafness. An antisense oligonucleotide (ASO) was used to correct defective pre-mRNA splicing of transcripts from the USH1C gene with the c.216G>A mutation, which causes human Usher syndrome, the leading genetic cause of combined deafness and blindness. Treatment of neonatal mice with a single systemic dose of ASO partially corrects Ush1c c.216G>A splicing, increases protein expression, improves stereocilia organization in the cochlea, and rescues cochlear hair cells, vestibular function and low-frequency hearing in mice. These effects were sustained for several months, providing evidence that congenital deafness can be effectively overcome by treatment early in development to correct gene expression and demonstrating the therapeutic potential of ASOs in the treatment of deafness. PMID: 23380860 [PubMed - indexed for MEDLINE]
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Related Articles Mesenchymal stem cells from trabecular meshwork become photoreceptor-like cells on amniotic membrane. Neurosci Lett. 2013 Apr 29;541:43-8 Authors: Nadri S, Yazdani S, Arefian E, Gohari Z, Eslaminejad MB, Kazemi B, Soleimani M Abstract Stem cell therapy is a promising approach for treatment of degenerative retinal disorders such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). In this study, human mesenchymal stem cells (MSCs) were isolated from the trabecular meshwork (TM), the major functional tissue of the anterior chamber angle in the eye, were characterized and differentiated into photoreceptor cells on amniotic membrane (AM). After isolation of trabecular meshwork and culture of the stromal segment of this tissue, fibroblast-like cells (CD105(+), CD90(+), CD44(+), CD166(+) cells) capable of differentiation toward mesenchymal and photoreceptor lineages were obtained. The isolated cells were seeded on amniotic membrane and were treated with induction medium. Immunocytochemistry and quantitative real time RT-PCR (qPCR) were used to detect expression of photoreceptor genes such as rhodopsin, recoverin, CRX, and peripherin; and the bipolar cell marker protein kinase C alpha (PKC-alpha). As a result, immunocytochemistry revealed that the differentiated TMMSCs expressed rhodopsin, CRX and PKC proteins. qPCR showed the expression of rhodopsin (rod like photoreceptor-specific marker), and CRX genes were significantly higher in TMMSCs differentiated on AM than those differentiated on tissue culture polystyrene (TCPS). In conclusion, our findings suggested that a combination of TMMSCs (as a new source) and basement membrane support from AM might be a suitable source of cells for subretinal transplantation in regenerative therapy for retinal disorders such as AMD and RP. PMID: 23403103 [PubMed - indexed for MEDLINE]
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Related Articles Developing rods transplanted into the degenerating retina of Crx-knockout mice exhibit neural activity similar to native photoreceptors. Stem Cells. 2013 Jun;31(6):1149-59 Authors: Homma K, Okamoto S, Mandai M, Gotoh N, Rajasimha HK, Chang YS, Chen S, Li W, Cogliati T, Swaroop A, Takahashi M Abstract Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age-related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild-type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with green fluorescent protein (GFP) driven by the promoter of rod differentiation factor, Nrl. GFP-tagged developing rods show Ca(2 +) responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization-activated current (Ih ) induced by the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. When transplanted into the subretinal space of wild-type or retinal degeneration mice, GFP-tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild-type mouse retina and exhibit Ca(2 +) responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin-positive outer segment-like structures within 2 weeks after transplantation into the retina of Crx-knockout mice and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP-positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod-specific phototransduction genes, and HCN-1 channels. We conclude that Nrl-promoter-driven GFP-tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell-derived rods in vitro may provide a renewable source for cell-replacement therapy. PMID: 23495178 [PubMed - indexed for MEDLINE]
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Related Articles Heme and FLVCR-related transporter families SLC48 and SLC49. Mol Aspects Med. 2013 Apr-Jun;34(2-3):669-82 Authors: Khan AA, Quigley JG Abstract Heme is critical for a variety of cellular processes, but excess intracellular heme may result in oxidative stress and membrane injury. Feline leukemia virus subgroup C receptor (FLVCR1), a member of the SLC49 family of four paralogous genes, is a cell surface heme exporter, essential for erythropoiesis and systemic iron homeostasis. Disruption of FLVCR1 function blocks development of erythroid progenitors, likely due to heme toxicity. Mutations of SLC49A1 encoding FLVCR1 are noted in patients with a rare neurodegenerative disorder: posterior column ataxia with retinitis pigmentosa. FLVCR2 is highly homologous to FLVCR1 and may function as a cellular heme importer. Mutations of SLC49A2 encoding FLVCR2 are observed in Fowler syndrome, a rare proliferative vascular disorder of the brain. The functions of the remaining members of the SLC49 family, MFSD7 and DIRC2 (encoded by the SLC49A3 and SLC49A4 genes), are unknown, although the latter is implicated in hereditary renal carcinomas. SLC48A1 (heme responsive gene-1, HRG-1), the sole member of the SLC48 family, is associated with the endosome and appears to transport heme from the endosome into the cytosol. PMID: 23506900 [PubMed - indexed for MEDLINE]
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Related Articles Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells. Cell Res. 2013 Jun;23(6):788-802 Authors: Li T, Lewallen M, Chen S, Yu W, Zhang N, Xie T Abstract Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptor-deficient mice, but there is still some concern of tumor formation. In this study, we have successfully cultured Nestin(+)Sox2(+)Pax6(+) multipotent retinal stem cells (RSCs) from the adult mouse retina, which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation. After they have been expanded for over 35 passages in the presence of FGF and EGF, the cultured RSCs still maintain stable proliferation and differentiation potential. Under proper differentiation conditions, they can differentiate into all the major retinal cell types found in the adult retina. More importantly, they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions. Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes, RSC-derived photoreceptor cells integrate into the retina, morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons. When transplanted into eyes of photoreceptor-deficient rd1 mutant mice, a RP model, RSC-derived photoreceptors can partially restore light response, indicating that those RSC-derived photoreceptors are functional. Finally, there is no evidence for tumor formation in the photoreceptor-transplanted eyes. Therefore, this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD. PMID: 23567557 [PubMed - indexed for MEDLINE]
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Related Articles High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds. Mol Biol Rep. 2013 Jun;40(6):3883-90 Authors: Nadri S, Kazemi B, Eslaminejad MB, Eeslaminejad MB, Yazdani S, Soleimani M Abstract Transplantation of stem cells using biodegradable and biocompatible nanofibrous scaffolds is a promising therapeutic approach for treating inherited retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. In this study, conjunctiva mesenchymal stem cells (CJMSCs) were seeded onto poly-L-lactic acid (PLLA) nanofibrous scaffolds and were induced to differentiate toward photoreceptor cell lineages. Furthermore, the effects of orientation of scaffold on photoreceptor differentiation were examined. Scanning electron microscopy (SEM) imaging, quantitative real time RT-PCR (qPCR) and immunocytochemistry were used to analyze differentiated cells and their expression of photoreceptor-specific genes. Our observations demonstrated the differentiation of CJMSCs to photoreceptor cells on nanofibrous scaffolds and suggested their potential application in retinal regeneration. SEM imaging showed that CJMSCs were spindle shaped and well oriented on the aligned nanofiber scaffolds. The expression of rod photoreceptor-specific genes was significantly higher in CJMSCs differentiated on randomly-oriented nanofibers compared to those on aligned nanofibers. According to our results we may conclude that the nanofibrous PLLA scaffold reported herein could be used as a potential cell carrier for retinal tissue engineering and a combination of electrospun nanofiber scaffolds and MSC-derived conjunctiva stromal cells may have potential application in retinal regenerative therapy. PMID: 23588957 [PubMed - indexed for MEDLINE]
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Related Articles Poly(ADP-ribose) glycohydrolase and poly(ADP-ribose)-interacting protein Hrp38 regulate pattern formation during Drosophila eye development. Gene. 2013 Sep 10;526(2):187-94 Authors: Ji Y, Jarnik M, Tulin AV Abstract Drosophila Hrp38, a homolog of human hnRNP A1, has been shown to regulate splicing, but its function can be modified by poly(ADP-ribosyl)ation. Notwithstanding such findings, our understanding of the roles of poly(ADP-ribosyl)ated Hrp38 on development is limited. Here, we have demonstrated that Hrp38 is essential for fly eye development based on a rough-eye phenotype with disorganized ommatidia observed in adult escapers of the hrp38 mutant. We also observed that poly(ADP-ribose) glycohydrolase (Parg) loss-of-function, which caused increased Hrp38 poly(ADP-ribosyl)ation, also resulted in the rough-eye phenotype with disrupted ommatidial lattice and reduced number of photoreceptor cells. In addition, ectopic expression of DE-cadherin, which is required for retinal morphogenesis, fully rescued the rough-eye phenotype of the hrp38 mutant. Similarly, Parg mutant eye clones had decreased expression level of DE-cadherin with orientation defects, which is reminiscent of DE-cadherin mutant eye phenotype. Therefore, our results suggest that Hrp38 poly(ADP-ribosyl)ation controls eye pattern formation via regulation of DE-cadherin expression, a finding which has implications for understanding the pathogenic mechanisms of Hrp38-related Fragile X syndrome and PARP1-related retinal degeneration diseases. PMID: 23711619 [PubMed - indexed for MEDLINE]
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Related Articles Stem cells in retinal regeneration: past, present and future. Development. 2013 Jun;140(12):2576-85 Authors: Ramsden CM, Powner MB, Carr AJ, Smart MJ, da Cruz L, Coffey PJ Abstract Stem cell therapy for retinal disease is under way, and several clinical trials are currently recruiting. These trials use human embryonic, foetal and umbilical cord tissue-derived stem cells and bone marrow-derived stem cells to treat visual disorders such as age-related macular degeneration, Stargardt's disease and retinitis pigmentosa. Over a decade of analysing the developmental cues involved in retinal generation and stem cell biology, coupled with extensive surgical research, have yielded differing cellular approaches to tackle these retinopathies. Here, we review these various stem cell-based approaches for treating retinal diseases and discuss future directions and challenges for the field. PMID: 23715550 [PubMed - indexed for MEDLINE]
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Related Articles Targeted exome sequencing identified novel USH2A mutations in Usher syndrome families. PLoS One. 2013;8(5):e63832 Authors: Huang XF, Xiang P, Chen J, Xing DJ, Huang N, Min Q, Gu F, Tong Y, Pang CP, Qu J, Jin ZB Abstract Usher syndrome (USH) is a leading cause of deaf-blindness in autosomal recessive trait. Phenotypic and genetic heterogeneities in USH make molecular diagnosis much difficult. This is a pilot study aiming to develop an approach based on next-generation sequencing to determine the genetic defects in patients with USH or allied diseases precisely and effectively. Eight affected patients and twelve unaffected relatives from five unrelated Chinese USH families, including 2 pseudo-dominant ones, were recruited. A total of 144 known genes of inherited retinal diseases were selected for deep exome resequencing. Through systematic data analysis using established bioinformatics pipeline and segregation analysis, a number of genetic variants were released. Eleven mutations, eight of them were novel, in the USH2A gene were identified. Biparental mutations in USH2A were revealed in 2 families with pseudo-dominant inheritance. A proband was found to have triple mutations, two of them were supposed to locate in the same chromosome. In conclusion, this study revealed the genetic defects in the USH2A gene and demonstrated the robustness of targeted exome sequencing to precisely and rapidly determine genetic defects. The methodology provides a reliable strategy for routine gene diagnosis of USH. PMID: 23737954 [PubMed - indexed for MEDLINE]
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Related Articles Cell-based therapies for retinal degenerative diseases: a thousand strategies. J Glaucoma. 2013 Jun-Jul;22 Suppl 5:S42-5 Authors: Lewallen M, Xie T Abstract Retinal neuronal death causes a severe and irreversible loss of visual function in the patients of retinitis pigmentosa, age-related macular degeneration and glaucoma, but these degenerative diseases currently still lack effective medical treatments. The restorative properties of stem cells hold the promise in the treatment of these retinal degenerative diseases. The exciting progress has been made on stem cell research in the last decade. Many different stem cell types have been explored for their potential in treating the retinal degenerative diseases, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells and retinal stem cells. This review will summarize the recent progress in this exciting area. PMID: 23733127 [PubMed - indexed for MEDLINE]
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Related Articles Functional endothelial progenitor cells selectively recruit neurovascular protective monocyte-derived F4/80(+) /Ly6c(+) macrophages in a mouse model of retinal degeneration. Stem Cells. 2013 Oct;31(10):2149-61 Authors: Fukuda S, Nagano M, Yamashita T, Kimura K, Tsuboi I, Salazar G, Ueno S, Kondo M, Kunath T, Oshika T, Ohneda O Abstract Retinitis pigmentosa is a group of inherited eye disorders that result in profound vision loss with characteristic retinal neuronal degeneration and vasculature attenuation. In a mouse model of retinitis pigmentosa, endothelial progenitor cells (EPC) from bone marrow rescued the vasculature and photoreceptors. However, the mechanisms and cell types underlying these protective effects were uncertain. We divided EPC, which contribute to angiogenesis, into two subpopulations based on their aldehyde dehydrogenase (ALDH) activity and observed that EPC with low ALDH activity (Alde-Low) had greater neuroprotection and vasoprotection capabilities after injection into the eyes of an rd1 mouse model of retinitis pigmentosa compared with EPC with high ALDH activity (Alde-High). Of note, Alde-Low EPC selectively recruited F4/80(+) /Ly6c(+) monocyte-derived macrophages from bone marrow into retina through CCL2 secretion. In addition, the mRNA levels of CCR2, the neurotrophic factors TGF-β1 and IGF-1, and the anti-inflammatory mediator interleukin-10 were higher in migrated F4/80(+) /Ly6c(+) monocyte-derived macrophages as compared with F4/80(+) /Ly6c(-) resident retinal microglial cells. These results suggest a novel therapeutic approach using EPC to recruit neuroprotective macrophages that delay the progression of neural degenerative disease. PMID: 23843337 [PubMed - indexed for MEDLINE]
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Related Articles Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. Elife. 2013;2:e00824 Authors: Tucker BA, Mullins RF, Streb LM, Anfinson K, Eyestone ME, Kaalberg E, Riker MJ, Drack AV, Braun TA, Stone EM Abstract Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient's keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient's mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient's other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1 (-/-) mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI:http://dx.doi.org/10.7554/eLife.00824.001. PMID: 23991284 [PubMed]
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Related Articles Stem cells set their sights on retinitis pigmentosa. Elife. 2013;2:e01291 Authors: Bennicelli JL, Bennett J Abstract Skin cells from a patient with a form of inherited blindness have been reprogrammed into retinal cells and successfully transplanted into mice. PMID: 23991287 [PubMed]
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Related Articles Genetically modified neural stem cells for a local and sustained delivery of neuroprotective factors to the dystrophic mouse retina. Stem Cells Transl Med. 2013 Dec;2(12):1001-10 Authors: Jung G, Sun J, Petrowitz B, Riecken K, Kruszewski K, Jankowiak W, Kunst F, Skevas C, Richard G, Fehse B, Bartsch U Abstract A continuous intraocular delivery of neurotrophic factors (NFs) is being explored as a strategy to rescue photoreceptor cells and visual functions in degenerative retinal disorders that are currently untreatable. To establish a cell-based intraocular delivery system for a sustained administration of NFs to the dystrophic mouse retina, we used a polycistronic lentiviral vector to genetically modify adherently cultivated murine neural stem (NS) cells. The vector concurrently encoded a gene of interest, a reporter gene, and a resistance gene and thus facilitated the selection, cloning, and in vivo tracking of the modified cells. To evaluate whether modified NS cells permit delivery of functionally relevant quantities of NFs to the dystrophic mouse retina, we expressed a secretable variant of ciliary neurotrophic factor (CNTF) in NS cells and grafted the cells into the vitreous space of Pde6b(rd1) and Pde6b(rd10) mice, two animal models of retinitis pigmentosa. In both mouse lines, grafted cells attached to the retina and lens, where they differentiated into astrocytes and some neurons. Adverse effects of the transplanted cells on the morphology of host retinas were not observed. Importantly, the CNTF-secreting NS cells significantly attenuated photoreceptor degeneration in both mutant mouse lines. The neuroprotective effect was significantly more pronounced when clonally derived NS cell lines selected for high expression levels of CNTF were grafted into Pde6b(rd1) mice. Intravitreal transplantations of modified NS cells may thus represent a useful method for preclinical studies aimed at evaluating the therapeutic potential of a cell-based intraocular delivery of NFs in mouse models of photoreceptor degeneration. PMID: 24167317 [PubMed - indexed for MEDLINE]
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Related Articles Transplantation of human bone marrow mesenchymal stem cells as a thin subretinal layer ameliorates retinal degeneration in a rat model of retinal dystrophy. Exp Eye Res. 2014 Jan;118:135-44 Authors: Tzameret A, Sher I, Belkin M, Treves AJ, Meir A, Nagler A, Levkovitch-Verbin H, Barshack I, Rosner M, Rotenstreich Y Abstract Vision incapacitation and blindness associated with retinal degeneration affect millions of people worldwide. Cell based therapy and specifically transplantation of human adult bone marrow-derived stem cells (hBM-MSCs) present possible treatment strategy. Subretinal transplantation of human or rat BM-MSCs was shown previously to improve retinal function in Royal College Surgeons (RCS) rats. In those studies cells were transplanted via a transscleral-transchoroidal approach, creating a localized subretinal bleb. Limited number of cells could be injected and photoreceptor rescue was restricted to areas in proximity to the injection site. Here we describe a new surgical method for subretinal transplantation that facilitates uniform distribution of transplanted cells as a thin layer along most of the subretinal space. We assessed the therapeutic effect of hBM-MSCs on RCS rats when transplanted either subretinally or intravitreally. We also examined whether a second transplantation can prolong the therapeutic effect. A cell suspension of 2.5 × 10(6) cells in 5 μl was injected subretinally or intravitreally in RCS rats at 28 days postnatal. In the subretinal group, hBM-MSCs were transplanted posterior to the limbus in the superotemporal part of the eye through a longitudinal triangular scleral tunnel reaching the choroid. In the intravitreal group, the cells were injected into the superotemporal part of the vitreous cavity. In cross sections of subretinally transplanted eyes, removed 2 h following transplantation, hBM-MSCs were distributed as a near-homogenous thin layer along most of the subretinal space. In some animals the cells were also detected in the choroid. In the intravitreal injection group, hBM-MSCs were clustered in the vitreous cavity. Transplanted cells could be detected up to 2 weeks after transplantation but not at later time points. Retinal function and structure were assessed by electroretinogram (ERG) and histology analysis, respectively. Six weeks post transplantation, the mean maximal scotopic ERG b-wave amplitude response recorded in RCS control eyes was 1.2 μV. By contrast, in transplanted eyes mean responses of 56.4 μV and 66.2 μV were recorded in the intravitreally and subretinally transplanted eyes, respectively. In the subretinal group, retinal function was significantly higher in transplanted compared with control eyes up to 20 weeks following transplantation. By contrast, in the intravitreal group, rescue of retinal function persisted only up to 12 weeks following transplantation. Histological analysis revealed that 8 weeks following subretinal transplantation, the retinas of control eyes were dystrophic, with outer nuclear layer (ONL) containing a single cell layer. An extensive photoreceptor rescue was demonstrated in transplanted eyes at this time point, with 3-4 cell layers in the ONL along the entire retina. A second subretinal transplantation at 70 days postnatal did not enhance or prolong the therapeutic effect of hBM-MSCs. No immunosuppressants were used and long-term safety analysis demonstrated no gross or microscopic adverse effects. Taken together our findings suggest that transplantation of hBM-MSCs as a thin subretinal layer enhances the therapeutic effect and the safety of cell transplantation. PMID: 24239509 [PubMed - indexed for MEDLINE]
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Related Articles Retinal repair with induced pluripotent stem cells. Transl Res. 2014 Apr;163(4):377-86 Authors: Al-Shamekh S, Goldberg JL Abstract Retinal degeneration such as age-related macular degeneration and other inherited forms, such as Stargardt's disease and retinitis pigmentosa, and optic neuropathies including glaucoma and ischemic optic neuropathy are major causes of vision loss and blindness worldwide. Damage to retinal pigment epithelial cells and photoreceptors in the former, and to retinal ganglion cell axons in the optic nerve and their cell bodies in the retina in the latter diseases lead to the eventual death of these retinal cells, and in humans there is no endogenous replacement or repair. Cell replacement therapies provide 1 avenue to restore function in these diseases, particularly in the case of retinal repair, although there are considerable issues to overcome, including the differentiation and integration of the transplanted cells. What stem cell sources could be used for such therapies? One promising source is induced pluripotent stem cells (iPSCs), which could be drawn from an individual patient needing therapy, or generated and banked from select donors. We review developing research in the use of iPSCs for retinal cell replacement therapy. PMID: 24291154 [PubMed - indexed for MEDLINE]
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Related Articles Efficient Production of Photoreceptor Precursor Cells from Human Embryonic Stem Cells. Methods Mol Biol. 2013 Dec 4; Authors: Yanai A, Laver C, Joe AW, Gregory-Evans K Abstract Transplantation of photoreceptor precursor cells (PPCs) differentiated from human embryonic stem cells (hESCs) is a promising approach to treat common blinding diseases such as age-related macular degeneration and retinitis pigmentosa. However, existing PPC generation methods are inefficient. To enhance differentiation protocols for rapid and high-yield production of PPCs, we focused on optimizing the handling of the cells by including feeder-independent growth of hESCs, using size-controlled embryoid bodies (EBs), and addition of triiodothyronine (T3) and taurine to the differentiation medium, with subsequent removal of undifferentiated cells via negative cell-selection. Our novel protocol produces higher yields of PPCs than previously reported while reducing the time required for differentiation, which will help understand retinal diseases and facilitate large-scale preclinical trials. PMID: 24301073 [PubMed - as supplied by publisher]
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Related Articles Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells. Int J Nanomedicine. 2013;8:4641-58 Authors: Bhise NS, Wahlin KJ, Zack DJ, Green JJ Abstract BACKGROUND: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. METHODS: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. RESULTS: 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. CONCLUSION: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming. PMID: 24348039 [PubMed - indexed for MEDLINE]
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Related Articles Survival and migration of pre-induced adult human peripheral blood mononuclear cells in retinal degeneration slow (rds) mice three months after subretinal transplantation. Curr Stem Cell Res Ther. 2014 Mar;9(2):124-33 Authors: Peng Y, Zhang Y, Huang B, Luo Y, Zhang M, Li K, Li W, Wen W, Tang S Abstract INTRODUCTION: Retinitis pigmentosa (RP), an inherited disease characterized by progressive loss of photoreceptors and retinal pigment epithelium, is a leading genetic cause of blindness. Cell transplantation to replace lost photoreceptors is a potential therapeutic strategy, but technical limitations have prevented clinical application. Adult human peripheral blood mononuclear cells (hPBMCs) may be an ideal cell source for such therapies. This study examined the survival and migration of pre-induced hPBMCs three months after subretinal transplantation in the retinal degeneration slow (rds) mouse model of RP. MATERIALS AND METHODS: Freshly isolated adult hPBMCs were pre-induced by co-culture with neonatal Sprague-Dawley (SD) rat retinal tissue for 4 days in neural stem cell medium. Pre-induced cells were labeled with CMDiI for tracing and injected into the right subretinal space of rds mice by the trans-scleral approach. After two and three months, right eyes were harvested and transplanted cell survival and migration examined in frozen sections and wholemount retinas. Immunofluorescence in whole-mount retinas was used to detect the expression of human neuronal and photoreceptors protein markers by transplanted cells. RESULTS: Pre-induced adult hPBMCs could survive in vivo and migrate to various parts of the retina. After two and three months, transplanted cells were observed in the ciliary body, retinal outer nuclear layer, inner nuclear layer, ganglion cell layer, optic papilla, and within the optic nerve. The neuronal and photoreceptor markers CD90/Thy1, MAP-2, nestin, and rhodopsin were expressed by subpopulations of CM-DiI-positive cells three months after subretinal transplantation. CONCLUSION: Pre-induced adult hPBMCs survived for at least three months after subretinal transplantation, migrated throughout the retina, and expressed human protein markers. These results suggest that hPBMCs could be used for cell replacement therapy to treat retinal degenerative diseases. PMID: 24350910 [PubMed - in process]
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Related Articles Proteomic survey reveals altered energetic patterns and metabolic failure prior to retinal degeneration. J Neurosci. 2014 Feb 19;34(8):2797-812 Authors: Griciuc A, Roux MJ, Merl J, Giangrande A, Hauck SM, Aron L, Ueffing M Abstract Inherited mutations that lead to misfolding of the visual pigment rhodopsin (Rho) are a prominent cause of photoreceptor neuron (PN) degeneration and blindness. How Rho proteotoxic stress progressively impairs PN viability remains unknown. To identify the pathways that mediate Rho toxicity in PNs, we performed a comprehensive proteomic profiling of retinas from Drosophila transgenics expressing Rh1(P37H), the equivalent of mammalian Rho(P23H), the most common Rho mutation linked to blindness in humans. Profiling of young Rh1(P37H) retinas revealed a coordinated upregulation of energy-producing pathways and attenuation of energy-consuming pathways involving target of rapamycin (TOR) signaling, which was reversed in older retinas at the onset of PN degeneration. We probed the relevance of these metabolic changes to PN survival by using a combination of pharmacological and genetic approaches. Chronic suppression of TOR signaling, using the inhibitor rapamycin, strongly mitigated PN degeneration, indicating that TOR signaling activation by chronic Rh1(P37H) proteotoxic stress is deleterious for PNs. Genetic inactivation of the endoplasmic reticulum stress-induced JNK/TRAF1 axis as well as the APAF-1/caspase-9 axis, activated by damaged mitochondria, dramatically suppressed Rh1(P37H)-induced PN degeneration, identifying the mitochondria as novel mediators of Rh1(P37H) toxicity. We thus propose that chronic Rh1(P37H) proteotoxic stress distorts the energetic profile of PNs leading to metabolic imbalance, mitochondrial failure, and PN degeneration and therapies normalizing metabolic function might be used to alleviate Rh1(P37H) toxicity in the retina. Our study offers a glimpse into the intricate higher order interactions that underlie PN dysfunction and provides a useful resource for identifying other molecular networks that mediate Rho toxicity in PNs. PMID: 24553922 [PubMed - indexed for MEDLINE]
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Related Articles Developing cellular therapies for retinal degenerative diseases. Invest Ophthalmol Vis Sci. 2014 Feb;55(2):1191-202 Authors: Bharti K, Rao M, Hull SC, Stroncek D, Brooks BP, Feigal E, van Meurs JC, Huang CA, Miller SS Abstract Biomedical advances in vision research have been greatly facilitated by the clinical accessibility of the visual system, its ease of experimental manipulation, and its ability to be functionally monitored in real time with noninvasive imaging techniques at the level of single cells and with quantitative end-point measures. A recent example is the development of stem cell-based therapies for degenerative eye diseases including AMD. Two phase I clinical trials using embryonic stem cell-derived RPE are already underway and several others using both pluripotent and multipotent adult stem cells are in earlier stages of development. These clinical trials will use a variety of cell types, including embryonic or induced pluripotent stem cell-derived RPE, bone marrow- or umbilical cord-derived mesenchymal stem cells, fetal neural or retinal progenitor cells, and adult RPE stem cells-derived RPE. Although quite distinct, these approaches, share common principles, concerns and issues across the clinical development pipeline. These considerations were a central part of the discussions at a recent National Eye Institute meeting on the development of cellular therapies for retinal degenerative disease. At this meeting, emphasis was placed on the general value of identifying and sharing information in the so-called "precompetitive space." The utility of this behavior was described in terms of how it could allow us to remove road blocks in the clinical development pipeline, and more efficiently and economically move stem cell-based therapies for retinal degenerative diseases toward the clinic. Many of the ocular stem cell approaches we discuss are also being used more broadly, for nonocular conditions and therefore the model we develop here, using the precompetitive space, should benefit the entire scientific community. PMID: 24573369 [PubMed - indexed for MEDLINE]
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Related Articles Stem cell therapy: a novel approach for vision restoration in retinitis pigmentosa. Med Hypothesis Discov Innov Ophthalmol. 2013;2(2):52-5 Authors: Uy HS, Chan PS, Cruz FM Abstract Unfortunately, at present, degenerative retinal diseases such as retinitis pigmentosa remains untreatable. Patients with these conditions suffer progressive visual decline resulting from continuing loss of photoreceptor cells and outer nuclear layers. However, stem cell therapy is a promising approach to restore visual function in eyes with degenerative retinal diseases such as retinitis pigmentosa. Animal studies have established that pluripotent stem cells when placed in the mouse retinitis pigmentosa models have the potential not only to survive, but also to differentiate, organize into and function as photoreceptor cells. Furthermore, there is early evidence that these transplanted cells provide improved visual function. These groundbreaking studies provide proof of concept that stem cell therapy is a viable method of visual rehabilitation among eyes with retinitis pigmentosa. Further studies are required to optimize these techniques in human application. This review focuses on stem cell therapy as a new approach for vision restitution in retinitis pigmentosa. PMID: 24600643 [PubMed]
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Related Articles Stem cells for investigation and treatment of inherited retinal disease. Hum Mol Genet. 2014 Sep 15;23(R1):R9-R16 Authors: Tucker BA, Mullins RF, Stone EM Abstract Vision is the most important human sense. It facilitates every major activity of daily living ranging from basic communication, mobility and independence to an appreciation of art and nature. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through some type of drug, gene or cell-based transplantation approach. In this review, we will discuss the current literature pertaining to retinal transplantation. We will focus on the use of induced pluripotent stem cells for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell replacement. PMID: 24647603 [PubMed - as supplied by publisher]
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Related Articles Progress of mesenchymal stem cell therapy for neural and retinal diseases. World J Stem Cells. 2014 Apr 26;6(2):111-9 Authors: Ng TK, Fortino VR, Pelaez D, Cheung HS Abstract Complex circuitry and limited regenerative power make central nervous system (CNS) disorders the most challenging and difficult for functional repair. With elusive disease mechanisms, traditional surgical and medical interventions merely slow down the progression of the neurodegenerative diseases. However, the number of neurons still diminishes in many patients. Recently, stem cell therapy has been proposed as a viable option. Mesenchymal stem cells (MSCs), a widely-studied human adult stem cell population, have been discovered for more than 20 years. MSCs have been found all over the body and can be conveniently obtained from different accessible tissues: bone marrow, blood, and adipose and dental tissue. MSCs have high proliferative and differentiation abilities, providing an inexhaustible source of neurons and glia for cell replacement therapy. Moreover, MSCs also show neuroprotective effects without any genetic modification or reprogramming. In addition, the extraordinary immunomodulatory properties of MSCs enable autologous and heterologous transplantation. These qualities heighten the clinical applicability of MSCs when dealing with the pathologies of CNS disorders. Here, we summarize the latest progress of MSC experimental research as well as human clinical trials for neural and retinal diseases. This review article will focus on multiple sclerosis, spinal cord injury, autism, glaucoma, retinitis pigmentosa and age-related macular degeneration. PMID: 24772238 [PubMed]
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Related Articles Gene Therapy in Patient-specific Stem Cell Lines and a Preclinical Model of Retinitis Pigmentosa With Membrane Frizzled-related Protein Defects. Mol Ther. 2014 Jun 4; Authors: Li Y, Wu WH, Hsu CW, Nguyen HV, Tsai YT, Chan L, Nagasaki T, Maumenee IH, Yannuzzi LA, Hoang QV, Hua H, Egli D, Tsang SH Abstract Defects in Membrane Frizzled-related Protein (MFRP) cause autosomal recessive retinitis pigmentosa (RP). MFRP codes for a retinal pigment epithelium (RPE)-specific membrane receptor of unknown function. In patient-specific induced pluripotent stem (iPS)-derived RPE cells, precise levels of MFRP, and its dicistronic partner CTRP5, are critical to the regulation of actin organization. Overexpression of CTRP5 in naïve human RPE cells phenocopied behavior of MFRP-deficient patient RPE (iPS-RPE) cells. AAV8 (Y733F) vector expressing human MFRP rescued the actin disorganization phenotype and restored apical microvilli in patient-specific iPS-RPE cell lines. As a result, AAV-treated MFRP mutant iPS-RPE recovered pigmentation and transepithelial resistance. The efficacy of AAV-mediated gene therapy was also evaluated in Mfrp(rd6)/Mfrp(rd6) mice-an established preclinical model of RP-and long-term improvement in visual function was observed in AAV-Mfrp-treated mice. This report is the first to indicate the successful use of human iPS-RPE cells as a recipient for gene therapy. The observed favorable response to gene therapy in both patient-specific cell lines, and the Mfrp(rd6)/Mfrp(rd6) preclinical model suggests that this form of degeneration caused by MFRP mutations is a potential target for interventional trials.Molecular Therapy (2014); doi:10.1038/mt.2014.100. PMID: 24895994 [PubMed - as supplied by publisher]
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Related Articles Stem cells as source for retinal pigment epithelium transplantation. Prog Retin Eye Res. 2014 Sep;42C:130-144 Authors: Bertolotti E, Neri A, Camparini M, Macaluso C, Marigo V Abstract Inherited maculopathies, age related macular degeneration and some forms of retinitis pigmentosa are associated with impaired function or loss of the retinal pigment epithelium (RPE). Among potential treatments, transplantation approaches are particularly promising. The arrangement of RPE cells in a well-defined tissue layer makes the RPE amenable to cell or tissue sheet transplantation. Different cell sources have been suggested for RPE transplantation but the development of a clinical protocol faces several obstacles. The source should provide a sufficient number of cells to at least recover the macula area. Secondly, cells should be plastic enough to be able to integrate in the host tissue. Tissue sheets should be considered as well, but the substrate on which RPE cells are cultured needs to be carefully evaluated. Immunogenicity can also be an obstacle for effective transplantation as well as tumorigenicity of not fully differentiated cells. Finally, ethical concerns may represent drawbacks when embryo-derived cells are proposed for RPE transplantation. Here we discuss different cell sources that became available in recent years and their different properties. We also present data on a new source of human RPE. We provide a protocol for RPE differentiation of retinal stem cells derived from adult ciliary bodies of post-mortem donors. We show molecular characterization of the in vitro differentiated RPE tissue and demonstrate its functionality based on a phagocytosis assay. This new source may provide tissue for allogenic transplantation based on best matches through histocompatibility testing. PMID: 24933042 [PubMed - as supplied by publisher]
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Related Articles The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa. Mol Brain. 2014;7:45 Authors: Yoshida T, Ozawa Y, Suzuki K, Yuki K, Ohyama M, Akamatsu W, Matsuzaki Y, Shimmura S, Mitani K, Tsubota K, Okano H Abstract BACKGROUND: Retinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds. RESULTS: We generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient's iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5 weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient's iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes. CONCLUSION: The use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches. PMID: 24935155 [PubMed - in process]
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Related Articles Recent Advances of Stem Cell Therapy for Retinitis Pigmentosa. Int J Mol Sci. 2014;15(8):14456-14474 Authors: He Y, Zhang Y, Liu X, Ghazaryan E, Li Y, Xie J, Su G Abstract Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive loss of photoreceptors and eventually leads to retina degeneration and atrophy. Until now, the exact pathogenesis and etiology of this disease has not been clear, and many approaches for RP therapies have been carried out in animals and in clinical trials. In recent years, stem cell transplantation-based attempts made some progress, especially the transplantation of bone marrow-derived mesenchymal stem cells (BMSCs). This review will provide an overview of stem cell-based treatment of RP and its main problems, to provide evidence for the safety and feasibility for further clinical treatment. PMID: 25141102 [PubMed - as supplied by publisher]
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