65 research outputs found
Pluripotent Stem Cell-Based Approaches to Explore and Treat Optic Neuropathies
Sight is a major sense for human and visual impairment profoundly affects quality of life, especially retinal degenerative diseases which are the leading cause of irreversible blindness worldwide. As for other neurodegenerative disorders, almost all retinal dystrophies are characterized by the specific loss of one or two cell types, such as retinal ganglion cells, photoreceptor cells, or retinal pigmented epithelial cells. This feature is a critical point when dealing with cell replacement strategies considering that the preservation of other cell types and retinal circuitry is a prerequisite. Retinal ganglion cells are particularly vulnerable to degenerative process and glaucoma, the most common optic neuropathy, is a frequent retinal dystrophy. Cell replacement has been proposed as a potential approach to take on the challenge of visual restoration, but its application to optic neuropathies is particularly challenging. Many obstacles need to be overcome before any clinical application. Beyond their survival and differentiation, engrafted cells have to reconnect with both upstream synaptic retinal cell partners and specific targets in the brain. To date, reconnection of retinal ganglion cells with distal central targets appears unrealistic since central nervous system is refractory to regenerative processes. Significant progress on the understanding of molecular mechanisms that prevent central nervous system regeneration offer hope to overcome this obstacle in the future. At the same time, emergence of reprogramming of human somatic cells into pluripotent stem cells has facilitated both the generation of new source of cells with therapeutic potential and the development of innovative methods for the generation of transplantable cells. In this review, we discuss the feasibility of stem cell-based strategies applied to retinal ganglion cells and optic nerve impairment. We present the different strategies for the generation, characterization and the delivery of transplantable retinal ganglion cells derived from pluripotent stem cells. The relevance of pluripotent stem cell-derived retinal organoid and retinal ganglion cells for disease modeling or drug screening will be also introduced in the context of optic neuropathies
Dynamic full-field optical coherence tomography: 3D live-imaging of retinal organoids
Optical coherence tomography offers astounding opportunities to image the
complex structure of living tissue, but lacks functional information. We
present dynamic full-field optical coherence tomography to image living human
induced pluripotent stem cell-derived retinal organoids non-invasively. Colored
images with an endogenous contrast linked to organelle motility are generated,
with sub-micrometer spatial resolution and millisecond temporal resolution,
opening an avenue to identify specific cell types in living tissue via their
function.Comment: 14 pages, 5 figures, 1 table, 6 video
Expression of Rod-Derived Cone Viability Factor: Dual Role of CRX in Regulating Promoter Activity and Cell-Type Specificity
International audienceBACKGROUND: RdCVF and RdCVF2, encoded by the nucleoredoxin-like genes NXNL1 and NXNL2, are trophic factors with therapeutic potential that are involved in cone photoreceptor survival. Studying how their expression is regulated in the retina has implications for understanding both their activity and the mechanisms determining cell-type specificity within the retina. METHODOLOGY/PRINCIPAL FINDINGS: In order to define and characterize their promoters, a series of luciferase/GFP reporter constructs that contain various fragments of the 5'-upstream region of each gene, both murine and human, were tested in photoreceptor-like and non-photoreceptor cell lines and also in a biologically more relevant mouse retinal explant system. For NXNL1, 5'-deletion analysis identified the human -205/+57 bp and murine -351/+51 bp regions as having promoter activity. Moreover, in the retinal explants these constructs drove expression specifically to photoreceptor cells. For NXNL2, the human -393/+27 bp and murine -195/+70 bp regions were found to be sufficient for promoter activity. However, despite the fact that endogenous NXNL2 expression is photoreceptor-specific within the retina, neither of these DNA sequences nor larger upstream regions demonstrated photoreceptor-specific expression. Further analysis showed that a 79 bp NXNL2 positive regulatory sequence (-393 to 315 bp) combined with a 134 bp inactive minimal NXNL1 promoter fragment (-77 to +57 bp) was able to drive photoreceptor-specific expression, suggesting that the minimal NXNL1 fragment contains latent elements that encode cell-type specificity. Finally, based on bioinformatic analysis that suggested the importance of a CRX binding site within the minimal NXNL1 fragment, we found by mutation analysis that, depending on the context, the CRX site can play a dual role. CONCLUSIONS/SIGNIFICANCE: The regulation of the Nucleoredoxin-like genes involves a CRX responsive element that can act as both as a positive regulator of promoter activity and as a modulator of cell-type specificity
Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF-α
Optogenetic Light Sensors in Human Retinal Organoids
Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules
Analyse du fonctionnement des recepteurs aux prostaglandines et muscariniques dans le muscle uterin de rate. Modulation au cours de la gestation des proteines G et de la generation des messagers intracellulaires
SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : TD 80548 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc
Caractérisation de l'expression et du rÎle de Bclaf1 au cours de la différenciation des progéniteurs de la rétine des vertébrés
La diffĂ©renciation des progĂ©niteurs rĂ©tiniens se fait sous le contrĂŽle de facteurs endogĂšnes et exogĂšnes. Dans le but d identifier des gĂšnes rĂ©gulant le dĂ©veloppement rĂ©tinien, notre Ă©quipe avait analysĂ© le programme transcriptionnel des progĂ©niteurs d une part au cours du dĂ©veloppement normal de la rĂ©tine et d'autre part dans des conditions oĂč la formation des photorĂ©cepteurs est profondĂ©ment altĂ©rĂ©e. Cette approche a permis de rĂ©vĂ©ler plusieurs facteurs candidats dont Bclaf1 (Bcl-2 associated transcription factor), dĂ©crit comme un inducteur potentiel de la mort cellulaire et un rĂ©presseur gĂ©nĂ©ral de la transcription et identifiĂ© dans des complexes ribonuclĂ©oprotĂ©iques. L analyse immunohistochimique de son profil d expression spatio-temporel montre que Bclaf1 est exprimĂ© dans les progĂ©niteurs rĂ©tiniens prĂ©coces et les prĂ©curseurs post-mitotiques exprimant le facteur de transcription Pax6, puis son expression se restreint aux cellules ganglionnaires et dans les cellules amacrines (CAs) et horizontales (CHs). L invalidation gĂ©nĂ©tique de ce facteur in vivo (souris Bclaf1 KO) et in vitro (Ă©lectroporation shRNA sur explants de rĂ©tines) mĂšne, aux stades prĂ©coces (E14-E16), Ă une rĂ©duction importante du nombre de progĂ©niteurs mitotiques, de prĂ©curseurs post-mitotiques et de CAs et CHs. En parallĂšle une augmentation du nombre de photorĂ©cepteurs (PRs) est observĂ©e en absence de Bclaf1. La surexpression de Bclaf1 dans les progĂ©niteurs rĂ©tiniens tardifs (P0) n est pas suffisante pour gĂ©nĂ©rer des CAs et des CHs, mais entraine une diminution du nombre de PRs. Nous avons proposĂ© un modĂšle dans lequel Bclaf1 serait nĂ©cessaire mais pas suffisant pour le maintien des progĂ©niteurs rĂ©tiniens prĂ©coces dans le cycle cellulaire et pour leur diffĂ©renciation en CAs et CHs. Dans ce contexte, l extinction de son expression permettrait la transition des progĂ©niteurs rĂ©tiniens vers un Ă©tat de compĂ©tence dit tardif afin de produire les PRs et les cellules bipolaires et gliales de MĂŒller. L utilisation de mutants fonctionnels de Bclaf1 nous a permis d Ă©claircir les mĂ©canismes molĂ©culaires qui sous-tendent ses effets sur les progĂ©niteurs rĂ©tiniens. Nous avons montrĂ© que le domaine nĂ©cessaire Ă son interaction avec Bcl-2 n Ă©tait pas requis mais que la partie N-terminale de Bclaf1 (portant un domaine riche en RS ainsi qu un motif de type bZIP) Ă©tait responsable des effets observĂ©s. La capacitĂ© de Bclaf1 Ă favoriser l activitĂ© transcriptionnelle de Pax6 via son domaine bZIP pourrait ĂȘtre un des mĂ©canismes mis en jeu en plus d une implication dans la rĂ©gulation de la biogenĂšse des ARN, via son domaine riche en arginine et sĂ©rine (RS), dans les effets biologiques de Bclaf1PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF
Identification de gÚnes régulés par le Ciliary neurotrophic factor au cours de la différenciation des progéniteurs rétiniens de vertébrés
LE KREMLIN-B.- PARIS 11-BU MĂ©d (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF
Les mini-cerveaux vous observent-ils ?
International audienceLes organoĂŻdes cĂ©rĂ©braux, comme les organoĂŻdes rĂ©tiniens dĂ©rivĂ©s de cellules souches de type iPS, sont des modĂšles en trois dimensions (3D) biologiquement pertinents. Une Ă©tude rĂ©cente du laboratoire de Jay Gopalakrishnan (universitĂ© de DĂŒsseldorf), en collaboration avec un groupe de lâuniversitĂ© de Bonn et notre Ă©quipe de lâInstitut de la vision Ă Paris, a montrĂ© la capacitĂ© des cellules iPS humaines Ă dĂ©velopper spontanĂ©ment des organoĂŻdes cĂ©rĂ©braux incluant des structures oculaires rudimentaires bilatĂ©rales et symĂ©triques. Cette innovation aboutissant Ă la formation dâorganoĂŻdes toujours plus complexes et proches des organes modĂ©lisĂ©s constitue une Ă©tape majeure pour comprendre comment lâĆil humain se dĂ©veloppe de concert avec le cerveau pour crĂ©er un systĂšme visuel fonctionnel
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