Gotowa bibliografia na temat „Neuroretinal organoids”

The proneural transcription factor atonal basic helix–loop–helix transcription factor 7 (ATOH7) is expressed in early progenitors in the developing neuroretina. In vertebrates, this is crucial for the development of retinal ganglion cells (RGCs), as mutant animals show an almost complete absence of RGCs, underdeveloped optic nerves, and aberrations in retinal vessel development. Human mutations are rare and result in autosomal recessive optic nerve hypoplasia (ONH) or severe vascular changes, diagnosed as autosomal recessive persistent hyperplasia of the primary vitreous (PHPVAR). To better understand the role of ATOH7 in neuroretinal development, we created ATOH7 knockout and eGFP-expressing ATOH7 reporter human induced pluripotent stem cells (hiPSCs), which were differentiated into early-stage retinal organoids. Target loci regulated by ATOH7 were identified by Cleavage Under Targets and Release Using Nuclease with sequencing (CUT&RUN-seq) and differential expression by RNA sequencing (RNA-seq) of wildtype and mutant organoid-derived reporter cells. Additionally, single-cell RNA sequencing (scRNA-seq) was performed on whole organoids to identify cell type-specific genes. Mutant organoids displayed substantial deficiency in axon sprouting, reduction in RGCs, and an increase in other cell types. We identified 469 differentially expressed target genes, with an overrepresentation of genes belonging to axon development/guidance and Notch signaling. Taken together, we consolidate the function of human ATOH7 in guiding progenitor competence by inducing RGC-specific genes while inhibiting other cell fates. Furthermore, we highlight candidate genes responsible for ATOH7-associated optic nerve and retinovascular anomalies, which sheds light to potential future therapy targets for related disorders.

AbstractOrganoids are transformative in vitro model systems that mimic features of the corresponding tissue in vivo. However, across tissue types and species, organoids still often fail to reach full maturity and function because biochemical cues cannot be provided from within the organoid to guide their development. Here we introduce nanoengineered DNA microbeads with tissue mimetic tunable stiffness for implementing spatio-temporally controlled morphogen gradients inside of organoids at any point in their development. Using medaka retinal organoids and early embryos, we show that DNA microbeads can be integrated into embryos and organoids by microinjection and erased in a non-invasive manner with light. Coupling a recombinant surrogate Wnt to the DNA microbeads, we demonstrate the spatio-temporally controlled morphogen release from the microinjection site, which leads to morphogen gradients resulting in the formation of retinal pigmented epithelium while maintaining neuroretinal cell types. Thus, we bioengineered retinal organoids to more closely mirror the cell type diversity of in vivo retinae. Owing to the facile, one-pot fabrication process, the DNA microbead technology can be adapted to other organoid systems for improved tissue mimicry.

The telencephalon and eye in mammals are originated from adjacent fields at the anterior neural plate. Morphogenesis of these fields generates telencephalon, optic-stalk, optic-disc, and neuroretina along a spatial axis. How these telencephalic and ocular tissues are specified coordinately to ensure directional retinal ganglion cell (RGC) axon growth is unclear. Here, we report self-formation of human telencephalon-eye organoids comprising concentric zones of telencephalic, optic-stalk, optic-disc, and neuroretinal tissues along the center-periphery axis. Initially-differentiated RGCs grew axons towards and then along a path defined by adjacent PAX2+ VSX2+ optic-disc cells. Single-cell RNA sequencing of these organoids not only confirmed telencephalic and ocular identities but also identified expression signatures of early optic-disc, optic-stalk, and RGCs. These signatures were similar to those in human fetal retinas. Optic-disc cells in these organoids differentially expressed FGF8 and FGF9; FGFR inhibitions drastically decreased early RGC differentiation and directional axon growth. Through the RGC-specific cell-surface marker CNTN2 identified here, electrophysiologically excitable RGCs were isolated under a native condition. Our findings provide insight into the coordinated specification of early telencephalic and ocular tissues in humans and establish resources for studying RGC-related diseases such as glaucoma.

Abstract Background All-trans retinoic acid (ATRA) plays an essential role during human eye development, being temporally and spatially adjusted to create gradient concentrations that guide embryonic anterior and posterior axis formation of the eye. Perturbations in ATRA signaling can result in severe ocular developmental diseases. Although it is known that ATRA is essential for correct eye formation, how ATRA influences the different ocular tissues during the embryonic development of the human eye is still not well studied. Here, we investigated the effects of ATRA on the differentiation and the maturation of human ocular tissues using an in vitro model of human-induced pluripotent stem cells-derived multiocular organoids. Methods Multiocular organoids, consisting of the retina, retinal pigment epithelium (RPE), and cornea, were cultured in a medium containing low (500 nM) or high (10 µM) ATRA concentrations for 60 or 90 days. Furthermore, retinal organoids were cultured with taurine and T3 to further study photoreceptor modulation during maturation. Histology, immunochemistry, qPCR, and western blot were used to study gene and protein differential expression between groups. Results High ATRA levels promote the transparency of corneal organoids and the neuroretinal development in retinal organoids. However, the same high ATRA levels decreased the pigmentation levels of RPE organoids and, in long-term cultures, inhibited the maturation of photoreceptors. By contrast, low ATRA levels enhanced the pigmentation of RPE organoids, induced the opacity of corneal organoids—due to an increase in collagen type IV in the stroma— and allowed the maturation of photoreceptors in retinal organoids. Moreover, T3 promoted rod photoreceptor maturation, whereas taurine promoted red/green cone photoreceptors. Conclusion ATRA can modulate corneal epithelial integrity and transparency, photoreceptor development and maturation, and the pigmentation of RPE cells in a dose-dependent manner. These experiments revealed the high relevance of ATRA during ocular tissue development and its use as a potential new strategy to better modulate the development and maturation of ocular tissue through temporal and spatial control of ATRA signaling.

Le syndrome d'Alström (SA) est une maladie monogénique récessive multi-systémique, caractérisée notamment par une perte de l'audition et de la vue, une obésité, un diabète de type 2, une cardiomyopathie et une insuffisance hépatique et rénale progressive. Les symptômes affectant la vision se développent dès les premières semaines après la naissance et mènent progressivement à une perte totale de la vue. À l'heure actuelle, aucun traitement ne permet de soigner cette maladie et seules des solutions permettant de réduire les effets des symptômes peuvent être proposées.L'objet de la thèse est de mettre au point un modèle cellulaire du SA dans le but de comprendre les mécanismes moléculaires entrainant la maladie et d'identifier des cibles thérapeutiques.Nous avons obtenu différents clones présentant des mutations pathologiques ou de novo à l'aide de systèmes d'édition génomique associés à CRISPR/Cas9. Nous avons caractérisé ces clones modèles en cherchant à identifier des marqueurs phénotypiques particuliers au sein des CSPih. Les mutations engendrées ne provoquent pas de changement des propriétés de ces cellules.Dans un second temps, toujours dans le but d'identifier un phénotype pathologique, nous avons différencié les lignées de CSPih modèles en cellules de l'EPR. Là encore, nous n'avons pas identifié de marqueur phénotypique spécifique. Enfin, nous avons différencié les lignées de CSPih modèles en organoïdes neuro-rétiniens afin d'étudier le développement des cellules rétiniennes au sein de ces structures avec une attention particulière apportée aux photorécepteurs. Nous avons ainsi pu constater une absence ou une réduction d'expression des opsines caractéristiques des cônes et des bâtonnets dans les organoïdes issus de CSPih mutées dans ALMS1. De plus, ces organoïdes présentent une mortalité cellulaire accrue par rapport aux organoïdes issus des lignées de CSPih saines. Ces éléments laissent penser que les photorécepteurs dégénèrent durant leur différenciation au sein des organoïdes. Les mécanismes par lesquels les mutations dans ALMS1 entrainent cette dégénérescence ne sont pas élucidés à l'heure actuelle.Les modèles cellulaires du SA présentés dans cette thèse reproduisent donc un phénotype pathologique et seront des outils précieux pour la compréhension des mécanismes responsables des symptômes visuels de la maladie et pavent la voie à des stratégies de criblage visant à identifier de nouvelles cibles thérapeutiques
Alström syndrome (AS) is a monogenic recessive multi-systemic disease characterized by hearing and vision loss, obesity, type 2 diabetes, cardiomyopathy and progressive liver and kidney failure. Symptoms affecting vision develop in the first few weeks after birth and gradually lead to total loss of sight. At present, there is no cure for this disease, and only solutions that reduce the effects of the symptoms can be proposed.The aim of this thesis is to develop a cellular model of AS with a view to understanding the molecular mechanisms driving the disease and identifying therapeutic targets.We obtained different clones with pathological or de novo mutations using genome-editing systems associated with CRISPR/Cas9. We characterized these model clones by seeking to identify specific phenotypic markers within the hiPSCs. The mutations generated did not change the properties of these cells.In a second step, still with the aim of identifying a pathological phenotype, we differentiated the model iPSC lines into RPE cells.Again, no specific phenotypic marker was identified. Finally, we differentiated our model hiPSC lines into neuroretinal organoids to study retinal cells development within these structures with a particular focus on photoreceptors. We were able to observe the absence or reduced expression of opsins characteristic of cones and rods in organoids derived from ALMS1-mutant hiPSCs. In addition, these organoids showed increased cell death compared with organoids derived from healthy hiPSC lines. This suggests that photoreceptors degenerate during differentiation within organoids. The mechanisms by which mutations in ALMS1 lead to this degeneration remain unclear.The cellular models of AS presented in this thesis therefore reproduce a pathological phenotype and will be invaluable tools for understanding the mechanisms responsible for the visual symptoms of the disease, and pave the way for screening strategies aimed at identifying new therapeutic targets