Retina-Specific Laminin 523 Drives Differentiation of Human Pluripotent Stem Cells to Photoreceptors [single cell_RNAseq]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE161416 | GEO | 2023/03/26
REPOSITORIES: GEO
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Project description:Inherited retinal diseases and aged related macular degeneration are main causes of blindness that involves irreversible photoreceptor loss. Gene therapy approaches do not prevent photoreceptor degeneration during disease progression, and to date protocols for generating photoreceptors from pluripotent stem cells do not exist. Therefore, transplantation of photoreceptors containing retinal organoids is considered a potential option. However, in vitro formation of organoids requires animal-derived materials and they vary considerably in cell composition between batches, which strongly limits their applications in therapy. Here, we show that human recombinant retina-specific laminin isoform LN523, normally present in the extra cellular matrix ECM surrounding photoreceptors, supports differentiation of pluripotent embryonic stem cells to photoreceptor progenitors in vitro. Using a rabbit macular degeneration model, the transplanted and engrafted cells mature in vivo and form synaptic connectivity with the host retina. Furthermore, addition of a rod-derived cone viability factor increased the formation of cone photoreceptors. These results may pave the way for cell therapy treatment of macular degeneration.
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Project description:Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor NRL. The loss of Nrl in mice (Nrl-/-) results in a retina with predominantly S-opsin containing cones that exhibit molecular and functional characteristics of WT cones. Here we report that Nrl-/- retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by four months of age, resulting in a thinned but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic ERG. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of stress response and inflammation genes, implying their involvement in cone death. The Nrl-/- retina illustrates the long-term viability of cones in the absence of rods and may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula. Targets were generated from a pair of retinas (one Nrl-/- mouse) per biological replicate. Four biological replicates were generated for each of the five aging timepoints (1, 2, 4, 6, and 10 months post natal).
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2019-04-01 | GSE112507 | GEO
Project description:The initial aim of this work was to understand the pathophysiology of Enhanced S-cone Syndrome (ESCS) that leads to retinal degeneration. Although ESCS was identified in humans decades ago and since then the causative genes have been elucidated, our understanding of the accompanying retinal degeneration is still poorly understood. Knockout of the Nrl transcription factor in mice produces a retina overpopulated with S-cone like photoreceptors along with absence of rod photoreceptors and recapitulates many of the phenotypic features seen in human ESCS patients. We wanted to study this murine model through a combinatorial genetic and structural approach to improve understanding of the disease process that leads to photoreceptor degeneration and blindness, potentially guiding future therapies. By using RNA-Sequencing (RNA-Seq) to examine mature wild type and Nrl-/- ocular tissues, we were able to determine global changes in their transcriptomes. The massively parallel RNA-sequencing experiment revealed new insight into the transcriptional mis-regulation in the ESCS murine model and revealed a change in gene expression in putative proteins involved in photoreceptor phagocytosis. Key photoreceptor ligands necessary for phagocyotsis, Tub and Tulp1, were down-regulated in the Nrl-/- retina. Down regulation of key retinoid metabolic genes, coupled with down-regulation of Tub and Tulp1, suggested a potential mechanism involving defective phagocytosis underlies the photoreceptor degeneration seen in ESCS. We report RNA-Seq experiments of whole eye and retinal tissues from wild type and Nrl transcription factor knockout mice on the C57BL/6 background. Examine two different ocular tissues in two mouse models of varying photoreceptor populations
2011-06-07 | E-GEOD-29752 | biostudies-arrayexpress
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2022-04-13 | GSE183206 | GEO
Project description:Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor NRL. The loss of Nrl in mice (Nrl-/-) results in a retina with predominantly S-opsin containing cones that exhibit molecular and functional characteristics of WT cones. Here we report that Nrl-/- retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by four months of age, resulting in a thinned but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic ERG. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of stress response and inflammation genes, implying their involvement in cone death. The Nrl-/- retina illustrates the long-term viability of cones in the absence of rods and may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.
2011-11-15 | GSE33674 | GEO
Project description:The loss of cone photoreceptor cells, which are critical for optimal daylight vision, have the great impact on vision during retinal degenerations. Retinal differentiation of human induced pluripotent stem cell (hiPSC) sources could provide a renewable source of cone photoreceptors towards developing a cone cell replacement therapy to treat blindness. Demonstration of comparable gene expression profiles between human foetal and stem cell-derived cones at equivalent stages is required to progress the cell transplantation approach into the patient, as it is hypothesised stem cell-derived cones are required to show high levels of developmental recapitulation of the in vivo generated cones. In this study, the AAV2/9.pR2.1.GFP reporter was used to specifically label L/M-opsin cone photoreceptors in human foetal retinal samples, obtained from the MRC-Wellcome Trust Human Developmental Biology Resource, at a range of developmental stages. The L/M-opsin cone population represent the majority cone cell types in the adult human retina and are the only photoreceptors present within the fovea. Using fluorescence activated cell sorting, L/M-opsin GFP+ cones and GFP- retinal populations, alongside total foetal retinal samples containing all retinal cell tytpes, were isolated and processed for bulk RNA sequencing and downstream comparative analysis. Using DESeq2 differential gene expression analyses, statistically significant genes enriched within the GFP+ human foetal LM-opsin cone populations were determined which led to the identification of a cone enriched gene signature of human L/M-opsin cone photoreceptors. The AAV2/9.pR2.1.GFP reporter was applied to hiPSC-derived retinal cultures to isolate and process cone-like cell populations for RNA sequencing using the same strategy developed within the human foetal retina. Applying the cone enriched gene signature to the transcriptome of hiPSC-derived GFP+ samples at equivalent developmental stages revealed some expression similarities in genes found to be enriched within the late foetal L/M-opsin cone photoreceptors. This analysis overall revealed an intermediate stage of cone differentiation was achieved within the hiPSC-derived samples and the comparison to human foetal L/M-opsin gene express profiles suggesting further differentiation of hiPSC-derived sample is required.
2018-04-20 | E-MTAB-6057 | biostudies-arrayexpress
Project description:Methods: Profiles of individual human retinal organoids (HRO) at 150, 200 and 250 days of development days were treated for 10 days with HBEGF and TNF, and compared to solvent controls (CTRL). N=6 HROs per timepoint and variable. Single-end sequencing with 30 Mio reads per sample was performed at a length of 75 bases on HiSeq2500 (Illumina). The sequence reads that passed quality filters were analyzed at the transcripts level. Results: Our data independently confirms that this protocol provides HROs with macular-like characteristics and shows low baseline variabilities for rods, cones and Müller glia. These features might be key for disease modeling, since most animal models lack a macula, and most other organoid protocols have low cone cell numbers. We found a significant reduction in the number of rod and cone photoreceptor cells after 10 days after HBEGF-TNF (HT) treatment. Specifically, some photoreceptors show pathologic changes, and some are apically displaced, with their nuclei protruding into or passing out of the retina. Histopathologic changes indicate photoreceptor extrusion of viable or dying cones and rods as a pathomechanism. Notably, photoreceptor displacement through the apical retinal border, and thus ectopy in the region of photoreceptor inner and outer segments, has been described in patients and animal models. In controls, photoreceptors were highly ordered, interspersed and interconnected with each other and with Müller glia processes, and these cell connections make up the outer limiting membrane. Upon HT, glial processes expanded in size, extended laterally along the retinal circumference, and replaced or covered photoreceptors over large areas – forming a seal-like glial scar. Taken together, HT induces photoreceptor degeneration via cell extrusion in conjunction with reactive gliosis, glial proliferation, retinal thickening/dyslamination, and glial seal-like scar formation, which reproduces a complex outer retinal atrophy. Interestingly, our transcriptome data revealed distinct gene expression patterns supporting our histological phenotype, specifically, cone and rod degeneration, reactive gliosis, glial proliferation, and photoreceptor cell extrusion. Conclusions: We show that combined application of HBEGF and TNF, two neuropathology associated factors, is sufficient to induce a complex human retinal pathology. We discovered cone and rod cell extrusion as a pathomechanism for photoreceptor degeneration and a potential interrelationship with glial pathologies. Pharmacological inhibitor experiments support this, and PIEZO1 and MAPK signaling as untapped therapeutic targets, even for complex pathologies. Thus, our model provides mechanistic access to several pathologic processes as well as pathogenic functions of inflammation and biomechanics.
2022-09-05 | GSE146641 | GEO
Project description:Photoreceptor disorders are collectively known as retinal degeneration (RD), and include retinitis pigmentosa (RP), cone-rod dystrophy and age related macular degeneration (AMD). These disorders are largely genetic in origin; individual mutations in any one of >200 genes cause RD, making mutation specific therapies prohibitively expensive. A better treatment plan, particularly for late stage disease, may involve stem cell transplants into the photoreceptor or ganglion cell layers of the retina. Stem cells from young mouse retinas can be transplanted, and can form photoreceptors in adult retinas. These cells can be grown in tissue culture, but can no longer form photoreceptors. We have used microarrays to investigate differences in gene expression between cultured retinal progenitor cells (RPCs) that have lost photoreceptor potential, postnatal day 1 (pn1) retinas and the postnatal day 5 (pn5) retinas that contain transplantable photoreceptors. We have also compared FACS sorted Rho-eGFP expressing rod photoreceptors from pn5 retinas with Rho-eGFP negative cells from the same retinas. We have identified over 300 genes upregulated in rod photoreceptor development in multiple comparisons, 37 of which have been previously identified as causative of retinal disease when mutated. It is anticipated that this research should bring us closer to growing photoreceptors in culture and therefore better treatments for RD. This dataset is also a resource for those seeking to identify novel retinopathy genes in RD patients.
2015-01-20 | GSE59201 | GEO