Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis
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ABSTRACT:
PROVIDER: PRJNA573847 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10172146_1.fastq.gz | Fastqsanger.gz | |||
| SRR10172146_2.fastq.gz | Fastqsanger.gz | |||
| SRR10172147_1.fastq.gz | Fastqsanger.gz | |||
| SRR10172147_2.fastq.gz | Fastqsanger.gz | |||
| SRR10172148_1.fastq.gz | Fastqsanger.gz |
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Project description:X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.
| S-EPMC6895767 | biostudies-literature
Project description:The goals of this study are to analyze the effects of RS1 mutation on retinal transcriptome profiling during retina differentiation. We used next generation sequencing to analyze the retinal mRNA profiles in a panel of time-course differentiated 3D retinal cups (RCs) from control donor- and XLRS patient-derived iPSCs, as well as 150-day differentiated RCs from CRISPR/Cas9-reparied patient-derived iPSCs. The transcriptomes of control-RCs and patient-RCs considerably diverged along the time course of differentiation, with the difference becoming more pronounced at the later time points. In contrast, analysis of repaired RCs revealed transcriptomes that were highly similar to those of control-RCs and significantly different from those of patient-RCs. Because RS1 is only expressed in later stage of differentiation, we focued on days 90, 120, and 150 post-induction to identify the genes whose expression was reduced at least 2-fold in patient-RCs. Among all of these genes, 326 genes were overexpressed in normal RCs at all of these stages, emphasizing their importance. According to GSEA annotation, nine out of these 326 genes were implicated in eye development. Among these nine genes, the expression of RS1, IQCB1, and OPA1 was rescued in CRISPR repaired RCs. The relative loss of expression became even more prominent in later stages of retinal differentiation. Our transcriptome analysis revealed significant changes in gene expression between patient-derived and control-RCs that become more pronounced at the later stages of development. This implies that RS1 is essential for normal retinal development at the systemic level. These results reflect the progressive nature of the disease phenotypes observed in RS-1-mutant RCs, mirror the observed ciliary phenotype, and reveal potential links of XLRS to other hereditary retinopathies.
2022-09-03 | GSE137910 | GEO
Project description:An organoid culture system can better recapitulate the cellular structure, function, and interaction between cells and the extracellular matrix (ECM) than the traditional two-dimensional (2D) culture system. We here constructed a condylar cartilage organoid and utilized it to explore the regulatory role of primary cilia at the organoid level. RNA sequencing unveiled the differences of transcriptomics between the condylar cartilage organoid and 2D culture chondrocytes.
2023-05-01 | GSE230630 | GEO
Project description:BACKGROUND: Although reossification of large calvarial defects is possible in children, adults lack this tissue engineering capacity. In this study, the authors compared the differences in gene expression between juvenile and adult dura mater using a mouse cDNA microarray with 42,000 unique elements. METHODS: Non-suture-associated parietal bone was harvested from 6-day-old and 60-day-old mice. The dura mater was carefully dissected from the calvarial disk and snap-frozen. RNA was extracted from pooled dura mater for microarray analysis. The 25 most differentially expressed genes were listed, as were selected bone-related genes. In addition, quantitative real-time reverse-transcriptase polymerase chain reaction confirmation of selected genes-BMP-2, BMP-4, and BMP-7; and osteopontin (OP), osteocalcin (OC), and FGFR-1-was performed. RESULTS: Juvenile dura mater expressed significantly greater amounts of BMP-2 and OP. Minimal difference in OC expression was observed between juvenile and adult dura mater. Extracellular matrix proteins (Col3a1, 5a1, 6a1, and fibronectin 1), osteoblast differentiation markers (Runx2/Cbfa1, Itm2a, and FGFR-1), and the growth factor Ptn were among other genes with greater expression in juvenile dura mater. Markers of osteoclasts (Acp5, MMP9, Ctsk) and the multiple candidate gene Ntrk2 were also expressed at higher levels in the juvenile dura mater. CONCLUSIONS: These findings suggest a more differentiated osteoprogenitor population to exist along with a greater presence of osteoclasts in the juvenile dura mater relative to adults. In addition to establishing a baseline difference in gene expression between juvenile and adult dura mater, new genes potentially critical to the regenerative potential of juvenile calvaria were identified.
2006-11-06 | E-SMDB-3845 | biostudies-arrayexpress
Project description:Background: It has widely been observed that young children are capable of reossifying large calvarial defects, while adults lack this endogenous tissue-engineering capacity. The ability of juvenile animals to regenerate calvarial defects has been investigated in multiple animal models, including mice. In this study, the authors used cDNA microarrays to investigate the expression of osteogenesis-associated genes upstream and downstream of Runx2 in juvenile and adult mouse calvaria. Methods: Nonsuture-associated parietal bone discs were harvested from 6-day-old (n = 50) and 60-day-old (n = 35) male CD-1 mice. After separation of the underlying dura mater and overlying pericranium, the calvarial discs were snap-frozen and RNA was extracted from pooled samples of calvaria for microarray analysis. Genes analyzed included cytokines, receptors, and cell-surface and matrix proteins both upstream and downstream of Runx2. Results: Genes associated with the Runx2 pathway had notably higher levels in the juvenile versus adult calvaria. All genes except for osteocalcin were expressed at least twofold higher in the juvenile calvaria. This pattern was validated with quantitative real-time polymerase chain reaction. In addition, mRNA for potent osteoinductive growth factors was present at higher levels in the juvenile compared with the adult calvaria. Conclusions: These findings reflect a genomic environment of active osteoblast differentia-tion and ossification in the juvenile calvaria compared with the adult aquiescent calvarial tissue. These data suggest that a decreased osteogenic potential of adult calvarial osteoblasts may, in part, explain the inability of adult animals to heal calvarial defects.
2005-07-05 | E-SMDB-3396 | biostudies-arrayexpress
Project description:In Rspondin-based three-dimensional cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. Here we report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely resemble the original tumor. The spectrum of genetic changes within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. Self-renewal of the intestinal epithelium is driven by Lgr5 stem cells located in crypts. We have recently developed a long-term culture system that maintains basic crypt physiology. Wnt signals are required for the maintenance of active crypt stem cells. Indeed, the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo. R-spondin-1 is the ligand for Lgr5. Epidermal growth factor (EGF) signaling is associated with intestinal proliferation, while transgenic expression of Noggin induces a dramatic increase in crypt numbers. The combination of R-spondin-1, EGF, and Noggin in Matrigel sustains ever-expanding small intestinal organoids, which display all hallmarks of the original tissue in terms of architecture, cell type composition, and self-renewal dynamics. We adapted the culture condition for long-term expansion of human colonic epithelium and primary colonic adenocarcinoma, by adding nicotinamide, A83-01 (Alk inhibitor), Prostaglandin E2 and the p38 inhibitor SB202190. Of note, a two-dimensional culture method for cells from normal and malignant primary tissue has been described by Schlegel and colleagues. Here, we explore organoid technology to routinely establish and phenotypically annotate ‘paired organoids’ derived from adjacent tumor and healthy epithelium from CRC patients.
2015-05-07 | E-GEOD-65253 | biostudies-arrayexpress
Project description:Persistent or recurrent retinoblastoma (RB) is associated with the presence of vitreous or/and subretinal seeds in advanced RB and represents a major cause of therapeutic failure. This necessitates the development of novel therapies and thus requires a model of advanced RB for testing candidate therapeutics. To this aim, we established and characterized a three-dimensional, self-organizing organoid model derived from chemotherapy-naïve tumors. The responses of organoids to drugs were determined and compared to relate organoid model to advanced RB, in terms of drug sensitivities. We found that organoids had histological features resembling retinal tumors and seeds and retained DNA copy-number alterations as well as gene expression profile and protein expression of the parental tissue. Cone signal circuitry (M/L+ cells) and glial tumor microenvironment (GFAP+ cells) were primarily present in organoids. Topotecan alone or the combined drug regimen of topotecan and melphalan effectively targeted proliferative tumor cones (RXRg+ Ki67+) in organoids after 24-h drug exposure, blocking mitotic entry. In contrast, methotrexate showed the least efficacy against tumor cells. The drug responses of organoids were consistent with those of tumor cells in advanced disease. Patient-derived organoids enable the creation of a faithful model to use in examining novel therapeutics for RB.
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