Molecular pathology of Usher 1B patient-derived retinal organoids at single cell resolution
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ABSTRACT: Compare single cell transcriptomes of control and USH1B patient iPSC-derived retinal organoids to elucidate disease mechanisms of Usher syndrome type IB (USH1B). USH1B patient fibroblasts were collected at Great Ormond Street Hospital (GOSH) and reprogrammed to iPSCs. Control and patient iPSCs differentiated in vitro to generate retinal organoids and collected at 35wks. Sequencing was performed at GENEWIZ (Azenta life sciences) on a Illumina NovaSeq system. Data aligned to the human genome UCSC hg38 using cellranger package.
Project description:We show that Retinal pigment epithelium (RPE) secreted-factor, pigment epithelium derived factor (PEDF) secreted/derived from primary or iPSC-derived retinal pigment epithelium (RPE)RPE, dramatically inhibitsed the cell growth of iPSCs. PEDF was detected abundantly in culture supernatant media of primary and iPSC-derived RPE. We examined the gene expression in primary RPE and iPS-derived RPE. Two samples: RPE derived from 253G1 iPSC, Primary RPE.
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.
Project description:Retinitis Pigmentosa is a group of inherited eye disorders characterized by progressive degeneration of photoreceptor cells in the retina, leading to vision loss and eventual blindness. One of the known genetic mutations associated with RP is the c.6926A>C mutation in the RPE (retinal pigment epithelium) cells. The dataset involves multiple experimental approaches and cell types, providing a comprehensive understanding of the disease and potential corrective strategies.
Project description:To assess the geome-wide similarities between primary fetal retinal pigmented epithelium (RPE) and stem-cell derived RPE, we performed whole genome microarray expression on primary RPE and both embryonic stem cell (ESC) derived RPE and induced pluripotent stem cell (iPSC) derived RPE. We found ES-derived RPE better resembles fetal RPE than iPS-derived RPE. Gene expression was measured in primary fetal RPE, ES-derived RPE, iPS-derived RPE. ES cells and BJ fibroblasts were used as controls.
Project description:Ocular pigment epithelium (PE) cells promote the generation of T regulators (PE-induced Treg cells). Moreover, T cells exposed to PE acquire the capacity to suppress the activation of bystander T cells via TGFbeta. Membrane-bound TGFbeta on iris PE cells interacts with TGFbeta receptors on T cells, leading to the conversion of T cells to CD8(+) Treg cells via a cell contact-dependent mechanism. Conversely, soluble forms of TGFbeta produced by retinal PE cells can convert CD4(+) T cells into Treg cells in a manner that is independent of cell contact. In this study, we looked at the expression of immunoregulatory factors (TGFbeta, thrombospondins, CD59, IL-1 receptor antagonist, etc.) in PE cells as identified via an oligonucleotide microarray. Several thrombospondin-binding molecules were detected, and thus we focused subsequent analyses on thrombospondins. Via the conversion of latent TGFbeta to an active form that appears to be mediated by thrombospondin 1 (TSP-1), cultured iris PE and retinal PE cells induce a PE-induced Treg cell fate. After conversion, both ocular PE and PE-induced Treg cells express TSP-1. Regulatory T cell generation was amplified when the T cells also expressed TSP-1. In addition, PE-induced Treg cells significantly suppressed activation of bystander T cells via TSP-1. These results strongly suggest that the ability of ocular PE and PE-induced Treg cells to suppress bystander T cells depends on their capacity to produce TSP-1. Thus, intraocular TSP-1 produced by both ocular parenchymal cells and regulatory T cells is essential for immune regulation in the eye. Experiment Overall Design: Comparison of gene expression pattern in Mouse Primary Cultured Cell Lines (RPE, IPE, CBPE). Experiment Overall Design: Adult (6- to 8-week-old) C57BL/6 purchased from CLEA Japan Inc. (Tokyo, Japan) were used as donors ocular pigment epithelium (PE). To cultivate iris PE (IPE) and ciliary body PE (CBPE), iris and ciliary body tissues in the eyes were dissected out, and then incubated in 1 mg/ml Dispase and 0.05 mg/ml DNase I (both from Boehringer-Mannheim, Mannheim, Germany) for 1 hr. To cultivate retinal PE (RPE) Eyes were cut into two parts along a circumferential line posterior to the ciliary process, creating a ciliary body-deficient posterior eyecup. The eyecup was then incubated in 0.2% trypsin (Biowhitaker) for 1 hr. These tissues were triturated to make a single cell suspension, and then re-suspended in the medium.
Project description:To investigate the effects of ZIKV infection or ZIKV-NS4B-transduction on the global proteome scale at early stages of hNPC differentiation into neurons, hNPC cells were infected with ZIKV (Asian strain: H/PF/2013; MOI=0.01) or transduced with ZIKV-NS4B or HCV-NS4B and one day later cells were either left under proliferative conditions or neuronal differentiation was induced with ROCK inhibitors treatment and growth factors withdrawals. Five days later samples were harvested and processed for quantitative label-free proteomics.
Project description:Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin-free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment of heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined DMEM/F-12 based medium on either Matrigel or defined matrices like Vitronectin and Synthemax. One of heparinâs main functions was to act as a WNT modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost-effective method for cardiomyocyte derivation from hPSCs that can be used for potential large-scale drug screening, disease modeling, and future cellular therapies. 12 human pluripotent stem cells (hPSCs) at three different cardiac differentiation times (0 Days, 3 Days, 6 Days, 10 Days) under different culture conditions (+/- Heparin, +/- IWP2).
Project description:Our purpose was to investigate genes and molecular mechanisms involved in patients with Leber congenital amaurosis (LCA). Fibroblasts from two unrelated clinically-identified patients (Coriell) were reprogrammed to pluripotency by retroviral transduction. These human induced Pluripotent Stem Cells (hiPSCs) were differentiated into neural stem cells (NSC) that mimicked the neural tube stage and retinal pigmented epithelial (RPE) cells that could be targeted by the disease. A genome wide transcriptome analysis was performed with Affymetrix Exon Array GeneChipM-BM-., comparing LCA-hiPSCs derivatives to controls. The aim was to identify differentially expressed genes which may be associated with early developmental defect before the establishment of mature retinal circuitry. We analyzed iPSC-derived retinal pigmented epithelial (RPE) cells from LCA patient's fibroblast (n=2) and iPSC-derivedretinal pigmented epithelial (RPE) cells from healthy people fibroblast (n=2). A total of 13 samples were analyzed : 9 RPE cells derived from iPSC LCA and 4 RPE cells derived from wild-type iPSC.
Project description:This is a combined SWATH database of early myopic guinea pig retina. The retinal tissues were divided into lens induced myopia 4 days group (4-day LIM) and control group; 5 individual animals (10 retinas) were included. The potential biomarkers and underlying biochemical pathways during early myopia could be investigated using SWATH based proteomics approach.
Project description:Embryonic stem (ES) cells have a remarkable capacity to self-organize complex, multi-layered optic cups in vitro via a culture technique called SFEBq. During both SFEBq and in vivo optic cup development, Rax (Rx) expressing neural retina epithelial (NRE) tissues utilize Fgf and Wnt/β-catenin signalling pathways to differentiate into neural retina (NR) and retinal-pigmented epithelial (RPE) tissues, respectively. How these signaling pathways affect gene expression during optic tissue formation has remained largely unknown, especially at the transcriptome scale. We generated Day 10 Rx+ optic tissue using SFEBq, exposed these tissues to either Fgf or Wnt/β-catenin stimulation, and assayed their gene expression at Days 12 and 15 using RNA-Seq. We measured gene expression in these 5 sample groups in biological triplicate using RNA-seq (Illumina HiSeq) .