Project description:Retinal organoids samples that derived from human embryonic stem cells were analyzed by single-cell RNA sequencing. Two samples at different differentiation stages (day57 and day 171) were included in this study for cell type comparison.
Project description: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:This study investigates the differentiation of retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) to optimise and characterise in-vitro RGC generation. Retinal organoids were derived from a human embryonic stem cell line (H9) and a laboratory-established human induced pluripotent stem cell (hiPSC) line. For each stem cell line, two independent passages of cells were generated as biological replicates (n = 2 per line). To assess transcriptional diversity and differentiation efficiency, organoids were harvested at day 40 of differentiation, dissociated into single cells, and cultured for an additional 14 days to enrich for RGCs. Following enrichment, single-cell suspensions were processed using the 10x Genomics Chromium platform and sequenced on an Illumina NovaSeq X Plus. The resulting single-cell transcriptomes provide a molecular snapshot of RGC differentiation from hPSCs and enable comparison between hESC- and hiPSC-derived retinal lineages. The data contribute to refining differentiation protocols and improving reproducibility of stem-cell-derived retinal models. Raw BCL files were processed at the Australian Genome Research Facility (AGRF) using Illumina’s standard pipeline to generate FASTQ files. FASTQs were additionally processed with Cell Ranger v7.1.0 (10x Genomics) using the GRCh38/Ensembl v109 reference to produce unfiltered gene-by-cell UMI count matrices in standard 10x sparse format (barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz), which are provided as processed data in this submission. Raw FASTQ files are also archived in this ArrayExpress submission.
Project description:The macula of the retina has a high ratio of cones to rods and is critical for central vision and visual acuity. Here we report the generation, transcriptome profiling, and functional validation of single cells from cone-enriched human retinal organoids differentiated from hESCs. Single-cell RNA-seq of 8-month retinal organoids identified clusters of cone and rod photoreceptors and confirmed the cone enrichment initially revealed by immunostaining. Collectively, we have established cone-enriched retinal organoids and a reference of transcriptomes that are rich resources for retinal studies.
Project description:To study the development of human retina, we used single cell RNAseq at key fetal stages and followed the development of the major cell types, as well as populations of transitional cells. We also analyzed stem cell (hPSC)-derived retinal organoids; although organoids have a very similar cellular composition at equivalent ages to the fetal retina, there are some differences in gene expression of particular cell types. Moreover, the inner retinal lamination is disrupted in more advanced stages of organoids when compared with fetal retina. To determine whether the disorganization in the inner retina was due to the culture conditions, we analyzed retinal development in fetal retina maintained under similar conditions. These retinospheres develop for at least 6 months, displaying better inner retinal lamination than retinal organoids. Our scRNAseq comparisons between fetal retina, retinal organoids and retinospheres provide a new resource for developing better in vitro models for retinal disease.
Project description:hESC lines carrying deleterious mutations in the RB1 gene in heterozygous and homozygous state were generated by genome-editing based on CRISPR/Cas9. Parental cell line and genome-edited cell lines were differentiated into retinal organoids for 152 days based on the Protocol published by Döpper et al., Current protocols, PMID: 32956559. Briefly, single cells were reaggregated in presence of dual SMAD and WNT-inhibition; retinal tissue became visible from day 12 onward. BMP4-induction and addition of small molecules CHIR99021 and SU5402 directed differentiation towards retina and retinal pigment epithelium. Long-term differentiation was carried out in the presence of 10% FBS, taurine and retinoic acids. Organoids were collected at indicated time points and either embedded for cryosectioning and immunostaining or frozen at -80°C for RNA preparation.
Project description:To gain a deeper insight into roles of MECP2 in hESCs derived organoids, we performed RNA-seq to analyze the transcriptome changes after MECP2 deletion in hESCs-derived organoids.
Project description:Single-cell gene expression profiles were generated for human retinal organoids derived from the WA09 CRX-tdTomato human embryonic stem cell line. Fragmented organoids were transplanted into the subretinal space of normal canine retinas and analyzed three days post-transplantation. In parallel, fragmented organoids from the same batch, maintained in culture for the same duration, were used as controls. Comparative single-cell transcriptomic analysis provided insights into the early molecular changes associated with the transplanted environment versus in vitro culture conditions, shedding light on the cellular responses and adaptation of retinal organoids post-transplantation.
Project description:The macula of the retina has a high ratio of cones to rods and is critical for central vision and visual acuity. Macula degenerations affect vision the most and are incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-enriched human retinal organoids differentiated from hESCs. Transcriptome profiling using bulk RNA-seq demonstrated that retinal differentiation in vitro recapitulated retinogenesis in vivo in the temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-seq of 8-month retinal organoids identified clusters of cone and rod photoreceptors and confirmed the cone enrichment initially revealed by immunostaining. Notably, comparisons of single-cell transcriptomes demonstrated the similarity between retinal organoids and human macula in cones and rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-enriched retinal organoids and a reference of transcriptomes that are rich resources for retinal studies.