Mosaic perturbation of transcription factors in human retinal organoid development
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PROVIDER: PRJEB59921 | ENA |
REPOSITORIES: ENA
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Project description:To begin to understand how TFs regulate retinal cell type identity in human tissues, we established a pooled loss of function (LOF) experiment based on the CROP-seq protocol in developed retinal organoids. We targeted five TFs (OTX2, NRL, CRX, VSX2, and PAX6) that are important for retinal development and expressed dynamically over the organoid developmental time course.
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2023-03-12 | E-MTAB-12715 | biostudies-arrayexpress
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Project description:Single-cell perturbation assays such as Mosaic-seq enable highly multiplexed functional assessment of enhancers in their endogenous genomic context. By introducing a few computational and experimental improvements, we expanded the Mosaic-seq analysis to capture the secondary gene targets of enhancers. Our analysis of >500 putative enhancers in K562 cells demonstrates that many secondary hits are shared among enhancers targeting different transcriptional factors, which reveals an interwoven enhancer-driven gene regulatory network. Together, our data underscore the flexibility of manipulating gene transcription by modifying enhancer activity.
2019-10-31 | GSE129826 | GEO
Project description:Human pluripotent stem cell (hPSC)-derived retinal organoids are three-dimensional cellular aggregates that differentiate and self-organize to closely mimic the spatial and temporal patterning of the developing human retina. Retinal organoid models serve as reliable tools for studying human retinogenesis, yet limitations in the efficiency and reproducibility of current retinal organoid differentiation protocols have limited the use of these models for more high throughput applications such as disease modelling and drug screening. To address these shortcomings, the current study aimed to standardize prior differentiation protocols to yield a highly reproducible and efficient method for generating retinal organoids. Results demonstrated that through regulation of organoid size and shape using quick reaggregation methods, retinal organoids were highly reproducible compared to more traditional methods. Additionally, the timed activation of BMP signaling within developing cells generated pure populations of retinal organoids at 100% efficiency from multiple widely used cell lines, with the default forebrain fate resulting from the inhibition of BMP signaling. Furthermore, given the ability to direct retinal or forebrain fates at complete purity, mRNA-seq analyses were then utilized to identify some of the earliest transcriptional changes that occur during the specification of these two lineages from a common progenitor. These improved methods also yielded retinal organoids with expedited differentiation timelines when compared to traditional methods. Taken together, the results of this study demonstrates the development of a novel, highly reproducible and minimally variable method for generating retinal organoids suitable for analyzing the earliest stages of human retinal/forebrain cell fate specification.
2024-02-05 | GSE254830 | GEO
Project description:Recent advances in organoid and genome editing technologies are allowing for perturbation experiments at an unprecedented scale. However, before doing such experiments it is important to understand the gene expression profile in each of the organoid’s cells, as well as how much heterogeneity there is between individual organoids. Here we characterise an organoid model of mouse gastrulation called gastruloids using single cell RNA-sequencing of individual organoids at half-day intervals between day 3 and day 5 of differentiation (roughly corresponding to E6.5-E8.75 in vivo). Our study reveals multiple differentiation trajectories that have hitherto not been characterised in gastruloids. Intriguingly, we observe that individual gastruloids displayed a strong bias towards producing either mesodermal (largely somitic) or ectodermal (specifically neural) cell types. This bifurcation is already seen at the earliest sampled time point, and is characterised by increased activity of WNT-associated pathways in mesodermally-biased gastruloids as compared to neurally-biased gastruloids. Notably, at day 5, mesodermal gastruloids show an increase in the proportion of neural cells, while neural gastruloids do not produce notably more mesodermal cells. This is in line with previous studies on how the balance between these cell types is regulated. We demonstrate using in silico simulations that without proper understanding of the inter-organoid heterogeneity, perturbation experiments have either very high false positive or negative rates, depending on the statistical model used. Thus in future studies, modelling of inter-organoid heterogeneity will be crucial when designing organoid-based perturbation studies.
2022-08-30 | GSE212050 | GEO
Project description:Molecular information on the very early stages of human retinal development remains scarce due to limitations in obtaining human eye samples of less than six weeks old. Pluripotent stem cell derived retinal organoids provide an unprecedented opportunity for studying human retinal development; however their ability to fully recapitulate early retinal development has not been assessed as yet. Using a combination of scRNA-Seq and ST approaches, we present for the first time a genome wide, single cell spatio-temporal transcriptome of retinal organoid development. Our data demonstrate that retinal organoids recapitulate key events of retinogenesis including optic vesicle/cup formation, formation of a putative ciliary margin zone, emergence of RPCs and their precise and orderly differentiation to various types of retinal neurons. Combining the scRNA- with scATAC-Seq data, we were able to reveal cell type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal cell types. Our work provides the first integrated molecular and spatial atlas of human retinal organoid development that could be used to identify novel genes and key pathways that underpin human retinal development and function.
2024-02-15 | GSE235577 | GEO
Project description:Molecular information on the very early stages of human retinal development remains scarce due to limitations in obtaining human eye samples of less than six weeks old. Pluripotent stem cell derived retinal organoids provide an unprecedented opportunity for studying human retinal development; however their ability to fully recapitulate early retinal development has not been assessed as yet. Using a combination of scRNA-Seq and ST approaches, we present for the first time a genome wide, single cell spatio-temporal transcriptome of retinal organoid development. Our data demonstrate that retinal organoids recapitulate key events of retinogenesis including optic vesicle/cup formation, formation of a putative ciliary margin zone, emergence of RPCs and their precise and orderly differentiation to various types of retinal neurons. Combining the scRNA- with scATAC-Seq data, we were able to reveal cell type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal cell types. Our work provides the first integrated molecular and spatial atlas of human retinal organoid development that could be used to identify novel genes and key pathways that underpin human retinal development and function.
2024-02-15 | GSE235583 | GEO
Project description:Molecular information on the very early stages of human retinal development remains scarce due to limitations in obtaining human eye samples of less than six weeks old. Pluripotent stem cell derived retinal organoids provide an unprecedented opportunity for studying human retinal development; however their ability to fully recapitulate early retinal development has not been assessed as yet. Using a combination of scRNA-Seq and ST approaches, we present for the first time a genome wide, single cell spatio-temporal transcriptome of retinal organoid development. Our data demonstrate that retinal organoids recapitulate key events of retinogenesis including optic vesicle/cup formation, formation of a putative ciliary margin zone, emergence of RPCs and their precise and orderly differentiation to various types of retinal neurons. Combining the scRNA- with scATAC-Seq data, we were able to reveal cell type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal cell types. Our work provides the first integrated molecular and spatial atlas of human retinal organoid development that could be used to identify novel genes and key pathways that underpin human retinal development and function.
2024-02-15 | GSE235582 | GEO
Project description:Single-cell transcriptome atlas of human retinal organoid development
| PRJEB60391 | ENA