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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Rows show DEGs with the ten strongest decreased and increased expressions.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Columns show average expression in the AQP4 population of disease-modelling samples (first panel), the matched HNOCA metacells per sample (second panel), all predicted control astrocytes and all astrocytes in HNOCA.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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m, Volcano plot shows DE analysis between dorsal telencephalic cells in the FXS-2021 dataset and their matched HNOCA metacells.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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DEGs coloured in red (increased in FXS) and blue (decreased in FXS).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Encircled dots show DEGs annotated in SFARI database.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Top bars show the log-transformed odds ratio of SFARI gene enrichment in the increased (red) and decreased (blue) DEGs.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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GBM, glioblastoma.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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a, Overview of disease-modelling neural organoid atlas construction, and projection to primary atlas and HNOCA for downstream analysis.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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b–f, UMAP of integrated disease-modelling neural organoid atlas coloured by predicted cell type annotation (b), predicted regional identities of NPCs, intermediate progenitor cells and neurons (c), publications (d), disease status (e) and marker gene expression (f).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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g,h, Proportions (prop.)
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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of cells assigned to different cell classes (g) and regions (h).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Every stacked bar represents one biological sample.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Side bars show disease status and publication.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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i, Schematic of reconstructing matched HNOCA metacell for each cell in the disease-modelling neural organoid atlas.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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j, UMAP of disease-modelling neural organoid atlas, coloured by transcriptomic similarity with the matched HNOCA metacells.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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k, Violin plot indicates distribution of estimated transcriptomic similarities, split by publication.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Left, distribution in control cells and right, distribution in disease cells.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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l, Heatmap showing expression of top DEGs between the AQP4 population in the GBM-2019 dataset and their matched HNOCA metacells.
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PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
Rows show DEGs with the ten strongest decreased and increased expressions.
|
PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
Columns show average expression in the AQP4 population of disease-modelling samples (first panel), the matched HNOCA metacells per sample (second panel), all predicted control astrocytes and all astrocytes in HNOCA.
|
PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
m, Volcano plot shows DE analysis between dorsal telencephalic cells in the FXS-2021 dataset and their matched HNOCA metacells.
|
PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
DEGs coloured in red (increased in FXS) and blue (decreased in FXS).
|
PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
Encircled dots show DEGs annotated in SFARI database.
|
PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
Top bars show the log-transformed odds ratio of SFARI gene enrichment in the increased (red) and decreased (blue) DEGs.
|
PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
GBM, glioblastoma.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
|
We developed a wkNN-based strategy to generate matched HNOCA metacells for every cell in each disease model organoid scRNA-seq dataset (Fig. 5i), and quantified their transcriptomic similarity (Fig. 5j).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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The dataset of glioblastoma organoids showed substantially lower similarity to their primary counterpart than the other disease models (Fig. 5k).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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To assess these transcriptomic differences, we performed DE analysis between glioblastoma and matched control metacells.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Focusing on the AQP4 population (Extended Data Fig. 10), we identified 1,951 DEGs in glioblastoma cells compared to matched HNOCA metacells (Supplementary Table 9) and found increased expression of genes such as RBM25 (ref. )
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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CALD1 (ref. ),
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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HNRNPU and SPARC (Fig. 5l), all of which have been reported to be relevant to glioblastoma.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Next, we focused on the organoid model of FXS, in which NPCs and neurons in the control organoids were of non-telencephalic identities whereas the disease model organoids mainly contained telencephalic cells (Fig. 5h and Extended Data Fig. 10).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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The integrated HNOCA provides the opportunity to perform DE analysis for FXS neocortical neurons with matched HNOCA metacells, which identified 444 DEGs.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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DEGs higher expressed in FXS cells (122 genes) were enriched for autism-associated genes annotated in the Simons Foundation Autism Research Initiative (SFARI) database.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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One such gene, CHD2, was reported in the original publication as a key regulator of FXS with increased protein level, but its expression change on messenger RNA (mRNA) level change could not be detected in a bulk RNA-seq experiment.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We also detected decreased expression of FMR1, whose loss-of-function mutation causes FXS.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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New scRNA-seq datasets of human neural organoids continue to be generated, and it will be important to continuously extend and update the HNOCA with this extra data.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We therefore established a computational toolkit to project new scRNA-seq data to the HNOCA (Fig. 6a).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We demonstrate the use of the toolkit by incorporating scRNA-seq data from six more studies into the HNOCA (HNOCA-extended; Fig. 6b and Supplementary Table 10), using query-to-reference mapping.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We harmonized cell type annotations using wkNN-based label transfer, and placed the cells in the context of the existing organoid single-cell transcriptomic landscape as represented by the HNOCA (Fig. 6c–e).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Mapping further datasets to the HNOCA using our approach enhances the atlas by increasing its coverage over existing neural organoid protocols and neural cell types generated in organoids.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Fig. 6Extending the HNOCA by means of projection of extra datasets.a, Schematic of projecting further scRNA-seq data by the community to extend the HNOCA.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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b, UMAP shows the dataset composition of the current extended HNOCA.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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c, UMAP shows the projected cell type annotation of cells in the five extended datasets.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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NE, neuroepithelium; NC-D, neural crest derivatives; MC, mesenchymal cell; EC, endothelial cell.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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d, Dot plot shows the expression of selected cell type and regional markers across projected cell types in the extended HNOCA datasets.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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e, Dot plot shows cell type composition and average similarity to the matched HNOCA metacells of the extended datasets.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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f, Schematic shows the analytical pipelines and varied interfaces to facilitate analysing scRNA-seq data of neural organoids for the community.
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PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
a, Schematic of projecting further scRNA-seq data by the community to extend the HNOCA.
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PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
b, UMAP shows the dataset composition of the current extended HNOCA.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
|
c, UMAP shows the projected cell type annotation of cells in the five extended datasets.
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PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
|
NE, neuroepithelium; NC-D, neural crest derivatives; MC, mesenchymal cell; EC, endothelial cell.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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d, Dot plot shows the expression of selected cell type and regional markers across projected cell types in the extended HNOCA datasets.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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e, Dot plot shows cell type composition and average similarity to the matched HNOCA metacells of the extended datasets.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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f, Schematic shows the analytical pipelines and varied interfaces to facilitate analysing scRNA-seq data of neural organoids for the community.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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To enable researchers to use the HNOCA in their own analysis, we provide various options for exploration and interaction with the atlas (Fig. 6f).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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The HNOCA can be browsed through an online portal, enabling visualization of gene expression and discovery of marker genes.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We also provide the HNOCA through an online interface (http://www.archmap.bio/) for the interactive mapping of new datasets, enabling label transfer, presence score computation and metabolic scoring of cell states.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Finally, we have developed HNOCA-tools, a Python package implementing all central analysis approaches presented in this paper, such as annotation, reference mapping, label transfer and DE testing methods.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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In this study, we built a large-scale integrated cell atlas of human neural organoids, the HNOCA, by integrating 1.8 million cells spanning 36 scRNA-seq datasets generated by 15 different laboratories worldwide using 26 different differentiation protocols as well as diverse scRNA-seq technologies.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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The resulting atlas revealed the high complexity of neuronal, glial and non-neural cell types that can develop in neural organoids grown under existing protocol conditions.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Mapping the HNOCA data to various human developing brain cell reference atlases allowed comprehensive evaluation of neural organoid protocols to generate cell types of different brain regions.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We found that organoids in the first 3 months of culture best match to first-trimester primary data, whereas organoids around 3 months of culture and older best match second-trimester primary cell states.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We did not observe significant neuronal maturation and diversification signatures matching older developmental stages, suggesting a limitation of neuronal maturation in current neural organoid protocols.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We performed DE analysis between organoid neuron types and their primary counterparts to evaluate transcriptomic fidelity, and identified metabolic changes related to the glycolysis pathway as a main factor that distinguishes organoid and primary cell states, consistent with previous reports.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Despite the negative effects of metabolic stress on overall transcriptomic fidelity, the molecular identity of regional cell types is maintained as evidenced by transcription factor coexpression patterns that are highly consistent with primary counterparts.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We showcased the mapping of query data, a recently published single-cell transcriptomic neural organoid morphogen screen, to the HNOCA and the primary reference, which enabled a refined cell type annotation, as well as a compositional comparison with existing neural organoid datasets.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Our powerful framework will facilitate quantitative and comparative analysis of scRNA-seq data of human neural organoids, and for the benchmarking of new neural organoid protocols.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Consistent with earlier reports, we find that unguided protocols generate neural cells with high brain regional variability, which is useful when studying broader fate determination during neurodevelopment.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Guided protocols resulted in a strong enrichment of the targeted brain regions.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We also note that some guided protocols, particularly those targeting midbrain, show relatively low specificity and generate neural cells from the nearby brain regions.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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This issue may be due to a differential response of neural stem cells in the organoid to the same morphogen cue, or to the lack of a full understanding of the timing, concentration and combinations of morphogens required to precisely define cells of the deeper regions in the central nervous system.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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The integrated HNOCA is also an excellent resource for analysis of disease-modelling neural organoid data.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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It facilitates cell type annotation and provides a large control cohort of single-cell transcriptomes for comparison.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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For example, we observed discrepancy of cell type and regional composition between control and disease model samples in many studies.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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At the same time, the HNOCA provides the opportunity to identify disease-specific molecular features against a multi-line multi-protocol large-scale control cohort.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We demonstrate how the HNOCA can be extended and updated by projecting extra single-cell transcriptomic data of neural organoids to the atlas.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Further, we have developed a computational toolkit, HNOCA-tools, which will enable other researchers to recapitulate the analytic framework applied in our study.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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Together, we imagine that the HNOCA will be kept up to date and continue to reflect the landscape of human neural cell states generated in organoids in vitro, serving as a living resource for the neural organoid community that enables the assessment of organoid fidelity, the characterization of perturbed and diseased states and the development of new protocols.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We included 33 human neural organoid data from a total of 25 publications plus three unpublished datasets in our atlas (Supplementary Table 1).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We curated all neural organoid datasets used in this study through the sfaira framework (GitHub dev branch, 18 April 2023).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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For this, we obtained scRNA-seq count matrices and associated metadata from the location provided in the data availability section for every included publication or directly from the authors in case of unpublished data.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We harmonized metadata according to the sfaira standards (https://sfaira.readthedocs.io/en/latest/adding_datasets.html) and manually curated an extra metadata column organoid_age_days, which described the number of days the organoid had been in culture before collection.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We next removed any non-applicable subsets of the published datasets: diseased samples or samples expressing disease-associated mutations (refs. ),
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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fused organoids (ref. ),
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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primary fetal data (refs. ),
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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hormone-treated samples (ref. ),
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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data collected before neural induction (refs. )
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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and share-seq data (ref. ).
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We harmonized all remaining datasets to a common feature space using any genes of the biotype ‘protein_coding’ or ‘lncRNA’ from ensembl release 104 while filling any genes missing in a dataset with zero counts.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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On average, 50% of the full gene space (36,842 genes) was reported in each of the constituent datasets.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We then concatenated all remaining datasets to create a single AnnData object.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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All processing and analyses were carried out using scanpy (v.1.9.3) unless indicated otherwise.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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For quality control and filtering of HNOCA, we removed any cells with fewer than 200 genes expressed.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We next removed outlier cells in terms of two quality control metrics: the number of expressed genes and percentage mitochondrial counts.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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To define outlier cells on the basis of each quality control metric, z-transformation is first applied to values across all cells.
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PMC11578878
|
An integrated transcriptomic cell atlas of human neural organoids.
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Cells with any z-transformed metric less than −1.96 or greater than 1.96 are defined as outliers.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
|
For any dataset collected using the v.3 chemistry by 10X Genomics, which contains more than 500 cells after the filtering, we fitted a Gaussian distribution to the histogram denoting the number of expressed genes per cell.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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If a bimodal distribution was detected, we removed any cell with fewer genes expressed than defined by the valley between the two maxima of the distribution.
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PMC11578878
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An integrated transcriptomic cell atlas of human neural organoids.
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We then normalized the raw read counts for all Smart-seq2 data by dividing it by the maximum gene length for each gene obtained from BioMart.
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