Journal: Nature

Article Title: Developmental convergence and divergence in human stem cell models of autism

doi: 10.1038/s41586-025-10047-5

Figure Lengend Snippet: a , Schematic workflow going from hiPS cells to cortical organoids to sequencing data. The number of hiPS cell lines and individuals (in parentheses) for each form of ASD and controls is indicated. b , Schematic representation of hCO differentiations derived from each hiPS cell line for two forms of ASD (16p11.2 deletion and 22q13.3 deletion). The other forms of ASD can be found in Extended Data Fig. . c , Spearman’s correlation of gene expression between samples from the same time point and form of ASD that were derived either from different lines (red) or from the same line (blue). The sample sizes for each group (number of differentiations) are as follows: day 25: control lines, n = 46; 15q13.3del, n = 7; 16p11.2del, n = 5; 16p11.2dup, n = 4; 22q11.2del, n = 13; 22q13.3del, n = 11; idiopathic, n = 12; PCDH19, n = 2; SHANK3, n = 2; and Timothy syndrome, n = 3. Day 50: control lines, n = 53; 15q13.3del, n = 7; 16p11.2del, n = 6; 16p11.2dup, n = 4; 22q11.2del, n = 15; 22q13.3del, n = 12; idiopathic, n = 15; PCDH19 , n = 2; SHANK3, n = 2; and Timothy syndrome, n = 3. Day 75: control lines, n = 54; 15q13.3del, n = 7; 16p11.2del, n = 6; 16p11.2dup, n = 4; 22q11.2del, n = 23; 22q13.3del, n = 12; idiopathic, n = 15; PCDH19, n = 2; SHANK3, n = 2; and Timothy syndrome, n = 2. Day 100: control lines, n = 50; 15q13.3del, n = 6; 16p11.2del, n = 6; 16p11.2dup, n = 4; 22q11.2del, n = 15; 22q13.3del, n = 12; idiopathic, n = 15; PCDH19, n = 2; SHANK3, n = 2; and Timothy syndrome, n = 2. Boxplots show: centre, median; lower hinge, 25% quantile; upper hinge, 75% quantile; lower whisker, smallest observation greater than or equal to lower hinge −1.5× interquartile range; upper whisker, largest observation less than or equal to upper hinge +1.5× interquartile range. d , Genes within the CNVs are downregulated in deletions and upregulated in duplications, as exemplified by 16p11.2 deletion (del) and duplication (dup) and 15q13.3 deletion. 16p11.2del, 61.5% of genes; 16p11.2dup, 50% of genes and 15q13.3del, 52.9% of genes. *dreamlet P values of less than 0.005. Top illustration adapted from ref. , Springer Nature America; illustrations in the IP–MS and CRISPRi panels created in BioRender. Geschwind, D. (2025) ( https://biorender.com/m2jkj03 ).

Article Snippet: To control for technical variation due to the sequencing and library prep we calculated the principal components of the Picard sequencing metrics ( http://broadinstitute.github.io/picard/ ) using the CollectAlignmentSummaryMetrics, CollectRnaSeqMetrics and MarkDuplicates modules, and included them in our model. To infer genetic ancestry, we called single-nucleotide polymorphisms (SNPs) from the aligned reads using the GATK (v.3.3) Haplotype caller .

Techniques: Sequencing, Derivative Assay, Gene Expression, Control, Whisker Assay, Protein-Protein interactions

Journal: Nature

Article Title: Developmental convergence and divergence in human stem cell models of autism

doi: 10.1038/s41586-025-10047-5

Figure Lengend Snippet: Whole genome sequencing depth in ( a ) 16p11.2 ( b ) 15q13.3 ( c ) 22q11.2 and ( d ) 22q13.3 loci averaged over 10 kb windows. ( e ) Mutational forms not shown in main figure showing genes inside the CNV locus or genes carrying the point mutations (marked in blue). Genes significantly differentially expressed over all time points are denoted by asterisks (dreamlet p-values of p < 0.005). The percent of DE genes in the CNV across the 4 time points is as follows: 22q13del - 77.3% of genes, 22q11del - 88.9% of genes.

Article Snippet: To control for technical variation due to the sequencing and library prep we calculated the principal components of the Picard sequencing metrics ( http://broadinstitute.github.io/picard/ ) using the CollectAlignmentSummaryMetrics, CollectRnaSeqMetrics and MarkDuplicates modules, and included them in our model. To infer genetic ancestry, we called single-nucleotide polymorphisms (SNPs) from the aligned reads using the GATK (v.3.3) Haplotype caller .

Techniques: Sequencing

Journal: Nature

Article Title: Developmental convergence and divergence in human stem cell models of autism

doi: 10.1038/s41586-025-10047-5

Figure Lengend Snippet: ( a ) Overlaps between differentially expressed genes at all time points and risk genes for ASD from either SFARI or from large scale whole exome (WES) sequencing studies , as well as with neurodevelopmental disorders (NDDs) and intellectual disability (ID) risk genes . Colour represents the OR (two-sided Fishers exact test) and the size of the point represented the -log 10 FDR. Only positive significant overlaps (OR > 1 and FDR < 0.05) are shown. ( b ) Select gene ontology (GO) terms enriched in upregulated (red) or downregulated (blue) genes in each of the ASD forms at day 25 (top) or day 100 (bottom). Statistics derived from GSEA. ( c ) Distribution of normalized expression for meta analysis significant example genes at day 100. Boxplots show: centre, median, lower hinge – 25% quantile, upper hinge – 75% quantile, lower whisker – smallest observation greater than or equal to lower hinge –1.5× interquartile range, upper whisker – largest observation less than or equal to upper hinge +1.5× interquartile range. Number of samples (differentiations) for each form: Control – 50, 15q13.3del – 6, 16p11.2del – 6, 16p11.2dup – 4, 22q11.2del – 15, 22q13.3del – 12, Idiopathic – 15, PCDH19 – 2, SHANK3 – 2, Timothy Syndrome – 2. Asterisks denote dreamlet derived p-values: +p < 0.005,* FDR < 0.05, ** FDR < 0.01, *** FDR < 0.005. ( d ) Select GO terms enriched in upregulated or downregulated meta-analysis significant genes Colour corresponds to normalized enrichment score (NES). Point size corresponds to the level of significance (−log 10 (FDR)). Statistics derived from GSEA.

Article Snippet: To control for technical variation due to the sequencing and library prep we calculated the principal components of the Picard sequencing metrics ( http://broadinstitute.github.io/picard/ ) using the CollectAlignmentSummaryMetrics, CollectRnaSeqMetrics and MarkDuplicates modules, and included them in our model. To infer genetic ancestry, we called single-nucleotide polymorphisms (SNPs) from the aligned reads using the GATK (v.3.3) Haplotype caller .

Techniques: Sequencing, Derivative Assay, Expressing, Whisker Assay, Control

Journal: Nature

Article Title: Developmental convergence and divergence in human stem cell models of autism

doi: 10.1038/s41586-025-10047-5

Figure Lengend Snippet: ( a ) Schema of CROP-Seq vector. ( b ) Example of gating for flow cytometry. Left shows a set of hNPCs not expressing virus used for gating compared with hNPCs expressing both dCas9-Krab (GFP) and Crop-Seq-opti-dsRed (TdTomato). Box in the upper right shows double positive cells selected for downstream analysis (~20% of live cell fraction). ( c ) QC measures for CRISPRi single cell libraries organized by gRNA negative (no gRNA detected in sequencing), Control gRNA positive, or Target gRNA positive. ( d ) Number of gRNA UMIs per cell. X-axis shows cut-off of 10 UMIs used to consider a cell gRNA positive (left). Number of of unique gRNAs expressed in each cell (x-axis). Only cells expressing gRNA for a single gene-target are retained for downstream analyses. ( e ) Single cell UMAPs of markers used to differentiate cycling and non-cyling NPCs ( f ) Single cell UMAP coloured by Target-gene presence ( g ) Proportion of cells uniquely expressing gRNAs barcodes for each target within each experiment (n = 18 per Gene comprised of 6 technical replicates, 3 gRNAs barcodes per target, for Controls n = 108: 18 gRNAs across 6 technical replicates). TP53 show increased proportion compared to non-targeting controls (NTCs). Boxplots a-c show: centre, median, lower hinge – 25% quantile, upper hinge – 75% quantile, lower whisker – smallest observation greater than or equal to lower hinge –1.5× interquartile range, upper whisker – largest observation less than or equal to upper hinge +1.5× interquartile range. ***Family wise corrected p < 0.005, Dunnets post-hoc contrast of linear model.

Article Snippet: To control for technical variation due to the sequencing and library prep we calculated the principal components of the Picard sequencing metrics ( http://broadinstitute.github.io/picard/ ) using the CollectAlignmentSummaryMetrics, CollectRnaSeqMetrics and MarkDuplicates modules, and included them in our model. To infer genetic ancestry, we called single-nucleotide polymorphisms (SNPs) from the aligned reads using the GATK (v.3.3) Haplotype caller .

Techniques: Plasmid Preparation, Flow Cytometry, Expressing, Virus, Single Cell, Sequencing, Control, Whisker Assay

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, infected with Pcc, and treated with control (IgG; n = 1 biological replicate) or anti‐IL‐27 mAb (α‐IL‐27; n = 1 biological replicate) between −1 and 5 days of infection. PbT‐II cells were purified from these mice 7 days after infection and single‐cell RNA sequencing (scRNA‐seq) analysis was performed. Details of the experiments are shown in Fig . UMAP plots of PbT‐II cells from IgG control ( n = 4,030) and anti‐IL‐27 mAb‐treated mice ( n = 7,476) after unsupervised clustering of pooled single‐cell data from the two groups, with clusters colored by gene expression profiles. UMAP clustering of PbT‐II cells colored by cell cycle profiles. Summary graph of proportions of PbT‐II cells in each cluster for IgG and anti‐IL‐27 mAb‐treated mice in (A). Dot plots showing the expression of Th1‐, Tfh‐, Tcmp‐related genes (Ciucci et al , ), and other genes of interest in each UMAP cluster of PbT‐II cells from IgG and anti‐IL‐27 mAb‐treated mice. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. Violin plots showing the expression of Th1‐, Tfh‐, Tcmp‐, and proliferation‐associated genes in PbT‐II cells from IgG (light blue) and anti‐IL‐27 mAb (blue) treated mice. Ridge plots showing the expression of published Th1, Tfh, Tmem, and Tcmp CD4 + T cell signatures in each of the UMAP clusters in (A) based on (Ciucci et al , ). Source data are available online for this figure.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Control, Purification, RNA Sequencing, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, infected with Pcc, and were treated with either IgG or anti‐IL‐27 mAb between −1 and 7 days after infection ( n = 1 biological replicate per timepoint). PbT‐II cells were prepared from spleen at day 28 pi, stained for CD4/TCR/CD45.1 and for CD127, KLRG1, and CD49d with TotalSeq antibodies, sort purified, and processed for scRNA‐seq and CITE‐Seq analysis. Details of the experiment are found in Fig . A–G Comparative analysis of scRNA‐seq data from IgG and anti‐IL‐27 mAb‐treated PbT‐II cells. (A) UMAP plot colored of day 28 PbT‐II cells from IgG control ( n = 7,491) and anti‐IL‐27 mAb‐treated mice ( n = 4,944) after unsupervised clustering of pooled single cell data from the two groups, with clusters colored by gene expression profiles. Cluster labels were harmonized to reflect similar gene expression patterns in the clusters at day 7 pi (Fig ) and anti‐IL27 mAb day 7–28 PbT‐II analysis (Fig ). (B) UMAP clustering of PbT‐II cells colored by cell cycle profiles. (C) CITE‐seq analysis of PbT‐II cells for IgG2a (isotype control), CD127, KLRG1, and CD49d, shown in the same UMAP clustering as (A). (D) Proportions (%) of each cluster within PbT‐II cells, with bar graph sizes shown relative to the total number of PbT‐II cells in IgG (36.8 × 10 ) and anti‐IL‐27 mAb treated (265.7 × 10 ) mice. (E) Ridge plots of PbT‐II cells showing the expression of published CD4 + T cell signature genes (Ciucci et al , ). (F) Violin plots comparing the expression of the CD4 + T cell signature genes. (G) Dot plots showing the expression of Th1‐, Tfh‐, Tcmp‐, and proliferation‐associated genes in each cluster. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. H Volcano plot of differentially expressed genes between major clusters 1* and 1** within PbT‐II cells from anti‐IL‐27‐treated mice and corresponding Gene Ontology enrichment analysis for the upregulated genes in each group using Metascape. Source data are available online for this figure.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Staining, Purification, Control, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, treated with IgG or anti‐IL‐27 mAb on day −1, 2 and 5 for day 7 analysis, while mice were treated with anti‐IL‐27 mAb on day −1, 2, 5, and 7 for day 14 and 28 analysis ( n = 1 biological replicate per timepoint). PbT‐II cells were purified and subjected to single‐cell RNA sequencing (scRNA‐seq) and CITE‐seq analysis. The ProjecTILs algorithm (Andreatta et al , ) was used to analyze CD4 + T cell states of PbT‐II cells based on a published reference atlas (Andreatta et al , ). A Experimental scheme. B Gating strategy for the sorting of PbT‐II cells for the scRNA‐seq experiments: Spleen cells were stained for CD4, TCRβ, and CD45.1 to distinguish PbT‐II cells and for TotalSeq IgG2a, CD127, KLRG1, and CD49d for CITE‐seq analysis. C Flow cytometry profiles for each PbT‐II sample analyzed for single‐cell transcriptomics. D, E Predicted distribution of the projected PbT‐II cells in IgG and anti‐IL‐27 mAb‐treated mice on day 7 (D) and day 28 (E) after Pcc infection as density contours in a UMAP plot of a CD4 + T cell reference map (Andreatta et al , ). The bar graphs represent the proportions of the PbT‐II cells projected in the indicated reference subtype.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Purification, RNA Sequencing, Staining, Flow Cytometry, Single-cell Transcriptomics, Infection

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: scRNA‐seq data of PbT‐II cells from Pcc‐infected anti‐IL‐27 mAb‐treated mice (day7, 14, and 28) were pooled, and unsupervised clustering was performed. UMAP plot colored by gene expression clustering. Proportions (%) of each cluster for each time point. Feature plots of indicated genes across cell clusters as distributed in UMAP plots. Dot plots showing the expression of Th1‐, Tfh‐, Tmem‐, and proliferation‐associated genes in each cluster. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. Ridge plots of PbT‐II cell clusters showing the expression of published CD4 + T cell signature genes (Ciucci et al , ).

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet:

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Marker, Staining, FACS, Purification, Software, Cell Isolation