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control human ipsc line  (WiCell Research Institute Inc)
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WiCell Research Institute Inc control human ipsc line
Control Human Ipsc Line, supplied by WiCell Research Institute Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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clonal control ipsc lines sc102a 1  (System Biosciences Inc)
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( A , B ) Bright-field images (top rows in ( A , B )) and DAPI-stained sections (bottom rows in ( A , B )) of 30-day-old ( A ) and 50-day-old ( B ) cerebral organoids generated from two control iPSC lines <t>(c1,</t> <t>SC102A-1</t> and c2, CRTDi011-A; left two columns), from BWCFF-S ACTB Thr120Ile patient-derived iPSCs (second column from right), and from BWCFF-S ACTG1 Thr203Met patient-derived iPSCs (right column). Scale bars, 500 µm. ( C , D ) Quantification of the organoid area in control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoid sections at culture day 30 ( C ) and day 50 ( D ). ( C ) Data are the mean of 70 control (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 34 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 36 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 30 days-old cerebral organoids of two to eight independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: ** P = 0.0046). ( D ) Data are the mean of 56 control (generated from two different iPSC lines; indicated by circles and triangles as in ( C )), 46 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 42 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 50-day-old cerebral organoids of 2–8 independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: **** P < 0.0001). .
Clonal Control Ipsc Lines Sc102a 1, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human ipsc control lines 1210b2  (ReproCELL)
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( A , B ) Bright-field images (top rows in ( A , B )) and DAPI-stained sections (bottom rows in ( A , B )) of 30-day-old ( A ) and 50-day-old ( B ) cerebral organoids generated from two control iPSC lines <t>(c1,</t> <t>SC102A-1</t> and c2, CRTDi011-A; left two columns), from BWCFF-S ACTB Thr120Ile patient-derived iPSCs (second column from right), and from BWCFF-S ACTG1 Thr203Met patient-derived iPSCs (right column). Scale bars, 500 µm. ( C , D ) Quantification of the organoid area in control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoid sections at culture day 30 ( C ) and day 50 ( D ). ( C ) Data are the mean of 70 control (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 34 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 36 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 30 days-old cerebral organoids of two to eight independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: ** P = 0.0046). ( D ) Data are the mean of 56 control (generated from two different iPSC lines; indicated by circles and triangles as in ( C )), 46 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 42 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 50-day-old cerebral organoids of 2–8 independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: **** P < 0.0001). .
Human Ipsc Control Lines 1210b2, supplied by ReproCELL, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human ipsc control line rpc771  (ReproCELL)
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ReproCELL human ipsc control line rpc771
a Representative images of ATRX-KO HEK293T cells expressing EGFP-IDR2 and its poly-E deletion mutant. The nuclear area is shown as dashed white lines. Schematics of the constructs are shown in the upper right panel. Quantification is shown in the bottom right panel. Quantification was performed on 10 individual cells from 3–4 images per sample. Scale bars, 10 μm. b Schematics of genome editing targeting the human ATRX gene. The poly-E sequences in exon 15 of ATRX are underlined. Arrowheads indicate target sites of sgRNAs. c Sanger sequencing of cDNA <t>from</t> <t>ΔEx15</t> hiPSCs confirmed deletion of exon 15. d Immunostaining of ATRX in WT and ΔEx15 hiPSCs. Data are presented as in ( a ). Scale bars, 10 μm. e CUT&RUN qPCR for ATRX in WT and ΔEx15 hNPCs at SOX21 and RNA45SN1 genomic loci (n = 3 biological replicates in each condition). P-values were calculated by two-tailed Welch’s t-test (SOX21) or Student’s t-test (RNA45SN1). **p < 0.01, *p < 0.05. f Differentially expressed genes (DEGs) of ΔEx15 hNPCs were evaluated using GO analysis for biological processes. g Heatmap representation of correlations between gene expression levels in human <t>iPSC-derived</t> NPCs, NCCs, and MSCs and those in WT and ΔEx15 hNPCs. Colors indicate z-values regarding correlation significance. h Immunostaining of βIII-tubulin and NeuN in WT and ΔEx15 hNPC-derived cells after 14 day-neuronal differentiation. (Insets) DAPI nuclear staining of each field. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. i A schematic of the cerebral organoid differentiation protocol ( top panel). Bright-field observation of day-30 organoids is shown in the bottom panel. Similar results were obtained in 3 independent experiments. Scale bars, 500 μm. j Immunostaining of SOX2, ZO1, and NeuN in organoids is shown in the left panel. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. [Dot plot] n = 100 puncta sampled from 3 biologically independent experiments. Each dot represents the puncta volume, and the red crossbar indicates the mean value. P-values were calculated by a two-tailed Welch’s t-test. [Bar graph] n = 10 cells per condition, sampled from 3 biologically independent experiments. Data are presented as the mean ± SEM. P-values were calculated by two-tailed Student’s t-test ( a , h , j ) or Welch’s t-test ( d ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Human Ipsc Control Line Rpc771, supplied by ReproCELL, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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control ipsc line rpchips8023g1  (ReproCELL)
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ReproCELL control ipsc line rpchips8023g1
a Representative images of ATRX-KO HEK293T cells expressing EGFP-IDR2 and its poly-E deletion mutant. The nuclear area is shown as dashed white lines. Schematics of the constructs are shown in the upper right panel. Quantification is shown in the bottom right panel. Quantification was performed on 10 individual cells from 3–4 images per sample. Scale bars, 10 μm. b Schematics of genome editing targeting the human ATRX gene. The poly-E sequences in exon 15 of ATRX are underlined. Arrowheads indicate target sites of sgRNAs. c Sanger sequencing of cDNA <t>from</t> <t>ΔEx15</t> hiPSCs confirmed deletion of exon 15. d Immunostaining of ATRX in WT and ΔEx15 hiPSCs. Data are presented as in ( a ). Scale bars, 10 μm. e CUT&RUN qPCR for ATRX in WT and ΔEx15 hNPCs at SOX21 and RNA45SN1 genomic loci (n = 3 biological replicates in each condition). P-values were calculated by two-tailed Welch’s t-test (SOX21) or Student’s t-test (RNA45SN1). **p < 0.01, *p < 0.05. f Differentially expressed genes (DEGs) of ΔEx15 hNPCs were evaluated using GO analysis for biological processes. g Heatmap representation of correlations between gene expression levels in human <t>iPSC-derived</t> NPCs, NCCs, and MSCs and those in WT and ΔEx15 hNPCs. Colors indicate z-values regarding correlation significance. h Immunostaining of βIII-tubulin and NeuN in WT and ΔEx15 hNPC-derived cells after 14 day-neuronal differentiation. (Insets) DAPI nuclear staining of each field. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. i A schematic of the cerebral organoid differentiation protocol ( top panel). Bright-field observation of day-30 organoids is shown in the bottom panel. Similar results were obtained in 3 independent experiments. Scale bars, 500 μm. j Immunostaining of SOX2, ZO1, and NeuN in organoids is shown in the left panel. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. [Dot plot] n = 100 puncta sampled from 3 biologically independent experiments. Each dot represents the puncta volume, and the red crossbar indicates the mean value. P-values were calculated by a two-tailed Welch’s t-test. [Bar graph] n = 10 cells per condition, sampled from 3 biologically independent experiments. Data are presented as the mean ± SEM. P-values were calculated by two-tailed Student’s t-test ( a , h , j ) or Welch’s t-test ( d ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Control Ipsc Line Rpchips8023g1, supplied by ReproCELL, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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healthy control human ipsc line scti003-a  (STEMCELL Technologies Inc)
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STEMCELL Technologies Inc healthy control human ipsc line scti003-a
a Representative images of ATRX-KO HEK293T cells expressing EGFP-IDR2 and its poly-E deletion mutant. The nuclear area is shown as dashed white lines. Schematics of the constructs are shown in the upper right panel. Quantification is shown in the bottom right panel. Quantification was performed on 10 individual cells from 3–4 images per sample. Scale bars, 10 μm. b Schematics of genome editing targeting the human ATRX gene. The poly-E sequences in exon 15 of ATRX are underlined. Arrowheads indicate target sites of sgRNAs. c Sanger sequencing of cDNA <t>from</t> <t>ΔEx15</t> hiPSCs confirmed deletion of exon 15. d Immunostaining of ATRX in WT and ΔEx15 hiPSCs. Data are presented as in ( a ). Scale bars, 10 μm. e CUT&RUN qPCR for ATRX in WT and ΔEx15 hNPCs at SOX21 and RNA45SN1 genomic loci (n = 3 biological replicates in each condition). P-values were calculated by two-tailed Welch’s t-test (SOX21) or Student’s t-test (RNA45SN1). **p < 0.01, *p < 0.05. f Differentially expressed genes (DEGs) of ΔEx15 hNPCs were evaluated using GO analysis for biological processes. g Heatmap representation of correlations between gene expression levels in human <t>iPSC-derived</t> NPCs, NCCs, and MSCs and those in WT and ΔEx15 hNPCs. Colors indicate z-values regarding correlation significance. h Immunostaining of βIII-tubulin and NeuN in WT and ΔEx15 hNPC-derived cells after 14 day-neuronal differentiation. (Insets) DAPI nuclear staining of each field. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. i A schematic of the cerebral organoid differentiation protocol ( top panel). Bright-field observation of day-30 organoids is shown in the bottom panel. Similar results were obtained in 3 independent experiments. Scale bars, 500 μm. j Immunostaining of SOX2, ZO1, and NeuN in organoids is shown in the left panel. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. [Dot plot] n = 100 puncta sampled from 3 biologically independent experiments. Each dot represents the puncta volume, and the red crossbar indicates the mean value. P-values were calculated by a two-tailed Welch’s t-test. [Bar graph] n = 10 cells per condition, sampled from 3 biologically independent experiments. Data are presented as the mean ± SEM. P-values were calculated by two-tailed Student’s t-test ( a , h , j ) or Welch’s t-test ( d ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Healthy Control Human Ipsc Line Scti003 A, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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healthy control human ipsc line, scti003-a  (STEMCELL Technologies Inc)
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a Representative images of ATRX-KO HEK293T cells expressing EGFP-IDR2 and its poly-E deletion mutant. The nuclear area is shown as dashed white lines. Schematics of the constructs are shown in the upper right panel. Quantification is shown in the bottom right panel. Quantification was performed on 10 individual cells from 3–4 images per sample. Scale bars, 10 μm. b Schematics of genome editing targeting the human ATRX gene. The poly-E sequences in exon 15 of ATRX are underlined. Arrowheads indicate target sites of sgRNAs. c Sanger sequencing of cDNA <t>from</t> <t>ΔEx15</t> hiPSCs confirmed deletion of exon 15. d Immunostaining of ATRX in WT and ΔEx15 hiPSCs. Data are presented as in ( a ). Scale bars, 10 μm. e CUT&RUN qPCR for ATRX in WT and ΔEx15 hNPCs at SOX21 and RNA45SN1 genomic loci (n = 3 biological replicates in each condition). P-values were calculated by two-tailed Welch’s t-test (SOX21) or Student’s t-test (RNA45SN1). **p < 0.01, *p < 0.05. f Differentially expressed genes (DEGs) of ΔEx15 hNPCs were evaluated using GO analysis for biological processes. g Heatmap representation of correlations between gene expression levels in human <t>iPSC-derived</t> NPCs, NCCs, and MSCs and those in WT and ΔEx15 hNPCs. Colors indicate z-values regarding correlation significance. h Immunostaining of βIII-tubulin and NeuN in WT and ΔEx15 hNPC-derived cells after 14 day-neuronal differentiation. (Insets) DAPI nuclear staining of each field. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. i A schematic of the cerebral organoid differentiation protocol ( top panel). Bright-field observation of day-30 organoids is shown in the bottom panel. Similar results were obtained in 3 independent experiments. Scale bars, 500 μm. j Immunostaining of SOX2, ZO1, and NeuN in organoids is shown in the left panel. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. [Dot plot] n = 100 puncta sampled from 3 biologically independent experiments. Each dot represents the puncta volume, and the red crossbar indicates the mean value. P-values were calculated by a two-tailed Welch’s t-test. [Bar graph] n = 10 cells per condition, sampled from 3 biologically independent experiments. Data are presented as the mean ± SEM. P-values were calculated by two-tailed Student’s t-test ( a , h , j ) or Welch’s t-test ( d ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Healthy Control Human Ipsc Line, Scti003 A, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


( A , B ) Bright-field images (top rows in ( A , B )) and DAPI-stained sections (bottom rows in ( A , B )) of 30-day-old ( A ) and 50-day-old ( B ) cerebral organoids generated from two control iPSC lines (c1, SC102A-1 and c2, CRTDi011-A; left two columns), from BWCFF-S ACTB Thr120Ile patient-derived iPSCs (second column from right), and from BWCFF-S ACTG1 Thr203Met patient-derived iPSCs (right column). Scale bars, 500 µm. ( C , D ) Quantification of the organoid area in control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoid sections at culture day 30 ( C ) and day 50 ( D ). ( C ) Data are the mean of 70 control (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 34 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 36 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 30 days-old cerebral organoids of two to eight independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: ** P = 0.0046). ( D ) Data are the mean of 56 control (generated from two different iPSC lines; indicated by circles and triangles as in ( C )), 46 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 42 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 50-day-old cerebral organoids of 2–8 independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: **** P < 0.0001). .

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: ( A , B ) Bright-field images (top rows in ( A , B )) and DAPI-stained sections (bottom rows in ( A , B )) of 30-day-old ( A ) and 50-day-old ( B ) cerebral organoids generated from two control iPSC lines (c1, SC102A-1 and c2, CRTDi011-A; left two columns), from BWCFF-S ACTB Thr120Ile patient-derived iPSCs (second column from right), and from BWCFF-S ACTG1 Thr203Met patient-derived iPSCs (right column). Scale bars, 500 µm. ( C , D ) Quantification of the organoid area in control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoid sections at culture day 30 ( C ) and day 50 ( D ). ( C ) Data are the mean of 70 control (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 34 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 36 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 30 days-old cerebral organoids of two to eight independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: ** P = 0.0046). ( D ) Data are the mean of 56 control (generated from two different iPSC lines; indicated by circles and triangles as in ( C )), 46 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 42 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 50-day-old cerebral organoids of 2–8 independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: **** P < 0.0001). .

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: Staining, Generated, Control, Derivative Assay, Clone Assay

( A , B ) UMAP plot of single-cell RNA-seq data from control (generated from two different iPSC lines; SC102A-1 and CRTDi011-A)), BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; mut ACTB -1 and mut ACTB -2), BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; mut ACTG1 -1 and mut ACTG1 -2), and BWCFF-S–like ACTB Thr120Ile (generated from the CRISPR/Cas9-edited CRTDi011-A–derived iPSC clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, collectively referred to as cr. ACTB Thr120Ile). Each point represents a single cell, colored either by annotated cell type ( A ; specification on the right side of the panel) or by cell cycle phase ( B ; specification in the right lower corner of the panel). Data consists of 260,880 single cells of 80 control, 44 BWCFF-S ACTB Thr120Ile, 41 BWCFF-S ACTG1 Thr203Met, and 20 cr. ACTB Thr120Ile 30 and 50 days-old cerebral organoids of 1–4 independent batches. ( C ) Quantification of the cell type composition in percent of scRNA-seq data from control (c1, SC102A-1 and c2, CRTDi011-A;), BWCFF-S ACTB Thr120Ile, BWCFF-S ACTG1 Thr203Met, and cr. ACTB Thr120Ile cerebral organoids at culture day 30. Specification of the color-coded cell types on the right side of the panel. ( D ) Quantification of the ratio between basal progenitors and radial glia from scRNA-seq data of control (c1, SC102A-1 and c2, CRTDi011-A; blue bar), BWCFF-S ACTB Thr120Ile (red bar), BWCFF-S ACTG1 Thr203Met (green bar), and cr. ACTB Thr120Ile (violet bar) cerebral organoids at culture day 30. ( C , D ) Data consists of 144,458 single cells of 40 control, 20 BWCFF-S ACTB Thr120Ile, 23 BWCFF-S ACTG1 Thr203Met, and 10 cr. ACTB Thr120Ile 30-day-old cerebral organoids of 1–4 independent batches. .

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: ( A , B ) UMAP plot of single-cell RNA-seq data from control (generated from two different iPSC lines; SC102A-1 and CRTDi011-A)), BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; mut ACTB -1 and mut ACTB -2), BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; mut ACTG1 -1 and mut ACTG1 -2), and BWCFF-S–like ACTB Thr120Ile (generated from the CRISPR/Cas9-edited CRTDi011-A–derived iPSC clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, collectively referred to as cr. ACTB Thr120Ile). Each point represents a single cell, colored either by annotated cell type ( A ; specification on the right side of the panel) or by cell cycle phase ( B ; specification in the right lower corner of the panel). Data consists of 260,880 single cells of 80 control, 44 BWCFF-S ACTB Thr120Ile, 41 BWCFF-S ACTG1 Thr203Met, and 20 cr. ACTB Thr120Ile 30 and 50 days-old cerebral organoids of 1–4 independent batches. ( C ) Quantification of the cell type composition in percent of scRNA-seq data from control (c1, SC102A-1 and c2, CRTDi011-A;), BWCFF-S ACTB Thr120Ile, BWCFF-S ACTG1 Thr203Met, and cr. ACTB Thr120Ile cerebral organoids at culture day 30. Specification of the color-coded cell types on the right side of the panel. ( D ) Quantification of the ratio between basal progenitors and radial glia from scRNA-seq data of control (c1, SC102A-1 and c2, CRTDi011-A; blue bar), BWCFF-S ACTB Thr120Ile (red bar), BWCFF-S ACTG1 Thr203Met (green bar), and cr. ACTB Thr120Ile (violet bar) cerebral organoids at culture day 30. ( C , D ) Data consists of 144,458 single cells of 40 control, 20 BWCFF-S ACTB Thr120Ile, 23 BWCFF-S ACTG1 Thr203Met, and 10 cr. ACTB Thr120Ile 30-day-old cerebral organoids of 1–4 independent batches. .

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: RNA Sequencing, Control, Generated, Clone Assay, CRISPR, Derivative Assay

( A , B ) Quantification of the cell type composition in percent of scRNA-seq data from control (c1, SC102A-1 and c2, CRTDi011-A;), BWCFF-S ACTB Thr120Ile, BWCFF-S ACTG1 Thr203Met, and cr. ACTB Thr120Ile cerebral organoids at culture day 30 ( A ) and 50 ( B ). Specification of the color-coded cell types on the right side of the panel. ( C ) Quantification of the composition of the combined cell types (i.e., radial glia, basal progenitors, neurons, neuroectodermal-like cells, neural crest and mesenchymal-like cells) in percent of scRNA-seq data from control (c1, SC102A-1 and c2, CRTDi011-A;), BWCFF-S ACTB Thr120Ile, BWCFF-S ACTG1 Thr203Met, and cr. ACTB Thr120Ile cerebral organoids at culture day 50. Specification of the color-coded cell types on the right side of the panel.

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: ( A , B ) Quantification of the cell type composition in percent of scRNA-seq data from control (c1, SC102A-1 and c2, CRTDi011-A;), BWCFF-S ACTB Thr120Ile, BWCFF-S ACTG1 Thr203Met, and cr. ACTB Thr120Ile cerebral organoids at culture day 30 ( A ) and 50 ( B ). Specification of the color-coded cell types on the right side of the panel. ( C ) Quantification of the composition of the combined cell types (i.e., radial glia, basal progenitors, neurons, neuroectodermal-like cells, neural crest and mesenchymal-like cells) in percent of scRNA-seq data from control (c1, SC102A-1 and c2, CRTDi011-A;), BWCFF-S ACTB Thr120Ile, BWCFF-S ACTG1 Thr203Met, and cr. ACTB Thr120Ile cerebral organoids at culture day 50. Specification of the color-coded cell types on the right side of the panel.

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: Control

( A – J ) All cerebral organoids are at culture day 30. ( A , G ) SOX2 immunofluorescence (magenta), combined with DAPI staining (white), of sections containing ventricle-like structures of control (c1, SC102A-1, left image), BWCFF-S ACTB Thr120Ile (middle image), and BWCFF-S ACTG1 Thr203Met (right image) cerebral organoids. High magnification images of the same samples are shown in ( G ). White lines indicate the area of the VZ; magenta lines indicate the radial thickness of the VZ at four different positions per ventricle-like structure. Scale bars, 50 µm ( A ) and 20 µm ( G ). ( B – E , H , I ) Quantification of various parameters of the VZ of control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoids. ( B ) Quantification of the radial thickness of the VZ (magenta lines in ( A )). ( C ) Quantification of the VZ perimeter (outer white line in ( A )). ( D ) Quantification of the VZ area (area between the inner and outer white lines in ( A )). ( E ) Quantification of the length of the apical surface of a given ventricle-like structure (inner white line in ( A )). ( F ) Quantification of the number of ventricle-like structures. Data are the mean of 34 control organoids (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A), 345 ventricle-like structures in total), 40 BWCFF-S ACTB Thr120Ile organoids (generated from two different iPSC clones; indicated by circles and triangles, 201 ventricle-like structures in total) and 30 ACTG1 Thr203Met organoids (generated from two different iPSC clones; indicated by circles and triangles, 248 ventricle-like structures in total), each from two to four independent batches; error bars indicate SD; statistical significance was determined by Kruskal–Wallis test (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: ns, not significant). ( H ) Quantification of the cell density in a 100 µm 2 field of the VZ. ( I ) Quantification of VZ cells per ventricle-like structure. ( B – E , H , I ) Data are the mean of 35 control organoids (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A), 159 ventricle-like structures in total), 26 BWCFF-S ACTB Thr120Ile organoids (generated from two different iPSC clones; indicated by circles and triangles, 138 ventricle-like structures in total) and 22 ACTG1 Thr203Met organoids (generated from two different iPSC clones; indicated by circles and triangles, 132 ventricle-like structures in total), each from two to three independent batches; error bars indicate SD; statistical significance was determined by Kruskal–Wallis test ( B – E , I ) and one-way ANOVA ( H ) (c1 + c2 vs. ACTB Thr120Ile: ****, P < 0.0001 ( B – D , I ), ***, P = 0.0003 ( E ); c1 + c2 vs. ACTG1 Thr203Met: **** P < 0.0001 ( B – D , I ), *** P = 0.0004 ( E ); ns, not significant ( H )). ( J ) Quantification of the proportion of DAPI + cells that are also Ki-67 + in the VZ of control (c1, SC102A-1 and c2, CRTDi011-A; blue bar), BWCFF-S ACTB Thr120Ile (red bar), and BWCFF-S ACTG1 Thr203Met (green bar) cerebral organoids at culture day 30. Data are the mean of 68 control (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 23 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 34 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) VZs of 8–12 30-day-old cerebral organoids from 1–2 independent batches; error bars indicate SD; ns, not significant (one-way ANOVA). ( K ) Quantification of the proportion of DAPI + cells that are also PH3 + in the VZ of control (c1, SC102A- 1 and c2, CRTDi011-A; blue bar), BWCFF-S ACTB Thr120Ile (red bar), and BWCFF-S ACTG1 Thr203Met (green bar) cerebral organoids at culture day 30. Data are the mean of 59 ventricle-like structures of control organoids (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 48 ventricle-like structures of BWCFF-S ACTB Thr120Ile organoids (generated from two different iPSC clones; indicated by circles and triangles) and 38 ventricle-like structures of BWCFF-S ACTG1 Thr203Met organoids (generated from two different iPSC clones; indicated by circles and triangles), of 16–17 30-day-old cerebral organoids from two independent batches each; error bars indicate SD; ns, not significant (Kruskal–Wallis test). .

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: ( A – J ) All cerebral organoids are at culture day 30. ( A , G ) SOX2 immunofluorescence (magenta), combined with DAPI staining (white), of sections containing ventricle-like structures of control (c1, SC102A-1, left image), BWCFF-S ACTB Thr120Ile (middle image), and BWCFF-S ACTG1 Thr203Met (right image) cerebral organoids. High magnification images of the same samples are shown in ( G ). White lines indicate the area of the VZ; magenta lines indicate the radial thickness of the VZ at four different positions per ventricle-like structure. Scale bars, 50 µm ( A ) and 20 µm ( G ). ( B – E , H , I ) Quantification of various parameters of the VZ of control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoids. ( B ) Quantification of the radial thickness of the VZ (magenta lines in ( A )). ( C ) Quantification of the VZ perimeter (outer white line in ( A )). ( D ) Quantification of the VZ area (area between the inner and outer white lines in ( A )). ( E ) Quantification of the length of the apical surface of a given ventricle-like structure (inner white line in ( A )). ( F ) Quantification of the number of ventricle-like structures. Data are the mean of 34 control organoids (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A), 345 ventricle-like structures in total), 40 BWCFF-S ACTB Thr120Ile organoids (generated from two different iPSC clones; indicated by circles and triangles, 201 ventricle-like structures in total) and 30 ACTG1 Thr203Met organoids (generated from two different iPSC clones; indicated by circles and triangles, 248 ventricle-like structures in total), each from two to four independent batches; error bars indicate SD; statistical significance was determined by Kruskal–Wallis test (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: ns, not significant). ( H ) Quantification of the cell density in a 100 µm 2 field of the VZ. ( I ) Quantification of VZ cells per ventricle-like structure. ( B – E , H , I ) Data are the mean of 35 control organoids (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A), 159 ventricle-like structures in total), 26 BWCFF-S ACTB Thr120Ile organoids (generated from two different iPSC clones; indicated by circles and triangles, 138 ventricle-like structures in total) and 22 ACTG1 Thr203Met organoids (generated from two different iPSC clones; indicated by circles and triangles, 132 ventricle-like structures in total), each from two to three independent batches; error bars indicate SD; statistical significance was determined by Kruskal–Wallis test ( B – E , I ) and one-way ANOVA ( H ) (c1 + c2 vs. ACTB Thr120Ile: ****, P < 0.0001 ( B – D , I ), ***, P = 0.0003 ( E ); c1 + c2 vs. ACTG1 Thr203Met: **** P < 0.0001 ( B – D , I ), *** P = 0.0004 ( E ); ns, not significant ( H )). ( J ) Quantification of the proportion of DAPI + cells that are also Ki-67 + in the VZ of control (c1, SC102A-1 and c2, CRTDi011-A; blue bar), BWCFF-S ACTB Thr120Ile (red bar), and BWCFF-S ACTG1 Thr203Met (green bar) cerebral organoids at culture day 30. Data are the mean of 68 control (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 23 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 34 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) VZs of 8–12 30-day-old cerebral organoids from 1–2 independent batches; error bars indicate SD; ns, not significant (one-way ANOVA). ( K ) Quantification of the proportion of DAPI + cells that are also PH3 + in the VZ of control (c1, SC102A- 1 and c2, CRTDi011-A; blue bar), BWCFF-S ACTB Thr120Ile (red bar), and BWCFF-S ACTG1 Thr203Met (green bar) cerebral organoids at culture day 30. Data are the mean of 59 ventricle-like structures of control organoids (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 48 ventricle-like structures of BWCFF-S ACTB Thr120Ile organoids (generated from two different iPSC clones; indicated by circles and triangles) and 38 ventricle-like structures of BWCFF-S ACTG1 Thr203Met organoids (generated from two different iPSC clones; indicated by circles and triangles), of 16–17 30-day-old cerebral organoids from two independent batches each; error bars indicate SD; ns, not significant (Kruskal–Wallis test). .

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: Immunofluorescence, Staining, Control, Generated, Clone Assay

Triple immunofluorescence for Caspase 3 (green), SOX2 (magenta) and ZO-1 (orange), combined with DAPI staining (blue), of sections of control (c1, SC102A-1 and c2, CRTDi011-A; two left columns), BWCFF-S ACTB Thr120Ile (second column from right) and BWCFF-S ACTG1 Thr203Met (right column) 30-day-old cerebral organoids showing ventricle-like structures. Scale bars, 100 μm.

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: Triple immunofluorescence for Caspase 3 (green), SOX2 (magenta) and ZO-1 (orange), combined with DAPI staining (blue), of sections of control (c1, SC102A-1 and c2, CRTDi011-A; two left columns), BWCFF-S ACTB Thr120Ile (second column from right) and BWCFF-S ACTG1 Thr203Met (right column) 30-day-old cerebral organoids showing ventricle-like structures. Scale bars, 100 μm.

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: Immunofluorescence, Staining, Control

( A ) Triple immunofluorescence for Ki-67 (orange), SOX2 (magenta) and pan-cadherin (green) of sections of control (c1, SC102A-1 and c2, CRTDi011-A; two left columns), BWCFF-S ACTB Thr120Ile (second column from right) and BWCFF-S ACTG1 Thr203Met (right column) 30-day-old cerebral organoids showing ventricle-like structures. Scale bars, 100 μm. ( B ) Triple immunofluorescence for SOX2 (magenta), phosphohistone H3 (PH3, green) and ZO-1 (orange), combined with DAPI staining (blue), of sections of control (c1, SC102A-1 and c2, CRTDi011- A; two left columns), BWCFF-S ACTB Thr120Ile (second column from right) and BWCFF-S ACTG1 Thr203Met (right column) 30-day-old cerebral organoids showing ventricle-like structures. Scale bars, 100 μm.

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: ( A ) Triple immunofluorescence for Ki-67 (orange), SOX2 (magenta) and pan-cadherin (green) of sections of control (c1, SC102A-1 and c2, CRTDi011-A; two left columns), BWCFF-S ACTB Thr120Ile (second column from right) and BWCFF-S ACTG1 Thr203Met (right column) 30-day-old cerebral organoids showing ventricle-like structures. Scale bars, 100 μm. ( B ) Triple immunofluorescence for SOX2 (magenta), phosphohistone H3 (PH3, green) and ZO-1 (orange), combined with DAPI staining (blue), of sections of control (c1, SC102A-1 and c2, CRTDi011- A; two left columns), BWCFF-S ACTB Thr120Ile (second column from right) and BWCFF-S ACTG1 Thr203Met (right column) 30-day-old cerebral organoids showing ventricle-like structures. Scale bars, 100 μm.

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: Immunofluorescence, Control, Staining

( A ) Alpha-tubulin immunofluorescence (white) of 30-day-old human cerebral organoid sections showing mitotic microtubules, including astral microtubules, and counterstained with DAPI (blue). Upper images, vertical metaphase plate orientation, typical of mitotic APs in control organoids. Lower images, horizontal metaphase plate orientation, typical of mitotic APs in actin mutant organoids. Images are single 0.75 μm confocal sections. Apical surface is down. Scale bar, 5 μm. ( B ) Quantitation of the chromosome metaphase plate orientation in mitotic APs of control (left chart) and BCWFF-S (middle and right charts) cerebral organoids at culture day 30. Each dot represents a cell. 90 ̊ indicates a perfectly vertical metaphase plate orientation with respect to the local apical surface. Cells in the 90–61 ° bin have largely vertical orientations, cells in the 60–31 ° bin have largely oblique orientations, and cells in the 30–0 ° bin have largely horizontal orientations. ( C ) Quantification of apical plus basal (left) and central (right) astral microtubule fibers per mitotic AP of control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoids at culture day 30. Data are the mean of 30 control (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 20 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 27 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) mitotic APs; error bars indicate SD; ns, not significant (two-way ANOVA). ( D ) Double immunofluorescence for SOX2 (magenta) and ZO-1 (orange), combined with DAPI staining (blue), of sections of control (c2, CRTDi011-A) and BWCFF-S ACTB Thr120Ile 30-day-old cerebral organoids, as follows. Images show representative examples of interphase (left column, c2, CRTDi011-A) and metaphase (second column from left, c2, CRTDi011-A) APs, and APs in anaphase (two right columns) with vertical (c2, CRTDi011-A; second column from right) or horizontal ( ACTB Thr120Ile; right column) cleavage plane angle; Scale bars, 5 µm. The axis of the cleavage plane is marked with a white dotted line. ( E ) Schematic depiction of the determination of anaphase AP cleavage plane angles relative to the apical surface (created with biorender.com). Cleavage plane angles are compiled into three groups as indicated. These three groups also apply to ( F , G ). ( F , G ) Quantification ( F ) and percentage distribution ( G ) of cleavage plane angles of anaphase APs of (i) control cerebral organoids (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A) in ( F ) and by blue bars in ( G )); (ii) BWCFF-S ACTB Thr120Ile cerebral organoids (generated from two different iPSC clones; indicated by circles and triangles in ( F ) and by red bars in ( G )); and (iii) ACTG1 Thr203Met cerebral organoids (generated from two different iPSC clones; indicated by circles and triangles in ( F ) and by green bars in ( G )); all cerebral organoids are at culture day 30. Data consist of 49–114 anaphase APs, analyzed in (i) 23 control cerebral organoids (generated from 2 different iPSC lines; c1, SC102A-1: 10 cerebral organoids, 53 APs; c2, CRTDi011-A: 13 cerebral organoids, 61 APs); (ii) 21 ACTB Thr120Ile cerebral organoids (generated from two different iPSC clones; 33 and 21 APs per clone); and 23 ACTG1 Thr203Met cerebral organoids (generated from two different iPSC clones; 15 and 34 APs, respectively); in each of the three conditions, the cerebral organoids are of 2–3 independent batches per clone. ( G ) For each of the three conditions, the numbers of mitotic AP cleavage plane angles in each group are expressed as a percentage of the total (set to 100). Error bars indicate SD; statistical significance was determined by two-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: *** P = 0.001). Moreover, statistical analysis using the chi-squared test resulted in P < 0.0001. ( H ) Quantification of the number of TBR2 + cells in a 100 µm 2 field of the SVZ/NL of control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoid sections at culture day 30. Data are the mean of 24 control (generated from two different iPSC lines; indicated by circles and triangles), 18 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 16 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 30-day-old cerebral organoids of 1–2 independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: *** P = 0.0004; c1 + c2 vs. ACTG1 Thr203Met: ** P = 0.0043). ( I ) TBR2 immunofluorescence (yellow, top row), combined with DAPI staining (blue, middle row), of sections containing ventricle-like structures of control (c1, SC102A-1, left images), BWCFF-S ACTB Thr120Ile (middle images), and BWCFF-S ACTG1 Thr203Met (right images) cerebral organoids. Bottom row, high magnification views of the insets shown in the middle row. Scale bars, 100 µm. .

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: ( A ) Alpha-tubulin immunofluorescence (white) of 30-day-old human cerebral organoid sections showing mitotic microtubules, including astral microtubules, and counterstained with DAPI (blue). Upper images, vertical metaphase plate orientation, typical of mitotic APs in control organoids. Lower images, horizontal metaphase plate orientation, typical of mitotic APs in actin mutant organoids. Images are single 0.75 μm confocal sections. Apical surface is down. Scale bar, 5 μm. ( B ) Quantitation of the chromosome metaphase plate orientation in mitotic APs of control (left chart) and BCWFF-S (middle and right charts) cerebral organoids at culture day 30. Each dot represents a cell. 90 ̊ indicates a perfectly vertical metaphase plate orientation with respect to the local apical surface. Cells in the 90–61 ° bin have largely vertical orientations, cells in the 60–31 ° bin have largely oblique orientations, and cells in the 30–0 ° bin have largely horizontal orientations. ( C ) Quantification of apical plus basal (left) and central (right) astral microtubule fibers per mitotic AP of control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoids at culture day 30. Data are the mean of 30 control (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A)), 20 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 27 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) mitotic APs; error bars indicate SD; ns, not significant (two-way ANOVA). ( D ) Double immunofluorescence for SOX2 (magenta) and ZO-1 (orange), combined with DAPI staining (blue), of sections of control (c2, CRTDi011-A) and BWCFF-S ACTB Thr120Ile 30-day-old cerebral organoids, as follows. Images show representative examples of interphase (left column, c2, CRTDi011-A) and metaphase (second column from left, c2, CRTDi011-A) APs, and APs in anaphase (two right columns) with vertical (c2, CRTDi011-A; second column from right) or horizontal ( ACTB Thr120Ile; right column) cleavage plane angle; Scale bars, 5 µm. The axis of the cleavage plane is marked with a white dotted line. ( E ) Schematic depiction of the determination of anaphase AP cleavage plane angles relative to the apical surface (created with biorender.com). Cleavage plane angles are compiled into three groups as indicated. These three groups also apply to ( F , G ). ( F , G ) Quantification ( F ) and percentage distribution ( G ) of cleavage plane angles of anaphase APs of (i) control cerebral organoids (generated from two different iPSC lines; indicated by circles (c1, SC102A-1) and triangles (c2, CRTDi011-A) in ( F ) and by blue bars in ( G )); (ii) BWCFF-S ACTB Thr120Ile cerebral organoids (generated from two different iPSC clones; indicated by circles and triangles in ( F ) and by red bars in ( G )); and (iii) ACTG1 Thr203Met cerebral organoids (generated from two different iPSC clones; indicated by circles and triangles in ( F ) and by green bars in ( G )); all cerebral organoids are at culture day 30. Data consist of 49–114 anaphase APs, analyzed in (i) 23 control cerebral organoids (generated from 2 different iPSC lines; c1, SC102A-1: 10 cerebral organoids, 53 APs; c2, CRTDi011-A: 13 cerebral organoids, 61 APs); (ii) 21 ACTB Thr120Ile cerebral organoids (generated from two different iPSC clones; 33 and 21 APs per clone); and 23 ACTG1 Thr203Met cerebral organoids (generated from two different iPSC clones; 15 and 34 APs, respectively); in each of the three conditions, the cerebral organoids are of 2–3 independent batches per clone. ( G ) For each of the three conditions, the numbers of mitotic AP cleavage plane angles in each group are expressed as a percentage of the total (set to 100). Error bars indicate SD; statistical significance was determined by two-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: **** P < 0.0001; c1 + c2 vs. ACTG1 Thr203Met: *** P = 0.001). Moreover, statistical analysis using the chi-squared test resulted in P < 0.0001. ( H ) Quantification of the number of TBR2 + cells in a 100 µm 2 field of the SVZ/NL of control (c1, SC102A-1 and c2, CRTDi011-A; blue bars), BWCFF-S ACTB Thr120Ile (red bars), and BWCFF-S ACTG1 Thr203Met (green bars) cerebral organoid sections at culture day 30. Data are the mean of 24 control (generated from two different iPSC lines; indicated by circles and triangles), 18 BWCFF-S ACTB Thr120Ile (generated from two different iPSC clones; indicated by circles and triangles) and 16 BWCFF-S ACTG1 Thr203Met (generated from two different iPSC clones; indicated by circles and triangles) 30-day-old cerebral organoids of 1–2 independent batches; error bars indicate SD; statistical significance was determined by one-way ANOVA (c1 + c2 vs. ACTB Thr120Ile: *** P = 0.0004; c1 + c2 vs. ACTG1 Thr203Met: ** P = 0.0043). ( I ) TBR2 immunofluorescence (yellow, top row), combined with DAPI staining (blue, middle row), of sections containing ventricle-like structures of control (c1, SC102A-1, left images), BWCFF-S ACTB Thr120Ile (middle images), and BWCFF-S ACTG1 Thr203Met (right images) cerebral organoids. Bottom row, high magnification views of the insets shown in the middle row. Scale bars, 100 µm. .

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: Immunofluorescence, Control, Mutagenesis, Quantitation Assay, Generated, Clone Assay, Staining

( A ) Triple immunofluorescence for SOX2 (magenta), ZO-1 (orange) and pan-actin (green) of sections of control (c1, SC102A-1 and c2, CRTDi011-A; two left panels) and BWCFF-S ACTB Thr120Ile (second panel from right) and ACTG1 Thr203Met (right panel) 30-day-old cerebral organoids; Scale bars, 100 μm. ( B ) Exemplary images of ZO-1 immunofluorescence of sections of control (c1, SC102A-1 and c2, CRTDi011-A; two left panels), BWCFF-S ACTB Thr120Ile (second panel from right) and ACTG1 Thr203Met (right panel) 30-day-old cerebral organoids showing normal (left panel) and abnormal (second, third and fourth panel from left) adherent junction belt morphology. Scale bars, 100 μm.

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: ( A ) Triple immunofluorescence for SOX2 (magenta), ZO-1 (orange) and pan-actin (green) of sections of control (c1, SC102A-1 and c2, CRTDi011-A; two left panels) and BWCFF-S ACTB Thr120Ile (second panel from right) and ACTG1 Thr203Met (right panel) 30-day-old cerebral organoids; Scale bars, 100 μm. ( B ) Exemplary images of ZO-1 immunofluorescence of sections of control (c1, SC102A-1 and c2, CRTDi011-A; two left panels), BWCFF-S ACTB Thr120Ile (second panel from right) and ACTG1 Thr203Met (right panel) 30-day-old cerebral organoids showing normal (left panel) and abnormal (second, third and fourth panel from left) adherent junction belt morphology. Scale bars, 100 μm.

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: Immunofluorescence, Control

Triple immunofluorescence for alpha-tubulin (orange, shown in rows 1, 3 and 4), beta-tubulin (green, shown in rows 2–4) and SOX2 (magenta, shown in row 4) of control (c1, SC102A-1 and c2, CRTDi011-A; two left columns), BWCFF-S ACTB Thr120Ile (second column from right) and BWCFF-S ACTG1 Thr203Met (right column) 30-day-old cerebral organoids. Note the strong alpha-tubulin and beta-tubulin fluorescence signal at the apical cell cortex of BWCFF-S VZ progenitors (white arrows); Scale bars, 100 µm.

Journal: EMBO Reports

Article Title: Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly

doi: 10.1038/s44319-025-00647-7

Figure Lengend Snippet: Triple immunofluorescence for alpha-tubulin (orange, shown in rows 1, 3 and 4), beta-tubulin (green, shown in rows 2–4) and SOX2 (magenta, shown in row 4) of control (c1, SC102A-1 and c2, CRTDi011-A; two left columns), BWCFF-S ACTB Thr120Ile (second column from right) and BWCFF-S ACTG1 Thr203Met (right column) 30-day-old cerebral organoids. Note the strong alpha-tubulin and beta-tubulin fluorescence signal at the apical cell cortex of BWCFF-S VZ progenitors (white arrows); Scale bars, 100 µm.

Article Snippet: The clonal control iPSC lines SC102A-1 (System Biosciences) and CRTDi011-A (this study), the mutated CRTDi011-A-derived clones CRTDi011-A–mut ACTB -1 and CRTDi011-A–mut ACTB -2, and the BWCFF-S patient-derived iPSC lines BWCFF-S ACTB Thr120Ile (clones mut ACTB -1 and mut ACTB -2) and BWCFF-S ACTG1 Thr203Met (clones mut ACTG1 -1 and mut ACTG1 -2) (all this study) were subjected to an identical culture protocol under feeder-free conditions on Matrigel-coated plates (Corning) in mTeSRTM1 (StemCell Technologies) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air.

Techniques: Immunofluorescence, Control, Fluorescence

a Representative images of ATRX-KO HEK293T cells expressing EGFP-IDR2 and its poly-E deletion mutant. The nuclear area is shown as dashed white lines. Schematics of the constructs are shown in the upper right panel. Quantification is shown in the bottom right panel. Quantification was performed on 10 individual cells from 3–4 images per sample. Scale bars, 10 μm. b Schematics of genome editing targeting the human ATRX gene. The poly-E sequences in exon 15 of ATRX are underlined. Arrowheads indicate target sites of sgRNAs. c Sanger sequencing of cDNA from ΔEx15 hiPSCs confirmed deletion of exon 15. d Immunostaining of ATRX in WT and ΔEx15 hiPSCs. Data are presented as in ( a ). Scale bars, 10 μm. e CUT&RUN qPCR for ATRX in WT and ΔEx15 hNPCs at SOX21 and RNA45SN1 genomic loci (n = 3 biological replicates in each condition). P-values were calculated by two-tailed Welch’s t-test (SOX21) or Student’s t-test (RNA45SN1). **p < 0.01, *p < 0.05. f Differentially expressed genes (DEGs) of ΔEx15 hNPCs were evaluated using GO analysis for biological processes. g Heatmap representation of correlations between gene expression levels in human iPSC-derived NPCs, NCCs, and MSCs and those in WT and ΔEx15 hNPCs. Colors indicate z-values regarding correlation significance. h Immunostaining of βIII-tubulin and NeuN in WT and ΔEx15 hNPC-derived cells after 14 day-neuronal differentiation. (Insets) DAPI nuclear staining of each field. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. i A schematic of the cerebral organoid differentiation protocol ( top panel). Bright-field observation of day-30 organoids is shown in the bottom panel. Similar results were obtained in 3 independent experiments. Scale bars, 500 μm. j Immunostaining of SOX2, ZO1, and NeuN in organoids is shown in the left panel. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. [Dot plot] n = 100 puncta sampled from 3 biologically independent experiments. Each dot represents the puncta volume, and the red crossbar indicates the mean value. P-values were calculated by a two-tailed Welch’s t-test. [Bar graph] n = 10 cells per condition, sampled from 3 biologically independent experiments. Data are presented as the mean ± SEM. P-values were calculated by two-tailed Student’s t-test ( a , h , j ) or Welch’s t-test ( d ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Nature Communications

Article Title: Phase separated condensates of ATRX regulate neural progenitor identity

doi: 10.1038/s41467-025-61881-0

Figure Lengend Snippet: a Representative images of ATRX-KO HEK293T cells expressing EGFP-IDR2 and its poly-E deletion mutant. The nuclear area is shown as dashed white lines. Schematics of the constructs are shown in the upper right panel. Quantification is shown in the bottom right panel. Quantification was performed on 10 individual cells from 3–4 images per sample. Scale bars, 10 μm. b Schematics of genome editing targeting the human ATRX gene. The poly-E sequences in exon 15 of ATRX are underlined. Arrowheads indicate target sites of sgRNAs. c Sanger sequencing of cDNA from ΔEx15 hiPSCs confirmed deletion of exon 15. d Immunostaining of ATRX in WT and ΔEx15 hiPSCs. Data are presented as in ( a ). Scale bars, 10 μm. e CUT&RUN qPCR for ATRX in WT and ΔEx15 hNPCs at SOX21 and RNA45SN1 genomic loci (n = 3 biological replicates in each condition). P-values were calculated by two-tailed Welch’s t-test (SOX21) or Student’s t-test (RNA45SN1). **p < 0.01, *p < 0.05. f Differentially expressed genes (DEGs) of ΔEx15 hNPCs were evaluated using GO analysis for biological processes. g Heatmap representation of correlations between gene expression levels in human iPSC-derived NPCs, NCCs, and MSCs and those in WT and ΔEx15 hNPCs. Colors indicate z-values regarding correlation significance. h Immunostaining of βIII-tubulin and NeuN in WT and ΔEx15 hNPC-derived cells after 14 day-neuronal differentiation. (Insets) DAPI nuclear staining of each field. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. i A schematic of the cerebral organoid differentiation protocol ( top panel). Bright-field observation of day-30 organoids is shown in the bottom panel. Similar results were obtained in 3 independent experiments. Scale bars, 500 μm. j Immunostaining of SOX2, ZO1, and NeuN in organoids is shown in the left panel. Quantification is shown in the right panel (n = 3 biological replicates in each condition). Scale bars, 50 μm. [Dot plot] n = 100 puncta sampled from 3 biologically independent experiments. Each dot represents the puncta volume, and the red crossbar indicates the mean value. P-values were calculated by a two-tailed Welch’s t-test. [Bar graph] n = 10 cells per condition, sampled from 3 biologically independent experiments. Data are presented as the mean ± SEM. P-values were calculated by two-tailed Student’s t-test ( a , h , j ) or Welch’s t-test ( d ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The human iPSC control lines (1210B2 and RPC771 (ReproCELL)) and the genome-edited derivative of RPC771 (ΔEx15) were maintained as previously described , .

Techniques: Expressing, Mutagenesis, Construct, Sequencing, Immunostaining, Two Tailed Test, Gene Expression, Derivative Assay, Staining