Journal: Theranostics

Article Title: Yap1 modulates cardiomyocyte hypertrophy via impaired mitochondrial biogenesis in response to chronic mechanical stress overload

doi: 10.7150/thno.74563

Figure Lengend Snippet: YAP1 activation is responsible for mitochondrial perturbation and cardiomyocyte hypertrophy. (A) Normalized enrichment scores (NESs) of gene sets related to YAP signaling and mitochondria. At 1-, 2-, and 3-month post AAC surgery, ventricular tissues were subjected to RNA-seq and gene set enrichment analysis (GSEA). N = 3 for each group. (B) Immunofluorescence staining for activated YAP (aYAP) in cardiac tissue sections and quantification of aYAP positive cardiomyocytes. Scale bar, 50 µm. N = 6 hearts per group. (C) Immunoblotting analysis for aYAP in cardiac tissues. N = 3 hearts per group. (D) Immunostaining for YAP and ACTN2 on iPSC derived cardiomyocytes tissue bundles which are subjected to normal and exceeded stretch. (E) The quantitative result of the ratio of YAP+ cardiomyocytes on iPSC derived tissue bundles. N = 8, each dot presents the average results of 5 fields/slice. Bar, 50 µm. (F). Experimental design to determine the role of YAP1 in AAC-induced cardiac responses. (G) Echocardiographic measurement of ventricular wall thickness in AAC-treated Yap1 F/F mice. Mean ± SD. N = 4 hearts per group. (H) WGA-stained cardiac cryosections and quantification of cardiomyocyte transverse section area. In AAC and AAV treated hearts, GFP+ and GFP- cardiomyocytes were separately measured. Scale bar, 50 µm. N = 3 hearts per group. (I) Measurement of the oxygen consumption rates (OCRs) of isolated GFP positive cardiomyocytes after FACS sorting. OCRs were normalized by total cardiomyocyte number per group. N = 9 hearts per group. Mean ± SD. In statistical analysis, student's t-test or Mann-Whitney U-test was applied. **P < 0.01. ***P < 0.001.

Article Snippet: The WTC human induced pluripotent stem cell (WTC-iPSC), which was described in previous research , was maintained in mTeSR1 (STEMCELL Technologies, 85850) and passaged by versene solution (Gibco, 15040-066).

Techniques: Activation Assay, RNA Sequencing, Immunofluorescence, Staining, Western Blot, Immunostaining, Derivative Assay, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

doi: 10.1101/685057

Figure Lengend Snippet: (a) scRNA-seq was performed on iPSC- and ESC-derived cells at different time points during cerebral organoid differentiation from pluripotency. (b) Immunohistochemical staining for PAX6 (green) and BCL11B/CTIP2 (pink) of a 63 day human organoid from iPSC line 409b2 with a zoom into a cortical-like region (scale bars 100 μm). (c) All time points were combined and cell heterogeneity was assessed using t-distributed stochastic neighbor embedding (tSNE). See . (d) Pseudocells were constructed by pooling nearest neighbors and the entire differentiation trajectory was reconstructed using SPRING . Pseudocells are colored by time point or cell line (inset). (e) Tracking unspliced and spliced transcripts using RNA velocity supports differentiation of progenitor cells into distinct regions of the developing human brain. (f) Left, magenta colored, SPRING plot colored by reference similarity spectrum (RSS) to bulk RNA-seq data generated from diverse brain regions at different time points (Allen Brain Atlas). Shown are the tissues and time points with maximum correlation. Right, cyan-blue colored, SPRING plot colored by marker gene expression.

Article Snippet: We acquired 6 human induced pluripotent stem cell (iPSC) lines (Sojd3, Hoik1, Kucg2, Wibj2 from the HipSci resource ; h409b2 from the RIKEN BRC cell bank ; Sc102a1 from System Biosciences), one human ES cell line (H9, WiCell) , three chimpanzee iPSC lines (SandraA ; PR818-5 , originally generated by the Gage lab and kindly provided to us by the R. Livesey group; JoC, generated in this study), one bonobo iPSC line (Bokela, generated in this study) and one ES macaque cell line (MN1 , kindly provided through the R. Livesey group from Eliza Curnow).

Techniques: Derivative Assay, Immunohistochemical staining, Staining, Construct, RNA Sequencing, Generated, Marker, Gene Expression

Journal: bioRxiv

Article Title: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

doi: 10.1101/685057

Figure Lengend Snippet: (a) Phase contrast (iPSC stage – neuroepithelium, scale bar 200 μm; H9 for human, SandraA for chimpanzee) and bright field images (organoid stages, scale bar 1 mm; H9 and Wibj2 for human, JoC and SandraA for chimpanzee) of different stages of organoid development for human and chimpanzee organoid differentiation. (b) Immunohistochemical stainings of human (Sc102a1 and 409b2) and chimpanzee (all SandraA) organoids reveal proper formation of cortical-like regions (scale bar 200 μm).

Article Snippet: We acquired 6 human induced pluripotent stem cell (iPSC) lines (Sojd3, Hoik1, Kucg2, Wibj2 from the HipSci resource ; h409b2 from the RIKEN BRC cell bank ; Sc102a1 from System Biosciences), one human ES cell line (H9, WiCell) , three chimpanzee iPSC lines (SandraA ; PR818-5 , originally generated by the Gage lab and kindly provided to us by the R. Livesey group; JoC, generated in this study), one bonobo iPSC line (Bokela, generated in this study) and one ES macaque cell line (MN1 , kindly provided through the R. Livesey group from Eliza Curnow).

Techniques: Immunohistochemical staining

Journal: bioRxiv

Article Title: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

doi: 10.1101/685057

Figure Lengend Snippet: (a) Cells from different human cell lines were integrated using canonical correlation analysis and visualized using t-stochastic neighbor embedding (tSNE). (b) tSNE color coded based on cell line and batch. (c) tSNE colored based on time point. Heterogeneity analysis was performed on combined cells from day 0 of differentiation to 4 month old organoids for iPSC and ESC-derived cells. (d) Distribution of number of genes and UMIs for different time points and cell lines. (e) Clustering was performed using the top 20 principal components as input for tSNE and cluster names were assigned based on expression of cluster marker genes and known marker genes. SC stem cells, NEC – neuroectoderm-like cells, NSC – neural stem cells, (g/o)RGC – (gliogenic/outer) radial glia cells, G2M/S NPC – neural progenitor cells in G2M/S phase, G2M/S DP – dorsal progenitor cells in G2M/S phase, IP – intermediate progenitor, CN – cortical neurons, G2M/S vP – ventral progenitors in G2M/S phase, M/H – midbrain/hindbrain, CP – choroid plexus, M – mesenchymal-like cells. (f) tSNE plot colored with respect to expression of selected marker genes based on non-integrated expression values. (g) Heatmap showing averaged cluster expression for representative marker genes for clusters ordered according to their differentiation time from early to later stages and regional identity from dorsal to ventral forebrain and non-forebrain cells.

Article Snippet: We acquired 6 human induced pluripotent stem cell (iPSC) lines (Sojd3, Hoik1, Kucg2, Wibj2 from the HipSci resource ; h409b2 from the RIKEN BRC cell bank ; Sc102a1 from System Biosciences), one human ES cell line (H9, WiCell) , three chimpanzee iPSC lines (SandraA ; PR818-5 , originally generated by the Gage lab and kindly provided to us by the R. Livesey group; JoC, generated in this study), one bonobo iPSC line (Bokela, generated in this study) and one ES macaque cell line (MN1 , kindly provided through the R. Livesey group from Eliza Curnow).

Techniques: Derivative Assay, Expressing, Marker

Journal: bioRxiv

Article Title: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

doi: 10.1101/685057

Figure Lengend Snippet: (a) To assess the reproducibility of gene expression patterns in organoids, scRNA-seq was performed on 2-month-old human organoids from 6 iPSC lines and 1 ESC (H9) line. (b-c) SPRING reconstruction based on the reference similarity spectrum (RSS) of organoid cells with plots colored by (b) cell types and by (c) line. (d) Proportion of cells per organoid that are within the dorsal telencephalon, ventral telencephalon, or diencephalon, mesencephalon and rhombencephalon neuronal branches. The data shows that there is variation in the types of cells that form in each organoid. (e) Pseudotime along the dorsal telencephalon and ventral telencephalon branch. (f) Boxplots (interquartile range with minimum and maximum, outliers removed) showing expression of marker genes for major neuron populations that emerge in the human cerebral organoids. CGE/MGE, caudal/medial ganglionic eminence; LGE, lateral ganglionic eminence; Dien. ex., diencephalon excitatory; Dien. in., diencephalon inhibitory; Mesen. Rhom. ex., Mesencephalon / rhombencephalon excitatory; Mesen. Rhom. in., Mesencephalon / rhombencephalon inhibitory. (g) Pseudotemporal expression patterns of neuronal differentiation markers for the dorsal (cortex, upper) and ventral telencephalon trajectories (lower) for each line. (h) Correlations of pseudotime-dependent gene expression patterns between cells within dorsal (upper) or ventral (middle) telencephalon branches, and between dorsal and ventral cells from the same line (lower). (i) Dendrogram based on pairwise correlations between cells from different lines/branches/stages based on pseudotime-dependent gene expression patterns. The clustering shows that differences between progenitors and neurons, as well as the variation between those cell types in different brain regions, are larger than variation between cell lines.

Article Snippet: We acquired 6 human induced pluripotent stem cell (iPSC) lines (Sojd3, Hoik1, Kucg2, Wibj2 from the HipSci resource ; h409b2 from the RIKEN BRC cell bank ; Sc102a1 from System Biosciences), one human ES cell line (H9, WiCell) , three chimpanzee iPSC lines (SandraA ; PR818-5 , originally generated by the Gage lab and kindly provided to us by the R. Livesey group; JoC, generated in this study), one bonobo iPSC line (Bokela, generated in this study) and one ES macaque cell line (MN1 , kindly provided through the R. Livesey group from Eliza Curnow).

Techniques: Gene Expression, Expressing, Marker

Journal: bioRxiv

Article Title: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

doi: 10.1101/685057

Figure Lengend Snippet: (a) scRNA-seq was performed on 2-month old cerebral organoids from human ESC and six iPSC lines. (b) All data were combined and cell heterogeneity was assessed using t-distributed stochastic neighbor embedding (tSNE) with the top-20 principal components (PCs) as the input. Cells are also colored by marker gene expression and RSS. (c) tSNE plot with RSS against Brainspan fetal reference data as the input (RSS-tSNE), colored by cell lines. Cells from different lines are well integrated. (d) SPRING plot of 2-month old human organoid pseudocells, colored by neuronal trajectory branches and pseudotimes. (e) SPRING plot of 2-month old human organoid cells, colored by marker gene expression. (f) Correlations of expression trajectories of genes with pseudotime-dependent expression patterns between cortical cells from each line to the others (pink), ventral cells from each line to others (blue), and cortical and ventral cells from the same lines after or before aligning the cortical and ventral pseudotimes (purple). (g) Spatial location inference of neuron subtypes in human cerebral organoids. (Left) Barplots show proportion of cells of each cell type which show highest gene expression pattern similarity to the average expression patterns in different structures, based on the processed in situ hybridization image data (E13.5) provided in Developing Mouse Brain database of Allen Brain Atlas. Expression similarity was calculated based on highly variable genes of the scRNA-seq data (top), or regional markers defined with the ISH data (bottom left). (Right) Correlation patterns of average regional marker gene expression of each neuron subtype to voxels in five example sections (E13.5), as well as the structural annotation of the sections. (h) Expression of two marker genes of Diencephalon inhibitory neurons (PCP4, RSPO3) in the SPRING embeddings, and their spatial expression patterns in E13.5 mouse brain (data from Allen Brain Atlas).

Article Snippet: We acquired 6 human induced pluripotent stem cell (iPSC) lines (Sojd3, Hoik1, Kucg2, Wibj2 from the HipSci resource ; h409b2 from the RIKEN BRC cell bank ; Sc102a1 from System Biosciences), one human ES cell line (H9, WiCell) , three chimpanzee iPSC lines (SandraA ; PR818-5 , originally generated by the Gage lab and kindly provided to us by the R. Livesey group; JoC, generated in this study), one bonobo iPSC line (Bokela, generated in this study) and one ES macaque cell line (MN1 , kindly provided through the R. Livesey group from Eliza Curnow).

Techniques: Marker, Gene Expression, Expressing, In Situ Hybridization

Journal: bioRxiv

Article Title: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

doi: 10.1101/685057

Figure Lengend Snippet: (a) scRNA-seq was performed on chimpanzee iPSC-derived cells at different time points during cerebral organoid differentiation from pluripotency. (b) Immunohistochemical staining for PAX6 (green) and BCL11B/CTIP2 (pink) of a 63-day chimpanzee organoid from iPSC line SandraA with a zoom into a cortical-like region (scale bars 100 μm). (c) All time points were combined and cell heterogeneity was assessed using tSNE . Pseudocells were constructed by pooling nearest neighbors and the entire differentiation trajectory was reconstructed using SPRING. Cells and pseudocells are colored by time point. (d) SPRING plots of human (left) and chimpanzee (right), colored by stage and lineage pseudotimes. (e) Marker gene expression along pseudotime trajectories in chimpanzee (upper) and human (lower). (f) Alignment of human and chimpanzee pseudotimes after combining pseudocells from the early stages and the dorsal forebrain lineage. The later chimpanzee pseudotime points fail to align with human pseudocells. (g) SPRING plots of human (upper) and chimpanzee (lower) organoid development, colored by the aligned pseudotimes with chimpanzee pseudotime as the template. (h) Projection of human and chimpanzee organoid cells to human fetal brain data reveals higher similarity of chimpanzee organoid cells to later stages of development compared to human organoid cells. (i) Boxplots (interquartile range with minimum and maximum, outliers removed) showing neuron projection scores defined as the sum expression of genes related to neuron projection in human and chimpanzee along the unaligned cortical pseudotimes. (j) Number of astrocytes captured by scRNA-seq in organoids at different time points, normalized by the number of radial glia for each respective time point.

Article Snippet: We acquired 6 human induced pluripotent stem cell (iPSC) lines (Sojd3, Hoik1, Kucg2, Wibj2 from the HipSci resource ; h409b2 from the RIKEN BRC cell bank ; Sc102a1 from System Biosciences), one human ES cell line (H9, WiCell) , three chimpanzee iPSC lines (SandraA ; PR818-5 , originally generated by the Gage lab and kindly provided to us by the R. Livesey group; JoC, generated in this study), one bonobo iPSC line (Bokela, generated in this study) and one ES macaque cell line (MN1 , kindly provided through the R. Livesey group from Eliza Curnow).

Techniques: Derivative Assay, Immunohistochemical staining, Staining, Construct, Marker, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

doi: 10.1101/685057

Figure Lengend Snippet: (a) Heterogeneity analysis for iPSC-derived chimpanzee cells from day 0 of differentiation to 4 months of organoid development for one cell line. (b) Heatmap visualizing averaged cluster expression for marker genes with columns ordered based on differentiation progress from early to late time points and regional identity sorted from dorsal to ventral forebrain to non-forebrain cells and non-ectodermal-derived cells. (c) Cluster identification and t-stochastic neighbor embedding using the top 15 principal components for clustering. Cluster assignment was based on cluster markers as well as expression patterns of known marker genes. SC – stem cells, NSC - neural stem cells, RGC – radial glia cells, G2M/S DP – dorsal progenitors in G2M/S phase, IP – intermediate progenitors, CN – cortical neuron, dlN – deep layer neuron, ulN – upper layer neurons, vP/N – ventral progenitor/neuron, H – hindbrain, M – mesenchymal-like cells, OL – off lineage cells, MIC - microglia. (d) tSNE plots colored based on gene expression of representative marker genes used to assign cluster identities.

Article Snippet: We acquired 6 human induced pluripotent stem cell (iPSC) lines (Sojd3, Hoik1, Kucg2, Wibj2 from the HipSci resource ; h409b2 from the RIKEN BRC cell bank ; Sc102a1 from System Biosciences), one human ES cell line (H9, WiCell) , three chimpanzee iPSC lines (SandraA ; PR818-5 , originally generated by the Gage lab and kindly provided to us by the R. Livesey group; JoC, generated in this study), one bonobo iPSC line (Bokela, generated in this study) and one ES macaque cell line (MN1 , kindly provided through the R. Livesey group from Eliza Curnow).

Techniques: Derivative Assay, Expressing, Marker, Gene Expression

Journal: bioRxiv

Article Title: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

doi: 10.1101/685057

Figure Lengend Snippet: (a) scRNA-seq was performed on 2 to 4-month cerebral organoids from a macaque iPSC line. The SPRING plot of pseudocells was constructed with the top 20 PCs as the input. The heterogeneity analysis suggests multiple cell types in the macaque organoids, including cortical neurons, NPCs, astrocytes and other cell types such as retina and mesenchyme-like cells. (b) SPRING plot colored by pseudotimes of cortical pseudocells, which are the pseudocells’ quantiles of DC1 of the cortical pseudocells diffusion map. (c) SPRING plot colored by marker gene expression. (d) The onset of anti-correlation between SATB2 and BCL11B occurs earlier along the macaque pseudotime, relative to human and chimpanzee, when focusing on the 2-month cerebral organoids. (e) Boxplots (box, interquartile range (IQR); whisker, 1.5*IQR) showing the neuron projection scores in human, chimpanzee and macaque along the unaligned cortical pseudotimes.

Article Snippet: We acquired 6 human induced pluripotent stem cell (iPSC) lines (Sojd3, Hoik1, Kucg2, Wibj2 from the HipSci resource ; h409b2 from the RIKEN BRC cell bank ; Sc102a1 from System Biosciences), one human ES cell line (H9, WiCell) , three chimpanzee iPSC lines (SandraA ; PR818-5 , originally generated by the Gage lab and kindly provided to us by the R. Livesey group; JoC, generated in this study), one bonobo iPSC line (Bokela, generated in this study) and one ES macaque cell line (MN1 , kindly provided through the R. Livesey group from Eliza Curnow).

Techniques: Construct, Diffusion-based Assay, Marker, Gene Expression, Whisker Assay