High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures
- Department of Biomedical Engineering, Washington University, United States
- Department of Developmental Biology, Washington University School of Medicine, United States
- Department of Genetics, Washington University School of Medicine, United States
- Center of Regenerative Medicine, Washington University School of Medicine, United States
- Department of Computer Science & Engineering, Washington University, United States
- Division of Nephrology, Washington University School of Medicine, United States
- Institute for Informatics, Washington University School of Medicine, United States
- Department of Biomedical Engineering, University of California, Davis, United States
- Department of Bioengineering, University of Illinois, United States
Figures
Figure 1 with 2 supplements
BMP4 induces differentiation of H1 hESCs in Matrigel micro-discs into radially-arranged germ layers and ExE-like cells after 44 hr.
(A) Immunofluorescence images (top) and quantification of fluorescence intensity (bottom) for BMP4 downstream effector pSMAD1 relative to the DAPI fluorescence marking nuclei (N = 4 experiments; n = …
Figure 1—figure supplement 1
Protocol for microcontact printing method and micropatterned differentiation of gastruloids.
(A) Schematic of protocol for fabricating PDMS (poly-dimethylsiloxane) stamp used in microcontact printing. (B) An image of a typical stamp used in microcontact printing with a ruler and one-inch …
Figure 1—figure supplement 2
Immunofluorescence images of germ layer and TE markers in H9 hESC micropatterned gastruloid differentiation.
Scale bar is 100 µm.
Figure 2 with 2 supplements
Single-cell RNA-seq profiling reveals cellular complexity of BMP4-induced gastruloids.
(A) Schematic showing scRNA-seq workflow for gastruloids from two biological replicates. (B) UMAP display of seven cell clusters detected by unsupervised clustering in gastruloids replicate 1 …
Figure 2—figure supplement 1
Selected parameters of scRNA-seq analysis.
Figure 2—figure supplement 2
Comparison of gastruloid scRNA-seq datasets from two independent biological replicates.
(A) Bar plot indicating relative proportion of cells in each cluster of replicates 1 and 2. (B) Heatmap showing correlation of average expression of 23,271 total genes between scRNA-seq clusters …
Figure 3 with 4 supplements
Cross-species comparison with mouse and cyno monkey gastrulating embryo.
(A, B) UMAP displaying overlap of cells in gastruloids and mouse (A), and monkey (B) at indicated gastrulation stages. (C, D) Bar plots showing composition of predicted mouse (C) and monkey (D) cell …
Figure 3—figure supplement 1
Gastruloids exhibit similar cell type composition profile to early-mid mouse and cynomolgus monkey gastrula stages.
(A, B) Bar plots showing cellular composition of mouse (A) and cynomolgus monkey (B) embryos at indicated stages, and gastruloids with predicted cell types (each color bar represents a cell type).
Figure 3—figure supplement 2
Species comparison shows high similarity between the two gastruloid replicates.
(A, B) Dot plots showing predicted mouse (A) and cynomolgus monkey (B) cell types and stages in each cluster of gastruloid replicate 1 and replicate 2.
Figure 3—figure supplement 3
Species comparison between mouse and cynomolgus monkey gastrulating embryos.
(A) Violin plot showing predicted score of mouse development stages in 16 dpf cynomolgus monkey cells (**** indicates p<0.0001, using one-way ANOVA with Tukey’s multiple comparisons test; dash lines …
Figure 3—figure supplement 4
Heatmap of average gene expression correlation between gastruloids and cynomolgus monkey embryos from a separate study.
(Based on 492 shared highly variable genes between gastruloids and cynomolgus monkey dataset from Nakamura et al., 2016).
Figure 4 with 6 supplements
Characterization of gastruloid EPI-like, Ectoderm, Mesoderm, and Endoderm clusters.
(A) Average expression correlation of 434 shared highly variable genes between the gastruloid EPI-like cluster and epiblast-related cell types from in vitro cultured human embryos. (B) UMAP …
Figure 4—figure supplement 1
Pseudotime and transitional probability analyses in gastruloid clusters based on diffusion maps.
(A) 3D plot showing gastruloid clusters in the first three diffusion coordinates (arrow lines indicate plausible differentiation trajectories). (B) Chart illustrating proportion of cells from all …
Figure 4—figure supplement 2
Expression of neural and surface ectoderm markers in the gastruloid Ectoderm cluster.
Figure 4—figure supplement 3
Expression of selected markers in gastruloid mesoderm clusters.
(A) Violin plot showing expression of indicated mesoderm markers in the gastruloid Mesoderm-1 and -2 clusters (**** indicates p<0.0001 using Mann-Whitney test). (B) Violin plot showing expression of …
Figure 4—figure supplement 4
Expression of top 50 differentially expressed monkey EXMC markers in gastruloids.
Figure 4—figure supplement 5
Overlap of CDH2 expression with T and SOX17 expression in gastruloids.
(N = 2, 5, 6, 1, five experiments; n = 63, 84, 71, 21, 63 gastruloids, respectively for SOX2, T, SOX17, GATA3, CDH2; fluorescence intensity of each marker is normalized against that of DAPI for each …
Figure 4—figure supplement 6
Expression of definitive endoderm marker FOXA2 and primitive endoderm marker GATA6 in the SOX17+ gastruloid Endoderm cluster.
Figure 5 with 1 supplement
Characterization of gastruloid hPGCLCs.
(A) Violin plot showing expression of PGC markers NANOS3 and TFAP2C in gastruloid clusters (**** indicates p<0.0001; one-way ANOVA with Tukey’s multiple comparisons test; dash lines indicate 1st, …
Figure 5—figure supplement 1
Expression of primate PGC markers in gastruloids.
(A) Heatmap showing the expression of monkey early PGC signature in the gastruloid hPGCLC cluster. (B) Additional replicates of immunofluorescence images showing indicated markers (arrows indicate …
Figure 6
Characterization of the gastruloid ExE-like cluster.
(A, B) Immunofluorescence images of TE makers GATA3 and CDX2 (A), and amnion marker TFAP2A (B) in gastruloids. (C) UMAP showing expression of TE markers GATA3, CDX2, KRT7, and amnion markers TFAP2A, …
Figure 7 with 2 supplements
BMP4-differentiated germ layer and ExE-like cells exhibit cell sorting behaviors.
(A) Schema of protocol for assaying cell sorting behaviors from dissociated gastruloids. (B, C, D) Immunofluorescence images of indicated markers in reseeded gastruloid cells at indicated timepoints …
Figure 7—figure supplement 1
Visualization and quantification of reseeded cells.
(A) Still time-lapse images of dissociated gastruloid cells reseeded in micropattern after 3, 10, 17, and 24 hr (arrow indicates motile cells, arrowhead indicates non-motile cells, dotted line …
Figure 7—figure supplement 2
Reseeded cells retain transcriptional similarity to gastruloid cells.
(A) Gene Ontology analysis of downregulated and upregulated genes between reseeded cultures and gastruloids. (B) Heatmap illustrating gene expression correlation of 2000 shared highly variable genes …
Figure 8 with 5 supplements
Reseeded gastruloid cells exhibit selective cell sorting behaviors.
(A, B) Immunofluorescence images of indicated markers in 48 hr post-reseed cultures. (C) Cosine similarity analysis of expression domains between pairs of indicated markers (*, **, ***, **** …
Figure 8—figure supplement 1
Immunofluorescence analyses of cell sorting behavior in reseeded cells.
(A) Additional replicates of immunofluorescence staining showing indicated markers of dissociated and reseeded gastruloid cells after 48 hr (note mixing between SOX2+ and T+ cells, but segregation …
Figure 8—figure supplement 2
Cosine similarity or expression pattern overlap analysis between cell type markers in reseeded cells.
(A, B) Cosine value depicting overlap between expression domains of pairs of indicated markers at indicated timepoints and cell densities (*, **, ***, **** indicates p<0.05, 0.01, 0.001, 0.0001, …
Figure 8—figure supplement 3
ScRNA-seq analysis of protein markers used in immunofluorescence studies of reseeded cells.
(A) UMAP display of reseeded culture from dissociated gastruloids showing combined clusters with strong expression of SOX2 (EPI-like and Ectoderm clusters into reEcto), T (Mesoderm-1 and -2 clusters …
Figure 8—figure supplement 4
Differential expression of cell adhesion molecules in reseeded cells.
Figure 8—figure supplement 5
Ephrin-Eph interactions in reseeded cells.
(A) Dot plot showing predicted Ephrin-Eph interactions between indicated pairs of cell clusters. Arrows in x-axis labels indicate direction of signaling (signaling to receiving cluster). In each …
Figure 9
BMP4-differentiated gastruloids generate primate-relevant gastrulation cell types that show cell sorting behaviors when dissociated and reseeded in single-cell mixture.
(A) Immunofluorescence and scRNA-seq analyses identify seven major cell types relevant during primate gastrulation. (B) Gastruloids dissociated into single cells and reseeded onto micropatterns show …
Videos
Video 1
Dissociated and reseeded gastruloid cells show motility.
Time-lapse confocal series from 3 to 27 hr after reseeding of dissociated gastruloid cells in 500 µm ECM microdiscs.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Human) | WA01 (H1) human embryonic stem cell | WiCell | RRID:CVCL_9771, hPSCReg ID: WAe001-A | |
| Cell line (Human) | WA09 (H9) human embryonic stem cell | WiCell | RRID:CVCL_9773, hPSCReg ID: WAe009-A | |
| Antibody | anti-SOX2 (Mouse monoclonal) | Cell Signaling Technology | Cat# 4900, RRID:AB_10560516 | (1:200) |
| Antibody | anti-SOX2 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 3579, RRID:AB_2195767 | (1:200) |
| Antibody | anti-SOX1 (Goat polyclonal) | R and D Systems | Cat# AF3369, RRID:AB_2239879 | (10 μg/mL) |
| Antibody | anti-T (Goat polyclonal) | R and D Systems | Cat# AF2085, RRID:AB_2200235 | (2 μg/mL) |
| Antibody | anti-SOX17 (Goat polyclonal) | R and D Systems | Cat# AF1924, RRID:AB_355060 | (1 μg/mL) |
| Antibody | anti-CDX2 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 12306, RRID:AB_2797879 | (1:100) |
| Antibody | anti-pSMAD1 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 9516, RRID:AB_491015 | (1:200) |
| Antibody | anti-CDH1 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 3195, RRID:AB_2291471 | (1:400) |
| Antibody | anti-CDH2 (Mouse monoclonal) | Cell Signaling Technology | Cat# 14215, RRID:AB_2798427 | (1:800) |
| antibody | anti-GATA3 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 5852, RRID:AB_10835690 | (1:1600) |
| Antibody | anti-GATA3 (Mouse monoclonal) | R and D Systems | Cat# MAB6330, RRID:AB_10640512 | (1:500) |
| Antibody | anti-GATA6 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 5851, RRID:AB_10705521 | (1:1600) |
| Antibody | anti-FOXA2 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 8186, RRID:AB_10891055 | (1:800) |
| Antibody | anti-OCT3/4(POU5F1) (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-5279, RRID:AB_628051 | (1:200) |
| Antibody | anti-ZO-1 (Mouse monoclonal) | Thermo Fisher Scientific | Cat# 33–9100, RRID:AB_2533147 | (1:500) |
| Antibody | anti-KRT7 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 4465, RRID:AB_11178382 | (1:200) |
| Antibody | anti-AP-2α (Rabbit monoclonal) | Santa Cruz Biotechnology | Cat# sc-12726, RRID:AB_667767 | (1:200) |
| Antibody | anti-AP-2γ (Rabbit polyclonal) | Cell Signaling Technology | Cat# 2320, RRID:AB_2202287 | (1:800) |
| Chemical compound, drug | Gentle Cell Dissociation Reagent | Stemcell Technologies | Cat # 07174 | |
| Chemical compound, drug | Accutase solution | Sigma Aldrich | Cat # A6964 | |
| Chemical compound, drug | RPMI 1640 Medium | Thermo Fisher Scientific | Cat # 11879020 | |
| Chemical compound, drug | ROCK Inhibitor (Y-27632) | Millipore Sigma | Cat # SCM075 | (10 μM) |
| Chemical compound, drug | Recombinant Human BMP-4 Protein | R and D Systems | Cat # 314 BP | (50 ng/mL) |
| Chemical compound, drug | mTeSR1 | Stemcell Technologies | Cat # 85857 | |
| Commercial assay, kit | Chromium Single Cell 3’ Library and Gel Bead Kit v2 | 10xGenomics | Cat # 120237 | |
| Commercial assay, kit | Chromium Single Cell 3’ Chip Kit v2 | 10xGenomics | Cat # 120236 | |
| Commercial assay, kit | Chromium i7 Multiplex Kit | 10xGenomics | Cat # 120262 | |
| Software, algorithm | Seurat 3.1 | Stuart et al., 2019 Butler et al., 2018 | Seurat, RRID:SCR_016341 | scRNA-seq analysis |
| Software, algorithm | CellPhoneDB | Efremova et al., 2020 | Receptor-ligand interaction analysis | |
| Software, algorithm | R Bioconductor destiny | Angerer et al., 2016 (https://bioconductor.org/packages/release/bioc/html/destiny.html) | Pseudotime and transition probabilities | |
| Software, algorithm | ImageJ/FIJI | ImageJ/FIJI | RRID:SCR_002285 | Image analysis |
| Software, algorithm | FIJI plugin – Concentric Circles | https://imagej.nih.gov/ij/plugins/concentric-circles.html | Image analysis | |
| Software, algorithm | FIJI plugin – ClearVolume | Royer et al., 2015 (https://imagej.net/ClearVolume) | Image analysis | |
| Software, algorithm | Prism 8 | Graphpad | RRID:SCR_002798 | Statistics and graphs |
| Other | DAPI | Millipore Sigma | Cat# D9542 | (0.4 μg/mL) |
| Other | Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | Cat# A-21202, RRID:AB_141607 | (1:500) |
| Other | Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Thermo Fisher Scientific | Cat# A-21432, RRID:AB_2535853 | (1:500) |
| Other | Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Thermo Fisher Scientific | Cat# A-31573, RRID:AB_2536183 | (1:500) |
Additional files
-
Source data 1
Metadata for gastruloid cells including cluster annotation, replicate number, and predicted mouse and monkey cell types and stages.
Average gene expression correlation values between replicates 1 and 2.
- https://cdn.elifesciences.org/articles/59445/elife-59445-data1-v2.xlsx
-
Source data 2
Differentially expressed genes of gastruloid clusters.
- https://cdn.elifesciences.org/articles/59445/elife-59445-data2-v2.xlsx
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Source data 3
Shared highly variable genes (HVGs) between gastruloid and reference datasets used for average gene expression correlation, and correlation values.
- https://cdn.elifesciences.org/articles/59445/elife-59445-data3-v2.xlsx
-
Source data 4
Metadata for cyno monkey cells including monkey cell type and developmental stage, and predicted mouse cell type and developmental stage.
Shared HVGs between mouse and monkey dataset, and gene expression correlation values of developmental stages.
- https://cdn.elifesciences.org/articles/59445/elife-59445-data4-v2.xlsx
-
Source data 5
Metadata for reseed cells including cluster annotation, and prediction scores for gastruloid cell types.
- https://cdn.elifesciences.org/articles/59445/elife-59445-data5-v2.xlsx
-
Source data 6
Downregulated and upregulated genes, shared HVGs, and gene expression correlation values between reseed and gastruloids.
- https://cdn.elifesciences.org/articles/59445/elife-59445-data6-v2.xlsx
-
Source data 7
Predicted interactions between reseed clusters.
- https://cdn.elifesciences.org/articles/59445/elife-59445-data7-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/59445/elife-59445-transrepform-v2.docx
