An in vitro stem cell model of human epiblast and yolk sac interaction
- Mammalian Embryo and Stem Cell Group, University of Cambridge, Department of Physiology, Development and Neuroscience, United Kingdom
- National Laboratory for Embryonic Stem Cells (LaNCE), Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, Brazil
- MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, United Kingdom
- Centre for Trophoblast Research, University of Cambridge, United Kingdom
- Wellcome Sanger Institute, United Kingdom
- Wellcome – MRC Cambridge Stem Cell Institute, Cambridge Biomedical Campus, United Kingdom
- Synthetic Mouse and Human Embryology Group, California Institute of Technology (Caltech), Division of Biology and Biological Engineering, United States
Figures
Figure 1 with 5 supplements
Endoderm conversion of RUES2-GLR human pluripotent stem cells (hPSCs) in response to ACL treatment.
(A, E, and H) Immunofluorescence images of SOX2 (green) and SOX17 (red) in human extended pluripotent stem cells (hEPSCs), Rset human pluripotent stem cells (hPSCs), and human embryonic stem cells (h…
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Figure 1—source data 1
Fluorescence intensity and qPCR analysis of endoderm and pluripotency markers in RUES2 human pluripotent stem cells (hPSCs) in response to ACL.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
6-Day molecular screen using RUES2 human pluripotent stem cells (hPSCs).
(A) Diagram outlining the screen procedure. At the beginning of the screen human extended pluripotent stem cells (hEPSCs) harbour a predominantly SOX2+ population. After 6 days (6D), average levels …
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Figure 1—figure supplement 1—source data 1
Fluorescence intensity analysis of human extended pluripotent stem cell (hEPSC) screen.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Endoderm conversion of RUES2 and H9 human pluripotent stem cells (hPSCs) in response to ACL treatment.
(A) Relative expression levels (REL) (± SEM) of EOMES and POU5F1 in RUES2 human extended pluripotent stem cell (hEPSC), Rset hPSCs (n=6 samples, three independent experiments) or hESCs (n=4 samples, …
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Figure 1—figure supplement 2—source data 1
Fluorescence intensity and qPCR analysis of endoderm and pluripotency markers in RUES2 and H9 human pluripotent stem cells (hPSCs) in response to ACL.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
Prolonged culture of Rset-ACL.
(A) Immunofluorescence images of SOX2 (green) and SOX17 (red) in RUES2 Rset human pluripotent stem cells (hPSCs) on D0, D14, and D30 of culture in ACL (scale bar = 50 μm). (B) Bar chart of relative …
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Figure 1—figure supplement 3—source data 1
Fluorescence intensity and qPCR analysis of long-term culture of Rset-ACL.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig1-figsupp3-data1-v1.xlsx
Figure 1—figure supplement 4
Endoderm conversion of SHEF6 human pluripotent stem cells (hPSCs) in response to ACL treatment.
(A, D, and G) Immunofluorescence images of SOX2 (green) and GATA6 (red) in human extended pluripotent stem cells (hEPSCs), Rset hPSCs, and human embryonic stem cells (hESCs) after 6 days (6D) of ACL …
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Figure 1—figure supplement 4—source data 1
Fluorescence intensity and qPCR analysis of SHEF6 human pluripotent stem cells (hPSCs) in response to ACL.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig1-figsupp4-data1-v1.xlsx
Figure 1—figure supplement 5
Flow cytometry gating strategy for isolating the SOX17::tdTomato+ cells within human extended pluripotent stem cell (hEPSC)-ACL, Rset-ACL, and human embryonic stem cell (hESC)-ACL populations.
(A) Gating strategy for separating debris from cell population. (B) Gating strategy for isolating single cells from doublets. (C) Gating strategy to separate SOX17::tdTomato+ from SOX17::tdTomato- …
Figure 2
Transcriptional profiling of ACL-treated human pluripotent stem cells (hPSCs) relative to extra-embryonic endoderm and definitive endoderm expression signatures.
(A) Venn diagram depicting the number of shared genes between human extra-embryonic endoderm (E6–14) and human embryonic stem cell (hESC)-derived definitive endoderm (brown), of extra-embryonic …
Figure 3 with 3 supplements
Functional characterisation of ACL-treated human pluripotent stem cells (hPSCs).
(A) Line graph (± SEM) plotting the relative fluorescence intensity (RFI arbitrary units [a.u.]) of SOX2, SOX17, and BRACHYURY in ACL-treated population. Kruskal–Wallis test, ****p < 0.0001 (number …
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Figure 3—source data 1
Fluorescence intensity analysis of ACL-treated human pluripotent stem cells (hPSCs) and analysis of mouse-human pre-implantation chimeras.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Characterisation of mouse blastocysts cultured in interspecies chimera conditions.
(A) Characterisation of lineage allocation in E4.5 mouse blastocysts cultured in either KSOM or ACL-KSOM. Bar chart (± SEM) depicting the relative number of cells that were NANOG+, SOX17+, …
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Figure 3—figure supplement 1—source data 1
Analysis of mouse embryo culture condition effects and mouse-human chimeras.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Contribution of human extended pluripotent stem cell (hEPSC) and Rset human pluripotent stem cells (hPSCs) to interspecies chimeras.
(A) Immunofluorescence images of human nuclear antigen (HuNu) (red), SOX2 (green), GATA6 (grey) and DAPI (blue) in E4.5 human-mouse chimeric blastocysts demonstrating contribution of hEPSCs (scale …
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Figure 3—figure supplement 2—source data 1
Analysis of mouse-human blastocyst chimeras using human extended pluripotent stem cells (hEPSCs) and Rset human pluripotent stem cells (hPSCs).
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig3-figsupp2-data1-v1.xlsx
Figure 3—figure supplement 3
Contribution of ACL-treated human embryonic stem cells (hESCs) to interspecies chimeras.
(A) Brightfield image of E4.5 mouse blastocysts after aggregation with SOX17::H2B-tdTomato+ ACL-treated hESCs (scale bar = 100 μm). (B) Pie charts showing percentage contribution to the primitive …
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Figure 3—figure supplement 3—source data 1
Analysis of mouse-human blastocyst chimeras using human embryonic stem cell (hESC)-ACL.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig3-figsupp3-data1-v1.xlsx
Figure 4
Transcriptional profiling of T2iLGö-PrE and Rset-ACL relative to human pre- and post-implantation extra-embryonic endoderm expression signatures.
(A) Venn diagram depicting the number of shared genes between pre-implantation extra-embryonic endoderm (E6–7) and post-implantation extra-embryonic endoderm (E10–14) (brown), of pre-implantation …
Figure 5 with 1 supplement
Yolk sac-like cells (YSLCs) antagonise BMP and WNT signalling in human embryonic stem cells (hESCs).
(A) Bar chart plotting average expression levels of WNT, NODAL, and BMP inhibitors in pre-implantation (Pre, E6–7) and post-implantation human hypoblast (Post, E10–14) denoted as relative counts per …
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Figure 5—source data 1
Analysis of BMP/WNT/NODAL inhibitors in the human embryo and yolk sac-like cells (YSLCs) and analysis of human embryonic stem cell (hESC) response to YSLCs.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Yolk sac-like cells (YSLCs) prevent SOX2 and NANOG loss.
(A) Immunofluorescence of SOX2 (green), SOX17 (red), and DKK1 (grey) in Rset and YSLCs (after 6D ACL treatment) (scale bar = 50 μm). (B) Immunofluorescence of SOX2 (green), SOX17 (red), and CER1 …
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Figure 5—figure supplement 1—source data 1
Fluorescence intensity analysis of SOX2 and NANOG levels in human embryonic stem cells (hESCs) in the presence of yolk sac-like cells (YSLCs) and YSLC conditioned media (CM).
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig5-figsupp1-data1-v1.xlsx
Figure 6
Yolk sac-like cells (YSLCs) inhibit mesoderm and endoderm specification in human embryonic stem cells (hESCs) in 3D and 2D.
(A) Immunofluorescence of pSMAD1/5 (green) and GATA6 (grey), and a merge of the two, in hESCs cultured in 3D Geltrex in mTeSR (hESC) or YSLC conditioned media (+YSLC CM) (scale bar = 50 μm). (B) …
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Figure 6—source data 1
Fluorescence intensity and qPCR analysis of mesoderm and endoderm markers in human embryonic stem cells (hESCs) in the presence of yolk sac-like cell (YSLC) conditioned medium (CM).
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig6-data1-v1.xlsx
Figure 7 with 1 supplement
Yolk sac-like cells (YSLCs) induce the co-expression of anterior ectoderm markers and pluripotency markers in human embryonic stem cell (hESC) in 3D and 2D.
(A) Immunofluorescence of SOX2 (green), NANOG (grey), and OCT4 (red) in hESCs after 1, 3, 4, or 5 days cultured in mTeSR (hESC) or in YSLC conditioned media (+YSLC CM) in 3D Geltrex (scale bar = 50 …
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Figure 7—source data 1
Fluorescence intensity and qPCR analysis of anterior ectoderm and pluripotency markers in human embryonic stem cells (hESCs) in the presence of yolk sac-like cell (YSLC) conditioned medium (CM) in 2D and 3D.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Yolk sac-like cells (YSLCs) induce the co-expression of anterior ectoderm markers and pluripotency markers in human embryonic stem cells (hESCs) in 2D and 3D.
(A) Immunofluorescence of SOX1 (grey) in hESCs after 6 and 7 days cultured in mTeSR (hESC) or YSLC conditioned media (+YSLC CM) in 3D Geltrex (scale bar = 50 μm). (B) Bar charts (± SEM) depicting …
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Figure 7—figure supplement 1—source data 1
Fluorescence intensity and qPCR analysis of anterior ectoderm and pluripotency markers in human embryonic stem cells (hESCs) in the presence of yolk sac-like cell (YSLC) conditioned medium (CM) in 2D and 3D.
- https://cdn.elifesciences.org/articles/63930/elife-63930-fig7-figsupp1-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | RUES2-GLR | Brivanlou Lab (Rockefeller Institute, New York City, NY) | Human embryonic stem cell line | |
| Cell line (Homo sapiens) | H9 | Vallier Lab (Stem Cell Institute, Cambridge, UK) under agreement with WiCell | (RRID:CVCL_1240) | Human embryonic stem cell line |
| Cell line (Homo sapiens) | SHEF6 | Shef-6 (UK Stem Cell Bank, NIBSC) | Human embryonic stem cell line | |
| Peptide, recombinant protein | Recombinant human ACTIVIN-A | Stem Cell Institute, Cambridge, UK | 100 ng ml−1 | |
| Peptide, recombinant protein | Recombinant human LIF | Stem Cell Institute, Cambridge, UK | 10 ng ml−1 | |
| Peptide, recombinant protein | Recombinant human WNT3a | R and D Systems | 5036-WN-010 | 1 μg ml−1 |
| Peptide, recombinant protein | Recombinant human BMP4 | Peprotech | 120–05 | |
| Chemical compound, drug | CHIR99021 | Stem Cell Institute, Cambridge, UK | 3 μM | |
| Antibody | NANOG (goat polyclonal) | R and D Systems | AF1997 (RRID:AB_355097) | (1:200) |
| Antibody | SOX1 (goat polyclonal) | R and D Systems | AF3369-SP (RRID:AB_2239879) | (1:200) |
| Antibody | SOX2 (rabbit polyclonal) | Abcam | Ab59776 (RRID:AB_945584) | (1:200) |
| Antibody | T (goat polyclonal) | R and D Systems | AF2085 (RRID:AB_2200235) | (1:200) |
| Antibody | GATA6 (goat polyclonal) | R and D Systems | AF1700 (RRID:AB_2108901) | (1:200) |
| Antibody | LEF1 (rabbit monoclonal) | Cell Signaling Technology | 2230S (RRID:AB_823558) | (1:200) |
| Antibody | pSMAD1/5/9 (rabbit monoclonal) | Cell Signaling Technology | 13820S (RRID:AB_2493181) | (1:200) |
| Software, algorithm | ImageJ software | ImageJ (http://imagej.nih.gov/ij/) | RRID:SCR_003070 | |
| Software, algorithm | GraphPad Prism software | GraphPad Prism (https://graphpad.com) | RRID:SCR_015807 | |
| Other | 8-well µ-plates ibiTreat | Ibidi | 80826 | |
| Other | Rset (2-component) | StemCell Technologies | 05978 | |
| Other | mTeSR1 PLUS | StemCell Technologies | 05825 | |
| Other | Geltrex LDEV-free growth factor reduced | Thermo Fisher Scientific | A1413302 | |
| Other | Matrigel | BD Biosciences | 356230 |
Additional files
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Supplementary file 1
Transcriptional signature of human embryonic and extra-embryonic lineages.
- https://cdn.elifesciences.org/articles/63930/elife-63930-supp1-v1.xlsx
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Supplementary file 2
Transcriptional signature of human extra-embryonic endoderm and definitive endoderm.
- https://cdn.elifesciences.org/articles/63930/elife-63930-supp2-v1.xlsx
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Supplementary file 3
Transcriptional signature of human pre- and post-extra-embryonic endoderm.
- https://cdn.elifesciences.org/articles/63930/elife-63930-supp3-v1.xlsx
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Supplementary file 4
Supplementary materials and methods.
- https://cdn.elifesciences.org/articles/63930/elife-63930-supp4-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/63930/elife-63930-transrepform-v1.docx
