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 (hESCs) after 6 days (6D) of ACL treatment (scale bar = 50 μm). (B, F, and I) Bar chart of elative fluorescence intensity (RFI arbitrary units [a.u.]) of SOX17 and SOX2 (± SEM) before and after 6D of ACL treatment in hPSCs as measured via reporter protein immunostaining. Mann–Whitney U-test; ****p < 0.0001 (number of cells analysed: hEPSC control n=2736, hEPSC + 6D ACL n=2270, Rset control n=2749, Rset + 6D ACL n=3146, hESC control n=1939, hESC + 6D ACL n=2538, three independent experiments each with one sample). (C, G, and J) Quantification of SOX2 and SOX17 protein levels at the single cell level in hEPSC, Rset hPSC, and hESC 6D ACL-treated cells based on relative reporter fluorescence intensity. Each dot represents an individual cell. (D) Relative expression levels (REL) (± SEM) of GATA6, SOX17, NANOG (n=6 samples, three independent experiments) and SOX2 (n=4 samples, two independent experiments) in hEPSC before and after 6D ACL treatment, normalised to their respective control. Mann–Whitney U-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (K) Relative expression levels (REL) (± SEM) of GATA6, SOX17, SOX2, and NANOG in Rset hPSCs (n=6 samples, three independent experiments), or hESCs (n=4 samples, two independent experiments) after 6D ACL treatment, normalised to their respective control . Mann–Whitney U-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (L) Transcriptomic-signature score (gene set variation analysis ([GSVA]) positive marker score) when comparing ACL-treated cells to either the human hypoblast, trophoblast, or epiblast within the human embryo (E6–14), or to human definitive endoderm using sc-RNA-seq expression data (embryo and definitive endoderm) and bulk RNA-seq expression data (cell lines). The higher the value (orange), the more relatively similar to each lineage.
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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 of SOX17 and SOX2 were determined. Endoderm differentiation is indicated by a concomitant increase in SOX17 and decrease in SOX2 levels. (B) Table of conditions used within the screen. (C) Bar chart depicting the average fluorescence intensity (± SEM) of SOX2 and SOX17 as measured by SOX2::mCitrine and SOX17::H2B-tdTomato+ levels via fluorescent intensity arbitrary units (a.u.). hEPSC medium (LCDM) was supplemented with pairs of pathway activators, each combination annotated with a number (n=1 independent experiment with one sample). Table in panel B should be used as a key for each medium condition on the x-axis. (D) Table of conditions used in the second screen used to optimise basal media and chemokine concentration. (E) Bar chart depicting the average fluorescence intensity (± SEM) of SOX2 and SOX17 as measured by SOX2::mCitrine and SOX17::H2B-tdTomato+ levels via fluorescent intensity a.u. (n=1 independent experiment with one sample). Table in panel D should be used as a key for each medium condition on the x-axis. (F) Line graph depicting relative increase in cell numbers of hEPSC, Rset hPSC, or human embryonic stem cell (hESC) cultured in ACTIVIN-A, Chiron, and LIF (ACL), or hEPSC, Rset hPSC, or hESC cultured in ACTIVIN-A, Chiron, and JAK inhibitor (ACi) over 6 days of conversion (± SEM). Cell numbers were calculated every 24 hr and normalised to the first day of conversion. Tukey’s multiple comparisons test result for the comparison between day 6 experimental condition (ACi) and control (ACL) for hEPSC, Rset, and hESC; ****p < 0.0001 (n=3 independent experiments each with one sample).
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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, two independent experiments) before and after 6 days (6D) ACL treatment, normalised to their respective control. Mann–Whitney U-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B, E, and H) Immunofluorescence images of SOX2 (green) and GATA6 (red) in H9 hEPSCs, human embryonic stem cells (hESCs), and Rset hPSCs after 6D of ACL treatment (scale bar = 50 μm). (C, F, and I) Bar chart of relative fluorescence intensity (RFI) of GATA6 and SOX2 (± SEM) in H9 hEPSCs, Rset, and hESCs before and after 6D of ACL treatment as measured via protein immunostaining. Mann–Whitney U-test (number of cells analysed: hEPSC control n=2375, hEPSC + 6D ACL n=2139, Rset control n=2472, Rset + 6D ACL n=1739, hESC n=3227, hESC + 6D ACL n=3661 n = 3 independent experiments each with one sample). (D, G, and J) Quantification of SOX2 and GATA6 protein levels at the single cell level in H9 hEPSC, Rset hPSC, and hESC 6D ACL-treated cells measured by protein immunostaining. Each dot represents an individual cell. (K) REL (± SEM) of GATA6, SOX17, SOX2, EOMES, NANOG, and POU5F1 in H9 hEPSC, hESCs, or Rset hPSCs after 6D ACL treatment, normalised to their respective control (n=4 samples, two independent experiments). Mann–Whitney U-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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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 fluorescence intensity (RFI arbitrary units [a.u.]) of SOX17 and SOX2 (± SEM) in Rset hPSCs on D0, D14, and D30 of culture in ACL as measured via reporter protein immunostaining. Kruskal-Wallis test with Dunn's multiple comparisons test; ****p < 0.0001 (number of cells analysed: D0 n=5580, D14 n=1846, D30 n=3003, three independent experiments each with one sample). (C) Relative expression levels (REL) (± SEM) of SOX17, GATA6, EOMES, SOX2, POU5F1, and NANOG (n=4 samples, two independent experiments) after 30 days of culture in ACL normalised to D0 control. Mann–Whitney U-test; NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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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 treatment (scale bar = 50 μm). (B, E, and H) Bar chart of relative fluorescence intensity (RFI arbitrary units [a.u.]) of GATA6 and SOX2 (± SEM) before and after 6D of ACL treatment in hPSCs as measured via protein immunostaining. Mann–Whitney U-test; ****p < 0.0001 (number of cells analysed: hEPSC control n=3865, hEPSC + 6D ACL n=2454, Rset control n=4125, Rset + 6D ACL n=3274, hESC control n=7020, hESC + 6D ACL n=2986, three independent experiments each with one sample). (C, F, and I) Quantification of SOX2 and GATA6 protein levels at the single cell level in hEPSC, Rset hPSC, and hESC 6D ACL-treated cells based on protein immunostaining. Each dot represents an individual cell. (J) Relative expression levels (REL) (± SEM) of GATA6, SOX17, NANOG, SOX2, EOMES, and POU5F1 in hEPSCs, Rset hPSCs, and hESCs before and after 6D ACL treatment, normalised to their respective control (n=4 samples, two independent experiments) Mann–Whitney U-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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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- population of hEPSC-ACL and Rset-ACL and hESC-ACL populations. The percentage of the population which was SOX17::tdTomato+ is shown on the graphs.
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 endoderm differentially expressed genes (orange), and of definitive endoderm differentially expressed genes (green). (B) MA plot representing the differentially expressed genes between human definitive endoderm and extra-embryonic endoderm. Top 20 differentially expressed genes are labelled, along with key extra-embryonic endoderm markers NID2, PDGFRa, HNF4a, HNF1β, PITX2, PODXL, and HHEX (>10% of cell type of interest, log2FC>0.25, p<0.05). (C) Transcriptomic-signature comparison score of human extra-embryonic endoderm vs. definitive endoderm within ACL-treated hPSCs (gene set variation analysis ([GSVA]) score for negative markers subtracted from GVSA score for positive markers and values normalised to 1). A negative value represents definitive endoderm similarity and a positive value represents an extra-embryonic endoderm similarity. hEPSC-ACL: n=2 technical replicates, Rset-ACL: n=3 technical replicates, hESC-ACL: n=2 technical replicates. Sc-RNA-seq expression data (extra-embryonic endoderm and definitive endoderm) and bulk RNA-seq expression data (cell lines) were used for all relevant panels.
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 of cells analysed SOX17: human extended pluripotent stem cell (hEPSC): D0 n=2774, D1 n=3258, D2 n = 2932, D3 n=3292, D4 n=2459, D5 n=2293, D6 n=1883, Rset: D0 n=2108, D1 n=3580, D2 n=2738, D3 n=2586, D4 n=2149, D5 n=2698, D6 n = 2441, human embryonic stem cell (hESC): D0 n=1426, D1 n=1924, D2 n=1943, D3 n=2817, D4 n=2248, D5 n=1148, D6 n=1957 (n=2 independent experiments); SOX2: hEPSC: D0 n = 2774, D1 n=3258, D2 n=2932, D3 n=3292, D4 n=2459, D5 n=2293, D6 n=1883, Rset: D0 n=2342, D1 n=3634, D2 n=2794, D3 n=2671, D4 n=2068, D5 n=2293, D6 n=2918, hESC: D0 n=2353, D1 n=3082, D2 n=3129, D3 n=4174, D4 n=2989, D5 n=1787, D6 n=3114; BRACHYURY: hEPSC: D0 n = 3452, D1 n=4090, D2 n=3435, D3 n=4223, D4 n=2881, D5 n=2837, D6 n=2218, Rset: D0 n=2317, D1 n = 3739, D2 n=3141, D3 n=2881, D4 n=2467, D5 n=2951, D6 n=4400, hESC: D0 n=2685, D1 n=3433, D2 n=3997, D3 n=5325, D4 n=3490, D5 n=2155, D6 n = 3258. n = 3 independent experiments). (B) Immunofluorescence images of SOX2 (green), SOX17 (red), and BRACHYURY (grey) in hEPSCs, Rset hPSCs, and hESCs during 6 days of ACL treatment (scale bar = 50 μm). (C) Schematic of human-mouse chimera protocol using ACL-treated cells. Clumps of three to six SOX-17::H2B-tdTomato+ treated cells were aggregated with E2.5 mouse embryos and cultured for 24 hr in ACL. After this, embryos were moved into KSOM and cultured for another 24 hr. (D) Immunofluorescence images of SOX17::H2B-tdTomato (red), GATA6 (grey), and DAPI (blue) in E4.5 mouse blastocysts. hEPSC-ACL cells contribution denoted by SOX17::H2B-tdTomato+ (red) cells (white arrowheads) (scale bar = 50 μm). (E) Immunofluorescence images of SOX17::H2B-tdTomato (red), GATA6 (grey), and DAPI (blue) in E4.5 mouse blastocysts. Rset-ACL cells contribution denoted by SOX17::H2B-tdTomato+ (red) cells (white arrowheads). (F) Pie charts showing percentage of human cell contribution to the primitive endoderm, epiblast, or both. Rset (N = 67 embryos, n=3 independent experiments), hEPSC (N = 39 embryos, n=3 independent experiments).
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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+, NANOG+/SOX17+, and the relative number of total cells within the inner cell mass (ICM). Unpaired Student’s t-test, NS = p>0.05. (B) Bar chart depicting the proportion of the total ICM that consisted of NANOG+/SOX17+ cells (± SEM). Unpaired Student’s t-test, NS = p>0.05. (C) Bar chart depicting the ratio between SOX17+ cells and NANOG+ cells within the ICM (± SEM). Unpaired Student’s t-test, NS = p>0.05. For A-C: KSOM: n = 16 embryos, ACL-KSOM: n = 14 embryos, n=2 independent experiment. (D) Viability of E4.5 blastocysts within either KSOM (n = 14 embryos) or ACL-KSOM (n = 28 embryos) (n=1 independent experiment). Chi-squared test. NS p > 0.05.
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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 bar = 50 μm). (B) Pie charts showing percentage of hEPSC contribution to the primitive endoderm, epiblast, or both (N=18 embryos, n=2 independent experiments). (C) Immunofluorescence images of HuNu (red), SOX2 (green), GATA6 (grey) and DAPI (blue) in E4.5 human-mouse chimeric blastocysts demonstrating contribution of Rset hPSCs (scale bar = 50 μm). (D) Pie charts showing percentage of Rset hPSC contribution to the primitive endoderm, epiblast, or both (N=26 embryos, n=2 independent experiments).
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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 endoderm, epiblast, or both by SOX17::H2B-tdTomato+ ACL-treated hESCs (n = 48 embryos, n=3 independent experiments).
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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 extra-embryonic endoderm differentially expressed genes (green) and of post-implantation extra-embryonic endoderm differentially expressed genes (orange). (B) MA plot representing the differentially expressed genes between human pre-implantation and post-implantation extra-embryonic endoderm. Top 20 differentially expressed genes are labelled (>10% of cell type of interest, log2FC>0.25, p<0.05). (C) Transcriptomic-signature comparison score of pre-implantation extra-embryonic endoderm vs. post-implantation extra-embryonic endoderm for T2iLGö-Pre and Rset-ACL (gene set variation analysis [GSVA] score for pre-implantation extra-embryonic endoderm subtracted from GSVA score for post-implantation extra-embryonic endoderm and values normalised to 1). A negative value (green) represents pre-implantation extra-embryonic endoderm similarity and a positive value (orange) represents post-implantation extra-embryonic endoderm. T2iLGö-Pre: n=2 technical replicates (from published dataset), Rset-ACL: n=3 technical replicates. Sc-RNA-seq expression data (pre- and post-implantation extra-embryonic endoderm) and bulk RNA-seq expression data (cell lines) were used for all relevant panels.
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 million (RCPM) using published sc-RNA-seq data. (B) Bar chart (± SEM) plotting expression levels of BMP, NODAL, and WNT inhibitors in Rset hPSCs and YSLCs denoted as transcripts per million (TPM) using bulk RNA-seq data (YSLCs: n=3 technical replicates, Rset: n=2 technical replicates [from published dataset]). (C) Immunofluorescence images of SOX2 (green) and ACTIN (grey) in hESCs cultured in 3D Geltrex in either mTeSR (hESC) or mTeSR conditioned on YSLCs (+YSLC CM) (scale bar = 50 μm). (D) Percentage of structures that are columnar, squamous, or disorganised based on cell aspect ratio calculations in cells from panel C using ACTIN stainings. Chi-squared test. ****p < 0.0001 (number of structures analysed: control n=33, +YSLC CM n=30, n=3 independent experiments each with one sample). (E) Bar chart (± SEM) depicting SOX2 levels as inferred from relative fluorescence intensity (RFI arbitrary units [a.u.]) in cells from panel C. Mann–Whitney U-test; ****p < 0.0001 (number of cells analysed: SOX2: hESC n=2346, +YSLC CM n=596, n=3 independent experiments each with one sample). (F) Immunofluorescence of pSMAD1/5 (grey) in hESCs cultured in mTeSR (hESC) or mTeSR conditioned on YSLCs (+YSLC CM) in 3D Geltrex (scale bar = 50 μm). (G) Immunofluorescence of LEF1 (grey) in hESCs cultured in mTeSR (hESC) or mTeSR conditioned on YSLCs (+YSLC CM) in 3D Geltrex (scale bar = 50 μm). (H) Bar chart (± SEM) depicting the levels of pSMAD1/5 (nuclear) and LEF1 in hESCs cultured in mTeSR (hESC) or mTeSR conditioned on YSLCs (+YSLC CM) as inferred from RFI in cells from panels F and G, respectively. Mann–Whitney U-test, ****p <0.0001 (number of cells analysed: pSMAD1/5: hESC n=2563, +YSLC CM n=1660; LEF1: hESC n=1984, +YSLC CM n=1041. n=3 independent experiments each with one sample). (I) Immunofluorescence of SOX2 (green) and pSMAD1/5 (grey) in hESCs cultured in mTeSR (hESC), treated with BMP4 (+BMP4) or treated with BMP4 and YSLC conditioned media (+BMP4+YSLC CM) (scale bar = 50 μm). (J) Bar charts (± SEM) depicting the levels of SOX2 and pSMAD1/5 RFI in samples from panel I. Kruskal–Wallis test with Dunn’s multiple comparisons test, ****p<0.0001 (number of cells analysed: hESC n=1549, +BMP4 n = 1063, +BMP4+YSLC CM n=1544, n=3 independent experiments each with one sample). (K) Immunofluorescence of SOX2 (green) and LEF1 (grey) in hESCs cultured in mTeSR (hESC), treated with WNT3a (+WNT3a) or treated with WNT3a and YSLC conditioned media (+WNT3a+YSLC CM) (scale bar = 50 μm). (L) Bar chart (± SEM) depicting the levels of SOX2 and LEF1 RFI in samples from panel K. Kruskal–Wallis test with Dunn’s multiple comparisons test, ****p<0.0001 (number of cells analysed: hESC n=1604, +WNT3a n=1682, +WNT3a+YSLC CM n=1376, n=3 independent experiments each with one sample).
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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 (grey) in Rset and YSLCs (after 6D ACL treatment) (scale bar = 50 μm) (C) Immunofluorescence of SOX2 (green) and ACTIN (grey) within human embryonic stem cells (hESCs) cultured in 3D Geltrex for 72 hr (scale bar = 50 μm). (D) Bar chart (± SEM) depicting relative fluorescence intensity (RFI arbitrary units [a.u]) of SOX2 as studied via protein immunostaining in hESCs cultured in 3D Geltrex alone or in the presence of the WNT inhibitors XAV (+XAV) or IWR1 (+IWR1). Kruskal–Wallis test with Dunn’s multiple comparisons test. NS: p > 0.05, ****p < 0.0001 (number of cells analysed: control n=3273, +XAV n=2220, +IWR1 n=2234, n = 3 independent experiments each with one sample). (E) Immunofluorescence images of SOX2 (green), GATA6 (red), and ACTIN (grey) in hESCs and hESC-YSLC co-cultures after 72 hr in 3D Geltrex (scale bar = 50 μm). (F) Bar chart (± SEM) depicting SOX2 levels as calculated using relative fluorescence intensity (RFI a.u.) in cells from panel E. Mann–Whitney U-test. ****p < 0.0001 (number of cells analysed: control n=773, hESC+YSLC=1198, n=3 independent experiments each with one sample). (G) Percentage of structures that are columnar, squamous, or disorganised based on cell aspect ratio calculations in cells from panel E using ACTIN stainings. Chi-squared test; NS p >0.05 (number of structures analysed: hESC n = 34, hESC+YSLC n=28. n=3 independent experiments each with one sample). (H) Immunofluorescence of SOX2 (green) and NANOG (grey) in hESCs, hESCs treated with BMP4 (+BMP4) or treated with BMP4 and YSLC conditioned media (CM) (+BMP4+YSLC CM) (scale bar = 50 μm). (I) Bar charts (± SEM) depicting the levels of NANOG RFI in samples from panel H. Kruskal–Wallis test with Dunn’s multiple comparisons test, ****p<0.0001 (number of cells analysed: hESC n=6736, +BMP4 n=8659, +BMP4+YSLC CM n=7255, n=3 independent experiments each with one sample). (J) Immunofluorescence of SOX2 (green) and NANOG (grey) in hESCs, hESCs treated with WNT3a (+WNT3a) or treated with WNT3a and YSLC CM (+WNT3a+YSLC CM) (scale bar = 50 μm). (K) Bar chart (± SEM) depicting the levels of NANOG RFI in samples from panel J. Kruskal–Wallis test with Dunn’s multiple comparisons test. ****p<0.0001, *p < 0.05 (number of cells analysed: hESC n=3225, +WNT3a n=2702, +WNT3a+YSLC CM n=3723, n=3 independent experiments each with one sample).
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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) Immunofluorescence of LEF1 (green) and BRACHYURY (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). (C) Bar charts (± SEM) depicting the levels of GATA6 and BRACHYURY in hESCs cultured in 3D Geltrex in mTeSR (hESC) or YSLC conditioned media (+YSLC CM) as inferred from relative fluorescence intensity (RFI) arbitrary units [a.u.] in cells from panels A and B, respectively (number of cells analysed: GATA6: hESC n=2336, YSLC CM n=1074, BRACHURY: hESC n=2112, YSLC CM n=1261, n=3 independent experiments each with one sample) Mann–Whitney U-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Immunofluorescence of SOX2 (green) and GATA6 (grey), and a merge of the two in hESCs, hESCs in MesoI (mesoderm induction medium), or MesoI + YSLC CM (scale bar = 50 μm). (E) Immunofluorescence of SOX2 (green) and BRACHYURY (grey), and a merge of the two, in hESCs, hESCs in mesoderm induction medium (MesoI), or MesoI conditioned on YSLCs (MesoI + YSLC CM) (scale bar = 50 μm). (F) Bar chart (± SEM) depicting the levels of GATA6, BRACHYURY and SOX2 in hESCs cultured in basal medium (hESC), hESCs in mesoderm induction medium (MesoI), or MesoI + YSLC CM as measured by RFI. Kruskal–Wallis test with Dunn’s multiple comparisons test. ****p <0.0001 (number of cells analysed: GATA6: hESC n = 5583, +MesoI n=2554, +MesoI+YSLC CM n=2383, BRACHYURY: hESC n = 6859, +MesoI n=6741, +MesoI+YSLC CM n=2072; SOX2: hESC n=6859, +MesoI n=6741, +MesoI+ YSLC CM n=2072. n=3 independent experiments each with one sample). (G) Relative expression levels (REL) (± SEM) of EOMES, SOX17, MIXL1, FOXA2, and GSC in hESCs cultured in basal medium (hESC), hESCs in mesoderm induction medium (MesoI), or MesoI + YSLC CM as measured by RFI, normalised to their hESC control (n=4 samples, two independent experiments). Kruskal–Wallis test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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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 μm). (B) Bar charts (± SEM) depicting relative fluorescence intensity (RFI arbitrary units [a.u.]) of SOX2, NANOG, and OCT4 in hESCs cultured in YSLC CM (+YSLC CM) relative to hESCs cultured in mTeSR (hESCs) in 3D Geltrex for 1, 3, 4, and 5 days. Two-way ANOVA with Sidak’s multiple comparisons test comparing hESC vs. +YSLC CM for each day. ****p<0.0001, ** <0.01, NS p>0.05 (number of cells analysed: SOX2: day 1: hESC n=567, +YSLC CM n=662, day 3: hESC n=2318, +YSLC CM n=1870, day 4: hESC n=3755, +YSLC CM n=1249, day 5: hESC n=4946, +YSLC CM n=1897, OCT4: day 1: hESC n=450, +YSLC CM n=601, day 3: hESC=1929, +YSLC CM n=1902, day 4: hESC=3472, +YSLC CM n=1314, day 5: hESC n=4946, +YSLC CM n=1897, NANOG: day 1: hESC n=450, +YSLC CM n=601, day 3: hESC n=1929, +YSLC CM n=1902, day 4: hESC n=3472, +YSLC CM n=1314, day 5: hESC n=4946, +YSLC CM n=1897, n = 3 independent experiments each with one sample). (C) Immunofluorescence of OTX2 (green) and SOX1 (grey) in hESCs after 1, 3, 4, or 5 days cultured in mTeSR (hESC) or YSLC conditioned media (+YSLC CM) in 3D Geltrex (scale bar = 50 μm). (D) Bar charts (± SEM) depicting RFI of OTX2 and SOX1 in hESCs cultured in YSLC CM (+YSLC CM) relative to hESCs cultured in mTeSR (hESC) in 3D Geltrex for 1, 3, 4, and 5 days. Two-way ANOVA with Sidak’s multiple comparisons test comparing hESC vs. +YSLC CM for each day. ****p<0.0001 NS, p>0.05 (number of cells analysed: OTX2: day 1: hESC n=456, YSLC CM n=914, day 3: hESC n=4278, +YSLC CM n=2036, day 4: hESC n=2432, +YSLC CM n=1123, day 5: hESC n=3858, +YSLC CM n=3243, SOX1: day 1: hESC n=503, +YSLC CM n=735, day 3: hESC n=2933, +YSLC CM n=3023, day 4: hESC n=3426, +YSLC CM n=2507, day 5: hESC n=3764, +YSLC CM n=2620, n = 3 independent experiments each with one sample). (E) Diagram depicting the modified neural specification protocol in which YSLC conditioned media is used to replace the SB431542+NOGGIN treatment in the conventional protocol for 11 days, before culturing hESCs in the presence of just FGF2 for 4 days. (F) Immunofluorescence of SOX2 (green), SOX1 (grey), and a merge of both in hESCs cultured in mTeSR (hESC) and hESCs cultured in YSLC conditioned media (+YSLC CM) for the first 11 days of the neural protocol before being placed in FGF2 as outlined in panel E (scale bar = 50 μm). (G) Bar charts (± SEM) depicting RFI of SOX1 and SOX2 in cells from panel F. Mann–Whitney U-test; *p<0.05, ****p<0.0001 (number of cells analysed: SOX1: hESC n = 4949, +YSLC CM n=4736, SOX2: hESC n = 4947, +YSLC CM n=4736, n=3 independent experiments). (H) Relative expression levels (REL) (± SEM) of PAX6, NESTIN, and NOTCH1 in hESCs cultured as outlined in panel E normalised to hESC control. Mann-Whitney U-test ; *p < 0.05 (n=4 samples, two independent experiments).
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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 relative fluorescence intensity (RFI arbitrary units [a.u.]) of SOX1 in hESCs after 6 and 7 days cultured in YSLC conditioned media (+YSLC CM) relative to hESCs cultured in mTeSR (hESC) in 3D Geltrex. Two-way ANOVA with Sidak’s multiple comparisons test comparing hESC VS +YSLC CM for each day. ****p<0.0001 (number of cells analysed day 6: hESC n=3338, + YSLC CM n=2205, day 7: hESC n=2745, + YSLC CM n = 4390, n=3 independent experiments). (C) Quantification of OTX2 or SOX1 vs. OCT4 or SOX2 at the single cell level in hESCs cultured in mTeSR (hESC) or YSLC conditioned media (+YSLC CM) in 3D Geltrex for 5 days based on protein immunostaining. Each dot represents an individual cell (numbers of cells depicted: SOX1vsOCT4: day 5 hESC n=1184, day 5 +YSLC CM n=478, OTX2vsOCT4: day 5 hESC n=1364, day 5 +YSLC CM n=748, SOX1vsSOX2: day 5 hESC n=1184, day 5+YSLC CM n=478, OTX2vsSOX2: day 5 hESC n=1364, day 5 +YSLC CM n=748). (D) Relative expression levels (REL) (± SEM) of PAX6, NOTCH1, NESTIN, OCT4, and NANOG in hESCs cultured as outlined in Figure 7E or in a dual SMAD inhibition neural protocol where SB431542 and NOGGIN (SBN) replace YSLC CM for the first 11 days of culture. Samples were normalised to hESC control. Two-way ANOVA with statistical result representing the p-value calculated when comparing columns for all genes (hESC vs. SBN vs. YSLC CM values); **p < 0.001 (n=4 samples, two independent experiments).
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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
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An in vitro stem cell model of human epiblast and yolk sac interaction
eLife 10:e63930.
https://doi.org/10.7554/eLife.63930
