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af1997 nestin goat igg  (R&D Systems)


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    R&D Systems af1997 nestin goat igg
    Af1997 Nestin Goat Igg, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 465 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a) Immunocytochemistry for the hiPSC related markers, TRA-1-60 (upper panel, green), <t>NANOG</t> (upper panel, blue), SOX2 (lower panel, green), and OCT3/4 (lower panel, red) performed on expanded clone. b) Karyostat assay (ThermoFisher) demonstrating no abnormalities detected. The whole genome view displays all somatic and sex chromosomes in one frame with high level copy number. The smooth signal plot (right y-axis) is the smoothing of the log2 ratios which depict the signal intensities of probes on the microarray. A value of 2 represents a normal copy number state (CN = 2). A value of 3 represents chromosomal gain (CN = 3). A value of 1 represents a chromosomal loss (CN = 1). The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number. Referent to .
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    a) Immunocytochemistry for the hiPSC related markers, TRA-1-60 (upper panel, green), <t>NANOG</t> (upper panel, blue), SOX2 (lower panel, green), and OCT3/4 (lower panel, red) performed on expanded clone. b) Karyostat assay (ThermoFisher) demonstrating no abnormalities detected. The whole genome view displays all somatic and sex chromosomes in one frame with high level copy number. The smooth signal plot (right y-axis) is the smoothing of the log2 ratios which depict the signal intensities of probes on the microarray. A value of 2 represents a normal copy number state (CN = 2). A value of 3 represents chromosomal gain (CN = 3). A value of 1 represents a chromosomal loss (CN = 1). The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number. Referent to .
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    a) Immunocytochemistry for the hiPSC related markers, TRA-1-60 (upper panel, green), <t>NANOG</t> (upper panel, blue), SOX2 (lower panel, green), and OCT3/4 (lower panel, red) performed on expanded clone. b) Karyostat assay (ThermoFisher) demonstrating no abnormalities detected. The whole genome view displays all somatic and sex chromosomes in one frame with high level copy number. The smooth signal plot (right y-axis) is the smoothing of the log2 ratios which depict the signal intensities of probes on the microarray. A value of 2 represents a normal copy number state (CN = 2). A value of 3 represents chromosomal gain (CN = 3). A value of 1 represents a chromosomal loss (CN = 1). The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number. Referent to .
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    a) Immunocytochemistry for the hiPSC related markers, TRA-1-60 (upper panel, green), <t>NANOG</t> (upper panel, blue), SOX2 (lower panel, green), and OCT3/4 (lower panel, red) performed on expanded clone. b) Karyostat assay (ThermoFisher) demonstrating no abnormalities detected. The whole genome view displays all somatic and sex chromosomes in one frame with high level copy number. The smooth signal plot (right y-axis) is the smoothing of the log2 ratios which depict the signal intensities of probes on the microarray. A value of 2 represents a normal copy number state (CN = 2). A value of 3 represents chromosomal gain (CN = 3). A value of 1 represents a chromosomal loss (CN = 1). The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number. Referent to .
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    (a) IF staining for epiblast marker <t>NANOG</t> (red) and hypoblast <t>marker</t> <t>GATA6</t> (blue) of the human gastruloid (based on hiPSC) from day 1 to 3. Nuclei were counterstained with DAPI (grey). Scale bars,100 μm. (b) IF staining for epiblast marker OCT4 (red) and hypoblast/ anterior visceral endoderm marker OTX2 (blue) of the human gastruloid (based on hiPSC) on day 3. Nuclei were counterstained with DAPI (grey). Yellow asterisk, primary yolk sac. Scale bar, 100 μm (c) IF staining for epiblast marker OCT4 (red), hypoblast/anterior visceral endoderm marker OTX2 (blue) and secondary yolk sac marker PDGFRα (green) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, anterior visceral endoderm. Scale bar, 100 μm. (d) IF staining for epiblast marker OCT4 (blue), anterior visceral endoderm marker LHX1 (red) and hypoblast marker GATA6 (green) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, anterior visceral endoderm. Scale bar, 100 μm. (e) IF staining for hypoblast marker GATA6 (blue), secondary yolk sac marker PDGFRa (green) and extraembryonic mesoderm marker LAMC1 (red) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, secondary yolk sac. White triangle arrow, extraembryonic mesoderm. Scale bar, 100 μm. (f) TriMap showing 11 subtypes of hypoblast derivatives. hiPSC, human induced pluripotent stem cell; hPFC, human pluripotent founder cell; PYS-PE, primary yolk sac -parietal endoderm; PYS-VE, PYS-visceral endoderm; pSYS, precursor of secondary yolk sac; SYS-VE, SYS-visceral endoderm; SYS-PE, SYS-parietal endoderm; AVE, anterior visceral endoderm; HEP, haemato-endothelial progenitor; PYS-ExM, PYS-extraembryonic mesoderm, tExM, transition state of ExM. (g) TriMap projection of the VE/AVE and PE from the integrated human embryonic reference datasets onto this study (grey). (h-i) TriMaps of SYS-VE, SYS-PE and AVE in gastruloids on day 7 (h) integrated with human CS7 gastrula (i). (j) Bar plot showing the proportions of 11 subtypes in each sampling day. (k) The RNA velocity analysis of the hypoblast differentiation.
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    Image Search Results


    a) Immunocytochemistry for the hiPSC related markers, TRA-1-60 (upper panel, green), NANOG (upper panel, blue), SOX2 (lower panel, green), and OCT3/4 (lower panel, red) performed on expanded clone. b) Karyostat assay (ThermoFisher) demonstrating no abnormalities detected. The whole genome view displays all somatic and sex chromosomes in one frame with high level copy number. The smooth signal plot (right y-axis) is the smoothing of the log2 ratios which depict the signal intensities of probes on the microarray. A value of 2 represents a normal copy number state (CN = 2). A value of 3 represents chromosomal gain (CN = 3). A value of 1 represents a chromosomal loss (CN = 1). The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number. Referent to .

    Journal: bioRxiv

    Article Title: Reproducible differentiation of pure ovarian support cells from clinical-grade hiPSCs as a novel infertility treatment

    doi: 10.1101/2024.04.29.591741

    Figure Lengend Snippet: a) Immunocytochemistry for the hiPSC related markers, TRA-1-60 (upper panel, green), NANOG (upper panel, blue), SOX2 (lower panel, green), and OCT3/4 (lower panel, red) performed on expanded clone. b) Karyostat assay (ThermoFisher) demonstrating no abnormalities detected. The whole genome view displays all somatic and sex chromosomes in one frame with high level copy number. The smooth signal plot (right y-axis) is the smoothing of the log2 ratios which depict the signal intensities of probes on the microarray. A value of 2 represents a normal copy number state (CN = 2). A value of 3 represents chromosomal gain (CN = 3). A value of 1 represents a chromosomal loss (CN = 1). The pink, green and yellow colors indicate the raw signal for each individual chromosome probe, while the blue signal represents the normalized probe signal which is used to identify copy number. Referent to .

    Article Snippet: The primary antibodies used were mouse monoclonal antibody against OCT3/4 (1:200; sc5279, Santa Cruz Biotechnology), goat polyclonal antibody against SOX2 (1:50; AF2018, R&D systems), goat polyclonal antibody against NANOG (1:50; AF1997, R&D systems), and Alexa Fluor 488 mouse monoclonal antibody against TRA-1-60 (1:100; 560173, BD Biosciences).

    Techniques: Immunocytochemistry, Microarray

    a) Images of clinical grade OSCs (lot 90) grown on laminin on day 5 of hiPSC expansion and day 5 of OSC differentiation. Scale bar, 250 μm. b) Flow cytometry analysis of 4 markers: FOXL2, CD82, OCT4, and NANOG. Expression levels of these markers were tested against a control and CG-OSC-L. d) UMAP projection of the CG-OSC subset. e) UMAP projection of the individual lots found in the CG-OSC-L subset. f) Stacked bar plot depicting the amount of each cluster type found in each lot relative to the CG-OSC-L subset. Overall percentages per group are given to the right of the barplot. g) Dotplot representing the expression of granulosa cell markers in the CG-OSC subset. Scale represents ‘Mean expression in groups’ ranging from 0 to 2, and circles represent ‘Fraction of cells in group (%) ranging from 0 to 100. h) GO chord plot for differently regulated proteins in both RUO-OSC and CG-OSC versus hiPSC. i) Correlation curve for proteins detected in the secretome of RUO-OSC versus CG-OSC.

    Journal: bioRxiv

    Article Title: Reproducible differentiation of pure ovarian support cells from clinical-grade hiPSCs as a novel infertility treatment

    doi: 10.1101/2024.04.29.591741

    Figure Lengend Snippet: a) Images of clinical grade OSCs (lot 90) grown on laminin on day 5 of hiPSC expansion and day 5 of OSC differentiation. Scale bar, 250 μm. b) Flow cytometry analysis of 4 markers: FOXL2, CD82, OCT4, and NANOG. Expression levels of these markers were tested against a control and CG-OSC-L. d) UMAP projection of the CG-OSC subset. e) UMAP projection of the individual lots found in the CG-OSC-L subset. f) Stacked bar plot depicting the amount of each cluster type found in each lot relative to the CG-OSC-L subset. Overall percentages per group are given to the right of the barplot. g) Dotplot representing the expression of granulosa cell markers in the CG-OSC subset. Scale represents ‘Mean expression in groups’ ranging from 0 to 2, and circles represent ‘Fraction of cells in group (%) ranging from 0 to 100. h) GO chord plot for differently regulated proteins in both RUO-OSC and CG-OSC versus hiPSC. i) Correlation curve for proteins detected in the secretome of RUO-OSC versus CG-OSC.

    Article Snippet: The primary antibodies used were mouse monoclonal antibody against OCT3/4 (1:200; sc5279, Santa Cruz Biotechnology), goat polyclonal antibody against SOX2 (1:50; AF2018, R&D systems), goat polyclonal antibody against NANOG (1:50; AF1997, R&D systems), and Alexa Fluor 488 mouse monoclonal antibody against TRA-1-60 (1:100; 560173, BD Biosciences).

    Techniques: Flow Cytometry, Expressing, Control

    Bar plots depicting relative expression of hiPSC related markers, OCT4 and NANOG in OSCs after 5 days of differentiation (CG-OSCs). Clinical-grade (CG)-hiPSC are used as a positive control. Expression is normalized by expression of the housekeeping gene, GAPDH. Referent to .

    Journal: bioRxiv

    Article Title: Reproducible differentiation of pure ovarian support cells from clinical-grade hiPSCs as a novel infertility treatment

    doi: 10.1101/2024.04.29.591741

    Figure Lengend Snippet: Bar plots depicting relative expression of hiPSC related markers, OCT4 and NANOG in OSCs after 5 days of differentiation (CG-OSCs). Clinical-grade (CG)-hiPSC are used as a positive control. Expression is normalized by expression of the housekeeping gene, GAPDH. Referent to .

    Article Snippet: The primary antibodies used were mouse monoclonal antibody against OCT3/4 (1:200; sc5279, Santa Cruz Biotechnology), goat polyclonal antibody against SOX2 (1:50; AF2018, R&D systems), goat polyclonal antibody against NANOG (1:50; AF1997, R&D systems), and Alexa Fluor 488 mouse monoclonal antibody against TRA-1-60 (1:100; 560173, BD Biosciences).

    Techniques: Expressing, Positive Control

    (a) IF staining for epiblast marker NANOG (red) and hypoblast marker GATA6 (blue) of the human gastruloid (based on hiPSC) from day 1 to 3. Nuclei were counterstained with DAPI (grey). Scale bars,100 μm. (b) IF staining for epiblast marker OCT4 (red) and hypoblast/ anterior visceral endoderm marker OTX2 (blue) of the human gastruloid (based on hiPSC) on day 3. Nuclei were counterstained with DAPI (grey). Yellow asterisk, primary yolk sac. Scale bar, 100 μm (c) IF staining for epiblast marker OCT4 (red), hypoblast/anterior visceral endoderm marker OTX2 (blue) and secondary yolk sac marker PDGFRα (green) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, anterior visceral endoderm. Scale bar, 100 μm. (d) IF staining for epiblast marker OCT4 (blue), anterior visceral endoderm marker LHX1 (red) and hypoblast marker GATA6 (green) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, anterior visceral endoderm. Scale bar, 100 μm. (e) IF staining for hypoblast marker GATA6 (blue), secondary yolk sac marker PDGFRa (green) and extraembryonic mesoderm marker LAMC1 (red) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, secondary yolk sac. White triangle arrow, extraembryonic mesoderm. Scale bar, 100 μm. (f) TriMap showing 11 subtypes of hypoblast derivatives. hiPSC, human induced pluripotent stem cell; hPFC, human pluripotent founder cell; PYS-PE, primary yolk sac -parietal endoderm; PYS-VE, PYS-visceral endoderm; pSYS, precursor of secondary yolk sac; SYS-VE, SYS-visceral endoderm; SYS-PE, SYS-parietal endoderm; AVE, anterior visceral endoderm; HEP, haemato-endothelial progenitor; PYS-ExM, PYS-extraembryonic mesoderm, tExM, transition state of ExM. (g) TriMap projection of the VE/AVE and PE from the integrated human embryonic reference datasets onto this study (grey). (h-i) TriMaps of SYS-VE, SYS-PE and AVE in gastruloids on day 7 (h) integrated with human CS7 gastrula (i). (j) Bar plot showing the proportions of 11 subtypes in each sampling day. (k) The RNA velocity analysis of the hypoblast differentiation.

    Journal: bioRxiv

    Article Title: Establishment of a novel non-integrated human pluripotent stem cell-based gastruloid model

    doi: 10.1101/2023.06.28.546720

    Figure Lengend Snippet: (a) IF staining for epiblast marker NANOG (red) and hypoblast marker GATA6 (blue) of the human gastruloid (based on hiPSC) from day 1 to 3. Nuclei were counterstained with DAPI (grey). Scale bars,100 μm. (b) IF staining for epiblast marker OCT4 (red) and hypoblast/ anterior visceral endoderm marker OTX2 (blue) of the human gastruloid (based on hiPSC) on day 3. Nuclei were counterstained with DAPI (grey). Yellow asterisk, primary yolk sac. Scale bar, 100 μm (c) IF staining for epiblast marker OCT4 (red), hypoblast/anterior visceral endoderm marker OTX2 (blue) and secondary yolk sac marker PDGFRα (green) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, anterior visceral endoderm. Scale bar, 100 μm. (d) IF staining for epiblast marker OCT4 (blue), anterior visceral endoderm marker LHX1 (red) and hypoblast marker GATA6 (green) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, anterior visceral endoderm. Scale bar, 100 μm. (e) IF staining for hypoblast marker GATA6 (blue), secondary yolk sac marker PDGFRa (green) and extraembryonic mesoderm marker LAMC1 (red) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, secondary yolk sac. White triangle arrow, extraembryonic mesoderm. Scale bar, 100 μm. (f) TriMap showing 11 subtypes of hypoblast derivatives. hiPSC, human induced pluripotent stem cell; hPFC, human pluripotent founder cell; PYS-PE, primary yolk sac -parietal endoderm; PYS-VE, PYS-visceral endoderm; pSYS, precursor of secondary yolk sac; SYS-VE, SYS-visceral endoderm; SYS-PE, SYS-parietal endoderm; AVE, anterior visceral endoderm; HEP, haemato-endothelial progenitor; PYS-ExM, PYS-extraembryonic mesoderm, tExM, transition state of ExM. (g) TriMap projection of the VE/AVE and PE from the integrated human embryonic reference datasets onto this study (grey). (h-i) TriMaps of SYS-VE, SYS-PE and AVE in gastruloids on day 7 (h) integrated with human CS7 gastrula (i). (j) Bar plot showing the proportions of 11 subtypes in each sampling day. (k) The RNA velocity analysis of the hypoblast differentiation.

    Article Snippet: The primary antibodies included: mouse anti-OCT4 antibody (Santa Cruz Biotechnology, Sc-5279; 1:100), rabbit anti-OCT4 antibody (Abcam, ab181557; 1:200), rabbit anti-SOX2 antibody (Abcam, ab92494; 1:200), goat anti-SOX2 antibody (R&D system, AF2018; 1:200), rabbit anti-NANOG antibody (Cell signaling tech, 4903S; 1:200), goat anti-NANOG antibody (R&D system, AF1997; 1:200), goat anti-GATA6 antibody (R&D system, AF1700; 1:500), goat anti-SOX17 antibody (R&D system, AF1924; 1:500), goat anti-OTX2 antibody (R&D system, AF1979; 1:200), rabbit anti-CDX2 antibody (Cell signaling tech, 12306; 1:200), rabbit anti-PDGFRa antibody (Abcam, ab203491; 1:200), rabbit anti-LHX1 antibody (Developmental Studies Hybridoma Bank, 4F2; 1:200), mouse anti-LAMC1 antibody (R&D system, MAB2139; 1:200), goat anti-TBXT antibody (R&D system, AF2085; 1:200), mouse anti-EZRIN antibody (Sigma-Aldrich, E8897; 1:200), rabbit anti-ISL1 antibody (Invitrogen, PA5-27789; 1:200), rabbit anti-EOMES antibody (Sigma-Aldrich, HPA028896; 1:200), rabbit anti-MXIL1 antibody (Proteintech, 22772-1-AP; 1:200), mouse anti-AP2γ antibody (Santa Cruz Biotechnology, SC-12762; 1:100), rabbit anti-BLIMP1 antibody (Cell signaling tech, 9115; 1:200), mouse anti-AP2α antibody (Santa Cruz Biotechnology, SC-12726; 1:100), rabbit anti-KRT7 antibody (Zsbio, ZA-0573; 1:200), goat anti-VIM antibody (R&D system, AF2105; 1:200).

    Techniques: Staining, Marker, Sampling

    (a) Line plot showing expression of genes related to pluripotency and endoderm formation during hPFC development to PYS. (b) TriMap showing expression of pluripotent marker NANOG and sequential upregulation of hypoblast specifiers GATA6, SOX17 and GATA4. (c) IF staining for epiblast marker OCT4 (red), hypoblast marker GATA6 (blue) and GATA4 (green) of the human gastruloid (based on hiPSC) on day 2. Nuclei were counterstained with DAPI (grey). Scale bar, 100 μm. (d) Heatmaps showing expression of genes in TGF-β (ref: GO:0030511) (left) and WNT (ref: GO:0030177) (right) signaling pathways in indicated cell types.

    Journal: bioRxiv

    Article Title: Establishment of a novel non-integrated human pluripotent stem cell-based gastruloid model

    doi: 10.1101/2023.06.28.546720

    Figure Lengend Snippet: (a) Line plot showing expression of genes related to pluripotency and endoderm formation during hPFC development to PYS. (b) TriMap showing expression of pluripotent marker NANOG and sequential upregulation of hypoblast specifiers GATA6, SOX17 and GATA4. (c) IF staining for epiblast marker OCT4 (red), hypoblast marker GATA6 (blue) and GATA4 (green) of the human gastruloid (based on hiPSC) on day 2. Nuclei were counterstained with DAPI (grey). Scale bar, 100 μm. (d) Heatmaps showing expression of genes in TGF-β (ref: GO:0030511) (left) and WNT (ref: GO:0030177) (right) signaling pathways in indicated cell types.

    Article Snippet: The primary antibodies included: mouse anti-OCT4 antibody (Santa Cruz Biotechnology, Sc-5279; 1:100), rabbit anti-OCT4 antibody (Abcam, ab181557; 1:200), rabbit anti-SOX2 antibody (Abcam, ab92494; 1:200), goat anti-SOX2 antibody (R&D system, AF2018; 1:200), rabbit anti-NANOG antibody (Cell signaling tech, 4903S; 1:200), goat anti-NANOG antibody (R&D system, AF1997; 1:200), goat anti-GATA6 antibody (R&D system, AF1700; 1:500), goat anti-SOX17 antibody (R&D system, AF1924; 1:500), goat anti-OTX2 antibody (R&D system, AF1979; 1:200), rabbit anti-CDX2 antibody (Cell signaling tech, 12306; 1:200), rabbit anti-PDGFRa antibody (Abcam, ab203491; 1:200), rabbit anti-LHX1 antibody (Developmental Studies Hybridoma Bank, 4F2; 1:200), mouse anti-LAMC1 antibody (R&D system, MAB2139; 1:200), goat anti-TBXT antibody (R&D system, AF2085; 1:200), mouse anti-EZRIN antibody (Sigma-Aldrich, E8897; 1:200), rabbit anti-ISL1 antibody (Invitrogen, PA5-27789; 1:200), rabbit anti-EOMES antibody (Sigma-Aldrich, HPA028896; 1:200), rabbit anti-MXIL1 antibody (Proteintech, 22772-1-AP; 1:200), mouse anti-AP2γ antibody (Santa Cruz Biotechnology, SC-12762; 1:100), rabbit anti-BLIMP1 antibody (Cell signaling tech, 9115; 1:200), mouse anti-AP2α antibody (Santa Cruz Biotechnology, SC-12726; 1:100), rabbit anti-KRT7 antibody (Zsbio, ZA-0573; 1:200), goat anti-VIM antibody (R&D system, AF2105; 1:200).

    Techniques: Expressing, Marker, Staining, Protein-Protein interactions

    (a) IF staining for epiblast marker OCT4 (red), hypoblast marker GATA6 (blue) and epithelial polarization marker EZRIN (green) of the human gastruloid (based on hiPSC) on day 5. Nuclei were counterstained with DAPI (grey). Scale bar, 100 μm. (b) IF staining for epiblast marker SOX2 (blue), amnion cell marker AP2α (green) and KRT7 (red) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrow, amnion cell. White arrowhead, squamous amniotic epithelium. Scale bar, 100 μm. (c) IF staining for epiblast marker OCT4 (blue), primitive streak marker TBXT (red) and mesoderm marker MIXL1 (green) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White solid arrow, only OCT4 + epiblast. White arrowhead, OCT4 + TBXT + primitive streak. White arrow, TBXT + MIXL1 + cell. White triangle arrow, only MIXL1 + nascent mesoderm. Scale bar, 100 μm. (d) IF staining for epiblast marker (blue) OCT4, nascent mesoderm marker (green) MIXL1 and advanced mesoderm marker(red) VIM of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, only OCT4 + epiblast. White arrow, MIXL1 + nascent mesoderm. White triangle arrow, VIM + advanced mesoderm. Scale bar, 100 μm. (e) IF staining for primordial germ cell marker SOX17 (blue), AP2γ (green) and BLIMP1 (red) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White triangle arrow, primordial germ cells. Scale bar, 100 μm. (f) IF staining for epiblast marker SOX2 (green), amnion cell marker ISL1 (red), amnion cavity marker EZRIN (blue) (upper) and the epiblast marker SOX2 (blue), amnion cell marker AP2α (green) and KRT7 (red) (lower) of the human gastruloid (based on hESC) on day 7. Nuclei were counterstained with DAPI (grey). White arrow, amnion cell. White arrowhead, squamous amniotic epithelium. Scale bars, 100 μm. (g) Upper: IF staining for epiblast marker OCT4 (blue), primitive streak marker TBXT (red) and endoderm marker EOMES (green) of the human gastruloid (based on hESC) on day 7. Nuclei were counterstained with DAPI (grey). White solid arrow, only OCT4 + epiblast. White arrowhead, OCT4 + TBXT + primitive streak. White arrow, TBXT + EOMES + cell. White triangle arrow, only EOMES + definitive endoderm. Scale bar, 100 μm. Lower: IF staining for epiblast marker OCT4 (blue), primitive streak marker TBXT (red) and nascent mesoderm marker MIXL1 (green) of the human gastruloid (based on hESC) on day 7. Nuclei were counterstained with DAPI (grey). White solid arrow, only OCT4 + epiblast. White arrowhead, OCT4 + TBXT + primitive streak. White arrow, TBXT + MIXL1 + cell. White triangle arrow, only MIXL1 + nascent mesoderm. Scale bar, 100 μm. (h) IF staining for primordial germ cell marker SOX17 (blue), AP2γ (green) co-stained with NANOG (red) (upper) and BLIMP1 (red) (lower) of the human gastruloid (based on hESC) on day 7. Nuclei were counterstained with DAPI (grey). White triangle arrow, primordial germ cells. Scale bars, 100 μm.

    Journal: bioRxiv

    Article Title: Establishment of a novel non-integrated human pluripotent stem cell-based gastruloid model

    doi: 10.1101/2023.06.28.546720

    Figure Lengend Snippet: (a) IF staining for epiblast marker OCT4 (red), hypoblast marker GATA6 (blue) and epithelial polarization marker EZRIN (green) of the human gastruloid (based on hiPSC) on day 5. Nuclei were counterstained with DAPI (grey). Scale bar, 100 μm. (b) IF staining for epiblast marker SOX2 (blue), amnion cell marker AP2α (green) and KRT7 (red) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrow, amnion cell. White arrowhead, squamous amniotic epithelium. Scale bar, 100 μm. (c) IF staining for epiblast marker OCT4 (blue), primitive streak marker TBXT (red) and mesoderm marker MIXL1 (green) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White solid arrow, only OCT4 + epiblast. White arrowhead, OCT4 + TBXT + primitive streak. White arrow, TBXT + MIXL1 + cell. White triangle arrow, only MIXL1 + nascent mesoderm. Scale bar, 100 μm. (d) IF staining for epiblast marker (blue) OCT4, nascent mesoderm marker (green) MIXL1 and advanced mesoderm marker(red) VIM of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White arrowhead, only OCT4 + epiblast. White arrow, MIXL1 + nascent mesoderm. White triangle arrow, VIM + advanced mesoderm. Scale bar, 100 μm. (e) IF staining for primordial germ cell marker SOX17 (blue), AP2γ (green) and BLIMP1 (red) of the human gastruloid (based on hiPSC) on day 7. Nuclei were counterstained with DAPI (grey). White triangle arrow, primordial germ cells. Scale bar, 100 μm. (f) IF staining for epiblast marker SOX2 (green), amnion cell marker ISL1 (red), amnion cavity marker EZRIN (blue) (upper) and the epiblast marker SOX2 (blue), amnion cell marker AP2α (green) and KRT7 (red) (lower) of the human gastruloid (based on hESC) on day 7. Nuclei were counterstained with DAPI (grey). White arrow, amnion cell. White arrowhead, squamous amniotic epithelium. Scale bars, 100 μm. (g) Upper: IF staining for epiblast marker OCT4 (blue), primitive streak marker TBXT (red) and endoderm marker EOMES (green) of the human gastruloid (based on hESC) on day 7. Nuclei were counterstained with DAPI (grey). White solid arrow, only OCT4 + epiblast. White arrowhead, OCT4 + TBXT + primitive streak. White arrow, TBXT + EOMES + cell. White triangle arrow, only EOMES + definitive endoderm. Scale bar, 100 μm. Lower: IF staining for epiblast marker OCT4 (blue), primitive streak marker TBXT (red) and nascent mesoderm marker MIXL1 (green) of the human gastruloid (based on hESC) on day 7. Nuclei were counterstained with DAPI (grey). White solid arrow, only OCT4 + epiblast. White arrowhead, OCT4 + TBXT + primitive streak. White arrow, TBXT + MIXL1 + cell. White triangle arrow, only MIXL1 + nascent mesoderm. Scale bar, 100 μm. (h) IF staining for primordial germ cell marker SOX17 (blue), AP2γ (green) co-stained with NANOG (red) (upper) and BLIMP1 (red) (lower) of the human gastruloid (based on hESC) on day 7. Nuclei were counterstained with DAPI (grey). White triangle arrow, primordial germ cells. Scale bars, 100 μm.

    Article Snippet: The primary antibodies included: mouse anti-OCT4 antibody (Santa Cruz Biotechnology, Sc-5279; 1:100), rabbit anti-OCT4 antibody (Abcam, ab181557; 1:200), rabbit anti-SOX2 antibody (Abcam, ab92494; 1:200), goat anti-SOX2 antibody (R&D system, AF2018; 1:200), rabbit anti-NANOG antibody (Cell signaling tech, 4903S; 1:200), goat anti-NANOG antibody (R&D system, AF1997; 1:200), goat anti-GATA6 antibody (R&D system, AF1700; 1:500), goat anti-SOX17 antibody (R&D system, AF1924; 1:500), goat anti-OTX2 antibody (R&D system, AF1979; 1:200), rabbit anti-CDX2 antibody (Cell signaling tech, 12306; 1:200), rabbit anti-PDGFRa antibody (Abcam, ab203491; 1:200), rabbit anti-LHX1 antibody (Developmental Studies Hybridoma Bank, 4F2; 1:200), mouse anti-LAMC1 antibody (R&D system, MAB2139; 1:200), goat anti-TBXT antibody (R&D system, AF2085; 1:200), mouse anti-EZRIN antibody (Sigma-Aldrich, E8897; 1:200), rabbit anti-ISL1 antibody (Invitrogen, PA5-27789; 1:200), rabbit anti-EOMES antibody (Sigma-Aldrich, HPA028896; 1:200), rabbit anti-MXIL1 antibody (Proteintech, 22772-1-AP; 1:200), mouse anti-AP2γ antibody (Santa Cruz Biotechnology, SC-12762; 1:100), rabbit anti-BLIMP1 antibody (Cell signaling tech, 9115; 1:200), mouse anti-AP2α antibody (Santa Cruz Biotechnology, SC-12726; 1:100), rabbit anti-KRT7 antibody (Zsbio, ZA-0573; 1:200), goat anti-VIM antibody (R&D system, AF2105; 1:200).

    Techniques: Staining, Marker