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The Generation of Definitive Endoderm from Human Embryonic Stem Cells is Initially Independent from Activin A but Requires Canonical Wnt-Signaling

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Abstract

The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.

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Acknowledgments

This work has been supported by the Deutsche Forschungsgemeinschaft (German Research Foundation) within the framework of the Cluster of Excellence REBIRTH. The skillful technical assistance of J. Kresse and R. Strauss is gratefully acknowledged. We would like to acknowledge the assistance of the Cell Sorting Facility of Hannover Medical School supported by the Braukmann-Wittenberg-Herz-Stiftung and the Deutsche Forschungsgemeinschaft.

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The authors declare no potential conflicts of interest.

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  1. Institute of Clinical Biochemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Lower Saxony, Germany

    Ortwin Naujok, Ulf Diekmann & Sigurd Lenzen

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  1. Ortwin Naujok
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  2. Ulf Diekmann
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  3. Sigurd Lenzen
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Correspondence to Ortwin Naujok.

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This work has been supported by the German Research Foundation within the framework of the Cluster of Excellence REBIRTH.

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Fig. S1

Gene expression profile of primitive gut tube, foregut, and hindgut markers. Human ES cells were differentiated either randomly, with the combination of wnt3a and activin A (Wnt3a/ActA) for 3 days and a subsequent 3 day treatment with KGF or with 10 μM CHIR-99021 (Chir) for 6 days. As a comparison, the relative gene expression of undifferentiated human ES cells is depicted (d0). Gene expressions of marker genes were measured with TaqMan® array cards and calibrated normalized quantitates (CNRQ) were then calculated after normalization to four stably expressed housekeeping genes. a Relative gene expression of the primitive gut tube markers HNF1B and HNF1A, b of marker genes of the foregut, namely FOXA1, HHEX and HNF6, and c of the hindgut genes CDX1, CDX2, and CDX4. For a better comparison HNF1B, HNF4A, and CDX1/2 are presented with a two-segmented y-axis (GIF 60 kb)

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Fig. S2

CHIR-99021 treatment efficiently translocates active beta-catenin to the nucleus a-b Immunfluorescence staining of active beta-catenin in undifferentiated human ES cells, in cells after treatment with 25 ng/ml wnt3a, and in cells after treatment with 5 μM CHIR-99021 for 24 h. Depicted are a low magnification image a and a high magnification image b revealing the subcellular location of beta catenin (scale bars 20 and 100 μM, respectively). c Mean grey value of the staining of nuclear located beta catenin. Values are means ± SEM of 20 cells of two independent experiments. Data analysis was performed with Xcellence RT (Olympus, Hamburg, Germany) (GIF 232 kb)

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Fig. S3

FACS sorting of two cell populations after differentiation with Wnt3a/Act A. a Representative flow cytometry dot plot of human ES cells differentiated for three days with wnt3a/activin A and double-stained for CD49e (FITC) and CXCR4 (PE). Regions for the cell sorting are indicated and qRT-PCR analysis of T, MIXL1, GSC, SOX17, and FOXA2 of the sorted cells are depicted in b. Calibrated normalized quantitates (CNRQ) were calculated after normalization to three stably expressed housekeeping genes. Values are means ± SD of a triplicate measurement (GIF 37 kb)

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Fig. S4

Flow cytometry and qRT-PCR analysis of cell lineage selection after treatment of human ES cells with CHIR-99021, activin A and BMP4. a Flow cytometry dot plots of Hues8 cells double-stained for CD49e (FITC) and CXCR4 (PE) after a treatment with 5 μM CHIR-99021 for one day and either 100 ng/ml activin A or 25 ng/ml BMP4 for 2 days. The differentiated cells were stained initially (d0) at day two (24 h) and day three (48 h) of treatment/differentiation with activin A or BMP4. Of note is that BMP4-treated cells did not acquire CXCR4-positivity. Numbers in each quadrant denote the cell percentages of this particular experiment. b Relative gene expression of the pluripotency marker POU5F1 (OCT3/4), the primitive streak marker genes T and MIXL1, the definitive endoderm markers SOX17, FOXA2, and GSC, and the mesoderm markers genes FLK1, PDGFRa and MEOX1. Values are calibrated normalized relative quantities (CNRQ) after normalization to three stably expressed housekeeping genes and scaling to undifferentiated ES cells. Values are means ± SD of a triplicate measurement (GIF 100 kb)

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Fig. S5

Differentiation of Hues4 human ES cells with wnt3a, activin A and CHIR-99021 a Flow cytometry dot plots of Hues4 double-stained for CD49e (FITC) and CXCR4 (PE) after a 3 day treatment with Wnt3a/ActA and Chir+ActA. Numbers in each quadrant denote the cell percentages of the particular experiment. b Relative gene expression of the primitive streak marker genes T and MIXL1 and of the definitive endoderm related markers SOX17, FOXA2 and GSC. Values are calibrated normalized relative quantities (CNRQ) after normalization to three stably expressed housekeeping genes and scaling to undifferentiated ES cells. Values are means ± SD of two independent experiments (GIF 86 kb)

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Fig. S6

Immunofluorescence staining for SOX17, FOXA2, BRACHYURY and GSC after differentiation with CHIR-99021 followed by activin A. a Immunofluorescence staining for SOX17 (green) and FOXA2 (red) and b BRACHYURY (green) and FOXA2 (red) in Hues8 cells after 3 days of differentiation with Chir+ActA. A composite image was assembled from four individual images (each with 100x magnification). The image depicts a differentiated ES cell colony with distinct endodermal outgrowth (asterisk), a core region composed of FOXA2/SOX17-negative cells (arrowhead) with a rim of FOXA2-positive cells (arrow). This image shows the FOXA2-positive outgrowth (asterisk) and the core region with BRACHYURY-positive cells (arrows) and frequent FOXA2/BRACHYURY double-positive cells in the rim of the core region (merge = yellow). Scale bar = 200 μM. c The transition zone between the core region and the endodermal outgrowth was defined by a distinct fraction of GSC-positive cells. These cells were scarcely detected inside the core region. The outgrowth of differentiated cells was predominantly GSC-positive with a specific nuclear staining. Of note is the cytoplasmic distribution in dividing cells (arrowhead). Left and center image original magnification 100x, scale bar = 200 μM. Lower right image original magnification 200x, scale bar = 100 μM (GIF 392 kb)

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Fig. S7

Western blot analysis after treatment with wnt3a, activin A or CHIR-99,021. Western blot analyses showed the dynamics of wnt3a, activin A and CHIR-99021 on the protein expression of downstream effectors of the TGF-beta pathway and the canonical Wnt-pathway during 3 days of differentiation with the indicated treatment. a Presented is SMAD2/3 and phosphorylated SMAD2/3 to show activation of the nodal/TGF-beta pathway and beta catenin and ser33/37/thr41 phosporylated beta catenin to verify the activation of the canonical Wnt-pathway. GSK3beta was stably expressed in all samples without any notable changes. The GSK3beta activating tyr216 phosphorylation was reduced in CHIR-99021 induced cells and vice versa the inactivating ser9 phosphorylation was increased. After the one day treatment with CHIR-99021 and change to activin a supplemented medium, GSK3beta tyr216 and ser9 phosphorylation disappeared. b Quantification of the Western blot protein bands for SMAD2 and phosphorylated SMAD2/3 by densitometry and normalization against GAPDH (JPEG 253 kb)

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Fig. S8

Promoter region of the ITGA5. The figure shows the 1 kb upstream region of the transcription start site of the ITGA5 gene encoding CD49e. Three putative TCF1b binding sites are highlighted in green. TSS = transcription start site. CDS = coding sequence (GIF 194 kb)

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Naujok, O., Diekmann, U. & Lenzen, S. The Generation of Definitive Endoderm from Human Embryonic Stem Cells is Initially Independent from Activin A but Requires Canonical Wnt-Signaling. Stem Cell Rev and Rep 10, 480–493 (2014). https://doi.org/10.1007/s12015-014-9509-0

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