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First published online 30 July 2008
doi: 10.1242/dev.021121

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Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/β-catenin, Activin/Nodal and BMP signaling

¹ Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
² Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
³ Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, 69 Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8507, Japan.

View larger version (98K): [in this window] [in a new window]   Fig. 1. Temporal dynamics of the primitive streak and mesoderm progenitors in β-catenin-activated hES cells. (A) The Nβ-cateninER cells were cultured in defined medium with or without 4OHT (days 0-3), and the representative morphology at the indicated time periods was shown. Upper or lower panels shows low or high magnification images, respectively. Scale bars: 100 µm. (B) Immunostaining analysis of the day-3 Nβ-cateninER-activated cells. Cells were cultured with or without 4OHT for 3 days, and subjected to immunostaining with indicated antibodies, and nuclei were counterstained with diaminopimelic acid (DAPI). Scale bar: 100 µm. (C) The Nβ-cateninER cells were cultured with (days 1-3) or without (day 0) 4OHT for the indicated time periods, and subjected to immunostaining with anti-E-cadherin and anti-N-cadherin antibodies. Nuclei were counterstained with DAPI. Scale bar: 100 µm. (D) Quantitative real-time PCR (qPCR) analysis on RNA isolated from undifferentiated Nβ-cateninER cells (day 0) and β-catenin-activated cells harvested at daily intervals (days 1-5) was performed using specific primers for the genes indicated. (E) Immunoblot analysis of cell lysates from Nβ-cateninER cells. Cells were cultured for the indicated time periods with 4OHT, and cell lysates were subjected to SDS-PAGE and probed with specific antibodies as indicated.

View larger version (55K): [in this window] [in a new window]   Fig. 2. Characterization of β-catenin-activated hES cells. (A) The Nβ-cateninER cells cultured with or without 4OHT for the indicated time periods were subjected to immunostaining with anti-KDR and anti-CXCR4 antibodies, and analyzed by FACS. The gated region denoted the cells scored as antigen-positive (the percent cells scored positive is shown in each panel). ES, unstimulated Nβ-cateninER cells. (B) Developmental potentials of β-catenin-activated cells towards mesoderm lineage. RT-PCR analysis of endothelial cell-specific markers at different time points: the Nβ-cateninER cells were cultured with 4OHT for 3 days (day 3), and further cultured with vascular endothelial growth factor (VEGF) for additional 3 days (day 6). The transgenic cell lines derived from different ES cell lines are shown (K1βcatER derived from KhES-1 cells, K3βcatER derived from KhES-3 cells). (C) Morphology of endothelial-like cells derived from β-catenin-activated cells (day 6). (D) Capillary tube-like networks formed on matrigel for 24 hours after culturing endothelial-like cells derived from β-catenin-activated cells. Scale bar: 100 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 3. BMP signaling blockade changes cell fate from mesoderm to the anterior PS/endoderm. (A) The qPCR analysis on RNA isolated at the day 3 from unstimulated hES cells (vehicle: v) and β-catenin-activated cells (OHT) with or without Noggin (Nog; 250 ng/ml) or SB431542 (SB) was performed using specific primers for the genes indicated. (B) Expression of the anterior PS/endoderm markers in cells activated by β-catenin with Noggin. The Nβ-cateninER cells were cultured with 4OHT in the presence or absence Noggin for 3 days, and subjected to immunostaining with T, FOXA2 and SOX17. Nuclei were counterstained with DAPI. Scale bar: 100 µm. (C) Developmental potentials of β-catenin-activated cells toward definitive endoderm and cardiac lineage. The day 3 β-catenin-activated cells with or without Noggin were cultured with BMP4 and FGF2 for additional 4 days, and analyzed by RT-PCR. The transgenic cell lines derived from different ES cell lines are shown (K1βcatER derived from KhES-1 cells, K3βcatER derived from KhES-3 cells). ES, unstimulated Nβ-cateninER cells.

View larger version (126K): [in this window] [in a new window]   Fig. 4. Involvement of Activin/Nodal, PI3-kinase and MAPK signaling pathways in β-catenin-induced mesoderm and the anterior PS differentiation. (A,C) Immunoblot analysis of total cell lysates from Nβ-cateninER cells. Cells were cultured for 3 days with or without 4OHT, Noggin (Nog; 250 ng/ml), Activin A (25 ng/ml) or BMP4 (10 ng/ml). Cell lysates were subjected to SDS-PAGE, and probed with specific antibodies as indicated. (B) The Nβ-cateninER cells were cultured with 4OHT and Noggin in the presence or absence of SB431542 (SB) for 3 days, and immunostained with anti-N-cadherin and FOXA2 antibodies. Scale bar: 100 µm. (D) Localization of β-catenin in Nβ-cateninER cells. Cells were cultured with or without 4OHT and Noggin, and subjected to immunostaining with anti-β-catenin (total) and anti-ER antibodies (to recognize exogenous Nβ-cateninER). Nuclei were counterstained with DAPI. Scale bar: 50 µm.

View larger version (115K): [in this window] [in a new window]   Fig. 5. Inhibition of PI3-kinase or MAP-kinase signaling abolishes the anterior PS or mesoderm induction, respectively. The Nβ-cateninER cells were cultured with 4OHT and Noggin (Nog) in the presence or absence of U0126 (U) and LY294002 (LY) for 3 days, and then analyzed by qPCR (A), and stained with specific antibodies as indicated (B). v, vehicle control. Scale bar: 100 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 6. Generation of mesoderm and the anterior PS from genetically unmanipulated hES cell lines, KhES-3 and HES-3 cells. (A) Phase-contrast microscopy of KhES-3 cells, treated with BIO at different concentrations for 3 days. Scale bar: 100 µm. (B,C) The qPCR analysis on RNA isolated after 3 days from untreated KhES-3 cells (vehicle, v) or BIO-treated cells (BIO, 5 µM) with or without Noggin (Nog; 250 ng/ml) for 3 days was performed using specific primers for the genes indicated. (D) Expression of N-cadherin and FOXA2 in BIO-treated KhES-3 cells. The hES cells were cultured with BIO (5 µM) in the presence or absence Noggin for 3 days, and stained with specific antibodies as indicated. Scale bar: 100 µm. (E) Phase-contrast microscopy and localization of β-catenin in BIO-Acetoxime-treated HES-3 cells. Cells were cultured for 3 days with BIO-Acetoxime that exhibits greater selectivity for GSK3 than for BIO (left panels, phase-contrast images), and subjected to immunostaining with anti-β-catenin antibody (right panels). Scale bars: 100 µm. (F) qPCR analysis of RNA isolated from HES-3 cells, with or without BIO-Acetoxime (0-10 µM) for 3 days, was performed as described in B. (G) Expression of N-cadherin and FOXA2 in BIO-Acetoxime-treated HES-3 cells. The HES-3 cells were cultured with BIO-Acetoxime (10 µM) in the presence or absence Noggin for 3 days, and subjected to immunostaining with anti-N-cadherin and anti-FOXA2 antibodies. Scale bar: 100 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Synergistic interaction of Activin and canonical Wnt/β-catenin signaling pathways in the anterior PS/endoderm specification during hES cell differentiation. (A) qPCR analysis on RNA isolated from HES-3 cells, with or without Activin A (100 ng/ml) or DKK1 (100 ng/ml) for 3 days, was performed (v, vehicle control; A, Activin A treated). (B,C) The Nβ-cateninER cells were cultured with Activin A (100 ng/ml) in the presence or absence of 4OHT for 3 days, and then analyzed by qPCR (B), or cell lysates were subjected to SDS-PAGE and probed with specific antibodies as indicated (C) (OHT, 4OHT treated). (D) Proposed model for early lineage determination of hES cell differentiation by the canonical Wnt/β-catenin, Activin and BMP signaling pathways (see Discussion).

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