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First published online 29 May 2007
doi: 10.1242/jcs.004127

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Regulated Nodal signaling promotes differentiation of the definitive endoderm and mesoderm from ES cells

¹ Department of Gastroenterological Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
² 21st Century COE Program, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

View larger version (30K): [in this window] [in a new window]   Fig. 1. Generation of a tetracycline-regulated Nodal-expressing ES cell line. (A) The construct and strategy for the regulated expression of Nodal in ES cells (Tet-off system). (B) Outline of definitive endoderm and mesoderm differentiation using Nodal-expressing ES cells. (C) FACS analysis of cells with GFP expression during stages 1 and 2. (D) Bright-field and GFP immunofluorescent images of cells during stage 2. ES cells were plated on feeder-free, type IV collagen-coated dishes. Most ES cells expressed GFP throughout differentiation. (E) Western blot analysis of Nodal protein expression during stages 1 and 2. Both the mature form (13 kDa) and the precursor of Nodal protein are detected at stage 1 and is diminish at stage 2 in Tet-on/Nodal-off ES cells. (F) Nodal gene expression by real-time RT-PCR analysis during stages 1 and 2. The level of Nodal expression in Nodal-on ES cells is roughly 2000 times higher than that in the Nodal-off control cells. (G) The total cell number relative to the initial number during stages 1 and 2. Nodal expression shows an insignificant effect on cell growth at stage 2.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Nodal-expressing ES cells differentiate into definitive endoderm and mesoderm at the expense of neuroectoderm. (A) Western blot showing the phosphorylation of Smad2, an effector of Nodal signaling, and the phosphorylation of Smad1/5/8, in each condition. (B) The expression of downstream transcriptional targets of Nodal at stage 2 by RT-PCR analysis. (C) Quantitative real time RT-PCR analysis of transcriptional targets at stage 2. (D,E) RT-PCR analysis showing the expression of definitive endoderm and mesoderm gene markers (D), and anterior visceral endoderm and neuroectoderm markers (E) at stage 2. (F,G) Immunofluorescent microscopy image of pan-neuronal marker (β-tubulinIII) in Tet-on/Nodal-off ES cells (F) or Tet-off/Nodal-on ES cells (G). The default pathway to neuroectoderm from ES cells is repressed in the Nodal-expressing ES cells. The original magnification was 400x. (H) Flow cytometric analysis of Nodal-expressing ES cells for the definitive endoderm progenitor marker (CXCR4) and mesoderm progenitor markers (VEGFR2 and PDGFR). The CXCR4+GFP+, VEGF2+GFP+, and PDGFR+GFP+ cell population increased more in stage 2 Nodal-expressing ES cells than in Nodal-off ES cells. (I) The populations of progenitor cells in each culture condition. Notably, the largest populations of cells were definitive endoderm and mesoderm progenitor cells in Nodal-on ES cells at stage 2. *P<0.01 compared with wild-type ES cells, **P<0.05 compared with 100 ng/ml activin A-treated ES cells, ***P<0.05 compared with Nodal on ES cells.

View larger version (68K): [in this window] [in a new window]   Fig. 3. Maturation of definitive endoderm by the reduction of Nodal signaling. (A) Outline of the three-stage maturation protocol of definitive endoderm with the reduction of Nodal signaling and the formation of a 3D structure. (B) RT-PCR analysis of definitive endodermal gene expression by stages 2, 3a and 3b cells. (C) Immunofluorescence images of stage-3a cell aggregates were obtained by confocal microscopy and are representative of at least five samples for each probe (Foxa2, E-cadherin, panCK, TTF1, proSftpC, albumin and Pdx1). Original magnification was 400x; bars, 50 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Sustained Nodal expression inhibited the maturation of definitive endoderm. (A) RT-PCR showing expression of the pluripotent marker, Oct4, at stages 2, 3a and 3b. (B) Immunohistochemistry showing Oct4 and SSEA1 expression at stage 3. (C) Quantitative single cell analysis by FACS for definitive endoderm and mesoderm progenitor cells at stage 3. The CXCR4+GFP+, VEGFR2+GFP+ and PDGFR+GFP+ cell populations decreased in stage 3a ES cells. By contrast, the CXCR4+GFP+ cell population in stage 3b ES cells was unchanged in comparison to that of stage 2 Nodal upregulated ES cells.

View larger version (56K): [in this window] [in a new window]   Fig. 5. Nodal-expressing ES cells were tightly regulated and differentiated into definitive endoderm and mesoderm in vivo. Teratoma formation in each condition. (A) graft weight, *P<0.001 compared with wild-type ES cells transplants or Nodal on/on grafts. (B) Representative images of grafts and kidney. Nodal on/off grafts clearly had smaller tumors 3 weeks after engraftment in comparison to wild-type or Nodal on/on grafts. (C) The expression of the pluripotent marker, Oct4, in the grafts. (D) RT-PCR analysis showing gene expression in graft including the transcriptional targets of Nodal, and the mature definitive endoderm, mesoderm, and neuroectoderm. (E) Immunofluorescence images of Foxa2, proSftpC, albumin, Pdx1, glucagon and IFABP in Nodal on/off grafts were obtained by confocal microscopy. The original magnification was 400x; bars, 50 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Nodal-expressing ES cells promote the differentiation of definitive endoderm from genetically unmanipulated ES cells. (A) 1x104 Nodal-expressing ES cells were plated on an upper insert and 5x103 genetically unmanipulated ES cells were plated on a gelatin-coated lower chamber and cultured for 7 days. (B) The phosphorylation of Smad2, an effector of Nodal signaling, and the phosphorylation of Smad1/5/8 in each culture condition. CM; conditional medium of Nodal expressing ES cells. (C) The gene expression of Nodal signaling in genetically unmanipulated ES cells. Co-culture with Nodal-expressing ES cells or CM upregulated the gene expression of downstream transcriptional targets of Nodal in genetically unmanipulated ES cells. (D) Mature definitive endoderm markers were induced in the cells co-cultured with Nodal-expressing ES cells or CM. This effect is blocked by Cripto-neutralizing antibody. (E) Flow cytometric analysis of the expression of the definitive endoderm progenitor, CXCR4, in the cells co-cultured with Nodal-expressing ES cells or CM. FSC, forward scattered light. (F) Expression of definitive endoderm genes (Foxa2 and TTF-1) in genetically unmanipulated ES cells co-cultured with Nodal-expressing ES cells. The original magnification was 400x. (G) A flow cytometric analysis of the expression of GFP in the Pdx1-GFP knock-in ES cells co-cultured with Nodal-expressing ES cells.