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First published online 19 October 2004
doi: 10.1242/jcs.01489

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Aggregation of embryonic stem cells induces Nanog repression and primitive endoderm differentiation

¹ Department of Pathology, University of Florida College of Medicine, PO Box 100275, Gainesville, FL 32610, USA
² Laboratory of Animal Molecular Technology, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

View larger version (71K): [in a new window]   Fig. 1. (A) Visualization of visceral endoderm differentiation in ES cell aggregates by using GFP expression under the control of the promoter of the Afp gene (which encodes -fetoprotein). GFP expression was not detected when Afp-GFP ES cells were cultured as monolayer in the presence of LIF (ES, left). GFP-positive cells appeared on the outer layer of aggregates within 4 days regardless of the presence of LIF in the medium (LIF– or LIF+, middle and right). (top) Phase-contrast image under a light microscope; (bottom) pictures under a fluorescence microscope. Bars, 200 µm. (B) Expression of ES-cell-specific genes and differentiation markers upon ES-cell aggregation. Total RNA was isolated from undifferentiated ES cells (ES) and ES-cell aggregates (Day2, Day4). RNA was subjected to RT-PCR analysis and the PCR products were separated on a 2% agarose gel and visualized by ethidium-bromide staining. TTR, transthyretin. (C) The LIF/STAT3 pathway and BMP4 stimulation did not inhibit visceral endoderm differentiation upon ES-cell aggregation. Total RNA was isolated from STAT3ER ES cells, which were maintained as monolayer cells in the medium containing either LIF or 4HT (ES) and the cells, which were cultured as aggregates for 2 days in the same medium with or without BMP4 as indicated.

View larger version (58K): [in a new window]   Fig. 2. (A) Separation of GFP-positive visceral endoderm cells from ES-cell aggregates. Afp-GFP ES cells were aggregated for 4 days in the presence of LIF and subjected to FACS. The cells in R2 region were collected as GFP-positive cells and those in R3 as GFP-negative cells. The y axis indicates forwards scatter and the x axis indicates GFP expression (left). Total RNA was isolated from both GFP-positive and GFP-negative cells, and the RNA was subjected to RT-PCR analysis. PCR products were separated on a 2% agarose gel and visualized by ethidium-bromide staining (right). OPN, osteopontin; Ihh, Indian hedgehog; TTR, transthyretin; AFP, -fetoprotein. (B) Oct4 and osteopontin (OPN) expression were downregulated in the outer layer of ES-cell aggregates. After 4 days of aggregation of Afp-GFP ES cells in the presence of LIF, the aggregates were fixed with 3.7% formaldehyde and subjected to frozen section. The sections were incubated initially with anti-Oct4 antibody (1:100 dilution), anti-osteopontin antibody (1:500 dilution) or anti-HNF4 antibody (1:100 dilution), and subsequently with fluorescein-isothiocyanate-conjugated anti-mouse-IgG (1:200 dilution; for Oct4) or anti-goat-IgG (1:200 dilution; for osteopontin and HNF4) (left, red). Endogenous GFP expression derived from Afp-GFP ES cells was detected in the corresponding section (middle, green). Cells were co-stained with 4,6-diamidino-2-phenylindole to demonstrate nuclei (right, blue). Bars, 50 µm.

View larger version (75K): [in a new window]   Fig. 3. Downregulation of Nanog expression in ES cells upon aggregation. Whole-mount X-Gal staining of Nanog ßgeo ES cells that were aggregated for the indicated number of days in the presence of LIF (LIF+) or the absence of LIF (LIF–). Rosa26 ES cells were stained as a positive control for whole-mount X-gal staining (Rosa). The paraffin sections were counterstained with eosin Y (pink). Bars, 50 µm.

View larger version (72K): [in a new window]   Fig. 4. (A) TRE-Nanog ES cells were plated on gelatin-coated plate and cultivated for 3 days in the presence or absence of LIF and doxycycline (DOX, 1 µg ml–1). In the absence of DOX, Nanog transgene was overexpressed in the cells (tet-off system). Nanog-overexpressed ES cells maintained undifferentiated morphology regardless of the presence of LIF. Bars, 500 µm. (B) Morphology of TRE-Nanog ES-cell aggregates. TRE-Nanog ES cells were subjected to aggregation culture in the presence or absence of LIF and DOX. The phase-contrast images were captured under a light microscope after 4 days of aggregation. Bars, 200 µm. (C) Overexpression of Nanog inhibited visceral endoderm gene expression in the aggregates. Isolated total RNA from each condition in B was subjected to RT-PCR analysis. PCR products were separated on a 2% agarose gel and visualized by ethidium-bromide staining.

View larger version (21K): [in a new window]   Fig. 5. Primitive endoderm specification of mouse ES cells can be induced solely by cellular aggregation regardless of the presence of external factors (LIF and BMP4), which are sufficient to maintain self-renewal of ES cells in monolayer culture (top). ES-cell aggregation induces repression of the Nanog gene in the outer layer independent of the LIF/STAT3 and BMP4/Smads pathways (Cavaleri and Schöler, 2003; Ying et al., 2003). The repression of Nanog subsequently leads to ES cells differentiating into the primitive endoderm (bottom).

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