Dissociation, and not specific actions of Trypsin-EDTA, acts to suppress neurectoderm formation.

<p>(A). EPL cells, formed in aggregates in MEDII, were dissociated with Dispase, Dissociation buffer or Trypsin-EDTA, reaggregated and maintained for a further 4 days before being seeded individually into 48 tissue culture wells and allowed to differentiate. Aggregates were scored for the presence of beating cardiocytes and neural extensions and the peak score for each represented. Outcomes are compared to outcomes from EPL cells that were maintained in aggregates. n = 3 independent experiments. Error bars represent sem. (a) denotes a decrease compared to MEDII⁻/Dissociation⁻ where p<0.01. (B). EPL cells, formed in aggregates in MEDII, were either maintained in medium supplemented with 50% MEDII (control), cultured in unsupplemented medium (no addition) or dissociated with Dispase, Dissociation buffer or Trypsin-EDTA, reaggregated and maintained in suspension culture in unsupplemented medium for a further 6 days. Aggregates were collected and extracted RNA was analyzed for the presence of <i>Sox1</i>, and <i>GAPDH</i> by real-time PCR. n = 3 independent experiments. Error bars represent sem.</p

Characterisation of the endoderm populations seen in EBs and EPLEBs by comparison with the endoderm populations of the 6.5 and 7.5 d.p.c. embryo.

<p><b>A.</b> (<b>i</b>) A 1 μm transverse section across the distal tip of a 6.5 d.p.c mouse embryo showing the distinctive morphology of the visceral endoderm (ve) surrounding the inner pluripotent cell core (ep). Size bar represents 40 μm. (<b>ii</b>) 1 μm transverse section across the extraembryonic region of a 7.5 d.p.c embryo showing the visceral endoderm (ve) surrounding the extraembryonic mesoderm (eem) and extraembryonic ectoderm (eee). Parietal endoderm (pa) is indicated by an arrow. (<b>iii</b>) Transmission electron micrographs (TEM) of visceral endoderm from the extraembryonic region of a 7.5 d.p.c. embryo, showing the typical cuboidal cell morphology with large apical vacuoles and dense microvilli on the apical surface, which can be seen at the top of the figure. Size bar represents 10 μm. <b>B.</b> (<b>i</b>) Longitudinal section of a 7.5 d.p.c. late-streak stage embryo. Size bar represents 200 μm, posterior to the right, parietal endoderm (pa) indicated by an arrow. (<b>ii</b>) Detail of (i), showing the trilaminar structure of the egg cylinder, with an outer layer of definitive endoderm (de), middle layer of mesoderm (m) and inner layer of ectoderm (ec) Size bar represents 40 μm. (<b>iii, iv</b>) TEM of definitive endoderm, showing an outer, squamous, cell layer of endoderm with a sparse decoration of microvilli on the apical surface. Inset shows the surface of the cells at a higher magnification. Size bars represent 10 μm (inset 2 μm). (<b>v, vi</b>) TEM of parietal endoderm, showing a dispersed, squamous, cell population in close contact with Reichart's membrane (rm), indicated by an arrow. The surface of the parietal endoderm is devoid of microvilli. Inset shows the surface of the cells at a higher magnification. Size bars represent 10 μm (inset 2 μm). <b>C.</b> (<b>i, ii</b>) TEM of the surface populations of cells observed on a day 5 EB. Cells appear reminiscent of the visceral (<b>i</b>) and parietal (<b>ii</b>) endoderm populations of the embryo. Size bars represent 10 μm. <b>D</b>. (<b>i</b>) Toluidine blue-stained 1 μm section of an EPLEB at day 5 of differentiation. Size bar represents 200 μm. (<b>ii</b>) Detail of (<b>i</b>) showing the squamous outer cell layer. (<b>iii, iv</b>) TEM of the outer layer of cells of EPLEB on day 5 of differentiation, showing an outer, squamous, cell layer with sparse microvilli on the apical surface, reminiscent of the morphology of the embryonic definitive endoderm. Size bars represent 10 μm (<b>iii</b>) and 5 μm (<b>iv</b>).</p

Localisation of definitive endoderm and mesoderm marker expression in EPLEBs.

<p><b>A.</b> RNA isolated from EBs and EPLEBs on days 1–5 of differentiation was analysed by RT-PCR for the expression of <i>Fgf5, Brachyury, Trh, Sox17, Cdx2</i> and <i>Hand1. Gapdh</i> expression was used a loading control. Expression in undifferentiated ES cells (ES) and EPL cells (EPL) is shown for comparison. -RT (control, no reverse transcriptase) and a no template control (NTC) were included. n = 3, a representative result is shown. <b>B.</b> Wholemount <i>in situ</i> hybridisation of EPLEBs on day 5 of differentiation with a DIG-labelled probe complimentary to <i>Sox17</i> (<b>i</b>) or <i>Trh</i> (<b>ii, iii, iv</b>). Representative aggregates are shown sectioned into 10 µm slices. Size bars represent 50 µm (i, iii, iv) or 500 µm (ii). <b>C.</b> EBs, EBMs, EPLEBs and EPLEBs cultured in the presence of 30 ng/mL Activin A were analysed by flow cytometry for the presence of CXCR4 positive cells. n = 3. <b>D.</b> Low magnification image of EPLEBs (<b>i</b>) and EBs (<b>ii</b>) on day 5 and day 7 respectively, stained for the uptake of horse radish peroxidase (HRP). Open arrowheads indicate areas of staining. Representative EPLEBs with (<b>iii</b>) or without (<b>iv</b>) a foci of HRP activity are shown sectioned and counterstained with haematoxylin to illustrate cell morphology. Open arrowheads indicate areas of staining, closed arrowheads indicate the non-staining endoderm layer on the outside of the EPLEBs. Size bars represent 500 µm (i, ii,) or 50 µm (iii, iv). <b>E.</b> Wholemount <i>in situ</i> hybridisation of EPLEBs on day 5 of differentiation using a DIG-labelled probe complimentary to <i>Cdx2</i> (<b>i</b>) or <i>Hand1</i> (<b>ii, iii</b>). Representative aggregates are shown sectioned into 10 µm slices. Size bars represent 50 µm.</p

Primers used for RT-PCR and qRT-PCR analysis of gene expression.

<p>Primers used for RT-PCR and qRT-PCR analysis of gene expression.</p

The MEDII-associated mesoderm suppressing activity is a discrete moiety within the medium.

<p>(A). E was fractionated over a Superdex® peptide column. <i>Mixl1:GFP</i> ES cells were cultured as aggregates in MEDII for 3 days to form EPL cells. Aggregates were transferred to unsupplemented medium or medium supplemented with 50% MEDII, 50% E or a 50% MEDII equivalent volume of fractions 1–8 and cultured for a further 2 days before reduction to a single cell suspension. The proportion of GFP⁺ cells present was determined by flow cytometry. (B). EPL cells were prepared as for (A) and transferred to unsupplemented medium or medium supplemented with 50% MEDII, 50% E or fraction 4. Aggregates were maintained in culture for a further 4 days before being seeded individually into 48 tissue culture wells and allowed to differentiate. Aggregates were scored for the presence of visible red blood cells, beating cardiocytes and neural extensions and the peak score for each represented. (C). EPL cell aggregates were formed from <i>Mixl1:GFP</i> ES cells for 3 days in MEDII supplemented medium before being transferred to unsupplemented medium or medium supplemented with 50% MEDII or 200 µM l-proline. Aggregates were maintained in culture for a further 4 days before being seeded individually into 48 tissue culture wells and allowed to differentiate. Aggregates were scored for the presence of visible red blood cells, beating cardiocytes and neural extensions and the peak score for each represented. n = 3 independent experiments. Error bars represent sem.</p

Posterior primitive ectoderm and primitive streak markers are expressed in differentiating EPL cells.

<p><b>A.</b> q PCR analysis of RNA isolated from ES cells, EPL cells cultured for 2 days in MEDII (EPL) and EPLEBs formed from EPL cells and cultured for 4 days for the expression of <i>Brachyury, Mixl1</i>, <i>Eomesodermin</i>, and <i>Nanog</i>. Gene expression has been normalised to <i>actin</i> and is expressed relative to EPLEB² (<i>Brachyury, Mixl1</i>, <i>Eomesodermin</i>) or ES cells (<i>Nanog</i>). n = 3. Error bars represent standard error of the mean. <b>B.</b> RNA was extracted from EPLEBs and EBMs on day 12 and analysed by RT-PCR for the expression of a number of genes characteristic of definitive endoderm cell lineages. Reactions in which reverse transcriptase has been omitted (no RT) were included as controls.</p

Disruption of cell:cell contact promotes the establishment of the mesoderm lineage while MEDII has an opposing activity that promotes formation of neurectoderm.

<p>EPL cells were dissociated and reaggregated in unsupplemented tissue culture medium (○) or medium supplemented with 50% MEDII (▴). Alternatively, EPL cells were maintained in aggregates and transferred to unsupplemented tissue culture medium (□) or medium supplemented with 50% MEDII (♦). Aggregates were collected daily to day 7 and analyzed by real-time PCR for the expression of markers of primitive streak, EMT and differentiated lineages. Gene expression was normalized to <i>β-actin</i> (here) and <i>GAPDH</i> (data not shown); the relative gene expression seen with either housekeeping gene was comparable. n = 4 independent experiments, error bars represent sem. MEDII⁺/Dis⁻EBs were only included where changes in gene expression were detected.</p

The MEDII-associated mesoderm suppressing activity is found within the small molecular weight components of the medium.

<p>(A). <i>Mixl1:GFP</i> ES cells were cultured as aggregates in MEDII for 3 days to form EPL cells. Aggregates were transferred to unsupplemented medium or medium supplemented with 50% MEDII, 50% E or R and cultured for a further 2 days before reduction to a single cell suspension. The proportion of GFP⁺ cells present was determined by flow cytometry. n = 7 independent experiments (MEDII⁻Dis⁻R⁺ n = 3). Error bars represent sem. (a) represents a decrease when compared with MEDII-Dis- where p<0.001. (B). EPL cells were prepared as for (A) and transferred to unsupplemented medium or medium supplemented with 50% MEDII or 50% E. Aggregates were maintained in culture for a further 4 days before being seeded individually into 48 tissue culture wells and allowed to differentiate. Aggregates were scored for the presence of visible red blood cells, beating cardiocytes and neural extensions and the peak score for each represented. n = 3 independent experiments and includes data generated from both <i>Mixl1:GFP</i> and D3 ES cell lines. Error bars represent sem.</p

Cell dissociation and MEDII both impact on lineage choice during EPL cell differentiation.

<p>(A). EPL cells were formed in aggregates in MEDII for three days before being dissociated and reaggregated in unsupplemented tissue culture medium or medium supplemented with 50% MEDII (MEDII+). Alternatively, EPL cells were maintained in aggregates and transferred to unsupplemented tissue culture medium or medium supplemented with 50% MEDII. Aggregates were maintained for a further 4 days (to day 7) in suspension culture before being seeded individually into 48 tissue culture wells in unsupplemented medium and allowed to differentiate. Aggregates were scored for the presence of visible red blood cells, beating cardiocytes and neural extensions and the peak score for each represented. n≥3 independent experiments. Error bars represent standard error of the mean (sem). (a) denotes an increase compared to MEDII⁺/Dissociation⁻ where p<0.01. (b) denotes an increase compared to MEDII⁻/Dissociation⁺ where p<0.05. (B–D). Aggregates cultured as for A were collected and fixed on day 7 before paraffin embedding and sectioning. Sections were stained with haemotoxylin/eosin and viewed with a Ziess Axiophot microscope. (B). MEDII⁺/Dis⁻EB. (C). MEDII⁺/Dis⁺EB. (D). MEDII⁻/Dis⁻EB. (E). MEDII⁻/Dis⁺EB. Scale bar represents 50 µm. The white arrowhead in B indicates an area of cell necrosis. The black arrowheads in C and D denote areas of cell morphology similar to that found in E.</p

Formation of endoderm on EBs, EPLEBs, EBMs.

<p><b>A–F.</b> Scanning electron micrographs of EBs (A, D), EBMs (B, E) and EPLEBs (C, F) on days 2.5 (A–C) and 5 (D–F). Arrowheads mark the boundary of the prospective patch of primitive endoderm on EBs on day 2.5 (A) and the prospective endoderm population forming on the surface of EPLEBs at 2.5 days of differentiation (C). The dotted line on D demarcates the boundary of two distinct surface morphologies. Size bars represent 50 μm.</p