Distribution of cell cluster sizes during segregation.

<p>Clusters of EphB2/GFP expressing cells from the time course experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111803#pone-0111803-g001" target="_blank">Figure 1</a> were grouped according to their footprint into three groups as indicated; bar graphs illustrate frequency of clusters in each bin at indicated time points, (red bars), experimental microscopy data; (blue bars), simulated data. Error bars represent the upper and lower bounds, below which 75% and 25% of the data points are included. *Multivariate-ANOVA, p&gt;0.5.</p

A Mathematical Model for Eph/Ephrin-Directed Segregation of Intermingled Cells

<div><p>Eph receptors, the largest family of receptor tyrosine kinases, control cell-cell adhesion/de-adhesion, cell morphology and cell positioning through interaction with cell surface ephrin ligands. Bi-directional signalling from the Eph and ephrin complexes on interacting cells have a significant role in controlling normal tissue development and oncogenic tissue patterning. Eph-mediated tissue patterning is based on the fine-tuned balance of adhesion and de-adhesion reactions between distinct Eph- and ephrin-expressing cell populations, and adhesion within like populations (expressing either Eph or ephrin). Here we develop a stochastic, Lagrangian model that is based on Eph/ephrin biology: incorporating independent Brownian motion to describe cell movement and a deterministic term (the drift term) to represent repulsive and adhesive interactions between neighbouring cells. Comparison between the experimental and computer simulated Eph/ephrin cell patterning events shows that the model recapitulates the dynamics of cell-cell segregation and cell cluster formation. Moreover, by modulating the term for Eph/ephrin-mediated repulsion, the model can be tuned to match the actual behaviour of cells with different levels of Eph expression or activity. Together the results of our experiments and modelling suggest that the complexity of Eph/ephrin signalling mechanisms that control cell-cell interactions can be described well by a mathematical model with a single term balancing adhesion and de-adhesion between interacting cells. This model allows reliable prediction of Eph/ephrin-dependent control of cell patterning behaviour.</p></div

Increased cell-cell adhesion within one cell population is required for the formation of tightly packed cell clusters.

<p><b>A</b>) Simulation of cell-cell segregation using the same adhesion term in both cell types (<i>A_eph</i>  =  <i>A_ephrin</i>  = 100, left panel) vs. increased adhesion only in the green cell population (<i>A_eph</i>  = 110, <i>A_ephrin</i>  = 100, right panel). Both simulations started with the same number of Eph (green) and ephrin (black) expressing cells. In the “Equal adhesion” case, an ‘Islands-in-a-sea’ pattern is less apparent. <b>B</b>) Representative images from segregation assays of unlabelled ephrin-B1 cells co-cultured with Cell Tracker-green labelled (green staining) EphB2 cells, without (left) or with E-cadherin-cherry expression (red staining, right); scale bar, 75 µm. <b>C</b>) Quantitation of cell densities in the cell clusters shown in B (n = 10). <b>D</b>) Western blot analysis of lysates from parental and E-cadherin-cherry-transduced cells, using the indicated antibodies.</p

Eph expression level and signalling capacity regulate cell segregation: comparison of experimental versus modeling outcomes.

<p><b>A</b>) Increased Eph signalling capacity results in enhanced cell-cell segregation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111803#pone.0111803-Janes3" target="_blank">[44]</a>: HEK293 cells expressing cytoplasmic deleted (signalling-inactive), wild type EphB2 or co-expressing EphB2 and EphA3 were co-cultured with cells expressing low or high levels of ephrin-B1, as indicated. EphB2 cells were stained with Cell-Tracker green for ease of visualisation, images taken after 48 h co-culture when cell-cell segregation was regarded as complete. <b>B</b>) Simulation of the same experimental conditions, using parameter values of: <i>A_eph  =  A_ephrin  = </i>100, <i>R_eph  =  R_ephrin  = </i>250, <i>a</i> = 7.5, <i>r</i> = 5.8 and , apart from the right-most panel, where <i>R_eph  = 220.</i> The initial ratio of Eph: ephrin cells in all cases is 1∶1. <b>C</b>) Functions of the force potential, u(d) between two cells at distance d&gt;0, for the simulations illustrated in B. Unbroken black line,u_r,is the potential of the repulsion force at <i>R_eph  = 250</i>; Unbroken red line,u′_r,is the potential of the repulsion force at <i>R_eph  = 220</i>; Broken black line, u_a, is the potential of the attraction force; Unbroken blue line,u_r − u_a,is the potential of the total force between same type cells <i>R_eph  = 250</i>; Broken blue line,u′_r − u_a,is the potential of the total force between same type cells when <i>R_eph  = 220</i>; Broken green line, u_r − (1−C)u_a, is the potential of the total force between cells of different types; <b>D</b>) Statistical analysis of cluster characteristics from a minimum of 5 independent data sets: Comparison of the cellular densities that were observed experimentally by microscopy (green bars), or by simulation under the conditions detailed in panels C (blue bars; except for <i>C</i> = 1, <i>R_eph</i>  = 250, red bar). For microscopic images the number of cells in a cluster was estimated from the total fluoresence intensity of the cluster, divided by the average fluorescence intensity of a single cell as detailed previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111803#pone.0111803-Janes3" target="_blank">[44]</a>. Error bars represent the upper and lower bounds, below which 75% and 25% of the data points are included.</p

Time-lapse imaging and simulation of Eph/ephrin-driven cell-cell segregation.

<p><b>A</b>) Representative time-lapse microscopic images (taken every 20 minutes for indicated times) from co-cultured EphB2/GFP (green) and ephrin-B1 (unstained) expressing HEK293T cells. Bright-field micrographs (top panels), green-fluorescent images (middle panels) and merged images (bottom panels) are shown, scale bars, 100 µm). <b>B</b>) Simulation of the same experiment; the corresponding time points are shown, ephrin-B1 and EphB2/GFP-expressing cells are represented as black and green circles, respectively.</p

Karyotype analysis of ES cells and live offspring derived from vitrified oocytes.

<p>A) Karyotype analysis performed on ViO-ES9 cells revealed a number of abnormalities. The male cell line has a chromosome count of 46. B) Karyotype analysis performed on the mice that were generated from vitrified oocytes revealed normal karyotypes, a representative karyotype for one of the male mice shows a normal 40 XY chromosome count with no abnormalities.</p

Histology and Hematoxylin and Eosin staining of teratoma tissue from ViO-ES9 cells showing differentiation into tissues indicative of the three germ layers (A) including secretory epithelium (i), articular cartilage (ii) and keratinized epithelium (iii).

<p>Immunoflorescent analysis of differentiation into all three germ layers (B): AFP (i); GATA-4 (ii); and NESTIN (iii). Secondary antibodies were labelled with Alexa Fluro® 488 (green) except for GATA-4 which was labelled with Alexa Fluro® 594 (red). Nuclei are stained with DAPI (blue). RT-PCR for Flk-1, VE-Cadherin, PECAM, Vimentin and Nestin (C).</p

Survival, two-cell and blastocyst rates for vitrified mouse oocytes following IVF.

*<p>Fisher's test significantly lower than other treatments (P&lt;0.05);</p>**<p>Fisher's test significantly higher than other treatments within the same column (P&lt;0.001). IVF Data collected from 3 replicates.</p

Survival, vitrified two-cell embryo and live offspring rates from either fresh or vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05).</p

Following in vitro fertilization and culture, both fresh (A1) and vitrified oocytes in 0.1 M trehalose (B1) were capable of developing to the two-cell (A2; B2), four-cell (A3; B3) and blastocyst (A4; B4) stages, respectively.

<p>Furthermore, blastocysts were able to hatch (A5; B5) without chemical or mechanical assistance and some were used to derive embryonic stem cells.</p