Additional file 3: Table S1. of Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

Primer sequences. (DOCX 14 kb

Additional file 1: Figure S1. of Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

ES cells differentiated in the absence of LIF give rise to PS-like cells in vitro. (A) Time-course qPCR expression analysis of ES- and PS/organiser-specific markers in wild type E14 ES cells cultured in the presence of serum and absence of LIF for the indicated amount of time. (B) Immunocytochemistry of PS/organiser marker expression in wild type E14 ES cells cultured in the presence of serum and absence of LIF for 4 days. (JPEG 943 kb

Additional file 2: Figure S2. of Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

The induction of PS-like cells in vitro correlates with Venus upregulation in Tps/tb-Oct-Nanog-Venus ES cells. (A) Flow cytometry analysis of Venus expression in Tps/tb-Oct-Nanog-Venus ES cells after 4 days of differentiation in the presence of serum/Dox and absence of LIF. (B) Fluorescence microscopy of Venus expression Dox-treated and untreated Tps/tb-Oct-Nanog-Venus ES cells after 4 days of differentiation in the presence of serum/Dox and absence of LIF. (JPEG 607 kb

Mouse phagocytic screen.

<p><b>(a)</b> Schematic diagram illustrating design of the CombiCult screen. Ten different cell culture media were tested in each of four stages of differentiation by split-pool passaging cells seeded onto beads in differentiation media spiked with tags, on day 1 (D1), D2, D5 and D7. On D14 beads were assayed to identify beads bearing phagocytes or neuroectodermal precursors (‘hits’). A total of 300,000 beads were used in the experiment to test 10,000 protocols so that on average each protocol was sampled by 30 beads. <b>(b)</b> Fluorescence micrographs showing hits bearing (i) red fluorescent phagocytic cells or (ii) GFP-positive neuroectoderm cells amongst negative beads. Scale bars  =  100 µm. <b>(c)</b> Cummulative large particle flow sorter dot plots, showing parameters used to sort hits. (i) Gating of monomeric beads (gate 1) from higher order agreggates. (ii) Sorting of monomeric beads with high red (gate 2) and green (gate 3) fluorescent signal. <b>(d)</b> Micrograph images of sorted hits: (i) bead 31 (phagocyte), (ii) bead 131 (phagocyte), (iii) bead 76 (neuroectoderm), (iv) bead 34 (neuroectoderm) and (v) bead 34 (phagocyte and neuroectoderm). Scale bars  =  100 µm. <b>(e)</b> Deconvolution of cell culture history of bead 31 (pictured in d(i) above; one of a triple hit from the phagocyte screen) by FACS analysis of bound tags. A set of 30 unique tags distinguishable by size (small, medium, large) and fluorescence intensity (ten levels for each size set) was used to spike cell culture media in the first three stages of differentiation. The figure shows magenta peaks corresponding to quantitation of a reference sample of the 30 tags used to calibrate the flow cytometer (histograms and tag numbers shown in magenta) in order to set gates. Quantitation of tags released from bead 31 is superimposed in black, showing detection of tags SR10, MR1 and LR8, corresponding to media 1.10, 2.1 and 3.8. <b>(f)</b> Schematic diagram illustrating an overlay of all protocols deconvoluted from phagocyte hits. The height of boxes representing each cell culture medium is proportional to the number of hits generated by that medium (written at the bottom of each box). The opacity of the linkage lines is proportional to the number of hits generated by specific media combinations - the darkest line corresponds to 21 hits. <b>(g)</b> Hierarchical clustering analysis of 92 unique protocols derived from the phagocyte screen, showing protocol clusters A and B. Each node at the bottom of the dendrogram (leaf node) corresponds to a hit bead. The associated protocol is denoted by the column of four colours directly below the node, specifying the media sampled in splits 1–4. The legend at the bottom of the figure specifies the colour used to denote the cell culture media in each split. <b>(h)</b> Similarity matrix comprising a pair-wise comparison of all protocols. Each column and each row corresponds to a protocol. The brightness of each cell in the matrix is proportional to the number of identical cell culture media shared by the two protocols. The brightest cell corresponds to identical protocols, while a black cell corresponds to two protocols with no common media. The diagonal row of cells (from the top left to bottom right) corresponds to protocols being compared to themselves. Protocol families with high internal homology appear as bright red squares, e.g. Clusters A and B marked on the diagram, with Cluster A protocols being more conserved. <b>(i)</b> Efficiency of phagocyte and/or neuroectoderm cell generation from mES cells on PTC5000 beads using a selection of protocols discovered by CombiCult. Coloured bars represent the number of fluorescent colonies per square centimeter (64 fields of view). Red bars correspond to phagocytic colonies, green bars correspond to GFP-expressing (neuroectodermal) colonies, orange bars correspond to beads with both phagocytic and neuroectodermal colonies, black bars correspond to negative protocols. Hit bead numbers and protocols listed below the histograms are coloured to show whether they were derived from phagocyte- (red), neuroectoderm- (green) or phagocyte/neuroectoderm- (orange) bearing hits, whereas black corresponds to two negative protocols – i.e. protocols which did not return any hits, these represent a range of 12 protocols tested, all but one (shown) had 0 colonies. Each bar represents the average of 2 wells (each one containing 4000 beads) and the graph is representative of 3 separate experiments. Protocols with a statistically significant difference (p>0.05) to the negative control are marked with an asterisk (*).</p

Mouse and human TH positive neuronal screens.

<p><b>(a)</b> Fluorescence micrographs showing hits bearing (i) mouse and (ii) human TH+ cells amongst negative beads. Scale bars  =  100 µm. <b>(b)</b> Schematic diagram illustrating an overlay of all protocols deconvoluted from screens for TH+ neurons generated using (i) mES and (ii) hES cells. Histograms representing each cell culture medium are proportional to the number of hits generated (written at the bottom of each bar). The opacity of the linkage lines is proportional to the number of hits generated by specific media combinations - the darkest line in each plot corresponds to (i) 21 and (ii) 12 beads. <b>(c)</b> Microgrographs showing immunofluorescence images of differentiated ES cells in monolayer cultures. Differentiation protocols shown for mES cells are <b>(i)</b> 5929 and <b>(ii)</b> 4671, stained for TH (red), β-III tubulin (green) and DAPI (blue). Differentiation protocol shown for hES cells is 8976 (iii and iv) stained for TH (red), β-III tubulin (green) and DAPI (blue); except in (iv) where FOXA2 is stained blue. Scale bars  =  100 µm. Representative photographs from a field of view are shown. Monolayer differentiations were repeated at least twice for the protocols shown. <b>(d)</b> Efficiency of (i) mES (ii) hES differentiation to TH+ neurons on PTC5000 beads measured by COPAS. Experiments were performed in duplicate wells (containing 4000 beads/well) and the proportion of beads bearing TH+ neurons in each well is plotted separately (light and dark histograms) and ranked according to the average proportion of positive beads. The bead number(s) and the corresponding deconvoluted protocol are listed below the histogram.</p

Analysis of Cluster A protocol derived phagocytes following further maturation in MethoCult or on OP9 cells supplemented with IL7.

<p><b>(a)</b> Dot plots illustrating flow cytometry analysis of cells obtained after maturation in IL7 supplemented MethoCult for 2 weeks, showing the appearance of cells co-expressing B220/CD43 and B220/CD19; <b>(b)</b> Histogram showing the proportions of different cell populations present in MethoCult or OP9 derived cultures, measured by flow cytometry analysis. Blue bars represent cells taken off beads on D15 (prior to maturation), red bars represent cell isolated on D15 and cultured for a further 2 weeks on OP9 feeder layers and green bars represent cells isolated on D15 and cultured in MethoCult for 2 weeks. The experiment was carried out twice and in duplicate the bars show the average percentage of cells, the error bars show the standard deviation. There is a statistically significant difference between the cells taken from the beads at D15 and those cultured on MethoCult for 2 weeks for markers CD43 and CD19 *p<0.05.</p