The origin and properties of naïve and primed pluriopotent stem cells.

<p>The origin and properties of naïve and primed pluriopotent stem cells.</p

Protocol for the generation of oligodendrocytes from NS cells.

<p>NS cells propagated in NS-A medium plus N2 in the presence of EGF and FGF2 (A) were cultured in DMEM/F12 plus N2 in the presence of FGF2, PDGF and forskolin for 4 days on polyornithine/laminin coated plastic (B) before they were induced to differentiate by growth factor withdrawal in the presence of 3,3,5-tri-iodothyronine hormone (T3) and ascorbic acid (AA) (C,D). After four days, immunostaining for the O4 antigen revealed differentiation into oligodendrocytes (C). The differentiated cultures also contained GFAP-positive astrocytes and ß-III tubulin/TUJ1-positive neurons (C,D), demonstrating the tripotential differentiation capacity of these cells.</p

Tripotential differentiation of NS cells in vitro and generation of myelinating oligodendrocytes in vivo.

<p>(A–E) Quantitative marker expression and representative immunofluorescence images. The specific culture conditions used to differentiate NS cells resulted in the generation of oligodendrocytes (∼20%) positive for O4 (B), Rip (D) and PLP (E), GFAP-expressing astrocytes (∼40%; C–D) and neurons positive for ß-III tubulin/TUJ1 (∼10%; C). (F–H) NS cells cultured in N2 medium and proliferated for 4 days in the presence of FGF2, PDGF and forskolin were transplanted into the brain of 2- to 3-day-old myelin-deficient rats. Two weeks after transplantation, the engrafted cells had formed PLP-positive myelin internodes. Shown are representative pictures from septum (F) and corpus callosum (G–H). Scale bars B–D, 100 µm; F–H, 20 µm.</p

Dependency of stable Nanog states on Nanog autoregulation and noise.

<p>(A) The bifurcation diagram indicates the existence of Nanog states depending on the autoregulatory transcription rate s₄. The lower solid line shows the existence of Nanog-low (NL) states and the upper solid line shows the existence of Nanog-high (NH) states. Within the bistable region (shaded in grey) coexisting stable states are separated by unstable states (dashed line). (B) Simulating a cell population, the heat map illustrates the proportion of NL cells depending on the transcription rate s₄ and on the transcriptional noise σ_N at time point t = 4320min (i.e. 3 days of in silico culture). For any value of the background noise, an increase in the transcription rate s₄ reduces the proportion of NL cells.</p

Mechanistic explanation and simulation results for the 2i scenario.

<p>(A) Model scheme. In the 2i scenario Erk signalling is efficiently blocked (i.e. p = 0). (B) Bifurcation diagram. Keeping all regulatory rates and the transcriptional noise constant, the removal of the Erk repression in 2i shifts the Nanog level into a monostable region. (C) Single cell trajectories. The diagram shows simulated trajectories of Oct4-Sox2 (grey), Nanog (green) and Rex1 (blue) concentrations for the 2i scenario. (D) Simulated TF distributions of Nanog (green) and Oct4-Sox2 (grey) within mESC populations at time point t = 4320 (i.e. 3 days of in silico culture). The homogenous high expression levels under 2i result in unimodal TF distributions. (E) Comparison of the simulation result for Rex1 (blue line) with experimental data (grey histogram) obtained from flow cytometry analysis of Rex1GFPd2 mESCs maintained in 2i. The parameter set for these simulations is given in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092496#pone.0092496.s001" target="_blank">File S1</a>.</p

<i>piggyBac</i> mutagenesis and monolayer differentiation screen.

<p><b>A</b>. Binary <i>piggyBac</i> gene trap system composed of gene trap vector, <i>pGG85</i>, and transposase expressing helper plasmid, p<i>CAGG-PBase</i>. <b>B</b>. G418 resistant colonies produced by co-electroporation of 1 µg of <i>pGG85</i> and 3 µg of helper plasmid. <b>C</b>. Splinkerette PCR amplified genome junction flanking PB insertions indicating the number of PB inserts in each clone. <b>D</b>. Schematic representation of monolayer differentiation screen.</p

<i>Tcf3</i> deficiency suppresses serum-induced differentiation.

<p><b>A</b>. Parental NN97-5 cells differentiate after 4 days in serum without LIF while <i>Tcf3</i> gene trap mutant P1-2 cells remain undifferentiated and retain uniform Oct4 expression in serum. <b>B</b>. Flow cytometry analysis for Rex1-EGFP positive cells during monolayer differentiation in serum. P1-2, Tcf3 gene trap mutant; CreA12, heterozygous Tcf3 Cre-revertant; CreD10, homozygous Tcf3 Cre-revertant. Graph shown is a representative of two independent experiments. <b>C</b>. <i>Tcf3</i> mutant (P1-2) and the <i>Tcf3</i> reverted cells were plated at single cell density in serum with or without LIF for colony forming assay. Colonies were stained after 9 days for alkaline phosphatase (AP) activity and colony numbers were quantified manually. Undifferentiated colonies are showing in red in figure and partially differentiated showing in green and differentiated showing in yellow. <b>D</b>. Images show typical AP positive morphologically undifferentiated ES cell colonies generated by P1-2 cells in serum with or without LIF. The experiment has been repeated once.</p

<i>Tcf3</i> mutation has subtle molecular consequences.

<p><b>A</b>. Relative gene expression analysis by qRT-PCR in <i>Tcf3</i> mutant (P1-2) compared to Cre-reverted (CreD10)(Blue column) and wild type (NN97-5)(Red column). <b>B</b>. TOPFlash assay of Tcf-mediated transcriptional activation. None, N2B27 alone; Wnt, Wnt3A; Wnt+LIF, Wnt3A plus LIF. <b>C</b>. qRT-PCR analyses of Wnt target gene expression. S+L, Serum plus LIF. <b>D</b>. Immunoblotting analysis of Nanog and Oct4 protein expression in serum and LIF. <b>E</b>. NN97-5 and P1-2 cells cultured in serum and LIF immunostained for Oct4 and Nanog. Images show typical heterogeneous Nanog protein expression in NN97-5 cells compared to more uniform staining in P1-2 cells. <b>F</b>. Mean nuclear staining intensity of Oct4 and Nanog in individual cell was quantified using Cell profiler software and presented as a scatter plot using Microsoft Excel. 1600 cells were scored for NN97-5 and 2172 cells for P1-2. The experiment has been repeated three times.</p

Monolayer neural differentiation of individual gene trap clones.

<p>Monolayer neural differentiation of twenty clones from gene trap mutation pool 1 is presented. Clones with <i>Tcf3</i> mutation are labelled with “*”. Two clones from mutant pool 2 were also included as a control for monolayer differentiation assay. “D” represents clones showing extensive neural differentiation. “Non-D” represents cells showing predominantly undifferentiated ES cell morphology. P1-8 cells differentiated to flat non-neural cells.</p

The differentiation process of mESCs.

<p>(A) Experimental and simulated TF kinetics. mRNA levels of Nanog (green circles) and Rex1 (blue triangles) are measured after 2i withdrawal and used to adapt the velocity of the differentiation process in terms of the proposed network model. Solid lines depict the respective simulation results of average Nanog and Rex1 levels. (B) – (C) Single cell trajectories. The Nanog level (green) initially switches from the NH into the NL state. Rex1 expression levels gradually decrease (blue). The Oct4-Sox2 level (black) is suddenly downregulated if the intracellular activity Y_in becomes sufficiently strong. (D) Flow cytometry measurements of Rex1GFPd2 mESCs show intercellular differences in the differentiation dynamics of Rex1 (lower panel). The respective model results are consistent with the experimental findings (upper panel). The parameter set for these simulations is given in Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092496#pone.0092496.s001" target="_blank">File S1</a>.</p