Generation and characterisation of hNMPs.

<p>(A) Scheme describing the culture conditions employed for neural differentiation of hES cells treated for 72 h either with FGF/CHIR or subjected to dual SMAD inhibition (LDN, LDN193189; SB43, SB431542). (B) BRACHYURY/SOX2 immunocytochemistry in undifferentiated and FGF/CHIR-treated (48 h) hES cells. Corresponding graphs depict image analysis of BRACHYURY and SOX2 expression in the indicated culture conditions. Numbers: percentages of cells in each quadrant. (C) qPCR analysis for indicated markers in hES cells treated with FGF/CHIR for 72 h (D3) or 96 h (D4). Error bars = s.d. (n = 2). Results are represented as log₁₀ ratio of expression versus untreated hES cells. (D) qPCR analysis for indicated differentiation markers in hES cells differentiated in N2B27 following either an NM progenitor induction- (N_P) or a dual SMAD inhibition-intermediate step (N_A). Error bars = s.d. (n = 2). Anterior, anterior neural markers; PXM, paraxial mesoderm; n/d, not determined. (E) Immunocytochemistry for SOX2/HOXC8 in N_A and N_P culture conditions indicated in the scheme (A). (F) Quantitation of the coexpression of Hoxc8 with Sox2 in N_A and N_P conditions. All data used to generate the plots can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s013" target="_blank">Data S5</a>.</p

Transient Wnt and FGF signalling induce dual fated neuromesodermal progenitors.

<p>(A) Schematic of differentiation protocols used to generate mesoderm and neural cells from a common NM progenitor population. (B) mRNA-seq expression values of <i>Sox2</i>, <i>Brachyury</i>, <i>Tbx6</i> and <i>Cdx2</i> following exposure to bFGF alone or bFGF/CHIR for 12 h (D2.5) and 24 h (D3). Activation of Wnt signalling with CHIR upregulated Brachyury within 12 h. Expression of <i>Tbx6</i> and <i>Cdx2</i> was also upregulated in NMPs by D3, whereas <i>Sox2</i> transcript levels were decreased. (C) Immunostaining of cells treated with FGF/Wnt revealed the coexpression of Brachyury with Sox2 (NMPs). In the absence of Wnt, NPCs express Sox2 but the expression of Brachyury is only evident in a very small proportion of cells. (D) mRNA expression values of neural (<i>Sox1</i>, <i>Sox2</i>, <i>Sox3</i>) and mesodermal progenitors markers (<i>Tbx6</i>, <i>Bra</i>, <i>Msgn1</i>) in posterior neural (N_P) and mesodermal cells (Meso) at D5 show the generation of distinct populations depending on treatment after D3. Removal of Wnt at D3 results in the generation of neural cells expressing Sox1–3 whereas continued Wnt exposure induces expression of Tbx6, Brachyury and Msgn1, characteristic of paraxial mesodermal. (E) Immunostaining indicates that continued Wnt exposure generates paraxial mesodermal progenitors that express Tbx6 at D5 and Desmin and MyoD at D8. (F) Sketch of a chick embryo (HH8–9) showing the injection site (IS) of NMP or N_A cells. (G) NMP cells were labelled with DiI and transplanted in the CLE region. After 24 h the cells had incorporated into both the neural tube and somites. Whole-mount and transverse sections of HH17 chick embryos show the incorporation (asterisks) in the neural tube (H) and somites (I). (J) Table summarizing the number of chick embryos that were injected at stage HH8–9 and had engrafted cells in the neural tube, the somites or both 24 h later. Injection of N_A cells resulted in incorporation only in the neural tube (K, L). All data used to generate the plots of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio-1001937-g002" target="_blank">Figure 2</a> can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s010" target="_blank">Data S2</a>.</p

Brachyury is necessary for mesoderm formation but not posterior neural identity.

<p>(A) Schematic of the conditions used for mesoderm differentiation. (B) qRT-PCR analysis of the expression of <i>Tbx6</i>, <i>Cdx2</i> and <i>Hoxb1</i> relative to b-actin at D3 of differentiation in wild-type (wt) and Brachyury null cells (Bra^−/−) with and without CHIR. In wild-type cells activation of Wnt signalling induces the expression of these three genes. In the absence of Brachyury while <i>Cdx2</i> and <i>Hoxb1</i> continue to be induced by Wnt signalling, Tbx6 induction is lost. (C) qRT-PCR analysis of the expression of mesodermal, neural and posterior marker genes at D5 of differentiation in wt and Bra^−/− ESCs exposed to CHIR from D2–D5 (Meso conditions). Posterior Hox genes <i>Hoxc8</i> and <i>Hoxc9</i> are induced in both wt and Brachyury null cells. However, in contrast to wild-type cells neural markers <i>Sox1</i> and <i>Sox2</i> are expressed only in Bra^−/− cells exposed to Meso conditions. (D) Immunostaining of Tbx6 and Sox2 at D5 of Meso differentiation in Bra^−/− and wild-type ESCs. Wild-type cells efficiently differentiate to paraxial mesoderm and expresses Tbx6 but not Sox2. By contrast Bra^−/− cells differentiate to a neural identity exemplified by Sox2 expression in the absence of Tbx6. (E) At D8 wt cells cultured in CHIR express Desmin/MyoD but not β-Tubulin (Tuj1) whereas Bra^−/− cells fail to produce Desmin/MyoD and differentiate into neurons expressing β-Tubulin (Tuj1). (F) The time course of Cdx gene expression in posterior neural (N_P) and mesodermal inducing conditions (Meso). Cdx genes are transiently induced in posterior neural cells but continuously upregulated in mesodermal cells. (Note, log₂₊ scale). All data used for the plots can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s014" target="_blank">Data S6</a>. (G) Model for the generation of spinal cord and paraxial mesodermal tissue from ESCs. ESCs cultured in N2B27 with FGF generate anterior but not posterior neural tissue. The activation of Wnt signalling in differentiating ESCs results in the generation of a bipotential neuromesodermal progenitor, equivalent to those found in the CLE of the embryo, which generate spinal cord or paraxial mesodermal tissue. Wnt signalling activates homeodomain proteins of the Cdx family in these progenitors that could account for the posteriorisation. In addition, Wnt signalling activates the mesodermal specifier Brachyury (Bra) that is required for Tbx6 induction and the repression of Sox2. The induction of Brachyury induces the Brachyury-Wnt autoregulatory loop that is necessary for mesoderm induction. In the absence of this gene ESCs differentiate into posterior neural tissue even in the presence of continued Wnt signalling.</p

Generation of neural cells with specific AP identities from ESCs.

<p>(A) Schematic representation of differentiation conditions used for the generation of NPCs with specific Anterior (N_A), Hindbrain (N_H) and Spinal cord (N_P) identities. (B) Relative expression levels of the indicated genes from N_A, N_H and N_P cells at day 5 (D5) of differentiation indicate that N_A, N_H and N_P cells express distinct sets of genes. The standard scores (z-scores) of the indicated genes from mRNA-seq analysis reveals that N_A cells express high levels of forebrain markers including <i>Otx1</i> and <i>Otx2</i>; N_H cells express genes characteristic of hindbrain including <i>Mafb</i> and <i>Hoxa2</i> genes; N_P cells express high levels of posterior 5′ Hox genes including <i>Hoxc8</i> and <i>Hoxc9</i>. The individual Z-score for each replicate is indicated on the graph with circles, triangles and squares. (C) Time course of Hoxb and Hoxc cluster activation in cells cultured in N_H and N_P conditions showing fold change compared to D1. Posterior Hox genes are selectively activated only in the N_P conditions and show temporal colinearity with the induction of anterior Hox genes prior to posterior Hox genes <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937-Kmita1" target="_blank">[74]</a>. In N_H cells <i>Hoxb1</i> and <i>Hoxb2</i> are induced prior to <i>Hoxc4</i>. However, the more posterior Hox genes are not induced. By contrast, in N_P conditions the 5′ Hox genes <i>Hoxc6</i>, <i>Hoxc8</i> and <i>Hoxc9</i> are induced at D4 and their expression is maintained at day 5. (Note log₂ scale). (D) Immunohistochemistry indicates that N_H cells analysed at D8 differentiate into MNs of hindbrain identity coexpressing Hoxb4 and Phox2b. (E) N_P cells exposed to SAG generate spinal neurons coexpressing Hoxc6 and Hoxc9 with b-tubulin (Tuj1). These were not detected in N_H conditions. Coexpression of Hoxc6 and Hoxc9 with Islet1 indicates the generation of spinal MNs of forelimb and thoracic identity, respectively. These MNs also expressed Lim3/Raldh2 and HB9. (F) Graph showing the standard scores (z-scores) of Zfp42 (Rex1), Pou5F1 (Oct3/4) and Fgf5 from the mRNA-seq from D1 to D3. The kinetics of gene expression indicate that ESCs progressively lose their stem cell identity and acquire a transient epiblast identity at D2. (G) Hoxc6/Tuj1 and Hoxc9/Tuj1 positive cells were quantified in independent fields of D8 cells differentiated in N_P conditions. All data used to generate the plots of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio-1001937-g001" target="_blank">Figure 1</a> can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s009" target="_blank">Data S1</a>.</p

Generation of NMPs from EpiSCs.

<p>(A) Brachyury/Sox2 immunocytochemistry in EpiSC cultures treated with FGF/CHIR for 72 h. (B) qPCR analysis for indicated markers in mouse EpiSCs treated with FGF/CHIR. Error bars = s.d. (n = 3). n/d, not determined. Results are represented as log₁₀ ratio of expression versus untreated EpiSCs. The data used to generate the plot can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s012" target="_blank">Data S4</a>. (C) Combined fluorescence/brightfield microscopy showing donor cell incorporation of grafted GFP⁺ EpiSC differentiated for 48 h in FGF/CHIR after 48 h embryo culture. (D) Table summarizing the incorporation of grafted GFP⁺ EpiSC differentiated for 24 h or 48 h in Fgf/Wnt within host embryos. NT, neural tube; Som, somite; PSM, presomitic mesoderm; n/a, not applicable. (E) Representative examples of donor cell incorporation (green, GFP) and differentiation (red, immunofluorescence for indicated markers). Cell nuclei were stained with DAPI (blue). White boxes indicate the position of magnified images of GFP⁺ cells.</p

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

Supplementary Figures and Tables from A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and <i>in vivo</i> tracking of therapeutic cells

Figure S1: Production of SFCi55-ZsG iPSC line; Figure S2 and S3: Flow cytometry analysis of mature SFCi55 and SFCi55-ZsG- derived macrophages macrophages; Figure S4: Representative Images of phagocytosis assay; Supplementary Table 1: Primer sequences used in study; Supplementary Table 2: Antibodies used in study