Additional file 4: Figure S3. of Global gene expression profiling and senescence biomarker analysis of hESC exposed to H2O2 induced non-cytotoxic oxidative stress

Clustering analysis of the differentially expressed genes and functional gene ontology classification. Microarray data analysis with limma package software is displayed in a histogram representing the significantly enriched gene ontology categories (p = 0.05) involved in the oxidative stress response after the H2O2 non-cytotoxic treatment. (TIF 7774 kb

Neuronal differentiation of lt-NES cells.

<p>(<b>A–B</b>) Four weeks after growth factor removal, lt-NES cells differentiated predominantly into neurons expressing beta III-tubulin (90%), MAP2ab and the neurotransmitter GABA, plus a minor fraction of GFAP-positive astrocytes (10%). Error bars represent STD. (<b>C</b>) Following growth factor withdrawal the clonal line PKa-2 gave rise to glia and neurons of preferentially GABAergic phenotype, comparable to the parental line PKa. (<b>D</b>) Specific neuronal subtypes expressing TH, 5HT and HB9 could be observed after 4 weeks of differentiation; O4-positive oligodendrocytes were detected after 10 weeks of differentiation. (<b>E</b>) AF22 lt-NES cells stained for Nestin and beta III-tubulin at day 0, 5, 8, 11, 18, 22 of differentiation. (<b>F</b>) Proliferating AF22 cells expressing GFP under the control of the Nestin enhancer (Nestin-GFP) show double labeling with an antibody to the Nestin protein. (<b>G</b>) Nestin-GFP expression in AF22 cells at day 0, 5, 7, 9, 11, 13, 15 of differentiation under control conditions or after exposure to DAPT (2 µM). Scale bars: 100 µm.</p

Contrary to mESCs, EpiSCs are responsive to culture conditions controlling differentiation of hESCs into extra-embryonic tissues, neuroectoderm and mesendoderm.

<p>(A) Expression of pluripotency markers (Oct-4, Nanog) and neuroectoderm (Sox2, Sox1, Six3, Tuj1 and Gbx2), extra-embryonic markers (Cdx2, Hand1, Sox7, GATA6) and mesendoderm markers (Brachyury, Mixl1, Eomes, Sox17) during differentiation of mouse EpiSCs using the culture conditions developed with hESCs. EpiSCs were differentiated following the conditions described above for hESCs (SB+FGF for neuroectoderm, BMP4 for extra-embryonic tissues, three step protocol for mesendoderm). Following the ninth day after plating, RNAs were extracted and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. (B) Expression of pluripotency markers (Oct-4, Nanog, SSEA-1), extra-embryonic markers (CDX2, Sox7, GATA4), neuroectoderm markers (Sox1, Nestin, βIII tubulin) and mesendoderm markers (Brachyury and Sox17) in EpiSCs differentiated using the conditions developed for hESCs. Expression of the genes denoted was analysed by immuno-fluoresence analyses. Nuclei are shown by Hoechst staining. Scale Bar 100 µM. (C) Expression of pluripotency markers (Oct-4), extra-embryonic markers (CDX2, GATA6), neuroectoderm markers (Sox2, Six3) and mesendoderm markers (Brachyury and Sox17) in mESCs differentiated using the method developed with hESCs. mESCs were differentiated in CDM as described for hESCs and then the expression of the genes denoted was analysed by Real-Time PCR. Normalized expression is shown as the mean±SD from three experiments.</p

Generation of mesendoderm using a combination of Activin, FGF2 and BMP4.

<p>(A) Colony morphologies formed in response to the three-step protocol to differentiate hESCs into mesendoderm progeny. H9 cells were grown for 2 days in CDM/PVA+Activin 10 ng/ml+FGF2 12 ng/ml, then for 72 hours in CDM/PVA+SU5402 10 µM+Activin 5 ng/ml. The next 4 days, cells were grown in CDM/PVA+Activin 30 ng/ml+FGF2 20 ng/ml+BMP4 10 ng/ml. Images of the same colonies were captured every day for 9 days. Scale Bar 200 µM. (B) Effect of different combinations and doses of Activin, FGF2 and BMP4 on the differentiation of hESCs grown in CDM/PVA. Following the third day in CDM/PVA+SU5402 10 µM, H9 cells were induced to differentiated in CDM/PVA supplemented with different combination of growth factors. RNAs were extracted after 3 days and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. hESCs grown in CDM+Activin+FGF or differentiated in CDM+SB431542+FGF2 were used as negative controls. (C) Expression of specific markers for mesendoderm in hESCs differentiated in CDM/PVA supplemented with Activin, FGF2 and BMP4 in the three step protocol. Nuclei are shown by Hoechst staining. Scale Bar 100 µM. (D) Microarray gene expression heat map to compare human embryonic stem cells (ESC) grown in CDM supplemented with Activin and FGF and mesendoderm cells generated in CDM/PVA supplemented with Activin, FGF and BMP4 (LE). Up-regulation is coloured in shades of red and down-regulation in shades of blue according to the log z scale shown at the bottom of the heat map. Gene names marked with an asterisk denote genes that pass a significant differential regulation threshold. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006082#s4" target="_blank">Material and Methods</a>). (E) Fraction of cells expressing the definitive endoderm marker CXCR4 and the mesendoderm/mesoderm marker PDGFαR after induction of differentiation in CDM PVA in the presence of increasing doses of Activin. H9 cells were differentiated following the three step protocol described above in the presence of Activin (30 or 100 ng/ml). Fraction of cells expressing CXCR4 or PDGFαR was determined using FACS 8 days after plating. (F) RT-PCR analyses for the expression of liver markers (Albumin, HNF4, αFP), gut marker (CDX2) and cardiac markers (ANF, α Actinin, α-1 Channel) in endoderm progenitors grown in media containing serum. Endoderm progenitors generated using the three step protocol were differentiated in media containing three different FBS lots. RNAs were extracted after 5 and 10 days of differentiation and the expression of genes expressed was analysed using RT-PCR.</p

Induction of midbrain dopaminergic neurons using extrinsic factors.

<p>IPS cell derived lt-NES cells cultured for 7 days in the presence of sonic hedgehog (Shh) and FGF8 showed nuclear expression of Lmx1a and FoxA2 (<b>A</b>). (<b>B</b>) With prolonged (14 days) exposure to Shh and FGF8, the number of cells expressing FoxA2 gradually increased. Shh/FGF8-treated cells were also positive for the midbrain marker En1, whereas neither FoxA2 nor Lmx1a were expressed in control populations cultured in the absence of Shh/FGF8. (<b>C–D</b>) Growth factor removal from lt-NES cell lines grown in Shh/FGF8 for 14 days induced differentiation and appearance of cell clusters positive for FoxA2 and tyrosine hydroxylase (TH), with beta III-tubulin-positive neurons co-expressing Nurr1. In contrast, control cultures propagated in FGF2/EGF remained TH-negative. Abbreviations in upper right corners of the immunofluorescence photomicrographs denote lt-NES cell lines used. Scale bars: 50 µm.</p

Identification of culture conditions for inducing neuroectoderm and mesendoderm differentiation in CDM-BSA.

<p>H9 hESCs were grown for different periods in CDM-BSA supplemented with different growth factors (Activin, FGF2 or BMP4) or chemical inhibitors [SB431542 (SB) for Activin/Nodal, SU5402 (SU) for FGF or BIO for GSK3β/Wnt]. Then, immunostaining analyses were used to determine the fraction of cells expressing the pluripotency marker Oct-4, the neuroectoderm marker Sox2, the mesoderm marker Brachyury, and the endoderm marker Sox17. The levels of marker-expressing cells were divided into five arbitrary categories: 0 for absence of expression, Very Low (VL) for <1% expressing cells, Low (L) for <5% expressing cells, Moderate (M) for >10% expressing cells, High (H) for >50% expressing cells. Indicated in blue are conditions allowing the generation of neuroectoderm (expressing Sox2) and in red those inducing mesendoderm (cells expressing Brachyury or Sox17).</p

Gene expression analysis of lt-NES cells and fetal NS cells.

<p>(<b>A</b>) A total of six cell lines representing two cell types (lt-NES and fetal NS) of different origins were profiled using TLDAs. Hierarchical cluster visualization (Pearson) shows separation into two major clusters representing the two cell types analyzed. (<b>B</b>) Correlation plots based on CT gene expression values of the individual cell lines. The lt-NES cell lines AF24 and AF22 show a higher degree of correlation than the lt-NES cell line AF24 and the NS line CB660, although the latter carry the same genome (R = 0.95 vs. R = 0.76). Red dots represent the median aggregated CT expression value of the 18S endogenous control; blue dots indicate the median aggregated CT expression values of all other genes on the TLDA. (<b>C</b>) Relative levels of differently expressed genes (corrected p-value threshold 0.05) in 9 lt-NES cell lines and 3 fetal NS cell lines. (<b>D–H</b>) Panels depicting the expression of markers representing the categories neural stem cell (NSC) markers, ventricular zone (VZ) expressed transcription factors (TF), regional position, rosette genes, and cell cycle. Data in C–H are depicted at a Log10 scale with the mean expression in fetal NS cells set to 1. No significant differences in expression levels were detected in ESC- vs. iPSC-derived lt-NES cells.</p

Cell signalling pathways controlling cell fate specification of pluripotent cells in vitro.

<p>A) Pluripotency of hESCs and mEpiSCs relies on Activin signalling and to a lesser extent on FGF signalling to maintain their pluripotent status. BMP signalling inhibition may be required to avoid extra-embryonic differentiation, depending on the level of endogenous BMP signalling activity of each cell type. B) Inhibition of Activin/Nodal signalling induces differentiation toward neuroectoderm in the presence of FGF2. C) BMP4 induces differentiation toward extra-embryonic tissues which is blocked by Activin and FGF2. D) BMP alternatively induces mesendoderm in cooperation with Activin (high dose) and FGF2. This model summarises results from hESCs and mEpiSCs and distinguish them from mESCs which remain pluripotent in LIF+BMP.</p

Long-term self-renewing neuroepithelial stem cells (lt-NES cells) derived from pluripotent stem cells of different origins.

<p>(<b>A</b>) Representative pictures of the lt-NES cell lines PKa and AF22, derived from iPSCs generated from reprogrammed adult dermal fibroblasts. The iPSCs were induced to form neural rosettes, which were isolated and expanded into lt-NES cell lines. Lt-NES cells exhibit a rosette-like growth pattern, stain positive for Sox2, Dach1, Nestin and PLZF and show apical expression of the tight junction protein ZO-1. (<b>B</b>) Endpoint RT-PCR analysis (30 cycles) reveals expression of the neural progenitor markers SOX1, PAX6, HES5 and the neural rosette markers PLZF, DACH1, MMNR1 and PLAGL1. Lt-NES cells also express telomerase and high levels of the hindbrain marker GBX2. Fetal human cortex (FC) was used as control. (<b>C</b>) Lt-NES cells exhibit a stable karyotype over extensive passaging as assessed by G-banding (line AF22 at passage 30). (<b>D</b>) Growth kinetics of the three lt-NES cell lines AF22 (dark blue), AF23 (green), and AF24 (light blue) as measured by change in % confluence over time. (<b>E</b>) The clonal lt-NES line AF22:3, derived by single cell deposition into 96-well plates, displays a morphology indistinguishable from its parental line AF22 and stains positive for Sox2 and Nestin. (<b>F</b>) The cell surface expression of CD133/PROMININ by AF23 and AF22 lt-NES cells was analyzed using flow cytometry. Debris was excluded by use of TO-PRO-3, and both non-stained cells and cells stained with only the secondary antibody were used to set up the gate (negative control). (<b>G</b>) Early passage lt-NES cells (PKb, PKc) still express forebrain markers such as FOXG1 and OTX2. However, expression is lost at higher passages. P, passage number; white scale bars: 100 µm; red scale bars: 10 µm.</p

List of lt-NES cell lines, their origins, the reprogramming method used and the capacity of the pluripotent cells lines to form neural rosettes.

<p>(KSR) knock out serum replacement, (zfFGF2) zebrafish fibroblast growth factor 2, (BMP4) bone morphogenic protein 4, (LIF) leukemia inhibitory factor, (ADF) adult dermal fibroblasts, (hESC) human embryonic stem cells, (NSC) neural stem cells, (VSV-G) vesicular stomatitis virus G protein.</p