Additional file 1: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Daf1-positive cells are negative for Nanog expression. Mouse Nanog-iPS cells, in which GFP expression is driven by Nanog promoter [45], are differentiated into DE. Cxcr4+/E-cadherin + cells were sorted and analyzed for Daf1 and Nanog-GFP expression. Daf1-positive cells are negative for Nanog expression. (TIF 312 kb

Additional file 3: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Primer sequences used for RT-PCR analysis. Primer sequences used for detection of gene expression in Fig. 1, 2. (DOCX 15 kb

Immunofluorescent staining of SOX17 and FOXA2 proteins in DE differentiated SNL-201B7 and -253G1 or MEFP1-201B7 and -253G1 cells.

<p>The images of SOX17 (green) and FOXA2 (red), and DAPI (blue) are shown (A and B). White scale bars, 100 μm. The percentages of SOX17 (C and D, left panels) and FOXA2 (C and D, right panels)-positive cells per DAPI-positive cells (201B7, n = 10 each and 253G1, n = 8 each) were calculated based on DE marker-positive and DAPI-positive cell numbers. Data represent the mean ± SEM and are representative of three experiments. ***p < 0.001 versus SNL-201B7 and -253G1 cells.</p

Additional file 2: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Flow cytometric analyses of the cell cycle. Histograms of flow cytometric analyses of Daf1-DE and Daf1 + DE (n = 5) are shown. Cell cycle was analyzed by measuring DNA quantities using DyeCycle. (TIF 314 kb

Mapping of RNA-sequencing of SNL-201B7 and MEFP1-201B7 on the human genome, hg38.

<p>(A) RNA-sequencing of SNL-201B7 and MEFP1-201B7 (n = 6, each) was mapped on human genome, hg38, especially around the <i>hXIST</i> gene. The result was visualized by genome viewer (IGV_2.4.4). The vertical axis indicates number of reads of RNA-sequencing. (B) Mapping of RNA-sequencing on <i>hXIST</i> exon 4. The specific, restricted RNA-fragment was found to be expressed in MEFP1-201B7 at levels higher than in SNL-201B7.</p

Experimental procedure for DE differentiation from SNL- and MEFP1-201B7 and -253G1 cells.

<p>hiPSC line 201B7 or 253G1 colonies that had been passaged more than 20 times on SNLs (>P20) were passaged on SNLs (SNL-201B7 and -253G1) and MEFs (MEFP1-201B7 and -253G1) and then cultured for 6 days in basic fibroblast growth factor (bFGF)-supplemented stem cell medium. SNL- and MEFP1-201B7 and -253G1 cells were seeded in Matrigel-coated wells. The medium was changed to DE differentiation medium supplemented with 1% dimethyl sulfoxide (DMSO) and 100 ng/ml recombinant human activin A (rhActA) and the cells were incubated for 4 days. Medium without rhActA was used as a negative control.</p

Experimental procedure for DE differentiation from MEFP2- or MEFP1-SNL-201B7 cells.

<p>The MEFP1-201B7 colonies in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201239#pone.0201239.g001" target="_blank">Fig 1</a> were passaged on MEFs (MEFP2-201B7) or SNLs (MEFP1-SNL-201B7) and then cultured for 6 days. The MEFP2- and MEFP1-SNL-201B7 cells were seeded in Matrigel-coated wells. The medium was changed to differentiation medium supplemented with 1% DMSO and 100 ng/ml rhActA and the cells were incubated for 4 days. Medium without rhActA was used as a negative control.</p

Schematic summary of this study.

<p>This scheme includes the results and conclusions of the present study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201239#pone.0201239.ref010" target="_blank">10</a>].</p

Molecular network analysis of SNL- and MEFP1-201B7 cells by KeyMolnet program using the comprehensive RNA sequencing data.

<p>Using the comprehensive RNA sequencing data (deposited in the DNA Data Bank of Japan database (accession number DRA006179) and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201239#pone.0201239.t001" target="_blank">Table 1</a>) of SNL-201B7 and MEFP1-201B7 (n = 6 each), the molecular networks, pathways, and transcriptional regulators of altered gene-expression were analyzed by the KeyMolnet program. (A) Molecular network of the supposed transcription factors and the regulated genes of SNL- and MEFP1-201B7 cells. The upper part shows the molecules that regulate gene expression. The orange -filled and -framed circles in the upper part indicate SMAD1-5 and SMAD-related factors, respectively. The lower part shows those molecules whose gene expression was altered. The ratio of the reads per kilobase per million mapped reads (RPKM) values of expressed mRNA of MEFP1-201B7 cells, against those of SNL-201B7 cells, revealed by the comprehensive RNA analysis, are shown in red and blue colors that indicate up- and down-regulated molecules, respectively. (B) The supposed molecular pathways and their calculated scores in the altered genes-expression of SNL- and MEFP1-201B7 cells. The extracted molecular network shown in the panel (A) was compared with distinct canonical pathways of the KeyMolnet library. This makes it possible to identify the canonical pathway showing the most significant contribution to the extracted network. The score and score (p) were calculated as described in the experimental procedures. In the list of (B), several abbreviations were used, indicated below. SRC: steroid receptor coactivator, HIF: hypoxia-inducible factor, C/EBP: CCAAT-enhancer-binding protein, RUNX: Runt-related transcription factor, POU: Pit-1, Octamer transcription factor and Unc-86, RB: Retinoblastoma gene product, and MAPK: MAP kinase.</p

Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells

<div><p>The crosstalk between cells is important for differentiation of cells. Murine-derived feeder cells, SNL76/7 feeder cells (SNLs) or mouse primary embryonic fibroblast feeder cells (MEFs) are widely used for culturing undifferentiated human induced pluripotent stem cells (hiPSCs). It is still unclear whether different culture conditions affect the induction efficiency of definitive endoderm (DE) differentiation from hiPSCs. Here we show that the efficiency of DE differentiation from hiPSCs cultured on MEFs was higher than that of hiPSCs cultured on SNLs. The qPCR, immunofluorescent and flow cytometry analyses revealed that the expression levels of mRNA and/or proteins of the DE marker genes, SOX17, FOXA2 and CXCR4, in DE cells differentiated from hiPSCs cultured on MEFs were significantly higher than those cultured on SNLs. Comprehensive RNA sequencing and molecular network analyses showed the alteration of the gene expression and the signal transduction of hiPSCs cultured on SNLs and MEFs. Interestingly, the expression of non-coding h<i>XIST exon 4</i> was up-regulated in hiPSCs cultured on MEFs, in comparison to that in hiPSCs cultured on SNLs. By qPCR analysis, the mRNA expression of undifferentiated stem cell markers <i>KLF4</i>, <i>KLF5</i>, <i>OCT3/4</i>, <i>SOX2</i>, <i>NANOG</i>, <i>UTF1</i>, and <i>GRB7</i> were lower, while that of <i>hXIST exon 4</i>, <i>LEFTY1</i>, and <i>LEFTY2</i> was higher in hiPSCs cultured on MEFs than in those cultured on SNLs. Taken together, our finding indicated that differences in murine-feeder cells used for maintenance of the undifferentiated state alter the expression of pluripotency-related genes in hiPSCs by the signaling pathways and affect DE differentiation from hiPSCs, suggesting that the feeder cells can potentiate hiPSCs for DE differentiation.</p></div