Comparison of hematopoietic potential of iPSCs to mouse ESCs.

<p>Fractions of Lin⁻ cells (A), KLS cells (B), Kit+Lin-Sca-1- Progenitors (C), GMPs (D), CMPs (E), and MEPs (F) from iPSCs relative to mouse ESCs after 7 days of OP9 coculture (unsorted).</p

Generation of iPSC clones from a single mouse C57BL/6 male mouse.

<p><b>A.</b> Schematic of experimental approach (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120585#sec002" target="_blank">Material and Methods</a> for full protocol). <b>B.</b> Representative bright-field images (left) and alkaline-phosphastase stains (right) of B6/BLU ESCs (top) and a representative iPSC (bottom, Ax1-10). All images at 100x magnification. <b>C.</b> Images from the teratoma derived from Ax1-10 demonstrating ectoderm (neural tissue), mesoderm (cartilage) and endoderm (ciliated respiratory epithelium).</p

A typical discussion at BioStar.

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Validation array coverage.

<p>Validation array coverage.</p

Sequencing of iPSC clones.

<p><b>A.</b> Number of variants identified per iPSC clone. <b>B.</b> Variant allele frequencies of all validated mutations for each clone. Samples are ranked by the number of variants in decreasing order. No mutations were identified in Ax2-11, thus it is not listed in panel B.</p

Hematopoietic differentiation potential of the 24 iPSC clones.

<p>100,000 cells from OP9 cocultured mESCs (B6/BLU) or iPSCs were plated in methylcellulose media containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). <b>A.</b> CFUs were counted after 7 additional days of culture. The relative number of CFUs per 100,000 cells plated from Day7 iPSC-derived progenitors vs. Day7 ESC (B6/BLU)-derived progenitors are shown. iPSC clones are ranked from the highest to the lowest average of two independent experiments. Error bars represent the means +/− one standard deviation. <b>B.</b> Morphology of day 7 OP9 cocultured ESC-derived cells after 7–8 days of additional culture in MethoCult media containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). A scale bar of 20 μm is shown. <b>(C-E)</b>. Fractions of CD11b⁺ (<b>C</b>), CD34⁺Kit⁺ (<b>D</b>), and Ter119⁺ (<b>E</b>) cells obtained after 7 days of methylcellulose culture containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo), comparing iPSC-derived progenitors relative to ESC-derived progenitors, in the same order as panel A. <b>F.</b> Lack of correlation between the number of mutations and the hematopoietic differentiation potential of the iPSC clones (r² = 0.0006065).</p

Hematopoietic differentiation from murine ESCs.

<p><b>A.</b> Morphology of wild type ESC-derived cells after 7 days of OP9 coculture (unsorted) by Wright-Giemsa staining. A scale bar of 20 μm is shown. (B-D). Immunophenotyping of hematopoietic progenitor cells from wild-type mouse bone marrow cells (panel B), murine ESCs after 7 days of OP9 coculture (panel C), and iPSC clone Ax1-14 after 7 days of OP9 coculture (panel D). Lineage⁻ (Lin⁻), KLS (Lin⁻Kit⁺Sca-1⁺), progenitors (Lin⁻Kit⁺Sca-1⁻), CMPs (Lin⁻Kit⁺Sca-1⁻CD34⁺FCγ⁻), GMPs (Lin⁻Kit⁺Sca-1⁻CD34⁺FCγ⁺), and MEPs (Lin⁻Kit⁺Sca-1⁻CD34⁻FCγ-).</p

Whole exome sequencing coverage.

<p>Whole exome sequencing coverage.</p

Clinical history and distribution of metastases from patients A1 and A7, who both had clinically triple-negative and basal-like breast cancer.

<p>Clinical history and distribution of metastases from patients A1 and A7, who both had clinically triple-negative and basal-like breast cancer.</p

Molecular relatedness of matched primary and metastases.

<p>(A) Hierarchical clustering of patient A1 and A7’s tumors with 1,100 TCGA Primary samples and 98 normal breast samples analyzed using a breast cancer intrinsic gene list. The color bars under the dendrogram indicate (i) where A1 (red) and A7 (blue) specimens are clustered and (ii) the PAM50 subtype of each sample (basal-like, red; HER2-enriched, pink; luminal A, dark blue; luminal B, light blue; and normal-like, green). (B) The position of A1 (red) and the position of A7 (blue) within the basal-like cluster are highlighted. (C) The relationship of the primary and metastases for each patient based upon gene expression patterns.</p