Peri-infarct angiogenesis after treatment.

<p>Bar graph representing vessel density (mean±SD vs. contralateral) in peri-infarct regions of ischemic animals treated with vehicle (n = 10), EPCs (n = 9) or CM (n = 6); dashed line shows reference (mean) for sham (n = 4) animals (<b>A</b>); * indicates p<0.05 vs. vehicle and † indicates p<0.05 vs. sham. Graph showing CM content of PDGF-bb, FGF-b and VEGF in six different cultures of EPCs represented as black or grey circles, squares or triangles (<b>B</b>). Representative micrographs of lectin immunofluorescence; bars represent 100 µm (<b>C</b>). Representative micrographs of CD-31 (green) and BrdU (red) staining merged with DAPI showing proliferating endothelial cells (arrows) in peri-infarct areas; bars represent 20 µm (<b>D</b>).</p

Immunofluorescence images showing neurogenesis at two weeks in the SVZ.

<p>Composition of four immunofluorescence images for DCX (green) and DAPI (blue) showing neuroblast migration from the SVZ towards cortical peri-infarct thorough the corpus callosum external capsule in an EPC-treated animal (<b>A</b>). Representative images of double immunofluorescence for DCX (green), BrdU (red) and DAPI (blue) are shown for the different treatment groups (<b>B</b>). Bottom right inserts show a magnification of the dorsolateral SVZ. Box-plots representing the percentage of DCX+ or BrdU+ area in the SVZ (n = 11 for vehicle, n = 11 for EPCs, n = 11 for CM and n = 3 for shams) (<b>C</b>); data is represented as median (IQR).</p

Representative images of Bielschowsky’s staining.

<p>Ipsilateral and contralateral hemispheres of vehicle and EPC-treated brains (<b>A</b>); bars represent 500 µm. White arrows in (<b>A</b>) indicate the region of the corpus callosum (CC) with increased thickness, with emerging axonal tracks shown in detail in the bottom panels. Bar graph representing the ipsilateral CC axonal thickness expressed as a ratio vs. the contralateral hemisphere (n = 10 for vehicle, n = 9 for EPCs and n = 7 for CM), dashed line shows reference (mean) for sham (n = 4) animals (<b>B</b>). Data is presented as mean±SD. * indicates p<0.05 vs. sham.</p

Examination of Target Genes in Late-Onset AD Brains.

<p><i>A–B</i>. qPCR of NLRP2 (<i>A</i>) and <i>ASB9</i> (<i>B</i>) from mRNA from Brodmann's area (BA38) from control and AD brains. Black bars (1–5) are controls and red bars represent AD patients (6–16), which are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s005" target="_blank">Fig S5</a>. qPCR data was normalized internally to <i>GAPDH</i> expression and also to the average of 5 control lines. Statistical significance was determined by Student's t-Test and error bars reflect SEM. For control vs. AD, n = 5 for control, n = 11 for AD, p = 0.005. <i>C</i>. List of <i>PSEN1</i> NPC target genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s008" target="_blank">Table S2</a>) that have differential expression in independent microarray data of laser captured microdissected (LCM) cortical neurons from one of three brain areas (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s005" target="_blank">Fig S5</a>). All comparisons are either non-demented AD pathology (NDAD) or AD versus control samples. HIP refers to hippocampus, EC for entorhinal cortex, and MTG for middle temporal gyrus. Fold change and significance (FDR: false discover rate) reflect values for LCM neuron arrays. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s004" target="_blank">Figure S4</a>.</p

Characterization and Molecular Profiling of <i>PSEN1</i> Familial Alzheimer's Disease iPSC-Derived Neural Progenitors

<div><p>Presenilin 1 (<i>PSEN1</i>) encodes the catalytic subunit of γ-secretase, and <i>PSEN1</i> mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of <i>PSEN1</i>, we characterized neural progenitor cells (NPCs) derived from FAD mutant <i>PSEN1</i> subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying <i>PSEN1</i> mutations. <i>PSEN1</i> mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in <i>PSEN1</i> NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in <i>PSEN1</i> NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our <i>PSEN1</i> iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.</p></div

Gene Expression Profiling of Control vs. PS1 NPCs/Early Neurons.

<p>All 8 core iPSC lines were differentiated for 14 days in triplicate wells, lysed for RNA, amplified to generate cRNA, and ran on the Illumina HumanHT-12-14 BeadChip platform. <i>A</i>. Clustering of 8 core lines by correlation. UR stands for unrelated control. <i>B</i>. Scatter plot (log scale) of the correlation of gene expression between 4 control lines and 4 PSEN1 lines. The red lines indicate a 3-fold expression difference. <i>C</i>. Chart indicating the number of upregulated (shown in blue) and downregulated (shown in yellow) genes for each threshold of analysis. “DiffScore” refers to genes with a Diff Score of >13 (upregulated) or <13 (downregulated), which indicate a change in expression with a pValue of p≤0.05, without regard to the relative fold change. Criteria for fold change categories include the listed fold change as well as statistical significance. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s007" target="_blank">Table S1</a>.</p

Core set of iPSC Lines.

<p>See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s002" target="_blank">Fig S2</a>.</p

Aβ42/Aβ40 Ratio is Elevated in <i>PSEN1</i> Cells.

<p>All assays detected Aβ1-40 and 1-42 using ELISA (Wako) on conditioned media from the cell type indicated. Ratios were normalized against the first control line listed on each panel. Statistical significance was determined via Student's t-Test, error bars reflect SEM. Each n equals an individual cell line (averaged biological triplicates) in 1 independent experiment. <i>A</i>. Aβ42/Aβ40 ratio is increased in day 14 differentiated NPCs/early neurons. Control and <i>PSEN1</i> NPCs were generated from the core set of iPSC lines, and one of three independent experiments is shown. For control compared to PSEN1 NPCs (n = 4 for each genotype), p = 0.003. <i>B–C.</i> Aggregate data is shown from 3 independent fibroblast and 3 independent NPCs/early neuron experiments. N = 7 for each fibroblast genotype data point, and n = 12 for each NPC/early neuron genotype data point. <i>B</i>. Aβ 42/40 ratios are shown for both control and PSEN1 fibroblasts and NPCs. For control fibroblasts vs. <i>PSEN</i>1 fibroblast, p = 0.001; for control NPCs vs. <i>PSEN1</i> NPCs, p = 0.000005; for <i>PSEN1</i> fibroblasts vs. <i>PSEN1</i> NPCs, p = 0.036. <i>C</i>. Total Aβlevels (Aβ40 + Aβ42) are statistically similar between control and <i>PSEN1</i> fibroblasts and NPCs/early neurons. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s003" target="_blank">Figure S3</a>.</p

Validation of Target Genes in <i>PSEN1</i> NPCs.

<p>All qPCR data was normalized internally to <i>GAPDH</i> expression and also to cell line 7889O. Statistical significance was determined by Student's t-Test and error bars reflect SEM. <i>A</i>–<i>B</i>. <i>NLRP2</i> mRNA expression was assessed in undifferentiated iPSCs (<i>control vs. PSEN1</i>, n = 4 for each genotype, <i>p = 0.016</i>) and NPCs (<i>control vs.PSEN1, n = 4 for each genotype, p = 0.03</i>). <i>C</i>. Western blot analysis of NLRP2 protein expression in NPCs. α-Tubulin was used as a loading control <i>D</i>. Representative experiment showing <i>ASB9</i> mRNA expression in NPCs. For control vs. PSEN1, n = 4 for each genotype, p = 0.03. <i>E</i>. Representative experiment showing <i>NDP</i> mRNA expression in NPCs. For control vs. PSEN1, n = 4 for each genotype, p = 0.005. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s008" target="_blank">Table S2</a>.</p