Labeling of embryonic progenitor cell line using peptide targeted Qdot605.

<p>(A) Cell targeting by fluorescent Qdots. Qdot605-ITK-SA were complexed with an excess of chemically synthesized C-terminal biotinylated peptide; unbound peptide was removed by dialysis. W10 progenitor cells were incubated for 16 h at 37°C with 5 nM of Qdot complexes, washed and imaged using a fluorescence microscope. (B) Competition with free peptide or peptide-targeted Qdots. Cells were pre-incubated with 5nM peptide, peptide targeted Qdots, or untargeted Qdots, for 30 min at 4°C, followed by addition of peptide phage (2×10¹⁰pfu) for an additional 1 h at 4°C. After washing, the recovered phage was quantified by titration. The competition is shown as percentage of no-peptide control. Values are from triplicate experiments and shown as mean ± standard deviation. Competition by corresponding free peptide or peptide-Qdot complex at 5 nM was statistically significant. Competition by uncoupled Qdots was not statistically significant (ANOVA with Dunnett’s multiple comparison tests; p values: *: <0.05. **: <0.01 and ***: <0.001) (C) Flow cytometry analysis. Cells were labeled as in (A), dissociated from the tissue culture plate using TrypLE, resuspended in PBS and analyzed in LSRFortessa flow cytometer. 10,000 events were recorded for each sample; cells were excited using the 405 nm laser and fluorescence emission was detected with the 605/12 bandpass filter. Cells labeled with W10-R3-18 peptide-Qdot complexes (green) showed higher mean fluorescent intensity than cells labeled with untargeted Qdots (red) or unlabeled W10 cells (blue).</p

Binding of peptide display phages to W10 embryonic progenitor cell line.

<p>(A) Immunofluorescent detection of bound phages. Cells were incubated with 2×10¹⁰ phage particles for 2 h at 37°C; unbound phages were removed by washing and cells were fixed and permeabilized. Bound phages were detected by immunocytochemistry using rabbit anti-phage antibody and Alexa568-conjugated goat anti-rabbit antibody. Cell nuclei were stained using DAPI. (B) Quantitation of peptide phage cell binding. 2×10¹⁰ pfu of each candidate or controls (RGD, Gly12 and empty phage M13KE) phages were assessed for binding on 1×10⁵ W10 progenitor cells for 2 h at 37°C. Cell associated phages were recovered from cell lysates and quantified by titration. Protein in cell lysates was measured by microBCA assay. The relative binding factor (BF) is calculated as peptide phage recovery (percentage of input) relative to M13KE control phage recovery (percentage of input). Values are from triplicate experiments and shown as mean ± standard deviation. BFs for the 4 W10 peptide phage were statistical significant from the control M13KE phage (ANOVA with Dunnett’s multiple comparison tests; p values: *: <0.05 and **: <0.01). BFs for RGD and Gly12 were not statistically significant.</p

Phage binding competition with free peptide.

<p>Competition of the peptide phage with free peptide was measured using (A) Immunofluorescent detection of bound peptide phages. Chemically synthesized peptides were added to compete with binding of peptide phages to W10 progenitor cells. Cells were pre-incubated with different peptides at 100 µM or without peptide for 30 min at 4°C, followed by peptide phages (2×10¹⁰pfu) for an additional 1 h at 4°C. After washing, the bound peptide phages were detected by immunofluorescence. Peptide sequences are: W10-R2-11-biotin: GWVIDYDYYPMRGGGK(biotin); FITC-W10-R2-11: FITC-GWVIDYDYYPMRGGG and FITC-unrelated: FITC-NHVHRMHATPAY (B) Percentage of input phage recovered from cell lysate. Cells were pre-incubated with peptides at 5 µM or 5 nM, or without peptide for 30 min at 4°C, followed by peptide phages (2×10¹⁰pfu) for an additional 1h at 4°C. After washing, the recovered phage was quantified by titration. The competition is shown as percentage of no-peptide control. Values are from triplicate experiments shown as mean ± standard deviation. Competition by the corresponding free peptide was statistically significant at 5 nM and 5 µM with the exception of W10-R2-21 (only significant at 5 µM). Competition by scrambled or unrelated peptide was not statistically significant. (ANOVA with Dunnett’s multiple comparison tests; p values: *: <0.05. **: <0.01 and ***: <0.001). Peptide sequences are: peptide: X₁₂GGGK(biotin); unrelated: biotin-NHVHRMHATPAY; W10-R2-11-scrambled: DYWDVGPIYRMYGGGG; W10-R2-21-scrambled: LGTMDWFWPYNEGGGG; W10-R3-18-scrambled: VSDPFDNLWTAWGGGK.</p

Selectivity of Qdot peptide complexes.

<p>Embryonic progenitor cell lines were labeled with Qdot complexes in their corresponding growth media and analyzed by flow cytometry as in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058200#pone-0058200-g005" target="_blank">Figure 5C</a>). Percentage of labeled cells was calculated by setting up gates (allowing up to 1%) using the embryonic progenitor cell line labeled with untargeted Qdots and unlabeled cells. 10,000 events were recorded for each sample. Values are from triplicate experiments and shown as mean ± standard deviation.</p

Selection of a peptide phage display library against W10 embryonic progenitor cells.

<p>(A) Peptide phages that bind to W10 embryonic progenitor cell line were enriched by 3 rounds of biopanning. PhD-12 phage display peptide library (2×10¹¹ pfu, for round 1) or amplified recovered phage (2×10¹⁰ pfu, for rounds 2 and 3) were first adsorbed against human adult dermal fibroblasts cells and then incubated with adherent W10 cells. The phages were recovered from the cell lysate and sample phage clones were sequenced. The enriched library was amplified for further rounds of selection. (B) The percentage of input phages recovered increased with each round of selection. The percentage of input phages recovered was determined by titration of plaque forming units (pfu) in the cell lysate relative to the input pfu used for each panning round. (C) Frequency and multiple sequence alignment of peptides identified as candidate peptide phage in rounds 2 and 3 of panning generated by CLUSTAL W (2.10). (D) Phylogram based on (C) denoting peptide similarities.</p

Pro-fibrotic pathway activation in trabecular meshwork and lamina cribrosa is the main driving force of glaucoma

<p>While primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, it still does not have a clear mechanism that can explain all clinical cases of the disease. Elevated IOP is associated with increased accumulation of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) that prevents normal outflow of aqueous humor (AH) and has damaging effects on the fine mesh-like lamina cribrosa (LC) through which the optic nerve fibers pass. Applying a pathway analysis algorithm, we discovered that an elevated level of TGFβ observed in glaucoma-affected tissues could lead to pro-fibrotic pathway activation in TM and in LC. In turn, activated pro-fibrotic pathways lead to ECM remodeling in TM and LC, making TM less efficient in AH drainage and making LC more susceptible to damage from elevated IOP via ECM transformation in LC. We propose pathway targets for potential therapeutic interventions to delay or avoid fibrosis initiation in TM and LC tissues.</p

Assignment of Embryonic Index (EIndex).

<p>Entities in each embryonic level are assigned a local index, e.g. the Head Mesenchyme local index is HdM, and one of its anatomical compartments, Branchial Arch 1 local index is BA1. The full Embryonic index for Branchial Arch 1 is hence HdM.BA1. Similarly, Paraxial Mesoderm Cells contained in the Branchial Arch 1 are given a local index PMCs and a fully qualified EIndex of HdM.BA1.PMCs.</p

Branchial Arch 1 (HdM.BA1) anatomical compartment development card.

<p><b>A.</b> Anatomical compartment viewer demonstrating the contribution of ‘Head Mesoderm’ and ‘Cranial Neural Crest’ to the developing ‘Head Mesenchyme’, which give rise to Branchial Arch 1 and its functional derivatives (i.e., cartilage, bone and skeletal muscles), is depicted in this viewer (highlighted, orange colored boxes). <b>B.</b> Illustration showing the embryonic ontology and development of the two main cellular components (i.e., paraxial mesoderm and neural crest cells) which give rise to skeletal elements of the head (lateral view). Such anatomical illustrations are available in the database at the organ/tissue and anatomical compartment cards <b>C.</b> Examples of the display of selective gene markers of cells populating the anatomical compartment. Here the selective genes of CNC cells and Paraxial Mesoderm cells are shown in the BA1 anatomical compartment card. <b>Abbreviations</b>: mb, midbrain; fb, forebrain;r1–7, rhombomeres 1–7; HdM.BA1–4, branchial arch 1–4; OV, optic vesicle.</p

7PEND24 PureStem Progenitor Cell Card.

<p><b>A</b>. Cell description and available data summary. <b>B.</b> Gene expression list including expression pattern, assay type and links to external resources. <b>C.</b> Links to cell-related high-throughput experiments, available in the database. <b>D.</b> List of <i>in vivo</i> cells or anatomical compartments that were matched to the PureStem progenitor and the related genes for each match<b>. E.</b> List of culturing conditions and protocols related to the PureStem progenitor cell.</p

Cellular Development level and Cell Card Representation.

<p>This figure demonstrates some of the comprehensive data provided for a single cell in the database. <b>A.</b> The cellular filter list, available at LifeMap Discovery, showing all available cellular developmental paths currently available in the database. This example demonstrates results from search of skeletal muscle-related cells; two selected cells with their developmental path annotation (e.g., Skeletal Muscle) are shown for simplification. <b>B.</b> ‘Cranial Neural Crest Cell’ (CNCCs) card shows the available information (e.g., gene expression) for these cells, accompanied by the interactive clickable graphical development viewer on the right. <b>C.</b> An example for a specific signal display- with a description (SHH), signal source, associated gene cascade and biological cellular outcome.</p