anti dll4 r d systems  (R&D Systems)

Journal: EMBO Molecular Medicine

Article Title: Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling

doi: 10.15252/emmm.202114526

Figure Lengend Snippet: Principal Component Analysis (PCA) showing mMuSC‐derived myoblasts (left), human myoblasts (centre) and hiMPs (right). Four cell lines were analysed by RNAseq in treated and untreated conditions for each cell population. Each point on the PCA represents a cell population. Additional information in Appendix Tables and . Volcano plots showing differentially expressed genes between untreated and DLL4 and PDGFBB‐treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold‐change in expression upon treatment with DLL4 and PDGF‐BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05 to be considered significant. Heatmaps showing changes in expression of key myogenic ( MYOGENIN, MYOD1 ), perivascular ( PDGFRB , CD146 , NG2 , ALPL ) and NOTCH target ( HEY1 , HES1 ) genes upon treatment with DLL4 and PDGF‐BB in mMuSC‐derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z‐scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled. Validation of RNAseq data of panel (C) by real‐time PCR analysis of the same myogenic, perivascular and NOTCH target transcripts in treated and untreated hiMPs (experimental replicates = 3; error bars; SEM). Statistical analysis (paired t ‐test) was performed on ΔCt values whilst graphs show fold change relative to untreated controls. Curated dot plot Gene Ontology (GO; left), Kyoto Encyclopaedia of Genes and Genomes (KEGG; centre) and Reactome (right) enrichment analyses showing shared gene functions amongst the cell groups; numbers in brackets: genes analysed with a P value threshold set at 0.05; full lists in a dedicated spreadsheet available in Dataset . Source data are available online for this figure.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506‐D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma‐Aldrich; A9418‐10G) as a carrier protein.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: EMBO Molecular Medicine

Article Title: Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling

doi: 10.15252/emmm.202114526

Figure Lengend Snippet: Top cellular and molecular functions associated with DLL4 and PDGF‐BB modulation generated via ingenuity pathway analysis ( IPA ).

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506‐D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma‐Aldrich; A9418‐10G) as a carrier protein.

Techniques: Generated, Cell Function Assay

Journal: EMBO Molecular Medicine

Article Title: Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling

doi: 10.15252/emmm.202114526

Figure Lengend Snippet: A Trajectory plots for visualisation of the migratory paths of treated and untreated cells over the duration of the motility assay. Each line represents the path of a single cell. DLL4 and PDGF‐BB treatment was completed prior to motility assessment and all cells were imaged on plastic dishes in absence of the two inducing factors. B Visualisation of the motility state space of untreated and DLL4 and PDGF‐BB‐treated hiMPs using t‐SNE plots (perplexity = 35). C Unsupervised hierarchical clustering (Ward's method) visualised with a t‐SNE plot showing two clusters (Silhouette S i = 0.22). D, E Bar charts demonstrating normalised values for comparison of motility features between conditions (D; untreated and DLL4 and PDGF‐BB) and clusters (E) (experimental replicates = 3; total 408 cells; error bars: SEM). F Bar graph demonstrating proportions of control and DLL4 and PDGF‐BB‐treated cells within each cluster. Hypothesis testing was performed using the chi‐squared (χ 2 ) test. G Functional protein association network analysis ( https://string‐db.org ). The network view summarises predicted associations for proteins positively regulating cell migration common to all three datasets. The nodes are proteins and the edges represent the predicted functional associations. Red line: fusion evidence; Green line: neighbourhood evidence; Blue line: co‐occurrence evidence; Purple line: experimental evidence; Yellow line: text mining evidence; Light blue line: database evidence; Black line: co‐expression evidence. Blue nodes: GO:0030335 positive regulation of cell migration, Count in gene set: 8 of 452, false discovery rate: 0.0156. Source data are available online for this figure.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506‐D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma‐Aldrich; A9418‐10G) as a carrier protein.

Techniques: Motility Assay, Comparison, Control, Functional Assay, Migration, Expressing

Journal: EMBO Molecular Medicine

Article Title: Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling

doi: 10.15252/emmm.202114526

Figure Lengend Snippet: A Trajectory plots for visualisation of the migratory paths of untreated and treated cells that were exposed to either 1% BSA or DLL4 and PDGF‐BB, respectively, over the course of the motility assay. Each line depicts the path of an individual cell. B Visualisation of the motility state space of untreated and treated hiMPs using t‐SNE (perplexity = 35). C Hierarchical clustering of the first 30 principal components visualised with a t‐SNE plot showing two clusters (Silhouette S i = 0.19). D Bar charts displaying the normalised motility feature values for comparison between conditions: untreated and DLL4 and PDGF‐BB (left), cluster 1 and cluster 2 (right) (experimental replicates = 3; total 412 cells). E Bar graph showing proportions of untreated and treated hiMPs within the two clusters. Hypothesis testing was performed with a chi‐squared (χ 2 ) test. F Trajectory plots for visualisation of hiMP migration after 24 h of treatment (top row), or 72 h of treatment (bottom row). G, H t‐SNE plots (perplexity = 35) for visualisation of the motility state space of hiMPs in two‐dimensions (left). Cluster assignments after hierarchical clustering (S i = 0.13 (24 h); S i = 0.18 (72 h)). (H) Bar plots showing normalised motility features for both 24 h (top row) and 72 h (bottom row) conditions (experimental replicates = 3; total 876 cells and total 478 cells analysed for 24 and 72 h conditions, respectively.). I Bar graph displaying proportions of untreated and DLL4 and PDGF‐BB‐treated hiMPs treated for 24 and 72 h. Hypothesis testing was performed with a chi‐squared (χ 2 ) test. J Bar graphs depict quantification of parameters obtained from single cell tracking analysed using TrackMate. Motility statistics were calculated for untreated (grey bars) and treated (white bars) hiMPs (experimental replicates = 3; error bars: SD). P values within figure: t ‐test. Source data are available online for this figure.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506‐D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma‐Aldrich; A9418‐10G) as a carrier protein.

Techniques: Motility Assay, Comparison, Migration, Single Cell Tracking

Journal: EMBO Molecular Medicine

Article Title: Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling

doi: 10.15252/emmm.202114526

Figure Lengend Snippet: P value‐adjusted hierarchical clustering heatmap displaying hierarchical clustering of genes associated with leukocyte trans‐endothelial migration (KEGG pathway: hsa04670; P set at 0.05). Graphical representation of an individual chip of the OrganoPlate® (produced with BioRender, www.biorender.com ). Each chip consists of a top perfusion channel, central ECM channel and bottom perfusion channel. Phase guides between channels allows for generation of surface tension after deposition of collagen‐I within the ECM channel so that there is no physical barrier between the collagen gel and perfusion channels. This facilitates generation of a 3D blood vessel that is in direct contact with the ECM channel. Maximum intensity projections of the top perfusion channel, 48 h after seeding HUVECs, immunostained for CD31 and F‐actin. Scale bar: 100 μm. 3D projections of blood vessel‐like tubules of the top perfusion channel stained for F‐actin. Scale bar: 50 μm. Representative fluorescence images of 150 kDa TRITC‐conjugated dextran added to the top perfusion channel of OrganoPlate® chips with and without 3D endothelial monolayers generated by HUVECs. Chips were imaged every 3 min. See Appendix Fig for extended panel and quantification. Scale bar: 100 μm. Representative fluorescence images of CMFDA‐stained untreated and DLL4 and PDGF‐BB‐treated hiMPs within the top perfusion channel, 15 min after delivery and kept on the OrganoFlow®. Scale bar: 50 μm. Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t ‐test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 min within a single chip. Assessment of DLL4 and PDGF‐BB‐treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans‐well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 h. Bar graphs quantifying the average number of CFDA‐positive cells/ mm 2 , that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm 2 ) fields per condition was quantified (error bars: SEM). Scale bar: 250 μm. Bar graph showing fold‐change in trans‐endothelial migration (error bars: SEM). Statistical significance based on one‐way ANOVA with Bonferroni's multiple comparison. Source data are available online for this figure.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506‐D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma‐Aldrich; A9418‐10G) as a carrier protein.

Techniques: Migration, Produced, Staining, Fluorescence, Generated, Membrane, Comparison

Journal: EMBO Molecular Medicine

Article Title: Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling

doi: 10.15252/emmm.202114526

Figure Lengend Snippet: Schematic representation of the experimental setup (produced with BioRender, www.biorender.com ). Immunofluorescence images of 3D artificial muscles stained for myosin heavy chain (MyHC), after 15 min exposure to PBS (uninjured) or 10/100 μM cardiotoxin. Scale bar: 50 μm. Maximum intensity projections of fluorescence images of CMPTX‐labelled human 3D muscles after background subtraction, stained with CMPTX deposited with containing “transplanted” CMFDA‐labelled (green) hiMPs. Dotted lines demarcate the outline of the 3D construct. Scale bar: 100 μm. Time lapse video availabe in Movie . 3D trajectory plots for visualisation of single‐cell tracks of hiMPs on 3D muscles for 8 h for both untreated (left) and DLL4 and PDGF‐BB‐treated (right) conditions. Bar chart representing the total distances travelled of single‐cells tracked for DLL4 and PDGF‐BB and untreated hiMPs. Statistical testing was performed with an independent t ‐test with each experimental replicate as data points (experimental replicates = 3). Velocities of individual cells are displayed as single points to visualise the distribution of data. Bar chart displaying the velocity of cells within clusters generated using hierarchical clustering of cells using total distance travelled as a feature (S i = 0.67). Each point represents a single cell. Bar plots showing the proportions of untreated and DLL4 and PDGF‐BB‐treated cells within the two clusters shown in (F). Statistical test performed with a Chi‐squared (χ 2 ) test. Source data are available online for this figure.

Article Snippet: Recombinant human DLL4 (DLL4 fused with the Fc domain of human IgG; R&D Systems; 1506‐D4) was resuspended to a final concentration of 10 μg/ml in sterile PBS containing 1% wt/vol bovine serum albumin (BSA; Sigma‐Aldrich; A9418‐10G) as a carrier protein.

Techniques: Produced, Immunofluorescence, Muscles, Staining, Fluorescence, Construct, Generated