percp vio700 a  (Miltenyi Biotec)

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Single-cell transcriptomic analysis of developing aorta-gonad-mesonephros (AGM) collected from human embryos at Carnegie stages 14 and 15 enriched for CD31 + and CD34 + cells from , Nature. Arterial haemogenic endothelium, dashed line, is identified by the colocalisation of DLL4 and RUNX1 . ( B ) Gene expression analysis of the nine selected target genes in the developing human aorta, showing their expression in the haemogenic endothelium.

Article Snippet: Antibody , Anti-human CD34 Percp-Efluor710 (mouse monoclonal) , eBioscience , Clone 4H11 , Flow cytometry 1:100.

Techniques: Gene Expression, Expressing

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Schematic of the derivation of the inducible SAM (iSAM) cell line by ZNFs mediated targeting of the AAVS1 locus and the subsequent derivation of the iSAM_NT (containing the non-targeting control gRNA) and iSAM_AGM (containing the gRNAs for the target genes) by viral transduction of the gRNAs. ( B ) Schematic of the differentiation protocol with activation of the target genes, used for both the control line iSAM_NT and iSAM_AGM. ( C ) Schematic of the IGFBP2 functional validation experiment. ( D ) Expansion of the arterial population marker by membrane expression of DLL4+ following targets’ activation, quantified by flow cytometry at day 8 of differentiation (*p=0.0190, **p=0.0011, Sidak’s two-way ANOVA). ( E ) Flow cytometry analysis of the cell cycle analysis of suspension progenitor cells was obtained following the OP9 coculture of cells treated with IGFBP2 and control at day 13. ( F ) iSAM expressing cells at day 8 of differentiation upon doxycycline (DOX) treatment. ( G ) Target genes’ expression profile across the different libraries, the colour legend for the libraries is on the top right. ( H ) IGFBP2 expression profile across the cell clusters and in the cell lines and conditions indicated in the title (cluster legend same as in I). ( I ) Contribution of each of the libraries to the cell clusters, grouped by libraries (cluster legend at the bottom). Figure 3—figure supplement 1—source data 1. Spreadsheet source file containing the source data used for the plots in . Each tab is labelled to uniquely refer to a specific panel.

Article Snippet: Antibody , Anti-human CD34 Percp-Efluor710 (mouse monoclonal) , eBioscience , Clone 4H11 , Flow cytometry 1:100.

Techniques: Control, Transduction, Activation Assay, Functional Assay, Biomarker Discovery, Marker, Membrane, Expressing, Flow Cytometry, Cell Cycle Assay, Suspension

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Gene expression profile of target genes following target genes’ activation, heatmap shows the expression level of the target genes in the iSAM_NT and iSAM_AGM treated with doxycycline (DOX) following normalisation on the -DOX control. ( B ) Dimension reduction and clustering analysis of the scRNAseq data following activation, filtered on cells where the gRNA expression was detected. ( C ) Arterial ( GJA4 , DLL4 ), venous ( NRP2 , APLNR ), and haemogenic marker ( CD44 , RUNX1 ) expression distribution in the clusters indicated by the colour. ( D ) Expression distribution visualised on the UMAP plot showing the location of arterial cells marked by DLL4 , and haemogenic endothelium marked by CD44 and RUNX1 . ( E ) Heatmap of the top 15 marker genes for each of the clusters. ( F ) Contribution of the different libraries to the clusters showing that arterial cell cluster is overrepresented in the iSAM_AGM treated with DOX, compared to the other libraries. ( G ) Expansion of the arterial population assessed by the membrane marker expression of DLL4+ following targets’ activation, quantified by flow cytometry at day 8 of differentiation. (Data are normalised on the iSAM_NT+DOX sample, n=5 independent differentiations, *p=0.0417 paired t-test.) ( H ) Colony-forming potential of the suspension progenitor cells derived from the two lines treated with or without DOX following OP9 coculture activation, data show the colony obtained for 104 CD34+ input equivalent (n=3 from independent differentiations *p<0.05, Tukey’s two-way ANOVA). Figure 3—source data 1. Spreadsheet source file containing the source data used for the plots in . Each tab is labelled to uniquely refer to a specific panel.

Article Snippet: Antibody , Anti-human CD34 Percp-Efluor710 (mouse monoclonal) , eBioscience , Clone 4H11 , Flow cytometry 1:100.

Techniques: Gene Expression, Activation Assay, Expressing, Control, Marker, Membrane, Flow Cytometry, Suspension, Derivative Assay

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Violin plot of IGFBP2 expression profile in the arterial cells obtained from the different conditions, in the presence or absence of gRNAs and doxycycline (DOX). ( B ) Number of haematopoietic colonies obtained after coculture on OP9 in the presence or absence of IGFBP2 (n=3–4 from independent differentiations, **p=0.0080, Sidak’s two-way ANOVA). ( C ) Percentage of DLL4 + arterial cells differentiation within the CD34 + compartment analysed by flow cytometry in day 8 embryoid bodies (EBs) (n=4 from independent differentiations, two-way ANOVA, ns = p>0.99). ( D ) Expansion of haematopoietic progenitors analysed using markers’ expression on suspension progenitors derived after coculture of CD34+ cells onto OP9 support (data are expressed as fold over the CTR in the absence of IGFBP2; n=4 from independent differentiations, *p<0.02, Sidak’s two-way ANOVA). ( E ) Single-cell transcriptomic analysis of developing aorta-gonad-mesonephros (AGM) collected from human embryos at Carnegie stages14 and 15 enriched for CD31+ and CD34+ showing the IGFBP2 expression profile in vivo in the AGM. Figure 4—source data 1. Spreadsheet source file containing the source data used for the plots in . Each tab is labelled to uniquely refer to a specific panel.

Article Snippet: Antibody , Anti-human CD34 Percp-Efluor710 (mouse monoclonal) , eBioscience , Clone 4H11 , Flow cytometry 1:100.

Techniques: Expressing, Flow Cytometry, Suspension, Derivative Assay, In Vivo

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Dot plot showing the expression profile of the genes coding for the enzymes mediating glycolysis in CTR and IGFBP2-treated cells at day 10 and 13. ( B ) Dot plot showing the expression profile of the genes coding for the checkpoints of the glycolysis in CTR and IGFBP2-treated cells at days 10 and 13. ( C ) Percentage of cells expressing different markers following OP9 coculture of CD34+ cells (ns for all markers, Kruskall-Wallis one-way ANOVA). ( D ) OP9 cocultures of CD34+ DLL4+ and CD34+DLLL4- (scale bar indicates 50 μm), and in ( E ) their colony formation capacities following 1 week of OP9 coculture (mixed-effect analysis, Sidak’s post-test, ****p<0.0001, **p<0.01). ( F ) Cluster composition in cells treated with IGFBP2 data shows the composition at day 10 and day 13. Figure 5—figure supplement 1—source data 1. Spreadsheet source file containing the source data used for the plots in . Each tab is labelled to uniquely refer to a specific panel.

Article Snippet: Antibody , Anti-human CD34 Percp-Efluor710 (mouse monoclonal) , eBioscience , Clone 4H11 , Flow cytometry 1:100.

Techniques: Expressing

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human CD34 Percp-Efluor710 (mouse monoclonal) , eBioscience , Clone 4H11 , Flow cytometry 1:100.

Techniques: Cloning, Plasmid Preparation, Recombinant, Expressing, Sequencing, Flow Cytometry, Amplification

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Single-cell transcriptomic analysis of developing aorta-gonad-mesonephros (AGM) collected from human embryos at Carnegie stages 14 and 15 enriched for CD31 + and CD34 + cells from , Nature. Arterial haemogenic endothelium, dashed line, is identified by the colocalisation of DLL4 and RUNX1 . ( B ) Gene expression analysis of the nine selected target genes in the developing human aorta, showing their expression in the haemogenic endothelium.

Article Snippet: For flow cytometry 10 5 cells per test were stained in 50 μl of staining solution with the following antibodies: CD34 Percp-Efluor710 (4H11 eBioscience, 1:100), CD34 Pe (4H11 eBioscience, 1:200), CD43 APC (eBio84-3C1, 1:100), CD45 FITC (2D1 eBioscience, 1:100), DLL4 Pe (MHD4-46 BioLegend, 1:200), CD41 PE (HIP8 BioLegend, 1:200), CD144 APC (16B1 eBioscience, 1:100), CD235a FITC (HIR2 BD Bioscience, 1:250).

Techniques: Gene Expression, Expressing

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Schematic of the derivation of the inducible SAM (iSAM) cell line by ZNFs mediated targeting of the AAVS1 locus and the subsequent derivation of the iSAM_NT (containing the non-targeting control gRNA) and iSAM_AGM (containing the gRNAs for the target genes) by viral transduction of the gRNAs. ( B ) Schematic of the differentiation protocol with activation of the target genes, used for both the control line iSAM_NT and iSAM_AGM. ( C ) Schematic of the IGFBP2 functional validation experiment. ( D ) Expansion of the arterial population marker by membrane expression of DLL4+ following targets’ activation, quantified by flow cytometry at day 8 of differentiation (*p=0.0190, **p=0.0011, Sidak’s two-way ANOVA). ( E ) Flow cytometry analysis of the cell cycle analysis of suspension progenitor cells was obtained following the OP9 coculture of cells treated with IGFBP2 and control at day 13. ( F ) iSAM expressing cells at day 8 of differentiation upon doxycycline (DOX) treatment. ( G ) Target genes’ expression profile across the different libraries, the colour legend for the libraries is on the top right. ( H ) IGFBP2 expression profile across the cell clusters and in the cell lines and conditions indicated in the title (cluster legend same as in I). ( I ) Contribution of each of the libraries to the cell clusters, grouped by libraries (cluster legend at the bottom). Figure 3—figure supplement 1—source data 1. Spreadsheet source file containing the source data used for the plots in . Each tab is labelled to uniquely refer to a specific panel.

Article Snippet: For flow cytometry 10 5 cells per test were stained in 50 μl of staining solution with the following antibodies: CD34 Percp-Efluor710 (4H11 eBioscience, 1:100), CD34 Pe (4H11 eBioscience, 1:200), CD43 APC (eBio84-3C1, 1:100), CD45 FITC (2D1 eBioscience, 1:100), DLL4 Pe (MHD4-46 BioLegend, 1:200), CD41 PE (HIP8 BioLegend, 1:200), CD144 APC (16B1 eBioscience, 1:100), CD235a FITC (HIR2 BD Bioscience, 1:250).

Techniques: Control, Transduction, Activation Assay, Functional Assay, Biomarker Discovery, Marker, Membrane, Expressing, Flow Cytometry, Cell Cycle Assay, Suspension

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Gene expression profile of target genes following target genes’ activation, heatmap shows the expression level of the target genes in the iSAM_NT and iSAM_AGM treated with doxycycline (DOX) following normalisation on the -DOX control. ( B ) Dimension reduction and clustering analysis of the scRNAseq data following activation, filtered on cells where the gRNA expression was detected. ( C ) Arterial ( GJA4 , DLL4 ), venous ( NRP2 , APLNR ), and haemogenic marker ( CD44 , RUNX1 ) expression distribution in the clusters indicated by the colour. ( D ) Expression distribution visualised on the UMAP plot showing the location of arterial cells marked by DLL4 , and haemogenic endothelium marked by CD44 and RUNX1 . ( E ) Heatmap of the top 15 marker genes for each of the clusters. ( F ) Contribution of the different libraries to the clusters showing that arterial cell cluster is overrepresented in the iSAM_AGM treated with DOX, compared to the other libraries. ( G ) Expansion of the arterial population assessed by the membrane marker expression of DLL4+ following targets’ activation, quantified by flow cytometry at day 8 of differentiation. (Data are normalised on the iSAM_NT+DOX sample, n=5 independent differentiations, *p=0.0417 paired t-test.) ( H ) Colony-forming potential of the suspension progenitor cells derived from the two lines treated with or without DOX following OP9 coculture activation, data show the colony obtained for 104 CD34+ input equivalent (n=3 from independent differentiations *p<0.05, Tukey’s two-way ANOVA). Figure 3—source data 1. Spreadsheet source file containing the source data used for the plots in . Each tab is labelled to uniquely refer to a specific panel.

Article Snippet: For flow cytometry 10 5 cells per test were stained in 50 μl of staining solution with the following antibodies: CD34 Percp-Efluor710 (4H11 eBioscience, 1:100), CD34 Pe (4H11 eBioscience, 1:200), CD43 APC (eBio84-3C1, 1:100), CD45 FITC (2D1 eBioscience, 1:100), DLL4 Pe (MHD4-46 BioLegend, 1:200), CD41 PE (HIP8 BioLegend, 1:200), CD144 APC (16B1 eBioscience, 1:100), CD235a FITC (HIR2 BD Bioscience, 1:250).

Techniques: Gene Expression, Activation Assay, Expressing, Control, Marker, Membrane, Flow Cytometry, Suspension, Derivative Assay

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Violin plot of IGFBP2 expression profile in the arterial cells obtained from the different conditions, in the presence or absence of gRNAs and doxycycline (DOX). ( B ) Number of haematopoietic colonies obtained after coculture on OP9 in the presence or absence of IGFBP2 (n=3–4 from independent differentiations, **p=0.0080, Sidak’s two-way ANOVA). ( C ) Percentage of DLL4 + arterial cells differentiation within the CD34 + compartment analysed by flow cytometry in day 8 embryoid bodies (EBs) (n=4 from independent differentiations, two-way ANOVA, ns = p>0.99). ( D ) Expansion of haematopoietic progenitors analysed using markers’ expression on suspension progenitors derived after coculture of CD34+ cells onto OP9 support (data are expressed as fold over the CTR in the absence of IGFBP2; n=4 from independent differentiations, *p<0.02, Sidak’s two-way ANOVA). ( E ) Single-cell transcriptomic analysis of developing aorta-gonad-mesonephros (AGM) collected from human embryos at Carnegie stages14 and 15 enriched for CD31+ and CD34+ showing the IGFBP2 expression profile in vivo in the AGM. Figure 4—source data 1. Spreadsheet source file containing the source data used for the plots in . Each tab is labelled to uniquely refer to a specific panel.

Article Snippet: For flow cytometry 10 5 cells per test were stained in 50 μl of staining solution with the following antibodies: CD34 Percp-Efluor710 (4H11 eBioscience, 1:100), CD34 Pe (4H11 eBioscience, 1:200), CD43 APC (eBio84-3C1, 1:100), CD45 FITC (2D1 eBioscience, 1:100), DLL4 Pe (MHD4-46 BioLegend, 1:200), CD41 PE (HIP8 BioLegend, 1:200), CD144 APC (16B1 eBioscience, 1:100), CD235a FITC (HIR2 BD Bioscience, 1:250).

Techniques: Expressing, Flow Cytometry, Suspension, Derivative Assay, In Vivo

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet: ( A ) Dot plot showing the expression profile of the genes coding for the enzymes mediating glycolysis in CTR and IGFBP2-treated cells at day 10 and 13. ( B ) Dot plot showing the expression profile of the genes coding for the checkpoints of the glycolysis in CTR and IGFBP2-treated cells at days 10 and 13. ( C ) Percentage of cells expressing different markers following OP9 coculture of CD34+ cells (ns for all markers, Kruskall-Wallis one-way ANOVA). ( D ) OP9 cocultures of CD34+ DLL4+ and CD34+DLLL4- (scale bar indicates 50 μm), and in ( E ) their colony formation capacities following 1 week of OP9 coculture (mixed-effect analysis, Sidak’s post-test, ****p<0.0001, **p<0.01). ( F ) Cluster composition in cells treated with IGFBP2 data shows the composition at day 10 and day 13. Figure 5—figure supplement 1—source data 1. Spreadsheet source file containing the source data used for the plots in . Each tab is labelled to uniquely refer to a specific panel.

Article Snippet: For flow cytometry 10 5 cells per test were stained in 50 μl of staining solution with the following antibodies: CD34 Percp-Efluor710 (4H11 eBioscience, 1:100), CD34 Pe (4H11 eBioscience, 1:200), CD43 APC (eBio84-3C1, 1:100), CD45 FITC (2D1 eBioscience, 1:100), DLL4 Pe (MHD4-46 BioLegend, 1:200), CD41 PE (HIP8 BioLegend, 1:200), CD144 APC (16B1 eBioscience, 1:100), CD235a FITC (HIR2 BD Bioscience, 1:250).

Techniques: Expressing

Journal: eLife

Article Title: A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro

doi: 10.7554/eLife.94884

Figure Lengend Snippet:

Article Snippet: For flow cytometry 10 5 cells per test were stained in 50 μl of staining solution with the following antibodies: CD34 Percp-Efluor710 (4H11 eBioscience, 1:100), CD34 Pe (4H11 eBioscience, 1:200), CD43 APC (eBio84-3C1, 1:100), CD45 FITC (2D1 eBioscience, 1:100), DLL4 Pe (MHD4-46 BioLegend, 1:200), CD41 PE (HIP8 BioLegend, 1:200), CD144 APC (16B1 eBioscience, 1:100), CD235a FITC (HIR2 BD Bioscience, 1:250).

Techniques: Cloning, Plasmid Preparation, Recombinant, Expressing, Sequencing, Flow Cytometry, Amplification

Journal: bioRxiv

Article Title: Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states

doi: 10.1101/2024.07.31.606073

Figure Lengend Snippet: A. Identification of a myofibroblast gene expression program and its regulators. Top genes defining the program form the rows of the heatmap, and top perturbations repressing (left) or activating (right) the program form the columns. Green denotes canonical marker genes or perturbations while magenta marks transcription factor hits highlighted in further analysis. B. Morphological changes and ACTA2 induction by HES7 . C. Identification of an inflammatory fibroblast gene expression program and its regulators. In vivo inflammatory state marker genes, including COL1A1, COL1A2, COL3A1, COL5A2, SPARC , are colored yellow with highlighted perturbations sharing the same color. Genes exhibiting overlap with inflammatory state genes in references 53 and 54 respectively are indicated (left). D. To score the ability of perturbations to induce new expression states, we counted how many genes they activated that were unexpressed in control cells. Labeled points in the box plots show outlier perturbations for each cell type. E. Examples of genes newly activated by top Hs27 hits from this analysis. Plot is colored according to how many independent guides led to activation. Marker genes PI16 and CD34 , associated with the universal fibroblasts, are highlighted. F. Perturbations that induced markers of universal fibroblasts (see E) also activated a sparse gene expression program. Genes exhibiting overlap with universal state genes in references 53 and 54 respectively are indicated (left). G. Flow cytometry validation of PI16 induction by predicted regulators using CRISPRa. H. Flow cytometry validation of PI16 induction by predicted regulators using cDNA overexpression.

Article Snippet: Cells were stained with PI16-APC (Miltenyi, 130-111-177) and/or CD34-PerCP-Cy5.5 (BD, 347213) for 30 minutes on ice in the dark, washed, and analyzed on BD LSRFortessa or Thermo Fisher Attune flow cytometer.

Techniques: Expressing, Marker, In Vivo, Control, Labeling, Activation Assay, Flow Cytometry, Over Expression

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Patients With Coronary Microvascular Dysfunction Have Less Circulating α‐Klotho

doi: 10.1161/JAHA.123.031972

Figure Lengend Snippet: Baseline Characteristics Among Patients With and Without CMD

Article Snippet: Cell fluorescence (FACSCalibur, Becton Dickson) was measured immediately following immunofluorescent staining by CD34‐PerCP Cy 5.5 (Becton Dickson) and KDR‐APC (R&D Systems) by a laboratory technician blinded to patient study data using CellQuest software (Becton Dickson).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Patients With Coronary Microvascular Dysfunction Have Less Circulating α‐Klotho

doi: 10.1161/JAHA.123.031972

Figure Lengend Snippet: Association Between Higher Circulating Klotho and EPCs

Article Snippet: Cell fluorescence (FACSCalibur, Becton Dickson) was measured immediately following immunofluorescent staining by CD34‐PerCP Cy 5.5 (Becton Dickson) and KDR‐APC (R&D Systems) by a laboratory technician blinded to patient study data using CellQuest software (Becton Dickson).

Techniques: