t-sne maps  (BrainScope)

Journal: Nature Communications

Article Title: Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis

doi: 10.1038/s41467-019-09459-5

Figure Lengend Snippet: Gene and protein expression profiles of human valve progenitors. SSEA-1+-sorted human MESP1+ cells were treated with 10 ng/ml FGF8, 2 ng/ml FGF2 and 30 ng/ml VEGF for 6 days. a RNA was then collected and cDNAs of FGF8/VEGF-treated cells (HPVCs) were run in Real-Time PCR (mean ± SEM of 9 separate cell differentiation experiments). Data are normalized to 1 as the level of gene expression in SSEA1+MESP1+ cells. (**significantly different from 1; p ≤ 0.01). Boxes and whiskers (min to max) show the values lower than the 2.5th percentile and greater than the 97.5th percentile as circles. b cRNAs ( n = 3 separate cell differentiation experiments) were used for microarrays and normalized vs. MESP1+ cells from the same respective cell sorting. Heatmaps of transcriptomes of HPVCs, E9AVC (our data), AVC GDS3663 and MSCs (GDS1288). A few AVC-specific genes are highlighted in the inset. c Bright field image (top) and co-immunostaining of VEGF/FGF8/FGF2-treated SSEA-1+/MESP1+ derived colonies with anti-CD31 and anti-Flk1(KDR) or anti-VE-cadherin antibodies. Data are representative of 5 separate cell differentiation experiments. d Immunostaining of VEGF/FGF8-treated SSEA-1+/MESP1+ derived colonies with anti-Sox9, -Msx1, -Nfatc1,-Tbx3, -Tbx20 and -GATA5 antibodies (green) and DAPI (blue). The data are representative of 5 experiments. The scale bar indicates 50 μm. e HPVCs were further sorted using anti-CD31 conjugated beads and used in single-cell RNA sequencing. t-distributed stochastic neighbor embedding (t-SNE) 2D cell map 10X genomics ( n = 2440 cells) (upper panel). Highlight of cell populations expressing genes marking endothelial, hemogenic and early EMT cells (lower panel) and heatmap of graph-based Log2 fold changes in gene expression of cell cluster compared to all other cells (lower right panel). Source data are provided as a Source Data file

Article Snippet: Fig. 7 Single cell-sequencing of DCHS1 c.6988 C > T post-EMT valvular interstitial cells. a t-distributed stochastic neighbor embedding (t-SNE) 2D cell map 10X genomics ( n = 4316 cells). b PCA of wt versus DCHS1 c.6988 C > T post-EMT (BMP2 treated) HPVC cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Differentiation, Gene Expression, FACS, Immunostaining, Derivative Assay, RNA Sequencing

Journal: Nature Communications

Article Title: Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis

doi: 10.1038/s41467-019-09459-5

Figure Lengend Snippet: EMT of HPVC cells. a After 6 days of FGF8/FGF2/VEGF treatment on MEFs, valve progenitors (HPVCs) were recovered with trypsin, seeded on fibronectin-coated wells and treated with 100 ng/ml BMP2. After 2 days, RNA was recovered and cDNAs were run in real-time PCR for post EMT markers. BMP2 samples are normalized on control (before treatment) samples, showing an increase in the expression of post-EMT markers Data are representative of 5 cell differentiation and EMT- induction experiments.Boxes and whiskers (min to max) show the values lower than the 2.5th percentile and greater than the 97.5th percentile as circles. (*) significantly different p ≤ 0.01. b t-distributed stochastic neighbor embedding (t-SNE) 2D cell map 10X genomics ( n = 3700) cells (left panel). Highlight of cell populations expressing TAGLN and genes marking more specifically fibrosa ( COL1A1 ) or spongiosa ( CD9, TDGF1 ) (middle panel) and heatmap of graph-based Log2 fold changes in gene expression of cell cluster compared to all other cells (right panel). c HPVCs give rise to tendinous/chondrogenic cells. HPVCs were pelleted into a tube and treated with a chondrogenic medium as described in the methods. Real-Time PCR shows an upregulation of Scleraxis and Collagen 1a ( Col 1 ) genes versus non-treated cells following three weeks of treatment (two experiments in duplicate; (*) significantly different p ≤ 0.01). d The cell pellets were embedded in paraffin and 5 um sections stained with anti Sox9, anti-Collagen1a and anti-calcitonin antibodies. Data are representative of 3 experiments. The scale bar indicates 50 μm. Source data are provided as a Source Data file

Article Snippet: Fig. 7 Single cell-sequencing of DCHS1 c.6988 C > T post-EMT valvular interstitial cells. a t-distributed stochastic neighbor embedding (t-SNE) 2D cell map 10X genomics ( n = 4316 cells). b PCA of wt versus DCHS1 c.6988 C > T post-EMT (BMP2 treated) HPVC cells.

Techniques: Real-time Polymerase Chain Reaction, Control, Expressing, Cell Differentiation, Gene Expression, Staining

Journal: Nature Communications

Article Title: Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis

doi: 10.1038/s41467-019-09459-5

Figure Lengend Snippet: Single cell-sequencing of DCHS1 c.6988 C > T post-EMT valvular interstitial cells. a t-distributed stochastic neighbor embedding (t-SNE) 2D cell map 10X genomics ( n = 4316 cells). b PCA of wt versus DCHS1 c.6988 C > T post-EMT (BMP2 treated) HPVC cells. Inset: PCA of cell cycle genes expressed in wt versus DCHS1 c.6988 C > T . c Graph-based Log2 fold changes in gene expression of cell clusters compared to all other cells (left panel) and highlight of cell populations expressing genes marking fibrosa or spongiosa valve layers

Article Snippet: Fig. 7 Single cell-sequencing of DCHS1 c.6988 C > T post-EMT valvular interstitial cells. a t-distributed stochastic neighbor embedding (t-SNE) 2D cell map 10X genomics ( n = 4316 cells). b PCA of wt versus DCHS1 c.6988 C > T post-EMT (BMP2 treated) HPVC cells.

Techniques: Sequencing, Gene Expression, Expressing

Journal: Nature Communications

Article Title: Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis

doi: 10.1038/s41467-019-09459-5

Figure Lengend Snippet: Gene and protein expression profiles of human valve progenitors. SSEA-1+-sorted human MESP1+ cells were treated with 10 ng/ml FGF8, 2 ng/ml FGF2 and 30 ng/ml VEGF for 6 days. a RNA was then collected and cDNAs of FGF8/VEGF-treated cells (HPVCs) were run in Real-Time PCR (mean ± SEM of 9 separate cell differentiation experiments). Data are normalized to 1 as the level of gene expression in SSEA1+MESP1+ cells. (**significantly different from 1; p ≤ 0.01). Boxes and whiskers (min to max) show the values lower than the 2.5th percentile and greater than the 97.5th percentile as circles. b cRNAs ( n = 3 separate cell differentiation experiments) were used for microarrays and normalized vs. MESP1+ cells from the same respective cell sorting. Heatmaps of transcriptomes of HPVCs, E9AVC (our data), AVC GDS3663 and MSCs (GDS1288). A few AVC-specific genes are highlighted in the inset. c Bright field image (top) and co-immunostaining of VEGF/FGF8/FGF2-treated SSEA-1+/MESP1+ derived colonies with anti-CD31 and anti-Flk1(KDR) or anti-VE-cadherin antibodies. Data are representative of 5 separate cell differentiation experiments. d Immunostaining of VEGF/FGF8-treated SSEA-1+/MESP1+ derived colonies with anti-Sox9, -Msx1, -Nfatc1,-Tbx3, -Tbx20 and -GATA5 antibodies (green) and DAPI (blue). The data are representative of 5 experiments. The scale bar indicates 50 μm. e HPVCs were further sorted using anti-CD31 conjugated beads and used in single-cell RNA sequencing. t-distributed stochastic neighbor embedding (t-SNE) 2D cell map 10X genomics ( n = 2440 cells) (upper panel). Highlight of cell populations expressing genes marking endothelial, hemogenic and early EMT cells (lower panel) and heatmap of graph-based Log2 fold changes in gene expression of cell cluster compared to all other cells (lower right panel). Source data are provided as a Source Data file

Article Snippet: The scale bar indicates 50 μm. e HPVCs were further sorted using anti-CD31 conjugated beads and used in single-cell RNA sequencing. t-distributed stochastic neighbor embedding (t-SNE) 2D cell map 10X genomics ( n = 2440 cells) (upper panel).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Differentiation, Gene Expression, FACS, Immunostaining, Derivative Assay, RNA Sequencing