recombinant mouse fgf 10  (R&D Systems)

Journal: Stem Cells Translational Medicine

Article Title: FGF2 alters the calvarial suture niche homeostasis shifting skeletal stem cell/progenitors toward an osteo-angiogenic coupling fate

doi: 10.1093/stcltm/szag003

Figure Lengend Snippet: Morphology of FGF-treated calvarial suture explants. A, Schematic outlining the experimental procedure. B, Phase contrast images of a representative calvarial suture (COR suture) explant after 8 days in culture with recombinant FGF2 protein (100 ng/mL) revealing the presence of a tube-like structure (red arrows) sprouting from the edge (green arrow) of the suture (magnification at 10×). C, 20× magnification of the red dashed boxed area in panel B. Panel D highlights the presence of round cells; bottom panel D(i) is a magnification at 40× of the boxed area in panel D, and green arrows mark the small, round, and bright cells. E, Phase contrast image of FGF2 treated PF suture shows a morphology similar to FGF2 treated COR suture. Green arrows point to the suture edge. Red arrows mark tube-like structures and blue arrow a lacunae-like formation. COR calvarial suture explant control. Green arrows point the suture edge. Magnification at 20×. F, Phase contrast image of untreated COR calvarial suture explant control. G, Phase contrast image of untreated PF calvarial suture explant control. Green arrows point the suture edge. H, Cartoon depicting the following experimental steps. I-K, Representative phase contrast images of cells isolated by FACS sorting from FGF2 treated COR suture explants cultured in presence of FGF2 (100 ng/mL), followed by VEGF (150 ng/mL) addition at day 4 for additional 48 hours (total 6 days of culture). I, Cells display a foci formation (dashed green circle) and sprouting of tubular structures in panels J and K, (green arrows). Magnification at 20× and 40×. L, Control cells isolated from untreated calvarial suture explants grow in a monolayer without forming structures resembling either foci or tube-like structures (magnification at 20×). Experiments were performed at least 3 times. Scale bars: 100 µm in panel B; 50 µm panels (C, D, E, F, G, I, J, I, L); 20 µm in panel D(i) and K. Abbreviations: COR, coronal; FGF, fibroblast growth factor.

Article Snippet: Recombinant mouse FGF2 protein (#3139, R&D System) was added at concentration of 100 ng/mL.

Techniques: Recombinant, Control, Isolation, Cell Culture

Journal: Stem Cells Translational Medicine

Article Title: FGF2 alters the calvarial suture niche homeostasis shifting skeletal stem cell/progenitors toward an osteo-angiogenic coupling fate

doi: 10.1093/stcltm/szag003

Figure Lengend Snippet: Bulk RNA-Seq profiling of skeletal stem cell/progenitor population isolated from FGF2 treated and untreated sutures. A and B, Heatmaps of endothelial cell and endothelial progenitor cell marker genes, highlight a distinct upregulation of endothelial and endothelial progenitor cells (EPC) in FGF2 treated cells. Red color: upregulation (≥3-fold); Green color: downregulation. (−≥3-fold). The (+) symbol points to genes that are upregulated in the cell populations analyzed. C, Heatmap of osteogenic marker downregulation in cells as above. D, Validation of up- and down-regulation of key endothelial and osteogenic markers by RT-PCR analysis. E, Diagram/cartoon summarizing the trend in the expression profiles of key endothelial and osteogenic markers up- or downregulated by (≥3-fold) in cells isolated from FGF2 treat and untreated sutures.

Article Snippet: Recombinant mouse FGF2 protein (#3139, R&D System) was added at concentration of 100 ng/mL.

Techniques: RNA Sequencing, Isolation, Marker, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Stem Cells Translational Medicine

Article Title: FGF2 alters the calvarial suture niche homeostasis shifting skeletal stem cell/progenitors toward an osteo-angiogenic coupling fate

doi: 10.1093/stcltm/szag003

Figure Lengend Snippet: Chromatin accessibility between skeletal stem cells/progenitors derived from FGF2 treated and untreated calvarial sutures. A, Representation of enriched chromatin active motifs identified in skeletal stem cell/progenitor population isolated from FGF2 treated calvarial suture explants and predicted to function as activated transcription sites. Associated transcription factors and target genes are listed. B, Heatmap of peaks differentially expressed in cell isolated from FGF2 treated suture explants and untreated suture explants. C, Pie charts showing the proportion of chromatin open regions. D, Go-enrichment of biological processes in cells isolated from FGF2 treated suture explants unveiling blood vessel morphogenesis as major enriched process. E, KEGG enrichment analysis also reveals processes associated to angiogenesis, such as hemostasis, developmental biology, and extracellular matrix remodeling. F, Reactome enrichment analysis showing the gene ratio/intensity related to the biological processes listed in E.

Article Snippet: Recombinant mouse FGF2 protein (#3139, R&D System) was added at concentration of 100 ng/mL.

Techniques: Derivative Assay, Isolation

Journal: Stem Cells Translational Medicine

Article Title: FGF2 alters the calvarial suture niche homeostasis shifting skeletal stem cell/progenitors toward an osteo-angiogenic coupling fate

doi: 10.1093/stcltm/szag003

Figure Lengend Snippet: CITE sequencing analysis of skeletal stem cell/progenitor population isolated from FGF2 treated and untreated calvarial sutures. A, UMAP analysis showing changes in the representation between cells isolated from untreated (control) and FGF2 treated calvarial suture explants. B, Pies illustrating cluster-ratio overview of cell subpopulations isolated from untreated (control) and FGF2 treated suture explants (yellow lines mark clusters increased in cell representation; purple lines clusters with decreased cell representation) C, CITE-Seq Heatmap of the surface markers CD31 (PECAM-1), CD133 (Prominin-1), Sca-1 and CD200 reveals their increased levels in cells derived from FGF2 treated sutures (yellow indicates downregulation; purple upregulation). D, Violin plots showing the distribution of cells expressing the four CITE-Seq markers (top panel) and histogram of gene module scores expression (bottom panel) in control and FGF2 treated populations. E, Violin plots showing the distribution of cells expressing the four CITE-Seq markers (upper panel) in all 10 clusters and histogram of genes module scores (bottom panel). F, CITE-Seq heatmap of the surface markers CD31 (PECAM-1), CD133 (Prominin-1), Sca-1 and CD200 in all 10 clusters. G, Illustration of RNA clusters of cells expressing key markers of the osteogenic lineage by Violin plots (left panel), osteogenic modules (middle panel), and heatmap (right panel). H, Illustration of RNA clusters of cells expressing key markers of the endothelial/angiogenic lineage by Violin plots (left panels), angiogenic modules (top right panel), and heatmap (bottom right panel). I, Cartoon illustrates inferred trajectories/directions of lineages differentiation. J, Shows the velo latent time of differentiation for the different clusters. Yellow color indicates the more differentiated status of the cell populations.

Article Snippet: Recombinant mouse FGF2 protein (#3139, R&D System) was added at concentration of 100 ng/mL.

Techniques: Sequencing, Isolation, Control, Derivative Assay, Expressing

Journal: Stem Cells Translational Medicine

Article Title: FGF2 alters the calvarial suture niche homeostasis shifting skeletal stem cell/progenitors toward an osteo-angiogenic coupling fate

doi: 10.1093/stcltm/szag003

Figure Lengend Snippet: Skeletal stem cell/progenitor population isolated from FGF2 treated suture explants display an endothelial functional profile. A, Confocal microphotographs of an in vitro Matrigel assay performed on a pool of cells isolated from FGF2 treated PF and COR sutures and untreated sutures. Tube-like structures are observed after 18 hours. Staining dye (green color) shows the endothelial tube formation (left panel). Middle panel showing absence of tube-like structure formation in cells isolated from untreated calvarial sutures explants. Addition of the inhibitor vinblastine suppresses tube-like formation (right panel) in cells isolated from FGF2 treated suture explants. Experiments were performed three times. B, Quantification of in vitro tube-like formation by length measurement using ImageJ2 program. Results are presented as mean ± SD of tube-length formation. Analysis was performed on four independent assays. A significant difference in tube-formation length is observed in FGF2 treated cells. C, Acetylated LDL uptake by pool of cells isolated from FGF2 treated PF and COR sutures is confirmed by staining of DiI dye (left panel), whereas cells isolated from untreated calvarial suture explants do not uptake Acetylated LDL (middle panel). Likewise, in the presence of the inhibitor Protein-S (P-S), cells isolated from FGF2 treated sutures do not uptake acetylated LDL (right panel). D, Quantification of acetylated LDL uptake using ImageJ2 program (ImageJ 2, NIH) confirms a significant increased uptake by FGF2 treated cells as compared to untreated and P-S treated cells. Densitometric results are mean ± SD of the threshold levels from two independent experiments. E, H&E staining of OCT cryo-tissue sections from in vivo Matrigel plug assay showing the ability of cells isolated from FGF2 treated sutures to form vessels (left panel) which stained positive for CD31/PECAM-1 (green color) as assessed by immunofluorescence using specific antibodies (middle panel). Immunostaining using Sca-1 antibodies (red color), a marker associated with endothelial progenitor cells (EPC), reveals the presence of positive cells (right panel), and their partial colocalization with CD31/PECAM-1 positive cells as shown in panel F. G, Matrigel plug assay performed with cells isolated from untreated calvarial sutures does not reveal any vessel formation. Scale bars: 100 µm in panels A and C; 50 µm in panels E and G; 20 µm in panel F.

Article Snippet: Recombinant mouse FGF2 protein (#3139, R&D System) was added at concentration of 100 ng/mL.

Techniques: Isolation, Functional Assay, In Vitro, Matrigel Assay, Staining, In Vivo, Immunofluorescence, Immunostaining, Marker

Journal: Stem Cells Translational Medicine

Article Title: FGF2 alters the calvarial suture niche homeostasis shifting skeletal stem cell/progenitors toward an osteo-angiogenic coupling fate

doi: 10.1093/stcltm/szag003

Figure Lengend Snippet: Osteo-induction assay of skeletal stem cell/progenitor population isolated from FGF2 treated and untreated suture explants. A and B, Contrast-phase microphotographs of an osteogenic assay stained with von-Kossa procedure after 21 days in culture with osteogenic medium. Cells isolated from FGF2 treated suture explants (pool of PF and COR sutures) form a very dense and patchy mineralized areas as revealed by von Kossa staining (magnification at 20×). Right panels are a magnification at 40× of the red dashed boxed areas in panel A and B, green arrows mark the presence of tube-like structures. C, Von Kossa staining of cells derived from untreated suture explants showing a diffuse ECM mineralization and some bone nodules (yellow arrows) (magnification at 20×). Right panel is a magnification at 40× of the red dashed boxed area in panel C. D, RT-PCR analysis of osteogenic and endothelial markers profiles expression of both osteogenic and angiogenic markers in cells isolated from FGF2 treated suture explants (top panel), cells express all four markers. E, Cells isolated from untreated suture explants (pool of PF and COR sutures) express less of the osteogenic marker Runx2 and low levels of Bglap and Ve-cadherin, while no expression of the endothelial marker CD3 1 ( Pecam1 ) is detected (bottom panel). Experiments were performed two times. Scale bars: 100 µm in panels A-C (left side); 50 µm in panels A-C (right side). F, Densitometric analysis of the PCR bands performed by ImageJ2 program (ImageJ 2, NIH). The densitometric results were normalized to their respective loading control ( Gapdh -bands) and presented as percent increase (100%). Results are mean ± SD of three independent analyses. Statistical significance: * P <.05.

Article Snippet: Recombinant mouse FGF2 protein (#3139, R&D System) was added at concentration of 100 ng/mL.

Techniques: Isolation, Staining, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Control