fgf18v3  (ProCore Bio Med Ltd)

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: (A) Sox9 mRNA (n=4) at the end of 14 days of expansion (Exp. - d0) and at early chondrogenic differentiation in aggregate cultures (Diff. – d3) with and without FGF2. Statistical comparisons made against control-expanded data at d0 and d3 independently (left panel) and against d0 for both groups (right panel). (B) Sox9 protein at the end of 14 days of expansion in control and FGF2-rich medium. (C) FGFR1 and FGFR3 (n=3) gene expression profiles (assessed by qRT-PCR) evaluated after 14 days of expansion (d0) and subsequent aggregate culture for 21 days. (#): Statistical significance (p<0.0001) when data compared to control-expanded at d0; (&): Statistical significance (p<0.0001) when data compared to FGF2-expanded at d0. Statistical significances (p values) when Control and FGF2-expanded cells are compared at a particular time-point are shown in the graph for clarity. Gene expression analysis was performed for all four donors (Sox9) and three donors (FGFRs) using triplicate samples.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Control, Gene Expression, Quantitative RT-PCR

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: Control and FGF2-expanded cells (n=3) were subjected to FGF9 or FGF18 stimulation from d0 to d21 of differentiation induction in pellet cultures, where d0 data corresponds to expanded cells just before aggregate formation. Pellet cultures were harvested at day 5, 10, 15 and 21, and qRT-PCR performed to evaluate FGFR3 and FGFR1 gene expression (A & B) and FGFR3:FGFR1 ratio (C). Results with non-stimulated Control (black dots) and FGF2-expanded cells (white dots) are shown again as in Fig 1C for reference (statistical differences presented in Fig 1C). Data obtained at every time point with FGF9 and FGF18 stimulation were compared with Control-expanded and FGF2-expanded cells at the same point, and the statistical differences (p values) shown in the graph for clarity. All other comparison were p>0.1.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Control, Quantitative RT-PCR, Gene Expression, Comparison

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: FGF2-expanded cells were stimulated with ligands or mutant variants for 7 days starting at d14 (until d21). Receptor neutralizing antibodies were added 48 hours in advance (d12) to assure blocking before ligands were administered. Pellet shown in the insert (control) corresponds to black dots shown on B). (A) Histological assessment of representative pellets confirming the results with FGF ligands and mutants (as presented in Fig. 3 – d14), and the determinant role of FGFR3 in this effect, (B) GAG/DNA quantification and gene expression analysis of type 2 collagen and aggrecan, assessed by qRT-PCR performed at day 21. Statistical significance (#) was obtained comparing all values to control pellet (black dots) with the following p values: for GAG/DNA: β+9 (0.0007), β+9v1 (<0.0001), β+9+R1 (0.0004), β+9+R3 (0.0012), β+18 (0.0008), β+18v3 (0.0002), β+18+R1 (0.0091), β+18+R3 (0.0100). For Aggrecan expression: β+9v1 (<0.0013) and β+18v3 (0.0003). For Col2 expression β+9v1 (0.0002), β+18v3 (0.0001), β+18+R1 (0.0009). All other comparisons were p > 0.1. The effects of blocking FGFR1 and FGFR3 receptors when compared with respective growth factor alone are shown in the graph with significant differences (p values) displayed in color. N=4.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Mutagenesis, Blocking Assay, Control, Gene Expression, Quantitative RT-PCR, Expressing

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: (A) Gene expression profile of hypertrophy markers showing an early onset of terminal differentiation of FGF2-expanded cells compared with control-expanded cells. P values are shown for each gene in the graph for clarity. (B) Gene expression profile assessment of FGF2-expanded cells (n=4) stimulated with FGF9, FGF18 and variants along with blocking FGFR3 (+R3) or FGFR1 (+R1) antibodies for two weeks (d14 to d28). Compared to control pellets without stimulation (β), statistical significance (p<0.0001) was obtained for the majority of conditions (#) in all four genes. The effects of blocking FGFR1 and FGFR3 receptors when compared with respective growth factor alone are shown in the graph with significant differences (p values) displayed in color. N=4.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Gene Expression, Control, Blocking Assay

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: Induced terminal differentiation of FGF2-expanded hMSC evaluated cytomorphologically6,7 (top row) and by the activity of the hypertrophy-specific marker Alkaline Phosphatase (ALP) in sections (lower row) and quantified (dA/min = changes of Absorbance at A405 in time within the linear range) in the medium6 (insert). Compared to control, all conditions with hypertrophy induction are statistically different (p<0.0001). Compared to hypertrophy alone (H): the addition of FGF9 (p=0.0441) and FGF18 (p=0.0280) delayed the appearance of those enhanced terminal differentiation characteristics. Blocking FGFR3 prevented this effect (with FGF9: p=0.1975, and with FGF18: p=0.4863), while blocking FGFR1 did not (with FGF9: p=0.0459, and with FGF18: p=0.0478). This effect of blocking the receptors is further supported when compared to the growth factors alone: p values shown in graph for clarity and significant differences displayed in color. N=4.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Activity Assay, Marker, Control, Blocking Assay

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: FGF2 is used to increase cell number and prime the cells for chondrogenesis (via Sox9 early appearance) during expansion, followed by stimulation with TGF-β to start chondrogenesis and either FGF9, FGF18 or FGFR3-specific ligands at day 14 (high FGFR3:FGFR1 ratio) to induce ECM secretion and delay the appearance of a hypertrophic phenotype. The program is intended to induce hMSC differentiation into a more stable pre-hypertrophic phenotype, typical of articular cartilage chondrocytes. Figure made with images available at Servier Medical Art (www.servier.fr).

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: