Recombinant Human Fibroblast growth factor 9 (FGF9) (Active)

How is recombinant human FGF9 typically produced and what quality control parameters should researchers monitor?

Recombinant human FGF9 is predominantly produced using bacterial expression systems, most commonly in Escherichia coli, although some suppliers use insect cell expression systems such as Spodoptera frugiperda (Sf21) . For research applications, quality control should focus on:

When evaluating recombinant FGF9 preparations, researchers should confirm these parameters in the certificate of analysis and perform additional functional validation through bioactivity assays relevant to their experimental system .

What signaling pathways does FGF9 activate and how can researchers effectively assess its functional activity?

FGF9 binds and activates the 'c' splice isoforms of FGF receptors 1-3 (FGFR1-3), with particular affinity for FGFR3 (IIIb) . Upon receptor binding, FGF9 activates several downstream signaling cascades:

• ERK (Extracellular signal-regulated kinase) pathway

• JNK (c-Jun N-terminal kinase) pathway

• STAT (Signal transducer and activator of transcription) pathway

• PI3K (Phosphoinositide 3-kinase) pathway

• MAP kinase pathway

To assess FGF9 functional activity, researchers can employ several methodological approaches:

• Proliferation assays: The standard bioactivity assay measures cell proliferation in Balb/3T3 mouse embryonic fibroblast cells, with an expected ED50 of 1-5 ng/mL .

• Luciferase reporter assays: Serum response element (SRE) luciferase reporter assays in transfected HEK293T cells provide quantitative assessment of FGF9 activity (typical EC50 ≈ 246.2 pM or 5.7 ng/mL) .

• Phosphorylation analysis: Western blotting for phosphorylated ERK, JNK, and other pathway components following FGF9 stimulation .

• Clonogenicity assays: Measuring colony number and size in responsive cell lines (e.g., Hep3B, HepG2) .

• Migration assays: Transwell Boyden chamber assays to assess directed migration of cells in response to FGF9 .

How should researchers properly reconstitute and store recombinant FGF9 to maintain optimal activity?

Proper handling of recombinant FGF9 is critical for maintaining its biological activity:

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to bring the contents to the bottom.

• Reconstitute lyophilized FGF9 in deionized sterile water or sterile PBS to a concentration of 0.1-1.0 mg/mL.

• For long-term storage stability, add 5-50% glycerol (final concentration) .

• Allow the protein to sit for 10-15 minutes at room temperature to ensure complete solubilization.

Storage Recommendations:

• Store reconstituted protein at -20°C or -80°C in small aliquots to avoid repeated freeze-thaw cycles.

• For lyophilized protein, the recommended shelf life is 12 months at -20°C/-80°C.

• For reconstituted protein in liquid form, the shelf life is approximately 6 months at -20°C/-80°C .

Researchers should note that different commercial preparations may have specific reconstitution buffers optimized for stability, such as "20 mM PB, 150 mM NaCl, 5% Trehalos, pH 7.4" or "20 mM PB, 220 mM Sucrose, 0.02% Tween 80, pH 6.0" . Always refer to the product-specific Certificate of Analysis for optimal handling conditions.

How does FGF9 contribute to hepatocellular carcinoma (HCC) progression and what experimental models best recapitulate its effects?

FGF9 plays a significant role in hepatocellular carcinoma (HCC) progression through several mechanisms:

• Cellular source in HCC: FGF9 is primarily expressed by activated hepatic stellate cells (HSC) and cancer-associated myofibroblasts in the tumor microenvironment, not by HCC cells themselves .

• Clinical correlation: High expression levels of FGF9 significantly correlate with poor patient survival, and FGF9 expression positively correlates with alpha-smooth muscle actin (alpha-sma) expression in HCC tissues .

• Pro-tumorigenic effects: FGF9 enhances:

  • Cell proliferation through ERK and JNK pathway activation

  • Clonogenicity (reflecting stem cell properties and cell survival)

  • Migratory capacity of HCC cells

  • Therapeutic resistance, particularly to sorafenib

• Receptor specificity: FGF9's effects on HCC cells are mediated primarily through FGFR1/2/3, as the selective FGFR4 inhibitor BLU9931 had no significant effect, while the FGFR1/2/3 inhibitor BGJ398 abrogated FGF9's pro-tumorigenic effects .

Recommended experimental models:

For comprehensive analysis of FGF9's role in HCC, researchers should consider combining these models with analysis of signaling pathway activation (ERK, JNK) and therapeutic response assays .

What are the methodological considerations for using FGF9 in kidney organoid generation?

FGF9 is frequently used in the generation of kidney organoids due to its developmental roles. When incorporating FGF9 into kidney organoid protocols, researchers should consider:

• Concentration optimization: Titrate FGF9 concentrations (typically starting in the 50-200 ng/mL range) to determine optimal dosing for your specific protocol and cell lines.

• Timing of administration: FGF9 signaling is stage-specific during kidney development. Consider pulse treatments rather than continuous exposure to mimic developmental signaling dynamics.

• Receptor expression verification: Confirm expression of appropriate FGF receptors (especially FGFR1-3) in your stem cell population before FGF9 treatment using RT-PCR or western blotting.

• Combinatorial signaling: FGF9 often works in concert with other growth factors. Consider combining with:

  • WNT pathway modulators (e.g., CHIR99021)

  • BMP inhibitors (e.g., Noggin)

  • Activin/TGF-β pathway components

• Functional readouts: Validate organoid maturation using:

  • Immunohistochemistry for nephron segment markers

  • Single-cell RNA sequencing to confirm appropriate cell populations

  • Functional assays (albumin uptake, fluid transport)

• Stability considerations: Refresh FGF9-containing media every 1-2 days, as the protein may have limited stability at 37°C in complex media .

To enhance reproducibility, it's recommended to validate the bioactivity of each new lot of FGF9 using a standardized proliferation assay before application in organoid protocols.

How does FGF9 differ from other FGF family members in structure and function?

FGF9 belongs to a subfamily within the broader FGF family that includes FGF9, FGF16, and FGF20, which share 65-71% amino acid sequence identity . These key differences distinguish FGF9 from other FGF family members:

A unique feature of FGF9 is its constitutive dimerization, which regulates its activity and diffusion properties. Mutations that interfere with dimerization (as in the mouse Eks mutant) result in monomeric, more diffusible FGF9 that causes joint fusions (synostoses) . In humans, FGF9 mutations affecting receptor binding cause multiple synostoses syndrome (SYNS) .

FGF9 is particularly relevant in glial cell biology, kidney development, and sex determination, while other FGF family members may have more prominent roles in other developmental processes .

What are the common troubleshooting approaches when recombinant FGF9 shows reduced or no activity in experimental systems?

When researchers encounter reduced or absent FGF9 activity, systematic troubleshooting is essential:

• Protein Quality Issues:

  • Verification test: Analyze by SDS-PAGE under reducing and non-reducing conditions to check for degradation or aggregation.

  • Solution: Obtain a new lot of recombinant FGF9 with verified bioactivity.

• Improper Reconstitution:

  • Verification test: Measure protein concentration after reconstitution.

  • Solution: Ensure protein is properly solubilized; avoid vortexing; consider adding carrier proteins (0.1% BSA) for very dilute solutions.

• Receptor Expression:

  • Verification test: Perform RT-PCR or Western blot analysis for FGFR1-3 expression in your cell system.

  • Solution: Use a positive control cell line (e.g., Balb/3T3) to verify FGF9 activity in parallel.

• Heparan Sulfate Proteoglycan (HSPG) Availability:

  • Verification test: Add exogenous heparin (1-10 μg/mL) to your assay.

  • Solution: If activity increases with heparin, your system may lack sufficient HSPGs for proper FGF9 signaling.

• Downstream Signaling Pathway Integrity:

  • Verification test: Analyze ERK phosphorylation at 5-15 minutes after FGF9 treatment.

  • Solution: If no phosphorylation occurs, test other growth factors that utilize the same pathways to determine if the issue is FGF9-specific.

• Handling and Storage Problems:

  • Verification test: Test bioactivity of a freshly reconstituted aliquot versus stored protein.

  • Solution: Store in smaller aliquots to minimize freeze-thaw cycles; add stabilizers (e.g., 0.1% BSA) for dilute solutions.

• Experimental Design Factors:

  • Verification test: Review cell density, serum starvation conditions, and timing of FGF9 addition.

  • Solution: Optimize serum starvation (12-24h), cell density (50-70% confluence), and duration of FGF9 treatment.

Systematic testing of these parameters will help identify the source of reduced activity and guide appropriate corrective actions to restore experimental functionality.

How can FGF9 be utilized in investigating the tumor microenvironment and developing targeted cancer therapies?

FGF9's role in the tumor microenvironment, particularly in HCC, offers several research avenues for developing targeted cancer therapies:

• Targeting the HSC-Cancer Cell Axis:

  • FGF9 is expressed by activated hepatic stellate cells (HSC) and cancer-associated myofibroblasts but not by HCC cells themselves .

  • This creates an opportunity to disrupt the paracrine signaling between stromal cells and cancer cells.

  • Research methodology: Use co-culture systems of HSCs and HCC cells with selective inhibition of FGF9 production or signaling.

• FGF9 as a Prognostic Biomarker:

  • High FGF9 expression correlates with poor patient survival in HCC .

  • Research methodology: Analyze FGF9 expression in patient biopsies using immunohistochemistry and correlate with clinical outcomes.

• Combinatorial Therapy Approaches:

  • FGF9 contributes to sorafenib resistance in HCC .

  • Research methodology: Test combinations of FGFR inhibitors (particularly targeting FGFR1/2/3) with sorafenib in preclinical models.

• Novel Targeting Strategies:

  • FGF traps or anti-FGF9 antibodies could be developed to specifically target FGF9-mediated signaling .

  • Research methodology: Design and test FGF9-specific traps or antibodies in vitro and in animal models.

• MicroRNA-Based Regulation:

  • miR-140-5p has been identified as a tumor suppressor in HCC that may act partly through suppressing FGF9 .

  • Research methodology: Investigate miRNA-based therapies targeting FGF9 expression.

Experimental approaches should incorporate these methodologies with appropriate controls and validation in multiple model systems, progressing from in vitro studies to animal models and ultimately clinical samples.

What are the latest techniques for studying FGF9's role in developmental processes and tissue engineering applications?

Advanced methodologies for investigating FGF9's developmental roles and applications in tissue engineering include:

• CRISPR/Cas9 Gene Editing:

  • Precise modification of FGF9 or its receptors in model organisms and stem cells

  • Methodology: Design guide RNAs targeting specific FGF9 domains to create functional mutants that affect specific aspects of FGF9 activity.

• Spatiotemporal Control of FGF9 Signaling:

  • Optogenetic approaches to control FGF9 signaling with light-inducible systems

  • Methodology: Engineer light-responsive FGFR systems that activate upon illumination, allowing precise temporal and spatial control.

• Biomaterial-Based Delivery Systems:

  • Controlled release of FGF9 using advanced biomaterials

  • Methodology: Incorporate FGF9 into hydrogels, microspheres, or nanoparticles designed for sustained or triggered release in specific microenvironments.

• Organ-on-Chip Technologies:

  • Microfluidic systems mimicking developmental microenvironments

  • Methodology: Create multi-chamber devices with controlled gradients of FGF9 to study migration, differentiation, and morphogenesis in real-time.

• Single-Cell Analysis:

  • Profiling FGF9 effects on heterogeneous cell populations

  • Methodology: Apply single-cell RNA-seq to identify cell-type-specific responses to FGF9 signaling during development or disease progression.

• 3D Bioprinting with FGF9 Incorporation:

  • Spatial patterning of FGF9 in engineered tissues

  • Methodology: Develop bioinks containing FGF9 or FGF9-expressing cells for specific spatial distribution in printed constructs.

These advanced techniques allow researchers to better recapitulate the complexity of developmental processes and create more sophisticated tissue engineering applications leveraging FGF9's biological activities in cell proliferation, differentiation, and tissue patterning.

How can researchers optimize experiments involving FGF9's role in neuronal development and neuroprotection?

FGF9 plays important roles in neuronal development, differentiation, survival, and has been implicated in neuroprotection against neurodegenerative diseases . When designing experiments to study these functions, researchers should consider:

• Neural Cell Type Specificity:

  • FGF9 affects different neural cell populations distinctly (neurons, astrocytes, oligodendrocytes)

  • Methodology: Use cell type-specific markers (MAP2, GFAP, O4) in immunocytochemistry to distinguish effects on different neural populations.

• Developmental Timing:

  • FGF9's effects vary with developmental stage

  • Methodology: Create precise developmental timelines for FGF9 addition in neural differentiation protocols with specific markers for each stage.

• Receptor Expression Profiling:

  • Different neural cell types express varying levels of FGFR1-3

  • Methodology: Quantify receptor expression using qRT-PCR or flow cytometry before FGF9 treatment.

• Neuroprotection Assays:

  • For Parkinson's, Huntington's, or Alzheimer's disease models

  • Methodology: Pre-treat cultures with FGF9 before adding neurotoxic compounds (MPP+, amyloid-β, glutamate) and measure:

    • Cell viability (MTT, LDH release)

    • Apoptotic markers (cleaved caspase-3, TUNEL)

    • Oxidative stress (ROS production, GSH levels)

• Dose-Response Considerations:

  • Optimal concentrations for neuroprotection vs. differentiation may differ

  • Methodology: Perform comprehensive dose-response studies (1-200 ng/mL) with multiple readouts.

• Compound Experimental Models:

  Model System	Applications	Key Readouts
  Primary neuron cultures	Direct effects on neurons	Neurite outgrowth, synaptogenesis
  Neural organoids	3D developmental effects	Cytoarchitecture, layer formation
  Ex vivo brain slices	Circuit-level effects	Electrophysiology, connectivity
  In vivo models	Behavioral outcomes	Cognitive/motor testing, histology

• Co-factor Considerations:

  • FGF9 may require heparan sulfate proteoglycans as co-factors

  • Methodology: Include heparin (1-5 μg/mL) in experimental systems to optimize FGF9 activity.

Integrating these methodological considerations will enhance the rigor and reproducibility of neuronal FGF9 research while enabling more translational applications in neurodegenerative disease models.

What are the critical parameters to consider when using FGF9 in conjunction with other growth factors in complex developmental models?

When incorporating FGF9 into complex developmental models alongside other growth factors, several critical parameters must be considered to achieve optimal outcomes:

• Signaling Pathway Cross-talk:

  • FGF9 activates ERK, JNK, STAT, and PI3K pathways, which may interact with pathways activated by other factors

  • Methodology: Use specific pathway inhibitors (U0126 for MEK/ERK, SP600125 for JNK) to parse out individual contributions of each growth factor.

• Temporal Sequencing:

  • The order of growth factor exposure can dramatically affect cellular responses

  • Methodology: Design factorial experiments testing different sequences and durations of growth factor treatments (e.g., FGF9 before, after, or simultaneously with other factors).

• Concentration Ratios:

  • The relative concentrations of multiple growth factors are often more important than absolute concentrations

  • Methodology: Create response surface methodologies testing different concentration ratios rather than single-factor dose-response curves.

• Extracellular Matrix Context:

  • ECM components can modulate FGF9 binding to receptors and co-receptors

  • Methodology: Test FGF9 activity on cells cultured on different ECM substrates (laminin, fibronectin, Matrigel).

• Common Growth Factor Combinations with FGF9:

  Developmental Context	Growth Factor Combination	Concentration Ranges	Notes
  Kidney organoids	FGF9 + BMP7 + GDNF	FGF9: 50-200 ng/mL; BMP7: 10-50 ng/mL; GDNF: 50-100 ng/mL	Sequential addition often more effective than simultaneous
  Neural development	FGF9 + SHH + Noggin	FGF9: 10-50 ng/mL; SHH: 200-500 ng/mL; Noggin: 100-250 ng/mL	Position-dependent patterning requires gradient formation
  Testicular development	FGF9 + WNT4 inhibition	FGF9: 50-100 ng/mL; WNT inhibitor: context-dependent	Antagonistic relationship critical for sex determination

• Readout Selection:

  • Different combinations produce unique cellular responses requiring specific assays

  • Methodology: Include both short-term (signaling) and long-term (differentiation, morphogenesis) readouts.

• Supporting Technologies:

  • Microfluidic systems for precise spatial control

  • Methodology: Use gradient generators to create defined concentration gradients of multiple factors simultaneously.

• Statistical Design Considerations:

  • Multi-factor experiments require appropriate statistical approaches

  • Methodology: Employ factorial design and multivariate analysis rather than simple t-tests or ANOVA.

By systematically addressing these parameters, researchers can more effectively develop physiologically relevant models that recapitulate complex developmental processes involving FGF9 signaling.