Recombinant Human Fibroblast growth factor 4 (FGF4), partial (Active)

What is Recombinant Human FGF4 and what is its primary function in research?

Recombinant human Fibroblast Growth Factor 4 (FGF4) is a 14 kDa protein that plays an essential role in stem cell proliferation and differentiation. The recombinant form typically contains the bioactive domain (Ser54-Leu206) of the human FGF4 protein produced in expression systems such as E. coli .

In research settings, FGF4 is primarily used to:

• Promote proliferation and differentiation of embryonic stem cells (ESCs)

• Support induced-pluripotent stem cell (iPSC) maintenance

• Enhance mesenchymal stem cell proliferation

• Stimulate neural stem cell proliferation and differentiation

• Facilitate the development of organoid culture systems, particularly cardiac and intestinal organoids

The protein functions through activation of FGF receptors, triggering downstream signaling cascades that regulate gene expression patterns involved in cell fate decisions.

What are the optimal storage and handling conditions for recombinant FGF4?

For maximum stability and bioactivity retention, recombinant human FGF4 should be stored at -80°C for long-term preservation or -20°C for routine use. The protein is typically supplied in either carrier-free format or with bovine serum albumin (BSA) as a carrier protein. The latter enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations .

Methodological recommendations include:

• Minimize freeze-thaw cycles by aliquoting the stock solution before freezing

• When working with the protein, keep it on ice

• For cell culture applications, dilute in appropriate serum-free medium containing at least 0.1% BSA if using carrier-free format

• Use low-binding microcentrifuge tubes to prevent protein adherence to tube walls

What is the effective concentration range for FGF4 in experimental applications?

The effective dose (ED₅₀) of recombinant human FGF4 typically ranges from 0.25-1.25 ng/mL for basic biological activities . For more specific applications, bioactivity measurements show an EC₅₀ of approximately 306.5 pM (4.3 ng/mL) when measured using firefly luciferase reporter assays in stably transfected HEK293T cells .

Concentration recommendations vary by application:

• For stem cell maintenance: 5-10 ng/mL

• For differentiation protocols: 10-50 ng/mL (depending on cell type)

• For organoid culture: 25-100 ng/mL (often in combination with other growth factors)

Researchers should optimize concentrations through dose-response experiments for their specific cell lines and experimental conditions.

What is the species reactivity profile of recombinant human FGF4?

Recombinant human FGF4 exhibits cross-species reactivity due to the high conservation of this protein across mammals. Species similarity percentages include:

• Mouse: 91% sequence homology

• Rat: 80% sequence homology

• Porcine: 91% sequence homology

• Bovine: 90% sequence homology

This high degree of conservation enables researchers to use human FGF4 in experimental models involving these species, although activity titration is recommended when working with non-human cells.

How does FGF4 contribute to cell-cell communication in developmental contexts?

FGF4 functions as a critical mediator of local cell-cell communication during embryonic development. Recent research has revealed that FGF4 operates as a short-range signal that enables robust cell-type proportioning despite variable initial conditions in precursor cells .

The signaling mechanism works through:

• Paracrine secretion from specific cell populations

• Local diffusion to neighboring cells (primarily affecting nearest and second-nearest neighbors)

• Activation of ERK signaling in recipient cells

• Mutual regulation between FGF4 signaling and cell-intrinsic transcriptional circuits

This communication system allows differentiating cell populations to coordinate their fate decisions, resulting in reproducible proportions of specialized cell types even when starting conditions vary. The short-range nature of FGF4 signaling has been demonstrated through experiments showing that the spatial range primarily couples nearest and second-nearest neighbors (approximately 14.0±3.2 μm and 25.5±5.3 μm distance, respectively) .

What methodologies are recommended for measuring FGF4 signaling in experimental settings?

Several methodological approaches can be employed to quantify FGF4 signaling:

• Luciferase Reporter Assays

  • Stably transfect cells with an FGF-responsive element driving luciferase expression

  • Treat cells with serial dilutions of FGF4

  • Measure firefly luciferase activity and normalize to control (e.g., Renilla luciferase)

  • Determine EC₅₀ values (typically around 306.5 pM or 4.3 ng/mL for human FGF4)

• Transcriptional Reporters

  • Use of Sprouty4 H2B-Venus transcriptional reporter provides a quantitative readout for long-term FGF4 signaling

  • Analysis via flow cytometry or time-lapse microscopy enables dynamic monitoring

• In Situ mRNA Detection

  • Visualize Fgf4 expression patterns using hybridization techniques

  • Enables correlation with other markers (e.g., Gata6) to establish expression relationships

• Phospho-ERK Immunostaining

  • FGF4 activates the ERK pathway, making phospho-ERK a suitable proximal signaling readout

  • Quantitative imaging or Western blotting can measure signaling intensity

These complementary approaches allow researchers to characterize both FGF4 expression and downstream signaling events in their experimental systems.

How does FGF4 regulate cell-type proportioning in developmental systems?

FGF4 plays a fundamental role in establishing and maintaining proper proportions of cell types during development through a sophisticated feedback mechanism:

• Mutual Regulation Mechanism

  • GATA transcription factors (like GATA4 and GATA6) directly regulate Fgf4 transcription in embryonic stem cells

  • GATA factor expression leads to downregulation of Fgf4 mRNA within 2 hours

  • After 40 hours of differentiation, Fgf4 mRNA expression becomes mutually exclusive with Gata6 mRNA

• Robustness Through Local Communication

  • The proportion of differentiated cell types (epiblast-like and primitive endoderm-like) remains constant despite variable initial conditions

  • Cell-cell contacts in intact colonies support primitive endoderm-like differentiation

  • Disruption of cell-cell communication (through Fgf4 mutation) eliminates this robustness, making differentiation outcomes highly dependent on initial conditions

• Spatial Range of Influence

  • FGF4 acts locally rather than globally, as shown by:

    • Cell-type proportions changing negligibly with media volume

    • Disruption of cell-cell contacts reducing primitive endoderm-like differentiation

    • Differentiation outcomes correlated with cell density and proximity to FGF4-secreting cells

This system represents a novel function for FGF signaling in establishing reproducible proportions of discrete cell types, which may operate across various developing tissues.

What is the role of FGF4 in organoid development and maintenance?

Recombinant human FGF4 serves as a critical component in various organoid culture systems:

• Intestinal Organoids

  • FGF4 works synergistically with WNT signaling to form hindgut organoids from iPSC-derived human posterior gut endoderm cells

  • It mimics embryonic intestinal development during directed differentiation of pluripotent stem cells into intestinal organoids

• Cardiac Organoids

  • FGF4 promotes cardiomyocyte differentiation and organization in 3D cardiac organoid models

  • It helps establish proper tissue architecture and functional properties

• Neural Organoids

  • FGF4 enhances neural stem cell proliferation

  • Contributes to neuronal differentiation in the postnatal brain

Methodological considerations include:

• Concentration optimization (typically 25-100 ng/mL)

• Timing of FGF4 addition during differentiation protocols

• Combination with other growth factors (e.g., noggin, BMP-4, FGF-10) for synergistic effects

How can researchers distinguish between FGF4-specific effects and those of other FGF family members?

Distinguishing FGF4-specific effects from those of other FGF family members requires careful experimental design:

• Use of Genetic Models

  • Fgf4-knockout cells or organisms provide the cleanest system for specific attribution

  • Rescue experiments with recombinant FGF4 can confirm phenotype specificity

• Receptor Specificity Analysis

  • FGF4 acts through specific FGF receptors (primarily FGFR1c and FGFR2c)

  • Use of receptor-specific inhibitors can help distinguish signaling pathways

• Transcriptional Profiling

  • RNA-seq or qPCR analysis of FGF4-specific target genes

  • Comparison with transcriptional responses to other FGF family members

• Functional Assays

  • Measure biological responses unique to FGF4 (e.g., specific aspects of stem cell differentiation)

  • Compare dose-response relationships between different FGF family members

A particularly useful approach combines Fgf4-mutant cells with controlled addition of recombinant FGF4 at defined concentrations, allowing precise determination of FGF4-specific effects in a clean background .

What quality control measures should be implemented when working with recombinant FGF4?

Rigorous quality control is essential for reproducible results when working with recombinant FGF4:

• Purity Assessment

  • SDS-PAGE analysis under both reducing and non-reducing conditions

  • High-quality recombinant human FGF4 should appear as a single band at approximately 14 kDa

  • No contaminating protein bands should be visible

• Bioactivity Testing

  • Firefly luciferase reporter assays in stably transfected cells

  • Comparison with reference standards or alternative supplier proteins

  • Expected EC₅₀ values around 306.5 pM (4.3 ng/mL)

• Endotoxin Testing

  • Ensure low endotoxin levels (<1.0 EU/μg protein) for cell culture applications

  • Limulus Amebocyte Lysate (LAL) assay is commonly used for this purpose

• Functionality Verification

  • Test the ability to induce expected cellular responses in relevant model systems

  • For example, verify capacity to promote differentiation of primitive endoderm-like cells from embryonic stem cells

What are common issues encountered when using FGF4 in stem cell differentiation protocols?

Researchers frequently encounter several challenges when incorporating FGF4 into stem cell differentiation protocols:

• Variable Efficacy Between Lots

  • Solution: Perform bioactivity testing on each new lot using standardized assays

  • Maintain reference stocks of working preparations

• Protein Stability Concerns

  • Solution: Add carrier proteins (like BSA) for stabilization if using carrier-free preparations

  • Store as single-use aliquots to avoid freeze-thaw cycles

• Concentration Optimization

  • Solution: Perform dose-response experiments for each cell line and application

  • Remember that FGF4 works in concert with other factors, requiring balanced concentrations

• Background FGF4 Production

  • Solution: Use Fgf4-mutant cells for clean experimental systems when studying FGF4-specific effects

  • Consider that many stem cells produce endogenous FGF4, which may mask exogenous effects

• Insufficient Cellular Response

  • Solution: Verify FGF receptor expression in target cells

  • Consider adding heparin (1-10 μg/mL) to stabilize FGF4 and enhance receptor binding

How is FGF4 being utilized in regenerative medicine research?

Recent advances in regenerative medicine have expanded FGF4 applications beyond basic research:

• Enhanced Differentiation Protocols

  • Precise temporal and concentration control of FGF4 signaling improves differentiation efficiency and reproducibility

  • Integration into step-wise protocols for clinically relevant cell types

• Organoid Development for Disease Modeling

  • FGF4 helps establish physiologically relevant 3D tissue models

  • These organoids serve as platforms for testing therapeutic interventions

• Cell Therapy Manufacturing

  • FGF4 supplementation in bioreactor systems improves scalability of stem cell expansion

  • Contributes to standardized methods for producing clinical-grade cellular products

• Biomaterial Integration

  • Incorporation of FGF4 into hydrogels and scaffolds for sustained release

  • Creates instructive microenvironments for tissue engineering applications

As research progresses, understanding the precise mechanisms of FGF4 signaling will continue to refine these applications for therapeutic development.

What are the latest findings regarding FGF4's role in developmental patterning?

Recent research has revealed sophisticated aspects of FGF4 function in developmental patterning:

• Population-Level Robustness

  • FGF4 enables robust cell-type proportioning despite variable initial conditions

  • This system autonomously re-establishes proper proportions following perturbations

• Spatial Signaling Dynamics

  • FGF4 operates as a short-range signal primarily affecting cells within approximately 25.5±5.3 μm

  • This localized activity creates spatial patterns essential for tissue organization

• Transcriptional Circuit Integration

  • FGF4 signaling interfaces with cell-intrinsic transcriptional networks

  • GATA factors directly regulate Fgf4 transcription, creating feedback loops that stabilize developmental outcomes

These findings highlight FGF4's role not just as a growth factor, but as a critical coordinator of collective cellular behavior during development.

What are the differences between carrier-free and carrier-containing FGF4 preparations?

The choice between carrier-free and carrier-containing FGF4 preparations depends on specific research requirements:

Researchers should select the appropriate format based on experimental needs:

• Use carrier-free for applications requiring absolute purity

• Use carrier-containing for routine cell culture and most functional assays

How can FGF4 activity be quantitatively measured and compared between different sources?

To ensure reproducibility, researchers should employ standardized methods for quantitative comparison of FGF4 activity:

• Luciferase Reporter Assays

  • Stably transfected cells with FGF-responsive elements driving luciferase expression

  • Treat cells in triplicate with serial dilutions of FGF4 samples

  • Measure firefly luciferase activity and normalize to control (e.g., Renilla luciferase)

  • Compare EC₅₀ values between samples

• Cell Proliferation Assays

  • Measure dose-dependent effects on proliferation of FGF4-responsive cells

  • Quantify using MTT, XTT, or similar proliferation assays

  • Compare ED₅₀ values (typically 0.25-1.25 ng/mL for standard activity)

• Stem Cell Differentiation Quantification

  • Measure proportion of cells differentiating into specific lineages (e.g., primitive endoderm-like cells)

  • Use flow cytometry with appropriate markers

  • Compare effectiveness at standardized concentrations

For reliable comparisons, parallel testing of different FGF4 sources under identical conditions is essential, as shown in direct comparisons between supplier products where quantitative luciferase assays revealed comparable bioactivity profiles .