FGF 2 Human, Plant

What is the biological significance of FGF-2 in human cell proliferation?

FGF-2 plays a crucial role in stimulating the proliferation of mesenchyme-derived progenitor cells in both young and aging bone tissue. Research demonstrates that FGF-2 significantly enhances cell proliferation in a dose-dependent manner, with effects varying by tissue source, age, and species. For example, in human cells, FGF-2 increases and prevents the decline in cells expressing activated leukocyte cell adhesion molecule, a marker for early lineage osteoblasts .

For experimental purposes, researchers typically assess FGF-2 effects using proliferation assays such as MTS or thymidine incorporation, with verification through direct cell counting. Standard protocols typically involve treating cell cultures with FGF-2 concentrations ranging from 0.0016 to 1.6 ng/mL and measuring cellular responses at multiple time points (4, 24, 48, and 72 hours) .

How do FGF-2 responses differ between young and aging tissues?

Experimental evidence indicates age-dependent differences in FGF-2 responsiveness. Cells derived from younger patients demonstrate greater proliferative responses to FGF-2 compared to cells from older patients. For example, at 48 hours, young human cells treated with 0.016 ng/mL of FGF-2 showed a 13.9% increase in proliferation, while this effect was significantly diminished in cells from older individuals .

Additionally, cells from young human patients often exhibit a biphasic response, showing decreased proliferation at early time points with higher FGF-2 concentrations, but this effect diminishes with extended culture time. By 72 hours, all tested FGF-2 doses stimulate proliferation compared to vehicle-treated controls .

Which plant expression systems have been successful for producing recombinant human FGF-2?

Research has demonstrated successful production of recombinant human FGF-2 (rhFGF2) using both rice cell suspension cultures and Nicotiana benthamiana-based transient expression systems:

• Rice cell suspension culture system: The human FGF2 cDNA gene can be cloned into a plant expression vector driven by the rice α-amylase 3D promoter and introduced into rice calli (Oryza sativa L. cv. Dongjin). This system has achieved maximum accumulation of rhFGF2 protein at approximately 28 mg/L after 13 days post-incubation .

• Nicotiana benthamiana transient expression: Using geminiviral vector systems, researchers have successfully expressed bFGF with a C-terminus 8X-Histidine tag in N. benthamiana, with and without barley alpha amylase signal peptide .

Experimental protocols for both systems typically involve molecular cloning of the human FGF2 gene into appropriate plant expression vectors, followed by either stable transformation (rice) or transient expression (tobacco), with subsequent protein purification and biological activity testing.

What are the methodological considerations for optimizing plant-produced FGF-2 expression?

Several factors significantly impact the expression efficiency of recombinant human FGF-2 in plant systems:

• Signal peptide inclusion: Comparative studies have shown that the expression level of bFGF without a signal peptide (like barley alpha amylase signal peptide) can be approximately 5-fold higher than constructs containing signal peptides. This is particularly evident in N. benthamiana transient expression systems .

• Harvest timing: Expression kinetics vary based on the expression construct. For instance, in N. benthamiana:

  • Constructs without signal peptides (pBY-bFGF) show increased protein accumulation on days 3 and 4 post-infiltration, reaching 2.72 μg/g fresh weight .

  • Constructs with signal peptides (pBY-SP-bFGF) demonstrate earlier leaf necrosis (3 days post-infiltration) compared to constructs without signal peptides (4 days post-infiltration) .

• Purification methodology: A two-step purification process involving ammonium sulfate precipitation followed by Ni-affinity chromatography has been effective for isolating plant-produced FGF-2 while maintaining biological activity .

How does plant-produced FGF-2 compare to E. coli-produced FGF-2 in biological activity assays?

Rigorous comparative studies demonstrate that plant-produced recombinant human FGF-2 exhibits comparable biological activity to E. coli-derived FGF-2, but often at different effective concentrations:

• Bone regeneration: In calvarial defect mouse models, local delivery of plant-derived rhFGF2 (p-FGF2) protein impregnated in absorbable collagen sponge (ACS) enhanced bone healing to levels similar to E. coli-derived rhFGF2 (e-FGF2), both showing significant improvement compared to ACS-only controls .

• Cell proliferation assays:

  • In human periodontal ligament stem cells (hPDLSCs), plant-produced bFGF at 2 ng/mL showed comparable proliferation induction to E. coli-produced bFGF at 20 ng/mL, indicating potentially higher potency of the plant-derived protein (10-fold lower effective concentration) .

  • For human follicle dermal papilla cells (hFDPCs), both plant-produced and E. coli-produced bFGF significantly induced proliferation at concentrations of 50, 100, and 200 ng/mL .

• Osteogenic differentiation: In MC3T3-E1 cells, exogenous addition of plant-derived and E. coli-derived FGF-2 exhibited similar effects on proliferation, mineralization, and osteogenic marker expression .

These findings suggest that plant-produced FGF-2 maintains full biological functionality while potentially offering enhanced potency in certain applications.

What are the species-specific differences in FGF-2 responsiveness between human and mouse cells?

Research has revealed notable species-specific differences in FGF-2 responsiveness:

• Concentration sensitivity:

  • Human cells demonstrate responsiveness to significantly lower concentrations of FGF-2 (as low as 0.0016 ng/mL) compared to mouse cells .

  • Human cells from younger patients show greater proliferative responses to FGF-2 than cells from young mouse calvaria or femurs. For example, at 48 hours with 0.016 ng/mL FGF-2, young human cells showed a 13.9% increase in proliferation compared to only 7.5% in young mouse femur cells .

• Tissue-specific responses:

  • In young mice, femur-derived cells demonstrate higher sensitivity to FGF-2 (responding to 0.016, 0.16, and 1.6 ng/mL) and exhibit earlier responses (starting at 4 and 24 hours) compared to calvarial cells .

  • At later time points (48 and 72 hours), both higher concentrations of FGF-2 (0.16 and 1.6 ng/mL) stimulate proliferation in both tissue sources .

These differences are crucial considerations when designing experiments and interpreting results across species, particularly when using mouse models for human applications.

What are the recommended assays for quantifying FGF-2 effects on cell proliferation?

Several validated assays can be employed to quantify FGF-2 effects on cell proliferation:

• MTS assay: This metabolic activity assay correlates with cell proliferation and has been validated through comparison with more direct methods. Research confirms that the change in MTS assay values is proportional to the increase in cell number determined by direct microscopic counting .

• [³H]-thymidine incorporation: This method directly measures DNA synthesis by administering [³H]-thymidine for the last 4 hours of culture periods (typically 24, 48, and 72 hours). Comparative studies show that results from this assay are similar to those obtained with the MTS assay, confirming the latter's validity for measuring proliferation rates .

• Immunofluorescence microscopy: For phenotypic characterization of proliferating cells, immunostaining for markers such as alkaline phosphatase (expressed in >95% of mesenchyme-derived progenitor cells from both mouse and human bone) and activated leukocyte cell adhesion molecule (a marker for early lineage osteoblasts) provides valuable complementary data .

For comprehensive analysis, researchers should consider employing multiple assays and including appropriate time course analyses (4, 24, 48, and 72 hours) to capture both early and late responses to FGF-2 treatment.

What purification strategies yield the highest recovery of biologically active plant-produced FGF-2?

Optimized purification protocols for plant-produced FGF-2 typically employ a multi-step approach to maximize yield and biological activity:

• Two-step purification protocol: For His-tagged recombinant FGF-2 expressed in plants, a combination of ammonium sulfate precipitation followed by Ni-affinity chromatography has proven effective in isolating biologically active protein .

• Expression vector optimization: Including an 8X-Histidine tag at the C-terminus facilitates efficient purification using metal affinity chromatography, while the choice of signal peptide significantly impacts both expression level and purification efficiency. Research demonstrates that constructs without signal peptides yield approximately 5-fold higher expression compared to those with signal peptides .

• Harvest timing optimization: The optimal harvest time post-infiltration varies based on the expression construct. For N. benthamiana transient expression, maximum protein accumulation occurs at different times:

  • Day 2-3 for constructs with signal peptides (before leaf necrosis)

  • Day 4 for constructs without signal peptides (reaching 2.72 μg/g fresh weight)

Researchers should carefully balance maximizing protein expression with minimizing leaf necrosis, as tissue necrosis negatively affects protein expression and stability.

How effective is FGF-2 in enhancing bone regeneration in critical-sized defects?

FGF-2 demonstrates significant osteogenic potential in critical-sized bone defect models, with plant-produced FGF-2 showing comparable efficacy to E. coli-derived protein:

In mouse calvarial defect models, absorbable collagen sponges (ACS) impregnated with 5 μg of plant-derived rhFGF2 (p-FGF2) enhanced bone healing to significantly higher levels compared to ACS-only controls. The healing effect was comparable to that achieved with E. coli-derived rhFGF2 (e-FGF2) at the same concentration .

The mechanism of action appears to involve both:

• Stimulation of osteoprogenitor proliferation, as demonstrated by in vitro studies showing FGF-2 increases proliferation of mesenchyme-derived progenitor cells from bone .

• Enhancement of osteogenic differentiation, as evidenced by FGF-2's effect on mineralization and osteogenic marker expression in MC3T3-E1 cells .

These findings support the potential clinical application of plant-produced FGF-2 as a cost-effective alternative to traditional recombinant proteins for bone regeneration therapies.

What is the role of FGF-2 in maintaining stemness and promoting differentiation in human periodontal ligament stem cells?

FGF-2 plays a dual role in regulating human periodontal ligament stem cells (hPDLSCs), affecting both stemness maintenance and differentiation potential:

Experimental evidence demonstrates that plant-produced bFGF at concentrations as low as 2 ng/mL significantly induces proliferation of hPDLSCs compared to untreated controls. This effect is comparable to that of E. coli-produced bFGF at 20 ng/mL, suggesting higher potency of the plant-derived protein .

The biological mechanisms involve:

• Activation of proliferation pathways in hPDLSCs, which is essential for maintaining the stem cell population

• Regulation of differentiation factors, which is necessary for tissue engineering applications

These findings confirm that plant-produced bFGF can be effectively used for the maintenance of stemness in hPDLSCs, offering potential applications in periodontal tissue engineering and regenerative dentistry .

How might the differential response to FGF-2 between young and aging tissues inform personalized regenerative therapies?

The age-dependent differences in FGF-2 responsiveness present both challenges and opportunities for developing personalized regenerative therapies:

Research shows diminished proliferative responses to FGF-2 in cells from older individuals compared to younger ones. For example, human cells from younger patients exhibited a 13.9% increase in proliferation at 48 hours when treated with 0.016 ng/mL FGF-2, an effect that was significantly reduced in cells from older individuals .

This differential response suggests several research directions:

• Dose optimization strategies: Higher FGF-2 concentrations or modified delivery systems may be required to achieve therapeutic effects in aging tissues.

• Combination therapies: Investigating the synergistic effects of FGF-2 with other growth factors or signaling molecules could potentially overcome age-related declines in responsiveness.

• Mechanistic investigations: Determining the molecular basis for reduced FGF-2 sensitivity with age (potentially involving changes in receptor expression, signaling pathway efficiency, or downstream effector function) could identify additional therapeutic targets.

• Predictive biomarkers: Developing assays to predict individual patient responsiveness to FGF-2 therapy based on age, sex, or tissue-specific factors would facilitate more effective personalized treatment approaches.

What are the mechanistic differences explaining the higher potency of plant-produced FGF-2 compared to E. coli-derived protein in certain biological assays?

The observed higher potency of plant-produced FGF-2 in certain biological assays (effective at 10-fold lower concentrations than E. coli-derived protein in hPDLSC proliferation) raises important mechanistic questions :

Potential mechanisms requiring further investigation include:

• Post-translational modifications: Plant expression systems may provide specific modifications not present in bacterial systems, potentially enhancing protein stability, receptor binding, or signaling pathway activation.

• Protein folding differences: The eukaryotic protein folding machinery in plants may yield protein conformations with enhanced biological activity compared to those from prokaryotic systems.

• Reduced endotoxin contamination: Plant-produced proteins typically contain significantly lower endotoxin levels compared to E. coli-derived proteins, which could affect cellular responses, particularly in sensitive cell types.

• Differential receptor binding kinetics: Plant-produced FGF-2 may exhibit altered receptor binding kinetics or affinity, potentiating downstream signaling at lower concentrations.