Recombinant Human Fibroblast growth factor 17 (FGF17) (Active)

What is the molecular structure and basic characterization of recombinant human FGF17?

Recombinant human FGF17 is a protein with a predicted molecular weight of 22.6 kDa, though it typically migrates as 21.5 and 22.6 kDa bands on SDS-PAGE under reducing conditions. The protein begins at Thr23 (with or without an N-terminal Met) and contains conserved sequences highly similar (93% identity) to mouse FGF17 . It is typically produced in E. coli expression systems and purified to >95% purity as determined by SDS-PAGE with silver stain analysis. The recombinant protein is generally lyophilized from modified Dulbecco's phosphate buffered saline (1X PBS) with pH 7.2-7.3 without calcium or magnesium .

Through which receptors does FGF17 signal, and what are its primary biological activities?

FGF17 primarily signals through four FGF receptors: hFGFR1c, 2c, 3c, and 4 . Its biological activity can be measured through its ability to stimulate 3H-thymidine incorporation in NR6R-3T3 fibroblasts, with a typical ED50 of 15-60 ng/ml in the presence of 1 μg/ml heparin . FGF17 possesses broad mitogenic and cell survival activities and is involved in multiple biological processes including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion . Importantly, FGF17 plays crucial roles in organizing and inducing specific patterning at the midbrain/hindbrain junction and embryonic brain development .

How do storage and reconstitution conditions affect FGF17 activity?

While the search results don't directly address optimal storage and reconstitution conditions for FGF17 specifically, recombinant proteins of this nature typically require careful handling. Based on general practices for similar growth factors, lyophilized FGF17 should be stored at -20°C to -80°C. Upon reconstitution in sterile buffer solutions (typically phosphate-buffered saline with a carrier protein like BSA), aliquoting is recommended to avoid repeated freeze-thaw cycles which can compromise biological activity. For experimental work, it's essential to validate the activity of each new lot using established bioassays such as the 3H-thymidine incorporation assay with NR6R-3T3 fibroblasts .

How does FGF17 compare to FGF8 in ventral midbrain dopaminergic (VM DA) progenitor patterning?

Recent research has demonstrated that FGF17 induces expression of key VM DA progenitor markers at significantly higher levels than FGF8 . When comparing these two growth factors:

These findings suggest that FGF17 may be a more promising candidate for VM DA progenitor patterning in cell replacement therapy applications, particularly for conditions like Parkinson's disease .

What role does FGF17 play in mesenchymal stem cell proliferation under hypoxic conditions?

FGF17 has been identified as a key factor secreted by hypoxic human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs), especially at late passages . Research has demonstrated that:

• FGF17 secretion is significantly higher in hypoxic (1% O₂) compared to normoxic (21% O₂) conditions

• Knockdown of FGF17 in both hypoxic and normoxic hWJ-MSCs decreases cell proliferation

• Treatment with recombinant FGF17 increases proliferation in both conditions

• The signal transduction pathway involves ERK1/2 activation

• FGF17 affects expression of differentiation-related genes differently under normoxic versus hypoxic conditions

Specifically, FGF17 treatment of normoxic hWJ-MSCs increased cell proliferation in a dose-dependent manner up to 500 ng/ml, with optimal effects observed at this concentration. Higher concentrations (1,000 ng/ml) showed decreased efficacy . These findings suggest recombinant FGF17 could be valuable as a supplement in culture medium to enhance expansion of mesenchymal stem cells while maintaining stemness .

What are the mechanisms underlying FGF17's role in neuropsychiatric diseases?

FGF17 is required for several complex social behaviors, and disruptions in FGF17 signaling may contribute to neuropsychiatric diseases that affect such behaviors . At the molecular level, FGF17 plays important roles in organizing and inducing specific patterning at the midbrain/hindbrain junction, which is critical for proper brain development . Recent research using FGF17-patterned dopaminergic progenitors has shown that these cells can reverse motor deficits in rat models of Parkinson's disease, producing DA-rich and highly innervating grafts .

The signaling cascade involves activation of specific FGF receptors (hFGFR1c, 2c, 3c, and 4) and likely engages multiple downstream pathways including the ERK1/2 pathway, which has been confirmed in stem cell studies . Additionally, single-cell RNA sequencing has revealed that cyclic adenosine monophosphate (cAMP) signaling may be involved in FGF17-mediated upregulation of LMX1A in treated cells . These mechanisms together contribute to FGF17's influence on brain patterning and function, with implications for understanding and potentially treating neurological and psychiatric disorders.

How should researchers optimize FGF17 concentration for different experimental models?

Determining optimal FGF17 concentration requires careful titration for each experimental model. Based on available data:

For fibroblast models:

• The typical ED50 for stimulating 3H-thymidine incorporation in NR6R-3T3 fibroblasts is 15-60 ng/ml in the presence of 1 μg/ml heparin

For mesenchymal stem cells:

• Dose-dependent increases in proliferation are observed up to 500 ng/ml

• Higher concentrations (1,000 ng/ml) may show decreased efficacy

• Treatment duration of 48 hours has shown significant effects

For neural progenitor patterning:

• Comparison studies with FGF8 should be included as a reference point

• Assessment of key markers like FOXA2, LMX1A, OTX2, and EN1 is essential

A methodological approach would include:

• Initial broad range titration (10-1000 ng/ml)

• Narrower secondary titration around identified optimal range

• Time-course studies (15 min, 1h, 4h, 24h, and longer timepoints)

• Validation using multiple readouts (proliferation, marker expression, pathway activation)

• Inclusion of appropriate controls (untreated, related FGFs like FGF8)

What are the key considerations when designing transplantation studies using FGF17-patterned progenitors?

When designing transplantation studies using FGF17-patterned progenitors, researchers should consider:

• Cell preparation and characterization:

  • Ensure high purity of FOXA2+/LMX1A+ progenitors before transplantation

  • Validate expression levels of key markers through qRT-PCR and flow cytometry

  • Compare with FGF8-patterned cells as a reference

• Animal model selection:

  • Rat models of Parkinson's disease have been successfully used

  • Consider species differences in growth factor responsiveness

  • Document baseline motor deficits before transplantation

• Outcome measurements:

  • Motor function assessment using standardized tests

  • Histological analysis of graft survival and integration

  • Evaluation of dopamine production in transplanted cells

  • Assessment of innervation patterns

• Controls:

  • Include sham-operated controls

  • Compare with established protocols using other growth factors

  • Consider including FGF8-patterned cells as comparative controls

• Timeline:

  • Allow sufficient time for graft maturation and integration

  • Include both short-term and long-term assessment points

Based on published research, FGF17-derived VM DA progenitors have demonstrated ability to rescue motor deficits in rats and produce DA-rich and highly innervating grafts , suggesting this approach has significant therapeutic potential.

How can researchers address variability in FGF17 responsiveness across different cell types?

Variability in FGF17 responsiveness is a common challenge that can be addressed through several methodological approaches:

• Receptor profiling:

  • Verify expression of all relevant FGF receptors (FGFR1c, 2c, 3c, and 4) in your cell model

  • Consider quantitative assessment via qPCR or flow cytometry

• Co-factor optimization:

  • FGF17 is a heparin-binding growth factor; ensure appropriate heparin supplementation (typically 1 μg/ml)

  • Test different heparin concentrations if responsiveness is suboptimal

• Culture condition modifications:

  • Oxygen tension significantly affects FGF17 production and possibly responsiveness (compare 21% vs. 1% O₂)

  • Consider that passage number affects response, with late passages (7-10) showing different patterns than early passages

• Pathway validation:

  • Confirm ERK1/2 phosphorylation as a proximal readout of FGF17 signaling

  • Time-course studies (15 min to 24h) can help identify optimal assessment timepoints

• Combined approaches:

  • For neural progenitor patterning, consider combining FGF17 with cAMP pathway activators

  • Be aware that strong cAMP activation may come at the expense of EN1 expression

If troubleshooting a specific cell model, begin by establishing a positive control experiment using a cell type with well-documented FGF17 responsiveness, such as NR6R-3T3 fibroblasts or hypoxic hWJ-MSCs .

What are common pitfalls in analyzing FGF17 signaling pathways, and how can they be avoided?

When analyzing FGF17 signaling pathways, researchers should be aware of several common pitfalls:

• Overlooking temporal dynamics:

  • FGF17 signaling shows time-dependent effects ranging from 15 minutes to 24 hours and beyond

  • Solution: Implement comprehensive time-course experiments covering immediate (minutes), intermediate (hours), and long-term (days) responses

• Limited pathway analysis:

  • Focusing only on expected pathways (e.g., ERK1/2) may miss context-specific signaling

  • Solution: Use phospho-protein arrays or multiplex assays to simultaneously assess multiple pathways (ERK1/2, AKT, STAT3, etc.)

• Confounding by autocrine/paracrine factors:

  • Cells may respond to FGF17 by producing other factors that influence the observed outcome

  • Solution: Use conditioned media controls and specific pathway inhibitors to distinguish direct from indirect effects

• Ignoring receptor specificity and competition:

  • FGF17 signals through multiple receptors (FGFR1c, 2c, 3c, and 4) with potentially different outcomes

  • Solution: Consider receptor-specific blocking antibodies or siRNA approaches to identify the predominant signaling receptor in your model

• Neglecting cross-talk with other pathways:

  • The connection between FGF17 and cAMP signaling suggests important pathway cross-talk

  • Solution: Consider combinatorial treatment approaches and analyze pathway intersections

How might FGF17 be utilized in developing improved cell therapy products for Parkinson's disease?

FGF17 shows significant promise for developing improved cell therapy products for Parkinson's disease through several mechanisms:

Recent research has identified FGF17 as "a promising candidate for more robust VM DA progenitor patterning, with the potential to improve cell products for treatment of PD" , suggesting this is an important direction for future translational research.

What is the potential role of FGF17 in enhancing stem cell expansion protocols while maintaining stemness?

FGF17 shows promising potential for enhancing stem cell expansion protocols while maintaining stemness, particularly based on findings with mesenchymal stem cells:

• Enhanced proliferation:

  • Recombinant FGF17 treatment (up to 500 ng/ml) significantly increases proliferation of both normoxic and hypoxic human Wharton's Jelly-derived mesenchymal stem cells

  • This effect is mediated through the ERK1/2 pathway

• Preservation of phenotype:

  • Treatment with FGF17 does not alter the basic phenotypic characteristics of mesenchymal stem cells

  • This is crucial for maintaining cellular identity during expansion

• Differential effects on differentiation markers:

  • Under normoxic conditions, FGF17 treatment downregulates differentiation-related genes (adiponectin, Runx2, chondroadherin)

  • This suggests FGF17 may help maintain stemness during expansion

• Hypoxia interactions:

  • FGF17 is naturally upregulated under hypoxic conditions (1% O₂)

  • FGF17 plays a key role in maintaining the high proliferation rate of hypoxic MSCs at late passages (7-10)

  • This suggests potential for combining hypoxic culture with FGF17 supplementation for optimal results

• Application considerations:

  • Researchers developing expansion protocols should consider dose optimization (with 500 ng/ml showing optimal effects in MSCs)

  • The approach may be particularly valuable for late-passage expansion when proliferation typically declines

  • Verification in specific stem cell types of interest is essential

These findings provide "supportive evidence for the use of rFGF17 as a supplement in culture medium of human mesenchymal stem cells in order to enhance the expansion of cells with stemness" , representing an important advance for regenerative medicine applications.