Recombinant Mouse Thrombopoietin protein (Thpo), partial (Active)

What is recombinant mouse Thrombopoietin protein and what is its primary function?

Recombinant mouse Thrombopoietin (Thpo) is a lineage-specific cytokine that primarily affects the proliferation and maturation of megakaryocytes from their committed progenitor cells. It acts at a late stage of megakaryocyte development and is considered the major physiological regulator of circulating platelets . The recombinant partial (active) form typically encompasses amino acids 22-195 of the native protein and is expressed in expression systems such as Escherichia coli with high purity (>95%) .

Thrombopoietin belongs to the EPO/TPO family and is also known by several other names including C-mpl ligand, Megakaryocyte colony-stimulating factor, Megakaryocyte growth and development factor, and Myeloproliferative leukemia virus oncogene ligand . Beyond platelet regulation, TPO has also been demonstrated as a crucial cytokine supporting maintenance and self-renewal of hematopoietic stem cells (HSCs) .

How does mouse Thrombopoietin differ from human Thrombopoietin in research applications?

While mouse and human Thrombopoietin are both cross-reactive to their respective cognate receptors when used at supraphysiological doses in vitro, there are notable differences in their affinity and biological activity at physiological concentrations, particularly in vivo . These differences include:

• Physiological concentration: Human TPO serum concentrations are approximately 10-fold lower than mouse TPO, which is consistent with the respective physiological concentrations reported in healthy humans and mice .

• Post-transcriptional regulation: Human TPO expression is highly regulated at the post-transcriptional level through mechanisms involving alternative splicing and restriction of translation initiation. These mechanisms ensure that serum TPO concentrations remain at appropriate physiological levels .

• Cross-species activity: Although both proteins can activate the other species' receptor in supraphysiological conditions, their effectiveness at physiological concentrations may differ significantly, which can impact experimental outcomes when studying human cells in mouse models .

• Half-life and stability: The pharmacokinetic properties of mouse and human TPO differ, which can affect experimental design considerations, particularly for in vivo studies .

Understanding these differences is critical when designing experiments involving cross-species applications or when developing humanized mouse models for TPO-related research.

How should recombinant mouse Thrombopoietin protein be handled and stored for optimal activity?

For optimal maintenance of biological activity, recombinant mouse Thrombopoietin protein requires specific handling and storage conditions:

Handling Protocol:

• Upon receipt, briefly centrifuge the vial to ensure all material is at the bottom.

• Reconstitute lyophilized protein in sterile water or an appropriate buffer to a concentration of 0.1-1.0 mg/mL.

• Allow the protein to fully dissolve by gentle swirling or rotation for 10-20 minutes at room temperature.

• Avoid vigorous shaking or vortexing, which can cause protein denaturation.

• The reconstituted protein should be clear or slightly opalescent without visible particles.

Storage Recommendations:

• Store lyophilized protein at -20°C to -80°C.

• After reconstitution, aliquot the protein to minimize freeze-thaw cycles.

• For short-term storage (1-2 weeks), store at 4°C.

• For long-term storage, keep at -20°C to -80°C.

• Avoid more than 3 freeze-thaw cycles as this can significantly reduce biological activity .

Important Precautions:

• This product is an active protein and may elicit biological responses in vivo, so handle with appropriate caution .

• Maintain sterile conditions during handling to prevent contamination.

• Always use low-protein binding tubes and pipette tips to minimize protein loss due to adsorption.

What are the most effective methods for measuring platelet responses to Thrombopoietin in mouse models?

Measuring platelet responses to Thrombopoietin in mouse models requires careful methodological consideration. Based on research protocols, the following approaches are most effective:

Blood Collection Procedure:

• Collect peripheral blood (~50 μL) from mice via tail vein in EDTA tubes to prevent coagulation .

• Establish a baseline measurement before TPO administration, then collect at appropriate intervals (e.g., 2 weeks and 15 weeks post-administration) to track changes over time .

Analysis Methods:

• Complete Blood Count (CBC): Process samples using automated hematology systems such as the HEMAVET 950FS Hematology System for comprehensive blood cell analysis including platelet counts .

• Flow Cytometry: For detailed platelet population analysis, particularly to distinguish between reticulated (young) and mature platelets.

• Enzyme-Linked Immunosorbent Assay (ELISA): To measure serum TPO concentrations and correlate them with platelet responses.

Key Parameters to Measure:

• Total platelet count

• Reticulated platelet count (indicates new platelet production)

• Mean platelet volume (MPV)

• Platelet distribution width (PDW)

• Serum TPO concentration

Experimental Design Considerations:

• Include appropriate controls (vehicle-treated, baseline measurements)

• Consider the lag time between TPO administration and platelet response (typically 3-5 days)

• Account for the normal half-life of platelets (~5 days in mice)

• Plan sampling intervals to capture both acute and sustained effects

These methodologies provide comprehensive assessment of TPO activity on thrombopoiesis and platelets in mouse models.

How can Thrombopoietin function be assessed in in vitro experimental systems?

In vitro assessment of Thrombopoietin function can be performed through several established methodologies:

Cell-Based Functional Assays:

• Megakaryocyte Differentiation Assay:

  • Culture hematopoietic progenitor cells (CD34+ cells from bone marrow or cord blood) with recombinant TPO.

  • Assess megakaryocyte differentiation by flow cytometry analysis of CD41/CD61 expression.

  • Quantify megakaryocyte maturation by measuring ploidy through DNA content analysis.

  • Evaluate proplatelet formation using phase-contrast microscopy.

• Proliferation Assays with TPO-Dependent Cell Lines:

  • Use cell lines expressing the TPO receptor (c-Mpl) such as Ba/F3-mpl or UT-7/TPO.

  • Measure proliferation using colorimetric assays (MTT/XTT), cell counting, or incorporation of tritiated thymidine.

  • Generate dose-response curves to determine EC50 values.

• Signal Transduction Analysis:

  • Assess TPO-induced phosphorylation of JAK2, STAT3, STAT5, ERK, and AKT by Western blotting.

  • Use flow cytometry to measure phospho-protein levels at single-cell resolution.

  • Employ inhibitors of specific signaling pathways to determine their contribution to TPO-mediated effects.

Protein Interaction Studies:

• Receptor Binding Assays:

  • Measure binding affinity of TPO to c-Mpl using surface plasmon resonance (SPR).

  • Perform competitive binding assays with labeled TPO.

  • Assess receptor internalization following TPO binding.

• Protein Structure-Function Analysis:

  • Generate structure-based mutations to identify critical residues for activity.

  • Use circular dichroism to assess conformational changes.

These methodologies provide complementary approaches to characterize the functional activity of recombinant mouse Thrombopoietin protein in controlled experimental systems.

How can recombinant mouse Thrombopoietin be utilized in bone healing and regenerative medicine research?

Recombinant mouse Thrombopoietin has emerged as a significant factor in bone healing and regenerative medicine research, with applications in various experimental models:

Experimental Models for TPO in Bone Healing:

• Critical-Size Defect (CSD) Models:

  • TPO has been successfully utilized in rat, mouse, and pig critical-size defect models to enhance bone healing .

  • The delivery methods include incorporation into dicalcium phosphate dihydrate (DCPD) cement or collagen sponges placed directly in the defect site .

  • Assessment of healing typically involves radiographic analysis, micro-CT quantification of bone volume, and histological evaluation of new bone formation.

• Closed Fracture Models:

  • TPO administration has shown efficacy in enhancing healing in closed fracture models in mice .

  • This model more closely resembles clinical fractures and provides insights into TPO's effects on natural healing processes.

Methodological Considerations:

• Dosing and Administration:

  • Dose-dependent effects have been observed, requiring careful titration for optimal outcomes.

  • Local delivery at the fracture site versus systemic administration shows different efficacy profiles.

  • Timing of administration (pre-fracture, immediate post-fracture, or delayed) significantly impacts outcomes.

• Combination Therapies:

  • Synergistic effects have been observed when combining TPO with other growth factors or biomaterials.

  • TPO can be incorporated into various delivery systems including hydrogels, scaffolds, and microspheres for sustained release.

• Assessment Parameters:

  • Radiographic scoring of bone healing

  • Micro-CT analysis of bone volume and microarchitecture

  • Biomechanical testing of healed bone strength

  • Histological and immunohistochemical analysis of bone formation markers

  • Correlation between platelet counts and bone healing outcomes

This research area represents a promising frontier in translational medicine, potentially offering new therapeutic approaches for challenging bone healing scenarios such as non-unions or large bone defects.

What are the key considerations when using mRNA-based delivery of Thrombopoietin compared to recombinant protein?

The use of mRNA-based delivery of Thrombopoietin represents an advanced alternative to recombinant protein administration, with several important considerations:

Comparative Advantages of mRNA-TPO Delivery:

• Duration of Effect:

  • TPO mRNA-loaded lipid nanoparticles (LNPs) provide sustained protein production in vivo, potentially extending the therapeutic window compared to recombinant protein .

  • A single intravenous dose of TPO mRNA-LNPs can significantly increase both reticulated and total platelet counts for an extended period .

• Potency and Dose Requirements:

  • Submicrogram quantities of N1-methylpseudouridine-modified TPO mRNA have demonstrated similar effects in promoting thrombopoiesis as the TPO receptor agonist romiplostim .

  • mRNA-TPO overcomes the limitation of recombinant TPO's short half-life in vivo, which requires higher dosages to achieve similar effects .

• Physiological Regulation:

  • mRNA delivery allows for protein production within native cellular environments, potentially preserving important post-translational modifications.

  • Plasma TPO protein levels after mRNA delivery increase over 1000-fold in a dose-dependent manner compared to normal physiological values .

Methodological Considerations for mRNA-TPO:

Experimental Protocol Adjustments:

• For thrombocytopenia models using anti-GPIbα antibodies, mRNA-TPO should be administered 1-2 days before antibody treatment due to the lag time required for protein production and subsequent platelet generation .

• Monitoring should include both TPO protein levels (via ELISA) and platelet counts (via CBC) to correlate protein production with functional outcomes .

• The kinetics of mRNA-induced TPO production and subsequent platelet response differ from direct protein administration, requiring adjusted experimental timelines .

These considerations highlight the importance of selecting the appropriate TPO delivery method based on specific research questions and experimental models.

How does recombinant mouse Thrombopoietin affect hematopoietic stem cell (HSC) maintenance and self-renewal in research models?

Recombinant mouse Thrombopoietin plays a crucial role in hematopoietic stem cell (HSC) maintenance and self-renewal, beyond its well-known effects on platelet production:

Functional Impact on HSCs:

• Quiescence Regulation:

  • TPO signaling through its receptor c-Mpl promotes HSC quiescence, a critical state for maintaining long-term repopulating potential.

  • In the absence of TPO signaling, HSCs exit quiescence and enter cell cycle, eventually leading to HSC exhaustion.

• Self-Renewal Enhancement:

  • TPO supports asymmetric divisions that maintain the HSC pool while simultaneously generating committed progenitors.

  • Long-term exposure to TPO in culture systems enhances HSC expansion without compromising multipotency.

• Niche Interactions:

  • TPO produced by bone marrow stromal cells contributes to the HSC microenvironment.

  • The TPO-c-Mpl axis interacts with other niche-derived signals (e.g., CXCL12, SCF) to orchestrate HSC behavior .

Experimental Approaches to Study TPO Effects on HSCs:

• Long-Term Repopulation Assays:

  • Competitive transplantation experiments reveal the superior reconstitution ability of HSCs exposed to TPO.

  • Serial transplantation studies demonstrate the maintenance of self-renewal capacity through multiple rounds of reconstitution.

• In Vitro HSC Expansion Protocols:

  • TPO is a key component of cytokine cocktails used for ex vivo HSC expansion.

  • Optimization of TPO concentration and exposure timing is critical for maintaining HSC function during expansion.

• Genetic Models:

  • TPO or c-Mpl knockout mice show significant reduction in HSC numbers and function.

  • Humanized TPO knockin mice (TPO h/h) demonstrate how human TPO can support human HSC maintenance in mouse environments .

Cross-Species Considerations:

The affinity and biological activity of mouse TPO for human c-Mpl may differ at physiological doses in vivo, particularly in the microenvironment of the HSC niche in the bone marrow. This has led to the development of humanized TPO mice to better support human HSC maintenance in xenotransplantation models .

What are common pitfalls in Thrombopoietin experiments and how can they be addressed?

Researchers working with recombinant mouse Thrombopoietin may encounter several challenges that can affect experimental outcomes. Here are common pitfalls and their solutions:

Protein Stability and Activity Issues:

• Loss of Biological Activity:

  • Problem: Reduced efficacy after storage or handling.

  • Solution: Minimize freeze-thaw cycles (limit to ≤3), store in small single-use aliquots, and add carrier proteins (e.g., 0.1% BSA) to dilute working solutions .

• Protein Aggregation:

  • Problem: Formation of visible precipitates affecting dosing accuracy.

  • Solution: Centrifuge solutions before use, optimize buffer conditions (pH, ionic strength), and use low-protein binding tubes for storage.

Experimental Design Challenges:

• Timing Discrepancies:

  • Problem: Misalignment between TPO administration and biological readout.

  • Solution: Account for the lag time (3-5 days) between TPO administration and platelet response in experimental timelines .

• Dose-Response Variability:

  • Problem: Inconsistent responses to the same dose of TPO.

  • Solution: Establish dose-response curves for each new lot of protein, consider individual variations in baseline platelet counts, and include appropriate controls.

• Mouse Strain Differences:

  • Problem: Variable responses across different mouse strains.

  • Solution: Maintain strain consistency within experiments, establish strain-specific baselines, and consider strain-specific dose adjustments.

Technical and Analytical Challenges:

• Blood Collection Artifacts:

  • Problem: Platelet activation or clumping during collection affecting counts.

  • Solution: Use proper anticoagulants (EDTA), ensure appropriate needle size for blood collection, and process samples promptly .

• Assay Interference:

  • Problem: Components in sample matrix interfering with TPO detection.

  • Solution: Validate assays with spike-recovery experiments, use appropriate dilution series, and consider sample pre-treatment to remove interfering substances.

• Data Interpretation Complexities:

  • Problem: Distinguishing direct TPO effects from secondary physiological responses.

  • Solution: Include time-matched controls, measure multiple parameters (TPO levels, platelet counts, megakaryocyte numbers), and consider using genetic models (e.g., c-Mpl knockouts) as negative controls.

These troubleshooting approaches can significantly improve the reliability and reproducibility of experiments involving recombinant mouse Thrombopoietin.

How can researchers optimize the delivery methods for recombinant Thrombopoietin in different experimental models?

Optimizing delivery methods for recombinant Thrombopoietin requires careful consideration of the experimental model and research objectives:

Systemic Delivery Optimization:

• Intravenous (IV) Administration:

  • Advantages: Rapid distribution, immediate availability to target tissues.

  • Optimization: Use carrier-free formulations, deliver as a slow bolus to prevent hemodynamic effects, and consider using lipid nanoparticles (LNPs) for mRNA-TPO delivery to extend expression .

  • Best for: Acute thrombocytopenia models, studies requiring rapid TPO elevation.

• Subcutaneous (SC) Administration:

  • Advantages: Slower release, prolonged exposure, less invasive than IV.

  • Optimization: Adjust concentration and volume to prevent precipitation at injection site, use hyaluronidase pre-treatment for enhanced absorption of large volumes.

  • Best for: Studies requiring sustained TPO levels, chronic treatment protocols.

• Intraperitoneal (IP) Administration:

  • Advantages: Technical ease, intermediate absorption rate between IV and SC.

  • Optimization: Ensure proper injection technique to avoid organ damage, use warmed solutions to minimize discomfort.

  • Best for: High-throughput studies requiring frequent administration.

Local Delivery for Tissue-Specific Applications:

• Bone Defect Models:

  • Delivery Systems: DCPD (dicalcium phosphate dihydrate) cement or collagen sponges impregnated with TPO .

  • Optimization: Determine optimal protein loading capacity, test release kinetics in vitro before in vivo application, and consider combination with osteoconductive materials.

  • Assessment: Evaluate local TPO concentration in the defect site at various timepoints to confirm sustained release.

• Closed Fracture Models:

  • Delivery Systems: Injectable hydrogels with controlled release properties.

  • Optimization: Adjust crosslinking density to control release rate, incorporate protease-cleavable linkers for cell-responsive release.

  • Best for: Mimicking physiological fracture healing environments.

Comparative Delivery Efficiency:

These optimized delivery approaches enable researchers to tailor TPO administration to specific experimental requirements, improving both efficacy and reproducibility across different model systems.

How can researchers differentiate between physiological effects of Thrombopoietin on platelets versus its effects on other hematopoietic lineages?

Differentiating between the various effects of Thrombopoietin requires sophisticated experimental approaches that can isolate lineage-specific responses:

Methodological Approaches for Lineage Distinction:

• Temporal Analysis:

  • Rationale: Different hematopoietic lineages respond to TPO with distinct kinetics.

  • Method: Conduct time-course experiments monitoring multiple lineages simultaneously.

  • Analysis: Platelet responses typically occur within days (3-5 days), while HSC effects may take weeks to manifest fully.

  • Controls: Include lineage-specific cytokines (e.g., G-CSF, EPO) as comparators to establish response specificity.

• Cell-Specific Receptor Modulation:

  • Rationale: TPO effects are mediated through c-Mpl receptor, which has lineage-specific expression patterns.

  • Method: Use conditional knockout models with lineage-specific deletion of c-Mpl.

  • Analysis: Compare responses between wild-type and lineage-specific knockout animals to isolate direct effects.

• Competitive Transplantation Assays:

  • Rationale: Allows assessment of TPO effects on stem cell function versus mature lineages.

  • Method: Transplant mixed populations of wild-type and c-Mpl-deficient cells, then administer TPO.

  • Analysis: Track donor chimerism in different lineages over time to distinguish stem cell effects from direct lineage effects.

Analytical Framework for Distinguishing TPO Effects:

Advanced Approaches for Complex Models:

• Single-Cell Analysis:

  • Employ single-cell RNA sequencing to identify cell populations directly responding to TPO.

  • Track TPO receptor (c-Mpl) expression and downstream signaling at single-cell resolution.

  • Correlate molecular signatures with functional outcomes across lineages.

• Inducible Expression Systems:

  • Use tetracycline-inducible or tamoxifen-inducible systems to control the timing of TPO expression or c-Mpl activation.

  • This approach allows for precise temporal control to dissect immediate versus delayed effects.

• Phospho-Flow Cytometry:

  • Directly measure TPO-induced signaling in different hematopoietic populations simultaneously.

  • Quantify phosphorylation of STAT3, STAT5, ERK, and AKT in lineage-defined populations to identify direct responders.

These approaches provide robust frameworks for distinguishing the multifaceted effects of TPO across the hematopoietic system, enabling more precise interpretation of experimental outcomes.