Recombinant Human TPO

Recombinant Human Thrombopoietin (TPO) is widely used in research because it is a critical regulator of hematopoietic stem cell (HSC) proliferation, differentiation, and survival, as well as megakaryocyte maturation and platelet production. Its use enables precise control and reproducibility in studies involving hematopoiesis, thrombopoiesis, and related cellular therapies.

Key scientific reasons to use recombinant human TPO in research applications:

• Expansion and Maintenance of Hematopoietic Stem Cells: TPO promotes the survival, self-renewal, and expansion of HSCs and primitive multilineage progenitors, making it essential for ex vivo HSC culture, expansion, and transplantation studies.
• Megakaryocyte and Platelet Production: TPO is the principal cytokine driving megakaryocyte differentiation and maturation, leading to increased platelet production. This is crucial for in vitro models of thrombopoiesis and for generating platelets for research or therapeutic purposes.
• Modeling and Treating Thrombocytopenia: Recombinant TPO is used to model thrombocytopenia (low platelet count) and to study interventions for its treatment. It has been shown to accelerate platelet recovery and reduce the need for platelet transfusions in preclinical and clinical settings.
• Cellular Therapy and Immunotherapy Research: TPO, often in combination with other cytokines (e.g., SCF, Flt-3 ligand), is used to generate and expand immune cell populations such as induced natural killer (iNK) cells, which are being explored for cellular immunotherapies.
• Hematopoietic Recovery Post-Chemotherapy or Radiation: In animal models and clinical studies, recombinant TPO enhances hematological recovery after myelosuppressive treatments, supporting its use in research on bone marrow recovery and transplantation.
• Mechanistic Studies: Recombinant TPO allows for controlled mechanistic studies of the TPO receptor (c-MPL) signaling pathway, which is central to understanding hematopoietic and megakaryocytic biology.

Additional advantages:

• Reproducibility and Consistency: Recombinant proteins provide batch-to-batch consistency, which is critical for experimental reproducibility.
• Defined Activity: Recombinant TPO is available in animal-free and highly purified forms, reducing variability and the risk of confounding factors from serum or crude extracts.
• Safety and Specificity: Recombinant TPO acts specifically via the TPO receptor, with minimal off-target effects compared to some small-molecule TPO receptor agonists.

Typical research applications include:

• Ex vivo expansion of HSCs for transplantation studies.
• In vitro differentiation of megakaryocytes and production of platelets.
• Modeling hematopoietic disorders and testing new therapies.
• Investigating mechanisms of hematopoietic and immune cell regulation.

In summary, recombinant human TPO is a powerful and versatile tool for research in hematology, stem cell biology, and regenerative medicine due to its well-characterized, potent, and specific biological effects on hematopoietic and megakaryocytic lineages.

Yes, you can use recombinant human TPO as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative ELISA workflows, especially when a purified native protein standard is unavailable.

Key considerations and best practices:

• Purity and Formulation: Use a highly purified recombinant TPO protein. Some suppliers recommend using the BSA-containing formulation for ELISA standards, as it can help stabilize the protein during dilution and storage. Carrier-free formulations are also available if BSA may interfere with your assay.

• Standard Curve Preparation: Prepare a standard curve using serial dilutions of the recombinant TPO in the same buffer or matrix as your samples to ensure accurate quantification. The typical range for ELISA standard curves is from 0 to 1000 pg/mL, but this can be adjusted based on expected sample concentrations.

• Concentration Verification: The concentration of the recombinant protein should be verified, ideally by an independent method such as HPLC or absorbance at 280 nm, since discrepancies can occur between the mass reported on the vial and the immunologically detectable mass in the ELISA. Lot-to-lot variability and differences in glycosylation or folding can affect the apparent concentration in the assay.

• Value Assignment: When using a recombinant protein as a standard, it is best practice to assign its value based on its performance in the ELISA, rather than relying solely on the mass stated on the vial. This helps account for any differences in immunoreactivity.

• Compatibility: Ensure that the recombinant TPO you use as a standard is recognized by the antibodies in your ELISA kit. Most commercial ELISA kits are validated with both natural and recombinant TPO standards.

• Documentation: Follow the reconstitution and dilution instructions provided with your recombinant protein, as these can be lot-specific and affect the accuracy of your standard curve.

Limitations:

• Immunoreactivity Differences: Recombinant proteins may differ from native proteins in post-translational modifications (e.g., glycosylation), which can affect antibody recognition and quantification accuracy.
• Dilution Errors: Large dilution steps (from µg/mL to pg/mL) can introduce errors; careful pipetting and use of calibrated equipment are essential.

Summary Table: Recombinant TPO as ELISA Standard

In summary: Recombinant human TPO is suitable as a standard for ELISA quantification if you follow best practices for preparation, concentration verification, and compatibility with your assay system.

Recombinant Human Thrombopoietin (TPO) has been validated for a broad range of applications in published research, primarily focused on hematopoietic stem cell biology, megakaryocyte differentiation, platelet production, immunomodulation, and disease modeling.

Key validated applications include:

• Hematopoietic Stem and Progenitor Cell Expansion and Differentiation

  • Used to support the self-renewal, maintenance, and expansion of human hematopoietic stem cells (HSCs) in vitro.
  • Facilitates differentiation of HSCs into megakaryocytes and platelets.

• Megakaryocyte Maturation and Platelet Production

  • Promotes megakaryocyte proliferation and maturation, leading to increased platelet production.
  • Applied in bioassays to assess functional activity of TPO and its receptor c-MPL.

• Cell Culture and Disease Modeling

  • Used in the generation and maintenance of human induced pluripotent stem cells (iPSCs) and disease-specific iPSC lines.
  • Supports cell culture protocols for studying hematopoietic disorders, such as inherited thrombocytopenia, myelodysplastic syndromes, and severe aplastic anemia.

• Immunomodulation

  • Demonstrated to modulate immune cell populations, including enhancing CD4^+^ T cell and regulatory T cell (Treg) proliferation, and restoring immune homeostasis in severe aplastic anemia.
  • Shown to accelerate thymic T cell reconstitution and improve immune recovery post-irradiation.

• Functional and Analytical Assays

  • Validated for use in bioassays, ELISA (as control and standard), and Western blotting to measure TPO activity and detect TPO protein.
  • Used in differentiation assays to study lineage commitment and maturation of hematopoietic cells.

• Clinical and Translational Research

  • Evaluated for therapeutic efficacy in reducing thrombocytopenia and bleeding in patients undergoing chemotherapy, radiotherapy, or surgery.
  • Investigated as a radioprotectant and for ex vivo expansion of pluripotent stem cells for transplantation.

• Gene Editing and Stem Cell Engineering

  • Incorporated into protocols for CRISPR/Cas9-mediated gene editing in human iPSCs and HSCs to model hematologic diseases and study clonal evolution.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and are widely adopted in both basic and translational hematology research.

To reconstitute and prepare Recombinant Human TPO (Thrombopoietin) protein for cell culture experiments, follow these technical steps:

1. Preparation Before Opening:

   • Briefly centrifuge the lyophilized vial (microcentrifuge, 20–30 seconds) to ensure all protein is at the bottom and not on the cap or sides.

2. Reconstitution Buffer:

   • Use sterile 1× PBS (pH 7.4) containing 0.1% endotoxin-free recombinant human serum albumin (HSA) or bovine serum albumin (BSA) as a carrier protein.
   • Alternatively, some protocols recommend sterile water for reconstitution, but always check the specific product datasheet for buffer requirements.

3. Protein Concentration:

   • Typical reconstitution concentrations range from 50–200 μg/mL, but concentrations up to 0.1–1.0 mg/mL are also used depending on experimental needs.
   • For example, add 1 mL buffer to 100 μg protein for a 0.1 mg/mL solution.

4. Dissolution:

   • Gently add the buffer to the vial. Do not vortex or mix vigorously; gently pipette up and down or allow the protein to dissolve by gentle agitation.
   • If solubility is an issue, incubate the solution at 4°C overnight.

5. Aliquoting and Storage:

   • After reconstitution, aliquot the solution into working volumes (≥10 μL per aliquot) to avoid repeated freeze-thaw cycles.
   • Store aliquots at –20°C to –80°C for long-term use, or at 2–8°C for short-term (up to 1 week).
   • Avoid repeated freeze-thaw cycles, as this can denature the protein.

6. Quality Control:

   • Confirm protein presence and integrity by SDS-PAGE if needed (a band at expected molecular weight should be visible with as little as 10 ng loaded).

7. Working Solution for Cell Culture:

   • Dilute the stock solution to the desired working concentration in cell culture medium immediately before use.
   • Typical effective concentrations for cell stimulation range from 0.05–0.5 ng/mL for hematopoietic stem cell expansion or megakaryocyte differentiation.

Additional Notes:

• Always consult the product-specific Certificate of Analysis (CoA) or datasheet for any unique requirements regarding buffer composition, concentration, or storage.
• If the protein is supplied with acidic stabilizers (e.g., 4 mM HCl), ensure the final working solution is neutralized or diluted in culture medium to avoid cytotoxicity.
• Carrier proteins (HSA/BSA) help stabilize TPO and prevent adsorption to plasticware, especially at low concentrations.

Summary Table: Recombinant Human TPO Reconstitution

This protocol ensures optimal solubility, stability, and biological activity of recombinant human TPO for cell culture applications.