Recombinant Human LIF

Recombinant Human LIF (Leukemia Inhibitory Factor) is essential in research applications primarily for its ability to maintain the pluripotency and self-renewal of embryonic stem cells and induced pluripotent stem cells, making it indispensable for stem cell culture, regenerative medicine, and developmental biology studies.

LIF is a cytokine that activates the STAT3 signaling pathway, which is critical for keeping stem cells in an undifferentiated state and preventing spontaneous differentiation. This property is vital for:

• Stem Cell Culture: LIF is routinely used to maintain mouse embryonic stem cells (mESCs) and human pluripotent stem cells in an undifferentiated, proliferative state, supporting long-term culture and expansion for downstream applications.
• Pluripotency Maintenance: By preventing differentiation, LIF ensures high cell viability and consistent cell behavior, which is crucial for reproducible experimental results and for generating sufficient cell numbers for research or therapeutic use.
• Regenerative Medicine: LIF’s role in maintaining stem cell pluripotency underpins its use in tissue engineering, cell therapy development, and disease modeling, where undifferentiated stem cells are required for differentiation into specific cell types.
• Developmental Biology: LIF is involved in embryogenesis, hematopoietic differentiation, neuronal development, and implantation studies, making it a versatile tool for investigating developmental processes.
• Cancer and Disease Research: LIF can induce terminal differentiation of myeloid leukemia cells and is studied for its effects on bone metabolism, inflammation, and fertility treatments.

Advantages of Recombinant Human LIF:

• High Purity and Low Endotoxin: Recombinant production methods yield LIF with superior purity and minimal endotoxin contamination, reducing experimental variability and risk of unwanted immune responses.
• Animal-Origin-Free: Recombinant LIF is produced without animal-derived components, minimizing the risk of xenobiotic contamination and supporting clinical translation.
• Consistent Activity: Recombinant LIF from various expression systems (e.g., E. coli, rice, human cells) shows comparable biological activity, supporting reliable and reproducible results across experiments.

Summary of Key Applications:

• Maintenance of pluripotency in stem cell cultures
• Feeder-free culture systems for stem cells
• Regenerative medicine and tissue engineering
• Developmental and cancer biology research
• Functional assays and differentiation studies

Using recombinant human LIF ensures experimental consistency, safety, and scalability, which are critical for both basic research and translational applications in stem cell biology and regenerative medicine.

You can use recombinant human LIF as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately determined. Most commercial human LIF ELISA kits are specifically designed to quantify both recombinant and natural human LIF, and their standard curves are typically generated using recombinant LIF protein.

Key considerations:

• Parallelism: ELISA kits often validate that the dose-response curves for recombinant and natural LIF are parallel, ensuring that quantification is accurate for both forms. This means recombinant LIF is suitable as a standard for quantifying endogenous LIF in biological samples.
• Calibration: Some kits are calibrated against international standards (e.g., NIBSC/WHO Reference Reagent 93/562), and recombinant LIF standards are traceable to these references.
• Purity and Quantification: The recombinant LIF used as a standard should be highly purified, and its concentration should be determined by reliable methods such as HPLC or absorbance at 280 nm.
• Matrix Matching: Prepare your standard curve in the same matrix (e.g., serum, plasma, or buffer) as your samples to minimize matrix effects and ensure accuracy.
• Validation: Always validate that your recombinant LIF standard produces a standard curve with acceptable precision and parallelism to your sample matrix.

Best practices:

• Use recombinant LIF from a reliable source, with a certificate of analysis confirming purity and concentration.
• Prepare serial dilutions carefully, using the same diluent as your samples.
• Confirm that your ELISA kit or protocol is validated for recombinant LIF as a standard—most commercial kits are, but custom or in-house assays should be validated for this use.

In summary, recombinant human LIF is widely accepted and routinely used as a standard for ELISA quantification, as long as you follow best practices for standard preparation and assay validation.

Recombinant Human LIF (Leukemia Inhibitory Factor) has been validated in published research for several key applications, primarily in stem cell biology, cell differentiation, and immunology.

Validated Applications in Published Research:

• Maintenance of Pluripotency in Embryonic Stem Cells:
  Recombinant human LIF is widely used to maintain the undifferentiated, pluripotent state of mouse embryonic stem cells (mESCs) in culture by suppressing spontaneous differentiation. It supports the expression of pluripotency markers such as Oct4, Nanog, and SSEA-1.

• Induction of Differentiation in Leukemic and Hematopoietic Cells:
  LIF induces terminal differentiation in myeloid leukemia cell lines (e.g., M1 cells) and promotes hematopoietic differentiation in both normal and leukemic cells.

• Neuronal Cell Differentiation:
  LIF has been validated for inducing neuronal differentiation and is used in studies of neural development and neurobiology.

• Stem/Immune Cell Maintenance or Differentiation:
  LIF is used in protocols for the maintenance and differentiation of various stem and immune cells, including regulatory T cells and in studies of immune tolerance.

• Cell Culture and Reprogramming:
  It is applied in the reprogramming and maintenance of human induced pluripotent stem cells (iPSCs) to a naïve-like state.

• Functional Bioassays:
  LIF is validated in bioassays measuring its activity, such as inhibition of cell growth during LIF-dependent differentiation and apoptosis assays.

• Cell Migration/Motility and Apoptosis Assays:
  LIF has been used in cell migration/motility and apoptosis assays, particularly in cancer and developmental biology research.

• Investigation of Developmental Processes:
  LIF is used to study hematopoiesis, bone, and neural development, as well as mesenchymal-to-epithelial transition during kidney development.

• Immunomodulation:
  LIF promotes regulatory T cell differentiation, inhibits Th17 cell differentiation, and contributes to immune tolerance, especially during pregnancy and in the nervous system.

Summary Table of Validated Applications

These applications are supported by both primary research articles and product validation data from multiple independent sources.

To properly reconstitute and prepare recombinant human LIF protein for cell culture experiments, follow these general best practices, which are consistent across major suppliers and scientific protocols:

1. Preparation Before Reconstitution

• Centrifuge the vial briefly (20–30 seconds in a microcentrifuge) before opening to ensure all lyophilized powder is at the bottom.
• Warm the vial to room temperature before opening to prevent condensation.

2. Reconstitution Buffer

• Preferred buffer: Sterile 1x PBS (pH 7.4) is most commonly recommended.
• Carrier protein: For improved stability, especially for long-term storage or low-concentration use, add 0.1% endotoxin-free recombinant human serum albumin (HSA), bovine serum albumin (BSA), or use trehalose (for animal-free or in vivo applications).
• Alternative: Some protocols recommend sterile 10 mM acetic acid (AcOH) for initial reconstitution, followed by dilution into PBS or culture medium.

3. Reconstitution Procedure

• Add the recommended volume of buffer (e.g., sterile PBS or PBS + 0.1% BSA) to achieve a stock concentration of 0.1–0.2 mg/mL (100–200 µg/mL).
• Gently swirl or tap the vial to dissolve the protein. Do not vortex or shake vigorously to avoid foaming and protein denaturation.
• Allow the solution to sit at room temperature for 15–30 minutes with gentle agitation if needed.

4. Storage and Handling

• Short-term (≤1 week): Store the reconstituted protein at 2–8°C (refrigerator) under sterile conditions.
• Long-term: For storage beyond one week, aliquot the protein in small volumes, add carrier protein (e.g., 0.1% BSA or 5–10% trehalose), and freeze at –20°C to –80°C.
• Avoid repeated freeze-thaw cycles to maintain activity.

5. Use in Cell Culture

• Dilute the stock solution in culture medium just before use.
• Typical working concentrations range from 10–100 ng/mL, depending on the cell type and application (e.g., stem cell maintenance, differentiation, or signaling assays).

6. Additional Notes

• Always refer to the Certificate of Analysis (CoA) or product manual for specific recommendations, as formulations may vary by supplier.
• For serum-free or animal-free cultures, use trehalose instead of BSA or HSA.

Summary Protocol:

1. Centrifuge vial, warm to room temperature.
2. Reconstitute with sterile PBS (or PBS + 0.1% BSA/trehalose) to 0.1–0.2 mg/mL.
3. Gently mix, do not vortex.
4. Store short-term at 2–8°C; aliquot and freeze at –20°C for long-term.
5. Dilute in culture medium before use.

This approach ensures optimal activity and stability of recombinant human LIF for cell culture applications.