Generation of Natural Killer Cells

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system. Recent advances in regenerative medicine have enabled the derivation of functional NK cells from induced pluripotent stem cells (iPSCs). This protocol outlines a stepwise procedure for the efficient differentiation of human iPSCs into functional NK cells, covering feeder-based expansion, cytokine modulation, and phenotypic characterization.

Overview of the Generation of Natural Killer Cells

NK cells are key effector lymphocytes of the innate immune system, responsible for the rapid identification and elimination of virus-infected, transformed, and stressed cells. Unlike cytotoxic T lymphocytes, NK cells function without prior antigen sensitization and play crucial roles in immune surveillance, tumor immunology, and immunoregulatory balance. The generation of NK cells from iPSCs offers a transformative platform for both basic and applied research due to the inherent advantages of iPSCs—pluripotency, scalability, and the potential for autologous or universal donor-derived immune cell products.

Fig.1 Generation of iPSC-derived NK cell-based cell therapy.^1,2

In recent years, iPSC-derived NK (iPSC-NK) cells have gained significant attention as next-generation immune effectors for cell-based assays, disease modeling, and preclinical validation of immune-modulating therapies. Compared to peripheral blood- or cord blood-derived NK cells:

• iPSC-NK cells allow for clonal expansion, genome editing (e.g., CAR, TCR knock-in), and functional optimization through cytokine or checkpoint modulation.
• iPSC-NK models offer high batch-to-batch consistency, which is critical for reproducible and scalable studies.

At Creative Biolabs, we have established a robust and modular workflow for deriving functional NK cells from human iPSCs, supporting a variety of downstream applications.

Materials and Reagents

Protocol Steps

iPSC Maintenance

Maintain pluripotent stem cells under feeder-free, xeno-free conditions. First, plate iPSCs on Matrigel-coated dishes in mTeSR1 medium. Passage every 3–5 days. Confirm pluripotency markers (OCT4, NANOG, SSEA4) via flow cytometry or immunofluorescence.

Embryoid Body (EB) Formation and Mesoderm Induction

Use plates to generate uniform EBs. Culture EBs in RPMI-1640 + B27 supplement with BMP4, VEGF, and SCF. Induce mesodermal lineage markers (e.g., Brachyury, KDR).

Hematopoietic Differentiation

Transfer EBs to low-attachment dishes in hematopoietic induction medium containing IL-3, IL-6, TPO, SCF and Flt3L. Monitor the emergence of CD34⁺CD45⁺ progenitor populations using flow cytometry.

NK Cell Lineage Commitment

Culture hematopoietic progenitors with a defined cytokine cocktail: IL-15, IL-7, IL-21, SCF, and Flt3L. Optionally, co-culture with irradiated AFT024 or OP9-DL1 feeder cells to enhance NK development.

NK Cell Expansion and Maturation

Expand committed NK precursors in medium supplemented with IL-2 and IL-15. Assess for surface markers CD45⁺CD56⁺CD3⁻ and expression of NK functional receptors (e.g., NKG2D, NKp30, NKp44, NKp46).

Quality Control & Characterization

Robust quality control (QC) and characterization of iPSC-derived NK cells are essential for ensuring reproducibility, functionality, and suitability for downstream applications such as drug screening or immunotoxicity assays. At Creative Biolabs, we implement multi-parameter quality assessments encompassing phenotypic, functional, and genetic validation

Troubleshooting and Optimization Tips

Effective troubleshooting is crucial to avoid batch failures and ensure consistency. Below is a practical guide to resolving common technical challenges encountered during iPSC-NK differentiation.

To maximize efficiency and consistency in iPSC-derived NK cell production, the following strategies are recommended.

• Feeder cell management
• Use irradiated AFT024 or OP9-DL1 stromal cells to enhance hematopoietic differentiation and NK cell survival.
• Feeder-free alternatives
• Cytokine cocktail fine-tuning
• Avoid overuse of IL-2, which may cause premature differentiation or exhaustion.
• IL-21 has been shown to enhance cytotoxic capacity and proliferation, particularly in late-stage maturation.
• Incorporating SCF and Flt3L at early stages helps stabilize the CD34^⁺ hematopoietic progenitor pool.
• Timing and density control
• Track differentiation kinetics with flow cytometry to optimize protocol staging.
• Avoid over-confluence, which leads to nutrient depletion and stress response.
• Cryopreservation and thawing
• Freeze NK cells in DMSO-containing serum-free medium.
• Thaw quickly at 37°C, dilute slowly into prewarmed media with cytokine support, and assess viability post-thaw with trypan blue or Annexin V staining.

Related Services at Creative Biolabs

We offer fully integrated services to support your iPSC-to-NK workflow.

• iPSC Reprogramming & Characterization

Generation of GMP-grade iPSCs from PBMCs or fibroblasts

• iPSC Differentiation

Custom protocols for hematopoietic and NK cell generation

• NK Cell Phenotyping

Comprehensive flow cytometry and cytokine profiling

For further customization or large-scale NK cell production, contact our technical specialists to design a tailored solution.

References

1. Zhou, Yang, et al. "Engineering induced pluripotent stem cells for cancer immunotherapy." Cancers 14.9 (2022): 2266. https://doi.org/10.3390/cancers14092266
2. Distributed under Open Access license CC BY 4.0, without modification.

For Research Use Only. Not For Clinical Use.