How to scale induced pluripotent stem cell differentiation into natural killer cells

This article is based on a poster originally authored by Kun Shi, Na Zhang, Xing Zhang, Spencer Chiang, Haonan Li and Tianfu Zhang.

The immune system’s natural killer (NK) cells are large granular cells that constitute the third major lymphocyte subset, representing around 10-15% of circulating lymphocytes in the blood.

Natural killer cells are a vital part of the immune system. They play a central role in the anti-inflammatory response and tumor surveillance.

The ability to acquire high-quality NK cells in sufficient numbers represents a significant bottleneck in terms of NK cell application in adoptive immunotherapy.

Induced pluripotent stem cells (iPSCs) offer a new means of solving this dilemma, however, with iPSCs’ potential for self-renewal and pluridirectional differentiation meaning that they can be induced to differentiate into a variety of cell types in vitro, including NK cells.

ACROBiosystems has developed a robust, novel means of inducing NK cells from iPSCs under entirely serum-free conditions. Using this method, the proportion of CD3-CD56+ NK cells reached 85.9% after 25 days of induction.

Developed NK cells exhibited similar biological functionality to NK cells generated from other sources, highlighting their feasibility for scaling up NK cell manufacturing in an array of clinical applications.

iPSC to NK cell workflow

Image Credit: ACROBiosystems

Step 1: Expansion of iPSCs

iPSCs are regarded as a foundational resource, due to their capacity for indefinite self-renewal in culture while continuing to maintain their pluripotency. This highly useful characteristic allows researchers to generate the significantly high quantities of uniform, high-quality cells required for contemporary therapeutic applications.

Several aspects of cell production must be monitored at this step, in order to ensure high-quality iPSC culture. These are; expansion-fold, stemness, and genetic uniformity.

It is also important to use only well-defined raw materials, for example, Recombinant Laminin 521, which removed the need for animal-origin materials. The use of GMP-grade materials can also help ensure safety and efficacy.

Figure 1. Laminin 521 used as a coating substrate for the fast expansion of single cell human PSCs across 4 days. Image Credit: ACROBiosystems

Figure 2 . Stemness markers OCT4, SOX2, and Nanog are present after several passages, showing robust self- renewal of hPSCs. Image Credit: ACROBiosystems

Figure 3. Karyotype analysis after 10 passages reveals a normal karyotype in iPSCs. Image Credit: ACROBiosystems

Step 2: iPSC to HSC differentiation

Human-induced pluripotent stem cells were digested into single cells in order to form embryoid bodies. Embryoid bodies were cultured in a series of mediums for 14 days. Factors included: BMP4, VEGF, bFGF, SCF, TPO, and Flt-3L.

CD34 and CD45 expression were analyzed via flow cytometry to ensure the presence of a sufficient HSC population.

Figure 4. (A) Embroyoid bodies formed by human induced pluripotent stem cells. Scale bar, 250 μm. (B,C) CD34+ CD45 + hematopoietic cell acquisition by FACS. Image Credit: ACROBiosystems

Step 3: HSC to NK cell differentiation

This step involved the seeding of human CD34+ hematopoietic cells in precoated wells. These were then cultured for 21 days in a medium containing factors such as SCF, TPO, Flt-3L, and IL-7. CD5 and CD7 expression was analyzed via flow cytometry.

Next, selected Lymphoid Progenitor Cells (LPCs) were seeded into precoated wells before being cultured further in a medium containing SCF, FLT3L, TPO, IL-2, IL-3, IL-7, and IL-15, as well as either or both DLL4 and VCAM1.

It is important to note that adding small molecules may also be effective in NK expansion, considerably increasing cell viability.

Figure 5. CD7+ CD5 - Lymphoid Progenitor Cell (LPC) differentiated from HSC evaluation on Day 21. DLL4 and VCAM1 were used as a supportive environment to drive differentiation to NK cell lineage. Image Credit: ACROBiosystems

Figure 6. Hematopoietic cells differentiate to CD3- CD56+ NK Cells after 25 days of Culture (A) Flow cytometry assessment of the percentage of NK cells. (B) Statistics of the percentage of NK cells under three different conditions. The results indicate that VCAM1 and DLL4 promotes NK cell differentiation. Image Credit: ACROBiosystems

Cytotoxicity, degranulation, and secreted cytokine analysis of developed NK cells

NK cells were co-cultured with cells derived with K562. This was done at E:T ratios of 0.625:1, 1.25:1, 2.5:1, 5:1, and 10:1.

The cytolysis of target cells was measured using a combination of 7-AAD/CFSE staining and tested via flow cytometry testing. It was observed that NK cells’ cytotoxicity increased in line with the increment of E:T ratio. For example, more than 40% of target cells were lysed, even at a low ratio of E:T=0.625:1.

Levels of CD107a on the NK cells were analyzed prior to and following co-culturing with K562 cells, in order to further evaluate developed NK cells. CD107a expression increased after co-culturing with K562, signifying that the NK cells were secreting more perforin and granzyme.

NK and K562 cells were co-cultured for a total of 4 hours at an E:T ratio of 10:1. The supernatant was then measured for IFN-γ using a ClinMax ^™ Human IFN-γ ELISA kit, highlighting that NK cells co-cultured with K562 secreted a greater amount of IFN-γ than the blank group. 

Figure 7. Cytotoxicity, Degranulation marker expression and cytokine production of iNK cells after exposure to K562 cells. (A, B) degranulation marker CD107a in iNK cells after coculture with K562 cells detected by flow cytometry. (C) Cytolysis of K562 cells was done with 7-AAD/CFSE staining and tested by flow cytometry. Spontaneous death of target cells has been subtracted from all plots. (D) IFN -γ secreted by iNK cells after exposure to K562 were quantified through ELISA assay. Image Credit: ACROBiosystems

Conclusion

CAR-NK has significant potential for use in cell therapy, as well as innate advantages in safety and manufacturing. In order to take advantage of the higher patient efficacy of NK cell therapies, however, it is imperative that a scalable, sustainable, and uniform source of NK cells be sourced.

iPSCs are central to this work, due to their capacity for reproducible expansion and differentiation into NK cells, while ensuring an easily traceable source.

In the examples presented here, extensive validation of the final cell phenotype was performed at each step, highlighting that the presented protocol for iPSC to NK differentiation is sufficiently scalable for cell therapy manufacturing.

References and further reading

1. Fares, I., & Yzaguirre, P. (2018). Small molecules for human hematopoietic stem cell expansion and differentiation. Current Opinion in Hematology, 25(4), 318-324.
2. Bhatia, M., & Wang, J. C. (2016). Clinical translation of haematopoietic stem cell expansion: achievements and challenges. Nature Reviews Drug Discovery, 15(10), 709-723.
3. Small molecule regulators for the ex vivo expansion of hematopoietic stem and progenitor cells. Journal of Hematology & Oncology, 10(1), 1-11.
4. Boitano, A.E., et al. (2010). Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells. Science, (online) 329(5997), pp.1345–1348. https://doi.org/10.1126/science.1191536.
5. Cutler, C., & Antman, K. H. (2004). The use of hematopoietic growth factors and cytokines in cancer treatment. CA: A Cancer Journal for Clinicians, 54(3), 150-161.
6. Maskalenko, N.A., Zhigarev, D. and Campbell, K.S. (2022). Harnessing natural killer cells for cancer immunotherapy: dispatching the first responders. Nature Reviews Drug Discovery, 21(8), pp.559–577. https://doi.org/10.1038/s41573-022-00413-7.
7. Vivier, E., et al. (2024). Natural killer cell therapies. Nature, 626(8000), pp.727–736. https://doi.org/10.1038/s41586-023-06945-1.

Acknowledgements

Produced from materials originally authored by Kun Shi, Na Zhang, Xing Zhang, Spencer Chiang, Haonan Li, and Tianfu Zhang from ACROBiosystems.

About ACROBiosystems

ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.

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