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From Fibroblasts to Cancer-Attacking Cells: Insights from Recent Research on Natural Killer Cells Derived from Clinical Grade iPSCs

By Dayana Ivanova

In the past quarter-century, induced pluripotent stem cells (iPSCs) have emerged as a game-changer in medical research, offering groundbreaking opportunities for studying diseases and developing cutting-edge cell therapies. Their remarkable ability to self-renew indefinitely and differentiate into any cell type makes research, but especially clinical grade iPSCs, a versatile tool for regenerative medicine projects.

Among the variety of cell types iPSCs can be differentiated into, Natural Killer (NK) cells stand out for their impressive ability to target and destroy cancer cells, highlighting their potential for ‘’off-the-shelf’’ cancer immunotherapies 1. In this blog, we explore the general immune and protective role of NK cells against cancer in research and their promise as a cellular therapy. We will also summarize a recent research study conducted on NK cells derived from REPROCELL’s clinical iPSCs in collaboration with the Tokyo Metropolitan Institute of Medical Science in Japan.

Where Do NK Cells Come From?

Part of the innate lymphoid cell family, NK cells use their cytotoxic ability against cells endangering the host. Although the site of origin of NK cells is still debated, it is broadly recognized that CD34+ hematopoietic stem cells (HSCs) circulating in the blood differentiate to common lymphoid progenitors (CLPs) which further differentiate to NK cell progenitors. Upon maturation, NK cells begin to acquire self-tolerance–the ability to distinguish between self and non-self, including pathogens–which is critical for immune function 2. Functional NK cells are characterized by CD56 expression 3. CD56 plays a critical role in NK cell maturation by promoting migratory behavior on stromal cells, enabling the formation of developmental synapses. Its expression correlates with increased motility during maturation, and blocking CD56 disrupts both motility and maturation processes in NK cells 4. The expression of CD56, particularly in the CD56bright subset, is immature and primarily associated with cytokine release, while the CD56dim subset is considered more mature and predominantly releases cytotoxic granules 5.

 

NK cells: The Immune System’s First Responders

NK cells function as a first line of host immune defense against aberrant cells such as virally infected or transformed cells, including tumors. NK cells have cytotoxic abilities to kill either via the directed release of lytic granules towards the target cells or by inducing apoptosis through death receptor pathways 6. NK cells recognize target cells based on the presence or absence of major histocompatibility complex (MHC) Class I molecules. These molecules act as ligands for the inhibitory receptors present on NK cells. MHC Class I molecule binding suppresses cell death induced by NK cell-mediated cytotoxicity, whereas lack of these molecules triggers the “missing self-recognition” phenomenon associated with target cell lysis. The inhibitory receptors are not only essential for self-tolerance but also for refining cytotoxicity. The array of inhibitory receptors, such as KIRs, KLRs, and LILRs, as well as activation receptors like NKG2D and NKp46, play critical roles in fine-tuning the NK cell response 7. The mechanism of action of activating receptors on NK cells involves the recognition of stress-induced ligands and the subsequent engagement of immunoreceptor tyrosine-based activation motifs (ITAMs), which triggers a signaling cascade leading to NK cell activation, cytokine release, and cytotoxicity against target cells 8.

 

Why is the Focus Shifting to iPSC-derived NK Cells?

NK cells have a demonstrated ability to identify, target, and destroy cancer cells. The initial focus was concentrated on autologous NK cell-mediated immunotherapies as studies confirmed the safety and feasibility of these approaches. However, they often fail to elicit strong responses against hematological malignancies or solid tumors due to inhibitory interactions with self MHC‑I molecules. This led researchers to explore allogeneic NK cell therapies, sourcing NK cells from peripheral blood mononuclear cells or umbilical cord blood and clonal NK cell lines like NK-92. Allogeneic NK cells show higher cytotoxicity and proliferative potential, but each source has limitations, such as product heterogeneity, reduced cytotoxicity in UCB-NK cells, and the need for radiation in NK-92 cells due to genetic instability 1.

As a consequence, the scientific community's focus has shifted to allogeneic iPSC-derived NK (iNK) cells due to their high cytotoxicity, and consistent homogeneity 1. This trend can also be seen in several clinical trials exploring the immunotherapeutic potential of iNK cells 9. Moreover, the ease of genetic engineering in iPSCs enables the creation of iPSC-derived chimeric antigen receptor (CAR)-NK cells with improved safety, cytotoxicity, persistence, and proliferation, significantly boosting their therapeutic potential 1.

One approach to generating iNK cells is illustrated in Figure 1. In brief, iPSCs reprogrammed from fibroblasts are first differentiated into CD34+ hematopoietic progenitor cells, then to CD7+/CD45+ lymphoid progenitor cells, and ultimately into NK cells using a feeder-independent system.

 

UPDATED Fibroblast derived NK cells

Figure 1. Generation of iNK cells from fibroblasts. iPSCs derived from primary cells like fibroblasts are converted to embryoid bodies (EBs). Then, the EBs were differentiated into CD34+ hematopoietic progenitor cells which were further differentiated into CD45+ CD7+ lymphocyte progenitor cells. A feeder-free system was used for final differentiation to CD56+ NK cells.

 

Recent Research on NK cells differentiated from Clinical iPSCs

Using REPROCELL’s clinical StemRNA iPSCs, collaborators from the Tokyo Metropolitan Institute of Medical Science in Japan successfully generated functional human iNK cells using a feeder-free and serum-free system. By generating EB’s and culturing in a media containing a specific cytokine cocktail, CD34+ hematopoietic progenitors could be derived and isolated. These cells were further differentiated into CD45+ CD7+ lymphocyte progenitors, isolated by a cell sorter followed by differentiation into CD56+ NK. Flow analysis on cells isolated at day 36 of differentiation induction revealed expression of cell surface markers expressed by activating receptors on NK cells – CD45, CD56, CD335, CD336, CD314 (Figure 2). This expression pattern is indicative of mature NK cells 10.

 

iPSC-derived NK cells Figure

Figure 2. Expression of key NK cell surface markers on day 36 of differentiation induction from clinical iPSCs. CD45+CD56+ NK cells also expressed NKp44 (CD336), NKp46 (CD335), and NKG2D (CD314). 10

 

The potent cytotoxic activity of these functional human iNK cells is demonstrated by a coculture experiment. When ten thousand CD56+iNK cells were co-cultured with one thousand K562 cells (a human chronic myeloid leukemia cell line) they effectively targeted and destroyed the cancer cells. For a closer look at this process, take a look at Video 1 below, which shows live imaging of the iNK cells in action. In the footage, you will see green CFSE-labelled K562 cells being targeted and engulfed by the elongated iNK cells, confirming their Natural Killer ability 10.

 

 

Video 1. Live imaging of human iNK cells engulfing K562 cells. The elongated human iNK cells demonstrated cytotoxic properties when co-cultured with the cancer cell line K562, labeled with the cell staining reagent CFSE (green round cells) 10

 

A key insight from the described study is that hematopoietic cells such as NK cells can be effectively generated from iPSCs derived from cells of non-hematopoietic origin such as fibroblasts, and fibroblasts can generate functional NK cells with cytotoxic abilities.

 

In conclusion, this study demonstrates that these iNK cells show functions typical of native NK cells - the ability to attack and eliminate malignant cells—and phenotypical characteristics such as the expression of typical markers.  The development of NK cells derived from clinical-grade human iPSCs highlights the versatile differentiation capability of human iPSCs and marks a significant advancement in cancer immunotherapy. With their unique ability to target and destroy cancer cells effectively, these cells pave the way for personalized and off-the-shelf therapies. By adhering to Good Manufacturing Practice (GMP) in a xeno-free environment during iPSC production, iPSCs can ensure high safety and reliability for a range of applications in the cell therapy market.

 

Discover REPROCELL’s Ready-to-Use GMP grade iPSCs

Unlock the potential of cutting-edge cell therapy with REPROCELL’s 'off-the-shelf' clinical-grade human iPSCs, created using our advanced footprint-free StemRNA™ 5.0 Clinical Reprogramming Technology. Our GMP-grade human iPSCs are ready for immediate use, ensuring high quality and reliability for your research or therapeutic needs. In addition, we also offer personalized gene editing services. Whether you need to reprogram your cells into iPSCs or differentiate them into specific cell types, we have you covered.

Curious to learn more? Explore our offerings and get started by visiting our products and services pages:

GMP iPSC Production (reprocell.com)

Products for iPSC Research (reprocell.com)

StemEdit Clinical Gene Editing Service (reprocell.com)

 

 

References

  1. Lin et al. IPSC-derived CAR-NK cells for cancer immunotherapy. Biomedicine & Pharmacotherapy, 165, 115123 (2023).
  2. Portale and Di Mitri. NK Cells in Cancer: Mechanisms of Dysfunction and Therapeutic Potential. Int J Mol Sci, 24: 9521 (2023).
  3. Lanier et al. Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule. Journal of Experimental Medicine, 169, 2233–2238 (1989).
  4. Acker et al. CD56 in the Immune System: More Than a Marker for Cytotoxicity? Frontiers in Immunology 8 (2017).
  5. Seymour et al. NK cells CD56bright and CD56dim subset cytokine loss and exhaustion is associated with impaired survival in myeloma. Blood Advances 6, 5152–5159 (2022).
  6. Prager and Watzl. Mechanisms of natural killer cell-mediated cellular cytotoxicity. Journal of Leukocyte Biology, 105, 1319-1329 (2019).
  7. Pluripotent Stem Cell-Based Cell Therapy—Promise and Challenges. Cell Stem Cell, 27, 523-531 (2020).
  8. Paul and Lal. The Molecular Mechanism of Natural Killer Cells Function and Its Importance in Cancer Immunotherapy. Frontiers in Immunology, 8 (2017).
  9. ClinicalTrialGov- Search for: iPSC NK cells | Card Results | ClinicalTrials.gov
  10. Kenji Kitajima. Tokyo Metropolitan Institute of Medical Science (2024).

 

 

 

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