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Microbes & Immunity iPSC-derived NK cell immunotherapy

differentiating pluripotent stem cells, including embryonic differentiation of HSCs into NK cells. After approximately
stem cells and iPSCs, into HSCs. One such method involves 4 weeks of induced differentiation toward NK cells, the
culturing iPSCs on the top of feeder cells, which provides majority of the culture system will become CD45 CD56
+
+
a sufficient microenvironment for the differentiation NK cells. The differentiated NK cells can be expanded
into HSCs without the need for additional cytokines. For several hundred-fold with the support of IL-2 and artificial
instance, OP9 cells can directly facilitate the differentiation antigen-presenting cells, achieving increased purity during
of iPSCs into HSCs. In this process, iPSCs are digested the expansion process. In recent reports, 3D culture
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into small aggregates, and the cell suspension containing systems have been introduced in the preparation of NK
these iPSC aggregates is then seeded onto OP cells in cells derived from iPSCs, which will further enhance the
alpha minimum essential medium supplemented with scalability of NK cell production and improve the efficiency
fetal bovine serum. Within this culture setup, the optimal of clinical applications. 95
timeframe for HSC collection is typically between 7 and
8 days. For iPSC-derived NK cells intended for clinical 3.2. iPSC-derived vs. primary NK cells
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applications, it is preferable to establish a differentiation The killer-cell immunoglobulin-like receptor (KIR) is not
protocol that operates without the requirement for only pivotal for NK cells in establishing immune tolerance
feeder cells. This need led to the development of the spin but also plays a critical role in providing licensing signals
embryoid body (EB) method. In this approach, iPSCs are that are essential for NK cell development and maturation.
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first separated into single cells and seeded at a density of In the clinical context of leukemia treatment through HSC
approximately 4,000 cells per well into a low-attachment suppression, mismatches in KIR and KIR ligands between
96-well plate. The plate is then centrifuged to promote donors and recipients can enhance GVL effects and
the formation of a cell aggregate at the bottom of each reduce relapse rates, particularly in patients with AML.
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well. The culture medium is based on APEL medium, Although NK cell licensing is modulated by signals from
supplemented with cytokines such as stem cell factor KIR ligands in vivo, the expression and functionality of KIR
(SCF), bone morphogenetic protein-4, and vascular on NK cells following ex vivo expansion remain subjects of
endothelial growth factor, and includes the addition of debate. Some studies have observed that ex vivo expansion
a Rho-associated, coiled-coil containing protein kinase does not alter the expression state of KIRs on NK cells,
inhibitor during the first 2 – 3 days of EB formation. By thereby preserving the inhibitory impact mediated by
days 11 – 13 of hematopoietic induction, HSCs can be their ligands. Conversely, other studies have highlighted
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harvested for analysis or further differentiation. Before that ex vivo cytokine stimulation can license previously
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differentiating from HSCs into NK cells, the purity of the unlicensed NK cell subsets, which lacked high reactivity
HSCs is usually assessed. If the percentage of CD43 CD34 + in vivo, enabling them to become licensed. Once these ex
+
98
+
+
(or CD45 CD34 ) cells is around 30%, the process can vivo activated NK cells are reintroduced into the patients,
proceed to NK cell differentiation. It is generally believed they might become activated due to KIR and KIR ligand
that the presence of some non-hematopoietic stromal cells mismatches, thus activating previously tolerant NK cells
will not impede the differentiation of HSCs into NK cells. to target tumor cells more effectively. During the ex vivo
If the HSC ratio is too low, magnetic bead purification culturing and expansion process of NK cells, whether
can be employed to enrich the HSC population before derived from iPSCs or peripheral blood, a high cytokine
proceeding to NK cell differentiation. The second step concentration is essential. As a result, the cytotoxic capacity
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initiates the differentiation of HSCs into NK cells. The of ex vivo expanded NK cells might transcend the limitations
cytokines necessary for the maturation and differentiation imposed by KIRs or KIR ligands, effectively bypassing the
of NK cells have been extensively identified through KIR-mediated inhibition through KIR ligands. Notably,
prior basic research. Therefore, these cytokines are often research indicates that NK cells from different iPSC lines
employed in the process of differentiating HSCs into show varied KIR expression patterns. Among iPSC-derived
NK cells. While OP9 cells expressing delta-like 1 (DL1) NK cells, there is no significant difference in the levels of
are utilized in studies of HSC differentiation into NK activating receptor expression between KIR and KIR cells,
-
+
cells, methods that do not involve feeder cells may hold except for CD16, which is expressed at higher levels in
greater potential for clinical applications. In the feeder- KIR cells. Importantly, iPSC-derived KIR and KIR cells
-
+
+
free differentiation system, the EBs formed from HSC do not exhibit significant differences in tumor cytotoxicity,
differentiation steps are transferred to other cell culture underscoring the nuanced role of KIR in modulating NK
devices, such as 6-well plates, and the medium is replaced cell function. Therefore, maximizing the anti-tumor
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with one that facilitates NK cell production, containing capability mediated by KIR in iPSC-derived NK cells likely
IL-3, SCF, IL-7, and IL-15. These cytokines can induce the requires further research and more elegant designs.
Volume 2 Issue 1 (2025) 32 doi: 10.36922/mi.5653