Method for producing natural killer cells

Abstract

The present invention provides methods for producing, from pluripotent stem cells, natural killer (NK) cells or precursor cells thereof. The methods are: a method which is characterized by comprising a) a step for inducing hematopoietic progenitor cells (HPCs) from pluripotent stem cells in vitro, b) a step for amplifying the HPCs in vitro, and c) a step for inducing, from the HPCs, NK cells or precursor cells thereof in vitro, and which is characterized in that a histone methyltransferase and / or an allyl hydrocarbon receptor (AHR) is inhibited in one or more of steps a)-c); and a method which comprises (1) a step for forming, in a first culture medium, pluripotent stem cell spheres having an average particle size of 200 µm or more, (2) a step for inducing a cell population including HPCs through three dimensional culturing of the pluripotent stem cell spheres obtained in step (1) by using a second culture medium containing a histone methyltransferase inhibitor and / or an AHR antagonist, and (3) a step for inducing a cell population including NK cells or precursor cells thereof through three dimensional culturing of the cell population including the HPCs obtained in step (2) by using a third culture medium containing the histone methyltransferase inhibitor and / or the AHR antagonist, and in which one or more of steps (1)-(3) are performed through perfusion culturing, and preferably steps (1)-(3) are performed through continuous perfusion culturing.

Description

Method for producing natural killer cells

[0001] The present invention relates to a method for efficiently producing large amounts of natural killer cells (hereinafter sometimes abbreviated as "NK cells"), which are useful in the medical field, from pluripotent stem cells.

[0002] Numerous methods for inducing NK cells from pluripotent stem cells have been reported. For example, Patent Document 1 describes a method for inducing NK cells from pluripotent stem cell spheres via hematopoietic progenitor cells (hereinafter sometimes abbreviated as "HPCs"). The present inventors have discovered that by increasing the average particle size of pluripotent stem cell spheres formed in three-dimensional culture and combining this with perfusion culture, NK cells can be induced efficiently and rapidly, and have filed a patent application for this method (Patent Document 2).

[0003] Meanwhile, studies on medium additives have been conducted, for example, in Patent Document 3, which discloses a method for inducing NK cells using a Notch ligand. Patent Document 4 discloses a method for inducing lymphoid cells using a Notch ligand and an inhibitor of histone methyltransferase EZH1, a component of the Polycomb complex. Furthermore, a method for inducing T cell precursors using a Notch ligand and the aryl hydrocarbon receptor (AHR) antagonist StemRegenin 1 (SR1) (Patent Document 5) and a method for inducing NK cells from CD34+ cells using SR1 and thrombopoietin (TPO) (Patent Document 6) have also been reported. However, the NK cell induction effect of histone methyltransferase inhibition or AHR inhibition alone is unclear, and there have been no reports on the effect of combining histone methyltransferase inhibition and AHR inhibition. Against this background, there is a need for an NK cell induction method with improved induction efficiency.

[0004] WO 2020 / 086889WO 2023 / 145922US 8,518,397WO 2018 / 048828WO 2021 / 200901WO 2017 / 046142

[0005] Therefore, an object of the present invention is to provide a method for inducing NK cells from pluripotent stem cells more efficiently than conventional techniques. A further object of the present invention is to provide a novel method for inducing NK cells from pluripotent stem cells, which can further improve the induction efficiency of the method by combining it with a perfusion culture method.

[0006] As a result of extensive investigations to achieve the above-mentioned object, the present inventors have found that in a method for inducing NK cells in vitro from pluripotent stem cells via HPCs, NK cells can be induced efficiently and rapidly by inhibiting histone methyltransferase and / or AHR in one or more of the following steps: a) inducing HPCs from pluripotent stem cells; b) expanding HPCs; and c) inducing NK cells or their precursor cells from HPCs. Furthermore, the present inventors have succeeded in inducing NK cells more efficiently by performing steps a) to c) using three-dimensional culture, setting the average particle size of the pluripotent stem cell spheres formed in step a) to 200 μm or more, inducing a cell population containing HPCs from the spheres, and combining this with perfusion culture for at least a portion of the subsequent culture, thereby completing the present invention.

[0007] That is, the present invention is as follows: [Item 1] A method for producing natural killer (NK) cells or their precursor cells from pluripotent stem cells, comprising: a) in vitro inducing hematopoietic progenitor cells (HPCs) from pluripotent stem cells; b) in vitro expanding HPCs; and c) in vitro inducing NK cells or their precursor cells from HPCs, wherein the method is characterized in that histone methyltransferase and / or aryl hydrocarbon receptor (AHR) is inhibited in one or more of steps a) to c). [Item 2] The method of Item 1, wherein the histone methyltransferase catalyzes the addition of a methyl group to histone 3 lysine residue 27 (H3K27). [Item 3] The method of Item 1, wherein the histone methyltransferase is EZH1 (enhancer of zeste homolog 1) and / or EZH2 (enhancer of zeste homolog 2). [Item 4] The method of any one of Items 1 to 3, wherein the histone methyltransferase is inhibited by a small molecule inhibitor. [Item 5] The method of Item 4, wherein the small molecule inhibitor is GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55. [Item 6] The method according to any one of Items 1 to 5, wherein the AHR is inhibited by an inhibitor having antagonist activity against AHR. [Item 7] The method according to any one of Items 1 to 6, wherein the AHR is inhibited by a small molecule inhibitor. [Item 8] The method according to Item 7, wherein the small molecule inhibitor is SR1 (StemRegenin 1), CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059, or BAY 2416964.[Item 9] The method according to any one of Items 1 to 8, wherein one or more of steps a) to c) are performed by perfusion culture. [Item 10] A method for producing NK cells or their progenitor cells from pluripotent stem cells, comprising: (1) forming pluripotent stem cell spheres having an average particle size of 200 μm or more in a first medium; (2) inducing the pluripotent stem cell spheres obtained in step (1) into a cell population containing HPCs by three-dimensional culture using a second medium containing a histone methyltransferase inhibitor and / or an AHR antagonist; and (3) inducing the cell population containing HPCs obtained in step (2) into a cell population containing NK cells or their progenitor cells by three-dimensional culture using a third medium containing a histone methyltransferase inhibitor and / or an AHR antagonist, wherein one or more of steps (1) to (3) are performed by perfusion culture. [Item 11] The method of Item 10, wherein the histone methyltransferase catalyzes the addition of a methyl group to H3K27. [Item 12] The method of Item 10, wherein the histone methyltransferase is EZH1 and / or EZH2. [Item 13] The method of any one of Items 10 to 12, wherein the histone methyltransferase inhibitor is a small molecule inhibitor. [Item 14] The method of Item 13, wherein the small molecule inhibitor is GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55. [Item 15] The method of any one of Items 10 to 13, wherein the AHR antagonist is a small molecule inhibitor. [Item 16] The method according to Item 15, wherein the small molecule inhibitor is SR1, CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059, or BAY 2416964.[Item 17] The method of any one of Items 10 to 16, wherein steps (1) to (3) are performed without using a three-dimensional culture carrier or an extracellular matrix. [Item 18] The method of Item 17, wherein the perfusion culture in step (1) is performed using a separation membrane with a pore size of 15 to 75 μm. [Item 19] The method of Item 17 or 18, wherein the perfusion culture in step (2) is performed using a separation membrane with a pore size of 45 to 225 μm. [Item 20] The method of any one of Items 17 to 19, wherein the perfusion culture in step (3) is performed using a separation membrane with a pore size of 0.2 to 10 μm. [Item 21] The method of any one of Items 16 to 20, wherein steps (1) to (3) are performed by continuous perfusion culture. [Item 22] The method of any one of Items 16 to 21, wherein the first medium contains a ROCK inhibitor. [Item 23] The method of any one of Items 16 to 22, wherein the second culture medium comprises VEGF, BMP4, and a GSK3β inhibitor. [Item 24] The method of Items 23, wherein the second culture medium further comprises a ROCK inhibitor or bFGF. [Item 25] The method of any one of Items 16 to 22, wherein the second culture medium comprises SCF, a TGFβ / Smad inhibitor, and VEGF. [Item 26] The method of Items 25, wherein the second culture medium further comprises a ROCK inhibitor or bFGF. [Item 27] ​​The method of any one of Items 16 to 22, wherein the second culture medium comprises SCF and Flt3L. [Item 28] The method of Item 27, wherein the second culture medium further comprises at least one selected from a ROCK inhibitor, IL-3, and IL-7. [Item 29] The method of any one of Items 16 to 22, wherein the three-dimensional culturing in step (2) comprises: (2-1) a culturing step using a medium containing VEGF, BMP4, and a GSK3β inhibitor as the second medium; (2-2) a culturing step using a medium containing SCF, a TGFβ / Smad inhibitor, and VEGF as the second medium; and (2-3) a culturing step using a medium containing SCF and Flt3L as the second medium. [Item 30] The method of any one of Items 16 to 29, wherein the third medium contains IL-15 and SCF. [Item 31] The method of Item 30, wherein the third medium further contains IL-7 and Flt3L. [Item 32] The method of any one of Items 16 to 31, wherein the third medium does not require a step of separating NK cells.

[0008] The present invention enables the efficient simultaneous production of large quantities of NK cells from pluripotent stem cells by inhibiting histone methyltransferase and / or AHR signaling. By performing this method using three-dimensional culture combined with perfusion culture, NK cells can be produced extremely efficiently and rapidly. NK cells produced by this method exhibit high cytotoxic activity against various cancer cells, and this activity is maintained even after freezing, making large-scale clinical application possible.

[0009] 1 shows the change in live NK cell density over time when the hematopoietic progenitor cells obtained in step (2) are cultured in suspension under various conditions and induced to differentiate into NK cells. 2 shows the change in CD56-positive cells over time when the hematopoietic progenitor cells obtained in step (2) are cultured in suspension under various conditions and induced to differentiate into NK cells. 3 shows the results of FACS analysis showing the expression of CD56 and CD14 in cell populations 10, 17, 24, 32, and 39 days after induction of differentiation when the hematopoietic progenitor cells obtained in step (2) are cultured in suspension under various conditions and induced to differentiate into NK cells.

[0010] The present invention provides a method for producing NK cells or their precursor cells from pluripotent stem cells, comprising: a) in vitro inducing HPCs from pluripotent stem cells; b) in vitro expanding HPCs; and c) in vitro inducing NK cells or their precursor cells from HPCs, wherein histone methyltransferase and / or AHR are inhibited in one or more of steps a) to c) (hereinafter, this method may be referred to as the "method of the present invention").

[0011] Pluripotent stem cells used in the method of the present invention include, but are not limited to, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. For example, when iPS cells are used, the method for producing the iPS cells and the cells from which they are derived are not particularly limited. Furthermore, the method for culturing iPS cells is not particularly limited, and they may be two-dimensional or three-dimensional cultures. Furthermore, cryopreserved iPS cells can also be used.

[0012] The method of the present invention induces NK cells or their precursor cells in vitro from pluripotent stem cells via HPCs. That is, the method of the present invention comprises: a) inducing HPCs from pluripotent stem cells in vitro; b) expanding HPCs in vitro; and c) inducing NK cells or their precursor cells from HPCs in vitro. Here, steps a) and b) may be performed consecutively, from induction of differentiation into HPCs to maintenance and expansion of HPCs, using the same or a series of media. In this case, the entire process, including the maintenance and expansion phase, can be considered as a process of inducing HPCs from pluripotent stem cells. Alternatively, the HPCs obtained in step a) may be temporarily stocked, for example, by cryopreservation, and then stored for a certain period of time, after which the HPC stock is subjected to step b). For example, when steps a) and b) are performed in three-dimensional culture using continuous perfusion culture (e.g., in the case of a preferred embodiment of the present invention described below), the process of inducing a cell population containing HPCs from pluripotent stem cell spheres and further purifying and amplifying the HPCs can be performed as a single step (step (2) in a preferred embodiment of the present invention) using the same or a series of culture media.

[0013] Pluripotent stem cells can be induced to differentiate into NK cells via HPCs by known methods, such as those described in WO 2020 / 086889, Biochem Biophys Res Commun. 515(1): 1-8 (2019), and Methods Mol Biol. 2048: 107-119 (2019). For example, step a) can be performed by culturing pluripotent stem cells in a medium capable of inducing them into HPCs. Examples of such media include, but are not limited to, media containing vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), and a glycogen synthase 3β (GSK3β) inhibitor; media containing stem cell factor (SCF) and a transforming growth factor β (TGFβ) / Smad inhibitor; and media containing SCF and Flt3 ligand (Flt3L). For example, step b) can be performed by culturing HPCs in a medium capable of maintaining and amplifying HPCs. Examples of such a medium include, but are not limited to, a medium containing SCF and Flt3 ligand (Flt3L). For example, step c) can be performed by culturing HPCs in a medium capable of inducing HPCs into NK cells. Examples of such a medium include, but are not limited to, a medium containing IL-15 and SCF.

[0014] Examples of basal media used in the steps a) to c) include DMEM / F-12, HEPES (Thermo Fisher Scientific), Essential 6 medium (Thermo Fisher Scientific), Stem Pro-34 SFM (Thermo Fisher Scientific), AIM-V Medium (Thermo Fisher Scientific), Stemline (registered trademark) II (Sigma-Aldrich), and ALyS505N-0 (Cell Science Institute, Inc.). Those skilled in the art can select an appropriate basal medium for each step.

[0015] The culture period in steps a) and b) is usually 5 to 20 days, preferably about 10 to 18 days. The culture period in step c) is usually 20 to 60 days, preferably about 25 to 40 days.

[0016] The method of the present invention is characterized in that histone methyltransferase and / or AHR is inhibited in one or more of the steps a) to c).

[0017] The histone methyltransferase targeted in the method of the present invention is not particularly limited, but is preferably one that catalyzes the addition of a methyl group to histone 3 lysine residue 27 (H3K27), more preferably EZH1 (enhancer of zeste homolog 1) and / or EZH2 (enhancer of zeste homolog 2).

[0018] Histone methyltransferase can be inhibited by adding a substance that inhibits the enzyme to the medium. Here, "inhibition of histone methyltransferase" refers to any method that ultimately inhibits the methyl group transfer reaction to histones catalyzed by the enzyme, and may involve inhibiting not only the function but also the expression of the enzyme. Examples of substances that inhibit the function of histone methyltransferase include, for example, small molecule inhibitors, antibodies, and aptamers. Examples of substances that inhibit the expression of histone methyltransferase include, but are not limited to, nucleic acids complementary to the enzyme gene or its transcription product (e.g., siRNA, antisense nucleic acid, miRNA, etc.).

[0019] In a preferred embodiment, the histone methyltransferase inhibitor is a small molecule inhibitor. Known inhibitors are appropriately selected depending on the target histone methyltransferase. Examples of small molecule inhibitors include EZH1 and / or EZH2 inhibitors such as EPZ005687, GSK343, tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, and GSK126. Alternatively, examples of inhibitors that are expected to inhibit PRC1.1 and thereby suppress PRC2 recruitment, thereby inhibiting histone methylation, include FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, L55, etc. Among these, preferred are GSK503, EPZ005687, EI1, Tazemetostat (EPZ-6438), GSK126, etc., more preferably GSK126.

[0020] The concentration of the histone methyltransferase inhibitor added is not particularly limited, as long as it can efficiently and rapidly induce NK cells and does not adversely affect the cells, and a suitable concentration can be selected depending on the type of inhibitor, for example, from the range of about 10 nM to about 5 μM, preferably about 100 nM to about 2 μM, and more preferably about 500 nM to about 1 μM. Any one of the histone methyltransferase inhibitors may be used alone, or two or more of them may be used in combination.

[0021] Without wishing to be bound by any particular theory, it has been suggested that, for example, the use of an inhibitor of histone methyltransferase such as EZH1 and / or EZH2 (e.g., GSK126) accelerates the onset of NK cell differentiation when inducing differentiation of HPCs into NK cells, and that the longer the induction time for NK cell differentiation, the higher the cell proliferation efficiency.

[0022] The aryl hydrocarbon receptor (AHR) is a transcriptional regulatory factor that regulates the expression of target genes by translocating the complex into the nucleus upon binding with its ligand, an aromatic hydrocarbon compound. AHR inhibition can be achieved by adding a substance that inhibits the transcription factor to the culture medium. Here, "AHR inhibition" refers to any method that ultimately inhibits the transcriptional regulation of the target gene whose expression is regulated by the transcription factor, and may involve inhibiting not only the function but also the expression of the transcription factor. Substances that inhibit AHR function include, for example, small molecule inhibitors, antibodies, aptamers, decoy nucleic acids, etc., while substances that inhibit AHR expression include, but are not limited to, nucleic acids complementary to the AHR gene or its transcription product (e.g., siRNA, antisense nucleic acid, miRNA, etc.).

[0023] In a preferred embodiment, the AHR inhibitor (antagonist) is a small molecule inhibitor. The small molecule inhibitor may be appropriately selected from known inhibitors. In a preferred embodiment, examples include SR1 (StemRegenin 1), CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059, BAY 2416964, etc., preferably SR1, CH-223191, GNF-351, CB7993113, 6,2',4'-trimethoxyflavone, etc., more preferably SR1.

[0024] The concentration of the AHR inhibitor added is not particularly limited, as long as it can efficiently and rapidly induce NK cells and does not adversely affect the cells, and a suitable concentration can be selected depending on the type of inhibitor, for example, from the range of about 100 nM to about 10 μM, preferably about 500 nM to about 5 μM, and more preferably about 1 μM to about 5 μM. Any one of the AHR inhibitors may be used alone, or two or more may be used in combination.

[0025] Without wishing to be bound by any particular theory, it is suggested that an AHR inhibitor (e.g., SR1) may increase the efficiency of cell proliferation at an early stage during induction of differentiation of HPCs into NK cells.

[0026] Thus, histone methyltransferase inhibition and AHR inhibition suggest that their effective NK cell induction sites are different, and thus synergistic effects can be expected by inhibiting both. Therefore, in the methods of the present invention, a histone methyltransferase inhibitor and an AHR inhibitor may be used alone, but it is also preferable to use both in combination. When a histone methyltransferase inhibitor and an AHR inhibitor are used in combination, any of the above-mentioned combinations of histone methyltransferase inhibitor and AHR inhibitor may be used, but a combination of GSK126 and SR1 is particularly preferred.

[0027] The inhibition of histone methyltransferase and / or AHR is not particularly limited as long as it is carried out in at least one of steps a) to c), but in one embodiment, it can be carried out in all of steps a) to c). For example, a preferred timing for carrying out the inhibition is from the latter half of step a) to the first half of step c), which may further increase the efficiency of NK cell induction.

[0028] The method of the present invention is intended to efficiently and mass-produce NK cells useful in the medical field from pluripotent stem cells. Therefore, although the method can be performed using two-dimensional culture, it is preferable to perform it using three-dimensional culture, as this facilitates industrial scale-up. Furthermore, manual medium changes pose a risk of affecting the quality of the final product due to the artificial operation, and increased manufacturing costs due to increased labor are anticipated. Therefore, the method of the present invention is preferably performed using a perfusion culture method, which does not require manual medium changes.

[0029] Therefore, in a preferred embodiment, the method of the present invention is a method for producing NK cells or their precursor cells from pluripotent stem cells, comprising: (1) forming pluripotent stem cell spheres having an average particle size of 200 μm or more in a first medium; (2) inducing the pluripotent stem cell spheres obtained in step (1) into a cell population containing HPCs by three-dimensional culture using a second medium containing a histone methyltransferase inhibitor and / or an AHR antagonist; and (3) inducing the HPC-containing cell population obtained in step (2) into a cell population containing NK cells or their precursor cells by three-dimensional culture using a third medium containing a histone methyltransferase inhibitor and / or an AHR antagonist, wherein one or more of steps (1) to (3) are performed by perfusion culture. In a particularly preferred embodiment, steps (1) to (3) are performed by continuous perfusion culture. In this embodiment, steps (2) and (3) are not separated by a step of temporarily storing the cell population containing HPCs or HPCs purified from the cell population, for example, by cryopreservation. Instead, steps (2) and (3) are performed continuously in a single culture system using continuous perfusion culture. Therefore, in step (2), the differentiation of pluripotent stem cells into HPCs (step a)) and the expansion of HPCs (step b)) are performed continuously as a single step. Furthermore, in a preferred embodiment, pluripotent stem cells are cultured in three-dimensional culture, and step (1) is performed prior to step (2) (step a)) as a pre-step for three-dimensionally culturing the pluripotent stem cells to produce pluripotent stem cell spheres. The three-dimensional culture method and perfusion culture method are described in detail below.

[0030] (Three-dimensional culture method) The three-dimensional culture method of the present invention refers to a method in which cells are first formed into spheres (spheroids) and then cultured by suspending them in a medium (sometimes referred to as "three-dimensional suspension culture method" in this specification). Three-dimensional suspension culture is a cell culture method characterized by culturing suspended cells, spheres (spheroids), or three-dimensional culture carriers with cells attached thereto while expanding them three-dimensionally using a stirring blade or shaker. Therefore, compared to two-dimensional culture, in which cells are cultured only on the bottom of the culture vessel, three-dimensional suspension culture can maximize the number of cells per space. Furthermore, three-dimensional mixing using a stirring blade or shaker can homogenize the culture environment. Furthermore, three-dimensional culture can be combined with various detectors such as pH sensors and dissolved oxygen sensors to enable efficient and uniform culture.

[0031] These advantages, such as maximum utilization of the culture space and the ability to culture in a uniform environment, are also useful for scaling up cultures for commercial production. Furthermore, because suspension cell and spheroid culture does not require specially processed plastic carriers for cell culture or the extracellular matrix required for many cultured cell types, this culture method is cost-effective. Furthermore, compared to two-dimensional culture, in which cells are cultured on a flat surface, this method allows for culture in an environment closer to that found in vivo. This makes it a useful method for producing various cell products derived from pluripotent stem cells (e.g., ES cells, iPS cells, etc.) that require differentiation into various cell types.

[0032] (Perfusion culture method) The three-dimensional culture method of the present invention can be combined with perfusion culture to more effectively induce NK cells. Perfusion culture is a continuous culture method in which a constant amount of new medium is supplied to the culture solution in the culture vessel while simultaneously removing a constant amount of medium, thereby achieving the purpose of continuous medium exchange, namely, the supply of new nutrients and the removal of waste products.

[0033] Perfusion culture eliminates the need for medium changes, significantly reducing the workload. Furthermore, because this method is typically performed mechanically, it eliminates the environmental changes that occur with manual medium changes, such as changes in temperature, pH, dissolved oxygen concentration, and dissolved carbon dioxide concentration. This allows for longer-term culture in the same vessel, resulting in higher cell densities. Perfusion culture also maintains constant concentrations of various growth factors secreted by cells, which are removed by conventional medium changes. It also allows for culture in an environment similar to the in vivo blood circulation system.

[0034] The separation membrane used in perfusion culture desirably has a certain pore size and properties (e.g., hydrophobicity) that prevent the target cell population from escaping from the culture system. The pore size of the separation membrane can be appropriately selected depending on the target cell population. However, because various differentiated cells are induced inside and outside the spheres during the NK cell induction process, selecting the pore size generally requires extensive consideration, making optimization extremely difficult. Specific pore sizes for each process are described below. Suitable pore sizes for the perfusion culture of the present invention are 15-75 μm for process (1), 45-225 μm for process (2), and 0.2-10 μm for process (3).

[0035] The material of the separation membrane is not particularly limited, but a material that does not affect the cells or the components in the culture medium is preferably used. Examples of such materials include metal materials such as SUS304 and SUS316, natural fibers such as cellulose fibers, and chemical fibers such as polysulfone fibers and polyethersulfone fibers.

[0036] The three-dimensional culture method of the present invention is characterized by perfusion culture of cell populations, such as cell spheres or cells, present in a culture system in each of the steps described below. The conditions for each perfusion culture are as described below for each step. Furthermore, the perfusion culture of the present invention can be "continuous perfusion culture." Here, "continuous perfusion culture" refers to simply replacing the membrane between each step, without the washing step typically performed during medium exchange, and supplying the medium of the next step while withdrawing the medium from the previous step. For example, in step (1), continuous medium exchange is performed by supplying the first medium described below while withdrawing the medium in the same manner. When step (1) is replaced by step (2), continuous medium exchange is performed by simultaneously supplying the second medium described below while withdrawing the first medium. In step (2), continuous medium exchange is carried out by simultaneously supplying the second medium and withdrawing the medium, and when step (2) is switched to step (3), continuous medium exchange from the second medium to the third medium is carried out by simultaneously supplying the third medium described below and withdrawing the second medium. Furthermore, in step (3), continuous medium exchange is carried out by simultaneously supplying the third medium and withdrawing the medium.

[0037] (Regarding step (1) of forming pluripotent stem cell spheres) Step (1) in the preferred method of the present invention is a step of forming pluripotent stem cell spheres with an average particle size of 200 μm or more in a first culture medium.

[0038] Pluripotent stem cells used for forming pluripotent stem cells into spheres in the present invention include, but are not particularly limited to, ES cells and iPS cells as described above.

[0039] The formation of spheres is carried out under suspension culture conditions. The suspension culture conditions are not particularly limited as long as the culture medium environment can be homogenized by three-dimensional mixing using a stirring blade or a shaker. The vessel used for suspension culture is not particularly limited, and vessels with a stirring mechanism such as a rotating stirring blade type or an up-and-down shaking stirring blade type are used. The seeding density of pluripotent stem cells in the formation of spheres of the present invention is 1.0 × 104 cells / mL to 1.0 × 10 6 At a density higher than this, the spheres may become too large, which may affect the induction efficiency. 4 cells / mL to 2.0 × 10 5 It is more preferable to seed in the range of cells / mL.

[0040] The culture medium used for forming pluripotent stem cells into spheres in step (1) is not particularly limited as long as it is a culture medium used for the maintenance culture of pluripotent stem cells. Examples of such culture medium include StemFit (registered trademark) AK03N (Ajinomoto Healthy Supply Co., Ltd.), mTeSR TM A medium suitable for feeder-free culture, such as StemFit® AK03N (STEMCELL Technologies Inc.), is used. A preferred culture medium for use in the method of the present invention is a medium suitable for feeder-free culture containing a ROCK inhibitor. When using iPS cells as pluripotent stem cells, it is particularly preferable to use a medium such as StemFit® AK03N (Ajinomoto Healthy Supply Co., Ltd.). Examples of ROCK inhibitors used in the present invention include Y27632 and thiazovivin. In iPS cell culture, Y27632 is generally used as a ROCK inhibitor, and in the present invention, its concentration is preferably 1 to 20 μM, and it is preferably maintained in the added state for two days after cell seeding.

[0041] The culture period required for sphere formation is not limited as long as spheres are formed, but the spheres of the present invention are not formed by forcibly inserting pluripotent stem cells into dimples or the like and associating them, but rather are formed as the cells grow, and therefore require a certain period of time, for example, 2 to 10 days from cell seeding is preferred, and 4 to 7 days is more preferred.

[0042] The formation of spheres can be controlled by adjusting the stirring conditions, seeding density, and culture period. The average particle size of the spheres used in step (1) of the present invention is typically 200 μm or greater, specifically within the range of 200–600 μm. To further improve the production efficiency of NK cells, it is preferably adjusted to 200–500 μm, and even more preferably to 200–400 μm. An average particle size of less than 200 μm is undesirable because the culture period is insufficient, resulting in an insufficient number of cells, which affects step (2). Furthermore, an average particle size that is too large is undesirable because it prevents sufficient supply of nutrients and oxygen to the center of the sphere, leading to cell death such as necrosis (see, for example, Cells Tissues Organs 196.1 (2012): 34–47).

[0043] The spheroid formation step (1) is performed by perfusion culture. In step (1), perfusion culture may be performed from the time of seeding of pluripotent stem cells. However, it is preferably performed about one day after initial seeding of pluripotent stem cells, and more preferably, two days after initial seeding. This is because spheroids form after one to two days of culture, and under these conditions, the average particle size is 200 μm or greater. Performing perfusion culture before spheroid formation may result in insufficient spheroid formation. Furthermore, in step (1), the pore size of the separation membrane used in perfusion culture is preferably 15 to 75 μm, and more preferably 25 to 45 μm. Using a separation membrane with a pore size not exceeding the desired spheroid size enables perfusion culture while maintaining a certain number of spheroids within the culture vessel. Therefore, step (1) preferably comprises: (1-1) forming pluripotent stem cell spheres having an average particle size of 200 μm or more in a first medium; and (1-2) maintaining the pluripotent stem cell spheres having an average particle size of 200 μm or more obtained in step (1-1) by perfusion culture. That is, the pore size of the separation membrane used in the perfusion culture in step (1) (or step (1-2)) is preferably 15 to 75 μm, and more preferably 25 to 45 μm.

[0044] In perfusion culture, medium exchange is performed at a constant dilution rate. The sphere formation process of pluripotent stem cells takes 0.01 to 0.2 hours. -1 It is preferable to carry out the dilution at a dilution rate of 1000 kJ / ml, which can be adjusted with reference to the cell seeding density, the glucose concentration, the lactate concentration, the glutamine concentration, the glutamic acid concentration, etc. of the culture supernatant. The dilution rate can be adjusted by placing the culture vessel, the supply bottle, and the discharge bottle on a balance or a load cell, or by controlling the rotation speed of the peristaltic pump.

[0045] In step (1), temperature, pH, and dissolved oxygen concentration can be controlled to any desired value for sphere formation of pluripotent stem cells using a temperature sensor, pH sensor, dissolved oxygen sensor, etc. The culture temperature is preferably controlled between 35 and 39°C, more preferably between 36 and 38°C. pH is controlled by the inflow of sterile compressed air, sterile carbon dioxide gas, a pH adjuster, or the dilution rate of the perfusion culture medium, preferably between 6.8 and 8.0, more preferably between 7.0 and 7.4. Dissolved oxygen concentration is controlled by sterile compressed air, sterile nitrogen gas, or sterile oxygen gas, preferably between 0 and 6.86 mg / L, preferably maintained at a concentration of 2.00 mg / L or higher. The gas used to maintain pH and dissolved oxygen can be blown into the gas layer above the culture medium, blown into the culture medium, or exchanged through a gas-permeable membrane; there are no limitations. The pluripotent stem cell spheres thus obtained are subjected to the next step (2) to induce hematopoietic progenitor cells.

[0046] (Regarding step (2) of inducing pluripotent stem cell spheres into a cell population containing HPCs) Step (2) in a preferred method of the present invention is a step of inducing the spheres formed in step (1) into a cell population containing HPCs by three-dimensional culture using a second medium containing a histone methyltransferase inhibitor and / or an AHR antagonist. The histone methyltransferase inhibitor contained in the second medium is as described above. The AHR antagonist contained in the second medium is as described above.

[0047] Here, the "cell population containing HPCs" induced by this step refers to the cell population obtained by step (2) and includes HPC populations induced inside and outside the spheres. This cell population includes HPCs formed inside the spheres, HPCs that have escaped from the spheres after induction, and cells inside and outside the spheres that are in the process of being induced into HPCs.

[0048] Step (2) can also be performed by perfusion culture, as in step (1). The pore size of the separation membrane used in this step is preferably 45 to 225 μm, and more preferably 45 to 100 μm. The perfusion culture in this step is performed from immediately after the start of step (2) until the end of step (2). During this time, the "pluripotent stem cell spheres" are guided toward the "cell population containing HPCs." Using a separation membrane with the above-mentioned pore size during step (2) enables perfusion culture while maintaining a certain cell population containing HPCs within the culture vessel.

[0049] The medium exchange accompanying the perfusion culture in step (2) is also carried out at a constant dilution rate, similar to that described in step (1). Specifically, the medium exchange is carried out at a constant dilution rate of 0.01 to 0.2 hr, similar to step (1). -1 It is preferable to carry out the dilution at a dilution rate of 100 mg / mL, which can be adjusted with reference to the cell seeding density, the glucose concentration, the lactate concentration, the glutamine concentration, the glutamic acid concentration, etc. This dilution rate is also applied when the medium used in step (1) is replaced with the medium used in step (2).

[0050] The second medium used in step (2) is not particularly limited as long as it contains a histone methyltransferase inhibitor and / or an AHR antagonist and is capable of inducing pluripotent stem cells into HPCs. Examples of such media include a medium containing vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), and a glycogen synthase 3β (GSK3β) inhibitor (hereinafter referred to as medium (2-1)), a medium containing stem cell factor (SCF) and a transforming growth factor β (TGFβ) / Smad inhibitor (hereinafter referred to as medium (2-2)), and a medium containing SCF and Flt3 ligand (Flt3L) (hereinafter referred to as medium (2-3)). The combination and amount of each of these medium components can be appropriately adjusted as needed to design an appropriate induction medium. In particular, the timing of administration of the histone methyltransferase inhibitor and / or AHR antagonist (specifically, whether it is included in medium (2-1) to (2-3)) can be appropriately designed. As examples of such media, the media (2-1) to (2-3) will be described below.

[0051] The medium (2-1) contains VEGF, BMP4, and a GSK-3β inhibitor. Examples of GSK-3β inhibitors include CHIR99021 and SB216763, with CHIR99021 being preferred. The VEGF concentration is preferably 1 to 100 ng / mL, more preferably 50 to 100 ng / mL. The BMP4 concentration is preferably 1 to 100 ng / mL, more preferably 50 to 100 ng / mL. When using CHIR99021, the GSK-3β inhibitor concentration is preferably 1 to 10 μM, more preferably 1 to 5 μM.

[0052] Furthermore, medium (2-1) may contain a ROCK inhibitor or bFGF. When a ROCK inhibitor is added, examples of the ROCK inhibitor include those described in step (1), preferably Y27632. When Y27632 is used, the concentration of the ROCK inhibitor is preferably 1 to 20 μM, more preferably 1 to 10 μM. When bFGF is added, the concentration is preferably 1 to 100 ng / mL, more preferably 10 to 50 ng / mL.

[0053] The basal medium of the medium (2-1) is not particularly limited, but preferably used are, for example, DMEM / F-12, HEPES (Thermo Fisher Scientific), and Essential 6 medium (Thermo Fisher Scientific).

[0054] Medium (2-2) contains SCF, a TGFβ / Smad inhibitor, and VEGF. Examples of TGFβ / Smad inhibitors include SB431542, LY2157299, and LY2109761, with SB431542 being preferred. The SCF concentration is preferably 1 to 100 ng / mL, more preferably 20 to 50 ng / mL. When SB431542 is used, the TGFβ / Smad inhibitor concentration is preferably 1 to 10 μM, more preferably 1 to 5 μM. The VEGF concentration, similar to that of Medium (2-1), is preferably 1 to 100 ng / mL, more preferably 50 to 100 ng / mL.

[0055] Furthermore, the medium (2-2) may contain a ROCK inhibitor or bFGF. Specific examples and concentrations of each are the same as those described for the medium (2-1).

[0056] The basal medium for the medium (2-2) is not particularly limited, but the same basal medium as that described for the medium (2-1) can be used.

[0057] The medium (2-3) contains SCF and Flt3L. The SCF concentration is preferably 1 to 100 ng / mL, more preferably 20 to 50 ng / mL. The Flt3L concentration is preferably 1 to 100 ng / mL, more preferably 20 to 50 ng / mL.

[0058] Furthermore, the medium (2-3) may contain at least one selected from a ROCK inhibitor, interleukin (IL)-3, and IL-7. Examples of ROCK inhibitors include those mentioned above, preferably Y27632. When a ROCK inhibitor is added, its concentration is preferably 1 to 20 μM when Y27632 is used. When IL-3 is added, its concentration is preferably 1 to 100 ng / mL. When IL-7 is added, its concentration is preferably 1 to 100 ng / mL.

[0059] The basal medium for medium (2-3) is not particularly limited, and any medium suitable for HPC induction can be used. For example, a medium containing Stem Pro-34 SFM (Thermo Fisher Scientific) supplemented with L-glutamine or L-alanyl-L-glutamine to a final concentration of 1 to 10 mM is preferably used.

[0060] Although the above-mentioned media (2-1), (2-2), and (2-3) can be used individually, HPC induction can be achieved more efficiently by exchanging media (2-1) or (2-2) with (2-3) at appropriate times during step (2). For example, HPC induction can be achieved efficiently by culturing the first half of step (2) in media (2-1) or (2-2) and the second half in media (2-3). In this case, the duration of the first and second half of step (2) can be set appropriately. For example, HPC induction can be achieved efficiently by setting the first half to 2 to 6 days and the second half to 3 to 14 days. This exchange of media (2-1) or (2-2) with media (2-3) can be performed by continuous perfusion or by replacing the entire medium with fresh medium. It is more preferable to perform the medium exchange by continuous perfusion.

[0061] Furthermore, HPCs can be efficiently induced by exchanging the media (2-1) and (2-2) used in the first half of step (2) in this order. Specifically, the cells are first cultured in medium (2-1), followed by culture in medium (2-2). In this case, the culture time in each medium can be appropriately set. For example, HPCs can be efficiently induced by setting the culture time in medium (2-1) to 1 to 3 days and the culture time in medium (2-2) to 1 to 3 days. This exchange between medium (2-1) and medium (2-2) can be carried out by continuous perfusion or by replacing the entire volume with fresh medium. Continuous perfusion is more preferable.

[0062] Therefore, the three-dimensional culture in step (2) is preferably a three-dimensional culture including, for example, (2-1) a culture step using medium (2-1) as the second culture medium, (2-2) a culture step using medium (2-2) as the second culture medium, and (2-3) a culture step using medium (2-3) as the second culture medium. Each of steps (2-1) to (2-3) is preferably performed in this order.

[0063] In this step (2), similarly to the step (1), the temperature, pH, and dissolved oxygen concentration can be controlled to any desired values ​​for inducing hematopoietic progenitor cells using a temperature sensor, a pH sensor, a dissolved oxygen sensor, or the like.

[0064] The addition of the histone methyltransferase inhibitor and / or AHR antagonist to the medium in step (2) may be carried out throughout step (2) or for only part of the step. When the inhibitor (antagonist) is added only for part of step (2), it can be added, for example, only in the latter part of step (2). For example, when step (2) is carried out using media (2-1), (2-2), and (2-3) sequentially as the second medium, the inhibitor (antagonist) can be added to the medium only in the culture step using media (2-2) and (2-3), or only in the culture step using medium (2-3). However, addition only in the latter part of step (2) is not limited thereto. However, from the viewpoint of efficient and rapid induction of NK cells, it is preferable to add both the histone methyltransferase inhibitor and the AHR antagonist to the medium only in the culture step using medium (2-3).

[0065] When a histone methyltransferase inhibitor and / or an AHR antagonist is added to each medium in step (2), the inhibitor (antagonist) can be selected at any suitable concentration, as long as it can efficiently and rapidly induce NK cells and does not adversely affect the cells. The concentration of the histone methyltransferase inhibitor can generally be selected from the range of about 10 nM to about 5 μM, preferably about 100 nM to about 2 μM, and more preferably about 500 nM to about 1 μM. The concentration of the AHR antagonist can generally be selected from the range of about 100 nM to about 10 μM, preferably about 500 nM to about 5 μM, and more preferably about 1 μM to about 5 μM. The histone methyltransferase inhibitor may be used alone or in combination with two or more. The AHR inhibitor may also be used alone or in combination with two or more.

[0066] In the method of the present invention, an NK cell population can be obtained from step (1) to step (3) without any particular purification steps. Therefore, the cell population obtained in step (2) can be directly subjected to the next step (3), or the HPCs can be selected and then subjected to step (3). The present inventors have confirmed that the desired NK cells can be obtained in step (3) using either method. Therefore, the method of the present invention may include a step of removing cells other than HPCs from the "HPC-containing cell population" obtained in step (2), but it is preferable to subject the "HPC-containing cell population" obtained in step (2) directly to step (3). NK cells are induced by subjecting the "HPC-containing cell population" obtained in this manner to step (3).

[0067] (Regarding step (3) of inducing HPCs into NK cells) Step (3) in the method of the present invention is a step of inducing the "cell population containing HPCs" obtained in step (2) into a "cell population containing NK cells" by three-dimensional culture using a third medium containing a histone methyltransferase inhibitor and / or an AHR antagonist. The histone methyltransferase inhibitor contained in the third medium is as described above. The AHR antagonist contained in the third medium is as described above.

[0068] Here, the "cell population containing NK cells" induced by this step refers to a cell population containing NK cells obtained by step (3) and cells in the process of being induced to become NK cells. NK cells usually exist as single cells without forming spheres. Therefore, by step (3), the single NK cell population proliferates to become a cell population of many single NK cells. This cell population contains NK cells, which account for the majority, and some cells in the process of being induced to become NK cells.

[0069] Step (3) can also be performed by perfusion culture, as in steps (1) and (2). The pore size of the separation membrane used is preferably 0.1 to 10 μm, more preferably 0.2 to 5 μm. The perfusion culture in this step is performed continuously from immediately after the start of step (3) until the end of step (3). During this time, NK cells induced from the HPC spheres are generated, but the NK cells do not remain within the spheres but are individually filtered out. Therefore, by using a separation membrane that does not exceed the cell diameter of the NK cells during step (3), perfusion culture can be performed while maintaining a certain number of NK cells in the culture vessel.

[0070] The medium exchange in step (3) is also carried out at a constant dilution rate as described in step (1). Specifically, the medium exchange is carried out at a dilution rate of 0.01 to 0.2 hr, as in step (1). -1 It is preferable to carry out the dilution at a dilution rate of 1000 kJ / ml, which can be adjusted with reference to the cell seeding density, the glucose concentration, the lactate concentration, the glutamine concentration, the glutamic acid concentration, etc. of the culture supernatant. This dilution rate is also applied when the medium used in step (2) is replaced with the medium used in step (3).

[0071] The third medium used in step (3) is not particularly limited, as long as it contains a histone methyltransferase inhibitor and / or an AHR antagonist and is capable of inducing HPCs into NK cells. Examples of such a medium include a medium containing IL-15 and SCF. The IL-15 concentration is preferably 1-100 ng / mL, more preferably 20-50 ng / mL. The SCF concentration is preferably 1-100 ng / mL, more preferably 20-50 ng / mL. This medium may further contain one or more components selected from IL-7, Flt3L, a ROCK inhibitor, a GSK3β inhibitor, and a TGFβ receptor (TGFβR) inhibitor. Examples of ROCK inhibitors include Y27632 and thiazovivin, with Y27632 being preferred. Examples of GSK3β inhibitors include CHIR99021 and SB216763, with CHIR99021 being preferred. Examples of TGFβR inhibitors include LY2157299, SB431542, and LY2109761, with LY2157299 being preferred. In a preferred embodiment, the third culture medium further contains IL-7 and Flt3L in addition to IL-15 and SCF. When IL-7 is added, the concentration is preferably 1 to 100 ng / mL, more preferably 20 to 50 ng / mL. When Flt3L is added, the concentration is preferably 1 to 100 ng / mL, more preferably 20 to 50 ng / mL. When a ROCK inhibitor is added, the concentration is preferably 1 to 20 μM, more preferably 1 to 10 μM. When a GSK-3β inhibitor is added, the concentration is preferably 1 to 10 nM, more preferably 1 to 5 μM. When a TGFβR inhibitor is added, the concentration is preferably 0.1 to 100 μM, more preferably 0.1 to 5 μM.

[0072] The culture period in step (3) is usually 20 to 60 days, preferably about 25 to 40 days.

[0073] Furthermore, when IL-7, Flt3L, a ROCK inhibitor, a GSK3β inhibitor, or a TGFβR inhibitor is added in step (3), the induction efficiency can be improved by adjusting the timing of addition of these factors. For example, IL-7 and Flt3L are preferably removed after day 10 of culture in step (3), more preferably after day 20. Furthermore, ROCK inhibitors, GSK3β inhibitors, and TGFβR inhibitors are preferably added 4 to 7 days before the end of culture.

[0074] The basal medium used in step (3) is not particularly limited, but examples include AIM-V Medium (Thermo Fisher Scientific), Stemline® II (Sigma-Aldrich), ALyS505N-0 (Cell Science Institute, Inc.), and Stem Pro-34 SFM (Thermo Fisher Scientific), with AIM-V Medium being preferred. Furthermore, the medium may contain human serum, fetal bovine serum (FBS), or a serum substitute. The concentration of these is preferably 1 to 20%, more preferably 1 to 10%.

[0075] In step (3), it is preferable to further carry out a sphere removal step. The timing of removal can be, for example, before the start of this step, during this step, or at the end of this step, but is not particularly limited and can be carried out at any time. The spheres are preferably removed using a 20 to 100 μm cell strainer, more preferably a 20 to 40 μm cell strainer.

[0076] In this step (3), similarly to the step (1), it is possible to control the temperature, pH, and dissolved oxygen concentration to any desired values ​​for inducing NK cells using a temperature sensor, pH sensor, dissolved oxygen sensor, etc.

[0077] The histone methyltransferase inhibitor and / or AHR antagonist may be added to the medium in step (3) throughout step (3) or for only a portion of the step. When the inhibitor (antagonist) is added only for a portion of step (3), it can be added, for example, only in the first half of step (3) (e.g., until the time when IL-7 and Flt3L are removed from the medium). When the first half of step (3) is performed using a third medium containing IL-15, SCF, IL-7, and Flt3L, the inhibitor (antagonist) can be added to the medium, for example, until the time when IL-7 and Flt3L are removed from the medium, but this is not limiting. Furthermore, with regard to the addition of the histone methyltransferase inhibitor and / or AHR antagonist in step (3), there are no particular limitations on the selection of the inhibitor (antagonist) as long as it can efficiently and rapidly induce NK cells and does not adversely affect the cells, and a suitable concentration can be selected appropriately depending on the type of inhibitor. The concentration of the histone methyltransferase inhibitor can be selected from the range of usually about 10 nM to about 5 μM, preferably about 100 nM to about 2 μM, and more preferably about 500 nM to about 1 μM. The concentration of the AHR antagonist can be selected from the range of usually about 100 nM to about 10 μM, preferably about 500 nM to about 5 μM, and more preferably about 1 μM to about 5 μM. The histone methyltransferase inhibitors may be used alone or in combination of two or more. The AHR inhibitors may be used alone or in combination of two or more.

[0078] Furthermore, in the method of the present invention, a step of expanding and culturing the NK cells obtained in step (3) or a step of maturing the NK cells may be further carried out as step (4) after step (3). A known method can be appropriately applied for the expansion and culturing step or the maturation step.

[0079] The method of the present invention is characterized in that all of the above steps (1) to (3) or (1) to (4) are carried out without using a carrier for three-dimensional culture or an extracellular matrix.

[0080] The method of the present invention may further include a step of cryopreserving the obtained NK cells. After the NK cell production step is completed, the NK cells of the present invention can be frozen and preserved by a method known per se.

[0081] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and the present invention is not limited thereto.

[0082] Example 1: Induction of Differentiation from Pluripotent Stem Cells to NK Cells In this example, a human iPS cell line was used as the pluripotent stem cell, and the process from iPS cell sphere formation to NK cell differentiation was performed using a three-dimensional culture method. After the process of inducing pluripotent stem cell spheres into a cell population containing HPCs, the medium was supplemented with GSK126 or SR1, and the results were compared with those without GSK126 or SR1. In the perfusion culture process, the culture medium was mixed using a stirring blade, and sensors such as pH, temperature, and dissolved oxygen sensors were employed. Various control parameters were continuously monitored and controlled while the culture medium was being mixed. Furthermore, perfusion culture was managed by controlling the inflow and outflow rates per hour using a dedicated pump and balance.

[0083] Step (1): Formation of pluripotent stem cell spheres. The pluripotent stem cell spheres were formed by culturing iPS cells expanded in two dimensions in a three-dimensional culture vessel at a density of 0.25 × 10 5 The cells were seeded to a cell density of 1000 cells / mL. Other details of the procedure are as follows: The medium used in step (1) was StemFit (registered trademark) AK03N (Ajinomoto Healthy Supply Co., Ltd.) supplemented with the ROCK inhibitor Y27632 at a concentration of 10 μM.

[0084] The sphere formation process for pluripotent stem cells was performed using a 500 mL single-use bottle as the culture vessel. The culture medium was continuously mixed using an internal impeller. The pH was continuously monitored with a pH sensor and controlled to 7.00-7.40 by supplying sterile medium, sterile air, or sterile carbon dioxide. The dissolved oxygen concentration was continuously monitored with a dissolved oxygen sensor and controlled to maintain a concentration of 1.50 mg / L or higher by supplying sterile air or sterile oxygen gas. The culture temperature was continuously monitored with a temperature sensor and controlled to 37.0°C by heating the vessel externally with a heater. The culture volume was adjusted to 250 mL, and fresh medium was supplied and the culture medium was discharged at the same rate using a pump and balance. From days 3 to 5 of culture, the culture temperature was maintained at 0.04 h. -1 , 0.06 hr from the 5th to 10th day of culture -1 The dilution rate was controlled at 100%. The separation membrane was made of SUS316 and had a pore size of 25 μm.

[0085] On the 10th day of culture, the cell suspension containing the spheres was collected from the culture medium, and the average particle size and density of the spheres were measured using a microscope.

[0086] Step (2): Inducing the pluripotent stem cell spheres into a cell population containing hematopoietic progenitor cells. Hematopoietic progenitor cells were generated by seeding the iPS cell spheres generated in step (1) into a three-dimensional culture vessel at a density of 30 spheres / mL. Other details are as follows. For the first two days, the medium used was DMEM / F-12, HEPES (Thermo Fisher Scientific) supplemented with CHIR99021 at 2 μM, BMP4 at 80 ng / mL, VEGF165 at 80 ng / mL, bFGF at 50 ng / mL, and the ROCK inhibitor Y27632 at 10 μM. This was used as the basal condition (condition 1), and the test conditions were supplemented with SR1 at 2 μM (condition 2) or GSK126 at 1 μM (condition 3). From days 2 to 4, the basal condition (condition 1) was Essential 6 (Thermo Fisher Scientific) supplemented with 50 ng / mL SCF, 80 ng / mL VEGF165, 2 μM SB431542, 50 ng / mL bFGF, and 10 μM ROCK inhibitor Y27632. The experimental conditions were then supplemented with 2 μM SR1 (condition 2) or 1 μM GSK126 (condition 3). From days 4 to 14, the experimental conditions were standard hematopoietic progenitor differentiation medium supplemented with 50 ng / mL SCF and 50 ng / mL Flt3L. The experimental conditions were then supplemented with 2 μM SR1 (condition 2) or 1 μM GSK126 (condition 3).

[0087] This process was carried out using a single-use bottle with a total volume of 500 mL as the culture vessel, and the culture solution was continuously mixed using a stirring blade inside the vessel. The pH was continuously monitored with a pH sensor and controlled to 7.07 by supplying sterile medium, sterile air, or sterile carbon dioxide. The dissolved oxygen concentration was continuously monitored with a dissolved oxygen sensor and controlled to maintain a concentration of 1.50 mg / L or higher by supplying sterile air or sterile oxygen gas. The culture temperature was continuously monitored with a temperature sensor and controlled to 37.0°C by heating the vessel from outside with a heater. The culture volume was adjusted to 250 mL, and new medium was supplied and the culture solution was discharged at the same rate using a pump and balance. From the first to second day of culture, the culture temperature was 0.07 h -1 , 0.06 hr from the second to fourth day of culture -1 , 0.13 hr from day 5 to day 7 of culture -1 , 0.07 hr from day 7 to day 12 of culture -1 , 0.02 hr from day 12 to day 14 of culture -1 The dilution rate was controlled at 100%. The separation membrane used was made of SUS316 and had a pore size of 75 μm.

[0088] On day 14 of culture, the spheres were removed from the culture medium, and the floating cell suspension was collected. The cell surface markers (CD34, CD43, CD45, CD117) known to be expressed on hematopoietic progenitor cells were confirmed by flow cytometry. The production efficiency of hematopoietic progenitor cells was compared between the cultures with and without GSK126 and SR1, and no significant difference in the expression of the confirmed markers was found.

[0089] Step (3): Step of inducing hematopoietic progenitor cells to NK cells NK cells were produced by seeding the culture medium containing the hematopoietic progenitor cells prepared in step (2) (the culture medium prepared in step 2 on day 14) into a three-dimensional culture vessel at a concentration of 25%, including spheres. Other details are as follows:

[0090] The medium used for the basal conditions (condition 1) was AIM V Serum-Free Medium (Thermo Fisher Scientific) supplemented with 5% FBS and supplemented with IL-15, IL-7, SCF, and Flt3L to a final concentration of 50 ng / mL. SR1 was added to the medium at 2 μM (condition 2) or GSK126 at 1 μM (condition 3) for the test conditions. All culture vessels were 30 mL rotary shakers, and suspension culture was performed at 55 rpm in a 5% CO2 incubator at 37°C.

[0091] During the culture, 100 μL of the culture medium was sampled, and the density of the suspended cells was measured using NC-202 (ChemoMetec), and the proliferation rate from day 4 onward was calculated (Figure 1). Under conditions 2 and 3 (with the addition of SR1 and GSK126, respectively), the cells were cultured at a higher proliferation rate than the control after subculture.

[0092] The NK cell culture medium produced by the method of the present invention was collected over time, and the expression of CD56, a cell surface marker known to be expressed on NK cells, and the expression of CD14, known to be expressed on monocytes and macrophages to identify non-target cells, were confirmed by flow cytometry (Figures 2 and 3). As a result, the increase in CD56 expression was faster under condition 3 (with the addition of GSK126) than under condition 1, indicating that NK cells could be produced more efficiently.

[0093] As shown in Figure 1, SR1 conferred higher cell proliferation efficiency compared to basal conditions early in the NK cell differentiation induction process. On the other hand, GSK126 conferred higher cell proliferation efficiency late in the differentiation induction process, and the onset of differentiation into CD56-positive cells (NK cells or their precursor cells) was faster than under basal conditions. Therefore, the combined use of SR1 and GSK126 is expected to result in a synergistic or complementary effect of each drug, resulting in a faster onset of NK cell differentiation and increased proliferation efficiency throughout the differentiation induction process.

[0094] The method of the present invention for producing pluripotent stem cell-derived NK cells can be said to be a useful method for mass production of NK cells that can be used in cancer immunotherapy.

[0095] This application is based on Patent Application No. 2023-126651 filed in Japan on August 2, 2023, the entire contents of which are incorporated herein by reference.

Claims

1. A method for producing natural killer (NK) cells or their progenitor cells from pluripotent stem cells, a) a step of inducing hematopoietic progenitor cells (HPCs) from pluripotent stem cells in vitro; b) a step of amplifying HPCs in vitro; and The process of inducing NK cells or their progenitor cells from HPC in vitro. A method comprising inhibiting histone methyltransferase and / or allyl hydrocarbon receptor (AHR) in one or more of steps a) to c), and characterized in that one or more of steps a) to c) are performed by perfusion culture.

2. The method according to claim 1, wherein the histone methyltransferase catalyzes the addition of a methyl group to histone 3-lysine residue 27 (H3K27).

3. The method according to claim 1, wherein the histone methyltransferase is EZH1 (enhancer of zest homology 1) and / or EZH2 (enhancer of zest homology 2).

4. The method according to claim 1, wherein the inhibition of histone methyltransferase is performed by a small molecule inhibitor.

5. The method according to claim 4, wherein the small molecule inhibitor is GSK126, EPZ005687, GSK343, Tazemethostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Liramethostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55.

6. The method according to claim 1, wherein the inhibition of AHR has antagonist activity against AHR.

7. The method according to claim 1, wherein the inhibition of AHR is by a small molecule inhibitor.

8. The method according to claim 7, wherein the small molecule inhibitor is SR1 (StemRegenin 1), CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059, or BAY 2416964.

9. (delete)

10. A method for producing NK cells or their progenitor cells from pluripotent stem cells, (1) A step of forming pluripotent stem cell spheres with an average particle size of 200 μm or more in the first culture medium; (2) A step of inducing the pluripotent stem cell spheres obtained in step (1) into a cell population including HPCs by three-dimensional culture using a second medium containing a histone methyltransferase inhibitor and / or an AHR antagonist; and (3) A step of inducing a cell population containing NK cells or their progenitor cells from the cell population containing HPC obtained in step (2) by three-dimensional culture using a third medium containing a histone methyltransferase inhibitor and / or an AHR antagonist. A method comprising performing one or more of steps (1) to (3) by perfusion culture.

11. The method according to claim 10, wherein the histone methyltransferase catalyzes the addition of a methyl group to H3K27.

12. The method according to claim 10, wherein the histone methyltransferase is EZH1 and / or EZH2.

13. The method according to claim 10, wherein the histone methyltransferase inhibitor is a small molecule inhibitor.

14. The method according to claim 13, wherein the small molecule inhibitor is GSK126, EPZ005687, GSK343, Tazemethostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Liramethostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55.

15. The method according to claim 10, wherein the AHR antagonist is a small molecule inhibitor.

16. The method according to claim 15, wherein the small molecule inhibitor is SR1, CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059, or BAY 2416964.