Medium composition for inducing differentiation from pluripotent stem cells into natural killer cells and differentiation method using the same

Abstract

Regarding a culture medium composition for inducing differentiation from pluripotent stem cells into natural killer cells, according to the culture medium composition for inducing differentiation from pluripotent stem cells into natural killer cells according to one aspect of the present invention, it is possible to improve the differentiation efficiency into natural killer cells, and there is an effect that the expression of NK-specific markers can be improved without using artificial feeders and without gene recombination.

Description

Technical Field

[0001] The present invention relates to a culture medium composition for inducing differentiation of pluripotent stem cells into natural killer cells.

Background Art

[0002] Current common cancer treatment methods include surgery, radiation therapy, chemotherapy, etc., which are used alone or in combination depending on the type and stage of cancer. However, the fact is that they are accompanied by severe side effects and pain in patients. A perfect cancer treatment method can be said to be a method that selectively kills only cancer cells without damaging normal cells. However, conventional treatment methods cause some damage to normal cells. Cancer immunotherapy, which has recently attracted attention, is a treatment method that uses the body's innate immune system to minimize damage to normal cells and more specifically remove cancer cells. Research in many detailed fields (such as antibody therapy, immune cell therapy, viral immunotherapy, nanotechnology immunotherapy, etc.) is being actively carried out. Among these, immune cell therapy is a method of treating cancer by increasing the number of cells such as natural killer cells, natural killer T cells, T cells, B cells, dendritic cells, etc. among lymphocytes obtained from a patient's blood, enhancing their functions in vitro, and then returning them to the patient's body again. Such a treatment method using immune cells has shown good effects in immunomodulatory therapy and is evaluated to be excellent in terms of toxicity and safety. Among these, NK cells are important cells responsible for innate immunity. Different from T cells, they mature in the liver and bone marrow. In particular, they have the function of self-identifying and killing abnormal cells such as virus-infected cells and tumor cells. Over the past decade, tumor immunotherapy using the patient's immune system has been steadily developing, and "cell therapy products" using it are also being commercialized.A cell therapy agent is defined as a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention through a series of actions that change the biological properties of cells by a method of growing and selecting autologous, allogeneic, or xenogeneic cells in vitro (Article 2 of Notification No. 2003-26 of the Korea Food and Drug Administration).

[0003] On the other hand, despite the therapeutic advantages and safety of natural killer cells themselves, natural killer cells are known as cells that are difficult to culture and have limitations in that they are difficult to proliferate in large quantities. In addition, there have been attempts to differentiate natural killer cells using pluripotent stem cells, but in such cases, the differentiation efficiency into natural killer cells is low, the expression of specific markers of natural killer cells is low, and methods to enhance the expression have been attempted through artificial gene recombination. Furthermore, when inducing differentiation into natural killer cells, heterologous feeder cells are often used, and when applied to a therapeutic agent, there is a possibility that the feeder cells may contaminate the final product and cause problems with safety.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] One aspect of the present invention is a culture medium composition for inducing differentiation from pluripotent stem cells into natural killer cells, which comprises a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β inhibitor, PVA (polyvinyl alcohol), SCF (Stem Cell Factor) or a combination thereof, a first culture medium composition for inducing differentiation from pluripotent stem cells into hematopoietic stem cells, and a second culture medium composition for inducing differentiation from hematopoietic stem cells into natural killer cells, which comprises SCF (Stem Cell Factor), IL7 (Interleukin7), IL15 (Interleukin15), FLT3L (FMS-like tyrosine kinase 3 ligand) or a combination thereof.

[0006] Another aspect of the present invention is to provide a kit comprising the first culture medium composition and the second composition.

[0007] Still another aspect of the present invention is to provide a method for inducing differentiation from pluripotent stem cells into natural killer cells, which comprises a first step of inducing differentiation from pluripotent stem cells into hematopoietic stem cells by culturing pluripotent stem cells in the presence of the first culture medium composition, and a second step of inducing differentiation from hematopoietic stem cells into natural killer cells by culturing hematopoietic stem cells in the presence of the second culture medium composition.

[0008] Another aspect of the present invention is to provide natural killer cells or a cell population thereof differentiated from pluripotent stem cells by the above method.

[0009] A further aspect of the present invention is to provide a composition comprising the natural killer cells or a cell population thereof.

Means for Solving the Problems

[0010] One aspect of the present invention is a culture medium composition for inducing differentiation of pluripotent stem cells into natural killer cells, comprising a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β inhibitor, PVA (polyvinyl alcohol), SCF (Stem Cell Factor), or a combination thereof, a first culture medium composition for inducing differentiation of pluripotent stem cells into hematopoietic stem cells, and SCF (Stem Cell Factor), IL7 (Interleukin7), IL15 (Interleukin15), FLT3L (FMS-like tyrosine kinase 3 ligand), or a combination thereof, a second culture medium composition for inducing differentiation of hematopoietic stem cells into natural killer cells, and a culture medium composition comprising the same.

[0011] Another aspect of the present invention is to provide a kit comprising the first culture medium composition and the second composition.

[0012] Another aspect of the present invention is to provide a method for inducing differentiation of pluripotent stem cells into natural killer cells, comprising a first step of inducing differentiation of pluripotent stem cells into hematopoietic stem cells by culturing pluripotent stem cells in the presence of the first culture medium composition, and a second step of inducing differentiation of hematopoietic stem cells into natural killer cells by culturing hematopoietic stem cells in the presence of the second culture medium composition.

[0013] Another aspect of the present invention is to provide natural killer cells or a cell population thereof differentiated from pluripotent stem cells by the above method.

[0014] A further aspect of the present invention is to provide a composition comprising the natural killer cells or a cell population thereof.

Effects of the Invention

[0015] According to the medium composition for inducing differentiation from pluripotent stem cells to natural killer cells according to one aspect of the present invention, it is possible to improve the differentiation efficiency into natural killer cells, and there is an effect that the expression of NK-specific markers can be improved without using artificial feeders and without gene recombination.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0017] One aspect of the present invention is a culture medium composition for inducing differentiation of pluripotent stem cells into natural killer cells, comprising a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β (Transforming growth factor-beta) inhibitor, PVA (polyvinyl alcohol), SCF or a combination thereof, a first culture medium composition for inducing differentiation of pluripotent stem cells into hematopoietic stem cells, and SCF (Stem Cell Factor), IL7 (Interleukin7), IL15 (Interleukin15), FLT3L (FMS-like tyrosine kinase 3 ligand) or a combination thereof, a second culture medium composition for inducing differentiation of hematopoietic stem cells into natural killer cells. The first culture medium composition and the second culture medium composition are used separately for inducing differentiation of pluripotent stem cells into hematopoietic stem cells and for inducing differentiation of hematopoietic stem cells into natural killer cells, respectively.

[0018] In one embodiment, the first culture medium composition can comprise any one or more of the following compositions.

[0019] (i) A culture medium composition containing a GSK inhibitor; (ii) A culture medium composition containing BMP4, VEGF and bFGF; (iii) A culture medium composition containing VEGF, bFGF, a TGF-β inhibitor and retinoic acid; and (iv) A culture medium composition containing PVA, SCF and bFGF.

[0020] At this time, the culture medium compositions of (i) to (iv) can be used separately or together for inducing differentiation of hematopoietic stem cells into natural killer cells.

[0021] As used herein, the term "Natural Killer cells" or "NK cells" refers to cytotoxic lymphocytes that constitute a major component of the innate immune system, which are defined as large granular lymphocytes (LGLs) and are composed of the generation of common lymphoid progenitors (CLPs); a third cell differentiated from B lymphocytes and T lymphocytes. The "Natural Killer cells" or "NK cells" can include not only mature natural killer cells but also natural killer progenitor cells. The natural killer cells are activated in response to interferon or macrophage-derived cytokines, and the natural killer cells are labeled with "activating receptors" and "inhibitory receptors" and include two types of surface receptors that control the cytotoxic activity of the cells.

[0022] The activity of natural killer cells means that the action of the functions possessed by natural killer cells occurs or increases, and refers to the ability to sense pathogenic cells, pathogens of non-specific infections, cancer cells, etc., and remove or kill them.

[0023] In one embodiment, the GSK3 inhibitor can include the substances described in Table 1.

[0024] [Table 1]

[0025] In one embodiment, the TGF-β inhibitor is 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine, 4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, and 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate, 3-(6-methyl-2-pyridinyl)-N-phenyl-4(4-quinolinyl)-1H-pyrazole 1-carbothioamide, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol 4-yl)-1,5-Naphthyridine (2-(3-(6-Methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), 4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-quinoline (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), 2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine (2-[4-(1,3-Benzodioxol-5-yl)-2-(1,(1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine), 2-(5-chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine, 6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline, 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol, 3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide, or combinations thereof can be included.,

[0026] The term "CHIR99021" in the present invention means a compound having the structural formula of Chemical Formula 1 below.,

[0027] [Chemical Formula]

[0028] The term "SB-431542" in the present invention means a compound having the structural formula of the following Chemical Formula 2.

[0029] [Chemical formula]

[0030] The "medium" in the present invention varies depending on the purpose of use, but is a medium for animal cell culture that basically contains inorganic salts, a carbon source, amino acids, bovine serum albumin (BSA), cofactors, etc., and can include any common medium well known to those skilled in the art. Particularly preferably, the medium contains, as inorganic salts, NaCl, KCl, NaHCO3, etc., as a carbon source, glucose, sodium pyruvate, calcium lactate salt, etc., as amino acids, glutamine, essential amino acids and non-essential amino acids, and as cofactors, other trace elements, buffers, etc.

[0031] In addition, the medium can contain antibiotics. As the medium, various media known and commercialized for culturing animal cells, for example, DMEM (Dulbecco’s Modified Eagle’s Medium), EDM (Endothelial differentiation medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow’s Minimal Essential Medium), and IMDM (Iscove’s Modified Dulbecco’s Medium) can be used as they are, but are not limited thereto. In one embodiment, StemPro-34 SFM medium can be used.

[0032] In one embodiment, the concentration of the GSK inhibitor (e.g., CHIR99021) in the first culture medium composition may be 0.1 to 100 μM, 0.1 to 90 μM, 0.1 to 80 μM, 0.1 to 70 μM, 0.1 to 60 μM, 0.1 to 50 μM, 0.1 to 40 μM, 0.1 to 30 μM, 0.1 to 20 μM, 0.1 to 10 μM, 1 to 100 μM, 2 to 100 μM, 3 to 100 μM, 4 to 100 μM, 1 to 90 μM, 2 to 80 μM, 3 to 70 μM, 4 to 60 μM, 4 to 50 μM, 4 to 40 μM, 4 to 30 μM, 4 to 20 μM, 4 to 10 μM, or 1 to 10 μM.

[0033] In one embodiment, the concentration of the BMP4 in the first culture medium composition may be 1 to 1000 ng / ml, 1 to 900 ng / ml, 1 to 800 ng / ml, 1 to 700 ng / ml, 1 to 600 ng / ml, 1 to 500 ng / ml, 1 to 400 ng / ml, 1 to 300 ng / ml, 1 to 200 ng / ml, 1 to 100 ng / ml, 10 to 1000 ng / ml, 20 to 1000 ng / ml, 30 to 1000 ng / ml, 40 to 1000 ng / ml, 10 to 900 ng / ml, 20 to 800 ng / ml, 30 to 700 ng / ml, 40 to 600 ng / ml, 40 to 500 ng / ml, 40 to 400 ng / ml, 40 to 300 ng / ml, 40 to 200 ng / ml, 40 to 100 ng / ml or 10 to 100 ng / ml.

[0034] In one embodiment, the concentration of the VEGF in the first culture medium composition may be 1 to 1000 ng / ml, 1 to 900 ng / ml, 1 to 800 ng / ml, 1 to 700 ng / ml, 1 to 600 ng / ml, 1 to 500 ng / ml, 1 to 400 ng / ml, 1 to 300 ng / ml, 1 to 200 ng / ml, 1 to 100 ng / ml, 10 to 1000 ng / ml, 20 to 1000 ng / ml, 30 to 1000 ng / ml, 40 to 1000 ng / ml, 10 to 900 ng / ml, 20 to 800 ng / ml, 30 to 700 ng / ml, 40 to 600 ng / ml, 40 to 500 ng / ml, 40 to 400 ng / ml, 40 to 300 ng / ml, 40 to 200 ng / ml, 40 to 100 ng / ml or 10 to 100 ng / ml.

[0035] In one embodiment, the concentration of the bFGF in the first culture medium composition may be 1 to 1000 ng / ml, 1 to 900 ng / ml, 1 to 800 ng / ml, 1 to 700 ng / ml, 1 to 600 ng / ml, 1 to 500 ng / ml, 1 to 400 ng / ml, 1 to 300 ng / ml, 1 to 200 ng / ml, 10 to 1000 ng / ml, 20 to 1000 ng / ml, 30 to 1000 ng / ml, 40 to 1000 ng / ml, 10 to 900 ng / ml, 20 to 800 ng / ml, 30 to 700 ng / ml, 40 to 600 ng / ml, 40 to 500 ng / ml, 40 to 400 ng / ml, 40 to 300 ng / ml, 40 to 200 ng / ml or 10 to 200 ng / ml.

[0036] In one embodiment, the concentration of the retinoic acid in the first culture medium composition can be 0.05 to 50 μM, 0.07 to 50 μM, 0.09 to 50 μM, 0.1 to 50 μM, 0.3 to 50 μM, 0.5 to 50 μM, 0.7 to 50 μM, 0.9 to 50 μM, 0.05 to 40 μM, 0.05 to 30 μM, 0.05 to 20 μM, 0.05 to 10 μM, 0.05 to 5 μM, 0.07 to 40 μM, 0.09 to 30 μM, 0.1 to 20 μM, 0.3 to 20 μM, 0.5 to 10 μM, 0.7 to 5 μM or 0.1 to 10 μM.

[0037] In one embodiment, the concentration of the TGF-β inhibitor (e.g., SB-431542) in the first culture medium composition may be 0.1 to 500 μM, 0.1 to 400 μM, 0.1 to 300 μM, 0.1 to 200 μM, 0.1 to 100 μM, 1 to 500 μM, 5 to 500 μM, 5 to 400 μM, 5 to 300 μM, 5 to 200 μM, 5 to 100 μM, 5 to 50 μM or 1 to 100 μM.

[0038] In one embodiment, the concentration of the PVA in the first culture medium composition may be 0.002 to 5% (w / v), 0.004 to 2% (w / v), 0.01 to 1% (w / v), 0.02 to 0.8% (w / v), 0.02 to 0.5% (w / v), 0.04 to 0.3% (w / v), 0.05 to 0.2% (w / v), or 0.07 to 0.2% (w / v).

[0039] In one embodiment, the concentration of the SCF in the first culture medium composition may be 1 to 1000 ng / ml, 1 to 900 ng / ml, 1 to 800 ng / ml, 1 to 700 ng / ml, 1 to 600 ng / ml, 1 to 500 ng / ml, 1 to 400 ng / ml, 1 to 300 ng / ml, 1 to 200 ng / ml, 5 to 500 ng / ml, 10 to 1000 ng / ml, 10 to 500 ng / ml, 10 to 250 ng / ml, 10 to 100 ng / ml, 20 to 100 ng / ml, 30 to 80 ng / ml.

[0040] In one embodiment, the concentration of the GSK inhibitor in the first culture composition may be 1 to 10 μM, the concentration of BMP4 may be 10 to 100 ng / ml, the concentration of VEGF may be 10 to 100 ng / ml, the concentration of bFGF may be 10 to 200 ng / ml, the concentration of retinoic acid may be 0.1 to 10 μM, the concentration of the TGF-β inhibitor may be 1 to 100 μM, the concentration of PVA may be 0.01 to 1% (w / v) and the concentration of SCF may be 5 to 500 ng / ml.

[0041] In one embodiment, the concentration of the SCF in the second culture medium composition may be 1 to 100 ng / ml, 1 to 90 ng / ml, 1 to 80 ng / ml, 1 to 70 ng / ml, 1 to 60 ng / ml, 1 to 50 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, 3 to 100 ng / ml, 5 to 100 ng / ml, 7 to 100 ng / ml, 9 to 100 ng / ml, 11 to 100 ng / ml, 13 to 100 ng / ml, 15 to 100 ng / ml, 3 to 90 ng / ml, 5 to 80 ng / ml, 7 to 70 ng / ml, 9 to 60 ng / ml, 11 to 50 ng / ml, 13 to 40 ng / ml, 15 to 30 ng / ml or 1 to 50 ng / ml.

[0042] In one embodiment, the concentration of the IL7 in the second culture medium composition may be 1 to 100 ng / ml, 1 to 90 ng / ml, 1 to 80 ng / ml, 1 to 70 ng / ml, 1 to 60 ng / ml, 1 to 50 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, 3 to 100 ng / ml, 5 to 100 ng / ml, 7 to 100 ng / ml, 9 to 100 ng / ml, 11 to 100 ng / ml, 13 to 100 ng / ml, 15 to 100 ng / ml, 3 to 90 ng / ml, 5 to 80 ng / ml, 7 to 70 ng / ml, 9 to 60 ng / ml, 11 to 50 ng / ml, 13 to 40 ng / ml, 15 to 30 ng / ml, or 1 to 50 ng / ml.

[0043] In one embodiment, the concentration of the IL15 in the second culture medium composition may be 1 to 100 ng / ml, 1 to 90 ng / ml, 1 to 80 ng / ml, 1 to 70 ng / ml, 1 to 60 ng / ml, 1 to 50 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 3 to 100 ng / ml, 5 to 100 ng / ml, 7 to 100 ng / ml, 9 to 100 ng / ml, 3 to 90 ng / ml, 5 to 80 ng / ml, 7 to 70 ng / ml, 9 to 60 ng / ml, or 1 to 50 ng / ml.

[0044] In one embodiment, the concentration of the FLT3L in the second culture medium composition may be 1 to 100 ng / ml, 1 to 90 ng / ml, 1 to 80 ng / ml, 1 to 70 ng / ml, 1 to 60 ng / ml, 1 to 50 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 3 to 100 ng / ml, 5 to 100 ng / ml, 7 to 100 ng / ml, 9 to 100 ng / ml, 3 to 90 ng / ml, 5 to 80 ng / ml, 7 to 70 ng / ml, 9 to 60 ng / ml, or 1 to 50 ng / ml.

[0045] In one embodiment, the concentration of SCF in the second culture composition may be 1 to 50 ng / ml, the concentration of IL7 may be 1 to 50 ng / ml, the concentration of IL15 may be 1 to 50 ng / ml, and the concentration of FLT3L may be 1 to 50 ng / ml.

[0046] As used herein, "positive or +" may mean that, in relation to a cell label (marker), it is present in a greater amount or at a higher concentration when compared to other cells based on that label. A cell can be positive for a marker if the marker is present inside or on the surface of the cell and the cell can be distinguished from at least one other cell type using the label. Also, it can mean that the cell has the label in an amount sufficient to give a signal, for example, a signal from a cell measuring device, that is greater than the background value. For example, a cell can be detectably labeled with an antibody specific for CD56, and if the signal from the antibody is detectably greater than a control (e.g., the background value), the cell is "positive for CD56" or "CD56 + " can be represented. The term, "negative or -" can mean that, even when using an antibody specific for a marker on a particular cell surface, the marker cannot be detected when compared to the background value. For example, if a cell cannot be detectably labeled with an antibody specific for CD3, the cell is "negative for CD3" or "CD3 - " can be represented.

[0047] In one embodiment, in the total cell population of natural killer cells differentiated by the medium composition for inducing differentiation from pluripotent stem cells of the present invention, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or 99% or more of the cells may be negative for CD3 and positive for CD56.

[0048] In one embodiment, the natural killer cell population of the present application is NKG2D is CD3 - CD56 + expressed in 50%, 60%, or 64% or more of the cells, and / or NKp30 is CD3 - CD56 +expressed in cells at 50%, 70%, 80%, or at least 89%, and / or NKp44 is CD3 - CD56 + expressed in cells at 50%, 80%, 90%, or 94% or more, and / or NKp46 is CD3 - CD56 + expressed in cells at 50%, 60%, or 67% or more, and / or DNAM-1 is CD3 - CD56 + can be expressed in cells at 50%, 80%, 90%, or 94% or more.

[0049] In one embodiment, the natural killer cell population of the present application is NKG2A is CD3 - CD56 + expressed in cells at 5%, 2%, or 1% or less, and / or KIR2DL1 is CD3 - CD56 + expressed in cells at 5%, 2%, or 1% or less, and / or KIR2DL2 / 3 is CD3 - CD56 + expressed in cells at 20%, 15%, or 10% or less, and / or KIR3DL1 is CD3 - CD56 + can be expressed in cells at 10%, 6%, 5%, or 3% or less. In one embodiment, the natural killer cell population of the present application is Perforin is CD3 - CD56 + expressed in cells at 50%, 80%, 90%, or 98% or more, and / or Granzyme B is CD3 - CD56 + can be expressed in cells at 50%, 80%, 90%, or 98% or more.

[0050] In one embodiment, the pluripotent stem cells may be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Specifically, the embryonic stem cells and induced pluripotent stem cells may be of human origin, but are not limited thereto.

[0051] In the present invention, "pluripotent stem cells (PSCs)" refers to stem cells having pluripotent or totipotent self-renewal ability capable of differentiating into cells of all tissues of an individual, and can include embryonic stem cells and induced pluripotent stem cells. "Embryonic stem cells" are those obtained by extracting the inner cell mass from an early embryo immediately before the fertilized egg implants in the mother's uterus and culturing it in vitro, and have pluripotent or totipotent self-renewal ability capable of differentiating into cells of all tissues of an individual. In a broad sense, it also includes embryoid bodies derived from embryonic stem cells. "Induced pluripotent stem cells" refers to cells induced to have pluripotent differentiation ability through an artificial dedifferentiation process from differentiated cells, and are also called reverse-differentiated stem cells. The artificial dedifferentiation process is performed by introducing a virus-mediated or non-viral vector using retroviruses and lentiviruses, or non-viral-mediated dedifferentiation factors such as proteins and cell extracts, or can include a reverse-differentiation process using stem cell extracts, compounds, etc. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells. Specifically, they show a similar cell shape, have similar gene and protein expression patterns, have pluripotency in vitro and in vivo, form teratomas, form chimeric mice when inserted into a mouse blastocyst, and are capable of germline transmission of genes.

[0052] In one embodiment, the culture medium composition can further include a third culture medium composition for the maturation and proliferation of natural killer cells containing IL15, a ROCK (Rho-associated protein kinase) inhibitor, or a combination thereof.

[0053] In one embodiment, the ROCK inhibitor can include Fasudil, AT-13148, BA-210, β-Elemene, Belumosudil, Chroman 1, DJ4, GSK-576371, GSK429286A, H-1152, Hydroxyfasudil, Ibuprofen, LX-7101, Netarsudil, RKI-1447, Ripasudil, TCS-001, Thiazovivin, Verosudil (AR-12286), Y-27632, Y-30141, Y-33075, Y-39983, or a combination thereof.

[0054] In one embodiment, the concentration of the ROCK inhibitor (e.g., Fasudil) in the third culture medium composition can be 1 to 100 μM, 1 to 90 μM, 1 to 80 μM, 1 to 70 μM, 1 to 60 μM, 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 3 to 100 μM, 5 to 100 μM, 7 to 100 μM, 9 to 100 μM, 3 to 90 μM, 5 to 80 μM, 7 to 70 μM, 9 to 60 μM, or 1 to 50 μM.

[0055] In one embodiment, the concentration of the IL15 in the third culture medium composition can be 1 to 200 μg / ml, 1 to 100 μg / ml, 2 to 200 μg / ml, 5 to 80 μg / ml, 5 to 60 μg / ml, 5 to 40 μg / ml, 5 to 30 μg / ml, 10 to 80 μg / ml, 10 to 60 μg / ml, 10 to 40 μg / ml, or 10 to 30 μg / ml.

[0056] In one embodiment, the concentration of the IL-15 in the third culture solution composition can be 2 to 200 ng / ml, and the concentration of the ROCK inhibitor can be 1 to 100 μg / ml.

[0057] Another aspect provides a method for inducing differentiation of pluripotent stem cells into natural killer cells, including a first step of inducing differentiation of pluripotent stem cells into hematopoietic stem cells by culturing pluripotent stem cells in the presence of a first culture medium composition containing a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β inhibitor, PVA (polyvinyl alcohol), SCF (Stem Cell Factor), or a combination thereof, and a second step of inducing differentiation of hematopoietic stem cells into natural killer cells by culturing hematopoietic stem cells in the presence of a second culture medium composition containing SCF, IL7 (Interleukin7), IL15 (Interleukin15), FLT3L (FMS-like tyrosine kinase 3 ligand), or a combination thereof.

[0058] In one embodiment, during the induction of differentiation in the first step, the first culture medium composition can be replaced with a first culture medium composition having a different composition.

[0059] In one embodiment, the first culture medium composition can include any one or more of the following compositions.

[0060] (i) A culture medium composition containing a GSK inhibitor; (ii) A culture medium composition containing BMP4, VEGF, and bFGF; (iii) A culture medium composition containing VEGF, bFGF, a TGF-β inhibitor, and retinoic acid; and (iv) A culture medium composition containing PVA, SCF, and bFGF.

[0061] In one embodiment, the first step can include the following stages.

[0062] (1) Culturing the cells in a medium composition containing a GSK inhibitor; (2) Culturing the cells in a medium composition containing BMP4, VEGF, and bFGF; (3) Culturing the cells in a medium composition containing VEGF, bFGF, a TGF-β inhibitor, and retinoic acid; (4) Culturing the cells in a medium composition containing PVA, SCF, and bFGF.

[0063] At this time, the steps (1) to (4) can be performed in the forward order, reverse order, or simultaneously. When the steps are performed simultaneously, the cells can be cultured in a medium composition containing all the components of the medium compositions of those steps.

[0064] In one embodiment, bFGF may be included in the medium composition at different concentrations for each stage.

[0065] In one embodiment, the first step can be performed for at least 3 days, 3 to 25 days, 5 to 20 days, or 12 to 16 days.

[0066] In one embodiment, the step (1) can be performed for 1 to 4 days, the step (2) can be performed for 1 to 4 days, the step (3) can be performed for 0.5 to 2 days, and / or the step (4) can be performed for 2 days to 24 days.

[0067] In one embodiment, the composition of the second medium can include SCF, IL7, IL15, and FLT3L.

[0068] In one embodiment, the second stage can be carried out for at least 3 days, 3 to 25 days, 5 to 20 days, or 12 to 16 days. In one embodiment, the method of inducing differentiation further includes a third stage of maturing and proliferating natural killer cells by culturing natural killer cells in the presence of a third culture composition containing IL15, a ROCK inhibitor (such as fasudil), or a combination thereof.

[0069] In one embodiment, the third stage can be carried out for at least 1 day, 1 to 28 days, 2 to 25 days, 3 to 21 days, 5 to 20 days, or 7 to 14 days.

[0070] In one embodiment, the culture composition of the present application may contain each component in various concentration combinations.

[0071] Another aspect provides natural killer cells or a cell population thereof differentiated from pluripotent stem cells by the method of inducing differentiation described herein. The natural killer cells are the same as those described above.

[0072] Another aspect provides a composition containing natural killer cells or a cell population thereof differentiated from pluripotent stem cells by the method of inducing differentiation described herein.

[0073] In one embodiment, a pharmaceutical composition for preventing or treating cancer containing natural killer cells or a cell population thereof differentiated by the method of inducing differentiation as an active ingredient is provided.

[0074] The term "cancer" means including tumors, blood cancers or solid cancers, and includes those that impair the synergistic activity of NK cells of an individual or do not cause the synergistic activity of NK cells as target cells under specific conditions. In one embodiment, the cancer may be at least one selected from the group consisting of glioma, gastrointestinal stromal tumor, leukemia, breast cancer, uterine cancer, cervical cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, laryngeal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, fat cancer, fibroblast cancer, blood cancer, lymphoma, and multiple myeloma.

[0075] As used herein, the term "prevention" means all acts of suppressing or delaying the occurrence of cancer by administration of the composition of the present invention.

[0076] As used herein, the term "treatment" refers to or includes the alleviation, prevention of progression, or prevention of a disease, disorder, or condition, or one or more symptoms thereof, and the "active ingredient" or the term "pharmaceutically effective amount" can mean any amount of the composition used in the process of practicing the invention provided herein that is sufficient to alleviate, prevent the progression, or prevent a disease, disorder, or condition, or one or more symptoms thereof.

[0077] The composition may further include other known immune adjuvants, and as other immune adjuvants, preferably, it may include any one of monophosphoryl lipid A (MPL) and GLA-SE (Glucopyranosyl Lipid Adjuvant, formulated in a stable nano-emulsion of squalene oil in-water).

[0078] The method of administering the pharmaceutical composition is not particularly limited, but it can be administered parenterally or orally, such as intravenously, subcutaneously, intraperitoneally, by inhalation, or by topical application, depending on the intended method. The dosage range varies depending on factors such as the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage refers to the amount of the therapeutic substance according to one aspect that is sufficient to treat the alleviated condition when administered to an individual in need of treatment. The effective amount of the therapeutic substance varies depending on the specific compound, condition and its severity, and the individual in need of treatment, and this can be routinely determined by those skilled in the art. As a non-limiting example, the dosage of the composition according to one aspect for the human body can vary depending on the patient's age, weight, gender, dosage form, health status, and severity of the disease. When based on an adult patient weighing 70 kg, for example, it can be about 1,000 - 10,000 cells / time, 1,000 - 100,000 cells / time, 1,000 - 1,000,000 cells / time, 1,000 - 10,000,000 cells / time, 1,000 - 100,000,000 cells / time, 1,000 - 1,000,000,000 cells / time, 1,000 - 10,000,000,000 cells / time, and can be administered in divided doses 1 to several times a day at regular time intervals, or can be administered multiple times at regular time intervals.

[0079] The pharmaceutical composition can contain a pharmaceutically acceptable carrier and / or additive. For example, it can contain sterile water, physiological saline, well-known and commonly used buffers (such as phosphoric acid, citric acid, and other organic acids), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspending agents, isotonic agents, or preservatives. For topical administration, it can also include combinations with organic substances such as biopolymers and inorganic substances such as hydroxyapatite, specifically, collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and their chemical derivatives. When the pharmaceutical composition according to one embodiment is prepared in a dosage form suitable for injection, the immune cells, or substances that increase their activity, can be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution state.

[0080] Depending on the administration method and dosage form of the pharmaceutical composition, suspension agents, solubilizing agents, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, soothing agents, buffers, reducing agents, antioxidants, etc. can be appropriately included as needed. Pharmaceutical carriers and formulations suitable for the present invention, including those listed above, are described in detail in the literature [Remington’s Pharmaceutical Sciences, 19th ed., 1995]. The pharmaceutical composition is manufactured in the form of a unit dose by formulating it with a pharmaceutically acceptable carrier and / or excipient by a method that can be easily carried out by those having ordinary knowledge in the technical field to which the invention pertains, or it may be manufactured by charging it into a multi-dose container. At this time, the dosage form may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or in the form of powder, granules, tablets or capsules.

[0081] In one embodiment, a pharmaceutical composition for preventing or treating an infectious disease containing natural killer cells or a cell population thereof differentiated by the method of inducing differentiation as an active ingredient is provided.

[0082] As used herein, the term "disease" can mean a pathological condition, particularly cancer, infectious disease, inflammatory disease, metabolic disease, autoimmune disease, degenerative disease, cell death-related disease and graft rejection.

[0083] As used herein, the term "infectious disease" can generally refer to infectious diseases caused by organisms such as bacteria, viruses, fungi, etc. For example, in the case of viral infections such as HIV (Human Immunodeficiency Virus), EBV (Epstein-Barr virus), HHV (Human Herpes Virus), IAV (Influenza A virus), and COVID-19, activating receptors of natural killer cells such as NKp46, NKp44, or NKp30 can bind to and be activated by viral glycoproteins to induce the death of infected cells. Also, for example, in the case of viral infections such as CMV (Cytomegalovirus) and EMCV (Encephalomyocarditis virus), FASL, TRAIL, etc. of natural killer cells can bind to increased death receptors in virus-infected cells to induce the death of infected cells.

[0084] Another aspect provides a kit for inducing the differentiation of pluripotent stem cells into natural killer cells, which includes a first medium composition for inducing the differentiation of pluripotent stem cells into hematopoietic stem cells, containing a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β inhibitor, PVA (polyvinyl alcohol), SCF (Stem Cell Factor), or a combination thereof, and a second medium composition for inducing the differentiation of hematopoietic stem cells into natural killer cells, containing SCF, IL7 (Interleukin 7), IL15 (Interleukin 15), FLT3L (FMS-like tyrosine kinase 3 ligand), or a combination thereof. The first medium composition, the second medium composition, and each component are the same as those described above.

[0085] In this specification, the first culture medium composition and the second culture medium composition can be separately contained in the kit. Further, when the first culture medium composition contains the culture medium compositions of (i) to (iv), the culture medium compositions of (i) to (iv) can also be separately contained in the kit.

[0086] Another aspect is the use of inducing the differentiation of pluripotent stem cells into hematopoietic stem cells in a first culture medium composition containing a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β inhibitor, PVA (polyvinyl alcohol), SCF (Stem Cell Factor), or a combination thereof.

[0087] Another aspect is the use of inducing the differentiation of hematopoietic stem cells into natural killer cells in a second culture medium composition containing SCF, IL7 (Interleukin7), IL15 (Interleukin15), FLT3L (FMS-like tyrosine kinase 3 ligand), or a combination thereof.

[0088] Another aspect is the use of promoting the maturation and proliferation of natural killer cells in a third culture medium composition containing IL15, a ROCK (Rho-associated protein kinase) inhibitor, or a combination thereof.

[0089] The first culture medium composition, the second culture medium composition, the third culture medium composition, and each component, etc. are the same as those described above.

Examples

[0090] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0091] The terms and words used in the description and claims of the present invention shall not be construed as being limited to their ordinary or dictionary meanings. In accordance with the principle that the inventor can appropriately define the concept of the terms in order to explain his invention in the best way, they must be construed in a meaning and concept that conforms to the technical idea of the present invention.

[0092] Throughout the description of the present invention, when a certain part is said to "include" a certain component, this means that, unless otherwise stated to the contrary, it does not exclude other components and can further include other components.

[0093] In the general description of the present invention, "A and / or B" means A or B, or A and B.

[0094] Example 1: Natural killer cells derived from human pluripotent stem cells

[0095] A schematic diagram of the differentiation process from pluripotent stem cells to natural killer cells is shown in FIG. 1. Specifically, human pluripotent stem cells were cultured to differentiate into hematopoietic stem cells, and the hematopoietic stem cells were cultured to differentiate into natural killer cells. After differentiating into natural killer cells, they were matured and proliferated.

[0096] 1.1. Differentiation into hematopoietic stem cells (hematopoietic stem cells; HSCs)

[0097] Human pluripotent stem cells (Human pluripotent stem cells; hPSCs) were maintained in stemMACS-iPS brew media for 2 days. The basic differentiation culture medium was Stempro34-SFM (+stempro sup) + 200 μg / human transferrin + 2 mM L-glutamine + 0.5 mM L-ascorbic acid + 0.45 mM MTG (1-thioglycerol) + 1% penicillin / streptomycin.

[0098] The cells were treated with CHIR99021, a GSK inhibitor known to induce mesenchymal cells, alone at a concentration of 5 μM for 2 days. Further, the cells were treated with a differentiation culture medium supplemented with 50 ng / ml of BMP4, 50 ng / ml of VEGF, and 100 ng / ml of bFGF for 2 days. Thereafter, the cells were cultured in a culture medium supplemented with 50 ng / ml of VEGF, 50 ng / ml of bFGF, 10 μM of SB-431542, and 1 μM of retinoic acid overnight. From the 5th day to the 12th day after culturing, the cells were cultured while replacing the culture medium daily with a culture medium supplemented with 0.1% PVA (w / v, g / ml), 50 ng / ml of SCF, and 10 ng / ml of bFGF.

[0099] 1.2. Differentiation into natural killer cells

[0100] For the differentiation basic culture medium after HSC induction, the same culture medium as used for HSC induction was used. The basic culture medium was supplemented with 20 ng / ml of SCF, 20 ng / ml of IL-7, 10 ng / ml of IL-15, and 10 ng / ml of FLT3L, and cultured for 2 weeks while replacing the culture medium once every 2 days.

[0101] 1.3. Maturation and expansion of natural killer cells

[0102] Only the cells floating after differentiation into natural killer cells were collected and placed in a cell culture dish. Alys505NK-EX (containing 1000 IU / ml of IL-2) + 10% (w / v) FBS was used as the basic culture medium, 10 μM of fasudil, a ROCK inhibitor, and 20 μg / ml of IL-15 were added, and cultured for 1 to 2 weeks.

[0103] The NK cells of Example 1 were obtained by the above method.

[0104] Comparative Examples 1 to 4: Natural killer cells isolated from human peripheral blood

[0105] Natural killer cells were isolated according to the method disclosed in the literature [Jung, D., Baek, Y.S., Lee, I.J. et al. Ex vivo expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells through different receptor-ligand interactions. J Exp Clin Cancer Res 40, 333 (2021)].

[0106] [Isolation of NK cells]

[0107] Human peripheral blood mononuclear cells (PBMCs) were collected from four healthy donors via leukapheresis. Written consent was obtained from all healthy donors prior to proceeding with the study. CD3 - CD56 + Primary NK (pNK) cells were obtained by CD3 - / CD56 + selection using the Prodigy (manufactured by Miltenyi Biotec), a closed and automated platform, with an anti-CD3 antibody (manufactured by Miltenyi Biotec, Bergisch Gladbach, Germany) and an anti-CD56 antibody (manufactured by Miltenyi Biotec).

[0108] The isolated primary NK cells were used as Comparative Example 1. The cryopreserved isolated primary NK cells were used as Comparative Example 2.

[0109] [Activation, proliferation, and cryopreservation of NK cells]

[0110] The NK cells of Comparative Example 2 were inoculated into flasks coated with γ-globulin (manufactured by Green-cross, Yongin-si, Korea) and anti-NKp46 (manufactured by R&D Systems, Minneapolis, MN, USA), and cultured in Alys505NK-EX serum-free medium (manufactured by CSTI, Sendai-shi, Japan) supplemented with 1000 IU / mL of recombinant human IL-2 (manufactured by Novartis, Basel, Switzerland), 50 ng / mL of recombinant human IL-18 (manufactured by R&D system, Minneapolis, MN, USA) and 5% heat-inactivated autologous plasma. Fresh culture medium was added every 1-3 days according to the cell density (2×10 6 cells / mL). On the 6th day, the cells were transferred to a culture bag (manufactured by NIPRO, Osaka-shi, Japan)) and cultured for 14 days. The cultured NK cells were cryopreserved with Cryostor CS5 (manufactured by BioLife Solutions, Bothell, WA, USA). The cells transferred to the culture bag and cultured for 14 days were used as Comparative Example 3, and the cryopreserved cells of Comparative Example 3 were used as Comparative Example 4.

[0111] Comparative Example 5: NK-92MI

[0112] The human natural killer cell line NK-92MI was purchased from ATCC (American Type Culture Collection, USA) and used.

[0113] Experimental Example 1: Identification of specific markers of NK cells

[0114] To confirm whether the cells obtained in Example 1 had differentiated into NK cells, CD markers specific for NK cells were confirmed using FACS (fluorescence-activated cell sorting, BD FACSCalibur TM ). Specifically, on the 33rd day of all NK cell differentiation, all cells were collected and analyzed for CD markers. The results are shown in Figure 2.

[0115] As shown in Fig. 2, at least 97% of the analyzed cells differentiated into CD45-positive peripheral white blood cells, and CD3 - CD56 + cells were confirmed to be 97% or more. In addition, it was found that 56% of CD16, which induces the antibody-dependent cellular cytotoxicity (manufactured by ADCC) function of NK cells, was expressed.

[0116] From the above results, it was found that by using the composition according to the present invention, pluripotent stem cells can be effectively differentiated into NK cells.

[0117] Experimental Example 2: Identification of the phenotype of NK cells

[0118] The expression levels of cell surface receptors and cytoplasmic granules of the cells obtained in Example 1 were confirmed by flow cytometry. For comparison with primary NK cells, the cells of Comparative Example 3 and Comparative Example 5 were used for the same analysis. The results are shown in Figs. 3A and 3B.

[0119] As shown in Figs. 3A and 3B, the expression of the activating receptor of the cells of Example 1 was CD56 + The expression of NKG2D in 64% of the cells, NKp30 in 89%, NKp46 in 94%, and DNAM-1 in 94% was confirmed, and the expression of the inhibitory receptor was CD56 + The expression of NKG2A and KIR2DL1 in the cells was confirmed to be 1% or less, and the expression of 9% of KIR2DL2 / 3 and 3% of KIR3DL1 was confirmed.

[0120] In the case of perforin and granzyme B, which are cytoplasmic granules (cytolytic granules), the expression was confirmed to be 98%, 99% or more, respectively.

[0121] The cells of Example 1 had a somewhat lower expression of activating receptors at 64% - 94% compared to the activated primary NK cells of Comparative Example 3, but showed a relatively high expression rate compared to NK-92MI of Comparative Example 5 (NKp46, 4%; NKp44, 27%; DNAM-1, 4%). In the case of the inhibitory receptors of the cells of Example 1, it was found that their expression was much lower than that of primary NK cells. The cytoplasmic granules of the cells of Example 1 were highly expressed and were found to have the same characteristics as primary NK cells.

[0122] As a result, it was found that the NK cells of Example 1 had characteristics superior to those of the NK cells of Comparative Example 5 and were similar to or had characteristics superior to those of the activated NK cells of Comparative Example 3.

[0123] Experimental Example 3: Confirmation of cytotoxicity

[0124] 3-1. K562 cell line and A2780cis cell strain

[0125] Cytotoxicity was assayed against K562 cells (a cell line of human chronic myelogenous leukemia), which are highly sensitive to NK cells and are mainly used for measuring the activity of NK cells, and cisplatin-resistant ovarian cancer cells A2780cis (an ovarian cancer cell line). First, the target cancer cells (K562 or A2780cis) were collected, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Then the cell pellet was diluted and washed with DPBS, and the washed cell pellet was suspended in a culture solution containing 10% FBS in RPMI medium without phenol red. 1×10 per condition 5Cells were prepared and stained by allowing them to stand in an incubator under 5% CO2 conditions for 10 minutes at a concentration of 5 μM CFSE (manufactured by Life technologies). Subsequently, after washing the cells twice with DPBS, they were diluted with a culture solution supplemented with 10% FBS in RPMI medium without phenol red. Activated NK cells were prepared according to the E:T (Effector cell:Target cell) ratio (0:1, 1:1, 5:1, 10:1) with target cells, and dispensed and mixed together with the target cells in a 24-well plate. Next, the activated NK cells and target cells were reacted for 4 hours, and 7AAD (7-Aminoactinomycin D) was added to the cells 20 minutes before the end of the reaction. After the reaction ended, the cells were collected into a 5 ml FACS tube, and the cytotoxicity of the cells was analyzed by flow cytometry.

[0126] The results are shown in Figure 4 and Table 2.

[0127] As can be seen from Table 2, the NK cells of Example 1 showed 91% cytotoxicity against the K562 cell line, which is a blood cancer, at a ratio of 10:1, and 74% cytotoxicity against the A2780cis cell line, which is an anti-cancer drug-resistant ovarian cancer cell line. The activated primary NK cells of Comparative Example 3 and Comparative Example 4 showed 93.22% and 80.5% cytotoxicity, respectively, against the K562 cell line at a ratio of 10:1, and the primary NK cells of Comparative Example 2 showed 17.1% cytotoxicity against the K562 cell line at a ratio of 10:1. From the above results, it was found that the NK cells of Example 1 had similar or better cytotoxicity compared to the primary NK cells of Comparative Examples 2 to 4.

[0128] Also, as can be seen from Figure 4, it was shown that the cells of Example 1 had significantly higher cytotoxicity compared to the NK92MI cells of Comparative Example 5, and it was found that the cells of Example 1 had excellent cytotoxicity.

[0129]

Table 2

[0130] As a result, it was found that the NK cells of Embodiment 1 have characteristics superior to those of the other NK cells of Comparative Example 5 and have characteristics similar to or superior to those of the activated primary NK cells of Comparative Examples 3 and 4.

[0131] 3-2. SKOV3 cell line

[0132] The method for measuring the activity of NK cells (cytotoxicity assay) described in Experimental Example 3-1 was performed on SKOV3 cells (an ovarian cancer cell line). Specifically, activated NK cells and SKOV3 cells were reacted at an E:T ratio (0:1, 1:1, 5:1, 10:1) to analyze the killing ability of NK cells.

[0133] As a result, in the case of primary NK cells and NK92MI cells, the cytotoxic activity against the SKOV3 cell line was low, whereas the NK cells of Example 1 (ESC-NK) and iPSC-derived NK cells (CD34 + -derived NK cells) differentiated according to the method of the present invention showed very high cytotoxicity against the SKOV3 cell line, 65-82%, at an E:T ratio of 10:1 (Figure 5). Here, the CD34 + -derived NK cells are NK cells differentiated from iPSCs generated using CD34 + cells isolated from UCB by the method of Example 1. From such results, it can be seen that the NK cells differentiated from ESCs or iPSCs by the method of the present invention have cytotoxicity superior to that of other NK cells.

Claims

1. A kit for inducing differentiation of pluripotent stem cells into natural killer cells, a first medium composition for inducing differentiation of pluripotent stem cells into hematopoietic stem cells, comprising a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β inhibitor, PVA (polyvinyl alcohol), SCF (Stem Cell Factor) or a combination thereof, and a second medium composition for inducing differentiation of hematopoietic stem cells into natural killer cells, comprising SCF, IL7 (Interleukin7), IL15 (Interleukin15), FLT3L (FMS-like tyrosine kinase 3 ligand) or a combination thereof.

2. The kit according to claim 1, wherein the concentration of the GSK inhibitor in the first medium composition is 1 to 10 μM, the concentration of BMP4 is 10 to 100 ng / ml, the concentration of VEGF is 10 to 100 ng / ml, the concentration of bFGF is 10 to 200 ng / ml, the concentration of retinoic acid is 0.1 to 10 μM, the concentration of the TGF-β inhibitor is 1 to 100 μM, the concentration of PVA is 0.01 to 1% (w / v), and the concentration of SCF is 5 to 500 ng / ml.

3. The kit according to claim 1, wherein the concentration of SCF in the second medium composition is 1 to 50 ng / ml, the concentration of IL7 is 1 to 50 ng / ml, the concentration of IL15 is 1 to 50 ng / ml, and the concentration of FLT3L is 1 to 50 ng / ml.

4. The kit according to claim 1, wherein at least 90% of the total cell population of the differentiated natural killer cells is negative for CD3 and positive for CD56.

5. The kit according to claim 1, wherein the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

6. The kit according to any one of claims 1 to 5, wherein the first medium composition and the second medium composition are separately contained in the kit.

7. The kit according to claim 1, further comprising a third medium composition for the maturation and proliferation of natural killer cells, which contains IL15, a ROCK inhibitor, or a combination thereof.

8. The kit according to claim 7, wherein the concentration of the ROCK inhibitor in the third medium composition is 1 to 50 μM.

9. A first stage of inducing differentiation of pluripotent stem cells into hematopoietic stem cells by culturing pluripotent stem cells in the presence of a first medium composition containing a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β inhibitor, PVA (polyvinyl alcohol), SCF (Stem Cell Factor), or a combination thereof; A second stage of inducing differentiation of hematopoietic stem cells into natural killer cells by culturing hematopoietic stem cells in the presence of a second medium composition containing SCF (Stem Cell Factor), IL7 (Interleukin7), IL15 (Interleukin15), FLT3L (FMS-like tyrosine kinase 3 ligand), or a combination thereof; A method for inducing differentiation of pluripotent stem cells into natural killer cells, comprising:

10. The method according to claim 9, wherein the first stage is carried out for 5 to 20 days.

11. The method according to claim 9, wherein the second stage is carried out for 5 to 20 days.

12. The method according to claim 9, further comprising a third stage of maturing and proliferating natural killer cells by culturing natural killer cells in the presence of a third culture composition containing IL15, a Rock inhibitor, or a combination thereof.

13. The method according to claim 12, wherein the third stage is carried out for 5 to 20 days.

14. Natural killer cells or a cell population thereof differentiated from pluripotent stem cells by the method according to any one of claims 9 to 13.

15. A pharmaceutical composition for preventing or treating cancer, comprising natural killer cells or a cell population thereof differentiated from pluripotent stem cells by the method according to any one of claims 9 to 13 as an active ingredient.

16. The pharmaceutical composition according to claim 15, wherein the cancer is at least one selected from the group consisting of glioma, gastrointestinal stromal tumor, leukemia, breast cancer, uterine cancer, cervical cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, laryngeal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, fat cancer, fibroblast cancer, blood cancer, lymphoma, and multiple myeloma.

17. A pharmaceutical composition for preventing or treating infectious diseases, comprising natural killer cells or a cell population thereof differentiated from pluripotent stem cells by the method according to any one of claims 9 to 13 as an active ingredient.

18. A composition for inducing differentiation of pluripotent stem cells into natural killer cells, comprising A first culture medium composition for inducing differentiation of pluripotent stem cells into hematopoietic stem cells, comprising a GSK inhibitor, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, a TGF-β inhibitor, PVA (polyvinyl alcohol), SCF (Stem Cell Factor), or a combination thereof, and A composition comprising a second culture medium composition for inducing differentiation of hematopoietic stem cells into natural killer cells, comprising SCF (Stem Cell Factor), IL7 (Interleukin7), IL15 (Interleukin15), FLT3L (FMS-like tyrosine kinase 3 ligand), or a combination thereof.

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