Natural killer cells prepared from induced pluripotent stem cells, and preparation method and application thereof

Abstract

The present invention relates to natural killer cells which are prepared from induced pluripotent stem cells and proliferate in large quantities; and a pharmaceutical composition containing the natural killer cell as an active ingredient. The natural killer cells of the present invention exhibit oncolytic activity at cellular and animal levels. In addition, the expression of genes and cytokines involved in tumor death and immune cell activity is increased compared to natural killer cells isolated and activated from blood. It is also demonstrated that the method for induced differentiation of natural killer cells from induced pluripotent stem cells according to the present invention is a method for preparing a large number of cells in a short time using a simple method compared to a conventional induction method using induced pluripotent stem cells. Therefore, it can be believed that the method for induced differentiation of natural killer cells from induced pluripotent stem cells according to the present invention, and the natural killer cells prepared by the method can be used for treating various diseases including cancer.

Description

Technical Field

[0001] The present invention relates to natural killer cells prepared from induced pluripotent stem cells, a method for preparing the natural killer cells and uses thereof. Background Art

[0002] Natural killer cells (NK cells) are a type of cytotoxic lymphocyte that is very important for innate immunity. Natural killer cells are cytotoxic and can not only directly kill virus-infected cells, but also kill cancer cells. In general, immune cells detect the major histocompatibility complex (MHC) presented on the surface of infected cells, induce cytokine release, and induce infected cell death through cell lysis or apoptosis.

[0003] On the other hand, natural killer cells can recognize and destroy abnormal cells such as cancer cells without antibodies and MHC, so they can induce an immediate immune response. It is well known that natural killer cells can attack cancer cells, prevent the occurrence, proliferation and metastasis of cancer cells, and effectively control cancer stem cells that play an important role in cancer recurrence. Therefore, various studies are trying to use natural killer cells to treat cancer. Therefore, the technology of preparing a large number of allogeneic and autologous human natural killer cells has also become a key technology.

[0004] The main source of human natural killer cell resources is the method of isolating and proliferating natural killer cells from the blood. However, natural killer cells only account for about 10% of lymphocytes in the blood. In order to apply natural killer cells to various fields including anti-cancer therapy, it is necessary to proliferate natural killer cells in large quantities in vitro to achieve high yields, but it has not yet reached a level that can be applied clinically (Korean Patent Application Publication No. 10-2018-0117829). Recently, people are also actively studying the technology of preparing natural killer cells through differentiation induction culture from stem cells such as human hematopoietic stem cells, embryonic stem cells and induced pluripotent stem cells that have good differentiation potential. Summary of the invention

[0005] Technical issues

[0006] The present inventors attempted to develop a method for efficiently preparing human natural killer cells. The results confirmed that natural killer cells were prepared from isolated induced pluripotent stem cells (iPSCs), and the prepared natural killer cells were proliferated in large quantities, and their characteristics were analyzed. The results confirmed that the natural killer cells exhibited characteristics different from those of existing blood-derived natural killer cells. Based on the above, the present inventors completed the present invention.

[0007] Solutions to the problem

[0008] To achieve the above object, in one aspect of the present invention, provided are natural killer cells expressing any one protein selected from the group consisting of CD25, CD9, CD80 and a combination thereof; a pharmaceutical composition comprising the same as an active ingredient; and a method for preparing natural killer cells.

[0009] In another aspect of the present invention, there is provided use of natural killer cells in preventing or treating diseases.

[0010] In another aspect of the present invention, a method for preventing or treating a disease is provided, comprising: administering the natural killer cells.

[0011] Effects of the Invention

[0012] The natural killer cells of the present invention exhibit oncolytic activity at the cellular and animal levels. In addition, the expression of genes and cytokines involved in tumor death and immune cell activity is increased compared to natural killer cells isolated and activated from blood. In addition, compared to the conventional method of differentiating natural killer cells from iPSCs, the method of differentiating natural killer cells from iPSCs of the present invention can prepare a large number of cells in a short time. In addition, it has been confirmed that the activity of natural killer cells prepared by this method is excellent. Therefore, the method of differentiating natural killer cells from iPSCs of the present invention and the natural killer cells prepared by the method can be used to treat various diseases including cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of induced pluripotent stem cells (iPSCs) differentiating into natural killer cells in the presence of autologous feeder cells.

[0014] Figure 2 Methods for differentiating natural killer cells from induced pluripotent stem cells (iPSCs) in the presence of autologous feeder cells are outlined.

[0015] Figure 3 This is a schematic diagram of the results of confirming the morphological changes at each stage of iPSC differentiation into NK cells using an optical microscope.

[0016] Figure 4 The results were obtained by photographing the cell culture containers of each group, in which one group maintained autologous donor cells during the differentiation of iPSCs into NK cells, while the other group removed autologous donor cells after the hematopoietic stem cell differentiation stage.

[0017] Figure 5 This is a schematic diagram of the results of observing the differences between the groups using an optical microscope, in which one group maintained autologous donor cells during the differentiation of iPSCs into NK cells, while the other group removed autologous donor cells after the hematopoietic stem cell differentiation stage.

[0018] Figure 6 It is a schematic diagram of the results obtained by analyzing the number of natural killer cells (suspension cells) prepared at each differentiation stage, in which one group maintained autologous donor cells during the differentiation of iPSCs into NK cells, and the other group removed autologous donor cells after the hematopoietic stem cell differentiation stage.

[0019] Figure 7 Figure 1 is a schematic diagram of the results of flow cytometric analysis of major natural killer cell markers on cells differentiated into natural killer cells, in which one group maintained autologous donor cells during the differentiation of iPSCs into NK cells, while the other group removed autologous donor cells after the hematopoietic stem cell differentiation stage.

[0020] Figures 8a to 8c When differentiating iPSCs into natural killer cells, the number of iPSC colonies adhered to conditions 1 to 6 (8a), the total number of cells per condition / area of ​​adhered container (cm 2 )(8b), and a schematic diagram of the total number of iPSC colonies in each T-150 flask under each condition (8c).

[0021] Fig. 9 This is a schematic diagram showing the results of confirming the morphological changes of each cell differentiated in stages under conditions 1 to 6 when iPSCs were differentiated into NK cells using an optical microscope.

[0022] Fig.10 This is a graph showing the results of confirming the number of natural killer cells (suspension cells) differentiated under each condition.

[0023] Fig.11 This is a graph showing the results of analyzing the expression rates of cell surface proteins (markers) of differentiated natural killer cells under conditions 1 to 6 when iPSCs were differentiated into NK cells.

[0024] Fig.12 The diagram and graph are the results of analyzing the number of induced pluripotent stem cell colonies adhered to conditions 1 to 6 when iPSCs were differentiated into NK cells.

[0025] Fig.13 The images are the results of observing the morphological changes of the differentiated cells under different conditions (A to D) during the differentiation of iPSCs into NK cells using an optical microscope.

[0026] Fig.14 These are graphs showing the results of confirming the expression rates of cell surface proteins (markers) of differentiated natural killer cells under various conditions (A to D) by flow cytometry when iPSCs were differentiated into NK cells.

[0027] Fig.15Shows the results of flow cytometry analysis of cells at the mesodermal stage (differentiation day 4) during the differentiation of induced pluripotent stem cell colonies (iPSC colonies) into NK cells

[0028] Fig.16 The results of flow cytometric analysis of cells that differentiated into hematopoietic stem cells (differentiation day 12) after passing through the mesoderm cell stage during the differentiation of iPSC colonies into NK cells are shown.

[0029] Fig.17a and Fig.17b Shown are the results of flow cytometric analysis of cells differentiated from iPSC colonies through mesoderm and hematopoietic stem cell stages into NK cells (differentiation day 26).

[0030] Fig.18a and Fig.18b Shown are the results of flow cytometric analysis of cells differentiated from iPSC colonies through mesoderm and hematopoietic stem cell stages into NK cells (differentiation day 47).

[0031] Fig.19 Schematic diagram of the method for differentiating iPSCs into natural killer cells using Stempro34 medium.

[0032] Fig. 20 Shows the basis Fig.19 Flow cytometric analysis results of cells at the mesodermal cell stage (differentiation day 4) during the differentiation process from iPSC colonies to NK cells.

[0033] Fig.21 Shows the basis Fig.19 The results of flow cytometric analysis of cells that differentiated into hematopoietic stem cells (differentiation day 12) through the mesoderm cell stage during the differentiation process from iPSC colonies to NK cells.

[0034] Fig. 22 Shows the basis Fig.19 Results of flow cytometric analysis of cells that differentiated from iPSC colonies through mesoderm and hematopoietic stem cell stages into NK cells (differentiation day 26).

[0035] Fig.23 Shows the basis Fig.19 Results of flow cytometric analysis of cells that differentiated from iPSC colonies through mesoderm and hematopoietic stem cell stages into NK cells (differentiation day 30).

[0036] Fig.24 Shows the basis Fig.19 Results of flow cytometric analysis of cells that differentiated from iPSC colonies through mesoderm and hematopoietic stem cell stages into NK cells (differentiation day 34).

[0037] Fig.25 Shows the basis Fig.19 Results of flow cytometric analysis of cells that differentiated from iPSC colonies through mesoderm and hematopoietic stem cell stages into NK cells (differentiation day 42).

[0038] Fig.26 The results show that iPSC-derived NK cells (EiNK cells) were co-cultured with chronic myeloid leukemia cell line K562 cells to confirm the cytotoxicity.

[0039] Fig. 27 Schematic diagram of the feeder cell-based proliferation method of iPSC-derived NK cells (EiNK cells).

[0040] Fig.28 The graph shows the results of confirming the morphological changes of iPSC-derived NK cells (EiNK cells) at different time points during their massive proliferation using an optical microscope.

[0041] Fig.29 This is a graph showing the results of measuring the cell number at each time point during the massive proliferation of iPSC-derived natural killer cells (EiNK cells), along with the changes in cell growth rate and cell number.

[0042] Fig.30a and Fig.30b The graph shows the results of flow cytometry confirmation of NK cell activity after 20 days of massive proliferation of iPSC-derived NK cells (EiNK cells).

[0043] Fig.31 Comparison of the results of confirming the cytotoxicity of NK cells against tumor cells after 21 and 28 days of massive proliferation of iPSC-derived NK cells (EiNK cells).

[0044] Fig.32 This figure shows the results of confirming the expression of induced pluripotent stem cell marker proteins (TRA-1-60, SSEA4) by flow cytometry 21 days after iPSC-derived NK cells (EiNK cells) proliferated in large quantities.

[0045] Fig.33 The figure shows the results of confirming the cell proliferation rate of iPSC-derived NK cells (EiNK cells) during long-term culture.

[0046] Fig.34a , Fig.34b To confirm the expression levels of NK cell (CD3, CD45) or induced pluripotent stem cell (TRA-1-60, SSEA4) marker proteins ( Fig.35a, cultured for 63 days); and the tumor cell killing ability of iPSC-derived NK cells (EiNK) after different culture periods (20 days, 56 days and 63 days) ( Fig.35b ) result graph.

[0047] Fig.35a and 35b The graph shows the results of confirming the expression levels of cell surface proteins in iPSC-derived NK cells (EiNK cells, cultured for 21 days) and blood-derived NK cells (PBNK cells, cultured for 22 days) by flow cytometry.

[0048] Fig.36 The results show that the expression of genes related to tumor killing and NK cell activation in iPSC-derived NK cells (EiNK cells, cultured for 28 days) and blood-derived NK cells (PBNK cells, cultured for 22 days) was confirmed.

[0049] Fig.37a and 37b The results show that by measuring cytokines in NK cell culture medium of iPSC-derived NK cells (EiNK cells, cultured for 21 days) and blood-derived NK cells (PBNK cells, cultured for 21 days), cytokines whose concentrations in the culture medium of EiNK cells were 2 times or more (37a), or 1.3 to 1.9 times or more (37b) were selected.

[0050] Fig.38 The graph shows the results of confirming the expression levels of natural killer cell inhibitory receptors in iPSC-derived NK cells (EiNK cells, cultured for 21 days) and blood-derived NK cells (PBNK cells, cultured for 21 days) by flow cytometry.

[0051] Fig.39 The figure shows the results of flow cytometry to confirm the ability of iPSC-derived NK cells (EiNK cells, cultured for 21 or 28 days) and blood-derived NK cells (PBNK cells, cultured for 16 days) to kill tumor cells.

[0052] Figures 40a to 40e Figures 40a to 40d show the results obtained by administering an ovarian cancer (OVCAR3) cell line expressing luciferase to NSG mice, then treating them with iPSC-derived NK cells (EiNK cells, cultured for 21 days), and confirming the luciferase activity in mice at different time periods, as well as Figure 40e show the results obtained by quantifying the above activity.

[0053] Fig.41 This graph shows the results of confirming the survival rate of mice after NSG mice were administered with an ovarian cancer (OVCAR3) cell line expressing luciferase and then treated with iPSC-derived NK cells (EiNK cells, cultured for 21 days).

[0054] Figures 42a to 42c To confirm the cell yield obtained after CD34 sorting ( Fig.42a ), cell death( Fig.42a ), expression of NK cell markers (or activation markers) ( Figure 42b ) and cell proliferation rate ( Fig.42c ) Schematic diagram of the results obtained, Fig.42d This figure shows the results of confirming the expression of hematopoietic stem cell marker proteins (CD34 / CD45) during EiNK cell differentiation by flow cytometry without isolating the CD34 marker in one embodiment of the EiNK cell differentiation method of the present invention.

[0055] Fig.43 This is the result of measuring the size of embryoid bodies (EBs) formed in each culture cycle when iPSCs were cultured for the third time using the traditional EiNK cell preparation method.

[0056] Fig.44 This is a graph showing the results of batch measurement of the survival rate of isolated cells after cells were isolated from embryoid bodies (EBs) formed by the third culture of iPSCs using the traditional EiNK cell preparation method.

[0057] Fig.45 This is a graph showing the results obtained by comparing the cycle required for a conventional EiNK cell preparation method and the cycle required for an EiNK cell preparation method according to an embodiment of the present invention.

[0058] Fig.46 This is a graph showing the results of confirming by flow cytometry the expression levels of marker proteins of natural killer cells (CD56, CD45, NKp30, NKp44) and induced pluripotent stem cells (TRA-1-60, SSEA4) in EiNK cells prepared according to the method for preparing EiNK cells according to one embodiment of the present invention. DETAILED DESCRIPTION

[0059] Natural Killer Cells

[0060] In one aspect of the present invention, a natural killer cell expressing any one protein selected from the group consisting of CD25, CD9, CD80 and a combination thereof is provided. The natural killer cell may additionally express any one protein selected from the group consisting of CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, CD210 and a combination thereof.

[0061] The term "natural killer cell (NK cell)" as used herein is a type of cytotoxic lymphocyte, a major component of the innate immune system, defined as a large granular lymphocyte (LGL), which is a third type of cell differentiated from B lymphocytes and T lymphocytes produced by common lymphocyte progenitor cells (CLP). It is a type of lymphocyte that plays a role in immune response, accounting for about 10-15% in normal human blood, and has a strong killing ability when reacting with non-self. Natural killer cells can respond non-specifically and immediately to cells infected with various viruses, bacterial invasion, or the generation of abnormal cells to eliminate foreign substances. Natural killer cells are activated under the action of interferon or macrophage-derived cytokines, and natural killer cells contain two extracellular receptors that control cytotoxic activity, represented by "activation receptors" and "inhibition receptors", respectively.

[0062] The term "CD25" as used herein is the alpha subunit of the interleukin-2 receptor, which is known to regulate the activity of regulatory T cells (Tregs), inhibit the activity and proliferation of activated T cells, and thus regulate the immune response.

[0063] The term "CD80 (cluster of differentiation 80)" used in this article is also called B7-1. It is a cell surface protein that belongs to the B7 family of the immunoglobulin superfamily. CD80 consists of an Ig-like extracellular domain (a variable region and a constant region), a transmembrane region, and a cytoplasmic domain. The co-stimulatory ligand CD80 is expressed on antigen presenting cells (APCs) such as monocytes, dendritic cells, and activated B cells and T cells. CD80 binds to CD28 or CTLA-4 (cytotoxic T lymphocyte antigen-4) to regulate cell proliferation and IL-2 production. When CD80 binds to CD28, it activates T cells and promotes immune responses; when it binds to the immune checkpoint protein CTLA-4, it reduces the activity of T cells and inhibits immune responses.

[0064] The term "CD9" as used herein is a protein belonging to the transmembrane protein 4 superfamily, also known as the tetraspanin superfamily, which is involved in sperm-egg fusion associated with integrins, platelet activation and aggregation, cell adhesion and cell migration.

[0065] The term "CDld" as used herein is an antigen presenting protein that binds to self and non-self glycolipids and presents them to the T cell receptor of natural killer T cells (NKT cells).

[0066] The term "CD30" as used herein, also known as TNFRSF8, is a protein expressed in activated T cells and B cells. It is known to induce apoptosis by binding to TRAF or TRAF5 and activating the NF-κB signaling pathway. In addition, it suppresses autoimmunity by inhibiting the proliferation and activity of self-activated cytotoxic T cells.

[0067] The term "CD87" as used herein is also known as urokinase receptor or urokinase plasminogen activator surface receptor (uPAR), and plays a role in promoting the formation and localization of plasmin. Plasmin is a serine protease that has the function of dissolving fibrin clots. Cell surface-bound thrombin is known to promote ECM degradation by activating MMPs that degrade collagen and other proteins in the vessel wall, and is involved in the migration of neutrophils, monocytes, and macrophages.

[0068] The term "CDw327" as used herein is also known as SIGLEC6 (sialic acid-binding Ig-like lectin 6) or CD33LSIGLEC6, which is a cell adhesion protein involved in sialic acid-dependent cell binding.

[0069] The term "CD33" as used herein is also known as siglec-3, which is a receptor expressed on myeloid cells. CD33 binds to any molecule containing sialic acid residues, such as glycoproteins or glycolipids, and upon activation, induces a signaling pathway that inhibits phagocytosis.

[0070] The term "CD49a" as used herein is integrin alpha-1, which is a subunit of the integrin alpha1 beta1 receptor.

[0071] The term "CD49c" as used herein is integrin alpha-3, a subunit of the integrin alpha3beta1 receptor, involved in neural migration and cortical formation.

[0072] The term "CD49e" as used herein is integrin α-5, a subunit of the integrin α5β1 receptor. α5 is post-translationally cleaved in the extracellular domain and binds to β1 to form the fibronectin receptor.

[0073] The term "CD49f" as used herein is integrin α-6, which binds to integrin β4, called TSP180, or to β1 of integrin VLA-6, thereby mediating cell-cell adhesion and cell signaling.

[0074] The term "CD66a" used herein is also called CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), which belongs to the CEA (carcinoembryonic antigen) superfamily. This protein is both an inhibitory receptor and an activating receptor. For example, it acts as an inhibitory receptor in immune response and insulin action, and has the function of promoting angiogenesis. CEACAM1 is known to inhibit immune response by inhibiting T cell proliferation and cytokine production, inhibiting the activity of natural killer cells, and inhibiting neutrophil secretion of IL-1β.

[0075] The term "CD66c" used herein is also called CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6), which belongs to the CEA (carcinoembryonic antigen) superfamily. It is known to be involved in cell adhesion and tumor formation / progression.

[0076] The term "CD66d" as used herein is also known as CEACAM3 (carcinoembryonic antigen-related cell adhesion molecule 3), which belongs to the CEA (carcinoembryonic antigen) superfamily. It is expressed on granulocytes and is known to regulate phagocytosis of pathogens in innate immunity.

[0077] The term "CD66e" as used herein is also known as CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5), which belongs to the CEA (carcinoembryonic antigen) superfamily. It regulates cell adhesion, cell signaling, and tumor formation / progression.

[0078] The term "CD71" as used herein is also known as TFR1 (transferrin receptor protein 1), which is a protein involved in the entry of ions into cells via transferrin. It is known to be expressed only in normal hematopoietic bone marrow and spleen erythroid precursors.

[0079] The term "CD83" used herein is used as a marker protein for mature dendritic cells. It is known that it stabilizes the expression of MHC II, costimulatory molecules and CD28 on the membrane of dendritic cells by inhibiting the activity of E3 ubiquitin ligase.

[0080] The term "CDw93" as used herein is also referred to as C1q binding protein (C1qRp). Cdw93 is expressed in monocytes (macrophages) and neutrophils, but not in T cells and B cells. Cdw93 binds to C1q, the recognition subunit of the first component (C1) of the complement pathway, and promotes C1q-mediated phagocytosis. CDw93 is known to be highly expressed in stem cells (precursors) that will differentiate into hematopoietic cells and hepatocytes.

[0081] "CD106" as used herein is also referred to as VCAM-1 (vascular cell adhesion molecule 1), which mediates the adhesion of lymphocytes, monocytes, eosinophils and basophils to the vascular endothelium. In addition, it also mediates the interaction between leukocytes and endothelial cells through signal transduction. CD106 is the endothelial ligand of VLA-4 (delayed antigen-4 or integrin α4β1).

[0082] The term "CD108" used herein refers to SEMA7A (Semaphorin 7A), also known as the JMH (John-Milton-Hagen) blood group antigen. It is expressed on activated lymphocytes and erythrocytes, and is also present in the esophagus, heart, brain, spleen, testicles, lungs, ovaries, and uterus. It plays an important role in integrin-mediated signaling and regulates cell migration and immune responses.

[0083] The term "CD114" used herein is called GCSFR (granulocyte colony stimulating factor receptor), which is a cell surface receptor for GCSF. It is a type 1 cytokine receptor belonging to the hematopoietic receptor superfamily, expressed on progenitor cells present in the bone marrow, thereby mediating the proliferation and differentiation of progenitor cells into mature neutrophils and macrophages.

[0084] The term "CD123" as used herein is an interleukin-3 receptor that is expressed on basophils and dendritic cells and mediates immune responses through IL-3.

[0085] The term "CD124" used herein is an interleukin-4 receptor, which belongs to type 1 cytokine receptor. It regulates immune response by binding to IL-4 and IL-13, mediating the production of IgE antibodies, and promoting Th2 cell differentiation.

[0086] The term "CD146" used herein is called MCAM (melanoma cell adhesion molecule) and is used as a marker protein for the endothelial cell lineage. It is a receptor for laminin α4 and is expressed in large quantities in cells that constitute the vascular wall, such as vascular endothelial cells, smooth muscle cells, and perivascular cells. It is also known to be expressed in certain subsets of T cells and B cells.

[0087] The term "CD150" used herein is referred to as SLAMF1 (signaling lymphocyte activation molecule 1), which mediates interactions between homotypic or heterotypic cells, thereby regulating various immune cell activities and differentiation.

[0088] The term "CD164" used herein is also referred to as endolytic dimer protein or sialomucin core protein 24. Sialomucin is a protein involved in both cell adhesion inhibition and cell adhesion, while CD164 is a protein involved in cell adhesion.

[0089] The term "CD196" used in this article is also called chemokine receptor 6 (CCR6), which belongs to the G protein-coupled receptor superfamily. It is expressed in B cells, T cells (Th1, Th2, Th17, Treg), natural killer T cells (NKT cells), immature dendritic cells and neutrophils. It regulates immune responses by binding to CCL20 (MIP3α), mediating the maturation and differentiation of B cells, and mediating the migration of dendritic cells and T cells.

[0090] The term "CD210" used herein is a receptor for interleukin-10, which belongs to type 2 cytokine receptors. It consists of α and β subunits, and the α subunit is expressed in hematopoietic cells such as T cells, B cells, natural killer cells, mast cells and dendritic cells. On the other hand, the expression of the β subunit is mainly regulated by ubiquitin. The α subunit is the ligand binding site of the IL-10 receptor and is only present in the IL-10 receptor, but the β subunit also binds to the subunits of other type 2 cytokine receptors to form a receptor. It is known that the IL-10 receptor mainly mediates gastrointestinal immune responses in the mucosal layer.

[0091] In the present invention, the expression levels of the above-mentioned CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196 and CD210 proteins may be increased compared to natural killer cells isolated from blood. Specifically, compared with natural killer cells isolated from blood, the expression levels of the above-mentioned CD25, CD9, CD80, CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, and CD210 proteins can be increased by at least two times.

[0092] In this case, the natural killer cells separated from the blood can be from mammals, such as rats, mice, livestock and humans. The natural killer cells separated from the blood can be natural killer cells separated from the blood without additional modification, or can be natural killer cells induced and activated. Preferably, they are natural killer cells induced and activated.

[0093] The term "activated natural killer cells" used herein may refer to natural killer cells with enhanced cytotoxicity or enhanced natural killer cell intrinsic immunoregulatory capacity compared to natural killer cells isolated from blood without additional modification. The activated natural killer cells may overexpress receptors targeting cells or tissues to be treated.

[0094] The method of activating natural killer cells can be to culture natural killer cells separated from blood under specific conditions, genetically modify them, or treat them with substances that can enhance the activity of natural killer cells. In this case, the natural killer cells can use conventional animal cell culture media known in the art without limitation, for example, CellGro medium (Cellgenix), AIM-V medium, XVIVO 20, RPMI1640, etc.

[0095] The term "substance that increases the activity of natural killer cells" as used herein refers to any substance that can increase the activity of natural killer cells, such as increasing the cytotoxicity of natural killer cells, or generating, increasing or proliferating activated natural killer cells. The substance that increases the activity of natural killer cells may include immune cytokines, but is not limited thereto, as long as it is a substance that can increase the activity of natural killer cells.

[0096] The term "cytokine" as used herein refers to a signaling substance that controls the human defense system and stimulates the human body, and may also refer to a protein that regulates physiological activities. The cytokine regulates various biological activities, such as cell activation, differentiation, cell migration, senescence, and induction of apoptosis of various cells through autocrine and paracrine effects.

[0097] The cytokine can be of the interferon (IFN) family, the interleukin family, the tumor necrosis factor (TNF) family, or a combination thereof.

[0098] The interferon can be any one selected from the group consisting of type 1 interferon (e.g., interferon-α, interferon-β, interferon-κ, interferon-ω), type 2 interferon (e.g., interferon-γ), type 3 interferon, and combinations thereof. Interleukin can be any one selected from the group consisting of IL-2, IL-5, IL-8, IL-12, IL-15, IL-18, IL-21, and combinations thereof. In this case, TNF may include TNF-α, TNF-β, or TNF-γ, etc. The immunocytokine may include a cytokine-antibody complex. For example, it may include an interleukin-anti-interleukin antibody complex.

[0099] Specifically, the activated natural killer cells of the present invention can be obtained by culturing blood-derived natural killer cells for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or 22 days.

[0100] In addition to the above proteins, the natural killer cells of the present invention may also express any one natural killer cell marker protein selected from the group consisting of CD56, CD45, CD16, NKp30, NKp44, NKG2D, DNAM1 and a combination thereof, and may not express CD3, a marker protein of T cells, or express CD3 at a level lower than that of T cells. Here, the activation of natural killer cells is as described above, and whether the natural killer cells are activated can be assessed by detecting one or more function-related markers, such as CD56, CD45, CD16, NKp30, NKp44, NKG2D, DNAM1, etc.

[0101] In addition, the natural killer cells of the present invention may increase the expression of genes related to the tumor killing ability of natural killer cells and natural killer cell activation compared to natural killer cells isolated from blood. Specifically, the natural killer cells of the present invention may be characterized by an additional increase in the expression of any one gene selected from the group consisting of GZMB, CD226, RASGRP1, CD107a, CCR9, CD40LG, CD300c and a combination thereof compared to natural killer cells isolated from blood. In this case, the natural killer cells obtained from blood are as described above.

[0102] The term "GZMB (Granzyme B)" as used herein is one of the serine proteases expressed in cytotoxic T cells and natural killer cells. T cells and natural killer cells induce apoptosis of target cells through granzyme B.

[0103] The term "CD226" as used herein is an immunoglobulin-like transmembrane glycoprotein known as PTA1 (Platelet and T-cell Activation Antigen 1) or DNAM-1 (DNAX Accessory Molecule-1). CD226 is expressed on natural killer cells, natural killer T cells, B cells, dendritic cells, hematopoietic stem cells, platelets, monocytes and T cells. It binds to CD112 or CD155 and participates in cell adhesion. It mediates signal transduction of lymphocytes and regulates lymphokine secretion and cytotoxic effects of natural killer cells and cytotoxic T cells. In addition, it regulates the activation and aggregation of platelets, as well as the transendothelial migration of natural killer cells.

[0104] The term "RASGRP1 (RAS guanylate releasing protein 1)" as used herein is a protein belonging to the Ras superfamily, contains a guanine nucleotide exchange factor (GEF) domain, and regulates the development, homeostasis and differentiation of T cells and B cells.

[0105] The term "CD107a" used herein is referred to as LAMP-1 (lysosomal associated membrane protein 1 or lysosomal associated membrane glycoprotein 1), LGP120 and LAMPA, and is a degranulation marker for cytotoxic T cells and natural killer cells. It mediates the migration of cytotoxic granules to the cell surface and the adhesion of perforin to cytotoxic granules, thereby regulating the cytolysis of target cells. In addition, it can also protect natural killer cells from damage caused by the degranulation process.

[0106] The term "CCR9" used herein is a receptor for CCL25, also known as CDw199. It is known that CCR9 is involved in cancer proliferation and inflammatory diseases by binding to CCL25. The interaction of CCR9 / CCL25 promotes the proliferation and metastasis of tumor cells, inhibits cell death, and thus promotes the progression of cancer. In addition, it is known that it promotes the occurrence of inflammatory diseases (inflammatory bowel disease, cardiovascular disease, rheumatoid arthritis, asthma, etc.).

[0107] The term "CD40LG (or CD40L)" as used herein is also called CD154, and is a protein expressed on T cells. CD40LG binds to CD40 expressed on antigen presenting cells and is a co-stimulatory molecule that activates immune cells.

[0108] The term "CD300c" used herein is also referred to as CMRF-35 or CMRF-35A. The B7 family is a member of the Ig superfamily and is known to be expressed on the surface of antigen presenting cells such as B cells, macrophages and dendritic cells. Expression or inhibition of the activity of CD300c protein induces T cell activation and / or promotes the differentiation of M1 macrophages.

[0109] The natural killer cells of the present invention may be characterized by an increased secretion of any one protein selected from the group consisting of FLT3L, MIP1alpha / beta, PF4, IGFBP2, IGFBP3, MMP9, and a combination thereof, compared to natural killer cells isolated from blood. In this case, the protein is a secretory protein secreted outside the cell, and the secretion amount in the natural killer cells of the present invention may be increased by at least two times compared to natural killer cells isolated from blood. In this case, the natural killer cells obtained from blood are the same as described above.

[0110] The term "FLT3L (FMS-like tyrosine kinase 3 ligand)" used in this article refers to the protein encoded by the human FLT3LG gene. It is structurally homologous to stem cell factor (SCF) and colony stimulating factor 1 (CSF-1), and works with multiple regulatory factors to promote the proliferation and differentiation of hematopoietic stem cells.

[0111] The term "MIP1 (macrophage inflammatory protein 1)" used herein is a cc motif chemokine that has two forms in the human body, namely MIP-1α and MIP-1β. MIP-1α and MIP-1β are also known as CCL3 and CCL4, respectively, and are secreted by activated macrophages and monocytes. CCL3 is a ligand for CCR1, and CCL4 is a ligand for CCR1 and CCR5, thereby activating monocytes, T cells, dendritic cells, NK cells, platelets, etc.

[0112] The term "PF4 (platelet factor 4)" used herein is also called CXC4 (chemokine CXC ligand 4), which is a ligand of CXCR3-B. PF4 is released from the α granules of activated platelets during platelet aggregation and promotes blood coagulation.

[0113] The term "IGFBP2 (insulin-like growth factor binding protein 2)" as used herein is a protein that can bind to the IGF protein with high affinity. It inhibits the binding of IGF to the IGF receptor (IGFR) through the binding of IGFBP2 and IGF, thereby inhibiting cell proliferation and growth through the IGF / IGFR interaction.

[0114] The term "IGFBP3" used herein is a protein that can bind to IGF-1 or IGF-2 with high affinity. IGFBP3 is known to bind to IGF in the blood and play a role in prolonging the transport and half-life of IGF.

[0115] The term "MMP9 (matrix metallopeptidase 9)" used herein is also known as type 4 collagenase or gelatinase B (GELB), and has a molecular weight of 92 kDa. MMP9 is induced to express by inflammatory stimuli and promotes cell migration by degrading the matrix.

[0116] The natural killer cells of the present invention may be natural killer cells that do not express any protein selected from the group consisting of CD205, CD158b, CD243, γδTCR, NKB1, D158a, CDw328, TIM3, TIGIT, and a combination thereof, or that express it at a lower level than natural killer cells obtained from blood. Specifically, the natural killer cells may be natural killer cells that express any protein selected from the group consisting of CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM3, TIGIT, and a combination thereof at a level of at most 1 / 2 compared to natural killer cells obtained from blood. In this case, the natural killer cells obtained from blood are the same as described above.

[0117] The term "CD205" as used herein, also known as lymphocyte antigen 75 or DEC-205, is a receptor involved in the endocytosis of extracellular captured antigens. It is known to reduce the proliferation of B cells.

[0118] The term "CD158b" used herein is referred to as KIR2DL2 / DL3 (Killer Cell Immunoglobulin-like Receptor 2DL2 / DL3) or two Ig domains and long cytoplasmic tail 2, which is a receptor for HLA-C (HLA-Cw1, HLA-Cw3, HLA-Cw7) present in natural killer cells. This protein is known to inhibit the activity of natural killer cells and inhibit the lysis of cancer cells by natural killer cells.

[0119] The term "CD243 (cluster of differentiation 243)" used herein, also known as P-gp (permeability glycoprotein, P-glycoprotein 1, Pgp), MDR1 (multidrug resistance protein 1) or ABCB1 (ATP binding cassette subfamily B member 1), is a cell membrane protein that exports foreign substances (toxins or drugs) from the cell to the outside of the cell in an ATP-dependent manner. It is widely distributed in intestinal epithelial cells and is known to be highly expressed in certain cancer cells and leads to resistance to anticancer drugs.

[0120] The term "γδTCR" as used herein is a T cell antigen receptor (TCR) expressed on γδT cells. Although the antigen receptor (αβTCR) of αβT cells can recognize peptide antigens presented by major histocompatibility complex molecules (pMHC) on antigen presenting cells (APCs), it is known that γδTCR can also partially recognize stress-induced self-antigens, lipids or pyrophosphates secreted in a restricted manner by certain microorganisms or overproduced by tumor cells.

[0121] The term "NKB1" as used herein is also known as KIR3DL1 (Killer cell immunoglobulin-like receptor 3DL1), NKAT3 or MHC class I NK cell receptor. It is expressed on natural killer cells and is a receptor for HLA-Bw4. It is known to inhibit the activity of natural killer cells and inhibit the lysis of tumor cells by natural killer cells.

[0122] The term "CD158a" used herein is also known as KIR2DS1 (Killer Cell Immunoglobulin-like Receptor 2DS1), which is a receptor for HLA-C (HLA-Cw6) present in natural killer cells. Unlike CD158b, which inhibits the activity of natural killer cells, this protein does not inhibit the activity of natural killer cells.

[0123] The term "CDw328" as used herein, also known as SIGLEC7 (sialic acid-binding Ig-like lectin 7), is a cell adhesion protein involved in sialic acid-dependent cell binding.

[0124] The term "TIM-3 (T cell immunoglobulin mucin-3)" used herein, also known as HAVCR2 (hepatitis A virus cell receptor 2), is an immune checkpoint protein that inhibits immune responses. It is expressed in a variety of tumor cells, including T cells, monocytes, macrophages, dendritic cells, mast cells, and natural killer cells. The ligands of TIM-3 are known to be galectin-9, PtdSer (phosphatidylserine), HMGB1 (high mobility group protein 1), and CEACAM1.

[0125] The term "TIGIT (T cell immunoreceptor with Ig and ITIM domains)" used herein is also known as WUCAM and Vstm2, and is an immune receptor present on certain T cells and natural killer cells. It binds to CD155 present on cells such as dendritic cells and macrophages, inhibiting the activity of T cells and natural killer cells in the body, thereby reducing the immune response.

[0126] In the present invention, natural killer cells can be induced to differentiate from induced pluripotent stem cells. Specifically, the natural killer cells of the present invention can be prepared by a method for preparing natural killer cells, comprising: inoculating induced pluripotent stem cells (iPSCs) into a cell culture container so that 0.5 to 500 cells are formed per 1 square centimeter of the cell culture container, and performing adhesion culture to prepare iPSC colonies; differentiating the iPSC colonies into mesodermal cells to prepare mesodermal cells; differentiating the mesodermal cells into hematopoietic stem cells to prepare hematopoietic stem cells; differentiating the hematopoietic stem cells into natural killer cells to prepare natural killer cells.

[0127] Furthermore, in the present invention, natural killer cells can be prepared by culturing and proliferating induced pluripotent stem cells in large quantities.

[0128] Specifically, the natural killer cells of the present invention can be induced to differentiate from induced pluripotent stem cells through the following steps.

[0129] First, the method comprises seeding induced pluripotent stem cells (iPSCs) or iPSC spheroids into a cell culture medium. In the container, 0.5 to 20 colonies are formed per 1 cm2 of the cell culture container area, and adhesion culture is performed to prepare iPSC colonies.

[0130] The term "stem cell" as used herein is a relatively undifferentiated cell that has not yet developed, and refers to a cell that has the ability to differentiate into a specific tissue cell under appropriate conditions. The stem cell can self-renew, produce daughter cells that can maintain their own undifferentiated characteristics, and at the same time produce daughter cells that differentiate into specific types of cells. The marker proteins of the stem cell include Lgr5, Lrp4, Msi-1, Sca-1, SCF, SSEA-4, Tra-1-60, Tra-1-81, etc. The stem cell includes embryonic stem cells, adult stem cells and induced pluripotent stem cells.

[0131] The term "induced pluripotent stem cell (iPSC)" as used herein refers to a cell produced by inducing somatic cells that do not have pluripotency to dedifferentiate into cells with pluripotency (such as embryonic stem cells). The induced pluripotent stem cell may also be referred to as a dedifferentiated stem cell. The induced pluripotent stem cell may be prepared from somatic cells of a fetus, a newborn, a child or an adult. The induced pluripotent stem cell may be derived from fibroblasts, keratinocytes, blood cells or kidney epidermal cells. The induced pluripotent stem cell may be prepared by somatic cell reprogramming. The method for preparing induced pluripotent stem cells by somatic cell reprogramming may use a known method, such as the method described in Takahashi K, Yamanaka S (2006), Cell. 126: 663-676.

[0132] The term "differentiation" as used herein refers to the phenomenon that the structure or function of a cell becomes specialized when the cell divides, proliferates, and the entire organism grows. In other words, the term refers to the process by which cells, tissues, etc. of an organism change so that they have a morphology and function suitable for playing their respective roles. In the present invention, the above term includes the process of inducing pluripotent stem cells to transform into natural killer cells, and may also include the process by which progenitor cells express specific differentiation characteristics.

[0133] The number of cells or spheroids seeded above may be 0.5 to 20 per 1 cm2, specifically, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 iPSCs or iPSC spheroids per 1 cm2.

[0134] In this case, the iPSC spheroid may contain about 30 to about 600 iPSCs. In addition, the iPSC spheroid may contain about 50 to about 300 iPSCs. In addition, the iPSC spheroid may contain about 70 to about 200 iPSCs. In addition, the iPSC spheroid may contain about 80, about 90, about 100 or about 120 iPSCs.

[0135] Alternatively, another method can be used to calculate the number of inoculum cells. The number of cells present per milliliter of culture medium can be determined as the number of cells per square centimeter using the method for calculating the number of inoculum cells.

[0136] Specifically, the number of induced pluripotent stem cells seeded per 1 ml of culture solution may be about 0.5 to about 2500. In addition, the number of cells may be about 1 to about 2500, about 10 to about 2500, about 20 to about 2500, about 30 to about 2500, about 40 to about 2500, about 50 to about 2500, about 60 to about 2500, about 70 to about 2500, about 80 to about 2500, about 90 to about 2500, about 100 to about 2500, about 200 to about 2500, about 300 to about 2500, about 400 to about 2500, about 500 to about 2500, about 600 to about 2500, about 700 to about 2500, about 800 to about 2500, about 900 to about 2500, or about 1000 per 1 ml. Preferably, the number of cells may be from about 250 to about 2500.

[0137] On the differentiation initiation day, the total number of induced pluripotent stem cells seeded using the above method can be about 0.5 to about 1×10 per 1 cm2 of the cell culture container area. 7 Specifically, the total number of induced pluripotent stem cells seeded using the above method on the differentiation initiation day can be about 1 to about 5×10 per 1 square centimeter of the cell culture container area. 6 pcs, about 10 to about 2×10 6 pcs, about 1×10 2 About 1×10 6 pcs, about 1×10 3 About 5×10 5 or about 1×10 4 About 1×10 5 Preferably, on the differentiation start day, the total number of induced pluripotent stem cells seeded using the above method can be about 1×10 per 1 square centimeter of the cell culture container area. 3 About 1×10 6 indivual.

[0138] The term "culture" as used herein refers to culturing cells under appropriately controlled environmental conditions. The culture process of the present invention can be carried out in an appropriate culture medium and under appropriate culture conditions known in the art. A person of ordinary skill in the art can easily adjust and use this culture process according to the conditions of the selected cells. The above-mentioned "culture medium" refers to a known culture medium for culturing cells, including all known cell culture media or their modified culture media.

[0139] The iPSCs may be cultured in a medium containing a ROCK inhibitor. The ROCK inhibitor may be selected from the group consisting of Y-27632, Thiazoleviven, Fasudil, GSK429286A, RKI-1447, H-1152, and Azaindole 1. The iPSCs may be cultured in a medium containing a ROCK inhibitor for about 10 minutes to about 1 week, about 30 minutes to about 6 days, about 1 hour to about 5 days, about 2 hours to about 4 days, about 3 hours to about 3 days, about 6 hours to about 2 days, about 12 hours to about 2 days, or about 18 hours to about 1 day.

[0140] The iPSC can be cultured using an adhesion culture method. The single-cell iPSC can be cultured for about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days after adhering to the cell culture container.

[0141] The iPSC colony may be a cell group that proliferates after single-cell induced pluripotent stem cell adhesion culture. The iPSC colony may be cells with the same genetic information.

[0142] Next, the method comprises differentiating the iPSC colonies into mesodermal cells to prepare mesodermal cells.

[0143] In this case, the number of colonies on the starting day of differentiation of the iPSC colony into mesoderm cells may be 1 to 20. In addition, the total number of iPSC cells on the starting day of differentiation of the iPSC colony into mesoderm cells may be 0.5 to 1×10 per 1 square centimeter of the cell culture container area. 6 In this case, the total number of stem cells on the differentiation start day is as described above. Preferably, the total number of iPSC cells on the differentiation start day of the iPSC colony to mesoderm cells can be 1×10 per 1 square centimeter of the cell culture container area. 3 Up to 1×10 6 indivual.

[0144] The term "mesoderm" as used herein refers to a group of layered cells that exist between the ectoderm and endoderm produced during gastrulation and can be found in higher animals than flatworms. The mesoderm cells can differentiate into blood cells; vascular endothelial cells; muscles, including smooth muscle and cardiac muscle; connective tissues, such as bone, cartilage and fat.

[0145] In this case, the size of each colony of the iPSC may be about 10 μm to about 3000 μm.

[0146] Specifically, the size of each colony of the iPSC may be about 10 μm to about 3000 μm, about 50 μm to about 2500 μm, about 100 μm to about 2000 μm, about 300 μm to about 1200 μm, about 400 μm to about 800 μm, or about 500 μm to about 700 μm. Preferably, the size of each colony of the iPSC may be about 300 μm to 1200 μm, but is not limited thereto.

[0147] In addition, the iPSC colonies can be cultured in a serum-free medium. The iPSC colonies can be cultured in a medium capable of culturing stem cells.

[0148] The iPSC colony can be cultured in a medium containing at least one substance selected from the group consisting of a glycogen synthase kinase 3 (GSK3) inhibitor, an activin-like receptor kinase 5 (ALK5) inhibitor, a bone morphogenetic protein 4 (BMP4), a stem cell factor (SCF) and a vascular endothelial growth factor (VEGF). The GSK3 inhibitor can be selected from the group consisting of CHIR-99021, SB216763, AT7519, CHIR-98014, TWS119, Tideglusib, SB415286 and BIO (6-bromoindirubin-3-oxime, 6-bromoindirubin-3'-oxime). The ALK5 inhibitor can be selected from the group consisting of SB431542, Galunisertib, LY2109761, SB525334, SB505124, GW788388 and LY364947.

[0149] The iPSC colony can be cultured in a medium containing CHIR-99021, BMP4 and VEGF, and then cultured in a medium containing SB431542, SCF and VEGF. The iPSC colony can be cultured in a medium containing CHIR-99021, SB431542, BMP4, SCF and VEGF.

[0150] The iPSC colonies can be cultured in the above culture medium for about 1 hour to about 2 weeks, about 12 hours to about 2 weeks, about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days. Preferably, the iPSC colonies can be cultured for about 2 days to about 4 days, but are not limited thereto.

[0151] The mesodermal cells may be mesodermal stem cells.

[0152] Third, the method comprises differentiating the mesodermal cells into hematopoietic stem cells to prepare hematopoietic stem cells.

[0153] The term "hematopoietic stem cell (HSC)" as used herein can be used interchangeably with "hematopoietic precursor cell" or "hematopoietic progenitor cell", and refers to a progenitor cell that can differentiate into a hematopoietic cell. That is, hematopoietic stem cells (HSC) refer to multipotent stem cells that can produce all types of blood cells, including myeloid cells (monocytes and macrophages), granulocytes (neutrophils, basophils, eosinophils and mast cells), erythrocytes, megakaryocytes / platelets and dendritic cells) and lymphocytes (T cells, B cells, natural killer cells) (see [Doulatov et al., 2012; Notta et al., 2015]). The hematopoietic stem cell can express CD34 and CD133, and CD38 expression can be negative. The hematopoietic progenitor cell may include CD34+ / CD45+ hematopoietic progenitor cells and CD34+ / CD45+ / CD43+ hematopoietic progenitor cells.

[0154] In this case, the mesodermal cells may be cultured in a serum-free medium. In addition, the mesodermal cells may be cultured in a medium containing STF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand) or a combination thereof.

[0155] The mesoderm cells can be cultured for about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days. Preferably, the mesoderm cells can be cultured for about 2 days to about 8 days, but are not limited thereto.

[0156] The cells differentiated from the mesoderm cells into hematopoietic stem cells may express CD34. In this case, there is no need to use a separate sorting process to select cells expressing only CD34, and the expression rate of CD34 may be 80% or more of all cells induced to differentiate. Specifically, the CD34 expression rate of the cells differentiated from the mesoderm cells into hematopoietic stem cells may be about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more. Preferably, the CD34 expression rate of the cells differentiated from the mesoderm cells into hematopoietic stem cells is about 85% or more.

[0157] The above-mentioned "CD34" is a marker protein of hematopoietic stem cells and is involved in cell-cell adhesion. The sorting process of selecting cells expressing only CD34 can be performed by flow cytometry or a kit.

[0158] Fourthly, the method comprises differentiating the hematopoietic stem cells into natural killer cells to prepare natural killer cells.

[0159] In this case, the hematopoietic stem cells may be cultured in a serum-free medium. In addition, the hematopoietic stem cells may be cultured in a medium containing STF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand) or a combination thereof.

[0160] The hematopoietic stem cells may be cultured in a medium containing IL-7, IL-15 or a combination thereof. The hematopoietic stem cells may be cultured in a medium containing STF, FLT3L, IL-7, IL-15 or a combination thereof.

[0161] The hematopoietic stem cell can be cultured for about 1 day to about 3 months, about 5 days to about 2 months, about 1 week to about 2 months, about 2 weeks to about 2 months, about 3 weeks to about 2 months, about 1 month to about 2 months, about 1 month to about 7 weeks, about 1 month to about 6 weeks, about 1 month to about 5 weeks, or about 5 weeks to about 6 weeks (such as about 36 days). Preferably, the hematopoietic stem cell can be cultured for about 1 day to about 26 days, or about 1 day to about 48 days, but is not limited thereto.

[0162] In the present invention, the hematopoietic stem cells can be co-cultured with autologous feeder cells.

[0163] The term "feeder cell" as used herein refers to a cell that is co-cultured with the cell of the second type and provides growth factors and nutrients to allow the cell of the second type to grow. The above-mentioned feeder cells can be selectively derived from species that are homologous or heterologous to the cells they support. For example, a specific type of human cell including stem cells can be supported by cultivating mouse embryonic fibroblasts and non-apoptotic mouse embryonic fibroblasts. The feeder cells can be human-derived feeder cells, such as human skin fibroblasts or human embryonic stem cells. In this case, the feeder cells can be inactivated by anti-cell division agents such as mitomycin or irradiation. Through the above-mentioned inactivation, when co-cultured with the cell of the second type, the feeder cells can produce and secrete cell metabolites under the state of cell division arrest, thereby regulating the differentiation and growth of the second type of cells.

[0164] In this case, in the present invention, the feeder cells may be autologous feeder cells.

[0165] In one embodiment, the autologous feeder cells of the present invention may be cells that play a role in regulating the differentiation and growth of hematopoietic stem cells into natural killer cells. In the present invention, the feeder cells may be cells that play a role in regulating the proliferation and activity of differentiated natural killer cells.

[0166] The above-mentioned "autologous feeder cells" of the present invention include cells induced to differentiate into mesoderm cells from iPSCs. Preferably, it may include all cells that have not differentiated into hematopoietic stem cells and adhere to the container among the cells induced to differentiate into hematopoietic stem cells together in the iPSC colony, but is not limited thereto.

[0167] In this case, differentiation of induced pluripotent stem cells into natural killer cells can be performed in vitro.

[0168] Differentiation of induced pluripotent stem cells into natural killer cells can be performed by culturing for about 26 days. Specifically, differentiation of iPSCs into natural killer cells can be performed by culturing for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, or 26 days.

[0169] In addition, in the present invention, the induced pluripotent stem cell-derived natural killer cells can be cultured for an additional 21 days after differentiation is completed. Specifically, the natural killer cells can be cultured for an additional 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days.

[0170] also, In the present invention, natural killer cells differentiated from induced pluripotent stem cells can also be used. Large-scale culture and proliferation outside.

[0171] In this case, when the natural killer cells induced and differentiated from induced pluripotent stem cells are cultured and proliferated in large quantities, the natural killer cells can be cultured in a medium containing STF, FLT3L, IL-7, IL-15, IL-2 or a combination thereof. In addition, the natural killer cells induced and differentiated from induced pluripotent stem cells can be cultured in a medium further containing β-mercaptoethanol, sodium selenite, ethanolamine, L-ascorbic acid or a combination thereof. In this case, the natural killer cells differentiated from induced pluripotent stem cells can be cultured for about 1 day to about 9 weeks, about 1 day to about 8 weeks, about 1 day to about 7 weeks, about 1 day to about 6 weeks, about 1 day to about 5 weeks, about 1 day to about 4 weeks, or about 1 day to about 3 weeks for large-scale culture and proliferation.

[0172] In the present invention, when the natural killer cells induced and differentiated from induced pluripotent stem cells are cultured and proliferated in large quantities, the natural killer cells can be co-cultured with feeder cells. In this case, the feeder cells are as described above.

[0173] In this case, natural killer cells derived from induced pluripotent stem cells can be cultured and proliferated in large quantities in vitro.

[0174] In one embodiment of the present invention, the natural killer cells derived from induced pluripotent stem cells may not express induced pluripotent stem cell marker proteins TRA-1-60 and SSEA4. TRA-1-60 and SSEA4 are stem cell marker proteins, and the stem cells are as described above.

[0175] In one embodiment of the present invention, the natural killer cells exhibit killing ability against blood cancer cell lines ( Fig.26 , Fig.31 , Fig.34b , Fig.39 e and Table 10). In addition, it was confirmed that the natural killer cells were more capable of activating immune cells than natural killer cells obtained from blood, and the expression level of the receptor for natural killer cell activation inhibitory protein expressed in tumor cells was also reduced ( Fig.36 Figure 37 Fig.38 , Table 13, Table 14). In addition, it can be seen that the natural killer cells of the present invention are newly isolated natural killer cells, which have a high degree of natural killer cell activation and excellent killing ability against tumor cells compared with natural killer cells cultured by conventional known methods or natural killer cells isolated from the human body.

[0176] Cell therapy agent containing natural killer cells and pharmaceutical composition containing the same

[0177] In another aspect of the present invention, there is provided a cell therapeutic agent comprising the natural killer cells of the present invention as an active ingredient.

[0178] In another aspect of the present invention, a pharmaceutical composition for preventing or treating a disease is provided, which comprises the natural killer cells of the present invention as an active ingredient.

[0179] The term "cell therapeutic agent" as used herein is a drug used for treatment, diagnosis and prevention, and its tissues and cells are prepared by separation, cultivation and special manufacturing from a subject (US FDA regulations). It refers to a drug that restores cell or tissue function through a series of actions, such as in vitro proliferation or sorting of autologous, allogeneic or xenogeneic cells of a living body, or by changing the biological characteristics of cells in other ways, to achieve the purpose of treatment, diagnosis and prevention. The cell therapeutic agent is roughly divided into somatic cell therapeutic agents and stem cell therapeutic agents according to the degree of cell differentiation. In the present invention, the cell therapeutic agent can be a somatic cell therapeutic agent.

[0180] The pharmaceutical composition of the present invention can be used to prevent and treat infectious diseases, cancer, nervous system diseases, autoimmune diseases, etc.

[0181] The infectious disease may be an infectious disease caused by hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, influenza, and the like.

[0182] The cancer can be liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, etc.

[0183] The above-mentioned "neurological disease" may be a neurological disease caused by nerve damage or nerve abnormality, or may be a degenerative brain disease. Here, the nerve damage may be neurapraxia, axonal rupture or neurotruncation.

[0184] The nervous system disease caused by nerve damage or nerve abnormality may be a nervous system disease caused by nerve damage or nerve abnormality in the central nervous system, or may be a nervous system disease caused by nerve damage or nerve abnormality in the peripheral nervous system.

[0185] The nervous system diseases caused by nerve damage or nerve abnormality in the central nervous system can be organic diseases and functional disorders of the central nervous system, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Lewy body dementia, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, multiple system atrophy, cortical basal degeneration, spinocerebellar degeneration, cerebellar atrophy, post-traumatic stress disorder, amnesia, vascular dementia, cerebral infarction, etc.

[0186] The nervous system disease caused by nerve damage or nerve abnormality in the peripheral nervous system can be peripheral neuropathy, diabetic neuropathy, peripheral neuropathic pain, peripheral neuropathy caused by tumor chemotherapy, complex regional pain syndrome, optic neuropathy, mononeuropathy, multiple mononeuropathy (mononeuritis multiplex), polyneuropathy, Guillain-Barre syndrome (acute inflammatory demyelinating polyneuropathy), chronic inflammatory demyelinating polyneuropathy, hereditary neuropathy, plexus disorder, glaucoma, macular degeneration, amyotrophic lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, poliomyelitis, post-polio syndrome, stiff-man syndrome, Isaac syndrome, myasthenia gravis, neonatal myasthenia, botulism, Eaton-Lambert syndrome, thoracic outlet syndrome, hereditary neuromuscular atrophy, spinal muscular atrophy, etc.

[0187] The above-mentioned "degenerative brain disease" refers to a disease that occurs in the brain among the degenerative diseases that occur with age. The degenerative brain disease may include all diseases caused by the death of brain nerve cells, problems in the formation or function of synapses that transmit information between brain nerve cells, and abnormal increase or decrease in brain nerve electrical activity. The degenerative brain disease may include Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette syndrome, Friedrich's ataxia, Machado-Joseph disease, Lewy body dementia, dystonia, progressive supranuclear palsy and frontotemporal dementia, but is not limited to this.

[0188] The term "autoimmune disease" as used herein refers to a general term for diseases caused by antigen-antibody reactions caused by antibodies produced against one's own biological substances or body tissues. The autoimmune diseases may include Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guillain-Barré syndrome, Sjögren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic sclerosis, asthma, ulcerative colitis, etc., but are not limited thereto.

[0189] The term "prevention" used herein refers to any action of inhibiting or delaying the onset of senile diseases by administering the pharmaceutical composition. The term "treatment" refers to any action of improving or beneficially changing the symptoms of a disease by administering the pharmaceutical composition.

[0190] In the pharmaceutical composition of the present invention, the content (effective amount) of natural killer cells can be determined according to the purpose, formulation, mixing purpose, etc., as long as they can show activity. The pharmaceutical composition of the present invention can contain 1×10 2 Up to 1×10 16 , 1×10 2 Up to 1×10 15 , 1×10 2 Up to 1×10 14 or 1×10 2 Up to 1×10 13 Preferably, the pharmaceutical composition may contain 2×10 2 Up to 2×10 11 activated natural killer cells.

[0191] Here, "effective amount" refers to the amount of active ingredients that can induce anti-aging effects. Such an effective amount can be determined experimentally within the normal capabilities of ordinary technicians in the field.

[0192] The composition of the present invention can be used without freezing or frozen for later use. If frozen, a standard cryoprotectant (e.g., DMSO, glycerol, polyvinyl pyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, id erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride and Cell freezing medium (Ccascade Biologics).

[0193] The pharmaceutical composition comprising the natural killer cells of the present invention as an active ingredient can be formulated by further comprising a pharmaceutically acceptable carrier.

[0194] The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not significantly stimulate an organism and does not inhibit the biological activity and properties of the administered component. The pharmaceutical composition of the present invention can be used as a cell therapy agent, and the pharmaceutically acceptable carrier that can be included in the cell therapy agent can be used without restriction as long as it is known in the art, such as a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, a matrix, an excipient, a lubricant, etc. The pharmaceutical composition of the present invention can be prepared in the form of various preparations according to common techniques.

[0195] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount and can be administered by any route as long as it can induce migration to the diseased site. "Administration / administration" here refers to introducing a predetermined substance into the subject's body by an appropriate method. In some cases, it is contemplated that the pharmaceutical composition of the present invention is loaded into a carrier containing means for directing natural killer cells to lesions.

[0196] Thus, the pharmaceutical compositions of the present invention can be administered by several routes, including topical (including oral, sublingual, dermal and intraocular), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and cerebrospinal fluid) or transdermal administration, preferably, directly to the site of disease. In one embodiment, the natural killer cells can be suspended in a suitable diluent and administered to the subject; the diluent is used to protect and maintain the cells and facilitate their use when injected into the target tissue. The diluent can include a buffer solution, such as saline, phosphate buffered solution, HBSS, cerebrospinal fluid, etc. In addition, the composition can be administered by any device that allows the active substance to migrate to the target cells.

[0197] The preferred mode of administration and preparation is injection. The injection can use an aqueous solution, such as physiological saline, Ringer's solution, Hank's solution or sterile aqueous solution; vegetable oil, such as olive oil; higher fatty acid esters, such as ethyl oleate; and non-aqueous solvents, such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerol, etc. For mucosal penetration, a non-permeating agent suitable for passing through the barrier known in the art can be used, and a drug carrier as a stabilizer to prevent deterioration, such as ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA, etc.; and an emulsifier; a buffer for pH adjustment; a preservative for inhibiting microbial growth, such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol and benzyl alcohol. The method of preparing a pharmaceutical composition is known in the art, and specific references can be made to the literature [Remington's Pharmaceutical Sciences (19th edition, 1995)] and the like. The above-mentioned literature is considered to be part of this specification.

[0198] The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and without causing side effects. One of ordinary skill in the art can easily determine the effective dosage level based on the patient's sex, age, weight, health status, type and severity of the disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.

[0199] Depending on the patient's condition, weight, sex and age, the severity of the patient's condition and the route of administration, the preferred dosage of the pharmaceutical composition of the present invention may be about 1×10 / kg to about 1×10 cells per day. 12 / kg, about 1×10 / kg to about 1×10 11 / kg or about 1×10 / kg to about 1X10 10 / kg.

[0200] However, according to the factors such as preparation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, reaction sensitivity, etc., the dosage can be specified in various ways, and those of ordinary skill in the art can appropriately adjust the dosage by considering these factors. Within the range of clinically acceptable side effects, the administration frequency can be once or twice or more. The pharmaceutical composition of the present invention can be administered at a frequency of 1 to 3 times / day, 1 to 3 times / week or 1 to 10 times / month. For example, the pharmaceutical composition of the present invention can be administered at a frequency of 1 to 9 times / month, 1 to 8 times / month, 1 to 7 times / month, 1 to 6 times / month, 1 to 5 times / month, 1 to 4 times / month, 1 to 3 times / month or 1 to 2 times / month. For the administration site, it can be administered to 1 site or 2 or more sites. For animals other than humans, it can be administered at the same dosage per kilogram or per subject as humans, or it can be administered according to the volume ratio (e.g., mean value) of organs (heart, etc.) between, for example, target animals and humans, using the amount converted from the above dosage. Such dosage should not be understood as limiting the scope of the present invention in any way.

[0201] The term "subject" used herein refers to a subject who has or may suffer from a disease to which the composition of the present invention can be applied (prescribed), and can be a mammal, such as rats, mice or livestock, including humans. Preferably, it is a human, but not limited thereto.

[0202] If necessary, the pharmaceutical composition of the present invention may further contain known substances having preventive or therapeutic effects on diseases.

[0203] The pharmaceutical composition of the present invention may further contain the above-mentioned substance that increases the activity of natural killer cells. The pharmaceutical composition of the present invention may be administered in combination with conventional known substances for preventing or treating the disease. In this case, the natural killer cells, the substance that exhibits a preventive or therapeutic effect on the disease and / or the substance that increases the activity of natural killer cells may be administered simultaneously or sequentially.

[0204] In another aspect of the present invention, the use of the natural killer cells or a pharmaceutical composition containing the natural killer cells as an effective ingredient for preventing and treating diseases is provided. The natural killer cells, pharmaceutical composition, disease, prevention and treatment herein are as described above.

[0205] In another aspect of the present invention, a method for treating a disease is provided, comprising: administering the natural killer cell or a pharmaceutical composition comprising the natural killer cell as an active ingredient to a subject. In this case, the natural killer cell, pharmaceutical composition, disease, prevention and treatment are as described above.

[0206] The subject may be any animal, such as rats, mice or livestock, including humans who have or may have the disease, preferably humans.

[0207] The dosage may be about 1×10 / kg to about 1×10 / kg per day, depending on the patient's condition, weight, sex and age, the severity of the patient's condition and the route of administration. 12 / kg, about 1×10 / kg to about 1×10 11 / kg or about 1×10 / kg to about 1×10 10 / kg. However, the dosage can be specified in various ways according to factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, reaction sensitivity, etc., and those skilled in the art can appropriately adjust the dosage by considering these factors.

[0208] Method for preparing natural killer cells

[0209] In another aspect of the present invention, a method for preparing natural killer cells is provided, comprising: inoculating induced pluripotent stem cells in the form of iPSCs or iPSC spheroids into a cell culture container so that 0.5 to 20 colonies are formed per 1 square centimeter of the cell culture container area, and performing adhesion culture to prepare iPSC colonies; differentiating the iPSC colonies into mesodermal cells to prepare mesodermal cells; differentiating the mesodermal cells into hematopoietic stem cells to prepare hematopoietic stem cells; and differentiating the hematopoietic stem cells into natural killer cells to prepare natural killer cells.

[0210] In this case, the induced pluripotent stem cells can be cultured by an adherence culture method, and can be seeded as a single cell or a spheroid and cultured adherently. The number of cells included in the spheroid is as described above.

[0211] In addition, the number of the seeded iPSCs or iPSC spheroids may be 0.5 to 20 per 1 cm2. The seeded iPSCs or iPSC spheroids are as described above.

[0212] Alternatively, another method can be used to calculate the number of inoculum cells. The number of cells present per milliliter of culture medium can be determined as the number of cells per square centimeter using the method for calculating the number of inoculum cells.

[0213] In addition, the method for preparing natural killer cells of the present invention may further comprise culturing and proliferating the natural killer cells differentiated from induced pluripotent stem cells in large quantities.

[0214] In this case, the induced pluripotent stem cells, differentiation, natural killer cells and culture are as described above. In addition, the method for calculating the number of seeded iPSCs and the number of seeded cells per 1 square centimeter of cell culture container area in another different manner is as described above.

[0215] The iPSC can be cultivated in the culture medium containing ROCK inhibitor. In this case, the ROCK inhibitor is as described above. The iPSC can be cultivated in the culture medium containing ROCK inhibitor for about 10 minutes to about 1 week, about 30 minutes to about 6 days, about 1 hour to about 5 days, about 2 hours to about 4 days, about 3 hours to about 3 days, about 6 hours to about 2 days, about 12 hours to about 2 days or about 18 hours to about 1 day. In addition, the iPSC can be cultivated using adhesion culture method. Single cell iPSC can be cultivated for about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days or about 7 days to about 8 days after adhering to the cell culture container.

[0216] The iPSC colony may be a cell group that proliferates after single-cell induced pluripotent stem cell adhesion culture. The iPSC colony may be cells with the same genetic information.

[0217] The iPSC colony can be differentiated into mesodermal cells to prepare mesodermal cells. On the differentiation start day, the total number of induced pluripotent stem cells and the mesodermal cells are as described above. In this case, the total number of iPSC stem cells on the start day of differentiation of the iPSC colony into mesodermal cells can be 0.5 to 1×10 per 1 square centimeter of the cell culture container area. 6 In this case, the size of each colony of the iPSC may be about 10 μm to about 3000 μm. Specifically, the size of each colony of the iPSC may be about 10 μm to about 3000 μm, about 50 μm to about 2500 μm, about 100 μm to about 2000 μm, about 300 μm to about 1200 μm, about 400 μm to about 800 μm, or about 500 μm to about 700 μm. Preferably, the size of each colony of the iPSC may be about 300 μm to 1200 μm, but is not limited thereto.

[0218] In addition, the iPSC colonies can be cultured in serum-free medium. The iPSC colonies can be cultured in a medium capable of culturing stem cells. In addition, the iPSC colonies can be cultured in a medium comprising at least one selected from the group consisting of a GSK3 inhibitor, an ALK5 inhibitor, BMP4, SCF, and VEGF. The GSK3 inhibitor and the ALK5 inhibitor are as described above.

[0219] The iPSC colony can be first cultured in a culture medium containing CHIR-99021, BMP4 and VEGF, and then cultured in a culture medium containing SB431542, SCF and VEGF. The iPSC colony can be cultured in a culture medium containing CHIR-99021, SB431542, BMP4, SCF and VEGF. In this case, the iPSC colony can be cultured in the above-mentioned culture medium for about 1 hour to about 2 weeks, about 12 hours to about 2 weeks, about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days. Preferably, the iPSC colony can be cultured for about 2 days to about 4 days, but is not limited thereto.

[0220] The mesodermal cells may be mesodermal stem cells.

[0221] The mesoderm cells can be differentiated into hematopoietic stem cells to prepare hematopoietic stem cells. In this case, the hematopoietic stem cells are as described above.

[0222] The mesodermal cells can be cultured in a serum-free medium or in a medium containing STF, FLT3L or a combination thereof. In this case, the mesodermal cells can be cultured for about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days. Preferably, the mesodermal cells can be cultured for about 2 days to about 8 days, but are not limited thereto.

[0223] The cells differentiated from the mesoderm cells into hematopoietic stem cells may express CD34. In this case, there is no need to use a separate sorting process to select only cells expressing CD34, and the expression rate of CD34 may be 80% or more of all cells induced to differentiate. Specifically, the CD34 expression rate of all cells differentiated from the mesoderm cells into hematopoietic stem cells may be about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more. Preferably, the CD34 expression rate of all cells differentiated from the mesoderm cells into hematopoietic stem cells is about 85% or more.

[0224] The hematopoietic stem cells may be cultured in a medium containing IL-7, IL-15 or a combination thereof. The hematopoietic stem cells may be cultured in a medium containing STF, FLT3L, IL-7, IL-15 or a combination thereof.

[0225] The hematopoietic stem cell can be cultured for about 1 day to about 3 months, about 5 days to about 2 months, about 1 week to about 2 months, about 2 weeks to about 2 months, about 3 weeks to about 2 months, about 1 month to about 2 months, about 1 month to about 7 weeks, about 1 month to about 6 weeks, about 1 month to about 5 weeks, or about 5 weeks to about 6 weeks (such as about 36 days). Preferably, the hematopoietic stem cell can be cultured for about 1 day to about 26 days, or about 1 day to about 48 days, but is not limited thereto.

[0226] In the present invention, the hematopoietic stem cells can be co-cultured with autologous feeder cells.

[0227] The feeder cells are as described above. In this case, in the present invention, the feeder cells may be autologous feeder cells.

[0228] In one embodiment, the autologous feeder cells of the present invention may be cells that play a role in regulating the differentiation and growth of hematopoietic stem cells into natural killer cells. In the present invention, the feeder cells may be cells that play a role in regulating the proliferation and activity of differentiated natural killer cells.

[0229] The above-mentioned "autologous feeder cells" of the present invention include cells induced to differentiate into mesoderm cells from iPSCs. Preferably, it may include all cells that have not differentiated into hematopoietic stem cells and adhere to the container among the cells induced to differentiate into hematopoietic stem cells together in the iPSC colony, but is not limited thereto.

[0230] The method can be performed in vitro.

[0231] The above-mentioned medium may be a known medium for cell culture or a modified medium thereof.

[0232] In the method for preparing natural killer cells of the present invention, the induced differentiated natural killer cells can also be cultured and proliferated in large quantities.

[0233] In this case, during the mass culture proliferation, the natural killer cells induced and differentiated from induced pluripotent stem cells can be cultured in a culture medium containing STF, FLT3L, IL-7, IL-15, IL-2 or a combination thereof. In addition, the natural killer cells induced and differentiated from induced pluripotent stem cells can be cultured in a culture medium further containing β-mercaptoethanol, sodium selenite, ethanolamine, L-ascorbic acid or a combination thereof. In this case, the mass culture proliferation can be carried out for about 1 day to about 4 weeks, or about 1 day to about 3 weeks.

[0234] In the present invention, when the natural killer cells induced to differentiate from induced pluripotent stem cells are cultured and proliferated in large quantities, the natural killer cells can be co-cultured with feeder cells. In this case, the feeder cells are the same as those described above.

[0235] In this case, natural killer cells derived from induced pluripotent stem cells can be cultured and proliferated in large quantities in vitro.

[0236] The natural killer cells prepared by the above method may express any one protein selected from the group consisting of CD25, CD9, CD80 and a combination thereof. In addition, the natural killer cells may additionally express any one protein selected from the group consisting of CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, CD210 and a combination thereof.

[0237] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, or about 95% or more of the natural killer cell population prepared by the method of the present invention can express CD25.

[0238] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, or about 97% or more of the natural killer cell population prepared by the method of the present invention can express CD80.

[0239] Specifically, about 10% or more, about 20% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, or about 34% or more of the natural killer cell population prepared by the method of the present invention can express CD9.

[0240] Specifically, about 5% or more, about 6% or more, or about 7% or more of the natural killer cell population prepared by the method of the present invention may express CD1d.

[0241] Specifically, about 20% or more, about 30% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, or about 45% or more of the natural killer cell population prepared by the method of the present invention may express CD30.

[0242] Specifically, about 20% or more, about 30% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, about 45% or more, or about 46% or more of the natural killer cell population prepared by the method of the present invention can express CD87.

[0243] Specifically, about 10% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, or about 26% or more of the natural killer cell population prepared by the method of the present invention can express CDw327.

[0244] Specifically, about 10% or more, about 11% or more, about 12% or more, about 13% or more, or about 14% or more of the natural killer cell population prepared by the method of the present invention may express CD33.

[0245] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the natural killer cell population prepared by the method of the present invention can express CD49a.

[0246] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, or about 94% or more of the natural killer cell population prepared by the method of the present invention can express CD49c.

[0247] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, or about 96% or more of the natural killer cell population prepared by the method of the present invention can express CD49e.

[0248] Specifically, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, or about 15% or more of the natural killer cell population prepared by the method of the present invention can express CD49f.

[0249] Specifically, about 50% or more, about 60% or more, about 70% or more, about 74% or more, about 75% or more, about 76% or more, about 77% or more, about 78% or more, about 79% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, or about 84% or more of the natural killer cell population prepared by the method of the present invention can express CD66 (a / b / c / d / e).

[0250] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, or about 96% or more of the natural killer cell population prepared by the method of the present invention can express CD71.

[0251] Specifically, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, about 35% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, or about 40% or more of the natural killer cell population prepared by the method of the present invention can express CD83.

[0252] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, or about 95% or more of the natural killer cell population prepared by the method of the present invention can express CDw93.

[0253] Specifically, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 31% or more, or about 32% or more of the natural killer cell population prepared by the method of the present invention can express CD106.

[0254] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, or about 94% or more of the natural killer cell population prepared by the method of the present invention can express CD108.

[0255] Specifically, about 20% or more, about 30% or more, about 35% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, about 40% or more, about 41% or more, or about 42% or more of the natural killer cell population prepared by the method of the present invention can express CD114.

[0256] Specifically, about 10% or more, about 20% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, or about 35% or more of the natural killer cell population prepared by the method of the present invention can express CD123.

[0257] Specifically, about 5% or more, about 6% or more, about 7% or more, about 8% or more, or about 9% or more of the natural killer cell population prepared by the method of the present invention may express CD124.

[0258] Specifically, about 50% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, or about 65% or more of the natural killer cell population prepared by the method of the present invention can express CD146.

[0259] Specifically, about 10% or more, about 20% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 31% or more, or about 32% or more of the natural killer cell population prepared by the method of the present invention can express CD150.

[0260] Specifically, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, or about 97% or more of the natural killer cell population prepared by the method of the present invention can express CD164.

[0261] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, or about 95% or more of the natural killer cell population prepared by the method of the present invention can express CD196.

[0262] Specifically, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, or about 10% or more of the natural killer cell population prepared by the method of the present invention may express CD210.

[0263] Compared to natural killer cells isolated from blood, the expression levels of the above-mentioned CD25, CD9, CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, and CD210 proteins can be reduced to at most 1 / 5. Specifically, the expression level can be increased to about at least two times. Natural killer cells, CD25, CD9, CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, and CD210 obtained from blood were as described above.

[0264] The natural killer cells may express any one natural killer cell marker protein selected from the group consisting of CD56, CD45, CD16, NKp30, NKp44, NKG2D, DNAM1 and a combination thereof, and may not express the T cell marker protein CD3, or may express CD3 at a level lower than that of T cells.

[0265] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, or about 96% or more of the natural killer cell population prepared by the method of the present invention can express CD56, CD45, NKp30 or DNAM1.

[0266] Specifically, about 40% or more, about 50% or more, about 51% or more, about 52% or more, about 53% or more, about 54% or more, about 55% or more, about 56% or more, about 57% or more, or about 58% or more of the natural killer cell population prepared by the method of the present invention can express CD16.

[0267] Specifically, about 50% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, about 65% or more, about 66% or more, about 67% or more, about 68% or more, about 69% or more, about 70% or more, about 71% or more, or about 72% or more of the natural killer cell population prepared by the method of the present invention can express NKp44.

[0268] Specifically, about 70% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, or about 89% or more of the natural killer cell population prepared by the method of the present invention can express NKD2D.

[0269] In addition, about 90% or more, about 9% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, or about 99.4% or more of the natural killer cell population prepared by the method of the present invention may not express CD3.

[0270] In addition, compared with natural killer cells isolated from blood, the natural killer cells according to the method of the present invention may have an additional increased expression level of any one gene selected from the group consisting of GZMB, CD226, RASGRP1, CD107a, CCR9, CD40LG, CD300c and a combination thereof. The above-mentioned GZMB, CD226, RASGRP1, CD107a, CCR9, CD40LG, CD300c are as described above.

[0271] The natural killer cells of the present invention may be characterized by an increased secretion of any one protein selected from the group consisting of FLT3L, MIP1alpha / beta, PF4, IGFBP2, IGFBP3, MMP9 and a combination thereof compared to natural killer cells isolated from blood. In this case, the secretion of the above protein may be increased by at least 2 times compared to natural killer cells isolated from blood. In the natural killer cells obtained from blood, FLT3L, MIP1alpha / beta, PF4, IGFBP2, IGFBP3 and MMP9 are as described above.

[0272] The natural killer cells according to the present invention may not additionally express any one protein selected from the group consisting of CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM3, TIGIT and a combination thereof, or may express it at a lower level than natural killer cells obtained from blood. For natural killer cells obtained from blood, CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM3 and TIGIT are as described above.

[0273] Specifically, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 97.5% or more, about 97.6% or more, about 97.7% or more, about 97.8% or more, or about 97.9% or more of the natural killer cell population prepared by the method of the present invention may not express CD205.

[0274] In addition, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 97.1% or more, about 97.2% or more, about 97.3% or more, or about 97.4% or more of the natural killer cell population prepared by the method of the present invention may not express CD158b.

[0275] In addition, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, about 99.4% or more, about 99.5% or more, about 99.6% or more, or about 99.7% or more of the natural killer cell population prepared by the method of the present invention may not express CD243.

[0276] In addition, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, about 99.4% or more, about 99.5% or more, about 99.6% or more, or about 99.7% or more of the natural killer cell population prepared by the method of the present invention may not express γδTCR.

[0277] In addition, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, about 99.4% or more, or about 99.5% or more of the natural killer cell population prepared by the method of the present invention may not express NKB1.

[0278] In addition, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, about 99.4% or more, about 99.5% or more, or about 99.6% or more of the natural killer cell population prepared by the method of the present invention may not express CD158a.

[0279] In addition, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 98.1% or more, about 98.2% or more, about 98.3% or more, about 98.4% or more, about 98.5% or more, about 98.6% or more, or about 98.7% or more of the natural killer cell population prepared by the method of the present invention may not express CDw328.

[0280] In addition, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the natural killer cell population prepared by the method of the present invention may not express TIM-3.

[0281] In addition, about 80% or more, about 85% or more, about 90% or more, about 93% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the natural killer cell population prepared by the method of the present invention may not express TIGIT.

[0282] Compared with natural killer cells obtained from blood, the expression levels of the above CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM-3 and TIGIT proteins can be reduced to at most about 1 / 2. The natural killer cells obtained from blood are as described above.

[0283] Generally, when iPSCs were cultured in the third culture using Aggrewell, the size of embryoid bodies was confirmed to be larger than that in the second culture (adhesion culture) ( Fig.44 ). In addition, it was confirmed that there were differences in the size of embryoid bodies between different batches, and that cell survival rate decreased when single cells were obtained by degradative enzyme treatment ( Fig.45 ). On the other hand, the method for preparing natural killer cells according to the present invention may be simpler and more efficient than the third culture because iPSCs are subjected to the second culture (adhesion culture) and induced to differentiate.

[0284] In addition, when natural killer cells are prepared from induced pluripotent stem cells by conventional methods, only CD34-positive cells (hematopoietic stem cells) can be obtained by inducing differentiation from mesoderm cells to hematopoietic stem cells, which are then induced to differentiate into natural killer cells. In this case, the process of sorting only CD34-positive cells can use flow cytometry or commercially available sorting kits. For cells sorted for CD34-positive cells in this way, it was confirmed that the cell yield was significantly reduced, cell death increased, and the expression rate of natural killer cell marker protein (CD56) and activation marker protein (NKp30) was reduced in the sorted CD34-positive cells ( Fig.42a and 42b In addition, it was confirmed that the cell proliferation rate was also significantly reduced ( Figure 42c ). On the other hand, in one embodiment of the present invention, the method for preparing natural killer cells of the present invention can induce differentiation into natural killer cells without the need for a CD34-positive cell sorting process, because the expression rate of the differentiation marker CD34 of hematopoietic stem cells at the intermediate stage of NK differentiation is as high as 95% or more, thereby improving cell yield and activity ( FIG. 42 ).

[0285] In addition, the conventional method for preparing natural killer cells requires a total differentiation induction period of 48 days, while in one embodiment of the present invention, the method for preparing natural killer cells according to the present invention requires a differentiation induction period of 26 days. By confirming the natural killer cell marker proteins (CD56, CD45, NKp44) and activation proteins (CD56, NKp30) in EiNK cells induced for differentiation for 26 days, it was confirmed that the expression rate of NK marker proteins and activation proteins reached 66% or more. On the other hand, it was confirmed that the expression rate of iPSC marker proteins (TRA-1-60, SSEA4) was 0%, indicating that compared with the existing methods, the method for preparing natural killer cells according to the present invention is an efficient method for preparing natural killer cells in a short time ( Fig.45 and 46 ).

[0286] Stem cell culture methods

[0287] In one aspect of the present invention, a method for culturing stem cells is provided, comprising: inoculating stem cells in the form of iPSCs or iPSC spheroids into a cell culture container such that 1 to 20 iPSC colonies are formed per 1 square centimeter of the cell culture container area.

[0288] In this case, the stem cell can be an induced pluripotent stem cell (iPSC). In addition, in this case, the stem cell can be cultured by an adhesion culture method. In this case, the stem cell can be inoculated with a spheroid and cultured by adhesion. The number of cells included in the spheroid is as described above.

[0289] In addition, the number of the seeded iPSCs or iPSC spheroids may be 0.5 to 20 per 1 cm2. The seeded iPSCs or iPSC spheroids are as described above.

[0290] Alternatively, another method can be used to calculate the number of inoculum cells. The number of cells present per milliliter of culture medium can be determined as the number of cells per square centimeter using the method for calculating the number of inoculum cells.

[0291] The specific culture method is as described above.

[0292] Stem cell differentiation methods

[0293] In another aspect of the present invention, a method for differentiating stem cells is provided. Specifically, the method and the total number of stem cells on the differentiation start date are as described above. Preferably, the total number of stem cells on the differentiation start date can be 1×10 per 1 square centimeter of the cell culture container area. 3 Up to 1×10 6 The above stem cells can be differentiated in various ways according to the added culture medium. In one embodiment, the stem cells can be differentiated into mesodermal cells. The specific culture method is as described above.

[0294] Method for preparing natural killer cells from pluripotent stem cells

[0295] In another aspect of the present invention, a method for preparing natural killer cells from iPSCs is provided, comprising: inoculating induced pluripotent stem cells as iPSCs or iPSC spheroids into a cell culture container so that 0.5 to 20 colonies are formed per 1 square centimeter of the cell culture container area, and performing adhesion culture to prepare iPSC colonies; differentiating the iPSC colonies into mesodermal cells to prepare mesodermal cells; differentiating the mesodermal cells into hematopoietic stem cells to prepare hematopoietic stem cells; and differentiating the hematopoietic stem cells into natural killer cells to prepare natural killer cells.

[0296] First, the method comprises seeding induced pluripotent stem cells (iPSCs) as iPSCs or iPSC spheroids into a cell culture container to form 0.5 to 20 colonies per 1 square centimeter of the cell culture container area, and performing adhesion culture to prepare iPSC colonies. The number of seeded cells is as described above.

[0297] Next, the method comprises differentiating the iPSC colonies into mesodermal cells to prepare mesodermal cells.

[0298] Third, the method comprises differentiating the mesodermal cells into hematopoietic stem cells to prepare hematopoietic stem cells.

[0299] Fourthly, the method comprises differentiating the hematopoietic stem cells into natural killer cells to prepare natural killer cells.

[0300] The specific methods and conditions are as described above.

[0301] Method for culturing hematopoietic stem cells by co-culturing with autologous feeder cells

[0302] In the present invention, the hematopoietic stem cells can be co-cultured with autologous feeder cells.

[0303] The term "feeder cell" as used herein refers to a cell that is co-cultured with the cell of the second type and provides growth factors and nutrients to allow the cell of the second type to grow. The above-mentioned feeder cells can be selectively derived from species that are homologous or heterologous to the cells they support. For example, a specific type of human cell including stem cells can be supported by cultivating mouse embryonic fibroblasts and non-apoptotic mouse embryonic fibroblasts. The feeder cells can be human-derived feeder cells, such as human skin fibroblasts or human embryonic stem cells. In this case, the feeder cells can be inactivated by anti-cell division agents such as mitomycin or irradiation. Through the above-mentioned inactivation, when co-cultured with the cell of the second type, the feeder cells can produce and secrete cell metabolites under the state of cell division arrest, thereby regulating the differentiation and growth of the second type of cells.

[0304] In this case, in the present invention, the feeder cells may be autologous feeder cells.

[0305] The specific culture methods and conditions are as described above.

[0306] Embodiments of the present invention

[0307] Below, the present invention will be described in more detail by the following examples. However, these examples are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these examples.

[0308] I. Preparation of Natural Killer Cells

[0309] Example 1. Method for preparing natural killer cells from induced pluripotent stem cells

[0310] Schematic diagram of the method for obtaining natural killer cells from induced pluripotent stem cells Figure 1 In addition, the specific experimental methods are as follows Figure 2 and Fig.19The method can be roughly divided into the following steps: 1) inducing pluripotent stem cells (adherent cells) to produce induced pluripotent stem cell colonies (adherent cells); 2) inducing pluripotent stem cells (adherent cells) to differentiate into mesoderm cells (adherent cells); 3) mesoderm cells (adherent cells) to differentiate into hematopoietic stem cells (suspension cells) and autologous feeder cells (adherent cells); 4) hematopoietic stem cells (suspension cells) to differentiate into natural killer cells, while retaining or removing autologous feeder cells (adherent cells).

[0311] Example 1.1. Cultivation of induced pluripotent stem cells

[0312] The CMC-009 induced pluripotent stem cell (iPSC) cell line used in this example was provided by the National Stem Cell Bank of the National Center for Stem Cell and Regenerative Medicine of the United States. The cell line was cultured in a cell culture vessel using an extracellular matrix iMatrix-511 (Nippi, 892012) using StemFit Basic04 culture medium (Ajinomoto, AJBASIC04).

[0313] Example 1.2. Formation of homogeneous induced pluripotent stem cell colonies from induced pluripotent stem cells

[0314] Example 1.2.1. Formation of homogeneous induced pluripotent stem cell colonies from single induced pluripotent stem cells

[0315] As the first step in the differentiation of human induced pluripotent stem cells into natural killer cells, induced pluripotent stem cell colonies (iPSC colonies) were induced from single-cell induced pluripotent stem cells.

[0316] Specifically, for every 1 square centimeter of cell culture container, 1 μl of iMatrix-511 was diluted with 150 μl of DPBS (Cytiva, SH3002802), then applied to the cell culture container for at least 1 hour, and then replaced with StemFit Basic 04 medium containing 10 μM Y-27632 to prepare a cell culture container for adhering single-cell iPSCs. iPSCs were treated with TypLE (Gibco, 12604013) to separate them into single cells, and then inoculated into the prepared cell culture container at 0.5 to 20 single-cell iPSCs per 1 square centimeter of culture container area.

[0317] On the second day, the cell culture medium was replaced with fresh StemFit Basic 04 and cultured for another 9-12 days. When the diameter of the induced pluripotent stem cell colonies (iPSC colonies) was between 300 and 1600 μm (average diameter 300-700 μm), the stem cell culture medium was removed and natural killer cell differentiation was started. During the preparation of induced pluripotent stem cell colonies, the cell culture medium was changed every day.

[0318] Example 1.2.2. Formation of homogeneous induced pluripotent stem cell colonies from spheroid induced pluripotent stem cells

[0319] As the first step in differentiating human induced pluripotent stem cells into natural killer cells, induced pluripotent stem cell colonies (iPSC colonies) were induced by seeding spheroid-derived induced pluripotent stem cells.

[0320] Specifically, for every 1 square centimeter of cell culture container, 1 μl of iMatrix-511 was diluted with 150 μl of DPBS (Cytiva, SH3002802), then applied to the cell culture container for at least 1 hour, and then replaced with StemFit Basic 04 culture solution containing 10 μM Y-27632 to prepare a cell culture container for adhering spheroid iPSCs. Spheroid iPSCs are prepared by culturing single-cell iPSCs. At this point, a spheroid contains about 1 to about 600 iPSCs. Then, 0.5 to 20 iPSC spheroids are inoculated into the prepared cell culture container for every 1 square centimeter of container culture area.

[0321] On the second day, the cell culture medium was replaced with fresh StemFit Basic 04 and cultured for another 2-5 days. When the diameter of the induced pluripotent stem cell colonies (iPSC colonies) was between 300 and 1600 μm (average diameter 300-700 μm), the stem cell culture medium was removed and natural killer cell differentiation was started. During the preparation of induced pluripotent stem cell colonies, the cell culture medium was replaced every day.

[0322] Example 1.3. Preparation of natural killer cells from induced pluripotent stem cell colonies

[0323] Specifically, when the diameter of the induced pluripotent stem cell colonies (iPSC colonies) prepared in Example 1.2 above was between 300-1600 μm (average diameter 300-700 μm), the stem cell culture medium was removed and the cells were cultured in serum-free stem cell culture medium Essential 8 supplemented with 2 μm CHIR-99021 (Peprotech, 2520691), 80 ng / ml BMP4 (R&D Systems, 314-BP / CF) and 80 ng / ml VEGF (R&D Systems, 293-VE / CF). TM (Gibco, 1517001) and cultured for 2 days. After 2 days, the above medium was removed and the cells were cultured in serum-free cell culture medium Essential 6 containing 2 μM SB-431542 (Peprotech, 3014193), 50 ng / ml SCF (R&D Systems, 255-SC / CF) and 80 ng / ml VEGF. TM (Gibco, 1516401) to induce differentiation into mesoderm cells.

[0324] After 2 days, the above medium was removed and the cells were cultured in a serum-free hematopoietic stem cell medium Stemline II (Sigma, S0192) supplemented with 50 ng / ml SCF and 50 ng / ml FLT3L (R&D Systems, 308-FK / CF) to induce differentiation into hematopoietic stem cells. After 8 days, the above medium was removed and replaced with Stemline II medium, and 50 ng / ml SCF, 50 ng / ml FLT3L, IL-7 (R&D Systems, 207-IL-200 / CF) and IL-15 (R&D Systems, 247-ILB-500 / CF) were added and cultured for 36 days to finally induce differentiation into natural killer cells. At this time, half of all the culture media used for differentiation were replaced every 4 days.

[0325] Example 1.4. Preparation of natural killer cells from iPSC colonies under conditions that exclude autologous feeder cells

[0326] Specifically, when the diameter of the induced pluripotent stem cell colonies (iPSC colonies) prepared in Example 1.2 above was between 700 and 1200 μm, the stem cell culture medium was removed and the cells were cultured in serum-free stem cell culture medium Essential 8 supplemented with 2 μm CHIR-99021, 80 ng / ml BMP4 and 80 ng / ml VEGF. TM(Gibco, 1517001) and cultured for 2 days. After 2 days, the medium was removed and the cells were cultured in serum-free cell culture medium Essential 6 containing 2 μM SB-431542, 50 ng / ml SCF and 80 ng / ml VEGF. TM to induce differentiation into mesoderm cells.

[0327] After 2 days, the above medium was removed and the cells were cultured in a serum-free hematopoietic stem cell medium Stemline II supplemented with 50 ng / ml SCF and 50 ng / ml FLT3L to induce differentiation into hematopoietic stem cells. On the 12th day of differentiation, the suspended cells in the cell culture container except the autologous feeder cells were collected and transferred to a new cell culture container and replaced with Stemline II medium supplemented with 50 ng / ml SCF, 50 ng / ml FLT3L, IL-7 and IL-15, and cultured for 36 days to finally induce differentiation into natural killer cells. At this time, half of all the culture media used for differentiation were replaced every 4 days.

[0328] Example 1.5. Morphological verification of natural killer cells differentiated from iPSC colonies according to differentiation date

[0329] iPSCs were differentiated into NK cells by the methods of Examples 1.1 to 1.4 above, and the morphological changes of the differentiated cells were observed under an optical microscope. Specifically, an optical microscope (Olympus, Olympus-CKX53) was used to confirm that at each differentiation point, the morphology of the cells changed from pluripotent stem cells to mesoderm cells, hematopoietic stem cells, and then to natural killer cells according to different differentiation stages.

[0330] The morphological changes of cells at each differentiation stage were confirmed using an optical microscope. Figure 3 ).exist Figure 3 In the photo, the photo taken on day 0 shows induced pluripotent stem cell colonies (adherent cells) generated from induced pluripotent stem cells (adherent cells). At this time, the size of the colonies in the photo is between 700μm and 1200μm, and colonies of this size have differentiated into mesodermal cells. In addition, the photo taken on day 4 shows mesodermal cells (adherent cells) differentiated from induced pluripotent stem cell colonies. In addition, the photo taken on day 12 shows feeder cells (the part where cells adhere to form a mass) and hematopoietic stem cells (suspension cells in the form of single cells) differentiated from mesodermal cells (adherent cells). In addition, the photos taken on days 26 to 33 show natural killer cells differentiated from hematopoietic stem cells (autologous feeder cells are not included in the photo). Finally, the photo taken on day 48 shows natural killer cells (suspension cells) derived from induced pluripotent stem cells.

[0331] In addition, after changing the culture conditions, the morphological changes of differentiated cells at each differentiation stage under each condition (AD) were observed using an optical microscope, and the obtained results were confirmed ( Fig.13 ). At this point, take pictures under a microscope at each differentiation stage, from day 0 of differentiation until the day of final differentiation into natural killer cells.

[0332] Example 1.6. Confirmation of the production of natural killer cells based on the presence or absence of feeder cells derived from induced pluripotent stem cells, and confirmation of the properties of the produced natural killer cells

[0333] In the methods of Examples 1.1 to 1.4 above, depending on whether or not there are feeder cells derived from iPSCs ( Figure 4 ) to confirm whether mesoderm cells and natural killer cells are produced.

[0334] The image above shows a photograph of the entire culture flask of mesodermal cells derived from induced pluripotent stem cell colonies. At this time, because the size of the colonies at the beginning of induced pluripotent stem cell differentiation cannot be photographed with a normal camera, only the entire culture vessel can be photographed for 3 to 4 days during the mesodermal cell differentiation stage.

[0335] In addition, the middle picture is a picture of the whole culture flask after differentiation into hematopoietic stem cells (differentiation day 12) after only removing the autologous feeder cells and the group with the autologous feeder cells remaining, and after differentiation into natural killer cells (differentiation day 25). Finally, the bottom picture is a picture of the stage of autologous feeder cell differentiation completion.

[0336] In addition, hematopoietic stem cells and natural killer cells were photographed according to the presence or absence of feeder cells. Specifically, the images were taken from differentiation day 0 to day 45 when the final differentiation into natural killer cells was completed, and the group in which the autologous feeder cells were removed and the group in which the autologous feeder cells were retained were photographed and compared on differentiation day 12 (the stage when hematopoietic stem cell differentiation was completed).

[0337] In addition, flow cytometry was used to confirm the total amount of suspension cells obtained at each differentiation stage and the expression rate of natural killer cell surface marker protein (marker) in the group retaining autologous feeder cells and the group removing autologous feeder cells ( Figure 6 , Figure 7 ).

[0338] Example 1.7. Analysis of the yield of natural killer cells according to the number of iPSCs seeded

[0339] In order to confirm the proliferation ability of the inoculated iPSCs according to the number of inoculated iPSCs, the method of the above-mentioned Examples 1.1-1.4 was used to add different numbers of iPSCs to the culture medium for culture. The number of iPSCs at the time of inoculation can be determined by the number of colonies of the cultured iPSCs.

[0340] Specifically, the number of cells was measured using the following method. First, the culture medium in the T-150 culture flask was removed and then washed with 1 mL of DPBS. 10 mL of TrypLE Express was added and reacted in a 37°C incubator for 15 minutes. The colonies attached to the scraper were collected and placed in a 50 mL test tube. At this time, the colonies were collected after a thorough check that there were no remaining colonies. Wash with 15 ml of complete culture medium to collect all the cells remaining in the culture flask. The cells were then collected in a test tube. Repeat step 4 again to make a total of 40 ml.

[0341] Centrifuge at 300g for 5 minutes. Remove the supernatant, tap the cells to dislodge them, and resuspend the cells in 1ml of complete medium. Add 9ml of complete medium to make a total of 10ml. Then, count the number of cells.

[0342] Specifically, iPSCs were propagated under the conditions described in Table 1 below to prepare natural killer cells.

[0343]

Table 1

[0344]

[0345]

[0346] at this time, Figure 8a The results obtained by analyzing the number of iPSC colonies adhered under each of the above conditions are shown. Specifically, different induced pluripotent stem cell colonies were formed under each condition, and then the entire culture vessel was photographed at the mesodermal cell stage (differentiation day 3 to 4). The number and colony size of iPSC colonies were measured. As a result, the colonies of induced pluripotent stem cells on differentiation day 0 could not be photographed at the corresponding magnification, and there was no difference in the number of colonies on differentiation day 0 and differentiation day 4. However, it was confirmed that the size of the iPSC colonies on the fourth day was larger. For the above Figure 8a The measurement results were analyzed and expressed as total number of cells / adherence vessel area (cm 2 ) is a numerical representation of ( Figures 8b to 8c ).

[0347] In addition, the morphological changes of differentiated cells at each stage from condition 1 to condition 6 were confirmed under an optical microscope ( Fig. 9 ).

[0348] In addition, the number of differentiated NK cells (suspension cells, day 35) in each condition was confirmed ( Fig.10), and the yield of suspension cells, i.e., differentiated natural killer cells, under each condition was confirmed based on the yield of suspension cells under condition 2 (Table 2). In addition, based on the results of condition 2, the number of suspension cells under each condition was analyzed by "the number of early differentiated pluripotent cell colonies / medium volume" (Table 3) and "the number of early differentiated pluripotent cell colonies / differentiation area" (Table 4).

[0349]

Table 2

[0350]

[0351]

Table 3

[0352]

[0353]

Table 4

[0354]

[0355] In order to confirm the characteristics of the natural killer cells derived from iPSCs induced to differentiate under the above conditions 1 to 6, the expression rate of natural killer cell surface marker proteins (markers) was confirmed by flow cytometry. The results are as follows: Fig.11 When differentiation was induced, the total number of natural killer cells (suspension cells) obtained under the above conditions was confirmed. Smaller iPSC colonies were formed compared to the above conditions 1 to 6, and then these colonies were induced to differentiate into mesodermal cells. As a result, Fig.12 , Fig.13 As shown in Table 5, it was confirmed that even in iPSC colonies with a size of 300 μm to 400 μm, differentiation into mesoderm cells was possible ( Figures 12 to 14 and Table 5).

[0356]

Table 5

[0357] condition average number of colonies Culture medium Colony size Condition A 328 60 300-400 Condition B 513 60 300-400 Condition C 1037 60 300-400 Condition D 1015 180 300-400

[0358] Example 1.8. Reproducible Experiment of Natural Killer Cells Derived from Induced Pluripotent Stem Cell Colonies

[0359] Under the same conditions as in Condition 2 above, the differentiation of induced pluripotent stem cells into natural killer cells was repeated, and the experimental results were summarized ( Figure 15 to 1 8).

[0360] Example 1.9. Preparation of natural killer cells derived from induced pluripotent stem cell colonies according to culture medium changes

[0361] Natural killer cells were prepared from induced pluripotent stem cells by the differentiation method of Examples 1.1 to 1.4. In this case, the same method was used to prepare natural killer cells ( Fig.19 ).

[0362] The above method was used to induce iPSCs to differentiate into natural killer cells, and flow cytometry was used to identify the surface marker proteins (markers) of natural killer cells in the cells obtained at each stage. The results are as follows Figures 20 to 25 shown.

[0363] Example 1.10. Massive proliferation of natural killer cells derived from induced pluripotent stem cells

[0364] The natural killer cells obtained in the same manner as in Examples 1.1 to 1.4 above were proliferated by a mass culture process. The mass culture process was performed every 2-3 days. Specifically, it was performed on days 0, 3, 5, 7, 10, 12, 14, 17, 19 and 21, and the cells were finally harvested on day 21.

[0365] The medium used in the mass culture process is used after adding other components to the basic medium described in Table 6. Specifically, the basic medium prepared in step 1 is composed of DMEM, Ham's F-12 nutrient mixture, human AB serum, penicillin-streptomycin and L-glutamine. Then, cytokines and factors selected from β-mercaptoethanol, sodium selenite, ethanolamine, L-ascorbic acid, SCF, IL-7, IL-15, FLT-3 ligand and IL-2 are added and used in the basic medium. Hereinafter, the "culture medium" is referred to as the "culture solution".

[0366]

Table 6

[0367]

[0368] In the mass culture process, first, on the day when differentiation is completed (day 0), the differentiated natural killer cells are stimulated with irradiated feeder cells for the first time. Specifically, natural killer cells and irradiated feeder cells are co-cultured in a 6-well plate containing 2 ml of culture medium at a ratio of 1:2. On the third day of culture, half of the culture medium, i.e., 1 ml, is replaced. At this time, the cells in the replaced culture medium are collected and cultured again. On the fifth day of culture, half of the culture medium, i.e., 1 ml, is replaced, and then 3 ml of new culture medium is added to increase the volume to 4 ml. In addition, in the subsequent culture process, when all processes are carried out to the 14th day of culture, half of the culture medium is replaced, the cells are collected, and the volume of the culture medium is increased.

[0369] When changing the culture container to a T-25 culture flask, replace half of the culture medium, i.e. 2 ml. Then add 10 ml of new culture medium to increase the volume to 12 ml. At this time, use a 24 mm scraper (TPP, 99002) to collect the cells adhered to the bottom of the 6-well plate, count them, and transfer them to the T-25 culture flask.

[0370] On the 10th day of culture, the culture container was changed from a T-25 flask to a T-75 flask. At this time, half of the culture medium, i.e., 6 ml, was replaced, and then 24 ml of new culture medium was added to increase the volume of the culture medium to 30 ml. A 30 mm scraper (TPP, 99003) was used to collect and transfer the cells attached to the bottom of the T-25 flask. On the 12th day of culture, the culture container was changed from a T-75 flask to a T-150 flask. At this time, half of the culture medium, i.e., 15 ml, was replaced, and then 60 ml of new culture medium was added to increase the volume of the culture medium to 75 ml. When changing the culture container, the cells attached to the bottom of the T-75 flask were also collected with a 30 mm scraper (TPP, 99003) and transferred to the T-150 flask.

[0371] On the 14th day of culture, the culture container was replaced with a 350 ml carbon dioxide permeation bag. At this time, half of the culture solution, i.e., 38 ml, was replaced, and then 151 ml of new culture solution was added to increase the volume of the culture solution to 188 ml. In addition, cells adhered to the bottom of the T-150 culture flask were collected as described above using a 30 mm scraper (TPP, 99003), counted, and then transferred to a 350 ml carbon dioxide permeation bag. At this time, a 50 ml Luer lock syringe (NORM-JECT, 48500003000) was used to perform the above process.

[0372] On the 17th day of culture, the volume of the culture medium was increased to 470 ml using a 50 ml Luer lock syringe. From this point on, half of the culture medium was continuously added without replacement. On the 19th day of culture, the culture container was replaced with a 1 liter carbon dioxide permeable bag and the volume of the culture medium was increased to 1175 ml.

[0373] On the 21st day of culture, the cells cultured in a 1-liter carbon dioxide permeable bag were finally collected. In this specification, natural killer cells induced to differentiate from induced pluripotent stem cells are referred to as "EiNK cells."

[0374] II. Confirmation of the characteristics of prepared natural killer cells

[0375] Experimental Example 1. Verification of the expression of major markers of natural killer cells derived from induced pluripotent stem cells

[0376] The method of Example 1.7 was used to obtain the iPSC-derived natural killer cells prepared by the methods of Examples 1.1-1.4, and the total number of suspended cells was confirmed. Then, the expression of the main markers of natural killer cells was confirmed by flow cytometry.

[0377] Specifically, NK cells prepared by the methods of Examples 1.1 to 1.4 were obtained, and the cell pellet was resuspended in FACS buffer (Invitrogen, 00-4222-57). The total number of cells was calculated according to the method of Example 1.7, and then 1×10 5 Each cell was placed in a FACS tube, and 1 μl of the FACS antibody shown in Table 7 was added to each 100 μl of FACS buffer and suspended. The tube was wrapped with aluminum foil to block light, and the staining was performed at a refrigerated temperature for 1 hour.

[0378] Add 1 mL of FACS buffer to the stained cells, centrifuge at 300 g for 5 minutes, and then remove the supernatant. Add 1 mL of FACS buffer again to resuspend the cell pellet, centrifuge at 300 g for 5 minutes, and repeat twice. Finally, resuspend the cells in 500 μl of FACS buffer and use The expression of cell surface markers was detected by flow cytometry (Agilent, NovoCyte 3005) and analyzed using NovoExpress software (Agilent, Ver.1.5.0).

[0379]

Table 7

[0380]

[0381]

[0382] Experimental Example 2. Evaluation of cytotoxicity of natural killer cells derived from induced pluripotent stem cell colonies

[0383] The cytotoxicity function of the induced pluripotent stem cell-derived natural killer cells prepared by the methods of Examples 1.1 to 1.4 above was evaluated using Abcam's cytotoxicity assay kit (Abcam, ab270780). 5 K562 cells of chronic myeloid leukemia cell line were suspended in 180 μl of measurement buffer (Abcam, ab270780) + 20 μl of CSFE solution (Abcam, ab270780), and then reacted at room temperature for 15 minutes to perform CSFE staining.

[0384] After centrifugation at 1000 rpm for 3 minutes, the supernatant was removed and the CSFE-stained K562 cells and the induced pluripotent stem cell-derived natural killer cells (EiNK cells) prepared by the methods of Examples 1.1 to 1.4 above were mixed in 400 μl of RPMI1640 (Hyclone, SH30027.01) culture medium containing 10% FBS (Gibco, 10100147) at cell ratios of 1:10, 1:5, 2:1 and 1:1, respectively, and cultured in a cell culture incubator for 4 hours.

[0385] After the incubation, 20 μl of 7-AAD (Abcam, ab270780) solution was mixed with the cell suspension and reacted at 4°C for 10 minutes. The cytotoxicity level was confirmed by flow cytometry (Agilent, NovoCyte 3005) and analyzed using NovoExpress software (Agilent, Ver. 1.5.0). Fig.26 shown.

[0386] Experimental Example 3. Verification of the characteristics of natural killer cells that proliferate in a short period of time

[0387] Experimental Example 3.1. Morphological verification of prepared natural killer cells

[0388] The iPSC-derived natural killer cells (EiNK cells) prepared by the methods of Examples 1.1 to 1.4 were massively proliferated using the method of Example 1.10, and the morphological changes of the cultured natural killer cells were observed using an optical microscope. Specifically, according to the massive proliferation process of induced pluripotent stem cell-derived natural killer cells, the culture time after the first, second, and third co-culture with feeder cells and the morphology of natural killer cells during the massive proliferation process for 21 days were confirmed using an optical microscope (Olympus, Olympus-CKX53). Fig.28 ).

[0389] Experimental Example 3.2. Confirmation of production yield by inducing a large-scale proliferation process of natural killer cells derived from pluripotent stem cells

[0390] When the method of Example 1.10 was used for large-scale proliferation culture, the yield of induced pluripotent stem cell-derived natural killer cells (EiNK cells) prepared in the same manner as in Examples 1.1 to 1.4 was analyzed by the following method.

[0391] Specifically, during and after the mass culture of induced pluripotent stem cell-derived natural killer cells, all cells suspended in the cell culture container and the natural killer cell culture medium were obtained and centrifuged at 300 g for 5 minutes using a centrifuge (Labogene, 1580R). The cell pellet obtained by centrifugation was resuspended in the natural killer cell culture medium, and the total number of cells in the suspension was counted using an Incyto c-chip (Incyto, DHC-N01-5). The results are as follows: Fig.29 and as shown in Table 8.

[0392]

Table 8

[0393]

[0394]

[0395] Experimental Example 3.3. Verification of cell marker expression of massively proliferating natural killer cells derived from induced pluripotent stem cells

[0396] The method of Example 1.7 was used to obtain natural killer cells derived from induced pluripotent stem cells prepared in the same manner as in Examples 1.1-1.4, and the total number of suspended cells was confirmed. Then, the expression of major markers of natural killer cells was confirmed by flow cytometry.

[0397] Specifically, a large number of proliferating natural killer cells derived from induced pluripotent stem cells were obtained, and then the cell pellet was resuspended in FACS buffer (Invitrogen, 00-4222-57). 5 The resuspended cells were placed in FACS tubes, and then 1 μl of each antibody bound to a cell marker (activation marker protein) was added to each 100 μl staining buffer and suspended. The tubes were wrapped with aluminum foil to block light and stained at refrigerated temperature for 1 hour.

[0398] Add 1 mL of staining buffer to the stained cells, centrifuge at 300 g for 5 minutes, remove the supernatant, and resuspend in 1 mL of staining buffer. Centrifuge the resuspended solution at 300 g for 5 minutes. Repeat the resuspension and centrifugation process twice. Finally, resuspend the cells in 500 μl of staining buffer and use The expression of cell markers was detected by flow cytometry (Agilent, NovoCyte 3005), and the measured values ​​were analyzed using NovoExpress software (Agilent, Ver.1.5.0). At this time, the antibodies used are shown in Tables 7 and 9. In addition, anti-IgG1 antibody (BD, 555751) and anti-IgG2aκ antibody (BD, 559319) were also used.

[0399]

Table 9

[0400] Surface markers Supplier (Catalog Number) NKG2D BD(561815) DNAM1 BD(559789)

[0401] Results, such as Fig.30a and 30b As shown, the expression rate of NKG20, one of the natural killer cell activation receptors, is about 90% or more. NKG2D is an important receptor for recognizing heterogeneous tumor cells and is also an important receptor for combined treatment with anticancer drugs that increase the expression of NKG2D ligands in tumor cells. In addition, it was confirmed that the expression rates of DNAM1 and NKp30 were about 90%. The expression rate of CD16, which is involved in antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer cells, is about 50%. At this time, this receptor plays a role in mediating other forms of MHC-unrestricted killing.

[0402] Experimental Example 3.4. Verification of the characteristics of massively proliferating natural killer cells derived from induced pluripotent stem cells

[0403] In order to confirm the killing ability of induced pluripotent stem cell-derived natural killer cells (EiNK cells) prepared in the same manner as in Examples 1.1 to 1.4 on tumor cells, EiNK cells were obtained on the 21st and 28th days of massive proliferation using the method of Example 1.7. The obtained natural killer cells were counted and cultured with a blood cancer cell line (K562) stained with the fluorescent substance CFSE (Abcam, ab270780). At this time, the fluorescently stained blood cancer cell line was prepared by the following method. First, CFSE was diluted at a ratio of 1:250, and then K562 cells were treated with CFSE and reacted at room temperature for 15 minutes.

[0404] 1 mL of cell culture medium was added to the stained cells, centrifuged at 2000 rpm for 3 minutes, and then the supernatant was removed. 2 ml of cell culture medium was added to the cells again, reacted in a 37°C 5% CO2 incubator for 30 minutes, and then centrifuged at 2000 rpm for 3 minutes to obtain stained K562 cells. The K562 cells and natural killer cells prepared by the above method were counted, inoculated into each well according to the E:T ratio, and co-cultured for 4 hours.

[0405] After 4 hours of co-culture, all co-cultured cells were placed in a FACS tube and centrifuged at 2000 rpm for 3 minutes. After centrifugation, 1 mL of saline was added to resuspend the cells, and then centrifuged at 2000 rpm for 3 minutes, and the supernatant was removed. The above resuspension and centrifugation were repeated twice. Then, the cells were suspended in 200 μl of staining buffer, treated with 7-AAD (Abcam, ab270780), and then reacted on ice for 10 minutes.

[0406] After the reaction was completed, cells stained with both CFSE and 7-AAD were used The flow cytometer (Agilent, NovoCyte 3005) was used for analysis. The measured values ​​were analyzed using NovoExpress software (Agilent, Ver.1.5.0). Fig.31 As shown in Table 10.

[0407]

Table 10

[0408]

[0409] Experimental Example 3.5. Verification of the characteristics of massively proliferating natural killer cells derived from induced pluripotent stem cells

[0410] Using the same method as in Experimental Example 3.4 above, a large number of natural killer cells derived from induced pluripotent stem cells (EiNK cells) cultured for 26 days were obtained, the total number of suspended cells was confirmed, and the expression of iPSC markers (marker proteins, Table 11) in the obtained cells was confirmed by flow cytometry.

[0411] For flow cytometry, specifically, first obtain the natural killer cells prepared by the method of Example 1 above, and then resuspend the cell pellet in FACS buffer (Invitrogen, 00-4222-57). Count the total number of cells, and then 1×10 5 Place cells in a FACS tube. Add 1 μl of the antibody shown in Table 5 to every 100 μl of FACS buffer and suspend. Wrap the tube with aluminum foil to block light and react at refrigerated temperature for 1 hour to stain the cells. After staining, add 1 mL of FACS buffer to the cells and centrifuge at 300 g for 5 minutes. After removing the supernatant, resuspend the cell pellet in 1 mL of FACS buffer and centrifuge again at 300 g for 5 minutes to wash the cells. Repeat the washing process twice. Finally, add 500 μl of FACS buffer to resuspend the cells and use The expression of iPSC markers on the cell surface was detected by flow cytometry (Agilent, NovoCyte 3005), and the measured values ​​were analyzed using NovoExpress software (Agilent, Ver. 1.5.0).

[0412] Results, such as Fig.32 As shown, it was confirmed that the expression rate of induced pluripotent stem cell marker factors in natural killer cells derived from induced pluripotent stem cells cultured in large quantities was 1% or less. Based on the above results, it was confirmed that there were almost no induced pluripotent stem cells remaining in the cells after differentiation induction.

[0413]

Table 11

[0414] Antigen (clone) Manufacturer Catalog Number Tra-1-60(TRA-1-60) eBioscience 12-8863-82 SSEA4(MC-813-70) eBioscience 12-8843-42 Mouse IgM (G155-228) BD 555584 Mouse IgG3(B10) eBioscience 12-4742-42

[0415] Experimental Example 4. Verification of the characteristics of long-term and massively proliferating natural killer cells derived from induced pluripotent stem cells

[0416] Experimental Example 4.1. Verification of the yield of long-term and massively proliferating natural killer cells derived from induced pluripotent stem cells

[0417] The EiNK cells prepared by the methods of Examples 1.1 to 1.4 were cultured for up to 63 days, and then the proliferation rate of the EiNK cells was confirmed. At this time, β-mercaptoethanol (Gibco, 21985-023), sodium selenite (Sigma, S5261-100G), L-ascorbic acid (Sigma, A4544-100G) and IL-15 (R&D systems, 247-ILB-500 / CF) were added to RPMI1640 (Cytiva, SH30027.01) containing human AB serum (Gemcell, 100-512) and penicillin-streptomycin (Gibco, 15140-122) as EiNK cell culture medium.

[0418]

Table 12

[0419]

[0420]

[0421] EiNK cells were stimulated with irradiated feeder cells every 7 days for 63 days. Fig.33 As shown, the proliferation of EiNK cells cultured for 63 days was 6.5×10 of the initial cultured cells. 10 By confirming the results in logarithmic values, it can be confirmed that the expansion of cells follows a specific period of doubling at a constant rate.

[0422] Experimental Example 4.2. Verification of the cell killing ability of iPSC-derived, long-term, and massively proliferating natural killer cells

[0423] The phenotype of EiNK cells prepared by the same method as in Experimental Example 4.1 above, the phenotype of natural killer cells, and the killing ability against K562 cells were confirmed. The phenotype was confirmed in the same manner as in Experimental Example 3.3 and Experimental Example 3.5, and the killing ability was confirmed in the same manner as in Experimental Example 3.4.

[0424] Results, such as Fig.34aAs shown, it was confirmed that in EiNK cells cultured for 63 days, the expression rate of natural killer cell CD56+CD45 was 99%, and the expression rate of T cell phenotype CD56-CD3+ was 0.06%, indicating that there were almost no T cells. In addition, the induced pluripotent stem cell phenotype (TRA-1-60+, SSEA-4+) was confirmed in EiNK cells cultured for 63 days, and its expression rate was 1% or less, indicating that there were almost no induced pluripotent stem cells.

[0425] In addition, if Fig.34b As shown, the cell killing ability of EiNK cells cultured for 20 days, 56 days and 63 days against cancer cells was confirmed. The results show that a high level of cancer cell killing ability can be observed regardless of the length of culture time. Based on the above results, it is confirmed that the EiNK cells of the present invention still maintain a high cytotoxicity after long-term culture.

[0426] Experimental Example 5. Verification of the anticancer activity of iPSC-derived natural killer cells: in vivo

[0427] The anti-cancer activity of EiNK cells cultured in large quantities (cultured for 21 days) using the method of Example 1.10 above was confirmed at the animal level. This time, 6-week-old female NSG mice were used.

[0428] Specifically, luciferase was overexpressed in the ovarian cancer cell line OVCAR3 cells, and 2×10 6 EiNK cells were used as test substances, thawed 2 days before administration, stabilized in a T-75 culture flask for 2 days, and then cultured at 1×10 cells / 100 μl per mouse. 7 The cells were intraperitoneally administered at a concentration of 10 cells / 100 μl (PBS, vehicle). At this time, RPMI medium containing 5%-15% human AB serum, 1% L-glutamine, 50 IU / ml IL-2 and 10 ng / ml IL-15 was used as the culture medium for the thawed EiNK cells. The vehicle-treated group was used as the control group.

[0429] The test substance (EiNK) was administered on the 2nd day (Day 0), 4th day (Day 2), 6th day (Day 4), 9th day (Day 7), 11th day (Day 9), 13th day (Day 11), 16th day (Day 14), 18th day (Day 16), 20th day (Day 18), 23rd day (Day 21), 25th day (Day 23) and 27th day (Day 25) after tumor cell administration, for a total of 12 times. At the same time, IL-2 (10000 IU / mouse) and IL-15 (0.5 ug / mouse) were injected intraperitoneally every 2 to 3 days for 28 days. After the administration of the test substance, the weight of the mice was measured every 7 days from Day 0 for a total of 3 times. In addition, luciferin was injected into the right abdominal cavity of mice on days 0, 3, 7, 10, 14, 17, 21, 24, and 28, and the presence of tumor cells in the mice was confirmed using IVIS imaging equipment after 10 minutes of reaction.

[0430] Results, such as Figures 40a to 40e As shown, it was confirmed that in the control (vehicle) group, luciferase activity increased rapidly after the 17th day. The results showed that over time, the OVCAR3 cells in the peritoneal cavity gradually proliferated in vivo. On the other hand, in the test substance treatment group, tumor cells continued to decrease from the second day (day 0) of the test substance administration, and after the third day, almost no proliferated tumor cells were found in the peritoneal cavity. Based on the above results, it was confirmed that EiNK cells have anti-cancer activity at the animal level.

[0431] III. Comparison of iPSC-derived NK cells with blood-derived NK cells

[0432] Experimental Example 6. Comparison of cell surface marker expression between iPSC-derived, massively proliferating natural killer cells and blood-derived natural killer cells

[0433] EiNK cells cultured in large quantities by the method of Example 1.10 (cultured for 21 days) and natural killer cells (PBNK, cultured for 22 days) separated from blood and cultured by the method of Example 1.10 were counted. The expression of natural killer cell surface marker proteins (markers) was confirmed by flow cytometry.

[0434] At this time, the expression of cell surface markers was analyzed using the BD Lyoplate Human Cell Surface Screening Panel (BD, 560747). EiNK cells and PBNK cells were labeled with a panel of 242 human cell surface marker antibodies.

[0435] Specifically, EiNK cells were collected and counted on the 21st day of mass culture, and PBNK cells were collected and counted on the 22nd day of culture. Then, the cells were resuspended in staining buffer (FACS buffer) at a concentration of 2.5×106 cells / ml. The resuspended cells were concentrated to 2.5×10 5 The cells were seeded into each well of a 96-well plate at a density of 1000 cells / well.

[0436] Each inoculated cell was labeled with 242 primary antibodies for 30 minutes, then the cells were washed with staining buffer and centrifuged (300g, 4 minutes), and the supernatant was removed. The primary antibodies were washed, and then Alexa 647-labeled secondary antibodies (mouse or rat) were added to each primary antibody and reacted for 30 minutes. After the secondary antibody reaction, the cells were washed with staining buffer, centrifuged at 300g for 4 minutes, and the supernatant was removed. The stained cells were measured by flow cytometer (Agilent, NovoCyte 3005), and the measured values ​​were analyzed using NovoExpress software (Agilent, Ver. 1.5.0).

[0437] Among them, the results of cell surface markers showed the difference between EiNK cells proliferating for 21 days and PBNK cells cultured for 22 days, see Fig.35a and 35b , Table 13 and Table 14.

[0438]

Table 13

[0439]

[0440]

[0441]

Table 14

[0442] Cell surface markers PBNK E Supplier (clone, catalog number) CD243 63.88 0.28 BD(17F9,560747) γδTCR 46.44 0.28 BD(B1,560747) NKB1 56.14 0.42 BD(DX9,560747) CD205 82.8 2.02 BD(MG38,560747) CD158b 99.08 2.54 BD(HP-3E4,560747) CD158a 34.1 0.4 BD(HP-3E4,560747) CDw328 24.72 1.22 BD(F023-420,560747)

[0443] The results confirmed that among the 242 surface markers, 26 markers were expressed at higher rates in EiNK cells than in PBNK cells. It was confirmed that the markers that increased more than 20 times compared with PBNK cells were CD25, CD9, CDw327, and CD30, and the markers that increased more than 10 times were CD80 and CD114. In addition, compared with PBNK cells, the markers that increased more than 5 times were CD124, MIC A_B, CD210, CD87, CD123, CD83, and CD146, and the markers that increased more than 1.3 times were CD106, CD196, CD33, CD150, CD49e, CD108, CD49f, CD164, CD49c, CD66 (a, c, d, e), CD49a, CDw93, and CD71. On the other hand, there were 7 markers that were expressed only in blood-derived natural killer cells.

[0444] Experimental Example 7. Comparison of gene expression between induced pluripotent stem cell-derived natural killer cells and blood-derived natural killer cells

[0445] For EiNK cells cultured in large quantities by the method of Example 1.10 above (cultured for 28 days), and natural killer cells (PBNK, cultured for 22 days) isolated from blood and cultured by the method of Example 1.10, RNA of natural killer cells was extracted, and transcriptome sequencing and data analysis were performed using QuantSeq 3'mRNA-Seq. Whole genome RNA sequencing analysis was performed by outsourcing to Ebiogen (South Korea). ExDEGA (Excel-based differentially expressed gene tool) was used to analyze gene expression changes in sequencing results.

[0446] Results, such as Fig.36 As shown, it was confirmed that the expression of factors involved in tumor cell killing and factors involved in immune activation were increased in EiNK cells compared with PBNK cells.

[0447] Experimental Example 8. Comparative Analysis of Chemokines and Cytokines Secreted from Culture Medium of iPSC-derived Natural Killer Cells and Blood-derived Natural Killer Cells

[0448] For EiNK cells cultured in large quantities by the method of Example 1.10 above (cultured for 28 days), and natural killer cells (PBNK, cultured for 22 days) isolated from blood and cultured by the method of Example 1.10, the cytokines of natural killer cells in the culture medium were analyzed. Cytokine analysis was performed using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, catalog number: ARY022B) in accordance with the manufacturer's specifications. The measurement results of the cytokine array kit were analyzed using GelQuantNet software (BiochemLabSolutions) to obtain the relative point intensity with the reference control point. Table 15 lists the cytokines, chemokines, and growth factors used in the cytokine array.

[0449] Specifically, 102 cytokines, chemokines or cell growth factors were qualitatively analyzed according to the following process. After preparing all reagents and culture fluid samples of the provided products at room temperature, array buffer 6 and membrane were added to each well of the pre-designed four-well plate, and reacted on a shaker at room temperature for 1 hour. Then, the solution was discarded, and array buffer 6 was added to the culture fluid sample to make the final volume 1.5 ml (the culture fluid sample was diluted to 1 / 10 of the original concentration before use). After maintaining 2 to 8°C in a shaker and reacting for 12 hours or more, the sample was washed three times with a washing solution, each for 10 minutes. The detection antibody cocktail (60ul) was added to the mixture (6ml) of the provided array buffer 6 (4ml) and array buffer 4 (2ml), and 1.5ml was added to each well, and reacted for 1 hour at room temperature. Then, the wells were washed 3 times with a washing solution, each for 10 minutes, and Streptavidin-HRP was added to each well and reacted for 30 minutes. Then, the wells were washed three times with washing solution for 10 minutes each time, and then the Chemi reagent mixed solution was added, reacted for 1 minute, and the luminescence level was confirmed (Chemi-doc photography).

[0450] Results, such as Fig.37a and 37b As shown, it was confirmed that the concentrations of chemokines and cytokines released by activated natural killer cells in the culture medium of EiNK cells were higher than those of PBNK cells.

[0451]

Table 15

[0452]

[0453]

[0454] Experimental Example 9. Comparative Analysis of Inhibitory Receptor and Cell Surface Marker Expressions of iPSC-derived Natural Killer Cells and Blood-derived Natural Killer Cells

[0455] The expression of natural killer cell inhibitory receptors and cell surface markers was confirmed by flow cytometry for EiNK cells cultured in large quantities by the method of Example 1.10 (cultured for 21 days) and natural killer cells (PBNK, 21 days) separated from blood and cultured by the method of Example 1.10. The above experiment was repeated 4 times.

[0456] The methods of obtaining and using cells were the same as those in Experimental Example 3.4, and the FACS analysis method was the same as that in Experimental Example 3.5. At this time, the antibodies used for analysis are shown in Table 7, Table 11, and Table 16.

[0457]

Table 16

[0458]

[0459]

[0460] Results, such as Fig.38 As shown in the figure, the expression rate of NKG2A / CD94PBNK, a marker protein expressed in mature natural killer cells, is similar in PBNK cells and EiNK cells. On the other hand, TIM-3 and TIGIT, as inhibitory receptors with the function of inhibiting immune activity, have been confirmed to be lower in EiNK cells than in PBNK cells. Based on the above results, it can be considered that the immune activation ability of EiNK cells is better than that of PBNK cells.

[0461] Experimental Example 10. Comparison of the tumor cell killing ability of iPSC-derived natural killer cells and blood-derived natural killer cells

[0462] The tumor cell killing ability of natural killer cells was confirmed for EiNK cells cultured in large quantities by the method of Example 1.10 above (cultured for 21 days or 28 days), and natural killer cells (PBNK, cultured for 16 days) separated from blood and cultured by the method of Example 1.10. Each cell was obtained by the method of Example 3.4, and the tumor cell killing ability was confirmed by the method of Example 3.4. The above test to confirm the killing ability was repeated 7 times.

[0463] Results, such as Fig.39 As shown, it was confirmed that the killing ability of EiNK cells and PBNK cells against tumor cells was similar. However, the killing ability of PBNK cells showed differences due to different blood donors, while the killing ability of EiNK cells showed similar levels in different batches of culture.

[0464] IV. Comparison of the method for differentiating natural killer cells from induced pluripotent stem cells of the present invention with conventional methods for differentiating natural killer cells from induced pluripotent stem cells

[0465] Experimental Example 11. Comparison of cell yield and proliferation rate of CD34+ sorted cells

[0466] Fig.42a The results are shown in which changes in cell yield, cell surface marker expression, and cell proliferation rate were confirmed by sorting cells positive for CD34, a hematopoietic stem cell differentiation marker, during the conventional differentiation method for induced pluripotent stem cell-derived natural killer cells.

[0467] Specifically, the method for inducing natural killer cell differentiation from conventional iPSCs is as follows.

[0468] To induce differentiation of pluripotent stem cells (iPSCs), iPSCs were seeded on Aggrewell plates (aggrewell 400, STEMCELL, 34425) and then induced to form embryoid bodies (EBs) for 5 days, with EB-A medium (STEMdiff TM NK Cell Kit, STEMCELL, 100-0170). At this point, only 50% of the existing medium was removed, and new medium was added according to the amount removed. In order to collect only EBs larger than a certain size, the medium containing EBs was passed through a filter (reversible 37 μm, STEMCELL, 27215) to remove single cells or small EBs. After washing with medium to collect EBs, they were seeded into a 6-well plate that was not treated with tissue culture (STEMCELL, 38040), and the EB-B medium (STEMdiff) was replaced every 2 to 3 days. TM NK cell kit, STEMCELL, 100-0170), and cultured for 7 days. At this time, the culture medium was replaced as described above.

[0469] The cultured EBs were collected, reacted with collagenase (collagenase type IV, STEMCELL, 07909) for 20 minutes, and reacted with TrypLE Select (Gibco, A12563011) for 20 minutes (reacted with the degradation enzyme for a total of 40 minutes), and separated into single cells. The separated cells were sorted using the Easysep Human CD34 Positive Selection Kit II (STEMCELL, 17856), and only cells expressing CD34 were selected. At this time, the sorting method was to collect the suspended cells, mix them with anti-CD34 coupled magnetic bead antibodies (CD34 microbead kit, Miltenyi Biotec, 130-046-702), load them into the LC column attached to the MACS sorter (Miltenyi Biotec, 130-042-401), wash them several times with DPBS (Cytiva, SH3002802), and collect the remaining cells in the column.

[0470] The isolated cells were plated in 24-well plates (STEMCELL, 38042) that were not treated with tissue culture. The medium was changed every 3 to 4 days. The medium was changed as described above. From day 12 to day 19 after differentiation induction, lymphocyte differentiation medium (STEMdiff TM NK cell kit, STEMCELL, 100-0170), lymphocyte progenitor expansion medium (STEMdiff TMNK cell kit, STEMCELL, 100-0170). On the 26th day of differentiation induction, all cells were collected and counted, and the cells were re-seeded into 24-well plates that were not treated with tissue culture (STEMCELL, 38042) for culture. As described above, the culture medium was changed every 3 to 4 days. On the 41st day of differentiation induction, all cells were collected and subjected to flow cytometry analysis and cell counting to confirm the results of natural killer cell differentiation.

[0471] As described above, when inducing induced pluripotent stem cells to differentiate into natural killer cells using conventional methods, mesoderm cells are first induced to differentiate into hematopoietic stem cells, and then only CD34-positive cells (hematopoietic stem cells) are obtained and induced to differentiate into NK cells. When CD34-positive cells are sorted and used in this way, it is confirmed that the cell yield is significantly reduced, cell death is increased, and the expression rate of natural killer cell marker protein (CD56) and activation marker protein (NKp30) is reduced in the sorted CD34-positive cells ( Fig.42a and 42b In addition, it was confirmed that the cell proliferation rate was also significantly reduced ( Fig.42c ).

[0472] On the other hand, the EiNK preparation method according to the present invention can induce differentiation into NK cells without CD34 sorting, because the hematopoietic stem cell differentiation marker CD34, which represents the intermediate stage of NK differentiation, has an expression rate of more than 95%, thereby improving cell yield and activity (Figure 42).

[0473] Experimental Example 12. Comparison of cell survival rates between the third and second iPSC cultures

[0474] Fig.43 and Fig.44 These are the results obtained by confirming the cell survival rate when cells were obtained by culturing induced pluripotent stem cells using the third culture method.

[0475] Typically, induced pluripotent stem cells are cultured using the third culture method and induced to differentiate using the method described in Experimental Example 11.

[0476] like Fig.43 and Fig.44 As shown, it is confirmed that when iPSC is cultured in the third culture using Aggrewell, the size of the embryoid body is larger than that of the second culture. In addition, it is also confirmed that there are differences in the size of embryoid bodies in different batches, and when single cells are obtained by degradation enzyme treatment, the cell survival rate decreases. On the other hand, it can be considered that the method for differentiating EiNK cells according to the present invention is simpler and more effective than the third culture, because iPSC is subjected to the second culture and induced to differentiate.

[0477] Experimental Example 13. Comparison of the differentiation induction period of natural killer cells derived from induced pluripotent stem cells

[0478] Fig.45 and Fig.46 The comparison results of the cycles of preparing natural killer cells by the conventional EiNK differentiation method (general method) and the differentiation method according to the present invention (method of the present invention) are shown, as well as the confirmation results of the phenotype of EiNK prepared according to the present invention. The conventional EiNK differentiation process is the same as described in Experimental Example 11.

[0479] The traditional EiNK differentiation method requires a total of 48 days of differentiation induction period, while the EiNK differentiation method according to the present invention requires a differentiation induction period of 26 days. By verifying the natural killer cell marker proteins (CD56, CD45, NKp44) and activation proteins (CD56, NKp30) in EiNK cells induced for differentiation for 26 days, it was confirmed that the expression rate of NK marker proteins and activation proteins reached 66% or higher. On the other hand, it was found that the expression rate of iPSC marker proteins (TRA-1-60, SSEA4) was 0%, indicating that compared with the traditional method, the EiNK cell differentiation method according to the present invention is an efficient method for preparing natural killer cells in a short time.

Claims

1. A natural killer cell expressing any one protein selected from the group consisting of CD25, CD9, CD80 and a combination thereof.

2. The natural killer cell according to claim 1, wherein The natural killer cells additionally express any one protein selected from the group consisting of CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, CD210 and a combination thereof.

3. The natural killer cell according to claim 1, wherein The natural killer cells express any one natural killer cell marker protein selected from the group consisting of CD56, CD45, CD16, NKp30, NKp44, NKG2D, DNAM1 and a combination thereof.

4. The natural killer cell according to claim 1, wherein The natural killer cells have increased expression of a protein as defined in claim 1 or 2 compared to natural killer cells isolated from blood.

5. The natural killer cell according to claim 4, wherein The expression of the protein was increased at least 2-fold compared to natural killer cells isolated from blood.

6. The natural killer cell according to claim 1, wherein The natural killer cells additionally have increased expression of any one gene selected from the group consisting of GZMB, CD226, RASGRP1, CD107a, CCR9, CD40LG, CD300c and a combination thereof compared to natural killer cells isolated from blood.

7. The natural killer cell according to claim 1, wherein The natural killer cells secrete an increased amount of any one protein selected from the group consisting of FLT3L, MIP1alpha / beta, PF4, IGFBP2, IGFBP3, MMP9 and a combination thereof, compared to natural killer cells isolated from blood.

8. The natural killer cell according to claim 7, wherein The secretion amount of the protein was increased at least 2-fold compared to natural killer cells isolated from blood.

9. The natural killer cell according to claim 1, wherein Compared with natural killer cells isolated from blood, the natural killer cells do not additionally express or express at a low level any one protein selected from the group consisting of CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM3, TIGIT and a combination thereof.

10. The natural killer cell according to claim 9, wherein Compared with natural killer cells isolated from blood, the expression of the protein was reduced by at least 1 / 2.

11. The natural killer cell according to claim 1, wherein The natural killer cells do not express CD3, a marker protein of T cells.

12. The natural killer cell according to claim 1, wherein The natural killer cells are differentiated from induced pluripotent stem cells (iPSCs).

13. The natural killer cell according to claim 12, wherein The natural killer cells are prepared by a method for preparing natural killer cells, comprising: Induced pluripotent stem cells (iPSCs) are seeded into a cell culture container as iPSCs or iPSC spheroids to form 0.5 to 20 colonies per 1 square centimeter of the cell culture container area, and adhesion culture is performed to prepare iPSC colonies; differentiating the iPSC colonies into mesodermal cells to prepare mesodermal cells; differentiating the mesoderm cells into hematopoietic stem cells to prepare hematopoietic stem cells; The hematopoietic stem cells are differentiated into natural killer cells to prepare natural killer cells.

14. The natural killer cell according to claim 13, wherein The seeded iPSCs are seeded at a density of 1 to 8 iPSCs per 1 square centimeter of the cell culture container area.

15. The natural killer cell according to claim 13, wherein The seeded spheroids were seeded at a density of 1 to 8 spheroids per 1 cm2 of the cell culture vessel area.

16. The natural killer cell according to claim 13, wherein The total number of stem cells on the differentiation initiation day was 1×10 per 1 cm2 of the cell culture container area. 3 Up to 1×10 6 cells.

17. The natural killer cell according to claim 13, wherein During the differentiation of the mesodermal cells into hematopoietic stem cells, the cells adhered to the culture container are not removed.

18. The natural killer cell according to claim 13, wherein The cells differentiated from the mesodermal cells into hematopoietic stem cells have a CD34 protein expression rate of 85% or more without a CD34-positive cell sorting process.

19. The natural killer cell according to claim 13, wherein The natural killer cells are prepared by additionally culturing and proliferating the induced differentiated natural killer cells in a large amount.

20. The natural killer cell according to claim 19, wherein The large-scale culture proliferation refers to culturing the induced differentiated natural killer cells in a culture medium containing SCF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand), IL-7, IL-15, IL-2 or a combination thereof.

21. The natural killer cell according to claim 19, wherein The large-scale culture proliferation refers to culturing the induced differentiated natural killer cells in a culture medium containing β-mercaptoethanol, sodium selenite, ethanolamine, L-ascorbic acid or a combination thereof.

22. The natural killer cell according to claim 19, wherein The mass culture proliferation of the induced differentiated natural killer cells is carried out for 1 day to 9 weeks.

23. A cell therapeutic agent comprising the natural killer cell according to any one of claims 1 to 22 as an active ingredient.

24. A pharmaceutical composition for preventing and treating a disease, comprising the natural killer cell according to any one of claims 1 to 22 as an active ingredient.

25. The pharmaceutical composition for preventing and treating a disease according to claim 24, wherein: The disease is any one selected from the group consisting of infectious diseases, cancers, nervous system diseases and autoimmune diseases.

26. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein: The infectious disease is any one selected from the group consisting of hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease and influenza.

27. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein: The cancer is any one selected from the group consisting of liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma.

28. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein: The nervous system disease is a nervous system disease caused by nerve damage or nerve abnormality, or a degenerative brain disease.

29. The pharmaceutical composition for preventing and treating a disease according to claim 28, wherein: The nerve injury is any one selected from the group consisting of neurapraxia, axonotmesis and neurotmesis.

30. The pharmaceutical composition for preventing and treating a disease according to claim 28, wherein: The nervous system disease caused by nerve damage or nerve abnormality is a nervous system disease caused by nerve damage or nerve abnormality in the central nervous system, or a nervous system disease caused by nerve damage or nerve abnormality in the peripheral nervous system.

31. The pharmaceutical composition for preventing and treating a disease according to claim 30, wherein: The neurological disease caused by nerve damage or nerve abnormality in the central nervous system is any one selected from the group consisting of organic diseases and functional disorders of the central nervous system, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Lewy body dementia, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, multiple system atrophy, cortical basal degeneration, spinocerebellar degeneration, cerebellar atrophy, post-traumatic stress disorder, amnesia, vascular dementia, and cerebral infarction.

32. The pharmaceutical composition for preventing and treating a disease according to claim 30, wherein: The nervous system disease caused by nerve damage or nerve abnormality in the peripheral nervous system is any one selected from the group consisting of peripheral neuropathy, diabetic neuropathy, peripheral neuropathic pain, peripheral neuropathy caused by tumor chemotherapy, complex regional pain syndrome, optic neuropathy, mononeuropathy, multiple mononeuropathy (mononeuritis multiplex), polyneuropathy, Guillain-Barre syndrome (acute inflammatory demyelinating polyneuropathy), chronic inflammatory demyelinating polyneuropathy, hereditary neuropathy, plexus disorder, glaucoma, macular degeneration, amyotrophic lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, poliomyelitis, post-polio syndrome, stiff-man syndrome, Isaac syndrome, myasthenia gravis, neonatal myasthenia, botulism, Eaton-Lambert syndrome, thoracic outlet syndrome, hereditary neuromuscular atrophy, and spinal muscular atrophy.

33. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein: The nervous system disease is a degenerative brain disease.

34. The pharmaceutical composition for preventing and treating a disease according to claim 33, wherein: The degenerative brain disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette syndrome, Friedrich's ataxia, Machado-Joseph disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

35. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein: The autoimmune disease is any one selected from the group consisting of Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guillain-Barré syndrome, Sjögren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic sclerosis, asthma, and ulcerative colitis.

36. A method for preparing natural killer cells, comprising: Induced pluripotent stem cells (iPSCs) are seeded into a cell culture container as iPSCs or iPSC spheroids to form 0.5 to 20 colonies per 1 square centimeter of the cell culture container area, and adhesion culture is performed to prepare iPSC colonies; differentiating the iPSC colonies into mesodermal cells to prepare mesodermal cells; differentiating the mesoderm cells into hematopoietic stem cells to prepare hematopoietic stem cells; The hematopoietic stem cells are differentiated into natural killer cells to prepare natural killer cells.

37. The method for preparing natural killer cells according to claim 36, wherein: The preparation method further comprises culturing and proliferating the induced differentiated natural killer cells in large quantities.

38. The method for preparing natural killer cells according to claim 36, wherein: The total number of stem cells on the differentiation initiation day was 1×10 per 1 cm2 of the cell culture container area. 3 Up to 1×10 6 cells.

39. The method for preparing natural killer cells according to claim 36, wherein: During the differentiation of the mesodermal cells into hematopoietic stem cells, the cells adhered to the culture container are not removed.

40. Use of the natural killer cells according to claim 1 for preventing or treating a disease.

41. The method for preventing or treating a disease according to claim 39, wherein: The disease is any one selected from the group consisting of infectious diseases, cancers, nervous system diseases and autoimmune diseases.

42. A method for preventing or treating a disease, comprising: administering the natural killer cell according to claim 1.

43. The method for preventing or treating a disease according to claim 42, wherein: The disease is any one selected from the group consisting of infectious diseases, cancers, nervous system diseases and autoimmune diseases.

44. A method for culturing stem cells, comprising: inoculating stem cells in the form of iPSCs or iPSC spheroids into a cell culture container such that 0.5 to 20 colonies are formed per 1 square centimeter of the cell culture container area.

45. A method for preparing natural killer cells, comprising: differentiating hematopoietic stem cells into natural killer cells in the presence of feeder cells.

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