Method for induced differentiation of NK cells from iPSC

Abstract

The invention belongs to the field of cell culture, particularly relates to a method for inducing and differentiating NK cells from iPSC in vitro, and more particularly relates to a method for directionally inducing and differentiating NK cells from human iPSC by using a stimulant combination. The NK cells obtained by the technical scheme are high in purity and good in state.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture, and specifically relates to a method for inducing the differentiation of natural killer cells (NK cells) from induced pluripotent stem cells (iPSCs). More specifically, the present invention relates to a method for directed induction and differentiation of human NK cells from human iPSCs using a combination of stimulants. Background Art

[0002] NK cells are the core cells of the innate immune system and the first line of defense against tumors and viral infections. They also participate in the body's immune regulation function. Unlike the specific killing of the adaptive immune system, NK cells do not target any specific tumor markers, but identify through some common characteristics of tumors, such as downregulation of major histocompatibility complex (MHC-I) receptors and expression of damage-related proteins. Therefore, the cell therapy drugs developed by NK cells have broad-spectrum adaptability. After identifying tumor cells, NK cells can independently and quickly kill directly without the need for instructions from the immune system or the cooperation of other immune cells.

[0003] In the clinical studies that have been conducted, NK cells are most widely used in hematological tumors. Several clinical studies on acute myeloid leukemia have observed clear clinical effects, with an overall response rate of more than 50%, and some patients can achieve complete remission (Complete response, CR) (Cliona M. Rooney, Can Treg elimination enhance NK cell therapy for AML? Blood [J] 2014, Volume 123: 3848-3849; Romee et al. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Sci. Transl. Med. [J] 2016, 8 (357): 357ra123). Good clinical effects have also been observed in other hematological tumors, such as multiple myeloma, non-Hodgkin's lymphoma, and some solid tumors. More importantly, NK therapy is highly safe, and almost no graft-versus-host reaction (GVHD) and related toxicity have been observed.

[0004] Based on the above advantages, NK cells from various sources have been used in tumor immunotherapy, including autologous, allogeneic, cell line, umbilical cord blood, and in vitro induced pluripotent stem cells (iPSC), etc.

[0005] Human pluripotent stem cells, especially iPSCs, can effectively differentiate into immune cells (such as NK cells) and are considered to be a better way to produce immune cells. The receptor and gene expression profiles of iPSC-induced differentiated NK cells are similar to those of NK cells purified from peripheral blood or human umbilical cord blood, without the risk of chromosomal abnormalities, and have the advantages of homogeneity and being unaffected by donor variation. At the same time, NK cells derived from iPSCs can be produced on a scale suitable for clinical research on cancer immunotherapy, while primary NK cells are more difficult to isolate, purify and genetically modify. In addition, iPSC-induced differentiated NK cells are relatively easy to genetically engineer using viral vectors and CRISPR technology.

[0006] It has been reported that human iPSC-induced NK cells can effectively kill hematological malignancies, including acute myeloid leukemia (AML) and multiple myeloma, and can also effectively kill leukemia and ovarian cancer cells without further producing teratomas. In 2018, Li et al. reported in Cell Stem Cell (Li et al. Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity. Cell Stem Cell [J]. 2018, 23 (2): 181-192.) that iPSC-derived NK cells were modified with CAR to enhance the killing activity of NK cells against tumors expressing mesothelin, both in vitro and in vivo. Although safety issues and clinical effectiveness remain to be resolved, iPSC CAR-NK cells still provide an attractive option for CAR therapy. The CAR structure of CAR-NK is improved on the basis of CAR-T, which is more suitable for NK cell activation and has similar targets.

[0007] Many studies have reported the induction of mature NK cells from human iPSCs. The induction methods include the generation and proliferation of CD34+ hematopoietic stem cell precursors, while retaining the cytokines (IL-15, IL-3, IL-7, SCF, and FLT3 ligand) secreted by NK cells, and the use of cell lines expressing membrane-bound IL-21 as feeder cells to promote their development, thereby increasing the yield of NK cells to clinical levels.

[0008] Many domestic and foreign companies have deployed related products. In 2019, Fate Therapeutics' iPSC-derived NK cell product FT500 and iPSC-CAR-NK product FT596 obtained IND approval from the FDA for clinical trials with good safety. As of October 2021, 18 of 26 patients (69%) achieved objective remission on the 29th day after a single dose of FT596, and 12 patients (46%) achieved complete remission. The company's other products have also made good progress. The development of CAR-NK cell therapy is underway around the world. The vast majority of CAR-NK cell therapies are in Phase I clinical trials, accounting for about 68%, and projects in Phase II are about 32%. No CAR-NK cell therapy has entered Phase III clinical trials.

[0009] Therefore, the development of high-quality iPSC-derived NK cell culture technology has a broad market space and is crucial to improving the cell therapy effect of tumors. Summary of the invention

[0010] In order to solve the above problems, in one aspect, the object of the present invention is to provide a method for inducing differentiation of NK cells from iPSCs (preferably human iPSCs), comprising:

[0011] iPSCs are cultured in a first differentiation medium, a second differentiation medium, a third differentiation medium, a fourth differentiation medium, and a fifth differentiation medium in sequence,

[0012] The first differentiation medium was supplemented with CHIR99021,

[0013] The second differentiation medium is supplemented with VEGF and bFGF,

[0014] The third differentiation medium is supplemented with retinoic acid (RA),

[0015] The fourth differentiation medium is supplemented with IL-3, IL-7, IL-15, SCF and FLT3 ligand,

[0016] The fifth differentiation medium is supplemented with IL-7, IL-15, SCF and FLT3 ligand.

[0017] In some embodiments, in the above method of the present invention, the final concentration of CHIR99021 in the first differentiation medium is 1-12 μM; preferably, the final concentration of CHIR99021 is 6 μM.

[0018] In some embodiments, in the above method of the present invention, the final concentration of VEGF in the second differentiation medium is 50-500 ng / ml, and the final concentration of bFGF is 50-500 ng / ml; preferably, the final concentration of VEGF is 200 ng / ml, and the final concentration of bFGF is 400 ng / ml.

[0019] In some embodiments, in the above method of the present invention, the final concentration of RA in the third differentiation medium is 0.5-5 μM; preferably, the final concentration of RA is 2 μM.

[0020] In some embodiments, in the above method of the present invention, the final concentration of IL-3 in the fourth differentiation medium is 5-50ng / ml, the final concentration of IL-7 is 5-50ng / ml, the final concentration of IL-15 is 5-50ng / ml, the final concentration of SCF is 5-50ng / ml, and the final concentration of FLT3 ligand is 5-50ng / ml; preferably, the final concentration of IL-3 is 5ng / ml, the final concentration of IL-7 is 20ng / ml, the final concentration of IL-15 is 10ng / ml, the final concentration of SCF is 20ng / ml, and the final concentration of FLT3 ligand is 10ng / ml.

[0021] In some embodiments, in the above method of the present invention, the final concentration of IL-7 in the fifth differentiation medium is 5-50ng / ml, the final concentration of IL-15 is 5-50ng / ml, the final concentration of SCF is 5-50ng / ml, and the final concentration of FLT3 ligand is 5-50ng / ml; preferably, the final concentration of IL-7 is 20ng / ml, the final concentration of IL-15 is 10ng / ml, the final concentration of SCF is 20ng / ml, and the final concentration of FLT3 ligand is 10ng / ml.

[0022] In some embodiments, in the above method of the present invention, the seeding density of iPSC is 2-4×10 5 / ml.

[0023] In some embodiments, in the above method of the present invention, iPSCs are cultured using a first differentiation medium for 1 day, a second differentiation medium for 3 days, a third differentiation medium for 3 days, a fourth differentiation medium for 7 days, and a fifth differentiation medium for 21-46 days.

[0024] The method of the present invention comprises the following specific steps: placing iPSC cells in a 24-well cell culture plate under the conditions of 37° C., 5% CO2 and 100% humidity, culturing with a first differentiation medium for 1 day, culturing with a second differentiation medium for 3 days, culturing with a third differentiation medium for 3 days, culturing with a fourth differentiation medium for 7 days, culturing with a fifth differentiation medium for 21-46 days, during which time the suspended cells are transferred to a 6-well cell culture plate according to their growth conditions to obtain induced differentiated NK cells.

[0025] In another aspect, an object of the present invention is to provide a NK cell induced to differentiate from iPSC, which is obtained by the above method of the present invention.

[0026] In yet another aspect, an object of the present invention is to provide a kit for inducing differentiation of NK cells from iPSCs, comprising a differentiation basal medium, a first differentiation medium, a second differentiation medium, a third differentiation medium, a fourth differentiation medium and a fifth differentiation medium,

[0027] The first differentiation medium was supplemented with CHIR99021,

[0028] The second differentiation medium is supplemented with VEGF and bFGF,

[0029] The third differentiation medium is supplemented with RA,

[0030] The fourth differentiation medium is supplemented with IL-3, IL-7, IL-15, SCF and FLT3 ligand,

[0031] The fifth differentiation medium is supplemented with IL-7, IL-15, SCF and FLT3 ligand.

[0032] In some embodiments, in the above kit of the present invention, the final concentration of CHIR99021 in the first differentiation medium is 1-12 μM; preferably, the final concentration of CHIR99021 is 6 μM.

[0033] In some embodiments, in the above-mentioned kit of the present invention, the final concentration of VEGF in the second differentiation medium is 50-500 ng / ml, and the final concentration of bFGF is 50-500 ng / ml; preferably, the final concentration of VEGF is 200 ng / ml, and the final concentration of bFGF is 400 ng / ml.

[0034] In some embodiments, in the above kit of the present invention, the final concentration of RA in the third differentiation medium is 0.5-5 μM; preferably, the final concentration of RA is 2 μM.

[0035] In some embodiments, in the above-mentioned kit of the present invention, the final concentration of IL-3 in the fourth differentiation medium is 5-50ng / ml, the final concentration of IL-7 is 5-50ng / ml, the final concentration of IL-15 is 5-50ng / ml, the final concentration of SCF is 5-50ng / ml, and the final concentration of FLT3 ligand is 5-50ng / ml; preferably, the final concentration of IL-3 is 5ng / ml, the final concentration of IL-7 is 20ng / ml, the final concentration of IL-15 is 10ng / ml, the final concentration of SCF is 20ng / ml, and the final concentration of FLT3 ligand is 10ng / ml.

[0036] In some embodiments, in the above-mentioned kit of the present invention, the final concentration of IL-7 in the fifth differentiation medium is 5-50ng / ml, the final concentration of IL-15 is 5-50ng / ml, the final concentration of SCF is 5-50ng / ml, and the final concentration of FLT3 ligand is 5-50ng / ml; preferably, the final concentration of IL-7 is 20ng / ml, the final concentration of IL-15 is 10ng / ml, the final concentration of SCF is 20ng / ml, and the final concentration of FLT3 ligand is 10ng / ml.

[0037] The technical solution of the present invention has fewer additives and is more economical. It also adopts 2D (plane) step-by-step induction, which is simpler and easier to operate than conventional 3D (stereoscopic) culture. The obtained NK cells have good vitality and high purity, and the content of NK cells reaches 70% or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shows the cell morphology at different stages of cell differentiation observed under a microscope;

[0039] Figure 2 The results of the mesoderm cell ratio analysis on the third day after cell culture according to the method of Example 1 are shown;

[0040] Figure 3 The results of the hematopoietic stem cell ratio analysis on the 10th day after cell culture according to the method of Example 1 are shown;

[0041] Figure 4 The results of NK cell ratio analysis on the 50th and 63rd days after cell culture according to the method of Example 1 are shown. DETAILED DESCRIPTION

[0042] The present invention further illustrates the technical solution of the present invention through specific implementation methods and drawings, but it can be understood by those skilled in the art that the following specific implementation methods and embodiments are intended to illustrate the present invention and should not be construed as limiting the present invention in any way. It is well known to those skilled in the art that many modifications can be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention.

[0043] Laduviglusib (CHIR99021) is a potent, selective, orally active GSK-3α / β inhibitor. The hydrochloride used in the present invention has the following structure.

[0044]

[0045] Unless otherwise specified, the following experimental methods are all conventional experimental methods in the art, and the experimental materials used are all experimental materials that can be easily obtained from commercial companies unless otherwise specified.

[0046] Differentiation of NK cells from iPSCs

[0047] Example 1. NK cell differentiation induction

[0048] 1) Human iPSC cells (purchased from Beijing Saibei, catalog number CA4025106) were resuspended in differentiation basal medium (purchased from Nanjing Baishou, catalog number 400105) containing a final concentration of 2% additive (purchased from Nanjing Baishou, catalog number 600601) and cultured at 2-4×10 5 / ml / well was added into a 24-well cell culture plate, which was pre-coated with Rh vitronectin (purchased from Nanjing Baishou, catalog number 500125) for 2 hours and cultured at 37°C, 5% CO2 and 100% humidity for 1 day.

[0049] 2) On the next day, the cell culture plate was removed, the supernatant was discarded, and differentiation basal medium containing a final concentration of 2% additive supplemented with Laduviglusib (CHIR-99021) HCl (purchased from Selleck, catalog number S2924) at a final concentration of 6 μM was added, i.e., the first differentiation medium, and cultured at 37° C., 5% CO2 and 100% humidity for 1 day.

[0050] 3) On the third day, take out the cell culture plate and observe the cell morphology under a 10x microscope. Figure 1A and B in the figure, A is the cell control (0 day) without adding the first differentiation medium, B is the cell cultured with adding the first differentiation medium (3 days), the morphology has changed and is significantly different from the control. The supernatant was discarded and replaced with a differentiation basal medium containing a final concentration of 2% additives, and cultured for 1 day at 37°C, 5% CO2 and 100% humidity to obtain mesoderm cells.

[0051] 4) On the fourth day, the cell culture plate was removed, the cell morphology was observed under a 10x microscope, the supernatant was discarded, and differentiation basal medium containing a final concentration of 2% additives supplemented with VEGF (purchased from R&D, catalog number 293-VE-050) at a final concentration of 200 ng / ml and bFGF (purchased from Nanjing Baishou, catalog number 500201) at a final concentration of 400 ng / ml was added, i.e., the second differentiation medium, and cultured at 37°C, 5% CO2 and 100% humidity for 3 days.

[0052] 5) On the seventh day, the cell culture plate was removed, and the cell morphology was observed under a 10x microscope (no obvious changes in cell morphology were observed on days 4-7), the supernatant was discarded, and a differentiation basal medium containing a final concentration of 2% additives supplemented with retinoic acid (RA) (purchased from Sigma, item number R2625-50mg) at a final concentration of 1 μM, i.e., the third differentiation medium, was added, and cultured for 3 days at 37°C, 5% CO2, and 100% humidity. Half of the third differentiation medium was replaced on the eighth and ninth days to obtain hematopoietic stem cells.

[0053] 6) On the tenth day, take out the cell culture plate and observe the cell morphology under a 10x microscope. Figure 1 C, the suspension cell morphology began to appear. The cells were supplemented with IL-3 (purchased from Peprotech, catalog number 200-03) at a final concentration of 5 ng / ml, IL-7 (purchased from R&D, catalog number 207-IL-025) at a final concentration of 20 ng / ml, IL-15 (purchased from R&D, catalog number 247-ILB-025) at a final concentration of 10 ng / ml, SCF (purchased from R&D, catalog number 255-SC-050) at a final concentration of 20 ng / ml, FLT3 ligand (purchased from Peprotec, catalog number 255-SC-050) at a final concentration of 10 ng / ml, and FLT3 ligand (purchased from Peprotec, catalog number 255-SC-050) at a final concentration of 10 ng / ml. h, item number 300-19) containing a differentiation basal medium with a final concentration of 2% additives, i.e., the fourth differentiation medium, was cultured at 37°C, 5% CO2, and 100% humidity for 28-53 days, of which IL-3 was only added for 7 days, and half of the fourth medium was replaced on the 4th day. In the later stage, only the other 4 factors, i.e., the fifth medium, were used for 21-46 days. Half of the fifth differentiation medium was replaced twice a week to maintain normal cell growth. During this period, the cells were transferred to a 6-well cell culture plate according to the growth of the suspended cells to obtain induced differentiated NK cells. The cell morphology was observed under a 10x microscope, see Figure 1DF, the number of suspended cells gradually increased with the extension of culture time (D is 17 days, E is 37 days), and the adherent cells disappeared after being transferred into 6-well plates. On the 58th day, they had a typical NK cell morphology (F).

[0054] The cultured cells were counted separately, and no less than 5×10 5 The cells were prepared for subsequent flow cytometry analysis to perform qualitative and quantitative detection of the number and cell ratio of NK cells produced.

[0055] Cell ratio analysis

[0056] Example 2. Analysis of the proportion of cells differentiated into mesoderm

[0057] According to the culture method provided in Example 1, samples were taken on the third day after cell culture for flow cytometry analysis to evaluate the proportion of induced differentiated mesoderm cells.

[0058] Take the cells from three culture wells of the control group induced by Laduviglusib (CHIR-99021) HCl without addition and the experimental group cultured in the first differentiation medium (samples 1-3), add 200 μL Accutase cell digestion solution (purchased from Shanghai Maokang Biology, product number MX2001-100mL) to each well for digestion for 5 minutes, transfer to a 1.5mL centrifuge tube, centrifuge at 600g for 5 minutes, discard the supernatant, wash with 1ml of normal saline, centrifuge and discard the supernatant; resuspend with 200μL 4% tissue fixative (purchased from Beijing Solebow, product number P1110-500mL), fix at room temperature for 20 minutes, centrifuge at 600g for 5 minutes, and discard the supernatant; use 1mL 1× membrane permeabilization agent (purchased from Biolegend, product number 421002) to wash 3 times, centrifuge conditions 600g, 5 minutes; 50μL The pellet was resuspended with 1× membrane permeabilizer for intracellular staining; 0.5 reaction volume of PE-anti-h / mBrachyury antibody (purchased from R&D, IC2085P) was added to each parallel sample; incubated at room temperature in the dark for 20 minutes, and washed once with 1 mL 1× membrane permeabilizer after staining, centrifuged at 600g for 5 minutes, and the supernatant was discarded. Resuspended with 300 μL 4% tissue fixative, added to a flow tube (purchased from BD, 352052) and detected in a dual laser flow cytometer (purchased from BD, CantoII, the same below), and the data were analyzed with Flowjo.

[0059] The results are shown in Table 1 and Figure 2 shown. Figure 2The horizontal axis is Brachyury, and the positive represents mesoderm cells; A is the control group without the addition of Laduviglusib (CHIR-99021) HCl induction, and BD are the experimental groups cultured in the first differentiation medium (samples 1-3).

[0060] Table 1. Results of mesoderm cell ratio analysis (unit: percentage of this cell in all cells)

[0061] Sample 1 Sample 2 Sample 3 Brachyury 99.6％ 99.5％ 99.5％

[0062] The results showed that from the third day after culture using the method of Example 1, the proportion of Brachyury-positive mesoderm cells was close to 100%, with high purity.

[0063] Example 3. Analysis of the proportion of hematopoietic stem cells

[0064] According to the culture method provided in Example 1, samples were taken on the 10th day after cell culture for flow cytometry detection to evaluate the proportion of hematopoietic stem cells induced to differentiate after culture.

[0065] Three culture wells (samples 1-3) were taken from the control group without VEGF, bFGF and RA induction, the control group with VEGF and bFGF but without RA induction, and the experimental group cultured in the first to third differentiation medium in sequence. 200 μL Accutase cell digestion solution was added to each well for digestion for 5 minutes, transferred to a 1.5 mL centrifuge tube, washed once, and 1 reaction volume of PE-anti-CD34 antibody (purchased from Biolegend, 343506) and 7-AAD antibody (purchased from BD, 559925) were added to the sample tubes respectively, and incubated at room temperature for 15 minutes; 300 μL of physiological saline (containing 1% FBS) was added, and the cells were detected in a dual laser flow cytometer, and the data were analyzed using Flowjo.

[0066] The results are shown in Table 2 and Figure 3 As shown. Dead cells are excluded by 7-AAD (i.e., 7-AAD positive cells are dead cells). Figure 3 The expression of CD34 in living cells is shown (abscissa), and a positive expression represents hematopoietic stem cells; A and B are control groups without induction with VEGF, bFGF and RA and with induction with VEGF and bFGF but without RA, respectively; C, D, and E are three parallel experimental groups.

[0067] Table 2. Analysis results of hematopoietic stem cell ratio (the unit is the percentage of this cell in all living cells)

[0068] Sample 1 Sample 2 Sample 3 CD34+ 70.6％ 74.3％ 74.6％

[0069] The results showed that from the 10th day after being cultured using the method of Example 1, the proportion of CD34+ hematopoietic stem cells in the living cells was above 70%, and the purity was high.

[0070] Example 4. Analysis of NK cell ratio

[0071] According to the culture method provided in Example 1, samples were taken on the 50th and 63rd days after NK cell culture for flow cytometry analysis to evaluate the NK cell subpopulations induced to differentiate after culture.

[0072] On the 50th day and the 63rd day, 2×10 cells were cultured in each of two parallel samples (samples 1-2) from the uninduced control group and the experimental group cultured in the first to fifth differentiation medium. 6 , washed once, 1 reaction volume of each of PE-anti-CD56 antibody (purchased from Biolegend, 362508), APC-anti-NKG2D antibody (purchased from Biolegend, 320808), FITC-anti-CD45 (purchased from BD, 555482) and 7-AAD antibody (purchased from BD, 559925) was added to the sample tube, and incubated at room temperature for 15 minutes. 200 μL of physiological saline (containing 1% FBS) was added, and the samples were detected in a dual laser flow cytometer, and the data were analyzed by Flowjo.

[0073] The results are shown in Table 3 and Figure 4 shown. Figure 4 It shows that 7-AAD-living cells account for more than 98%; the proportion of NK cells is further analyzed in living cells, with CD45 on the ordinate and CD56 on the abscissa, and CD45+CD56+ positive represents NK cells; the NKG2D expression level of NK cells is shown in the CD45 and CD56 double positive gates, which is as high as more than 95%. A is a negative control, with basically no CD45+CD56+NK cells; B and C are the test results on the 50th day and the 63rd day respectively.

[0074] Table 3. NK cell ratio analysis results (unit is the percentage of this cell in all cells)

[0075] Day 50 Day 63 Sample 1 78.1％ 89.9％ Sample 2 74.9％ 92.8％

[0076] The results showed that from the 50th day after treatment and culture, the proportion of CD45+CD56+NK cells in living cells was above 70%, with high purity and good condition.

[0077] iPSC cells are widely available. Through the step-by-step induction method of the present invention, iPSC cells are gradually differentiated into mesoderm and hematopoietic stem cells, and then further induced to differentiate into NK cells. NK cells can also continue to proliferate, and products can be developed after the number is expanded to meet clinical application needs. The present invention has fewer additives and is more economical. At the same time, 2D step-by-step induction is adopted, which is simpler and easier to operate than conventional 3D culture.

Claims

1. A method for inducing differentiation of NK cells from iPSCs (preferably human iPSCs), comprising: The iPSCs are cultured in a first differentiation medium, a second differentiation medium, a third differentiation medium, a fourth differentiation medium, and a fifth differentiation medium in sequence, The first differentiation medium is supplemented with CHIR99021, The second differentiation medium is supplemented with VEGF and bFGF, The third differentiation medium is supplemented with RA, The fourth differentiation medium is supplemented with IL-3, IL-7, IL-15, SCF and FLT3 ligand, The fifth differentiation medium is supplemented with IL-7, IL-15, SCF and FLT3 ligand. 2 . The method according to claim 1 , wherein the final concentration of CHIR99021 in the first differentiation medium is 1-12 μM; preferably, the final concentration of CHIR99021 is 6 μM.

3. The method according to claim 1, wherein the final concentration of VEGF in the second differentiation medium is 50-500 ng / ml, and the final concentration of bFGF is 50-500 ng / ml; preferably, the final concentration of VEGF is 200 ng / ml, and the final concentration of bFGF is 400 ng / ml. 4 . The method according to claim 1 , wherein the final concentration of RA in the third differentiation medium is 0.5-5 μM; preferably, the final concentration of RA is 2 μM.

5. The method according to claim 1, wherein the final concentration of IL-3 in the fourth differentiation medium is 5-50 ng / ml, the final concentration of IL-7 is 5-50 ng / ml, the final concentration of IL-15 is 5-50 ng / ml, the final concentration of SCF is 5-50 ng / ml, and the final concentration of FLT3 ligand is 5-50 ng / ml; preferably, the final concentration of IL-3 is 5 ng / ml, the final concentration of IL-7 is 20 ng / ml, the final concentration of IL-15 is 10 ng / ml, the final concentration of SCF is 20 ng / ml, and the final concentration of FLT3 ligand is 10 ng / ml.

6. The method according to claim 1, wherein the final concentration of IL-7 in the fifth differentiation medium is 5-50 ng / ml, the final concentration of IL-15 is 5-50 ng / ml, the final concentration of SCF is 5-50 ng / ml, and the final concentration of FLT3 ligand is 5-50 ng / ml; preferably, the final concentration of IL-7 is 20 ng / ml, the final concentration of IL-15 is 10 ng / ml, the final concentration of SCF is 20 ng / ml, and the final concentration of FLT3 ligand is 10 ng / ml.

7. The method according to claim 1, wherein the seeding density of the iPSCs is 2-4×10 5 / ml.

8. The method according to claim 1, wherein the iPSCs are cultured using the first differentiation medium for 1 day, using the second differentiation medium for 3 days, using the third differentiation medium for 3 days, using the fourth differentiation medium for 7 days, and using the fifth differentiation medium for 21-46 days.

9. A NK cell induced to differentiate from iPSC, obtained by the method according to any one of claims 1 to 8.

10. A kit for inducing differentiation of NK cells from iPSCs, comprising a differentiation basal medium, a first differentiation medium, a second differentiation medium, a third differentiation medium, a fourth differentiation medium and a fifth differentiation medium, The first differentiation medium is supplemented with CHIR99021, The second differentiation medium is supplemented with VEGF and bFGF, The third differentiation medium is supplemented with RA, The fourth differentiation medium is supplemented with IL-3, IL-7, IL-15, SCF and FLT3 ligand, The fifth differentiation medium is supplemented with IL-7, IL-15, SCF and FLT3 ligand.

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