CN114507641A - Method for inducing and differentiating human embryonic stem cells into NK cells - Google Patents

Abstract

The invention relates to a method for inducing and differentiating human embryonic stem cells into NK cells, belonging to the technical field of genetic engineering; the method comprises the steps of recovering and passaging human embryonic stem cells, inducing to obtain mesodermal cells and inducing to obtain CD34⁺Hematopoietic stem cell, isolated and purified CD34⁺Hematopoietic stem cells, inducing to obtain NK cells, and performing amplification culture; the human embryonic stem cell is human embryonic stem cell line H9; recovering and passaging the human embryonic stem cells, namely recovering the human embryonic stem cells, then carrying out the passage, transferring to the 2 nd generation, and when the cell confluency is more than 80%, using the recovered human embryonic stem cells for induction to obtain mesodermal cells; the invention adopts human embryonic stem cells to induce and differentiate to obtain NK cells, and when the effective target ratio is 20:1, the killing rate to the HELA tumor cell line is 94.25 percent, and the killing rate to the LOVO tumor cell line is 91.24 percent.

Description

Method for inducing and differentiating human embryonic stem cells into NK cells

Technical Field

The invention relates to a method for inducing and differentiating human embryonic stem cells into NK cells, belonging to the technical field of genetic engineering.

Background

Cell-based therapies for treating relapsed or refractory cancers have attracted interest and interest. In addition to the study of CAR-T cells, clinical trials using NK cells isolated from peripheral blood or umbilical cord blood are also expanding rapidly, and this approach requires the collection of NK cells for each patient, resulting in donor variability and heterogeneity of NK cells. In contrast, human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) derived NK cells provide a more homogeneous population of cells that can be produced on a clinical scale. These properties make hESC or hiPSC-derived NK cells an ideal cell population for the development of standardized, "off-the-shelf" immunotherapeutic products.

CN109415699A is a method for preparing CD4CD8 double positive T cells applied by the japanese national university, which can be induced to differentiate into T cells by hESC/hiPSC cells, but does not involve culture of NK cells.

CN104711225A is induced and differentiated into NK cells by embryonic stem cells, the purity of the NK cells is more than 97%, but the effective target ratio is 40:1 when the killing efficiency reaches 92.21%.

As described above, the prior art has a technical problem of insufficient lethality in spite of high purity by inducing differentiation into NK cells using embryonic stem cells.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for inducing and differentiating human embryonic stem cells into NK cells, which realizes the following purposes:

the induced NK cells have higher purity, and the killing power of the NK cells is improved.

In order to solve the technical problems, the invention adopts the following technical scheme:

induced differentiation of human embryonic stem cells into NThe method for producing K cells comprises the steps of reviving and passaging human embryonic stem cells, inducing to obtain mesoderm cells, and inducing to obtain CD34⁺Hematopoietic stem cell, isolated and purified CD34⁺Hematopoietic stem cells, inducing to obtain NK cells, and performing amplification culture;

the human embryonic stem cell is human embryonic stem cell line H9;

recovering and passaging the human embryonic stem cells, namely recovering the human embryonic stem cells, then carrying out passage, and when the cells are transferred to the 2 nd generation and the cell confluence is more than 80%, inducing to obtain mesodermal cells;

the mesoderm cell induction method comprises adding factor-containing E3 culture medium into human embryonic stem cells with cell confluency of above 80% transferred to generation 2, and controlling cell density to 0.8 × 10⁶Stirring at 15 rpm per mL at 37 deg.C with 5% CO₂Culturing for 24h in the incubator to form cell aggregates; centrifuging cell aggregate, removing supernatant, adding into SFDM medium containing factor, and culturing at cell density of 1 × 10⁶one/mL, 5% CO at 37 ℃₂After 2 days of culture in the incubator of (1), the SFDM medium containing the factors was replaced and the temperature was continued at 37 ℃ with 5% CO₂Inducing for 2 days in the incubator to obtain mesodermal cells;

the factor-containing E3 culture medium is prepared by adding factors of L-ascorbic acid 2-magnesium phosphate with the final concentration of 50 mug/mL, 5ng/mL sodium selenite, 50ng/mL FGF2, 50ng/mL VEGF, 2 muM CHIR 99021, 10 muM Blebbistatin and 10 muM Y-27632 to the E3 culture medium;

the E3 culture medium is formed by mixing a DMEM culture medium with 75% Vol and a 25% Vol F-12 culture medium;

the SFDM culture medium containing the factors is added with the factors with the final concentrations of 25ng/mL BMP4, 50ng/mL VEGF and 50ng/mL FGF 2;

the preparation method of the SFDM medium comprises the following steps: mixing 50% Vol IMDM medium and 50% Vol F-12 medium, adding 100. mu.g/mL polyvinyl alcohol, 100. mu.g/mL HSA, 1 × MEM nonessential amino acid solution, 0.1 × chemically defined lipid concentrate, 125. mu.M magnesium L-ascorbate 2-phosphate, 10ng/mL IGF 1;

the induction resulted in CD34⁺The hematopoietic stem cells are obtained by centrifuging mesodermal cells, discarding the supernatant, adding SFDM medium containing hematopoietic supporting factor, and controlling the cell density to 0.8 × 10⁶one/mL, 5% CO at 37 ℃₂After 2 days of culture in the incubator, the culture medium of SFDM containing hematopoietic supporting factors is replaced, and the induction is continued for 2 days to obtain CD34⁺A hematopoietic stem cell;

the SFDM culture medium containing the hematopoietic support factors is prepared by adding the hematopoietic support factors with final concentrations of 50ng/mL SCF, 20ng/mL TPO, 10ng/mL FLT-3L, 20ng/mL IL-3 and 25ng/mL BMP4 into the SFDM culture medium;

the method for inducing the NK cells comprises the step of collecting separated and purified CD34⁺Hematopoietic stem cells, adding NK cell differentiation medium containing factor, and controlling cell density to 1 × 10⁶one/mL, at 37 ℃ and 5% O₂、5%CO₂Culturing for 2 weeks in the incubator, and replacing half of the NK cell differentiation medium containing the factors every three days;

the NK cell differentiation medium containing the factors is prepared by adding SCF with the final concentration of 50ng/mL, 50ng/mL TPO, 50ng/mL FLT-3L, 50ng/mL IL-7 and 2ng/mL IL-2 factors into the NK cell differentiation medium;

the NK cell differentiation medium is Stemspan SFEM added with 1% Vol glutamine, 0.5% Vol double antibody, 250 μ M magnesium ascorbyl phosphate and 2mM nicotinamide at final concentration.

The separation and purification of CD34⁺Hematopoietic stem cells, isolated and purified CD34⁺The purity of the hematopoietic stem cells was 96.9%.

The amplification culture method comprises collecting NK cells, transferring to a new culture flask for culture, adding factor-containing NK serum-free medium with cell density of 1 × 10⁶one/mL, at 37 ℃ and 5% O₂、5%CO₂Culturing in an incubator for 2 weeks, and replacing half amount of NK serum-free medium containing factors every three days;

the NK serum-free medium containing the factors is prepared by adding IL-2, IL-7, IL-15 and IL-21 factors with final concentration of 10ng/mL into the NK serum-free medium.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts human embryonic stem cells to induce and differentiate to obtain NK cells, when the effective target ratio is 20:1, the killing rate to HELA tumor cell line is 94.25%, the killing rate to LOVO tumor cell line is 91.24%, and the induced CD3 is^-CD56⁺ The purity of NK cells was 81.3%.

Drawings

FIG. 1 is a microscope photograph of embryonic stem cells after recovery according to the present invention;

FIG. 2 is a micrograph of mesodermal cells induced according to the invention;

FIG. 3 shows purified CD34 of the present invention⁺A flow chart of hematopoietic stem cells;

FIG. 4 is a micrograph of NK cells induced by the present invention;

FIG. 5 shows the CD3 induced by the present invention^-CD56⁺Flow scatter plots of NK cells;

FIG. 6 is a line graph of the kill rate of NK cells to HELA cells induced by the present invention;

FIG. 7 is a line graph showing the killing rate of LOVO cells by NK cells induced by the present invention.

Detailed Description

Example 1 recovery and passaging of human embryonic Stem cells

Human embryonic stem cell lines H9, Vitronectin (human Vitronectin), ROCK inhibitor Blebbistatin and EDTA digestive juice were all purchased from Shanghai-Dynasty Biotech Inc.

(1) Six-hole plate coating

Unfreezing the coated protein Vitronect at room temperature, subpackaging into 120 mu L/tube, adding 120 mu L Vitronect into 9 mL DMEM/F12 culture medium, gently mixing uniformly and diluting, subpackaging into six-hole plates according to 1.5 mL/hole, and gently shaking and mixing uniformly to obtain the six-hole plate coated with the Vitronect coating solution. The solution was allowed to stand at room temperature for 2 hours before use, and the six-well plate was tilted and the coating solution was completely aspirated with a pipette.

(2) Cell recovery

Preheating a water bath to 37 ℃, taking out 1 frozen human embryonic stem cell line H9 (1 mL), placing in the water bath at 37 ℃, slightly shaking by hand, thawing within 1 min, and taking out when ice crystals in the cell suspension are about to completely disappear by visual observation to obtain the cell suspension. Wiping the surface of the freezing storage tube by using 75% alcohol dust-free paper, and transferring the freezing storage tube into a super clean bench; the cell suspension was transferred to a 50mL centrifuge tube prepared in advance, followed by dropwise addition of 10 mL of DMEM/F12 medium while gently shaking the cells, and centrifugation was carried out at 850rpm for 5 min. And (4) sucking and discarding the supernatant, adding 4 mL ncEpic complete culture medium (pre-warmed to room temperature) containing Blebbistatin (the final concentration is 2.5 mu M), uniformly mixing the cells, and avoiding blowing to obtain a cell culture solution.

Absorbing and discarding Vitronectin coating solution of 2 holes in the six-hole plate coated in the step (1), inoculating the uniformly mixed cell culture solution into the 2 holes according to 2 mL/hole, horizontally and crossly shaking for three times, placing at 37 ℃ and 5% CO₂Culturing in an incubator with concentration. The ncEpic complete medium was replaced 24 hours later (2 mL per well) and the medium was replaced daily.

(3) Passage of cells

When the confluency of the cells reached about 85%, the cells were passaged (see FIG. 1) at a ratio of 1: 5.

And (5) carrying out passage at the ratio of 1:5, namely transferring the 1-hole cells to 5-hole cells to continue amplification culture.

The specific passage method comprises the following steps:

vitronectin-coated six-well plates were placed in an ultra-clean bench in advance for 1 hour to return to room temperature.

20mL of ncEpic complete medium was prepared and the volume ratio of 4000: 1, adding 5 mu L of 10mM ROCK inhibitor Blebbistatin, and returning to room temperature to obtain a pre-warmed ncEpic complete culture medium containing Blebbistatin. The final concentration of Blebbistatin in the ncEpic complete medium was 2.5 nM.

When the confluency of the recovered cells reached 85%, the medium in 2 wells was aspirated, 2 mL/well of DPBS (without calcium and magnesium) was added, gently shaken and aspirated. The solution was allowed to completely cover the bottom of the well by adding 2 mL/well of EDTA digest and incubated in an incubator at 37 ℃ for 7 min. Shaking the cells is avoided, EDTA digestive juice is obliquely absorbed, 2 mL/hole pre-warmed ncEpic complete culture medium containing Blebbistatin is added in time, and the six-hole plate is horizontally and crossly shaken to separate the cells from the matrix, so that 2-hole digested cells are obtained.

The Vitronectin coating solution in the coated six-well plate was discarded, 2 mL/well of prewarmed ncEpic complete medium containing Blebbistatin was added, and the 2-well digested cells were evenly distributed to 10 wells. Shaking horizontally for three times, standing at 37 deg.C and 5% CO₂Culturing in an incubator with concentration. The ncEpic complete medium was replaced 24 hours later and daily.

The cells passed to passage 2 were used in the following experiments when the degree of cell confluence was 80% or more.

Example 2 Induction of ESCs to CD34⁺Hematopoietic stem cell differentiation

(1) Inducing to obtain mesoderm cells

A. When the confluence ratio of the 2 nd generation embryonic stem cells obtained in example 1 was 80% or more, the medium was discarded, and E3 medium containing factors, in which the cell density was 0.8X 10, was added⁶piece/mL, noted day 0, in an Ultra Low Attachment (ULA) flask, under continuous stirring at 15 rpm on a rocker platform, 5% CO at 37 ℃₂The culture box is used for culturing for 24 hours to form cell aggregates.

The factor-containing E3 medium is prepared by adding 50. mu.g/mL of magnesium L-ascorbate 2-phosphate (ascorbyl acid 2-phosphate magnesium), 5ng/mL of Sodium selenite (Sodium selenite, available from Merck), 50ng/mL of FGF2 (human basic fibroblast growth factor), 50ng/mL of VEGF (vascular endothelial growth factor), 2. mu.M of CHIR 99021 (glycogen synthase kinase-3 inhibitor), 10. mu.M of Blebbistatin (non-muscle myosin II-type ATPase inhibitor), 10. mu.M of Y-27632 (ROCK inhibitor) (the factors are available from MCE except for the existing label) to E3 medium;

the E3 medium was a mixture of 75% Vol DMEM medium and 25% Vol F-12 medium (both from Gibco).

B. The cell aggregates after 1 day of induction were collected in a 50mL centrifuge tube, centrifuged at 400g for 5min, the supernatant was discarded, and 20mL of the factor-containing mixture was addedCulturing in SFDM medium at a cell density of 1 × 10⁶Per mL, 37 ℃, 5% CO₂After culturing for 2 days in the incubator (2), the SFDM medium containing the factors is replaced, and the temperature is kept at 37 ℃ and the content of 5% CO is kept₂The culture chamber (2) was used to induce mesodermal cells for 2 days (see FIG. 2), and the result was recorded as day 5.

The SFDM culture medium containing the factors is added with the factors with the final concentrations of 25ng/mL BMP4, 50ng/mL VEGF and 50ng/mL FGF 2;

the preparation method of the SFDM medium comprises the following steps: 50% Vol IMDM medium and 50% Vol F-12 medium were mixed and added to a final concentration of 100. mu.g/mL polyvinyl alcohol (from Merck), 100. mu.g/mL HSA (human serum albumin, from MCE), 1 × MEM non-essential amino acid solution (non-essential amino acid supplement, cat # 11140050, from Invitrogen), 0.1 × chemically defined lipid concentrate (chemical-defined lipid supplement, cat # 11905031, from Invitrogen), 125. mu.M magnesium L-ascorbate 2-phosphate (from MCE), 10ng/mL IGF1 (insulin-like growth factor-1, from MCE).

The 1 x is the dilution of the MEM nonessential amino acid solution by 1 time;

the 0.1 × ratio is 0.1 times the amount of the lipid concentrate having a definite chemical composition.

(2) Induced to obtain CD34⁺Hematopoietic stem cells

Collecting mesodermal cells obtained by inducing for 5 days into 50mL centrifuge tube, centrifuging at 400g for 5min, discarding supernatant, adding SFDM medium containing hematopoietic support factor, and controlling cell density to 0.8 × 10⁶And (2) the culture medium contains hematopoietic support factors of 50ng/mL SCF, 20ng/mL TPO, 10ng/mL FLT-3L, 20ng/mL IL-3 and 25ng/mL BMP4 in final concentration. At 37 deg.C, 5% CO₂After 2 days of culture in the incubator, the culture medium of SFDM containing hematopoietic supporting factors is replaced, and the induction is continued for 2 days to obtain CD34⁺Hematopoietic stem cells, day 9.

Example 3 immunomagnetic bead isolation and purification of CD34⁺Hematopoietic stem cells

(1) Collection of CDs 34⁺Hematopoietic stem cells, prepared in AccutasE.Box cell digestion solution, 37 ℃ digestion for 20 minutes, obtain single cell suspension. The single cell suspension was washed in MACs buffer (PBS containing 5 mg/mL BSA, 1 mM EDTA) and filtered through a 100 μm cell strainer to remove aggregates.

(2) CD34 labeled by CD34 paramagnetic microbead produced by Meitian whirlpool⁺Hematopoietic stem cells, manipulated according to kit instructions, harvested CD34⁺Cells were counted. Analysis of CD34 by flow cytometry⁺Cell purity, results show the CD34⁺The purity of the hematopoietic stem cells was 96.9% (see FIG. 3).

Example 4 Induction of CD34⁺Differentiation of hematopoietic Stem cells into NK cells

Collecting separated and purified CD34⁺Hematopoietic stem cells (96.9% pure CD34⁺Hematopoietic stem cells), transferring to a new culture flask for culture, and adding a factor-containing NK cell differentiation medium; cell density of 1X 10⁶Per mL, at 37 deg.C and low oxygen (5% O)₂)、5%CO₂The culture was carried out in an incubator for 2 weeks, and half-replacement of the factor-containing NK cell differentiation medium was carried out every three days.

The NK cell differentiation medium containing the factors is prepared by adding SCF with the final concentration of 50ng/mL, 50ng/mL TPO, 50ng/mL FLT-3L, 50ng/mL IL-7 and 2ng/mL IL-2 factors into the NK cell differentiation medium;

the NK cell differentiation medium was Stemspan SFEM (purchased from STEMCELL) supplemented with 1% Vol glutamine, 0.5% Vol diabody, 250. mu.M magnesium ascorbyl phosphate, 2mM nicotinamide at final concentration.

EXAMPLE 5 expansion culture of NK cells

Collecting differentiated NK cells, transferring to a new culture flask for culture, adding factor-containing NK serum-free medium, and culturing at a cell density of 1 × 10⁶Per mL, at 37 deg.C, and low oxygen (5% O)₂)、5%CO₂The culture was carried out in an incubator for 2 weeks (see FIG. 4), and half-replacement of factor-containing NK serum-free medium was carried out every three days.

The NK serum-free medium containing the factors is prepared by adding IL-2, IL-7, IL-15 and IL-21 factors with final concentration of 10ng/mL into the NK serum-free medium.

The NK serum-free medium is purchased from Hitachi bioscience, Inc., of Beijing Hokkiso.

The amplified NK cells were collected, and the cell markers CD3 and CD56 were detected using flow antibody, and the results are shown in FIG. 5, in which CD3^-CD56⁺The ratio of NK cells was 81.3%.

The induction time of the NK cells is 23 days, and the amplification time is 14 days.

Example 6 measurement of NK cell killing Activity

NK cells induced by the NK cell induction method in the CN104711225A patent are used as a control group, colon cancer cell line LOVO and cervical cancer cell line HELA are used as target cells, effector cells are NK cells induced by the invention (the purity is 81.3%) and NK cells induced by CN104711225A (the purity is 97%).

The effective target ratio is 20:1, 10:1, 5:1 and 1:1, the number of target cells is 1 × 10⁴Per well, corresponding effector cells according to different effector target ratios. Each group was set with 3 replicate wells, and the average of the 3 replicate wells was taken. The detection time is 4h after cell mixing.

Wherein each experimental group and each control group are as follows:

each experimental group: each target cell + a different NK cell;

control group 1: the target cells release LDH to the maximum extent, and a certain volume of cell lysate needs to be added;

control group 2: target cells spontaneously release LDH;

control group 3: the effector cells spontaneously release LDH;

control group 4: background of blank medium;

control group 5: volume calibrated background, blank medium is added to a volume of cell lysate.

The detection method comprises the following steps: the killing efficiency of effector cells to target cells was determined using the CytoTox96 non-radioactive cytotoxicity assay kit (Promega corporation). The method is a detection method based on a colorimetric method, and can replace a 51Cr release method. CytoTox detection quantitatively measures Lactate Dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released upon cell lysis in a manner substantially identical to that of 51Cr in a radioactive assay. The released LDH medium supernatant was detected by a 30-minute coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. The kit specifically refers to CytoTox96 non-radioactive cytotoxicity detection kit specifications.

The cytotoxicity is calculated by the formula:

as shown in fig. 6, fig. 7 and table 1, the induced NK cells of the present invention and the NK cells of the control group both showed significant specific cytotoxic effects on tumor cells HELA and LOVO, but the former exhibited higher cytotoxic effects than the latter, and exhibited stronger cytotoxic effects with higher effective target ratio than gradient-dependent effective target ratio. The induced NK cells have cytotoxicity on HELA of 94.25% and LOVO of 91.24% at a 20:1 effective target ratio, while the control group NK cells have cytotoxicity on HELA of 70.32% and cytotoxicity on LOVO of 64.35%.

TABLE 1 in vitro killing of tumor cell lines by NK cells of the invention and control group

。

Claims (3)

1. A method for inducing and differentiating human embryonic stem cells into NK cells is characterized in that:

the method comprises the steps of recovering and passaging human embryonic stem cells, inducing to obtain mesodermal cells and inducing to obtain CD34⁺Hematopoietic stem cell, isolated and purified CD34⁺Hematopoietic stem cells, inducing to obtain NK cells, and performing amplification culture;

the human embryonic stem cell is human embryonic stem cell line H9;

recovering and passaging the human embryonic stem cells, namely recovering the human embryonic stem cells, then carrying out the passage, transferring to the 2 nd generation, and when the cell confluency is more than 80%, using the recovered human embryonic stem cells for induction to obtain mesodermal cells;

the mesoderm cell induction method comprises adding factor-containing E3 culture medium into human embryonic stem cells with cell confluency of above 80% transferred to generation 2, and controlling cell density to 0.8 × 10⁶Stirring at 15 rpm/mL under 5% CO at 37 deg.C₂Culturing for 24h in the incubator to form cell aggregates; centrifuging cell aggregate, removing supernatant, adding into SFDM medium containing factor, and culturing at cell density of 1 × 10⁶one/mL, 5% CO at 37 ℃₂After 2 days of culture in the incubator of (1), the SFDM medium containing the factors was replaced and the temperature was continued at 37 ℃ with 5% CO₂Inducing for 2 days in the incubator to obtain mesodermal cells;

the factor-containing E3 culture medium is prepared by adding factors of L-ascorbic acid 2-magnesium phosphate with the final concentration of 50 mug/mL, 5ng/mL sodium selenite, 50ng/mL FGF2, 50ng/mL VEGF, 2 muM CHIR 99021, 10 muM Blebbistatin and 10 muM Y-27632 to the E3 culture medium;

the E3 culture medium is formed by mixing a DMEM culture medium with 75% Vol and a 25% Vol F-12 culture medium;

the SFDM culture medium containing the factors is added with the factors with the final concentrations of 25ng/mL BMP4, 50ng/mL VEGF and 50ng/mL FGF 2;

the preparation method of the SFDM medium comprises the following steps: mixing 50% Vol IMDM medium and 50% Vol F-12 medium, adding 100. mu.g/mL polyvinyl alcohol, 100. mu.g/mL HSA, 1 × MEM nonessential amino acid solution, 0.1 × chemically defined lipid concentrate, 125. mu.M magnesium L-ascorbate 2-phosphate, 10ng/mL IGF 1;

the induction resulted in CD34⁺The hematopoietic stem cells are obtained by centrifuging mesodermal cells, discarding the supernatant, adding SFDM medium containing hematopoietic supporting factor, and controlling the cell density to 0.8 × 10⁶one/mL, 5% CO at 37 ℃₂After 2 days of culture in the incubator, the culture medium of SFDM containing hematopoietic supporting factors is replaced, and the induction is continued for 2 days to obtain CD34⁺Hematopoietic stem cells;

the SFDM culture medium containing the hematopoietic support factors is prepared by adding the hematopoietic support factors with final concentrations of 50ng/mL SCF, 20ng/mL TPO, 10ng/mL FLT-3L, 20ng/mL IL-3 and 25ng/mL BMP4 into the SFDM culture medium;

the method for inducing the NK cells comprises the step of collecting separated and purified CD34⁺Hematopoietic stem cells, adding NK cell differentiation medium containing factor, and controlling cell density to 1 × 10⁶one/mL, at 37 ℃ and 5% O₂、5%CO₂Culturing for 2 weeks in the incubator, and replacing half of the NK cell differentiation medium containing the factors every three days;

the NK cell differentiation medium containing the factors is prepared by adding SCF with the final concentration of 50ng/mL, 50ng/mL TPO, 50ng/mL FLT-3L, 50ng/mL IL-7 and 2ng/mL IL-2 factors into the NK cell differentiation medium;

the NK cell differentiation medium is Stemspan SFEM added with 1% Vol glutamine, 0.5% Vol double antibody, 250 μ M magnesium ascorbyl phosphate and 2mM nicotinamide at final concentration.

2. The method for inducing differentiation of human embryonic stem cells into NK cells according to claim 1, wherein:

the separation and purification of CD34⁺Hematopoietic stem cell, isolated and purified CD34⁺The purity of the hematopoietic stem cells was 96.9%.

3. The method for inducing differentiation of human embryonic stem cells into NK cells according to claim 1, wherein:

the amplification culture method comprises collecting NK cells, transferring to a new culture flask for culture, adding factor-containing NK serum-free medium with cell density of 1 × 10⁶one/mL, at 37 ℃ and 5% O₂、5%CO₂Culturing in an incubator for 2 weeks, and replacing half amount of NK serum-free medium containing factors every three days;

the NK serum-free medium containing the factors is prepared by adding IL-2, IL-7, IL-15 and IL-21 factors with final concentration of 10ng/mL into the NK serum-free medium.

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