CN111235105A - Method for differentiating human pluripotent stem cells into natural killer cells and application - Google Patents

Abstract

The invention relates to the field of stem cell biology, in particular to a method and application for differentiating human pluripotent stem cells into natural killer cells. The invention discloses a natural killer cell derived from pluripotent stem cells, which expresses CD56, Nkp30, Nkp44 and Nkp46, and also expresses markers CD16 and CD94 of mature natural killer cells. The invention also discloses a method for preparing natural killer cells, comprising the following steps: S1: forming an embryoid body; S2: differentiating the embryoid body into hematopoietic progenitor cells; S3: differentiating hematopoietic progenitor cells into NK cells; S4: NK cells maturation and expansion. The differentiation method provided by the present invention, based on a well-defined medium and an optimized combination of cytokines, can rapidly, efficiently, simply and inexpensively differentiate induced pluripotent stem cells into natural killer cells.

Description

A kind of method and application of differentiating from human pluripotent stem cells to natural killer cells

technical field

The invention relates to the field of stem cell biology, in particular to the lineage-specific differentiation of pluripotent or pluripotent stem cells, and in particular to a method and application for differentiating human pluripotent stem cells into natural killer cells.

Background technique

The latest national cancer report in 2018 shows that in 2014, there were an estimated 3.804 million new cases of malignant tumors in the country, and an average of more than 10,000 people were diagnosed with cancer every day. The tumor incidence rate was 278.07/100,000, the cumulative incidence rate of 0-74 years old was 21.58%, and the cumulative mortality rate was 12.00%. In 2013, "Science" magazine listed tumor immunotherapy as the first of the ten scientific breakthroughs. Immunotherapy has become a new generation of tumor treatment methods following surgery, chemotherapy, radiotherapy and tumor targeted therapy, bringing great advantages to the treatment of tumor patients. new Hope.

Natural killer cells (NK) are the main force of the innate immune system and the body's first line of defense against infections and tumors. Stress-induced" (stress-induced) patterns recognize and kill diseased cells. Activation of NK cells depends on the balance of activating and inhibitory receptor signaling on their cell surfaces. Activated NK cells can kill tumor cells through various mechanisms: NK cells can release perforin and granzyme, which directly act on target cells to kill cancer cells; they can promote cancer cell apoptosis by secreting pro-inflammatory cytokines; they can recognize Monoclonal antibody drug-labeled cancer cells to complete the killing.

The recognition and killing of NK cells are characterized by MHC-I non-restrictive, pan-specific and rapid response, no graft-versus-host reaction (GVHD), allogeneic cell reinfusion can be used, short survival cycle in vivo, and no cytokine storm It can be used in combination with tumor-targeting antibodies to enhance the anti-cancer effect, and can be combined with gene editing technology to develop targeted NK cell products for different tumor targets. These characteristics make them show in tumor immunotherapy. huge application potential.

Previous studies have confirmed that NK cells in cancer patients are often reduced in number and impaired in function. Therefore, adoptive therapy of NK cells has the effect of restoring the patient's immune function to resist tumors. However, the results of clinical studies after reinfusion of autologous NK cells show that their anti-tumor effect is very limited. The main reasons may include: (1) the KIR receptor of autologous NK cells matches the HLA of tumor cells, and the "self" recognition signal inhibits the activation of NK cells; (2) the function of autologous NK cells in patients is impaired, and it is difficult to produce the ideal killing effect.

It is difficult to directly expand NK cells from peripheral blood, and the patient's cells may fail to expand, and usually the expanded cell mixture contains a certain proportion of T cells, which needs to be removed, otherwise it will cause transplantation. Germ-versus-host disease (GVHD). At the same time, the quality of NK cells from different donors varies greatly, which directly affects the efficacy and makes it difficult to form standardized products.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the ability to proliferate indefinitely and can be differentiated into Almost all functional cells, including NK cells. In vitro large-scale and standardized preparation of NK cells from human pluripotent stem cells is an effective solution for NK cell products. However, the differentiation process that has been reported so far has problems such as complex process, long cycle, feeder cell dependence, unclear medium composition, functional defects of differentiated cells, and low purity and viability, making the differentiation process unfavorable for large-scale production.

The patents closest to the present invention are: patent CN102388130A discloses a kind of differentiation of pluripotent cells, patent CN107429230A discloses a method and composition for inducing hematopoietic cell differentiation, patent CN102822332A discloses a Methods for the production of natural killer cells and dendritic cells from hemangioblasts.

Patent CN102388130A has at least the following problems: it only includes the differentiation of hPSCs to hematopoietic precursor cells, but does not include the stage of differentiation of hematopoietic precursor cells to NK cells. The combination of factors used did not bypass the primitive hematopoietic phase.

Patent CN107429230A has at least the following problems: (1) The efficiency of EB 3D culture is extremely low. (2) 2D differentiation conditions were employed from hPSCs to permanent pluripotent blood precursor cells. (3) The expensive commercial medium (such as Stempro 34), bovine serum, etc. with unclear components was used in the whole process, which is not conducive to the production of clinical grade cell products. (4) The enriched CD34+/CD45+ cells are used for iNK (iPSC-derived NK, NK cells induced to differentiate by iPSC) differentiation, and the process is cumbersome.

Patent CN102822332A has at least the following problems: (1) expensive commercial medium with unclear composition is used; (2) most of the obtained hematopoietic endothelial cells belong to primitive hematopoietic stage cells; (3) it involves the fourth day of differentiation The single cells obtained by EB digestion were inoculated into the Methylcellulose culture system for further differentiation to obtain vascular cells, which is not conducive to the large-scale production of clinical-grade NK cells; (4) The second and third steps in the method used human-containing serum medium.

In conclusion, the application of NK cells in the immunotherapy of cancer has unique advantages and broad application prospects, while peripheral blood-derived NK cells (PB-NK) have a limited source of NK cells, differ in quality and yield, and expand Posts often contain T cell contamination. Therefore, the preparation of NK cells from hPSC can solve these problems and achieve stable large-scale production. But there are many problems in the existing differentiation scheme: such as the use of serum-containing medium, and serum is an uncontrollable culture factor that brings unstable factors such as batch differences; the use of animal cell-derived feeder cells such as OP9 does not It meets the clinical production requirements; the use of commercial serum-free medium is expensive and not suitable for large-scale production; the differentiation process is complex, and the dosage and time of cytokine use are not fully optimized and streamlined, resulting in high costs; long differentiation cycle also increases production costs At the same time, it improves the uncontrollable process control; the problems of low purity and lack of function of differentiated NK cells.

Therefore, it is urgent to invent a stable, high-efficiency, low-cost, high-purity, and functional NK cell differentiation system to establish a foundation for the large-scale production and clinical application of NK cells.

SUMMARY OF THE INVENTION

Although the process of in vitro differentiation from pluripotent stem cells to natural killer cells is relatively clear in principle, and there are many methods for inducing differentiation, the existing differentiation methods may suffer from long differentiation cycle, low differentiation efficiency and purity, and poor differentiation. Due to the problems of unknown ingredients, feeder cell dependence, complicated operation and high cost, it is difficult to expand production and use in clinical practice. The differentiation procedure provided by the present invention is based on a well-defined medium and an optimized combination of cytokines, and can differentiate induced pluripotent stem cells into natural killer cells in a fast, efficient, simple and low-cost manner.

Its advantages are as follows: First, the present invention achieves stable and efficient differentiation of natural killer cells, and can obtain functional NK cells with a purity of more than 90% in about 40 days; second, we do not use serum-containing NK cells throughout the differentiation process. The culture system or feeder cells are suitable for scale-up production and clinical application; thirdly, we try to simplify and optimize the medium and cytokines used in the differentiation process, avoiding the use of expensive commercial medium such as Stemspan, etc., so The cost of the entire differentiation process is greatly reduced.

The key innovations of the present invention are: 1) The EB 3D differentiation method has low cost, short cycle and high efficiency. It only takes about 25 days to obtain a large number of cells with high purity, and cells similar to NK cells derived from peripheral blood NK cells with toxic effects are suitable for large-scale production; 2) The composition of the differentiation medium is clear, and NK cells can be successfully differentiated without the use of serum and trophoblast cells, which is suitable for clinical applications; 3) The expansion conditions are optimized, and the same The differentiated NK cells can be massively expanded without serum and trophoblast cells; 4) With optimized cryopreservation conditions, NK cells can be stored for a long time and maintain high cell viability after recovery.

Specifically, the technical scheme of the present invention is as follows:

The first aspect of the present invention discloses a natural killer cell derived from pluripotent stem cells, which expresses CD56, Nkp30, Nkp44 and Nkp46, and also expresses markers CD16 and CD94 of mature natural killer cells. In the present invention, NKp46, NKp30 and NKp44 all belong to natural cytotoxicity receptor (NCR), and all three are members of immunoglobulin superfamily (IgSF), but have no homology with each other. NCR is only expressed on the surface of NK cells and is a unique marker of NK cells. It usually plays a killing role when KIR/KLR loses the ability to recognize "self". NKp30 (NCR3) is an important member of the NCR family, expressed on the surface of all NK cells, and plays an important role in the process of NK cell activation and tumor killing. NKp44, namely CD336, is a member of IgSF, a natural cytotoxicity receptor (NCR2). It is expressed in activated NK cells, and its ligand is DAP12, which is involved in mediating the killing activity of NK cells. The extracellular domain of NKp46 contains two Ig-like domains, and the extracellular domain of NKp30 has only one V-type domain. The cytoplasmic domains of NKp46 and NKp30 are shorter, and both transmembrane domains contain positively charged arginine.

A second aspect of the present invention discloses a method for preparing natural killer cells, comprising the following steps:

S1: form embryoid body;

S2: embryoid body differentiates into hematopoietic progenitor cells;

S3: hematopoietic progenitor cells differentiate into NK cells;

S4: Maturation and expansion of NK cells.

Preferably, the S1 includes:

S11: The cell suspension of human pluripotent stem cells is placed on a shaker and shaken overnight to form an embryoid body.

Preferably, the S2 includes:

S21: remove the embryoid body supernatant, add fresh first-step differentiation medium for cell culture; wherein, the first-step differentiation medium is to add a small molecule GSK3β inhibitor to the basal differentiation medium, and at least one The following components: BMP signaling pathway activator, VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), Flt3L (FMS-like tyrosine kinase 3 ligand), IL3 (leukocyte interleukin 3), IL6 (interleukin 6), insulin (insulin), IGF-1 (insulin-like growth factor 1) and TPO (human thrombopoietin);

S22: Remove the first-step differentiation medium, and add the second-step differentiation medium for cell culture; wherein, the second-step differentiation medium is the addition of VEGF, bFGF, and at least one of the following components to the basal differentiation medium: Cytokines of BMP signaling pathway activator, Noda inhibitor, SCF, Flt3L, IL15, IL3, IL6, insulin, IGF-1 and TPO.

S23: remove the second-step differentiation medium, add the third-step differentiation medium for cell culture, and obtain hematopoietic progenitor cells; wherein, the third-step differentiation medium is the addition of growth factors and colony-stimulating factors to the basal differentiation medium .

More preferably, the growth factor is selected from one of EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), bFGF, insulin, IGF-1, PGF (platelet growth factor) and PDGF (platelet-derived growth factor). One or more; the colony-stimulating factor is selected from G-CSF (granulocyte colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor) , one or more of multi-CSF (multiple colony stimulating factor, also known as IL-3), EPO (erythropoietin), TPO, SCF and Flt-3L.

Preferably, in S3, the third-step differentiation medium is removed, the cells are inoculated into a cell culture vessel coated with matrix protein, and the fourth-step differentiation medium is added for cell culture; the matrix protein at least includes Notch pathway activator protein or one of integrins; wherein, the fourth step differentiation medium is to add colony stimulating factor and interleukin to the basal differentiation medium.

Culturing in a cell culture vessel coated with Notch pathway activator protein and integrin can not only increase the growth rate of cells, but also improve the differentiation efficiency of NK cells.

In some specific embodiments of the present invention, the cell culture vessel is a cell culture flask.

More preferably, the colony stimulating factor is selected from one or more of G-CSF, M-CSF, GM-CSF, multi-CSF(IL-3), EPO, TPO, SCF and Flt-3L; The interleukin is selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27.

More preferably, the Notch pathway activating protein is selected from one or more of DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein, Jagged-2 recombinant protein, and their variants, where "they" refers to DLL1 Recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein or Jagged-2 recombinant protein.

The integrin is selected from the group consisting of Fibronectin (fibronectin), Laminin (laminin), Vitronectin (vitronectin), MAdCAM-1 (adhesion cell adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule- 1) and ICAM (Intercellular Adhesion Molecule) and one or more of the variants of these integrins, where "they" refers to Fibronectin (fibronectin), Laminin (laminin), Vitronectin (vitronectin) ), MAdCAM-1 (adhesion cell adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1) or ICAM (intercellular adhesion molecule).

Preferably, the step S4 includes: removing the fourth step differentiation medium and adding the fifth step differentiation medium for cell culture; wherein, the fifth step differentiation medium is adding interleukin and promoting Substances by which NK cells mature and expand.

More preferably, the interleukin is selected from one or more of IL-2, IL-12, IL-18, IL-21, IL-27 and IL-15; the promotion of NK cell maturation and expansion. The substance to be increased is selected from one or more of human AB plasma, human platelet lysate, Vitamin A (vitamin A), nicotinamide, Vitamin E (vitamin E) and Heparin (heparin).

Preferably, it also includes step S5: freezing the differentiated NK cells using a cryopreservation solution; the cryopreservation solution includes: sodium chloride, sodium dextrose, sodium acetate, potassium chloride, magnesium chloride, human serum albumin and DMSO.

In some specific embodiments of the present invention, the components of the cryopreserved solution of NK cells are shown in Table 1 below.

Table 1

serial number Element Final concentration 1 Sodium chloride 1-10mg/ml 2 Sodium Gluconate 1-10mg/ml 3 Sodium acetate 1-10mg/ml 4 Potassium chloride 0-1mg/ml 5 Magnesium chloride 0-1mg/ml 6 HSA (Human Serum Albumin) 10-30% 7 DMSO (Dimethyl Sulfoxide) 5-20%

The steps of the method for preparing the above-mentioned natural killer cells in the present invention are as follows:

(1) Day-1～Day 0: Formation of embryoid body (EB)

Digest the well-undifferentiated iPSCs cultured to a degree of polymerization of 70-90% into a single-cell suspension, resuspend them in iPSC maintenance medium at a certain density, and place them in a 37°C incubator for overnight shaking with shaking. EBs with relatively uniform size and shape were formed.

Day-1～Day 0 experimental operation details and optimization

1) Culture of human iPSCs

The human iPSCs used in the experiments have been rigorously verified for pluripotency (expressing various pluripotency markers, and can form teratomas containing inner, middle and outer germ layers in immunodeficient mice). iPSCs were normally cultured in iPSC maintenance medium using E8 or TeSR or other similar medium.

2) Formation of EB

Embryoid body formation experiments were performed when iPSCs were cultured to a degree of polymerization of 70-90% as described above. The specific operation is as follows: using TrypLE or accutase to digest iPSCs into a complete single-cell suspension, resuspending them in iPSC maintenance medium, and adding Rock inhibitor to the medium. The cell suspension was placed in a 3D shaker in a 37°C incubator for shaking culture, and the rotating speed of the shaker was 10-100 rpm; the shaking culture in this step was 8-32 hours; at the end of the culture, the obtained size and shape were relatively uniform EB. For example, when using a T25 culture flask, the Rock inhibitor is Y-27632 at a concentration of 10 μM; the cell density is 0.1×10 ⁶ -5×10 ⁶ /mL; the rotating speed of the shaker is 10-20 rpm; the incubation time is 8-32 Hour.

(2) Day 0～Day 6-12: Differentiation of pluripotent stem cells into hematopoietic progenitor cells

The above-mentioned EBs from D0 were replaced with differentiation medium at different time points, and EBs containing highly expressing CD34+ cells were harvested on D6-12.

Day 0～Day 6-12 Experimental operation details and optimization

1) Day 0: Change to the first step differentiation medium

Remove the flask containing the D0 EBs from the incubator, tilt the flask to allow the EBs to sink to the bottom, remove the supernatant, and add fresh first-step differentiation medium. The first step of differentiation medium is to add a small molecule GSK3β inhibitor to the basal differentiation medium, and one or more selected from BMP signaling pathway activator, VEGF, bFGF, SCF, Flt3L, IL3, IL6, insulin, IGF- 1 and TPO cytokines. BMP signaling pathway activators are BMP2, BMP4, SB4, ventromorphins (SJ000291942, SJ000063181, SJ000370178), isoliquiritigenin, diosmetin (geranin), apigenin (apigenin) and biochanin (garbanin), among which, Isoliquiritigenin is derived from The flavonoids from Glycyrrhiza glabra root have antitumor activity; GSK3β inhibitors are NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314 and CHIR99021, etc. For example, when using a T25 culture flask for culture, the GSK3β inhibitor is CHIR99021 at a concentration of 0.5-20 μM; the BMP signaling pathway activator is BMP4 at a concentration of 0-200 ng/mL; cytokines include insulin, IGF-1, VEGF, bFGF, the concentrations were 0-10μg/mL, 0-100ng/mL, 5-100ng/mL and 0-100ng/mL, respectively.

The time that EBs were cultured in the first-step differentiation medium was (2-4) days.

The composition of the basal differentiation medium is shown in Table 2 below.

Table 2

2) Day 2-4: Change to the second step differentiation medium

Take out the culture flask containing EBs from the incubator, tilt the flask to make the EBs sink to the bottom, remove the supernatant of the first-step differentiation medium, and add fresh second-step differentiation medium. The second step of differentiation medium is to add VEGF, bFGF, and one or more selected from BMP signaling pathway activator, Noda inhibitor, SCF, Flt3L, IL15, IL3, IL6, insulin, IGF- 1 and TPO cytokines. The selection range of BMP signaling pathway activators is similar to that of D0; Noda inhibitors are selected from Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947 , LT580276, A83-01, and their derivatives. For example, when using T25 culture flasks for culture, the BMP signaling pathway activator is BMP4 at a concentration of 0-200ng/mL; Noda inhibitor is SB431542 at a concentration of 0-20μM; cytokines include insulin, IGF-1, VEGF, bFGF, IL3, and IL6 at concentrations of 0-10 μg/mL, 0-100 ng/mL, 5-100 ng/mL, 0-100 ng/mL, 0-20 ng/mL, and 0-20 ng/mL, respectively.

The medium exchange time point of the second step differentiation medium can be between Day2 and Day4.

3) Day 3-6: Change to the third step differentiation medium

Take out the culture flask containing the EBs from the incubator, tilt the flask to make the EBs sink to the bottom, remove the supernatant of the second-step differentiation medium, and add fresh third-step differentiation medium. The third step of differentiation medium is the addition of growth factors and colony-stimulating factors to the basal differentiation medium. The growth factor is one or more selected from the group consisting of EGF, VEGF, bFGF, insulin, IGF-1, PGF, PDGF, and the like. The colony stimulating factor is one or more selected from G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, Flt-3L and the like. For example, when cultured in T25 flasks, the growth factors are insulin, VEGF, IGF-1 and bFGF at concentrations of 0.5-10 μg/mL, 5-100 ng/mL, 0-100 ng/mL and 0.5-100 ng/mL, respectively ; The colony stimulating factors are TPO, SCF and Flt-3L at concentrations of 0-100ng/mL, 0-100ng/mL and 0-100ng/mL, respectively;

The medium exchange time point of the third step differentiation medium can be between Day3 and Day6.

4) Day 6-12: Detect the obtained hematopoietic progenitor cells

The diameter of the embryoid body was detected on the 0th day, the 5th day and the 12th day of the differentiation culture.

Choose an appropriate time point from Day6 to Day12, and use flow cytometry to detect the cell phenotype of the hematopoietic progenitor cells in the embryoid body. It is proved that the obtained cells contain hematopoietic progenitor cells expressing CD34, and CD34-positive hematopoietic progenitor cells account for the total number of hematopoietic progenitor cells. The percentage of cells should be between 20% and 80%.

Among them, the antibody information of flow is as follows:

APC Mouse Anti-Human CD34 Clone 581 (RUO), BD, #555824;

FITC anti-human CD45, Biolegend, #304006.

(3) 1-2 weeks after EB inoculation (Day6-12～Day20-26): hematopoietic progenitor cells differentiate into NK cells

The above Day6-12 EBs containing CD34+ hematopoietic progenitor cells were resuspended in the fourth step differentiation medium, seeded in culture flasks coated with cell matrix for 2 weeks, and replaced at the end of week 1 and 2 culture medium to maintain the different time periods of action of the following factors. NK cells containing high CD3-CD56+ were harvested at the end of week 2 (Day 20-26).

1-2 weeks after EB inoculation (Day6-12～Day20-26) experimental operation details:

1) Day 6-12: Change to the fourth step differentiation medium

Tilt the flask to allow the EBs to sink to the bottom, remove the third step differentiation medium supernatant, and resuspend the EBs in fresh fourth step differentiation medium. The fourth step of differentiation medium is to add colony-stimulating factor and interleukin to the basal differentiation medium. The colony stimulating factor is one or more selected from G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, Flt-3L and the like. Interleukin is one or more selected from IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27. For example, when cultured in T25 flasks, the colony stimulating factors are TPO, SCF, Flt-3L and IL-3 at concentrations of 0-100ng/mL, 5-100ng/mL, 5-100ng/mL and 5-100ng, respectively /mL, and the time period of effect is the first week, the second week or the 1-2 weeks after the inoculation. Interleukins are IL-2, L-7 and IL-15, the concentrations are 100-1000IU/mL, 5-100ng/mL and 5-100ng/mL, respectively, and the time period of action is the first week after EB inoculation, Week 2 or Weeks 1-2. The medium exchange time point of the fourth step differentiation medium can be between Day6 and Day12.

The EBs resuspended in the fourth step differentiation medium were seeded at the appropriate density in culture flasks coated with matrix protein. The matrix protein is at least one of the following components: Notch pathway activator protein and integrin. Notch pathway activating proteins are DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein, Jagged-2 recombinant protein, and variants of these Notch pathway activating proteins. The integrins are Fibronectin (fibronectin), Laminin (laminin), Vitronectin (vitronectin), MAdCAM-1 (adhesion cell adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1) and ICAM (Intercellular Adhesion Molecule), and variants of these integrins, etc. For example, when cultured in T25 flasks, the Notch pathway activating protein is the DLL4-Fc recombinant protein. The integrin is VCAM-1.

Change half of the fresh fourth step medium every 3-7 days depending on cell density.

2) Detect the NK cells obtained in the second week after EB inoculation (Day20-26)

At 2 weeks after EB seeding, cells in suspension in the wells were collected. The expression of cell surface related marker proteins was detected by flow cytometry. Detection indicators include: CD56, NKp30, NKp44, NKp46.

Among them, the antibody information of flow is as follows:

PE Mouse Anti-Human CD56 Clone B159 (RUO), BD, #555516;

647 Mouse anti-Human CD337(NKp30)，BD，#558408；Alexa

647 Mouse anti-Human CD337(NKp30), BD, #558408;

647 Mouse Anti-Human NKp44(CD336)，BD，#558564；Alexa

647 Mouse Anti-Human NKp44(CD336), BD, #558564;

APC Mouse Anti-Human CD335 (NKp46), BD, #558051.

3) 3-4 weeks after EB inoculation (Day27～Day40): Maturation and expansion of NK cells 3-4 weeks after EB inoculation (Day27～Day40) Experimental operation details:

After two weeks of EB differentiation, when the cell density reached (1-2)×10 ⁶ cells/mL, the cells were collected, centrifuged to remove the fourth-step differentiation medium, and resuspended at a cell density of (0.5-1)×10 ⁶ cells/mL in fresh fifth step differentiation medium. The fifth step of the differentiation medium is to add interleukins and other substances that promote the maturation and expansion of NK cells to the basal differentiation medium. Interleukin is one or more selected from IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27. Other substances that promote the maturation and expansion of NK cells are selected from human AB plasma, human platelet lysate, Vitamin A, nicotinamide (NAM, Vitamin B3), Vitamin E, Heparin and the like. For example, when cultured in T25 flasks, the interleukins are IL-2, IL-12, IL-18, IL-21 and IL-27 and IL-15 at concentrations of 100-1000IU/mL, 0-100ng, respectively /mL, 0-100ng/mL, 0-100ng/mL, 0-100ng/mL, and 5-100ng/mL; supplements to promote NK cell maturation and expansion are human platelet lysate, nicotinamide (NAM), vitamin E and Heparin at concentrations of 1-10%, 1-10 mmol/L, 0-10 mg/mL and 0-100 μg/mL, respectively.

The resulting NK cells were assayed at 3-4 weeks after EB inoculation (Day 27-40)

At 3-4 weeks of EB differentiation (Days 27-40), cells in suspension in the wells were collected. The expression of cell surface related marker proteins was detected by flow cytometry. Detection indicators include: CD56, CD94 and CD16.

Among them, the antibody information of flow is as follows:

PE Mouse Anti-Human CD56 Clone B159 (RUO), BD, #555516;

APC Mouse Anti-Human CD94, BD, #559876;

APC Mouse Anti-Human CD16 Clone B73.1 (RUO), BD, #561304.

In a specific embodiment of the present invention, the above-mentioned human pluripotent stem cells are prepared by the method disclosed in the patent CN 108085299A.

It should be understood that those skilled in the art can also select any commercial cell line or cell line of human pluripotent stem cells as required to complete the present invention, and all fall within the protection scope of the present invention.

In the present invention:

1. The components of the basal differentiation medium include: IMDM, F-12, rHSA (recombinant human serum albumin), MTG-Monothioglycerol (thioglycerol), Ascorbic Acid (ascorbic acid), Human Transferrin (human transferrin), Na Selenite (sodium selenite) and Ethanolamine (ethanolamine). Among them, IMDM and F12 can be used as the basis for the development of serum-free formula. This medium is suitable for mammalian cell culture under the condition of low serum content. In this medium formula, IMDM and F-12 are mixed in a 1:1 ratio. The combination and concentration of other ingredients is an optimized, serum-replacement supplement that maintains differentiation into blood progenitor cells and maintains proliferation of differentiated cells.

2. GSK-3 inhibitors: substances that block the kinase activity of GSK3β protein, such as GSK3β inhibitor IX (6-bromo indirubin 3'-oxime), SB216763, GSK3β inhibitor VII (4-dibromoacetophenone) , L803-mts, and CHIR99021 with high selectivity. CHIR99021 is preferred in the present invention.

The concentration of CHIR99021 in the medium is not particularly limited as long as it inhibits the kinase activity of the GSK3β protein. , 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, but not limited thereto. It is preferably 1 to 8 μM.

3. BMP signaling pathway activator: substances that activate the BMP signaling pathway, including BMP2, BMP4, SB4, ventromorphins (SJ000291942, SJ000063181, SJ000370178), isoliquiritigenin, diosmetin, apigenin, biochaninA, etc. The BMP activator used in the present invention is BMP4. The concentration of BMP4 in the medium is not particularly limited as long as it activates the BMP signaling pathway. ml, 80ng/ml, 90ng/ml, 100ng/ml, 110ng/ml, 120dng/ml, 130ng/ml, 140ng/ml, 150ng/ml, 160ng/ml, 180ng/ml, 200ng/ml, but not limited thereto . It is preferably 5 to 50 ng/ml.

4. Nodal inhibitors: substances that inhibit the Nodal signaling pathway. Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276, A83-01, and derivatives thereof can be selected. The Nodal inhibitor used in the present invention is SB431542. The concentration of SB431542 in the medium is not particularly limited as long as it inhibits the Nodal signaling pathway. , 20 μM, 25 μM, 30 μM, but not limited thereto. It is preferably 1-20 μM.

5. Colony stimulating factor: a cytokine that can stimulate the proliferation and differentiation of hematopoietic stem cells. G-CSF, M-CSF, GM-CSF, multi-CSF(IL-3), EPO, TPO, SCF and Flt-3L can be selected. Colony stimulating factors used in the present invention are G-CSF, GM-CSF, TPO, SCF and Flt-3L and multi-CSF. The concentrations of TPO, SCF and Flt-3L in the medium are not particularly limited as long as they can stimulate the proliferation and differentiation of hematopoietic stem cells. For example, the concentrations of TPO are 0ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml. ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, but not limited thereto. It is preferably 0 to 100 ng/ml. The concentrations of SCF are 0ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 80ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, but not limited to this. It is preferably 0 to 100 ng/ml. The concentrations of Flt-3L are 0ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 80ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, but not limited thereto. It is preferably 0 to 100 ng/ml.

6. Notch signaling pathway activator: a recombinant protein that activates the Notch signaling pathway. DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein, Jagged-2 recombinant protein, and variants of these Notch pathway activating proteins can be selected. The Notch activator used in the present invention is DLL4-Fc recombinant protein. The density of the coated DLL4-Fc recombinant protein is not particularly limited as long as it can activate the Notch signaling pathway. For example, the density of the DLL4-Fc recombinant protein coating is 0 μg/cm ² , 0.1 μg/cm ² , 0.2 μg /cm ² , 0.3 μg/cm ² , 0.4 μg/cm ² , 0.5 μg/cm ² , 0.6 μg/cm ² , 0.7 μg/cm ² , 0.8 μg/cm ² , 0.9 μg/cm ² , 1 μg/cm ² , 2 μg/cm ² , 3 μg/cm ² , 4 μg/cm ² , 5 μg/cm ² , but not limited thereto. It is preferably 0.5 to 2 μg/cm ² .

7. Growth factors: natural proteins that can stimulate cell proliferation and cell differentiation. EGF, VEGF, bFGF, insulin, IGF-1, PGF, PDGF, etc. can be selected. Growth factors used in the present invention are insulin, VEGF, IGF-1 and bFGF. The concentration of insulin, VEGF and bFGF in the medium is not particularly limited as long as it can stimulate cell proliferation and cell differentiation. For example, the concentration of insulin is 0 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml , 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 20 μg/ml and 30 μg/ml. Preferably it is 0-10 microgram/ml. VEGF concentrations are 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml ml, 150ng/ml, 200ng/ml, but not limited thereto. It is preferably 5 to 100 ng/ml. The concentrations of IGF-1 were 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 150ng/ml, 200ng/ml. Preferably it is 0-100 microgram/ml. The concentrations of bEGF are 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml ml, 150ng/ml, 200ng/ml, but not limited thereto. It is preferably 0 to 100 ng/ml.

8. Interleukins: cytokines that can mediate the activation, proliferation and differentiation of immune cells. One or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27 can be selected at different differentiation stages .

In the present invention, the interleukins used in D6-12 to D20-26 (that is, within two weeks after EB inoculation) are IL-2, IL-7 and IL-15. The concentration of IL-2, IL-7 and IL-15 in the medium is not particularly limited as long as it can mediate immune cell activation, proliferation and differentiation, for example, the concentration of IL-2 is 0IU/ml, 50IU/ml, 100IU /ml, 200IU/ml, 300IU/ml, 400IU/ml, 500IU/ml, 700IU/ml, 800IU/ml, 1000IU/ml, 2000IU/ml, but not limited thereto. It is preferably 0 to 1000 IU/ml. IL-7 concentrations were 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, but not limited thereto. It is preferably 5 to 100 ng/ml. IL-15 concentrations were 1ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 25ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 80ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, but not limited thereto. It is preferably 5 to 100 ng/ml.

9. ROCK inhibitor: a substance that inhibits the function of Rho kinase (ROCK). For example: Y-27632, HA100, HA1152, and Blebbistatin. Y-27632 is preferred in the present invention.

The concentration of Y-27632 in the medium is not particularly limited as long as it is a concentration that inhibits Rho kinase. μM, 7 μM, 7.5 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 30 μM, 50 μM, but not limited thereto. Preferably it is 10 μM.

The third aspect of the present invention discloses a natural killer cell, which is prepared by the above method.

A fourth aspect of the present invention discloses a cell population enriched with the above-mentioned natural killer cells.

A fifth aspect of the present invention discloses a medicament for preventing and/or treating tumors, the medicament comprising the above-mentioned natural killer cells.

The key points of the present invention and the points to be protected:

1) A fast, efficient, and low-cost EB 3D differentiation method based on the combined use of small molecule compounds and cytokines (including the types of small molecule compounds and cytokines and their combination, adding time, concentration, etc.), making the operation easier, The results are more stable and the cost is lower, which is conducive to large-scale iNK production.

2) Components of NK cell differentiation and expansion medium. Key innovations: The differentiation and expansion medium has well-defined components, which can successfully differentiate and expand NK cells without the use of serum and trophoblast cells, suitable for the production of large-scale cell preparations and clinical applications.

3) With optimized cryopreservation conditions, NK cells can be stored for a long time and maintain high cell viability after recovery.

On the basis of conforming to common knowledge in the art, the above preferred conditions can be combined arbitrarily without departing from the concept and protection scope of the present invention.

Compared with the prior art, the present invention has the following significant advantages and effects:

First, this method uses 3D suspension culture during differentiation induction, which can obtain NK cells larger than 1×10 ⁷ from 1×10 ⁵ hiPSCs in a short period of time (about 40 days), which is suitable for large-scale cell preparation production;

Second, through the combined use of small molecule compounds and cytokines, the differentiation process is finely regulated to achieve stable and efficient differentiation, and the proportion of CD56+ NK cells in the final culture system can reach more than 90%;

Third, the NK cells prepared by this method have similar cell phenotypes and functions to PB-NK cells, and the preparation cost is low, and has great potential for clinical and scientific research applications;

Fourth, the method uses serum-free medium and does not use feeder cells during induction of differentiation and expansion, which is suitable for the production and application of subsequent clinical-grade cell preparations.

Description of drawings

Figure 1 shows the flow chart of the preparation of natural killer cells disclosed in the present invention.

Figure 2 shows the process of inducing differentiation from pluripotent stem cells to mature NK cells in the method disclosed in the present invention (day -1, day 0, day 5, day 12, 2 weeks after EB inoculation and EB inoculation, respectively 4 weeks later).

Figure 3 uses flow cytometry to detect the specific indicators of D8 EB, showing that hematopoietic stem cells (CD34+) have reached a high level.

Figure 4. The specific indicators of iNK cells in the 2nd and 4th weeks after EB inoculation were detected by flow cytometry. High expression of Nkp30, Nkp44 and Nkp46. At the 4th week after EB inoculation, iNK cells simultaneously expressed CD94 and CD16, specific markers of mature NK cells, indicating that NK cells had matured.

Figure 5 shows that iNK cells collected at the 4th week after EB inoculation produced significant cytotoxic effect on target cell K562, and the cell killing ability was similar to that of PB-NK.

Figure 6 shows that the concentration of CHIR99021 in the first step differentiation medium affects the differentiation efficiency of CD34+ blood progenitor cells.

Figure 7 shows that the timing of second differentiation medium change affects the differentiation efficiency of CD34+ blood progenitor cells.

Figure 8 shows that the timing of the third differentiation medium change affects the differentiation efficiency of CD34+ blood progenitor cells.

Figure 9 shows that EB inoculation time points affect the differentiation efficiency of NK cells (CD3-CD56+).

Figure 10 shows that the effected concentration of BMP4 affects the differentiation efficiency of CD34+ blood progenitor cells.

Figure 11 shows that the effected concentration of SB affects the differentiation efficiency of CD34+ blood progenitor cells.

Figure 12 shows that the effected concentration of bFGF affects the differentiation efficiency of CD34+ blood progenitor cells.

Detailed ways

The technical solutions of the present invention will be described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the described embodiments.

The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description. The reagents and raw materials used in the present invention are all commercially available.

Example 1

The present embodiment discloses a method for preparing natural killer cells, comprising the following steps:

S1: form embryoid body;

S2: embryoid body differentiates into hematopoietic progenitor cells;

S3: hematopoietic progenitor cells differentiate into NK cells;

S4: Maturation and expansion of NK cells.

Specifically, the flowchart is shown in Figure 1, and the steps are as follows:

(1) Day-1～Day 0: Formation of embryoid body (EB)

1) Culture of human pluripotent stem cells (iPSC)

The human iPSCs used in the experiments have been rigorously verified for pluripotency (expressing various pluripotency markers, and can form teratomas containing inner, middle and outer germ layers in immunodeficient mice). iPSCs were normally cultured in iPSC maintenance medium using E8 or TeSR or other similar medium.

The human iPSCs were prepared by the method disclosed in patent CN 108085299A.

2) Formation of EB

Embryoid body formation experiments were performed when iPSCs were cultured to a degree of polymerization of 70-90% as described above. The specific operation is as follows: using TrypLE or accutase to digest iPSCs into a complete single-cell suspension, resuspending them in iPSC maintenance medium, and adding Rock inhibitor to the medium. The cell suspension was placed in a 3D shaker in a 37°C incubator for shaking culture, and the rotating speed of the shaker was 10-100 rpm; the shaking culture in this step was 8-32 hours; at the end of the culture, the obtained size and shape were relatively uniform EB. When using a T25 culture flask, the Rock inhibitor is Y-27632 at a concentration of 10 μM; the cell density is 0.1×10 ⁶ -5×10 ⁶ /mL; the rotating speed of the shaker is 10-20 rpm; the incubation time is 8-32 hours .

(2) Day 0～Day 6-12: Differentiation of pluripotent stem cells into hematopoietic progenitor cells

1) Day 0: Change to the first step differentiation medium

Remove the flask containing the D0 EBs from the incubator, tilt the flask to allow the EBs to sink to the bottom, remove the supernatant, and add fresh first-step differentiation medium. The first step of the differentiation medium is to add a small molecule GSK3β inhibitor to the basal differentiation medium, and one or more components selected from the following components: BMP signaling pathway activator, VEGF, bFGF, SCF, Flt3L, IL3, IL6, insulin , IGF-1 and TPO. BMP signaling pathway activators are BMP2, BMP4, SB4, ventromorphins (SJ000291942, SJ000063181, SJ000370178), isoliquiritigenin, diosmetin (geranin), apigenin (apigenin) and biochanin (garbanin), etc.; GSK3β inhibitor is NP031112 , TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314 and CHIR99021, etc. For example, when using a T25 culture flask for culture, the GSK3β inhibitor is CHIR99021 at a concentration of 0.5-20 μM; the BMP signaling pathway activator is BMP4 at a concentration of 0-200 ng/mL; cytokines include insulin, IGF-1, VEGF, bFGF at concentrations of 0-10 μg/mL, 0-100 ng/mL, 5-100 ng/mL and 0-100 ng/mL, respectively.

Among them, Isoliquiritigenin is a flavonoid compound isolated from Glycyrrhiza glabra root, which has antitumor activity.

The time that EBs were cultured in the first-step differentiation medium was (2-4) days.

The composition of the basal differentiation medium is shown in Table 2 below.

Table 2

2) Day 2-4: Change to the second step differentiation medium

Take out the culture flask containing EBs from the incubator, tilt the flask to make the EBs sink to the bottom, remove the supernatant of the first-step differentiation medium, and add fresh second-step differentiation medium. The second step of differentiation medium is to add VEGF, bFGF, and one or more selected from BMP signaling pathway activator, Noda inhibitor, SCF, Flt3L, IL15, IL3, IL6, insulin, IGF- 1 and TPO cytokines. The selection range of BMP signaling pathway activators is similar to that of D0; Noda inhibitors are selected from Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947 , LT580276, A83-01, and their derivatives. When cultured in T25 flasks, the BMP signaling pathway activator is BMP4 at a concentration of 0-200ng/mL; Noda inhibitor is SB431542 at a concentration of 0-20μM; cytokines include insulin, IGF-1, VEGF, bFGF , IL3 and IL6 at concentrations of 0-10 μg/mL, 0-100 ng/mL, 5-100 ng/mL, 0-100 ng/mL, 0-20 ng/mL and 0-20 ng/mL, respectively.

The medium exchange time point of the second step differentiation medium can be between Day2 and Day4.

3) Day 3-6: Change to the third step differentiation medium

Take out the culture flask containing the EBs from the incubator, tilt the flask to make the EBs sink to the bottom, remove the supernatant of the second-step differentiation medium, and add fresh third-step differentiation medium. The third step of differentiation medium is the addition of growth factors and colony-stimulating factors to the basal differentiation medium. The growth factor is one or more selected from the group consisting of EGF, VEGF, bFGF, insulin, IGF-1, PGF, PDGF, and the like. The colony stimulating factor is one or more selected from G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, Flt-3L and the like. For example, when cultured in T25 flasks, the growth factors are insulin, VEGF, IGF-1 and bFGF at concentrations of 0.5-10 μg/mL, 5-100 ng/mL, 0-100 ng/mL and 0.5-100 ng/mL, respectively ; The colony stimulating factors are TPO, SCF and Flt-3L at concentrations of 0-100ng/mL, 0-100ng/mL and 0-100ng/mL, respectively;

The medium exchange time point of the third step differentiation medium can be between Day3 and Day6.

4) Day 6-12: Detect the obtained hematopoietic progenitor cells

On day 0, day 5, and day 12 of differentiation culture, the diameters of embryoid bodies were 50-120 μM, 300-400 μM, and 400-650 μM, respectively. The specific cell morphology is shown in Figure 3.

Choose an appropriate time point from Day6 to Day12, and use flow cytometry to detect the cell phenotype of the hematopoietic progenitor cells in the embryoid body. It is proved that the obtained cells contain hematopoietic progenitor cells expressing CD34, and CD34-positive hematopoietic progenitor cells account for the total number of hematopoietic progenitor cells. The percentage of cells should be between 20% and 80%. The specific data are shown in Figure 5.

Among them, the antibody information of flow is as follows:

APC Mouse Anti-Human CD34 Clone 581 (RUO), BD, #555824;

FITC anti-human CD45, Biolegend, #304006.

(3) 1-2 weeks after EB inoculation (Day6-12～Day20-26): hematopoietic progenitor cells differentiate into NK cells

The above Day6-12 EBs containing CD34+ hematopoietic progenitor cells were resuspended in the fourth step differentiation medium, seeded in culture flasks coated with cell matrix for 2 weeks, and replaced at the end of week 1 and 2 culture medium to maintain the different time periods of action of the following factors. NK cells containing high CD3-CD56+ were harvested at the end of week 2 (Day 20-26).

1-2 weeks after EB inoculation (Day6-12～Day20-26) experimental operation details:

1) Day 6-12: Change to the fourth step differentiation medium

Tilt the flask to allow the EBs to sink to the bottom, remove the third step differentiation medium supernatant, and resuspend the EBs in fresh fourth step differentiation medium. The fourth step of differentiation medium is to add colony-stimulating factor and interleukin to the basal differentiation medium. The colony stimulating factor is one or more selected from G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, Flt-3L and the like. Interleukin is one or more selected from IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27. When cultured in T25 flasks, the colony stimulating factors are TPO, SCF, Flt-3L and IL-3 at concentrations of 0-100ng/mL, 5-100ng/mL, 5-100ng/mL and 5-100ng/mL, respectively , the time period of action is the 1st week, the 2nd week or the 1st to 2nd week after vaccination. Interleukins are IL-2, L-7 and IL-15, the concentrations are 100-1000IU/mL, 5-100ng/mL and 5-100ng/mL, respectively, and the time period of action is the first week after EB inoculation, Week 2 or Weeks 1-2. The medium exchange time point of the fourth step differentiation medium can be between Day6 and Day12.

The EBs resuspended in the fourth step differentiation medium were seeded at the appropriate density in culture flasks coated with matrix protein. The matrix protein is at least one of the following components: Notch pathway activator protein and integrin. Notch pathway activating proteins are DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein, Jagged-2 recombinant protein, and variants of these Notch pathway activating proteins. The integrins are Fibronectin (fibronectin), Laminin (laminin), Vitronectin (vitronectin), MAdCAM-1 (adhesion cell adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1) and ICAM (Intercellular Adhesion Molecule), and variants of these integrins, etc. For example, when cultured in T25 flasks, the Notch pathway activating protein is the DLL4-Fc recombinant protein. The integrin is VCAM-1.

Change half of the fresh fourth step medium every 3-7 days depending on cell density.

2) Detect the NK cells obtained in the second week after EB inoculation (Day20-26)

At 2 weeks after EB seeding, cells in suspension in the wells were collected. The expression of cell surface related marker proteins was detected by flow cytometry. Detection indicators include: CD56, NKp30, NKp44, NKp46.

Among them, the antibody information of flow is as follows:

PE Mouse Anti-Human CD56 Clone B159 (RUO), BD, #555516;

647 Mouse anti-Human CD337(NKp30)，BD，#558408；Alexa

647 Mouse anti-Human CD337(NKp30), BD, #558408;

647 Mouse Anti-Human NKp44(CD336)，BD，#558564；Alexa

647 Mouse Anti-Human NKp44(CD336), BD, #558564;

APC Mouse Anti-Human CD335 (NKp46), BD, #558051.

3) 3-4 weeks after EB inoculation (Day27～Day40): Maturation and expansion of NK cells

3-4 weeks after EB inoculation (Day27～Day40) experimental operation details:

After two weeks of EB differentiation, when the cell density reached (1-2)×10 ⁶ cells/mL, the cells were collected, centrifuged to remove the fourth-step differentiation medium, and resuspended at a cell density of (0.5-1)×10 ⁶ cells/mL in fresh fifth step differentiation medium. The fifth step of the differentiation medium is to add interleukins and other substances that promote the maturation and expansion of NK cells to the basal differentiation medium. Interleukin is one or more selected from IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27. Other substances that promote the maturation and expansion of NK cells are selected from human AB plasma, human platelet lysate, Vitamin A, nicotinamide (NAM, Vitamin B3), Vitamin E, Heparin and the like. For example, when cultured in T25 flasks, the interleukins are IL-2, IL-12, IL-18, IL-21 and IL-27 and IL-15 at concentrations of 100-1000IU/mL, 0-100ng, respectively /mL, 0-100ng/mL, 0-100ng/mL, 0-100ng/mL, and 5-100ng/mL; supplements to promote NK cell maturation and expansion are human platelet lysate, nicotinamide (NAM), vitamin E and Heparin at concentrations of 1-10%, 1-10 mmol/L, 0-10 mg/mL and 0-100 μg/mL, respectively.

The resulting NK cells were assayed at the 4th week (Day27-40) after EB inoculation

At 4 weeks of EB differentiation (Days 27-40), cells in suspension in the wells were collected. The expression of cell surface related marker proteins was detected by flow cytometry. Detection indicators include: CD56, CD94 and CD16.

Among them, the antibody information of flow is as follows:

PE Mouse Anti-Human CD56 Clone B159 (RUO), BD, #555516;

APC Mouse Anti-Human CD94, BD, #559876;

APC Mouse Anti-Human CD16 Clone B73.1 (RUO), BD, #561304.

Figure 2 shows the specific cell morphology on the -1st day, the 0th day, the 5th day, the 12th day, the 2nd week after EB inoculation, and the 4th week after EB inoculation.

Example 2

This example studies the effect of different EB culture methods on the EB differentiation rate. In the experimental group, 3D suspension culture was used to form embryoid bodies during the entire induction and differentiation process, that is, iPSCs were directly formed into EBs of smaller volume on a 3D shaker, and then replaced with different special differentiation media at different differentiation stages ( For specific steps, see Example 1). During the culture process, the volume of EBs gradually increased and the cells proliferated continuously. Compared with the 2D differentiation scheme (see patent CN107429230A), the 3D differentiation conditions greatly save the culture space and culture volume, and the number of cells obtained under the same culture system is significantly increased, which is conducive to the scale of hPSC to pluripotent blood precursor cells. Production. Compared with the differentiation scheme of Spin EB (US20180072992A1) and the method for sorting and purifying hematopoietic progenitor cells in EB cells before further differentiation, the 3D differentiation scheme of the present invention is simpler and more efficient. , which is more suitable for clinical scale production. At the same time, this protocol directly used EBs on days 6-12 for differentiation, and obtained a high differentiation efficiency (the proportion of CD56-positive NK cells reached more than 90% at the fourth week of EB differentiation). The results are shown in Figure 4.

Example 3

The present invention finds that in the 3D induction culture, the use of small molecule reagents, cytokines and cell matrix can replace serum and trophoblast cells, accelerate the differentiation process, improve the differentiation efficiency, and facilitate the production of subsequent clinical grade cell preparations.

In our differentiation method, in addition to using 3D to improve differentiation efficiency, the combined use of small molecule reagents, cytokines and cell matrix not only avoids the use of serum and trophoblast cells, but also further increases the proportion of blood precursor cells. GSK3β inhibitor selected CHIR99021, BMP signaling pathway activator BMP4, Nodal inhibitor SB431542, Notch signaling pathway activator in cell matrix using DLL4-Fc recombinant protein. On days 6-12, the proportion of blood progenitor cells (CD34+) obtained can be as high as 20-80%, which is higher than the proportion of positive cells reported in the literature using serum-containing medium and trophoblast differentiation methods (such as In patent CN102822332A, the proportion of CD56+ cells only reaches 20-30%).

Example 4

The present inventors found that the cell density and culture time of the starting human iPSCs can affect the size of EB formation, thereby affecting the efficiency of 3D differentiation. On the 0th day of differentiation, we used accutase (cell digestion solution) to digest human iPSCs into single cells, the initial cell density was controlled at 0.1-5 million cells/T25, and hPSC maintenance medium was cultured for 8-32 hours.

Example 5

The present invention finds that optimizing the concentration of CHIR99021 in the first-step differentiation medium can significantly improve the efficiency of differentiation of pluripotent stem cells into blood progenitor cells.

When using T25 culture flasks for EB differentiation, other conditions are as described in Example 1, and the concentration of CHIR99021 in the first step differentiation medium is optimized by gradient, that is, using 0 μM, 1 μM, 2 μM, 4 μM, 6 μM, Cells were treated with 8 μM or 10 μM CHIR99021, and CD34, an indicator of blood progenitor cells, was measured by flow cytometry on D12. The results are shown in Figure 6.

It can be seen that the addition of optimized CHIR99021 will improve the differentiation efficiency of blood progenitor cells; at lower concentrations, the differentiation ratio of blood progenitor cells is low, and at higher concentrations, the differentiation of blood progenitor cells is inhibited; optimized CHIR99021 concentration can Improving the purity of blood progenitor cells at D6-12 increased the proportion of CD34+ cells; the optimal concentration of CHIR99021 should be 1-8 μM.

Example 6

The present invention finds that optimizing the medium exchange time point of the second-step differentiation medium can significantly improve the differentiation efficiency of pluripotent stem cells to blood progenitor cells.

When using T25 culture flasks for EB differentiation, other conditions and methods are as described in Example 1, and the time points for changing to the second-step differentiation medium are optimized, that is, at D1, D2, D2.5, D3, D3.5, D4 or D5 was replaced with the second-step differentiation medium to continue differentiation, and at D12, the indicator CD34 of blood progenitor cells was measured by flow cytometry, and the results are shown in Figure 7. It can be seen that optimizing the time point of medium exchange in the second step of differentiation can significantly increase the proportion of blood progenitor cells; if the medium is exchanged at an earlier or later time point, the proportion of blood progenitor cells differentiated is low; the second step of differentiation culture The optimal choice of the liquid change time point of the base should be D2~D4.

Example 7

The present invention finds that optimizing the medium exchange time point of the third step differentiation medium can significantly improve the differentiation efficiency of pluripotent stem cells to blood progenitor cells.

When using T25 culture flasks for EB differentiation, other conditions and methods are as described in Example 1, and the time point of changing to the third-step differentiation medium is optimized, that is, changing to the third-step differentiation medium at D3, D4, D5, D6, D7 or D8. The four-step differentiation medium continued to differentiate, and on D12, the indicator CD34 of blood progenitor cells was measured by flow cytometry, and the results are shown in Figure 8. It can be seen that optimizing the time point of changing to the third-step differentiation medium can significantly increase the proportion of blood progenitor cells; replacing at a later time point, the differentiation ratio of blood progenitor cells is low; the third-step differentiation medium exchange medium The preferred time point should be D3 to D6.

Example 8

The present invention finds that the optimized EB inoculation time point can significantly improve the differentiation efficiency of NK cells.

When using T25 culture flasks for EB differentiation, other conditions and methods are as described in Example 1, and the time point of EB inoculation is optimized, that is, after forming EBs according to the above differentiation method, on the 4th, 6th, and 8th day of differentiation, respectively. EBs were removed on day 10, day 12 and day 14 and seeded in culture plates coated with matrix protein for subsequent differentiation. The proportion of differentiated NK cells (CD56+) was detected two weeks after inoculation, and the results are shown in Figure 9 .

It can be seen that optimizing the time point of EB inoculation will improve the differentiation efficiency of NK cells; the differentiation efficiency of NK cells of EBs at the early time point (day 4) and the EB at the late time point (day 14) is lower; the better The time point of EB inoculation should be from D6 to D12.

Example 9

The present invention finds that optimizing the action concentration of BMP4 can significantly increase the proportion of blood progenitor cells obtained.

When using T25 culture flask for EB differentiation, other conditions and methods are as described in Example 1, and the concentration of BMP4 in the first-step differentiation medium and the second-step differentiation medium is optimized by gradient, that is, 0ng/ml, 0ng/ml, Cells were treated with BMP4 at 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, or 100ng/ml and on D12 using flow The blood progenitor cell marker CD34 was determined by cytometry, and the results are shown in Figure 10. It can be seen that the addition of optimized BMP4 will improve the differentiation efficiency of blood progenitor cells; at lower concentrations, the differentiation ratio of blood progenitor cells is low, and at higher concentrations, the differentiation of blood progenitor cells is inhibited; optimized BMP4 concentration can Improving the purity of blood progenitor cells at D12 means increasing the proportion of CD34+ cells; the optimal concentration of BMP4 should be 10-50ng/ml.

Example 10

The present invention finds that optimizing the action concentration of SB431542 can significantly increase the proportion of blood progenitor cells obtained.

When using T25 culture flasks for EB differentiation, other conditions are as described in Example 1, and the concentration of SB431542 in the second step differentiation medium is optimized by gradient, that is, 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM are used respectively. , 20 μM, 30 μM, 40 μM or 50 μM of SB431542 to treat cells, and use flow cytometry to measure blood progenitor cell index CD34 on D12. The results are shown in Figure 11 .

It can be seen that the addition of optimized SB431542 will improve the differentiation efficiency of blood progenitor cells, but at higher concentrations, the differentiation of blood progenitor cells will be inhibited; optimizing the concentration of SB431542 can improve the purity of blood progenitor cells at D6-12, that is, increased The proportion of CD34+ cells; the optimal concentration of SB431542 should be 0-20μM.

Example 11

The present invention finds that optimizing the action concentration of bFGF can significantly increase the proportion of blood progenitor cells obtained.

When using T25 culture flasks for EB differentiation, other conditions are as described in Example 1, and the concentration of bFGF in the first-step differentiation medium, the second-step differentiation medium, and the third-step differentiation medium is optimized by gradient, that is, Cells were treated with bFGF at 0 ng/ml, 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 2.5 ng/ml, 5 ng/ml, 10 ng/ml or 20 ng/ml, respectively, and at D6-12 using flow CD34, an indicator of blood progenitor cells, was determined by cytometry, and the results are shown in Figure 12.

It can be seen that the addition of optimized bFGF will improve the differentiation efficiency of blood progenitor cells, but at higher concentrations, the differentiation of blood progenitor cells will be inhibited; optimizing the concentration of bFGF can improve the purity of blood progenitor cells at D6-12, that is, increased The proportion of CD34+ cells; the optimal concentration of bFGF should be 0.1～5ng/ml.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (14)

What is claimed is: 1. A natural killer cell derived from pluripotent stem cells, which expresses CD56, Nkp30, Nkp44 and Nkp46, and also expresses CD16 and CD94, markers of mature natural killer cells. 2. a method for preparing natural killer cells, is characterized in that, comprises the following steps: S1: form embryoid body; S2: embryoid body differentiates into hematopoietic progenitor cells; S3: hematopoietic progenitor cells differentiate into NK cells; S4: Maturation and expansion of NK cells. 3. The method according to claim 2, wherein the S1 comprises: S11: The cell suspension of human pluripotent stem cells is placed on a shaker and shaken overnight to form an embryoid body. 4. The method according to claim 2, wherein the S2 comprises: S21: remove the embryoid body supernatant, add fresh first-step differentiation medium for cell culture; wherein, the first-step differentiation medium is to add a small molecule GSK3β inhibitor to the basal differentiation medium, and at least one The following components: BMP signaling pathway activator, VEGF, bFGF, SCF, Flt3L, IL3, IL6, insulin, IGF-1 and TPO; S22: Remove the first-step differentiation medium, and add the second-step differentiation medium for cell culture; wherein, the second-step differentiation medium is the addition of VEGF, bFGF, and at least one of the following components to the basal differentiation medium: Cytokines of BMP signaling pathway activator, Noda inhibitor, SCF, Flt3L, IL15, IL3, IL6, insulin, IGF-1 and TPO; S23: remove the second-step differentiation medium, add the third-step differentiation medium for cell culture, and obtain hematopoietic progenitor cells; wherein, the third-step differentiation medium is the addition of growth factors and colony-stimulating factors to the basal differentiation medium . 5. The method according to claim 4, wherein the growth factor is selected from one or more of EGF, VEGF, bFGF, insulin, IGF-1, PGF and PDGF; One or more of G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF and Flt-3L. 6. The method according to claim 2, wherein in S3, the third step differentiation medium is removed, the cells are inoculated in a cell culture vessel coated with matrix protein, and the fourth step differentiation medium is added to carry out the cell culture. nourish; The matrix protein includes at least one of Notch pathway activating protein or integrin; wherein, the fourth step differentiation medium is to add colony-stimulating factor and interleukin to the basal differentiation medium. 7. The method of claim 6, wherein the colony stimulating factor is selected from the group consisting of G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF and Flt - one or more of 3L; the interleukins are selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL - one or more of 27. 8. The method according to claim 6, wherein the Notch pathway activating protein is selected from one of DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein, Jagged-2 recombinant protein and their variants. one or more; The integrin is selected from the group consisting of Fibronectin (fibronectin), Laminin (laminin), Vitronectin (vitronectin), MAdCAM-1 (adhesion cell adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule- 1) and one or more of ICAM (Intercellular Adhesion Molecule) and their variants. 9. The method according to claim 2, wherein said S4 comprises: removing the fourth step differentiation medium, and adding the fifth step differentiation medium for cell culture; wherein, the fifth step differentiation medium is The basal differentiation medium is supplemented with interleukins and substances that promote NK cell maturation and expansion. 10. The method according to claim 9, wherein the interleukin is selected from one of IL-2, IL-12, IL-18, IL-21, IL-27 and IL-15 or A variety of; the substances that promote the maturation and expansion of NK cells are selected from one or more of human AB plasma, human platelet lysate, Vitamin A, nicotinamide, Vitamin E and Heparin. 11. The method according to claim 2, further comprising step S5: freezing the differentiated NK cells using a cryopreservation solution; the cryopreservation solution comprises: sodium chloride, sodium dextrose, sodium acetate , potassium chloride, magnesium chloride, human albumin and DMSO. 12. A natural killer cell prepared by the method of any one of claims 2-11. 13. A cell population characterized by enriching the natural killer cells of claim 1 or 12. 14. A medicament for preventing and/or treating tumors, characterized in that the medicament comprises the natural killer cells described in claim 1 or 12.

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