A method for NK cell differentiation

Abstract

The present invention discloses a method for NK cell differentiation, in which hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem cells (HSPC) are differentiated into NK cells under the condition of co-culture with human mesenchymal-like cells. The advantage of the method of the present invention is that it avoids the use of xenogeneic stromal cells in the early stage of NK cell differentiation, greatly improves the yield of NK cells, and enables a single pluripotent stem cell to produce an average of about 1,500 NK cells, which is 15 to 150 times higher than the existing differentiation system, thereby eliminating the need to use engineered K562 tumor cells to further amplify NK cells to meet the usage quantity in the later stage. Without the use of xenogeneic stromal cells, the entire operation is simple and reproducible, and the risk of animal-derived contamination is avoided. In addition, compared to traditional NK cell differentiation, which takes 2 to 3 months, this method shortens the differentiation time to about 1 month.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a method for differentiating NK cells. Background Art

[0002] Natural killer cells (NK) are an important type of immune cell involved in anti-tumor, anti-viral infection and immune regulation. Unlike allogeneic T cells, allogeneic NK cells are safe for adoptive cell therapy, and they usually do not develop graft-versus-host disease (GVHD). In addition, NK cells only secrete small amounts of IFN-γ and GM-CSF, and do not secrete IL-1 and IL-6, so there are no side effects such as high-level cytokine storms or neurotoxicity. Secondly, NK cells have a natural broad-spectrum killing mechanism that does not rely on specific targets and can overcome the problem of heterogeneity of solid tumor targets. Therefore, in recent years, there have been more and more basic research and clinical trials on the use of NK cells for anti-tumor treatment, and how to generate NK cells in large quantities has also become a hot topic of current research.

[0003] In the prior art, the methods for preparing NK cells based on human pluripotent stem cells mainly include 2D culture and 3D culture. The 3D culture method based on embryoid body (EB) formation is currently the most commonly used NK induction differentiation method. In recent years, a 3D differentiation scheme based on organoid culture has also been gradually used for hematopoietic differentiation of human pluripotent stem cells. However, the NK cell differentiation system in the prior art has the following disadvantages: 1) It is inseparable from the co-culture of xenogeneic stromal cells or xenogeneic tumor cells. For example, in the early hematopoietic induction stage, it is necessary to co-culture with mouse OP9 and other cells to support the differentiation of hematopoietic progenitor cells. For example, in the late differentiation stage, engineered K562 tumor cells are used to expand NK cells, etc.; 2) The entire NK cell differentiation cycle is long, and it takes about 2 to 3 months to complete differentiation; the yield is low, and the number of NK cells produced by a single hPSC is only 10 to 100. The existence of the above problems has greatly hindered the large-scale production and application of NK cells. Therefore, solving the above problems has become a top priority. Summary of the Invention Summary of the invention:

[0005] One aspect of the present invention is to provide a method for differentiating NK cells in order to address the shortcomings of existing NK cell differentiation methods, which require support from xenogeneic cells or tumor cells, have a long cycle, and have a low yield.

[0006] Specifically, the purpose of the present invention is to utilize mesenchymal-like cells to replace xenogeneic stromal cells and engineered K562 tumor cells during the NK cell differentiation process, thereby shortening the entire NK cell differentiation cycle and increasing the NK cell yield.

[0007] The technical solution provided by the present invention is:

[0008] A method for differentiating NK cells, wherein hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) are differentiated into NK cells under conditions of co-culture with human mesenchymal-like cells.

[0009] In certain embodiments of the present invention, the human mesenchymal-like cells can be derived from an allogeneic source or from the subject / patient in whom the NK cells are intended. When the human mesenchymal-like cells are derived from an allogeneic source, industrialized production of mesenchymal-like cells can be effectively achieved. For example, the mesenchymal-like cells can be prepared, enriched, sorted, or screened using the methods of the present invention.

[0010] In order to better achieve the purpose of the present invention, preferably, in certain embodiments of the present invention, the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) are differentiated into NK cells under the condition of co-culture only with autologous mesenchymal-like cells.

[0011] The phrase "co-cultured only with autologous mesenchymal-like cells" can be understood as meaning that the co-culture system contains only the hemogenic endothelial cells (HECs), hemogenic endothelial progenitor cells (HEPCs), or hematopoietic stem and progenitor cells (HSPCs) and the autologous mesenchymal-like cells, as well as the necessary culture medium, and does not contain, for example, xenogeneic stromal cells, tumor cells, or other cells that play a supporting or promoting role.

[0012] In the present invention, the mesenchymal-like cells may be characterized by the following markers:

[0013] i) the surface marker CD34 is negative, and one or more of the following genes or proteins (stromal-related genes) are expressed: ACTA2, SNAI2, HAND1, COL1A1, IGF2, BMP4, PDGFRB, ITGAV, ACTC1, COL3A1, COL11A1, HMGA2, BASP1, LOXL2, MSX1, GLIPR2, THY1 (CD90), or ENG (CD105); and

[0014] ii) negative for the surface marker CD34 and no expression of the following genes or proteins (endothelial-related genes): PECAM1 (CD31), CDH5, SOX18, TIE1, HEY2, GJA4, NOTCH1, or GJA5; and

[0015] iii) Negative for the surface marker CD34 and no expression of the following genes or proteins (hematopoiesis-related genes): CD40, RUNX1, PTPRC (CD45), SPN (CD43), or TAL1.

[0016] The mesenchymal-like cells can be obtained by sorting and screening cells with the above characteristics or other suitable methods in the prior art. Similarly, in the present invention, the mesenchymal-like cells can also be obtained by the following method: performing hematopoietic differentiation on pluripotent stem cells, and when the cells differentiate into hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC), sorting CD34-negative cells from all cells, and these cells are the mesenchymal-like cells.

[0017] Preferably, in certain embodiments of the present invention, the pluripotent stem cells are human pluripotent stem cells.

[0018] More preferably, in one embodiment of the present invention, the cells differentiate into hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) on the 4th to 10th day after the start of differentiation.

[0019] Further preferably, in certain embodiments of the present invention, the human pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells (iPSCs).

[0020] Further preferably, in certain embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cells, for example, derived from a commercial human embryonic stem cell bank.

[0021] Further preferably, in one embodiment of the present invention, the human embryonic stem cells are H1 cells.

[0022] Preferably, in one embodiment of the present invention, the mesenchymal-like cells are mesenchymal stem cells (MSCs). The inventors have found that the mesenchymal-like cells obtained by the above method also include the mesenchymal stem cells (MSCs), and the mesenchymal stem cells (MSCs) also have the characteristics of the above markers.

[0023] Preferably, in one embodiment of the present invention, the mesenchymal stem cells (MSCs) are autologous or allogeneic mesenchymal stem cells.

[0024] In the present invention, the hematopoietic stem and progenitor cells (HSPCs) include hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs). Hematopoietic progenitor cells are progenitor cells that proliferate and differentiate into various blood cells under the regulation of a certain microenvironment and certain factors. They are also called hematopoietic progenitor cells (HPCs). They are also relatively primitive cells with the ability to proliferate, but have lost their multidirectional differentiation ability and can only proliferate and differentiate into one or several blood cell lineages. Therefore, they are also called committed stem cells. Human hematopoietic stem and progenitor cells typically express the CD34 surface marker.

[0025] In the present invention, conventional methods can be used to separate, purify, and enrich CD34 + For example, the CD34 cells were analyzed using a MicroBead Kit (Miltenyi Biotec) as described in Wenetal.Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.Genome Biology, 2021. + Acquisition of cells.

[0026] In the present invention, the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) (CD34 +Cells) can have a variety of different sources. For example, in certain embodiments of the present invention, the hematopoietic stem and progenitor cells can be derived from peripheral blood or umbilical cord blood, and the hematopoietic stem and progenitor cells can also be derived from bone marrow. In other embodiments of the present invention, the hematopoietic endothelial cells (HEC), hematopoietic endothelial progenitor cells (HEPC) or hematopoietic stem and progenitor cells (HSPC) can be hematopoietic endothelial cells (HEC), hematopoietic endothelial progenitor cells (HEPC) or hematopoietic stem and progenitor cells (HSPC) obtained by induced differentiation. For example, hematopoietic endothelial cells (HEC), hematopoietic endothelial progenitor cells (HEPC) or hematopoietic stem and progenitor cells (HSPC) are obtained by differentiation from induced pluripotent stem cells (iPSC). In other embodiments of the present invention, the hematopoietic endothelial cells (HEC), hematopoietic endothelial progenitor cells (HEPC) or hematopoietic stem and progenitor cells (HSPC) can also be hematopoietic endothelial cells (HEC), hematopoietic endothelial progenitor cells (HEPC) or hematopoietic stem and progenitor cells (HSPC) produced by reprogramming of certain specific cells. Preferably, in one embodiment of the present invention, the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) are derived from pluripotent stem cells.

[0027] Further preferably, in certain embodiments of the present invention, the pluripotent stem cells are human pluripotent stem cells.

[0028] Further preferably, in certain embodiments of the present invention, the human pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells (iPSCs).

[0029] Further preferably, in certain embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cells.

[0030] Further preferably, in one embodiment of the present invention, the human embryonic stem cells are H1 cells.

[0031] Preferably, in certain embodiments of the present invention, the method for obtaining the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem and progenitor cells (HSPC) derived from pluripotent stem cells may include:

[0032] i) culturing the pluripotent stem cells in a medium containing a Rho kinase inhibitor and / or a Wnt signaling pathway activator to obtain mesodermal cells;

[0033] ii) culturing the mesodermal cells obtained in i) in a culture medium containing bFGF and VEGF to obtain the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC).

[0034] More preferably, in certain embodiments of the present invention, the culture medium in i) further comprises a histone deacetylase (HDAC) inhibitor.

[0035] In the present invention, the Rho kinase inhibitor can be any suitable Rho kinase inhibitor known in the art. Preferably, in certain embodiments of the present invention, the Rho kinase inhibitor is one or more selected from Y-27632, Y-230141, Y-39983, GSK429286A, GSK269962, RKI-1447, Thiazovivin, ZINC00881524, KD025, Fasudil, Hydroxyfasudil, Netarsudil, or Ripasudil hydrochloride dihydrate. More preferably, in one embodiment of the present invention, the Rho kinase inhibitor is Y-27632.

[0036] In the present invention, the Wnt signaling pathway activator may include a GSK-3β inhibitor. Preferably, in certain embodiments of the present invention, the GSK-3β inhibitor is one or more selected from CHIR99021, LY2090314, SB216763, and SB415286. More preferably, in one embodiment of the present invention, the GSK-3β inhibitor is CHIR99021.

[0037] In the present invention, the histone deacetylase (HDAC) inhibitor is an important anti-tumor compound. It can cause cell cycle blocking and tumor cell selective apoptosis, and has been shown to have significant anti-tumor effects in in vitro cell culture and animals. The histone deacetylase inhibitor includes but is not limited to, for example, trichostatin A, Vorinostat, trapoxin B, MS-275, valproic, romidepsin, Pracinostat, Resminostate. Preferably, in one embodiment of the present invention, the histone deacetylase inhibitor is one or more selected from Vorinostat or its derivatives, Pracinostat or its derivatives, Resminostate or its derivatives. More preferably, in one embodiment of the present invention, the histone deacetylase inhibitor is Vorinostat or its derivatives.

[0038] Preferably, in one embodiment of the present invention, the culture medium in i) is: the basal culture medium is STEMdiff APEL 2 (STEMCELL Technologies), supplemented with 2-10 ng / ml Activin A, 10-40 ng / ml BMP4, 2-10 μM Wnt signaling pathway activator CHIR99021 and 10 μM Y-27632.

[0039] Preferably, in one embodiment of the present invention, the culture medium in ii) is: STEMdiffAPEL2 basal culture medium supplemented with 10-100 ng / ml VEGF and 10-100 ng / ml bFGF.

[0040] The enriched or differentiated CD34 + Cells are expanded. In certain embodiments of the present invention, the basal medium used in the expansion medium can be, for example, SFEM2 (StemCell Technologies, 09655). In addition, other substances such as cytokines, cell differentiation mobilization agents, etc. can be added to the basal medium, for example, including but not limited to SCF, TPO, IGF-2, FGF-1, FLT-3L, IL-6, SR1, UM171, IL-3, Notch ligands, other small molecule compounds of the aryl hydrocarbon receptor antagonist family, G-CSF, GM-CSF, LIF, MIP-1α, or a combination thereof. Preferably, in one embodiment of the present invention, the expansion medium comprises SFEM2 (StemCell Technologies, 09655) + 100ng / mlTPO + 100ng / ml SCF + 100ng / ml FLT-3L + 50ng / mlIL-6 + 750nM SR1 + 50nM UM171.

[0041] In certain embodiments of the present invention, the expansion medium may further comprise heparin, preferably low molecular weight heparin (LMHW). The heparin may be modified, for example, acylated, desulfurized, or phosphorylated.

[0042] In certain embodiments of the present invention, the amplification may also be performed in a solid tissue culture substrate, which is pre-coated with Notch ligand and / or vitronectin or a fragment thereof.

[0043] In the present invention, the above-mentioned amplification process can generally last for multiple days, for example, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days or at least 10 days.

[0044] In the present invention, after the above expansion is completed, the cells can be transferred to a new culture container such as a culture flask, culture dish or culture plate to prepare for the NK cell differentiation stage.

[0045] In an embodiment of the present invention, the NK cell differentiation stage, i.e., the process of differentiating the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC), or hematopoietic stem progenitor cells (HSPC) into NK cells under conditions of co-culture with human mesenchymal-like cells, can be a two-dimensional or three-dimensional cell culture method. As an advantage of the present invention, the co-culture can be carried out without the support of cells such as xenogeneic stromal cells or tumor cells.

[0046] Preferably, in certain embodiments of the present invention, the co-culture is a three-dimensional cell culture method.

[0047] More preferably, in one embodiment of the present invention, the co-culture is a three-dimensional organoid cell culture method.

[0048] In certain embodiments of the present invention, the above-mentioned co-culture to obtain NK cells can be performed using cell differentiation conditions in the prior art, for example, the conditions described in Chinese Patent Publication CN114621920A.

[0049] Preferably, in certain embodiments of the present invention, the culture medium for co-culturing to obtain NK cells may contain one or more selected from SCF, FLT3L, VEGF, IL2, IL3, IL6, IL7, IL15, BMP activators or Rho kinase inhibitors.

[0050] Preferably, in certain embodiments of the present invention, the co-culture ratio of the mesenchymal-like cells to the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem and progenitor cells (HSPC) is 0.5:1 to 4:1. For example, 0.5:1, 0.7:1, 0.9:1, 1.1:1, 1.3:1, 1.5:1, 1.7:1, 1.9:1, 2.1:1, 2.3:1, 2.5:1, 2.7:1, 2.9:1, 3.1:1, 3.3:1, 3.5:1, 3.7:1, 3.9:1 or 4.0:1.

[0051] Preferably, in certain embodiments of the present invention, the cell number ratio is 1:1 to 2:1.

[0052] More preferably, in one embodiment of the present invention, the cell number ratio is 1:1.

[0053] In order to achieve continuous operation and / or shorten the time to obtain NK cells, in one embodiment of the present invention, pluripotent stem cells can be directly differentiated into NK cells using the following method. The method comprises: performing hematopoietic differentiation on pluripotent stem cells, and when the cells differentiate to simultaneously produce hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC), and mesenchymal-like cells, directly aggregating the differentiated cells and performing three-dimensional organoid differentiation culture, without adding other supporting cells to the culture system, to obtain the NK cells.

[0054] In the present invention, the simultaneous is when the cells differentiate to produce hemogenic endothelial cells (HEC) and mesenchymal-like cells at the same time; or when the cells differentiate to produce hemogenic endothelial progenitor cells (HEPC) and mesenchymal-like cells at the same time; or when the cells differentiate to produce hematopoietic stem progenitor cells (HSPC) and mesenchymal-like cells at the same time.

[0055] Preferably, in certain embodiments of the present invention, the pluripotent stem cells are human pluripotent stem cells.

[0056] More preferably, in certain embodiments of the present invention, the pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells (iPSCs).

[0057] Further preferably, in certain embodiments of the present invention, the human embryonic stem cells are commercial human embryonic stem cells.

[0058] Further preferably, in certain embodiments of the present invention, the human embryonic stem cells are H1 cells.

[0059] Further preferably, in certain embodiments of the present invention, the cells differentiate into hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) on the 4th to 10th day after the start of differentiation.

[0060] Specifically, in one embodiment of the present invention, the three-dimensional organoid differentiation culture method may include:

[0061] i) The differentiated cells were cultured in the first NK differentiation medium at a rate of 1×10 6 ~3×10 6 Resuspend at a density of 6 to 10 μl;

[0062] ii) dropping the cell suspension obtained in i) into a cell culture container previously placed in the first NK differentiation medium, and continuing the culture until the 11th day after the start of differentiation;

[0063] iii) after day 11, the culture medium in ii) was replaced with a second NK differentiation medium, and the culture was continued until day 25 after the start of differentiation;

[0064] iv) digesting the three-dimensional organoid cell cluster obtained in iii) into single cells and then seeding them into a new cell culture container, continuing to culture in the second NK differentiation medium until day 32 after the start of differentiation, and collecting the NK cells.

[0065] Preferably, in one embodiment of the present invention, the basal culture medium of the first NK differentiation medium is EGM2 (Lonza), supplemented with 10 μM SB431542, 20-100 ng / ml SCF, 5-20 ng / ml FLT3L and 5-20 ng / ml TPO; the basal culture medium of the second NK differentiation medium is X-VIVO15 (Lonza), supplemented with 20 ng / ml SCF, 10 ng / ml FLT3L, 30 ng / ml IL-7, 20 ng / ml IL15 and 20 ng / ml IL3.

[0066] In certain embodiments of the present invention, the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) may be modified hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC); the pluripotent stem cells may be modified pluripotent stem cells.

[0067] In certain embodiments of the present invention, the above-mentioned modification can be performed before the NK cell differentiation stage. Preferably, in certain embodiments of the present invention, the modification is to introduce a polynucleotide expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR) into the cell. The CAR or TCR can specifically bind to viral antigens, bacterial antigens or tumor-specific antigens or tumor-associated antigens. The viral antigens include, for example, human immunodeficiency virus (HIV), herpes simplex virus (HSV), hepatitis virus, Zika virus, influenza virus or coronavirus. The herpes simplex virus (HSV) includes HSV1, HSV2; the hepatitis virus includes hepatitis A, B or C virus; the coronavirus includes SARS-CoV or SARS-CoV-2. The tumor-specific antigens include blood tumor surface antigens, for example, CD7, CD10, CD19, CD20, CD22, CD34, BCMA, CD123; and also include solid tumor surface antigens, for example, Her2, PSMA, PSCA, GPC3, EGFRvIII, IL13Rα2, L1CAM.

[0068] The polynucleotide encoding CAR can also encode a transmembrane region, an intracellular signal transduction region, and a costimulatory molecule. The transmembrane region can be a transmembrane domain selected from the following proteins or a sequence having more than 90% identity with the protein and having the same biological function as the protein: α, β or ζ chain of T cell receptor, CD2, CD3ε, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD28, CD29, CD30, CD40, CD48, CD49a, CD49d, CD49f, CD66a, CD66b, CD66c, CD66d, CD66e, CD69, CD79A, CD79B, CD84, CD96, CD100, CD10 3. CD134, CD137, CD150, CD158A, CD158B1, CD158B2, CD158C, CD158D, CD158F1, CD158F2, CD158K, CD160, CD162, CD226, CD229, CD244, CD247, CD258 , CD268, CD270, CD272, CD276, CD279, CD314, CD319, CD335, CD336, CD337, CD352, CD353, CD355, CD357, LFA-1, NKG2C, DAP-10, ICAM-1, NKp80, IL-2R beta, IL-2Rgamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS, SLP-76, PAG1 / CBP, CD83 ligand, Fc gamma receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or a combination thereof.The intracellular signal transduction region can be selected from the following proteins or sequences having more than 90% identity with the proteins and having the same biological function as the proteins: 4-1BB, B7-H3, BAFFR, BLAME, BTLA, CD100, CD103, CD160, CD18, CD19, CD19a, CD2, CD247, CD27, CD276, CD28, CD29, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96, CDS, CEACAM1, CRTAM, DAP-10, DNAM1, Fc gamma receptor, GADS, GITR, HVEM, IA4, ICAM-1, ICAM-1, Ig alpha, IL2R beta, IL2Rgamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT, LTBR, ​​Ly9, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80, OX-40, PAG / Cbp, PD-1, PSGL1, SELPLG, SLAMF4, SLAMF6, SLAMF7, SLP-76, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or a combination thereof.The co-stimulatory molecule can be one or more functional signaling domains selected from the following proteins or sequences having more than 90% identity with the proteins and having the same biological function as the proteins: integrin, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, 4-1BB, B7-H3, CD278, GITR, BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49α, IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11α, ITGAM, CD11b, ITGAX, CD11c, CD29, ITGB1, ITGB2, CD18, I TGB7, NKG2D, NKG2C, TNFR2, CD226, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, CD162, LTBR, ​​LAT, GADS or SLP-76.

[0069] Another aspect of the present invention provides NK cells prepared by the above method. The NK cells prepared in the present invention can be used in any suitable application, including, but not limited to, tumor treatment, anti-infection treatment, scientific research, etc.

[0070] Another aspect of the present invention provides a composition, which may include: i) the above-mentioned mesenchymal-like cells, which are human-derived allogeneic mesenchymal-like cells or human-derived somatic mesenchymal-like cells; and ii) a culture medium.

[0071] In certain embodiments of the present invention, the composition may further include a cryoprotectant, and the composition may be frozen for long-term storage.

[0072] Another aspect of the present invention is to provide use of the composition in preparing NK cells.

[0073] Another aspect of the present invention provides a method of administering NK cells to a subject, comprising:

[0074] i) preparing NK cells using the above method;

[0075] ii) administering an effective dose of NK cells to the subject.

[0076] Preferably, in certain embodiments of the present invention, the step of modifying the NK cells prepared in i) is further included before ii). Preferably, in certain embodiments of the present invention, the modification is to introduce into the cells a polynucleotide expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0077] Another aspect of the present invention is to provide a method for differentiating NK cells, wherein hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) are differentiated into NK cells under conditions of co-culture with human mesenchymal stem cells (MSC).

[0078] Preferably, in certain embodiments of the present invention, the mesenchymal stem cells are human-derived or allogeneic mesenchymal stem cells.

[0079] More preferably, in certain embodiments of the present invention, the hemogenic endothelial cells (HEC), hemogenic endothelial progenitor cells (HEPC) or hematopoietic stem progenitor cells (HSPC) differentiate into NK cells under the condition of co-culture only with the mesenchymal stem cells.

[0080] Preferably, in certain embodiments of the present invention, the co-culture is a two-dimensional or three-dimensional cell culture method.

[0081] Preferably, in certain embodiments of the present invention, the co-culture is a three-dimensional cell culture method.

[0082] More preferably, in certain embodiments of the present invention, the co-culture is a three-dimensional organoid cell culture method.

[0083] The beneficial effects of the present invention are:

[0084] The advantage of the NK cell differentiation method mediated by mesenchymal-like cells provided by the present invention is that it avoids the use of xenogeneic stromal cells in the early stage of NK cell differentiation, thereby greatly improving the NK yield, so that the average number of NK cells produced by a single pluripotent stem cell can reach about 1,500, which is 15 to 150 times higher than the existing system in terms of NK cell production. The direct result of this is that there is no need to use engineered K562 tumor cells to further expand NK cells in the later stage to meet the usage quantity.

[0085] The lack of xenogeneic stromal cells simplifies the entire procedure, making it highly reproducible and avoiding the risk of animal-derived contamination, facilitating future clinical translation. Furthermore, compared to traditional NK cell differentiation, which takes 2 to 3 months, this method shortens the differentiation time to approximately 1 month. The higher NK cell yield also eliminates the need for the additional use of xenogeneic K562 tumor cells, eliminating potential safety risks such as tumorigenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1A This is a flow chart of the differentiation of mesenchymal-like cells and hemogenic endothelial progenitor cells in an embodiment of the present invention;

[0087] Figure 1B and Figure 1C This is a graph showing the percentage of mesenchymal-like cells and hemogenic endothelial progenitor cells on the fourth day of differentiation in an embodiment of the present invention;

[0088] Figure 1D and Figure 1E This is a graph showing the results of single-cell transcriptome identification of mesenchymal-like cells and hemogenic endothelial progenitor cells on day 4 of differentiation in an embodiment of the present invention;

[0089] Figure 1F This is a diagram showing the results of phenotypic identification of mesenchymal-like cells on day 5 of differentiation in an embodiment of the present invention;

[0090] Figure 2A CD34 in the embodiment of the present invention - cells, CD34 + cells, CD34 - cells and CD34 + Comparison of NK cell differentiation ability after cell mixing;

[0091] Figure 2B CD34 in the embodiment of the present invention - cells, CD43 + CD45 + cells, CD34 - cells and CD43 + CD45 + Comparison of NK cell differentiation ability after cell mixing;

[0092] Figure 3A This is a graph showing the effects of different ratios of mesenchymal-like cells on NK cell differentiation in an embodiment of the present invention;

[0093] Figure 3B This is a graph showing the effect of whether or not to separate mesenchymal-like cells on NK cell differentiation in an embodiment of the present invention;

[0094] Figure 4A Flowchart of NK cell differentiation in an embodiment of the present invention;

[0095] Figure 4B and Figure 4C This is a diagram showing the identification results of NK cells prepared in an embodiment of the present invention;

[0096] Figure 4D Graph showing the identification results of other phenotypes of NK cells prepared in the examples of the present invention;

[0097] Figure 4E This is a graph showing the counting results of NK cell production prepared in an embodiment of the present invention;

[0098] Figure 5A and Figure 5B This is a diagram showing the results of evaluating the tumor killing ability of NK cells prepared in an embodiment of the present invention;

[0099] Figure 6 This figure shows the results of human mesenchymal stem cells supporting NK cell differentiation in an embodiment of the present invention. DETAILED DESCRIPTION

[0100] The present invention discloses a method for NK cell differentiation. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. It is obvious that relevant persons can modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0101] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "comprise" or variations thereof such as "comprises" or "comprising" will be understood to include the elements or components recited without excluding other elements or other components. The terms "such as" and "for example" are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0102] definition:

[0103] The term "NK cells" refers to natural killer cells, which have CD16 + CD56 + and / or CD57 + TCR -NK cells are differentiated lymphocytes with a phenotype. NK cells are characterized by their ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activating specific cytolytic enzymes, their ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or suppress immune responses.

[0104] The term "hemogenic endothelial cells" or "hemogenic endothelial progenitor cells" refers to a population of endothelial cells or their precursors with hematopoietic potential. Under specific conditions, hemogenic endothelial cells can gradually transform into hematopoietic cells. Hemogenic endothelial cells or hemogenic endothelial progenitor cells possess both endothelial and hematopoietic molecular characteristics. They typically express the surface markers CD34 and CD144, as well as RUNX1 and / or GATA2, and lack CD43 and / or CD45.

[0105] The term "hematopoietic stem and progenitor cells" (HSPC) refers to hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs). Hematopoietic progenitor cells (HPCs) are progenitors of hematopoietic stem cells that, under the regulation of a specific microenvironment and certain factors, proliferate and differentiate into various blood cell types. These cells are also called committed stem cells. Human hematopoietic stem and progenitor cells typically express the CD34 surface marker.

[0106] The term "cell differentiation" or "differentiation" refers to the process by which cells from the same origin gradually give rise to cell populations with distinct morphological structures and functional characteristics. This results in spatial differentiation of cells and temporal differences between the same cell and its previous state. The essence of cell differentiation is the selective expression of the genome in both time and space, ultimately producing signature proteins through the switching on and off of gene expression.

[0107] The term "pluripotent" refers to a cell that has the ability to differentiate into more than one differentiated cell type under different conditions, and is preferably differentiated into a cell type with all three germ cell layer characteristics. The main feature of pluripotent stem cells is that they use, for example, nude mouse teratoma formation tests to differentiate into a variety of cell types, preferably the ability to differentiate into all three germ layers. This type of cell includes human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC), human embryo-derived cells (hEDC) and adult-derived stem cells. Pluripotent stem cells can be genetically modified. In some embodiments, the pluripotent stem cells are without genetic modification. The genetically modified cells can include markers, such as fluorescent proteins, so that they can be identified. Pluripotency is also demonstrated by the expression of embryonic stem cell (ESC) markers.

[0108] The terms "iPSC" or "induced pluripotent stem cell" are used interchangeably and refer to pluripotent stem cells that are artificially derived (e.g., induced or by complete reversion) from non-pluripotent stem cells (typically adult somatic cells), e.g., by inducing forced expression of one or more genes.

[0109] The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into that individual.

[0110] The term "organoid" refers to three-dimensional (3D) cell cultures that incorporate some of the key characteristics of the organs they represent. These in vitro culture systems comprise a self-renewing stem cell population that can differentiate into multiple organ-specific cell types, share a similar spatial organization with the corresponding organ, and can recapitulate some of the organ's functions, thus providing a highly physiologically relevant system.

[0111] The term "aggregation" refers to the use of natural gravity or intercellular affinity to aggregate multiple single cells together to form cell clusters, and also includes the use of materials such as cell scaffolds or microcarriers to aggregate multiple single cells together to form cell clusters.

[0112] The term "chimeric antigen receptor" or the abbreviation "CAR" refers to an artificial receptor protein or chimeric immune receptor, and comprises an engineered receptor that is artificially specific and transplanted onto a specific immune effector cell. CAR can be used to confer the specificity of a monoclonal antibody to NK cells, thereby producing a large number of specific NK cells, for example for adoptive cell therapy. CAR typically includes an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. CAR can combine the specificity of an antibody-based antigen with the intracellular domain that activates the NK cell receptor to produce a chimeric protein with specific cellular immune activity (e.g., anti-tumor cell immune activity). In some cases, molecules can be co-expressed with CAR, including costimulatory molecules, reporter genes for imaging, gene products that conditionally remove NK cells after the addition of prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0113] The term "effective dose" refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug employed, the mode of administration, and the like.

[0114] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0115] In this specific embodiment, the NK cell differentiation method adopts a 3D organoid culture method. Similarly, the technical solution of the present invention is also applicable to 2D and conventional 3D embryoid body culture methods. The 3D organoid culture method has been gradually used for the hematopoietic differentiation of human pluripotent stem cells. However, the current scheme based on organoid culture cannot do without the support of xenogeneic stromal cells in the early stage of differentiation, such as mouse bone marrow stromal cells OP9. The major improvement in this specific embodiment is to replace the co-culture of xenogeneic stromal cells and use human pluripotent stem cells to synchronously differentiate into mesenchymal-like cells and hemogenic endothelial progenitor cells, among which mesenchymal-like cells can replace xenogeneic stromal cells to support the efficient differentiation of hemogenic endothelial progenitor cells into NK cells. In this specific embodiment, the cells differentiated to the hemogenic endothelial stage are removed without the help of xenogeneic stromal cells, but are directly aggregated into 3D organoids for culture. Analysis of cells in the hemogenic endothelial stage found that this stage contains two cell components: CD34 + hemogenic endothelial progenitor cells and CD34 - Through separation and re-culture, it was found that CD34 - Mesenchymal-like cells can support and enhance CD34+ Hemogenic endothelial progenitor cells differentiate into NK cells. Through different matching ratios (stromal-like cells: hemogenic endothelial progenitor cells), it was found that a matching ratio of 0.5:1 to 4:1 can achieve better NK differentiation effects. Furthermore, in this specific embodiment, it was found that under continuous differentiation conditions, in the hemogenic endothelial stage, the ratio of mesenchymal-like cells and hemogenic endothelial progenitor cells was maintained at about 1:1. Verification has shown that in specific operations, it is not necessary to separate mesenchymal-like cells and endothelial progenitor cells first, but the hemogenic endothelial stage cells can be directly aggregated and cultured. Finally, this specific embodiment shows that the NK cells obtained with the support of mesenchymal-like cells have normal phenotypes and functions. Furthermore, in this specific embodiment, in addition to mesenchymal-like cells derived from pluripotent stem cells, human mesenchymal stem cells can also support the efficient differentiation of hemogenic endothelial progenitor cells into NK.

[0116] Example:

[0117] Example 1: Induction of mesenchymal-like cells and hemogenic endothelial progenitor cells

[0118] A monolayer step-by-step endothelial-hematopoietic differentiation system was used to simultaneously induce the differentiation of mesenchymal-like cells and hemogenic endothelial progenitor cells. The induction time for this stage was 4 days (see the process for details). Figure 1A The first two days of hematopoietic differentiation (day 0 to day 2) are the mesoderm formation stage (see Figure 1A On day 0 of differentiation, human pluripotent stem cells were digested into single cells and seeded into 12-well plates pre-coated with vitronectin at a density of 5,000 to 10,000 cells per well. The basal culture medium used from day 0 to day 2 was STEMdiff APEL 2 (STEMCELL Technologies), supplemented with 2 to 10 ng / ml Activin A (PeproTech), 10 to 40 ng / ml BMP4 (PeproTech), 2 to 10 μM Wnt signaling pathway activator CHIR99021 (Applied Biological Materials), and 10 μM Y27632 (STEMCELL Technologies) (see Figure 1A ). Differentiation day 2 to day 4 is the endothelial hematopoietic specialization stage. In this stage, by adding 10-100ng / ml VEGF (PeproTech) and 10-100ng / ml bFGF (PeproTech) to the STEMdiff APEL2 basal medium, the mesodermal progenitor cells can be induced to gradually transform into endothelial and hematopoietic cells (see Figure 1A On the 4th day of differentiation, the expression of CD43 and CD45 was almost undetectable (see flow cytometry results). Figure 1B), indicating that CD34+CD43+CD45+ hematopoietic stem and progenitor cells were not generated at this stage. At this stage, about 50% of CD34+ hemogenic endothelial progenitor cells and about 50% of CD34- mesenchymal cells were detected, and the ratio of CD34+ cells to CD34- cells was maintained at about 1:1 (see the results). Figure 1B and Figure 1C To further clarify the cell properties of CD34+ and CD34-, we performed single-cell transcriptome sequencing on cells on day 4 of differentiation. We found that CD34- cells mainly expressed mesenchymal cell-related genes, such as ACTA2, SNAI2, HAND1, COL1A1, IGF2, BMP4, PDGFRB, ITGAV, ACTC1, COL3A1, COL11A1, HMGA2, BASP1, LOXL2, MSX1, or GLIPR2; and did not express endothelial-related genes, such as PECAM1, CDH5, SOX18, TIE1, HEY2, GJA4, NOTCH1, or GJA5; and did not express hematopoietic-related genes, such as CD40, RUNX1, or TAL1 (see results). Figure 1D In contrast to CD34- cells, CD34+ cells expressed endothelial and hematopoietic related genes, but not mesenchymal cell related genes (see results). Figure 1D Further functional enrichment analysis of CD34- cells and CD34+ cells revealed that CD34- cells were mainly enriched in pathways related to mesenchymal development, while CD34+ cells were mainly enriched in pathways related to vascular and endothelial development (see results). Figure 1E In addition, we also performed flow cytometry on cells at day 5 of differentiation and found that CD34- cells were consistent with the surface markers of mesenchymal cells, such as not expressing CD31, CD43 and CD45, but expressing CD90 and CD105 (see the results). Figure 1F These results indicate that CD34- cells belong to mesenchymal-like cells and CD34+ cells belong to endothelial hematopoietic progenitor cells, and that the ratio of the two is maintained at approximately 1:1 on day 4 of differentiation.

[0119] Example 2: Mesenchymal-like cells support and enhance NK cell differentiation

[0120] To investigate whether the differentiated mesenchymal-like cells can support the differentiation of hemogenic endothelial cells and hemogenic endothelial progenitor cells into NK cells, CD34+ and CD34- cells were sorted out on day 4 of differentiation and cultured separately or co-cultured with each other. We found that under the conditions of single culture, CD34- cells did not have the potential to differentiate into NK cells (see the results). Figure 2A ), although CD34+ cells have the potential to differentiate into NK cells, the differentiation efficiency is low, maintaining at around 20% (see Figure 2AWhen CD34+ and CD34- cells were mixed in a 1:1 ratio and then cultured, it was found that the differentiation efficiency of NK cells was significantly increased to more than 80% (see the results). Figure 2A These results indicate that human pluripotent stem cell-derived CD34- mesenchymal-like cells can effectively support the differentiation of CD34+ hemogenic endothelial progenitor cells into NK cells.

[0121] To investigate whether the differentiated mesenchymal-like cells can support the differentiation of hematopoietic stem and progenitor cells into NK cells, we collected CD34- cells on day 4 of differentiation and CD34+CD43+CD45+ hematopoietic stem and progenitor cells on day 8 of differentiation, and then cultured them separately or co-cultured with each other. We found that under the conditions of single culture, CD34- cells did not have the potential to differentiate into NK cells (see the results). Figure 2B ), although CD34+CD43+CD45+ hematopoietic stem and progenitor cells have the potential to differentiate into NK cells, the differentiation efficiency is low, maintaining at around 10% (see Figure 2B After mixing CD34+ and CD34+CD43+CD45+ cells in a 1:1 ratio and then culturing them, it was found that the differentiation efficiency of NK cells was significantly increased to about 60% (see the results). Figure 2B These results indicate that human pluripotent stem cell-derived CD34- mesenchymal-like cells can effectively support the differentiation of CD34+CD43+CD45+ hematopoietic stem and progenitor cells into NK cells.

[0122] The specific experimental procedures are as follows: 1. All cells on day 4 or day 8 of differentiation were digested and collected. CD34+ hemogenic endothelial progenitor cells on day 4, CD34- mesenchymal-like cells on day 4, and CD34+CD43+CD45+ hematopoietic stem and progenitor cells on day 8 were separated by flow cytometry for later use. 2. CD34+, CD34-, or CD34+CD43+CD45+ cells were resuspended in NK-1 medium and centrifuged. The basal medium of NK-1 medium consists of EGM2 (Lonza) supplemented with 10 μM SB431542 (Selleck Chemicals), 20-100 ng / ml SCF (PeproTech), 5-20 ng / ml FLT3L (PeproTech), and 5-20 ng / ml TPO (PeproTech). 3. Resuspend the centrifuged CD34+ or CD34- or CD34+CD43+CD45+ cells at a density of 1x10^6-3x10^6 / 6-10 μL in NK-1 medium. 4. Take 6-10 μL of the CD34+ or CD34- or CD34+CD43+CD45+ cell suspension and drop it onto a 0.4-mm Transwell insert (EMD Millipore) placed in a 6-well plate containing NK-1 medium. 5. For co-culture of CD34+ or CD34+CD43+CD45+ cells and CD34- cells, mix equal numbers of CD34+ or CD34+CD43+CD45+ cells and CD34- cells in a 1:1 ratio to a density of 1-3 x 10^6 / 6-10 μl. Drop the cells in a volume of 6-10 μl onto a 0.4-mm Transwell insert placed in a 6-well plate containing NK-1 medium. 6. From day 4 to day 11, the culture medium is NK-1 medium. After day 11, the medium is changed to NK-2 medium. Change the medium every 2-3 days. The basal medium of NK-2 culture medium was X-VIVO15 (Lonza), supplemented with 20 ng / ml SCF (PeproTech), 10 ng / ml FLT3L (PeproTech), 30 ng / ml IL-7 (PeproTech), 20 ng / ml IL15 (PeproTech), and 20 ng / ml IL3 (PeproTech). 7. After 14 days of culture in NK-2 culture medium (day 25), the formed 3D organoids were digested into single cells and seeded into new six-well plates. Differentiation culture was continued for another 7 days (day 32) using NK-2 culture medium. 8. On day 32 of differentiation, cells were harvested for subsequent analysis.

[0123] Example 3: Effects of different ratios of mesenchymal-like cells on NK cell differentiation

[0124] Under the above differentiation conditions, by adjusting the different matching ratios between CD34- mesenchymal-like cells and CD34+ hemogenic endothelial progenitor cells, it was found that the ratio of mesenchymal-like cells to hemogenic endothelial progenitor cells was maintained at 0.5:1 to 4:1, especially 1:1 or 2:1, which could achieve the best NK differentiation effect. At 1:1, the NK differentiation efficiency was about 80%, at 2:1, the NK differentiation efficiency was 60% to 80%, and at 0.5:1 and 4:1, the NK differentiation efficiency was maintained between 20% and 40% (see the results). Figure 3A ).

[0125] Considering that in all cells on the 4th day of differentiation, CD34+ cells and CD34- cells each accounted for about 50%, that is, the ratio of CD34+ cells to CD34- cells was maintained at about 1:1 (see the results). Figure 1C ), so it is not necessary to separate CD34- cells and CD34+ cells first. Instead, all cells on the 4th day of differentiation can be directly aggregated for 3D organoid differentiation. The experimental results further showed that in the specific operation, it is not necessary to separate mesenchymal-like cells and hemogenic endothelial progenitor cells first. Instead, cells on the 4th day of differentiation can be directly aggregated for organoid culture, and the differentiation effect is similar to the result of 1:1 separation (see the results). Figure 3B ).

[0126] The specific procedure for direct 3D organoid culture without cell separation is as follows: 1. Digest and harvest all cells on day 4 of differentiation, resuspend in NK-1 medium, and centrifuge. 2. Resuspend the centrifuged day 4 cells at a density of 1x10^6-3x10^6 / 6-10 μl in NK-1 medium. 3. Drop 6-10 μl of the cell suspension onto a 0.4-mm Transwell insert (EMD Millipore) placed in a 6-well plate containing NK-1 medium. 3. From day 4 to day 11, culture medium is NK-1 medium; after day 11, the medium is switched to NK-2 medium. Medium changes are performed every 2-3 days. 4. After 14 days of culture in NK-2 medium (day 25), the 3D organoids are digested into single cells and seeded into new 6-well plates for further differentiation (day 32) in NK-2 medium. 5. Collect cells on day 32 of differentiation for subsequent testing.

[0127] Example 4: NK cell phenotype identification

[0128] On the 4th day of differentiation, cells were directly cultured into 3D organoids without cell separation. Cells were collected on the 25th and 32nd days of differentiation for flow cytometry phenotyping (see the results). Figure 4AOn the 25th day of differentiation, about 80% of the cells were NK cells, with the main phenotype being CD3-CD45+CD56+ (see the results). Figure 4B and Figure 4C On the 32nd day of differentiation, more than 90% of the cells showed CD3-CD45+CD56+ phenotype (see the results). Figure 4B and Figure 4C On day 32 of differentiation, we also analyzed other phenotypes of NK cells: natural killer receptor expression: NKP30 > 90%, NKP44 about 30%, NKP46 about 20%; tumor necrosis factor-related apoptosis-inducing ligand TRAIL 20% to 60%, apoptosis protein FasL 20% to 60%; granzyme B about 100%, perforin about 100%; inhibitory receptor NKG2A 10% to 20%, CD94 20% to 30%; activation-related molecule NKG2D about 90%, CD319 60% to 90%, CD69 > 90%, CD96 > 90%; CD16 about 30%, CD7 > 90% (results see Figure 4D Finally, we calculated the absolute production of NK cells: on the 25th day of differentiation, one pluripotent stem cell produced an average of about 200 NK cells, and on the 32nd day of differentiation, one pluripotent stem cell produced an average of about 1500 NK cells (see the results). Figure 4E ).

[0129] Example 5: Identification of tumor killing ability of NK cells

[0130] The NK cells differentiated on day 32 were co-cultured with erythroleukemia K562 cells (with red fluorescence) at effector-target ratios (NK:K562) of 0:1, 1:4, 1:2, 1:1, 2:1, and 4:1. After 24 hours of co-culture with K562 cells, cytotoxicity began to be produced at an effector-target ratio of 1:1 (see results). Figure 5A and Figure 5B ), K562 cells were almost completely killed under the conditions of 2:1 and 4:1 (see the results). Figure 5B Under the conditions of effector-target ratio of 1:4 and 1:2, the proliferation of K562 cells was significantly inhibited (see the results). Figure 5B ).

[0131] Example 6: Human mesenchymal stem cells support NK differentiation

[0132] Considering the great clinical application value of umbilical cord blood mesenchymal stem cells (MSCs), we also tested whether umbilical cord blood mesenchymal stem cells can support NK cell differentiation. The results showed that: umbilical cord blood MSCs alone cannot differentiate into NK cells; compared with CD34+ hemogenic endothelial progenitor cells alone, umbilical cord blood MSC co-culture can support CD34+ hemogenic endothelial progenitor cells to differentiate into NK cells more effectively (see the results). Figure 6 ).

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for differentiating NK cells, characterized in that: The method further comprises: performing hematopoietic differentiation on the pluripotent stem cells, and on the 4th to 10th day after the start of differentiation, when hemogenic endothelial progenitor cells and mesenchymal-like cells are simultaneously generated, directly aggregating all the differentiated cells and performing three-dimensional organoid differentiation culture, without adding other supporting cells to the culture system, to obtain the NK cells; Wherein, the pluripotent stem cells are human pluripotent stem cells, and the mesenchymal-like cells are: i) negative for the surface marker CD34 and expressing one or more of the following genes or proteins: ACTA2, SNAI2, HAND1, COL1A1, IGF2, BMP4, PDGFRB, ITGAV, ACTC1, COL3A1, COL11A1, HMGA2, BASP1, LOXL2, MSX1, GLIPR2, THY1 (CD90), or ENG (CD105); and ii) negative for the surface marker CD34 and no expression of the following genes or proteins: PECAM1 (CD31), CDH5, SOX18, TIE1, HEY2, GJA4, NOTCH1, or GJA5; and iii) Negative for the surface marker CD34 and no expression of the following genes or proteins: CD40, RUNX1, PTPRC (CD45), SPN (CD43), or TAL1.

2. The method according to claim 1, characterized in that The human pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells (iPSCs).

3. The method according to claim 2, characterized in that The human embryonic stem cells are commercial human embryonic stem cells.

4. The method according to claim 3, characterized in that The human embryonic stem cells are H1 cells.

5. The method according to claim 1, wherein The method for three-dimensional organoid differentiation culture comprises: i) The differentiated cells were cultured in the first NK differentiation medium at a rate of 1×10 6 ~3×10 6 Resuspend at a density of 6 to 10 μl; ii) dropping the cell suspension obtained in i) into a cell culture container previously placed in the first NK differentiation medium, and continuing the culture until the 11th day after the start of differentiation; iii) after day 11, the culture medium in ii) was replaced with the second NK differentiation medium, and the culture was continued until day 25 after the start of differentiation; iv) digesting the three-dimensional organoid cell cluster obtained in iii) into single cells and seeding them into a new cell culture container, continuing to culture in a second NK differentiation medium until day 32 after the start of differentiation, and collecting the NK cells; Among them, the basal culture medium of the first NK differentiation medium is EGM2, supplemented with 10μM SB431542, 20-100 ng / ml SCF, 5-20 ng / ml FLT3L and 5-20ng / ml TPO; the basal culture medium of the second NK differentiation medium is X-VIVO15, supplemented with 20 ng / ml SCF, 10 ng / ml FLT3L, 30ng / ml IL-7, 20ng / ml IL15 and 20ng / ml IL3.

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