Method and application of obtaining NK cells from human pluripotent stem cells by rapid and efficient in vitro differentiation

Abstract

The present invention relates to a culture medium for obtaining natural killer cells (NK cells) derived from human pluripotent stem cells through rapid and efficient in vitro differentiation, and its use and a method for preparing NK cells. The culture medium includes: basal culture medium, vitamin C, IGF-1, serum, glutamine, double antibody, IL2, IL7, IL15, SCF and Flt3-L; the culture medium is used in promoting the differentiation of hematopoietic stem / progenitor cells derived from human pluripotent stem cells into NK cells; the method for preparing NK cells includes: culturing hematopoietic stem / progenitor cells derived from human pluripotent stem cells using the above-mentioned culture medium to obtain the NK cells. The culture medium can effectively promote the differentiation of hematopoietic stem / progenitor cells into NK cells, and has the advantages of short culture cycle and high purity of the obtained NK cells.

Description

Technical Field

[0001] The present invention relates to the field of biology. Specifically, the present invention relates to a method for rapidly and efficiently differentiating human pluripotent stem cell-derived NK cells in vitro and its application. More specifically, the present invention relates to a culture medium, its use, and a method for preparing NK cells. Background Art

[0002] Immune cell therapy is of great significance in tumor treatment. Natural killer cells (NK) are the main cells that eliminate cancer cells in healthy human bodies, and have significant anti-cancer effects, which are of great significance for hematological tumors such as acute lymphoblastic leukemia and solid tumors such as liver cancer.

[0003] Currently, the relatively mature chimeric antigen receptor T cell (CAR-T) therapy has serious side effects, such as neurotoxicity and "cytokine release syndrome" (CRS). The patient's condition is critical and the prognosis is poor. Compared with T cells, NK cells have higher safety. Currently, no severe side effects such as graft-versus-host disease (GvHD) and CRS have been reported in adoptive NK cell therapy in clinical trials. At the same time, the function of NK cells is not restricted by MHC, which determines that the future application scope of NK is wider than that of T cells. Currently, NK cells isolated and amplified from healthy human peripheral blood mononuclear cells are used in clinical trials, which have problems such as insufficient donor sources, and NK cells provided by one donor can only treat one patient, and there are individual differences, making it difficult to achieve large-scale and standardization, and there are great difficulties in evaluating the prognosis of patients. The above problems pose new requirements for providing NK cells with sufficient dose and anti-tumor activity.

[0004] Currently, by inducing human induced pluripotent stem cells (hiPSC) to differentiate into hematopoietic stem / progenitor cells (HSPCs), a large number of CD34+CD43+HSPCs are obtained. HSPCs have the potential for lymphoid and myeloid differentiation; then, HSPCs are induced to differentiate into NK cells to obtain functionally mature NK cells (iPS-NK); and then the NK cells are amplified to the clinical application scale.

[0005] Therefore, there is an urgent need to develop a new method for obtaining NK cells. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a culture medium, its use, and a method for preparing NK cells. The culture of the present invention can enable hematopoietic stem / progenitor cells to differentiate into NK cells, and this culture medium can effectively promote the differentiation of hematopoietic stem / progenitor cells into NK cells, having the advantages of a short culture period and high purity of the obtained NK cells.

[0007] The present invention is completed based on the following findings of the inventors:

[0008] Currently, in the culture methods for differentiating hematopoietic stem / progenitor cells into NK cells, it may be necessary to change the culture medium according to the differentiation state of hematopoietic stem / progenitor cells, and the operation is cumbersome; moreover, in traditional culture methods, the time for differentiating hematopoietic stem / progenitor cells into NK cells in vitro is generally 14 - 30 days, and the culture period is long.

[0009] To solve the above problems, the inventors adjusted the components and addition amounts of the culture medium through a large number of experiments, and finally found that adding vitamin C and IGF-1 to the culture medium simultaneously can effectively promote the differentiation of hematopoietic stem / progenitor cells into NK cells. This culture process only takes 8 - 10 days, and the purity of the obtained NK cells is more than 90%, having the advantages of high purity of NK cells and a short culture period.

[0010] Based on this, in one aspect of the present invention, the present invention provides a culture medium. According to an embodiment of the present invention, the culture medium includes: a basal medium, vitamin C, IGF-1, serum, glutamine, double antibody, IL2, IL7, IL15, SCF, and Flt3-L. The inventors obtained the above-mentioned superior culture medium through a large number of experiments. This culture medium can promote the differentiation of hematopoietic stem / progenitor cells into NK cells, having the advantages of high purity of NK cells and a short culture period.

[0011] In another aspect of the present invention, the present invention provides a use of the foregoing culture medium in promoting the differentiation of hematopoietic stem / progenitor cells into NK cells. The inventors found through experiments that by using the foregoing culture medium, it can promote the differentiation of hematopoietic stem / progenitor cells into NK cells, having the advantages of high purity of NK cells and a short culture period.

[0012] In yet another aspect of the present invention, the present invention provides a method for preparing NK cells. According to an embodiment of the present invention, the method includes: culturing hematopoietic stem / progenitor cells using the foregoing culture medium to obtain the NK cells. The method for preparing NK cells according to the embodiment of the present invention can promote the differentiation of hematopoietic stem / progenitor cells into NK cells, having the advantages of high purity of NK cells and a short culture period.

[0013] In yet another aspect of the present invention, the present invention provides a culture medium. According to an embodiment of the present invention, the culture medium comprises: HDM medium, a BMP signaling pathway activator, Activin A, bFGF, and a GSK3 inhibitor. Through a large number of experiments, the inventors obtained the above-mentioned superior culture medium, which can promote the differentiation of pluripotent stem cells into early mesoderm cells.

[0014] In yet another aspect of the present invention, the present invention provides a culture medium. According to an embodiment of the present invention, the culture medium comprises: HDM medium, a BMP signaling pathway activator, a TGF-β pathway inhibitor, and a WNT pathway inhibitor. Through a large number of experiments, the inventors obtained the above-mentioned superior culture medium, which can promote the differentiation of early mesoderm cells into lateral mesoderm (LM) cells.

[0015] In yet another aspect of the present invention, the present invention provides a culture medium. According to an embodiment of the present invention, the culture medium comprises: HDM medium, VEGF, bFGF, a TGF-β receptor blocker, thrombopoietin, IL-6, SCF, and IL-3. Through a large number of experiments, the inventors obtained the above-mentioned superior culture medium, which can promote the differentiation of hematopoietic endothelial cells into hematopoietic stem / progenitor cells.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0018] Figure 1 is a schematic diagram of the culture process from hematopoietic stem cells to NK cells according to an embodiment of the present invention;

[0019] Figure 2 are cell morphology diagrams on the 2nd, 4th, 6th, and 8th days of the culture process according to Example 1 of the present invention;

[0020] Figure 3 are the detection results of the differentiation efficiency of early mesoderm cells, lateral mesoderm cells, and hematopoietic endothelial cells according to Example 1 of the present invention;

[0021] Figure 4 is the detection result of the generation of NK cells on the 8th day of differentiation according to Example 1 of the present invention;

[0022] Figure 5 is the situation of NK cells generating CD45 and CD56 obtained according to Example 2 of the present invention and their morphological comparison with NK cells in peripheral blood;

[0023] Figure 6 is based on the expressions of CD45, CD56, CD16, CD94, NKG2D and NKP46 of NK cells obtained in Example 2 of the present invention;

[0024] Figure 7 is based on the detection of the secretion of INF-γ by NK cells after K562 stimulation or PMA stimulation in Example 3 of the present invention;

[0025] Figure 8 is based on the detection of the killing of tumor cells by NK cells in Example 4 of the present invention;

[0026] Figure 9 is based on the detection of the killing ability of NK cells against different tumor cells under different effector-to-target ratios in Example 4 of the present invention;

[0027] Figure 10 is based on the detection results of the generation of NK cells on the 10th day of differentiation in different groups in Example 5 of the present invention. Detailed implementation manners

[0028] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0029] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0030] The endpoints and any values disclosed in this document are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.

[0031] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0032] In this text, the term "pluripotent stem cell" or "PSC" refers to a cell that can differentiate into a wide variety of specialized cell types under appropriate conditions and can self-renew and remain in a substantially undifferentiated pluripotent state under other appropriate conditions. Pluripotent stem cells have the potential to differentiate into a variety of cell tissues, but have lost the ability to develop into a complete individual, and their developmental potential is subject to certain limitations.

[0033] In this text, the term "human induced pluripotent stem cell" or "hiPSC" refers to a stem cell generated from induced or altered differentiated adult / newborn or fetal cells, that is, a cell capable of differentiating into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. The produced hiPSCs do not refer to cells found in nature.

[0034] In this text, the term "early mesoderm" refers to one of the three germ layers that appears during early embryogenesis and gives rise to various specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, bone, and other supportive and connective tissues. The term "early mesoderm cell" refers to a cell in the early stage that can give rise to mesoderm.

[0035] In this text, the term "lateral plate mesoderm" refers to the region of the mesoderm that is farthest from the notochord and can form the heart, blood vessels, blood cells of the circulatory system, the inner wall of the body cavity, other mesodermal components in the limb except for muscle, and can also help form a series of extraembryonic membranes to enable the transfer of nutrients from the mother to the fetus. The term "lateral plate mesoderm cell" refers to a cell that can give rise to lateral plate mesoderm. It should be noted that the developmental sequence of the blood lineage is: pluripotent stem cell - early mesoderm cell - lateral plate mesoderm cell - hematopoietic endothelial cell - hematopoietic stem and progenitor cell. For details, please refer to the literature Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types. Cell, 2016, 166, 451 - 467.

[0036] In this text, the term "hematopoietic endothelial cell" or "HEC" refers to a subset of endothelial cells that have the ability to generate hematopoietic stem / progenitor cells during the endothelial-to-hematopoietic transition.

[0037] As used herein, the term "hematopoietic stem / progenitor cell" or "HPSC" refers to a pluripotent cell with the potential to differentiate into blood lineages, capable of giving rise to all blood cell types, including myeloid (such as monocytes and macrophages, granulocytes (such as neutrophils, basophils, eosinophils, and mast cells), erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (e.g., T cells, B cells, and NK cells) (see, e.g., Doulatov et al., 2012; Notta et al., 2015).

[0038] As used herein, the term "differentiation" refers to the process by which non-specific ("free") or less specific cells acquire the characteristics of specific cells (such as blood cells or muscle cells).

[0039] The present invention provides a culture medium, its uses, and a method for preparing NK cells, which will be described in detail below respectively.

[0040] Culture medium

[0041] In a first aspect of the present invention, the present invention provides a culture medium. According to an embodiment of the present invention, the culture medium comprises: a basal medium, vitamin C, IGF-1, serum, glutamine, double antibody, IL2, IL7, IL15, SCF, and Flt3-L.

[0042] The inventors adjusted the components and addition amounts of the culture medium through a large number of experiments, and finally found that when vitamin C and IGF-1 are simultaneously added to the culture medium, vitamin C and IGF-1 have a synergistic effect, which can effectively promote the differentiation of hematopoietic stem / progenitor cells into NK cells, and the purity of the obtained NK cells is more than 90%; the culture process only takes 8-10 days, and the culture period is short. In addition, the inventors found through experiments that when vitamin C and / or IGF-1 are not added to the culture medium, the culture period will be increased and the purity of NK cells will be reduced.

[0043] In some preferred embodiments, the basal medium is selected from α-MEM medium.

[0044] In some preferred embodiments, the double antibody is penicillin and streptomycin.

[0045] In some preferred embodiments, the final concentration of vitamin C in the culture medium is 0.1-100 mg / ml, preferably 40-60 mg / ml. The inventors obtained the above preferred dosage through a large number of experiments. At the above dosage, the culture medium can further improve the efficiency of differentiating hematopoietic stem / progenitor cells into NK cells.

[0046] In some preferred embodiments, the final concentration of IL2 in the medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of hematopoietic stem / progenitor cells differentiating into NK cells.

[0047] In some preferred embodiments, the final concentration of IL7 in the medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of hematopoietic stem / progenitor cells differentiating into NK cells.

[0048] In some preferred embodiments, the final concentration of IL15 in the medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of hematopoietic stem / progenitor cells differentiating into NK cells.

[0049] In some preferred embodiments, the final concentration of SCF in the medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of hematopoietic stem / progenitor cells differentiating into NK cells.

[0050] In some preferred embodiments, the final concentration of Flt3-L in the medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of hematopoietic stem / progenitor cells differentiating into NK cells.

[0051] In some preferred embodiments, the final concentration of serum in the medium is 5 - 25%, preferably 15 - 25%. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of hematopoietic stem / progenitor cells differentiating into NK cells.

[0052] In some preferred embodiments, the final concentration of glutamine in the medium is 0.5 - 1.5%. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of hematopoietic stem / progenitor cells differentiating into NK cells.

[0053] In some preferred embodiments, the double antibody is provided in the form of a solution. Exemplarily, the double antibody is selected from the double antibody solution with the manufacturer Hyclone and the product number SV30010.

[0054] In some preferred embodiments, the final concentration of the bispecific antibody in the medium is 0.5-1.5%. Through a large number of experiments, the inventors obtained the above-mentioned optimal dosage. At the above dosage, the medium can further improve the efficiency of hematopoietic stem / progenitor cells differentiating into NK cells.

[0055] In a second aspect of the present invention, the present invention provides a medium. According to the embodiments of the present invention, the medium comprises: HDM medium, a BMP signaling pathway activator, Activin A, bFGF, and a GSK3 inhibitor. Through a large number of experiments, the inventors obtained the above-mentioned optimal medium, which can promote the differentiation of pluripotent stem cells into early mesoderm cells, and the purity of the finally obtained early mesoderm cells can be as high as 100%. More importantly, in the present invention, by adding bFGF and a GSK3 inhibitor, the differentiation of pluripotent stem cells into early mesoderm cells can be further promoted.

[0056] In some preferred embodiments, the final concentration of the BMP signaling pathway activator in the first differentiation medium is 0.1-100 ng / ml, preferably 40-60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned optimal dosage. At the above dosage, the medium can further improve the efficiency of pluripotent stem cells differentiating into early mesoderm cells.

[0057] In some preferred embodiments, the final concentration of Activin A in the first differentiation medium is 0.1-100 ng / ml, preferably 40-60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned optimal dosage. At the above dosage, the medium can further improve the efficiency of pluripotent stem cells differentiating into early mesoderm cells.

[0058] In some preferred embodiments, the final concentration of bFGF in the first differentiation medium is 0.1-100 ng / ml, preferably 40-60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned optimal dosage. At the above dosage, the medium can further improve the efficiency of pluripotent stem cells differentiating into early mesoderm cells.

[0059] In some preferred embodiments, the final concentration of the GSK3 inhibitor in the first differentiation medium is 0.1-100 μM, preferably 40-60 μM. Through a large number of experiments, the inventors obtained the above-mentioned optimal dosage. At the above dosage, the medium can further improve the efficiency of pluripotent stem cells differentiating into early mesoderm cells.

[0060] In some preferred embodiments, the BMP signaling pathway activator is selected from BMP4.

[0061] In some preferred embodiments, the GSK3 inhibitor is selected from CHIR99021.

[0062] In some preferred embodiments, the HDM medium comprises: DMEM / F12, ITS and vitamin C.

[0063] In some preferred embodiments, based on 100 mL of the DMEM / F12, the dosage of the ITS is 1 mL, and the dosage of the vitamin C is not less than 1 mg, preferably 7 mg.

[0064] In a third aspect of the present invention, the present invention provides a medium. According to an embodiment of the present invention, the medium comprises: an HDM medium, a BMP signaling pathway activator, a TGF-β pathway inhibitor, and a WNT pathway inhibitor. Through a large number of experiments, the inventors obtained the above-mentioned preferred medium, which can promote the differentiation of early mesoderm cells into lateral plate mesoderm cells, and the purity of the finally obtained lateral plate mesoderm cells can be as high as 100%.

[0065] In some preferred embodiments, the final concentration of the BMP signaling pathway activator in the second differentiation medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of the differentiation of early mesoderm cells into lateral plate mesoderm cells.

[0066] In some preferred embodiments, the final concentration of the TGF-β pathway inhibitor in the second differentiation medium is 0.1 - 10 μM, preferably 4 - 6 μM. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of the differentiation of early mesoderm cells into lateral plate mesoderm cells.

[0067] In some preferred embodiments, the final concentration of the WNT pathway inhibitor in the second differentiation medium is 0.1 - 10 μM, preferably 4 - 6 μM. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the medium can further improve the efficiency of the differentiation of early mesoderm cells into lateral plate mesoderm cells.

[0068] In some preferred embodiments, the BMP signaling pathway activator is selected from BMP4.

[0069] In some preferred embodiments, the TGF-β pathway inhibitor is selected from A-83-01.

[0070] In some preferred embodiments, the WNT pathway inhibitor is selected from IWR-1-endo.

[0071] In some preferred embodiments, the HDM medium comprises: DMEM / F12, ITS and vitamin C.

[0072] In some preferred embodiments, based on 100 mL of the DMEM / F12, the dosage of the ITS is 1 mL, and the dosage of vitamin C is not less than 1 mg, preferably 7 mg.

[0073] In a fourth aspect of the present invention, the present invention provides a culture medium. According to embodiments of the present invention, the culture medium comprises: HDM medium, VEGF, bFGF, TGF-β receptor blocker, thrombopoietin (TPO), IL-6, SCF, and IL-3. Through a large number of experiments, the inventors obtained the above-mentioned preferred culture medium, which can promote the differentiation of hematopoietic endothelial cells into hematopoietic stem / progenitor cells, shorten the acquisition time of hematopoietic stem / progenitor cells (48 hours), and improve the acquisition efficiency of hematopoietic stem / progenitor cells (more than 90%).

[0074] In some preferred embodiments, the final concentration of VEGF in the fourth differentiation medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the culture medium can further improve the efficiency of differentiating hematopoietic endothelial cells into hematopoietic stem / progenitor cells.

[0075] In some preferred embodiments, the final concentration of bFGF in the fourth differentiation medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the culture medium can further improve the efficiency of differentiating hematopoietic endothelial cells into hematopoietic stem / progenitor cells.

[0076] In some preferred embodiments, the final concentration of the TGF-β receptor blocker in the fourth differentiation medium is 0.1 - 100 μM, preferably 4 - 6 μM. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the culture medium can further improve the efficiency of differentiating hematopoietic endothelial cells into hematopoietic stem / progenitor cells.

[0077] In some preferred embodiments, the final concentration of thrombopoietin in the fourth differentiation medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the culture medium can further improve the efficiency of differentiating hematopoietic endothelial cells into hematopoietic stem / progenitor cells.

[0078] In some preferred embodiments, the final concentration of IL-6 in the fourth differentiation medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned preferred dosage. At the above dosage, the culture medium can further improve the efficiency of differentiating hematopoietic endothelial cells into hematopoietic stem / progenitor cells.

[0079] In some preferred embodiments, the final concentration of IL-3 in the fourth differentiation medium is 0.1-100 ng / ml, preferably 40-60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned optimal dosage. At the above dosage, the medium can further improve the efficiency of the differentiation of hemogenic endothelial cells into hematopoietic stem / progenitor cells.

[0080] In some preferred embodiments, the final concentration of SCF in the fourth differentiation medium is 0.1-100 ng / ml, preferably 40-60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned optimal dosage. At the above dosage, the medium can further improve the efficiency of the differentiation of hemogenic endothelial cells into hematopoietic stem / progenitor cells.

[0081] In some preferred embodiments, the TGF-β receptor blocker is selected from SB43154.

[0082] In some preferred embodiments, the HDM medium comprises: DMEM / F12, ITS and vitamin C.

[0083] In some preferred embodiments, based on 100 mL of the DMEM / F12, the dosage of ITS is 1 mL, and the dosage of vitamin C is not less than 1 mg, preferably 7 mg.

[0084] Use

[0085] In a fifth aspect of the present invention, the present invention provides a use of the medium described in the first aspect in promoting the differentiation of hematopoietic stem / progenitor cells into NK cells. The inventors found through experiments that using the medium described in the first aspect can promote the differentiation of hematopoietic stem / progenitor cells into NK cells, and the obtained NK cells have high purity and a short differentiation cycle.

[0086] Those skilled in the art can understand that the characteristics and advantages described for the medium in the first aspect also apply to this use and will not be elaborated herein.

[0087] In a sixth aspect of the present invention, the present invention provides a use of the medium described in the second aspect in promoting the differentiation of pluripotent stem cells into early mesoderm cells. The inventors found through experiments that using the medium described in the second aspect can promote the differentiation of pluripotent stem cells into early mesoderm cells, and the obtained early mesoderm cells have high purity.

[0088] Those skilled in the art can understand that the characteristics and advantages described for the medium in the second aspect also apply to this use and will not be elaborated herein.

[0089] In the seventh aspect of the present invention, the present invention provides a use of the culture medium described in the third aspect in promoting the differentiation of early mesoderm cells into lateral plate mesoderm cells. Through experiments, the inventors found that the culture medium described in the second aspect can promote the differentiation of early mesoderm cells into lateral plate mesoderm cells, and the obtained lateral plate mesoderm cells have high purity.

[0090] Those skilled in the art can understand that the characteristics and advantages described for the culture medium in the third aspect also apply to this use and will not be elaborated here.

[0091] In the eighth aspect of the present invention, the present invention provides a use of the culture medium described in the fourth aspect in promoting the differentiation of hematopoietic endothelial cells into hematopoietic stem / progenitor cells. Through experiments, the inventors found that the culture medium described in the second aspect can promote the differentiation of hematopoietic endothelial cells into hematopoietic stem / progenitor cells, and the obtained hematopoietic stem / progenitor cells have high purity.

[0092] Those skilled in the art can understand that the characteristics and advantages described for the culture medium in the fourth aspect also apply to this use and will not be elaborated here.

[0093] Method

[0094] Currently, the induction protocol for differentiating hiPSCs into NK cells established in domestic and foreign laboratories is the embryoid body (EB) differentiation method. Among them, the differentiation of EB into NK cells better simulates the cellular and physicochemical microenvironments required for NK differentiation in vivo. In vitro experiments show that NK cells induced by EB have good cytotoxicity against various tumor cells. However, during the EB differentiation process, a complex variety of cells are generated, which is not conducive to the large-scale acquisition of NK cells and subsequent clinical applications. In addition, the differentiation cycle of EB into NK cells is long. Generally, hiPSCs need 1 week to induce EB, and then another 4 - 5 weeks to differentiate into NK cells. Moreover, the technology is difficult and the cost is high.

[0095] To solve the above problems, the inventors first induced hiPSCs to differentiate into CD34+CD43+HSPCs, and then co-cultured the suspended HSPCs with mouse bone marrow stromal cells to obtain functional NK cells. Compared with the EB differentiation method, the differentiation and culture cycle of the method of the present invention is significantly shortened, and the obtained NK cells have a higher purity.

[0096] Based on this, in the ninth aspect of the present invention, the present invention proposes a method for preparing hematopoietic stem / progenitor cells. According to an embodiment of the present invention, the method includes: differentiating and culturing pluripotent stem cells in a pluripotent stem cell differentiation medium to obtain the hematopoietic stem / progenitor cells; the pluripotent stem cell differentiation medium includes a first differentiation medium, a second differentiation medium, a third differentiation medium, and a fourth differentiation medium. Through a large number of experiments, the inventors obtained the above four media. When differentiating and culturing pluripotent stem cells, different media can be selected according to the differentiation situation of pluripotent stem cells to better simulate the in vivo development environment of pluripotent stem cells. Therefore, this method can promote the differentiation of pluripotent stem cells (especially human pluripotent stem cells) into hematopoietic stem / progenitor cells, and has the advantages of stable and high efficiency, short cycle, and high purity of the obtained hematopoietic stem / progenitor cells.

[0097] In some preferred embodiments, the differentiating and culturing process includes: performing a first culturing process on the pluripotent stem cells in the first differentiation medium; performing a second culturing process on the product of the first culturing process in the second differentiation medium; performing a third culturing process on the product of the second culturing process in the third differentiation medium; performing a fourth culturing process on the product of the fourth culturing process in the fourth differentiation medium to obtain the hematopoietic stem / progenitor cells. The inventors found through experiments that the first differentiation medium can promote the differentiation of pluripotent stem cells into early mesoderm cells, the second differentiation medium can promote the differentiation of early mesoderm cells into lateral plate mesoderm cells, the third differentiation medium can promote the differentiation of lateral plate mesoderm cells into hemogenic endothelial cells, and the fourth differentiation medium can promote the differentiation of hemogenic endothelial cells into hematopoietic stem / progenitor cells.

[0098] It should be noted that for the specific composition of the first differentiation medium, please refer to the medium described in the second aspect; for the specific composition of the second differentiation medium, please refer to the medium described in the third aspect; for the specific composition of the fourth differentiation medium, please refer to the medium described in the fourth aspect.

[0099] In some preferred embodiments, the third differentiation medium includes: HDM medium, VEGF, and bFGF.

[0100] In some preferred embodiments, the final concentration of VEGF in the third differentiation medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above preferred dosage. At the above dosage, the medium can further improve the efficiency of differentiating lateral plate mesoderm cells into hemogenic endothelial cells.

[0101] In some preferred embodiments, the final concentration of bFGF in the third differentiation medium is 0.1 - 100 ng / ml, preferably 40 - 60 ng / ml. Through a large number of experiments, the inventors obtained the above-mentioned optimal dosage. At the above dosage, the medium can further improve the efficiency of differentiating lateral plate mesoderm cells into hematopoietic endothelial cells.

[0102] In some preferred embodiments, the time of the first culture treatment is 22 - 24 h. The inventors found through experiments that the purity of the early mesoderm cells differentiated at the above culture time can be as high as 100%.

[0103] In some preferred embodiments, the time of the second culture treatment is 22 - 24 h. The inventors found through experiments that the purity of the lateral plate mesoderm cells differentiated at the above culture time can be as high as 100%.

[0104] In some preferred embodiments, the time of the third culture treatment is 45 - 48 h. The inventors found through experiments that the purity of the hematopoietic endothelial cells differentiated at the above culture time can reach more than 70%.

[0105] In some preferred embodiments, the time of the fourth culture treatment is 4 - 5 days. The inventors found through experiments that the purity of the hematopoietic stem / progenitor cells differentiated at the above culture time can reach more than 90%.

[0106] Those skilled in the art can understand that the features and advantages described for the media in the second, third, and fourth aspects also apply to the method for preparing hematopoietic stem / progenitor cells, and will not be elaborated herein.

[0107] In the tenth aspect of the present invention, the present invention provides a method for preparing NK cells. According to an embodiment of the present invention, the method includes: culturing hematopoietic stem / progenitor cells using the medium described in the first aspect to obtain the NK cells. The method for preparing NK cells according to the embodiment of the present invention can promote the differentiation of hematopoietic stem / progenitor cells into NK cells, and the purity of the obtained NK cells is more than 90%. Moreover, the culture process only requires 8 - 10 days, and the culture period is short. This method has the advantages of being stable, efficient, short in cycle, and the obtained NK cells having perfect functions and high purity.

[0108] In some preferred embodiments, as Figure 1 shown, the method further includes: before culturing the hematopoietic stem / progenitor cells, adding mouse bone marrow stromal cells to the medium in advance. The inventors found that when mouse bone marrow stromal cells are co-cultured with hematopoietic stem / progenitor cells, the mouse bone marrow stromal cells can serve as nutritional cells for hematopoietic stem / progenitor cells, and can rapidly induce the differentiation of hematopoietic stem / progenitor cells into NK cells, thereby improving the differentiation efficiency of hematopoietic stem / progenitor cells.

[0109] In some preferred embodiments, the culturing time is 8 to 10 days. Thus, this method has the advantage of a short culturing period.

[0110] In some preferred embodiments, the hematopoietic stem / progenitor cells are obtained by differentiating pluripotent stem cells.

[0111] In some preferred embodiments, the hematopoietic stem / progenitor cells are differentiated from pluripotent stem cells in a pluripotent stem cell differentiation medium.

[0112] It should be noted that for the specific pluripotent stem cell differentiation medium and the method for differentiating pluripotent stem cells in the pluripotent stem cell differentiation medium, refer to the method for preparing hematopoietic stem / progenitor cells described in the ninth aspect.

[0113] Those skilled in the art can understand that the features and advantages described in the medium of the first aspect and the method for preparing hematopoietic stem / progenitor cells of the ninth aspect also apply to this method for preparing NK cells, and will not be elaborated here.

[0114] The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, follow the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0115] In the examples of the present invention, the bispecific antibody is selected from the bispecific antibody solution with the manufacturer Hyclone and the catalog number SV30010.

[0116] Example 1: Monolayer Hematopoietic Differentiation

[0117] First, differentiate hiPSCs into HSPCs using the constructed monolayer hematopoietic differentiation system. Specifically: On day 0, culture hiPSCs in the first differentiation medium (HDM medium + 50 ng / ml BMP4 + 50 ng / ml Activin A + 50 ng / ml bFGF + 50 μM CHIR99021) to induce the differentiation of hiPSCs into early mesoderm cells. On day 1, replace the first differentiation medium with the second differentiation medium (HDM medium + 50 ng / ml BMP4 + 5 μM A - 83 - 01 + 5 μM IWR - 1 - endo) to induce the early mesoderm cells into lateral mesoderm (LM) cells. On day 2, replace the second differentiation medium with the third differentiation medium (HDM medium + 50 ng / ml VEGF + 50 ng / ml bFGF) to induce the lateral mesoderm cells to differentiate into hemogenic endothelial cells. After day 4, replace the third differentiation medium with the fourth differentiation medium (HDM medium + 50 ng / ml VEGF + 50 ng / ml bFGF + 50 μM SB43154 + 50 ng / ml thrombopoietin (abbreviated as TPO) + 50 ng / ml IL6 + 50 ng / ml SCF + 50 ng / ml IL3) to induce the hemogenic endothelial cells to transform into HSPCs. Moreover, take microscope photos and observe the cell morphology diagrams on days 2, 4, 6, and 8 during the differentiation of hiPSCs into HSPCs. For details, see Figure 2 ; Use a flow cytometer to detect the differentiation efficiency of early mesoderm cells, lateral mesoderm cells, and hemogenic endothelial cells respectively. For details, see Figure 3 ; Use a flow cytometer to detect the suspended cells on day 8. For details, see Figure 4 . As Figure 2 shown, HSPCs start to be produced on day 6, and a large number of suspended HSPCs can be collected on day 8. As Figure 3 shown, the differentiation efficiency of T + early mesoderm cells is close to 100%, the differentiation efficiency of HAND1 + lateral mesoderm cells is also close to 100%, and the differentiation efficiency of CD34 + hemogenic endothelial cells reaches more than 80%. As Figure 4 shown, most of the cells on day 8 are CD34 + CD43 + CD44 + hematopoietic stem / progenitor cells.

[0118] The HDM medium of this application includes DMEM / F12 (Hyclone), ITS (insulin transferrin - selenium, GIBCO), and vitamin C (Vc, GIBCO). Among them, based on 100 mL of the DMEM / F12, the dosage of ITS is 1 mL, and the dosage of vitamin C is 7 mg.

[0119] Example 2: Preparation of NK cells

[0120] Differentiate the HSPCs obtained in Example 1 into NK cells. Specifically: One day in advance, mouse bone marrow stromal cells (OP9-DLL1) were seeded in a culture medium (α-MEM medium + 20% FBS + 1% GlutaMAXTM-1 + 1% P / S + 50 mg / ml ascorbic acid + 50 ng / ml IL-2 + 50 ng / ml IL-7 + 50 ng / ml IL-15 + 50 ng / ml SCF + 50 ng / ml Flt-3L + 50 ng / ml IGF-1). The next day, the HSPCs obtained in Example 1 were collected, and the HSPCs were co-cultured with the culture medium seeded with mouse bone marrow stromal cells. After culturing for 10 days, the suspended cells were collected, and then cell morphology observation, flow cytometry detection, and immunofluorescence technique detection were performed. For details, see Figures 5 - 6 .

[0121] As Figure 5 shown, the iPS-NK cells on the 10th day of culture were functionally mature (i.e., terminally differentiated). The iPS-NK cells differentiated from human pluripotent stem cells were morphologically similar to NK cells (PB-NK cells) in peripheral blood, highly expressed the NK marker genes CD45 and CD56, and the purity of the obtained iPS-NK cells was above 90%.

[0122] Figure 6 respectively in Figure 6 , the expression of CD45 and CD56 in the differentiated iPS-NK cells was detected by immunofluorescence technique ( Figure 6 on the left), and the expression of NK maturation-related genes such as CD16 / CD94 / NKG2D / NKP46 in the differentiated iPS-NK cells was detected by flow cytometry (

[0123] Example 3: Detection of the secretion function of NK cells

[0124] The secretion function of the iPS-NK cells obtained in Example 2 was detected by flow cytometry. The secretion of INF-γ by the iPS-NK cells after 4 hours of stimulation with K562 or PMA was detected by flow cytometry. For details, see Figure 7 , and the results showed that the differentiated iPS-NK cells had a perfect interferon secretion function.

[0125] Example 4: Detection of the tumor killing function of NK cells

[0126] The iPS-NK cells obtained in Example 2 were co-cultured with leukemia cells (K562) at a ratio of 1:1 for 24 hours, and then the cell morphology was observed under a microscope and the ability of killing tumor cells was detected by flow cytometry. For details, see Figure 8 , and the results showed that the iPS-NK cells could significantly kill K562 tumor cells (AnnexinV was apoptotic cells).

[0127] The iPS-NK cells obtained in Example 2 were co-cultured with different tumor cells expressing luciferase gene at different effector-to-target ratios. After 24 hours, a fluorescent substrate was added and the fluorescence intensity was detected by a microplate reader and the killing activity was calculated. For details, see Figure 9 , and the results showed that the iPS-NK cells had a strong killing effect on different tumor cells.

[0128] Example 5: Preparation of NK cells with different culture media

[0129] In this example, by comparing the experimental group, the control group, the +Vc group and the +IGF-1 group, it was found that adding vitamin C and IGF-1 could significantly improve the differentiation efficiency of NK. Specifically, the culture medium of the experimental group (i.e., "+Vc+IGF-1") was α-MEM medium + 20% FBS + 1% GlutaMAXTM-1 + 1% P / S + 50 mg / ml ascorbic acid + 50 ng / ml IL-2 + 50 ng / ml IL-7 + 50 ng / ml IL-15 + 50 ng / ml SCF + 50 ng / ml Flt-3L + 50 ng / ml IGF-1, and vitamin C and IGF-1 were added simultaneously in this experimental group; the difference between the control group and the experimental group was only that vitamin C and IGF-1 were not added in the culture medium; the difference between the +Vc group and the experimental group was only that IGF-1 was not added in the culture medium; the difference between the +IGF-1 group and the experimental group was only that vitamin C was not added in the culture medium. Then, HSPCs in the experimental group, the control group, the +Vc group and the +IGF-1 group were differentiated into NK cells. For the specific differentiation culture conditions, see Example 2. The cells were collected on the 10th day of differentiation, and CD45+CD56+ NK cells were detected by flow cytometry. For details, see Figure 10 . The results showed that adding vitamin C and IGF-1 simultaneously could significantly improve the differentiation efficiency of NK.

[0130] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc., mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0131] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of culture medium in promoting differentiation of hematopoietic stem / progenitor cells into NK cells; The culture medium comprises a basic culture medium, vitamin C, IGF-1, serum, glutamine, double antibody, IL2, IL7, IL15, SCF and Flt3-L; The dual antibodies are penicillin and streptomycin, and the dual antibodies are provided in the form of a solution, and the final concentration of the dual antibodies in the culture medium is 0.5-1.5%; The final concentration of the vitamin C in the culture medium is 40-60 mg / ml; The final concentration of IGF-1 in the culture medium is 40-60 mg / ml; The final concentration of IL2 in the culture medium is 40-60 ng / ml; The final concentration of IL7 in the culture medium is 40-60 ng / ml; The final concentration of IL15 in the culture medium is 40-60 ng / ml; The final concentration of SCF in the culture medium is 40-60 ng / ml; The final concentration of Flt3-L in the culture medium is 40-60 ng / ml; The final concentration of the serum in the culture medium is 15-25%; The final concentration of glutamine in the culture medium is 0.5-1.5%; The basic culture medium is selected from α-MEM culture medium.

2. A method for preparing NK cells, characterized in that: include: Cultivating hematopoietic stem / progenitor cells using a culture medium to obtain the NK cells; The culture medium comprises a basic culture medium, vitamin C, IGF-1, serum, glutamine, double antibody, IL2, IL7, IL15, SCF and Flt3-L; The dual antibodies are penicillin and streptomycin, and the dual antibodies are provided in the form of a solution, and the final concentration of the dual antibodies in the culture medium is 0.5-1.5%; The final concentration of the vitamin C in the culture medium is 40-60 mg / ml; The final concentration of IGF-1 in the culture medium is 40-60 mg / ml; The final concentration of IL2 in the culture medium is 40-60 ng / ml; The final concentration of IL7 in the culture medium is 40-60 ng / ml; The final concentration of IL15 in the culture medium is 40-60 ng / ml; The final concentration of SCF in the culture medium is 40-60 ng / ml; The final concentration of Flt3-L in the culture medium is 40-60 ng / ml; The final concentration of the serum in the culture medium is 15-25%; The final concentration of glutamine in the culture medium is 0.5-1.5%; The basic culture medium is selected from α-MEM culture medium.

3. The method according to claim 2, characterized in that Further including: Before culturing the hematopoietic stem / progenitor cells, mouse bone marrow stromal cells are added to the culture medium.

4. The method according to claim 2, characterized in that: The culture time is 8 to 10 days.

5. The method according to claim 2, characterized in that: The hematopoietic stem / progenitor cells are obtained by differentiating and culturing pluripotent stem cells.

6. The method according to claim 2, characterized in that The hematopoietic stem / progenitor cells are obtained by subjecting pluripotent stem cells to differentiation and culture in a pluripotent stem cell differentiation medium.

7. The method according to claim 6, characterized in that The pluripotent stem cell differentiation medium includes a first differentiation medium, a second differentiation medium, a third differentiation medium and a fourth differentiation medium.

8. The method according to claim 7, characterized in that The differentiation culture process comprises: subjecting the pluripotent stem cells to a first culture treatment in the first differentiation medium; subjecting the product of the first culture treatment to a second culture treatment in the second differentiation medium; subjecting the product of the second culture treatment to a third culture treatment in the third differentiation medium; The product of the third culture treatment is subjected to a fourth culture treatment in the fourth differentiation medium to obtain the hematopoietic stem / progenitor cells.

9. The method according to claim 8, characterized in that The first differentiation medium comprises: HDM medium, BMP signaling pathway activator, Activin A, bFGF and GSK3 inhibitor.

10. The method according to claim 9, characterized in that The final concentration of the BMP signaling pathway activator in the first differentiation medium is 0.1-100 mg / ml.

11. The method according to claim 9, characterized in that The final concentration of Activin A in the first differentiation medium is 0.1-100 ng / ml.

12. The method according to claim 9, characterized in that The final concentration of bFGF in the first differentiation medium is 0.1-100 ng / ml.

13. The method according to claim 9, characterized in that The final concentration of the GSK3 inhibitor in the first differentiation medium is 0.1-100 μM.

14. The method according to claim 9, characterized in that The BMP signaling pathway activator is selected from BMP4.

15. The method according to claim 9, characterized in that The GSK3 inhibitor is selected from CHIR99021.

16. The method according to claim 9, characterized in that The first culture treatment time is 22-24 hours.

17. The method according to claim 8, characterized in that The second differentiation medium comprises: HDM culture medium, BMP signaling pathway activator, TGF-β pathway inhibitor and WNT pathway inhibitor.

18. The method according to claim 17, characterized in that The final concentration of the BMP signaling pathway activator in the second differentiation medium is 0.1-100 ng / ml.

19. The method according to claim 17, characterized in that The final concentration of the WNT pathway inhibitor in the second differentiation medium is 0.1-10 μM.

20. The method according to claim 17, characterized in that The BMP signaling pathway activator is selected from BMP4.

21. The method according to claim 17, characterized in that The TGF-β pathway inhibitor is selected from A-83-01.

22. The method according to claim 21, characterized in that The final concentration of A-83-01 in the second differentiation medium is 0.1-10 μM.

23. The method according to claim 17, characterized in that The WNT pathway inhibitor is selected from IWR-1-endo.

24. The method according to claim 17, characterized in that The second culture treatment time is 22-24 hours.

25. The method according to claim 8, characterized in that The third differentiation medium comprises: HDM medium, VEGF and bFGF.

26. The method according to claim 25, characterized in that The final concentration of the VEGF in the third differentiation medium is 0.1-100 ng / ml.

27. The method according to claim 25, characterized in that The final concentration of bFGF in the third differentiation medium is 0.1-100 ng / ml.

28. The method according to claim 25, characterized in that The third culture treatment time is 45-48 hours.

29. The method according to claim 8, characterized in that The fourth differentiation medium comprises: HDM medium, VEGF, bFGF, TGF-β receptor blocker, thrombopoietin, IL-6, SCF and IL-3.

30. The method according to claim 29, characterized in that The final concentration of the VEGF in the fourth differentiation medium is 0.1-100 ng / ml.

31. The method according to claim 29, characterized in that The final concentration of bFGF in the fourth differentiation medium is 0.1-100 ng / ml.

32. The method according to claim 29, characterized in that The final concentration of the TGF-β receptor blocker in the fourth differentiation medium is 0.1-100 μM.

33. The method according to claim 29, characterized in that The final concentration of thrombopoietin in the fourth differentiation medium is 0.1-100 ng / ml.

34. The method according to claim 29, characterized in that The final concentration of IL-6 in the fourth differentiation medium is 0.1-100 ng / ml.

35. The method according to claim 29, characterized in that The final concentration of the IL-3 in the fourth differentiation medium is 0.1-100 ng / ml.

36. The method according to claim 29, characterized in that The final concentration of the SCF in the fourth differentiation medium is 0.1-100 ng / ml.

37. The method according to claim 29, characterized in that The TGF-β receptor blocker is selected from SB43154.

38. The method according to claim 29, characterized in that The duration of the fourth culture treatment is 4-5 days.

39. The method of claim 9, 17, 25 or 29, wherein: The HDM culture medium comprises: DMEM / F12, ITS, and vitamin C.

40. The method according to claim 39, characterized in that Based on 100 mL of the DMEM / F12, the amount of the ITS is 1 mL, and the amount of the vitamin C is not less than 1 mg.

41. The method according to claim 39, characterized in that Based on 100 mL of the DMEM / F12, the amount of the vitamin C is 7 mg.

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