A medium combination that induces differentiation of pluripotent stem cells into CD34+ hematopoietic stem / progenitor cells

Abstract

CD34 from pluripotent stem cells + A method for inducing differentiation into hematopoietic stem / progenitor cells and a medium combination therefor are provided. + We provide a method for inducing differentiation into hematopoietic stem / progenitor cells or NK cells and a culture medium combination for the same. The NK cells obtained using the provided culture method have high purity, good in vitro expansion effect, and high iNK cell production. A single iPSC can be differentiated to obtain approximately 2,000 NK cells. The obtained iNK cells highly express CD16 (over 70%), solving the problem of low iNK CD16 expression in conventional technologies and the need to use gene modification methods to address the low CD16 expression.

Description

[Technical Field]

[0001] The present disclosure relates to the field of cell engineering, specifically to the generation of CD34 from pluripotent stem cells. + The present invention relates to a method for inducing differentiation into hematopoietic stem / progenitor cells and a medium combination therefor. [Background technology]

[0002] Pluripotent stem cells (PSCs) possess the potential for self-renewal and multilineage differentiation, enabling them to differentiate into almost all cell types, including hematopoietic stem / progenitor cells (HSPCs) and mature hematopoietic cells. HSPCs can further differentiate in vitro and in vivo to produce various blood cells, such as myeloid (My), erythrocytes (Er), and megakaryocytes (Mk), as well as important immune cells, such as NK cells and T cells, thereby demonstrating their potential for disease treatment. Recently, immune cells such as NK cells, cytotoxic T cells, Treg cells, macrophages, and chimeric antigen receptors (CARs) have been combined to produce artificial immune cells, such as CAR-NK, CAR-T, CAR-Treg, and CAR-macrophage, which can be used to treat tumors, infectious diseases, and autoimmune diseases. Therefore, pluripotent stem cells can be combined with CAR and directed differentiation technology to obtain HSPCs through directed differentiation, which can then be further differentiated into artificial immune cells such as CAR-NK, CAR-T, CAR-Treg, and CAR-macrophage.

[0003] At the same time, HSPC transplantation and hematopoietic cell infusion have also been successful in curing some patients, especially those with malignant hematologic diseases. Due to donor shortages and limited cell numbers, HSPCs derived from pluripotent stem cells are also useful as an alternative source of hematopoietic stem cells for transplantation.

[0004] However, conventional methods for inducing differentiation of pluripotent stem cells into HSPCs and NK cells have a low percentage of CD34+ cell colonies, are not suitable for industrialized production because they do not induce a 3D state, and still use mesenchymal trophoblast (MEF) cells, making them unsuitable for clinical applications. Furthermore, the differentiation efficiency and production volume of HSPCs differentiated from pluripotent stem cells are relatively low.

[0005] Therefore, there is a need for improved medium components and methods for inducing differentiation of pluripotent stem cells into HSPCs and NK cells. Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure aims to solve at least to some extent one of the technical problems in the related art. Therefore, one object of the present disclosure is to provide a method for extracting CD34 from pluripotent stem cells. + The present invention proposes a method for inducing differentiation into hematopoietic stem / progenitor cells or NK cells, and a culture medium combination for the same. The NK cells obtained by the culture method disclosed herein have high purity, good ex vivo expansion efficiency, and high iNK cell production. A single iPSC can be differentiated to obtain approximately 2,000 NK cells. The obtained iNK cells highly express CD16 (over 70%), solving the problem of low iNK CD16 expression in conventional methods, which requires the use of gene modification methods to address the low CD16 expression. [Means for solving the problem]

[0007] Therefore, the first aspect of the present disclosure is a method for extracting CD34 from pluripotent stem cells. + A medium combination for inducing differentiation into hematopoietic stem / progenitor cells is provided. According to some embodiments of the present disclosure, the medium combination comprises a first differentiation medium, a second differentiation medium, and a third differentiation medium, and the first differentiation medium, the second differentiation medium, and the third differentiation medium are added in the first differentiation stage, the second differentiation stage, and the third differentiation stage, respectively. (1) the first differentiation medium comprises a first basal medium, BMP4, and a GSK-3β inhibitor; (2) the second differentiation medium comprises a second basal medium and, optionally, UM171, and does not contain IL-3; (3) The third differentiation medium contains a third basal medium, VEGF, bFGF, SCF, FLT3L, and TPO, but does not contain IL-3.

[0008] Conventional culture conditions for inducing differentiation of pluripotent stem cells into CD34+ hematopoietic stem / progenitor cells are complicated (e.g., they require low-oxygen culture conditions during the differentiation process, require an additional selection and enrichment step to obtain highly pure HSCs, include 2D culture steps, and are not suitable for closed culture systems), resulting in relatively low differentiation efficiency and yield of HSPCs differentiated from pluripotent stem cells. The inventors discovered that adding BMP4 or a GSK-3β inhibitor, or a combination thereof, to the first differentiation medium significantly accelerates the progression of HSPC differentiation and increases CD34 expression. Preferably, this can be combined with orbital shaker spheroid formation or the AggreWell spheroid formation method to obtain highly pure CD34+ hematopoietic stem / progenitor cells. + UM171 produced the highest CD34 cells when added only to the second differentiation medium. + This results in efficient differentiation and maximum HSPC production.

[0009] According to some embodiments of the present disclosure, the pluripotent stem cells are iPSC cells or commercial embryonic stem cells.

[0010] According to some embodiments of the present disclosure, the first basal medium is mTeSR TM 1. TeSR TM 2. TeSR TM -AOF, Essential 8 TM Culture medium, NutriStem @ hESC XF, StemFit @ The present invention includes at least one selected from the group consisting of Feeder-Free Stem Cell Culture Media.

[0011] According to some embodiments of the present disclosure, the first basal medium is mTeSR TM1 or TeSR TM -AOF medium.

[0012] According to some embodiments of the present disclosure, the second basal medium is StemPro TM -34 SFM complete medium.

[0013] According to some embodiments of the present disclosure, the third basal medium is StemPro TM -34 SFM complete medium.

[0014] According to some embodiments of the present disclosure, the first differentiation medium further comprises a Rock inhibitor.

[0015] According to some embodiments of the present disclosure, the Rock inhibitor includes at least one selected from Y27632 and HB-100.

[0016] According to some embodiments of the present disclosure, the second differentiation medium further contains VEGF, bFGF, and BMP4.

[0017] According to some embodiments of the present disclosure, the second differentiation medium further comprises an induction enhancer.

[0018] According to some embodiments of the present disclosure, the induction enhancer includes a GSK-3β inhibitor selected from SR1.

[0019] According to some embodiments of the present disclosure, the GSK-3β inhibitors include CHIR99021, NP031112, AT7519, TWS119, SB216763, CHIR-98014, AZD1080, SB415286, LY2090314, (E / Z)-GSK-3β inhibitor1, KY19382, Alsterpaullone, BIO-acetoxime, IM-12, 1-Azakenpaullone, and Indirubin.

[0020] According to some embodiments of the present disclosure, the second differentiation medium further comprises ITS-X, β-mercaptoethanol, ascorbic acid, and GlutaMAX.

[0021] According to some embodiments of the present disclosure, the third differentiation medium further comprises ITS-X, β-mercaptoethanol, ascorbic acid, and GlutaMAX.

[0022] According to some embodiments of the present disclosure, the concentration of BMP4 used is 5 ng / mL-100 ng / mL.

[0023] According to some embodiments of the present disclosure, the GSK-3β inhibitor is CHIR99021, and the concentration of CHIR99021 used is 1 μM-20 μM.

[0024] According to some embodiments of the present disclosure, the Rock inhibitor is Y27632, and the concentration of Y27632 used is 1 μM-20 μM.

[0025] According to some embodiments of the present disclosure, the VEGF is used at a concentration of 5 ng / mL-100 ng / mL.

[0026] According to some embodiments of the present disclosure, the bFGF is used at a concentration of 5 ng / mL-100 ng / mL.

[0027] According to some embodiments of the present disclosure, the SCF is used at a concentration of 5 ng / mL-100 ng / mL.

[0028] According to some embodiments of the present disclosure, the TPO concentration used is 1 ng / mL-100 ng / mL.

[0029] According to some embodiments of the present disclosure, the concentration of FLT3L used is 1-200 ng / mL, preferably 1-50 ng / mL.

[0030] According to some embodiments of the present disclosure, the UM171 is used at a concentration of 0 nM-1 μM.

[0031] According to some embodiments of the present disclosure, the induction enhancer is SR1, and the concentration of SR1 used is 0 μM-2 μM.

[0032] A second aspect of the present disclosure provides a medium combination for inducing differentiation of pluripotent stem cells into NK cells. According to some implementations of the present disclosure, the medium combination comprises a fourth differentiation medium and the first, second, and third differentiation media of the medium combination described in the first aspect; the fourth differentiation medium comprises a fourth basal medium, IL-7, IL-15, FLT3L, SCF, and optionally IL-3; and adding UM171 to the second differentiation medium; Optionally, an SPHK2 inhibitor is added to the second or third differentiation medium.

[0033] According to some embodiments of the present disclosure, the pluripotent stem cells are iPSC cells or commercial embryonic stem cells.

[0034] According to some embodiments of the present disclosure, the fourth basal medium is DMEM / F12.

[0035] According to some embodiments of the present disclosure, the SPHK2 inhibitor is ABC294640.

[0036] According to some embodiments of the present disclosure, the ABC294640 is used at a concentration of 1-50 μM.

[0037] According to some embodiments of the present disclosure, the IL-3 is used at a concentration of 0-10 ng / mL.

[0038] According to some embodiments of the present disclosure, the concentration of IL-7 used is 0.1-30 ng / mL.

[0039] According to some embodiments of the present disclosure, the concentration of IL-15 used is 1-10 ng / mL.

[0040] According to some embodiments of the present disclosure, the concentration of FLT3L used is 1-200 ng / mL, preferably 1-50 ng / mL.

[0041] According to some embodiments of the present disclosure, the SCF is used at a concentration of 5 ng / mL-100 ng / mL.

[0042] According to some embodiments of the present disclosure, the fourth differentiation medium further comprises at least one of serum replacement, human AB serum, FBS, and human blood albumin.

[0043] According to some embodiments of the present disclosure, the fourth basal medium further comprises NAD + , HLA-C, SB203580, and IL-2.

[0044] According to some implementations of the present disclosure, the NAD + The working concentration of is 1-500 μM.

[0045] According to some embodiments of the present disclosure, the HLA-C concentration used is 0.05 ng / mL-1 ng / mL.

[0046] According to some embodiments of the present disclosure, the concentration of SB203580 used is 1 μM-50 μM.

[0047] According to some embodiments of the present disclosure, the concentration of IL-2 used is 100-5000 IU.

[0048] A third aspect of the present disclosure provides a method for producing CD34 from the pluripotent stem cells according to the first aspect. + The present invention provides a use of a medium combination for inducing differentiation into hematopoietic stem / progenitor cells and a medium combination for inducing differentiation from pluripotent stem cells into NK cells according to the second aspect in the preparation of NK cells.

[0049] A fourth aspect of the present disclosure is a method for detecting CD34 + A method for preparing hematopoietic stem / progenitor cells is provided. According to some embodiments of the present disclosure, the method comprises: A. Obtaining pluripotent stem cells in a single cell state; B. Culturing the single-cell pluripotent stem cells under first culture conditions to obtain a first cell sphere; C. culturing the first cell sphere under second culture conditions to obtain a second cell sphere; D. Culturing the second cell sphere under a third culture condition to identify CD34 + obtaining hematopoietic stem / progenitor cells; the first culture conditions include differentiating and culturing the pluripotent stem cells using a first differentiation medium; the second culture conditions include differentiating and culturing the first cell sphere using a second differentiation medium; the third culture condition comprises differentiating and culturing the second cell sphere using a third differentiation medium; The first differentiation medium, the second differentiation medium, and the third differentiation medium are used to differentiate CD34 from the pluripotent stem cells according to the first aspect. + A first differentiation medium, a second differentiation medium, and a third differentiation medium are included in a medium combination that induces differentiation into hematopoietic stem / progenitor cells; The pluripotent stem cells are iPSC cells or commercial embryonic stem cells.

[0050] The present disclosure provides a method for differentiating pluripotent stem cells to express CD34 + The present invention provides a method for obtaining HSPCs, which employs a 3D continuous culture method, and +The cells co-express CD43 and CD44. This method begins with digested pluripotent stem cells (PSCs). The resulting spheroids are then transferred to a first differentiation medium to form primary spheroids. The primary spheroids are then cultured sequentially using a second and third differentiation medium to form secondary and tertiary spheroids. These spheroids can be cultured in a sealed system. These steps take a total of 6-14 days, and HSPCs are obtained from the suspended cells that drop from the tertiary spheroids. NK cells (iNK) can be obtained by continuing to culture the HSPCs and tertiary spheroids. This method is suitable for culturing under normal CO2 conditions, does not require hypoxic culture conditions, and achieves CD34 spheroids with over 90% purity without an intermediate selection or enrichment step. + CD43 + , CD34 + CD44 + A cell population can be obtained, and no trophoblast cells are present during the differentiation process, making it suitable for preparing cells for clinical use.

[0051] A fifth aspect of the present disclosure is a method for detecting CD34 + providing hematopoietic stem / progenitor cells, and + The hematopoietic stem / progenitor cells are obtained by the method according to the fourth aspect.

[0052] A sixth aspect of the present disclosure provides a method for preparing NK cells, which, according to some implementations of the present disclosure, comprises: a. Using the method described in the fourth aspect, CD34 + Obtaining hematopoietic stem / progenitor cells; b. CD34 + Culturing the hematopoietic stem / progenitor cells under a fourth culture condition to obtain NK cells; The fourth culture condition is to differentiate the CD34 + Differentiating and culturing hematopoietic stem / progenitor cells, The fourth differentiation medium is a fourth differentiation medium among the medium combinations that induce differentiation from pluripotent stem cells to NK cells, and adding UM171 to the second differentiation medium used in the method according to the fourth aspect; Optionally, an SPHK2 inhibitor is added to the second or third differentiation medium.

[0053] The traditional iNK differentiation process relies on trophoblast cells, making it unsuitable for clinical application. The iNK cell CD16 expression level is low (generally 20-30%) (Zhu, Huang et al. “Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.” Blood vol. 135,6 (2020): 399-410), limiting iNK antibody-dependent cellular cytotoxicity (ADCC). The purpose of this method is to establish a method for efficiently obtaining HSPCs, which can then be further differentiated into NK cells. The method also has the following advantages: 1) simple culture conditions (normoxic culture, no additional selection or enrichment steps required, and 3D culture throughout), 2) serum-free medium, and no need for trophoblast cells during iNK differentiation, making it suitable for clinical application; and 3) high purity and yield of HSPCs (more than 99% pure CD34 expression). + 4) CD16 expression is significantly improved, reaching over 70%. 5) iNK production is high, with up to 2000-fold expansion over iPSCs at the end of the differentiation process. Furthermore, the inventors discovered that adding UM171 to the second differentiation medium and an SPHK2 inhibitor to the second or third differentiation medium significantly improved subsequent iNK production. Adding nicotinamide adenine dinucleotide (NAD) to the fourth differentiation medium significantly improved iNK production. + ), or HLA-C, or SB203580, or IL-2, + or CD45 + It is possible to increase iNK production without affecting the proportion of cells.

[0054] According to some embodiments of the present disclosure, the UM171 is used at a concentration of 1 nM-1 μM.

[0055] According to some embodiments of the present disclosure, the concentration of UM171 used is 35 nM.

[0056] According to some embodiments of the present disclosure, the SPHK2 inhibitor comprises ABC294640.

[0057] According to some embodiments of the present disclosure, the ABC294640 is used at a concentration of 1-50 μM.

[0058] A seventh aspect of the present disclosure provides NK cells. According to some embodiments of the present disclosure, the NK cells are obtained by the method according to the sixth aspect.

[0059] An eighth aspect of the present disclosure provides a method for producing CD34 from the pluripotent stem cells according to the first aspect. + A medium combination for inducing differentiation into hematopoietic stem / progenitor cells, a medium combination for inducing differentiation from pluripotent stem cells into NK cells according to the second aspect, a CD34 medium combination for inducing differentiation into NK cells according to the fourth aspect, + CD34 prepared by the method for preparing hematopoietic stem / progenitor cells + Hematopoietic stem / progenitor cells, CD34 according to the fifth aspect + The present invention provides use of hematopoietic stem / progenitor cells, NK cells prepared by the method for preparing NK cells according to the sixth aspect, and NK cells according to the seventh aspect in the preparation of CAR-NK cells.

[0060] A ninth aspect of the present disclosure provides CAR-NK cells. According to some embodiments of the present disclosure, the CAR-NK cells are obtained by the following method: modifying or gene-editing pluripotent stem cells to obtain pluripotent stem cells expressing a CAR, and differentiating and culturing the CAR-modified pluripotent stem cells by the method for preparing NK cells described in the sixth aspect to obtain CAR-NK cells.

[0061] According to some embodiments of the present disclosure, the pluripotent stem cells are iPSC cells or commercial embryonic stem cells.

[0062] A tenth aspect of the present disclosure provides a cell population. According to some embodiments of the present disclosure, the cell population is obtained from the NK cells according to the seventh aspect and / or the CAR-NK cells according to the ninth aspect.

[0063] An eleventh aspect of the present disclosure provides a drug for preventing and / or treating a tumor. According to some embodiments of the present disclosure, the drug comprises the NK cells according to the seventh aspect and / or the CAR-NK cells according to the ninth aspect and / or the cell population according to the tenth aspect.

[0064] The technical solutions of the present disclosure have the following beneficial effects: 1) The culture conditions are simple, normal oxygen conditions are sufficient, and hypoxic culture is not required. 2) CD34 with approximately 94% purity + The cells can be obtained 3) CD34 levels 70 times higher than those of primary pluripotent stem cells + Obtaining cells 4) The NK cells are further differentiated and have high purity, and the effect of in vitro expansion is good. 5) It is suitable for 3D culture and closed culture systems throughout the differentiation process. 6) The production of iNK cells is high, and a single iPSC can differentiate to obtain approximately 2,000 NK cells. 7) The high iNK CD16 expression (over 70%) obtained by this method solves the problem of low iNK CD16 expression in the prior art and low CD16 expression through gene modification methods; 8) The resulting iNKs can be used to treat solid tumors and hematological tumors, 9) iNKs can be loaded with CARs and perform specific killing.

[0065] Additional aspects and advantages of the present disclosure will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the present disclosure. [Brief explanation of the drawings]

[0066] The above and / or additional aspects and advantages of the present disclosure will become apparent and understood from the following description of the embodiments in conjunction with the drawings. [Figure 1] FIG. 1 is a schematic diagram showing the process of differentiation from iPSCs to HSPCs according to one embodiment of the present disclosure, in which the culture flask and culture bag in the figure both refer to culture vessels. [Figure 2] FIG. 1 is a schematic diagram showing the process of differentiation from iPSCs to iNKs according to one embodiment of the present disclosure. The dotted lines indicate steps that allow flexible selection of different culture systems according to production capacity needs. The culture flasks or culture bags in the diagram both refer to culture vessels. [Figure 3]

[0023] Figure 1 shows the percentage of differentiation of iPSCs into CD34+CD43+ cells in HSPC colonies in Example 1 of the present disclosure. In a typical implementation, the first differentiation medium is not supplemented with BMP4 or CHIR99021, and the second differentiation medium is not supplemented with UM171, CHIR99021, or SR1. [Figure 4] 1 shows the rate of differentiation of iPSCs into CD34+CD43+ cells in HSPC colonies in Example 1 of the present disclosure. [Figure 5] 1 shows statistics of cell purity in the differentiation process from five iPSC lines to CD34+ (HSPC) in Example 1 of the present disclosure, with iPSC-1 comprising two independent experiments. [Figure 6] 1 shows the cell expansion fold statistics in the differentiation process from five iPSC lines to CD34+ (HSPC) in Example 1 of the present disclosure, and iPSC-1 includes two independent experiments. [Figure 7] 1 shows the purity (>Day 40) of iNK obtained by the method of the present disclosure as determined by FACS detection in Example 1 of the present disclosure. [Figure 8] 1 shows CD16 expression (>Day 40) of iNK obtained by the method of the present disclosure by FACS detection in Example 1 of the present disclosure. [Figure 9]This shows the percentage (>Day 40) of activating receptors or costimulatory factors expressed by iNK cells obtained by the method of the present disclosure, as determined by FACS detection in Example 1 of the present disclosure. Along the horizontal axis, the peak on the left represents the same type control, and the peak on the right represents the activating receptor or costimulatory factor. [Figure 10] 1 shows the differentiation fold increase curve from iPSC to iNK in Example 1 of the present disclosure. [Figure 11] 1 shows the expression status of OCT4 on day 3 of the differentiation process from pluripotent stem cells to HSPCs in Example 2 of the present disclosure. [Figure 12] 1 shows the expression status of the early mesodermal marker MIXL1 and the mesodermal marker T on day 3 of the differentiation process from pluripotent stem cells to HSPCs in Example 2 of the present disclosure. [Figure 13] 1 shows the expression status of hemogenic endothelial progenitor cell markers KDR and SCL on day 3 of the differentiation process from pluripotent stem cells to HSPCs in Example 2 of the present disclosure. [Figure 14] 1 shows the expression status of the HSPC marker CD34 on day 3 of the differentiation process from pluripotent stem cells to HSPCs in Example 2 of the present disclosure. [Figure 15] FIG. 1 shows the effect of adding CHIR99021 and / or SR1 to the second differentiation medium in Example 3 of the present disclosure on the proportion of CD34+ or CD43+ cells and cell production volume. A. The proportion of CD34+ cells after two weeks of differentiation when CHIR99021 is added to the second differentiation medium. B. The expansion fold of cell colonies after two weeks of differentiation when CHIR99021 is added to the second differentiation medium. C. The proportion of CD34+ and CD43+ cells after two weeks of differentiation when SR1 is added to the second differentiation medium. [Figure 16] 1 shows the effect of adding UM171 to the second and / or third differentiation medium on the percentage of CD34+ and CD56+ cells and the cell expansion fold during the differentiation process in Example 3 of the present disclosure. A. The percentage of CD34+ cells at week 2 and the percentage of CD56+ cells at week 5. B. The cell expansion fold at weeks 2 and 5. [Figure 17]In Example 4 of the present disclosure, the CD56+ cell purity and cell expansion fold of iNK obtained using serum replacement, human AB serum, or fetal bovine serum (FBS) in the fourth differentiation medium were compared, and each triangle, block, or circle represents an independent experiment. [Figure 18] 1 shows the effect of adding an SPHK2 inhibitor to the second and third differentiation media in Example 5 of the present disclosure on the purity and production of HSPCs and iNK cells. [Figure 19] 1 shows the effect of adding NAD+ to the fourth differentiation medium in Example 5 of the present disclosure on iNK differentiation efficiency and iNK production. [Figure 20] 11 shows the effect of adding HLA-C to the fourth differentiation medium in Example 5 of the present disclosure on the iNK differentiation efficiency and iNK production amount. [Figure 21] 1 shows the effect of adding IL-2 or SB203580 to the fourth differentiation medium in Example 5 of the present disclosure on the iNK differentiation efficiency and iNK production amount. [Figure 22] Cord blood-derived NK (CB-NK) or iNK and iNK-derived iPSCs in Example 6 of the present disclosure were co-incubated for 4 hours or 24 hours, and the morphology of adherent iPSCs was observed under a light microscope. [Figure 23] The iNK obtained in Example 10 of the present disclosure was cryopreserved in several cryopreservation media, and the cell activity rate after resuscitation is shown. [Figure 24] The iNK obtained in Example 10 of the present disclosure was cryopreserved in cryopreservation solution 3, and then the killing of hematologic tumor cells (K562, Forage, U937) and solid tumor cells (A549) by the resuscitated cells under a microscope was demonstrated. K562 is a chronic myeloid leukemia cell line, Forage is a diffuse large cell non-Hodgkin's lymphoma cell line, U937 is a human histopathological lymphoma cell line, and A549 is a human non-small cell lung cancer cell line. [Figure 25]The iNK obtained in Example 10 of the present disclosure was cryopreserved in cryopreservation solution 3, and then the resuscitated cells were co-incubated with U-87 MG-GFP (a brain glioma cell line) using Incucyte for 40 hours or more. The number of remaining target cells (measured by the fluorescence intensity of the target cells) was monitored in real time, and the killing activity of iNK against U-87 MG cells could be obtained. [Figure 26] In Example 11 of the present disclosure, CAR-iNK differentiated from iPSCs can specifically kill tumor cells. Figure A shows the preparation process of CAR-iNK, in which the CAR is introduced at the iPSC stage. Figure B shows the killing test of CB-NK, WT iNK, and CAR-BCMA iNK against K562 (which does not express BCMA). Figure C shows a comparison of the killing ability of CAR-BCMA iNK against H929 (a tumor cell line that highly expresses BCMA) and two other types of NK cells. Figure D shows the relationship between the killing ability of CAR-BCMA iNK against H929 and dose. DETAILED DESCRIPTION OF THE INVENTION

[0067] The present disclosure will now be described with reference to specific examples, which are for illustrative purposes only and are not intended to limit the present disclosure in any way.

[0068] Reagents used in the experiments in the examples are commercially available unless otherwise specified.

[0069] It should be noted that the terms "first" and "second" are for descriptive purposes only and cannot be considered to indicate or imply relative importance or the number of technical features. Thus, a feature qualified as "first" or "second" can expressly or imply the inclusion of at least one of the feature. In the description of this disclosure, the concept of "plurality" refers to at least two, e.g., two, three, etc., unless otherwise clearly and specifically limited.

[0070] The endpoints of ranges and any value disclosed herein should be understood to be not limited to such exact ranges or values, but to include values ​​close to those ranges or values. In the case of ranges of numerical values, values ​​between the endpoints of each range, between the endpoints of each range and any single point value, and between any single point value can be combined with each other to create one or more new numerical ranges, and these numerical ranges are considered to be specifically disclosed in the specification.

[0071] In order to more readily understand this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this specification, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0072] As used herein, the terms "comprise" or "include" are open-ended expressions, i.e., include the content set forth in the present disclosure but do not exclude the content of other embodiments.

[0073] term: Closed System: A system designed, operated, and used so that products or materials are not exposed to the room environment. When products or materials are transferred into the closed system, they must be transferred without exposure (e.g., sterile connectors or closed transfer systems) to prevent exposure of the products or materials to the room environment. If the closed system must be opened (e.g., to install or connect a filter), it must be disinfected or sterilized before it can be returned to its closed state or used.

[0074] 3D continuous culture: Cells are cultured in a non-adherent manner, typically in suspension, without cryopreservation or resuscitation procedures between each culture step.

[0075] Shaking culture: A culture method in which the cell culture medium is moved regularly or irregularly using a specific device or culture vessel during the cell culture process.

[0076] Static culture: A culture method in which the medium is essentially stationary relative to the culture vessel during the cell culture process.

[0077] Pluripotent stem cells: A type of stem cell that has the ability to self-renew and the potential for multilineage differentiation, and expresses OCT4, NANOG, SSEA-4, and Tra-1-60. Representative examples include human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs). The embryonic stem cells referred to in this disclosure refer to commercial embryonic stem cells.

[0078] Pluripotent stem cells in single-cell state: After being dissociated by specific digestive enzymes, pluripotent stem cells change from a clonal or cell sphere state to a single-cell state.

[0079] CD34 + Cells or HSPCs: These two types of cells refer to hematopoietic stem / progenitor cells, which can be obtained by primary isolation or by differentiation from pluripotent stem cells such as human iPSCs or ESCs. These cells have the characteristics of hematopoietic stem / progenitor cells.

[0080] Spheroids: Cell aggregates formed by multiple cells under agitated or static culture conditions. They have a relatively dense structure and require specific digestive enzymes to dissociate them into single cells.

[0081] Pluripotent stem cell maintenance medium: A medium that maintains the pluripotency of pluripotent stem cells and allows them to proliferate.

[0082] Inhibitor: A substance that can specifically block a specific signal pathway, generally a small molecule compound, nucleic acid, protein, etc.

[0083] Serum replacement: A serum-free formulation additive that, as a component of cell culture media, supports cell survival or growth.

[0084] Chimeric antigen receptor (CAR) consists of an extracellular antigen-binding domain, a single-chain antibody formed by linking a flexible hinge domain in the middle, a transmembrane domain, and an intracellular signaling domain.

[0085] According to one specific implementation of the present disclosure, the present disclosure provides a method for detecting CD34+ A method for preparing hematopoietic stem / progenitor cells is provided, as shown in Figure 1. A. Obtaining pluripotent stem cells in a single cell state; B. Culturing the single-cell pluripotent stem cells under first culture conditions to obtain a first cell sphere; C. culturing the first cell sphere under second culture conditions to obtain a second cell sphere; D. Culturing the second cell sphere under a third culture condition to identify CD34 + obtaining hematopoietic stem / progenitor cells; the first culture conditions include differentiating and culturing the pluripotent stem cells using a first differentiation medium; the second culture conditions include differentiating and culturing the first cell sphere using a second differentiation medium; the third culture condition comprises differentiating and culturing the second cell sphere using a third differentiation medium; The pluripotent stem cells are iPSC cells or commercial embryonic stem cells.

[0086] According to one specific implementation of the present disclosure, (1) the first differentiation medium contains a first basal medium, BMP4, and a GSK-3β inhibitor; (2) the second differentiation medium comprises a second basal medium and, optionally, UM171, and does not contain IL-3; (3) The third differentiation medium contains a third basal medium, VEGF, bFGF, SCF, FLT3L, and TPO, but does not contain IL-3.

[0087] According to one specific embodiment of the present disclosure, the pluripotent stem cells are iPSC cells or commercial embryonic stem cells.

[0088] According to one specific embodiment of the present disclosure, the first basal medium is mTeSR TM 1. TeSR TM 2. TeSR TM -AOF, Essential 8 TM Culture medium, NutriStem @ hESC XF, StemFit@ Feeder-Free Stem Cell Culture Media, and preferably the first basal medium is mTeSR. TM 1 or TeSR TM -AOF medium, wherein the second basal medium is StemPro TM -34 SFM complete medium, and the third basal medium is StemPro TM -34 SFM complete medium.

[0089] According to a specific embodiment of the present disclosure, the first differentiation medium further comprises a Rock inhibitor, The Rock inhibitors include, but are not limited to, Y27632 and HB-100, and may be types of Rock inhibitors other than Y27632 and HB-100.

[0090] According to one specific embodiment of the present disclosure, the second differentiation medium further contains VEGF, bFGF, and BMP4, and the second differentiation medium further contains an induction enhancer, which includes at least one selected from a GSK-3β inhibitor and SR1, and the GSK-3β inhibitor may be CHIR99021, NP031112, AT7519, TWS119, SB216763, CHIR-98014, AZD1080, SB415286, LY2090314, (E / Z)-GSK-3β inhibitor1, KY19382, Alsterpaullone, BIO-acetoxime, IM-12, 1-Azakenpaullone, or Indirubin.

[0091] According to one specific embodiment of the present disclosure, the second differentiation medium further contains ITS-X, β-mercaptoethanol, ascorbic acid, and GlutaMAX.

[0092] According to one specific embodiment of the present disclosure, the third differentiation medium further contains ITS-X, β-mercaptoethanol, ascorbic acid, and GlutaMAX.

[0093] According to one specific embodiment of the present disclosure, the concentration of BMP4 used is 5 ng / mL-100 ng / mL; The concentration of CHIR99021 used is 1 μM-20 μM, The VEGF concentration used is 5 ng / mL-100 ng / mL; The concentration of the bFGF used is 5 ng / mL to 100 ng / mL, The SCF concentration used is 5 ng / mL-100 ng / mL; The TPO concentration used is 1 ng / mL to 100 ng / mL, The concentration of FLT3L used is 1-200 ng / mL, preferably 1-50 ng / mL; The concentration of UM171 used is 0 nM-1 uM, The concentration of SR1 used is 0 μM-2 μM.

[0094] The "working concentration" referred to in this disclosure is the final concentration used during cell culture.

[0095] There are several methods for obtaining pluripotent stem cells in a single cell state, which can be used to aggregate and form spheroids. For example, spheroid formation can be achieved by shaking using an orbital shaker, or by using an AggreWell TM After centrifugation, the microplate may be left to stand to allow spheroid formation. A single-cell pluripotent stem cell suspension can be obtained by referring to the method disclosed in the patent "Method and Application of Human Induced Pluripotent Stem Cells" (Patent No.: ZL201310276246.7). According to one specific implementation of the present disclosure, the method for obtaining single-cell pluripotent stem cells is as follows: 1) In the case of iPSCs or ESCs in culture, when the cell confluence reaches 75%-85% or the cell sphere culture time in 3D culture reaches 4-7 days, wash the cells once with DMEM / F12 medium, add Accutase digestion enzyme or TrypLE digestion enzyme, and digest the cells in a 37°C, 5% CO2 incubator. When the cells are essentially digested, add pluripotent stem cell maintenance medium (mTeSR). TM1) Addition of the following steps to stop the digestion: 2) Centrifugation, discarding the supernatant, and then adding mTeSR supplemented with 5 μM ROCK inhibitor, 50 ng / mL BMP4, and 10 μM CHIR99021. TM 1 or TeSR TM - Resuspending the cells in AOF medium, i.e., generating iPSCs or ESCs in a single-cell state.

[0096] According to a specific embodiment of the present disclosure, the present disclosure provides a method for culturing pluripotent stem cells by transfecting them with CD34 + The present invention provides a method for differentiation of cells, which takes a total of approximately 6-14 days and includes the following sequential steps:

[0097] 1) In a pluripotent stem cell maintenance medium (first differentiation medium) containing BMP4, a GSK-3β inhibitor (e.g., CHIR99021), and a Rock inhibitor, multiple single-cell pluripotent stem cells are aggregated for 12-48 hours to obtain first spheroids. The spheroid formation process of pluripotent stem cells can be performed by culturing in a multi-well plate with shaking or by culturing in an AggreWell® plate. TM This can be completed by static culture in a microplate. 2) The cells are cultured for 1-7 days in a second differentiation medium containing BMP4, VEGF, bFGF, UM171, and an induction enhancer, but without IL-3, to obtain second spheroids. During this period, the cells can be optionally transferred to a closed system. The closed system can be a culture flask or a culture bag, and the culture method can be shaking or static culture. The induction enhancer includes at least one component of a GSK-3β inhibitor, SR1, or a combination thereof. 3) The second spheroids are cultured for 2-8 days in a third differentiation medium containing VEGF, bFGF, SCF, FLT3L, and TPO, but not containing IL-3, to obtain third spheroids and suspended cells.

[0098] According to a specific embodiment of the present disclosure, the pluripotent stem cell maintenance medium is a medium capable of maintaining the pluripotency of pluripotent stem cells, the GSK-3β inhibitor may be CHIR99021, and the Rock inhibitor may be Y27632, HB-100, or other substances capable of supporting the survival of pluripotent stem cells in a single-cell state.

[0099] According to one specific implementation of the present disclosure, the concentration of BMP4 used is 5ng / mL-100ng / mL, the concentration of CHIR99021 used is 1uM-20uM, the concentration of VEGF used is 5ng / mL-100ng / mL, the concentration of bFGF used is 5ng / mL-100ng / mL, the concentration of UM171 used is 0nM-1uM, the concentration of SR1 used is 0μM-2μM, the concentration of SCF used is 5ng / mL-100ng / mL, the concentration of TPO used is 1ng / mL-100ng / mL, and the concentration of FLT3L used is 1-200ng / mL; Using the above method, on days 10-14 of differentiation, the CD34+ cell count reached 99-fold (average approximately 72-fold) higher than the starting pluripotent stem cell count, and CD34 + Suspended HSPCs with purity reaching 99% (average 94%) can be obtained. Furthermore, these HSPCs simultaneously express CD43 and CD44. A recent study (Zhu, Yanling et al. "Characterization and generation of human definitive multipotent hematopoietic stem / progenitor cells." Cell discovery vol. 6,1 89.1 Dec. 2020) demonstrated that CD44 can be used as a marker to distinguish two different hematopoietic stem cell types (primitive hematopoiesis and permanent hematopoiesis) in the early stages of human hematopoietic development and to identify permanent hematopoietic HSPCs produced from human pluripotent stem cells. CD44 produced from human pluripotent stem cells +Multipotential hematopoietic stem cells have multiple potentials and can produce various blood cells, such as myeloid (My), erythrocytes (Er), and megakaryocytes (Mk), in vitro and in vivo, as well as more important immune cells, such as NK cells and T cells, which have potential for disease treatment. + The cell colonies have the characteristics of permanent HSPCs derived from human pluripotent stem cells.

[0100] In some embodiments, the above-described medium combination can promote differentiation of pluripotent stem cells into HSPCs. The first differentiation medium contains BMP4, a GSK-3β inhibitor, and a Rock inhibitor, and a medium capable of maintaining pluripotency of pluripotent stem cells. The medium for maintaining pluripotency of pluripotent stem cells is mTeSR, excluding animal-derived components. TM 1. TeSR TM 2. TeSR TM -AOF, Essential 8 TM Culture medium, NutriStem @ hESC XF, StemFit @ Any of the above media or their equivalents, including but not limited to Feeder-Free Stem Cell Culture Media, can be used in this method. For the production of clinically applicable products, a medium free of animal-derived components can be selected from the above media. Here, "equivalent" means that pluripotent stem cells can maintain their pluripotent state even when used alone or with a Rock inhibitor alone and continuously cultured for three days or more. Specifically, OCT4 expression in cultured cell colonies was measured by flow cytometry. + The percentage change of cells was detected to be less than 20%.

[0101] In some embodiments, adding at least one of BMP4 and a GSK-3β inhibitor during the initial spheroid formation process of pluripotent stem cells significantly accelerates the differentiation of pluripotent stem cells into HSPCs during the subsequent differentiation process. On day 3 of differentiation, OCT4 expression significantly decreased, expression of early mesoderm and mesoderm markers (MIXL1 and T) also began to decrease, and expression of hemogenic endothelial lineage-associated markers (KDR, SCL) significantly increased. More notably, expression of HSPC marker (CD34) significantly increased. Adding a GSK-3β inhibitor and BMP4 to the pluripotent stem cell maintenance medium at the beginning of this stage significantly accelerated the progression of differentiation, and on day 3 of differentiation, CD34 expression was detectable, and expression of hematopoietic system markers was significantly higher than in control conditions.

[0102] In some embodiments, the concentrations of ITS-X, β-mercaptoethanol, ascorbate vitamin C, and 1x GlutaMAX contained in the second and third differentiation media may be within the range of concentrations typically used in media for differentiating pluripotent stem cells into HSPCs known in the art. In some embodiments, both the second and third differentiation media contain StemPro TM -34 SFM complete medium, 0.1-5% ITS-X, 0.1-10uM β-mercaptoethanol, 5-100ug / mL ascorbic acid, 1x GlutaMAX, none of which contain IL-3. The second differentiation medium contains UM171, which enhances the differentiation of CD34 cells. + The cell yield can be significantly improved. While traditional HSPC differentiation processes have been supplemented with different compounds (Li, Xuejia et al. "Pyrimidoindole derivative UM171 enhances derivation of hematopoietic progenitor cells from human pluripotent stem cells." Stem cell research vol. 21 (2017): 32-39), in the differentiation system disclosed herein, UM171 can be added to the second differentiation medium to produce good CD34 expression. +In some embodiments, the second medium further comprises at least one component of an induction enhancer, which enhances the differentiation efficiency and yield of HSPCs. + cell count, or higher CD34 + In some embodiments, the duration of the three stages of the culture medium is determined by the percentage of CD34 cells. + Affects cell production and rate.

[0103] For the avoidance of doubt, CD34 + The terms "cell," "HSPC," or "HSPCs" refer to a type of cell with characteristics of hematopoietic stem / progenitor cells or cells differentiated from pluripotent stem cells (e.g., human iPSCs or ESCs). This type of cell can be further differentiated into various hematopoietic lineage cells, such as myeloid (My), erythroid (Er), and megakaryocyte (Mk), as well as lymphoid cells, such as NK cells, T cells, and B cells (plasma cells). Pluripotent stem cells, as described herein, express OCT4, NANOG, SSEA-4, and Tra-1-60, and are typified by human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs).

[0104] In addition, single or multiple CD34 + Cells or HSPCs can differentiate into myeloid (My), erythroid (Er), and megakaryocyte (Mk) cells, as well as lymphoid cells. Myeloid cells include myeloid progenitor cells, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, and dendritic cells. Lymphoid cells include NK cells, T cells, and B cells (plasma cells).

[0105] According to a specific embodiment of the present disclosure, the present disclosure provides a method for preparing NK cells, as shown in FIG. a. Using the above method, CD34 + Obtaining hematopoietic stem / progenitor cells; b. CD34 + and culturing the hematopoietic stem / progenitor cells under a fourth culture condition to obtain NK cells; The fourth culture condition is a fourth differentiation medium for the CD34 + Differentiating and culturing hematopoietic stem / progenitor cells, In addition, an SPHK2 inhibitor is added to the second and / or third differentiation medium.

[0106] According to one specific embodiment of the present disclosure, the fourth differentiation medium comprises a fourth basal medium, IL-7, IL-15, FLT3L, SCF, and optionally IL-3.

[0107] According to one specific embodiment of the present disclosure, the pluripotent stem cells are iPSC cells or commercial embryonic stem cells.

[0108] According to one specific implementation of the present disclosure, the fourth basal medium may be DMEM / F12, the SPHK2 inhibitor is ABC294640, and the concentration of ABC294640 used is 1-50 μM.

[0109] Optionally, the concentration of IL-3 used is 0-10 ng / mL; The concentration of IL-7 used is 0.1-30 ng / mL, The concentration of IL-15 used is 1-10 ng / mL; The concentration of FLT3L used is 1-200 ng / mL, preferably 1-50 ng / mL; The concentration of the SCF used is 5 ng / mL-100 ng / mL.

[0110] According to a specific embodiment of the present disclosure, the fourth differentiation medium further comprises at least one of serum replacement, human AB serum, FBS, and human blood albumin.

[0111] In some embodiments, the fourth differentiation medium further comprises serum replacement and human serum albumin. The dosage of serum replacement and human serum albumin may be within the conventional dosage range of serum replacement and human serum albumin contained in a differentiation medium used for inducing differentiation of HSPCs into NK cells, for example, 2% serum replacement-3% + 0.2%-1% human serum albumin. According to a specific embodiment of the present disclosure, the serum replacement may be any brand of serum replacement known in the art, for example, BIT 9500, SR3, T-NK Xeno-Free Serum Substitute, Knockout TM It may be a serum replacement, a CTS™ immune cell serum replacement, or the like.

[0112] According to a specific embodiment of the present disclosure, the fourth differentiation medium further comprises NAD + , HLA-C, SB203580, and IL-2; Optionally, the NAD + The working concentration of is 1-500 μM, The HLA-C concentration used is 0.05 ng / mL to 1 ng / mL, The concentration of SB203580 used is 1 μM to 50 μM. The concentration of IL-2 used is 100-5000 IU.

[0113] In some embodiments, the CD34 obtained by this method + The cells can be cultured and differentiated into innate NK cells (iNK) using a fourth differentiation medium. The iNK cells express CD56, CD45, CD16, NKG2D, NKp30, NKp44, NKp46, CD226, and 2B4, and highly express CD16. The fourth differentiation medium contains a basal medium and cytokines. The basal medium is DMEM / F12 + serum replacement + human serum albumin, and the serum replacements are BIT 9500, SR3, T-NK Xeno-Free Serum Substitute, and Knockout. TM Serum replacement, CTS TMThe serum replacement in the fourth differentiation medium can be selected from at least one of the immune cell serum replacements, or any combination thereof. In some embodiments, the serum replacement in the fourth differentiation medium can also be replaced with human AB serum or FBS. The iNK CD56 expression obtained using the serum replacement is similar to that obtained with AB serum and superior to that obtained with FBS. However, the iNK production yield obtained is more stable, more reproducible, and superior to that obtained with AB serum and FBS. In some embodiments, the cytokines in the fourth differentiation medium include IL-3, IL-7, IL-15, FLT3L, and SCF. In some embodiments, the cytokines in the fourth differentiation medium may not include IL-3. During this period, cells are cultured in 3D, which may be in cell culture flasks or cell culture bags, or in shaking or static culture. Industrially acceptable closed culture systems, such as wave bioreactors, may also be used. The concentrations of the cytokines in the fourth differentiation medium are IL3: 0-10 ng / mL; IL-7: 0.1-30 ng / mL; IL-15: 1-10 ng / mL; FLT3L: 1-200 ng / mL; and SCF: 5-100 ng / mL. + CD45 + The percentage of iNK cells can reach over 99%, with CD16 expression exceeding 70%, and high expression of multiple activating receptors and costimulatory factors. Approximately five weeks after differentiation, the cell population can double approximately 2000-fold. In some embodiments, iNK cells obtained by this method are co-cultured with donor iPSCs for 24 hours. Under conditions of an effective target ratio of 5:1, nearly all cells are killed. In some embodiments, the inventors cryopreserved iNK cells obtained by this method. Under all tested cryopreservation conditions, the vitality of resuscitated iNK cells was over 80%, and under optimal cryopreservation conditions, the viable cell rate after resuscitation could reach 96%. More importantly, the resuscitated cells still have excellent killing ability against solid tumors and hematologic tumors.

[0114] In some embodiments, the second differentiation medium contains UM171, and adding UM171 can not only increase the production of HSPCs but also significantly increase the production of iNK cells. In some embodiments, adding an SPHK2 inhibitor to the second and third differentiation media reduces the production of HSPCs but significantly increases the production of iNK cells differentiated from these HSPCs. The SPHK2 inhibitor may be ABC294640, and its effective concentration may be 1-50 μM. In some embodiments, the fourth differentiation medium contains nicotinamide adenine dinucleotide (NAD + ), or HLA-C, or SB203580, or IL-2, + or CD45 + It can increase the production of iNK without affecting the cell ratio. + The concentration of is 1-500 μM, the concentration of HLA-C is 0.05-1 ng / mL, the concentration of SB203580 is 1-50 μM, and the concentration of IL-2 is 100-5000 IU.

[0115] In some embodiments, iPSCs were loaded with a chimeric antigen receptor (CAR-BCMA), and these iPSCs were further differentiated into anti-BCMA CARNK cells using this method, which then specifically killed target cells H929 (H929 expressing BCMA antigen). The CAR comprises an extracellular domain of an antigen recognition region, a transmembrane domain connected to the extracellular domain, and an intracellular domain connected to the transmembrane domain. In some embodiments, as the number of suspension cells released from the culture system increases during the differentiation of HSPCs into iNK cells, a larger-capacity culture vessel can be used and the medium volume can be increased. Those skilled in the art can flexibly determine this depending on the number of suspension cells released to ensure that the cultured cells receive sufficient nutrients.

[0116] The present invention provides a method for preparing HSPC cells, and the HSPC cells can be further differentiated into NK cells, which have the ability to kill hematologic and solid tumors. Furthermore, by loading CARs at the iPSC stage, the iNK cells differentiated from the iPSCs have the ability to kill tumor cells expressing specific antigens.

[0117] In another aspect, the present invention describes pharmaceutical compositions of HSPCs (and / or iPSCs) obtained by differentiating pluripotent stem cells according to any of the embodiments.

[0118] In another aspect, the present invention describes pharmaceutical compositions of NK cells (and / or iPSCs) obtained by differentiating pluripotent stem cells or HSPCs according to any of the embodiments.

[0119] In another aspect, the present invention describes pharmaceutical compositions of NK cells (and / or iPSCs) obtained by differentiating CAR-loaded pluripotent stem cells according to any of the embodiments.

[0120] In other aspects, the disclosure provides methods of potential immune cell therapy for a diseased subject, typically comprising administering to the subject iNK or CAR-iNK cells as referred to above, where INK cells are capable of killing hematologic or solid tumors, and CAR-iNK cells are capable of specifically killing tumor cells expressing a specific antigen.

[0121] The means provided by the present disclosure is that, at the pseudo-embryonic body formation stage, CD34 + , CD43 + and CD44 + The purity of the cells can be significantly improved, and CD44 + Cell purity is close to 100%, with no intermediate sorting or enrichment steps, and stably differentiated iPSCs within 2 weeks without sorting, resulting in over 90% CD34 + It can produce cells with up to >99% purity of CD34 +cells, CD43 cells, CD44 + CD34 cells can be obtained from HSPCs differentiated from iPSCs. + / CD43 + Compared with the initial number of pluripotent stem cells, the number of CD34 + On average, cell numbers can be expanded by more than 70-fold (maximum 99-fold). Furthermore, the differentiated iNK cells have high purity and high expression levels of activating receptors and costimulatory factors. The iNK cells obtained by this method express high levels of CD16 (over 70%), resolving the issue of low iNK CD16 levels (only 20%-30%) in conventional technologies, which requires genetic modification to address the need for low CD16 expression. The differentiation process involves 3D culture, which is suitable for a closed culture system. High iNK cell yields can be achieved, with a single iPSC capable of differentiating into 2,000 NK cells. The resulting iNK cells can be used to treat hematologic and solid tumors. iNK cells can be loaded with CARs to specifically kill tumor cells.

[0122] The following examples are used in conjunction with the present disclosure to illustrate the present disclosure. Those skilled in the art can understand that the following examples are intended to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. If specific techniques or conditions are not specified in the examples, they should be carried out according to the techniques or conditions described in the literature of the field or according to the product specifications. If the manufacturer of the reagents or equipment used is not specified, they are conventional products that can be purchased commercially.

[0123] Example 1 Differentiation of iPSCs into HSPCs and iNKs

[0124] 1. Obtaining pluripotent stem cells in a single cell state To prepare pluripotent stem cells in a single cell state, refer to the method disclosed in the patent "Method and application of human induced pluripotent stem cells" (patent number: ZL201310276246.7) to obtain a suspension of pluripotent stem cells in a single cell state.

[0125] Specifically, the method involves the following steps: 1) For cultured iPSCs, when the cell confluence reaches 75%-85% or the 3D culture cell sphere culture time reaches 4-7 days, wash once with DMEM / F12 medium, add Accutase digestion enzyme or TrypLE digestion enzyme, and digest in a 37°C, 5% CO2 incubator. When the cells are almost digested, add pluripotent stem cell maintenance medium (mTeSR). TM 1) was added to terminate the digestion. 2) After centrifugation, the supernatant was discarded and the mixture was resuspended in mTeSR containing 5 μM Y27632, 50 ng / mL BMP4, and 10 μM CHIR99021. TM The cells were resuspended using medium 1 to produce single-cell iPSCs.

[0126] 2. Differentiation of iPSCs into HSPCs (1) First-stage differentiation of HSPCs iPSC spheroid formation uses two methods: static spheroid formation or shaking spheroid formation. 1) Static spheroid formation. An appropriate number of single-cell iPSCs were cultured in AggreWell®. TM Transfer to a microplate and AggreWell TM Complete subsequent procedures according to the 400 or 800 instructions. Briefly, adjust the concentration of the single-cell suspension and use AggreWell. TM 400 or AggreWell TM Add enough volume to each well so that each well contains approximately 500,000 single-cell iPSCs. Then, add mTeSR TM 1 or TeSR TM The AggreWell AOF medium was replenished and 5 μM Y27632 was added to reach 5 mL / well. Next, the AggreWell AOF medium was replenished and 5 μM Y27632 was added to reach 5 mL / well. TM Prepare a centrifuge balance plate to match the weight and position of the microplate. Next, gently pipette the cells up and down several times to ensure they are evenly distributed throughout the wells. Immediately place the balance plate prepared in the previous step in the AggreWell well. TMThe plate was centrifuged at 100 g for 3 minutes to press the cells into the microwells. The plate was observed under a microscope to confirm that the cells were evenly distributed in the microwells. The plate was then incubated at 37°C, 5% CO2, and 95% humidity for 1–2 days. 2) Shaking spheroid formation. The concentration of the single-cell suspension was adjusted, and 1.5 million single-cell iPSCs were transferred to a 6-well plate. Each well was filled with mTeSR supplemented with 5 μM Y27632, 50 ng / mL BMP4, and 10 μM CHIR99021, so that the total volume reached 3 mL per well. TM The culture plate was placed on a shaking shaker at rotation speeds of 60, 70, 80, and 90 rpm, and wells with uniformly sized spheroids were selected and cultured.

[0127] CD34 obtained by the above two methods + The cell percentages are shown in Table 1 below.

[0128] Table 1: Orbital shaker and AggreWell spheroid formation method using CD34 + Effect of cell population purity [Table 1]

[0129] As can be seen from the results in Table 1, both the orbital shaker and AggreWell spheroid formation methods produced highly pure CD34 + A cell population can be obtained.

[0130] (2) HSPC second stage differentiation After 1-2 days of cell spheroid formation, the cells were cultured in the second differentiation medium, which was then changed every two days for 4-5 days. The second differentiation medium consisted of StemPro-34 complete medium supplemented with 1% ITS-X, 1 μM β-mercaptoethanol, 50 μg / mL ascorbic acid, 1x GlutaMAX, 50 ng / mL VEGF, 50 ng / mL bFGF, 50 ng / mL BMP4, and 35 nM UM171. At this stage, the cells were transferred to a closed system and cultured either statically or with shaking.

[0131] (3) Third-stage differentiation of HSPCs After culturing for 3 days in the second differentiation medium, the medium was changed to the third differentiation medium, which contained StemPro-34 TM SFM is a complete medium supplemented with 1% ITS-X, 1 μM β-mercaptoethanol, 50 μg / mL ascorbic acid, 1x GlutaMAX, 50 ng / mL VEGF, 50 ng / mL bFGF, 50 ng / mL SCF, 10 ng / mL FLT3L, and 30 ng / mL TPO. FACS was used to detect the expression of CD34, CD43, and CD44. CD34, CD43, and CD44 antibodies were purchased from Biolegend. The FACS detection steps can be performed using the operating instructions commonly used with the relevant equipment. The results are shown in Figures 3 and 4. CD34 cells differentiated using the method disclosed herein were analyzed. + The cells simultaneously expressed CD43 and CD44. At the same time, the typical method was used as a control, i.e., the first differentiation medium was not supplemented with BMP4 or CHIR99021, and the second differentiation medium was not supplemented with CHIR99021, UM171, or SR1. Using this method, five iPSC cell lines, iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-5, were selected and differentiated into HSPCs. At five time points, i.e., Day 8, Day 9, Day 10, Day 11, and Day 12, respectively, CD34 + The percentage of cells and cell expansion fold were detected. iPSC-1 included two independent tests (iPSC-1-1, iPSC-1-2), and the results are shown in Figures 5 and 6. CD34 cells obtained on day 12 of differentiation were +Cell numbers increased 100-180 fold over the starting pluripotent stem cell numbers, and CD34 + It can be seen that the cell purity can reach 96%-99% on Day 8. This result demonstrates the general applicability of the present disclosure.

[0132] (4) Differentiation of HSPCs into NK cells After the three stages of culture, typically on day 10-14 of differentiation, depending on the FACS results, the fourth differentiation medium was changed to further differentiate the HSPCs into NK cells, which typically lasts 3-5 weeks. During the differentiation and expansion process, the expression of iNK markers CD45 and CD56 gradually increased, reaching a purity of over 99% after 40 days (Figure 7). During this period, CD16 expression levels also gradually increased, reaching over 70% (Figure 8). The fourth differentiation medium contains basal medium and cytokines. For example, the basal medium contains DMEM / F12 + 2% serum replacement-3 (Sigma-Aldrich) + 0.2%-1% human serum albumin. Cytokines used included IL-3 (used in the first week), 20 ng / mL IL-7, 10 ng / mL IL-15, 10 ng / mL FLT3L, and 50 ng / mL SCF. FACS was used to detect CD56, CD45, CD16, NKG2D, NKp30, NKp44, NKp46, CD226, and 2B4. The antibodies (including isotype controls) were purchased from Biolegend. The FACS detection steps can be performed according to the operating specifications of the relevant equipment. The results are shown in Figures 7-9. iNK (>Day 40) CD56 generated by the culture method of the present disclosure. + CD45 + The cell population reached over 99% (Figure 7), CD16 expression exceeded 70% (Figure 8), and multiple activating receptors and costimulatory factors were highly expressed (Figure 9). Approximately 5 weeks after differentiation, the cell population doubled approximately 2000-fold (Figure 10).

[0133] Example 2 Optimization of the first differentiation medium

[0134] After harvesting single-cell iPSCs, 1) mTeSR TM1) 5 μM Y27632, 2) mTeSR TM 1) 5 μM Y27632 + 50 ng / mL BMP4, 3) mTeSR TM 1+5μM Y27632 +10μM CHIR99021, 4)mTeSR TM The cells were resuspended in four media containing 1 μM Y27632, 50 ng / mL BMP4, and 10 μM CHIR99021, and then cultured according to the method described in Example 1. After differentiation, the cells were lysed on day 3 to extract mRNA and then purified to obtain cDNA. The primers listed in Table 2 were used to measure the expression levels of OCT4, MIXL1, T, KDR, SCL, and CD34 in HSPCs under the culture conditions of the above media up to day 3. The results are shown in Figures 11-14. On day 3 of differentiation, OCT4 expression significantly decreased (Figure 11), and the expression of early mesoderm and mesoderm markers (MIXL1 and T) also began to decrease (Figure 12). The expression of hemogenic endothelial lineage-related markers (KDR and SCL) significantly increased (Figure 13). More significantly, the expression of the HSPC marker (CD34) significantly increased (Figure 14). During the spheroid formation stage of pluripotent stem cells, adding a GSK-3β inhibitor and BMP4 to the pluripotent stem cell culture medium significantly accelerated the differentiation process, and CD34 expression could be detected on the third day of differentiation. The expression of hematopoietic system markers was significantly higher than that under control conditions, and CD34 expression was significantly improved.

[0135] Table 2: qPCR primers [Table 2]

[0136] Example 3. Analysis of the effects of adding CHIR99021, SR1, or UM171 on HSPCs or iNKs during differentiation

[0137] Furthermore, CD34 + To further increase the cell production and positive rate, 10 μM CHIR99021 or 0.75 μM SR1 was added to the second differentiation medium in Example 1, and the CD34+ The percentage of CD34 cells in the cell colonies was detected, and the results are shown in Figure 15. CHIR99021 and SR1 were CD34 cells in the cell colonies on Day 12. + The proportion of cells can be increased.

[0138] 35 nM UM171 was added to the second differentiation medium and / or the third differentiation medium of Example 1, and CD34 in the cell colonies on Day 12 was detected by FACS. + The percentage of CD56+ cells in the cell colonies on Day 35 was detected, and the results for these two time points are shown in Figure 16. Adding UM171 to the second differentiation medium further improved the productivity of iPSC differentiation into HSPCs and further differentiation into iNK cells.

[0139] Example 4. Optimization of serum replacement in NK cell induction medium

[0140] BIT 9500, SR3, T-NK Xeno-Free Serum Substitute, Knockout TM Serum replacement, CTS TM Immune cell serum substitutes or combinations thereof (in Figure 17, each box in the serum substitute group represents a serum substitute or combination from a different manufacturer, with concentrations ranging from 2% to 20%. Specific concentrations are determined based on each manufacturer's product specifications) were tested as controls, and NK cell induction was tested using 15% human AB serum or 10% fetal bovine serum (FBS). The results are shown in Figure 17, and show that various serum substitutes support efficient induction of HSPCs into NK cells, with iNK production slightly higher and more stable than AB serum.

[0141] Example 5. Optimization of iNK purity and production yield

[0142] To further improve the differentiation efficiency and toxicity of the resulting NK cells, UM171 was added to the second differentiation medium, and then either 1) 10 μM of the SPHK2 inhibitor ABC294640 was added to the second and third differentiation media, or 2) 75 μM of nicotinamide adenine dinucleotide (NAD +), 0.1 ng / mL HLA-C, 1000 IU IL-2, or 15 μM SB203580 was added to the fourth differentiation medium, and the CD56 and CD45 expression levels and cell proliferation fold of the NK cells obtained were measured by flow cytometry. The results are shown in Figures 18-21. Figure 18 shows that adding an SPHK2 inhibitor (ABC294640) to the second and third differentiation media reduces the production of HSPCs, but increases the production of iNK cells differentiated from these HSPCs. Figures 19-21 show that adding nicotinamide adenine dinucleotide (NAD + ), or HLA-C, or SB203580, or IL-2, to enhance CD56 + or CD45 + The results showed that iNK production can be increased without affecting the cell ratio. + , HLA-C, IL-2, and SB203580 can all increase the proliferation fold of iNK.

[0143] Example 6. Measurement of killing ability of iNK against donor iPSCs

[0144] Using conventional methods, iPSCs were plated onto Matrigel-coated 6-well plates at a density of 250,000 per well, using the Matrigel concentration recommended in the specifications. TM 1 medium, supplemented with 5 μM Y27632, and the next day and thereafter, fresh mTeSR TM The liquid was replaced with medium 1. After the cells had grown to 60-70% confluence, the iPSCs from one well were harvested and counted. Next, depending on the number of iPSCs counted, 5x the amount of CBNK or iNK was added to each well containing the iPSCs. After 4 and 24 hours of co-culture, images were taken using an inverted microscope. The results are shown in Figure 22, demonstrating that the iNKs obtained by this method can eliminate their derived iPSCs within 24 hours at an effective target ratio of 5:1.

[0145] Example 7. Cell doubling calculations

[0146] The cell doublings at the HSPC stage and the NK stage were both calculated by dividing the number of suspension cells obtained at the time of sampling by the amount of iPSC cells at the time of initial spheroid formation. TM 2. Use an automatic cell counter. For specific operation methods, please refer to the instrument's instruction manual.

[0147] Example 8. Measurement of tumor cell killing by NK cells - Luciferase method

[0148] Using tumor cells K562-Luc (target cells) as an example, first prepare a target cell suspension (target cell activity rate of 90% or higher). After counting, place the cells into wells of a 96-well plate at the desired effective target ratio (e.g., 10:1, 5:1, 2:1, 1:1). Set up at least three wells for each effective target ratio, adjusting the medium to 100 μL per well. Next, prepare the required number of iNK cells or control NK cells according to the desired effective target ratio. Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for the desired time (e.g., 4 or 24 hours). Then, take the fluorescein substrate and dilute it 100x with cell medium. Add 50 μl of the diluted solution containing the fluorescein substrate to each well and allow to react for 10 minutes at room temperature, away from light. Set the microplate reader light source to Lumen and the time to 1000 ms, and read the data on the machine. Killing efficiency %=(control group-killed group) / cell control group*100.

[0149] Example 9. Tumor cell killing assay - Incucyte assay

[0150] Using tumor cells U-87 MG-GFP (target cells) as an example, first prepare a target cell suspension (target cell activity rate of 90% or higher), count the cells, and then place them into wells of a 96-well plate at a designated effective target ratio (e.g., 10:1, 5:1, 2:1, 1:1). For each effective target ratio, at least three wells are set up. Each well is filled with 100 μL of medium. After 24-48 hours, observe the cell adhesion status. Then, add the required number of iNK cells or control NK cells according to the designated effective target ratio. The 96-well plate is incubated in an Incucyte incubator at 37°C and 5% CO2. @ Place the cells in a live cell analysis system (Sartorius) and collect images once every 45 minutes or every hour. After 24 or 48 hours, remove the cells, copy the image data, and analyze them in an Incucyte @ The killing efficiency was calculated using software.

[0151] Example 10. Cryopreservation and resuscitation of iNK

[0152] iNK cryopreservation: iNK cells were collected, centrifuged, and the supernatant removed. The cells were resuspended in CS10 cryopreservation medium or other commercial cryopreservation medium (cryopreservation medium 1-3) or a homemade cryopreservation medium (iNK medium + 10% DMSO, cryopreservation medium 4) to a cell density of 25 million cells / mL. Then, the cells were aliquoted into cryopreservation tubes, 1 mL per tube. The cryopreservation tubes containing the cells were quickly transferred to a programmable cooling box and placed in a -80°C refrigerator overnight. The next day, the cryopreservation tubes were transferred to liquid nitrogen. iNK resuscitation: Remove the cryopreservation tube from liquid nitrogen, rub it with both hands several times, then quickly transfer it to a 37°C water bath or metal bath and shake until only small pieces of ice remain. Quickly wipe the exterior with 70% alcohol. In a safety cabinet, gently open the cell freezing tube, aspirate the cells with a pipette, and gently transfer (by shaking while dropping) the cells into the centrifuge tube from step 1 and mix well. After mixing thoroughly, the cell suspension was centrifuged (200g for 5 min). The supernatant was removed, and 10 mL of iNK medium was added to resuspend the cells, mix well, and count. Then, the cells were centrifuged. Cell seeding: Remove the supernatant (taking care not to aspirate the cells). Depending on the cell number, NK medium was added at a density of 1.0-1.6 million / mL, mixed well, and seeded into culture flasks or culture plates. Figure 23 shows the cell viability of iNK cells cryopreserved in several cryopreservation media, demonstrating that they were resuscitated and their vitality was well maintained after cryopreservation. The killing of hematopoietic or solid tumor cells is shown in Figures 24-25, which show that iNK after cryopreservation and resuscitation has killing activity against hematopoietic and solid tumor cells, and iNK has killing activity against U-87 MG cells.

[0153] Example 11. Genetic modification of iPSCs and introduction of a CAR

[0154] The anti-BCMA CAR was cloned into a piggybac vector, and then the piggybac plasmid expressing the anti-BCMA CAR and the transposase-expressing plasmid were co-transcribed into iPSCs derived from healthy humans using nuclear transcription. CAR-BCMA iPSCs were differentiated and expanded into iNK cells using the method described in Example 1 above (Figure 26A). The complete amino acid sequence of CAR-BCMA is shown in SEQ ID NO:15: QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSSLINTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSNLFVASWIAVMIIFRIGMAVAIFCCFFFPSWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR After day 40, iNK cells and target cells (K562-luc or H929-luc) were mixed at effective target ratios of 8:1, 4:1, 2:1, or 1:1, respectively, and cultured at 37°C, 5% CO for 4 hours. The luciferase assay was used to measure iNK cell target cell killing (Figure 26, panels B-D). Panel B shows the killing of K562 cells (which do not express BCMA) by CB-NK, WT iNK, and CAR-BCMA iNK. At different effective target ratios, the specific killing ability of CAR-BCMA-iNK cells against K562 cells exhibited a dose effect, decreasing with decreasing effective target ratio. Panel C shows that CAR-BCMA iNK cells exhibited significantly superior killing ability against H929 cells (a tumor cell line that highly expresses BCMA) compared with the other two NK cell types, demonstrating that CAR-BCMA mediates iNK-specific killing of tumor cells. Figure D shows that the killing of H929 cells by CAR-BCMA iNK cells exhibits a dose-dependent effect. The above explanation shows that after loading the iPSC cells with a chimeric antigen receptor (CAR-BCMA), these iPSC cells can be differentiated into anti-BCMA CAR-NK cells using the culture method disclosed herein, which can then specifically kill target cells H929 (H929 expressing the BCMA antigen).

[0155] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, general references to such terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described herein and the features of the various embodiments or examples without mutual contradiction.

[0156] Although the embodiments of the present disclosure have been presented and described, the above embodiments are illustrative and should not be construed as limiting the present disclosure, and it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present disclosure.

[0157] This application claims priority to and benefits from patent application number 202310292114.7, filed with the State Intellectual Property Office of China on March 17, 2023, the entire contents of which are incorporated herein by reference.

Claims

1. CD34 from pluripotent stem cells ＋ A medium combination for inducing differentiation into hematopoietic stem / progenitor cells, comprising a first differentiation medium, a second differentiation medium, and a third differentiation medium, wherein the first differentiation medium, the second differentiation medium, and the third differentiation medium are added in the first differentiation stage, the second differentiation stage, and the third differentiation stage, respectively; (1) The first differentiation medium contains a first basal medium, BMP4, and a GSK-3β inhibitor; (2) The second differentiation medium comprises a second basal medium and optionally UM171, and does not contain IL-3; (3) The third differentiation medium comprises a third basal medium, VEGF, bFGF, SCF, FLT3L, TPO, and does not contain IL-3, and is used to differentiate CD34 cells from pluripotent stem cells. ＋ A medium combination that induces differentiation into hematopoietic stem / progenitor cells.

2. the pluripotent stem cells are iPSC cells or commercial embryonic stem cells; Optionally, the first basal medium is mTeSR TM 1. TeSR TM 2. TeSR TM -AOF, Essential 8 TM Culture medium, NutriStem ＠ hESC XF, StemFit ＠ and at least one selected from the group consisting of: Feeder-Free Stem Cell Culture Media; Optionally, the first basal medium is mTeSR TM 1 or TeSR TM - AOF medium, Optionally, the second basal medium is StemPro TM -34 SFM complete medium, Optionally, the third basal medium is StemPro TM The medium combination according to claim 1, which is -34 SFM complete medium.

3. the first differentiation medium further comprises a Rock inhibitor; 2. The medium combination according to claim 1, wherein the Rock inhibitor optionally comprises at least one selected from the group consisting of Y27632 and HB-100.

4. the second differentiation medium further comprises VEGF, bFGF, and BMP4; Optionally, the second differentiation medium further comprises an induction enhancer; Optionally, the induction enhancer comprises at least one selected from a GSK-3β inhibitor, an SR1; Optionally, the GSK-3β inhibitor comprises at least one selected from CHIR99021, NP031112, AT7519, TWS119, SB216763, CHIR-98014, AZD1080, SB415286, LY2090314, (E / Z)-GSK-3β inhibitor 1, KY19382, Alsterpaullone, BIO-acetoxime, IM-12, 1-Azakenpaullone, and Indirubin; Optionally, the second differentiation medium further comprises ITS-X, β-mercaptoethanol, ascorbic acid, and GlutaMAX.

5. The medium combination according to claim 1, wherein the third differentiation medium further comprises ITS-X, β-mercaptoethanol, ascorbic acid, and GlutaMAX.

6. The concentration of BMP4 used is 5 ng / mL to 100 ng / mL; Optionally, the GSK-3β inhibitor is CHIR99021 and the concentration of CHIR99021 used is 1 μM-20 μM; Optionally, the Rock inhibitor is Y27632, and the concentration of Y27632 used is 1 μM-20 μM; Optionally, the VEGF is used at a concentration of 5 ng / mL-100 ng / mL; Optionally, the bFGF is used at a concentration of 5 ng / mL-100 ng / mL; Optionally, the SCF is used at a concentration of 5 ng / mL-100 ng / mL; Optionally, the TPO concentration used is 1 ng / mL-100 ng / mL; Optionally, the concentration of FLT3L used is 1-200 ng / mL, preferably 1-50 ng / mL; Optionally, the concentration of UM171 used is 0 nM-1 μM; 6. The medium combination of any one of claims 1-5, wherein optionally, the induction enhancer is SR1, and the concentration of SR1 used is 0 μM-2 μM.

7. A medium combination for inducing differentiation of pluripotent stem cells into NK cells, the medium combination comprising a fourth differentiation medium and the first, second, and third differentiation media of the medium combination according to any one of claims 1 to 6; the fourth differentiation medium comprises a fourth basal medium, and IL-7, IL-15, FLT3L, SCF, and optionally IL-3; and adding UM171 to the second differentiation medium; Optionally, a medium combination for inducing differentiation of pluripotent stem cells into NK cells, wherein an SPHK2 inhibitor is added to the second differentiation medium or the third differentiation medium.

8. the pluripotent stem cells are iPSC cells or commercial embryonic stem cells; Optionally, the fourth basal medium is DMEM / F12; Optionally, the concentration of UM171 used is 10 nM-1 μM; Optionally, the SPHK2 inhibitor is ABC294640; Optionally, the concentration of ABC294640 used is 1-50 μM; Optionally, the concentration of IL-3 used is 0-10 ng / mL; Optionally, the IL-7 is used at a concentration of 0.1-30 ng / mL; Optionally, the concentration of IL-15 used is 1-10 ng / mL; Optionally, the concentration of FLT3L used is 1-200 ng / mL, preferably 1-50 ng / mL; Optionally, the SCF is used at a concentration of 5 ng / mL-100 ng / mL; 8. The medium combination of claim 7, wherein optionally, the fourth differentiation medium further comprises at least one of serum replacement, human AB serum, FBS, and human blood albumin.

9. The fourth differentiation medium further comprises NAD ＋ , HLA-C, SB203580, and IL-2; Optionally, the NAD ＋ The concentration used is 1-500 μM, Optionally, the HLA-C concentration used is 0.05 ng / mL-1 ng / mL; Optionally, the concentration of SB203580 used is 1 μM-50 μM; Optionally, the medium combination of claim 7, wherein the concentration of IL-2 used is 100-5000 IU.

10. The pluripotent stem cells according to any one of claims 1 to 6 are isolated from CD34. ＋ Use of a medium combination for inducing differentiation into hematopoietic stem / progenitor cells, or a medium combination for inducing differentiation from pluripotent stem cells into NK cells according to any one of claims 7 to 9, in the preparation of NK cells.

11. CD34 ＋ 1. A method for preparing hematopoietic stem / progenitor cells, comprising: A. Obtaining pluripotent stem cells in a single cell state; B. Culturing the pluripotent stem cells in a single cell state under first culture conditions to obtain a first cell sphere; C. Culturing the first cell sphere under second culture conditions to obtain a second cell sphere; D. Culturing the second cell sphere under a third culture condition to induce CD34 ＋ obtaining hematopoietic stem / progenitor cells; the first culture conditions include differentiating and culturing the pluripotent stem cells using a first differentiation medium; the second culture conditions include differentiating and culturing the first cell sphere using a second differentiation medium; the third culture condition comprises differentiating and culturing the second cell sphere using a third differentiation medium; The first differentiation medium, the second differentiation medium, and the third differentiation medium are used to differentiate CD34 from the pluripotent stem cells according to any one of claims 1 to 6. ＋ A first differentiation medium, a second differentiation medium, and a third differentiation medium are included in a combination of media that induce differentiation into hematopoietic stem / progenitor cells; The pluripotent stem cells are iPSC cells or commercially available embryonic stem cells, CD34 ＋ A method for preparing hematopoietic stem / progenitor cells.

12. CD34 obtained by the method of claim 11 ＋ Hematopoietic stem / progenitor cells.

13. 1. A method for preparing NK cells, comprising: a. CD34 using the method of claim 11 ＋ Obtaining hematopoietic stem / progenitor cells; b. the CD34 ＋ Culturing the hematopoietic stem / progenitor cells under a fourth culture condition to obtain NK cells; The fourth culture condition is a fourth differentiation medium for the CD34 ＋ Differentiating and culturing hematopoietic stem / progenitor cells, The fourth differentiation medium is the fourth differentiation medium in the medium combination for inducing differentiation of pluripotent stem cells into NK cells according to any one of claims 7 to 9, and adding UM171 to the second differentiation medium used in the method of claim 11; Optionally, the method for preparing NK cells further comprises adding an SPHK2 inhibitor to the second or third differentiation medium.

14. The concentration of UM171 used is 1 nM-1 μM, Optionally, the concentration of UM171 used is 35 nM; Optionally, the SPHK2 inhibitor is ABC294640; Optionally, the ABC294640 is used at a concentration of 1-50 μM.

15. NK cells obtained by the method of claim 13 or 14.

16. The pluripotent stem cells according to any one of claims 1 to 6 are isolated from CD34. ＋ A medium combination for inducing differentiation into hematopoietic stem / progenitor cells, a medium combination for inducing differentiation from pluripotent stem cells to NK cells according to any one of claims 7 to 9, and CD34 according to claim 11. ＋ CD34 obtained by the method for preparing hematopoietic stem / progenitor cells ＋ Hematopoietic stem / progenitor cells, CD34 according to claim 12 ＋ Use of hematopoietic stem / progenitor cells, NK cells prepared by the method for preparing NK cells according to claim 13 or 14, or the NK cells according to claim 15, in the preparation of CAR-NK cells.

17. CAR-NK cells obtained by a method comprising modifying or gene-editing pluripotent stem cells to obtain pluripotent stem cells that express a CAR, and then differentiating and culturing the CAR-modified pluripotent stem cells using the method for preparing NK cells according to claim 13 or 14 to obtain CAR-NK cells.

18. The CAR-NK cell according to claim 17, wherein the pluripotent stem cells are iPSC cells or commercial embryonic stem cells.

19. A cell population comprising the NK cell according to claim 15 and / or the CAR-NK cell according to claims 17 and 18.

20. A drug for preventing and / or treating a tumor, comprising the NK cells of claim 15 and / or the CAR-NK cells of claims 17 and 18 and / or the cell group of claim 19.

21. CD34 according to claim 11 ＋ CD34 obtained by the method for preparing hematopoietic stem / progenitor cells ＋ Hematopoietic stem / progenitor cells, CD34 according to claim 12 ＋ Use of hematopoietic stem / progenitor cells, NK cells prepared by the method for preparing NK cells according to claim 13 or 14, NK cells according to claim 15, CAR-NK cells according to claim 17 or 18, or the group of cells according to claim 19 in the prevention and / or treatment of tumors.

22. The use of claim 21, wherein the tumor comprises a hematological tumor or a solid tumor.

23. The use of claim 22, wherein the hematological tumors include chronic myeloid leukemia, lymphoma, and myeloma, and the solid tumors include non-small cell lung cancer and glioblastoma.

24. A method for preventing and / or treating a tumor, comprising a step of administering to a subject in need thereof at least one of the NK cells of claim 15, the CAR-NK cells of claim 17 or 18, or the group of cells of claim 19.

25. 25. The method of claim 24, wherein the tumor comprises a hematological tumor or a solid tumor.

26. 26. The method of claim 25, wherein the hematological tumors include chronic myeloid leukemia, lymphoma, and myeloma, and the solid tumors include non-small cell lung cancer and glioblastoma.

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