Methods and compositions for differentiation of pluripotent stem cells and derived natural killer cells

Abstract

The present disclosure provides, inter alia, methods and compositions for the differentiation of pluripotent stem cells and derived hematopoietic cells, including hemogenic endothelial cells, hematopoietic progenitor cells, and natural killer cells. The efficiency of differentiation of hemogenic endothelial cells, hematopoietic progenitor cells, and natural killer cells can be improved by using the methods and compositions of the present disclosure described herein.

Description

[Technical Field]

[0001] The present disclosure relates generally to the field of stem cell technology, and more particularly to methods and compositions for promoting the directed differentiation of pluripotent stem cells and derived hematopoietic cells. [Background technology]

[0002] Natural killer (NK) cells are innate lymphocytes that play important roles in defense against, for example, viral infections and cancer, as well as immune regulation. Due to their inherent properties, NK cells hold promise for immune-based therapies against various diseases and disorders. However, procuring NK cells (e.g., for therapeutic or biomedical research) presents several challenges. These challenges include the number of primary NK cells that can be isolated (e.g., during apheresis), significant variability in the quantity and / or quality of primary NK cells between donors, and / or the efficient production of safe NK cells with well-understood phenotypes and / or functionality (e.g., phenotypes and / or functionality similar to primary NK cells) by alternative methods, such as differentiation of pluripotent cells (e.g., human pluripotent stem cells [hPSCs]) into NK cells (iNK cells).

[0003] Pluripotent stem cell technologies, including human PSC (hPSC) technology, are extremely promising and represent a potentially limitless source of therapeutically viable cells. However, current protocols for directed differentiation (e.g., differentiation into hemogenic endothelial (HE) cells, hematopoietic progenitor (HP) cells, and iNK cells) and subsequent expansion typically lack high efficiency. Furthermore, these protocols typically require the use of serum. The use of such components poses potential risks of contamination and can lead to cell lot dependency, making them unsuitable for the production of clinical and therapeutic cells. For example, cells cultured in environments contaminated with animal-derived components are generally considered unsuitable for human use because exposure to these components can pose significant risks, such as immune rejection, transmission of unidentified pathogens to treated subjects, and reactivation of animal retroviruses. Producing large quantities of HP or iNK cells with consistent and reproducible phenotype and / or functionality (e.g., phenotype and / or functionality equivalent to or better than primary NK cells) also remains a challenge with traditional methods of directed differentiation. Summary of the Invention [Problem to be solved by the invention]

[0004] To advance the technology for directed differentiation of pluripotent cells (e.g., hPSCs), it is important to be able to efficiently, safely, and / or reproducibly generate not only PSCs and partially differentiated cells (e.g., hematopoietic progenitor cells), but also immune effector populations, including iNK cells. Thus, there remains a need for improved methods and compositions for the directed differentiation of pluripotent stem cells (e.g., hPSCs) into hematopoietic lineage cells, including HE, HP, or iNK cells (e.g., immature iNK cells, functional iNK cells). [Means for solving the problem]

[0005] The present disclosure provides, inter alia, methods and compositions for promoting the directed differentiation of pluripotent cells (e.g., hPSCs) or cell populations thereof into hematopoietic cells, such as non-pluripotent cells (e.g., iNK cells) or partially differentiated cells, including, for example, HE cells and / or HP cells. The present disclosure also relates to cell populations, cell lines, and / or clonal cells produced using the methods and compositions described herein.

[0006] In a first aspect, the present disclosure relates to a method for promoting directed differentiation of pluripotent stem cells (PSCs), comprising the steps of contacting PSCs with a maintenance culture medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation culture medium, or sequentially with a first differentiation culture medium and a second differentiation culture medium, to form mesodermal cells; contacting the mesodermal cells with a third differentiation culture medium to form hemogenic endothelial (HE) cells; contacting the HE cells with a fourth differentiation culture medium to form hematopoietic progenitor (HP) cells; and contacting the HP cells with a fifth differentiation culture medium to obtain immature iNK cells; wherein a basal medium supplemented with a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate is used as the basal medium of the fourth differentiation culture medium and / or the basal medium of the fifth differentiation culture medium.

[0007] In a second aspect, the present disclosure relates to a method for promoting directed differentiation of pluripotent stem cells (PSCs), comprising the steps of contacting PSCs with a maintenance culture medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation culture medium supplemented with a Wnt signaling pathway activator, or sequentially with a first differentiation culture medium supplemented with a Wnt signaling pathway activator and a second differentiation culture medium supplemented with a Wnt signaling pathway activator, to form mesodermal cells; and contacting the mesodermal cells with a third differentiation culture medium supplemented with a Wnt signaling pathway inhibitor to obtain hemogenic endothelial (HE) cells.

[0008] In a third aspect, the present disclosure relates to a culture medium for promoting directed differentiation of pluripotent stem cells (PSCs) into hematopoietic cells, the culture medium comprising a basal medium to which an inhibitor of the Wnt signaling pathway has been added.

[0009] In a fourth aspect, the present disclosure relates to a kit comprising the culture medium of the third aspect of the present disclosure.

[0010] In a fifth aspect, the present disclosure relates to a method for producing iNK cells, comprising a method for promoting directed differentiation of pluripotent stem cells (PSCs) according to the first aspect of the disclosure and expanding and maturing immature iNK cells.

[0011] In a sixth aspect, the present disclosure relates to a cell population produced by a method according to the first, second, or fifth aspect of the disclosure.

[0012] In a seventh aspect, the present disclosure relates to a cell population in which greater than 90% of the cells in the unenriched and unpurified population are mature CD56+CD3- iNK cells.

[0013] In an eighth aspect, the present disclosure relates to a pharmaceutical composition comprising a cell population according to the seventh aspect of the present disclosure and a pharmaceutically acceptable carrier.

[0014] In a ninth aspect, the present disclosure relates to the use of a cell population according to the seventh aspect of the disclosure in the manufacture of a medicament for treating or preventing cancer.

[0015] Various objects and advantages of the reagents, compositions and methods provided herein will become apparent from the following description taken in conjunction with the accompanying drawings, in which are set forth, by way of example, certain embodiments of the disclosure. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a diagram illustrating the steps for generating iNK cells from hPSCs according to one illustrative example of the disclosed method. [Figure 2] FIG. 2 shows the effect of nicotinamide (NAM) concentration in an exemplary basal medium on PBNK cell proliferation, including NK cell fold expansion (FIG. 2A) and the percentage of CD56+CD3- NK cells (FIG. 2B), according to Example 1 of the present disclosure. [Figure 3] FIG. 3 illustrates the effect of heparin sodium concentration in an exemplary NKSFM basal medium on PBNK cell proliferation, including fold expansion of NK cells (FIG. 3A), percentage of CD56+CD3- NK cells (FIG. 3B), and lysis of K562 tumor cells (FIG. 3C), according to Example 2 of the present disclosure. [Figure 4] FIG. 4 shows the effect of PLT concentration in an exemplary NKSFM basal medium on the proliferation of PBNK cells from donors 1 and 2, including fold expansion of NK cells (FIG. 4A), percentage of CD56+CD3- NK cells (FIG. 4B), and lysis of K562 tumor cells (FIG. 4C), according to Example 3 of the present disclosure. [Figure 5]Figure 5 shows a comparison of the effect of an exemplary NKSFM basal medium and a commercial kit on the proliferation of PBNK cells, including fold NK cell expansion (Figure 5A), the percentage of CD56+CD3- NK cells (Figure 5B), and lysis of K562 tumor cells (Figure 5C), according to Example 4 of the present disclosure, where 1 represents NKSFM (feeder-free), 2 represents commercial kit (feeder-free), 3 represents NKSFM (feeder-present), and 4 represents commercial kit (feeder-present). [Figure 6] FIG. 6 shows the effect of different basal media used during Stage 2 of the method on the differentiation efficiency into iNK cells (CD56+ % (FIG. 6A) and CD56+ cell number (FIG. 6B)) according to Example 6 of the present disclosure, where 1, 2, 3, 4, 5, 6, and 7 represent #1, #2, #3, #4, #5, #6, and #7, respectively. [Figure 7] FIG. 7 shows the effect of plate coating in Stage 2 of the method on iNK differentiation according to Example 7 of the present disclosure, where FIGS. 7A and 7C show representative morphology and flow cytometry diagrams, respectively, of cells differentiated using exemplary NKSFM basal medium on a cell culture surface without a coating matrix, and FIGS. 7B and 7D show representative morphology and flow cytometry diagrams, respectively, of cells differentiated using exemplary NKSFM basal medium on a cell culture surface coated with DLL4 and VCAM1. [Figure 8] 8A and 8B show a comparison of the effects of XAV939 and / or SB431542 used during stages 1-4 of the method on the differentiation efficiency into HP cells (FIG. 8A) or iNK cells (FIGS. 8B and 8C) according to Example 8 of the present disclosure, where None represents when neither XAV939 nor SB431542 is used during stages 1-4, XAV represents when XAV939 is used during stages 1-4, SB represents when SB431542 is used during stages 1-4, and SB+XAV represents when both XAV939 and SB431542 are used during stages 1-4. [Figure 9]FIG. 9 shows the effect of the concentration of CHIR99021 used in stages 1-2 of the method on the efficiency of differentiation into HP cells (FIG. 9A) or iNK cells (FIGS. 9B and 9C) according to Example 9 of the present disclosure. [Figure 10] FIG. 10 shows the effect of the concentration of CHIR99021 used in stages 1 to 3 of the method on the efficiency of differentiation into HP cells ( FIG. 10A ) or iNK cells ( FIG. 10B and FIG. 10C ) according to Example 10 of the present disclosure, where iPSC line 1 represents the case where the hPSCs for differentiation are iPSC line 1, and iPSC line 2 represents the case where the hPSCs for differentiation are iPSC line 2. [Figure 11] Figure 11 shows the effect of different VEGF concentrations in stage 1 (stage 1-2, stage 1-3, stage 1-4, and stage 1-5) of the method on the differentiation efficiency into HE cells (KDR+%, Figure 11A), HP cells (CD34+%, Figure 11B), or iNK cells (outputs shown by cell morphology, Figure 11C) according to Example 11 of the present disclosure, where 1 to 10 represent #1 to #10, respectively. [Figure 12] FIG. 12 shows the effect of adjusting the VEGF concentration in stage 1 (stages 1-2, 1-3, 1-4, and 1-5) of the method on the differentiation efficiency into HE cells (FIG. 12A), HP cells (FIG. 12B), or iNK cells (FIGS. 12C and 12D) according to Example 12 of the present disclosure, where 1 to 4 represent #1 to #4, respectively. [Figure 13]Figure 13 shows the effect of IL-10 and / or IL-18 added during stage 3 of the method on specific lysis of K562 tumor cells by iNK cells (Figure 13A), and the effect of IL-18 added during stage 3 of the method on iNK cell fold expansion, according to Examples 13-14 of the present disclosure. In Figure 13A, None represents when neither IL-10 nor IL-18 is added during stage 3, IL-10 represents when IL-10 is added during stage 3, IL-18 represents when IL-18 is added during stage 3, and IL-10 + IL-18 represents when both IL-10 and IL-18 are added during stage 3. In Figure 13B, None represents the case where only IL-2 is added during stage 3, S3-wk1 represents the case where IL-2 is added during stage 3 and IL-18 is added in the first week of stage 3, and S3-wk2 represents the case where IL-2 is added during stage 3 and IL-18 is added in the second week of stage 3. [Figure 14] FIG. 14 shows the morphology of hematopoietic cells formed at various stages (day -1, day 0, stages 1-4, stages 1-5, stage 2, and stage 3) of the method for producing iNK cells from hPSCs according to Example 15 of the present disclosure. [Figure 15] FIG. 15 shows flow cytometry diagrams of hematopoietic cells formed at various stages (stages 1-4, stages 1-5, stage 2, and stage 3) of the method for producing iNK cells from hPSCs according to Example 15 of the present disclosure. [Figure 16] FIG. 16 shows the long-term proliferation capacity of immature iNK cells according to Example 16 of the present disclosure. [Figure 17] FIG. 17 shows a comparison of the expression of surface receptors NKG2D, NKp30, NKG2A, KIRe1 and CCR6 on mature iNK cells and PBNK cells according to Example 17 of the present disclosure. [Figure 18] FIG. 18 shows global gene expression profiling of mature iNK, PBNK, and CBNK cells according to Example 18 of the present disclosure. [Figure 19] FIG. 19 shows the cytotoxicity of mature iNK cells against a broad range of tumor cell lines, according to Example 19 of the present disclosure. [Figure 20] FIG. 20 shows the levels of inflammatory cytokines secreted by mature iNK cells according to Example 20 of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0017] It will be appreciated that certain aspects, modes, embodiments, variations and features of the present disclosure are described below at varying levels of detail in order to provide a substantial understanding of the technology.

[0018] Throughout this specification, references to "first," "second," "third," "fourth," "fifth," "sixth," "seventh," "eighth," or "ninth" do not imply any order or sequence of the features, structures (e.g., media or compositions), or properties described in connection with the reference, but are used for distinguishing purposes only.

[0019] Throughout this specification, reference to a "first aspect," "second aspect," "third aspect," "fourth aspect," "fifth aspect," "sixth aspect," "seventh aspect," "eighth aspect," or "ninth aspect" means that the particular feature, structure, or characteristic described in connection with that aspect is included in at least one or more aspects of the disclosure. Also, particular feature(s), structure(s), characteristic(s) or embodiment(s) in one aspect can be combined in any suitable manner with those in one or more other aspects.

[0020] Throughout this specification, references to "one embodiment," "some embodiments," "preferred embodiment(s)," "particular embodiment(s)," or "some embodiment(s)" mean that the particular feature, structure, or characteristic described in connection with the embodiment(s) is included in at least one or more embodiments of the present disclosure. Also, particular feature(s), structure(s), or characteristic(s) in one embodiment may be combined in any suitable manner with feature(s) in one or more other embodiments.

[0021] It is to be understood that this disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary, and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0022] The present disclosure is based, at least in part, on the discovery of a basal medium supplemented with a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) a human platelet lysate, which can improve the efficiency of differentiation into immature NK cells by stage-specific (post-HE cell formation) use during directed differentiation of pluripotent stem cells (PSCs). The present disclosure is also based, at least in part, on the discovery of a differentiation culture medium supplemented with a Wnt signaling pathway inhibitor, which can improve the efficiency of differentiation into HE cells, HP cells, or immature NK cells during directed differentiation of pluripotent stem cells (PSCs) by using the medium alone or in combination with another differentiation culture medium supplemented with a Wnt signaling pathway activator.

[0023] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the following meanings unless otherwise specified. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the disclosure.

[0024] Unless otherwise specified, "a" or "an" means "one or more."

[0025] As used herein, "about" means plus or minus 10%, or plus or minus 5%, or plus or minus 4%, or plus or minus 3%, or plus or minus 2%, or plus or minus 1%, and the specified numerical value.

[0026] As used herein, the term "comprising" is intended to mean that compositions and methods include the recited elements but do not exclude other elements. When used in defining compositions or methods, "consisting essentially of" is intended to mean excluding other elements essential to the composition or method. "Consisting of" is intended to mean, for the claimed compositions and substantial method steps, excluding more than trace elements of other components. Embodiments defined by each of these transitional terms are within the scope of this disclosure. Thus, methods and compositions may comprise additional steps and components, or alternatively, may comprise insignificant steps and compositions, or alternatively, may contemplate only the recited method steps or compositions. Furthermore, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0027] As used herein, the term "pluripotent stem cells (PSCs)" refers to cells derived from the inner cell mass of a blastocyst. Pluripotent stem cells are multipotent and can generate derivatives of all three primary germ layers (ectoderm, endoderm, and mesoderm) during development. Pluripotent stem cells can be human (e.g., human PSCs, or hPSCs). Pluripotent stem cells can be induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). ESCs (e.g., hESCs) and iPSCs (e.g., hiPSCs) are known in the art and can be readily obtained using conventional methods, e.g., methods described in existing technology or commercially available products. Suitable methods for generating iPSCs from somatic or pluripotent stem cells are well known to those skilled in the art. For example, iPSCs can be reliably generated from somatic cells by conventional reprogramming techniques.

[0028] As used herein, the terms "pluripotency" or "multipotency" refer to cells that have the developmental potential to differentiate into cells of all three germ cell layers (ectoderm, mesoderm, and endoderm). Pluripotency can be determined, at least in part, by assessing the pluripotency characteristics of the cells. Characteristics of pluripotency include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers, including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50; (iv) the ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation composed of the three somatic lineages; and (vi) the formation of embryoid bodies composed of cells of the three somatic lineages.

[0029] As used herein, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a small, round shape, a high nuclear to cytoplasm ratio, prominent nucleoli, and / or typical intercellular spacing.

[0030] As used herein, the term "reprogramming" refers to a method of increasing the potency of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased potency can have greater developmental plasticity (i.e., the ability to differentiate into more cell types) than the same cell in its unreprogrammed state. In other words, a reprogrammed cell refers to a cell that is in a less differentiated state than the same cell in its unreprogrammed state. "Reprogramming" can refer to the dedifferentiation of somatic cells or pluripotent stem cells into pluripotent stem cells, also known as induced pluripotent stem cells (iPSCs).

[0031] As used herein, the term "differentiation" refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as blood cells or immune cells. In certain embodiments, differentiated or differentiation-induced cells are cells that are at a more specialized ("committed") position within a cell's lineage. For example, human pluripotent stem cells (hPSCs) can be differentiated into various differentiated cell types, such as neural or hematopoietic progenitor cells, lymphocytes, cardiomyocytes, immune cells (e.g., natural killer cells), and other cell types, by treatment with appropriate differentiation factors in cell culture medium. In certain embodiments, the term "committed" is applied to the differentiation process to refer to cells that have progressed along a differentiation pathway to a point where, under normal circumstances, they will differentiate or continue to differentiate into a specific cell type or subset of cell types and cannot, under normal circumstances, differentiate into a different cell type (other than the specific cell type or subset of cell types) or revert to a less differentiated cell type.

[0032] As used herein, the term "hematopoietic cells" refers to cells differentiated in vitro from PSCs and / or their progeny, and may include one or more of hemangioblasts, hemogenic endothelial cells (HECs), hematopoietic stem cells, hematopoietic progenitor cells (HPCs), erythroid / megakaryocyte progenitor cells, erythrocytes, megakaryocytes, platelets, and lymphoid cells.

[0033] As used herein, the term "lymphoid cells" includes one or more of lymphoid progenitor cells, lymphocytes (such as T lymphocytes), natural killer (NK) cells, myeloid progenitor cells, granulocytic progenitor cells, monocytes, macrophages, and dendritic cells.

[0034] As used herein, the term "embryoid body" (EB) refers to three-dimensional clusters that have been shown to give rise to multiple lineages within their three-dimensional space, mimicking embryonic development.

[0035] As used herein, the term "mesoderm" or "mesodermal cells" refers to one of the three germ layers, or cells thereof, that emerge early in embryonic development and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0036] As used herein, the terms "hemogenic endothelium" (HE), "hemogenic endothelial cells," or "hemogenic endothelial cells" refer to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called the endothelial-to-hematopoietic transition. Embryonic hematopoietic cell development progresses sequentially from lateral plate mesoderm through hemangioblasts to definitive hemogenic endothelium and hematopoietic progenitor cells.

[0037] As used herein, the term "hematopoietic progenitor" (HP) or "hematopoietic progenitor cell" (HPC) refers to cells present in the blood and bone marrow that can form mature blood cells such as red blood cells, platelets, and immune cells.

[0038] As used herein, the term "immune cell" refers to any cell that plays a role in the immune response of a subject.Immune cells are derived from the hematopoietic system and include lymphocytes such as B cells and T cells, natural killer cells, monocytes, macrophages, dendritic cells, myeloid cells such as eosinophils, neutrophils, mast cells, basophils, and granulocytes.

[0039] As used herein, the term "lymphocyte" refers to all immature, mature, undifferentiated, and differentiated white blood cell populations derived from lymphoid progenitors, including tissue-specific and specialized types, including, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells. In certain embodiments, lymphocytes include all B cell lineages, including pre-B cells, precursor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-1 cells, B-2 cells, and the anergic AN1 / T3 cell population.

[0040] As used herein, "natural killer cells" or NK cells refer to lymphoid cells defined by their marker expression and function / activity. For example, in humans, NK cells express CD56. For example, such NK cells can be CD56+CD3- cells. NK cells can express varying levels of CD56. NK cells include primary NK cells or induced NK (iNK) cells.

[0041] As used herein, "primary NK cells" refers to naturally occurring natural killer cells, which can be sourced, for example, from blood (e.g., cord blood or peripheral blood collected by apheresis), bone marrow, or frozen primary NK cells (e.g., commercially available). Examples of primary NK cells include PBNK (peripheral blood-derived NK) cells and CBNK (cord blood-derived NK) cells.

[0042] As used herein, the term "iNK cells" refers to natural killer cells that have been differentiated, proliferated, and matured from pluripotent stem cells (e.g., hPSCs). iNK cells can be, for example, iPSC-derived iNK cells or ESC-derived iNK cells. These iNK cells can be used interchangeably with mature iNK cells. Mature iNK cells express higher levels of specific markers, such as CD56, than immature iNK cells and have cytokine-releasing and cytotoxic properties similar to primary NK cells.

[0043] As used herein, the term "immature iNK cells" refers to natural killer cells differentiated from pluripotent cells (e.g., hPSCs) that have not yet expanded or matured. Immature iNK cells can be, for example, iPSC-derived immature iNK cells or ESC-derived immature iNK cells. Compared to mature iNK cells, immature iNK cells express lower levels of specific markers such as CD56, and also have lower cytokine release and cytotoxicity.

[0044] As used herein, the term "culture medium" refers to a culture medium capable of supporting the survival, growth, proliferation, maintenance and / or differentiation of cells in an in vitro environment. The culture medium may have a basal medium and one or more supplements.

[0045] As used herein, the term "maintenance culture medium" refers to a culture medium capable of supporting the survival, growth, proliferation or maintenance of cells in an in vitro environment.

[0046] As used herein, the term "differentiation culture medium" (singular) or "differentiation culture media" (plural) refers to a culture medium(s) capable of supporting the differentiation of cells in an in vitro environment.

[0047] As used herein, the term "basal medium" refers to the base components of a culture medium (e.g., a differentiation culture medium or a proliferation culture medium) relative to its supplements. Generally, the basal medium comprises approximately 95% to 99% by volume of the culture medium (e.g., a differentiation culture medium, a proliferation culture medium, etc.). The basal medium of a cell maintenance culture medium serves as a source of nutrients, hormones, and / or other factors useful for cell growth and / or maintenance. The basal medium of a cell differentiation culture medium serves as a source of nutrients, hormones, and / or other factors useful for cell differentiation.

[0048] As used herein, the term "supplement(s)" refers to additional component(s) to the basal medium of a culture medium (e.g., a differentiation culture medium, or an proliferation culture medium).

[0049] As used herein, the term "supplemented" refers to adding a supplement for a culture medium (e.g., a differentiation culture medium) to its basal medium. The supplement(s) can be added to the basal medium of the culture medium before or at the time of use of the culture medium.

[0050] As used herein, the term "in vitro" generally refers to activities performed outside a living organism.

[0051] As used herein, the term "in vivo" generally refers to activities that take place inside a living organism.

[0052] As used herein, the term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside of the body, preferably with minimal alteration of natural conditions. In particular embodiments, "ex vivo" procedures involve culturing live cells or tissues removed from an organism, usually under sterile conditions, in a laboratory setting, typically for a few hours up to about 24 hours, but in some circumstances up to 48 or 72 hours or longer. In certain embodiments, such tissues or cells can be harvested and frozen, and later thawed for ex vivo treatment. While tissue culture experiments or procedures using living cells or tissues lasting several days or more are typically considered "in vitro," in certain embodiments, the term can be used interchangeably with ex vivo.

[0053] As used herein, the term "feeder cells" or "feeders" refers to a type of cell that is co-cultured with cells of a second cell type to provide an environment in which the cells of the second cell type can grow, such that the feeder cells provide growth factors and nutrients for the support of the second cell type. Feeder cells may optionally be from a different species than the supporting cells. For example, certain human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. When co-cultured with other cells, feeder cells are typically inactivated by irradiation or treatment with antimitotic agents such as mitomycin to prevent them from destroying the supporting cells. Feeder cells can include, for example, endothelial cells, stromal cells (e.g., epithelial cells and fibroblasts), and leukemia cells. Without limitation, one specific feeder cell type is a human feeder cell, such as human dermal fibroblasts. Another feeder cell type would be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be used, in part, to maintain pluripotency, induce differentiation into specific lineages, and promote maturation into specialized cell types such as effector cells.

[0054] As used herein, a "feeder-free" (FF) environment refers to culture conditions, cell cultures, culture media, etc. that are essentially free of feeder cells or stromal cells and / or that have not been pre-conditioned by the culture of feeder cells.

[0055] As used herein, the term "cell population" or "population of cells" refers to a group of at least two cells that express similar or different phenotypes. In non-limiting examples, a cell population may include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1 x 10 cells, or at least about 1 x 10 cells that express similar or different phenotypes. 6 cells, at least approximately 1 x 10 7 cells, at least approximately 1 x 10 8 cells, at least approximately 1 x 10 9 cells, at least approximately 1 x 10 10 cells, at least approximately 1 x 10 11 cells, at least approximately 1 x 10 12 It may contain one or more cells.

[0056] As used herein, the term "effective amount" refers to an amount of a drug sufficient to produce a beneficial or desired result upon administration. The amount of a drug administered to a subject may depend on individual characteristics, such as general health, age, sex, weight, the effective concentration of administered cells (e.g., iNK cells), and tolerance to the drug. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors. An effective amount can be administered to a subject in one or more doses.

[0057] As used herein, the term "administration" of an agent to a subject includes any route by which an agent is introduced or delivered to a subject to perform its intended function. Administration can be by any suitable route, including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes described herein. Administration can be self-administration or administration by another.

[0058] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to an individual organism, vertebrate, or mammal, and may include humans, non-human primates, rodents, etc. (e.g., those that will be the recipient of a particular medical intervention or from which cells are harvested). In certain embodiments, the individual, patient, or subject is human.

[0059] As used herein, the terms "treatment," "treat," and "treating," as used herein, refer to a clinical intervention intended to reverse, alleviate, delay the onset or inhibit the progression of, ameliorate, reduce the severity of, prevent, or delay the recurrence of, a disease, disorder, and / or condition, or one or more symptoms thereof, and / or ameliorate one or more symptoms of a disease, disorder, and / or condition. Treatment, e.g., in the form of iNK cells or populations of iNK cells described herein, may be administered to a subject after the onset of one or more symptoms and / or after a disease has been diagnosed. Treatment may also be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or inhibit the onset or progression of a disease. For example, treatment may be administered to susceptible individuals before the onset of symptoms (taking into account genetic and other susceptibility factors). Treatment may also be continued after symptoms have subsided, e.g., to prevent or delay recurrence. Treatment results in the improvement and / or elimination of one or more symptoms of a disease, disorder, and / or condition.

[0060] As used herein, the terms "prophylaxis," "preventing," and "prevention" refer to reducing the chance of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition, but who is at risk of or susceptible to developing the disease, disorder, or condition.

[0061] First differentiation method A first aspect of the present disclosure relates to a method for promoting directed differentiation (e.g., hematopoietic differentiation) of pluripotent stem cells (PSCs) (e.g., into iNK cells), comprising the steps of: contacting PSCs with a maintenance culture medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation culture medium, or sequentially with a first differentiation culture medium and a second differentiation culture medium, to form mesodermal cells; contacting the mesodermal cells with a third differentiation culture medium to form hemogenic endothelial (HE) cells; and contacting the HE cells with a fourth differentiation culture medium to form hematopoietic progenitor (HE) cells. forming immature iNK (progenitor; HP) cells; and contacting the HP cells with a fifth differentiation culture medium to obtain immature iNK cells; wherein a basal medium supplemented with a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate is used as the basal medium of the fourth differentiation culture medium and / or the basal medium of the fifth differentiation culture medium.

[0062] According to the first aspect of the present disclosure, by using a basal medium supplemented with a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate (hereinafter referred to as SFM basal medium) in a stage-specific manner (after hemogenic endothelial (HE) cell formation), the above method can improve the differentiation efficiency into immature iNK (CD56+) cells, or HP cells and immature iNK (CD56+) cells. The above method can be achieved within approximately 20 to 40 days and can produce a large number of immature iNK cells. For example, 1 x 10 6 3 x 10 hPSCs 8 Immature iNK cells are obtained.

[0063] Figure 1 is a diagram illustrating the process of producing iNK cells from hPSCs according to an illustrative example of the disclosed method. As shown in Figure 1, hPSCs first differentiate into hematopoietic progenitor cells (Stage 1) and then differentiate into immature NK cells (Stage 2). Stage 1 is further divided into five substages: Stages 1-1, 1-2, 1-3, 1-4, and 1-5. Stage 1-1 is the hPSC maintenance stage. Stages 1-2 and 1-3 are substages for the differentiation of hPSCs into mesodermal cells, Stages 1-4 are substages for the differentiation of mesodermal cells into HE cells, and Stages 1-5 are substages for the differentiation of HE cells into HP cells. Stage 3 is the stage in which iNK cells proliferate and mature, as described below.

[0064] According to the present disclosure, SFM basal medium can be obtained by adding (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate to a basal medium commonly used in the art or any other suitable basal medium, such as IF-4 basal medium or CD34A basal medium (e.g., those used in the Examples). Examples of common basal media can include IMDM / F12, Ham's F12, IMDM, BME, DMEM, RPMI-1640, α-MEM (all commercially available), and any combination thereof.

[0065] In certain embodiments, SFM basal medium is used continuously throughout the step of contacting the HE cells with the fourth differentiation culture medium to form HP cells, and SFM basal medium is used discontinuously throughout the step of contacting the HP cells with the fifth differentiation culture medium to obtain immature iNK cells.

[0066] In certain embodiments, during the step of contacting HP cells with the fifth differentiation culture medium to obtain immature iNK cells, SFM basal medium and another different basal medium are used separately, in any order, as the basal medium of the fifth differentiation culture medium.

[0067] In a preferred embodiment, SFM basal medium is used continuously throughout the step of contacting HE cells with the fourth differentiation culture medium to form HP cells, and SFM basal medium is used continuously throughout the step of contacting HP cells with the fifth differentiation culture medium to obtain immature iNK cells. According to the above embodiment, the differentiation efficiency into HP cells and immature iNK (CD56+) cells can be further improved.

[0068] In certain embodiments, the first through third differentiation basal media comprise different basal media.

[0069] In certain embodiments, the first through third basal differentiation media comprise the same basal medium, which is useful for simplifying the differentiation protocol of the present disclosure.

[0070] When an SFM basal medium and another different basal medium are used separately in the step of contacting HP cells with the fifth differentiation culture medium to obtain immature iNK cells, the another different basal medium may be the same as, similar to, or different from the first to third differentiation culture media. In a preferred embodiment, the another different basal medium is a basal medium supplemented with only one or two of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate. In a more preferred embodiment, the another different basal medium is a basal medium supplemented with (i) a nicotinamide-based compound and (ii) a heparin-based compound and (iii) human platelet lysate. In a most preferred embodiment, the another different basal medium is a basal medium supplemented with (i) a nicotinamide-based compound and (ii) human platelet lysate but not with (iii) a heparin-based compound.

[0071] In a preferred embodiment, the SFM basal medium is used consecutively as one of the basal medium for the fourth differentiation culture medium and the basal culture medium for the fifth differentiation culture medium, and a basal medium supplemented with (i) a nicotinamide-based compound and (ii) a heparin-based compound, but not (iii) human platelet lysate, is used consecutively as the other of the basal medium for the fourth differentiation culture medium and the basal medium for the fifth differentiation culture medium.

[0072] Nicotinamide compounds, heparin compounds, and human platelet lysates are described in more detail below.

[0073] Nicotinamide compounds As used herein, "nicotinamide-based compounds" refers to nicotinamide, as well as analogs thereof, metabolic products of nicotinamide or nicotinamide analogs, such as NAD, NADH, NADPH, and products derived from these compounds.

[0074] According to embodiments of the present disclosure, the nicotinamide-based compound may be selected from the group consisting of nicotinamide, nicotinamide analogs, nicotinamide metabolites, nicotinamide analog metabolites, and derivatives thereof.

[0075] Nicotinamide is the amide form of niacin, and both belong to the vitamin B3 family. They are precursors of nicotinamide adenine dinucleotide (NAD), which functions as a coenzyme in multiple cellular processes, including energy metabolism and DNA repair. Nicotinamide can be converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT) and further to NAD+ by nicotinamide mononucleotide adenylyltransferase (NMNAT).

[0076] As used herein, "nicotinamide analogue" refers to a molecule known to function similarly to nicotinamide. Examples of nicotinamide analogues include, but are not limited to, nicotinic acid, nicotinic acid thiol analogues, and the like. Examples of nicotinamide derivatives include, but are not limited to, substituted nicotinamide compounds and nicotinic acid thiol analogues, and N-substituted nicotinamide compounds and nicotinic acid thiol analogues.

[0077] Heparin-based compounds As used herein, "heparin-based compound" refers to heparin, its derivatives, or its salts.

[0078] According to embodiments of the present disclosure, the heparin-based compound may be selected from the group consisting of heparin, a derivative thereof, or a salt thereof.

[0079] Heparin, a highly sulfated variant of heparin glycosaminoglycan primarily produced and stored by mast cells, possesses the highest net negative charge density of any known biomolecule. Its negative charge binds to positively charged heparin-binding domains present in many extracellular proteins, including fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), bone morphogenetic proteins (BMPs), and large extracellular structural molecules such as fibronectin and laminin.

[0080] Examples of derivatives of heparin include, but are not limited to, substituted heparins. Examples of salts of heparin or its derivatives include, but are not limited to, sodium heparin, lithium heparin, and salts of substituted heparins.

[0081] Human platelet lysate (PLT) PLTs for use in accordance with the present disclosure are commercially available. Human platelet lysate (PLT) can be derived from human platelets from healthy donors and is rich in growth factors.

[0082] According to the present disclosure, the concentration of the nicotinamide compound in a composition of the present disclosure (e.g., SFM basal medium or a basal medium supplemented with only one or two of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) a human platelet lysate) is not particularly limited. In some embodiments, the concentration of the nicotinamide compound in a composition of the present disclosure (e.g., SFM basal medium or a basal medium supplemented with only one or two of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) a human platelet lysate) is about 0.5 to about 20 mM, preferably about 1 to about 10 mM, and more preferably about 1 to about 5 mM.

[0083] In certain embodiments, compositions of the present disclosure (e.g., SFM basal medium or basal medium supplemented with only one or two of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate) may comprise a nicotinamide-based compound at a concentration of about 0.1 mM or more, 0.2 mM or more, 0.5 mM or more, 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, 10 mM or more).

[0084] According to the present disclosure, the concentration of the heparin-based compound in a composition of the present disclosure (e.g., SFM basal medium or a basal medium supplemented with only one or two of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) a human platelet lysate) is not particularly limited. In some embodiments, the concentration of the heparin-based compound in a composition of the present disclosure (e.g., SFM basal medium or a basal medium supplemented with only one or two of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) a human platelet lysate) is preferably about 0.1 to about 100 μg / mL, more preferably about 0.5 to about 50 μg / mL.

[0085] In certain embodiments, compositions of the present disclosure (e.g., SFM basal medium or basal medium supplemented with only one or two of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate) may comprise a heparin-based compound at a concentration of about 0.1 μg / ml or more, 0.2 μg / ml or more, 0.3 μg / ml or more, 0.4 μg / ml or more, 0.5 μg / ml or more, 1 μg / ml or more, 2 μg / ml or more, 3 μg / ml or more, 4 μg / ml or more, 5 μg / ml or more, 6 μg / ml or more, 7 μg / ml or more, 8 μg / ml or more, 9 μg / ml or more, 10 μg / ml or more, 20 μg / ml or more, 30 μg / ml or more, 40 μg / ml or more, 50 μg / ml or more, or 100 μg / ml or more.

[0086] According to the present disclosure, the concentration of human platelet lysate in a composition of the present disclosure (e.g., SFM basal medium or a basal medium supplemented with only one or two of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) a human platelet lysate) is not particularly limited. In some embodiments, the concentration of human platelet lysate in a composition of the present disclosure (e.g., SFM basal medium or a basal medium supplemented with only one or two of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) a human platelet lysate) is about 0.1% to about 20% by volume, preferably about 0.1% to about 10% by volume, and more preferably about 0.1% to about 5% by volume.

[0087] In certain embodiments, compositions of the present disclosure (e.g., SFM basal medium or basal medium supplemented with only one or two of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate) may contain PLTs at about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10% or more (v / v) of PLTs.

[0088] In addition, when a nicotinamide compound, a heparin compound, or a PLT is present in a basal medium other than SFM basal medium (e.g., a basal medium supplemented with only one or two of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) a human platelet lysate), the examples and concentrations of the nicotinamide compound, the heparin compound, and the PLT in the medium are the same as those in SFM basal medium.

[0089] Optionally, the SFM basal medium may contain glutamine or a derivative thereof. Examples of glutamine or a derivative thereof may include glutamine, GlutaMAX-1, L-glutamine, and L-alanyl-L-glutamine.

[0090] In certain embodiments, glutamine or a derivative thereof may be present in SFM basal medium at a concentration of about 0.1 to about 5% (v / v), or at a concentration of about 0.5% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v).

[0091] Optionally, the SFM basal medium may contain one or more antioxidants, such as ascorbic acid or its salts or derivatives, such as magnesium ascorbate, sodium ascorbate, ascorbyl glucoside, 3-ethyl ascorbic acid, ascorbyl tetraisopalmitate, ascorbyl phosphate, and ascorbyl palmitate.

[0092] In certain embodiments, ascorbic acid or a salt or derivative thereof may be present in SFM basal medium at a concentration of about 10 to about 200 μg / mL, preferably about 50 to about 150 μg / mL, or about 20 μg / mL, about 40 μg / mL, about 60 μg / mL, about 80 μg / mL, or about 100 μg / mL.

[0093] Optionally, the SFM basal medium may contain human serum albumin (HSA). In certain embodiments, HSA may be present in the SFM basal medium at a concentration of about 0.1 to about 20 mg / mL. In certain embodiments, HSA may be present at a concentration of about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, or about 20 mg / mL.

[0094] Optionally, the SFM basal medium may contain monothioglycerol (MTG). In certain embodiments, MTG may be present in the SFM basal medium at a concentration of about 1 to about 400 μM. In certain embodiments, MTG may be present at a concentration of about 10 μM, about 30 μM, about 50 μM, about 70 μM, about 90 μM, about 120 μM, about 150 μM, or about 200 μM.

[0095] Optionally, the SFM basal medium may contain transferrin. In certain embodiments, transferrin may be present in the SFM basal medium at a concentration of about 1 to about 200 μg / mL, preferably about 50 to about 150 μg / mL. In certain embodiments, transferrin may be present at a concentration of about 20 μg / mL, about 40 μg / mL, about 60 μg / mL, about 80 μg / mL, or about 100 μg / mL.

[0096] Optionally, the SFM basal medium can contain a selenite, such as sodium selenite. In certain embodiments, the selenite can be present in the SFM basal medium at a concentration of about 1 to about 50 ng / mL, preferably about 5 to about 40 ng / mL. In certain embodiments, the selenite can be present at a concentration of about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, or about 30 ng / mL.

[0097] Optionally, the SFM basal medium may contain ethanolamine. In certain embodiments, ethanolamine may be present in the SFM basal medium at a concentration of about 1 to about 100 μM, preferably about 5 to about 50 μM. In certain embodiments, ethanolamine may be present at a concentration of about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 60 μM, about 80 μM, or about 100 μM.

[0098] Optionally, the SFM basal medium can include a pyruvate, such as sodium pyruvate. In certain embodiments, pyruvate can be present in the SFM basal medium at a concentration of about 10 to about 500 μg / mL, preferably about 50 to about 200 μg / mL. In certain embodiments, pyruvate can be present at a concentration of about 20 μg / mL, about 40 μg / mL, about 60 μg / mL, about 80 μg / mL, or about 100 μg / mL.

[0099] Optionally, the SFM basal medium may contain insulin. In certain embodiments, insulin may be present in the SFM basal medium at a concentration of about 0.1 to about 20 μg / mL. In certain embodiments, insulin may be present at a concentration of about 1 μg / mL, about 3 μg / mL, about 5 μg / mL, about 8 μg / mL, about 12 μg / mL, or about 15 μg / mL.

[0100] In addition to the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) a human platelet lysate, the basal medium and the other different basal medium of the first to third differentiation culture media may contain the same optional components as those in the SFM basal medium. In certain embodiments, the concentrations of these components in the basal medium and the other different basal medium of the first to third differentiation culture media may be the same as those in the SFM basal medium.

[0101] In certain embodiments, the basal medium and / or another different basal medium of the first to third differentiation culture media may be an IF-4 or CD34A basal medium, preferably an IF-4 basal medium.

[0102] In certain embodiments, the nicotinamide-based compound comprises nicotinamide, and the heparin-based compound comprises sodium heparin. In a preferred embodiment, the SFM basal medium comprises IF-4 basal medium in addition to a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate. In a more preferred embodiment, the SFM basal medium comprises IF-4 basal medium in addition to a combination of (i) nicotinamide, (ii) sodium heparin, and (iii) human platelet lysate. In a most preferred embodiment, the SFM basal medium comprises NKSFM basal medium.

[0103] In certain embodiments, the other different basal medium may be NKM or NKSFM-EP basal medium, preferably NKSFM-EP basal medium.

[0104] The SFM basal medium, the basal medium of the first to third differentiation culture media, and other different basal media may also contain other components, such as mercaptoethanol, non-essential amino acids (NEAA), bovine serum albumin (BSA), sulfate, nitrate, trace elements, CD lipid concentrate, and human serum. The concentrations of these components can be easily determined. In certain embodiments, mercaptoethanol may be included in these basal media at a concentration of 1 to 100 μM. In certain embodiments, NEAA may be included in these basal media at a concentration of 0.1 to 5% (v / v). In certain embodiments, BSA may be included in these basal media at a concentration of 0.1 to 20 mg / mL. In certain embodiments, sulfate may be included in these basal media at a concentration of 0.1 to 10 ng / mL. In certain embodiments, nitrate may be included in these basal media at a concentration of 0.1 to 10 μg / mL. In certain embodiments, trace elements may be included in these basal media at a concentration of 0-1% (v / v). In certain embodiments, CD lipid concentrate may be included in these basal media at a concentration of 0-1% (v / v). In certain embodiments, human serum may be included in these basal media at a concentration of 1-20% (v / v).

[0105] In certain embodiments, the cell culture surface can be coated with a coating matrix. In certain embodiments, the cell culture surface may not be coated with any coating matrix. In a preferred embodiment, the method further comprises seeding HP cells onto a cell culture surface coated with a Notch pathway activator and an adhesion molecule. This embodiment can further improve the efficiency of differentiation into immature iNK (CD56+) cells compared to an embodiment in which the cell culture surface is not coated with any coating matrix.

[0106] In a more preferred embodiment, the Notch pathway activator is selected from DLL4, DLL1, Jagged-1, Jagged-2, variants thereof, and any combination thereof, and the adhesion molecule is selected from VCAM1, fibronectin, laminin, vitronectin, MAdCAM-1, ICAM, variants thereof, and any combination thereof. In a most preferred embodiment, the cell culture surface is coated with DLL4 and VCAM1. Techniques for coating cell culture surfaces are conventionally known in the art and can be easily determined by one skilled in the art.

[0107] In a specific embodiment, a Wnt signaling pathway inhibitor is further added to the third differentiation culture medium. According to this embodiment, the differentiation efficiency into HP cells and immature NK cells can be further improved.

[0108] Wnt signaling pathway inhibitors The third differentiation culture medium of the present disclosure can be supplemented with a Wnt signaling pathway inhibitor. The Wnt signaling pathway inhibitor refers to an antagonist of the Wnt signaling pathway (e.g., an agent that can downregulate the activity and / or amount of components involved in the Wnt signaling pathway).

[0109] Examples of Wnt signaling pathway inhibitors include agents that antagonize one or more human FZD proteins, FZD-binding agents, which are antibodies or polypeptides.

[0110] Examples of Wnt signaling pathway inhibitors include, but are not limited to, one or more of the following: a polypeptide comprising the amino acid sequence of a Wnt antagonist, a small organic molecule that inhibits Wnt / β-catenin signaling, a small organic molecule that inhibits the expression or activity of a Wnt antagonist, an antibody that binds to and inhibits the activity of a Wnt antagonist, preferably a small organic molecule that inhibits Wnt / β-catenin signaling, and a small organic molecule that inhibits the expression or activity of a Wnt antagonist.

[0111] Non-limiting examples of Wnt signaling pathway inhibitors include, more preferably, one or more of the following: iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, ciclopirox, cardamonin, diethyl benzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, XAV-939, fosenvivint (ICG-001), capmatinib, isoquercitrin, gigantol, JW55, MSAB, IWR-1-endo, KY02111, FH535, WIKI4, CCT251545, prodigiosin, KYA1797K, NCB-0846, LF3, and iCRT14. , Adavivint, Triptonide, M435-1279, and XAV939, more preferably CRT3, IWP-O1, IWP-2, IWP-3, IWP-4, ciclopirox, cardamonin, diethyl benzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, isoquercitrin, gigantol, JW55, MSAB, IWR-1-endo, FH535, WIKI4, CCT251545, KYA1797K, NCB-0846, iCRT14, Adavivint, M435-1279, and XAV939, most preferably XAV939.

[0112] According to the present disclosure, the concentration of the Wnt signaling pathway inhibitor in the third differentiation culture medium is not particularly limited. In a preferred embodiment, the concentration of the Wnt signaling pathway inhibitor in the third differentiation culture medium is 1 to 30 μM.

[0113] In certain embodiments, the third differentiation culture medium of the present disclosure contains a Wnt signaling pathway inhibitor at a concentration of about 0.1 μM or more, about 0.5 μM or more, for example, about 1 to 30 μM, preferably about 1 to 20 μM, more preferably 1 to 10 μM, and most preferably 2 to 8 μM.

[0114] In certain embodiments, the third differentiation culture medium does not include a TGF-β signaling pathway inhibitor as a supplement.

[0115] When a Wnt signaling pathway inhibitor is further added to the third differentiation culture medium, EBs can be contacted only with the first differentiation culture medium further containing a Wnt signaling pathway activator. According to the above embodiment, the differentiation efficiency into HP cells and immature NK cells can be further improved by fine-tuning Wnt signaling during the hematopoietic differentiation process (first activating and then suppressing Wnt signaling in cells, or by gradually downregulating Wnt signaling).

[0116] When a Wnt signaling pathway inhibitor is further added to the third differentiation culture medium, the EBs can be sequentially contacted with a first differentiation culture medium further supplemented with a Wnt signaling pathway activator and a second differentiation culture medium further supplemented with a Wnt signaling pathway activator, and the Wnt signaling pathway activator in the second differentiation culture medium can be the same as or different from the Wnt signaling pathway activator in the first differentiation culture medium, and can be at the same concentration or a lower concentration.

[0117] In a preferred embodiment, the second differentiation culture medium has the same composition as the first differentiation culture medium, except that the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is lower than that in the first differentiation culture medium. This embodiment allows for a smoother transition from activation to inhibition of Wnt signaling, thereby further promoting differentiation into HP (CD34+) cells and iNK (CD56+) cells.

[0118] According to the present disclosure, the concentration of the Wnt signaling pathway activator in the first and second differentiation culture media is not particularly limited. In certain embodiments, the first or second differentiation culture medium of the present disclosure contains a Wnt signaling pathway activator at a concentration of 0.5 μM or more, 1 μM or more, 1.5 μM or more, 2 μM or more, 2.5 μM or more, 3 μM or more, 3.5 μM or more, 4 μM or more, 4.5 μM or more, 5 μM or more, 5.5 μM or more, 6 μM or more, 6.5 μM or more, 7 μM or more, 7.5 μM or more, 8 μM or more, 8.5 μM or more, 9 μM or more, 9.5 μM or more, or 10 μM or more. In certain embodiments, the first or second differentiation culture medium of the present disclosure contains a Wnt signaling pathway activator at a concentration of about 1 to 20 μM, preferably about 1 to 10 μM, and more preferably about 1 to 5 μM.

[0119] In certain embodiments, the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is at least 50% lower (e.g., 55% lower, 60% lower, 65% lower, 70% lower, 75% lower, 80% lower, 85% lower, 90% lower, 95% lower, 99% lower, or 100% lower) than the concentration of the Wnt signaling pathway activator in the first differentiation culture medium.

[0120] In a preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation culture medium is 4 to 8 μM. In a more preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation culture medium is 5 to 8 μM.

[0121] Wnt signaling pathway activators The technology of the present disclosure can utilize a Wnt signaling pathway activator as a supplement.

[0122] A Wnt signaling pathway activator refers to an agonist of the Wnt signaling pathway (eg, an agent that can upregulate the activity and / or amount of a component involved in the Wnt signaling pathway).

[0123] Non-limiting examples of Wnt signaling pathway activators include one or more of the following: a polypeptide comprising the amino acid sequence of a Wnt polypeptide, a polypeptide comprising the amino acid sequence of an activated Wnt receptor, a small organic molecule that promotes Wnt / β-catenin signaling, a small organic molecule that inhibits the expression or activity of a Wnt antagonist, an antibody that binds to and inhibits the activity of a Wnt antagonist, a polypeptide comprising the amino acid sequence of a β-catenin polypeptide, and a polypeptide comprising the amino acid sequence of a Lef-1 polypeptide, preferably a small organic molecule that promotes Wnt / β-catenin signaling and a small organic molecule that inhibits the expression or activity of a Wnt antagonist.

[0124] Wnt signaling pathway activators also include GSK3 inhibitors, including, but not limited to, polynucleotides, polypeptides, and small molecules.Exemplary GSK3 inhibitors include, but are not limited to, kenpaullone, 1-azakempaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, TDZD-8, BIO, BIO-Acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine Pyridocarbazole-cyclopenadienyl ruthenium complex, TDZD-8, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole, OTDZT. α-4-dibromoacetophenone, AR-AO144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-pyrazin-2-yl-pyrrole-2,5-dione, TWS119, pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, GF109203X, RO318220, TDZD-8, TIBPO, and OTDZT, preferably Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, SB216763, AR-A014418, BIO- Acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, 2-thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole, α-4-dibromoacetophenone, AR-AO144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-ylpyrrole-2,5-dione, 2-chloro-1-(4,5-dibromothiophen-2-yl)-ethanone, and GF109203X.

[0125] In a preferred embodiment, the Wnt signaling pathway activator is Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, TDZD-8, BIO, BIO-Acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopenadienyl ruthenium complex, TDZD-8, 4-benzyl-2-methyl-1,2,4-thiadiazolium The Wnt signaling pathway activator is selected from the group consisting of lysine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole, OTDZT, α-4-dibromoacetophenone, AR-AO144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-pyrazin-2-yl-pyrrole-2,5-dione, TWS119, pyrrolopyrimidine compound, L803H-KEAPPAPPQSpP-NH2 or its myristoylated form, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, GF109203X, RO318220, TDZD-8, TIBPO, and OTDZT. In a most preferred embodiment, the Wnt signaling pathway activator is CHIR99021.

[0126] Additional Reagents In addition to the basal medium, the compositions of the present disclosure (e.g., the first to fifth differentiation culture media) may have or contain one or more additional reagents as supplements. Depending on the requirements of the present disclosure, one or more additional reagents may be added independently to the basal medium of the first to fifth differentiation culture media before or during use of the culture medium. Examples of additional reagents include one or more cytokines (e.g., cytokines that stimulate hematopoietic differentiation). The concentration of one or more cytokines in the medium is not particularly limited, as long as it stimulates differentiation of pluripotent stem cells into hematopoietic cells, including HE cells, HP cells, or iNK cells.

[0127] cytokines Cytokines are a group of cell signaling molecules that include, for example, growth factors, interleukins, colony-stimulating factors, chemokines, interferons, lymphokines, tumor necrosis factors, etc. Examples of interleukins include, but are not limited to, IL-1, IL-2, IL-3, IL-6, IL-7, IL-9, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, and IL-27. Examples of colony-stimulating factors include, but are not limited to, stem cell factor (SCF), Flt-3 ligand (Flt3L), thrombopoietin (TPO), G-CSF, GM-CSF, etc. For example, examples of tumor necrosis factors include, but are not limited to, tumor necrosis factor alpha (TNFα) and tumor necrosis factor beta (TNF-β). For example, examples of interferons include, but are not limited to, IFN-γ, IFN-κ, IFN-1, IFN-2, IFN-3, and IFN-4.

[0128] growth factors Growth factors are molecules that can stimulate various cellular processes, including cell proliferation, cell migration, differentiation, and multicellular morphogenesis, e.g., in development and tissue healing. Examples of growth factors include, but are not limited to, bone morphogenetic factors (BMPs), epidermal growth factors (EGFs), endothelial cell growth factors (ECGFs), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), nerve growth factors (NGFs), platelet-derived growth factors (PDGFs), and vascular endothelial growth factors (VEGFs).

[0129] In a preferred embodiment, the first to third differentiation culture media of the present disclosure are supplemented with one or more growth factors. In a specific embodiment, the first and second differentiation culture media are each supplemented with BMP4, VEGF, and bFGF. In a specific embodiment, the third and fourth differentiation culture media are each supplemented with VEGF and bFGF.

[0130] The concentration of the growth factor in the medium is not particularly limited, but may be, for example, 1 to 200 ng / ml.

[0131] In a preferred embodiment, the first and second differentiation culture media of the present disclosure are supplemented with one or more BMP signaling pathway activators. Preferred examples of BMP signaling pathway activators include BMP2, BMP4, SB4, ventromorphin (SJ000291942, SJ000063181, SJ000370178), isoliquiritigenin, diosmetin, apigenin, biochanin, etc. The most preferred embodiment of the BMP signaling pathway activator used in the present disclosure is BMP4. The concentration of BMP4 in the culture medium is not particularly limited as long as it activates the BMP signaling pathway, and examples include, but are not limited to, 1 to 200 ng / ml, for example, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 110 ng / ml, 120 ng / ml, 130 ng / ml, 140 ng / ml, 150 ng / ml, 160 ng / ml, 180 ng / ml, and 200 ng / ml.

[0132] In a preferred embodiment, VEGF is added to each of the first to fourth differentiation culture media of the present disclosure. In a preferred embodiment, the first to fourth differentiation culture media are each independently further supplemented with VEGF at a concentration of 15 to 100 ng / mL. In a specific embodiment, since a higher concentration of VEGF in the first to third differentiation culture media promotes differentiation into HP cells and immature iNK cells, the first to third differentiation culture media are each independently further supplemented with VEGF at a concentration of 25 to 100 ng / mL, preferably 50 to 100 ng / mL.

[0133] In certain embodiments, the first to fourth differentiation culture media of the present disclosure are each supplemented with VEGF at a concentration of about 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 ng / mL, or in certain embodiments, the first to fourth differentiation culture media of the present disclosure are each supplemented with VEGF at a concentration of about 20 to 50 ng / mL.

[0134] In a preferred embodiment, the first to fourth differentiation culture media of the present disclosure are each supplemented with bFGF. In a preferred embodiment, the first to fourth differentiation culture media are each independently further supplemented with bFGF at a concentration of 0.1 to 20 ng / mL.

[0135] In certain embodiments, each of the first to fourth differentiation culture media of the present disclosure is supplemented with bFGF at a concentration of about 0.5 ng / mL. In certain embodiments, each of the first to fourth differentiation culture media of the present disclosure is supplemented with bFGF at a concentration of about 5 ng / mL. In certain embodiments, each of the first to fourth differentiation culture media of the present disclosure is supplemented with bFGF at a concentration of about 10 ng / mL. In certain embodiments, each of the first to fourth differentiation culture media of the present disclosure is supplemented with bFGF at a concentration of about 0.1 to 15 ng / mL. In certain embodiments, each of the first to fourth differentiation culture media of the present disclosure is supplemented with bFGF at a concentration of about 1 to 10 ng / mL. In certain embodiments, each of the first to fourth differentiation culture media of the present disclosure is supplemented with bFGF at a concentration of about 5 to 10 ng / mL.

[0136] Colony-stimulating factors Colony-stimulating factors (CSFs) are cytokines that can stimulate the differentiation and proliferation of hematopoietic stem or progenitor cells. Examples of CSFs include, but are not limited to, stem cell factor (SCF), Flt-3 ligand (Flt3L), thrombopoietin (TPO), G-CSF, GM-CSF, and multi-CSF.

[0137] In a preferred embodiment, the fourth and fifth differentiation culture media of the present disclosure each contain one or more colony-stimulating factors. The concentration of the colony-stimulating factor in the medium is not particularly limited as long as it stimulates the differentiation and proliferation of hematopoietic stem cells or progenitor cells, and can be, for example, 1 to 200 ng / ml. Examples of such factors include TPO at concentrations of 1 to 100 ng / ml, for example, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, and 100 ng / ml; TPO at concentrations of 1 to 200 ng / ml, for example, 1 ng / ml; SCF at concentrations of 10ng / ml, 20ng / ml, 30ng / ml, 40ng / ml, 50ng / ml, 60ng / ml, 80ng / ml, 100ng / ml, 150ng / ml, 200ng / ml; Flt-3L at concentrations of 1 to 200ng / ml, for example, 1ng / ml, 5ng / ml, 10ng / ml, 20ng / ml, 30ng / ml, 40ng / ml, 50ng / ml, 80ng / ml, 100ng / ml, 150ng / ml, 200ng / ml, and the like, but are not limited to these.

[0138] In certain embodiments, the fourth differentiation culture medium is supplemented with SCF, Flt3L, TPO, VEGF, and bFGF.

[0139] Interleukin Interleukins are cytokines that can stimulate the differentiation and proliferation of hematopoietic stem or progenitor cells. Examples of interleukins include, but are not limited to, IL-1, IL-2, IL-3, IL-6, IL-7, IL-9, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, and IL-27. In a preferred embodiment, the fifth differentiation culture medium of the present disclosure is supplemented with one or more interleukins. In a most preferred embodiment, the fifth differentiation culture medium is supplemented with IL-7, IL-3, IL-2, and IL-15. The concentration of interleukins in the medium is not particularly limited and may be, for example, 1 to 200 ng / ml.

[0140] In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-7 at a concentration of about 10 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-7 at a concentration of about 25 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-7 at a concentration of about 50 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-7 at a concentration of about 1 to 50 ng / mL.

[0141] In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-3 at a concentration of about 5 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-3 at a concentration of about 10 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-3 at a concentration of about 20 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-3 at a concentration of about 1 to 20 ng / mL.

[0142] In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-2 at a concentration of about 100 IU / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-2 at a concentration of about 400 IU / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-2 at a concentration of about 700 IU / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-2 at a concentration of about 10 to 700 IU / mL.

[0143] In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-15 at a concentration of about 10 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-15 at a concentration of about 20 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-15 at a concentration of about 50 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-15 at a concentration of about 1 to 50 ng / mL.

[0144] In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-10 at a concentration of about 10 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-10 at a concentration of about 20 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-10 at a concentration of about 50 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-10 at a concentration of about 1 to 50 ng / mL.

[0145] In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-18 at a concentration of about 20 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-18 at a concentration of about 50 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-18 at a concentration of about 100 ng / mL. In certain embodiments, the fifth differentiation culture medium of the present disclosure is supplemented with IL-18 at a concentration of about 20 to 100 ng / mL.

[0146] In certain embodiments, the fifth differentiation culture medium is supplemented with SCF, Flt3L, TPO, IL-7, IL-3, IL-2, and IL-15.

[0147] In certain embodiments, the first to fifth differentiation culture media are chemically defined serum-free and animal-derived component-free differentiation culture media. According to the above embodiment, such media can avoid the potential risk of animal-derived component contamination, reduce batch-to-batch variations, and be more suitable for clinical and therapeutic use.

[0148] In a preferred embodiment, the step of contacting the PSCs with a maintenance culture medium to form embryoid bodies (EBs) comprises forming EBs from the PSCs by suspension maintenance culture, hanging drop EB formation, or spin EB formation. In a more preferred embodiment, the EBs are formed by suspension maintenance culture.

[0149] In a preferred embodiment, the maintenance medium contains a ROCK inhibitor. The maintenance medium can be, for example, E8 or mTeSR, or other similar medium. In a more preferred embodiment, the ROCK inhibitor is selected from the group consisting of Y27632, blebbistatin, HA100, HA1152, HA-1077, and any combination thereof. The concentration of the ROCK inhibitor in the maintenance medium can be 1 to 20 μM, such as 10 μM.

[0150] In a preferred embodiment, the method of the present disclosure is carried out under 3D culture conditions. According to the above embodiment, the 3D differentiation system significantly saves culture space and volume, is simpler and easier, and significantly increases the number of cells obtained, making it useful for mass production of hematopoietic cells, such as HE cells, HP cells, and iNK cells, from hPSCs.

[0151] Second differentiation method A second aspect of the present disclosure relates to a method for promoting directed differentiation (e.g., hematopoietic differentiation) of pluripotent stem cells (PSCs) (e.g., into hematopoietic cells), comprising the steps of contacting PSCs with a maintenance culture medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation culture medium supplemented with a Wnt signaling pathway activator, or sequentially with a first differentiation culture medium supplemented with a Wnt signaling pathway activator and a second differentiation culture medium supplemented with a Wnt signaling pathway activator, to form mesodermal cells; and contacting the mesodermal cells with a third differentiation culture medium supplemented with a Wnt signaling pathway inhibitor to obtain hemogenic endothelial (HE) cells. According to the second aspect of the present disclosure, the efficiency of differentiation into HE cells can be improved by using a Wnt signaling pathway inhibitor in combination with a Wnt signaling pathway activator in a stage-specific manner during the directed differentiation of pluripotent stem cells (PSCs). The above method can be accomplished within approximately 3 days and can produce large numbers of HE cells.

[0152] In certain embodiments, the disclosed method further comprises contacting HE cells with a fourth differentiation culture medium to obtain hematopoietic progenitor (HP) cells. According to the above embodiment, the efficiency of differentiation into HP cells can be improved by using a Wnt signaling pathway inhibitor in combination with a Wnt signaling pathway activator in a stage-specific manner during the directed differentiation process of pluripotent stem cells (PSCs) into HP cells. The above method can be accomplished within approximately 6 to 12 days and can produce a large number of HP cells, for example, 1 x 10 6 hPSCs to 9 x 10 6 HP cells are obtained.

[0153] The step of contacting HE cells with the fourth differentiation culture medium to obtain hematopoietic progenitor (HP) cells can be carried out according to conventional methods in the art or any other suitable method, such as a corresponding method described elsewhere in this specification (e.g., the first differentiation method or the method described in the first aspect of this specification), and descriptions of the same will be omitted herein for the sake of brevity.

[0154] In certain embodiments, the method of the present disclosure further comprises contacting HP cells with a fifth differentiation culture medium to obtain immature iNK cells. According to the above embodiment, the efficiency of differentiation into immature iNK cells can be improved by using a Wnt signaling pathway inhibitor in a stage-specific manner in combination with a Wnt signaling pathway activator during the process of directed differentiation of pluripotent stem cells (PSCs) into iNK cells.

[0155] The step of contacting HP cells with the fifth differentiation culture medium to obtain immature iNK cells can be carried out according to conventional methods in the art or any other suitable method, such as a corresponding method described elsewhere in this specification (e.g., the first differentiation method or the method described in the first aspect of this specification), and descriptions of the same will be omitted herein for the sake of brevity.

[0156] The step of contacting PSCs with a maintenance culture medium to form embryoid bodies (EBs) can be carried out according to corresponding methods described elsewhere herein (e.g., the first differentiation method or the method described in the first aspect of the present specification), and for the sake of brevity, descriptions of the same will be omitted herein.

[0157] In a preferred embodiment, EBs are sequentially contacted with a first differentiation culture medium and a second differentiation culture medium to form mesoderm cells, where the Wnt signaling pathway activator in the second differentiation culture medium may be the same as or different from the Wnt signaling pathway activator in the first differentiation culture medium, and may be at an equal or lower concentration. According to the above embodiment, the differentiation efficiency into HE cells, HP cells, or immature NK cells can be further improved by fine-tuning Wnt signaling during the hematopoietic differentiation process (first activating and then suppressing Wnt signaling in cells, or stepwise downregulating Wnt signaling).

[0158] In a preferred embodiment, the second differentiation culture medium has the same composition as the first differentiation culture medium, except that the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is lower than that in the first differentiation culture medium. This embodiment allows for a smoother transition from activation to inhibition of Wnt signaling, thereby further promoting differentiation into HE cells, HP (CD34+) cells, or iNK (CD56+) cells.

[0159] Examples of Wnt signaling pathway activators and their concentrations in the first and second differentiation culture media are described elsewhere herein (e.g., as described in the first differentiation method or first embodiment herein), and these same descriptions are omitted herein for the sake of brevity.

[0160] In a preferred embodiment, the Wnt signaling pathway activator is selected from the group consisting of Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, SB216763, AR-A014418, BIO-Acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, 2-thio(3-iodobenzyl)-5-(1- pyridyl)[1,3,4]-oxadiazole, α-4-dibromoacetophenone, AR-AO144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, GF109203X, and combinations thereof. In a most preferred embodiment, the Wnt signaling pathway activator is CHIR99021.

[0161] In a preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation culture medium is 4 to 8 μM. In a more preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation culture medium is 5 to 8 μM.

[0162] Examples of Wnt signaling pathway inhibitors and their concentrations in the third differentiation culture medium are described elsewhere herein (e.g., in the first differentiation method or first embodiment herein).

[0163] In a preferred embodiment, the Wnt signaling pathway inhibitor is selected from the group consisting of iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, ciclopirox, cardamonin, diethylbenzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, isoquercitrin, Gigantol, JW55, MSAB, IWR-1-endo, FH535, WIKI4, CCT251545, KYA1797K, NCB-0846, iCRT14, Adavivint, M435-1279, XAV939, and any combination thereof. In a most preferred embodiment, the Wnt signaling pathway inhibitor is XAV939.

[0164] In a preferred embodiment, the concentration of the Wnt signaling pathway inhibitor in the third differentiation culture medium is 1 to 30 μM.

[0165] In a preferred embodiment, the third differentiation culture medium is further supplemented with a TGF-β signaling pathway inhibitor, which can further improve the efficiency of differentiation into HE cells or HP (CD34+) cells.

[0166] In certain embodiments, the third differentiation culture medium of the present disclosure is supplemented with a TGF-β signaling pathway inhibitor at a concentration of about 1 to 20 μM, 1 to 15 μM, or 1 to 10 μM, such as about 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, or 20 μM.

[0167] TGF-β signaling pathway inhibitors Transforming growth factor β (TGF-β) is part of a large superfamily of secreted dimeric multifunctional proteins, which also includes activins and bone morphogenetic proteins. TGF-β plays important roles in multiple cellular functions, including embryogenesis, tissue homeostasis in multicellular organisms, and tumor suppression.

[0168] A TGF-β signaling pathway inhibitor (also referred to as a "TGF-β inhibitor") refers to an antagonist of the TGF-β signaling pathway (e.g., an agent capable of downregulating the activity and / or amount of a component involved in the TGF-β signaling pathway), including, but not limited to, one or more antagonists of TGF-β1, TGF-β2, TGF-β3, TGF-β receptor type I (TβRI), TGF-β receptor type II (TβRII), and TGF-β receptor type III (TβRIII). Non-limiting examples of TβRIs include ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7. Non-limiting examples of TβRIIs include TGFβR2, BMPR2, ACVR2A, ACVR2B, AMHR2, etc. Examples of TβRIIIs include TGFBR3.

[0169] Examples of TGF-β signaling pathway inhibitors include, but are not limited to, RepSox (2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine), A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB525334, SB505124, SD208, GW6604, and GW788388. In a preferred embodiment, the TGF-β signaling pathway inhibitor comprises SB431542.

[0170] Examples of the basal media for the first to third differentiation culture media are described elsewhere in this specification (e.g., in the first differentiation method or first aspect of this specification), and for the sake of brevity, the same descriptions will be omitted here.

[0171] In certain embodiments, the first to third basal differentiation media contain the same basal medium. According to the above embodiment, the differentiation protocol of the present disclosure can be simplified.

[0172] In certain embodiments, the basal medium of the first to third differentiation culture media may be an IF-4 or CD34A basal medium, preferably an IF-4 basal medium.

[0173] In certain embodiments, the basal medium of the fourth differentiation culture medium is an IF-4 or CD34A basal medium.

[0174] When the method of the present disclosure further comprises contacting HE cells with a fourth differentiation culture medium to obtain HP cells, the basal medium of the fourth differentiation culture medium may be a basal medium supplemented with (i) a nicotinamide-based compound and (ii) a heparin-based compound, and preferably is a basal medium supplemented with a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate. According to the above embodiment, the efficiency of differentiation into HP (CD34+) cells can be further improved.

[0175] Examples and concentrations of nicotinamide-based compounds, heparin-based compounds, and human platelet lysate in the basal medium of the fourth differentiation culture medium are described elsewhere herein (e.g., in the first differentiation method or first aspect herein), and the same descriptions are omitted here for the sake of brevity.

[0176] In a specific embodiment, the concentration of the nicotinamide-based compound in the fourth differentiation culture medium is 0.5 to 20 mM.

[0177] In a specific embodiment, the concentration of the heparin-based compound in the fourth differentiation culture medium is 0.1 to 100 μg / mL.

[0178] In certain embodiments, the concentration of human platelet lysate in the fourth differentiation culture medium is 0.1% to 20% by volume.

[0179] In a preferred embodiment, the nicotinamide-based compound comprises nicotinamide and the heparin-based compound comprises sodium heparin.

[0180] In a preferred embodiment, the basal medium of the fourth differentiation culture medium comprises IF-4 basal medium in addition to a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate. In a more preferred embodiment, the basal medium of the fourth differentiation culture medium comprises IF-4 basal medium in addition to a combination of (i) nicotinamide, (ii) sodium heparin, and (iii) human platelet lysate. In a most preferred embodiment, the basal medium of the fourth differentiation culture medium comprises NKSFM basal medium.

[0181] When the method of the present disclosure further comprises a step of contacting HP cells with a fifth differentiation culture medium to obtain immature iNK cells, the basal medium of the fifth differentiation culture medium may be an IF-4 or CD34A basal medium, preferably an IF-4 basal medium.

[0182] Additional Reagents In addition to the basal medium, the compositions of the second embodiment of the present disclosure (e.g., the first to third differentiation culture media when HE cells are produced, the first to fourth differentiation culture media when HP cells are produced, and the first to fifth differentiation culture media when immature cells are produced) may have or contain one or more additional reagents as supplement(s). Depending on the requirements of the present disclosure, one or more additional reagents may be added independently to the basal medium of the first to fifth differentiation culture media before or during use of the culture medium. The additional reagent may include, for example, one or more cytokines (e.g., cytokines that stimulate hematopoietic differentiation). The concentration of the one or more cytokines in the medium is not particularly limited, as long as it stimulates differentiation of pluripotent stem cells into hematopoietic cells, including HE cells, HP cells, or iNK cells.

[0183] Examples and concentrations of corresponding additional reagents are described elsewhere herein (e.g., as described in the first differentiation method or first embodiment herein), and these same descriptions are omitted here for the sake of brevity.

[0184] In certain embodiments, the first to fourth differentiation culture media are each independently further supplemented with VEGF at a concentration of 15 to 100 ng / mL. In certain embodiments, the first to third differentiation culture media are each independently further supplemented with VEGF at a concentration of 25 to 100 ng / mL, preferably 50 to 100 ng / mL, because a higher concentration of VEGF in the first to third differentiation culture media promotes differentiation into HP cells and immature iNK cells.

[0185] In certain embodiments, the first to fifth differentiation culture media are chemically defined serum-free and animal-derived component-free differentiation culture media. According to the above embodiment, such media can avoid the potential risk of animal-derived component contamination, reduce batch-to-batch variations, and be more suitable for clinical and therapeutic use.

[0186] In a preferred embodiment, the method is carried out under 3D culture conditions. According to the above embodiment, the 3D differentiation system significantly saves culture space and volume, is simpler and easier, and significantly increases the number of cells obtained, making it beneficial for the large-scale production of hematopoietic cells such as hPSCs, HE cells, and other hematopoietic cells such as HP cells, NK cells, or T cells.

[0187] Culture medium A third aspect of the present disclosure relates to a culture medium for promoting the directed differentiation of pluripotent stem cells (PSCs) into hematopoietic cells, the culture medium comprising a basal medium to which a Wnt signaling pathway inhibitor has been added. According to the third aspect of the present disclosure, by using a Wnt signaling pathway inhibitor in the process of promoting the directed differentiation of pluripotent stem cells (PSCs) into hematopoietic cells, the culture medium can improve the differentiation efficiency into HE (KDR+) cells, HP (CD34+) cells, or immature iNK (CD56+) cells.

[0188] The basal medium used in the third aspect of the present disclosure may be a basal medium commonly used in the art or other basal medium, as long as it does not inhibit the promotion of directed differentiation of pluripotent stem cells (PSCs) into hematopoietic cells. The basal medium may be commercially available or may be prepared as required, for example, by adding one or more additives to a basal medium commonly used in the art. Examples of basal media commonly used in the art are described elsewhere herein (e.g., as described in the first differentiation method or first aspect of the present disclosure), and for the sake of brevity, the same description will be omitted herein. Examples of the basal medium may include, for example, IF-4 or CD34A basal medium.

[0189] Examples and concentrations of Wnt signaling pathway inhibitors are described elsewhere herein (e.g., as described in the first and second aspects of the present specification), and these same descriptions are omitted here for the sake of brevity.

[0190] In a preferred embodiment, the Wnt signaling pathway inhibitor is selected from the group consisting of iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, ciclopirox, cardamonin, diethylbenzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, isoquercitrin, Gigantol, JW55, MSAB, IWR-1-endo, FH535, WIKI4, CCT251545, KYA1797K, NCB-0846, iCRT14, Adavivint, M435-1279, XAV939, and any combination thereof. In a most preferred embodiment, the Wnt signaling pathway inhibitor comprises XAV939.

[0191] In a preferred embodiment, the concentration of the Wnt signaling pathway inhibitor in the culture medium is 1 to 30 μM.

[0192] In a preferred embodiment, the culture medium is further supplemented with a TGF-β signaling pathway inhibitor. According to the above embodiment, the efficiency of differentiation into HE cells or HP (CD34+) cells can be further improved.

[0193] Examples and concentrations of TGF-β signaling pathway inhibitors are described elsewhere herein (e.g., as described in the first and second aspects of the present specification), and these same descriptions are omitted here for the sake of brevity.

[0194] In a preferred embodiment, the culture medium is further supplemented with one or more growth factors, examples and concentrations of which are described elsewhere herein (e.g., as described in the first and second aspects of the present specification), and for the sake of brevity, these same descriptions are omitted herein.

[0195] In a preferred embodiment, the growth factor is selected from bFGF and / or VEGF. In a most preferred embodiment, the growth factor is selected from bFGF and VEGF.

[0196] In a preferred embodiment, the culture medium is further supplemented with 15 to 100 ng / mL, preferably 15 to 50 ng / mL, of VEGF.

[0197] In a preferred embodiment, the culture medium is a chemically defined serum-free and animal-derived component-free differentiation culture medium.

[0198] kit A fourth aspect of the present disclosure relates to a kit comprising the culture medium (eg, the culture medium of the third aspect described herein).

[0199] In a preferred embodiment, the kit further comprises a first differentiation culture medium supplemented with a Wnt signaling pathway activator.

[0200] The first differentiation culture medium and Wnt signaling pathway activators have been described elsewhere herein (e.g., as described in the first and second aspects of the present specification), and the same description will be omitted herein for the sake of brevity.

[0201] In a preferred embodiment, the kit further comprises a second differentiation culture medium supplemented with a Wnt signaling pathway activator, wherein the Wnt signaling pathway activator in the second differentiation culture medium may be the same as or different from the Wnt signaling pathway activator in the first differentiation culture medium, and is at an equal or lower concentration.

[0202] The second differentiation culture medium and Wnt signaling pathway activators have been described elsewhere herein (e.g., as described in the first and second aspects of the present specification), and the same description will be omitted here for the sake of brevity.

[0203] In a preferred embodiment, the second differentiation culture medium has the same composition as the first differentiation culture medium, except that the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is lower than the concentration of the Wnt signaling pathway activator in the first differentiation culture medium.

[0204] In a preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation culture medium is 4 to 8 μM. In a more preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation culture medium is 5 to 8 μM.

[0205] In a preferred embodiment, the Wnt signaling pathway activators in the first and second differentiation culture media are each independently selected from the group consisting of Kenpaullone, 1-azakempaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, SB216763, AR-A014418, BIO-Acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylindole-1,3-diol), 2-phenylindole-1,3-diol, ... 2-(4,5-dibromo-1-nazolin-4-yl)amine, 2-thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole, α-4-dibromoacetophenone, AR-AO144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, GF109203X, and combinations thereof.

[0206] The first and second differentiation culture media may each be supplemented with one or more growth factors.

[0207] Examples and concentrations of growth factors are described elsewhere herein (e.g., as described in the first and second aspects of the present specification), and these same descriptions are omitted here for the sake of brevity.

[0208] In a preferred embodiment, the first and second differentiation culture media are each supplemented with 15 to 100 ng / mL, preferably 15 to 50 ng / mL, of VEGF.

[0209] In a preferred embodiment, all culture media in the kit are chemically defined serum-free and animal-derived component-free differentiation culture media containing the same basal medium.

[0210] Methods for producing iNK cells The fifth aspect of the present disclosure relates to a method for producing iNK cells, comprising the above-described method for promoting directed differentiation of pluripotent stem cells (PSCs) according to the first aspect, and a step of expanding and maturing immature iNK cells (stage 3 in FIG. 1). The above-described method can be accomplished within approximately 27 to 69 days and can produce large numbers of iNK cells, for example, 1×10 6 3 x 10 hPSCs 10 More than 10 functional iNK cells can be obtained.

[0211] The above method according to the first aspect has been described elsewhere in this specification (e.g., described in the first differentiation method or first aspect of this specification), and for the sake of brevity, similar descriptions will be omitted here.

[0212] The step of expanding and maturing immature iNK cells can be carried out by conventional methods in the art or by any other suitable method.

[0213] In a preferred embodiment, the step of expanding and maturing immature iNK cells may include contacting the immature iNK cells with a proliferation and maturation culture medium containing a basal medium supplemented with a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate, in the presence or absence of feeder cells. According to the above embodiment, the NK cell proliferation fold, the proportion of mature iNK cells (CD56+CD3- cells), and the cytotoxicity against cancer cells can be improved compared to commercially available kits.

[0214] The basal medium of the proliferation / maturation culture medium containing a nicotinamide-based compound, a heparin-based compound, and human platelet lysate is described in the same manner as the SFM basal medium described elsewhere in this specification (e.g., in the first aspect of this specification), and therefore, for the sake of brevity, the same description will be omitted here.

[0215] In a preferred embodiment, the concentration of the nicotinamide-based compound in the growth and maturation culture medium is 0.5 to 20 mM.

[0216] In a preferred embodiment, the concentration of the heparin-based compound in the proliferation and maturation culture medium is 0.1 to 100 μg / mL.

[0217] In a preferred embodiment, the concentration of human platelet lysate in the proliferation and maturation culture medium is between 0.1% and 20% by volume.

[0218] In a preferred embodiment, the proliferation and maturation culture medium is further supplemented with one or more of IL-2, IL-10, IL-18, and SB431542. According to the above embodiment, the cytotoxicity of the proliferated and matured iNK cells against cancer cells can be improved. In a more preferred embodiment, the proliferation and maturation culture medium is further supplemented with IL-10 and / or IL-18 to further improve the proliferation rate of NK cells. In a most preferred embodiment, the proliferation and maturation culture medium contains IL-18.

[0219] The concentration of interleukins such as IL-2, IL-10, and IL-18 in the medium is not particularly limited.

[0220] In certain embodiments, the proliferation and maturation culture medium of the present disclosure is supplemented with IL-2 at a concentration of about 10-700 IU / mL, such as about 100 IU / mL.

[0221] In certain embodiments, the proliferation and maturation culture medium of the present disclosure is supplemented with IL-10 at a concentration of about 5-100 ng / mL, preferably 5-50 ng / mL, such as 20 ng / mL.

[0222] In certain embodiments, the proliferation and maturation culture medium of the present disclosure is supplemented with IL-18 at a concentration of about 5-100 ng / mL, preferably 5-60 ng / mL, such as 50 ng / mL.

[0223] In certain embodiments, the proliferation and maturation culture medium of the present disclosure is supplemented with SB431542 at a concentration of about 1-20 μM.

[0224] In a preferred embodiment, the nicotinamide-based compound comprises nicotinamide and the heparin-based compound comprises sodium heparin.

[0225] In a preferred embodiment, the basal medium of the proliferation and maturation medium comprises IF-4 basal medium in addition to a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate. In a more preferred embodiment, the basal medium of the proliferation and maturation medium comprises IF-4 basal medium in addition to a combination of (i) nicotinamide, (ii) sodium heparin, and (iii) human platelet lysate. In a most preferred embodiment, the basal medium of the proliferation and maturation medium comprises NKSFM basal medium.

[0226] In certain embodiments, the step of expanding and maturing the immature iNK cells further comprises co-culturing the immature iNK cells with feeder cells. In some such embodiments, multiple rounds (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds) of feeder stimulation are utilized to expand and / or mature the iNK cells.

[0227] Immature iNK cells can optionally be cryopreserved prior to expansion and maturation. Cryopreserved iNK cells can be thawed at a later date to continue the expansion and maturation process. Functional or mature iNK cells can optionally be cryopreserved at the end of the expansion and maturation steps of immature iNK cells.

[0228] In a preferred embodiment, the cryopreservation medium comprises a basal medium supplemented with human serum albumin (HSA, 20-40 mg / mL) and DMSO (5-10%, v / v). In a more preferred embodiment, the basal medium is a multiple electrolyte injection solution or a dextran injection solution.

[0229] In a preferred embodiment, the growth and maturation culture medium is a chemically defined serum-free and animal-derived component-free culture medium.

[0230] cell population The present disclosure provides hematopoietic cells or cell populations thereof, including HE cells, hematopoietic progenitor (HP) cells, immature natural killer (NK) cells, and mature NK cells, as well as compositions comprising them ("cell compositions").

[0231] The present disclosure provides cell populations, wherein at least 50% of the cells in the unenriched and unpurified population are KDR+HE cells. In a preferred embodiment, at least 60% of the cells in the unenriched and unpurified population are KDR+HE cells. In a more preferred embodiment, at least 70% of the cells in the unenriched and unpurified population are KDR+HE cells. In a most preferred embodiment, at least 80% of the cells in the unenriched and unpurified population are KDR+HE cells. Such cell populations with a high proportion of HE cells are relatively homogeneous, have good differentiation potential, and can be used for subsequent differentiation (e.g., HP differentiation) without purification or enrichment. Such cell populations can be produced by methods described elsewhere herein (e.g., the methods described in the first and / or second differentiation methods herein).

[0232] The present disclosure also provides a cell population in which at least 40% of the cells in the unenriched and unpurified population are CD34+CD43-HP cells. In a preferred embodiment, at least 50% of the cells in the unenriched and unpurified population are CD34+CD43-HP cells. In a more preferred embodiment, at least 60% of the cells in the unenriched and unpurified population are CD34+CD43-HP cells. In an even more preferred embodiment, at least 70% of the cells in the unenriched and unpurified population are CD34+CD43-HP cells. In a most preferred embodiment, at least 80% of the cells in the unenriched and unpurified population are CD34+CD43-HP cells. In a specific embodiment, at least 40% of the cells in the unenriched and unpurified population are CD34+CD43-CD73-HP cells. The above-described cell populations with a high proportion of HP cells are relatively homogeneous, have good differentiation potential, and can be used for subsequent differentiation (e.g., NK differentiation) without purification or enrichment. The above cell populations can be produced by methods elsewhere herein (eg, methods described in the first and / or second differentiation methods herein).

[0233] The present disclosure further provides cell populations, wherein at least 40% of the cells in the unenriched and unpurified population are immature CD56+CD3- iNK cells. In certain embodiments, at least 50% of the cells in the unenriched and unpurified population are immature CD56+CD3- iNK cells. In further embodiments, at least 60% of the cells in the unenriched and unpurified population are immature CD56+CD3- iNK cells. In preferred embodiments, at least 70% of the cells in the unenriched and unpurified population are immature CD56+CD3- iNK cells. In more preferred embodiments, at least 80% of the cells in the unenriched and unpurified population are immature CD56+CD3- iNK cells. The above cell populations enriched in iNK cells are homogenous populations, have high expansion potential, and can be used for subsequent expansion and maturation without further purification or enrichment. The above cell populations can be produced by methods described elsewhere herein (e.g., the methods described in the first and / or second differentiation methods herein).

[0234] In particular, a sixth aspect of the present disclosure provides a cell population produced by the first differentiation method described herein or the method according to the first aspect, or the second differentiation method described herein or the method according to the second aspect.

[0235] In particular, the sixth aspect of the present disclosure also provides a cell population produced by the method of producing iNK cells described herein or the method of the fifth aspect.

[0236] In particular, the seventh aspect of the present disclosure further provides a cell population, wherein greater than 90% of the cells in the unenriched and unpurified population are mature CD56+CD3- iNK cells. In a preferred embodiment, at least 95% of the cells in the unenriched and unpurified population are mature CD56+CD3- iNK cells. In a more preferred embodiment, at least 98% of the cells in the unenriched and unpurified population are mature CD56+CD3- iNK cells. In an even more preferred embodiment, at least 99% of the cells in the unenriched and unpurified population are mature CD56+CD3- iNK cells. In an even more preferred embodiment, at least 99.5% of the cells in the unenriched and unpurified population are mature CD56+CD3- iNK cells. In a most preferred embodiment, at least 99.8% (e.g., 99.9%, 100%) of the cells in the unenriched and unpurified population are mature CD56+CD3- iNK cells. The above cell populations, which have a high proportion of functional or mature iNK cells, are highly homogeneous (highly pure) and have improved functions (e.g., high cytotoxicity against tumor cells, improved homing to tissues and targets, and high levels of inflammatory cytokine secretion), making them more suitable for clinical and therapeutic applications without purification or enrichment. The above cell populations can be produced by methods described elsewhere herein (e.g., the methods for producing iNK cells described herein or the methods described in the fifth aspect).

[0237] The functional or mature iNK cells and cell populations of the present disclosure have improved surface receptor expression patterns and exhibit superior functionality compared to primary NK cells (e.g., higher expression of chemokine receptors such as CCR6 and lower expression of inhibitory receptors such as NKG2A and KIRe1 compared to primary NK cells) or iNK cells reported in the literature.

[0238] Functional iNK cells or mature iNK cells and cell populations thereof disclosed herein have lower expression of inhibitory receptors (e.g., NKG2A and KIRe1) compared to primary NK cells, indicating superior function (e.g., activation of target cells). In certain embodiments, functional iNK cells or mature iNK cells and cell populations thereof disclosed herein have half or less expression of inhibitory receptors (e.g., NKG2A and KIRe1) compared to primary NK cells, indicating significantly improved function (e.g., activation of target cells). In certain embodiments, functional iNK cells or mature iNK cells and cell populations thereof disclosed herein have three or less fold or less expression of inhibitory receptors (e.g., NKG2A and KIRe1) compared to primary NK cells.

[0239] As an example, less than 20% of the cells contained in iNK cells are NKG2A+ cells. In a preferred embodiment, less than 17% of the cells contained in iNK cells are NKG2A+ cells.

[0240] As an example, less than 20% of the cells contained in iNK cells are KIRe1+ cells. In a preferred embodiment, less than 12% of the cells contained in iNK cells are KIRe1+ cells.

[0241] The functional iNK cells or mature iNK cells and cell populations disclosed herein have higher expression of chemokine receptors compared to primary NK cells, and exhibit superior functions (such as homing to tissues and targets) than primary NK cells.

[0242] In certain embodiments, functional or mature iNK cells and cell populations thereof disclosed herein have at least 19-fold higher expression of chemokine receptors (e.g., CCR6) compared to primary NK cells, indicating significantly improved function (e.g., homing to tissues and targets). In certain embodiments, functional or mature iNK cells and cell populations thereof disclosed herein have at least 20-fold higher expression of chemokine receptors (e.g., CCR6) compared to primary NK cells.

[0243] As an example, at least 70% of the cells contained in the iNK cells are CCR6+ cells. In a preferred embodiment, at least 80% of the cells contained in the iNK cells are CCR6+ cells.

[0244] In preferred embodiments, functional or mature iNK cells and cell populations of the present disclosure have comparable expression of activating receptors such as NKG2D and NKp30 compared to primary NK cells.

[0245] As an example, at least 60% of the cells contained in iNK cells are NKG2D+ cells. In a preferred embodiment, at least 70% of the cells contained in iNK cells are NKG2D+ cells.

[0246] As an example, at least 80% of the cells contained in the iNK cells are NKp30+ cells. In a preferred embodiment, at least 90% of the cells contained in the iNK cells are NKp30+ cells.

[0247] In certain embodiments, greater than 90% of the cells in the iNK cells are CD45+ iNK cells. In more preferred embodiments, greater than 95% of the cells in the iNK cells are CD45+ iNK cells. In most preferred embodiments, greater than 99%, including 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and 100%, of the cells in the iNK cells are CD45+ iNK cells.

[0248] In certain embodiments, less than 20%, including 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0%, of the iNK cells are NKG2A+. In certain embodiments, less than 20%, including 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% of the iNK cells are KIRe1+. In certain embodiments, at least 70%, including 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the iNK cells are CCR6+.

[0249] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising any of the cell populations described herein in the cell populations section and a pharmaceutically acceptable carrier. The cells in the cell population can be in an effective amount suitable for use in treating a condition or disease in a subject.

[0250] In particular, an eighth aspect of the present disclosure provides a pharmaceutical composition comprising the cell population according to the seventh aspect of the present disclosure and a pharmaceutically acceptable carrier. The cells in the cell population can be in an effective amount suitable for use in treating a condition or disease in a subject.

[0251] In certain embodiments, the cell population may be prepared as a pharmaceutical composition (e.g., with a pharmaceutically acceptable carrier or excipient). The cell population may be emulsified or presented as a liposomal composition, provided that the emulsification procedure does not adversely affect the viability of the cells. The cells, or the cells and other active ingredients, may be mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient in an effective amount suitable for use in the therapeutic methods described herein.

[0252] Pharmaceutically acceptable carriers are well known in the art. Exemplary pharmaceutically acceptable carriers are sterile aqueous solutions containing no materials other than the active ingredient and water, or buffers such as sodium phosphate, saline, or both at physiological pH values, as in phosphate-buffered saline. Additionally, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to or in addition to water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of cell population used in a pharmaceutical composition effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0253] Methods of administration include, but are not limited to, injection and infusion. In certain embodiments, injections include, but are not limited to, intravenous, intrathecal, intraperitoneal, intraspinal, intracerebrospinal, and intrasternal injection. In certain embodiments, the route is intravenous. In certain embodiments, the cells described herein are administered as a bolus or by continuous infusion over a period of time (e.g., intravenous infusion). In certain embodiments, the cells described herein are administered in multiple doses over a period of time (e.g., multiple infusions over a period of time). The cells described herein can also be administered in a single dose or in multiple doses (or infusions) of 2, 3, 4, 5, 6, or more.

[0254] In certain embodiments, the pharmaceutical composition comprises a cell population allogeneic to the subject. In certain embodiments, the pharmaceutical composition comprises a cell population described herein that is autologous to the subject.

[0255] use The hematopoietic cells or cell populations of the present disclosure can be widely used for the treatment or prevention of various diseases or conditions, such as cancer, autoimmune diseases, and blood disorders. In particular, the cell population of the seventh aspect of the present disclosure has improved functions (e.g., high cytotoxicity against tumor cells, improved homing to tissues and targets, and high levels of secretion of inflammatory cytokines), and is therefore more applicable to clinical and therapeutic use in cancer. The cell population of the seventh aspect of the present disclosure can also be used to kill various microorganisms, such as viruses and bacteria, and senescent cells.

[0256] The present disclosure also provides the use of any of the cell populations described herein in the manufacture of a medicament for treating or preventing cancer, an autoimmune disease, or a hematological disorder.

[0257] In particular, a ninth aspect of the present disclosure provides the use of the cell population of the seventh aspect in the manufacture of a medicament for treating or preventing cancer.

[0258] A wide range of cancers may be treated or prevented by administering the cell populations or pharmaceutical compositions of the present disclosure to a subject in need thereof.

[0259] Examples of cancer include, but are not limited to, adrenal gland cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, carcinoma, cervical cancer, colon cancer, colorectal cancer, uterine cancer, ear, nose and throat (ENT) cancer, endometrial cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, Hodgkin's disease, intestinal cancer, kidney cancer, laryngeal cancer, leukemia, liver cancer, lymph node cancer, lymphoma, mesothelioma, myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, pancreatic cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, sarcoma, seminoma, skin cancer, stomach cancer, teratoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, vascular tumors, chronic myeloid leukemia, acute myeloid leukemia, myelomonocytic leukemia, melanoma, large cell lung cancer, ovarian cancer, non-small cell lung cancer, or small cell lung cancer, and metastases thereof. In a preferred embodiment, the cancer comprises chronic myeloid leukemia, acute myeloid leukemia, myelomonocytic leukemia, melanoma, large cell lung cancer, ovarian cancer, non-small cell lung cancer, or small cell lung cancer. In a more preferred embodiment, the cancer is acute myeloid leukemia, melanoma, small cell lung cancer, large cell lung cancer, ovarian cancer, or non-small cell lung cancer. The cell population or pharmaceutical composition of the present disclosure exhibits significantly superior efficacy in treating or preventing these cancers compared to primary NK cells.

[0260] In a further aspect, the present disclosure provides a method of treating a subject in need thereof by administering to the subject a cell population (e.g., according to the seventh aspect) or pharmaceutical composition (e.g., according to the eighth aspect) described herein. The pharmaceutical composition, cell composition or cell population of the present disclosure can be administered before, during, and / or after the onset of a disease, disorder, and / or condition (e.g., cancer).

[0261] In certain embodiments, the subject has a disease, disorder, or condition that can be cured with a cell-based therapy. In certain embodiments, the subject in need of a cell-based therapy is a subject having a disease, disorder, and / or condition, whereby a cell-based therapy is administered to the subject, e.g., a therapy in which a pharmaceutical composition or cell population described herein is administered to the subject, whereby the cell-based therapy treats at least one symptom associated with the disease, disorder, and / or condition.

[0262] Characterization Method Methods for characterizing hematopoietic cells, including HE cells, HP cells, and NK cells (e.g., iNK cells described herein), including characterization of cell phenotype and / or functionality, are known to those of skill in the art. Such methods include, but are not limited to, morphological analysis, flow cytometry, and / or gene expression profiling. One or more cellular markers can also be determined using one or more characterization methods to determine the composition, phenotype, and / or functionality of one or more cells and / or cell populations produced by the compositions and / or methods described herein. For example, in certain embodiments, cells of a particular population are characterized using gene expression profiling. In some such embodiments, a sample of a population of cells or cell composition is evaluated for transcriptional signatures characteristic of a particular cell type (e.g., primary NK cells).

[0263] In additional examples, cells of a particular population or cellular composition can be characterized by flow cytometry. In certain embodiments, a sample of a population of cells can be evaluated for the presence and / or proportion of one or more cell surface markers and / or one or more intracellular markers. As will be understood by one of skill in the art, cell surface markers can indicate different lineages. For example, pluripotent cells can also be identified by one or more of any number of markers associated with such cells, including, for example, CD34. Cells (e.g., cell compositions described herein) can be identified by markers indicating a degree of differentiation (e.g., partial differentiation). For example, markers for differentiated cells can include markers associated with hematopoietic progenitor cells, such as CD45 and CD34. In certain embodiments, markers for differentiated cells can be associated with NK cells, such as CD56, CD3, CD45, natural killer group 2 member A (NKG2A), killer immunoglobulin-like receptor (KIR) (e.g., KIRe1), CC motif chemokine receptor 6 (CCR6), NKG2D, and NKp30.

[0264] General Method In practicing the present disclosure, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA will be used. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. al.(1995) PCR 2:A Practical Approach;Harlow and Lane eds.(1999)Antibodies,A Laboratory Manual;Freshney(2005)Culture of Animal Cells:A Manual of Basic Technique,5th edition;Gait ed.(1984)Oligonucleotide Synthesis;USPatent No.4,683,195;Hames and Higgins eds.(1984)Nucleic Acid Hybridization;Anderson(1999)Nucleic Acid Hybridization;Hames and Higgins eds.(1984)Transcription and Translation;Immobilized Cells and Enzymes(IRL Press(1986));Perbal(1984)A Practical Guide to Molecular Cloning;Miller and Calos eds.(1987)Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory);Makrides ed.(2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology. [Example]

[0265] material and method All reagents related to culture media and devices utilized throughout the examples of this disclosure are commercially available. Each step of the method utilized throughout the examples will be described with reference to FIG.

[0266] Examples 1 to 4: Development of NKSFM This example demonstrates the development of an exemplary serum-free and animal-derived component-free basal SFM medium, NKSFM, which began with the use of nicotinamide (NAM), sodium heparin, and human platelet lysate for the expansion and maturation of PBNK cells as surrogate cells.

[0267] Example 1 The effect of nicotinamide (NAM) concentration in an exemplary basal medium on NK cell proliferation was evaluated. The basal medium contained the following components: IMDM (Sigma):RPMI 1640 (Gibco) (50%:50%), approximately 1.3 ng / mL copper sulfate, approximately 3 μM ferric sulfate, approximately 0.432 μg / mL zinc sulfate, approximately 1-50 ng / mL sodium selenite, approximately 110 μg / mL sodium pyruvate, 0.1-20 μg / mL insulin, 1-200 μg / mL transferrin, approximately 1% (v / v) GlutaMAX-1, 0.1-20 mg / mL HSA, 1-400 μM MTG, approximately 80 μg / mL ascorbic acid, and the indicated concentrations of NAM. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque. On day 0, PBMCs were plated at 1 × 10 in 48-well plates in basal medium supplemented with NAM (0, 1, 2, 2.5, 3, 3.5, 4, or 5 mM). 6 Plate at 100 cells / mL and the following reagents: immobilized CD16 antibody (Biolegend, 302014) (135 ng / cm 2 The cells were stimulated with IL-2 (700 IU / mL), IL-15 (10 ng / mL), and OK432 (0.01 KE / mL) for 3 days. On day 3, the cells were resuspended in the above basal medium supplemented with IL-2 (700 IU / mL) and IL-15 (10 ng / mL). The cell density was 2 × 10 6 Fresh medium was added every other day to maintain the cell density below 100 cells / mL. On day 14, cells were harvested and stained with CD56-PE and CD3-PerCP-Cy5.5 antibodies, and the percentage of CD56+CD3-NK cells was analyzed by flow cytometry. The results in Figure 2 suggest that the NAM concentration in the basal medium significantly affects the fold expansion of NK cells (Figure 2A), but only minimally affects the percentage of CD56+CD3-NK cells (Figure 2B).

[0268] Example 2 The effect of heparin sodium concentration in an exemplary NKSFM on NK cell proliferation was also evaluated. NKSFM contained the following components: IMDM (Sigma):DMEM / F12 (Gibco) (50%:50%); approximately 20 μM ethanolamine, approximately 1-50 ng / mL sodium selenite, approximately 110 μg / mL sodium pyruvate, 0.1-20 μg / mL insulin, 1-200 μg / mL transferrin, approximately 1% (v / v) GlutaMAX-1, 0.1-20 mg / mL human serum albumin, 1-400 μM MTG, approximately 80 μg / mL ascorbic acid, approximately 1-5 mM NAM, 4% (v / v) PLT, and the indicated concentrations of heparin sodium. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque. On day 0, PBMCs were plated at 1 × 10 in 48-well plates in NKSFM supplemented with heparin sodium (0, 0.5, 1, 3, 10, 30, or 50 μg / mL). 6 Plated at 135 ng / cm cells / mL and treated with immobilized CD16 antibody (Biolegend, 302014) (135 ng / cm 2 The cells were stimulated for 3 days with the following reagents: IL-12 (10 ng / mL), OK432 (0.01 KE / mL), NeoIL-2 (50 ng / mL), IL-12 (10 ng / mL), and IL-18 (50 ng / mL). On day 3, the cells were resuspended in NKSFM supplemented with NeoIL-2 (10 ng / mL). The cell density was 2 × 10 6 Fresh medium was added every other day to maintain the cell density below 100 cells / mL. On day 13, cells were harvested and stained with CD56-PE and CD3-PerCP-Cy5.5 antibodies. CD56 was detected by flow cytometry. + CD3 - The percentage of NK cells was analyzed. Specific lysis of K562 tumor cells by NK cells was assessed by a CFSE / 7-AAD cytotoxicity assay. Briefly, K562 tumor cells were labeled with CFSE and then co-cultured with NK cells at an E:T ratio of 3:1. After 4 hours of culture, the cells were stained with 7-AAD (live / dead staining) and analyzed by flow cytometry. The results in Figure 3 show that the heparin sodium concentration in NKSFM significantly affected the fold expansion of NK cells (Figure 3A), but not CD56. + CD3 -It is shown that there is minimal effect on the percentage of NK cells (Fig. 3B) and specific lysis of K562 tumor cells (Fig. 3C).

[0269] Example 3 The effect of the ratio (v / v) of PLTs in an exemplary NKSFM on NK cell proliferation was evaluated. NKSFM contained the following components: IMDM (Sigma):DMEM / F12 (Gibco) (50%:50%); approximately 20 μM ethanolamine, approximately 1-50 ng / mL sodium selenite, approximately 110 μg / mL sodium pyruvate, 0.1-20 μg / mL insulin, 1-200 μg / mL transferrin, approximately 1% (v / v) GlutaMAX-1, 0.1-20 mg / mL human serum albumin, 1-400 μM MTG, approximately 80 μg / mL ascorbic acid, approximately 0.5-50 μg / mL sodium heparin, approximately 1-5 mM NAM, and the indicated concentrations of PLTs. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque. On day 0, PBMCs were plated at 1 × 10 in 48-well plates in NKSFM supplemented with PLT (0, 0.5, 1, 2, 3, or 4%). 6 Plate at 100 cells / mL and the following reagents: immobilized CD16 antibody (135 ng / cm 2 The cells were stimulated with IL-12 (10 ng / mL), OK432 (0.01 KE / mL), IL-12 (10 ng / mL), IL-15 (50 ng / mL), and IL-18 (50 ng / mL) for 3 days. On day 3, the cells were resuspended in basal medium supplemented with IL-2 (700 IU / mL) and IL-15 (10 ng / mL). The cell density was 2 × 10 6Fresh medium was added every other day to maintain the PLT concentration below 100 cells / mL. On day 14, cells were harvested and stained with CD56-PE and CD3-PerCP-Cy5.5 antibodies, and the percentage of CD56+CD3-NK cells was analyzed by flow cytometry. Specific lysis of K562 tumor cells by NK cells was assessed using a CFSE / 7-AAD cytotoxicity assay. Briefly, K562 tumor cells were labeled with CFSE and then cocultured with NK cells at a 1:1 E:T ratio. After 4 hours of culture, the cells were stained with 7-AAD (live / dead staining) and analyzed by flow cytometry. As shown in Figure 4, the PLT concentration in NKSFM increased with increasing CD56 levels. + CD3 - It has minimal effect on the proportion of NK cells (Fig. 4B) and specific lysis of K562 tumor cells (Fig. 4C), but does have some effect on NK cell proliferation (Fig. 4A).

[0270] Example 4 Finally, the exemplary NKSFM was compared with commercially available feeder-free and feeder-dependent NK cell expansion systems. The NKSFM contained the following components: IMDM (Sigma):DMEM / F12 (Gibco) (50%:50%); approximately 20 μM ethanolamine, approximately 1-50 ng / mL sodium selenite, approximately 110 μg / mL sodium pyruvate, 0.1-20 μg / mL insulin, 1-200 μg / mL transferrin, approximately 1% (v / v) GlutaMAX-1, 0.1-20 mg / mL human serum albumin, 1-400 μM MTG, approximately 80 μg / mL ascorbic acid, approximately 1-5 mM NAM, 1% (v / v) PLT, and 0.5-50 μg / mL sodium heparin. Four conditions were tested.

[0271] Condition 1. NKSFM (no feeder) PBMCs were isolated by density gradient centrifugation using Ficoll-Paque. On day 0, PBMCs were plated at 1 × 10 in 6-well plates in NKSFM. 6 Plate at 100 cells / mL and the following reagents: immobilized CD16 antibody (Biolegend, 302014) (135 ng / cm 2The cells were stimulated with IL-12 (10 ng / mL), OK432 (0.01 KE / mL), NeoIL-2 (50 ng / mL), IL-12 (10 ng / mL), and IL-18 (50 ng / mL) for 3 days. On day 3, the cells were resuspended in NKSFM supplemented with NeoIL-2 (10 ng / mL). The cell density was 2 × 10 6 Fresh medium was added every other day to maintain a cell density below 100 cells / mL. On day 14, cells were harvested and stained with CD56-PE and CD3-PerCP-Cy5.5 antibodies, and the percentage of CD56+CD3-NK cells was analyzed by flow cytometry. Specific lysis of K562 tumor cells by NK cells was assessed using a CFSE / 7-AAD cytotoxicity assay. Briefly, K562 tumor cells were labeled with CFSE and then cocultured with NK cells at a 3:1 E:T ratio. After incubation for the indicated times, cells were stained with 7-AAD (live / dead staining) and analyzed by flow cytometry.

[0272] Condition 2: Commercial kit (without feeder) For condition 2, a feeder-free commercial kit (Baso, 3.0A) was used. Feeder-free expansion of PBNK cells was performed according to the manufacturer's protocol. During expansion, 5% (v / v) autoplasmic plasma was added.

[0273] Condition 3. NKSFM (with feeder) Feeder-dependent NKSFM was utilized as condition 3. Feeder-dependent expansion of PBNK cells was performed according to the manufacturer's protocol, except that the plasma-containing medium was replaced with NKSFM.

[0274] Condition 4: Commercially available kit (feeder included) Finally, a commercially available feeder-dependent kit (The Life ARK, ZY-NKZ-0104) was used as condition 4. Feeder-dependent expansion of PBNK cells was performed according to the manufacturer's protocol (The Life ARK, CN). During expansion, 10% (v / v) autoplasmic plasma was added.

[0275] The results in Figure 5 show that NKSFM significantly outperformed the commercial kit in the absence of feeder cells in terms of fold expansion of NK cells (Figure 5A), percentage of CD56+CD3 cells (Figure 5B), and lysis of K562 cells (Figure 5C). This indicates that NKSFM also improved NK cell proliferation in the presence of feeder cells compared to the commercial kit.

[0276] Taken together, the above experiments demonstrate that NKSFM is useful for the production of NK cells (e.g., for clinical use). The key components of NKSFM, NAM, sodium heparin, and PLT, were found to be crucial for NK cell proliferation and function. Most commercially available NK cell expansion kits require serum and / or plasma to support NK cell proliferation, or require the use of CD3+ in PBMCs. + To avoid unwanted proliferation of T cells, CD56 + CD3 - NK cells require selection. However, the use of NKSFM does not require serum or plasma, nor does it require sorting for preferential NK cell expansion. Furthermore, NKSFM can be used for both feeder-free and feeder-dependent NK cell expansion, and in both cases, it was significantly superior to commercially available kits in terms of both cell number and cytolytic activity. NKSFM can be used not only for primary NK cells but also for the expansion of iNK cells. Furthermore, we have found that the above-mentioned NKSFM can also be used for the directed differentiation process of human pluripotent stem cells (hPSCs).

[0277] Exemplary basal media and their components utilized throughout the examples of this disclosure are summarized in Table 1.1.

[0278] [Table 1-1] + means "yes", - means "no", and +- means "yes" or "no".

[0279] Composition of IF-4: IMDM (Sigma):DMEM / F12 (Gibco) (50%:50%); 0.1–20 mg / mL human serum albumin (HSA, Sinopharm, CN), 1–400 μM monothioglycerol (MTG), approximately 80 μg / mL ascorbic acid (Sigma), 1–200 μg / mL transferrin (Sigma), 1–50 ng / mL sodium selenite (Sigma), approximately 20 μM ethanolamine (Sigma), and 0.1–20 μg / mL insulin (Baiying, CN).

[0280] Composition of NKSFM: IMDM (Sigma):DMEM / F12 (Gibco) (50%:50%); approximately 1% (v / v) GlutaMAX-1 (Invitrogen); 0.1–20 mg / mL human serum albumin (HSA, Sinopharm, CN); 1–400 μM monothioglycerol (MTG); approximately 80 μg / mL ascorbic acid (Sigma); 1–200 μg / mL transferrin (Sigma); approximately 1–50 ng / mL sodium selenite (Sigma); approximately 20 µM ethanolamine (Sigma); approximately 110 µg / mL sodium pyruvate (Sigma); 0.1–20 µg / mL insulin (Baiying, CN); 1–5 mM nicotinamide (NAM, Sigma); 0.5–50 µg / mL sodium heparin (Thermo); and 0.5–4% (v / v) human platelet lysate (PLT, BI).

[0281] Composition of NKM: IMDM (Sigma) (100%); approximately 1% (v / v) NEAA (Invitrogen); 0.1–20 mg / mL human serum albumin (HSA, Sinopharm, CN); 1–5 mM nicotinamide (Sigma); approximately 50 µM mercaptoethanol (β-ME, Sigma); and 10% (v / v) human serum.

[0282] The composition of NKSFM-EP is: IMDM (Sigma) (100%); approximately 1% (v / v) GlutaMAX-1 (Invitrogen); 0.1–20 mg / mL human serum albumin (HSA, Sinopharm, CN); approximately 80 μg / mL ascorbic acid (Sigma); 1–200 μg / mL transferrin (Sigma); approximately 3 μM ferric sulfate (Sigma); approximately 0.65 μg / mL ferric nitrate (Sigma); approximately 0.1% (v / v) CD Lipid Concentrate (Invitrogen); 0.1–20 μg / mL insulin (Baiying, CN); 1–5 mM nicotinamide (Sigma); and 0.5–4% (v / v) human platelet lysate (PLT, BI).

[0283] Composition of CD34A: IMDM (Sigma) (100%); approximately 1% (v / v) GlutaMAX-1 (Invitrogen); approximately 1% (v / v) NEAA (Invitrogen); 0.1–20 mg / mL bovine serum albumin (BSA, Sigma); 1–400 μM monothioglycerol (MTG); approximately 80 μg / mL ascorbic acid (Sigma); 1–200 μg / mL transferrin (Sigma); 0.1% (v / v) trace element A (Mediatech), 0.1% (v / v) trace element B (Mediatech), 0.1% (v / v) trace element C (Mediatech), approximately 0.1% (v / v) CD Lipid Concentrate (Invitrogen).

[0284] Examples 5 to 7: Novel basal cell culture medium that promotes cell differentiation This example demonstrates that the use of an exemplary NKSFM at specific stages promotes efficient iNK cell differentiation from hPSCs.

[0285] Example 5 hiPSCs were prepared as described in CN108373998B. On day -1, individualized hiPSCs were cultured overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a belly dancer to form embryoid bodies (EBs) (stage 1-1). On day 0, lateral mesoderm differentiation (stage 1-2) was initiated by changing the medium to stage 1-2 differentiation medium containing the indicated basal medium (see Table 2.1) supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) for 2 days. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing the indicated basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (1 μM). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing the indicated basal medium supplemented with VEGF (50 ng / mL), bFGF (5 ng / mL), and XAV939 (5 ​​μM) for 1 day. On day 4 (end of stages 1-4), EBs were dissociated into single cells and stained with KDR-APC antibody to identify KDR. + The percentage of cells was detected by flow cytometry. HP differentiation (stages 1-5) was initiated by adding stage 1-5 differentiation medium containing the indicated basal medium supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL) for 3 days. On day 7 (the end of stages 1-5), EBs were dissociated into single cells and stained with CD34-APC antibody to identify CD34. +The percentage of HP cells was detected by flow cytometry. To initiate differentiation of HP cells into iNK cells (stage 2), EBs were collected and seeded onto 6-well plates coated with matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL, PeproTech), IL-3 (5 ng / mL, PeproTech), IL-2 (700 IU / mL), and IL-15 (10 ng / mL, PeproTech) for 14 days. On day 21 (stage 2-wk2), induced cells were counted and stained with CD56-PE antibody to detect CD56. + The percentage of iNK cells was detected by flow cytometry. Comparing #2 with #1, the results in Table 2.1 show that replacing conventional IF-4 with NKSFM improves the differentiation efficiency (CD34+%, CD56+%, and CD56+ cell count) into HE cells, HP cells, or iNK cells in stages 1–5 (Table 2.1). This improvement is due to the addition of NAM, PLT, and heparin sodium to NKSFM compared with IF-4. Furthermore, comparing #3 (control experiment) with #2, we find that NKSFM results in a very low KDR+% and is therefore not applicable to stages 1–2, 1–3, and 1–4.

[0286] [Table 2-1]

[0287] Example 6 hiPSCs were prepared as described in CN108373998B. The basal medium used for stage 2 of differentiation was also evaluated. On day -1, embryoid body (EB) formation (stage 1-1) was performed by culturing individualized hiPSCs overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a shaker. On day 0, the medium was changed to stage 1-2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (15 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1-2) for 2 days. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (15 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF (15 ng / mL), bFGF (5 ng / mL), and SB431542 (6 μM) for 1 day. On day 4 (the end of stages 1-4), EBs were dissociated into single cells and stained with KDR-APC antibody. The percentage of KDR+ cells was detected by flow cytometry. HP differentiation (stages 1-5) was initiated by adding stage 1-5 differentiation medium containing NKSFM supplemented with SCF (50 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL) for 5 days. To initiate differentiation of HP cells into iNK cells (stage 2), day 9 EBs were harvested and plated onto 24-well plates coated with the matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium containing the indicated basal medium (see Table 2.2) supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), IL-2 (700 IU / mL), and IL-15 (10 ng / mL) for 13 days. On day 22 (stage 2-wk2), the induced cells were counted and stained with CD56-PE antibody, and the percentage of CD56+ iNK cells was detected by flow cytometry.Figure 6 shows that NKSFM used discontinuously or continuously during stage 2 significantly promoted the differentiation efficiency into iNK cells (% CD56+ cells (Figure 6A) and number of CD56+ cells (Figure 6B)) compared to other basal media used during stage 2.

[0288] [Table 2-2]

[0289] Example 7 hiPSCs were prepared as described in CN108373998B. The effect of plate coating at stage 2 on iNK differentiation was further evaluated. On day -1, individualized hiPSCs were plated in E8 medium containing 10 μM blebbistatin in a T25 flask on a belly dancer and cultured overnight to form embryoid bodies (EBs) (stage 1-1). On day 0, the medium was changed to stage 1-2 differentiation medium containing CD34A basal medium supplemented with BMP4 (25 ng / mL), VEGF (15 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1-2) for 2 days. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing CD34A basal medium supplemented with BMP4 (25 ng / mL), VEGF (15 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing CD34A basal medium supplemented with insulin (5 μg / mL), VEGF (15 ng / mL), bFGF (5 ng / mL), and SB431542 (6 μM) for 1 day. On day 4 (end of stages 1-4), HP differentiation (stages 1-5) was initiated by adding stage 1-5 differentiation medium containing CD34A basal medium supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL). To initiate differentiation of HP cells into iNK cells (stage 2), day 11 EBs were collected and seeded onto matrix-free or DLL4- and VCAM1-coated 24-well plates in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), and IL-15 (10 ng / mL) for 14 days. On day 25 (stage 2-wk2), induced cells were counted and stained with CD56-PE antibody, and the percentage of CD56+ iNK cells was detected by flow cytometry.The results show that coating the plate with DLL4 and VCAM1 during stage 2 significantly promoted iNK differentiation compared to iNK differentiation without plate coating (Figure 7). Differentiation of hPSCs into immature iNK cells was achieved in just 26 days. Furthermore, when differentiation was performed using NKSFM and DLL4 / VCAM1, the percentage of CD56+CD3- iNK cells at the end of stage 2 was as high as 80.56%.

[0290] Examples 8-10: Activation and inhibition of the Wnt pathway promotes cell differentiation This example demonstrates that Wnt activation and inhibition at specific substages of hematopoietic differentiation is important for both hematopoietic differentiation and subsequent NK differentiation.

[0291] Example 8 hiPSCs were prepared as described in CN108373998B. On day -1, individualized hiPSCs were cultured overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a belly dancer to initiate embryoid body (EB) formation (stage 1-1). On day 0, the medium was changed to stage 1-2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1-2) over a 2-day period. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (1 μM). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF (50 ng / mL), bFGF (5 ng / mL), without or with XAV939 (5 ​​μM), and / or SB431542 (6 μM) for 1 day. On day 4, stage 1-5 differentiation medium containing NKSFM and supplemented with SCF (50 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL) was added for 6 days to initiate HP differentiation (stages 1-5). On day 10 (the end of stage 1), EBs were dissociated into single cells and stained with CD34-APC antibody (BD, #555824) to identify CD34. + The percentage of HP cells was detected by flow cytometry. To initiate differentiation of HP cells into iNK cells (stage 2), EBs were collected and seeded onto 24-well plates coated with matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), IL-2 (700 IU / mL), and IL-15 (10 ng / mL) for 15 days. On day 25 (the end of stage 2), induced cells were counted and stained with CD56-PE antibody (BD, #555516) to detect CD56. +The percentage of iNK cells was detected by flow cytometry. The results in Figure 8 show that when XAV939 inhibited the Wnt pathway during HE differentiation (stages 1-4), the CD34 expression was significantly increased compared to differentiation without both XAV939 and SB431542 (control experiment) and differentiation with SB431542 alone. + HP cells (Figure 8A) and CD56 + The results show that the production of both iNK cells (Figures 8B and 8C) is significantly enhanced. Furthermore, when compared to differentiation without XAV939 or SB431542 (control), differentiation with SB431542 significantly enhanced the production of CD34 + The only improvement observed was a moderate increase in HP cell production, but no improvement was observed in iNK cell differentiation. Furthermore, when SB431542 was used in combination with XAV939, the CD34 + The production of HP cells can be further promoted (Fig. 8A).

[0292] Example 9 HiPSCs were prepared as described in CN108373998B. The concentration of the Wnt signaling pathway activator CHIR99021 used in stages 1-2 was optimized. On day -1, individualized hiPSCs were cultured overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a belly dancer to perform embryoid body (EB) formation (stage 1-1). On day 0, lateral mesoderm differentiation (stage 1-2) was initiated by changing the medium to stage 1-2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL, Peprotech), and CHIR99021 at either 5, 6, 7, or 8 μM for 2 days. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with CHIR99021 (1 μM), BMP4 (25 ng / mL), VEGF (50 ng / mL), and bFGF (0.5 ng / mL). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF (50 ng / mL), bFGF (5 ng / mL), and XAV939 (5 ​​μM) for 1 day. On day 4, HP differentiation (stages 1-5) was initiated by adding stage 1-5 differentiation medium containing NKSFM supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL) for 3 days. On day 7 (end of stage 1), EBs were dissociated into single cells and stained with CD34-APC antibody (BD, #555824) to identify CD34 + The percentage of HP cells was detected by flow cytometry. To initiate differentiation of HP cells into iNK cells (stage 2), EBs were collected and seeded into T182 flasks coated with the matrix protein DLL4 in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), IL-2 (400 IU / mL), and IL-15 (10 ng / mL) for 17 days. On day 24 (the end of stage 2), cells were counted and stained with CD56-PE antibody (BD, #555516) to identify CD56.+ The percentage of iNK cells was detected by flow cytometry. The results in Figure 9 show that a higher concentration of CHIR99021 at stages 1-2 generally favored differentiation into CD34+ HP cells (Figure 9A), but slightly disadvantaged differentiation into CD56+ iNK cells (Figures 9B and 9C).

[0293] Example 10 HiPSCs were prepared as described in CN108373998B. The concentration of the Wnt signaling pathway activator CHIR99021 used in stages 1–3 was optimized. On day −1, embryoid body (EB) formation (stage 1–1) was performed by culturing individualized hiPSCs overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a shaker. On day 0, the medium was changed to stage 1–2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1–2) over a 2-day period. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 at 0, 1, 2, 3, or 4 μM. On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF (50 ng / mL), bFGF (5 ng / mL), and XAV939 (5 ​​μM) for 1 day. On day 4, HP differentiation (stages 1-5) was initiated by adding stage 1-5 differentiation medium containing NKSFM supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL) for 3 days. On day 7 (end of stage 1), EBs were dissociated into single cells and stained with CD34-APC antibody (BD, #555824) to identify CD34 +The percentage of HP cells was detected by flow cytometry. To initiate differentiation of HP cells into iNK cells (stage 2), EBs were harvested and seeded onto 6-well plates coated with matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL, Peprotech), IL-3 (5 ng / mL), IL-2 (400 IU / mL), and IL-15 (10 ng / mL) for 17 days. On day 24 (the end of stage 2), induced cells were counted and stained with CD56-PE antibody (BD, #555516), and the percentage of CD56+ iNK cells was detected by flow cytometry. The results in Figure 10 show that lower concentrations of CHIR99021 at stages 1-3 were slightly detrimental to differentiation into CD34+ HP cells (Figure 10A), but generally favored differentiation into CD56+ iNK cells (Figures 10B and 10C).

[0294] Thus, this example demonstrates that highly activating Wnt signaling in the early stages of lateral mesoderm differentiation (stages 1-2) and less activating Wnt signaling in the later stages (stages 1-3), followed by inhibition of Wnt signaling during HE differentiation, is important for highly efficient differentiation of hPSCs into HE cells, HP cells, and iNK cells, particularly HP cells and iNK cells.

[0295] Examples 11-12: Cell differentiation is promoted by adjusting the VEGF concentration in stage 1 This example demonstrates that modulation of VEGF concentration at stage 1 of differentiation is beneficial for the efficient differentiation of hPSCs into HE cells, HP cells, or iNK cells.

[0296] Example 11 hiPSCs were prepared as described in CN108373998B. The effect of varying VEGF concentrations on the substages of hPSC differentiation to HE or HP cells was evaluated. On day -1, embryoid body (EB) formation (stage 1-1) was performed by culturing individualized hiPSCs overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a shaker. On day 0, the medium was changed to stage 1-2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF at the concentrations shown in Table 3.1, bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1-2) over a 2-day period. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF at the concentrations shown in Table 3.1, and bFGF (0.5 ng / mL) and CHIR99021 (1 μM). On day 3, HE differentiation (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF, bFGF (5 ng / mL), XAV939 (5 ​​μM), and SB431542 (6 μM) at the concentrations shown in Table 3.1. On day 4 (end of stages 1-4), EBs were dissociated into single cells and stained with KDR-APC antibody (BD, #560495) to identify KDR. + The percentage of cells was detected by flow cytometry. Stage 1-5 differentiation medium containing NKSFM and supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (5 ng / mL) at the concentrations shown in Table 3.1, and bFGF (5 ng / mL) was added for 6 days to initiate HP differentiation (stages 1-5). On day 10 (the end of stage 1), EBs were dissociated into single cells and stained with CD34-APC antibody (BD, #555824) to detect CD34. +The percentage of HP cells was detected by flow cytometry. To initiate differentiation of HP cells into iNK cells (stage 2), EBs were collected and seeded onto 24-well plates coated with matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), IL-2 (700 IU / mL), and IL-15 (10 ng / mL) for 14 days. On day 24 (the end of stage 2), the morphology of iNK cells under different conditions was recorded as shown in Figure 11C. As shown in Figures 11A and 11B, different VEGF concentrations have minimal effects on the percentage of KDR+ HE cells but significantly affect the percentage of CD34+ HP cells and the efficiency of further differentiation into iNK cells. Specifically, higher VEGF concentrations in stages 1-2 and 1-3 generally favor differentiation into HP cells and iNK cells, whereas higher VEGF concentrations in stages 1-5 generally favor differentiation into HP cells but are unfavorable for differentiation into iNK cells.

[0297] [Table 3-1]

[0298] Example 12 hiPSCs were prepared as described in CN108373998B. The effects of varying VEGF concentrations on the substages of hPSC differentiation into HE, HP, and iNK cells were evaluated. On day -1, individualized hiPSCs were cultured overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a belly dancer to initiate embryoid body (EB) formation (stage 1-1). On day 0, the medium was changed to stage 1-2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF at the concentrations shown in Table 3.2, bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1-2) over a 2-day period. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF and bFGF (0.5 ng / mL) at the concentrations shown in Table 3.2, and CHIR99021 (1 μM). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF, bFGF (5 ng / mL), and XAV939 (5 ​​μM) at the concentrations shown in Table 3.2 for 1 day. On day 4 (the end of stages 1-4), EBs were dissociated into single cells and stained with KDR-APC antibody (BD, #560495), and the percentage of KDR+ cells was detected by flow cytometry. HP differentiation (stages 1-5) was initiated by adding stage 1-5 differentiation medium containing NKSFM and supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (5 ng / mL) at the concentrations shown in Table 3.2, and bFGF (5 ng / mL) for 3 days. On day 7 (the end of stage 1), EBs were dissociated into single cells and stained with CD34-APC antibody (BD, #555824), and the percentage of CD34+ HP cells was detected by flow cytometry.To initiate differentiation of HP cells into iNK cells (stage 2), EBs were harvested and seeded onto 24-well plates coated with the matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), IL-2 (700 IU / mL), and IL-15 (10 ng / mL) for 14 days. On day 21 (the end of stage 2), induced cells were counted and stained with CD56-PE antibody (BD, #555516), and the percentage of CD56+ iNK cells was analyzed by flow cytometry. The results in Figure 12 demonstrate that during stage 1, and especially during differentiation of hPSCs into HE cells (stages 1-2, 1-3, and 1-4), higher concentrations of VEGF significantly improved differentiation of HP cells and iNK cells.

[0299] [Table 3-2]

[0300] Examples 13-14: Expansion and maturation of immature iNK cells with NKSFM This example demonstrates that the use of NKSFM can promote the proliferation and maturation of NK cells, particularly immature iNK cells.

[0301] Example 13 hiPSCs were prepared as described in CN108373998B. On day -1, individualized hiPSCs were cultured overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a belly dancer to initiate embryoid body (EB) formation (stage 1-1). On day 0, the medium was changed to stage 1-2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (15 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1-2) over a 2-day period. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (1 μM). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF (15 ng / mL), bFGF (5 ng / mL), and XAV939 (5 ​​μM) for 1 day. On day 4 (the end of stages 1-4), HP differentiation (stages 1-5) was initiated by adding stage 1-5 differentiation medium containing IF-4 basal medium supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL). To initiate differentiation of HP cells into iNK cells (stage 2), day 10 EBs were collected and seeded onto DLL4- and / or VCAM1-coated 24-well plates in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), IL-2 (700 IU / mL), and IL-15 (10 ng / mL, Peprotech) for 14 days. On day 24 (the end of stage 2), immature iNK cells were cultured at 50 × 10 in multiple electrolyte injection solution (Zhendong Health, H20113035) supplemented with HSA (40 mg / mL) and DMSO (10%). 6Thawed iNK cells were cryopreserved in liquid nitrogen at 1000 cells / mL. Thawed day 24 iNK cells were co-cultured with feeder cells (Nuwacell, feeder:iNK cells = 1:1) in 6-well plates in stage 3 proliferation and maturation medium containing NKSFM, supplemented with or without one or more reagents selected from IL-10 (20 ng / mL) and IL-18 (50 ng / mL). The feeder cells were K562 cells (Procell) genetically engineered to co-express mbIL-21 (membrane-bound IL-21), 41BBL, CD19, and CD64. The feeder cells were inactivated by gamma irradiation before co-culture with iNK cells. On day 11 of co-culture, iNK cells were harvested and stained with CD56-PE antibody (BD, #555516), and the percentage of CD56+ cells was analyzed by flow cytometry. The cytotoxicity of stage 3 iNK cells against K562 cells was detected by CFSE / 7AAD assay after 4 hours of co-culture at an E:T ratio of 3:1. As shown in Figure 13A, IL-10 (20 ng / mL) and / or IL-18, especially IL-18, can significantly improve the cytotoxicity of iNK cells.

[0302] Example 14 hiPSCs were prepared as described in CN108373998B. On day -1, individualized hiPSCs were cultured overnight in E8 medium containing 10 μM blebbistatin in a T25 flask on a belly dancer to initiate embryoid body (EB) formation (stage 1-1). On day 0, the medium was changed to stage 1-2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (15 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1-2) over a 2-day period. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (15 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (1 μM). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF (15 ng / mL), bFGF (5 ng / mL), and XAV939 (5 ​​μM) for 1 day. On day 4 (the end of stages 1-4), HP differentiation (stages 1-5) was initiated by adding stage 1-5 differentiation medium containing IF-4 basal medium supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL). To initiate differentiation of HP cells into iNK cells (stage 2), day 10 EBs were collected and seeded onto DLL4- and VCAM1-coated 24-well plates in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), IL-2 (700 IU / mL), and IL-15 (10 ng / mL) for 14 days. At D24 (the end of stage 2), immature iNK cells were cultured at 50 × 10 cells / well in dextran injection solution (SJZ No. 4 Pharmaceuticals, H13022493) supplemented with HSA (40 mg / mL) and DMSO (10%). 6iNK cells were cryopreserved in liquid nitrogen at 100 cells / mL. Thawed day 25 iNK cells were cocultured with feeder cells (feeder cells:iNK cells = 0.5:1) in 6-well plates for 2 weeks in stage 3 expansion / maturation medium containing NKSFM basal medium supplemented with IL-2 (100 IU / mL) and IL-18 (10-50 ng / mL, added to S3-wk1 or S3-wk2 as indicated) with or without IL-18. iNK cells were harvested on day 14 of coculture and stained with CD56-PE antibody (BD, #555516), and the percentage of CD56+ cells was analyzed by flow cytometry. As shown in Figure 13B, the combination of IL-18 and IL-2, especially when added early in stage 3, can improve iNK cell expansion fold compared to IL-2 alone.

[0303] Examples 15-20: Exemplary characterization of hematopoietic cells formed during the generation of iNK cells from hPSCs Example 15 hiPSCs were prepared as described in CN108373998B. On day -1, individualized hiPSCs were placed in E8 medium containing 10 μM blebbistatin in a T25 flask on a belly dancer and cultured overnight to form embryoid bodies (EBs) (stage 1-1). Day -1 and day 0 cells were analyzed microscopically. On day 0, the medium was changed to stage 1-2 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) to initiate lateral mesoderm differentiation (stage 1-2) for 2 days. On day 2, lateral mesoderm differentiation (stages 1-3) continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (1 μM). On day 3, HE specification (stages 1-4) was initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF (50 ng / mL), bFGF (5 ng / mL), and XAV939 (5 ​​μM) for 1 day. At the end of stages 1-4, cells were analyzed by microscopy and FACS. On day 4 (the end of stages 1–4), stage 1–5 differentiation medium containing NKSFM supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL), and bFGF (5 ng / mL) was added to initiate HP differentiation (stages 1–5). At the end of stages 1–5, cells were analyzed by microscopy and FACS. To initiate differentiation of HP cells into iNK cells (stage 2), day 7 EBs were collected and seeded onto DLL4- and VCAM1-coated 24-well plates in stage 2 differentiation medium containing NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL-7 (25 ng / mL), IL-3 (5 ng / mL), IL-2 (700 IU / mL), and IL-15 (10 ng / mL) for 17 days. At the end of stage 2, cells were analyzed by microscopy and FACS. On day 24 (end of stage 2), immature iNK cells were cultured at 50 × 10 in dextran injection solution (SJZ No. 4 Pharmaceuticals, H13022493) supplemented with HSA (40 mg / mL) and DMSO (10%).6 iNK cells were cryopreserved in liquid nitrogen at 100 cells / mL. Thawed iNK cells on day 24 were cocultured with feeder cells in 6-well plates (feeder cells:iNK cells = 1:1) in stage 3 proliferation and maturation medium containing NKSFM basal medium supplemented with IL-2 (100 IU / mL) and IL-18 (10 ng / mL). On day 11 of coculture, iNK cells were harvested and stained with CD56-PE antibody (BD, #555516). CD56+ cells were analyzed by microscopy and FACS. Figure 14 shows the morphology of hematopoietic cells formed at different stages of iNK cell differentiation from hPSCs. As a result, EB size gradually increased during stage 1 of iNK cell differentiation. After EB plating, iNK cells formed small cell clusters around the attached EBs during stage 2. During stage 3, iNK cells actively proliferated and tended to aggregate into cell clumps in a quiescent state. As shown in Figure 15A, at the end of stages 1-4, cells exhibit efficient lateral mesoderm and HE differentiation. As shown in Figure 15B, at the end of stages 1-5, the percentage of CD34+CD43-HP cells was high at 61.23%, indicating efficient HP differentiation. As shown in Figure 15C, at the end of stage 2, the percentage of CD56+CD3-iNK cells was high at 81.30%, indicating efficient iNK cell specification. As shown in Figure 15D, at the end of stage 3, the percentage of CD56+CD3-iNK cells was high at 99.32%, indicating the production of highly pure iNK cells.

[0304] Example 16 To test the expansion potential of our iNK cells, immature iNK cells obtained at stage 2 in Example 15 were cocultured with feeder cells (Nuwacell) in a 6-well plate in stage 3 expansion maturation medium containing NKSFM basal medium supplemented with IL-2 (100 IU / mL) and IL-18 (10 ng / mL) (feeder cells:iNK cells = 1:1). iNK cells were continuously expanded for 7 weeks with weekly stimulation with feeder cells. The results showed that the iNK cells obtained by this method had high expansion potential and could be continuously expanded by repeated stimulation for at least 7 weeks (Figure 16). The expansion fold observed here was much higher than that observed by other groups in the continuous long-term expansion of hPSC-derived NK cells repeatedly stimulated with aAPCs (mbIL-21 artificial antigen-presenting cells) (Knorr DA et al., Stem Cells Transl Med. 2013 Apr;2(4):274-283). Therefore, the immature iNK cells thus obtained exhibit high proliferation potential.

[0305] Example 17 To compare the phenotype of primary NK cells with that of our iNK cells, we analyzed the expression of NK cell surface receptors on PBNK cells and the iNK cells obtained in Example 15 by flow cytometry. Stage 3 iNK cells were stained with CD56-PE (BD, #555516), NKG2D-APC (BD, #558071), Nkp30-APC (BD, #558408), NKG2A-APC (Biolegend, #375107), KIRe1-APC (Biolegend, #312716), and CCR6-APC (Biolegend, #353416) antibodies. The percentages of NKG2D+, NKp30+, NKG2A+, KIRe1+, and CCR6+ cells in the CD56+ population were analyzed by flow cytometry. As shown in Figure 17, the expression of the activating receptors NKG2D and NKp30 was comparable between PBNK and iNK cells. However, iNK cells showed significantly lower expression of the inhibitory receptors NKG2A and KIRe1 compared to PBNK cells, indicating less inhibition from target cells. Furthermore, iNK cells showed significantly higher expression of the chemokine receptor CCR6 compared to PBNK cells, indicating improved tissue and target homing (Figure 17). Interestingly, the cell surface marker expression of iNK cells generated using the method described herein also differed from that reported in the literature. Thus, iNK cells displayed a different surface receptor expression pattern than primary NK cells, indicating significantly improved function.

[0306] Example 18 To analyze the correlation between primary NK cells and our iNK cells, we evaluated the global gene expression of stage 3 iNK cells, PBNK cells, and CBNK cells obtained in Example 17 using microarray and dendrogram clustering analysis. These results indicate that the iNK cells resemble primary NK cells but are a distinct cell type distinct from both PBNK and CBNK cells. Thus, the iNK cells are similar to primary NK cells in gene expression but cluster into a unique cell type (Figure 18).

[0307] Example 19 To evaluate the cytolytic function of iNK cells compared with PBNK cells, iNK cells or PBNK cells were co-cultured with different types of tumor cells and their cytotoxicity was analyzed. Cryopreserved stage 3 iNK cells obtained in Example 17 were thawed and subjected to cytotoxicity assays. Various tumor cell lines were labeled with CFSE before co-culture with NK cells at an E:T ratio of 3:1 (E:T is the effector cell to target cell ratio, where effector cells are iNK cells or PBNK cells and target cells are tumor cells). After incubation for the indicated times, cells were stained with 7-AAD (live / dead staining) and analyzed by flow cytometry. Compared with PBNK cells, iNK cells exhibited comparable cytotoxicity against K562, Kasumi, MV-4-11, H69, and H146 cells, and significantly higher cytotoxicity against H460, SKOV3, A549, SKMEL2, DMS114, MOLM13, and H82 cells. These results indicate that iNK cells are cytotoxic against various tumor cell lines and have comparable or even greater activity than primary NK cells (Figure 19).

[0308] Example 20 To further evaluate the function of iNK cells, the iNK cells obtained in Example 17 were stimulated with 50 ng / mL phorbol myristate acetate (PMA) and 1 μg / mL ionomycin for 4 hours, and the production of TNF-α and IFN-γ was analyzed by enzyme-linked immunosorbent assay (ELISA). The results show that iNK cells can secrete high levels of pro-inflammatory cytokines, TNF-α and IFN-γ, in response to stimulation (Figure 20).

[0309] Those skilled in the art will readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended to limit the scope of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the disclosure disclosed herein without departing from the scope and spirit of the invention.

[0310] All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0311] The present disclosure is not limited in terms of the specific embodiments described in this application, which are intended as examples of individual aspects of the present disclosure. Not all various embodiments of the present disclosure are described herein. The terms and expressions employed are used as terms of description and are not intended to be limiting. There is no intention in the use of such terms and expressions to exclude equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present disclosure as claimed. Thus, while the present disclosure has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

Claims

1. A method for promoting directed differentiation of pluripotent stem cells (PSCs), comprising: contacting the PSCs with a maintenance culture medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation culture medium, or sequentially with a first differentiation culture medium and a second differentiation culture medium, to form mesodermal cells; contacting the mesodermal cells with a third differentiation culture medium to form hemogenic endothelial (HE) cells; contacting the HE cells with a fourth differentiation culture medium to form hematopoietic progenitor (HP) cells; contacting the HP cells with a fifth differentiation culture medium to obtain immature iNK cells; A method in which a basal medium supplemented with a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate is used as the basal medium of the fourth differentiation culture medium and / or the basal medium of the fifth differentiation culture medium.

2. 2. The method of claim 1, wherein the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate is used continuously throughout the step of contacting the HE cells with the fourth differentiation culture medium to form the HP cells, and throughout the step of contacting the HP cells with the fifth differentiation culture medium to obtain the immature iNK cells.

3. 2. The method of claim 1, wherein the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) a human platelet lysate is used continuously throughout the step of contacting the HE cells with the fourth differentiation culture medium to form the HP cells, and the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) a human platelet lysate is used discontinuously during the step of contacting the HP cells with the fifth differentiation culture medium to obtain the immature iNK cells.

4. The method of claim 3, wherein the basal medium of the fifth differentiation culture medium is individually and in any order the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate, and another different basal medium, throughout the step of contacting the HP cells with the fifth differentiation culture medium to obtain the immature iNK cells.

5. 5. The method of claim 4, wherein the other different basal medium is supplemented with one selected from a nicotinamide-based compound, a heparin-based compound, and human platelet lysate.

6. The method according to any one of claims 1 to 5, wherein the first to third differentiation basal media comprise the same basal medium.

7. The method of claim 1, wherein the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate is continuously used as one of the basal medium for the fourth differentiation culture medium and the basal medium for the fifth differentiation culture medium, and the other of the basal medium for the fourth differentiation culture medium and the fifth differentiation culture medium is continuously used as a basal medium supplemented with (i) a nicotinamide-based compound and (ii) a heparin-based compound, but not (iii) human platelet lysate.

8. The method according to any one of claims 1 to 7, wherein the concentrations of the nicotinamide-based compound in the fourth and fifth differentiation culture media are each 0.5 to 20 mM.

9. The method according to any one of claims 1 to 7, wherein the concentrations of the heparin-based compound in the fourth and fifth differentiation culture media are each 0.1 to 100 µg / mL.

10. The method according to any one of claims 1 to 7, wherein the concentration of the human platelet lysate in the fourth and fifth differentiation culture media is 0.1 to 20% by volume, respectively.

11. The method of any one of claims 1 to 10, further comprising seeding the HP cells onto a cell culture surface coated with a Notch pathway activator and an adhesion molecule.

12. 12. The method of claim 11, wherein the Notch pathway activator is selected from DLL4, DLL1, Jagged-1, Jagged-2, mutants thereof, and any combination thereof, and the adhesion molecule is selected from VCAM1, fibronectin, laminin, vitronectin, MAdCAM-1, ICAM, mutants thereof, and any combination thereof.

13. The method according to any one of claims 1 to 12, wherein the third differentiation culture medium is further supplemented with a Wnt signaling pathway inhibitor.

14. The method of claim 13, wherein the concentration of the Wnt signaling pathway inhibitor in the third differentiation culture medium is 1 to 30 μM.

15. 15. The method of any one of claims 13 to 14, wherein the Wnt signaling pathway inhibitor is selected from the group consisting of iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, ciclopirox, cardamonin, diethylbenzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, isoquercitrin, gigantol, JW55, MSAB, IWR-1-endo, FH535, WIKI4, CCT251545, KYA1797K, NCB-0846, iCRT14, adavivint, M435-1279, XAV939, and any combination thereof.

16. The method according to any one of claims 13 to 15, wherein the third differentiation culture medium is not supplemented with a TGF-β signaling pathway inhibitor.

17. 17. The method according to any one of claims 1 to 16, wherein the first and second differentiation culture media are further supplemented with a Wnt signaling pathway activator, and the Wnt signaling pathway activator in the second differentiation culture medium may be the same as or different from the Wnt signaling pathway activator in the first differentiation culture medium, and may be at an equal or lower concentration.

18. The Wnt signaling pathway activator is selected from the group consisting of Kenpaullone, 1-azakempaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, SB216763, AR-A014418, BIO-Acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, 2-thio(3-iodobenzyl)amine, and 2-methyl-1H-pyrazol-3-yl.

18. The method of claim 17, wherein the compound is selected from the group consisting of 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, GF109203X, and any combination thereof.

19. 19. The method of any one of claims 17 to 18, wherein the second differentiation culture medium has the same composition as the first differentiation culture medium, except that the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is lower than the concentration of the Wnt signaling pathway activator in the first differentiation culture medium.

20. 18. The method of claim 17, wherein the concentration of the Wnt signaling pathway activator in the second differentiation culture medium is 0-4 μM and the concentration of the Wnt signaling pathway activator in the first differentiation culture medium is 4-8 μM.

21. 21. The method of any one of claims 1 to 20, wherein the nicotinamide-based compound comprises nicotinamide and the heparin-based compound comprises sodium heparin.

22. 22. The method of claim 21, wherein the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate comprises IF-4 basal medium in addition to the combination of (i) nicotinamide, (ii) heparin sodium, and (iii) human platelet lysate.

23. 22. The method of claim 21, wherein the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate comprises NKSFM basal medium.

24. The method according to any one of claims 1 to 23, wherein the first to fourth differentiation culture media are each independently further supplemented with VEGF at a concentration of 15 to 100 ng / mL.

25. The method according to any one of claims 1 to 24, wherein the first to fifth differentiation culture media are chemically defined serum-free and animal-derived component-free differentiation culture media.

26. The method according to any one of claims 1 to 25, wherein the method is carried out under three-dimensional culture conditions.

27. A method for producing iNK cells, comprising the method of any one of claims 1 to 26 and a step of expanding and maturing the immature iNK cells.

28. The step of expanding and maturing the immature iNK cells comprises:

28. The method of claim 27, comprising contacting the immature iNK cells with a proliferation and maturation culture medium comprising a basal medium supplemented with a combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate, in the presence or absence of feeder cells.

29. 29. The method of claim 28, wherein the proliferation and maturation culture medium is further supplemented with one or more of IL-2, IL-10, IL-18, and SB431542.

30. 30. The method of claim 28 or 29, wherein the concentration of the nicotinamide-based compound in the growth and maturation culture medium is 0.5 to 20 mM.

31. 30. The method of claim 28 or 29, wherein the concentration of the heparin-based compound in the proliferation and maturation culture medium is 0.1 to 100 μg / mL.

32. 30. The method of claim 28 or 29, wherein the concentration of the human platelet lysate in the proliferation and maturation culture medium is between 0.1% and 20% by volume.

33. 33. The method of any one of claims 28 to 32, wherein the nicotinamide-based compound comprises nicotinamide and the heparin-based compound comprises sodium heparin.

34. 34. The method of claim 33, wherein the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate comprises IF-4 basal medium in addition to the combination of (i) nicotinamide, (ii) sodium heparin, and (iii) human platelet lysate.

35. 34. The method of claim 33, wherein the basal medium supplemented with the combination of (i) a nicotinamide-based compound, (ii) a heparin-based compound, and (iii) human platelet lysate comprises NKSFM basal medium.

36. 36. The method of any one of claims 28 to 35, wherein the growth and maturation culture medium is a chemically defined serum-free and animal-derived component-free culture medium.

37. A cell population produced by the method of any one of claims 1 to 36.

38. A cell population, wherein greater than 90% of the cells in said population without enrichment or purification are mature CD56+CD3- iNK cells.

39. 39. The cell population of claim 38, wherein the iNK cells have lower expression of inhibitory receptors and higher expression of chemokine receptors compared to primary NK cells.

40. 40. The cell population of claim 39, wherein less than 20% of the cells in the iNK cells are NKG2A+ cells.

41. 40. The cell population of claim 39, wherein less than 20% of the cells in the iNK cells are KIRe1+ cells.

42. 40. The cell population of claim 39, wherein at least 70% of the cells in the iNK cells are CCR6+ cells.

43. 40. The cell population of claim 39, wherein at least 60% of the cells in the iNK cells are NKG2D+ cells.

44. 40. The cell population of claim 39, wherein at least 80% of the cells in the iNK cells are NKp30+ cells.

45. A pharmaceutical composition comprising the cell population of any one of claims 38 to 44 and a pharmaceutically acceptable carrier.

46. Use of the cell population of any one of claims 38 to 44 in the manufacture of a medicament for treating or preventing cancer.

47. 47. The use of claim 46, wherein the cancer is acute myeloid leukemia, melanoma, small cell lung cancer, large cell lung cancer, ovarian cancer, or non-small cell lung cancer.

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