Methods and compositions for differentiating pluripotent stem cells and derived natural killer cells

Abstract

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

Description

Technical Field

[0001] The present disclosure generally relates 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 lineage cells. Background Art

[0002] Natural killer (NK) cells are innate lymphocytes and play important roles in defense (e.g., viral infections and cancer) and immune regulation. Due to their innate properties, NK cells have been widely considered for immune-based therapies against various diseases and / or conditions. However, there are multiple challenges in obtaining NK cells (e.g., NK cells for therapeutic use and biomedical research). These challenges include, for example, the number of primary NK cells that can be isolated (e.g., during apheresis), significant differences in the quantity and / or quality of primary NK cells between donors, and / or the efficient production of safe NK cells with a well-characterized phenotype and / or function (e.g., a phenotype and / or function similar to that of primary NK cells) by alternative methods (e.g., differentiating pluripotent cells (e.g., hPSCs) into NK cells (iNK cells)).

[0003] Pluripotent stem cell technology, including human PSC (hPSC) technology, is a very promising and potentially limitless source of therapeutic living cells. However, current protocols for directed differentiation (e.g., into hematopoietic endothelium (HE) cells, hematopoietic progenitors (HP), or iNK cells) and subsequent expansion are generally inefficient. In addition, these protocols typically require the use of serum. The use of such components poses potential contamination risks, can cause lot dependency of the cells, and may not be suitable for generating cells for clinical and therapeutic use. For example, cells cultured in such an environment contaminated with animal-derived components are generally considered unsuitable for humans because exposure to animal components can pose serious risks such as immune rejection, transmission of unknown pathogens to the treated subject, and reactivation of animal retroviruses. Using current directed differentiation methods, it remains a challenge to generate large numbers of HP or iNK cells with a consistent and reproducible phenotype and / or function (e.g., a phenotype and / or function similar to or superior to that of primary NK cells).

[0004] To advance techniques related to the directed differentiation of pluripotent cells (e.g., hPSCs), it is important to be able to efficiently, safely, and / or reproducibly generate PSCs and partially differentiated cells (e.g., hematopoietic progenitor cells), and also to be able to efficiently, safely, and / or reproducibly generate a population of immune effector cells, including iNK cells. Accordingly, there remains a need for improved methods and compositions for directing the 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)).

[0005] Overview

[0006] The present disclosure provides methods and compositions for promoting the directed differentiation of pluripotent cells (e.g., hPSCs) or a population of cells thereof into hematopoietic lineage cells such as, for example, 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 populations of cells, cell lines, and / or clonal cells produced using the methods and compositions described herein.

[0007] In a first aspect, the present disclosure relates to a method for promoting the directed differentiation of pluripotent stem cells (PSCs), the method comprising the steps of: contacting the PSCs with a maintenance medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation medium or sequentially with a first differentiation medium and a second differentiation medium to form mesoderm cells; contacting the mesoderm cells with a third differentiation medium to form hematopoietic endothelial (HE) cells; contacting the HE cells with a fourth differentiation medium to form hematopoietic progenitors (HP); and contacting the HP cells with a fifth differentiation medium to obtain immature iNK cells, wherein a basal medium supplemented with a combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate is used as the basal medium of the fourth differentiation medium and / or the basal medium of the fifth differentiation medium.

[0008] In a second aspect, the present disclosure relates to a method for promoting the directed differentiation of pluripotent stem cells (PSCs), the method comprising the steps of: contacting the PSCs with a maintenance medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation medium supplemented with a Wnt signaling pathway activator, or sequentially with a first differentiation medium supplemented with a Wnt signaling pathway activator and a second differentiation medium supplemented with a Wnt signaling pathway activator, to form mesoderm cells; and contacting the mesoderm cells with a third differentiation medium supplemented with a Wnt signaling pathway inhibitor to obtain hematopoietic endothelial (HE) cells.

[0009] In a third aspect, the present disclosure relates to a culture medium for promoting the directed differentiation of pluripotent stem cells (PSCs) into hematopoietic lineage cells, which comprises a basal medium supplemented with a Wnt signaling pathway inhibitor.

[0010] In a fourth aspect, the present disclosure relates to a kit comprising the culture medium of the third aspect of the present disclosure.

[0011] In a fifth aspect, the present disclosure relates to a method for producing iNK cells, which includes a method for promoting the directed differentiation of pluripotent stem cells (PSCs) according to the first aspect of the present disclosure, and steps of amplifying and maturing immature iNK cells.

[0012] In a sixth aspect, the present disclosure relates to a cell population produced by the method according to the first aspect, second aspect or fifth aspect of the present disclosure.

[0013] In a seventh aspect, the present disclosure relates to a cell population, wherein more than 90% of the cells in the population are mature CD56+CD3− iNK cells without any enrichment or purification.

[0014] In an eighth aspect, the present disclosure relates to a pharmaceutical composition comprising the cell population of the seventh aspect of the present disclosure and a pharmaceutical carrier.

[0015] In a ninth aspect, the present disclosure relates to the use of the cell population of the seventh aspect of the present disclosure in the preparation of a drug for treating or preventing cancer.

[0016] By the following description in conjunction with the accompanying drawings, various objects and advantages of the reagents, compositions and methods provided herein will become apparent, wherein certain embodiments of the present disclosure are illustrated by way of illustration and example. Brief Description of the Drawings

[0018] Figure 1 A diagram showing the process of producing iNK cells from hPSCs in an exemplary example according to the method of the present disclosure.

[0019] Figure 2 Shows the effect of the concentration of nicotinamide (NAM) in an exemplary basal medium on the expansion of PBNK cells according to Example 1 of the present disclosure, including the fold expansion of NK cells ( Figure 2 A) and the percentage of CD56+CD3− NK cells ( Figure 2 B).

[0020] Figure 3 Shows the effect of the concentration of sodium heparin in an exemplary NKSFM basal medium on the expansion of PBNK cells according to Example 2 of the present disclosure, including the fold expansion of NK cells ( Figure 3A), percentage of CD56+CD3− NK cells ( Figure 3 B) and lysis rate of K562 tumor cells ( Figure 3 C).

[0021] Figure 4 Shows the effect of the concentration of PLT in an exemplary NKSFM basal medium on the expansion of PBNK cells from donors 1 and 2 according to Example 3 of the present disclosure, including the fold expansion of NK cells ( Figure 4 A), percentage of CD56+CD3− NK cells ( Figure 4 B) and lysis rate of K562 tumor cells ( Figure 4 C).

[0022] Figure 5 Shows the comparison of the effect of an exemplary NKSFM basal medium and a commercial kit on the expansion of PBNK cells according to Example 4 of the present disclosure, including the fold expansion of NK cells ( Figure 5 A), percentage of CD56+CD3− NK cells ( Figure 5 B) and lysis rate of K562 tumor cells ( Figure 5 C), where 1 represents NKSFM (without feeder cells), 2 represents a commercial kit (without feeder cells), 3 represents NKSFM (with feeder cells), and 4 represents a commercial kit (with feeder cells).

[0023] Figure 6 Shows the effect of different basal media used in the second stage of the method on the differentiation efficiency of iNK cells (CD56+% ( Figure 6 A) and number of CD56+ cells ( Figure 6 B), where 1, 2, 3, 4, 5, 6, and 7 represent #1, #2, #3, #4, #5, #6, and #7, respectively.

[0024] Figure 7 Shows the effect of plate coating in the second stage of the method on iNK differentiation, where Figure 7 A and 7C respectively show the representative morphology and flow cytometry plots of cells differentiated using an exemplary NKSFM basal medium on a cell culture surface without any coating matrix; and Figure 7 B and 7D respectively show the representative morphology and flow cytometry plots of cells differentiated using an exemplary NKSFM basal medium on a cell culture surface coated with DLL4 and VCAM1.

[0025] Figure 8Shows the effect of XAV939 and / or SB431542 used in the first to fourth stages of the method on the differentiation efficiency of HP cells ( Figure 8 A) or iNK cells ( Figure 8 B and 8C) according to Example 8 of the present disclosure, where none represents the case where neither XAV939 nor SB431542 was used in the first to fourth stages, XAV represents the case where XAV939 was used in the first to fourth stages, SB represents the case where SB431542 was used in the first to fourth stages, and SB + XAV represents the case where both XAV939 and SB431542 were used in the first to fourth stages.

[0026] Figure 9 Shows the effect of the concentration of CHIR99021 used in the first to second stages of the method on the differentiation efficiency of HP cells ( Figure 9 A) or iNK cells ( Figure 9 B and 9C) according to Example 9 of the present disclosure.

[0027] Figure 10 Shows the effect of the concentration of CHIR99021 used in the first to third stages of the method on the differentiation efficiency of HP cells ( Figure 10 A) or iNK cells ( Figure 10 B and 10C) according to Example 10 of the present disclosure, where iPSC line 1 represents the case where the hPSC used for differentiation is iPSC line 1, and iPSC line 2 represents the case where the hPSC used for differentiation is iPSC line 2.

[0028] Figure 11 Shows the effect of different VEGF concentrations in the first stage (the first to second, first to third, first to fourth, and first to fifth stages) of the method on the differentiation efficiency of HE cells (KDR+%, Figure 11 A), HP cells (CD34+%, Figure 11 B) or iNK cells (output represented by cell morphology, Figure 11 C) according to Example 11 of the present disclosure, where 1 - 10 represent #1 - 10 respectively.

[0029] Figure 12 Shows the effect of the regulation of VEGF concentration in the first stage (the first to second, first to third, first to fourth, and first to fifth stages) of the method on the differentiation efficiency of HE cells ( Figure 12 A), HP cells ( Figure 12 B) or iNK cells ( Figure 12 C and 12D) according to Example 12 of the present disclosure, where 1 - 4 represent #1 - 4 respectively.

[0030] Figure 13Shows the effects of IL-10 and / or IL-18 added in the third stage of the present method on the specific lysis of K562 tumor cells by iNK cells according to Embodiments 13-14 of the present disclosure ( Figure 13 A), and the effect of IL-18 added in the third stage of the present method on the amplification fold of iNK cells, wherein, in Figure 13 A, no represents the case where neither IL-10 nor IL-18 was added in the third stage, IL-10 represents the case where IL-10 was added in the third stage, IL-18 represents the case where IL-18 was added in the third stage, and IL-10 + IL-18 represents the case where both IL-10 and IL-18 were added in the third stage; and in Figure 13 B, no represents the case where only IL-2 was added in the third stage, S3-wk1 represents the case where IL-2 was added in the third stage and IL-18 was added in the first week of the third stage, and S3-wk2 represents the case where IL-2 was added in the third stage and IL-18 was added in the second week of the third stage.

[0031] Figure 14 Shows the morphology of hematopoietic lineage cells formed at different stages (D-1, D0, the first to fourth stages, the first to fifth stages, the second stage, and the third stage) of the method for generating iNK cells from hPSCs according to Embodiment 15 of the present disclosure.

[0032] Figure 15 Shows the flow cytometry plots of hematopoietic lineage cells formed at different stages (the first to fourth stages, the first to fifth stages, the second stage, and the third stage) of the method for generating iNK cells from hPSCs according to Embodiment 15 of the present disclosure.

[0033] Figure 16 Shows the long-term expansion potential of immature iNK cells according to Embodiment 16 of the present disclosure.

[0034] Figure 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 Embodiment 17 of the present disclosure.

[0035] Figure 18 Shows the total gene expression profiles of mature iNK cells, PBNK cells, and CBNK cells according to Embodiment 18 of the present disclosure.

[0036] Figure 19 Shows the cytotoxicity of mature iNK cells against a wide range of tumor cell lines according to Embodiment 19 of the present disclosure.

[0037] Figure 20 Shows the levels of pro-inflammatory cytokines secreted by mature iNK cells according to Example 20 of the present disclosure.

[0038] Detailed description

[0039] It should be understood that certain aspects, modes, embodiments, variations, and features of the present disclosure are described below in various levels of detail in order to provide an understanding of the essence of the technology.

[0040] The "first", "second", "third", "fourth", "fifth", "sixth", "seventh", "eighth", or "ninth" mentioned throughout this specification does not denote the order or sequence of the features, structures (e.g., culture media or compositions), or properties related to the mentioned description, but may be for the purpose of distinction only.

[0041] The "first aspect", "second aspect", "third aspect", "fourth aspect", "fifth aspect", "sixth aspect", "seventh aspect", "eighth aspect", or "ninth aspect" mentioned throughout this specification indicates that the specific features, structures, or properties described in relation to the aspect are included in at least one or more aspects of the present disclosure. In addition, the specific features, structures, properties, or embodiments in one aspect can be combined with the specific features, structures, properties, or embodiments in one or more other aspects in any appropriate manner.

[0042] The "one embodiment", "some embodiments", "preferred embodiment", "certain embodiments", or "a certain embodiment" mentioned throughout this specification indicates that the specific features, structures, or properties described in relation to the embodiment are included in at least one or more embodiments of the present disclosure. In addition, the specific features, structures, or properties in one embodiment can be combined with the specific features, structures, or properties in one or more other embodiments in any suitable manner.

[0043] It should be understood that the present disclosure is not limited to specific uses, methods, reagents, compounds, compositions, or biological systems, although these can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive.

[0044] The present disclosure is at least in part based on the discovery of a basal medium supplemented with a combination of (i) a niacinamide compound, (ii) a heparin compound, and (iii) human platelet lysate, which can be used to improve the differentiation efficiency of immature NK cells through its stage-specific use (after hematopoietic endothelial (HE) cell formation) during the directed differentiation of pluripotent stem cells (PSCs). The present disclosure is also at least in part based on the discovery of a differentiation medium supplemented with a Wnt signaling pathway inhibitor, which, alone or in combination with another differentiation medium supplemented with a Wnt signaling pathway activator, can improve the differentiation efficiency of HE cells, HP cells, or immature NK cells during the directed differentiation of pluripotent stem cells (PSCs).

[0045] Definitions

[0046] Unless otherwise defined, as used herein, all technical and scientific terms have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. The following references provide a general definition of many of the terms used in this disclosure to those skilled in the art. 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, unless otherwise indicated, the following terms have the meanings given to them below. As used herein, the terms are for the purpose of describing particular embodiments only and are not intended to limit the present disclosure.

[0047] Unless otherwise indicated, "a" or "an" means "one or more" or "at least one".

[0048] 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 number.

[0049] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not to exclude other elements. "Consisting essentially of" when used to define compositions and methods shall mean excluding other elements of any substantial significance to the composition or method. "Consisting of" shall mean excluding other ingredients in excess of trace amounts for the claimed compositions and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of the present disclosure. Thus, it is intended that the methods and compositions may include additional steps and components (comprising), or alternatively may include insignificant steps and compositions (consisting essentially of), or alternatively are intended to consist only of the recited method steps or compositions (consisting of). Further, in each instance herein, any one of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced by any of the other two terms.

[0050] As used herein, the term "pluripotent stem cell" (PSC) refers to a cell derived from the inner cell mass of an embryonic blastocyst. Pluripotent stem cells are pluripotent and give rise to all derivatives of the three primary germ layers during development: ectoderm, endoderm, and mesoderm. Pluripotent stem cells can be of human origin (e.g., human PSC or hPSC). Pluripotent stem cells can be induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC). ESCs (e.g., hESC) and iPSCs (e.g., hiPSC) are known in the art and can be readily obtained using conventional methods (e.g., those described in the prior art) or commercially available products. Suitable methods for generating iPSCs from somatic or adult pluripotent stem cells are well known to those skilled in the art. For example, iPSCs can be reliably generated from somatic cells by traditional reprogramming techniques.

[0051] As used herein, the term "pluripotency" or "pluripotent" refers to a cell's developmental potential to differentiate into cells of all three germ layers (ectoderm, mesoderm, and endoderm). Pluripotency can be determined at least in part by assessing the pluripotency characteristics of the cell. Pluripotency characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) the 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 consisting of the three somatic lineages; and (vi) embryoid body formation consisting of cells from the three somatic lineages.

[0052] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology can be characterized as small and round in shape, having a high nucleus-to-cytoplasm ratio, a distinct nucleolus, and / or typical cell spacing.

[0053] As used herein, the term "reprogramming" refers to methods for increasing a cell's potency or de-differentiating a cell into a less differentiated state. For example, a cell with increased cell potency can have greater developmental plasticity (i.e., be able to differentiate into more cell types) compared to the same cell in an un-reprogrammed state. That is, a reprogrammed cell is a cell in a less differentiated state compared to the same cell in a non-reprogrammed state. "Reprogramming" can refer to de-differentiating somatic or adult pluripotent stem cells into pluripotent stem cells, also known as induced pluripotent stem cells or iPSCs.

[0054] As used herein, the term "differentiation" refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells (e.g., blood cells or immune cells). In certain embodiments, the differentiated cells or cells induced to differentiate are cells that have occupied a more specialized ("committed") position within a cell lineage. For example, human pluripotent stem cells (hPSCs) can be differentiated into various more differentiated cell types, such as neural or hematopoietic progenitor cells, lymphocytes, cardiomyocytes, immune cells (e.g., natural killer cells), and other cell types, after being treated with appropriate differentiation factors in cell culture. In certain embodiments, the term "committed" is applied to the differentiation process to mean that a cell has progressed to a certain extent along a differentiation pathway such that, in a normal environment, the cell will or will continue to differentiate into a specific cell type or subtype of cell type and cannot differentiate into a different cell type (other than the specific cell type or subtype of cell type) or revert to a less differentiated cell type in a normal environment.

[0055] As used herein, the term "hematopoietic lineage cells" refers to cells differentiated from PSCs in vitro and / or their progeny, and may include one or more of the following: angioblasts, hematopoietic endothelial cells (HECs), hematopoietic stem cells, hematopoietic progenitor cells (HPCs), erythroid / megakaryocyte progenitor cells, erythrocytes, megakaryocytes, platelets, and lymphoid lineage cells.

[0056] As used herein, the term "lymphoid lineage cells" includes one or more of the following: lymphoid progenitor cells, lymphocytes (e.g., T lymphocytes), natural killer (NK) cells, myeloid progenitor cells, granulomonocytic progenitor cells, monocytes, macrophages, and dendritic cells.

[0057] As used herein, the term "embryoid body" (EB) refers to a three-dimensional cluster that has been shown to mimic embryonic development in that it gives rise to many lineages within its three-dimensional volume.

[0058] As used herein, the term "mesoderm" or "mesodermal cells" refers to one of the three germ layers that arise during early embryogenesis or cells derived therefrom, which give rise to various specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, bone, and other supportive and connective tissues.

[0059] As used herein, the terms "hematopoietic endothelium" (HE), "hemogenic endothelium cell", or "hemogenic endothelial cell" refer to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells during a process called the endothelial-to-hematopoietic transition. The development of hematopoietic cells in the embryo proceeds sequentially from the lateral plate mesoderm through angioblasts to committed hematopoietic endothelium and hematopoietic progenitor cells.

[0060] As used herein, the terms "hematopoietic progenitor" (HP) or "hematopoietic progenitor cell" (HPC) refer to cells present in the blood and bone marrow that are capable of giving rise to mature blood cells, such as red blood cells, platelets, and immune cells.

[0061] 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 of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes.

[0062] As used herein, the term "lymphocyte" refers to all populations of immature, mature, undifferentiated, and differentiated white blood cells derived from lymphoid progenitors, including tissue-specific and specialized types, and by non-limiting example includes B cells, T cells, NKT cells, and NK cells. In certain embodiments, lymphocytes include all B cell lineages, including pre-B cells, pro-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.

[0063] As used herein, "natural killer cell" or NK cell refers to a lymphoid cell defined by its 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 different levels of CD56. NK cells can include primary NK cells or induced NK (iNK) cells.

[0064] As used herein, "primary NK cell" refers to a naturally occurring natural killer cell that can be derived from, for example, blood (e.g., cord blood or peripheral blood collected by apheresis), bone marrow, or cryopreserved 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.

[0065] As used herein, the term "iNK cell" refers to natural killer cells differentiated, expanded, and matured from pluripotent stem cells (e.g., hPSC). iNK cells can be, for example, iPSC-derived iNK cells or ESC-derived iNK cells. iNK cells can be used interchangeably with mature iNK cells. The expression level of specific markers such as CD56 in mature iNK cells is higher than that in immature iNK cells, and it has cytokine release function and cytotoxicity similar to those of primary NK cells.

[0066] As used herein, the term "immature iNK cell" refers to natural killer cells differentiated from pluripotent cells (e.g., hPSC) that have not been expanded and matured. Immature iNK cells can be, for example, iPSC-derived immature iNK cells or ESC-derived immature iNK cells. Compared with mature iNK cells, the expression level of specific markers such as CD56 in immature iNK cells is lower, and the cytokine release function and cytotoxicity are also lower.

[0067] As used herein, the term "culture medium" refers to a culture medium that can support the survival, growth, proliferation, maintenance, and / or differentiation of cells in an in vitro environment. The culture medium can have a basal medium and one or more supplements.

[0068] As used herein, the term "maintenance medium" refers to a culture medium that can support the survival, growth, proliferation, or maintenance of cells in an in vitro environment.

[0069] As used herein, the term "differentiation medium" or "differentiation culture medium" refers to a culture medium that can support the differentiation of cells in an in vitro environment.

[0070] As used herein, the term "basal medium" refers to the basal component of a culture medium (e.g., differentiation medium or expansion medium) relative to its supplements. Generally, the basal medium accounts for about 95% to 99% of the volume of the culture medium (e.g., differentiation medium, or expansion medium) by volume. The basal medium of a cell maintenance medium serves as a source of nutrients, hormones, and / or other factors that contribute to the proliferation and / or maintenance of cells. The basal medium of a cell differentiation medium serves as a source of nutrients, hormones, and / or other factors that contribute to the differentiation of cells.

[0071] As used herein, the term "supplement" refers to one or more added components of a culture medium (e.g., differentiation medium or expansion medium) relative to its basal medium.

[0072] As used herein, the term "supplementing" refers to adding the supplements of a culture medium (e.g., differentiation medium) to its basal medium. One or more supplements can be added to the basal medium of the culture medium before or during the use of the medium.

[0073] As used herein, the term "in vitro" generally refers to activities that occur outside of a living organism.

[0074] As used herein, the term "in vivo" generally refers to activities that occur inside of a living organism.

[0075] As used herein, the term "ex vivo" generally refers to activities that are performed outside of a living organism, e.g., experiments or measurements performed within or on living tissue in an artificial environment outside of a living organism, preferably with minimal alteration of natural conditions. In certain embodiments, an "ex vivo" procedure involves removing living cells or tissue from a living organism and culturing them in laboratory equipment, typically under sterile conditions, and generally for several hours or up to about 24 hours, but including up to 48 or 72 hours or longer, depending on the circumstances. In some embodiments, such tissue or cells can be collected and frozen and then thawed for ex vivo processing. Tissue culture experiments or procedures using living cells or tissue that last longer than a few days are generally considered "in vitro", although in certain embodiments the term can be used interchangeably with ex vivo.

[0076] As used herein, the term "feeder cells" or "feeders" refers to a type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, because the feeder cells provide growth factors and nutrients to support the second cell type. The feeder cells are optionally from a different species than the cells they support. For example, certain types of human cells (including stem cells) can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. When feeder cells are co-cultured with other cells, the feeder cells can typically be inactivated by irradiation or treatment with an anti-mitotic agent (e.g., mitomycin) to prevent them from growing beyond the cells they support. Feeder cells can include, for example, endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one specific type of feeder cell can be human feeder cells, such as human skin fibroblasts. Another type of feeder cell can be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation into a particular lineage, and promote maturation into specialized cell types, such as effector cells.

[0077] As used herein, a "feeder-free" (FF) environment refers to an environment that is substantially free of feeder cells or stromal cells and / or has not been pre-conditioned by a culture of feeder cells, such as culture conditions, cell cultures, or media.

[0078] 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 can 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 1,000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1×10 6 cells, at least about 1×10 7 cells, at least about 1×10 8 cells, at least about 1×10 9 cells, at least about 1×10 10 cells, at least about 1×10 11 cells, at least about 1×10 12 cells or more cells, wherein the cells express similar or different phenotypes.

[0079] As used herein, the term "effective amount" refers to the amount of a reagent that is sufficient to achieve a beneficial or desired result after administration. The amount of the reagent administered to a subject can depend on characteristics of the individual, such as general health, age, sex, weight, effective concentration of the administered cells (e.g., iNK cells), and tolerance to the drug. Those skilled in the art will be able to determine an appropriate dose based on these and other factors. An effective amount can be administered to a subject in one or more doses.

[0080] As used herein, the term "administering" a reagent to a subject includes any route by which the reagent is introduced or delivered to the subject to perform its intended function. Administration can be carried out by any suitable route, including but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes described herein. Administration includes self-administration and administration by another person.

[0081] As used herein, the terms "subject", "individual", or "patient" are used interchangeably and refer to a single organism, vertebrate, or mammal, and can include humans, non-human primates, rodents, etc. (e.g., which is the recipient of a particular medical intervention, or from which cells are harvested). In certain embodiments, the individual, patient, or subject is a human.

[0082] As used herein, the terms "treatment", "treat", and "treating" refer to a clinical intervention that is intended to reverse, alleviate, delay the onset of, or inhibit the progression of a disease, disorder, and / or condition or one or more of its symptoms, reduce the severity thereof, prevent or delay its recurrence, and / or improve one or more of the symptoms of the disease, disorder, and / or condition as described herein. After one or more symptoms have developed and / or after a disease has been diagnosed, a treatment, such as, for example, a treatment in the form of iNK cells or a population of iNK cells as described herein, can be administered to a subject. A treatment can be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of a disease. For example, a treatment can be administered to a susceptible individual (e.g., based on genetic or other predisposing factors) prior to the onset of symptoms. Treatment can also be continued after symptoms have resolved, for example, to prevent or delay their recurrence. A treatment can result in the improvement and / or resolution of one or more of the symptoms of a disease, disorder, and / or condition.

[0083] As used herein, the terms "prevent", "preventing", and "prevention" refer to reducing the probability of the occurrence of a disease, disorder, or condition in a subject who does not have but is at risk of developing or is predisposed to a disease, disorder, or condition.

[0084] The first differentiation method

[0085] A first aspect of the present disclosure relates to a method for promoting the directed differentiation (e.g., hematopoietic differentiation) (e.g., into iNK cells) of pluripotent stem cells (PSCs), the method comprising the steps of: contacting the PSCs with a maintenance medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation medium, or sequentially with a first differentiation medium and a second differentiation medium, to form mesoderm cells; contacting the mesoderm cells with a third differentiation medium to form hematopoietic endothelial (HE) cells; contacting the HE cells with a fourth differentiation medium to form hematopoietic progenitor (HP) cells; and contacting the HP cells with a fifth differentiation medium to obtain immature iNK cells, wherein a basal medium supplemented with a combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate is used as the basal medium for the fourth differentiation medium and / or the basal medium for the fifth differentiation medium.

[0086] According to a first aspect of the present disclosure, due to the stage-specific use (after the formation of hematopoietic endothelial (HE) cells) of a basal medium supplemented with a combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate (hereinafter referred to as the SFM basal medium), the above method can improve the differentiation efficiency of immature iNK (CD56+) cells, or HP cells and immature iNK (CD56+) cells. The above method can be completed in about 20 - 40 days and produce a large number of immature iNK cells. For example, 3×10 8 immature iNK cells can be derived from 1×10 6 hPSCs.

[0087] Figure 1 A diagram showing the process of generating iNK cells from hPSCs in an exemplary example of the method according to the present disclosure is shown. As Figure 1 shown, hPSCs first differentiate into hematopoietic progenitor cells (stage 1), and then further differentiate into immature NK cells (stage 2). Stage 1 is further divided into 5 sub-stages, namely 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 sub-stages in which hPSCs differentiate into mesoderm cells, stage 1-4 is the sub-stage in which mesoderm differentiates into HE cells, and stage 1-5 is the sub-stage in which HE differentiates into HP cells. Stage 3 is the stage of iNK cell expansion and maturation and will be described below.

[0088] According to the present disclosure, the SFM basal medium can be obtained by adding (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate to a commonly used basal medium in the art or any other suitable basal medium such as the IF-4 basal medium or the CD34A basal medium (for example, 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 of which are commercially available) and any combination thereof.

[0089] In certain embodiments, the SFM basal medium is used continuously throughout the step of contacting HE cells with a fourth differentiation medium to form HP cells, and the SFM basal medium is used intermittently throughout the step of contacting HP cells with a fifth differentiation medium to obtain immature iNK cells.

[0090] In certain embodiments, during the entire step of contacting HP cells with a fifth differentiation medium to obtain immature iNK cells, an SFM basal medium and another different basal medium are used separately as the basal medium of the fifth differentiation medium in any order.

[0091] In a preferred embodiment, the SFM basal medium is continuously used during the entire step of contacting HE cells with a fourth differentiation medium to form HP cells and during the entire step of contacting HP cells with a fifth differentiation medium to obtain immature iNK cells. According to the above embodiments, the differentiation efficiency of HP cells and immature iNK (CD56+) cells can be further improved.

[0092] In certain embodiments, the first to third differentiation basal media comprise different basal media.

[0093] In certain embodiments, the first to third differentiation basal media comprise the same basal medium, which helps to simplify the differentiation protocol of the present disclosure.

[0094] In the case where an SFM basal medium and another different basal medium are separately used during the step of contacting HP cells with a fifth differentiation medium to obtain immature iNK cells, the another different basal medium may be the same as, similar to, or different from the basal medium in the first to third differentiation media. In a preferred embodiment, a basal medium supplemented with only one or two of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate is used as the another different basal medium. In a more preferred embodiment, a basal medium supplemented with (i) a nicotinamide compound and only one of (ii) a heparin compound and (iii) human platelet lysate is used as the another different basal medium. In a most preferred embodiment, a basal medium supplemented with (i) a nicotinamide compound and (ii) human platelet lysate without (iii) a heparin compound is used as the another different basal medium.

[0095] In a preferred embodiment, the SFM basal medium is continuously used as one of the basal media of the fourth differentiation medium and the fifth differentiation medium, and a basal medium supplemented with (i) a nicotinamide compound and (ii) a heparin compound without (iii) human platelet lysate is continuously used as the other of the basal media of the fourth differentiation medium and the fifth differentiation medium.

[0096] The nicotinamide compound, heparin compound, and human platelet lysate will be described in detail below.

[0097] Nicotinamide compound

[0098] As used herein, "nicotinamide compounds" refers to nicotinamide and its analogs and metabolites of nicotinamide or nicotinamide analogs, such as, for example, NAD, NADH, and NADPH, and products derived from these compounds.

[0099] According to an embodiment of the present invention, the nicotinamide compound is selected from the group consisting of nicotinamide, nicotinamide analogs, nicotinamide metabolites, nicotinamide analog metabolites, and derivatives thereof.

[0100] Nicotinamide is the amide form of niacin, and both belong to the vitamin B3 family. They are precursors of nicotinamide adenine dinucleotide (NAD), which acts as a coenzyme in a variety of cellular processes, including energy metabolism and DNA repair. Nicotinamide can be converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT), which is then converted to NAD by nicotinamide mononucleotide adenylyltransferase (NMNAT). + 。

[0101] As used herein, "nicotinamide analogs" refers to any molecule known to act in a manner similar to nicotinamide. Examples of nicotinamide analogs include, but are not limited to, nicotinethioamides (thiol analogs of nicotinamide) and niacin. Examples of nicotinamide derivatives include, but are not limited to, substituted nicotinamide compounds and nicotinethioamides, as well as N-substituted nicotinamide compounds and nicotinethioamides.

[0102] Heparinoid compounds

[0103] As used herein, "heparinoid compounds" refers to heparin, its derivatives, or its salts.

[0104] According to an embodiment of the present disclosure, the heparinoid compound is selected from the group consisting of heparin, its derivatives, or its salts.

[0105] Heparin (a highly sulfated heparan sulfate glycosaminoglycan variant mainly produced and stored by mast cells) is considered to have the highest net negative charge density among all known biomolecules. Its negative charge binds to positively charged heparin-binding domains present in a large number of extracellular proteins. These proteins include, for example, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), and large extracellular structural molecules such as fibronectin and laminin.

[0106] Examples of heparin derivatives include, but are not limited to, substituted heparins. Examples of salts of heparin or its derivatives include, but are not limited to, sodium heparin and lithium heparin, and salts of substituted heparins.

[0107] Human platelet lysate (PLT)

[0108] The PLT used according to the present disclosure is commercially available. Human platelet lysate (PLT) can be derived from healthy donor human platelets and is rich in growth factors.

[0109] According to the present disclosure, the concentration of the niacinamide compound in the compositions of the present disclosure (e.g., SFM basal medium, or a basal medium supplemented with only one or two of (i) a niacinamide compound, (ii) a heparin compound, and (iii) human platelet lysate) is not particularly limited. In some embodiments, the concentration of the niacinamide compound in the compositions of the present disclosure (e.g., SFM basal medium, or a basal medium supplemented with only one or two of (i) a niacinamide compound, (ii) a heparin compound, and (iii) human platelet lysate) is from about 0.5 to about 20 mM, preferably from about 1 to about 10 mM, more preferably from about 1 to about 5 mM.

[0110] In certain embodiments, the compositions of the present disclosure (e.g., SFM basal medium, or a basal medium supplemented with only one or two of (i) a niacinamide compound, (ii) a heparin compound, and (iii) human platelet lysate) can contain a niacinamide compound at a concentration of about 0.1 mM or higher, 0.2 mM or higher, 0.5 mM or higher, 1 mM or higher, 2 mM or higher, 3 mM or higher, 4 mM or higher, 5 mM or higher, 6 mM or higher, 7 mM or higher, 8 mM or higher, 9 mM or higher, 10 mM or higher.

[0111] According to the present disclosure, the concentration of the heparin compound in the compositions of the present disclosure (e.g., SFM basal medium, or a basal medium supplemented with only one or two of (i) a niacinamide compound, (ii) a heparin compound, and (iii) human platelet lysate) is not particularly limited. In some embodiments, the concentration of the heparin compound in the compositions of the present disclosure (e.g., SFM basal medium, or a basal medium supplemented with only one or two of (i) a niacinamide compound, (ii) a heparin compound, and (iii) human platelet lysate) is preferably from about 0.1 to about 100 μg / mL, more preferably from about 0.5 to about 50 μg / mL.

[0112] In certain embodiments, the compositions 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 heparinoid compound, and (iii) human platelet lysate) can contain a heparinoid compound at a concentration of about 0.1 μg / ml or higher, 0.2 μg / ml or higher, 0.3 μg / ml or higher, 0.4 μg / ml or higher, 0.5 μg / ml or higher, 1 μg / ml or higher, 2 μg / ml or higher, 3 μg / ml or higher, 4 μg / ml or higher, 5 μg / ml or higher, 6 μg / ml or higher, 7 μg / ml or higher, 8 μg / ml or higher, 9 μg / ml or higher, 10 μg / ml or higher, 20 μg / ml or higher, 30 μg / ml or higher, 40 μg / ml or higher, 50 μg / ml or higher, or 100 μg / ml or higher.

[0113] According to the present disclosure, the concentration of human platelet lysate in the compositions 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 heparinoid compound, and (iii) human platelet lysate) is not particularly limited. In some embodiments, the concentration of human platelet lysate in the compositions 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 heparinoid compound, and (iii) human platelet lysate) is from about 0.1% to about 20% by volume, preferably from about 0.1% to about 10% by volume, more preferably from about 0.1% to about 5% by volume.

[0114] In certain embodiments, the compositions 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 heparinoid compound, and (iii) human platelet lysate) can contain 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 higher PLT by percentage (v / v).

[0115] In the case where a nicotinamide compound, a heparinoid compound, or PLT is present in a basal medium other than the SFM basal medium (e.g., a basal medium supplemented with only one or two of (i) a nicotinamide compound, (ii) a heparinoid compound, and (iii) human platelet lysate), examples of the nicotinamide compound, the heparinoid compound, and PLT and their concentrations in the medium are the same as those in the SFM basal medium.

[0116] Optionally, the SFM basal medium may contain glutamine or its derivatives. Examples of glutamine or its derivatives may include glutamine, GlutaMAX-1, L-glutamine, and L-alanyl-L-glutamine.

[0117] In certain embodiments, glutamine or its derivatives may be present in the SFM basal medium at a concentration of about 0.1 to about 5% (v / v). In certain embodiments, glutamine or its derivatives may be present 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).

[0118] Optionally, the SFM basal medium may contain one or more antioxidants. Examples of antioxidants may include ascorbic acid or its salts or derivatives, such as magnesium ascorbate, sodium ascorbate, ascorbyl glucoside, 3-ethyl ascorbic acid, ascorbyl tetraisopalmitate, ascorbic acid phosphate, and ascorbyl palmitate.

[0119] In certain embodiments, ascorbic acid or its salts or derivatives may be present in the SFM basal medium at a concentration of about 10 to about 200 μg / mL, preferably about 50 to about 150 μg / mL. In certain embodiments, ascorbic acid or its salts or derivatives 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.

[0120] 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.

[0121] Optionally, the SFM basal medium may contain mercaptoethanol (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.

[0122] 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, and preferably at a concentration of 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.

[0123] Optionally, the SFM basal medium may contain selenite, e.g., sodium selenite. In certain embodiments, selenite may be present in the SFM basal medium at a concentration of about 1 to about 50 ng / mL, and preferably at a concentration of about 5 to about 40 ng / mL. In certain embodiments, selenite may 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.

[0124] 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, and preferably at a concentration of 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.

[0125] Optionally, the SFM basal medium may contain pyruvate, e.g., sodium pyruvate. In certain embodiments, pyruvate may be present in the SFM basal medium at a concentration of about 10 to about 500 μg / mL, and preferably at a concentration of about 50 to about 200 μg / mL. In certain embodiments, pyruvate 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.

[0126] 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.

[0127] Except for the combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate, the basal media of the first to third differentiation media and the other different basal media may contain the same components as the above optional components in the SFM basal media. In certain embodiments, the concentrations of these components in the basal media of the first to third differentiation media and the other different basal media may be the same as those in the SFM basal media.

[0128] In certain embodiments, the basal media of the first to third differentiation media and / or the other different basal media may be IF-4 or CD34A basal media, and preferably IF-4 basal media.

[0129] In certain embodiments, the nicotinamide compound includes nicotinamide, and the heparin compound includes sodium heparin. In a preferred embodiment, the SFM basal media contains the IF-4 basal media in addition to the combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate. In a more preferred embodiment, the SFM basal media contains the IF-4 basal media in addition to the combination of (i) nicotinamide, (ii) sodium heparin, and (iii) human platelet lysate. In a most preferred embodiment, the SFM basal media includes the NKSFM basal media.

[0130] In certain embodiments, the other different basal media may be NKM or NKSFM-EP basal media, and preferably NKSFM-EP basal media.

[0131] The basal media of the SFM basal medium, the first to third differentiation media, and the 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, these basal media may contain mercaptoethanol at a concentration of 1 to 100 μM. In certain embodiments, these basal media may contain NEAA at a concentration of 0.1 to 5% (v / v). In certain embodiments, these basal media may contain BSA at a concentration of 0.1 to 20 mg / mL. In certain embodiments, these basal media may contain sulfate at a concentration of 0.1 to 10 ng / mL. In certain embodiments, these basal media may contain nitrate at a concentration of 0.1 to 10 μg / mL. In certain embodiments, these basal media may contain trace elements at a concentration of 0 to 1% (v / v). In certain embodiments, these basal media may contain CD lipid concentrate at a concentration of 0 to 1% (v / v). In certain embodiments, these basal media may contain human serum at a concentration of 1 to 20% (v / v).

[0132] In certain embodiments, the cell culture surface may 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 includes inoculating HP cells on a cell culture surface coated with a Notch pathway activator and an adhesion molecule. According to the above embodiments, compared with the embodiment in which the cell culture surface is not coated with any coating matrix, the differentiation efficiency of immature iNK (CD56+) cells can be further improved.

[0133] In a more preferred embodiment, the Notch pathway activator is selected from DLL4, DLL1, Jagged-1, Jagged-2, their variants, and any combination thereof, and the adhesion molecule is selected from VCAM1, fibronectin, laminin, vitronectin, MAdCAM-1, ICAM, their variants, and any combination thereof. In a most preferred embodiment, the cell culture surface is coated with DLL4 and VCAM1. The techniques for coating the cell culture surface are conventional in the art, and those skilled in the art can easily determine them.

[0134] In certain embodiments, the third differentiation medium is further supplemented with a Wnt signaling pathway inhibitor. According to the above embodiments, the differentiation efficiency of HP cells and immature NK cells can be further improved.

[0135] Wnt signaling pathway inhibitor

[0136] The third differentiation medium of the present disclosure can utilize a Wnt signaling pathway inhibitor as a supplement. A Wnt signaling pathway inhibitor refers to an antagonist of the Wnt signaling pathway (e.g., a reagent capable of downregulating the activity and / or amount of components involved in the Wnt signaling pathway).

[0137] The Wnt signaling pathway inhibitor can include, for example, a reagent that antagonizes one or more human FZD proteins, i.e., an FZD binder. The FZD binder can be an antibody or a polypeptide.

[0138] 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 agonist, an antibody that binds and inhibits the activity of a Wnt agonist and 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 agonist.

[0139] Non-limiting examples of Wnt signaling pathway inhibitors further preferably include one or more of the following: iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, Ciclopirox, Cardamonin, Diethyl benzylphosphonate, Disodium pamidronate hydrate, Ginsenoside Rh4, KY-05009, XAV-939, Foscenvivint (ICG-001), Capmatinib, Isoquercitrin, Macrophyllol, JW55, MSAB, IWR-1-endo, KY02111, FH535, WIKI4, CCT251545, Prodigiosin, KYA1797K, NCB-0846, LF3, iCRT14, Adavivint, Triptonide, M435-1279, and XAV939, more preferably CRT3, IWP-O1, IWP-2, IWP-3, IWP-4, Ciclopirox, Cardamonin, Diethyl benzylphosphonate, Disodium pamidronate hydrate, Ginsenoside Rh4, KY-05009, Isoquercitrin, Macrophyllol, JW55, MSAB, IWR-1-endo, FH535, WIKI4, CCT251545, KYA1797K, NCB-0846, iCRT14, Adavivint, M435-1279, and XAV939, and most preferably XAV939.

[0140] According to the present disclosure, the concentration of the Wnt signaling pathway inhibitor in the third differentiation medium is not particularly limited. In a preferred embodiment, the concentration of the Wnt signaling pathway inhibitor in the third differentiation medium is 1 to 30 μM.

[0141] In certain embodiments, the third differentiation medium of the present disclosure comprises a Wnt signaling pathway inhibitor at a concentration of about 0.1 μM or higher, about 0.5 μM or higher, e.g., about 1 - 30 μM, preferably about 1 - 20 μM, more preferably 1 - 10 μM, and most preferably 2 - 8 μM.

[0142] In certain embodiments, the third differentiation medium does not contain a TGF-β signaling pathway inhibitor as a supplement.

[0143] In the case where the third differentiation medium is further supplemented with a Wnt signaling pathway inhibitor, the EB can be contacted only with the first differentiation medium further supplemented with a Wnt signaling pathway activator. According to the above embodiments, by finely regulating the Wnt signaling during the hematopoietic differentiation process (first activating and then inhibiting the Wnt signaling of the cells, or downregulating the Wnt signaling), the differentiation efficiency of HP cells and immature NK cells can be further improved.

[0144] In the case where the third differentiation medium is further supplemented with a Wnt signaling pathway inhibitor, the EB can be sequentially contacted with the first differentiation medium further supplemented with a Wnt signaling pathway activator and the second differentiation medium further supplemented with a Wnt signaling pathway activator, wherein the Wnt signaling pathway activator in the second differentiation medium can be the same or different from the Wnt signaling pathway activator in the first differentiation medium and has an equal or lower concentration.

[0145] In a preferred embodiment, the second differentiation medium has the same composition as the first differentiation medium except that the concentration of the Wnt signaling pathway activator in the second differentiation medium is lower than that in the first differentiation medium. According to the above embodiments, since the transition of the Wnt signaling from activation to inhibition is smoother, the differentiation into HP (CD34+) cells and iNK (CD56+) cells can be further promoted.

[0146] According to the present disclosure, the concentration of the Wnt signaling pathway activator in the first and second differentiation media is not particularly limited. In certain embodiments, the first differentiation medium or the second differentiation medium of the present disclosure contains a Wnt signaling pathway activator at a concentration of 0.5 μM or higher, 1 μM or higher, 1.5 μM or higher, 2 μM or higher, 2.5 μM or higher, 3 μM or higher, 3.5 μM or higher, 4 μM or higher, 4.5 μM or higher, 5 μM or higher, 5.5 μM or higher, 6 μM or higher, 6.5 μM or higher, 7 μM or higher, 7.5 μM or higher, 8 μM or higher, 8.5 μM or higher, 9 μM or higher, 9.5 μM or higher, or 10 μM or higher. In certain embodiments, the first differentiation medium or the second differentiation 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, or more preferably about 1 to 5 μM.

[0147] In certain embodiments, the concentration of the Wnt signaling pathway activator in the second differentiation medium is at least 50% lower (including, for example, 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 medium.

[0148] In a preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation medium is 4 to 8 μM. In a more preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation medium is 5 to 8 μM.

[0149] Wnt signaling pathway activator

[0150] The technology of the present disclosure can utilize a Wnt signaling pathway activator as a supplement.

[0151] A Wnt signaling pathway activator refers to an agonist of the Wnt signaling pathway (e.g., a reagent capable of upregulating the activity and / or amount of components involved in the Wnt signaling pathway).

[0152] 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 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, and 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.

[0153] The Wnt signaling pathway activator further includes a GSK3 inhibitor. The GSK3 inhibitor can include, for example but not limited to, polynucleotides, polypeptides, and small molecules. Exemplary GSK3 inhibitors include, for example but not limited to, Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418, TDZD-8, BIO, BIO-Acetoxime (Acetoxime), (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, Pyridocarbazole-cyclopenadienylruthenium 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-AO 144-18, 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, and GF109203X.

[0154] 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, CT 99021, CT 20026, SB216763, AR-A014418, TDZD-8, BIO, BIO-Acetoxime, (5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, Pyrido-carbazole-cyclopentadienyl ruthenium complex, TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-Thioxo(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]oxadiazole, OTDZT, α-4-Dibromoacetophenone, AR-AO 144-18, 3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, TWS1 19 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 or OTDZT. In a most preferred embodiment, the Wnt signaling pathway activator is CHIR99021.

[0155] Additional reagents

[0156] In addition to the basal medium, the compositions of the present disclosure (e.g., the first to fifth differentiation media) can have or contain one or more additional reagents as supplements. One or more additional reagents can be independently added to the basal medium of the first to fifth differentiation media before or at the time of use of the medium, as required by the present disclosure. The additional reagents can include, for example, 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 they stimulate the differentiation of pluripotent stem cells into hematopoietic lineage cells, including HE, HP, or iNK cells.

[0157] Cytokines

[0158] Cytokines are a class of cell signaling molecules, including, for example, growth factors, interleukins, colony-stimulating factors, chemokines, interferons, lymphokines, and tumor necrosis factors. For example, 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. For example, examples of colony-stimulating factors include, but are not limited to, stem cell factor (SCF), Flt-3 ligand (Flt3L), thrombopoietin (TPO), G-CSF, and GM-CSF. For example, examples of tumor necrosis factors include, but are not limited to, tumor necrosis factor α (TNFα) and tumor necrosis factor β (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.

[0159] Growth factor

[0160] Growth factors are molecules that can stimulate a variety of cellular processes, including, for example, cell proliferation, cell migration, differentiation, and multicellular morphogenesis during development and tissue healing processes. Examples of growth factors include, but are not limited to, bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), endothelial cell growth factor (ECGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF).

[0161] In a preferred embodiment, the first to third differentiation media of the present disclosure are supplemented with one or more cell growth factors. In certain embodiments, the first and second differentiation media are each supplemented with BMP4, VEGF, and bFGF. In certain embodiments, the third and fourth differentiation media are each supplemented with VEGF and bFGF.

[0162] The concentration of the growth factor in the medium is not particularly limited and can be, for example, 1 to 200 ng / ml.

[0163] In a preferred embodiment, the first and second differentiation 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, Ventromorphins (SJ000291942, SJ000063181, SJ000370178), Isoliquiritigenin, Diosmetin, Apigenin, Biochanin. The most preferred embodiment of the BMP signaling pathway activator used in the present disclosure is BMP4. The concentration of BMP4 in the medium is not particularly limited as long as it activates the BMP signaling pathway, and examples thereof include but are not limited to 1 to 200 ng / ml, such as 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, 200 ng / ml.

[0164] In a preferred embodiment, the first to fourth differentiation media of the present disclosure are each supplemented with VEGF. In a preferred embodiment, the first to fourth differentiation 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 media are each independently further supplemented with VEGF at a concentration of 25 to 100 ng / mL, and preferably 50 to 100 ng / mL, because a higher concentration of VEGF in the first to third differentiation media can promote differentiation into HP and immature iNK cells.

[0165] In certain embodiments, the first to fourth differentiation 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. In certain embodiments, the first to fourth differentiation media of the present disclosure are each supplemented with VEGF at a concentration of about 20 - 50 ng / mL.

[0166] In a preferred embodiment, the first to fourth differentiation media of the present disclosure are each supplemented with bFGF. In a preferred embodiment, the first to fourth differentiation media are each independently further supplemented with bFGF at a concentration of 0.1 to 20 ng / mL.

[0167] In certain embodiments, each of the first to fourth differentiation 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 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 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 media of the present disclosure is supplemented with bFGF at a concentration of about 0.1 - 15 ng / mL. In certain embodiments, each of the first to fourth differentiation media of the present disclosure is supplemented with bFGF at a concentration of about 1 - 10 ng / mL. In certain embodiments, each of the first to fourth differentiation media of the present disclosure is supplemented with bFGF at a concentration of about 5 - 10 ng / mL.

[0168] Colony-stimulating factor

[0169] Colony-stimulating factors are cytokines capable of stimulating the differentiation and proliferation of hematopoietic stem cells or progenitor cells. 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, and multi-CSF.

[0170] In preferred embodiments, each of the fourth to fifth differentiation media of the present disclosure is supplemented with one or more colony-stimulating factors. The concentration of colony-stimulating factors in the medium is not particularly limited as long as they stimulate the differentiation and proliferation of hematopoietic stem cells or progenitor cells, and can be, for example, 1 to 200 ng / ml, and examples thereof include, but are not limited to: TPO at a concentration of 1 to 100 ng / ml, such as 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, 100 ng / ml; SCF at a concentration of 1 to 200 ng / ml, such as 1 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 80 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml; Flt-3L at a concentration of 1 to 200 ng / ml, such as 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 80 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml.

[0171] In certain embodiments, the fourth differentiation medium is supplemented with SCF, Flt3L, TPO, VEGF, and bFGF.

[0172] Interleukin

[0173] Interleukins are cytokines capable of stimulating the differentiation and proliferation of hematopoietic stem cells 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 medium of the present disclosure is supplemented with one or more interleukins. In a most preferred embodiment, the fifth differentiation 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 can be, for example, 1 to 200 ng / ml.

[0174] In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-7 at a concentration of about 10 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-7 at a concentration of about 25 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-7 at a concentration of about 50 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-7 at a concentration of about 1 - 50 ng / mL.

[0175] In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-3 at a concentration of about 5 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-3 at a concentration of about 10 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-3 at a concentration of about 20 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-3 at a concentration of about 1 - 20 ng / mL.

[0176] In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-2 at a concentration of about 100 IU / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-2 at a concentration of about 400 IU / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-2 at a concentration of about 700 IU / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-2 at a concentration of about 10 - 700 IU / mL.

[0177] In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-15 at a concentration of about 10 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-15 at a concentration of about 20 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-15 at a concentration of about 50 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-15 at a concentration of about 1-50 ng / mL.

[0178] In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-10 at a concentration of about 10 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-10 at a concentration of about 20 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-10 at a concentration of about 50 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-10 at a concentration of about 1-50 ng / mL.

[0179] In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-18 at a concentration of about 20 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-18 at a concentration of about 50 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-18 at a concentration of about 100 ng / mL. In certain embodiments, the fifth differentiation medium of the present disclosure is supplemented with IL-18 at a concentration of about 20-100 ng / mL.

[0180] In certain embodiments, the fifth differentiation medium is supplemented with SCF, Flt3L, TPO, IL-7, IL-3, IL-2, and IL-15.

[0181] In certain embodiments, the first to fifth differentiation media are chemically defined, serum-free, and xeno-free differentiation media. According to the above embodiments, such media can avoid the potential risk of xeno contamination, have reduced variability between batches, and can be more suitable for clinical and therapeutic use.

[0182] In a preferred embodiment, the step of contacting PSCs with a maintenance medium to form embryoid bodies (EBs) includes forming EBs from PSCs by suspension maintenance culture, hanging drop EB formation, or rotating EB formation. In a more preferred embodiment, EBs are formed by suspension maintenance culture.

[0183] In a preferred embodiment, the maintenance medium contains a ROCK inhibitor. The maintenance medium can be, for example, E8 or mTeSR or other similar media. In a more preferred embodiment, the ROCK inhibitor is selected from 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.

[0184] In a preferred embodiment, the method of the present disclosure is carried out under 3D culture conditions. According to the above embodiments, the 3D differentiation system greatly saves the culture space and culture volume, is more simple and easy to implement, and significantly increases the number of cells obtained, thereby facilitating the large-scale generation of hematopoietic lineage cells from hPSCs, such as HE cells, HP cells, and iNK cells.

[0185] The second differentiation method

[0186] A second aspect of the present disclosure relates to a method for promoting the directed differentiation (e.g., hematopoietic differentiation) (e.g., into hematopoietic lineage cells) of pluripotent stem cells (PSCs), which comprises the steps of: contacting the PSCs with a maintenance medium to form embryoid bodies (EBs); contacting the EBs with a first differentiation medium supplemented with a Wnt signaling pathway activator, or sequentially contacting the EBs with a first differentiation medium supplemented with a Wnt signaling pathway activator and a second differentiation medium supplemented with a Wnt signaling pathway activator to form mesoderm cells; and contacting the mesoderm cells with a third differentiation medium supplemented with a Wnt signaling pathway inhibitor to obtain hematopoietic endothelial (HE) cells. According to the second aspect of the present disclosure, since a Wnt signaling pathway inhibitor is used in a stage-specific manner in combination with a Wnt signaling pathway activator during the directed differentiation of pluripotent stem cells (PSCs), the differentiation efficiency of HE cells can be improved. The above method can be completed in about 3 days and produces a large number of HE cells.

[0187] In certain embodiments, the method of the present disclosure further comprises the step of contacting the HE cells with a fourth differentiation medium to obtain hematopoietic progenitor (HP) cells. According to the above embodiments, since a Wnt signaling pathway inhibitor is used in a stage-specific manner in combination with a Wnt signaling pathway activator during the directed differentiation of pluripotent stem cells (PSCs) into HP cells, the differentiation efficiency of HP cells can be improved. The above method can be completed in about 6 - 12 days and produces a large number of HP cells. For example, 9×10 6 HP cells can be derived from 1×10 6 hPSCs.

[0188] The step of contacting HE cells with a fourth differentiation medium to obtain hematopoietic progenitor cells (HP) can be carried out according to conventional methods in the art, or any other suitable method, for example, the corresponding methods described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0189] In certain embodiments, the method of the present disclosure further includes the step of contacting HP cells with a fifth differentiation medium to obtain immature iNK cells. According to the above embodiments, due to the stage-specific use of a Wnt signaling pathway inhibitor in combination with a Wnt signaling pathway activator during the directed differentiation of pluripotent stem cells (PSC) into iNK cells, the differentiation efficiency of immature iNK cells can be improved.

[0190] The step of contacting HP cells with a fifth differentiation medium to obtain immature iNK cells can be carried out according to conventional methods in the art, or any other suitable method, for example, the corresponding methods described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0191] The step of contacting PSC with a maintenance medium to form embryoid bodies (EB) can be carried out according to the corresponding methods described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0192] In a preferred embodiment, the EB is sequentially contacted with a first differentiation medium and a second differentiation medium to form mesoderm cells, wherein the Wnt signaling pathway activator in the second differentiation medium can be the same or different from that in the first differentiation medium and has an equal or lower concentration. According to the above embodiments, by finely regulating the Wnt signaling during hematopoietic differentiation (first activating and then inhibiting the Wnt signaling of cells, or gradually downregulating the Wnt signaling), the differentiation efficiency of HE cells, HP cells, or immature NK cells can be further improved.

[0193] In a preferred embodiment, the second differentiation medium has the same composition as the first differentiation medium, except that the concentration of the Wnt signaling pathway activator in the second differentiation medium is lower than that in the first differentiation medium. According to the above embodiments, since the transition of Wnt signaling from activation to inhibition is smoother, the differentiation into HE cells, HP (CD34+) cells, or iNK (CD56+) cells can be further promoted.

[0194] Examples of Wnt signaling pathway activators in the first and second differentiation media and their concentrations have been described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0195] In a preferred embodiment, the Wnt signaling pathway activator is selected from 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-thioxo(3-iodobenzyl)-5-(1-pyridinyl)[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 any combination thereof. In a most preferred embodiment, the Wnt signaling pathway activator is CHIR99021.

[0196] In a preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation medium is from 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation medium is from 4 to 8 μM. In a more preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation medium is from 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation medium is from 5 to 8 μM.

[0197] Examples of Wnt signaling pathway inhibitors in the third differentiation medium and their concentrations have been described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein).

[0198] In a preferred embodiment, the Wnt signaling pathway inhibitor is selected from iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, ciclopirox, myristicin, diethyl benzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, isoquercitrin, grandiflorol, 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.

[0199] In a preferred embodiment, the concentration of the Wnt signaling pathway inhibitor in the third differentiation medium is 1 to 30 μM.

[0200] In a preferred embodiment, the third differentiation medium is further supplemented with a TGF-β signaling pathway inhibitor. According to the above embodiments, the differentiation efficiency of HE cells or HP(CD34+) cells can be further improved.

[0201] In certain embodiments, the third differentiation medium of the present disclosure is supplemented with a TGF-β signaling pathway inhibitor at a concentration of about 1-20 μM, 1-15 μM, 1-10 μM, for example, 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.

[0202] TGF-β signaling pathway inhibitor

[0203] Transforming growth factor β (TGF-β) is part of a larger superfamily of secreted dimeric multifunctional proteins that also includes, for example, activins and bone morphogenetic proteins. TGF-β plays important roles in a variety of cellular functions, including embryogenesis, maintenance of tissue homeostasis in multicellular organisms, and tumor suppression.

[0204] Inhibitors of the TGF-β signaling pathway (also referred to as "TGF-β inhibitors") refer to antagonists of the TGF-β signaling pathway (e.g., reagents capable of downregulating the activity and / or amount of components involved in the TGF-β signaling pathway), including but not limited to antagonists of one or more of TGF-β1, TGF-β2, TGF-β3, type I TGF-β receptor (TβRI), type II TGF-β receptor (TβRII), and type III TGF-β receptor (TβRIII). Non-limiting examples of TβRI include ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7. Non-limiting examples of TβRII include TGFβR2, BMPR2, ACVR2A, ACVR2B, AMHR2. TβRIII includes, for example, TGFBR3.

[0205] Examples of inhibitors of the TGF-β signaling pathway also 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 inhibitor of the TGF-β signaling pathway comprises SB431542.

[0206] Examples of the basal media of the first to third differentiation media have been described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0207] In certain embodiments, the first to third differentiation basal media comprise the same basal media. According to the above embodiments, the differentiation protocol of the present disclosure can be simplified.

[0208] In certain embodiments, the basal media of the first to third differentiation media can be IF-4 or CD34A basal media, and preferably IF-4 basal media.

[0209] In certain embodiments, the basal media of the fourth differentiation media can be IF-4 or CD34A basal media.

[0210] In the case where the method of the present disclosure further includes the step of contacting HE cells with a fourth differentiation medium to obtain HP cells, the basal medium of the fourth differentiation medium can be a basal medium supplemented with (i) a nicotinamide compound and (ii) a heparin compound, and preferably, the basal medium of the fourth differentiation medium can be a basal medium supplemented with a combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate. According to the above embodiments, the differentiation efficiency of HP (CD34+) can be further improved.

[0211] Examples and concentrations of the nicotinamide compound, heparin compound, and human platelet lysate in the basal medium of the fourth differentiation medium have been described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0212] In certain embodiments, the concentration of the nicotinamide compound in the fourth differentiation medium is 0.5 to 20 mM.

[0213] In certain embodiments, the concentration of the heparin compound in the fourth differentiation medium is 0.1 to 100 μg / mL.

[0214] In certain embodiments, the concentration of human platelet lysate in the fourth differentiation medium is 0.1% to 20% by volume.

[0215] In a preferred embodiment, the nicotinamide compound includes nicotinamide, and the heparin compound includes sodium heparin.

[0216] In a preferred embodiment, the basal medium of the fourth differentiation medium contains, in addition to the combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate, an IF-4 basal medium. In a more preferred embodiment, the basal medium of the fourth differentiation medium contains, in addition to the combination of (i) nicotinamide, (ii) sodium heparin, and (iii) human platelet lysate, an IF-4 basal medium. In a most preferred embodiment, the basal medium of the fourth differentiation medium comprises an NKSFM basal medium.

[0217] In the case where the method of the present disclosure further includes the step of contacting HP cells with a fifth differentiation medium to obtain immature iNK cells, the basal medium of the fifth differentiation medium can be an IF-4 or CD34A basal medium, and preferably an IF-4 basal medium.

[0218] Additional reagents

[0219] In addition to the basal medium, the compositions of the second aspect of the present disclosure (e.g., the first to third differentiation media in the case of producing HE cells, the first to fourth differentiation media in the case of producing HP cells, and the first to fifth differentiation media in the case of producing immature cells) can have or contain one or more additional reagents as supplements. Depending on the requirements of the present disclosure, one or more additional reagents can be independently added to the basal medium of the first to fifth differentiation media before or during use of the medium. The additional reagents can include, for example, 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 they stimulate the differentiation of pluripotent stem cells into hematopoietic lineage cells, including HE, HP, or iNK cells.

[0220] Examples and concentrations of the corresponding additional reagents have been described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0221] In certain embodiments, the first to fourth differentiation 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 media are each independently further supplemented with VEGF at a concentration of 25 to 100 ng / mL, and preferably 50 to 100 ng / mL, because a higher concentration of VEGF in the first to third differentiation media can promote differentiation into HP and immature iNK cells.

[0222] In certain embodiments, the first to fifth differentiation media are chemically defined, serum-free, and animal component-free differentiation media. According to the above embodiments, such media can avoid the potential risk of animal component contamination, have reduced variability between batches, and can be more suitable for clinical and therapeutic use.

[0223] In a preferred embodiment, the method is carried out under 3D culture conditions. According to the above embodiments, the 3D differentiation system greatly saves culture space and culture volume, is more simple and easy to perform, and significantly increases the number of cells obtained, thereby facilitating the large-scale production of hematopoietic lineage cells from hPSCs, such as HE cells, and subsequent HP cells, NK cells, or other hematopoietic lineage cells, such as T cells.

[0224] Medium

[0225] The third aspect of the present disclosure relates to a culture medium for promoting the directed differentiation of pluripotent stem cells (PSCs) into hematopoietic lineage cells, which comprises a basal medium supplemented with a Wnt signaling pathway inhibitor. According to the third aspect of the present disclosure, by using a Wnt signaling pathway inhibitor during the process of promoting the directed differentiation of pluripotent stem cells (PSCs) into hematopoietic lineage cells, the above-mentioned culture medium can improve the differentiation efficiency of HE (KDR+) cells, HP (CD34+) cells or immature iNK (CD56+) cells.

[0226] The basal medium used in the third aspect of the present disclosure can be a common basal medium in the art or any other basal medium, as long as it does not prevent the promotion of the directed differentiation of pluripotent stem cells (PSCs) into hematopoietic lineage cells. The above basal medium can be commercially available or can be formulated as needed, for example, by adding one or more additives to a common basal medium in the art. Examples of common basal media in the art have been described elsewhere herein (for example, as described in the first differentiation method or the first aspect herein), and for the purpose of simplicity, these same descriptions are omitted herein. Examples of the above basal medium can include, for example, IF-4 and CD34A basal media.

[0227] Examples and concentrations of Wnt signaling pathway inhibitors have been described elsewhere herein (for example, as described in the first and second aspects herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0228] In a preferred embodiment, the Wnt signaling pathway inhibitor is selected from iCRT3, IWP-O1, IWP-2, IWP-3, IWP-4, ciclopirox, myristicin, diethyl benzylphosphonate, pamidronate disodium hydrate, ginsenoside Rh4, KY-05009, isoquercitrin, maclurin, 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.

[0229] In a preferred embodiment, the concentration of the Wnt signaling pathway inhibitor in the culture medium is 1 to 30 μM.

[0230] In a preferred embodiment, the culture medium is further supplemented with a TGF-β signaling pathway inhibitor. According to the above embodiment, the differentiation efficiency of HE cells or HP (CD34+) cells can be further improved.

[0231] Examples and concentrations of TGF-β signaling pathway inhibitors have been described elsewhere herein (e.g., as described in the first and second aspects herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0232] In a preferred embodiment, the culture medium is further supplemented with one or more growth factors. Examples and concentrations of growth factors have been described elsewhere herein (e.g., as described in the first and second aspects herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0233] 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.

[0234] In a preferred embodiment, the culture medium is further supplemented with VEGF at 15 - 100 ng / mL, and preferably 15 - 50 ng / mL.

[0235] In a preferred embodiment, the culture medium is a chemically defined serum-free and animal component-free differentiation medium.

[0236] Kit

[0237] A fourth aspect of the present disclosure relates to a kit comprising the above culture medium (e.g., the culture medium of the third aspect described herein).

[0238] In a preferred embodiment, the kit further comprises a first differentiation medium supplemented with a Wnt signaling pathway activator.

[0239] The first differentiation medium and the Wnt signaling pathway activator have been described elsewhere herein (e.g., as described in the first and second aspects herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0240] In a preferred embodiment, the kit further comprises a second differentiation medium supplemented with a Wnt signaling pathway activator, wherein the Wnt signaling pathway activator in the second differentiation medium may be the same or different from the Wnt signaling pathway activator in the first differentiation medium and has an equal or lower concentration.

[0241] The second differentiation medium and the Wnt signaling pathway activator have been described elsewhere herein (e.g., as described in the first and second aspects herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0242] In a preferred embodiment, the second differentiation medium has the same composition as the first differentiation medium, except that the concentration of the Wnt signaling pathway activator in the second differentiation medium is lower than that in the first differentiation medium.

[0243] In a preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation medium is 4 to 8 μM. In a more preferred embodiment, the concentration of the Wnt signaling pathway activator in the second differentiation medium is 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation medium is 5 to 8 μM.

[0244] In a preferred embodiment, the Wnt signaling pathway activators in the first and second differentiation media are each independently selected from 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-Thioxo(3-iodobenzyl)-5-(1-pyridinyl)[1,3,4]oxadiazole, α-4-Dibromoacetophenone, AR-AO 144-18, 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 any combination thereof.

[0245] The first and second differentiation media may each be supplemented with one or more growth factors.

[0246] Examples and concentrations of growth factors have been described elsewhere herein (e.g., as described in the first and second aspects herein), and for the purpose of simplicity, these same descriptions are omitted herein.

[0247] In a preferred embodiment, the first and second differentiation media are each supplemented with 15 - 100 ng / mL, and preferably 15 - 50 ng / mL, of VEGF.

[0248] In a preferred embodiment, all media in the kit are chemically defined serum-free and animal component-free differentiation media, which contain the same basal medium.

[0249] Method for producing iNK cells

[0250] The fifth aspect of the present disclosure relates to a method for producing iNK cells, which includes the above-described method for promoting the directed differentiation of pluripotent stem cells (PSCs) according to the first aspect, and steps of amplifying and maturing immature iNK cells ( Figure 1 phase 3 in 6 ). The above method can be completed within about 27 - 69 days and produce a large number of iNK cells. For example, more than 3×10 10 functional iNK cells can be derived from 1×10

[0251] The above method according to the first aspect has been described elsewhere herein (e.g., as described in the first differentiation method or the first aspect herein), and for the purpose of simplification, these same descriptions are omitted herein.

[0252] The steps for amplifying and maturing immature iNK cells can be carried out by conventional methods in the art or any other suitable method.

[0253] In a preferred embodiment, the steps for amplifying and maturing immature iNK cells can include contacting the immature iNK cells with an amplification and maturation medium in the presence or absence of feeder cells, the amplification and maturation medium comprising a basal medium supplemented with a combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate. According to the above embodiment, compared with commercial kits, the NK cell expansion fold, the percentage of mature iNK cells (CD56+CD3− cells), and the cytotoxicity against cancer cells can be improved.

[0254] For the basal medium of the amplification and maturation medium containing a nicotinamide compound, a heparin compound, and human platelet lysate, its description is the same as that of the SFM basal medium described elsewhere herein (e.g., as described in the first aspect herein), and for the purpose of simplification, these same descriptions are omitted herein.

[0255] In a preferred embodiment, the concentration of the nicotinamide compound in the amplification and maturation medium is 0.5 to 20 mM.

[0256] In a preferred embodiment, the concentration of the heparin compound in the amplification and maturation medium is 0.1 to 100 μg / mL.

[0257] In a preferred embodiment, the concentration of human platelet lysate in the amplification and maturation medium is 0.1% to 20% by volume.

[0258] In a preferred embodiment, the amplification and maturation 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 amplified and matured iNK cells against cancer cells can be enhanced. In a more preferred embodiment, the amplification and maturation medium is further supplemented with IL-10 and / or IL-18 because the NK cell amplification fold can be further increased. In the most preferred embodiment, the amplification and maturation medium contains IL-18.

[0259] There is no particular limitation on the concentration of interleukin such as IL-2, IL-10, IL-18 in the medium.

[0260] In certain embodiments, the amplification and maturation medium of the present disclosure is supplemented with IL-2 at a concentration of about 10 - 700 IU / mL, for example, about 100 IU / mL.

[0261] In certain embodiments, the amplification and maturation medium of the present disclosure is supplemented with IL-10 at a concentration of about 5 - 100 ng / mL, preferably 5 - 50 ng / mL, for example 20 ng / mL.

[0262] In certain embodiments, the amplification and maturation medium of the present disclosure is supplemented with IL-18 at a concentration of about 5 - 100 ng / mL, preferably 5 - 60 ng / mL, for example 50 ng / mL.

[0263] In certain embodiments, the amplification and maturation medium of the present disclosure is supplemented with SB431542 at a concentration of about 1 - 20 μM.

[0264] In a preferred embodiment, the nicotinamide compound includes nicotinamide, and the heparin compound includes sodium heparin.

[0265] In a preferred embodiment, the basal medium of the amplification and maturation medium contains IF-4 basal medium in addition to the combination of (i) nicotinamide compound, (ii) heparin compound, and (iii) human platelet lysate. In a more preferred embodiment, the basal medium of the amplification and maturation medium contains IF-4 basal medium in addition to the combination of (i) nicotinamide, (ii) sodium heparin, and (iii) human platelet lysate. In the most preferred embodiment, the basal medium of the amplification and maturation medium comprises NKSFM basal medium.

[0266] In certain embodiments, the steps of expanding and maturing the immature iNK cells further comprise 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 cell stimulation are utilized to expand and / or mature the iNK cells.

[0267] Optionally, the immature iNK cells can be cryopreserved prior to expansion and maturation. The cryopreserved iNK cells can be thawed later to continue the expansion and maturation process. Optionally, the functional or mature iNK cells can be cryopreserved at the end of the steps of expanding and maturing the immature iNK cells.

[0268] 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 Multiple Electrolytes injection or dextran injection.

[0269] In a preferred embodiment, the expansion and maturation medium is a chemically defined serum-free and animal component-free medium.

[0270] Cell population

[0271] The present disclosure provides hematopoietic lineage cells or cell populations thereof, including HE cells, hematopoietic progenitor cells (HP), immature natural killer (NK) cells, mature NK cells, and compositions comprising these cells (“cell compositions”).

[0272] The present disclosure provides a cell population in which at least 50% of the cells in the population are KDR+HE cells without any enrichment or purification. In a preferred embodiment, at least 60% of the cells in the population are KDR+HE cells without any enrichment or purification. In a more preferred embodiment, at least 70% of the cells in the population are KDR+HE cells without any enrichment or purification. In a most preferred embodiment, at least 80% of the cells in the population are KDR+HE cells without any enrichment or purification. The above cell population with a high proportion of HE cells is a relatively homogeneous population, has better differentiation potential, and can be used for later differentiation (e.g., HP differentiation) without any purification or enrichment. The above cell population can be produced by the methods elsewhere herein (e.g., the methods described in the first and / or second differentiation methods herein).

[0273] The present disclosure also provides a cell population, wherein at least 40% of the cells in the population are CD34+CD43-HP cells without any enrichment or purification. In a preferred embodiment, at least 50% of the cells in the population are CD34+CD43-HP cells without any enrichment or purification. In a more preferred embodiment, at least 60% of the cells in the population are CD34+CD43-HP cells without any enrichment or purification. In a further preferred embodiment, at least 70% of the cells in the population are CD34+CD43-HP cells without any enrichment or purification. In a most preferred embodiment, at least 80% of the cells in the population are CD34+CD43-HP cells without any enrichment or purification. In certain embodiments, at least 40% of the cells in the population are CD34+CD43-CD73-HP cells without any enrichment or purification. The above cell population with a high proportion of HP cells is a relatively homogeneous population, has better differentiation potential, and can be used for subsequent differentiation (e.g., NK differentiation) without any purification or enrichment. The above cell population can be produced by the methods elsewhere in this document (e.g., the methods described in the first and / or second differentiation methods herein).

[0274] The present disclosure also provides a cell population, wherein at least 40% of the cells in the population are immature CD56+CD3-iNK cells without any enrichment or purification. In certain embodiments, at least 50% of the cells in the population are immature CD56+CD3-iNK cells without any enrichment or purification. In a further embodiment, at least 60% of the cells in the population are immature CD56+CD3-iNK cells without any enrichment or purification. In a preferred embodiment, at least 70% of the cells in the population are immature CD56+CD3-iNK cells without any enrichment or purification. In a more preferred embodiment, at least 80% of the cells in the population are immature CD56+CD3-iNK cells without any enrichment or purification. The above cell population with a high proportion of iNK cells is a homogeneous population, has better differentiation potential, and can be used for subsequent expansion and maturation without any purification or enrichment. The above cell population can be produced by the methods elsewhere in this document (e.g., the methods described in the first and / or second differentiation methods herein).

[0275] In particular, the sixth aspect of the present disclosure provides a cell population produced by the first differentiation method described herein or the method described in the first aspect, or by the second differentiation method described herein or the method described in the second aspect.

[0276] In particular, a sixth aspect of the present disclosure also provides a cell population produced by the method for producing iNK cells described herein or the method described in the fifth aspect.

[0277] In particular, a seventh aspect of the present disclosure also provides a cell population in which more than 90% of the cells in the population are mature CD56+CD3− iNK cells without any enrichment or purification. In a preferred embodiment, at least 95% of the cells in the population are mature CD56+CD3− iNK cells without any enrichment or purification. In a more preferred embodiment, at least 98% of the cells in the population are mature CD56+CD3− iNK cells without any enrichment or purification. In a further preferred embodiment, at least 99% of the cells in the population are mature CD56+CD3− iNK cells without any enrichment or purification. In an even further preferred embodiment, at least 99.5% of the cells in the population are mature CD56+CD3− iNK cells without any enrichment or purification. In a most preferred embodiment, at least 99.8% (e.g., 99.9% and 100%) of the cells in the population are mature CD56+CD3− iNK cells without any enrichment or purification. The above cell populations with a high proportion of functional or mature iNK cells are highly homogeneous populations (high purity), have improved functions (e.g., higher cytotoxicity against tumor cells, improved homing to tissues and targets, secretion of high levels of pro-inflammatory cytokines), and can be better applied to clinical and therapeutic uses without any purification or enrichment. The above cell populations can be produced by the methods elsewhere herein (e.g., the method for producing iNK cells described herein or the method described in the fifth aspect).

[0278] The functional or mature iNK cells and their cell populations of the present disclosure have an improved surface receptor expression profile compared to primary NK cells or iNK cells reported in the literature (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), indicating better function.

[0279] The functional or mature iNK cells and cell populations of the present disclosure have lower expression of inhibitory receptors (e.g., NKG2A and KIRe1) compared to primary NK cells, indicating better function (e.g., activation against target cells). In certain embodiments, the functional or mature iNK cells and cell populations of the present disclosure have at least 2-fold lower expression of inhibitory receptors (e.g., NKG2A and KIRe1) compared to primary NK cells, indicating a great improvement in function (e.g., activation against target cells). In certain embodiments, the functional or mature iNK cells and cell populations of the present disclosure have at least 3-fold lower expression of inhibitory receptors (e.g., NKG2A and KIRe1) compared to primary NK cells.

[0280] For example, fewer than 20% of the cells in iNK cells are NKG2A+ cells. In a preferred embodiment, fewer than 17% of the cells in iNK cells are NKG2A+ cells.

[0281] For example, fewer than 20% of the cells in iNK cells are KIRe1+ cells. In a preferred embodiment, fewer than 12% of the cells in iNK cells are KIRe1+ cells.

[0282] The functional or mature iNK cells and cell populations of the present disclosure have higher expression of chemokine receptors compared to primary NK cells, indicating better function than primary NK cells (e.g., homing to tissues and targets).

[0283] In certain embodiments, the functional or mature iNK cells and cell populations of the present disclosure have at least 19-fold higher expression of chemokine receptors (e.g., CCR6) compared to primary NK cells, indicating a great improvement in function (e.g., homing to tissues and targets). In certain embodiments, the functional or mature iNK cells and cell populations of the present disclosure have at least 20-fold higher expression of chemokine receptors (e.g., CCR6) compared to primary NK cells.

[0284] For example, at least 70% of the cells in iNK cells are CCR6+ cells. In a preferred embodiment, at least 80% of the cells in iNK cells are CCR6+ cells.

[0285] In a preferred embodiment, the 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.

[0286] For example, at least 60% of the cells in iNK cells are NKG2D+ cells. In a preferred embodiment, at least 70% of the cells in iNK cells are NKG2D+ cells.

[0287] For example, at least 80% of the cells in the iNK cells are NKp30+ cells. In a preferred embodiment, at least 90% of the cells in the iNK cells are NKp30+ cells.

[0288] In certain embodiments, more than 90% of the cells in the iNK cells are CD45+ iNK cells. In a more preferred embodiment, more than 95% of the cells in the iNK cells are CD45+ iNK cells. In a most preferred embodiment, more than 99% of the cells in the iNK cells, including 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and 100% of the cells are CD45+ iNK cells.

[0289] 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+.

[0290] Pharmaceutical composition

[0291] The present disclosure provides a pharmaceutical composition comprising any cell population as described in the cell populations herein and a pharmaceutically acceptable carrier. The cells in the cell population can be in an effective amount suitable for treating a condition or disease of a subject.

[0292] In particular, the eighth aspect of the present disclosure provides a pharmaceutical composition, the pharmaceutical composition comprising the cell population as described in 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 treating a condition or disease of a subject.

[0293] In certain embodiments, the cell population can be formulated into a pharmaceutical composition (e.g., comprising a pharmaceutically acceptable carrier or excipient). The cell population can also be emulsified or presented as a liposomal composition, provided that the emulsification process does not adversely affect cell viability. The cells, or the cells and any other active ingredient, can be mixed with a pharmaceutically acceptable and compatible excipient of the active ingredient in an effective amount suitable for the treatment methods described herein.

[0294] Pharmaceutical carriers are well known in the art. Exemplary pharmaceutical carriers are sterile aqueous solutions that contain nothing other than the active ingredient and water, or contain a buffer at physiological pH (e.g., sodium phosphate), physiological saline, or both (e.g., phosphate buffered saline). Further, the aqueous carrier may contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. The liquid composition may also contain a liquid phase in addition to not containing water. Examples of such additional liquid phases are glycerol, vegetable oils (e.g., cottonseed oil), and water-oil emulsions. The amount of the cell population used in a pharmaceutical composition effective in treating a particular disease or condition may depend on the nature of the disease or condition and can be determined by standard clinical techniques.

[0295] Routes of administration include, but are not limited to, injection and infusion. In certain embodiments, injection includes, but is not limited to, intravenous, intrathecal, intraperitoneal, intraspinal, intracerebrospinal, and intrasternal infusion. In certain embodiments, the route is intravenous. In certain embodiments, the cells described herein are administered as a bolus or by continuous infusion (e.g., intravenous infusion) over a period of time. In certain embodiments, the cells described herein are administered in several doses over a period of time (e.g., several infusions over a period of time). The cells described herein can be administered in a single dose or in 2, 3, 4, 5, 6, or more doses (or infusions).

[0296] In certain embodiments, the pharmaceutical composition comprises a cell population allogeneic to the subject. In certain embodiments, the pharmaceutical composition comprises the cell population described herein autologous to the subject.

[0297] Use

[0298] The hematopoietic lineage cells or cell populations of the present disclosure can be widely used for treating or preventing a variety of disorders or diseases, including cancer, autoimmune diseases, and blood diseases. In particular, the cell populations of the seventh aspect of the present disclosure have improved functions (e.g., higher cytotoxicity against tumor cells, improved homing to tissues and targets, secretion of high levels of pro-inflammatory cytokines), and thus can be better applied to the clinical and therapeutic uses of cancer. The cell populations of the seventh aspect of the present disclosure can also be used to kill a variety of microorganisms, such as viruses, bacteria, etc., as well as senescent cells, etc.

[0299] The present disclosure also provides the use of any of the cell populations described herein in the preparation of a medicament for treating or preventing cancer, autoimmune diseases, or blood diseases.

[0300] In particular, the ninth aspect of the present disclosure provides the use of the cell population of the seventh aspect in the preparation of a medicament for treating or preventing cancer.

[0301] Administration of the cell populations or pharmaceutical compositions of the present disclosure to a subject in need thereof can treat or prevent a variety of cancers.

[0302] Examples of cancers include, but are not limited to, adrenal cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, malignant epithelial tumors, cervical cancer, colon cancer, colorectal cancer, corpus 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 myelogenous leukemia, acute myeloid leukemia, myelomonocytic leukemia, melanoma, large cell membrane lung cancer, ovarian cancer, non-small cell lung cancer, or small cell lung cancer and their metastases. In a preferred embodiment, the cancers include chronic myelogenous leukemia, acute myeloid leukemia, myelomonocytic leukemia, melanoma, large cell membrane lung cancer, ovarian cancer, non-small cell lung cancer or small cell lung cancer. In a more preferred embodiment, the cancers are acute myeloid leukemia, melanoma, small cell lung cancer, large cell membrane lung cancer, ovarian cancer or non-small cell lung cancer. The cell populations or pharmaceutical compositions of the present disclosure have much better effects for treating or preventing these cancers compared to primary NK cells.

[0303] 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 a pharmaceutical composition (e.g., according to the eighth aspect) as described herein. The pharmaceutical compositions, cell compositions or populations of the present disclosure can be administered before, during and / or after the onset of a disease, disorder and / or condition (e.g., cancer).

[0304] In certain embodiments, the subject has a disease, disorder or condition that can be treated by 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 (e.g., a therapy in which the 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.

[0305] Characterization method

[0306] Methods for characterizing hematopoietic lineage cells, including HE cells, HP cells, and NK cells (e.g., iNK cells as described herein), including characterizing cell phenotype and / or functionality, are known to those of skill in the art. These methods include, but are not limited to, morphological analysis, flow cytometry, and / or gene expression profiling. One or more characterization methods can be used to determine one or more cell markers 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, gene expression profiling is used to characterize a particular population of cells. In some such embodiments, the transcriptional signatures characteristic of a particular cell type (e.g., primary NK cells) in a sample of a cell population or cell composition are evaluated.

[0307] In additional instances, cells of a particular population or cell composition can be characterized by flow cytometry. In certain embodiments, the presence and / or proportion of one or more cell surface markers and / or one or more intracellular markers in a sample of a cell population can be evaluated. Those of skill in the art will understand that cell surface markers can represent different lineages. For example, pluripotent cells can be identified by one or more of any number of markers associated with such cells, including, for example, CD34. Cells (e.g., cell compositions as described herein) can be identified by markers indicative of a certain degree of differentiation (e.g., partial differentiation). For example, markers of differentiated cells can include those associated with hematopoietic progenitor cells, such as, for example, CD45, CD34. In certain embodiments, markers of differentiated cells can be associated with NK cells, such as, for example, CD56, CD3, CD45, natural killer group 2 member A (NKG2A), killer immunoglobulin-like receptor (KIR) (e.g., KIRe1), C-C motif chemokine receptor 6 (CCR6), NKG2D, and NKp30.

[0308] General methods

[0309] In practicing the present disclosure, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology, and recombinant DNA are used. See, for example, Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; the series Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et 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 ed.; Gait, ed. (1984) Oligonucleotide Synthesis; U.S.Patent 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; Maye 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.

[0310] Working Examples

[0311] Materials and Methods

[0312] All culture media reagents and devices used in all examples of the present disclosure are commercially available. The steps of the methods used throughout the examples will be described with reference to Figure 1 for description.

[0313] Examples 1-4: Development of NKSFM

[0314] This example demonstrates the process of developing NKSFM, an exemplary serum-free and animal component-free SFM basal medium. The development originated from the exploration of nicotinamide (NAM), sodium heparin, and human platelet lysate in the expansion and maturation of PBNK cells as surrogate cells.

[0315] Example 1

[0316] The effect of nicotinamide (NAM) concentration in an exemplary basal medium on NK cell expansion was evaluated. The above basal medium contained the following components: IMDM (Sigma): RPMI 1640 (Gibco) (50%:50%); about 1.3 ng / mL of copper sulfate, about 3 μM of iron sulfate, about 0.432 μg / mL of zinc sulfate, about 1 - 50 ng / mL of sodium selenite, about 110 μg / mL of sodium pyruvate, 0.1 - 20 μg / mL of insulin, 1 - 200 μg / mL of transferrin, about 1% (v / v) of GlutaMAX-1, 0.1 - 20 mg / mL of HSA, 1 - 400 μM of MTG, about 80 μg / mL of ascorbic acid, and NAM at the indicated concentrations. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using Ficoll-Paque. On day 0, PBMCs were plated at 1×10 6 cells / mL in the basal medium with NAM (0, 1, 2, 2.5, 3, 3.5, 4, or 5 mM) in 48-well plates and stimulated for 3 days with the following reagents: immobilized CD16 antibody (Biolegend, 302014) (135 ng / cm 2 ), IL-2 (700 IU / mL), IL-15 (10 ng / mL), and OK432 (0.01 KE / mL). 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). Fresh medium was added every other day to maintain the cell density below 2×10 6 cells / mL. Cells were collected on day 14 and stained with CD56-PE and CD3-PerCP-Cy5.5 antibodies, and the percentage of CD56 + CD3 - NK cells was analyzed by flow cytometry. Figure 2 The results showed that the concentration of NAM in the basal medium significantly affected the NK cell expansion fold ( Figure 2 A), while having a minimal effect on the percentage of CD56 + CD3 - NK cells ( Figure 2 B).

[0317] Example 2

[0318] The effect of sodium heparin concentration on NK cell expansion in an exemplary NKSFM was also evaluated. The NKSFM contains the following components: IMDM (Sigma): DMEM / F12 (Gibco) (50%:50%); about 20 μM ethanolamine, about 1 - 50 ng / mL sodium selenite, about 110 μg / mL sodium pyruvate, 0.1 - 20 μg / mL insulin, 1 - 200 μg / mL transferrin, about 1% (v / v) GlutaMAX-1, 0.1 - 20 mg / mL human serum albumin, 1 - 400 μM MTG, about 80 μg / mL ascorbic acid, about 1 - 5 mM NAM, 4% (v / v) PLT, and sodium heparin at the indicated concentrations. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque. On day 0, PBMCs were plated at 1×10 6 cells / mL in NKSFM with sodium heparin (0, 0.5, 1, 3, 10, 30, or 50 μg / mL) in 48-well plates and stimulated for 3 days with the following reagents: immobilized CD16 antibody (Biolegend, 302014) (135 ng / cm 2 ), OK432 (0.01 KE / mL), NeoIL-2 (50 ng / mL), IL-12 (10 ng / mL), IL-18 (50 ng / mL). On day 3, the cells were resuspended in NKSFM supplemented with NeoIL-2 (10 ng / mL). Fresh medium was added every other day to maintain the cell density below 2×10 6 cells / mL. Cells were collected on day 13 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 NK cells against K562 tumor cells was evaluated by CFSE / 7-AAD cytotoxicity assay. Briefly, K562 tumor cells were labeled with CFSE before co-culture with NK cells at an E:T of 3:1. After incubation for 4 hours, the cells were stained with 7-AAD (live / dead staining) and analyzed by flow cytometry. Figure 3 The results showed that the concentration of sodium heparin in NKSFM significantly affected the fold expansion of NK cells ( Figure 3 A), while having minimal effect on the percentage of CD56 + CD3 - NK cells ( Figure 3 B) and specific lysis of K562 tumor cells ( Figure 3 C).

[0319] Example 3

[0320] The effect of the percentage (v / v) of PLT in the exemplary NKSFM on NK cell expansion was evaluated. 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 0.5 - 50 μg / mL heparin sodium, approximately 1 - 5 mM NAM, and PLT at the indicated concentrations. PBMC were separated by density gradient centrifugation using Ficoll-Paque. On day 0, PBMC were plated at 1×10 6 cells / mL in NKSFM with PLT (0, 0.5, 1, 2, 3, or 4%) in 48-well plates and stimulated for 3 days with the following reagents: immobilized CD16 antibody (135 ng / cm 2 ), OK432 (0.01 KE / mL), IL-12 (10 ng / mL), IL-15 (50 ng / mL), IL-18 (50 ng / mL). On day 3, the cells were resuspended in basal medium supplemented with IL-2 (700 IU / mL) and IL-15 (10 ng / mL). Fresh medium was added every other day to maintain the cell density below 2×10 6 cells / mL. Cells were collected on day 14 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 evaluated by CFSE / 7-AAD cytotoxicity assay. Briefly, K562 tumor cells were labeled with CFSE before co-culture with NK cells at an E:T of 1:1. After incubation for 4 hours, the cells were stained with 7-AAD (live / dead staining) and analyzed by flow cytometry. As Figure 4 shown, the PLT concentration in NKSFM affected NK cell expansion to some extent ( Figure 4 A), while having minimal effect on the percentage of CD56 + CD3 - NK cells ( Figure 4 B) and specific lysis of K562 tumor cells ( Figure 4 C).

[0321] Example 4

[0322] Finally, the exemplary NKSFM was compared with commercial feeder-free or feeder cell-dependent NK cell expansion systems. NKSFM contains the following components: IMDM (Sigma): DMEM / F12 (Gibco) (50%:50%); about 20 μM ethanolamine, about 1 - 50 ng / mL sodium selenite, about 110 μg / mL sodium pyruvate, 0.1 - 20 μg / mL insulin, 1 - 200 μg / mL transferrin, about 1% (v / v) GlutaMAX-1, 0.1 - 20 mg / mL human serum albumin, 1 - 400 μM MTG, about 80 μg / mL ascorbic acid, about 1 - 5 mM NAM, 1% (v / v) PLT, and 0.5 - 50 μg / mL heparin sodium. Four conditions were tested.

[0323] Condition 1. NKSFM (feeder-free)

[0324] PBMC were separated by density gradient centrifugation using Ficoll-Paque. On day 0, PBMC were plated at 1×10 6 cells / mL in NKSFM in 6-well plates and stimulated for 3 days with the following reagents: immobilized CD16 antibody (Biolegend, 302014) (135 ng / cm 2 ), OK432 (0.01 KE / mL), NeoIL-2 (50 ng / mL), IL-12 (10 ng / mL), IL-18 (50 ng / mL). On day 3, the cells were resuspended in NKSFM supplemented with NeoIL-2 (10 ng / mL). Fresh medium was added every other day to maintain the cell density below 2×10 6 cells / mL. Cells were harvested on day 14 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 NK cells against K562 tumor cells was evaluated by CFSE / 7-AAD cytotoxicity assay. Briefly, K562 tumor cells were labeled with CFSE before co-culture with NK cells at an E:T of 3:1. After incubation for the specified time, the cells were stained with 7-AAD (live / dead staining) and analyzed by flow cytometry.

[0325] Condition 2. Commercial kit (feeder-free)

[0326] Use the feeder-free commercial kit (Baso, 3.0A) as Condition 2. The feeder-free expansion of PBNK cells was carried out according to the manufacturer's protocol. 5% (v / v) autoplasma was added throughout the expansion process.

[0327] Condition 3. NKSFM (with feeder cells)

[0328] Use the feeder cell-dependent NKSFM as Condition 3. The feeder cell-dependent expansion of PBNK cells was carried out according to the manufacturer's protocol, except that the plasma-containing medium was changed to NKSFM.

[0329] Condition 4. Commercial kit (with feeder cells)

[0330] Finally, use the feeder cell-dependent commercial kit (The Life ARK, ZY-NKZ-0104) as Condition 4. The feeder cell-dependent expansion of PBNK cells was carried out according to the manufacturer's protocol (The Life ARK, CN). 10% (v / v) autoplasma was added throughout the expansion process.

[0331] Figure 5 The results showed that in terms of the fold expansion of NK cells ( Figure 5 A), the percentage of CD56+CD3− cells ( Figure 5 B), and the lysis rate of K562 cells ( Figure 5 C), without feeder cells, NKSFM was significantly superior to the commercial kit, and in the presence of feeder cells, it also led to increased NK cell expansion compared to the commercial kit.

[0332] In summary, the above experiments demonstrated that NKSFM can be used to produce NK cells (e.g., for clinical use). It was found that the key components of NKSFM, NAM, sodium heparin, and PLT, are crucial for the expansion and function of NK cells. Most commercial NK cell expansion kits require serum and / or plasma to support NK cell expansion, or require sorting of CD56 + CD3 - NK cells to avoid unwanted CD3 + T cell expansion in PBMC. However, the use of NKSFM does not require serum or plasma or sorting to preferentially expand NK cells. In addition, NKSFM can be used for both feeder-free and feeder cell-dependent NK cell expansion, which are both significantly superior to the commercial kit in terms of cell number and cell lysis activity. NKSFM can be used not only to expand primary NK cells but also to expand iNK cells. In addition, the present inventors further found that the above NKSFM can also be used in the directed differentiation process of human pluripotent stem cells (hPSC).

[0333] Table 1.1 summarizes the exemplary basal media and their components utilized in all embodiments of the present disclosure.

[0334] Table 1.1: Components of the exemplary basal media.

[0335]

[0336]

[0337] + indicates present, - indicates absent, and +- indicates present or absent.

[0338] Composition of IF-4: IMDM (Sigma): DMEM / F12 (Gibco) (50%:50%); 0.1 - 20 mg / mL of human serum albumin (HSA, Sinopharm, CN); 1 - 400 μM of mercaptoethanol (MTG); approximately 80 μg / mL of ascorbic acid (Sigma); 1 - 200 μg / mL of transferrin (Sigma); 1 - 50 ng / mL of sodium selenite (Sigma); approximately 20 μM of ethanolamine (Sigma); and 0.1 - 20 μg / mL of insulin (Baiying, CN).

[0339] Composition of NKSFM: IMDM (Sigma): DMEM / F12 (Gibco) (50%:50%); approximately 1% (v / v) of GlutaMAX-1 (Invitrogen); 0.1 - 20 mg / mL of human serum albumin (HSA, Sinopharm, CN); 1 - 400 μM of mercaptoethanol (MTG); approximately 80 μg / mL of ascorbic acid (Sigma); 1 - 200 μg / mL of transferrin (Sigma); approximately 1 - 50 ng / mL of sodium selenite (Sigma); approximately 20 μM of ethanolamine (Sigma); approximately 110 μg / mL of sodium pyruvate (Sigma); 0.1 - 20 μg / mL of insulin (Baiying, CN); 1 - 5 mM of nicotinamide (NAM, Sigma); 0.5 - 50 μg / mL of heparin sodium (Thermo); and 0.5 - 4% (v / v) of human platelet lysate (PLT, BI).

[0340] Composition of NKM: IMDM (Sigma) (100%); approximately 1% (v / v) non-essential amino acids (NEAA, Invitrogen); 0.1 - 20 mg / mL human serum albumin (HSA, Sinopharm, CN); 1 - 5 mM nicotinamide (Sigma); approximately 50 μM 2-mercaptoethanol (β-ME, Sigma); and 10% (v / v) human serum.

[0341] Composition of NKSFM-EP: 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 iron sulfate (Sigma); approximately 0.65 μg / mL iron 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).

[0342] Composition of CD34A: IMDM (Sigma) (100%); approximately 1% (v / v) GlutaMAX-1 (Invitrogen); approximately 1% (v / v) non-essential amino acids (NEAA, Invitrogen); 0.1 - 20 mg / mL bovine serum albumin (BSA, Sigma); 1 - 400 μM 2-mercaptoethanol (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).

[0343] Examples 5 - 7: Novel cell-based media promote cell differentiation

[0344] This example demonstrates that the exemplary NKSFM promotes efficient iNK cell differentiation from hPSCs when used at a specific stage.

[0345] Example 5

[0346] Prepare hiPSCs as described in CN108373998B. On day -1, embryoid body (EB) formation (stage 1-1) is carried out by placing single-cell hiPSCs in E8 medium containing 10 μM Bretfeldin in a T25 flask on a bellydancer and culturing overnight. On day 0, lateral mesoderm differentiation (stage 1-2) is initiated by replacing the medium with 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 (stage 1-3) continues 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) for 1 day. On day 3, HE specification (stage 1-4) is 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 stage 1-4), the EBs are dissociated into single cells, stained with KDR-APC antibody, and the percentage of KDR + cells is detected by flow cytometry. HP differentiation (stage 1-5) is 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 (end of stage 1-5), the EBs are dissociated into single cells, stained with CD34-APC antibody, and the percentage of CD34 + HP cells is detected by flow cytometry. To initiate the differentiation of HP cells into iNK cells (stage 2), the EBs are collected and seeded in a 6-well plate coated with matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium for 14 days. The stage 2 differentiation medium contains 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). On day 21 (week 2 of stage 2), the derived cells are counted and stained with CD56-PE antibody, and the percentage of CD56 +Percentage of iNK cells. By comparing #2 with #1, the results in Table 2.1 show that when NKSFM is substituted for conventional IF-4 during Stages 1-5, the differentiation efficiency (CD34+%, CD56+%, and CD56+ cell count) of HE cells, HP cells, or iNK cells is increased (Table 2.1). This increase is attributed to the addition of NAM, PLT, and sodium heparin in NKSFM compared to IF-4. In addition, by comparing #3 (control experiment) with #2, the results show that NKSFM is not suitable for Stages 1-2, 1-3, and 1-4 because it results in very low KDR+%.

[0347] Table 2.1: Effects of different basal media during Stage 1 and Stage 2.

[0348]

[0349] Example 6

[0350] hiPSCs were prepared as described in CN108373998B. The basal medium used during the second stage of differentiation was also evaluated. On day -1, embryoid body (EB) formation (stage 1-1) was carried out by placing single-cell hiPSCs in E8 medium containing 10 μM Bretfeldin in a T25 flask on an orbital shaker and culturing overnight. 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 (15 ng / mL), bFGF (0.5 ng / mL), and CHIR99021 (4 μM) for 2 days. On day 2, lateral mesoderm differentiation (stage 1-3) continued 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) for 1 day. On day 3, HE specification (stage 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 (end of stage 1-4), the EBs were dissociated into single cells, stained with KDR-APC antibody, and the percentage of KDR+ cells was detected by flow cytometry. HP differentiation (stage 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 the differentiation of HP cells into iNK cells (stage 2), EBs on day 9 were collected and seeded in a 24-well plate coated with matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium for 13 days. The stage 2 differentiation medium contained 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). On day 22 (week 2 of stage 2), the derived cells were counted and stained with CD56-PE antibody, and the percentage of CD56+ iNK cells was detected by flow cytometry. Figure 6 showed that NKSFM used intermittently or continuously during the second stage significantly promoted the iNK cell differentiation efficiency (percentage of CD56+ cells ( Figure 6 A) and number of CD56+ cells ( Figure 6 B)) compared to other basal media used during the second stage.

[0351] Table 2.2: Basal medium used during the second stage process.

[0352]

[0353] Example 7

[0354] hiPSCs were prepared as described in CN108373998B. Further, the effect of plate coating during the second stage on iNK differentiation was evaluated. On day - 1, embryoid body (EB) formation (stage 1 - 1) was carried out by placing single - cell hiPSCs in E8 medium containing 10 μM Bretfeldin in a T25 flask on a bellydancer and culturing overnight. On day 0, lateral mesoderm differentiation (stage 1 - 2) was initiated by changing the medium 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) for 2 days. On day 2, lateral mesoderm differentiation (stage 1 - 3) continued 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) for 1 day. On day 3, HE specialization (stage 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), SB431542 (6 μM) for 1 day. On day 4 (end of stage 1 - 4), HP differentiation (stage 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 the differentiation of HP cells into iNK cells (stage 2), EBs on day 11 were collected and seeded in 24 - well plates without matrix or coated with DLL4 and VCAM1 in stage 2 differentiation medium for 14 days. The stage 2 differentiation medium contained NKSFM supplemented with SCF (20 ng / mL), Flt3L (10 ng / mL), IL - 7 (25 ng / mL), IL - 3 (5 ng / mL), IL - 15 (10 ng / mL). On day 25 (stage 2 - wk2), the derived cells were counted and stained with CD56 - PE antibody, and the percentage of CD56 + iNK cells was detected by flow cytometry. The results showed that compared with iNK differentiation without plate coating, plates coated with DLL4 and VCAM1 during the second stage significantly promoted iNK differentiation ( Figure 7 ). The differentiation from hPSCs to immature iNK cells was completed within only 26 days. In addition, when differentiating with NKSFM and DLL4 / VCAM1, the percentage of CD56 + CD3 - iNK cells at the end of stage 2 was as high as 80.56%.

[0355] Examples 8 - 10: Activation and inhibition of the Wnt pathway promote cell differentiation

[0356] This example shows that both the activation and inhibition of Wnt during specific sub - stages of the hematopoietic differentiation process are crucial for both hematopoietic differentiation and subsequent NK differentiation.

[0357] Example 8

[0358] hiPSCs were prepared as described in CN108373998B. On day - 1, embryoid body (EB) formation (stage 1 - 1) was carried out by placing single - cell hiPSCs in E8 medium containing 10 μM Bretfeldin in a T25 flask on a bellydancer and culturing overnight. 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), and CHIR99021 (4 μM) for 2 days. On day 2, lateral mesoderm differentiation (stage 1 - 3) continued 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) for 1 day. On day 3, HE specification (stage 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 with or without XAV939 (5 μM) and / or SB431542 (6 μM) for 1 day. On day 4, HP differentiation (stage 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 6 days. On day 10 (end of stage 1), the EBs were dissociated into single cells, stained with CD34 - APC antibody (BD, #555824), and CD34 was detected by flow cytometry +Percentage of HP cells. To initiate the differentiation of HP cells into iNK cells (Phase 2), EBs were collected and seeded in a 24-well plate coated with the stromal proteins DLL4 and VCAM1 in Phase 2 differentiation medium for 15 days. The Phase 2 differentiation medium contained 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). On day 25 (end of Phase 2), the derived cells were counted and stained with CD56-PE antibody (BD, #555516), and the percentage of CD56+ iNK cells was detected by flow cytometry. Figure 8 The results showed that compared with differentiation without XAV939 and SB431542 (control experiment) or differentiation using only SB431542, inhibition of the Wnt pathway by XAV939 during HE differentiation (Phases 1-4) significantly promoted CD34 + HP cells ( Figure 8 A) and CD56 + iNK cells ( Figure 8 B and 8C) output. In addition, compared with differentiation without XAV939 and SB431542 (control), although differentiation using only SB431542 showed a certain degree of improvement in the production of CD34 + HP cells, it did not show any improvement in the differentiation of iNK cells. In addition, the combination of SB431542 and XAV939 could further promote the production of CD34 + HP cells ( Figure 8 A).

[0359] Example 9

[0360] hiPSCs were prepared as described in CN108373998B. The concentration of the Wnt signaling pathway activator CHIR99021 used in the stage 1-2 process was optimized. On day -1, embryoid body (EB) formation (stage 1-1) was carried out by placing single-cell hiPSCs in E8 medium containing 10 μM Bretfeldin in a T25 flask on a bellydancer and culturing overnight. 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 any concentration of 5, 6, 7, or 8 μM) for 2 days. On day 2, lateral mesoderm differentiation (stage 1-3) continued 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) for 1 day. On day 3, HE specialization (stage 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), XAV939 (5 μM) for 1 day. On day 4, HP differentiation (stage 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), the EBs were dissociated into single cells, stained with CD34-APC antibody (BD, #555824), and the percentage of CD34 + HP cells was detected by flow cytometry. To initiate the differentiation of HP cells into iNK cells (stage 2), the EBs were collected and seeded in a T182 flask coated with the stromal protein DLL4 in stage 2 differentiation medium for 17 days. The stage 2 differentiation medium contained 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), IL-15 (10 ng / mL). On day 24 (end of stage 2), the cells were counted and stained with CD56-PE antibody (BD, #555516), and the percentage of CD56+iNK cells was detected by flow cytometry. Figure 9 The results showed that higher concentrations of CHIR99021 during the stage 1-2 process were generally more favorable for differentiation into CD34+ HP cells (Figure 9 A), but it is a bit disadvantageous for differentiation into CD56+iNK cells ( Figure 9 B and 9C).

[0361] Example 10

[0362] Prepare hiPSCs as described in CN108373998B. The concentration of the Wnt signaling pathway activator CHIR99021 used in the processes of the first to third stages was optimized. On day -1, embryoid body (EB) formation (the first to first stage) was carried out by placing single - cell hiPSCs in E8 medium containing 10 μM Bretazenib in a T25 flask on an oscillator and culturing overnight. On day 0, lateral mesoderm differentiation (the first to second stage) was initiated by replacing the medium with the first - to - second - stage 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) for 2 days. On day 2, lateral mesoderm differentiation (the first to third stage) continued for 1 day in the first - to - third - stage 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 any concentration of 0, 1, 2, 3, or 4 μM). On day 3, HE specialization (the first to fourth stage) was initiated by adding the first - to - fourth - stage differentiation medium containing IF - 4 basal medium supplemented with VEGF (50 ng / mL), bFGF (5 ng / mL), XAV939 (5 μM) for 1 day. On day 4, HP differentiation (the first to fifth stage) was initiated by adding the first - to - fifth - stage 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 (the end of the first stage), the EBs were dissociated into single cells, stained with CD34 - APC antibody (BD, #555824), and CD34 was detected by flow cytometry +Percentage of HP cells. To initiate the differentiation of HP cells into iNK cells (Stage 2), EBs were collected and seeded in a 6-well plate coated with the stromal proteins DLL4 and VCAM1 in Stage 2 differentiation medium for 17 days. The Stage 2 differentiation medium contained 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). On day 24 (end of Stage 2), the derived cells were counted and stained with a CD56-PE antibody (BD, #555516), and the percentage of CD56+ iNK cells was detected by flow cytometry. Figure 10 The results showed that a lower concentration of CHIR99021 during Stages 1-3 was somewhat unfavorable for differentiation into CD34+ HP cells ( Figure 10 A), but was generally more favorable for differentiation into CD56+ iNK cells ( Figure 10 B and 10C).

[0363] Thus, this example demonstrates that higher Wnt signaling activation during the initial stages (Stages 1-2) of lateral mesoderm differentiation, lower Wnt signaling activation during the later stages (Stages 1-3), and subsequent Wnt signaling inhibition during HE differentiation are crucial for the efficient differentiation of hPSCs into HE, HP, and iNK cells, particularly HP and iNK cells.

[0364] Examples 11-12: Regulating the Concentration of VEGF in Stage 1 Promotes Cell Differentiation

[0365] This example shows that regulating the concentration of VEGF during Stage 1 of differentiation is beneficial for the efficient differentiation of hPSCs into HE, HP, or iNK cells.

[0366] Example 11

[0367] Prepare hiPSC as described in CN108373998B. The effect of different VEGF concentrations on HE or HP cells in the substages of hPSC differentiation was evaluated. On day -1, embryoid body (EB) formation (stage 1-1) was performed by placing the single-cell hiPSC in E8 medium containing 10 μM Blebbistatin in a T25 flask on a shaker and culturing overnight. On day 0, the lateral mesoderm differentiation (stage 1-2) was started by replacing the culture medium with a stage 1-2 differentiation medium containing IF-4 basal medium and supplemented with BMP4 (25 ng / mL), VEGF, bFGF (0.5 ng / mL) and CHIR99021 (4 μM) at concentrations as shown in Table 3.1 for 2 days. On day 2, lateral mesoderm differentiation (stage 1-3) was continued for 1 day in stage 1-3 differentiation medium containing IF-4 basal medium and supplemented with BMP4 (25 ng / mL), VEGF and bFGF (0.5 ng / mL) at concentrations as shown in Table 3.1, and CHIR99021 (1 μM). On day 3, HE differentiation (stage 1-4) was started by adding stage 1-4 differentiation medium containing IF-4 basal medium and supplemented with VEGF, bFGF (5 ng / mL), XAV939 (5 ​​μM), and SB431542 (6 μM) at concentrations as shown in Table 3.1 for 1 day. On day 4 (end of stage 1-4), EBs were dissociated into single cells and stained with KDR-APC antibody (BD, #560495) to detect KDR by flow cytometry. + 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 and bFGF (5 ng / mL) at concentrations as shown in Table 3.1 for 6 days. On day 10 (end of Stage 1), EBs were dissociated into single cells and stained with CD34-APC antibody (BD, #555824) to detect CD34 by flow cytometry. + Percentage of HP cells. To start the differentiation of HP cells into iNK cells (stage 2), EBs were collected and seeded in 24-well plates coated with matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium for 14 days, wherein the stage 2 differentiation medium contained NKSFM and 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), IL-15 (10 ng / mL). On day 24 (end of stage 2), the morphology of iNK cells under different conditions was recorded, as shown in Figure 2. Figure 11 As shown in C.Figure 11 As shown in FIGS. 11A and 11B, different VEGF concentrations had minimal effect on the percentage of KDR+HE cells, but had significant effects on the percentage of CD34+HP cells and the efficiency of further differentiation into iNK cells. Specifically, higher VEGF concentrations during the 1-2 and 1-3 phases generally favored differentiation into HP cells and iNK cells, and higher VEGF concentrations during the 1-5 phase generally favored differentiation into HP cells but not into iNK cells.

[0368] Table 3.1: Effects of modulating VEGF concentration during the 1st phase on HE and HP cells.

[0369]

[0370] Example 12

[0371] hiPSCs were prepared as described in CN108373998B. The effects of different VEGF concentrations on HE, HP, or iNK cells during the sub-stages of hPSC differentiation were evaluated. On day -1, embryoid body (EB) formation (phase 1-1) was carried out by placing single-cell hiPSCs in E8 medium containing 10 μM Bretfeldin in a T25 flask on a bellydancer and culturing overnight. On day 0, lateral mesoderm differentiation (phase 1-2) was initiated by changing the medium to phase 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) for 2 days. On day 2, lateral mesoderm differentiation (phase 1-3) continued in phase 1-3 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 (1 μM) for 1 day. On day 3, HE specification (phase 1-4) was initiated by adding phase 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF at the concentrations shown in Table 3.2, bFGF (5 ng / mL), and XAV939 (5 μM) for 1 day. On day 4 (end of phase 1-4), the EBs were dissociated into single cells, stained with KDR-APC antibody (BD, #560495), and KDR was detected by flow cytometry. +Percentage of cells. 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 and bFGF (5 ng / mL) at the concentrations shown in Table 3.2 for 3 days. On day 7 (end of stage 1), the EBs were dissociated into single cells, stained with CD34 - APC antibody (BD, #555824), and CD34 was detected by flow cytometry. + Percentage of HP cells. To initiate the differentiation of HP cells into iNK cells (stage 2), the EBs were collected and seeded in 24 - well plates coated with matrix proteins DLL4 and VCAM1 in stage 2 differentiation medium for 14 days. The stage 2 differentiation medium contained 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), IL - 15 (10 ng / mL). On day 21 (end of stage 2), the derived cells were counted and stained with CD56 - PE antibody (BD, #555516), and the percentage of CD56 + iNK cells was analyzed by flow cytometry. Figure 12 The results showed that during stage 1, especially during the differentiation of hPSCs into HE cells (stages 1 - 2, 1 - 3, and 1 - 4), higher concentrations of VEGF significantly enhanced HP and iNK cell differentiation.

[0372] Table 3.2: Effects of modulating VEGF concentration during stage 1 on HE, HP, and iNK cells.

[0373]

[0374] Examples 13 - 14: Expansion and maturation of immature iNK cells through NKSFM

[0375] This example shows that the use of NKSFM can promote the expansion and maturation of NK cells, especially immature iNK cells.

[0376] Example 13

[0377] Prepare hiPSC as described in CN108373998B.On the -1 day, embryoid body (EB) formation (stage 1-1) is performed by placing the hiPSC of a single cell in the E8 medium containing 10 μM Blebbistatin in a T25 flask on a bellydancer and culturing overnight.On the 0th day, lateral mesoderm differentiation (stage 1-2) is started by replacing the culture medium with the 1-2 stage differentiation medium containing IF-4 basal medium and supplemented with BMP4 (25 ng / mL), VEGF (15 ng / mL), bFGF (0.5 ng / mL) and CHIR99021 (4 μM), and continuing for 2 days.On the 2nd day, lateral mesoderm differentiation (stage 1-3) is continued for 1 day in the 1-3 stage differentiation medium containing IF-4 basal medium and supplemented with BMP4 (25 ng / mL), VEGF (50 ng / mL) and bFGF (0.5 ng / mL) and CHIR99021 (1 μM). On day 3, HE specification (stage 1-4) was started by adding stage 1-4 differentiation medium containing IF-4 basal medium and supplemented with VEGF (15 ng / mL), bFGF (5 ng / mL), XAV939 (5 ​​μM) for 1 day. On day 4 (end of stage 1-4), HP differentiation (stage 1-5) was started by adding stage 1-5 differentiation medium containing IF-4 basal medium and supplemented with SCF (10 ng / mL), Flt3L (10 ng / mL), VEGF (15 ng / mL) and bFGF (5 ng / mL). To start the differentiation of HP cells into iNK cells (Phase 2), EBs on day 10 were collected and seeded in 24-well plates coated with DLL4 and / or VCAM1 for 14 days in Phase 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), IL-15 (10 ng / mL, Peprotech). On day 24 (end of Phase 2), EBs were added to a 50×10-well plate in a compound electrolyte injection solution (Zhendong Health, H20113035) supplemented with HSA (40 mg / mL) and DMSO (10%). 6Immature iNK cells at cells / mL were cryopreserved in liquid nitrogen. The thawed D24 iNK cells were co-cultured with feeder cells (Nuwacell, feeder cells:iNK cells = 1:1) in a 6-well plate in a stage 3 expansion and maturation medium, which contained NKSFM and was 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) that were genetically engineered to co-express mbIL-21 (membrane-bound IL-21), 41BBL, CD19, and CD64. The feeder cells were inactivated by gamma irradiation prior to co-culture with the iNK cells. On day 11 of co-culture, the iNK cells were collected and stained with a CD56-PE antibody (BD, #555516), and the percentage of CD56+ cells was analyzed by flow cytometry. After co-culture at an E:T of 3:1 for 4 hours, the cytotoxicity of the stage 3 iNK cells against K562 cells was detected by CFSE / 7AAD assay. As Figure 13 shown in A, IL-10 (20 ng / mL) and / or IL-18, especially IL-18, can significantly enhance the cytotoxicity of iNK cells.

[0378] Example 14

[0379] Prepare hiPSCs as described in CN108373998B. On day -1, embryoid body (EB) formation (stage 1-1) is carried out by placing single-cell hiPSCs in E8 medium containing 10 μM Bretfeldin in a T25 flask on a bellydancer and culturing overnight. On day 0, lateral mesoderm differentiation (stage 1-2) is initiated by replacing the medium with 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) for 2 days. On day 2, lateral mesoderm differentiation (stage 1-3) continues 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) for 1 day. On day 3, HE specification (stage 1-4) is initiated by adding stage 1-4 differentiation medium containing IF-4 basal medium supplemented with VEGF (15 ng / mL), bFGF (5 ng / mL), XAV939 (5 μM) for 1 day. On day 4 (end of stage 1-4), HP differentiation (stage 1-5) is 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 the differentiation of HP cells into iNK cells (stage 2), EBs on day 10 are collected and seeded in stage 2 differentiation medium in a 24-well plate coated with DLL4 and VCAM1 for 14 days. The stage 2 differentiation medium contains 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), IL-15 (10 ng / mL). On day 24 (end of stage 2), in dextran injection solution (SJZ No.4 Pharmaceutical, H13022493) supplemented with HSA (40 mg / mL) and DMSO (10%) at 50×10 6Immature iNK cells at cells / mL were cryopreserved in liquid nitrogen. Thawed D25 iNK cells were co-cultured with feeder cells (feeder cells:iNK cells = 0.5:1) in a 6-well plate in a stage 3 expansion and maturation medium for 2 weeks. The stage 3 expansion and maturation medium contained NKSFM basal medium and was supplemented with IL-2 (100 IU / mL) and with or without IL-18 (10 - 50 ng / mL, added during S3-wk1 or S3-wk2 as indicated). On day 14 of co-culture, iNK cells were collected, stained with CD56-PE antibody (BD, #555516), and the percentage of CD56+ cells was analyzed by flow cytometry. As Figure 13 shown in B, the combination of IL-18 and IL-2, especially the combination of IL-18 and IL-2 added during the initial stage of stage 3, could increase the amplification fold of iNK cells compared to IL-2 alone.

[0380] Examples 15 - 20: Exemplary characterization of hematopoietic lineage cells formed during the production of iNK cells from hPSCs

[0381] Example 15

[0382] Prepare hiPSCs as described in CN108373998B. On day -1, embryoid body (EB) formation (stage 1-1) was carried out by placing single-cell hiPSCs in E8 medium containing 10 μM Bretfeldin in a T25 flask on a bellydancer and culturing overnight. D-1 and D0 cells were analyzed by microscopy. On day 0, lateral mesoderm differentiation (stage 1-2) was initiated by replacing the medium with 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) for 2 days. On day 2, lateral mesoderm differentiation (stage 1-3) continued 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) for 1 day. On day 3, HE specification (stage 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), XAV939 (5 μM) for 1 day. At the end of stage 1-4, cells were analyzed by microscopy and FACS. On day 4 (end of stage 1-4), HP differentiation (stage 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). At the end of stage 1-5, cells were analyzed by microscopy and FACS. To initiate the differentiation of HP cells into iNK cells (stage 2), EBs on day 7 were collected and seeded in a 24-well plate coated with DLL4 and VCAM1 in stage 2 differentiation medium for 17 days. The stage 2 differentiation medium contained 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), IL-15 (10 ng / mL). At the end of stage 2, cells were analyzed by microscopy and FACS. On day 24 (end of stage 2), in a dextran injection solution (SJZ No.4 Pharmaceutical, H13022493) supplemented with HSA (40 mg / mL) and DMSO (10%) at 50×10 6Immature iNK cells at [number of cells] / mL were cryopreserved in liquid nitrogen. The thawed D24 iNK cells were co-cultured with feeder cells (feeder cells:iNK cells = 1:1) in a 6-well plate in the stage 3 expansion and maturation medium, which contained NKSFM basal medium supplemented with IL-2 (100 IU / mL) and IL-18 (10 ng / mL). On day 11 of co-culture, the iNK cells were collected and stained with CD56-PE antibody (BD, #555516), and CD56+ cells were examined by microscopy and FACS analysis. Figure 14 Shows the morphology of hematopoietic lineage cells formed during different differentiation stages of generating iNK cells from hPSCs. The results showed that during the first stage of iNK cell differentiation, the EB size gradually increased. After EB plating, small cell clusters formed around the EBs to which iNK cells attached during the second stage. During the third stage, iNK cells proliferated vigorously and were prone to aggregate into cell masses under static conditions. As Figure 15 shown in Figure 15 A, at the end of stages 1 - 4, the cells showed a high efficiency of lateral mesoderm and HE differentiation. As Figure 15 shown in Figure 15 B, at the end of stages 1 - 5, the percentage of CD34+CD43-HP cells was as high as 61.23%, indicating a high efficiency of HP differentiation. As

[0383] Example 16

[0384] To test the proliferation potential of our iNK cells, the immature iNK cells obtained from the second stage in Example 15 were co-cultured with feeder cells (Nuwacell) (feeder cells:iNK cells = 1:1) in a 6-well plate in the stage 3 expansion and maturation medium, which contained NKSFM basal medium supplemented with IL-2 (100 IU / mL) and IL-18 (10 ng / mL). The iNK cells were continuously expanded for 7 weeks with feeder cell stimulation performed weekly. The results showed that the iNK cells obtained by this method had a high proliferation potential and could be repeatedly stimulated for continuous expansion for at least 7 weeks ( Figure 16)。Compared to the repeated stimulation of hPSC-derived NK cells by aAPC (mbIL-21 artificial antigen-presenting cells) from other groups for sustained long-term expansion (Knorr DA et al., Stem Cells Transl Med. 2013 Apr;2(4):274-283), the amplification fold observed in the present invention is much higher. Therefore, the resulting immature iNK cells confirmed high amplification potential.

[0385] Example 17

[0386] To compare the phenotypes of primary NK cells and our iNK cells, the expression of NK cell surface receptors on PBNK cells and the iNK cells obtained in Example 15 was analyzed by flow cytometry and compared. The 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 Figure 17 shown, the expression of the activating receptors NKG2D and NKp30 is comparable in PBNK cells and iNK cells. However, compared to PBNK cells, iNK cells have much lower expression of the inhibitory receptors NKG2A and KIRe1, indicating less inhibition from target cells. In addition, compared to PBNK cells, iNK has much higher expression of the chemokine receptor CCR6, indicating improved tissue and target homing ( Figure 17 ). Interestingly, the cell surface marker expression on the iNK cells generated by the method described herein is also different from those in the literature. Therefore, compared to primary NK cells, iNK cells show a different surface receptor expression profile, indicating greatly improved function.

[0387] Example 18

[0388] To analyze the correlation between primary NK cells and our iNK cells, the total gene expression of the stage 3 iNK cells, PBNK cells and CBNK cells obtained in Example 17 was evaluated by microarray and dendrogram clustering analysis. The results demonstrated that iNK cells are similar to primary NK cells, but also represent an independent cell type distinct from PBNK cells and CBNK cells. Therefore, iNK cells are similar to primary NK cells in gene expression, but cluster as a unique cell type ( Figure 18)。

[0389] Example 19

[0390] To evaluate the cytolytic function of iNK cells compared to 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 from Example 17 were thawed for cytotoxicity assays. Different tumor cell lines were labeled with CFSE before co-culturing with NK cells at an E:T (E:T is the ratio of the number of effector cells to the number of target cells, where the effector cells are iNK cells or PBNK cells and the target cells are tumor cells) of 3:1. After incubation for the specified time, the cells were stained with 7-AAD (live / dead staining) and analyzed by flow cytometry. Compared to PBNK cells, iNK cells exhibited comparable cytotoxicity against K562 cells, Kasumi cells, MV-4-11 cells, H69 cells, and H146 cells, and much higher cytotoxicity against H460 cells, SKOV3 cells, A549 cells, SKMEL2 cells, DMS114 cells, MOLM13 cells, and H82 cells. The results showed that iNK cells were cytotoxic to a wide range of tumor cell lines and had comparable or enhanced activity compared to primary NK cells( Figure 19 )。

[0391] Example 20

[0392] To further evaluate the function of iNK cells, iNK cells obtained from 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 demonstrated that iNK cells could respond to the stimulation to secrete high levels of pro-inflammatory cytokines: TNF-α and IFN-γ( Figure 20 )。

[0393] Those skilled in the art will readily understand that the methods, compositions, and products described herein represent exemplary embodiments and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various alternatives and modifications can be made to the present disclosure herein without departing from the scope and spirit of the present invention.

[0394] All patents and publications mentioned in the specification indicate the level of those skilled in the art to which the present disclosure pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0395] The present disclosure is not limited to the specific embodiments described in this application, which are intended as separate illustrations of various aspects of the present disclosure. All various embodiments of the present disclosure will not be described herein. The terms and expressions employed are used as descriptive rather than restrictive terms, and in using such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications can be made within the scope of the claimed present disclosure. Accordingly, it should be understood that although the present disclosure has been specifically disclosed by the preferred embodiments and optional features, those skilled in the art can make modifications and variations to the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the invention defined by the appended claims.

Claims

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

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

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

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

5. The method according to claim 4, wherein the another different basal medium is supplemented with a nicotinamide compound and one selected from a heparin compound and human platelet lysate.

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

7. The method according to claim 1, wherein the basal medium supplemented with the combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate is continuously used as one of the basal media for the fourth differentiation medium and the fifth differentiation medium, and the basal medium supplemented with (i) a nicotinamide compound and (ii) a heparin compound without (iii) human platelet lysate is continuously used as the other of the basal media for the fourth differentiation medium and the fifth differentiation medium.

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

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

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

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

12. The method according to claim 11, wherein 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.

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

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

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

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

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

18. The method according to claim 17, wherein 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-Thioxo(3-iodobenzyl)-5-(1-pyridinyl)[1,3,4]oxadiazole, α-4-Dibromoacetophenone, AR-AO 144-18, 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 any combination thereof.

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

20. The method according to claim 17, wherein the concentration of the Wnt signaling pathway activator in the second differentiation media is from 0 to 4 μM, and the concentration of the Wnt signaling pathway activator in the first differentiation media is from 4 to 8 μM.

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

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

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

24. The method according to any one of claims 1-23, wherein the first to fourth differentiation 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 1-24, wherein the first to fifth differentiation media are chemically defined serum-free and animal component-free differentiation media.

26. The method according to any one of claims 1-25, wherein the method is carried out under 3D culture conditions.

27. A method for producing iNK cells, which comprises the method according to any one of claims 1-26, and steps for amplifying and maturing immature iNK cells.

28. The method according to claim 27, wherein the steps for amplifying and maturing immature iNK cells comprise: contacting the immature iNK cells with an amplification and maturation medium in the presence or absence of feeder cells, the amplification and maturation medium comprising a basal medium supplemented with a combination of (i) a nicotinamide compound, (ii) a heparin compound, and (iii) human platelet lysate.

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

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

31. The method according to claim 28 or 29, wherein the concentration of the heparin compound in the amplification and maturation medium is 0.1 to 100 μg / mL.

32. The method according to claim 28 or 29, wherein the concentration of the human platelet lysate in the amplification and maturation medium is 0.1% to 20% by volume.

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

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

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

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

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

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

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

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

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

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

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

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

45. A pharmaceutical composition comprising the cell population according to any one of claims 38 - 44, and a pharmaceutical carrier.

46. Use of the cell population according to any one of claims 38 - 44 in the preparation of a medicament for the treatment or prevention of cancer.

47. The use according to 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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