Compositions and methods for NK cell differentiation

Abstract

Methods and compositions for producing heterologous-free genes of hematopoietic progenitor cells and natural killer (NK) cells are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 350,755, filed on June 9, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Background Art

[0002] Natural killer (NK) cells are a type of cytotoxic innate lymphocytes that are usually identified as being positive for the cell surface protein CD56 (CD56+) and other markers and possessing cytotoxic activity.

[0003] NK cells for immunotherapy can be obtained from primary sources such as peripheral blood or umbilical cord blood. Artificial sources of NK cells include pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs), which are cells obtained from somatic cells (usually fibroblasts or peripheral blood mononuclear cells [PBMCs]), either of which are induced to proliferate indefinitely and differentiate into other types of cells under appropriate differentiation conditions. NK cells can be obtained from iPSCs by sequentially differentiating iPSCs into hematopoietic progenitor cells (HPCs), also known as hematopoietic stem cells (HSCs).

[0004] Once obtained, the original NK cells or iPSC-NK cells can be expanded in vitro before being administered to the patient. The method of differentiating iPSC into NK cells generally involves the use of feeder cells or a culture medium containing serum. In order to provide cells suitable for in vivo administration, it is still necessary to produce NK cells without using heterologous factors (animal-derived raw materials such as fetal bovine serum (FBS) and / or feeder cells). Compositions and methods. Summary of the invention

[0005] The present disclosure is based, at least in part, on the discovery of a method for differentiating stem cells into hematopoietic progenitor cells and NK cells that is free of xenologous genes. Without being bound by theory, the xenologous gene-free methods described herein produce cells suitable for in vivo administration.

[0006] In some embodiments, the present disclosure provides a method for producing a CD34+ / CD43+ / CD45+ cell population, the method comprising contacting a stem cell population with a differentiation medium for a period of time sufficient to produce the CD34+ / CD43+ / CD45+ cell population from the stem cell population, the differentiation medium comprising a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).

[0007] In some embodiments, the present disclosure provides a method for differentiating a stem cell population into a hematopoietic progenitor cell population, the method comprising contacting the stem cell population with a differentiation medium for a period of time sufficient to differentiate the stem cell population into the hematopoietic progenitor cell population, the differentiation medium comprising a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF).

[0008] In some embodiments, the population of hematopoietic progenitor cells comprises CD34+ / CD43+ / CD45+ cells.

[0009] In some embodiments, the BMP pathway activator is BMP4. In some embodiments, the FGF is FGF2. In some embodiments, the VEGF is VEGF-165. In some embodiments, the differentiation medium comprises a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor. In some embodiments, the ROCK inhibitor is Y27632.

[0010] In some embodiments, the differentiation medium comprises stem cell factor (SCF). In some embodiments, the differentiation medium comprises thrombopoietin (TPO). In some embodiments, the differentiation medium comprises low-density lipoprotein (LDL). In some embodiments, the differentiation medium comprises a phosphatidylinositol 3-kinase (PI3K) inhibitor.

[0011] In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, the differentiation medium comprises a pyrimido-[4,5-b]-indole derivative. In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729. In some embodiments, the differentiation medium comprises an aryl hydrocarbon receptor (AhR) antagonist. In some embodiments, the AhR antagonist is StemRegenin 1 (SR1).

[0012] In some embodiments, the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, and the ROCK inhibitor.

[0013] In some embodiments, the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, and the PI3K inhibitor.

[0014] In some embodiments, the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimido-[4,5-b]-indole derivative, and the AhR antagonist.

[0015] In some embodiments, the method comprises contacting the stem cell population with the differentiation medium for 1-5 days, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, and optionally the ROCK inhibitor.

[0016] In some embodiments, the method comprises (i) contacting the stem cell population with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesoderm cells, and (ii) contacting the embryoid bodies or mesoderm cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL and the PI3K inhibitor for 1-15 days.

[0017] In some embodiments, the method comprises (i) contacting the stem cell population with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesodermal cells, and (ii) contacting the embryoid bodies or mesodermal cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimido-[4,5-b]-indole derivative and the AhR antagonist for 1-15 days.

[0018] In some embodiments, the differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, 1-50 ug / mL LDL, 0.1-100 PI3K inhibitor, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

[0019] In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC).

[0020] In some embodiments, the stem cells are human embryonic stem cells (hESC).

[0021] In some embodiments, the present disclosure provides a method for generating a CD43+ / CD45+ / CD56+ / LFA1+ cell population, the method comprising contacting a CD34+ / CD43+ / CD45+ cell population with a culture medium for a period of time sufficient to generate the CD43+ / CD45+ / CD56+ / LFA1+ cell population from the CD34+ / CD43+ / CD45+ cell population, the culture comprising SCF, interleukin-7 (IL-7), IL-12, IL-15, FMS-like tyrosine kinase 3 ligand (FLT3L), a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

[0022] In some embodiments, the present disclosure provides a method for differentiating a population of hematopoietic progenitor cells into a natural killer (NK) cell population, the method comprising contacting the population of hematopoietic progenitor cells with a differentiation medium for a period of time sufficient to differentiate the hematopoietic progenitor cells into the NK cell population, the differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

[0023] In some embodiments, the present disclosure provides a method for differentiating a common lymphoid progenitor (CLP) population into a natural killer (NK) cell population, the method comprising contacting the hematopoietic progenitor cell population with a differentiation medium for a period of time sufficient to differentiate the hematopoietic progenitor cells into the NK cell population, the differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

[0024] In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729. In some embodiments, the AhR inhibitor is SR1.

[0025] In some embodiments, the culture medium comprises 1-100 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

[0026] In some embodiments, the time period is 11-25 days.

[0027] In some embodiments, the method comprises maturing the NK cell population with a maturation medium comprising (i) IL-12, IL-15, and IL-18 or (ii) IL-12, IL-2, and IL-18.

[0028] In some embodiments, the differentiation medium and / or maturation medium is serum-free.

[0029] In some embodiments, the methods are free of heterologous genes.

[0030] In some embodiments, the present disclosure provides a method of generating a NK cell population, the method comprising: (a) obtaining a stem cell population; (b) contacting the stem cell population with a first culture medium comprising a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor for a period of time sufficient to generate embryoid bodies; (c) contacting the embryoid bodies with a first differentiation medium for a period of time sufficient to generate a population of hematopoietic progenitor cells, wherein the first differentiation medium comprises a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and optionally a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor; (d) contacting the population of hematopoietic progenitor cells with a second differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor for a period of time sufficient to produce the population of NK cells.

[0031] In some embodiments, the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, the ROCK inhibitor is Y27632, the PI3K inhibitor is LY294002, and the pyrimido-[4,5-b]-indole derivative is UM729.

[0032] In some embodiments, each culture medium in steps (b)-(d) is serum-free.

[0033] In some embodiments, the methods are free of heterologous genes.

[0034] In some embodiments, the first culture medium, the first differentiation medium, and the second differentiation medium each comprise the same basal medium.

[0035] In some embodiments, the first culture medium, the first differentiation medium, and the second differentiation medium each comprise a different basal culture medium.

[0036] In some embodiments, the first differentiation medium and the second differentiation medium each comprise the same basal medium, and the first medium comprises a basal medium that is different from the first and second differentiation medium.

[0037] In some embodiments, the first differentiation medium and the second differentiation medium each comprise a basal medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

[0038] In some embodiments, the time period of step (b) is 1-5 days, the time period of step (c) is 3-15 days, and the time period of step (d) is 11-25 days.

[0039] In some embodiments, steps (a)-(d) occur within 35-45 days.

[0040] In some embodiments, the method comprises (e) expanding the NK cell population with a maturation medium comprising (i) IL-12, IL-15, and IL-18 or (ii) IL-12, IL-2, and IL-18.

[0041] In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

[0042] In some embodiments, the population of hematopoietic progenitor cells comprises about 30% to about 50% CD34+ / CD43+ / CD45+ cells.

[0043] In some embodiments, the NK cell population comprises about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

[0044] In some embodiments, the method comprises expanding the NK cell population, wherein the NK cell population is expanded about 10 to about 350 fold.

[0045] In some embodiments, the stem cell population is genetically engineered or edited.

[0046] In some embodiments, the NK cell population is genetically engineered or edited.

[0047] In some embodiments, the present disclosure provides a cell population comprising hematopoietic progenitor cells produced by the methods of the present disclosure.

[0048] In some embodiments, the hematopoietic progenitor cells are CD34+ / CD43+ / CD45+.

[0049] In some embodiments, the population of cells comprises 30-50% hematopoietic progenitor cells.

[0050] In some embodiments, the present disclosure provides a cell population comprising NK cells, which is produced by the methods of the present disclosure.

[0051] In some embodiments, the NK cells are CD43+ / CD45+ / CD56+ / LFA1+ cells.

[0052] In some embodiments, the cell population comprises 60-100% NK cells.

[0053] In some embodiments, the present disclosure provides a hematopoietic progenitor cell differentiation medium comprising a serum-free basal medium, a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, and a PI3K inhibitor.

[0054] In some embodiments, the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, and the PI3K inhibitor is LY294002.

[0055] In some embodiments, the culture medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, 1-50 ug / mL LDL, 0.1-100 PI3K inhibitor, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

[0056] In some embodiments, the present disclosure provides a NK cell differentiation medium comprising a serum-free basal medium, SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

[0057] In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729 and the AhR inhibitor is SR1.

[0058] In some embodiments, the culture medium comprises 1-100 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

[0059] In some embodiments, the present disclosure provides a kit comprising a hematopoietic progenitor cell differentiation medium and instructions for contacting a population of stem cells with the hematopoietic progenitor cell differentiation medium for a period of time sufficient to generate a population of cells comprising hematopoietic progenitor cells.

[0060] In some embodiments, the time period is 1-15 days.

[0061] In some embodiments, the present disclosure provides a kit comprising a NK cell differentiation medium and instructions for contacting hematopoietic progenitor cells with the NK cell differentiation medium for a period of time sufficient to generate a cell population comprising NK cells.

[0062] In some embodiments, the time period is 11-25 days.

[0063] In some embodiments, the present disclosure provides a kit comprising a hematopoietic progenitor cell differentiation medium and a NK cell differentiation medium, and instructions for contacting a stem cell population with the hematopoietic progenitor cell differentiation medium for a first time period sufficient to produce a cell population comprising hematopoietic progenitor cells and contacting the cell population comprising hematopoietic progenitor cells with the NK cell differentiation medium for a second time period sufficient to produce a cell population comprising NK cells.

[0064] In some embodiments, the first period of time is 1-15 days and the second period of time is 11-25 days.

[0065] In some embodiments, the kit further comprises a maturation medium comprising a basal medium and (i) IL-12, IL-15, and IL-18, or (ii) IL-12, IL-2, and IL-18, and instructions for contacting a cell population comprising NK cells for a period of time sufficient to allow the NK cells to mature.

[0066] In some embodiments, the present disclosure provides a composition for improving the yield ratio of hematopoietic progenitor cells from a stem cell population, the composition comprising a bone morphogenetic protein (BMP) pathway activator, a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), and a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor. In some embodiments, the present disclosure provides a composition for improving the yield ratio of hematopoietic progenitor cells from stem cells, the composition comprising a bone morphogenetic protein (BMP) pathway activator, a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), and a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor.

[0067] In some embodiments, the hematopoietic progenitor cells comprise CD34+ / CD43+ / CD45+ cells.

[0068] In some embodiments, the BMP pathway activator is BMP4. In some embodiments, the FGF is FGF2. In some embodiments, the VEGF is VEGF-165. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the composition further comprises stem cell factor (SCF). In some embodiments, the composition further comprises thrombopoietin (TPO). In some embodiments, the composition further comprises low-density lipoprotein (LDL). In some embodiments, the composition further comprises a phosphatidylinositol 3-kinase (PI3K) inhibitor. In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, the composition further comprises a pyrimido-[4,5-b]-indole derivative. In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729. In some embodiments, the composition further comprises an aryl hydrocarbon receptor (AhR) antagonist. In some embodiments, the AhR antagonist is StemRegenin 1 (SR1).

[0069] In some embodiments, the composition comprises the BMP pathway activator, the FGF, the VEGF, and the ROCK inhibitor.

[0070] In some embodiments, the composition comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, and the PI3K inhibitor.

[0071] In some embodiments, the composition comprises the BMP pathway activator, the FGF, the VEGF, the SCF, the TPO, the LDL, the PI3K inhibitor, the pyrimido-[4,5-b]-indole derivative, and the AhR antagonist.

[0072] In some embodiments, the composition comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, 1-50 ug / mL LDL, 0.1-100 PI3K inhibitor, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

[0073] In some embodiments, the stem cell population is an induced pluripotent stem cell (iPSC). In some embodiments, the stem cell population is a human embryonic stem cell (hESC).

[0074] In some embodiments, the yield ratio of hematopoietic progenitor cells (HP) to stem cells (StC) (HP / StC) is about 2: 1 to about 10: 1. In some embodiments, the yield ratio of hematopoietic progenitor cells (HP) to stem cells (StC) (HP / StC) is about 5:1.

[0075] In some embodiments, the present disclosure provides a method for increasing the yield ratio of hematopoietic progenitor cells from a stem cell population, the method comprising contacting the stem cell population with a differentiation medium for a time period sufficient to differentiate the stem cell population into the hematopoietic progenitor cells, the differentiation medium comprising a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor. In some embodiments, the present disclosure provides a method for increasing the yield ratio of hematopoietic progenitor cells from stem cells, the method comprising contacting the stem cells with a differentiation medium for a time period sufficient to differentiate the stem cell population into the hematopoietic progenitor cells, the differentiation medium comprising a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor.

[0076] In some embodiments, the hematopoietic progenitor cells comprise CD34+ / CD43+ / CD45+ cells.

[0077] In some embodiments, the BMP pathway activator is BMP4. In some embodiments, the FGF is FGF2. In some embodiments, the VEGF is VEGF-165. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the differentiation medium comprises stem cell factor (SCF). In some embodiments, the differentiation medium comprises thrombopoietin (TPO). In some embodiments, the differentiation medium comprises low-density lipoprotein (LDL). In some embodiments, the differentiation medium comprises a phosphatidylinositol 3-kinase (PI3K) inhibitor. In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, the differentiation medium comprises a pyrimido-[4,5-b]-indole derivative. In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729. In some embodiments, the differentiation medium comprises an aryl hydrocarbon receptor (AhR) antagonist. In some embodiments, the AhR antagonist is StemRegenin 1 (SR1).

[0078] In some embodiments, the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, and the ROCK inhibitor.

[0079] In some embodiments, the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, and the PI3K inhibitor.

[0080] In some embodiments, the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimido-[4,5-b]-indole derivative, and the AhR antagonist.

[0081] In some embodiments, the method comprises contacting the stem cell population with the differentiation medium for 1-5 days, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, and optionally the ROCK inhibitor.

[0082] In some embodiments, the method comprises (i) contacting the stem cell population with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesoderm cells, and (ii) contacting the embryoid bodies or mesoderm cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL and the PI3K inhibitor for 1-15 days.

[0083] In some embodiments, the method comprises (i) contacting the stem cell population with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesoderm cells, and (ii) contacting the embryoid bodies or mesoderm cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimidine-[4,5-b]-indole derivative and the AhR antagonist for 1-15 days.

[0084] In some embodiments, the differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, 1-50 ug / mL LDL, 0.1-100 PI3K inhibitor, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

[0085] In some embodiments, the stem cell population is an induced pluripotent stem cell (iPSC) population. In some embodiments, the stem cell population is a human embryonic stem cell (hESC) population.

[0086] In some embodiments, the yield ratio of hematopoietic progenitor cells from a stem cell population is about 2: 1 to about 10: 1. In some embodiments, the yield ratio of hematopoietic progenitor cells from a stem cell population is about 5:1.

[0087] In some embodiments, the present disclosure provides a kit to increase the yield ratio of NK cells from a stem cell population, wherein the kit comprises instructions for differentiating the stem cell population into NK cells, and: (a) a first culture medium comprising a BMP pathway activator, FGF, VEGF and optionally a ROCK inhibitor; (b) a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and optionally a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor; (c) a second differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor.

[0088] In some embodiments, the present disclosure provides a kit to increase the yield ratio of NK cells from a stem cell population, wherein the kit comprises instructions for differentiating the stem cell population into NK cells, and: (a) a first culture medium comprising a BMP pathway activator, FGF, VEGF and optionally a ROCK inhibitor; (b) a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and optionally a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor; (c) a second differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

[0089] In some embodiments, the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, the ROCK inhibitor is Y27632, the PI3K inhibitor is LY294002, and the pyrimido-[4,5-b]-indole derivative is UM729.

[0090] In some embodiments, each of the culture media in (a)-(c) is serum-free. In some embodiments, the culture media does not contain a heterologous gene.

[0091] In some embodiments, the first culture medium, the first differentiation medium, and the second differentiation medium each comprise the same basal medium.

[0092] In some embodiments, the first culture medium, the first differentiation medium, and the second differentiation medium each comprise a different basal culture medium.

[0093] In some embodiments, the first differentiation medium and the second differentiation medium each comprise the same basal medium, and the first medium comprises a basal medium that is different from the first and second differentiation medium.

[0094] In some embodiments, the first differentiation medium and the second differentiation medium each comprise a basal medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

[0095] In some embodiments, the kit further comprises a maturation medium comprising (i) IL-12, IL-15, and IL-18, or (ii) IL-12, IL-2, and IL-18.

[0096] In some embodiments, the stem cell population is an induced pluripotent stem cell (iPSC) population or a human embryonic stem cell (hESC) population.

[0097] In some embodiments, the NK cells comprise about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

[0098] In some embodiments, the stem cell population is genetically engineered or edited

[0099] In some embodiments, the NK cells are genetically engineered or edited.

[0100] In some embodiments, the yield ratio of NK cells in the stem cell population is about 2: 1 to about 100: 1. In some embodiments, the yield ratio of NK cells in the stem cell population is about 35:1.

[0101] In some embodiments, the present disclosure provides a method for increasing the yield ratio of NK cells from a stem cell population, the method comprising: (a) obtaining a stem cell population; (b) contacting the stem cell population with a first culture medium comprising a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor for a period of time sufficient to generate embryoid bodies; (c) contacting the embryoid bodies with a first differentiation medium for a period of time sufficient to generate a population of hematopoietic progenitor cells, wherein the first differentiation medium comprises a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and optionally a pyrimido[4,5-b]indole derivative and an AhR inhibitor; (d) contacting the population of hematopoietic progenitor cells with a second differentiation medium for a period of time sufficient to produce the NK cells, wherein the second differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor. 137. The method of claim 136, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, the ROCK inhibitor is Y27632, the PI3K inhibitor is LY294002, and the pyrimido-[4,5-b]-indole derivative is UM729.

[0102] In some embodiments, the present disclosure provides a method for increasing the yield ratio of NK cells from stem cells, the method comprising: (a) obtaining stem cells; (b) contacting the stem cells with a first culture medium comprising a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor for a period of time sufficient to generate embryoid bodies; (c) contacting the embryoid bodies with a first differentiation medium for a period of time sufficient to generate a population of hematopoietic progenitor cells, wherein the first differentiation medium comprises a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and optionally a pyrimido[4,5-b]indole derivative and an AhR inhibitor; (d) contacting the population of hematopoietic progenitor cells with a second differentiation medium for a period of time sufficient to produce the NK cells, wherein the second culture medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor. 137. The method of claim 136, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, the ROCK inhibitor is Y27632, the PI3K inhibitor is LY294002, and the pyrimido-[4,5-b]-indole derivative is UM729.

[0103] In some embodiments, each culture medium in steps (b)-(d) is serum-free. In some embodiments, the method is free of heterologous genes.

[0104] In some embodiments, the first culture medium, the first differentiation medium, and the second differentiation medium each comprise the same basal medium.

[0105] In some embodiments, the first culture medium, the first differentiation medium, and the second differentiation medium each comprise a different basal culture medium.

[0106] In some embodiments, the first differentiation medium and the second differentiation medium each comprise the same basal medium, and the first medium comprises a basal medium that is different from the first and second differentiation medium.

[0107] In some embodiments, the first differentiation medium and the second differentiation medium each comprise a basal medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

[0108] In some embodiments, the time period of step (b) is 1-5 days, the time period of step (c) is 3-15 days, and the time period of step (d) is 11-25 days.

[0109] In some embodiments, steps (a)-(d) occur within 35-45 days.

[0110] In some embodiments, the method comprises (e) expanding the NK cells with a maturation medium comprising (i) IL-12, IL-15, and IL-18 or (ii) IL-12, IL-2, and IL-18.

[0111] In some embodiments, the stem cell population is induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

[0112] In some embodiments, the population of hematopoietic progenitor cells comprises about 30% to about 50% CD34+ / CD43+ / CD45+ cells.

[0113] In some embodiments, the NK cells comprise about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

[0114] In some embodiments, the method comprises expanding NK cells, wherein the NK cells are expanded about 10 to about 350 fold.

[0115] In some embodiments, the stem cell population is genetically engineered or edited.

[0116] In some embodiments, the NK cells are genetically engineered or edited.

[0117] In some embodiments, the yield ratio of NK cells from the stem cell population is about 2:1 to about 100:1.

[0118] In some embodiments, the yield ratio of NK cells from the stem cell population is about 35:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 A schematic diagram of an exemplary protocol for differentiating stem cells into hematopoietic progenitor cells and NK cells is provided.

[0120] Figures 2A-2D Shows the Figure 1 The provided protocol characterizes hematopoietic progenitor cells (HP) differentiated from stem cells. HPs were characterized on day 15. Figure 2A Shown is a flow cytometric analysis by sorting cells to quantify the percentage of cells triple positive for the HP markers CD34 / CD43 / CD45. Figure 2BGraph showing HP purity (29%-46%) of all CD34 / CD43 / CD45 triple positive cells cultured in Stemline II, StemSpan SFEM II or other media. Figure 2C Shown is a graph of the expansion of HPs at day 15 relative to iPSCs plated at day 0 in StemSpan SFEMII, Stemline II, or alternative media. Figure 2D Representative bright-field microscopy images of day 15 harvested pre-HP EBs are provided.

[0121] Figures 3A-3D Shows the basis Figure 1 Characterization of induced pluripotent stem cell-derived NK (iPSC-NK) cells differentiated from stem cells using the protocol provided in. Figure 3A Shown is a flow cytometry analysis by gating cells to quantify the quadruple positivity of four NK markers: CD43 / CD45 / CD56 / LFA1. Figure 3B It is a graph showing the purity of NK cells using Stemline II, StemSpanSFEMII or other culture media. Figure 3C is a graph showing the expansion of iNKs at day 40 relative to iPSCs seeded at day 0 in StemSpan SFEMII, StemlineII, or other media. Figure 3D Representative bright-field microscopy images of day 39 cells of iNK differentiation are shown.

[0122] Figure 4A -D shows the process of iNK differentiation to generate highly functional iNKs. Figure 4A Shown are the percentages of CD45+CD5-CD56+LFA1+ cells in differentiated NK cells on day 40 or mature NK cells on day 47 in the presence or absence of LY294002. Figure 4B Immunophenotyping results showed improved purity of NK cells after maturation, as well as increased activation markers NKp46, NKG2D, LFA1, and CD16 and decreased inhibitory markers CD161 and CD73. Figure 4C and Figure 4D Differentiated iNKs at day 40 or mature iNKs at day 47 were shown to be incubated with breast cancer MDA-MB231 cells expressing nuclear fluorescent protein at different T:E ratios. iNK cells reduced the growth of MDA in a dose-responsive manner.

[0123] Figures 5A-5D Shown are the fold expansion results of iPSCs cultured in SFEMII or Stemline medium containing different combinations of differentiation factors. Figure 5AHP purity of all CD34 / CD43 / CD45 triple positive cells is shown. Figure 5B The fold expansion of iPSCs on day 15 relative to iPSCs plated on day 0 is shown. Figure 5C The purity of iNKs at day 40, defined as CD43+ / CD45+ / CD56+, is shown. Figure 5D The iNK fold expansion from iPSC to iNK at day 40 is shown. DETAILED DESCRIPTION

[0124] In some aspects, the disclosure provides compositions and methods for producing hematopoietic progenitor cells, common lymphoid progenitor cells, pre-NK progenitor cells, NK progenitor cells, immature NK cells and / or NK cells.In some embodiments, the compositions and methods described herein are free of heterologous genes. definition

[0125] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0126] Unless the context dictates otherwise, the various features described herein may be used in any combination with any feature or combination of features described herein, and each feature may be excluded or omitted from a combination.

[0127] As used herein, the singular forms "a", "an" and "an" include the plural forms as well, unless the context indicates otherwise. The conjunction "and / or" refers to all possible combinations of one or more of the listed elements.

[0128] As used herein, "subject" refers to a recipient of a NK cell population generated by the methods of the present disclosure. The term includes mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep or pigs, preferably humans.

[0129] As used herein, "treating" or "treatment" refers to any type of action or administration that produces a benefit to a subject suffering from a disease or disorder, including improvement of the patient's condition (i.e., amelioration, alleviation or improvement of one or more symptoms, as well as partial or complete response to treatment).

[0130] The term "effective amount" refers to an amount effective to produce a desired biochemical, cellular or physiological response. The term "therapeutically effective amount" refers to an amount, dose or therapeutic dosage regimen effective to produce a desired therapeutic effect.

[0131] As used herein, "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently bound by an ester bond between the phosphate group of one nucleotide and the hydroxyl group of the sugar component of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

[0132] As used herein, "polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds, which forms a part (or all) of a protein.

[0133] It will be appreciated by those skilled in the art that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it will be appreciated that, without affecting the polypeptide sequence encoded by the polynucleotides described herein, those skilled in the art can use conventional techniques to make nucleotide substitutions to reflect the codon usage of any particular host organism to be expressed polypeptide.

[0134] Nucleic acids can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides containing synthetic or modified nucleotides. As known in the art, there are many different types of modifications of oligonucleotides. These include methylphosphonates and thiophosphate backbones, the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the uses described herein, it should be understood that polynucleotides can be modified by any method available in the art. Such modifications can be performed in order to increase the activity or lifespan of the polynucleotide of interest in vivo.

[0135] The term "variant" refers to a polynucleotide or polypeptide having at least one substitution, insertion or deletion in its sequence compared to a reference polynucleotide or polypeptide. A "functional variant" refers to a variant that retains one or more functions of a reference polynucleotide or polypeptide.

[0136] The term "inactivating mutation" refers to a mutation in a genomic sequence that destroys the function of a gene. Inactivating mutations can occur in any sequence region that contributes to gene expression (such as a coding region or a non-coding region). Examples include, but are not limited to, cis-acting elements (enhancers) or sequences that undergo transcription (such as mRNA transcript sequences). Inactivating mutations include mutations that render a gene or its encoded protein inoperable or that reduce the function of a gene or its encoded protein.

[0137] As used herein, the term "sequence identity" or "identity", with respect to polynucleotide or polypeptide sequences, refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences match at each position of the alignment over the entire length of the reference sequence. "Percentage of identity" is the number of the matching positions in the optimal alignment divided by the length of the reference sequence plus the sum of the lengths of any gaps in the reference sequence in the alignment. The optimal alignment refers to the alignment that results in the maximum percentage of identity. The alignment of sequences can be accomplished by many known methods, including, for example, using mathematical algorithms, such as the mathematical algorithms in the BLAST component or the Clustal Omega sequence analysis program. Unless otherwise indicated, the term "sequence identity" in the claims refers to the sequence identity calculated by BLAST version 2.12.0 using default parameters. And, unless otherwise indicated, the alignment is the alignment of all or part of the polynucleotide or polypeptide sequence of interest over the full length of the reference sequence.

[0138] As used herein, the term "engineered" refers to cells that have been stably transduced with a heterologous polynucleotide, or cells that have been subjected to gene editing to introduce, delete or modify a polynucleotide in the cell, or cells that have been transiently transduced with a polynucleotide in a manner that results in a stable phenotypic change in the cell.

[0139] As used herein, the term "stem cell" is used to describe a cell having an undifferentiated phenotype, such as a cell capable of differentiating into a hematopoietic progenitor cell and / or a NK cell.

[0140] As used herein, the term "pluripotency" means that a stem cell is capable of forming substantially all differentiated cell types of an organism, at least in culture. For example, embryonic stem cells are a type of pluripotent stem cell that is capable of forming cells from each of the three germ layers, ectoderm, mesoderm, and endoderm.

[0141] As used herein, the terms "induced pluripotent stem cells" and "iPSCs" are used to refer to cells of somatic cell origin that have been reprogrammed to a pluripotent state and are capable of proliferation, selective differentiation, and maturation. iPSCs are stem cells generated from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed, to become cells capable of differentiating into tissues of all three germ layers or dermis (mesoderm, endoderm, and ectoderm). The iPSCs generated do not refer to cells that exist in nature.

[0142] As used herein, the term "hematopoietic stem cell," "hematopoietic progenitor," or "hematopoietic progenitor cell" refers to a stem cell capable of generating mature myeloid and lymphoid cell types, including natural killer cells, T cells, and B cells. Hematopoietic stem cells are typically characterized by CD34+.

[0143] The term "progenitor cell" refers to a cell that is partially differentiated into a desired cell type. Progenitor cells retain a certain degree of multipotency and can differentiate into a variety of cell types.

[0144] As used herein, "differentiation" or "differentiated" refers to the process and conditions by which undifferentiated or immature (e.g., unspecialized) cells acquire the characteristics of mature (specialized) cells, thereby acquiring a specific form and function. Stem cells (unspecialized) are typically exposed to different conditions (e.g., growth factors and morphogenetic factors) to induce a specific lineage commitment or differentiation of the stem cells.

[0145] As used herein, "expand" or "expansion" refers to increasing the number and / or purity of a cell type in a cell population by mitosis of cells with limited proliferation capacity (such as NK cells).

[0146] As used herein, "activity," "activation," or "activation" refers to stimulation of activating receptors on cytotoxic innate lymphocytes, resulting in cell division, cytokine secretion (such as IFNγ and / or TNFα), and / or release of cytolytic granules to modulate or assist an immune response.

[0147] As used herein, "xeno-free" refers to compositions and methods that are free of animal-derived materials (eg, fetal bovine serum). In some embodiments, the xeno-free methods described herein do not include feeder cells.

[0148] As used herein, "source cell" refers to any progenitor cell known in the art.

[0149] As used herein, "yield ratio" refers to a measure that quantifies the amount of cells produced from a single source cell. For example, the yield ratio of NK / iPSC is 20: 1, meaning that 20 NK cells are produced from one iPSC. Similarly, if 20,000 NK cells are obtained from an initial population of 1,000 iPSCs, the NK / iPSC yield ratio of NK from iPSC will be one iPSC producing 20 NK cells, or 20: 1. In some embodiments, the yield ratio is determined by cell counting. In some embodiments, cell counting is performed at the beginning of the process, throughout the process, and at the end of the process to calculate the amplification of cells over time according to the change in viable cell density. In some embodiments, cells are diluted (batch feeding) over time. Differentiation and Expansion Media

[0150] In some embodiments, the disclosure provides a culture medium for differentiating stem cells into hematopoietic progenitor cells. In some embodiments, the disclosure provides a culture medium for differentiating hematopoietic progenitor cells into NK cells. In some embodiments, the disclosure provides a culture medium for amplifying NK cells. In some embodiments, the differentiation and / or expansion culture medium described herein comprises a serum-free basal culture medium having at least one exogenous factor for driving differentiation and / or amplification. Heterologous gene-free medium

[0151] In some embodiments, the differentiation and / or expansion medium described herein is a defined media. As used herein, "defined media" refers to a growth medium suitable for culturing human or animal cells in vitro, in which all chemical components are known. In some embodiments, the differentiation and / or expansion medium comprises a basal medium. In some embodiments, the basal medium comprises Iscove's Modified Dulbecco's Medium, serum albumin, human insulin, human transferrin, and 2-mercaptoethanol. In some embodiments, the basal medium comprises human serum albumin. In some embodiments, the basal medium does not contain animal-derived raw materials.

[0152] In some embodiments, the basal medium is selected from StemSpan SFEMII medium (STEMCELL Technologies; serum-free), Stemline II (Sigma-Aldrich; fully defined, serum-free and animal component-free, GMP produced), CTS NK Xpander medium (Gibco; serum-free and animal component-free medium), STEMdiff Hematopoietic-EB basal medium (STEMCELL Technologies; serum-free), STEMdiff APEL 2 medium (STEM CELL Technologies; serum-free and animal component-free) or Hematopoietic Progenitor Cell Expansion Medium XF (PromoCell; serum-free and heterologous component-free medium). In some embodiments, the basal medium is StemSpan SFEMII medium. In some embodiments, the basal medium is Stemline II medium. In some embodiments, the basal medium is STEMdiff APEL 2 medium. In some embodiments, the hematopoietic progenitor cell differentiation medium and the NK cell differentiation medium have the same basal medium. In some embodiments, the hematopoietic progenitor cell differentiation medium and the NK cell differentiation medium have different basal mediums. Exogenous factors

[0153] In some embodiments, differentiation and / or expansion culture medium described herein include exogenous factors. In some embodiments, the method disclosed herein includes contacting different cell populations with various exogenous factors in a culture medium without heterologous genes to drive cell differentiation into, for example, hematopoietic progenitor cells and / or NK cells. In some embodiments, the exogenous factors include, but are not limited to, cytokines, such as interleukins, fibroblast growth factor (FGF), stem cell factor (SCF), phosphatidylinositol 3-kinase (PI3K) inhibitors, FMS-like tyrosine kinase 3 ligands (FLT3L), bone morphogenetic protein (BMP) pathway activators, pyrimidine-indole derivatives, and aryl hydrocarbon receptor antagonists. In some embodiments, exogenous factors described herein can be commercially available.

[0154] In some embodiments, the exogenous factors suitable for differentiation and / or expansion culture medium are cytokines. Cytokines include interferons, interleukins and growth factors, which are small molecule proteins that play an important role in cell signaling. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is selected from IL-2, IL-7, IL-12, IL-15, IL-18 and any combination thereof. In some embodiments, the cytokine is a growth factor. In some embodiments, the growth factor is selected from fibroblast growth factor, vascular endothelial growth factor and any combination thereof.

[0155] In some embodiments, the exogenous factor is interleukin 2 (IL-2). IL-2 is a secretory cytokine produced by activated CD4+ and CD8+ T lymphocytes, which is very important for the proliferation of T lymphocytes and B lymphocytes. IL-2 is a member of the interleukin 2 (IL2) cytokine subfamily, which includes IL-4, IL-7, IL-9, IL-15, IL-21, erythropoietin and thrombopoietin.

[0156] In some embodiments, the exogenous factor is interleukin 7 (IL-7). IL-7 is a member of the interleukin 2 (IL2) cytokine subfamily. Lymphoid differentiation and activation are critically dependent on IL-7 signaling.

[0157] In some embodiments, the exogenous factor is interleukin 12 (IL-12). IL-12 is a cytokine that acts on T cells and natural killer cells and has a wide range of biological activities. NK cells can acquire memory-like properties after being briefly stimulated by IL-12.

[0158] In some embodiments, the exogenous factor is interleukin 15 (IL-15). IL-15 is a member of the interleukin 2 (IL2) cytokine subfamily. IL-15 regulates NK cell activation and proliferation.

[0159] In some embodiments, the exogenous factor is interleukin 18 (IL-18). IL-18 is a proinflammatory cytokine of the IL-1 family that exists as a precursor in the cytoplasm of a variety of immune cells. IL-18 has been shown to effectively activate NK cells.

[0160] In some embodiments, the exogenous factor is low-density lipoprotein (LDL). LDL induces enhancement of NK cell proliferation and cytotoxic activity.

[0161] In some embodiments, the exogenous factor is fibroblast growth factor (FGF). FGF family members are cell signaling proteins produced by macrophages. The FGF family includes 23 members. In some embodiments, the exogenous factor is FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 or FGF23. In some embodiments, the exogenous factor is FGF2. In some embodiments, the FGF2 is commercially available. In some embodiments, the FGF2 can be purchased commercially from Peprotech. In some embodiments, the FGF2 can be purchased commercially from Gibco.

[0162] In some embodiments, the exogenous factor is FMS-like tyrosine kinase 3 ligand (FLT3L). FLT3L is an essential growth factor for NK cells and has been shown to play an important role in the expansion of early hematopoietic progenitor cells and the expansion of mature peripheral NK cells.

[0163] In some embodiments, the exogenous factor is stem cell factor (SCF).SCF plays an important role in the survival of stem cells and the self-renewal and maintenance of stem cells.

[0164] In some embodiments, the exogenous factor is vascular endothelial growth factor (VEGF). The VEGF family is a subfamily of growth factors, namely the platelet-derived growth factor family of cystine knot growth factors. The VEGF family includes five family members. In some embodiments, the exogenous factor is VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. In some embodiments, the exogenous factor is VEGF-165. VEGF165 is a 38.2kDa disulfide bond-linked homodimeric protein consisting of two polypeptide chains of 165 amino acids.

[0165] In some embodiments, the exogenous factor is an aryl hydrocarbon inhibitor. The aryl hydrocarbon receptor is a transcription factor that regulates gene expression. The aryl hydrocarbon receptor plays a role in regulating immunity, stem cell maintenance, and cell differentiation. It has been shown that antagonism of the aryl hydrocarbon receptor promotes the renewal and expansion of stem cells. In some embodiments, the exogenous factor is an aryl hydrocarbon receptor antagonist selected from PD98059, StemRegenin 1 (SR1), GNF351, BAY 2416964, CH-223191, perillaldehyde, PDM-11, and BAY-218. In some embodiments, the exogenous factor is SR1.

[0166] In some embodiments, the exogenous factor is an inhibitor of phosphatidylinositol 3-kinase (PI3K). PI3K comprises a family of lipid and serine / threonine kinases that catalyze the transfer of phosphates to the D-3′ position of inositol lipids to produce phosphoinositide-3-phosphate (PIP), phosphoinositide-3,4-bisphosphate (PIP2), and phosphoinositide-3,4,5-triphosphate (PIP3), which in turn serve as secondary messengers in signaling cascades by docking proteins containing pleckstrin-homology, FYVE, Phox, and other phospholipid binding domains into various signaling complexes typically located on the plasma membrane.

[0167] PI3K inhibitors include, but are not limited to, Idelalisib, Copanlisib, Duvelisib, Alpelisib, Umbralisib, Buparlisib, Copanlisib, Dactolisib, Duvelisib, Idelalisib, Leniolisib, Parsaclisib, Paxalisib, Taselisib, Zandeli sib, Inavolisib, Apitolisib, Bimiralisib, Eganelisib, Fimepinostat, Gedatolisib, Linperlisib, Nemiralisib, Pictilisib, Pilaralisib, Samotolisib, Seletalisib, Serabelisib, Sonolisib, Tenalisib, Voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, Acalisib, Omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, Hibiscone C, IC87114, LY294002, and PI-103.In some embodiments, the exogenous factor is Idelalisib, Copanlisib, Duvelisib, Alpelisib, Umbralisib, Buparlisib, Copanlisib, Dactolisib, Duvelisib, Idelalisib, Leniolisib, Parsaclisib, Paxalisib, Taselisib, Zandel isib, Inavolisib, Apitolisib, Bimiralisib, Eganelisib, Fimepinostat, Gedatolisib, Linperlisib, Nemiralisib, Pictilisib, Pilaralisib, Samotolisib, Seletalisib, Serabelisib, Sonolisib, Tenalisib, Voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, Acalisib, Omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, Hibiscone C, IC87114, LY294002, PI-103, or any combination thereof. In some embodiments, the exogenous factor is LY294002.

[0168] In some embodiments, the exogenous factor is an activator of the BMP pathway. Bone morphogenetic protein (BMP) is produced in the form of a large precursor molecule, which is proteolytically hydrolyzed into a mature polypeptide after translation. BMP exerts its effects through specific transmembrane receptors located on the surface of target cells. BMP receptors are serine-threonine kinases, similar to TGF-β receptors, and are divided into two subgroups: type I receptors and type II receptors. BMP can only bind tightly to the heterotetrametric complexes of these receptors. The formation of this complex is crucial for BMP signal transduction. In target cells, BMP signals are transmitted to the nucleus through specific signaling molecules called Smads, which are also responsible for the inhibition of BMP signals.

[0169] BMP is a multifunctional cytokine that belongs to a member of the transforming growth factor-β superfamily. BMP receptors mediate BMP signaling by activating Smad. BMP ligands bind to BMP receptors BMPRI and BMPRII. Phosphorylated BMPRII activates BMPRI. Phosphorylated BMPRI then phosphorylates receptor-activated Smad protein (R-Smad), which binds to co-mediator-Smad (co-Smad) and enters the nucleus, where they regulate gene expression. BMP pathway activators include agents disclosed in WO2014011540, WO 2014062138, and WO 2005117994, which are incorporated herein by reference. BMP pathway activators include, but are not limited to, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, and BMP-4. In some embodiments, the BMP pathway activator is BMP-4. In some embodiments, the exogenous factor is BMP-4. In some embodiments, the BMP-4 is commercially available. In some embodiments, the BMP-4 is commercially available from Peprotech. In some embodiments, the BMP-4 is commercially available from Invitrogen. In some embodiments, the BMP-4 is commercially available from Biolegend.

[0170] In some embodiments, the exogenous factor is an inhibitor of ROCK. Rho-associated kinase (ROCK) is a serine / threonine kinase that is a downstream effector of Rho kinase (of which there are three isozymes, RhoA, RhoB and RhoC). ROCK inhibitors include, but are not limited to, polynucleotides, polypeptides and small molecules. ROCK inhibitors encompassed herein can reduce ROCK expression and / or ROCK activity. Exemplary examples of ROCK inhibitors encompassed herein include, but are not limited to, anti-ROCK antibodies, dominant negative ROCK variants, siRNA, shRNA, miRNA and antisense nucleic acids targeting ROCK.

[0171] Exemplary ROCK inhibitors contemplated herein include, but are not limited to, Thiazovivin, Y27632, Fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxy-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridinyl)-N′-(2,4,6-trichlorophenyl)urea, 3-(4-pyridinyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridinyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, as well as the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety. In some embodiments, the ROCK inhibitor is Thiazovivin, Y27632, or Pyrintegrin. In some embodiments, the ROCK inhibitor is Y27632. Mesoderm / embryoid body differentiation medium

[0172] In some embodiments, the present disclosure provides a differentiation medium for producing mesoderm and / or embryoid bodies by stem cells. In some embodiments, mesodermal cells are produced by iPSC or hESC. As stem cells begin to differentiate, three different germ layers are formed: ectoderm, mesoderm and endoderm. Immune cells (such as NK cells) are differentiated from mesodermal cells. Embryoid bodies are three-dimensional aggregates that can differentiate into cells of all three germ layers. In some embodiments, mesodermal cells are produced by embryoid bodies. In some embodiments, the mesodermal cells produced by the compositions and methods of the present disclosure are further differentiated into hematopoietic progenitor cells. In some embodiments, the mesodermal cells produced by the compositions and methods of the present disclosure are further differentiated into NK cells.

[0173] In some embodiments, at least one exogenous factor is used to culture a stem cell population (such as iPSC or hESC) to form mesoderm and / or embryoid body cells. In some embodiments, the exogenous factor is a bone morphogenetic protein (BMP) activator. In some embodiments, the exogenous factor is FGF. In some embodiments, the exogenous factor is VEGF. In some embodiments, the exogenous factor is a ROCK inhibitor. In some embodiments, the exogenous factor is selected from BMP pathway activators, FGF, VEGF, ROCK inhibitors, and any combination thereof. In some embodiments, the exogenous factor comprises a BMP pathway activator and FGF. In some embodiments, the exogenous factor comprises a BMP pathway activator and VEGF. In some embodiments, the exogenous factor comprises a BMP pathway activator and a ROCK inhibitor. In some embodiments, the exogenous factor comprises FGF and VEGF. In some embodiments, the exogenous factor comprises FGF and a ROCK inhibitor. In some embodiments, the exogenous factor comprises VEGF and a ROCK inhibitor. In some embodiments, the exogenous factor comprises a BMP pathway activator, FGF, and VEGF. In some embodiments, the exogenous factors include BMP pathway activators, FGF, and ROCK inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, and ROCK inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, and ROCK inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, and ROCK inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, and ROCK inhibitors.

[0174] In some embodiments, the exogenous factors include BMP4 and FGF2. In some embodiments, the exogenous factors include BMP4 and VEGF-165. In some embodiments, the exogenous factors include BMP4 and Y27632. In some embodiments, the exogenous factors include FGF2 and VEGF-165. In some embodiments, the exogenous factors include FGF2 and Y27632. In some embodiments, the exogenous factors include VEGF-165 and Y27632. In some embodiments, the exogenous factors include BMP4, FGF2 and VEGF-165. In some embodiments, the exogenous factors include BMP4, FGF2 and Y27632. In some embodiments, the exogenous factors include FGF, VEGF-165 and Y27632. In some embodiments, the exogenous factors include BMP4, FGF2 and Y27632. In some embodiments, the exogenous factors comprise BMP4, FGF2, VEGF-165, and Y27632.

[0175] In some embodiments, BMP4, FGF2, VEGF and / or ROCK inhibitor are used in the mesoderm formation step. For example, the mesoderm formation step may include contacting the cell population with BMP4 and FGF2; contacting with BMP4, FGF2 and ROCK inhibitor; contacting with BMP4 and VEGF; contacting with BMP4, VEGF and ROCK inhibitor; contacting with FGF2 and VEGF; contacting with FGF2, VEGF and ROCK inhibitor; contacting with BMP4, FGF2 and VEGF; contacting with BMP4, FGF2, VEGF and ROCK inhibitor; or contacting with any one of BMP4, FGF2, VEGF and ROCK inhibitor alone without using the others.

[0176] In some embodiments, the concentration of the bone morphogenetic protein (BMP) activator in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1-50 ng / ml, or any range thereof. In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1-50 ng / ml.

[0177] In some embodiments, the BMP pathway activator is BMP4. In some embodiments, the concentration of BMP4 in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 31 ng / ml, about 32 ng / ml, about 33 ng / ml, about 34 ng / ml, about 35 ng / ml, about 36 ng / ml, about 37 ng / ml, about 38 ng / ml, about 39 ng / ml, about 40 ng / ml, about 41 ng / ml, about 42 ng / ml, about 43 ng / ml, about 44 ng / ml In some embodiments, BMP4 is present in the differentiation medium at about 1-50 ng / ml, or any range thereof. In some embodiments, BMP4 is present in the differentiation medium at about 1-50 ng / ml, or any range thereof.

[0178] In some embodiments, the concentration of FGF2 in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml In some embodiments, FGF2 is present in the differentiation medium at about 1-50 ng / ml, or any range therein. In some embodiments, FGF2 is present in the differentiation medium at about 1-50 ng / ml.

[0179] In some embodiments, the concentration of VEGF in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml In some embodiments, VEGF is present in the differentiation medium at about 5-100 ng / ml, or any range derived therefrom. In some embodiments, VEGF is present in the differentiation medium at about 5-100 ng / ml.

[0180] In some embodiments, the concentration of the ROCK inhibitor in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM. In some embodiments, the ROCK inhibitor is present in the differentiation medium at about 0.1-20 μM.

[0181] In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the concentration of Y27632 is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 62 μM, about 6 In some embodiments, Y27632 is present in the differentiation medium at about 0.1-100 μM.

[0182] In some embodiments, the mesodermal differentiation medium comprises a BMP pathway activator, FGF and VEGF. In some embodiments, the mesodermal differentiation medium comprises a BMP pathway activator, FGF, VEGF and a ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises a defined xenogeneic-free basal medium, a BMP pathway activator, FGF and VEGF. In some embodiments, the mesodermal differentiation medium comprises a defined xenogeneic-free basal medium, a BMP pathway activator, FGF, VEGF and a ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises BMP4, FGF2 and VEGF-165. In some embodiments, the mesodermal differentiation medium comprises BMP4, FGF, VEGF-165 and a ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises BMP4, FGF, VEGF-165 and a ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises BMP4, FGF, VEGF-165 and Y27632.

[0183] In some embodiments, the mesodermal differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF2, and 1-100ng / mL VEGF-165. In some embodiments, the mesodermal differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM Y27632. Hematopoietic Progenitor Cell Differentiation Medium

[0184] In some embodiments, the present disclosure provides a differentiation medium for generating HP cells from mesodermal cells and embryoid body cells. In some embodiments, the mesodermal cells and embryoid body cells generated by the compositions and methods of the present disclosure are further differentiated into hematopoietic progenitor cells.

[0185] In some embodiments, at least one exogenous factor is used to culture a HP cell population to form differentiated NK cells. In some embodiments, the exogenous factor is a BMP pathway activator. In some embodiments, the exogenous factor is FGF. In some embodiments, the exogenous factor is VEGF. In some embodiments, the exogenous factor is SCF. In some embodiments, the exogenous factor is TPO. In some embodiments, the exogenous factor is LDL. In some embodiments, the exogenous factor is a PI3K inhibitor. In some embodiments, the exogenous factor is a pyrimidine-indole derivative. In some embodiments, the exogenous factor is an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor is a TGF-β receptor inhibitor. In some embodiments, the exogenous factor is selected from BMP pathway activators, FGF, VEGF, SCF, TPO, LDL, PI3K inhibitors, and any combination thereof. In some embodiments, the exogenous factor is selected from the group consisting of BMP pathway activators, FGF, VEGF, SCF, TPO, LDL, PI3K inhibitors, pyrimidine-indole derivatives, aryl hydrocarbon receptor antagonists, TGF-β receptor inhibitors, and any combination thereof.

[0186] In some embodiments, the exogenous factors include BMP pathway activators and FGF. In some embodiments, the exogenous factors include BMP pathway activators and VEGF. In some embodiments, the exogenous factors include BMP pathway activators and SCF. In some embodiments, the exogenous factors include BMP pathway activators and TPO. In some embodiments, the exogenous factors include BMP pathway activators and LDL. In some embodiments, the exogenous factors include BMP pathway activators and PI3K inhibitors. In some embodiments, the exogenous factors include FGF and VEGF. In some embodiments, the exogenous factors include FGF and SCF. In some embodiments, the exogenous factors include FGF and TPO. In some embodiments, the exogenous factors include FGF and LDL. In some embodiments, the exogenous factors include FGF and PI3K inhibitors. In some embodiments, the exogenous factors include VEGF and SCF. In some embodiments, the exogenous factors include VEGF and TPO. In some embodiments, the exogenous factors include VEGF and LDL. In some embodiments, the exogenous factors include VEGF and a PI3K inhibitor. In some embodiments, the exogenous factors include SCF and TPO. In some embodiments, the exogenous factors include SCF and LDL. In some embodiments, the exogenous factors include SCF and a PI3K inhibitor. In some embodiments, the exogenous factors include TPO and LDL. In some embodiments, the exogenous factors include TPO and a PI3K inhibitor. In some embodiments, the exogenous factors include LDL and a PI3K inhibitor.

[0187] In some embodiments, the exogenous factors include BMP pathway activators, FGF and VEGF. In some embodiments, the exogenous factors include BMP pathway activators, FGF and SCF. In some embodiments, the exogenous factors include BMP pathway activators, FGF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, FGF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and SCF. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF and SCF. In some embodiments, the exogenous factors include FGF, VEGF and TPO. In some embodiments, the exogenous factors include FGF, VEGF and LDL. In some embodiments, the exogenous factors include FGF, VEGF and LDL. In some embodiments, the exogenous factors include FGF, SCF and TPO. In some embodiments, the exogenous factors include FGF, SCF and LDL. In some embodiments, the exogenous factors include FGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, TPO and LDL. In some embodiments, the exogenous factors include FGF, TPO and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, LDL and a PI3K inhibitor. In some embodiments, the exogenous factors include VEGF, SCF and TPO. In some embodiments, the exogenous factors include VEGF, SCF and LDL. In some embodiments, the exogenous factors include VEGF, SCF and a PI3K inhibitor. In some embodiments, the exogenous factors include VEGF, TPO and LDL. In some embodiments, the exogenous factors include VEGF, TPO and a PI3K inhibitor.

[0188] In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and SCF. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF and TPO. In some embodiments, the exogenous factors include FGF, VEGF, SCF and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, TPO and LDL. In some embodiments, the exogenous factors include FGF, VEGF, TPO and PI3K inhibitors.In some embodiments, the exogenous factors include FGF, VEGF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, TPO, LDL and PI3K inhibitors.

[0189] In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL and PI3K inhibitors.In some embodiments, the exogenous factors include FGF, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include VEGF, SCF, TPO, LDL and PI3K inhibitors.

[0190] In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO, LDL and PI3K inhibitors.

[0191] In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF, TPO, LDL, and PI3K inhibitors.

[0192] In some embodiments, the exogenous factors include BMP4 and FGF2. In some embodiments, the exogenous factors include BMP4 and VEGF-165. In some embodiments, the exogenous factors include BMP4 and SCF. In some embodiments, the exogenous factors include BMP4 and TPO. In some embodiments, the exogenous factors include BMP4 and LDL. In some embodiments, the exogenous factors include BMP4 and LY294002. In some embodiments, the exogenous factors include FGF2 and VEGF-165. In some embodiments, the exogenous factors include FGF2 and SCF. In some embodiments, the exogenous factors include FGF2 and TPO. In some embodiments, the exogenous factors include FGF2 and LDL. In some embodiments, the exogenous factors include FGF2 and LY294002. In some embodiments, the exogenous factors include VEGF-165 and SCF. In some embodiments, the exogenous factors include VEGF-165 and TPO. In some embodiments, the exogenous factors include VEGF-165 and LDL. In some embodiments, the exogenous factors include VEGF-165 and LY294002. In some embodiments, the exogenous factors include SCF and TPO. In some embodiments, the exogenous factors include SCF and LDL. In some embodiments, the exogenous factors include SCF and LY294002. In some embodiments, the exogenous factors include TPO and LDL. In some embodiments, the exogenous factors include TPO and LY294002. In some embodiments, the exogenous factors include LDL and LY294002.

[0193] In some embodiments, the exogenous factors include BMP4, FGF2 and VEGF-165. In some embodiments, the exogenous factors include BMP4, FGF2 and SCF. In some embodiments, the exogenous factors include BMP4, FGF2 and TPO. In some embodiments, the exogenous factors include BMP4, FGF2 and LDL. In some embodiments, the exogenous factors include BMP4, FGF2 and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165 and SCF. In some embodiments, the exogenous factors include BMP4, VEGF-165 and TPO. In some embodiments, the exogenous factors include BMP4, VEGF-165 and LDL. In some embodiments, the exogenous factors include BMP4, VEGF-165 and LY294002. In some embodiments, the exogenous factors include BMP4, SCF and TPO. In some embodiments, the exogenous factors include BMP4, SCF and LDL. In some embodiments, the exogenous factors include BMP4, SCF and LY294002. In some embodiments, the exogenous factors include BMP4, TPO and LDL. In some embodiments, the exogenous factors include BMP4, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, LDL and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165 and SCF. In some embodiments, the exogenous factors include FGF2, VEGF-165 and TPO. In some embodiments, the exogenous factors include FGF2, VEGF-165 and LDL. In some embodiments, the exogenous factors include FGF2, VEGF-165 and LDL. In some embodiments, the exogenous factors include FGF2, SCF and TPO. In some embodiments, the exogenous factors include FGF2, SCF and LDL. In some embodiments, the exogenous factors include FGF2, SCF and LY294002. In some embodiments, the exogenous factors include FGF2, TPO and LDL. In some embodiments, the exogenous factors include FGF2, TPO and LY294002. In some embodiments, the exogenous factors include FGF2, LDL and LY294002. In some embodiments, the exogenous factors include VEGF-165, SCF and TPO. In some embodiments, the exogenous factors include VEGF-165, SCF and LDL. In some embodiments, the exogenous factors include VEGF-165, SCF and LY294002. In some embodiments, the exogenous factors include VEGF-165, TPO and LDL.In some embodiments, the exogenous factors include VEGF-165, TPO, and LY294002.

[0194] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165 and SCF. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165 and TPO. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165 and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165 and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, SCF and TPO. In some embodiments, the exogenous factors include BMP4, FGF2, SCF and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, SCF and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, TPO and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF and TPO. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF and LDL. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF and LDL. In some embodiments, the exogenous factors include BMP4, VEGF-165, TPO and LDL. In some embodiments, the exogenous factors include BMP4, VEGF-165, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP4, SCF, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, SCF, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, TPO, LDL and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF and TPO. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF and LDL. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, TPO and LDL. In some embodiments, the exogenous factors include FGF2, VEGF-165, TPO and LY294002.In some embodiments, the exogenous factors include FGF2, VEGF-165, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, SCF, TPO, and LDL. In some embodiments, the exogenous factors include FGF2, SCF, TPO, and LY294002. In some embodiments, the exogenous factors include FGF2, SCF, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, TPO, LDL, and LY294002.

[0195] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF and TPO. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, TPO and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, SCF, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, SCF, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, TPO, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, TPO, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, SCF, TPO, LDL and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, LDL and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, LDL and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO and LY294002.In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, SCF, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include VEGF-165, SCF, TPO, LDL, and LY294002.

[0196] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, TPO, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, TPO, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, TPO, LDL and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO, LDL, and LY294002.

[0197] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, TPO, LDL, and LY294002.

[0198] In some embodiments, the concentration of the bone morphogenetic protein (BMP) activator in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1-50 ng / ml, or any range thereof. In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1-50 ng / ml.

[0199] In some embodiments, the bone morphogenetic protein (BMP) activator is BMP4. In some embodiments, the concentration of BMP4 in the differentiation medium is about 0.1-500ng / ml, about 1-250ng / ml, about 1-150ng / ml, about 5-100ng / ml, about or about 0.1ng / ml, about 1ng / ml, about 2ng / ml, about 3ng / ml, about 4ng / ml, about 5ng / ml, about 6ng / ml, about 7ng / ml, about 8ng / ml, about 9ng / ml, about 10ng / ml, about 11ng / ml, about 12ng / ml, about 13ng / ml, about 14ng / ml, about 15ng / ml, about 16ng / ml, about 17ng / ml, about 18ng / ml, about 19ng / ml. In some embodiments, BMP4 is present in the differentiation medium at about 1-50 ng / ml, or any range thereof. In some embodiments, BMP4 is present in the differentiation medium at about 1-50 ng / ml, or any range thereof.

[0200] In some embodiments, the concentration of FGF2 in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml In some embodiments, FGF2 is present in the differentiation medium at about 1-50 ng / ml, or any range therein. In some embodiments, FGF2 is present in the differentiation medium at about 1-50 ng / ml.

[0201] In some embodiments, the concentration of VEGF in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml In some embodiments, VEGF is present in the differentiation medium at about 1-100 ng / ml, or any range derived therefrom. In some embodiments, VEGF is present in the differentiation medium at about 1-100 ng / ml.

[0202] In some embodiments, the concentration of SCF in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the SCF is present in the differentiation medium at about 1-100 ng / ml, or any range derived therefrom. In some embodiments, the SCF is present in the differentiation medium at about 1-100 ng / ml.

[0203] In some embodiments, the concentration of TPO in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the TPO is present in the differentiation medium at about 1-100 ng / ml, or any range derived therefrom. In some embodiments, the TPO is present in the differentiation medium at about 1-100 ng / ml.

[0204] In some embodiments, the concentration of LDL in the differentiation medium is about 0.1-500 μg / ml, about 1-250 μg / ml, about 1-150 μg / ml, about 5-100 μg / ml, about or about 0.1 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 μg / ml, about 19 μg / ml. In some embodiments, the LDL is present in the differentiation medium at about 1-50 μg / ml, or any range derived therefrom. In some embodiments, the LDL is present in the differentiation medium at about 1-50 μg / ml.

[0205] In some embodiments, the concentration of the PI3K inhibitor in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM. In some embodiments, the PI3K inhibitor is present in the differentiation medium at about 0.1-100 μM.

[0206] In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, the concentration of LY294002 is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about In some embodiments, LY294002 is present in the differentiation medium at about 0.1-100 μM.

[0207] In some embodiments, the concentration of the pyrimidine-indole derivative in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM. In some embodiments, the pyrimidine-indole derivative is present in the differentiation medium at about 0.1-10 μM.

[0208] In some embodiments, the pyrimidine-indole derivative is UM729. In some embodiments, the concentration of UM729 is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 62 μM, In some embodiments, UM729 is present in the differentiation medium at about 0.1-10 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derived therefrom. In some embodiments, UM729 is present in the differentiation medium at about 0.1-10 μM.

[0209] In some embodiments, the concentration of the aryl hydrocarbon receptor antagonist in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM. In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at about 0.1-10 μM.

[0210] In some embodiments, the aryl hydrocarbon receptor antagonist is StemRegenin 1 (SR1). In some embodiments, the concentration of SR1 is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM In some embodiments, SR1 is present in the differentiation medium at about 0.1-10 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derived therefrom. In some embodiments, SR1 is present in the differentiation medium at about 0.1-10 μM.

[0211] In some embodiments, the concentration of the TGF-β receptor inhibitor in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM In some embodiments, the TGF-β receptor inhibitor is present in the differentiation medium at about 0.1-20 μM.

[0212] In some embodiments, the TGF-β receptor inhibitor is GW788388. In some embodiments, the concentration of GW788388 in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM In some embodiments, GW788388 is present in the differentiation medium at about 0.1-20 μM.

[0213] In some embodiments, the TGF-β receptor inhibitor is SB431542. In some embodiments, the concentration of GW788388 in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM In some embodiments, SB431542 is present in the differentiation medium at about 0.1-20 μM.

[0214] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, and VEGF. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor. In some embodiments, the HP differentiation medium comprises BMP4, FGF2, and VEGF-165. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and a ROCK inhibitor. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and a ROCK inhibitor.

[0215] In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 5-50ng / mL FGF2, and 1-100ng / mL VEGF-165. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 1-20μM ROCK inhibitor. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 1-20μM Y27632.

[0216] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and a pyrimidine-indole derivative. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a pyrimidine-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0217] In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and pyrimidine-indole derivatives. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, a pyrimidine-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, a pyrimidine-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and SR1. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and UM729. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, UM729, and SR1.

[0218] In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, and 0.1-10 μM UM729. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, and 0.1-10 μM SR1. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM UM729, and 0.1-10 μM SR1.

[0219] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a pyrimidine-indole derivative, and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, an aryl hydrocarbon receptor antagonist, and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a pyrimidine-indole derivative, an aryl hydrocarbon receptor antagonist, and a TGF-β receptor inhibitor.

[0220] In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165 and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165 and GW788388. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, UM729 and GW788388. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, SR1 and GW788388. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, UM729, SR1 and GW788388.

[0221] In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM GW788388. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, 0.1-10μM UM729, and 0.1-20μM GW788388. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM SR1, and 0.1-20 μM GW788388. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM UM729, 0.1-10 μM SR1, and 0.1-20 μM GW788388.

[0222] In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165 and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165 and SB431542. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, UM729 and SB431542. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, SR1 and SB431542. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, UM729, SR1 and SB431542.

[0223] In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM SB431542. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, 0.1-10μM UM729, and 0.1-20μM SB431542. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM SR1, and 0.1-20 μM SB431542. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM UM729, 0.1-10 μM SR1, and 0.1-20 μM SB431542. NK cell differentiation medium

[0224] In some embodiments, the present disclosure provides a differentiation medium for producing NK cells from HP cells. In some embodiments, NK cells are produced by HP cells. In some embodiments, HP cells produced by the compositions and methods of the present disclosure are further differentiated into NK cells.

[0225] In some embodiments, at least one exogenous factor is used to culture HP cell colonies to form differentiated NK cells. In some embodiments, the exogenous factor is stem cell factor (SCF). In some embodiments, the exogenous factor is IL-7. In some embodiments, the exogenous factor is IL-15. In some embodiments, the exogenous factor is IL-12. In some embodiments, the exogenous factor is FLT3L. In some embodiments, the exogenous factor is a pyrimidine-indole derivative. In some embodiments, the exogenous factor is an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor is selected from SCF, IL-7, IL-15, IL-12, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists.

[0226] In some embodiments, the exogenous factors include SCF and IL-7. In some embodiments, the exogenous factors include SCF and IL-15. In some embodiments, the exogenous factors include SCF and IL-12. In some embodiments, the exogenous factors include SCF and FLT3L. In some embodiments, the exogenous factors include SCF and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7 and IL-15. In some embodiments, the exogenous factors include IL-7 and IL-12. In some embodiments, the exogenous factors include IL-7 and FLT3L. In some embodiments, the exogenous factors include IL-7 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-15 and IL-12. In some embodiments, the exogenous factors include IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-12 and FLT3L. In some embodiments, the exogenous factors include IL-12 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-12 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists.

[0227] In some embodiments, the exogenous factors include SCF, IL-7, and IL-12. In some embodiments, the exogenous factors include SCF, IL-7, and IL-15. In some embodiments, the exogenous factors include SCF, IL-7, and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, and aryl hydrocarbon receptor antagonists. . In some embodiments, the exogenous factors include SCF, IL-12, and IL-15. In some embodiments, the exogenous factors include SCF, IL-12, and FLT3L. In some embodiments, the exogenous factors include SCF, IL-12, and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-12, and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15, and FLT3L. In some embodiments, the exogenous factors include SCF, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-7, IL-12 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-12, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-12, IL-15 and aryl hydrocarbon receptor antagonists.In some embodiments, the exogenous factors include IL-12, FLT3L, and a pyrimidine-indole derivative. In some embodiments, the exogenous factors include IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist.

[0228] In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L and pyrimidine-indole derivatives.In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-15, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists.

[0229] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, and pyrimidine-indole derivatives.In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-12, IL-15, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists.

[0230] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L, pyrimidine-indole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, pyrimidine-indole derivatives, and aryl hydrocarbon receptor antagonists.

[0231] In some embodiments, the exogenous factors include SCF, IL-7 and IL-12. In some embodiments, the exogenous factors include SCF, IL-7 and IL-15. In some embodiments, the exogenous factors include SCF, IL-7 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-7 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12 and IL-15. In some embodiments, the exogenous factors include SCF, IL-12 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-12 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-12 and SR1. In some embodiments, the exogenous factors include SCF, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include SCF, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, pyrimidine-indole derivatives and SR1. In some embodiments, the exogenous factors include IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-7, IL-12 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-15 and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15 and SR1. In some embodiments, the exogenous factors include IL-7, FLT3L and pyrimidine-indole derivatives. In some embodiments, the exogenous factors include IL-7, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, UM729 and SR1. In some embodiments, the exogenous factors include IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-12, IL-15 and UM729. In some embodiments, the exogenous factors include IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include IL-12, FLT3L and UM729.In some embodiments, the exogenous factors include IL-12, FLT3L, and SR1.

[0232] In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and UM729. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-12, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and UM729. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L and UM729. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, UM729 and SR1.In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, IL-15, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, FLT3L, UM729 and SR1.

[0233] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, UM729 and SR1.In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include IL-12, IL-15, FLT3L, UM729 and SR1.

[0234] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, UM729 and SR1.

[0235] In some embodiments, the concentration of SCF in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the SCF is present in the differentiation medium at about 1-50 ng / ml, or any range derived therefrom. In some embodiments, the SCF is present in the differentiation medium at about 1-50 ng / ml.

[0236] In some embodiments, the concentration of IL-7 in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the IL-7 is present in the differentiation medium at about 1-50 ng / ml, or any range derived therefrom. In some embodiments, the IL-7 is present in the differentiation medium at about 1-50 ng / ml.

[0237] In some embodiments, the concentration of IL-12 in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the IL-12 is present in the differentiation medium at about 1-100 ng / ml, or any range derived therefrom. In some embodiments, the IL-12 is present in the differentiation medium at about 1-100 ng / ml.

[0238] In some embodiments, the concentration of IL-15 in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the IL-15 is present in the differentiation medium at about 1-100 ng / ml, or any range derived therefrom. In some embodiments, the IL-15 is present in the differentiation medium at about 1-100 ng / ml.

[0239] In some embodiments, the concentration of FLT3L in the differentiation medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the FLT3L is present in the differentiation medium at about 1-100 ng / ml, or any range therein. In some embodiments, the FLT3L is present in the differentiation medium at about 1-100 ng / ml.

[0240] In some embodiments, the concentration of the pyrimidine-indole derivative in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM. In some embodiments, the pyrimidine-indole derivative is present in the differentiation medium at about 0.1-10 μM.

[0241] In some embodiments, the pyrimidine-indole derivative is UM729. In some embodiments, the concentration of UM729 is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 62 μM, In some embodiments, UM729 is present in the differentiation medium at about 0.1-10 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derived therefrom. In some embodiments, UM729 is present in the differentiation medium at about 0.1-10 μM.

[0242] In some embodiments, the concentration of the aryl hydrocarbon receptor antagonist in the differentiation medium is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM. In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at about 0.1-10 μM.

[0243] In some embodiments, the aryl hydrocarbon receptor antagonist is StemRegenin 1 (SR1). In some embodiments, the concentration of SR1 is about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM In some embodiments, the StemRegenin 1 (SR1) is present in the differentiation medium at about 0.1-10 μM.

[0244] In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15 and FLT3L. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L and pyrimidine-indole derivatives. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L and an aryl hydrocarbon receptor antagonist. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, pyrimidine-indole derivatives and an aryl hydrocarbon receptor antagonist.

[0245] In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L and SR1. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L and UM729. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, SR1 and UM729.

[0246] In some embodiments, the NK cell differentiation medium comprises 1-50ng / ml SCF, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15 and 1-100ng / ml FLT3L. In some embodiments, the NK cell differentiation medium comprises 1-50ng / ml SCF, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml FLT3L and 0.1-10μΜ pyrimidine-indole derivatives. In some embodiments, the NK cell differentiation medium comprises 1-50ng / ml SCF, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml FLT3L, and 0.1-10μΜ aryl hydrocarbon receptor antagonist. In some embodiments, the NK cell differentiation medium comprises 1-50ng / ml SCF, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml FLT3L, 0.1-10μΜ pyrimidine-indole derivatives, and 0.1-10μΜ aryl hydrocarbon receptor antagonist.

[0247] In some embodiments, the NK cell differentiation medium comprises 1-50ng / ml SCF, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml FLT3L and SR1. In some embodiments, the NK cell differentiation medium comprises 1-50ng / ml SCF, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml FLT3L and 0.1-10μΜ UM729. In some embodiments, the NK cell differentiation medium comprises 1-50 ng / ml SCF, 1-50 ng / ml IL-7, 1-100 ng / ml IL-12, 1-100 ng / ml IL-15, 1-100 ng / ml FLT3L, 1-10 μΜ SR1, and 0.1-10 μΜ UM729.

[0248] In some embodiments, the NK cell differentiation medium comprises a defined xeno-free basal medium, 1-50ng / ml SCF, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml FLT3L and 0.1-10μΜ SR1. In some embodiments, the NK cell differentiation medium comprises a defined xeno-free basal medium, 1-50ng / ml SCF, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml FLT3L and 0.1-10μΜ UM729. In some embodiments, the NK cell differentiation medium comprises a defined xeno-free basal medium, 1-50 ng / ml SCF, 1-50 ng / ml IL-7, 1-100 ng / ml IL-12, 1-100 ng / ml IL-15, 1-100 ng / ml FLT3L, 0.1-10 μΜ SR1, and 0.1-10 μΜ UM729. NK Cell Expansion Medium

[0249] In some embodiments, the present disclosure provides an expansion medium for producing mature NK cells from differentiated NK cells. In some embodiments, differentiated NK cells are produced by HP cells. In some embodiments, the differentiated NK cells produced by the compositions and methods of the present disclosure are further expanded into mature NK cells.

[0250] In some embodiments, at least one exogenous factor is used to culture a differentiated NK cell population to form mature NK cells. In some embodiments, the exogenous factor is IL-2. In some embodiments, the exogenous factor is an exogenous factor IL-7. In some embodiments, the exogenous factor is IL-12. In some embodiments, the exogenous factor is IL-15. In some embodiments, the exogenous factor is IL-18. In some embodiments, the exogenous factor is LDL. In some embodiments, the exogenous factor is an activated bead. In some embodiments, the exogenous factor is selected from IL-2, IL-7, IL-12, IL-15, IL-18 and an activated bead.

[0251] In some embodiments, the exogenous factors include IL-2 and IL-7. In some embodiments, the exogenous factors include IL-2 and IL-12. In some embodiments, the exogenous factors include IL-2 and IL-15. In some embodiments, the exogenous factors include IL-2 and IL-18. In some embodiments, the exogenous factors include IL-2 and activation beads. In some embodiments, the exogenous factors include IL-7 and IL-12. In some embodiments, the exogenous factors include IL-7 and IL-15. In some embodiments, the exogenous factors include IL-7 and IL-18. In some embodiments, the exogenous factors include IL-7 and activation beads. In some embodiments, the exogenous factors include IL-12 and IL-15. In some embodiments, the exogenous factors include IL-12 and IL-18. In some embodiments, the exogenous factors include IL-12 and activation beads. In some embodiments, the exogenous factors include IL-15 and IL-18. In some embodiments, the exogenous factors include IL-15 and activation beads. In some embodiments, the exogenous factors include IL-18 and activation beads.

[0252] In some embodiments, the exogenous factors include IL-2, IL-7 and IL-12. In some embodiments, the exogenous factors include IL-2, IL-7 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-7 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-12 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-7, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-7, IL-12 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-7, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-15, IL-18 and activation beads.

[0253] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-15, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-15, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-12, IL-15, IL-18 and activation beads.

[0254] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, IL-18 and activation beads.

[0255] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads.

[0256] In some embodiments, the exogenous factors include IL-2 and IL-7. In some embodiments, the exogenous factors include IL-2 and IL-12. In some embodiments, the exogenous factors include IL-2 and IL-15. In some embodiments, the exogenous factors include IL-2 and IL-18. In some embodiments, the exogenous factors include IL-2 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7 and IL-12. In some embodiments, the exogenous factors include IL-7 and IL-15. In some embodiments, the exogenous factors include IL-7 and IL-18. In some embodiments, the exogenous factors include IL-7 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12 and IL-15. In some embodiments, the exogenous factors include IL-12 and IL-18. In some embodiments, the exogenous factors include IL-12 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-15 and IL-18. In some embodiments, the exogenous factors include IL-15 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-18 and activated beads coated with anti-CD2 / anti-NKp46.

[0257] In some embodiments, the exogenous factors include IL-2, IL-7 and IL-12. In some embodiments, the exogenous factors include IL-2, IL-7 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-7 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7 and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-12 and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-15 and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-18, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-12, and IL-15. In some embodiments, the exogenous factors include IL-7, IL-12, and IL-18. In some embodiments, the exogenous factors include IL-7, IL-12, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-15, and IL-18. In some embodiments, the exogenous factors include IL-7, IL-15, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-18, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12, IL-15, and IL-18. In some embodiments, the exogenous factors include IL-12, IL-15, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

[0258] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-7, IL-18 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12, IL-18, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-15, IL-18, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, and IL-18. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-12, IL-18, and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

[0259] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, IL-18 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, IL-18 and activated beads coated with anti-CD2 / anti-NKp46.

[0260] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

[0261] In some embodiments, the concentration of IL-2 in the expansion medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the IL-2 is present in the expansion medium at about 1-50 ng / ml, or any range derived therefrom. In some embodiments, the IL-2 is present in the expansion medium at about 1-50 ng / ml.

[0262] In some embodiments, the concentration of IL-7 in the expansion medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml In some embodiments, the IL-7 is present in the expansion medium at about 1-50 ng / ml, or any range derived therefrom. In some embodiments, the IL-7 is present in the expansion medium at about 1-50 ng / ml.

[0263] In some embodiments, the concentration of IL-12 in the expansion medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the IL-12 is present in the expansion medium at about 1-100 ng / ml, or any range derived therefrom. In some embodiments, the IL-12 is present in the expansion medium at about 1-100 ng / ml.

[0264] In some embodiments, the concentration of IL-15 in the expansion medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the IL-15 is present in the expansion medium at about 1-100 ng / ml, or any range derived therefrom. In some embodiments, the IL-15 is present in the expansion medium at about 1-100 ng / ml.

[0265] In some embodiments, the concentration of IL-18 in the expansion medium is about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml. In some embodiments, the IL-18 is present in the expansion medium at about 1-100 ng / ml, or any range derived therefrom. In some embodiments, the IL-18 is present in the expansion medium at about 1-100 ng / ml.

[0266] In some embodiments, activation beads are present in the expansion medium.

[0267] In some embodiments, the activation beads coated with anti-CD2 / anti-NKp46 are present in the expansion medium in a ratio based on the number of differentiated NK cells. For example, there is one activation bead for each NK cell, or the ratio of activation beads to NK cells is 1:1.

[0268] In some embodiments, the ratio of activated beads to NK cells is at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, or at least 50:1.

[0269] In some embodiments, the ratio of activated beads to NK cells is 1:1 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 6:1, 6:1 to 7:1, 7:1 to 8:1, 8:1 to 9:1, 9:1 to 10:1, 10:1 to 15:1, 15:1 to 20:1, 20:1 to 25:1, 25:1 to 30:1, 30:1 to 35:1, 35:1 to 40:1, 40:1 to 45:1, or 45:1 to 50:1.

[0270] In some embodiments, the ratio of NK cells to activated beads is at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, or at least 50:1.

[0271] In some embodiments, the ratio of NK cells to activation beads is 1:1 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 6:1, 6:1 to 7:1, 7:1 to 8:1, 8:1 to 9:1, 9:1 to 10:1, 10:1 to 15:1, 15:1 to 20:1, 20:1 to 25:1, 25:1 to 30:1, 30:1 to 35:1, 35:1 to 40:1, 40:1 to 45:1, or 45:1 to 50:1.

[0272] In some embodiments, the NK cell expansion medium comprises IL-2, IL-7, IL-12, IL-15, IL-18 and activation beads. In some embodiments, the NK cell expansion medium comprises a defined xenogeneic gene-free basal medium, IL-2, IL-7, IL-12, IL-15, IL-18 and activation beads.

[0273] In some embodiments, the NK cell expansion medium comprises IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the NK cell expansion medium comprises a defined xenogeneic-free basal medium, IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

[0274] In some embodiments, the NK cell expansion medium comprises 1-50 ng / ml IL-2, 1-50 ng / ml IL-7, 1-100 ng / ml IL-12, 1-100 ng / ml IL-15, 1-100 ng / ml IL-18, and activation beads at a cell: bead ratio of 1: 1. In some embodiments, the NK cell expansion medium comprises a defined xeno-free basal medium comprising 1-50 ng / ml IL-2, 1-50 ng / ml IL-7, 1-100 ng / ml IL-12, 1-100 ng / ml IL-15, 1-100 ng / ml IL-18, and activation beads at a cell: bead ratio of 1: 1.

[0275] In some embodiments, the NK cell expansion medium comprises 1-50ng / ml IL-2, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml IL-18, and activation beads coated with anti-CD2 / anti-NKp46 at a cell: bead ratio of 1: 1. In some embodiments, the NK cell expansion medium comprises a defined xenogeneic gene-free basal medium, comprising 1-50ng / ml IL-2, 1-50ng / ml IL-7, 1-100ng / ml IL-12, 1-100ng / ml IL-15, 1-100ng / ml IL-18, and activation beads coated with anti-CD2 / anti-NKp46 at a cell: bead ratio of 1: 1. Differentiation method

[0276] In some aspects, the present disclosure provides a method for producing hematopoietic progenitor cells from stem cells. In some aspects, the present disclosure provides a method for producing NK cells from stem cells. In some aspects, the present disclosure provides a method for producing NK cells from hematopoietic progenitor cells. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells, and differentiating the hematopoietic progenitor cells into NK cells.

[0277] In some aspects, the present disclosure provides a method for producing common lymphoid progenitor cells (CLP) from stem cells. In some embodiments, the method includes differentiating stem cells into hematopoietic progenitor cells, and differentiating the hematopoietic progenitor cells into CLP. CLP refers to a cell that is a precursor of a lymphocyte. CLP is a cell that can be transformed into hematopoiesis to a hematopoietic cell type. In some embodiments, CLP is CD45+CD7+CD5+ / lo CD3-CD56-. In some embodiments, CLP is CD45+CD5+ / lo CD7+. In some aspects, the present disclosure provides a method for producing NK cells from CLP. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells, differentiating the hematopoietic progenitor cells into CLP, and differentiating the CLP into NK cells.

[0278] In some aspects, the present disclosure provides a method for generating pre-NK cell progenitors (preNKPs) from stem cells. In some embodiments, the method comprises differentiating stem cells into hematopoietic progenitors, differentiating the hematopoietic progenitors into CLPs, and differentiating the CLPs into PreNKPs. PreNKPs are intermediate cells between CLPs and NKPs. In some embodiments, PreNKPs are Lin- / CD244+ / c-Kit low / IL-7Ra+ / FLT3- / CD122-. In some aspects, the present disclosure provides a method for producing NK cells from preNKP. In some embodiments, the method for producing NK cells comprises differentiating stem cells into hematopoietic progenitor cells, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into preNKPs, and differentiating the preNKPs into NK cells.

[0279] In some aspects, the present disclosure provides a method for producing NK cell precursors (NKP) from stem cells. In some embodiments, the method includes differentiating stem cells into hematopoietic progenitor cells, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into PreNKPs, and differentiating the PreNKPs into NKPs. NKP is the last cell before the final NK lineage is finalized. In some embodiments, NKP is Lin- / NK1.1-DX5- / IL-7Ra+ / CD122+ / NKG2D+. In some aspects, the present disclosure provides a method for producing NK cells from NKP. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into preNKPs, differentiating the preNKPs into NKPs, and differentiating the NKPs into NK cells.

[0280] In some aspects, the present disclosure provides a method for producing immature NK (iNK) cells from stem cells. In some embodiments, the method includes differentiating stem cells into hematopoietic progenitor cells, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into PreNKPs, differentiating the PreNKPs into NKPs, and differentiating the NKPs into iNK cells. In some aspects, the present disclosure provides a method for producing NK cells from iNK cells. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into preNKPs, differentiating the preNKPs into NKPs, differentiating the NKPs into iNK cells, and differentiating the iNK cells into NK cells.

[0281] In some aspects, the present disclosure provides a method for producing mature NK (mNK) cells from stem cells. In some aspects, the present disclosure provides a method for producing mature NK cells from immature NK cells. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into preNKPs, differentiating the preNKPs into NKPs, differentiating the NKPs into iNK cells, and differentiating the iNK cells into mNK cells.

[0282] In some embodiments, the methods provided herein are free of xenologous genes. In some embodiments, the methods provided herein are free of animal-derived raw materials. Expression markers

[0283] Differentiation of source cells to NK cells can be assessed by detecting markers (such as CD56, CD94, CD117, NKG2D, DNAM-1 and NKp46), for example, by flow cytometry. Differentiation can also be assessed by the morphological characteristics of NK cells, such as large volume, high protein synthesis activity in the abundant endoplasmic reticulum (ER) and / or pre-granules. The maturation of NK cells can be assessed by detecting one or more functionally related markers, such as CD94, CD161, NKp44, DNAM-1, 2B4, NKp46, CD94, KIR and NKG2 family activation receptors (such as NKG2D). The maturity of NK cells can also be assessed by detecting specific markers at different developmental stages. For example, in one embodiment, preNKP cells are CD34+, CD45RA+, CD10+, CD117- and / or CD161-. In another embodiment, immature NK cells are CD34-, CD117+, CD161+, NKp46- and / or CD94 / NKG2A-. In another embodiment, CD56bright NK cells are CD117+, NKp46+, CD94 / NKG2A+, CD16- and / or KIR+ / -. In another embodiment, CD56dim NK cells are CD117-, NKp46+, CD94 / NKG2A+ / -, CD16+ and / or KIR+. In a specific embodiment, the maturation of NK cells (such as TSNK cells) is determined by the percentage of NK cells (such as TSNK cells) of CD161-, CD94+ and / or NKp46+. In a more specific embodiment, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65% or 70% of mature NK cells (such as TSNK cells) are NKp46+. In another more specific embodiment, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of mature NK cells (such as TSNK cells) are CD94+. In another more specific embodiment, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of mature NK cells (such as TSNK cells) are CD161-.

[0284] In some embodiments, the differentiation of source cells into NK cells is assessed by detecting, for example, the expression level of CD3, CD7 or CD127, CD10, CD14, CD15, CD16, CD33, CD34, CD56, CD94, CD117, CD161, NKp44, NKp46, NKG2D, DNAM-1, 2B4 or TO-PRO-3, for example, using antibodies against one or more of these cell markers. Such antibodies can be combined with a detectable label, such as a fluorescent label, such as FITC, R-PE, PerCP, PerCP-Cy5.5, APC, APC-Cy7 or APC-H7. Source cells

[0285] In some embodiments, NK cells are generated from source cells.Any progenitor cell known in the art can be used as a source cell in the methods of the present disclosure.

[0286] In some embodiments, the source cell is hESC. In some embodiments, the source cell is iPSC. NK cells derived from iPSC may also be referred to as iPSC-derived NK cells.

[0287] In immunotherapy, the source cells can be allogeneic or autologous, meaning from a donor or from a subject, respectively. In some embodiments, the source cells are allogeneic. In some embodiments, the source cells are autologous.

[0288] In some embodiments, the source cell is a peripheral blood cell. As used herein, the term "peripheral blood cell" refers to a cell derived from circulating blood, and includes hematopoietic stem cells that can proliferate, selectively differentiate, and mature. Therefore, peripheral blood NK cells may also be referred to as differentiated hematogenous NK cells (bdNK).

[0289] In some embodiments, the source cells include hematopoietic stem cells characterized by being CD34+ and / or CD45+.

[0290] In some embodiments, the source cells include common lymphoid progenitor cells characterized as CD45+CD7+CD56-.

[0291] In some embodiments, NK cells can be produced by induced pluripotent stem cells (iPSC). iPSC is a type of pluripotent stem cell derived from adult somatic cells, and the adult somatic cells have been genetically reprogrammed to a state similar to embryonic stem cells by forced expression of genes and factors important for maintaining the defining characteristics of embryonic stem cells. iPSC can be produced from tissues containing somatic cells, including but not limited to skin, dental tissue, peripheral blood and urine. To produce iPSC, somatic cells can be reprogrammed by the following methods, including but not limited to transient expression of reprogramming factors, virus-free methods, adenovirus, plasmid, minicircle vector (minicircle vector), exon vector, Sendai virus, synthetic mRNA, self-replicating RNA, retrovirus, slow virus, PhiC31 integrase, excisable transposon, gene editing or recombinant protein based on CRISPR, etc. Methods for generating iPSCs are disclosed in US 9315779 B2, US10370452 B2, US11319555B2 and US20210015859A1, the entire contents of which are incorporated herein by reference. Mesoderm / embryoid body formation

[0292] In some embodiments, the methods described herein include generating mesodermal cells from iPSC and / or hESC. As stem cells begin to differentiate, three different germ layers are formed: ectoderm, mesoderm, and endoderm. Immune cells (such as NK cells) differentiate from mesodermal cells. In some embodiments, the mesodermal cells generated by the disclosed methods further differentiate into NK cells.

[0293] The mesoderm formation step may comprise contacting the iPSC or hESC cell population with one or more factors in a defined expansion medium for a specific period of time. In some embodiments, the mesoderm cells are formed from embryoid bodies.

[0294] In some embodiments, stem cells are contacted with differentiation medium as described herein for a period of time to produce mesodermal cells and / or embryoid bodies. In some embodiments, the time period sufficient to produce mesodermal cells from stem cells is at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours or at least 120 hours.

[0295] In some embodiments, the duration of the mesoderm formation step is 12 hours to 24 hours, 24 hours to 48 hours, 48 ​​hours to 72 hours, 72 hours to 96 hours, or 96 hours to 120 hours.

[0296] In some embodiments, cells derived from source cells are placed in a container to induce cell aggregation and cluster formation. In some embodiments, the container is a plate with holes or microwells, such as a 96-well plate and / or AggrewellTM Plates (microplates; STEMCELL Technologies Inc., Vancouver, Canada) In some embodiments, for example, Aggrewell TM Plate, prepare cell clusters on a plate with micropores to form cell aggregates of uniform size and shape. In some embodiments, at least 1 cell, at least 10 cells, at least 100 cells, at least 1000 cells, at least 10000 cells, or at least 50000 cells are inoculated in each well. In some embodiments, 1 cell to 10 cells, 10 cells to 100 cells, 100 cells to 1000 cells, 1000 cells to 10000 cells, or 10000 cells to 50000 cells are inoculated in each well. Differentiation into hematopoietic progenitor cells

[0297] In some aspects, the disclosure provides methods for producing NK cells from hematopoietic progenitor cells. In some embodiments, the method for producing NK cells includes differentiating hematopoietic progenitor cells into NK cells. In some embodiments, the methods provided herein are free of heterologous genes.

[0298] One aspect of the present disclosure is that the method for producing NK cells may include a hematopoietic progenitor cell differentiation step. The hematopoietic progenitor cell differentiation step may include contacting an embryoid body cell population with one or more factors in a defined differentiation medium for a specific period of time, thereby inducing the formation of hematopoietic progenitor cells in the cell population. Then, the hematopoietic progenitor cells are defined by expressing a combination of multiple markers.

[0299] In some embodiments, mesoderm and / or embryoid body cells are contacted with differentiation medium as described herein for a period of time to produce hematopoietic progenitor cells. In some embodiments, the time period sufficient to produce hematopoietic progenitor cells from mesoderm and / or embryoid body cells is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days or at least 20 days.

[0300] In some embodiments, the duration of the step of differentiating into hematopoietic progenitor cells is 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, 14 days to 15 days, 15 days to 16 days, 16 days to 17 days, 17 days to 18 days, 18 days to 19 days, or 19 days to 20 days.

[0301] In some embodiments, the hematopoietic progenitor cells express CD34, CD43, and CD45. In some embodiments, the methods of the present disclosure increase the percentage of CD34+CD43+CD45+ triple positive cells.

[0302] In some embodiments, the methods of the present disclosure produce a cell population from iPSCs having a purity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of CD34+CD43+CD45+ triple positive cells.

[0303] In some embodiments, the methods of the present disclosure produce cell populations from iPSCs having a purity of 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% CD34+CD43+CD45+ triple positive cells.

[0304] In some embodiments, the methods and compositions disclosed herein increase the yield ratio of hematopoietic progenitor cells (HP) from source cells (SC), such as hESC or iPSC. In some embodiments, the ratio of HP to source cells (SC) is expressed as HP / SC. In some embodiments, the ratio of HP to hESC is expressed as HP / hESC. In some embodiments, the ratio of HP to iPSC is expressed as HP / iPSC.

[0305] In some embodiments, the yield ratio of HP / SC is about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, or about 10: 1. In some embodiments, the yield ratio of HP / SC is about 1: 1 to about 2: 1, about 2: 1 to about 3: 1, about 3: 1 to about 4: 1, about 4: 1 to about 5: 1, about 5: 1 to about 6: 1, about 6: 1 to about 7: 1, about 7: 1 to about 8: 1, about 8: 1 to about 9: 1, or about 9: 1 to about 10: 1.

[0306] In some embodiments, the methods and compositions of the present disclosure improve the yield of HP / SC. For example, the methods and compositions can improve HP / SC from about 2:1 to about 4:1, or improve by about 2 times. In some embodiments, the methods and compositions of the present disclosure can improve the yield of HP / SC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 times. In some embodiments, the yield ratio of HP / SC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10.

[0307] In some embodiments, the yield ratio of HP to stem cells (StC) (HP / StC) is about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, or about 10: 1. In some embodiments, the yield ratio of HP / StC is about 1: 1 to about 2: 1, about 2: 1 to about 3: 1, about 3: 1 to about 4: 1, about 4: 1 to about 5: 1, about 5: 1 to about 6: 1, about 6: 1 to about 7: 1, about 7: 1 to about 8: 1, about 8: 1 to about 9: 1, or about 9: 1 to about 10: 1.

[0308] In some embodiments, the methods and compositions of the present disclosure improve the yield ratio of HP to stem cells (StC) (HP / StC). For example, the methods and compositions can increase HP / StC from about 2:1 to about 4:1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can increase the yield ratio of HP / StC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times. In some embodiments, the yield ratio of HP / StC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10.

[0309] In some embodiments, the yield ratio of HP to iPSC (HP / iPSC) is about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, or about 10: 1. In some embodiments, the yield ratio of HP / iPSC is about 1: 1 to about 2: 1, about 2: 1 to about 3: 1, about 3: 1 to about 4: 1, about 4: 1 to about 5: 1, about 5: 1 to about 6: 1, about 6: 1 to about 7: 1, about 7: 1 to about 8: 1, about 8: 1 to about 9: 1, or about 9: 1 to about 10: 1.

[0310] In some embodiments, the method and composition of the present disclosure improves the yield ratio of HP to iPSC (HP / iPSC). For example, the method and composition can improve HP to iPSC (HP / iPSC) from about 2: 1 to about 4: 1, or improve about 2 times. In some embodiments, the method and composition of the present disclosure can improve the yield ratio of HP to iPSC (HP / iPSC) by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 times. In some embodiments, the yield ratio of HP to iPSC (HP / iPSC) is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9 or about 9 to about 10.

[0311] In some embodiments, the yield ratio of HP to hESC (HP / hESC) is about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, or about 10: 1. In some embodiments, the yield ratio of HP / hESC is about 1: 1 to about 2: 1, about 2: 1 to about 3: 1, about 3: 1 to about 4: 1, about 4: 1 to about 5: 1, about 5: 1 to about 6: 1, about 6: 1 to about 7: 1, about 7: 1 to about 8: 1, about 8: 1 to about 9: 1, or about 9: 1 to about 10: 1.

[0312] In some embodiments, the methods and compositions of the present disclosure improve the yield ratio of HP to hESC (HP / hESC). For example, the methods and compositions can increase the HP to hESC (HP / hESC) from about 2: 1 to about 4: 1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can increase the yield ratio of HP to hESC (HP / hESC) by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 times. In some embodiments, the yield ratio of HP to hESC (HP / hESC) is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10. Differentiation into NK cells

[0313] In some aspects, the disclosure provides a method for producing NK cells from differentiated NK cells. In some embodiments, the method for producing NK cells includes differentiating the NK cells. In some embodiments, the method provided herein is free of heterologous genes.

[0314] One aspect of the present disclosure is that the method for producing NK cells may include an NK differentiation step. The NK differentiation step may include contacting a HP cell population with one or more factors in a defined differentiation medium for a specific period of time, thereby inducing the formation of NK cells in the cell population. NK cells are then defined by expressing a combination of multiple markers.

[0315] In some embodiments, hematopoietic progenitor cells are contacted with a differentiation medium as described herein for a period of time to produce NK cells. In some embodiments, the time period sufficient to produce NK cells from hematopoietic progenitor cells is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days or at least 40 days,

[0316] In some embodiments, the duration of the NK differentiation step is 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, 14 days to 15 days, 15 days to 16 days, 16 days to 17 days, 17 days to 18 days, 18 days to 19 days, or 19 days to 20 days, 21 days to 22 days, 23 days to 24 days, 25 days to 26 days, 27 days to 28 days, 29 days to 30 days, 31 days to 31 days, 32 days to 33 days, 34 days to 35 days, 36 days to 37 days, 38 days to 39 days, 39 days to 40 days, 40 days to 41 days, 41 days to 42 days, 42 days to 43 days, 43 days to 44 days 0 days to 21 days, 21 days to 22 days, 22 days to 23 days, 23 days to 24 days, 24 days to 25 days, 25 days to 26 days, 26 days to 27 days, 27 days to 28 days, 28 days to 29 days, 29 days to 30 days, 30 days to 31 days, 31 days to 32 days, 32 days to 33 days, 33 days to 34 days, 34 days to 35 days, 35 days to 36 days, 36 days to 37 days, 37 days to 38 days, 38 days to 39 days or 39 days to 40 days.

[0317] In some embodiments, the differentiated NK cells comprise the markers CD34, CD43, CD45 and LFA1. In some embodiments, the methods of the present disclosure increase the percentage of CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0318] In some embodiments, the methods of the present disclosure produce a NK cell population from hematopoietic progenitor cells having a purity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of CD34+CD43+CD45+LFA1+ quad-positive cells.

[0319] In some embodiments, the methods of the present disclosure produce a NK cell population from hematopoietic progenitor cells having a purity of 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% of CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0320] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of NK cells from source cells (SC) (such as hESC or iPSC). In some embodiments, the ratio of NK to source cells (SC) is expressed as NK / SC. In some embodiments, the ratio of NK to hESC is expressed as NK / hESC. In some embodiments, the ratio of NK to iPSC is expressed as NK / iPSC.

[0321] In some embodiments, the yield ratio of NK / SC is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the yield ratio of NK / SC is about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 30:1, about 30:1 to about 35:1, about 35:1 to about 40:1, about 40:1 to about 45:1, about 45:1 to about 50:1, about 50:1 to about 60:1, about 60:1 to about 70:1, about 70:1 to about 80:1, about 80:1 to about 90:1, or about 90:1 to about 100:1.

[0322] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of NK / SC. For example, the methods and compositions may increase the yield ratio of NK / SC from about 2:1 to about 4:1, or by about 2 times. In some embodiments, the methods and compositions of the present disclosure may increase the yield ratio of NK / SC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, or about 100 times. In some embodiments, the yield ratio of NK / SC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, or about 90 to about 100.

[0323] In some embodiments, the production ratio of NK cells to stem cells (StC) (NK / StC) is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In some embodiments, the yield ratio of NK / StC is about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 30:1, about 30:1 to about 35:1, about 35:1 to about 40:1, about 40:1 to about 45:1, about 45:1 to about 50:1, about 50:1 to about 60:1, about 60:1 to about 70:1, about 70:1 to about 80:1, about 80:1 to about 90:1, or about 90:1 to about 100:1.

[0324] In some embodiments, the methods and compositions of the present disclosure improve the yield ratio of NK / StC. For example, the methods and compositions can increase NK / StC from about 2:1 to about 4:1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can increase the yield ratio of NK / StC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, or about 100 times. In some embodiments, the yield ratio of NK / StC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, or about 90 to about 100.

[0325] In some embodiments, the yield ratio of NK cells to hESCs (NK / hESCs) is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In some embodiments, the yield ratio of NK / hESC is about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 30:1, about 30:1 to about 35:1, about 35:1 to about 40:1, about 40:1 to about 45:1, about 45:1 to about 50:1, about 50:1 to about 60:1, about 60:1 to about 70:1, about 70:1 to about 80:1, about 80:1 to about 90:1, or about 90:1 to about 100:1.

[0326] In some embodiments, the methods and compositions of the present disclosure improve the yield ratio of NK / hESC. For example, the methods and compositions can increase NK / hESC from about 2: 1 to about 4: 1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can increase the yield ratio of NK / hESC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90 or about 100 times. In some embodiments, the yield ratio of NK / hESC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, or about 90 to about 100.

[0327] In some embodiments, the yield ratio of NK cells to iPSCs (NK / iPSCs) is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In some embodiments, the yield ratio of NK / iPSC is about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 30:1, about 30:1 to about 35:1, about 35:1 to about 40:1, about 40:1 to about 45:1, about 45:1 to about 50:1, about 50:1 to about 60:1, about 60:1 to about 70:1, about 70:1 to about 80:1, about 80:1 to about 90:1, or about 90:1 to about 100:1.

[0328] In some embodiments, the methods and compositions of the present disclosure improve the yield ratio of NK / iPSC. For example, these methods and compositions can increase NK / iPSC from about 2: 1 to about 4: 1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can increase the yield ratio of NK / iPSC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90 or about 100 times. In some embodiments, the yield ratio of NK / iPSC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, or about 90 to about 100. NK cell maturation

[0329] In some aspects, the disclosure provides a method for producing mature NK cells from differentiated NK cells. In some embodiments, the method for producing mature NK cells includes differentiating the NK cells. In some embodiments, the method provided herein is free of heterologous genes.

[0330] One aspect of the present disclosure is that the method for producing NK cells may include an NK maturation step. The NK maturation step may include contacting a differentiated NK cell population with one or more factors in a defined differentiation medium for a specific period of time, thereby inducing NK cell maturation in the cell population. Mature NK cells are then defined by expressing a combination of multiple markers.

[0331] In some embodiments, the differentiated NK cells are contacted with a maturation medium as described herein for a period of time to produce mature NK cells. In some embodiments, the time period sufficient to produce mature NK cells from hematopoietic progenitor cells is at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 168 hours, at least 192 hours, at least 216 hours, or at least 240 hours.

[0332] In some embodiments, the duration of the maturation step is 12 hours to 24 hours, 24 hours to 48 hours, 48 ​​hours to 72 hours, 72 hours to 96 hours, 96 hours to 120 hours, 120 hours to 144 hours, 144 hours to 168 hours, 168 hours to 192 hours, 192 hours to 216 hours, or 216 hours to 240 hours.

[0333] In some embodiments, the mature NK cells comprise the markers CD34, CD43, CD45 and LFA1. In some embodiments, the methods of the present disclosure increase the percentage of CD34+CD43+CD45+LFA1+ four-positive cells. In some embodiments, the methods increase the expression of activation markers. In some embodiments, the activation markers include NKp46, NKG2D, LFA1 and / or CD16. In some embodiments, the methods reduce the expression of inhibitory markers. In some embodiments, the inhibitory markers include CD161 and CD73.

[0334] In some embodiments, the methods of the present disclosure produce a mature NK cell population from differentiated NK cells, wherein the purity of CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.

[0335] In some embodiments, the methods of the present disclosure produce a mature NK cell population from differentiated NK cells, wherein the purity of CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells is 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

[0336] In some embodiments, the maturation step reduces the CD56- cell population.

[0337] In some embodiments, the methods of the present disclosure produce a mature NK cell population from differentiated NK cells having a purity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of CD56- cells.

[0338] In some embodiments, the methods of the present disclosure produce a mature NK cell population from differentiated NK cells having a CD56- cell purity of 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

[0339] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of mature NK cells from source cells (SC) (such as hESC or iPSC). In some embodiments, the ratio of mature NK to source cells (SC) is expressed as mature NK / SC. In some embodiments, the ratio of mature NK to hESC is expressed as mature NK / hESC. In some embodiments, the ratio of mature NK to iPSC is expressed as mature NK / iPSC.

[0340] In some embodiments, the yield ratio of NK / SC is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the yield ratio of mature NK / SC is about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 30:1, about 30:1 to about 35:1, about 35:1 to about 40:1, about 40:1 to about 45:1, about 45:1 to about 50:1, about 50:1 to about 60:1, about 60:1 to about 70:1, about 70:1 to about 80:1, about 80:1 to about 90:1, or about 90:1 to about 100:1.

[0341] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of mature NK / SC. For example, the methods and compositions can increase the yield ratio of mature NK / SC from about 2:1 to about 4:1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can increase the yield ratio of mature NK / SC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, or about 100 times. In some embodiments, the yield ratio of mature NK / SC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, or about 90 to about 100.

[0342] In some embodiments, the yield ratio of mature NK cells to stem cells (StC) (mature NK / StC) is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In some embodiments, the yield ratio of mature NK / StC is about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 30:1, about 30:1 to about 35:1, about 35:1 to about 40:1, about 40:1 to about 45:1, about 45:1 to about 50:1, about 50:1 to about 60:1, about 60:1 to about 70:1, about 70:1 to about 80:1, about 80:1 to about 90:1, or about 90:1 to about 100:1.

[0343] In some embodiments, the methods and compositions of the present disclosure improve the yield ratio of mature NK / StC. For example, these methods and compositions can improve the yield ratio of mature NK / StC from about 2:1 to about 4:1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can improve the yield ratio of mature NK / StC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90 or about 100 times. In some embodiments, the yield ratio of mature NK / StC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, or about 90 to about 100.

[0344] In some embodiments, the yield ratio of mature NK cells to hESCs (mature NK / hESCs) is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In some embodiments, the yield ratio of mature NK / hESC is about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 30:1, about 30:1 to about 35:1, about 35:1 to about 40:1, about 40:1 to about 45:1, about 45:1 to about 50:1, about 50:1 to about 60:1, about 60:1 to about 70:1, about 70:1 to about 80:1, about 80:1 to about 90:1, or about 90:1 to about 100:1.

[0345] In some embodiments, the methods and compositions of the present disclosure improve the yield ratio of mature NK / hESC. For example, the methods and compositions can improve the yield ratio of mature NK / hESC from about 2: 1 to about 4: 1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can improve the yield ratio of mature NK / hESC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90 or about 100 times. In some embodiments, the yield ratio of mature NK / hESC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, or about 90 to about 100.

[0346] In some embodiments, the yield ratio of mature NK cells to iPSCs (mature NK / iPSCs) is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In some embodiments, the yield ratio of mature NK / iPSC is about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 30:1, about 30:1 to about 35:1, about 35:1 to about 40:1, about 40:1 to about 45:1, about 45:1 to about 50:1, about 50:1 to about 60:1, about 60:1 to about 70:1, about 70:1 to about 80:1, about 80:1 to about 90:1, or about 90:1 to about 100:1.

[0347] In some embodiments, the methods and compositions of the present disclosure improve the yield ratio of mature NK / iPSC. For example, the methods and compositions can increase the yield ratio of mature NK / iPSC from about 2: 1 to about 4: 1, or increase by about 2 times. In some embodiments, the methods and compositions of the present disclosure can increase the yield ratio of mature NK / iPSC by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90 or about 100 times. In some embodiments, the yield ratio of mature NK / iPSC is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, or about 90 to about 100. Exemplary Differentiation Methods

[0348] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a heterologous gene-free differentiation medium, the medium comprising BMP4, FGF2 and VEGF. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a heterologous gene-free differentiation medium, the medium comprising 1-50ng / mL BMP4, 1-50ng / mL FGF2 and 5-100ng / mL VEGF. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a heterologous gene-free differentiation medium for 12 to 120 hours, the medium comprising BMP4, FGF2 and VEGF. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a heterologous gene-free differentiation medium for 12 to 120 hours, the medium comprising 1-50ng / mLBMP4, 1-50ng / mL FGF2 and 5-100ng / mL VEGF.

[0349] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a heterologous gene-free differentiation medium, the medium comprising BMP4, FGF2, VEGF and a ROCK inhibitor. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a heterologous gene-free differentiation medium, the medium comprising 1-50ng / mL BMP4, 1-50ng / mL FGF2, 5-100ng / mL VEGF and 0.1-20μM ROCK inhibitor. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a heterologous gene-free differentiation medium for 12-120 hours, the medium comprising BMP4, FGF2, VEGF and a ROCK inhibitor. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a population of stem cells with a xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM ROCK inhibitor for 12 to 120 hours.

[0350] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a serum-free differentiation medium, the medium comprising BMP4, FGF2, VEGF and Y27632. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a heterologous gene-free differentiation medium, the medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF and 0.1-20 μM Y27632. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a heterologous gene-free differentiation medium for 12-120 hours, the medium comprising BMP4, FGF2, VEGF and Y27632. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a population of stem cells with a xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM Y27632 for 12 to 120 hours.

[0351] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, and 5-100 ng / mL VEGF to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM of a PI3K inhibitor to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, and 5-100 ng / mL VEGF for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM of a PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells.

[0352] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM ROCK inhibitor to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM PI3K inhibitor to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor for 12-120 hours to produce a population of mesoderm cells; and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM ROCK inhibitor for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells.

[0353] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM Y27632 to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM of a PI3K inhibitor to produce hematopoietic progenitor cells. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 for 12-120 hours to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and a PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM Y27632 for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells.

[0354] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, and 5-100 ng / mL VEGF to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM LY294002 to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, and 5-100 ng / mL VEGF for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM LY294002 for 2-20 days to produce hematopoietic progenitor cells.

[0355] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM ROCK inhibitor to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM LY294002 to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor for 12-120 hours to produce a population of mesoderm cells; and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM ROCK inhibitor for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM LY294002 for 2-20 days to produce hematopoietic progenitor cells.

[0356] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM Y27632 to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM LY294002 to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM Y27632 for 12-120 hours to produce a population of mesoderm cells, and (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM LY294002 for 2-20 days to produce hematopoietic progenitor cells.

[0357] In some embodiments, the hematopoietic progenitor cell formation step is followed by a NK cell differentiation step. In some embodiments, the method of differentiating HP into NK comprises contacting an HP cell population with a heterologous gene-free differentiation medium, the culture comprising SCF, IL-7, IL-15, IL-12, FLT3L, pyrimidine-indole derivatives, and an aryl hydrocarbon receptor antagonist. In some embodiments, the method of differentiating HP into NK comprises contacting an HP cell population with a heterologous gene-free differentiation medium for 15-25 days, the culture medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, pyrimidine-indole derivatives, and an aryl hydrocarbon receptor antagonist. In some embodiments, the method of differentiating HP into NK comprises contacting a population of HP cells with a xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μM pyrimidine-indole derivatives, and 1-10 μM aryl hydrocarbon receptor antagonists. In some embodiments, the method of differentiating HP into NK comprises contacting a population of HP cells with a xeno-free differentiation medium for 15 to 25 days, wherein the medium comprises 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μM pyrimidine-indole derivatives, and 1-10 μM aryl hydrocarbon receptor antagonists.

[0358] In some embodiments, the method of differentiating HP into NK comprises contacting an HP cell population with a heterologous gene-free differentiation medium, the medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729 and SR1. In some embodiments, the method of differentiating HP into NK comprises contacting an HP cell population with a heterologous gene-free differentiation medium for 15-25 days, the medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729 and SR1. In some embodiments, the method of differentiating HP into NK comprises contacting an HP cell population with a heterologous gene-free differentiation medium, the medium comprising 5-50ng / mL SCF, 5-50ng / mL IL-7, 5-100ng / mL IL-15, 5-100ng / mL IL-12, 5-100ng / mL FLT3L, 1-10μΜ UM729 and 1-10μΜ SR1. In some embodiments, the method of differentiating HP into NK cells comprises contacting a stem cell population with a xeno-free differentiation medium for 15 to 25 days, wherein the medium comprises 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μM UM729, and 1-10 μM SR1.

[0359] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimidine-indole derivative, and an aryl hydrocarbon receptor antagonist to produce NK cells.

[0360] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, and 5-100 ng / mL VEGF to produce a population of mesoderm cells, (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM of a PI3K inhibitor to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ of pyrimidine-indole derivatives, and 1-10 μΜ of aryl hydrocarbon receptor antagonists.

[0361] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2 and VEGF for 12-120 hours to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and a PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimidine-indole derivative and an aryl hydrocarbon receptor antagonist for 15-25 days to produce NK cells.

[0362] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting the stem cell population with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, and 5-100 ng / mL VEGF for 12-120 hours to produce a mesodermal cell population, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM of a PI3K inhibitor, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF for 2-20 days to produce hematopoietic progenitor cells. IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ of pyrimidine-indole derivatives, and 1-10 μΜ of aryl hydrocarbon receptor antagonists.

[0363] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1 to produce NK cells.

[0364] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, and 5-100 ng / mL VEGF to produce a population of mesoderm cells, (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM LY294002 to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ UM729, and 1-10 μΜ SR1.

[0365] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF for 12-120 hours to produce a mesodermal cell population, wherein the medium comprises BMP4, FGF2, and VEGF, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1 for 15-25 days to produce NK cells.

[0366] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, and 5-100 ng / mL VEGF for 12-120 hours to produce a population of mesoderm cells, (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM LY294002 for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ of UM729, and 1-10 μΜ of SR1.

[0367] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimidine-indole derivative, and an aryl hydrocarbon receptor antagonist to produce NK cells.

[0368] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 M ROCK inhibitor to produce a population of mesoderm cells, (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM PI3K inhibitor to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL IL-7, and 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ of pyrimidine-indole derivatives, and 1-10 μΜ of aryl hydrocarbon receptor antagonists.

[0369] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor for 12-120 hours to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimidine-indole derivative, and an aryl hydrocarbon receptor antagonist for 15-25 days to produce NK cells.

[0370] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting the stem cell population with a first xeno-free differentiation medium for 12-120 hours to produce a mesodermal cell population, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM ROCK inhibitor, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium for 2-20 days to produce hematopoietic progenitor cells, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM PI3K inhibitor, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium for 15-25 days to produce NK cells. SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ of pyrimidine-indole derivatives, and 1-10 μΜ of aryl hydrocarbon receptor antagonists.

[0371] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1 to produce NK cells.

[0372] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM ROCK inhibitor to produce a population of mesoderm cells, (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM LY294002 to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL IL-75-50ng / mL IL-7, 5-100ng / mL IL-15, 5-100ng / mL IL-12, 5-100ng / mL FLT3L, 1-10μΜ of UM729, and 1-10μΜ of SR1.

[0373] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor for 12-120 hours to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1 for 15-25 days to produce NK cells.

[0374] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium for 12-120 hours to produce a population of mesoderm cells, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM ROCK inhibitor, (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium for 2-20 days to produce hematopoietic progenitor cells, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO and 5-100 μM LY294002, and (iii) contacting the hematopoietic progenitor cell population with a third xenogeneic gene-free differentiation medium for 15-25 days to produce NK cells, wherein the medium comprises 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μM UM729 and 1-10 μM SR1.

[0375] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and a PI3K inhibitor to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimidine-indole derivative and an aryl hydrocarbon receptor antagonist to produce NK cells.

[0376] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM Y27632 to produce a population of mesoderm cells, (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM PI3K inhibitor to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ of pyrimidine-indole derivatives, and 1-10 μΜ of aryl hydrocarbon receptor antagonists.

[0377] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 for 12-120 hours to produce a population of mesodermal cells, (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and a PI3K inhibitor for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimidine-indole derivative and an aryl hydrocarbon receptor antagonist for 15-25 days to produce NK cells.

[0378] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting the stem cell population with a first xeno-free differentiation medium for 12-120 hours to produce a mesodermal cell population, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF and 1-20 μM Y27632, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium for 2-20 days to produce hematopoietic progenitor cells, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO and 5-100 μM of a PI3K inhibitor, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium for 15-25 days to produce NK cells. SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ of pyrimidine-indole derivatives, and 1-10 μΜ of aryl hydrocarbon receptor antagonists.

[0379] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and LY294002 to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729 and SR1 to produce NK cells.

[0380] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM Y27632 to produce a population of mesoderm cells, (ii) contacting the population of mesoderm cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO, and 5-100 μM LY294002 to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μΜ of UM729, and 1-10 μΜ of SR1.

[0381] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 for 12-120 hours to produce a mesodermal cell population; (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and LY294002 for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729 and SR1 for 15-25 days to produce NK cells.

[0382] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises: (i) contacting a stem cell population with a first xeno-free differentiation medium for 12-120 hours to produce a mesodermal cell population, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM Y27632, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium for 2-20 days to produce hematopoietic progenitor cells, wherein the medium comprises 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 ug / mL LDL, about 100 ng / mL TPO and 5-100 μM LY294002, and (iii) contacting the hematopoietic progenitor cell population with a third xenogeneic gene-free differentiation medium for 15-25 days to produce NK cells, wherein the medium comprises 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μM UM729 and 1-10 μM SR1. Characteristics of NK cells

[0383] In some embodiments, NK cells produced by the methods of the present disclosure have improved or enhanced properties compared to NK cells produced by other methods.

[0384] In some embodiments, the NK cells produced by the disclosed methods have enhanced expansion. In some embodiments, the NK cells have at least 50-fold expansion, at least 100-fold expansion, at least 150-fold expansion, at least 200-fold expansion, at least 250-fold expansion, at least 300-fold expansion, at least 350-fold expansion, at least 400-fold expansion, at least 450-fold expansion, at least 500-fold expansion, at least 550-fold expansion, or at least 600-fold expansion compared to NK cells produced by other methods.

[0385] In some embodiments, the NK cells have a 50-fold expansion to a 100-fold expansion, a 100-fold expansion to a 150-fold expansion, a 150-fold expansion to a 200-fold expansion, a 200-fold expansion to a 250-fold expansion, a 250-fold expansion to a 300-fold expansion, a 300-fold expansion to a 350-fold expansion, a 350-fold expansion to a 400-fold expansion, a 400-fold expansion to a 450-fold expansion, a 450-fold expansion to a 500-fold expansion, a 500-fold expansion to a 550-fold expansion, or a 550-fold expansion to a 600-fold expansion compared to NK cells produced by other methods.

[0386] In some embodiments, the differentiated NK cells produced by the methods of the present disclosure reduce tumor cell growth. In some embodiments, the differentiated NK cells reduce tumor cell growth by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0387] In some embodiments, the differentiated NK cells reduce tumor cell growth by 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

[0388] In certain embodiments, the mature NK cells produced by the methods of the present disclosure reduce tumor cell growth. In some embodiments, the mature NK cells reduce tumor cell growth by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0389] In some embodiments, the mature NK cells reduce tumor cell growth by 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

[0390] In some embodiments, the methods of the present disclosure produce a differentiated NK cell population. In some embodiments, the methods of the present disclosure produce a differentiated NK cell population that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0391] In some embodiments, the methods of the present disclosure produce a differentiated NK cell population that is 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0392] In some embodiments, the methods of the present disclosure produce a mature NK cell population. In some embodiments, the methods of the present disclosure produce a mature NK cell population that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells.

[0393] In some embodiments, the methods of the present disclosure produce a mature NK cell population that is 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells. Engineered cells

[0394] In some embodiments, the cell colonies described herein are genetically engineered. In some embodiments, the source cells are genetically engineered. In some embodiments, the mesoderm cells are genetically engineered. In some embodiments, the embryoid body cells are genetically engineered. In some embodiments, the hematopoietic progenitor cells are genetically engineered. In some embodiments, the differentiated NK cells are genetically engineered. In some embodiments, the mature NK cells are genetically engineered. In some embodiments, genetic engineering reduces the expression of endogenous genes. In some embodiments, genetic engineering increases the expression of endogenous genes.

[0395] In some embodiments, the genetically engineered cell comprises introducing foreign DNA into the cell. In some embodiments, the foreign DNA is a gene. In some embodiments, the foreign DNA changes the expression of an endogenous gene.

[0396] In some embodiments, genetic engineering comprises introducing RNA into the cell, such as interfering RNA (RNAi), double-stranded RNA (dsRNA), small interfering RNA (siRNA), and / or micro RNA (miRNA).

[0397] In some embodiments, genetic engineering involves introducing DNA, such as a plasmid or a bacterial artificial chromosome (BAC), into the cell.

[0398] In some embodiments, genetic engineering includes the introduction of: (a) a fusion protein including a DNA targeting protein and a nuclease or (b) an RNA-guided nuclease. For example, in some embodiments, the DNA targeting protein or RNA-guided nuclease includes a zinc finger protein (ZFP), a TAL protein, or a gene-specific clustered regularly interspaced short palindromic nucleic acid (CRISPR). In some embodiments, disruption includes the introduction of a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas9 combination that can specifically bind to, recognize, or hybridize with a gene. In some embodiments, the introduction is performed by introducing a nucleic acid into a cell, the nucleic acid comprising a sequence encoding a DNA binding protein, a DNA binding nucleotide, and / or a complex comprising a DNA binding protein or a DNA binding nucleotide. In some embodiments, the nucleic acid is a viral vector.

[0399] In some embodiments, the genetically engineered cells described herein include a chimeric antigen receptor (CAR). In some embodiments, genetically engineered stem cells include CAR. In some embodiments, genetically engineered hematopoietic progenitor cells include CAR. In some embodiments, genetically engineered NK cells include CAR.

[0400] In some embodiments, the genetically engineered cells described herein comprise a rapamycin-activated cytokine receptor (RACR). In some embodiments, genetically engineered stem cells comprise RACR. In some embodiments, genetically engineered hematopoietic progenitor cells comprise RACR. In some embodiments, genetically engineered NK cells comprise RACR.

[0401] In some embodiments, the genetically engineered cells described herein comprise CAR and RACR. In some embodiments, genetically engineered stem cells comprise CAR and RACR. In some embodiments, genetically engineered hematopoietic progenitor cells comprise CAR and RACR. In some embodiments, genetically engineered NK cells comprise CAR and RACR.

[0402] In some embodiments, the genetically engineered NK cells may comprise an inactivating mutation. In some embodiments, the inactivating mutation is a nonsense mutation. In some embodiments, the nonsense mutation is a premature stop codon. In some embodiments, the inactivating mutation is a missense mutation. Synthetic cytokine receptor complex

[0403] In some embodiments, the cells described herein are genetically engineered to express a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor comprises a synthetic γ chain and a synthetic β chain, each chain including a dimerization domain. In the presence of a non-physiological ligand, the dimerization domain can controllably dimerize, thereby activating the signaling of the synthetic cytokine receptor.

[0404] The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG). The dimerization domain can be an extracellular domain (N-terminal to the transmembrane domain) or an intracellular domain (C-terminal to the transmembrane domain), and N-terminal or C-terminal to the intracellular domain of IL-2G.

[0405] The synthesized β chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain, wherein the intracellular domain is selected from an intracellular domain of interleukin-2 receptor subunit β (IL-2RB), an intracellular domain of interleukin-7 receptor subunit β (IL-7RB) or an intracellular domain of interleukin-21 receptor subunit β (IL-21RB). The synthesized γ chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an intracellular domain of interleukin-2 receptor subunit γ (IL-2RG). The dimerization domain can be an extracellular domain (N-terminal to transmembrane domain) or an intracellular domain (C-terminal to transmembrane domain, N-terminal or C-terminal to IL-2RB or IL-7RB intracellular domain).

[0406] The non-physiological ligand can activate the synthetic cytokine receptor in the cytotoxic innate lymphocyte to induce the expansion and / or activation of the engineered cytotoxic innate lymphocyte. In a preferred embodiment, the non-physiological ligand is rapamycin or a rapamycin analog (rapalog), and such synthetic cytokine receptor is called rapamycin activated cytokine receptor (RACR).

[0407] In some embodiments, non-physiological ligands activate synthetic cytokine receptors in NK cells to induce expansion of NK cells. In some embodiments, activation of synthetic cytokine receptors results in an increase in the number of NK cells by at least about 10 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, or at least about 4000 times, compared to uninduced cells.

[0408] In some embodiments, NK cells are increased by about 10-fold to about 100-fold, about 50-fold to about 200-fold, about 100-fold to about 300-fold, about 200-fold to about 400-fold, about 300-fold to about 500-fold, about 400-fold to about 1000-fold, about 500-fold to about 1500-fold, about 1000-fold to about 2000-fold, about 1500-fold to about 2500-fold, about 2000-fold to about 3000-fold, about 2500-fold to about 3500-fold, about 3000-fold to about 4000-fold, or any value between these ranges. Intracellular domain

[0409] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain. In some embodiments, the IL2Rg domain comprises the sequence shown in SEQ ID NO: 1. In some embodiments, the IL2Rg common gamma chain intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 1.

[0410] The sequence of the IL2RG common γ chain intracellular domain is shown in SEQ ID NO: 1: ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET.

[0411] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain and a second transmembrane receptor protein comprising an IL-2RB intracellular domain and a second dimerization domain.

[0412] In some embodiments, the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL2RB) intracellular domain. In some embodiments, the IL2RB intracellular domain comprises the sequence shown in SEQ ID NO: 2. In some embodiments, the IL2RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 2.

[0413] The sequence of the intracellular domain of IL2RB is shown in SEQ ID NO: 2: NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0414] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain and a second transmembrane receptor protein comprising an IL-7RB intracellular domain and a second dimerization domain.

[0415] In some embodiments, the synthetic beta chain comprises an interleukin-7 receptor subunit beta (IL7RB) intracellular domain. In some embodiments, the IL7RB intracellular domain comprises the sequence shown in SEQ ID NO: 3. In some embodiments, the IL7RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 3.

[0416] The sequence of the intracellular domain of IL7RB is shown in SEQ ID NO:3: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0417] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain and a second transmembrane receptor protein comprising an IL-21RB intracellular domain and a second dimerization domain.

[0418] In some embodiments, the synthetic β chain comprises an interleukin-21 receptor subunit β (IL21RB) intracellular domain. In some embodiments, the IL21RB intracellular domain comprises the sequence shown in SEQ ID NO: 4. In some embodiments, the IL21RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 4.

[0419] The sequence of the intracellular domain of IL21RB is shown in SEQ ID NO:4: SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVLDAEGPCTWPCSCE DDGYPALDLDAGLEEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS Dimerization domain

[0420] The dimerization domain may be a heterologous dimerization domain, including but not limited to a FK506 binding protein (FKBP) of 12 kD in size and a FKBP12-rapamycin binding (FRB) domain, which dimerizes in the presence of rapamycin or a rapamycin analog known in the art. The FRB domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. The FKBP domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5.

[0421] The sequence of an exemplary FKBP domain is shown in SEQ ID NO:5: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0422] The sequence of an exemplary FRB domain is shown in SEQ ID NO:6: ILWHEMWEGLEASRLYFGERNVKMFEVLEPLHMERGPQTLKETSFNQAYGRDLMEAQEWCRKMKSGNVKDLTQAWDLYYHVFRRISK

[0423] The sequence of the variant FRB domain (FRB mutant domain) is shown in SEQ ID NO:7: ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK

[0424] Alternatively, the first dimerization domain and the second dimerization domain may be a 12 kD FK506 binding protein (FKBP) and a calcineurin domain, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0425] In some embodiments, the dimerization domain is a homodimerization domain selected from: i) 12kD FK506 binding protein (FKBP); ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); The corresponding non-physiological ligands are i) FK1012, AP1510, AP1903 or AP20187; ii) Cyclosporine-A (CsA); or iii) coumermycin or its analogs.

[0426] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a cyclophilin domain.

[0427] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0428] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0429] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1). Transmembrane domain

[0430] The transmembrane domain is a sequence of a synthetic cytokine receptor that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0431] The sequence of the transmembrane (TM) domain is shown in SEQ ID NO:8: VVISVGSMGLIISLLCVYFWL

[0432] The sequence of the TM domain is shown in SEQ ID NO:9: VAVAGCVFLLISVLLLSGL

[0433] The sequence of the TM domain is shown in SEQ ID NO: 10: PILLTISILSFFSVALLVILACVLW

[0434] The sequence of the TM domain is shown in SEQ ID NO: 11: GWNPHLLLLLLLVIVFIPAFW

[0435] The sequence of the CD8a signal sequence is shown in SEQ ID NO: 12: MALPVTALLLPLALLLHAARP Non-physiological ligands

[0436] In various embodiments of the compositions and methods of the present disclosure, the system includes a non-physiological ligand. Exemplary small molecules that can be used as ligands include, but are not limited to, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazine-2-yl)oxy]benzoic acid (aMPOB), folic acid, rhodamine, acetazolamide, and CA9 ligand.

[0437] In some embodiments, the synthetic cytokine receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0438] In some embodiments, the non-physiological ligand is a rapamycin analog.

[0439] In some embodiments, the non-physiological ligand is rapamycin.

[0440] In some embodiments, the non-physiological ligand is AP21967.

[0441] In some embodiments, the non-physiological ligand is FK506.

[0442] In some embodiments, the non-physiological ligand is FK1012. In some embodiments, the non-physiological ligand is AP1510. In some embodiments, the non-physiological ligand is AP1903. In some embodiments, the non-physiological ligand is AP20187. In some embodiments, the non-physiological ligand is cyclosporine-A (CsA). In some embodiments, the non-physiological ligand is kumarin.

[0443] In some embodiments, the synthetic cytokine receptor complex is activated by folic acid, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, rapalog (analog of rapamycin), CD28 ligand, poly(his) tag, Strep tag, FLAG tag, VS tag, Myc tag, HA tag, NE tag, biotin, digoxigenin, dinitrophenol or its derivatives.

[0444] In some embodiments, the non-physiological ligand may be an inorganic or organic compound less than 1000 Daltons.

[0445] In some embodiments, the ligand can be rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapamycin analog comprises a rapamycin variant with one or more modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy group at C7, C42 and / or C29; elimination, derivatization or replacement of the hydroxyl group at C13, C43 and / or C28; reduction, elimination or derivatization of the ketone at C14, C24 and / or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring.

[0446] Thus, in some embodiments, the rapamycin analog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, Temsirolimus (CCI-779), C20-methylrapamycin, C16-(S)-3-methylindorapamycin, C16-(S)-3-methylindorapamycin (C16-iRap), AP21967 (A / C heterodimer, Takara ), mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573 (Ridaforolimus), AP1903 (Rimiducid), or their metabolites, derivatives and / or combinations.

[0447] In some embodiments, the ligand includes FK1012 (a semisynthetic dimer of K506), tacrolimus (FK506), FKCsA (a composite of FK506 and cyclosporine), rapamycin, kumarin, gibberellin, HaXS dimer (a chemical dimer of Halo tag and SNAP tag), TMP-H tag (trimethoprim halogenase protein dimer), or ABT-737 or a functional derivative thereof.

[0448] In some embodiments, the non-physiological ligand is present or provided in an amount of 0 nM to 1000 nM, for example: 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 5.0 nM, 10.0 nM, 15.0 nM, 20.0 nM, 25.0 nM, 30.0 nM, 35.0 nM, 40.0 nM, 45.0 nM, 50.0 nM, 5 5.0 nM, 60.0 nM, 65.0 nM, 70.0 nM, 75.0 nM, 80.0 nM, 90.0 nM, 95.0 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1000 nM, or an amount within the range defined by any two of the above amounts.

[0449] In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 100 nM.

[0450] In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 50 nM.

[0451] In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 100 nM.

[0452] In some embodiments, the non-physiological ligand is present or provided at 1 nM. In some embodiments, the non-physiological ligand is present or provided at 10 nM. In some embodiments, the non-physiological ligand is present or provided at 100 nM. In some embodiments, the non-physiological ligand is present or provided at 1000 nM. Cytoplasmic FRB

[0453] The FRB domain is a domain of approximately 100 amino acids derived from the mTOR protein kinase. It can be expressed in the cytoplasm as a freely diffusible soluble protein. Advantageously, the FRB domain can reduce the inhibitory effect of rapamycin on mTOR in transduced cells and promote the sustained activation of transduced cells, giving the cells a proliferation advantage over native cells.

[0454] In some embodiments, the synthetic cytokine receptor complex comprises a cytoplasmic polypeptide bound to a ligand or a complex comprising a ligand.

[0455] In some embodiments, the cytoplasmic polypeptide comprises a FRB domain. In some embodiments, the cytoplasmic polypeptide comprises a FRB domain and the ligand is rapamycin. The cytoplasmic FRB domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. The FRB domain may comprise a naked FRB domain that is primarily composed of a polypeptide having a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. Advantageously, the cytoplasmic FRB confers resistance to the immunosuppressive effects of non-physiological ligands such as rapamycin or rapamycin analogs. Chimeric Antigen Receptor

[0456] In some embodiments, the cells described herein are genetically engineered to express a chimeric antigen receptor (CAR).

[0457] In some embodiments, the present disclosure encompasses a CAR system for treating cancer patients.In some embodiments, the NK cells of the present disclosure comprise a CAR sequence (CAR-NK cells).

[0458] In some embodiments, by transfecting a cell population with an expression vector encoding a CAR construct, NK cells are genetically engineered to express a CAR construct. Exemplary examples of transfectable cell populations include HSC, blood progenitor cells, common lymphocyte progenitor cells, or NK cells. Suitable methods for preparing NK cell transduction populations expressing selected CAR constructs are well known to those skilled in the art, and include examples such as retroviruses, slow viruses (virus-mediated CAR gene delivery systems), Sleeping Beauty, and PiggyBac (including transposons / transposase systems of non-viral-mediated CAR gene delivery systems). In some embodiments, any transduction method encompassed by the present disclosure can be used to produce NK cells expressing CAR. CAR targeted agents

[0459] Typically, if desired, CAR is produced by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding a transmembrane and intracellular domain, and transducing cells with the polynucleotide and the corresponding VH or VL. Many CAR variants are known in the art, and the present disclosure encompasses the use of any known variants. In addition, many hapten VL / VH pairs and scFv are known in the art, or can generally be produced by conventional methods. Therefore, the present disclosure encompasses the use of any known hapten binding domain.

[0460] Various methods for targeting CAR and cells expressing CAR have been described in the art, including, for example, US 2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, fluorescein or fluorescein isothiocyanate (FITC) moieties can be conjugated to agents that bind to desired target cells (such as cancer cells), so that CAR-NK cells expressing anti-fluorescein / FITC chimeric antigen receptors are selectively targeted to target cells labeled by the conjugate. In different cases, other haptens recognized by CAR can also be used instead of fluorescein / FITC. CAR can be produced using various scFv sequences known in the art or scFv sequences produced by conventional and customary methods. For example, further exemplary scFv sequences for fluorescein / FITC and for other antigens are provided in WO 2021 / 076788, the disclosure of which has been incorporated herein by reference.

[0461] In some embodiments, the CAR system of the present disclosure uses CAR, which targets a portion that is not produced or expressed by the cells of the subject to be treated. Therefore, the CAR system can focus NK cells on target cells, such as cancer cells. By administering small conjugate molecules together with NK cells expressing CAR, NK cell responses can only target cells that express tumor receptors, thereby reducing off-target toxicity, and due to the rapid clearance of small conjugate molecules, NK cell activation is more easily controlled. Another advantage is that NK cells expressing CAR can be used as "universal" cytotoxic cells to target a variety of tumors without the need to prepare separate CAR constructs. The targeting portion recognized by CAR can also remain unchanged. By simply changing the ligand portion of the small conjugate molecule, the system can be targeted to cancer cells of different characteristics (identity).

[0462] In one embodiment, the present disclosure provides examples of the conjugate molecule / CAR system.

[0463] In some embodiments, the CAR system of the present disclosure utilizes a conjugate molecule as a bridge between cells expressing CAR and targeted cancer cells. The conjugate molecule is a conjugate comprising a hapten and a targeted cell portion (such as any suitable tumor cell-specific ligand). Schematic haptens that can be recognized and bound by CAR include small molecular weight organic molecules, such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxin, and fluorescein and its derivatives, including FITC (fluorescein isothiocyanate), NHS-fluorescein, pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, desmin, Centyrin and DARPin. Suitable targeting cell moieties that can themselves serve as haptens for CARs include desmin (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), Centyrin and DARPin (see Reichert, JMM Abs 2009. 1(3):190-209).

[0464] In some embodiments, the targeting cell moiety is DUPA (DUPA-(99m)Tc), which is a ligand bound by PSMA-positive human prostate cancer cells with nanomolar affinity (KD = 14 nM; see Kularatne, SA et al., Mol Pharm. 2009. 6(3): 780-9). In one embodiment, a DUPA derivative can be a ligand of a small molecule ligand attached to a targeting moiety, and DUPA derivatives are described in WO 2015 / 057852, which is incorporated herein by reference.

[0465] In some embodiments, the targeting cell moiety is a CCK2R ligand, which is a ligand bound by CCK2R-positive cancer cells (such as thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal cancer, and colon cancer; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0466] In some embodiments, the targeting cell molecule is folate, folic acid, or an analog thereof, which is a ligand bound by folate receptors on cancer cells including ovarian, cervical, endometrial, lung, kidney, brain, breast, colon, and head and neck cancers; see Sega, EI et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

[0467] In some embodiments, the targeting cell moiety is a NK-1R ligand. For example, receptors for the ligand NK-1R are found in colon cancer and pancreatic cancer. In certain embodiments, the NK-1R ligand can be synthesized according to the method disclosed in International Patent Application No. PCT / US2015 / 044229, which has been incorporated herein by reference.

[0468] In some embodiments, the targeting cell moiety can be a peptide ligand, for example, the ligand can be a peptide ligand as an endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide, which belongs to the tachykinin family of targeting tachykinin receptors. Such regulatory peptides include substance P (SP), neurokinin A (substance K) and neurokinin B (neuromediatin K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0469] In some embodiments, the targeting cell moiety is a CAIX ligand. For example, receptors for the CAIX ligand are found in renal cancer, ovarian cancer, vulvar cancer, and breast cancer. The CAIX ligand may also be referred to herein as CA9.

[0470] In some embodiments, the targeting cell moiety is a ligand for gamma glutamyl transpeptidase, which is overexpressed in, for example, ovarian cancer, colon cancer, liver cancer, astrocytoma, melanoma, and leukemia.

[0471] In some embodiments, the targeting cell moiety is a CCK2R ligand. Receptors for CCK2R ligands are found in cancers such as thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal cancer, and colon cancer.

[0472] In one embodiment, the mass of the targeted cell portion may be less than about 10,000 Daltons, less than about 9,000 Daltons, less than about 8,000 Daltons, less than about 7,000 Daltons, less than about 6,000 Daltons, less than about 5,000 Daltons, less than about 4,500 Daltons, less than about 4,000 Daltons, less than about 3,500 Daltons, less than about 3,000 Daltons, less than about 2,500 Daltons, less than about 2,000 Daltons, less than about 1,500 Daltons, less than about 1,000 Daltons, or less than about 500 Daltons. In another embodiment, the small molecule ligand may have a mass of about 1 to about 10,000 Daltons, about 1 to about 9,000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7,000 Daltons, about 1 to about 6,000 Daltons, about 1 to about 5,000 Daltons, about 1 to about 4,500 Daltons, about 1 to about 4,000 Daltons, about 1 to about 3,500 Daltons, about 1 to about 3,000 Daltons, about 1 to about 2,500 Daltons, about 1 to about 2,000 Daltons, about 1 to about 1,500 Daltons, about 1 to about 1,000 Daltons, or about 1 to about 500 Daltons.

[0473] In an exemplary embodiment, the connection in the conjugate described herein can be a direct connection (e.g., a reaction between the isothiocyanate group of FITC and the free amine group of the small molecule ligand), or the connection can be through an intermediate linker. In one embodiment, if present, the intermediate linker can be any biocompatible linker known in the art, such as a divalent linker. In an exemplary embodiment, the divalent linker can contain about 1 to about 30 carbon atoms. In another exemplary embodiment, the divalent linker can contain about 2 to about 20 carbon atoms. In other embodiments, a divalent linker with a lower molecular weight (i.e., a linker with a molecular weight of about 30 to about 300 Da) is used. In another embodiment, suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 atoms or more.

[0474] In some embodiments, the hapten and the targeting cell moiety can be directly conjugated by means such as reaction between the isothiocyanate group of FITC and the free amine group of a small ligand (e.g., folate, DUPA, and CCK2R ligand). However, the use of a linker domain to connect the two molecules may be helpful as it can provide flexibility and stability. Examples of suitable linker domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) carbohydrates; 5) non-natural peptidoglycans; 6) polyvinyl pyrrolidone; 7) Pluronic F-127. Suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 atoms or more.

[0475] In some embodiments, the linker may be a bivalent linker, which may comprise one or more spacers.

[0476] An exemplary conjugate of the present disclosure is FITC-folate.

[0477] An exemplary conjugate of the present disclosure is FITC-CA9.

[0478] Exemplary conjugates of the present disclosure include the following molecules: FITC-(PEG) 12 -Folic acid, FITC-(PEG) 20 -Folic acid, FITC-(PEG) 108 -Folic acid, FITC-DUPA, FITC-(PEG) 12 -DUPA, FITC-CCK2R ligand, FITC-(PEG) 12 -CCK2R ligand, FITC-(PEG) 11 -NK1R ligand and FITC-(PEG)2-CA9.

[0479] Although the affinity with which a ligand binds to a cancer cell receptor will vary, and in some cases, low affinity binding (e.g., about 1 μM) is preferred, the binding affinity of a ligand to a cancer cell receptor is typically at least about 100 μM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0480] Examples of conjugates and methods of making them are provided in U.S. Patent Application Nos. US2017 / 0290900, US2019 / 0091308, and US2020 / 0023009, all of which are incorporated herein by reference. CAR constructs

[0481] In some embodiments, the binding portion of the CAR can be a single chain fragment variable region (scFv), Fab, Fv, Fc or F(ab')2 fragment of an antibody, etc. It is not within the scope of the present invention to use unaltered (ie, full size) antibodies such as IgG, IgM, IgA, IgD or IgE in CAR or to use them as CAR.

[0482] In some embodiments, the role of the costimulatory domain is to enhance the proliferation and survival of lymphocytes after CAR is combined with the targeting moiety. The identity of the costimulatory domain is limited to its ability to enhance cell proliferation and survival activation after CAR is combined with the targeting moiety. Suitable costimulatory domains include, but are not limited to: CD28 (see, e.g., Alvarez-Vallina, L. et al., Eur J Immunol. 1996. 26 (10): 2304-9); CD137 (4-1BB), which is a member of the tumor necrosis factor (TNF) receptor family (see, e.g., Imai, C. et al., Leukemia. 2004. 18: 676–84); and CD134 (OX40), which is a member of the TNFR receptor superfamily (see, e.g., Latza, U. et al., Eur. J. Immunol. 1994. 24: 677). It will be appreciated by those skilled in the art that sequence variants of these costimulatory domains can be used, wherein the variants have the same or similar activity as the domains modeled therefor. In various embodiments, such variants have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.

[0483] In some embodiments, the CAR construct comprises two co-stimulatory domains. Although specific combinations include all possible variants of the above four domains, specific examples include: 1) CD28+CD137 (4-1BB) and 2) CD28+CD134 (OX40).

[0484] In some embodiments, the role of the activation signaling domain is to activate cells after CAR binds to the targeting portion. The properties of the activation signaling domain are limited to its ability to induce selected cell activation after CAR binds to the target molecule. Suitable activation signaling domains include CD3 ζ chain and Fc receptor γ. It should be understood by those skilled in the art that sequence variants of these mentioned activation signaling domains can be used without adversely affecting the present invention, wherein the variants have the same or similar activity as the domain modeled for this. Such variants may have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which it is derived.

[0485] In some embodiments, CARs may include additional elements such as a signal peptide that ensures proper export of the fusion protein to the cell surface, a transmembrane domain that ensures the fusion protein remains an integral membrane protein, and a hinge domain that confers flexibility to the recognition region and allows strong binding to the targeting moiety.

[0486] Exemplary CAR constructs suitable for CAR-NK cells are provided below: (1) scFv-CD8TM-4-1BBIC-CD3ζs (see, e.g., Liu E, Tong Y, Dotti G, et al., Leukemia. 2018; 32: 520-531); (2) scFv-CD28TM+IC-CD3ζs (see, e.g., Han J, Chu J, Keung CW et al., Sci Rep. 2015; 5: 11483; Kruschinski A, Moosmann A, Poschke I et al., Proc Natl Acad Sci US A. 2008; 105: 17481-17486; and Chu J, Deng Y, Benson DM et al., Leukemia. 2014; 28: 917-927); (3) scFv-DAP12TM+IC (see, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015; 38: 197-210); (4) scFv-CD8TM-2B4IC-CD3ζs (see, e.g., Liu E, Tong Y, Dotti G, et al., J Hematol Oncol. 2019; 12-49); (5) scFv-2B4TM+IC-CD3ζs (see Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866); (6) scFv-CD28TM+IC-4-1BBIC-CD3ζs (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017; 28: 897-913); (7) scFv-CD16TM-2B4IC-CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (8) scFv-NKp44TM-DAP10IC-CD3ζs (see, e.g., Li Y, Hermanson DL, MoriarityBS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (9) scFv-NKp46TM-2B4IC-CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (10) scFv-NKG2DTM-2B4IC-CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (11) scFv-NKG2DTM-4-1BBIC-CD3ζs (see, e.g., Li Y, Hermanson DL, MoriarityBS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (12) scFv-NKG2DTM-2B4IC-DAP12IC-CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (13) scFv-NKG2DTM-2B4IC-DAP10IC-CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (14) scFv-NKG2DTM-4-1BBIC-2B4IC-CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (15) scFv-NKG2DTM-CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192).

[0487] Figure 8 shows an exemplary CAR of the present disclosure, wherein the fusion protein is encoded by a lentiviral expression vector, and wherein "SP" is a signal peptide, the CAR is an anti-FITC CAR, the CD8α hinge is present, the transmembrane domain ("TM") is present, the co-stimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ.

[0488] An exemplary nucleotide sequence encoding a CAR may comprise SEQ ID NO: 13:

[0489] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTAGAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO:13)

[0490] The exemplary CAR amino acid sequence may comprise SEQ ID NO:14:

[0491] MALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:14)

[0492] Exemplary nucleotide insert fragments may comprise SEQ ID NO:15:

[0493]

[0493] In various embodiments, a CAR-expressing cell comprising a nucleic acid of SEQ ID NO: 13 or 15 is provided. In some embodiments, a chimeric antigen receptor polypeptide comprising SEQ ID NO: 14 is contemplated. In some embodiments, a vector comprising SEQ ID NO: 13 or 15 is contemplated. In some embodiments, a lentiviral vector comprising SEQ ID NO: 13 or 15 is contemplated. In some embodiments, SEQ ID NO: 14 may comprise or consist of a human or humanized amino acid sequence.

[0494] In some embodiments, variant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15 are encompassed.

[0495] Although the affinity of CARs expressed by lymphocytes to bind to targeting moieties may vary, and in some cases low affinity binding (e.g., about 50 nM) may be preferred, the binding affinity of CARs to targeting ligands is typically at least about 100 nM, 1 pM, or 10 pM, preferably at least about 100 pM, 1 fM, or 10 fM, and more preferably at least about 100 fM. Therapeutic compositions

[0496] In some embodiments, the present disclosure provides compositions comprising one or more cell populations.

[0497] In some embodiments, the composition comprises a differentiated NK cell population. In some embodiments, the composition comprises a differentiated NK cell population that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0498] In some embodiments, the composition comprises a differentiated NK cell population that is 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0499] In some embodiments, the composition comprises a mature NK cell population. In some embodiments, the composition comprises a mature NK cell population that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells.

[0500] In some embodiments, the composition comprises a mature NK cell population that is 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells. Treatment

[0501] The present disclosure provides methods of treating a subject in need thereof with a composition, therapeutic composition, or cell disclosed herein. In some embodiments, the present disclosure provides methods of treating cancer in a subject and / or killing cancer cells in a subject, the method comprising administering to the subject a therapeutically effective amount of a cell of the present disclosure.

[0502] In some embodiments, the cancer is a solid tumor, a sarcoma, a carcinoma, a lymphoma, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), T cell lymphoma, one or more B cell acute lymphocytic leukemia ("BALL"), T cell acute lymphocytic leukemia ("TALL"), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell The invention relates to a type 2 B cell neoplasm, a Burkitt's lymphoma, a diffuse large B-cell lymphoma, a follicular lymphoma, a hairy cell leukemia, a small cell or large cell follicular lymphoma, a malignant lymphoproliferative disorder, a MALT lymphoma, a mantle cell lymphoma, a marginal zone lymphoma, a myelodysplastic disorder and a myelodysplastic syndrome, a plasmablastic lymphoma, a plasmacytoid dendritic cell neoplasm, a Waldenstrom macroglobulinemia, a plasma cell proliferative disorder (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), a monoclonal gammopathy of undetermined significance (MGUS), a plasma cell neoplasm (e.g., a plasma cell abnormality, a solitary myeloma, a solitary plasmacytoma, an extramedullary plasmacytoma, and a multiple plasmacytoma), a systemic amyloid light chain amyloidosis, a POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), or a combination thereof.

[0503] In some embodiments, the methods disclosed herein can be used to treat cancer in a subject and / or kill cancer cells in a subject by administering a therapeutically effective amount of a cell according to any one of the above embodiments.

[0504] The present disclosure also provides a method of treating cancer in a subject and / or killing cancer cells in a subject, the method comprising administering to the subject the composition according to any one of the above embodiments.

[0505] In some embodiments, the present disclosure provides a method for treating cancer with any of the compositions provided herein. When reading the specification, "cancer" has a common and commonly used meaning, and may include, but is not limited to, for example, a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Subjects treatable using the methods described herein include subjects with cancer identified or selected, including but not limited to colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer and brain cancer, etc. Such identification and / or selection can be performed by clinical or diagnostic evaluation. In some embodiments, tumor-associated antigens or molecules are known, such as melanoma, breast cancer, brain cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma and / or prostate cancer. Examples include but are not limited to B cell lymphoma, breast cancer, brain cancer, prostate cancer and / or leukemia. In some embodiments, one or more oncogenic polypeptides are associated with renal cancer, uterine cancer, colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, brain cancer, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia or leukemia. In some embodiments, a method of treating, ameliorating or inhibiting cancer in a subject is provided. In some embodiments, the cancer is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, renal cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colon cancer, skin cancer (including melanoma), bone cancer or brain cancer.

[0506] In some embodiments, additional cancer therapies are provided, such as small molecules, such as compounds; antibody therapies, such as humanized monoclonal antibodies conjugated with or without radionuclides, toxins, or drugs; surgery and / or radiation therapy.

[0507] In some embodiments, the subject is selected to receive additional cancer therapy, which may include cancer therapeutic agents, radiation therapy, chemotherapy, or drugs to treat cancer. In some embodiments, the drugs include Abiraterone, Alemtuzumab, Anastrozole, Aprepitant, Arsenic trioxide, trioxide), Atezolizumab, Azacitidine, Bevacizumab, Bleomycin, Bortezomib, Cabazitaxel, Capecitabine, Carboplatin, Cetuximab, Chemotherapy drug combinations, Cisplatin, Crizotinib, Cyclophosphamide, Cytarabine, Denosumab, Docetaxel, Doxorubicin, Eribulin, Erlotinib, Etoposide, Everolimus, Exemestane, Filgrastim, Fluorouracil, Fulvestrant, Gemcitabine, Imatinib, Imiquimod, Ipilimumab, Ixabepilone, Lapatinib, Lenalidomide, Letrozole, Leuprolide, Mesna, Methotrexate, Nivolumab, Oxaliplatin, Paclitaxel, Palonosetron, Pembrolizumab, Pemetrexed,Prednisone, Radium-223, Rituximab, Sipuleucel-T, Sorafenib, Sunitinib, Talc Intrapleural, Tamoxifen, Temozolomide, Temsirolimus, Thalidomide, Trastuzumab, Vinorelbine, or Zoledronic acid. Mode of administration and dosage

[0508] In some embodiments, NK cells can be grown under conditions suitable for the cell population to be introduced into a subject (e.g., a human). Specific considerations include using a culture medium that does not contain any animal products (e.g., bovine serum). Other considerations include sterilization conditions to avoid bacterial, fungal, and mycoplasma contamination.

[0509] In some embodiments, after transfection, the cells can be administered to a patient immediately, or the cells can be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 days or more, or about 5 to about 12 days, about 6 to about 13 days, about 7 to about 14 days, or about 8 to about 15 days, for example, to allow time for the cells to recover from the transfection. Suitable culture conditions can be similar to the conditions under which the cells were cultured for activation, and agents that promote activation may or may not be used.

[0510] The disclosed cells can be administered in a variety of ways as desired for local or systemic treatment.

[0511] In the case of adoptive cell therapy, methods of administering cells for adoptive cell therapy are known and can be administered in combination with the methods and compositions provided herein.

[0512] Typically, administration can be topical, parenteral or enteral. The compositions of the present disclosure are generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any of the following routes of administration, characterized in that the tissue of the subject is physically damaged, and the pharmaceutical composition is administered through the damaged part of the tissue, thereby generally resulting in direct administration into the bloodstream, muscle or internal organs. Therefore, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection, administering the composition by surgical incision, administering the composition by non-surgical wounds penetrating the tissue, etc. Specifically, parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, enteral, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, intrasynovial injection or infusion; and renal dialysis infusion technology. In one embodiment, the parenteral administration of the compositions of the present disclosure includes intravenous administration.

[0513] Pharmaceutical composition preparations suitable for parenteral administration generally include a combination of an active ingredient and a pharmaceutically acceptable carrier (such as sterile water or sterile isotonic saline). Such preparations can be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Injectable preparations can be prepared, packaged or sold in unit dose form, such as in ampoules or in multi-dose containers containing preservatives. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, and pastes in oily or aqueous vehicles. Such preparations may also further include one or more additional ingredients, including, but not limited to, suspending agents, stabilizers or dispersants. In one embodiment of a preparation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable vehicle (such as sterile pyrogen-free water) before the composition reconstructed for parenteral administration. Parenteral formulations also include aqueous solutions, which may contain excipients such as salts, carbohydrates and buffers (preferably pH 3 to 9), but for some applications, they may be more suitable for being formulated into sterile non-aqueous solutions or dried forms with suitable vehicles (such as sterile pyrogen-free water). Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, propylene glycol aqueous solutions or glucose aqueous solutions. If necessary, such dosage forms can be appropriately buffered. Other preparations that can be used for parenteral administration include preparations or liposome preparations containing active ingredients in microcrystalline form. Preparations for parenteral administration can be formulated into immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, directional release and programmed release.

[0514] In addition, the composition of the present invention can also contain other common auxiliary components in the pharmaceutical composition.Therefore, for example, the composition can contain additional, compatible, pharmaceutically active materials, for example, antipruritic, astringent, local anesthetic or anti-inflammatory, or can contain the additional materials that can be used for the various dosage forms of the physical preparation of the composition of the present ...

Claims

1. A method for generating a CD34+ / CD43+ / CD45+ cell population, the method comprising contacting a stem cell population with a differentiation medium comprising a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) for a period of time sufficient to generate the CD34+ / CD43+ / CD45+ cell population from the stem cell population.

2. A method for differentiating a stem cell population into a hematopoietic progenitor cell population, the method comprising contacting the stem cell population with a differentiation medium comprising a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) for a period of time sufficient to differentiate the stem cell population into a hematopoietic progenitor cell population.

3. The method of claim 2, wherein the hematopoietic progenitor cell population comprises CD34+ / CD43+ / CD45+ cells.

4. The method according to any one of claims 1-3, wherein the BMP pathway activator is BMP4.

5. The method according to any one of claims 1-4, wherein the FGF is FGF2.

6. The method of any one of claims 1-5, wherein the VEGF is VEGF-165.

7. The method of any one of claims 1-6, wherein the differentiation medium comprises a Rho-associated coiled-coil-forming protein serine / threonine kinase (ROCK) inhibitor.

8. The method of claim 7, wherein the ROCK inhibitor is Y27632.

9. The method of any one of claims 1-8, wherein the differentiation medium comprises stem cell factor (SCF).

10. The method of any one of claims 1-9, wherein the differentiation medium comprises thrombopoietin (TPO).

11. The method of any one of claims 1-10, wherein the differentiation medium comprises low-density lipoprotein (LDL).

12. The method of any one of claims 1-11, wherein the differentiation medium comprises a phosphatidylinositol 3-kinase (PI3K) inhibitor.

13. The method of claim 12, wherein the PI3K inhibitor is LY294002.

14. The method of any one of claims 1-13, wherein the differentiation medium comprises a pyrimido-[4,5-b]-indole derivative.

15. The method of claim 14, wherein the pyrimido-[4,5-b]-indole derivative is UM729.

16. The method of any one of claims 1-15, wherein the differentiation medium comprises an aryl hydrocarbon receptor (AhR) antagonist.

17. The method of claim 16, wherein the AhR antagonist is StemRegenin 1 (SR1).

18. The method according to any one of claims 1-17, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor.

19. The method of any one of claims 1-17, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, and the PI3K inhibitor.

20. The method of any one of claims 1-17, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimido-[4,5-b]-indole derivative, and the AhR antagonist.

21. The method of any one of claims 1-6, comprising contacting the stem cell population with the differentiation medium for 1-5 days, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, and optionally the ROCK inhibitor.

22. The method according to any one of claims 1-13, comprising (i) contacting the stem cell population with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesodermal cells, and (ii) contacting the embryoid bodies or mesodermal cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL and the PI3K inhibitor for 1-15 days.

23. The method according to any one of claims 1-17, comprising (i) contacting the stem cell population with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesodermal cells, and (ii) contacting the embryoid bodies or mesodermal cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimido-[4,5-b]-indole derivative and the AhR antagonist for 1-15 days.

24. The method of any one of claims 1-23, wherein the differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, 1-50 μg / mL LDL, 0.1-100 PI3K inhibitor, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

25. The method of any one of claims 1-24, wherein the stem cell is an induced pluripotent stem cell (iPSC).

26. The method of any one of claims 1-24, wherein the stem cells are human embryonic stem cells (hESC).

27. A method for producing a CD43+ / CD45+ / CD56+ / LFA1+ cell population, the method comprising contacting a CD34+ / CD43+ / CD45+ cell population with a culture medium for a period of time sufficient to produce the CD43+ / CD45+ / CD56+ / LFA1+ cell population from the CD34+ / CD43+ / CD45+ cell population, the culture medium comprising SCF, interleukin-7 (IL-7), IL-12, IL-15, FMS-like tyrosine kinase 3 ligand (FLT3L), a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

28. A method for differentiating a population of hematopoietic progenitor cells and / or common lymphoid progenitor cells into a natural killer (NK) cell population, the method comprising contacting the hematopoietic progenitor cell population with a differentiation medium for a period of time sufficient to differentiate the hematopoietic progenitor cells into the NK cell population, the differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

29. The method of claim 27 or 28, wherein the pyrimido-[4,5-b]-indole derivative is UM729.

30. The method of any one of claims 27-29, wherein the AhR inhibitor is SR1.

31. The method according to any one of claims 28 to 30, wherein the culture medium comprises 1-100 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

32. The method of any one of claims 27-31, wherein the time period is 11-25 days.

33. The method of any one of claims 27-32, further comprising maturing the NK cell population with a maturation medium comprising (i) IL-12, IL-15 and IL-18 or (ii) IL-12, IL-2 and IL-18.

34. The method of any one of claims 1-33, wherein the differentiation medium and / or maturation medium is serum-free.

35. The method of any one of claims 1-34, wherein the method is xenologous gene-free.

36. A method for producing a NK cell population, the method comprising: (a) obtaining a stem cell population; (b) contacting the stem cell population with a first culture medium comprising a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor for a period of time sufficient to generate embryoid bodies; (c) contacting the embryoid bodies with a first differentiation medium for a period of time sufficient to generate a population of hematopoietic progenitor cells, wherein the first differentiation medium comprises a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, optionally a pyrimido[4,5-b]indole derivative, and an AhR inhibitor; (d) contacting the population of hematopoietic progenitor cells with a second differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor for a period of time sufficient to produce the population of NK cells.

37. The method of claim 36, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, the ROCK inhibitor is Y27632, the PI3K inhibitor is LY294002, and the pyrimido-[4,5-b]-indole derivative is UM729.

38. The method of claim 36 or 37, wherein each culture medium of steps (b)-(d) is serum-free.

39. The method of any one of claims 36-38, wherein the method is xenogeneic.

40. The method of any one of claims 36-39, wherein the first culture medium, the first differentiation medium, and the second differentiation medium each comprise the same basal medium.

41. The method of any one of claims 36-39, wherein the first culture medium, the first differentiation medium, and the second differentiation medium each comprise a different basal culture medium.

42. The method of any one of claims 36-39, wherein the first differentiation medium and the second differentiation medium each comprise the same basal medium, and the first medium comprises a basal medium different from the first and second differentiation medium.

43. The method of any one of claims 36-39, wherein the first differentiation medium and the second differentiation medium each comprise a basal medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

44. The method of any one of claims 36-43, wherein the time period of step (b) is 1-5 days, the time period of step (c) is 3-15 days, and the time period of step (d) is 11-25 days.

45. The method of any one of claims 36-44, wherein steps (a)-(d) occur within 35-45 days.

46. ​​The method of any one of claims 36-45, comprising (e) expanding the NK cell population with a maturation medium comprising (i) IL-12, IL-15 and IL-18 or (ii) IL-12, IL-2 and IL-18.

47. The method of any one of claims 36-46, wherein the stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

48. The method of any one of claims 36-47, wherein the population of hematopoietic progenitor cells comprises about 30% to about 50% CD34+ / CD43+ / CD45+ cells.

49. The method of any one of claims 36-48, wherein the NK cell population comprises about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

50. The method of any one of claims 36-49, comprising expanding the NK cell population, wherein the NK cell population is expanded by about 10 to about 350 fold.

51. The method of any one of claims 1-26 and 36-50, wherein the stem cell population is genetically engineered or edited.

52. The method of any one of claims 27-51, wherein the NK cell population is genetically engineered or edited.

53. A cell population comprising hematopoietic progenitor cells produced by the method according to any one of claims 1-26.

54. The cell population of claim 53, wherein the hematopoietic progenitor cells are CD34+ / CD43+ / CD45+.

55. The cell population of claim 53 or 54, comprising 30-50% hematopoietic progenitor cells.

56. A cell population comprising NK cells produced by the method according to any one of claims 27-52.

57. The cell population of claim 56, wherein the NK cells are CD43+ / CD45+ / CD56+ / LFA1+.

58. The cell population of claim 56 or 57, comprising 60-100% NK cells.

59. A hematopoietic progenitor cell differentiation medium, comprising a serum-free basal medium, a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL and a PI3K inhibitor.

60. The hematopoietic progenitor cell differentiation medium of claim 59, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, and the PI3K inhibitor is LY294002.

61. The hematopoietic progenitor cell differentiation medium of claim 59 or 60, comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, 1-50 ug / mL LDL, 0.1-100 PI3K inhibitor, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

62. A NK cell differentiation medium, comprising serum-free basal medium, SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor.

63. The NK cell differentiation medium of claim 62, wherein the pyrimido-[4,5-b]-indole derivative is UM729 and the AhR inhibitor is SR1.

64. The NK cell differentiation medium according to claim 62 or 63, comprising 1-100 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist and any combination thereof.

65. A kit comprising a hematopoietic progenitor cell differentiation medium according to any one of claims 59-61, and instructions for contacting a stem cell population with the hematopoietic progenitor cell differentiation medium for a period of time sufficient to produce a cell population comprising hematopoietic progenitor cells.

66. The kit of claim 65, wherein the time period is 1-15 days.

67. A kit comprising the NK cell differentiation medium of any one of claims 62-64, and instructions for contacting a population of hematopoietic progenitor cells with the NK cell differentiation medium for a period of time sufficient to produce a cell population comprising NK cells.

68. The kit of claim 67, wherein the time period is 11-25 days.

69. A kit comprising a hematopoietic progenitor cell differentiation medium according to any one of claims 59-61 and a NK cell differentiation medium according to any one of claims 62-64, and instructions for contacting a stem cell population with the hematopoietic progenitor cell differentiation medium for a first time period sufficient to produce a cell population comprising hematopoietic progenitor cells, and instructions for contacting a cell population comprising hematopoietic progenitor cells with the NK cell differentiation medium for a second time period sufficient to produce a cell population comprising NK cells.

70. The kit of claim 69, wherein the first period of time is 1-15 days and the second period of time is 11-25 days.

71. A kit according to any one of claims 67-70, further comprising a maturation medium, wherein the maturation medium comprises a basal medium and (i) IL-12, IL-15 and IL-18 or (ii) IL-12, IL-2 and IL-18, and instructions for contacting a cell population comprising NK cells for a period of time sufficient to mature the NK cells.

72. A composition for increasing the yield ratio of hematopoietic progenitor cells from a stem cell population, the composition comprising a bone morphogenetic protein (BMP) pathway activator, a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), and a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor.

73. The composition of claim 72, wherein the hematopoietic progenitor cells comprise CD34+ / CD43+ / CD45+ cells.

74. The composition of any one of claims 72-73, wherein the BMP pathway activator is BMP4.

75. A composition according to any one of claims 72-74, wherein the FGF is FGF2.

76. The composition of any one of claims 72-75, wherein the VEGF is VEGF-165.

77. The composition of claim 76, wherein the ROCK inhibitor is Y27632.

78. The composition of any one of claims 72-77, wherein the composition further comprises stem cell factor (SCF).

79. The composition of any one of claims 72-78, wherein the composition further comprises thrombopoietin (TPO).

80. The composition of any one of claims 72-79, wherein the composition further comprises low-density lipoprotein (LDL).

81. The composition of any one of claims 72-80, wherein the composition further comprises a phosphatidylinositol 3-kinase (PI3K) inhibitor.

82. The composition of claim 81, wherein the PI3K inhibitor is LY294002.

83. The composition of any one of claims 72-82, wherein the composition further comprises a pyrimido-[4,5-b]-indole derivative.

84. The composition of claim 83, wherein the pyrimido-[4,5-b]-indole derivative is UM729.

85. The composition of any one of claims 72-84, wherein the composition further comprises an aryl hydrocarbon receptor (AhR) antagonist.

86. The composition of claim 85, wherein the AhR antagonist is StemRegenin 1 (SR1).

87. The composition of any one of claims 72-86, wherein the composition comprises the BMP pathway activator, the FGF, the VEGF, and the ROCK inhibitor.

88. The composition of any one of claims 72-87, wherein the composition comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, and the PI3K inhibitor.

89. The composition of any one of claims 72-88, wherein the composition comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimido-[4,5-b]-indole derivative, and the AhR antagonist.

90. The composition of any one of claims 72-89, wherein the composition comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, 1-50 ug / mL LDL, 0.1-100 PI3K inhibitor, 0.1-10 μM pyrimidine-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

91. The composition of any one of claims 72-90, wherein the stem cell population is induced pluripotent stem cells (iPSCs).

92. The composition of any one of claims 72-91, wherein the stem cell population is human embryonic stem cells (hESC).

93. A composition according to any one of claims 72-92, wherein the yield ratio (HP / StC) of hematopoietic progenitor cells (HP) to stem cells (StC) is about 2:1 to about 10:

1.

94. The composition of claim 93, wherein the yield ratio (HP / StC) of hematopoietic progenitor cells (HP) to stem cells (StC) is about 5:

1.

95. A method for increasing the yield of hematopoietic progenitor cells from a stem cell population, the method comprising contacting the stem cell population with a differentiation medium for a period of time sufficient to differentiate the stem cell population into the hematopoietic progenitor cells, the differentiation medium comprising a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor.

96. The method of claim 95, wherein the hematopoietic progenitor cells comprise CD34+ / CD43+ / CD45+ cells.

97. The method of any one of claims 95-96, wherein the BMP pathway activator is BMP4.

98. The method of any one of claims 95-97, wherein the FGF is FGF2.

99. The method of any one of claims 95-98, wherein the VEGF is VEGF-165.

100. The method of claim 99, wherein the ROCK inhibitor is Y27632.

101. The method of any one of claims 95-100, wherein the differentiation medium comprises stem cell factor (SCF).

102. The method of any one of claims 95-101, wherein the differentiation medium comprises thrombopoietin (TPO).

103. The method of any one of claims 95-102, wherein the differentiation medium comprises low-density lipoprotein (LDL).

104. The method of any one of claims 95-103, wherein the differentiation medium comprises a phosphatidylinositol 3-kinase (PI3K) inhibitor.

105. The method of claim 104, wherein the PI3K inhibitor is LY294002.

106. The method of any one of claims 95-105, wherein the differentiation medium comprises a pyrimido-[4,5-b]-indole derivative.

107. The method of claim 106, wherein the pyrimido-[4,5-b]-indole derivative is UM729.

108. The method of any one of claims 95-107, wherein the differentiation medium comprises an aryl hydrocarbon receptor (AhR) antagonist.

109. The method of claim 108, wherein the AhR antagonist is StemRegenin 1 (SR1).

110. The method of any one of claims 95-109, wherein the differentiation medium comprises a BMP pathway activator, the FGF, the VEGF, and the ROCK inhibitor.

111. The method of any one of claims 95-110, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, and the PI3K inhibitor.

112. The method of any one of claims 95-111, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimido-[4,5-b]-indole derivative, and the AhR antagonist.

113. The method of any one of claims 95-112, comprising contacting the stem cell population with the differentiation medium for 1-5 days, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF and optionally the ROCK inhibitor.

114. The method according to any one of claims 95-113, comprising (i) contacting the stem cell population with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesoderm cells, and (ii) contacting the embryoid bodies or mesoderm cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL and the PI3K inhibitor for 1-15 days.

115. The method according to any one of claims 95-114, comprising (i) contacting the stem cell population with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesoderm cells, and (ii) contacting the embryoid bodies or mesoderm cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, the LDL, the PI3K inhibitor, the pyrimidine [4,5-b] indole derivative and the AhR antagonist for 1-15 days.

116. The method according to any one of claims 95-115, wherein the differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, 1-50 ug / mL LDL, 0.1-100 PI3K inhibitor, 0.1-10 μM pyrimidine-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist and any combination thereof.

117. The method of any one of claims 95-116, wherein the stem cell population is an induced pluripotent stem cell (iPSC) population.

118. The method of any one of claims 95-117, wherein the stem cell population is a human embryonic stem cell (hESC) population.

119. The method of any one of claims 95-118, wherein the yield ratio of hematopoietic progenitor cells from the stem cell population is about 2:1 to about 10:

1.

120. The method of any one of claims 95-119, wherein the yield ratio of the hematopoietic progenitor cell population from the stem cells is about 5:

1.

121. A kit for increasing the yield of NK cells from a stem cell population, wherein the kit comprises instructions for differentiating the stem cell population into NK cells, and: (a) a first culture medium comprising a BMP pathway activator, FGF, VEGF and optionally a ROCK inhibitor; (b) a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and optionally a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor; (c) a second differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor.

122. The kit of claim 121, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, the ROCK inhibitor is Y27632, the PI3K inhibitor is LY294002, and the pyrimido-[4,5-b]-indole derivative is UM729.

123. A kit according to claim 121 or 122, wherein each culture medium in (a)-(c) is serum-free.

124. The kit of any one of claims 121-123, wherein the culture medium is xeno-free.

125. The kit of any one of claims 121-124, wherein the first culture medium, the first differentiation medium, and the second differentiation medium each comprise the same basal culture medium.

126. The kit of any one of claims 121-125, wherein the first culture medium, the first differentiation medium, and the second differentiation medium each comprise a different basal culture medium.

127. The kit of any one of claims 121-126, wherein the first differentiation medium and the second differentiation medium each comprise the same basal medium, and the first medium comprises a basal medium different from the first and second differentiation medium.

128. The kit according to any one of claims 121-127, wherein the first differentiation medium and the second differentiation medium each comprise a basal medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin and 2-mercaptoethanol.

129. The kit of any one of claims 121-128, further comprising a maturation medium comprising (i) IL-12, IL-15 and IL-18 or (ii) IL-12, IL-2 and IL-18.

130. The kit of any one of claims 121-129, wherein the stem cell population is an induced pluripotent stem cell (iPSC) population or a human embryonic stem cell (hESC) population.

131. The kit of any one of claims 121-130, wherein the NK cells comprise about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

132. The kit of any one of claims 121-131, wherein the stem cell population is genetically engineered or edited.

133. A kit according to any one of claims 121-132, wherein the NK cells are genetically engineered or edited.

134. The kit of any one of claims 121-133, wherein the yield ratio of NK cells from the stem cell population is about 2:1 to about 100:

1.

135. A kit according to any one of claims 121-134, wherein the yield ratio of NK cells from the stem cell population is about 35:

1.

136. A method for increasing the yield of NK cells from a stem cell population, the method comprising: (a) obtaining a stem cell population; (b) contacting the stem cell population with a first culture medium comprising a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor for a period of time sufficient to generate embryoid bodies; (c) contacting the embryoid bodies with a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and optionally a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor for a period of time sufficient to generate a population of hematopoietic progenitor cells; (d) contacting the population of hematopoietic progenitor cells with a second differentiation medium for a period of time sufficient to produce said NK cells, wherein said second culture medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

137. The method of claim 136, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, the ROCK inhibitor is Y27632, the PI3K inhibitor is LY294002, and the pyrimido-[4,5-b]-indole derivative is UM729.

138. The method of claim 136 or 137, wherein each culture medium of steps (b)-(d) is serum-free.

139. The method of any one of claims 136-138, wherein the method is xenogeneic.

140. The method of any one of claims 136-139, wherein the first culture medium, the first differentiation medium, and the second differentiation medium each comprise the same basal culture medium.

141. The method of any one of claims 136-139, wherein the first culture medium, the first differentiation medium, and the second differentiation medium each comprise a different basal culture medium.

142. The method of any one of claims 136-139, wherein the first differentiation medium and the second differentiation medium each comprise the same basal medium, and the first medium comprises a basal medium different from the first and second differentiation medium.

143. The method of any one of claims 136-139, wherein the first differentiation medium and the second differentiation medium each comprise a basal medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

144. The method of any one of claims 136-143, wherein the time period of step (b) is 1-5 days, the time period of step (c) is 3-15 days, and the time period of step (d) is 11-25 days.

145. The method of any one of claims 136-144, wherein steps (a)-(d) occur within 35-45 days.

146. The method of any one of claims 136-145, comprising (e) expanding NK cells with a maturation medium comprising (i) IL-12, IL-15 and IL-18 or (ii) IL-12, IL-2 and IL-18.

147. The method of any one of claims 136-146, wherein the stem cell population is induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

148. The method of any one of claims 136-147, wherein the hematopoietic progenitor cell population comprises about 30% to about 50% CD34+ / CD43+ / CD45+ cells.

149. The method of any one of claims 136-148, wherein the NK cells comprise about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

150. The method of any one of claims 136-149, comprising expanding the NK cells, wherein the NK cells are expanded from about 10 to about 350 fold.

151. The method of any one of claims 136-150, wherein the stem cell population is genetically engineered or edited.

152. The method of any one of claims 136-151, wherein the NK cells are genetically engineered or edited.

153. The method of any one of claims 136-152, wherein the yield ratio of NK cells from the stem cell population is about 2:1 to about 100:

1.

154. The method of any one of claims 136-153, wherein the yield ratio of NK cells from the stem cell population is about 35:1.

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