Compositions and methods for NK cell differentiation

Abstract

Xenogeneic-free methods and compositions for generating hematopoietic precursors and natural killer (NK) cells are provided. TIFF2025524353000033.tif130159

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 350,755, filed on June 9, 2022, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Background Art

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

[0003] NK cells for use in 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) derived from somatic cells (generally fibroblasts or peripheral blood mononuclear cells [PBMC]) that are induced to be capable of unlimited proliferation and differentiation into other cell types when subjected to appropriate differentiation conditions, and human embryonic stem cells (hESCs). NK cells can be induced from iPSCs by sequentially differentiating iPSCs into hematopoietic progenitor cells (HPCs), also called hematopoietic stem cells (HSCs).

[0004] Primary NK cells or iPSC - NK cells can be expanded ex vivo after being obtained and before administration to a patient. Methods for differentiating iPSCs into NK cells often involve the use of feeder cells or media containing serum. There remains a need for compositions and methods related to generating NK cells without using xenogeneic factors, such as animal - derived materials like fetal bovine serum (FBS) and / or feeder cells, to provide cells suitable for in vivo administration.

Summary of the Invention

[0005] Summary The present disclosure is based at least in part on the discovery of a xeno-free method for differentiating stem cells into hematopoietic precursors and NK cells. Without wishing to be bound by theory, the xeno-free methods described herein result in cells suitable for in vivo administration.

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

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

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

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

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

[0011] In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, the differentiation medium contains 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 contains an aryl hydrocarbon receptor (AhR) antagonist. In some embodiments, the AhR antagonist is StemRegenin 1 (SR1).

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

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

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

[0015] In some embodiments, the method includes contacting a population of stem cells with a differentiation medium for 1 to 5 days, the differentiation medium containing a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor.

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

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

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

[0019] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

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

[0021] In some embodiments, the present disclosure provides a method for generating a population of CD43+ / CD45+ / CD56+ / LFA1+ cells, comprising contacting a population of CD34+ / CD43+ / CD45+ cells with a 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 for a period sufficient to generate a population of CD43+ / CD45+ / CD56+ / LFA1+ cells from the population of CD34+ / CD43+ / CD45+ cells.

[0022] In some embodiments, the present disclosure provides a method for differentiating a population of hematopoietic precursors into a population of natural killer (NK) cells, comprising contacting the population of hematopoietic precursors with a 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 sufficient to differentiate the population of hematopoietic precursors into a population of NK cells.

[0023] In some embodiments, the present disclosure provides a method for differentiating a population of common lymphoid progenitors (CLPs) into a population of natural killer (NK) cells, comprising contacting the population of CLPs with a 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 sufficient to differentiate the population of CLPs into a population of NK cells.

[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 medium comprises SCF at 1-100 ng / mL, IL-7 at 1-50 ng / mL, IL-12 at 1-100 ng / mL, IL-15 at 1-100 ng / mL, FLT3L at 1-100 ng / mL, a pyrimido-[4,5-b]-indole derivative at 0.1-10 μM, an AhR antagonist at 0.1-10 μM, and any combination thereof.

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

[0027] In some embodiments, the method comprises the step of maturing a population of NK cells using 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 the maturation medium is serum-free.

[0029] In some embodiments, the method is xeno-free.

[0030] In some embodiments, the present disclosure provides a method for generating a population of NK cells, comprising: (a) obtaining a population of stem cells; (b) contacting the population of stem cells with a first medium comprising a BMP pathway activator, FGF, VEGF, and optionally an ROCK inhibitor for a period 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, an inhibitor of PI3K, and optionally a pyrimido-[4,5-b]-indole derivative and an AhR inhibitor for a period sufficient to generate a population of hematopoietic precursors; (d) contacting the population of hematopoietic precursors 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 sufficient to generate the population of NK cells. The present disclosure provides a method as described above.

[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 of the media in steps (b) to (d) is serum-free.

[0033] In some embodiments, the method is xenogeneic-free.

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

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

[0036] In some embodiments, the first differentiation medium and the second differentiation medium each contain the same basal medium, and the first medium contains a basal medium different from those of the first and second differentiation media.

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

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

[0039] In some embodiments, steps (a) to (d) are performed within 35 to 45 days.

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

[0041] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

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

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

[0044] In some embodiments, the method includes expanding a population of NK cells, and the population of NK cells expands and proliferates by about 10 to about 350 - fold.

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

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

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

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

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

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

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

[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, an FGF, a VEGF, an SCF, a TPO, an 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 medium comprises 1 - 50 ng / mL of BMP4, 1 - 50 ng / mL of FGF2, 5 - 100 ng / mL of VEGF, 0.1 - 20 μM of a ROCK inhibitor, 1 - 200 ng / mL of SCF, 1 - 100 ng / mL of TPO, 1 - 50 μg / mL of LDL, 0.1 - 100 of a PI3K inhibitor, 0.1 - 10 μM of a pyrimido-[4,5-b]-indole derivative, 0.1 - 10 μM of an AhR antagonist, and any combination thereof.

[0056] In some embodiments, the present disclosure provides an NK cell differentiation medium comprising a serum-free basal medium, an SCF, an IL-7, an IL-12, an IL-15, an 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 medium comprises 1-100 ng / mL of SCF, 1-50 ng / mL of IL-7, 1-100 ng / mL of IL-12, 1-100 ng / mL of IL-15, 1-100 ng / mL of FLT3L, 0.1-10 μM of pyrido-[4,5-b]-indole derivative, 0.1-10 μM of AhR antagonist, and any combination thereof.

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

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

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

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

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

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

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

[0066] In some embodiments, the present disclosure provides a composition for increasing the yield ratio of hematopoietic precursors from a population of stem cells, the composition comprising an osteogenic 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 increasing the yield ratio of hematopoietic precursors from stem cells, the composition comprising an osteogenic 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 precursors 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 phosphoinositide 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 a BMP pathway activator, an FGF, a VEGF, and a ROCK inhibitor.

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

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

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

[0073] In some embodiments, the population of stem cells is induced pluripotent stem cells (iPSCs). In some embodiments, the population of stem cells is human embryonic stem cells (hESCs).

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

[0075] In some embodiments, the present disclosure provides a method of increasing the yield ratio of hematopoietic progenitors from a population of stem cells, comprising contacting the population of stem cells with a differentiation medium 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 for a period of time sufficient to differentiate the population of stem cells into hematopoietic precursors. In some embodiments, the present disclosure provides a method of increasing the yield ratio of hematopoietic progenitors from stem cells, comprising contacting the stem cells with a differentiation medium 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 for a period of time sufficient to differentiate the stem cells into hematopoietic precursors.

[0076] In some embodiments, the hematopoietic precursors 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 contains stem cell factor (SCF). In some embodiments, the differentiation medium contains thrombopoietin (TPO). In some embodiments, the differentiation medium contains low density lipoprotein (LDL). In some embodiments, the differentiation medium contains a phosphoinositide 3-kinase (PI3K) inhibitor. In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, the differentiation medium contains 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 contains an aryl hydrocarbon receptor (AhR) antagonist. In some embodiments, the AhR antagonist is StemRegenin 1 (SR1).

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

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

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

[0081] In some embodiments, the method includes contacting a population of stem cells with a differentiation medium for 1 to 5 days, the differentiation medium containing a BMP pathway activator, an FGF, a VEGF, and optionally a ROCK inhibitor.

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

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

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

[0085] In some embodiments, the population of stem cells is a population of induced pluripotent stem cells (iPSCs). In some embodiments, the population of stem cells is a population of human embryonic stem cells (hESCs).

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

[0087] In some embodiments, the present disclosure provides a kit for increasing the yield ratio of NK cells from a population of stem cells, the kit comprising instructions for differentiating a population of stem cells into NK cells, and (a) a first medium comprising a BMP pathway activator, FGF, VEGF, and optionally an inhibitor of ROCK, (b) a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, an inhibitor of PI3K, 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 and comprising.

[0088] In some embodiments, the present disclosure provides a kit for increasing the yield ratio of NK cells from stem cells, the kit comprising instructions for differentiating stem cells into NK cells, and (a) a first medium comprising a BMP pathway activator, FGF, VEGF, and optionally an inhibitor of ROCK, (b) a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, an inhibitor of PI3K, 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 and comprising.

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

[0090] In some embodiments, each of the media (a)-(c) is serum-free. In some embodiments, the medium is xenogeneic-free.

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

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

[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 population of stem cells is a population of induced pluripotent stem cells (iPSCs) or a population of human embryonic stem cells (hESCs).

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

[0098] In some embodiments, the population of stem cells 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 from the population of stem cells is from about 2:1 to about 100:1. In some embodiments, the yield ratio of NK cells from the population of stem cells is about 35:1.

[0101] In some embodiments, the present disclosure provides a method for increasing the yield ratio of NK cells from a population of stem cells, comprising: (a) obtaining a population of stem cells; (b) contacting the population of stem cells with a first medium comprising a BMP pathway activator, FGF, VEGF, and optionally an inhibitor of ROCK for a period 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, an inhibitor of PI3K, and optionally a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor for a period sufficient to generate a population of hematopoietic precursors; (d) contacting the population of hematopoietic precursors 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 sufficient to generate NK cells. The method is provided. 137. The method of claim 136, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, the inhibitor of ROCK is Y27632, the inhibitor of PI3K 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, comprising: (a) obtaining stem cells; (b) contacting the stem cells with a first medium comprising a BMP pathway activator, FGF, VEGF, and optionally an inhibitor of ROCK for a period 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, an inhibitor of PI3K, and optionally a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor for a period sufficient to generate a population of hematopoietic precursors; (d) Contacting a population of hematopoietic precursors 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 sufficient to generate NK cells A method is provided that includes the above. 137. The method according to 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 of the media in steps (b)-(d) is serum-free. In some embodiments, the method is xenogeneic-free.

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

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

[0106] In some embodiments, the first differentiation medium and the second differentiation medium each contain the same basal medium, and the first medium contains a basal medium different from the first and second differentiation media.

[0107] In some embodiments, the first differentiation medium and the second differentiation medium each contain 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 period of step (b) is 1 to 5 days, the period of step (c) is 3 to 15 days, and the period of step (d) is 11 to 25 days.

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

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

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

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

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

[0114] In some embodiments, the method includes the step of expanding and proliferating NK cells, and the NK cells expand and proliferate by about 10 to about 350-fold.

[0115] In some embodiments, the population of stem cells 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 population of stem cells is from about 2:1 to about 100:1.

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

[0119]

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BRIEF DESCRIPTION OF THE DRAWINGS

[0120] DETAILED DESCRIPTION In some aspects, the present disclosure provides compositions and methods for generating hematopoietic precursors, common lymphoid progenitors, pre-NK precursors, NK precursors, immature NK cells and / or NK cells. In some embodiments, the compositions and methods described herein are xenogeneic-free.

[0121] DEFINITIONS All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0122] Unless otherwise indicated in the context, the various features described herein can be used arbitrarily in combination with any feature or combination of features described herein, and each feature can be excluded or omitted from the combination.

[0123] As used herein, the singular forms "a", "an" and "the" include the plural forms unless otherwise indicated in the context. The phrase "and / or" indicates any possible combination of one or more of the listed items.

[0124] As used herein, "subject" refers to a recipient of a population of NK cells 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.

[0125] As used herein, "treat", "treating" or "treatment" refers to any type of action or administration that confers a benefit on a subject having a disease or disorder, including amelioration of the patient's condition (i.e., improvement, reduction or amelioration of one or more symptoms, and partial or complete response to treatment).

[0126] The term "effective amount" refers to an amount effective to bring about a desired biochemical, cellular, or physiological response. The term "therapeutically effective amount" refers to an amount, dosage, or dosing regimen of a treatment effective to cause a desired therapeutic effect.

[0127] As used herein, "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently linked through phosphodiester bonds between the phosphoryl group of one nucleotide in the chain and the hydroxyl group of the sugar component of the next nucleotide. DNA and RNA are non-limiting examples of polynucleotides.

[0128] As used herein, "polypeptide" refers to a polymer consisting of amino acid residues linked together by peptide bonds, which form part (or all) of a protein.

[0129] It will be understood by those skilled in the art that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, it should be understood that those skilled in the art may make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein, using routine techniques, to reflect the codon usage frequency of any particular host organism in which the polypeptide is to be expressed.

[0130] Nucleic acids can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides that contain synthetic or modified nucleotides therein. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for the uses described herein, polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0131] The term "variant" means a polynucleotide or polypeptide that has at least one substitution, insertion, or deletion in its sequence as compared to a reference polynucleotide or reference polypeptide. A "functional variant" is a variant that retains one or more functions of the reference polynucleotide or reference polypeptide.

[0132] The term "inactivating mutation" refers to a mutation within a genomic sequence that disrupts the function of a gene. An inactivating mutation can be in any sequence region that contributes to gene expression (e.g., coding or non-coding). Examples include, without limitation, cis-acting elements (enhancers), or sequences that are transcribed (e.g., mRNA transcript sequences). Inactivating mutations include mutations that render the gene or its encoded protein non-functional, or mutations that reduce the function of the gene or its encoded protein.

[0133] As used herein, the terms "sequence identity" or "identity" with respect to a polynucleotide or polypeptide sequence refer to the degree to which two optimally aligned polynucleotide or polypeptide sequences match at each position in the alignment over the full length of the reference sequence. The "percent identity" is the number of matching positions in the optimal alignment divided by the sum of the length of the reference sequence + the length of any gaps in the reference sequence within the alignment. The optimal alignment is the alignment that yields the highest percent identity. Alignment of sequences for determining percent identity can be achieved by several well-known methods including, for example, using mathematical algorithms such as those within the BLAST suite or the Clustal Omega sequence analysis program. Unless otherwise specified, the term "sequence identity" in the claims refers to sequence identity calculated by BLAST version 2.12.0 using default parameters. Also, unless otherwise specified, the alignment is an alignment of all or a portion of the polynucleotide or polypeptide sequence of interest over the full length of the reference sequence.

[0134] As used herein, the term "engineered" refers to a cell in which a heterologous polynucleotide has been stably transfected, or a cell that has been subjected to gene editing to introduce, delete, or modify an intracellular polynucleotide, or a cell in which a polynucleotide has been transiently transfected to cause a stable phenotypic change within the cell.

[0135] As used herein, the term "stem cell" is used to represent a cell having an undifferentiated phenotype that can differentiate, for example, into hematopoietic precursors and / or NK cells.

[0136] As used herein, the term "pluripotent" means that a stem cell is capable of forming substantially all of the differentiated cell types of an organism, at least in culture. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, namely, the ectoderm, mesoderm, and endoderm.

[0137] As used herein, the terms "induced pluripotent stem cell" and "iPSC" are used to refer to cells derived from somatic cells that have been reprogrammed into a pluripotent state and are capable of proliferation, selectable differentiation, and maturation. iPSCs are derived from differentiated adult, neonatal, or fetal cells that have been induced or changed, i.e., reprogrammed, into cells that can differentiate into tissues of all three germ layers or cortical layers, namely, mesoderm, endoderm, and ectoderm. The resulting iPSCs do not refer to cells as found in nature.

[0138] As used herein, the terms "hematopoietic stem cell", "hematopoietic precursor", or "hematopoietic progenitor cell" refer to stem cells that can give rise to both mature myeloid cell types and mature lymphoid cell types, including natural killer cells, T cells, and B cells. Hematopoietic stem cells are typically characterized by CD34+.

[0139] The term "precursor" refers to a cell that has partially differentiated into a desired cell type. Precursor cells retain some degree of pluripotency and can differentiate into multiple cell types.

[0140] As used herein, "differentiating" or "differentiated" are used to refer to the process and conditions by which an undifferentiated or immature (e.g., unspecialized) cell acquires the characteristics of a mature (specialized) cell, thereby acquiring a particular morphology and function. Stem cells (unspecialized) are often exposed to various conditions (e.g., growth factors and morphogens) to induce a specific lineage commitment or differentiation of the stem cell.

[0141] As used herein, "expanding" or "expansion" refers to an increase in the number and / or purity of cell types within a cell population by mitosis of cells having limited proliferative capacity, such as NK cells.

[0142] As used herein, "activation", "activate" or "activated" refers to the stimulation of activating receptors on cytotoxic natural lymphocyte cells that results in cell division, cytokine secretion (e.g., IFNγ and / or TNFα), and / or release of cytolytic granules to regulate or assist in the immune response.

[0143] As used herein, "xeno-free" refers to compositions and methods lacking animal-derived raw materials (e.g., fetal bovine serum). In some embodiments, the xeno-free methods described herein are feeder cell-free.

[0144] As used herein, "source cell" refers to any progenitor cell known in the art.

[0145] As used herein, "yield ratio" refers to a measurement that quantifies the amount of cells produced from a single source cell. For example, an NK / iPSC yield ratio of 20:1 means that 20 NK cells were produced from 1 iPSC. Similarly, if 20,000 NK cells are obtained from an initial population of 1,000 iPSCs, the NK / iPSC yield ratio of NKs from iPSCs is 20 NKs per single iPSC, i.e., 20:1. In some embodiments, the yield ratio is determined by cell counting. In some embodiments, cell counting is performed at the start of the process, throughout the process, and at the end of the process to calculate how cells expand over time based on changes in viable cell density. In some embodiments, dilution of the cells is performed over time (batch feeding).

[0146] Differentiation and expansion medium In some embodiments, the present disclosure provides a medium for differentiating stem cells into hematopoietic precursors. In some embodiments, the present disclosure provides a medium for differentiating hematopoietic precursors into NK cells. In some embodiments, the present disclosure provides a medium for expanding and proliferating NK cells. In some embodiments, the differentiation medium and / or expansion and proliferation medium described herein comprises a serum-free basal medium containing at least one exogenous factor that promotes differentiation and / or expansion and proliferation.

[0147] Xenogeneic-free medium In some embodiments, the differentiation medium and / or expansion and proliferation medium described herein is a defined medium. As used herein, "defined medium" refers to a growth medium suitable for in vitro culture of human or animal cells, the total chemical composition of which is known. In some embodiments, the differentiation medium and / or expansion and proliferation 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.

[0148] In some embodiments, the basal medium is selected from StemSpan SFEM II Medium (STEMCELL Technologies; serum-free), Stemline II (Sigma-Aldrich; fully defined, serum-free and animal component-free, GMP manufactured), 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 Expansion Medium XF (PromoCell; serum-free and xenofree medium). In some embodiments, the basal medium is StemSpan SFEM II 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 differentiation medium and the NK cell differentiation medium have the same basal medium. In some embodiments, the hematopoietic progenitor differentiation medium and the NK cell differentiation medium have different basal media.

[0149] Exogenous factors In some embodiments, the differentiation medium and / or expansion medium described herein contains exogenous factors. In some embodiments, the methods of the disclosure include contacting different cell populations with various exogenous factors in a xenogeneic-free medium to promote, for example, the differentiation of cells into hematopoietic progenitors and / or NK cells. In some embodiments, exogenous factors include, without limitation, cytokines such as interleukins, fibroblast growth factor (FGF), stem cell factor (SCF), phosphatidylinositol 3-kinase (PI3K) inhibitors, FMS-like tyrosine kinase 3 ligand (FLT3L), bone morphogenetic protein (BMP) pathway activators, pyrimido-indole derivatives, and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors described herein are commercially available.

[0150] In some embodiments, the exogenous factors suitable for use in the differentiation medium and / or the expansion medium are cytokines. Cytokines include interferons, interleukins and growth factors, which are small proteins that play important roles 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.

[0151] In some embodiments, the exogenous factor is interleukin 2 (IL-2). IL-2 is a secreted cytokine produced by activated CD4+ T lymphocytes and CD8+ T lymphocytes and is important for the proliferation of T and B lymphocytes. IL-2 is a member of the interleukin 2 (IL2) cytokine subfamily that includes IL-4, IL-7, IL-9, IL-15, IL-21, erythropoietin and thrombopoietin.

[0152] In some embodiments, the exogenous factor is interleukin 7 (IL-7). IL-7 is a member of the interleukin 2 (IL2) cytokine subfamily. Lymphocyte differentiation and activation are critically dependent on IL-7 signaling.

[0153] 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 short-term stimulation with IL-12.

[0154] 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 the activation and proliferation of NK cells.

[0155] In some embodiments, the exogenous factor is interleukin 18 (IL-18). IL-18 is a pro-inflammatory cytokine of the IL-1 family that is constitutively found as a precursor within the cytoplasm of various immune cells. IL-18 has been shown to strongly activate NK cells.

[0156] In some embodiments, the exogenous factor is low density lipoprotein (LDL). LDL induces the proliferation of NK cells and an increase in cytotoxic activity.

[0157] 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, FGF2 is commercially available. In some embodiments, FGF2 is commercially available from Peprotech. In some embodiments, FGF2 is commercially available from Gibco.

[0158] 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 precursors and the generation of mature peripheral NK cells.

[0159] In some embodiments, the exogenous factor is stem cell factor (SCF). SCF plays an important role in the survival of stem cells, as well as in the self-renewal and maintenance of stem cells.

[0160] In some embodiments, the exogenous factor is vascular endothelial growth factor (VEGF). The VEGF family is a subfamily of growth factors and is a platelet-derived growth factor family of cysteine-knot growth factors. The VEGF family includes five family members. In some embodiments, the exogenous factors are 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 38.2 kDa disulfide-linked homodimer protein consisting of two 165 amino acid polypeptide chains.

[0161] 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 the regulation of immunity, stem cell maintenance, and cell differentiation. Antagonism of the aryl hydrocarbon receptor has been shown to promote the regeneration and expansion of stem cells. In some embodiments, the exogenous factor is an antagonist of the aryl hydrocarbon receptor selected from PD98059, StemRegenin 1 (SR1), GNF351, BAY 2416964, CH-223191, perilla aldehyde, PDM-11, and BAY-218. In some embodiments, the exogenous factor is SR1.

[0162] In some embodiments, the exogenous factor is an inhibitor of phosphatidylinositol 3-kinase (PI3K). PI3K is a family of lipids and serine / threonine kinases that catalyze the transfer of phosphate to the D-3' position of inositol lipids to produce phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3,4-bisphosphate (PIP2), and phosphatidylinositol-3,4,5-trisphosphate (PIP3), and phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3,4-bisphosphate (PIP2), and phosphatidylinositol-3,4,5-trisphosphate (PIP3) often act as second messengers of the signaling cascade by docking proteins containing pleckstrin homology domains, FYVE domains, Phox domains and other phospholipid-binding domains to various signaling complexes at the plasma membrane.

[0163] Inhibitors of PI3K include, without limitation, idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, duvelisib, idelalisib, leniolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimidostatin, 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 agent is idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, duvelisib, idelalisib, leniolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimidostatin, 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 agent is LY294002.

[0164] In some embodiments, the exogenous factor is an activator of the BMP pathway. Bone morphogenetic protein (BMP) is produced as a large precursor molecule that is proteolytically processed into a mature peptide after translation. BMP acts through specific transmembrane receptors located on the cell surface of target cells. BMP receptors are serine-threonine kinases similar to TGF-β receptors and are divided into two subgroups, namely, type I receptors and type II receptors. BMP can bind strongly only to heterotetrameric complexes of these receptors. This complex formation is essential for BMP signaling. Inside the target cells, the BMP signal is transmitted to the nucleus via specific signal molecules called Smads, which are also involved in the suppression of BMP signals.

[0165] BMP is a multifunctional cytokine that is a member of the transforming growth factor-beta superfamily. BMP receptors mediate BMP signaling by activating Smads. BMP ligands bind to BMP receptors BMPRI and BMPRII. Phosphorylated BMPRII activates BMPRI. Phosphorylated BMPRI then phosphorylates receptor-activated Smad proteins (R-Smads), and the receptor-activated Smad proteins (R-Smads) associate with common mediator Smads (co-Smads), enter the nucleus, and regulate gene expression there. BMP pathway activators include the agents disclosed in International Publication Nos. 2014011540, 2014062138, and 2005117994, which are incorporated herein by reference. BMP pathway activators include, without limitation, 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, BMP-4 is commercially available. In some embodiments, BMP-4 is commercially available from Peprotech. In some embodiments, BMP-4 is commercially available from Invitrogen. In some embodiments, BMP-4 is commercially available from Biolegend.

[0166] In some embodiments, the exogenous factor is an inhibitor of ROCK. Rho-associated kinase (ROCK) is a serine / threonine kinase that serves as a downstream effector of Rho kinases (there are three isoforms thereof - RhoA, RhoB, and RhoC). ROCK inhibitors include, without limitation, polynucleotides, polypeptides, and small molecules. ROCK inhibitors contemplated herein can reduce ROCK expression and / or ROCK activity. Exemplary examples of ROCK inhibitors contemplated herein include, without limitation, anti-ROCK antibodies, dominant negative ROCK mutants, siRNA, shRNA, miRNA, and antisense nucleic acids that target ROCK.

[0167] Exemplary ROCK inhibitors contemplated herein include, without limitation, thiazovivin, Y27632, fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, as well as the ROCK inhibitors disclosed in U.S. Patent No. 8,044,201, which is hereby incorporated by reference in its entirety. In some embodiments, the ROCK inhibitor is thiazovivin, Y27632 or pyrintegrin. In some embodiments, the ROCK inhibitor is Y27632.

[0168] Mesoderm / embryoid body differentiation medium In some embodiments, the present disclosure provides a differentiation medium for generating mesoderm and / or embryoid bodies from stem cells. In some embodiments, the mesoderm cells are generated from iPSCs or hESCs. When stem cells begin to differentiate, three different germ layers are formed: the ectoderm, mesoderm, and endoderm. Immune cells such as NK cells differentiate from mesoderm cells. An embryoid body is a three-dimensional aggregate that can differentiate into cells of all three germ layers. In some embodiments, the mesoderm cells are produced from embryoid bodies. In some embodiments, the mesoderm cells produced by the compositions and methods of the present disclosure are further differentiated into hematopoietic precursors. In some embodiments, the mesoderm cells produced by the compositions and methods of the present disclosure are further differentiated into NK cells.

[0169] In some embodiments, a population of stem cells (e.g., iPSCs or hESCs) is cultured with at least one exogenous factor 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 factor comprises a BMP pathway activator, FGF, and a ROCK inhibitor. In some embodiments, the exogenous factor comprises FGF, VEGF, and a ROCK inhibitor. In some embodiments, the exogenous factor comprises a BMP pathway activator, FGF, and a ROCK inhibitor. In some embodiments, the exogenous factor comprises a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor.

[0170] 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 include BMP4, FGF2, VEGF-165, and Y27632.

[0171] In some embodiments, BMP4, FGF2, VEGF, and / or a ROCK inhibitor are used in the mesoderm formation process. For example, the mesoderm formation process can include contacting a cell population with BMP4 and FGF2; BMP4, FGF2, and a ROCK inhibitor; BMP4 and VEGF; BMP4, VEGF, and a ROCK inhibitor; FGF2 and VEGF; FGF2, VEGF, and a ROCK inhibitor; BMP4, FGF2, and VEGF; BMP4, FGF2, VEGF, and a ROCK inhibitor; or individually, any one of BMP4, FGF2, VEGF, and a ROCK inhibitor.

[0172] In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable concentration range therein. In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1 to 50 ng / ml.

[0173] In some embodiments, the BMP pathway activator is BMP4. In some embodiments, BMP4 is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable concentration range therein. In some embodiments, BMP4 is present in the differentiation medium at about 1 to 50 ng / ml.

[0174] In some embodiments, FGF2 is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, FGF2 is present in the differentiation medium at about 1 to 50 ng / ml.

[0175] In some embodiments, VEGF is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about 100 ng / ml, or any derivable concentration range therein. In some embodiments, VEGF is present in the differentiation medium at about 5 to 100 ng / ml.

[0176] In some embodiments, the ROCK inhibitor is present in the differentiation medium at a concentration of about 0.1 to 500 μΜ, about 1 to 250 μΜ, about 1 to 150 μΜ, about 5 to 100 μΜ, or about or about 0.1 μΜ, about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, about 10 μΜ, about 11 μΜ, about 12 μΜ, about 13 μΜ, about 14 μΜ, about 15 μΜ, about 16 μΜ, about 17 μΜ, about 18 μΜ, about 19 μΜ, about 20 μΜ, about 21 μΜ, about 22 μΜ, about 23 μΜ, about 24 μΜ, about 25 μΜ, about 26 μΜ, about 27 μΜ, about 28 μΜ, about 29 μΜ, about 30 μΜ, about 35 μΜ, about 40 μΜ, about 45 μΜ, about 50 μΜ, about 55 μΜ, about 60 μΜ, about 65 μΜ, about 70 μΜ, about 75 μΜ, about 80 μΜ, about 85 μΜ, about 90 μΜ, about 95 μΜ, or about 100 μΜ, or any derivable concentration range therein. In some embodiments, the ROCK inhibitor is present in the differentiation medium at about 0.1 to 20 μM.

[0177] In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, Y27632 is present at a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 100 μM, or 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 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, or about or about 100 μM, or any derivable range of concentrations therein. In some embodiments, Y27632 is present in the differentiation medium at about 0.1 to 100 μM.

[0178] In some embodiments, the mesoderm differentiation medium comprises a BMP pathway activator, an FGF, and a VEGF. In some embodiments, the mesoderm differentiation medium comprises a BMP pathway activator, an FGF, a VEGF, and a ROCK inhibitor. In some embodiments, the mesoderm differentiation medium comprises a defined xeno-free basal medium, a BMP pathway activator, an FGF, and a VEGF. In some embodiments, the mesoderm differentiation medium comprises a defined xeno-free basal medium, a BMP pathway activator, an FGF, a VEGF, and a ROCK inhibitor. In some embodiments, the mesoderm differentiation medium comprises BMP4, FGF2, and VEGF-165. In some embodiments, the mesoderm differentiation medium comprises BMP4, an FGF, VEGF-165, and a ROCK inhibitor. In some embodiments, the mesoderm differentiation medium comprises BMP4, an FGF, VEGF-165, and Y27632.

[0179] In some embodiments, the mesoderm differentiation medium comprises 1 to 50 ng / mL of BMP4, 1 to 50 ng / mL of FGF2, and 1 to 100 ng / mL of VEGF-165. In some embodiments, the mesoderm differentiation medium comprises 1 to 50 ng / mL of BMP4, 1 to 50 ng / mL of FGF, 1 to 100 ng / mL of VEGF-165, and 0.1 to 20 μM of a ROCK inhibitor. In some embodiments, the mesoderm differentiation medium comprises 1 to 50 ng / mL of BMP4, 1 to 50 ng / mL of FGF, 1 to 100 ng / mL of VEGF-165, and 0.1 to 20 μM of Y27632.

[0180] Hematopoietic progenitor differentiation medium In some embodiments, the present disclosure provides a differentiation medium for generating HP cells from mesoderm cells and embryoid body cells. In some embodiments, the mesoderm cells and embryoid body cells produced by the compositions and methods of the present disclosure are further differentiated into hematopoietic progenitors.

[0181] In some embodiments, to form differentiated NK cells, a population of HP cells is cultured with at least one exogenous factor. 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 pyrimido-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 a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and any combination thereof. In some embodiments, the exogenous factor is selected from a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, a pyrimido-indole derivative, an aryl hydrocarbon receptor antagonist, a TGF-β receptor inhibitor, and any combination thereof.

[0182] In some embodiments, the exogenous factors include a BMP pathway activator and FGF. In some embodiments, the exogenous factors include a BMP pathway activator and VEGF. In some embodiments, the exogenous factors include a BMP pathway activator and SCF. In some embodiments, the exogenous factors include a BMP pathway activator and TPO. In some embodiments, the exogenous factors include a BMP pathway activator and LDL. In some embodiments, the exogenous factors include a BMP pathway activator and a PI3K inhibitor. 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 a PI3K inhibitor. 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.

[0183] In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, and a VEGF. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, and an SCF. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, and a TPO. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, and an SCF. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, and a TPO. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, an SCF, and a TPO. In some embodiments, the exogenous factors include a BMP pathway activator, an SCF, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, an SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, a TPO, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, a TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, an LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include an FGF, a VEGF, and an SCF. In some embodiments, the exogenous factors include an FGF, a VEGF, and a TPO. In some embodiments, the exogenous factors include an FGF, a VEGF, and an LDL. In some embodiments, the exogenous factors include an FGF, a VEGF, and an LDL. In some embodiments, the exogenous factors include an FGF, an SCF, and a TPO. In some embodiments, the exogenous factors include an FGF, an SCF, and an LDL. In some embodiments, the exogenous factors include an FGF, an SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include an FGF, a TPO, and an LDL. In some embodiments, the exogenous factors include an FGF, a 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.

[0184] In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, a VEGF, and an SCF. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, a VEGF, and a TPO. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, a VEGF, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, a VEGF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, an SCF, and a TPO. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, an SCF, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, an SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, a TPO, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, a TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, an FGF, an LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, an SCF, and a TPO. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, an SCF, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, an SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, a TPO, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, a TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, a VEGF, an LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, an SCF, a TPO, and an LDL. In some embodiments, the exogenous factors include a BMP pathway activator, an SCF, a TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, an SCF, a TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, a TPO, an LDL, and a PI3K inhibitor.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 a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, TPO, and LDL. In some embodiments, the exogenous factors include FGF, VEGF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors include FGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, SCF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, TPO, LDL, and a PI3K inhibitor.

[0185] In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, and TPO. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, TPO, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, VEGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, VEGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, VEGF, SCF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, VEGF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, SCF, TPO, LDL, and a PI3K inhibitor. 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 a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL, and a PI3K inhibitor.In some embodiments, the exogenous factors include FGF, VEGF, TPO, LDL, and a PI3K inhibitor. 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 a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, SCF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include VEGF, SCF, TPO, LDL, and a PI3K inhibitor.

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

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

[0188] 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.

[0189] 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.

[0190] 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, 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.

[0191] 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, 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 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, 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, 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 LY294Q02.

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

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

[0194] In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable concentration range therein. In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1 to 50 ng / ml.

[0195] In some embodiments, the bone morphogenetic protein (BMP) activator is BMP4. In some embodiments, BMP4 is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable concentration range therein. In some embodiments, BMP4 is present in the differentiation medium at about 1 to 50 ng / ml.

[0196] In some embodiments, FGF2 is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, FGF2 is present in the differentiation medium at about 1 to 50 ng / ml.

[0197] In some embodiments, VEGF is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, VEGF is present in the differentiation medium at about 1 to 100 ng / ml.

[0198] In some embodiments, SCF is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, SCF is present in the differentiation medium at about 1 to 100 ng / ml.

[0199] In some embodiments, TPO is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, TPO is present in the differentiation medium at about 1 to 100 ng / ml.

[0200] In some embodiments, LDL is present in the differentiation medium at a concentration of about 0.1 to 500 μg / ml, about 1 to 250 μg / ml, about 1 to 150 μg / ml, about 5 to 100 μg / ml, or 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, about 20 μg / ml, about 21 μg / ml, about 22 μg / ml, about 23 μg / ml, about 24 μg / ml, about 25 μg / ml, about 26 μg / ml, about 27 μg / ml, about 28 μg / ml, about 29 μg / ml, about 30 μg / ml, about 35 μg / ml, about 40 μg / ml, about 45 μg / ml, about 50 μg / ml, about 55 μg / ml, about 60 μg / ml, about 65 μg / ml, about 70 μg / ml, about 75 μg / ml, about 80 μg / ml, about 85 μg / ml, about 90 μg / ml, about 95 μg / ml, or about 100 μg / ml, or any derivable concentration range therein. In some embodiments, LDL is present in the differentiation medium at about 1 to 50 μg / ml.

[0201] In some embodiments, the PI3K inhibitor is present in the differentiation medium at a concentration of about 0.1 to 500 μΜ, about 1 to 250 μΜ, about 1 to 150 μΜ, about 5 to 100 μΜ, or about or about 0.1 μΜ, about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, about 10 μΜ, about 11 μΜ, about 12 μΜ, about 13 μΜ, about 14 μΜ, about 15 μΜ, about 16 μΜ, about 17 μΜ, about 18 μΜ, about 19 μΜ, about 20 μΜ, about 21 μΜ, about 22 μΜ, about 23 μΜ, about 24 μΜ, about 25 μΜ, about 26 μΜ, about 27 μΜ, about 28 μΜ, about 29 μΜ, about 30 μΜ, about 35 μΜ, about 40 μΜ, about 45 μΜ, about 50 μΜ, about 55 μΜ, about 60 μΜ, about 65 μΜ, about 70 μΜ, about 75 μΜ, about 80 μΜ, about 85 μΜ, about 90 μΜ, about 95 μΜ, or about 100 μΜ, or any derivable concentration range therein. In some embodiments, the PI3K inhibitor is present in the differentiation medium at about 0.1 to 100 μM.

[0202] In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, LY294002 is present at a concentration of about 0.1 - 500 μM, about 1 - 250 μM, about 1 - 150 μM, about 5 - 100 μM, or 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 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, or about or 100 μM, or any derivable concentration range therein. In some embodiments, LY294002 is present in the differentiation medium at about 0.1 - 100 μM.

[0203] In some embodiments, the pyrimido - indole derivative is present in the differentiation medium at a concentration of about 0.1 - 500 μM, about 1 - 250 μM, about 1 - 150 μM, about 5 - 100 μM, or 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 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, or about or 100 μM, or any derivable concentration range therein. In some embodiments, the pyrimido - indole derivative is present in the differentiation medium at about 0.1 - 10 μM.

[0204] In some embodiments, the pyrimido-indole derivative is UM729. In some embodiments, UM729 is present at a concentration of about 0.1 to 500 μΜ, about 1 to 250 μΜ, about 1 to 150 μΜ, about 5 to 100 μΜ, or about or about 0.1 μΜ, about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, about 10 μΜ, about 11 μΜ, about 12 μΜ, about 13 μΜ, about 14 μΜ, about 15 μΜ, about 16 μΜ, about 17 μΜ, about 18 μΜ, about 19 μΜ, about 20 μΜ, about 21 μΜ, about 22 μΜ, about 23 μΜ, about 24 μΜ, about 25 μΜ, about 26 μΜ, about 27 μΜ, about 28 μΜ, about 29 μΜ, about 30 μΜ, about 35 μΜ, about 40 μΜ, about 45 μΜ, about 50 μΜ, about 55 μΜ, about 60 μΜ, about 65 μΜ, about 70 μΜ, about 75 μΜ, about 80 μΜ, about 85 μΜ, about 90 μΜ, about 95 μΜ, or about or 100 μΜ, or any derivable range of concentrations therein. In some embodiments, UM729 is present in the differentiation medium at about 0.1 to 10 μM.

[0205] In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at a concentration of about 0.1 to 500 μΜ, about 1 to 250 μΜ, about 1 to 150 μΜ, about 5 to 100 μΜ, or about or about 0.1 μΜ, about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, about 10 μΜ, about 11 μΜ, about 12 μΜ, about 13 μΜ, about 14 μΜ, about 15 μΜ, about 16 μΜ, about 17 μΜ, about 18 μΜ, about 19 μΜ, about 20 μΜ, about 21 μΜ, about 22 μΜ, about 23 μΜ, about 24 μΜ, about 25 μΜ, about 26 μΜ, about 27 μΜ, about 28 μΜ, about 29 μΜ, about 30 μΜ, about 35 μΜ, about 40 μΜ, about 45 μΜ, about 50 μΜ, about 55 μΜ, about 60 μΜ, about 65 μΜ, about 70 μΜ, about 75 μΜ, about 80 μΜ, about 85 μΜ, about 90 μΜ, about 95 μΜ, or about or 100 μΜ, or any derivable range of concentrations therein. In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at about 0.1 to 10 μM.

[0206] In some embodiments, the aryl hydrocarbon receptor antagonist is StemRegenin 1 (SR1). In some embodiments, SR1 is present at a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 100 μM, or 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 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, or about or about 100 μM, or any derivable range of concentrations therein. In some embodiments, SR1 is present in the differentiation medium at about 0.1 to 10 μM.

[0207] In some embodiments, the TGF-β receptor inhibitor is present in the differentiation medium at a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 100 μM, or 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 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, or about or about 100 μM, or any derivable range of concentrations therein. In some embodiments, the TGF-β receptor inhibitor is present in the differentiation medium at about 0.1 to 20 μM.

[0208] In some embodiments, the TGF-β receptor inhibitor is GW788388. In some embodiments, GW788388 is present in the differentiation medium at a concentration of about 0.1 - 500 μM, about 1 - 250 μM, about 1 - 150 μM, about 5 - 100 μM, or 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 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, or about 100 μM, or any derivable concentration range therein. In some embodiments, GW788388 is present in the differentiation medium at about 0.1 - 20 μM.

[0209] In some embodiments, the TGF-β receptor inhibitor is SB431542. In some embodiments, GW788388 is present in the differentiation medium at a concentration of about 0.1 - 500 μM, about 1 - 250 μM, about 1 - 150 μM, about 5 - 100 μM, or 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 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, or about 100 μM, or any derivable concentration range therein. In some embodiments, SB431542 is present in the differentiation medium at about 0.1 - 20 μM.

[0210] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, an FGF, and a VEGF. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, an FGF, a 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, an FGF, VEGF-165, and a ROCK inhibitor. In some embodiments, the HP differentiation medium comprises BMP4, an FGF, VEGF-165, and Y27632.

[0211] In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL of BMP4, 5 to 50 ng / mL of FGF2, and 1 to 100 ng / mL of VEGF-165. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL of BMP4, 1 to 50 ng / mL of an FGF, 1 to 100 ng / mL of VEGF-165, and 1 to 20 μM of a ROCK inhibitor. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL of BMP4, 1 to 50 ng / mL of an FGF, 1 to 100 ng / mL of VEGF-165, and 1 to 20 μM of Y27632.

[0212] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, an FGF, a VEGF, and a pyrimido-indole derivative. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, an FGF, a VEGF, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, an FGF, a VEGF, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0213] In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and a pyrimido-indole derivative. 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 pyrimido-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.

[0214] In some embodiments, the HP differentiation medium comprises 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF, 1-100 ng / mL of VEGF-165, and 0.1-10 μM of UM729. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF, 1-100 ng / mL of VEGF-165, and 0.1-10 μM of SR1. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF, 1-100 ng / mL of VEGF-165, 0.1-10 μM of UM729, and 0.1-10 μM of SR1.

[0215] 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 pyrimido-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 pyrimido-indole derivative, an aryl hydrocarbon receptor antagonist, and a TGF-β receptor inhibitor.

[0216] 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.

[0217] In some embodiments, the HP differentiation medium comprises 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF, 1-100 ng / mL of VEGF-165, and 0.1-20 μM of a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF, 1-100 ng / mL of VEGF-165, and 0.1-20 μM of GW788388. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF, 1-100 ng / mL of VEGF-165, 0.1-10 μM of UM729, and 0.1-20 μM of GW788388. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF, 1-100 ng / mL of VEGF-165, 0.1-10 μM of SR1, and 0.1-20 μM of GW788388. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF, 1-100 ng / mL of VEGF-165, 0.1-10 μM of UM729, 0.1-10 μM of SR1, and 0.1-20 μM of GW788388.

[0218] 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.

[0219] In some embodiments, the HP differentiation medium comprises 1 - 50 ng / mL of BMP4, 1 - 50 ng / mL of FGF, 1 - 100 ng / mL of VEGF-165, and 0.1 - 20 μM of a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises 1 - 50 ng / mL of BMP4, 1 - 50 ng / mL of FGF, 1 - 100 ng / mL of VEGF-165, and 0.1 - 20 μM of SB431542. In some embodiments, the HP differentiation medium comprises 1 - 50 ng / mL of BMP4, 1 - 50 ng / mL of FGF, 1 - 100 ng / mL of VEGF-165, 0.1 - 10 μM of UM729, and 0.1 - 20 μM of SB431542. In some embodiments, the HP differentiation medium comprises 1 - 50 ng / mL of BMP4, 1 - 50 ng / mL of FGF, 1 - 100 ng / mL of VEGF-165, 0.1 - 10 μM of SR1, and 0.1 - 20 μM of SB431542. In some embodiments, the HP differentiation medium comprises 1 - 50 ng / mL of BMP4, 1 - 50 ng / mL of FGF, 1 - 100 ng / mL of VEGF-165, 0.1 - 10 μM of UM729, 0.1 - 10 μM of SR1, and 0.1 - 20 μM of SB431542.

[0220] NK cell differentiation medium In some embodiments, the present disclosure provides a differentiation medium for generating NK cells from HP cells. In some embodiments, NK cells are generated from HP cells. In some embodiments, HP cells produced by the compositions and methods of the present disclosure are further differentiated into NK cells.

[0221] In some embodiments, to form differentiated NK cells, a population of HP cells is cultured with at least one exogenous factor. 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 pyrimido-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, pyrimido-indole derivatives, and aryl hydrocarbon receptor antagonists.

[0222] In some embodiments, the exogenous factor includes SCF and IL-7. In some embodiments, the exogenous factor includes SCF and IL-15. In some embodiments, the exogenous factor includes SCF and IL-12. In some embodiments, the exogenous factor includes SCF and FLT3L. In some embodiments, the exogenous factor includes SCF and a pyrimido-indole derivative. In some embodiments, the exogenous factor includes SCF and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor includes IL-7 and IL-15. In some embodiments, the exogenous factor includes IL-7 and IL-12. In some embodiments, the exogenous factor includes IL-7 and FLT3L. In some embodiments, the exogenous factor includes IL-7 and a pyrimido-indole derivative. In some embodiments, the exogenous factor includes IL-7 and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor includes IL-15 and IL-12. In some embodiments, the exogenous factor includes IL-15 and FLT3L. In some embodiments, the exogenous factor includes IL-15 and a pyrimido-indole derivative. In some embodiments, the exogenous factor includes IL-15 and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor includes IL-12 and FLT3L. In some embodiments, the exogenous factor includes IL-12 and a pyrimido-indole derivative. In some embodiments, the exogenous factor includes IL-12 and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor includes FLT3L and a pyrimido-indole derivative. In some embodiments, the exogenous factor includes FLT3L and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor includes a pyrimido-indole derivative and an aryl hydrocarbon receptor antagonist.

[0223] 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, and an aryl hydrocarbon receptor antagonist. 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-12, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-15, and FLT3L. In some embodiments, the exogenous factors include SCF, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include IL-7, IL-12, and a pyrimido-indole derivative. 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 a pyrimido-indole derivative.In some embodiments, the exogenous factor comprises IL-7, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-7, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises IL-7, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-7, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factor comprises IL-12, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-12, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist.

[0224] 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, and an aryl hydrocarbon receptor antagonist. 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-12, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist.In some embodiments, the exogenous factor comprises SCF, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises SCF, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-7, IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factor comprises IL-7, IL-12, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises IL-7, IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-7, IL-12, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises IL-7, IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-7, IL-12, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-7, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises IL-7, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-7, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-7, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0225] 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-12, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0226] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-7, and an aryl hydrocarbon receptor antagonist. 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 a pyrimido-indole derivative. 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, IL-15, and SR1. In some embodiments, the exogenous factors include SCF, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors include SCF, FLT3L, and SR1. In some embodiments, the exogenous factors include SCF, a pyrimido-indole derivative, 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include IL-7, IL-12, and a pyrimido-indole derivative. 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 a pyrimido-indole derivative. In some embodiments, the exogenous factors include IL-7, IL-15, and SR1. In some embodiments, the exogenous factors include IL-7, FLT3L, and a pyrimido-indole derivative.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.

[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 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, 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, UM729, and SR1. In some embodiments, the exogenous factors include IL-7, FLT3L, UM729, and SR1.

[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 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, 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, UM729, and SR1. In some embodiments, the exogenous factors include SCF, IL- twelve, FLT3L, and 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 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.

[0230] 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, 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-7, 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.

[0231] In some embodiments, SCF is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, SCF is present in the differentiation medium at about 1 to 50 ng / ml.

[0232] In some embodiments, IL-7 is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, IL-7 is present in the differentiation medium at about 1 to 50 ng / ml.

[0233] In some embodiments, IL-12 is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, IL-12 is present in the differentiation medium at about 1 to 100 ng / ml.

[0234] In some embodiments, IL-15 is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, IL-15 is present in the differentiation medium at about 1 to 100 ng / ml.

[0235] In some embodiments, FLT3L is present in the differentiation medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about 100 ng / ml, or any derivable concentration range therein. In some embodiments, FLT3L is present in the differentiation medium at about 1 to 100 ng / ml.

[0236] In some embodiments, the pyrimido-indole derivative is present in the differentiation medium at a concentration of about 0.1 to 500 μΜ, about 1 to 250 μΜ, about 1 to 150 μΜ, about 5 to 100 μΜ, or about or about 0.1 μΜ, about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, about 10 μΜ, about 11 μΜ, about 12 μΜ, about 13 μΜ, about 14 μΜ, about 15 μΜ, about 16 μΜ, about 17 μΜ, about 18 μΜ, about 19 μΜ, about 20 μΜ, about 21 μΜ, about 22 μΜ, about 23 μΜ, about 24 μΜ, about 25 μΜ, about 26 μΜ, about 27 μΜ, about 28 μΜ, about 29 μΜ, about 30 μΜ, about 35 μΜ, about 40 μΜ, about 45 μΜ, about 50 μΜ, about 55 μΜ, about 60 μΜ, about 65 μΜ, about 70 μΜ, about 75 μΜ, about 80 μΜ, about 85 μΜ, about 90 μΜ, about 95 μΜ, or about 100 μΜ, or any derivable concentration range therein. In some embodiments, the pyrimido-indole derivative is present in the differentiation medium at about 0.1 to 10 μM.

[0237] In some embodiments, the pyrimido-indole derivative is UM729. In some embodiments, UM729 is present at a concentration of about 0.1 - 500 μM, about 1 - 250 μM, about 1 - 150 μM, about 5 - 100 μM, or 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 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, or about or about 100 μM, or any derivable concentration range therein. In some embodiments, UM729 is present in the differentiation medium at about 0.1 - 10 μM.

[0238] In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at a concentration of about 0.1 - 500 μM, about 1 - 250 μM, about 1 - 150 μM, about 5 - 100 μM, or 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 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, or about or about 100 μM, or any derivable concentration range therein. In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at about 0.1 - 10 μM.

[0239] In some embodiments, the aryl hydrocarbon receptor antagonist is StemRegenin 1 (SR1). In some embodiments, SR1 is present at a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 100 μM, or 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 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, or about or about 100 μM, or any derivable range of concentrations therein. In some embodiments, StemRegenin 1 (SR1) is present in the differentiation medium at about 0.1 to 10 μM.

[0240] 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 a pyrimido-indole derivative. 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, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0241] 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.

[0242] In some embodiments, the NK cell differentiation medium comprises 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, and 1 - 100 ng / ml of FLT3L. In some embodiments, the NK cell differentiation medium comprises 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, and 0.1 - 10 μM of a pyrimido-indole derivative. In some embodiments, the NK cell differentiation medium comprises 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, and 0.1 - 10 μM of an aryl hydrocarbon receptor antagonist. In some embodiments, the NK cell differentiation medium comprises 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, 0.1 - 10 μM of a pyrimido-indole derivative, and 0.1 - 10 μM of an aryl hydrocarbon receptor antagonist.

[0243] In some embodiments, the NK cell differentiation medium comprises 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, and SR1. In some embodiments, the NK cell differentiation medium comprises 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, and 0.1 - 10 μM of UM729. In some embodiments, the NK cell differentiation medium comprises 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, 1 - 10 μM of SR1, and 0.1 - 10 μM of UM729.

[0244] In some embodiments, the NK cell differentiation medium comprises a xeno-free basal medium, 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, and 0.1 - 10 μM of SR1. In some embodiments, the NK cell differentiation medium comprises a xeno-free basal medium, 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, and 0.1 - 10 μM of UM729. In some embodiments, the NK cell differentiation medium comprises a xeno-free basal medium, 1 - 50 ng / ml of SCF, 1 - 50 ng / ml of IL-7, 1 - 100 ng / ml of IL-12, 1 - 100 ng / ml of IL-15, 1 - 100 ng / ml of FLT3L, 0.1 - 10 μM of SR1, and 0.1 - 10 μM of UM729.

[0245] NK cell expansion medium In some embodiments, the present disclosure provides an expansion medium for generating mature NK cells from differentiated NK cells. In some embodiments, the differentiated NK cells are generated from 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.

[0246] In some embodiments, to form mature NK cells, a population of differentiated NK cells is cultured with at least one exogenous factor. In some embodiments, the exogenous factor is IL-2. In some embodiments, the exogenous factor is 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 activated beads. In some embodiments, the exogenous factor is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and activated beads.

[0247] In some embodiments, the exogenous factor comprises IL-2 and IL-7. In some embodiments, the exogenous factor comprises IL-2 and IL-12. In some embodiments, the exogenous factor comprises IL-2 and IL-15. In some embodiments, the exogenous factor comprises IL-2 and IL-18. In some embodiments, the exogenous factor comprises IL-2 and activated beads. In some embodiments, the exogenous factor comprises IL-7 and IL-12. In some embodiments, the exogenous factor comprises IL-7 and IL-15. In some embodiments, the exogenous factor comprises IL-7 and IL-18. In some embodiments, the exogenous factor comprises IL-7 and activated beads. In some embodiments, the exogenous factor comprises IL-12 and IL-15. In some embodiments, the exogenous factor comprises IL-12 and IL-18. In some embodiments, the exogenous factor comprises IL-12 and activated beads. In some embodiments, the exogenous factor comprises IL-15 and IL-18. In some embodiments, the exogenous factor comprises IL-15 and activated beads. In some embodiments, the exogenous factor comprises IL-18 and activated beads.

[0248] 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 activated 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 activated 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 activated beads. In some embodiments, the exogenous factors include IL-2, IL-18, and activated 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 activated 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 activated beads. In some embodiments, the exogenous factors include IL-7, IL-18, and activated 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 activated beads. In some embodiments, the exogenous factors include IL-12, IL-18, and activated beads. In some embodiments, the exogenous factors include IL-15, IL-18, and activated beads.

[0249] 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. 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. In some embodiments, the exogenous factors include IL-2, IL-7, IL-18, and activated 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 activated beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-18, and activated beads. In some embodiments, the exogenous factors include IL-2, IL-15, IL-18, and activated 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 activated beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-18, and activated beads. In some embodiments, the exogenous factors include IL-7, IL-15, IL-18, and activated beads. In some embodiments, the exogenous factors include IL-12, IL-15, IL-18, and activated beads.

[0250] In some embodiments, the exogenous factor includes IL-2, IL-7, IL-12, IL-15, and IL-18. In some embodiments, the exogenous factor includes IL-2, IL-7, IL-12, IL-15, and activated beads. In some embodiments, the exogenous factor includes IL-2, IL-7, IL-12, IL-18, and activated beads. In some embodiments, the exogenous factor includes IL-2, IL-7, IL-15, IL-18, and activated beads. In some embodiments, the exogenous factor includes IL-2, IL-12, IL-15, IL-18, and activated beads. In some embodiments, the exogenous factor includes IL-7, IL-12, IL-15, IL-18, and activated beads.

[0251] In some embodiments, the exogenous factor includes IL-2, IL-7, IL-12, IL-15, IL-18, and activated beads.

[0252] 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.

[0253] 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 activated 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 activated 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 activated 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 activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12, IL-18, and activated beads coated with anti-CD2 / anti-NKp46.In some embodiments, the exogenous factors include IL-15, IL-18, and activated beads coated with anti-CD2 / anti-NKp46.

[0254] 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-!2, 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 activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12, IL-15, IL-18, and activated beads coated with anti-CD2 / anti-NKp46.

[0255] 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-15, 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.

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

[0257] In some embodiments, IL-2 is present in the expansion growth medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, IL-2 is present in the expansion growth medium at about 1 to 50 ng / ml.

[0258] In some embodiments, IL-7 is present in the expansion growth medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, IL-7 is present in the expansion growth medium at about 1 to 50 ng / ml.

[0259] In some embodiments, IL-12 is present in the expansion growth medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, IL-12 is present in the expansion growth medium at about 1 to 100 ng / ml.

[0260] In some embodiments, IL-15 is present in the expansion growth medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, IL-15 is present in the expansion growth medium at about 1 to 100 ng / ml.

[0261] In some embodiments, IL-18 is present in the expansion culture medium at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, or 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 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about or 100 ng / ml, or any derivable range of concentrations therein. In some embodiments, IL-18 is present in the expansion culture medium at about 1 to 100 ng / ml.

[0262] In some embodiments, activated beads are present in the expansion culture medium.

[0263] In some embodiments, activated beads coated with anti-CD2 / anti-NKp46 are present in the expansion culture medium at a ratio based on the number of differentiated NK cells. For example, there is one activated bead per NK cell, i.e., a 1:1 activated bead to NK cell ratio.

[0264] 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.

[0265] In some embodiments, the ratio of activated beads to NK cells is from 1:1 to 2:1, from 2:1 to 3:1, from 3:1 to 4:1, from 4:1 to 5:1, from 5:1 to 6:1, from 6:1 to 7:1, from 7:1 to 8:1, from 8:1 to 9:1, from 9:1 to 10:1, from 10:1 to 15:1, from 15:1 to 20:1, from 20:1 to 25:1, from 25:1 to 30:1, from 30:1 to 35:1, from 35:1 to 40:1, from 40:1 to 45:1, or from 45:1 to 50:1.

[0266] 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.

[0267] In some embodiments, the ratio of NK cells to activated beads is from 1:1 to 2:1, from 2:1 to 3:1, from 3:1 to 4:1, from 4:1 to 5:1, from 5:1 to 6:1, from 6:1 to 7:1, from 7:1 to 8:1, from 8:1 to 9:1, from 9:1 to 10:1, from 10:1 to 15:1, from 15:1 to 20:1, from 20:1 to 25:1, from 25:1 to 30:1, from 30:1 to 35:1, from 35:1 to 40:1, from 40:1 to 45:1, or from 45:1 to 50:1.

[0268] In some embodiments, the NK cell expansion and proliferation medium comprises IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads. In some embodiments, the NK cell expansion and proliferation medium comprises a xeno-free basal medium and IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads.

[0269] In some embodiments, the NK cell expansion and proliferation 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 and proliferation medium comprises a xeno-free basal medium and IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

[0270] In some embodiments, the NK cell expansion and proliferation medium comprises 1 to 50 ng / ml of IL-2, 1 to 50 ng / ml of IL-7, 1 to 100 ng / ml of IL-12, 1 to 100 ng / ml of IL-15, 1 to 100 ng / ml of IL-18, and activation beads at a cell:bead ratio of 1:1. In some embodiments, the NK cell expansion and proliferation medium comprises a xeno-free basal medium and 1 to 50 ng / ml of IL-2, 1 to 50 ng / ml of IL-7, 1 to 100 ng / ml of IL-12, 1 to 100 ng / ml of IL-15, 1 to 100 ng / ml of IL-18, and activation beads at a cell:bead ratio of 1:1.

[0271] In some embodiments, the NK cell expansion and proliferation medium comprises IL-2 at 1-50 ng / ml, IL-7 at 1-50 ng / ml, IL-12 at 1-100 ng / ml, IL-15 at 1-100 ng / ml, IL-18 at 1-100 ng / ml, and activation beads coated with anti-CD2 / anti-NKp46 at a cell:bead ratio of 1:1. In some embodiments, the NK cell expansion and proliferation medium comprises a xeno-free basal medium and IL-2 at 1-50 ng / ml, IL-7 at 1-50 ng / ml, IL-12 at 1-100 ng / ml, IL-15 at 1-100 ng / ml, IL-18 at 1-100 ng / ml, and activation beads coated with anti-CD2 / anti-NKp46 at a cell:bead ratio of 1:1.

[0272] Differentiation method In some aspects, the present disclosure provides a method for generating hematopoietic precursors from stem cells. In some aspects, the present disclosure provides a method for generating NK cells from stem cells. In some aspects, the present disclosure provides a method for generating NK cells from hematopoietic precursors. In some embodiments, the method for generating NK cells comprises differentiating stem cells into hematopoietic precursors and differentiating the hematopoietic precursors into NK cells.

[0273] In some aspects, the present disclosure provides a method for generating common lymphoid progenitors (CLPs) from stem cells. In some embodiments, the method comprises differentiating stem cells into hematopoietic precursors and differentiating the hematopoietic precursors into CLPs. A CLP refers to a cell that is a precursor of lymphoid cells. A CLP is a cell capable of hematopoietic conversion into hematopoietic cell types. In some embodiments, a CLP is CD45+CD7+CD5+ / lo CD3-CD56-. In some embodiments, a CLP is CD45+CD5+ / lo CD7+. In some aspects, the present disclosure provides a method for generating NK cells from CLPs. In some embodiments, the method for generating NK cells comprises differentiating stem cells into hematopoietic precursors, differentiating the hematopoietic precursors into CLPs, and differentiating the CLPs into NK cells.

[0274] In some aspects, the present disclosure provides a method for generating pre-NK cell precursors (pre-NKPs) from stem cells. In some embodiments, the method includes differentiating a stem cell into a hematopoietic precursor, differentiating the hematopoietic precursor into a CLP, and differentiating the CLP into a pre-NKP. A pre-NKP is an intermediate cell between a CLP and an NKP. In some embodiments, the pre-NKP is Lin- / CD244+ / c-Kit low / IL-7Ra+ / FLT3- / CD122-. In some aspects, the present disclosure provides a method for generating NK cells from pre-NKPs. In some embodiments, the method for generating NK cells includes differentiating a stem cell into a hematopoietic precursor, differentiating the hematopoietic precursor into a CLP, differentiating the CLP into a pre-NKP, and differentiating the pre-NKP into an NK cell.

[0275] In some aspects, the present disclosure provides a method for generating NK cell precursors (NKPs) from stem cells. In some embodiments, the method includes differentiating a stem cell into a hematopoietic precursor, differentiating the hematopoietic precursor into a CLP, differentiating the CLP into a pre-NKP, and differentiating the pre-NKP into an NKP. An NKP is the last cell before the final NK differentiation lineage determination. In some embodiments, the NKP is Lin- / NK1.1-DX5- / IL-7Ra+ / CD122+ / NKG2D+. In some aspects, the present disclosure provides a method for generating NK cells from NKPs. In some embodiments, the method for generating NK cells includes differentiating a stem cell into a hematopoietic precursor, differentiating the hematopoietic precursor into a CLP, differentiating the CLP into a pre-NKP, differentiating the pre-NKP into an NKP, and differentiating the NKP into an NK cell.

[0276] In some aspects, the present disclosure provides a method for generating immature NK (iNK) cells from stem cells. In some embodiments, the method includes differentiating a stem cell into a hematopoietic progenitor, differentiating the hematopoietic progenitor into a CLP, differentiating the CLP into a pre-NKP, differentiating the pre-NKP into an NKP, and differentiating the NKP into an iNK cell. In some aspects, the present disclosure provides a method for generating NK cells from iNK cells. In some embodiments, the method for generating NK cells includes differentiating a stem cell into a hematopoietic progenitor, differentiating the hematopoietic progenitor into a CLP, differentiating the CLP into a pre-NKP, differentiating the pre-NKP into an NKP, differentiating the NKP into an iNK cell, and differentiating the iNK cell into an NK cell.

[0277] In some aspects, the present disclosure provides a method for generating mature NK (mNK) cells from stem cells. In some aspects, the present disclosure provides a method for generating mature NK cells from immature NK cells. In some embodiments, the method for generating NK cells includes differentiating a stem cell into a hematopoietic progenitor, differentiating the hematopoietic progenitor into a CLP, differentiating the CLP into a pre-NKP, differentiating the pre-NKP into an NKP, differentiating the NKP into an iNK cell, and differentiating the iNK cell into an mNK cell.

[0278] In some embodiments, the methods provided herein are xeno-free. In some embodiments, the methods provided herein do not include animal-derived materials.

[0279] Expression marker Differentiation of source cells into NK cells can be evaluated, for example, by detecting markers, such as CD56, CD94, CD117, NKG2D, DNAM-1, and NKp46, by flow cytometry. Differentiation can also be evaluated by morphological features of NK cells, such as large size, high protein synthesis activity in the abundant endoplasmic reticulum (ER), and / or pre-formed granules. Maturation of NK cells can be evaluated by detecting one or more functionally related makers, such as CD94, CD161, NKp44, DNAM-1, 2B4, NKp46, CD94, KIR, and the NKG2 family of activating receptors (e.g., NKG2D). Maturation of NK cells can also be evaluated by detecting specific markers during various developmental stages. For example, in one aspect, pre-NKP cells are CD34+, CD45RA+, CD10+, CD117-, and / or CD161-. In another aspect, immature NK cells are CD34-, CD117+, CD161+, NKp46-, and / or CD94 / NKG2A-. In another aspect, CD56bright NK cells are CD117+, NKp46+, CD94 / NKG2A+, CD16-, and / or KIR+ / - . In another aspect, CD56dim NK cells are CD117-, NKp46+, CD94 / NKG2A+ / - , CD16+, and / or KIR+ . In a specific aspect, maturation of NK cells (e.g., TSNK cells) is determined by the percentage of NK cells (e.g., TSNK cells) that are CD161-, CD94+, and / or NKp46+ . In a more specific aspect, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70% of mature NK cells (e.g., TSNK cells) are NKp46+ . In another more specific aspect, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% of mature NK cells (e.g., TSNK cells) are CD94+ .In another more specific embodiment, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% of the mature NK cells (e.g., TSNK cells) are CD161-.

[0280] In some embodiments, the differentiation of the source cells into NK cells is evaluated, for example, by using an antibody against one or more of these cell markers to detect the expression levels of, for example, CD3, CD7, or CD127, CD10, CD14, CD15, CD16, CD33, CD34, CD56, CD94, CD117, CD161, NKp44, NKp46, NKG2D, DNAM-1, 2B4, or TO-PRO-3. Such antibodies can be conjugated to a detectable label, such as a fluorescent label, such as FITC, R-PE, PerCP, PerCP-Cy5.5, APC, APC-Cy7, or APC-H7.

[0281] Source cells In some embodiments, the NK cells are generated from source cells. Any precursor cell known in the art can be used as the source cell in the methods of the present disclosure.

[0282] In some embodiments, the source cell is an hESC. In some embodiments, the source cell is an iPSC. NK cells derived from iPSCs can be alternatively referred to as iPSC-derived NK cells.

[0283] In immunotherapy, the source cells are allogeneic or autologous, which means they are derived from a donor or the subject, respectively. In some embodiments, the source cells are allogeneic. In some embodiments, the source cells are autologous.

[0284] In some embodiments, the source cells are peripheral blood cells. As used herein, the term "peripheral blood cells" is used to refer to cells derived from circulating blood and including hematopoietic stem cells capable of proliferation, selectable differentiation, and maturation. Thus, peripheral blood NK cells may also be alternatively referred to as differentiated blood-derived NK cells (bdNK).

[0285] In some embodiments, the source cells include hematopoietic stem cells characterized by being CD34+ and / or CD45+.

[0286] In some embodiments, the source cells include common lymphoid progenitor cells characterized by being CD45+CD7+CD56-.

[0287] In some embodiments, NK cells can be generated from induced pluripotent stem cells (iPSCs). iPSCs are a type of pluripotent stem cells derived from adult somatic cells that have been genetically reprogrammed into an embryonic stem cell-like state by forced expression of genes and factors important for maintaining the distinct characteristics of embryonic stem cells. iPSCs can be generated from tissues having somatic cells, including, without limitation, skin, dental tissue, peripheral blood, and urine. To generate iPSCs, somatic cells can be reprogrammed by, without limitation, transient expression of reprogramming factors, virus-free methods, adenoviruses, plasmids, minicircle vectors, episomal vectors, Sendai virus, synthetic mRNA, self-replicating RNA, retroviruses, lentiviruses, PhiC31 integrase, excisable transposons, CRISPR-based gene editing, or methods involving recombinant proteins. Methods for generating iPSCs are disclosed in U.S. Patent No. 9,315,779, U.S. Patent No. 10,370,452, U.S. Patent No. 11,319,555, and U.S. Patent Application Publication No. 2021 / 0015859, which are incorporated herein by reference in their entireties.

[0288] Mesoderm / embryoid body formation In some embodiments, the methods described herein include the step of generating mesodermal cells from iPSCs and / or hESCs. When the stem cells begin to differentiate, three different germ layers are formed: the ectoderm, mesoderm, and endoderm. Immune cells, such as NK cells, differentiate from mesodermal cells. In some embodiments, the mesodermal cells produced by the methods of the disclosure are further differentiated into NK cells.

[0289] The mesoderm formation step can include contacting a cell population of iPSCs or hESCs with one or more factors for a specified period of time in a limited expansion growth medium. In some embodiments, the mesodermal cells are formed from embryoid bodies.

[0290] In some embodiments, the stem cells are contacted with the differentiation medium described herein for a period of time to generate mesodermal cells and / or embryoid bodies. In some embodiments, a period of time sufficient to generate mesodermal cells from the stem cells is at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

[0291] In some embodiments, the mesoderm formation step has a duration of 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.

[0292] In some embodiments, cells derived from a source cell are placed in a container to induce the cells to aggregate and form clusters. In some embodiments, the container is a plate having wells or microwells, such as a 96-well plate and / or an Aggrewell™ plate (microwell plate; STEMCELL Technologies Inc., Vancouver, Canada). In some embodiments, for example, an Aggrewell™ plate is used to prepare cell clusters in a plate having microwells to form aggregates of cells of uniform size and shape. In some embodiments, at least 1 cell, at least 10 cells, at least 100 cells, at least 1,000 cells, at least 10,000 cells, or at least 50,000 cells are seeded into each well. In some embodiments, 1 cell to 10 cells, 10 cells to 100 cells, 100 cells to 1,000 cells, 1,000 cells to 10,000 cells, or 10,000 cells to 50,000 cells are seeded into each well.

[0293] Differentiation into hematopoietic precursors In some aspects, the present disclosure provides a method of generating NK cells from hematopoietic progenitor cells. In some embodiments, the method of generating NK cells includes differentiating hematopoietic precursors into NK cells. In some embodiments, the methods provided herein are xeno-free.

[0294] One aspect of the present disclosure is that a method of producing NK cells can include a hematopoietic precursor differentiation step. The hematopoietic precursor differentiation step can include contacting an embryoid body cell population with one or more factors for a specified period of time in a defined differentiation medium, thereby inducing the formation of hematopoietic precursors within the cell population. The hematopoietic precursors are then defined by expressing a combination of markers.

[0295] In some embodiments, mesoderm and / or embryoid body cells are contacted with the differentiation medium described herein for a period of time to generate hematopoietic progenitor cells. In some embodiments, a period of time sufficient to generate 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.

[0296] In some embodiments, the differentiation process into hematopoietic progenitors has a duration of 1 to 2 days, 2 to 3 days, 3 to 4 days, 4 to 5 days, 5 to 6 days, 6 to 7 days, 7 to 8 days, 8 to 9 days, 9 to 10 days, 10 to 11 days, 11 to 12 days, 12 to 13 days, 13 to 14 days, 14 to 15 days, 15 to 16 days, 16 to 17 days, 17 to 18 days, 18 to 19 days, or 19 to 20 days.

[0297] In some embodiments, hematopoietic progenitor cells express CD34, CD43 and CD45. In some embodiments, the methods of the disclosure increase the proportion of CD34+CD43+CD45+ triple positive cells.

[0298] In some embodiments, the methods of the disclosure generate a population of cells from iPSCs with a purity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% CD34+CD43+CD45+ triple positive cells.

[0299] In some embodiments, the method of the present disclosure generates a population of cells from iPSCs with a purity of 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% CD34+CD43+CD45+ triple-positive cells.

[0300] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of hematopoietic precursors (HP) from source cells (SC), such as hESCs or iPSCs. In some embodiments, the ratio of HP to source cell (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.

[0301] 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.

[0302] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of HP / SC. For example, the methods and compositions increase HP / SC from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure increase the yield ratio 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. 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.

[0303] In some embodiments, the yield ratio of hepatocytes (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 from 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.

[0304] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of hepatocytes (HP) to stem cells (StC) (HP / StC). For example, the methods and compositions increase HP / StC from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure 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. In some embodiments, the yield ratio of HP / StC is from 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.

[0305] In some embodiments, the yield ratio of hepatocytes (HP) to induced pluripotent stem cells (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 from 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 increase the yield ratio of HP to iPSC (HP / iPSC). For example, the methods and compositions increase HP to iPSC (HP / iPSC) from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure increase 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. In some embodiments, the yield ratio of HP to iPSC (HP / iPSC) is from 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 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 from 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 increase the yield ratio of HP to hESC (HP / hESC). For example, the methods and compositions increase HP to hESC (HP / hESC) from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure 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. In some embodiments, the yield ratio of HP to hESC (HP / hESC) is from 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] Differentiation into NK cells In some aspects, the present disclosure provides a method of generating NK cells from differentiated NK cells. In some embodiments, the method of generating NK cells includes the step of differentiating NK cells. In some embodiments, the methods provided herein are xeno-free.

[0310] One aspect of the present disclosure is that the method of producing NK cells can include an NK differentiation step. The NK differentiation step can include contacting a HP cell population with one or more factors in a defined differentiation medium for a specified period of time, thereby inducing the formation of NK cells within the cell population. The NK cells are then defined by expressing a combination of markers.

[0311] In some embodiments, hematopoietic progenitor cells are contacted with the differentiation media described herein for a period of time to generate NK cells. In some embodiments, a period of time sufficient to generate 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.

[0312] In some embodiments, the NK differentiation process has a duration of 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, 20 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.

[0313] In some embodiments, the differentiated NK cells include the markers CD34, CD43, CD45, and LFA1. In some embodiments, the method of the present disclosure increases the proportion of CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0314] In some embodiments, the method of the present disclosure generates a population of NK cells from hematopoietic progenitor cells with 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+ quadruple positive cells.

[0315] In some embodiments, the method of the present disclosure generates a population of NK cells from hematopoietic progenitor cells with 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.

[0316] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of NK cells from a source cell (SC), such as hESC or iPSC. In some embodiments, the ratio of NK to source cell (SC) is denoted as NK / SC. In some embodiments, the ratio of NK to hESC is denoted as NK / hESC. In some embodiments, the ratio of NK to iPSC is denoted as NK / iPSC.

[0317] 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 from 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.

[0318] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of NK / SC. For example, the methods and compositions increase the NK / SC from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure 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. In some embodiments, the yield ratio of NK / SC is from 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.

[0319] In some embodiments, the yield ratio of NK cells to stem cells (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 from 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.

[0320] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of NK / StC. For example, the methods and compositions increase NK / StC from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure 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. In some embodiments, the yield ratio of NK / StC is from 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.

[0321] In some embodiments, the yield ratio of NK cells to hESCs (NK / 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, 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 from 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 / hESCs. For example, the methods and compositions increase the NK / hESC yield ratio from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of NK / hESCs 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. In some embodiments, the yield ratio of NK / hESCs is from 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 yield ratio of NK cells to iPSCs (NK / 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, 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 / iPSCs is from 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 aspects, the methods and compositions of the present disclosure increase the NK / iPSC yield ratio. For example, the methods and compositions increase the NK / iPSC from about 2:1 to about 4:1, or by about 2. In some aspects, the methods and compositions of the present disclosure increase the NK / iPSC yield ratio 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. In some aspects, the NK / iPSC yield ratio is from 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] NK cell maturation In some aspects, the present disclosure provides a method for generating mature NK cells from differentiated NK cells. In some aspects, the method for generating mature NK cells includes the step of differentiating NK cells. In some aspects, the methods provided herein are xeno-free.

[0326] One aspect of the present disclosure is that the method of producing NK cells can include an NK maturation step. The NK maturation step can include contacting a differentiated NK cell population with one or more factors for a specified period of time in a limited expansion growth medium, thereby inducing NK cell maturation within the cell population. Mature NK cells are then defined by expressing a combination of markers.

[0327] In some embodiments, the differentiated NK cells are contacted with the maturation medium described herein for a period of time to generate mature NK cells. In some embodiments, a period of time sufficient to generate 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.

[0328] In some embodiments, the maturation process has a duration of 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.

[0329] In some embodiments, the mature NK cells include the markers CD34, CD43, CD45, and LFA1. In some embodiments, the methods of the disclosure increase the proportion of CD34+CD43+CD45+LFA1+ quadruple positive cells. In some embodiments, the method increases the expression of activation markers. In some embodiments, the activation markers include NKp46, NKG2D, LFA1, and / or CD16. In some embodiments, the method decreases the expression of inhibitory markers. In some embodiments, the inhibitory markers include CD161 and CD73.

[0330] In some embodiments, the methods of the disclosure generate a population of mature NK cells from differentiated NK cells with a purity of 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.

[0331] In some embodiments, the method of the present disclosure generates a population of mature NK cells from differentiated NK cells with a purity of CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells of 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100%.

[0332] In some embodiments, the maturation process reduces the population of CD56- cells.

[0333] In some embodiments, the method of the present disclosure generates a population of mature NK cells from differentiated NK cells with 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.

[0334] In some embodiments, the method of the present disclosure generates a population of mature NK cells from differentiated NK cells with a purity of CD56- cells of 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100%.

[0335] 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 hESCs or iPSCs. In some embodiments, the ratio of mature NK to source cell (SC) is denoted as mature NK / SC. In some embodiments, the ratio of mature NK to hESC is denoted as mature NK / hESC. In some embodiments, the ratio of mature NK to iPSC is denoted as mature NK / iPSC.

[0336] In some embodiments, the yield ratio of mature 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 from 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.

[0337] 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 increase mature NK / SC from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure 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. In some embodiments, the yield ratio of mature NK / SC is from 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.

[0338] 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.

[0339] In some embodiments, the methods and compositions of the present disclosure increase the yield ratio of mature NK / StC. For example, the methods and compositions increase mature NK / StC from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure increase 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. 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.

[0340] In some embodiments, the yield ratio of mature NK cells to hESCs (mature NK / 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, 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 from 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 / hESC. For example, the methods and compositions increase mature NK / hESC from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure increase 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. In some embodiments, the yield ratio of mature NK / hESC is from 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 iPSCs (mature NK / 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, 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 from 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 increase the yield ratio of mature NK / iPSC. For example, the methods and compositions increase the mature NK / iPSC from about 2:1 to about 4:1, or by about 2. In some embodiments, the methods and compositions of the present disclosure 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. In some embodiments, the yield ratio of mature NK / iPSC is from 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] Exemplary differentiation methods In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising BMP4, FGF2, and VEGF. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, and 5-100 ng / mL of VEGF. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising BMP4, FGF2, and VEGF for 12-120 hours. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, and 5-100 ng / mL of VEGF for 12-120 hours.

[0345] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of a ROCK inhibitor. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor for 12-120 hours. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of a ROCK inhibitor for 12-120 hours.

[0346] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a serum-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of Y27632. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 over a period of 12-120 hours. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises contacting a population of stem cells with a xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of Y27632 over a period of 12-120 hours.

[0347] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, and VEGF to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, and 5-100 ng / mL VEGF to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xenogeneic-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 μg / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM PI3K inhibitor to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, and VEGF for 12-120 hours to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor for 2-20 days to generate hematopoietic precursors.In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, and 5-100 ng / mL VEGF for 12-120 hours, and (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-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 μg / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM PI3K inhibitor for 2-20 days.

[0348] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-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 generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising ~50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 μg / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM PI3K inhibitor to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor for 12-120 hours to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor for 2-20 days to generate hematopoietic precursors.In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of a ROCK inhibitor for 12-120 hours, and (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 1-100 ng / mL of SCF, about 1-50 μg / mL of LDL, about 1-100 ng / mL of TPO, and 0.1-100 μM of a PI3K inhibitor for 2-20 days.

[0349] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of Y27632 to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising ~50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 1-100 ng / mL of SCF, about 1-50 μg / mL of LDL, about 1-100 ng / mL of TPO, and 0.1-100 μM of a PI3K inhibitor to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 for 12-120 hours to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor for 2-20 days to generate hematopoietic precursors.In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of Y27632 for 12-120 hours, and (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising ~50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 1-100 ng / mL of SCF, about 1-50 μg / mL of LDL, about 1-100 ng / mL of TPO, and 0.1-100 μM of a PI3K inhibitor for 2-20 days.

[0350] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, and VEGF to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1-50 ng / mL BMP4, 1-50 ng / mL FGF2, and 5-100 ng / mL VEGF to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising ~50 ng / mL BMP4, 1-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 μg / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM LY294002 to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, and VEGF for 12-120 hours to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 for 2-20 days to generate hematopoietic precursors.In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, and 5-100 ng / mL of VEGF for 12-120 hours, and (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 1-100 ng / mL of SCF, about 1-50 μg / mL of LDL, about 1-100 ng / mL of TPO, and 0.1-100 μM of LY294002 for 2-20 days.

[0351] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of a ROCK inhibitor to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 1-100 ng / mL of SCF, about 1-50 μg / mL of LDL, about 1-100 ng / mL of TPO, and 0.1-100 μM of LY294002 to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors 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 generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 for 2-20 days to generate hematopoietic precursors.In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 0.1-20 μM of a ROCK inhibitor for 12-120 hours, and (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 1-50 ng / mL of BMP4, 1-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 1-100 ng / mL of SCF, about 1-50 μg / mL of LDL, about 1-100 ng / mL of TPO, and 0.1-100 μM of LY294002 for 2-20 days.

[0352] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-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 generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-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 μg / mL LDL, about 1-100 ng / mL TPO, and 0.1-100 μM LY294002 to generate hematopoietic precursors. In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 for 12-120 hours to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 for 2-20 days to generate hematopoietic precursors.In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 1 to 50 ng / mL of BMP4, 1 to 50 ng / mL of FGF2, 5 to 100 ng / mL of VEGF, and 0.1 to 20 μM of Y27632 for 12 to 120 hours, and (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 1 to 50 ng / mL of BMP4, 1 to 50 ng / mL of FGF2, 5 to 100 ng / mL of VEGF, about 1 to 100 ng / mL of SCF, about 1 to 50 μg / mL of LDL, about 1 to 100 ng / mL of TPO, and 0.1 to 100 μM of LY294002 for 2 to 20 days.

[0353] In some embodiments, the NK cell differentiation step follows the hematopoietic progenitor cell formation step. In some embodiments, the method of differentiating HP into NK involves contacting a population of HP cells with a xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the method of differentiating HP into NK involves contacting a population of HP cells with a xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist for 15 to 25 days. In some embodiments, the method of differentiating HP into NK involves contacting a population of HP cells with a xenogeneic-free differentiation medium comprising 5 to 50 ng / mL of SCF, 5 to 50 ng / mL of IL-7, 5 to 100 ng / mL of IL-15, 5 to 100 ng / mL of IL-12, 5 to 100 ng / mL of FLT3L, 1 to 10 μM of a pyrimido-indole derivative, and 1 to 10 μM of an aryl hydrocarbon receptor antagonist. In some embodiments, the method of differentiating HP into NK involves contacting a population of HP cells with a xenogeneic-free differentiation medium comprising 5 to 50 ng / mL of SCF, 5 to 50 ng / mL of IL-7, 5 to 100 ng / mL of IL-15, 5 to 100 ng / mL of IL-12, 5 to 100 ng / mL of FLT3L, 1 to 10 μM of a pyrimido-indole derivative, and 1 to 10 μM of an aryl hydrocarbon receptor antagonist for 15 to 25 days.

[0354] In some embodiments, a method of differentiating HPs into NKs comprises contacting a population of HP cells with a xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1. In some embodiments, a method of differentiating HPs into NKs comprises contacting a population of HP cells with a xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1 over a period of 15 to 25 days. In some embodiments, a method of differentiating HPs into NKs comprises contacting a population of HP cells with a xeno-free differentiation medium comprising 5 - 50 ng / mL of SCF, 5 - 50 ng / mL of IL-7, 5 - 100 ng / mL of IL-15, 5 - 100 ng / mL of IL-12, 5 - 100 ng / mL of FLT3L, 1 - 10 μM of UM729, and 1 - 10 μM of SR1. In some embodiments, a method of differentiating HPs into NK cells comprises contacting a population of stem cells with a xeno-free differentiation medium comprising 5 - 50 ng / mL of SCF, 5 - 50 ng / mL of IL-7, 5 - 100 ng / mL of IL-15, 5 - 100 ng / mL of IL-12, 5 - 100 ng / mL of FLT3L, 1 - 10 μM of UM729, and 1 - 10 μM of SR1 over a period of 15 to 25 days.

[0355] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF to generate 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 to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xeno-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist to generate NK cells.

[0356] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, and 5-100 ng / mL of VEGF to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of a PI3K inhibitor to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of a pyrimido-indole derivative, and 1-10 μM of an aryl hydrocarbon receptor antagonist to generate NK cells.

[0357] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, and VEGF for 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor for 2-20 days to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist for 15-25 days to generate NK cells.

[0358] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, and 5-100 ng / mL of VEGF for 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of a PI3K inhibitor for 2-20 days to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of a pyrimido-indole derivative, and 1-10 μM of an aryl hydrocarbon receptor antagonist for 15-25 days to generate NK cells.

[0359] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2 and VEGF to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and LY294002 to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729 and SR1 to generate NK cells.

[0360] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, and 5-100 ng / mL of VEGF to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of LY294002 to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of UM729, and 1-10 μM of SR1 to generate NK cells.

[0361] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2 and VEGF for 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and LY294002 for 2-20 days to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729 and SR1 for 15-25 days to generate NK cells.

[0362] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium containing 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, and 5-100 ng / mL of VEGF for 12-120 hours; (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium containing 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of LY294002 for 2-20 days; and (iii) generating NK cells by contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium containing 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of UM729, and 1-10 μM of SR1 for 15-25 days.

[0363] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium containing BMP4, FGF2, VEGF, and a ROCK inhibitor; (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium containing BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor; and (iii) generating NK cells by contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium containing SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0364] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 1-20 μM of a ROCK inhibitor to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of a PI3K inhibitor to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of a pyrimido-indole derivative, and 1-10 μM of an aryl hydrocarbon receptor antagonist to generate NK cells.

[0365] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor over a period of 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor over a period of 2-20 days to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist over a period of 15-25 days to generate NK cells.

[0366] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-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 for 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-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 μg / mL LDL, about 100 ng / mL TPO, and 5-100 μM PI3K inhibitor for 2-20 days to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-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 pyrimido-indole derivative, and 1-10 μM aryl hydrocarbon receptor antagonist for 15-25 days to generate NK cells.

[0367] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and ROCK inhibitor to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1 to generate NK cells.

[0368] In some embodiments, a method of differentiating a stem cell into a hematopoietic progenitor comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 1-20 μM of a ROCK inhibitor to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of LY294002 to generate a hematopoietic progenitor; and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of UM729, and 1-10 μM of SR1 to generate NK cells.

[0369] In some embodiments, a method of differentiating a stem cell into a hematopoietic progenitor comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and a ROCK inhibitor over 12-120 hours to generate 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 LY294002 over 2-20 days to generate a hematopoietic progenitor; 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, UM729 and SR1 over 15-25 days to generate NK cells.

[0370] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 1-20 μM of a ROCK inhibitor for 12-120 hours; (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, approximately 100 ng / mL of SCF, approximately 50 μg / mL of LDL, approximately 100 ng / mL of TPO, and 5-100 μM of LY294002 for 2-20 days; and (iii) generating NK cells by contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of UM729, and 1-10 μM of SR1 for 15-25 days.

[0371] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632; (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor; and (iii) generating NK cells by contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0372] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 1-20 μM of Y27632 to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of a PI3K inhibitor to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of a pyrimido-indole derivative, and 1-10 μM of an aryl hydrocarbon receptor antagonist to generate NK cells.

[0373] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 for 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and a PI3K inhibitor for 2-20 days to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative and an aryl hydrocarbon receptor antagonist for 15-25 days to generate NK cells.

[0374] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 1-20 μM of Y27632 for 12 to 120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of a PI3K inhibitor for 2 to 20 days to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of a pyrimido-indole derivative, and 1-10 μM of an aryl hydrocarbon receptor antagonist for 15 to 25 days to generate NK cells.

[0375] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and LY294002 to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1 to generate NK cells.

[0376] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 1-20 μM of Y27632 to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, about 100 ng / mL of SCF, about 50 μg / mL of LDL, about 100 ng / mL of TPO, and 5-100 μM of LY294002 to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of UM729, and 1-10 μM of SR1 to generate NK cells.

[0377] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 for 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and LY294002 for 2-20 days to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising SCF, IL-7, IL-15, IL-12, FLT3L, UM729 and SR1 for 15-25 days to generate NK cells.

[0378] In some embodiments, a method of differentiating stem cells into hematopoietic precursors comprises: (i) generating a population of mesodermal cells by contacting a population of stem cells with a first xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, and 1-20 μM of Y27632 for 12-120 hours; (ii) generating hematopoietic precursors by contacting the population of mesodermal cells with a second xenogeneic-free differentiation medium comprising 5-50 ng / mL of BMP4, 5-50 ng / mL of FGF2, 5-100 ng / mL of VEGF, approximately 100 ng / mL of SCF, approximately 50 μg / mL of LDL, approximately 100 ng / mL of TPO, and 5-100 μM of LY294002 for 2-20 days; and (iii) generating NK cells by contacting the population of hematopoietic precursor cells with a third xenogeneic-free differentiation medium comprising 5-50 ng / mL of SCF, 5-50 ng / mL of IL-7, 5-100 ng / mL of IL-15, 5-100 ng / mL of IL-12, 5-100 ng / mL of FLT3L, 1-10 μM of UM729, and 1-10 μM of SR1 for 15-25 days.

[0379] Characteristics of NK cells In some embodiments, NK cells produced by the methods of the disclosure have improved or enhanced properties compared to NK cells produced by other methods.

[0380] In some embodiments, expansion of NK cells produced by the methods of the disclosure is enhanced. In some embodiments, the NK cells have an expansion fold of at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, or at least 600 compared to NK cells produced by other methods.

[0381] In some embodiments, the NK cells have an expansion fold change of 50 - fold to 100 - fold, 100 - fold to 150 - fold, 150 - fold to 200 - fold, 200 - fold to 250 - fold, 250 - fold to 300 - fold, 300 - fold to 350 - fold, 350 - fold to 400 - fold, 400 - fold to 450 - fold, 450 - fold to 500 - fold, 500 - fold to 550 - fold, or 550 - fold to 600 - fold, as compared to NK cells produced by other methods.

[0382] In some embodiments, the differentiated NK cells produced by the methods of the present disclosure reduce tumor cell proliferation. In some embodiments, the differentiated NK cells reduce tumor cell proliferation by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0383] In some embodiments, the differentiated NK cells reduce tumor cell proliferation by 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100%.

[0384] In some embodiments, the mature NK cells produced by the methods of the present disclosure reduce tumor cell proliferation. In some embodiments, the mature NK cells reduce tumor cell proliferation by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0385] In some embodiments, the mature NK cells reduce tumor cell proliferation by 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100%.

[0386] 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.

[0387] In some embodiments, the methods of the present disclosure produce a differentiated NK cell population that is 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0388] 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.

[0389] In some embodiments, the methods of the present disclosure produce a mature NK cell population that is 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161 - CD73 - cells.

[0390] engineered cells[[ID=********]] In some embodiments, the cell populations described herein are genetically engineered. In some embodiments, the source cells are genetically engineered. In some embodiments, the mesodermal 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, the genetic engineering decreases the expression of an endogenous gene. In some embodiments, the genetic engineering increases the expression of an endogenous gene.

[0391] In some embodiments, genetically engineering the cells comprises introducing foreign DNA into the cells. In some embodiments, the foreign DNA is a gene. In some embodiments, the foreign DNA alters the expression of an endogenous gene.

[0392] In some embodiments, the genetic engineering comprises introducing RNA such as interfering RNA (RNAi), double-stranded RNA (dsRNA), small interfering RNA (siRNA) and / or microRNA (miRNA) into the cells.

[0393] In some embodiments, the genetic engineering comprises introducing DNA such as a plasmid or bacterial artificial chromosome (BAC) into the cells.

[0394] In some embodiments, genetic manipulation comprises introducing (a) a fusion protein comprising 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 comprises a zinc finger protein (ZFP), TAL protein, or clustered regularly interspaced short palindromic nucleic acid (CRISPR) that is specific for a gene. In some embodiments, disruption comprises introducing a zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), or a combination of and CRISPR-Cas9 that specifically binds to, specifically recognizes, or specifically hybridizes to a gene. In some embodiments, introduction is effected by introducing into the cell a nucleic acid comprising a DNA-binding protein, a DNA-binding nucleotide, and / or a sequence encoding a complex comprising a DNA-binding protein or a DNA-binding nucleotide. In some embodiments, the nucleic acid is a viral vector.

[0395] In some embodiments, the genetically engineered cells described herein comprise a chimeric antigen receptor (CAR). In some embodiments, the genetically engineered stem cells comprise a CAR. In some embodiments, the genetically engineered hematopoietic precursors comprise a CAR. In some embodiments, the genetically engineered NK cells comprise a CAR.

[0396] In some embodiments, the genetically engineered cells described herein comprise a rapamycin-activated cytokine receptor (RACR). In some embodiments, the genetically engineered stem cells comprise a RACR. In some embodiments, the genetically engineered hematopoietic precursors comprise a RACR. In some embodiments, the genetically engineered NK cells comprise a RACR.

[0397] In some embodiments, the genetically engineered cells described herein comprise a CAR and a RACR. In some embodiments, the genetically engineered stem cells comprise a CAR and a RACR. In some embodiments, the genetically engineered hematopoietic precursors comprise a CAR and a RACR. In some embodiments, the genetically engineered NK cells comprise a CAR and a RACR.

[0398] 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.

[0399] Synthetic cytokine receptor complex 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 gamma chain and a synthetic beta chain, each comprising a dimerization domain. The dimerization domain dimerizes controllably in the presence of a non-physiological ligand, thereby activating signaling of the synthetic cytokine receptor.

[0400] The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain), and N-terminal or C-terminal to the IL-2G intracellular domain.

[0401] The synthetic beta-chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the intracellular domain of interleukin-2 receptor subunit beta (IL-2RB), the intracellular domain of interleukin-7 receptor subunit beta (IL-7RB), or the intracellular domain of interleukin-21 receptor subunit beta (IL-21RB). The synthetic gamma-chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and the intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG). The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2RB intracellular domain or the IL-7RB intracellular domain).

[0402] The non-physiological ligand can activate a synthetic cytokine receptor in cytotoxic natural lymphocyte cells to induce the expansion and / or activation of engineered cytotoxic natural lymphocyte cells. In a preferred embodiment, the non-physiological ligand is rapamycin or a rapalog, such a synthetic cytokine receptor called the rapamycin-activated cytokine receptor (RACR).

[0403] In some embodiments, the non-physiological ligand activates a synthetic cytokine receptor in NK cells to induce the expansion of NK cells. In some embodiments, the activation of the synthetic cytokine receptor results in at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, at least about 2500-fold, at least about 3000-fold, at least about 3500-fold, or at least about 4000-fold the number of NK cells compared to uninduced cells.

[0404] In some embodiments, the NK cells increase 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.

[0405] Intracellular domain In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises the interleukin-2 receptor subunit gamma (IL2Rγ) domain. In some embodiments, the IL2Rγ domain comprises the sequence set forth in SEQ ID NO:1. In some embodiments, the IL2Rγ 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 to SEQ ID NO:1.

[0406] The sequence of the IL2RG common gamma chain intracellular domain is set forth in SEQ ID NO:1. TIFF2025524353000001.tif11159

[0407] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-2RB intracellular domain, and a second dimerization domain.

[0408] In some embodiments, the synthetic beta chain comprises the intracellular domain of the interleukin-2 receptor subunit beta (IL2RB). In some embodiments, the IL2RB intracellular domain comprises the sequence set forth 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 to SEQ ID NO:2.

[0409] The sequence of the IL2RB intracellular domain is set forth in SEQ ID NO:2. TIFF2025524353000002.tif24159

[0410] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-7RB intracellular domain, and a second dimerization domain.

[0411] In some embodiments, the synthetic beta chain comprises the intracellular domain of the interleukin-7 receptor subunit beta (IL7RB). In some embodiments, the IL7RB intracellular domain comprises the sequence set forth 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 to SEQ ID NO:3.

[0412] The sequence of the IL7RB intracellular domain is set forth in SEQ ID NO:3. TIFF2025524353000003.tif19159

[0413] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-21RB intracellular domain, and a second dimerization domain.

[0414] In some embodiments, the synthetic beta chain comprises an interleukin-21 receptor subunit beta (IL21RB) intracellular domain. In some embodiments, the IL21RB intracellular domain comprises the sequence set forth 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 to SEQ ID NO:4.

[0415] The sequence of the IL21RB intracellular domain is set forth in SEQ ID NO:4. TIFF2025524353000004.tif24159

[0416] Dimerization domain The dimerization domain can be a heterodimerization domain including, but not limited to, a 12 kD FK506-binding protein (FKBP) that is known in the art to dimerize in the presence of rapamycin or a rapalog, and an FKBP12-rapamycin binding (FRB) domain. The FRB domain can 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 can 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.

[0417] The sequence of the exemplary FKBP domain is set forth in SEQ ID NO:5. TIFF2025524353000005.tif9157

[0418] The sequence of the exemplary FRB domain is set forth in SEQ ID NO:6. TIFF2025524353000006.tif11158

[0419] The sequence of the mutant FRB domain (FRB mutant domain) is set forth in SEQ ID NO:7. TIFF2025524353000007.tif9158

[0420] Alternatively, the first dimerization domain and the second dimerization domain can 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.

[0421] In some embodiments, the dimerization domain is as follows: i) a 12 kD FK506-binding protein (FKBP); ii) cyclophilin A (CypA); or iii) gyroase B (CyrB); which is a homodimerization domain selected from: and the corresponding non-physiological ligands are, respectively: i) FK1012, AP1510, AP1903 or AP20187; ii) cyclosporin-A (CsA); or iii) coumermycin or an analog thereof. That is.

[0422] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a cyclophilin domain.

[0423] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the FKBP domain and the bacterial dihydrofolate reductase (DHFR) domain.

[0424] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the calcineurin domain and the cyclophilin domain.

[0425] 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).

[0426] Transmembrane domain The transmembrane domain is the sequence of the synthetic cytokine receptor that spans the membrane. The transmembrane domain may include a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0427] The sequence of the transmembrane (TM) domain is shown as SEQ ID NO:8. TIFF2025524353000008.tif3128

[0428] The sequence of the TM domain is shown as SEQ ID NO:9. TIFF2025524353000009.tif3128

[0429] The sequence of the TM domain is shown as SEQ ID NO:10. TIFF2025524353000010.tif3128

[0430] The sequence of the TM domain is shown as SEQ ID NO:11. TIFF2025524353000011.tif3128

[0431] The sequence of the CD8a signal sequence is shown as SEQ ID NO:12. TIFF2025524353000012.tif3128

[0432] Non - physiological ligand In various aspects of the compositions and methods of the present disclosure, the system includes a non - physiological ligand. Exemplary small molecules useful as ligands include, without limitation, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4 - [(6 - methylpyrazin - 2 - yl)oxy]benzoic acid (aMPOB), folate, rhodamine, acetazolamide, and CA9 ligand.

[0433] In some aspects, the synthetic cytokine receptor is activated by a ligand. In some aspects, the ligand is a non - physiological ligand.

[0434] In some aspects, the non - physiological ligand is a rapalog.

[0435] In some aspects, the non - physiological ligand is rapamycin.

[0436] In some aspects, the non - physiological ligand is AP21967.

[0437] In some aspects, the non - physiological ligand is FK506.

[0438] In some aspects, the non - physiological ligand is FK1012. In some aspects, the non - physiological ligand is AP1510. In some aspects, the non - physiological ligand is AP1903. In some aspects, the non - physiological ligand is AP20187. In some aspects, the non - physiological ligand is cyclosporin - A (CsA). In some aspects, the non - physiological ligand is coumermycin.

[0439] In some embodiments, a synthetic cytokine receptor complex activated by folate, fluorescein, aMPOB, acetazolamide, a CA9 ligand, tacrolimus, rapamycin, a rapalog (rapamycin analog), a CD28 ligand, a poly(his) tag, a Strep-tag, a FLAG-tag, a VS-tag, a Myc-tag, an HA-tag, an NE-tag, biotin, digoxigenin, dinitrophenol, or a derivative thereof.

[0440] In some embodiments, the non-physiological ligand can be an inorganic or organic compound having a molecular weight of less than 1000 daltons.

[0441] In some embodiments, the ligand can be rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapalog includes a variant of rapamycin having one or more of the following modifications to rapamycin: demethylation, elimination or substitution of the methoxy groups at C7, C42 and / or C29; elimination, derivatization or substitution of the hydroxy groups at C13, C43 and / or C28; reduction, elimination or derivatization of the ketones at C14, C24 and / or C30; substitution of the six-membered pipecolate ring by a five-membered prolyl ring; and alternative substitution on the cyclohexyl ring, or substitution of the cyclohexyl ring by a substituted cyclopentyl ring.

[0442] Thus, in some embodiments, the rapalog is everolimus, sirolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, temsirolimus (CCI-779), C20-methylallyl rapamycin, C16-(S)-3-methylindole rapamycin, C16-(S)-3-methylindole rapamycin (C16-iRap), AP21967 (A / C Heterodimerizer, Takara Bio (registered trademark)), mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573 (ridaforolimus), AP1903 (limuside), or a metabolite, derivative and / or combination thereof.

[0443] In some embodiments, the ligand includes FK1012 (a semi-synthetic dimer of FK506), tacrolimus (FK506), FKCsA (a complex of FK506 and cyclosporine), rapamycin, coumermycin, gibberellin, HaXS dimerizer (a chemical dimerizer of HaloTag and SNAP-tag), TMP-HTag (a trimethoprim haloenzyme protein dimerizer), or ABT-737 or a functional derivative thereof.

[0444] In some embodiments, the non-physiological ligand is present or provided in an amount of 0 nM to 1000 nM, such as 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, 55.0 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 in an amount within a range defined by any two of the above amounts.

[0445] 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.

[0446] 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.

[0447] In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 1 nM. In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 10 nM. In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 20 nM. In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 50 nM. In some embodiments, the non - physiological ligand is a rapalog and is present or provided at 100 nM.

[0448] 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.

[0449] Cytosolic FRB The FRB domain is a domain of about 100 amino acids derived from the mTOR protein kinase. The FRB domain can be expressed in the cytosol as a freely diffusible soluble protein. Advantageously, the FRB domain reduces the inhibitory effect of rapamycin on mTOR in transfected cells, promotes a certain activation of the transfected cells, and confers a growth advantage over natural cells to the cells.

[0450] In some embodiments, the synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds a ligand, or a complex that includes the ligand.

[0451] In some embodiments, the cytosolic polypeptide comprises an FRB domain. In some embodiments, the cytosolic polypeptide comprises an FRB domain and the ligand is rapamycin. The cytosolic FRB domain can 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 can be a naked FRB domain consisting essentially 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 cytosolic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalogs).

[0452] Chimeric antigen receptor In some embodiments, the cells described herein are genetically engineered to express a chimeric antigen receptor (CAR).

[0453] In some embodiments, the present disclosure contemplates a CAR system for use in the treatment of a subject having cancer. In some embodiments, the NK cells of the present disclosure comprise a CAR sequence (CAR-NK cells).

[0454] In some embodiments, NK cells are engineered to express a CAR construct by transfecting a cell population with an expression vector encoding the CAR construct. Exemplary examples of cell populations that can be transfected include HSCs, hematopoietic progenitor cells, common lymphoid progenitor cells, or NK cells. Suitable means for preparing a transfected population of NK cells that express the selected CAR construct are well known to those of skill in the art and include, by way of example, retroviruses, lentiviruses (viral-mediated CAR gene delivery systems), sleeping beauty, and piggyback (transposon / transposase systems including non-viral-mediated CAR gene delivery systems). In some embodiments, CAR-expressing NK cells may be generated using any of the transduction methods contemplated in the present disclosure.

[0455] Targeting agents for CARs Conventionally, a CAR is generated by fusing a polynucleotide encoding a VL, VH, or scFv to the 5' end of a polynucleotide encoding a transmembrane domain and an intracellular domain, and optionally transfecting the cell with the polynucleotide and the corresponding VH or VL. Numerous variations of CARs are well known in the art, and the present disclosure contemplates using any of the known variations. Additionally, VL / VH pairs and scFvs for numerous haptens are known in the art or can be routinely generated by conventional methods. Accordingly, the present disclosure contemplates using any known hapten-binding domain.

[0456] For example, various methods targeting CARs and CAR-expressing cells have been described in the art, including U.S. Patent No. 2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, a fluorescein or fluorescein isothiocyanate (FITC) moiety can be conjugated to an agent that binds to a desired target cell (such as a cancer cell), such that CAR-NK cells expressing an anti-fluorescein / FITC chimeric antigen receptor can selectively target the target cells labeled by the conjugate. In a variant, other haptens recognized by the CAR may be used instead of fluorescein / FITC. The CAR can be generated using various scFv sequences known in the art, or scFv sequences generated by conventional routine methods. Further exemplary scFv sequences for fluorescein / FITC, and other haptens, are provided, for example, in International Publication No. 2021 / 076788, the disclosure of which is incorporated herein by reference.

[0457] In some aspects, the CAR system of the present disclosure utilizes a CAR that targets a moiety not produced or expressed by the cells of the subject being treated. Thus, this CAR system enables the focused targeting of NK cells to target cells such as cancer cells. By administering a small conjugate molecule together with the CAR-expressing NK cells, the NK cell response can target only the cells expressing the tumor receptor, thereby reducing off-target toxicity, and the activation of the NK cells can be more readily controlled due to the rapid clearance of the small conjugate molecule. As an additional advantage, the CAR-expressing NK cells can be used as "universal" cytotoxic cells that target a wide variety of tumors without the need to prepare separate CAR constructs. The target moiety recognized by the CAR can also remain constant. The only part that needs to be changed to enable the system to target cancer cells of different identities is the ligand part of the small conjugate molecule.

[0458] In one aspect, the present disclosure provides an exemplification of this conjugate molecule / CAR system.

[0459] In some embodiments, the CAR systems of the present disclosure utilize conjugate molecules as a bridge between CAR-expressing cells and target cancer cells. The conjugate molecule is a conjugate comprising a hapten and a cell targeting moiety, such as any suitable tumor cell specific ligand. Exemplary haptens that can be recognized and bound by the CAR include FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) derivatives and tetrafluorophenyl ester (TFP) derivatives, notchins, centyrins and DARPins, along with fluorescein and its derivatives, and low molecular weight organic molecules such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxigenin. Suitable cell targeting moieties that can themselves act as haptens for the CAR include notchins (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centyrins and DARPins (see Reichert, J.M. MAbs 2009. 1(3):190-209).

[0460] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), a ligand that is bound by PSMA-positive human prostate cancer cells with nanomolar affinity (KD = 14 nM; see Kularatne, S.A. et al., Mol Pharm. 2009. 6(3):780-9). In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand linked to the targeting moiety, and the DUPA derivative is described in International Publication No. WO 2015 / 057852, which is incorporated herein by reference.

[0461] In some embodiments, the cell targeting moiety is a CCK2R ligand, a ligand that is bound by CCK2R positive cancer cells (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua.C.et al., Molecular Pharmaceutics. 2013.ePublication).

[0462] In some embodiments, the cell targeting moiety is a folate, folic acid, or an analog thereof, a ligand that is bound by a folate receptor on the cells of cancers including cancers of the ovary, cervical, endometrial, lung, kidney, brain, breast, colon, and head and neck (see Sega,E.I.et al., Cancer Metastasis Rev. 2008.27(4):655-64).

[0463] In some embodiments, the cell targeting moiety is an NK-1R ligand. Receptors for NK-1R ligands are found, for example, on cancers of the colon and pancreas. In some embodiments, the NK-1R ligand can be synthesized according to the method disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0464] In some embodiments, the cell targeting moiety can be a peptide ligand. For example, the ligand can be a peptide ligand that is an endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide belonging to the tachykinin family that targets the tachykinin receptor. Such regulatory peptides include substance P (SP), neurokinin A (substance K), and neurokinin B (neuromedin K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0465] In some embodiments, the cell targeting moiety is a CAIX ligand. For example, the receptor for the CAIX ligand, which is found in renal cancer, ovarian cancer, vulvar cancer, and breast cancer. The CAIX ligand may also be referred to herein as CA9.

[0466] In some embodiments, the cell targeting moiety is a ligand of gamma-glutamyl transpeptidase. Transpeptidase is overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, astrocytoma, melanoma, and leukemia.

[0467] In some embodiments, the cell targeting moiety is a CCK2R ligand. In particular, the receptor for the CCK2R ligand, which is found in cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon.

[0468] In one embodiment, the cell targeting moiety can have a mass of less than about 10,000 daltons, less than about 9000 daltons, less than about 8,000 daltons, less than about 7000 daltons, less than about 6000 daltons, less than about 5000 daltons, less than about 4500 daltons, less than about 4000 daltons, less than about 3500 daltons, less than about 3000 daltons, less than about 2500 daltons, less than about 2000 daltons, less than about 1500 daltons, less than about 1000 daltons, or less than about 500 daltons. In another embodiment, the small molecule ligand can have a mass of about 1 to about 10,000 daltons, about 1 to about 9000 daltons, about 1 to about 8,000 daltons, about 1 to about 7000 daltons, about 1 to about 6000 daltons, about 1 to about 5000 daltons, about 1 to about 4500 daltons, about 1 to about 4000 daltons, about 1 to about 3500 daltons, about 1 to about 3000 daltons, about 1 to about 2500 daltons, about 1 to about 2000 daltons, about 1 to about 1500 daltons, about 1 to about 1000 daltons, or about 1 to about 500 daltons.

[0469] In an exemplary aspect, the linkage within the conjugate described herein can be a direct linkage (e.g., a reaction between the isothiocyanate group of FITC and the free amine group of a small molecule ligand), or the linkage can be through an intermediate linker. In one aspect, when present, the intermediate linker can be any biocompatible linker known in the art, such as a divalent linker. In an exemplary aspect, the divalent linker can contain from about 1 to about 30 carbon atoms. In another exemplary aspect, the divalent linker can contain from about 2 to about 20 carbon atoms. In other aspects, divalent linkers of even lower molecular weight (i.e., those having an approximate molecular weight of about 30 to about 300 Da) are used. In another aspect, suitable linker lengths include, without limitation, 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 or more atoms.

[0470] In some aspects, the hapten and the cell targeting moiety can be directly conjugated by means such as a 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 linking domain to connect the two molecules can be useful as it can provide flexibility and stability. Examples of suitable linking domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) non-natural peptidoglycans; 6) polyvinylpyrrolidone; 7) pluronic F-127. Suitable linker lengths include, without limitation, 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 or more atoms.

[0471] In some embodiments, the linker can be a bivalent linker that can include one or more spacers.

[0472] Exemplary conjugates of the present disclosure are FITC-folate: TIFF2025524353000013.tif39128.

[0473] Exemplary conjugates of the present disclosure are FITC-CA9: TIFF2025524353000014.tif31128.

[0474] Exemplary conjugates of the present disclosure include the following molecules: FITC-(PEG)-12-folate, FITC-(PEG)-20-folate, FITC-(PEG)-108-folate, FITC-DUPA, FITC-(PEG)12-DUPA, FITC-CCK2R ligand, FITC-(PEG)12-CCK2R ligand, FITC-(PEG)11-NK1R ligand, and FITC-(PEG)2-CA9.

[0475] The affinity with which the ligand and the cancer cell receptor bind can vary and in some cases low affinity binding may be preferred (such as about 1 μM), but the binding affinity of the ligand and the cancer cell receptor is generally at least about 100 μM, at least about 1 nM, at least about 10 nM or at least about 100 nM, preferably at least about 1 pM or at least about 10 pM, and even more preferably at least about 100 pM.

[0476] Examples of conjugates and methods of making them are provided in U.S. Patent Applications US 2017 / 0290900, US 2019 / 0091308 and US 2020 / 0023009, which are hereby incorporated by reference in their entirety.

[0477] CAR construct In some embodiments, the binding portion of the CAR can be, for example, a single-chain variable fragment (scFv), Fab, Fv, Fc, or F(ab')2 fragment of an antibody, etc. The use of an intact (i.e., full-size) antibody such as IgG, IgM, IgA, IgD or IgE in or as a CAR is excluded from the scope of the present invention.

[0478] In some embodiments, the co-stimulatory domain serves to enhance lymphocyte proliferation and survival when the CAR binds to the target moiety. The identity of the co-stimulatory domain is limited only in terms of having the ability to enhance cell proliferation and survival activation upon binding of the target moiety by the CAR. Suitable co-stimulatory domains include, without limitation, 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 superfamily of receptors (see, e.g., Latza, U. et al., Eur. J. Immunol. 1994. 24:677). Those skilled in the art will be able to use sequence variants of these co-stimulatory domains, and it will be understood that the variants have the same or similar activity as the domain they are modeled after. 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.

[0479] In some embodiments, the CAR construct includes two co-stimulatory domains. Specific combinations include any possible permutations of the four described domains, and specific examples include 1) CD28 + CD137 (4-1BB), and 2) CD28 + CD134 (OX40).

[0480] In some embodiments, the activation signaling domain serves to activate the cell when the CAR binds to the target moiety. The identity of the activation signaling domain is limited only in that it has the ability to induce activation of the selected cells upon binding of the target moiety by the CAR. Suitable activation signaling domains include the CD3ζ chain and the Fc receptor γ. Those skilled in the art will understand that sequence variants of these described activation signaling domains can be used without adversely affecting the present invention, and that the variants have the same or similar activity as the domains from which they are modeled. Such variants can 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.

[0481] In some embodiments, the CAR can include additional elements, such as a signal peptide that ensures proper transport of the fusion protein to the cell surface, a transmembrane domain that ensures the fusion protein is maintained as an integral membrane protein, and a hinge domain that confers flexibility to the recognition region and allows for strong binding to the target moiety.

[0482] Exemplary CAR constructs suitable for CAR-NK cells are provided below. TIFF2025524353000015.tif151170

[0483] An exemplary CAR of the present disclosure is shown in FIG. 8, in which the fusion protein is encoded by a lentiviral expression vector, "SP" is the signal peptide, the CAR is an anti-FITC CAR, the CD8α hinge is present, the transmembrane domain is present ("TM"), the co-stimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ.

[0484] An exemplary nucleotide sequence encoding the CAR can include SEQ ID NO:13. TIFF2025524353000016.tif209159

[0485] Exemplary CAR amino acid sequences may include SEQ ID NO:14. TIFF2025524353000017.tif70159

[0486] Exemplary nucleotide inserts may include SEQ ID NO:15. TIFF2025524353000018.tif140159

[0487] In various aspects, CAR-expressing cells comprising the nucleic acid of SEQ ID NO:13 or 15 are provided. In some aspects, chimeric antigen receptor polypeptides comprising SEQ ID NO:14 are contemplated. In some aspects, vectors comprising SEQ ID NO:13 or 15 are contemplated. In some aspects, lentiviral vectors comprising SEQ ID NO:13 or 15 are contemplated. In some aspects, SEQ ID NO:14 may comprise or consist of a human amino acid sequence or a humanized amino acid sequence.

[0488] In some aspects, variant nucleic acid sequences or variant 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 contemplated.

[0489] The affinity with which a CAR expressed by a lymphocyte binds to a target moiety can vary, and in some cases low affinity binding may be preferred (such as about 50 nM), but the binding affinity of the CAR for a target ligand is generally at least about 100 nM, at least about 1 pM or at least about 10 pM, preferably at least about 100 pM, at least about 1 fM or at least about 10 fM, and even more preferably at least about 100 fM.

[0490] Therapeutic composition In some embodiments, the present disclosure provides a composition comprising one or more cell populations.

[0491] In some embodiments, the composition comprises a population of differentiated NK cells. In some embodiments, the composition comprises a population of differentiated NK cells that are 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.

[0492] In some embodiments, the composition comprises a population of differentiated NK cells that are 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0493] In some embodiments, the composition comprises a population of mature NK cells. In some embodiments, the composition comprises a population of mature NK cells that are 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.

[0494] In some embodiments, the composition comprises a population of mature NK cells that are 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161 - CD73 - cells.

[0495] Therapeutic method The present disclosure provides a method of treating a subject in need thereof using the compositions, therapeutic compositions or cells disclosed herein. In some embodiments, the present disclosure provides a method of treating cancer and / or killing cancer cells in a subject, comprising administering to the subject a therapeutically effective amount of the disclosed cells.

[0496] In some embodiments, the cancer is a solid tumor, sarcoma, carcinoma, lymphoma, multiple myeloma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), primary mediastinal B-cell large 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, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), one or more of acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myelodysplasia and myelodysplastic syndromes, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström's macroglobulinemia, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma (e.g., plasmacytosis, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain amyloidosis, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease and PEP syndrome), or combinations thereof.

[0497] In some embodiments, the methods disclosed herein can be used to treat cancer and / or kill cancer cells in a subject by administering a therapeutically effective amount of the cells according to any of the foregoing embodiments.

[0498] The present disclosure also provides a method of treating cancer and / or killing cancer cells in a subject, comprising the step of administering to the subject a composition of any of the foregoing aspects.

[0499] In some aspects, the present disclosure provides a method of treating cancer using any of the compositions provided herein. "Cancer" has its plain ordinary meaning as read in the context of this specification, and may include, without limitation, a group of diseases involving abnormal cell growth that has the potential to invade or spread to other parts of the body. Subjects that can be addressed using the methods described herein include, without limitation, subjects identified or selected as having cancer, such as colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, and brain cancer. Such identification and / or selection can be performed by clinical or diagnostic evaluation. In some aspects, tumor-associated antigens or tumor-associated molecules are known, such as for melanoma, breast cancer, brain cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma, and / or prostate cancer. Examples include, without limitation, B cell lymphoma, breast cancer, brain cancer, prostate cancer, and / or leukemia. In some aspects, one or more oncogenic polypeptides are associated with kidney 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 aspects, methods of treating, ameliorating, or inhibiting cancer in a subject are provided. In some aspects, the cancer is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, kidney cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colon cancer, skin cancer (including melanoma), bone cancer, or brain cancer.

[0500] In some aspects, additional cancer treatments are provided, such as small molecules, e.g., chemical compounds, antibody therapies, e.g., conjugation to radionuclides, toxins, or drugs, surgery, and / or humanized monoclonal antibodies with or without radiation.

[0501] In some embodiments, the subject is selected to receive an additional cancer treatment that may include a cancer therapeutic agent, radiation, chemotherapy, or a drug for treating cancer. In some embodiments, the drug is abiraterone, alemtuzumab, anastrozole, aprepitant, arsenic trioxide, atezolizumab, azacitidine, bevacizumab, bleomycin, bortezomib, cabazitaxel, capecitabine, carboplatin, cetuximab, a combination of chemotherapeutic agents, cisplatin, crizotinib, cyclophosphamide, cytarabine, denosumab, docetaxel, doxorubicin, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fluorouracil, fulvestrant...

Claims

1. A method for generating a population of CD34+ / CD43+ / CD45+ cells, comprising the step of contacting the population of stem cells with a differentiation medium containing 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 population of CD34+ / CD43+ / CD45+ cells from the population of stem cells.

2. A method for differentiating a population of stem cells into a population of hematopoietic precursors, comprising the step of contacting the population of stem cells with a differentiation medium containing 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 population of stem cells into a population of hematopoietic precursors.

3. The method according to claim 2, wherein the population of hematopoietic precursors includes CD34+ / CD43+ / CD45+ cells.

4. The BMP pathway activator is BMP4; and / or The above FGF is FGF2. The method according to claim 1.

5. The method according to claim 1, wherein the differentiation medium comprises one or more of the following: a Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor, a stem cell factor (SCF), and thrombopoietin (TPO).

6. The method according to claim 1, wherein the differentiation medium comprises one or more of low-density lipoprotein (LDL), phosphoinositide 3-kinase (PI3K) inhibitors, pyrimido-[4,5-β]-indole derivatives, and aryl hydrocarbon receptor (AhR) antagonists.

7. The differentiation medium is The BMP pathway activator, the FGF, the VEGF, and the Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor; The BMP pathway activator, the FGF, the VEGF, stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), and phosphoinositide 3-kinase (PI3K) inhibitors; or The BMP pathway activator, the FGF, the VEGF, SCF, TPO, LDL, PI3K inhibitor, pyrimido-[4,5-β]-indole derivative, and AhR antagonist The method according to claim 1, including the method described in claim 1.

8. The method according to claim 1, comprising the step of contacting the population of stem cells with the differentiation medium for 1 to 5 days, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, and optionally a Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor.

9. The method according to claim 1, comprising: (i) contacting the population of stem cells with the differentiation medium containing the BMP pathway activator, the FGF, the VEGF, and a Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor for 1 to 5 days in order to generate embryoid bodies or mesoderm cells; and (ii) contacting the embryoid bodies or mesoderm cells with the differentiation medium containing the BMP pathway activator, the FGF, the VEGF, stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), and a phosphoinositide 3-kinase (PI3K) inhibitor for 1 to 15 days.

10. The method according to claim 1, comprising: (i) contacting the population of stem cells with the differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, and a Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor for 1 to 5 days in order to generate embryoid bodies or mesoderm cells; and (ii) contacting the embryoid bodies or mesoderm cells with the differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), and a phosphoinositide 3-kinase (PI3K) inhibitor, a pyrimido-[4,5-b]-indole derivative, and an AhR antagonist for 1 to 15 days.

11. The method according to claim 1, wherein the stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

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

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

14. The method according to claim 13, wherein the pyrimido-[4,5-b]-indole derivative is UM729 and / or the AhR inhibitor is SR1.

15. The method according to claim 13, further comprising the step of maturing the population of NK cells using a maturation medium comprising (i) IL-12, IL-15 and IL-18 or (ii) IL-12, IL-2 and IL-18.

16. The method according to claim 1, wherein the differentiation medium is serum-free.

17. The method according to claim 1, wherein the differentiation medium is heterogeneous.

18. A method for generating a population of NK cells, (a) A process for obtaining a population of stem cells, (b) Contacting the population of stem cells with a first medium containing bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF), and optionally a Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor for a period of time sufficient to generate embryoid bodies. (c) Contacting the embryoid body with a first differentiation medium comprising BMP pathway activator, FGF, VEGF, stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), phosphoinositide 3-kinase (PI3K) inhibitor, and optionally pyrimido-[4,5-β]-indole derivative, and AhR inhibitor for a period of time sufficient to generate a population of hematopoietic precursors. (d) The step of contacting the population of hematopoietic precursors with a second differentiation medium containing SCF, IL-7, IL-12, IL-15, FLT3L, pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor for a period of time sufficient to generate the population of NK cells. Methods that include...

19. The method according to claim 1, wherein the population of stem cells is genetically manipulated or edited.

20. A population of cells comprising a hematopoietic precursor produced by any one of claims 1 to 11.

21. A population of cells comprising NK cells produced by any one of claims 12 to 19, wherein the NK cells are optionally CD43+ / CD45+ / CD56+ / LFA1+.

22. A hematopoietic precursor differentiation medium comprising serum-free basal medium, bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), stem cell factor (SCF), and thrombopoietin (TPO), low-density lipoprotein (LDL), and a phosphoinositide 3-kinase (PI3K) inhibitor.

23. An NK cell differentiation medium comprising serum-free basal medium, stem cell factor (SCF), IL-7, IL-12, IL-15, FLT3L, pyrimide-[4,5-β]-indole derivative, and an AhR inhibitor.

24. A hematopoietic precursor differentiation medium according to claim 22, and instructions for contacting a population of stem cells with the hematopoietic precursor differentiation medium for a period of time sufficient to generate a population of cells containing hematopoietic precursors; or The NK cell differentiation medium according to claim 23, and instructions for contacting a population of hematopoietic precursors with the NK cell differentiation medium for a period of time sufficient to generate a population of cells containing NK cells. A kit that includes this.

25. The hematopoietic precursor differentiation medium according to claim 22 and the NK cell differentiation medium according to claim 23, Instructions for contacting a population of stem cells with a hematopoietic precursor differentiation medium for a first period sufficient to generate a population of cells containing hematopoietic precursors, and for contacting the population of cells containing hematopoietic precursors with an NK cell differentiation medium for a second period sufficient to generate a population of cells containing NK cells. A kit that includes this.

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

27. The composition according to claim 26, wherein the hematopoietic precursor comprises CD34+ / CD43+ / CD45+ cells.

28. The composition according to claim 26, further comprising stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), phosphoinositide 3-kinase (PI3K) inhibitor, pyrimide-[4,5-b]-indole derivative, and / or aryl hydrocarbon receptor (AhR) antagonist.

29. The composition according to claim 26, further comprising a phosphoinositide 3-kinase (PI3K) inhibitor and / or a pyrimido-[4,5-b]-indole derivative.

30. The BMP pathway activator, the FGF, the VEGF, and the ROCK inhibitor; The BMP pathway activator, the FGF, the VEGF, stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), phosphoinositide 3-kinase (PI3K) inhibitor, pyrimido-[4,5-β]-indole derivative, and AhR antagonist; or The BMP pathway activator, the FGF, the VEGF, SCF, TPO, LDL, PI3K inhibitor, pyrimido-[4,5-β]-indole derivative, and AhR antagonist The composition according to claim 26, comprising:

31. The composition according to any one of claims 26 to 30, wherein the population of stem cells is induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

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

33. A kit for increasing the yield ratio of NK cells from a population of stem cells, comprising instructions for differentiating the population of stem cells into NK cells, (a) A first medium comprising bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and optionally, a Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor; (b) A first differentiation medium comprising BMP pathway activator, FGF, VEGF, stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), phosphoinositide 3-kinase (PI3K) inhibitor, and optionally pyrimido-[4,5-β]-indole derivative, and an AhR inhibitor; and (c) A second differentiation medium comprising SCF, IL-7, IL-12, IL-15, FLT3L, pyrimide-[4,5-b]-indole derivative, and an AhR inhibitor. A kit that includes this.

34. A method for increasing the yield ratio of NK cells from a population of stem cells, (a) The process of obtaining a population of stem cells; (b) Contacting the population of stem cells with a first medium containing bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and optionally a Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor for a period of time sufficient to generate embryoid bodies; (c) Contacting the embryoid body with a first differentiation medium comprising BMP pathway activator, FGF, VEGF, stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), phosphoinositide 3-kinase (PI3K) inhibitor, and optionally pyrimido-[4,5-β]-indole derivative, and AhR inhibitor for a period of time sufficient to generate a population of hematopoietic precursors; and (d) The step of contacting the population of hematopoietic precursors with a second differentiation medium containing SCF, IL-7, IL-12, IL-15, FLT3L, pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor for a period of time sufficient to generate the NK cells. Methods that include...

35. A method for generating a population of hematopoietic progenitor cells, comprising the step of contacting a population of stem cells with a differentiation medium containing bone morphogenetic protein (BMP) pathway activator, phosphoinositide 3-kinase (PI3K) inhibitor, and fibroblast growth factor (FGF) for a period of time sufficient to generate a population of hematopoietic progenitor cells from the population of stem cells.

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