Differentiation of stem cells in suspension culture

Abstract

The present application provides heterogeneity-free methods and compositions for producing hematopoietic progenitor cells and natural killer (NK) cells in 3D suspension culture.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 392,760, filed on July 27, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Background Art

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

[0003] NK cells for immunotherapy can be obtained from primary sources such as peripheral blood or umbilical cord blood. The artificial sources of NK cells include pluripotent stem cells, including induced pluripotent stem cells (iPSC), which are cells derived from somatic cells (usually fibroblasts or peripheral blood mononuclear cells (PBMC)), and human embryonic stem cells (hESC), either of which is induced to become able to infinitely proliferate and differentiate into other cell types when subjected to appropriate differentiation conditions. By sequentially differentiating iPSC into hematopoietic progenitor cells (HPC) (also referred to as hematopoietic stem cells (HSC)), NK cells can be derived from iPSC.

[0004] Once obtained, primary NK or iPSC-NK cells can be expanded in vitro before being administered to a patient. The method of differentiating iPSC into NK cells generally includes the use of feeder cells or a culture medium containing serum. In order to provide cells suitable for in vivo administration, there is still a need for compositions and methods related to the production of NK cells without the use of heterologous gene factors, such as raw materials of animal origin, such as fetal bovine serum (FBS) and / or feeder cells, and allowing the scale of production to be expanded. Summary of the invention

[0005] The present disclosure is based at least in part on the discovery that stem cells are differentiated into hematopoietic progenitor cells and NK cells in suspension. As provided herein, relative to the two-dimensional culture system for differentiation, the hematopoietic progenitor cells and NK cells produced in the three-dimensional suspension culture system have higher purity and show higher frequency of desired cell markers. Without wishing to be bound by theory, the suspension culture method described herein is suitable for large-scale production of hematopoietic progenitor cells and / or NK cells. In addition, as demonstrated herein, the suspension culture method is xeno-free. Without wishing to be bound by theory, the xeno-free method described herein produces cells suitable for in vivo administration.

[0006] In some embodiments, the present disclosure provides a method for generating a CD34+ / CD43+ / CD45+ cell population, the method comprising: (i) culturing the progenitor cell population in a two-dimensional (2D) culture system for a period of time sufficient to form progenitor cell aggregates; (ii) passaging the progenitor cell aggregates from the 2D culture system to a three-dimensional (3D) suspension culture system; (iii) contacting the progenitor cell aggregates with a differentiation medium in the 3D suspension culture system for a period of time sufficient to generate the CD34+ / CD43+ / CD45+ cell population.

[0007] In some embodiments, the present disclosure provides a method for differentiating a stem cell population into a hematopoietic progenitor cell population, the method comprising: (i) culturing the stem cell population in a 2D culture system for a period of time sufficient to form stem cell aggregates; (ii) passage the stem cell aggregates from the 2D culture system to a 3D suspension culture system; (iii) contacting the stem cell aggregates in the 3D suspension culture system with a differentiation medium for a period of time sufficient to differentiate the stem cell population into the hematopoietic progenitor cell population, wherein the differentiation medium comprises a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).

[0008] In some embodiments, the 3D suspension culture has a volume between 50-50,000 ml.

[0009] In some embodiments, the 3D suspension culture is stirred. In some embodiments, the 3D suspension culture is stirred at a rate of 10 revolutions per minute (RPM) to 100 RPM. In some embodiments, the 3D suspension culture is stirred at a rate of 70 RPM.

[0010] In some embodiments, the population of hematopoietic progenitor cells comprises CD34+ / CD43+ / CD45+ cells.

[0011] In some embodiments, the BMP pathway activator is BMP4. In some embodiments, the FGF is FGF2. In some embodiments, the VEGF is VEGF-165. In some embodiments, the differentiation medium comprises a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor. In some embodiments, wherein the ROCK inhibitor is Y27632.

[0012] In some embodiments, the differentiation medium comprises stem cell factor (SCF). In some embodiments, the differentiation medium comprises thrombopoietin (TPO). In some embodiments, the differentiation medium comprises low-density lipoprotein (LDL).

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

[0014] In some embodiments, (iii) comprises contacting the population of stem cell aggregates with a differentiation medium for 1-5 days, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, and optionally the ROCK inhibitor.

[0015] In some embodiments, (iii) comprises (a) contacting the stem cell aggregates with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesoderm cells, and (b) contacting the embryoid bodies or mesoderm cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO and the LDL for 1-15 days.

[0016] In some embodiments, the differentiation medium comprises 1-50 ng / mL BMP, 1-50 ng / mL FGF, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, and 1-50 μg / ml LDL, or any combination thereof.

[0017] In some embodiments, the progenitor cell or stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the progenitor cell or stem cell is a human embryonic stem cell (hESC).

[0018] In some embodiments, the differentiation medium is serum-free. In some embodiments, the method is xeno-free.

[0019] In some embodiments, the present disclosure provides a method of generating a population of NK cells, the method comprising: (a) culturing a stem cell population in a 2D culture system for a period of time sufficient to form stem cell aggregates; (b) passage the stem cell aggregates from the 2D culture system to a 3D suspension culture system; (c) contacting the stem cell aggregates in the 3D suspension culture system with a first culture medium comprising a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor for a period of time sufficient to generate embryoid bodies; (d) contacting the embryoid bodies with a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL for a period of time sufficient to generate a population of hematopoietic progenitor cells; (e) contacting the population of hematopoietic progenitor cells with a second differentiation medium for a period of time sufficient to produce the population of NK cells.

[0020] In some embodiments, the second differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

[0021] In some embodiments, the culture medium comprises 1-100 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

[0022] In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729 and the AhR inhibitor is SR1.

[0023] In some embodiments, the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, and the ROCK inhibitor is Y27632.

[0024] In some embodiments, each culture medium of steps (b)-(e) is serum-free. In some embodiments, the method is xeno-free.

[0025] In some embodiments, the first culture medium, the first differentiation culture medium, and the second differentiation culture medium each comprise the same basal culture medium. In some embodiments, the first differentiation culture medium and the second differentiation culture medium each comprise a different basal culture medium. In some embodiments, the first differentiation culture medium and the second differentiation culture medium each comprise the same basal culture medium, and the first culture medium comprises a basal culture medium different from the first and second differentiation culture mediums. In some embodiments, the first differentiation culture medium and the second differentiation culture medium each comprise a basal culture medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

[0026] In some embodiments, the time period of step (b) is 2-8 days, the time period of step (c) is 1-5 days, the time period of step (d) is 3-15 days, and the time period of step (e) is 11-25 days. In some embodiments, steps (a)-(e) occur within 40-50 days.

[0027] In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

[0028] In some embodiments, the hematopoietic progenitor cell population comprises about 50% to about 100% CD34+ / CD43+ / CD45+ cells. In some embodiments, the NK cell population comprises about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells. In some embodiments, the NK cell population is expanded by about 1,000 to about 10,000 fold.

[0029] In some embodiments, the stem cell population is genetically engineered or edited.In some embodiments, the NK cell population is genetically engineered or edited.

[0030] In some embodiments, the disclosure provides a cell population comprising hematopoietic progenitor cells produced by the methods described herein. In some embodiments, the hematopoietic progenitor cells are CD34+ / CD43+ / CD45+. In some embodiments, the cell population comprises 30-50% hematopoietic progenitor cells.

[0031] In some embodiments, the disclosure provides a cell population comprising NK cells produced by the methods described herein. In some embodiments, the NK cells are CD45+ / CD56+ / LFA1+. In some embodiments, the cell population comprises 60-100% NK cells.

[0032] In some embodiments, the present disclosure provides pharmaceutical compositions comprising a cell population of the present disclosure.

[0033] In some embodiments, the present disclosure provides a method of generating a population of hematopoietic progenitor cells, the method comprising: (a) genetically engineering a stem cell population to express synthetic cytokine receptors for non-physiological ligands, Wherein the cytokine receptors include: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain; (b) culturing the stem cell population in a 2D culture system for a period of time sufficient to form stem cell aggregates; (c) passage the stem cell aggregates from the 2D culture system to a 3D suspension culture system; and (d) contacting the stem cell aggregates in the 3D suspension culture system with a differentiation medium for a period of time sufficient to generate hematopoietic progenitor cells.

[0034] In some embodiments, the present disclosure provides a method of generating a population of NK cells, the method comprising: (a) genetically engineering a stem cell population to express synthetic cytokine receptors for non-physiological ligands, Wherein the cytokine receptors include: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain; (b) culturing the stem cell population in a 2D culture system for a period of time sufficient to form stem cell aggregates; (c) passage the stem cell aggregates from the 2D culture system to a 3D suspension culture system; (d) contacting the stem cell aggregates in the 3D suspension culture system with a first differentiation medium for a period of time sufficient to generate hematopoietic progenitor cells; and (e) contacting the population of hematopoietic progenitor cells with a second differentiation medium for a period of time sufficient to produce the population of NK cells.

[0035] In some embodiments, the intracellular domain of the synthetic β chain polypeptide is selected from the intracellular domain of interleukin-2 receptor subunit β (IL-2RB), the intracellular domain of interleukin-7 receptor subunit β (IL-7RB) and / or the intracellular domain of interleukin-21 receptor subunit β (IL-21RB).

[0036] In some embodiments, the nucleotide sequence is inserted by homology directed repair (HDR).

[0037] In some embodiments, the vector comprises a nucleic acid comprising, from 5' to 3', (a) a nucleotide sequence homologous to a region located upstream of a target site, (b) a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, and (c) a nucleotide sequence homologous to a region located downstream, wherein a double-strand break occurs at the target site of an endogenous gene and the nucleic acid is exchanged with the homologous nucleotide sequence of the endogenous gene.

[0038] In some embodiments, the nucleotide sequence is inserted by non-homologous end joining (NHEJ).

[0039] In some embodiments, the cell is engineered with an RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease is selected from Cas endonucleases, Mad endonucleases, and Cpf1 endonucleases. In some embodiments, the RNA-guided endonuclease is Cas9 or Mad7.

[0040] In some embodiments, the method comprises disrupting a target gene and inserting a nucleotide sequence into the disrupted target gene, wherein disrupting the target gene comprises contacting a stem cell population with (i) a gRNA targeted to a target site in the target gene and (ii) an RNA-guided endonuclease. In some embodiments, the target gene is selected from B2M, TRAC, and SIRPA.

[0041] In some embodiments, the stem cell population is engineered to be resistant to rapamycin. In some embodiments, engineering the stem cell population to be resistant to rapamycin comprises knocking out the FKBP12 gene.

[0042] In some embodiments, the differentiation medium comprises a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor. In some embodiments, the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, and the ROCK inhibitor is Y27632.

[0043] In some embodiments, the differentiation medium comprises SCF, TPO, and LDL.

[0044] In some embodiments, (d) comprises contacting the population of stem cell aggregates with a differentiation medium for 1-5 days, wherein the differentiation medium comprises a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor.

[0045] In some embodiments, (d) comprises (i) contacting the stem cell aggregates with a differentiation medium comprising a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor for 1-5 days to produce embryoid bodies or mesoderm cells, and (ii) contacting the embryoid bodies or mesoderm cells with a differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL for 1-15 days.

[0046] In some embodiments, the differentiation medium comprises 1-50 ng / mL BMP, 1-50 ng / mL FGF, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO, and 1-50 μg / ml LDL, or any combination thereof.

[0047] In some embodiments, the first differentiation medium comprises a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor.

[0048] In some embodiments, (d) comprises contacting the population of stem cell aggregates with a first differentiation medium for 1-5 days, wherein the first differentiation medium comprises a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor.

[0049] In some embodiments, (d) comprises (i) contacting the stem cell aggregates with a medium comprising a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor for 1-5 days to produce embryoid bodies or mesoderm cells, and (ii) contacting the embryoid bodies or mesoderm cells with a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL for 1-15 days.

[0050] In some embodiments, the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, and the ROCK inhibitor is Y27632.

[0051] In some embodiments, the second differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

[0052] In some embodiments, the second differentiation medium comprises 1-100 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

[0053] In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729 and the AhR inhibitor is SR1.

[0054] In some embodiments, the first differentiation medium and the second differentiation medium are serum-free.

[0055] In some embodiments, the methods are xeno-free.

[0056] In some embodiments, the first differentiation medium and the second differentiation medium each comprise the same basal medium. In some embodiments, the first differentiation medium and the second differentiation medium each comprise a different basal medium. 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.

[0057] In some embodiments, the time period of step (b) is 2-8 days, the time period of step (c) is 1-5 days, the time period of step (d) is 3-15 days, and the time period of step (e) is 11-25 days. In some embodiments, steps (a)-(e) occur within 40-50 days.

[0058] In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

[0059] In some embodiments, the population of hematopoietic progenitor cells comprises about 50% to about 100% CD34+ / CD43+ / CD45+ cells.

[0060] In some embodiments, the NK cell population comprises about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

[0061] In some embodiments, the method comprises expanding a population of NK cells, wherein the population of NK cells is expanded about 1,000 to about 10,000 fold.

[0062] In some embodiments, the stem cell population is genetically engineered or edited.In some embodiments, the NK cell population is genetically engineered or edited.

[0063] In some embodiments, the stem cells are iPSCs.

[0064] In some embodiments, the present disclosure provides a cell population produced by a method of the present disclosure. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a cell population of the present disclosure.

[0065] In some embodiments, this includes administering an effective amount of a cell population or a pharmaceutical composition to a subject.

[0066] In some embodiments, the present disclosure provides a kit comprising the cell population and instructions for administering the cell population to a subject in need thereof.

[0067] In some embodiments, the subject has cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 A schematic diagram showing an exemplary method for differentiating stem cells into hematopoietic progenitor cells and NK cells in suspension culture ("Method #1") is provided.

[0069] Figure 2 A schematic diagram showing an exemplary method for differentiating stem cells into hematopoietic progenitor cells in suspension culture ("Method #2") is provided.

[0070] Figures 3A-3E Display based on Figure 1 and Figure 2 Characterization of hematopoietic progenitor cells (HP) differentiated from stem cells according to the protocol provided in . HP were characterized at day 15 unless otherwise stated. Figure 3A Shown is flow cytometry analysis performed by gating cells to quantify the percentage of cells triple positive for HP markers CD34 / CD43 / CD45. Figure 3B is a graph showing the average HP purity of all cells that were triple positive for CD34 / CD43 / CD45 (range 60-80%). Figure 3C is a graph showing the mean values ​​of HP expansion relative to plated iPSCs at day 0. Figure 3D is a graph showing the expansion of HPs generated at day 12 using the indicated methods compared to a standard 2D differentiation protocol. Figure 3E Representative bright field microscopy images of EBs prior to HP harvest are provided.

[0071] Figures 4A-4E Shown is the characterization of natural killer (NK) cells differentiated from stem cells using method 1. NK cells were characterized at day 40. Figure 4A Shown is flow cytometry analysis performed by gating cells to quantify the percentage of CD45+CD56+LFA1+ cells. Figure 4B It is a graph showing the NK purity of all cells positive for CD34 / CD45 / CD56 / LFA1. Figure 4C is a graph showing the expansion of NK relative to iPSC plated on day 0. Figure 4D is a graph showing the expansion of NKs generated using Method #1 compared to the standard 2D differentiation protocol. Figure 4E Representative bright-field microscopy images of NK are provided.

[0072] Figure 5A and Figure 5BD40 differentiated iNK cells incubated with breast cancer MDA-MB231 cells at different T:E ratios are shown. NK cells and MDA-MB231 cells were in the absence (unstimulated, Figure 5A ) or existence (stimulus, Figure 5B ) were incubated in the presence of IL-2 and IL-15. iNK cells reduced the growth of MDA in a dose-responsive manner.

[0073] Figure 6 A schematic diagram showing an exemplary experimental design for differentiating genetically engineered stem cells into hematopoietic progenitor cells and NK cells in suspension culture is provided.

[0074] Figure 7 Graph showing the ratio of hematopoietic progenitor cells (HP) to iPSCs in parental wild-type cells encoding RACR or engineered FKBP12 knockout iPSCs (B2M-EF1A-RACR and FKBP12 KO cells) after 14 days of 3D suspension culture. Cells were transferred to 3D suspension culture using gentle cell dissociation reagent (GCDR) or EDTA. DETAILED DESCRIPTION

[0075] In some aspects, the present disclosure provides compositions and methods for producing hematopoietic progenitor cells, common lymphoid progenitor cells, pre-NK progenitor cells, NK progenitor cells, immature NK cells and / or NK cells. In some embodiments, the methods described herein are in a three-dimensional culture system. In some embodiments, the compositions and methods described herein are xeno-free. definition

[0076] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0077] Unless the context dictates otherwise, the various features described herein may be used in any combination with any feature or combination of features described herein, and each feature may be excluded or omitted from that combination.

[0078] As used herein, the singular forms "a", "an" and "the" include the plural forms as well, unless the context indicates otherwise. The conjunction "and / or" refers to all possible combinations of one or more of the listed items.

[0079] As used herein, "subject" refers to a recipient of a NK cell population generated by the methods of the present disclosure. The term includes mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep or pigs, preferably humans.

[0080] As used herein, "treat," "treating," or "treatment" refers to any type of action or administration that confers a benefit to a subject suffering from a disease or disorder, including improvement of the patient's condition (i.e., amelioration, alleviation, or improvement of one or more symptoms, as well as partial or complete response to treatment).

[0081] The term "effective amount" refers to an amount effective to produce a desired biochemical, cellular or physiological response. The term "therapeutically effective amount" refers to an amount, dosage or dosage regimen effective to elicit a desired therapeutic effect.

[0082] As used herein, "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently bound by an ester bond between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar component of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

[0083] As used herein, "polypeptide" refers to a polymer composed of amino acid residues linked together by peptide bonds, which forms part (or all) of a protein.

[0084] It will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids may encode the same polypeptide. In addition, it will be appreciated that, without affecting the polypeptide sequence encoded by the polynucleotides described herein, the skilled artisan may use conventional techniques to make nucleotide substitutions to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed.

[0085] Nucleic acids may include DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides, including synthetic or modified nucleotides. Many different types of oligonucleotide modifications are known in the art. These include methylphosphonate and phosphorothioate backbones, with acridine or polylysine chains added to the 3' and / or 5' ends of the molecule. For the purposes described herein, it will be understood that polynucleotides may be modified by any method available in the art. Such modifications may be performed to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0086] The term "variant" refers to a polynucleotide or polypeptide having at least one substitution, insertion or deletion in its sequence compared to a reference polynucleotide or polypeptide. A "functional variant" is a variant that retains one or more functions of a reference polynucleotide or polypeptide.

[0087] The term "inactivating mutation" refers to a mutation in a genomic sequence that destroys gene function. Inactivating mutations can be in any sequence region (e.g., coding region or non-coding region) that contributes to gene expression. Examples include, but are not limited to, cis-acting elements (enhancers) or sequences that undergo transcription (e.g., mRNA transcript sequences). Inactivating mutations include mutations that render a gene or its encoded protein nonfunctional or reduce the function of a gene or its encoded protein.

[0088] As used herein, the term "sequence identity" or "identity" related to a polynucleotide or polypeptide sequence refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences match at each position in the full-length reference sequence alignment. "Percentage of identity" is the number of matching positions in the optimal alignment divided by the length of the reference sequence plus the sum of the lengths of any gaps in the reference sequence in the alignment. The optimal alignment is the alignment that produces the maximum percentage identity. Sequence alignment to determine the percentage of identity can be accomplished by many well-known methods, including, for example, by using mathematical algorithms, such as algorithms in the BLAST suite or Clustal Omega sequence analysis program. Unless otherwise indicated, the term "sequence identity" in the claims refers to sequence identity calculated using default parameters by BLAST version 2.1 2.0. And, unless otherwise indicated, the alignment is the alignment of all or part of the polynucleotide or polypeptide sequence of interest over the full length of the reference sequence.

[0089] As used herein, the term "engineered" refers to cells that have been stably transduced with a heterologous polynucleotide or genetically edited to introduce, delete or modify a polynucleotide in the cell, or cells that have been transiently transduced with a polynucleotide in a manner that causes a stable phenotypic change in the cell.

[0090] As used herein, the term "stem cell" is used to describe a cell with an undifferentiated phenotype that is capable of differentiating into, for example, hematopoietic progenitor cells and / or NK cells.

[0091] As used herein, the term "pluripotency" means that a stem cell is capable of forming essentially all differentiated cell types of an organism, at least in culture. For example, an embryonic stem cell is a pluripotent stem cell that is capable of forming cells from each of the three germ layers (ectoderm, mesoderm, and endoderm).

[0092] As used herein, the terms "induced pluripotent stem cells" and "iPSCs" are used to refer to cells derived from somatic cells that have been reprogrammed back to a pluripotent state and are capable of proliferation, selective differentiation, and maturation. iPSCs are stem cells produced from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed, to become cells capable of differentiating into all three germ layers or dermal (mesoderm, endoderm, and ectoderm) tissues. The induced pluripotent stem cells produced do not refer to cells found in nature.

[0093] As used herein, the term "hematopoietic stem cell", "hematopoietic progenitor" or "hematopoietic progenitor cell" refers to a stem cell that is capable of generating mature myeloid and lymphoid cell types, including natural killer cells, T cells and B cells. Hematopoietic stem cells are typically characterized by CD34+.

[0094] The term "progenitor cell" refers to a cell that is partially differentiated into a desired cell type. Progenitor cells retain a certain degree of multipotency and can differentiate into a variety of cell types.

[0095] As used herein, "differentiation" or "differentiated" is used to refer to the process and conditions by which undifferentiated or immature (e.g., non-specialized) cells acquire the characteristics of mature (specialized) cells to acquire specific form and function. Stem cells (non-specialized) are typically exposed to different conditions (e.g., growth factors and morphogenetic factors) to induce specific lineage commitment or differentiation of the stem cells.

[0096] As used herein, "expand" or "expansion" refers to increasing the number and / or purity of cell types in a cell population by mitosis of cells with limited proliferation capacity (eg, NK cells).

[0097] As used herein, "activity," "activation," or "activation" refers to stimulation of activating receptors on cytotoxic innate lymphoid cells, leading to cell division, cytokine secretion (e.g., IFNγ and / or TNFα), and / or release of cytolytic granules to modulate or assist an immune response.

[0098] As used herein, "xeno-free" refers to compositions and methods that lack animal-derived materials (eg, fetal bovine serum). In some embodiments, the xeno-free methods described herein do not include feeder cells.

[0099] As used herein, "2D culture" refers to growing a cell culture on a flat surface (eg, the bottom of a culture dish or flask), wherein the cells are in contact with the flat surface.

[0100] As used herein, "3D suspension culture" refers to an artificially created environment in which cells are allowed to grow or interact with their surroundings in all three dimensions. 3D suspension cultures allow cells to grow in all directions in vitro, similar to how they grow in vivo. These three-dimensional cultures are typically grown in bioreactors, small capsules, or three-dimensional cell colonies in which cells can grow as spheroids.

[0101] The term "bioreactor" refers to any manufactured device or system that supports a biologically active environment. In some embodiments, a bioreactor is a container in which a process that allows the growth of organisms is carried out. 3D suspension culture

[0102] In some embodiments, the present disclosure provides a method for differentiating stem cells into hematopoietic progenitor cells in 3D suspension cultures. In some embodiments, the method includes passage cells from 2D cultures to 3D suspension cultures. In some embodiments, the present disclosure provides a method for differentiating hematopoietic progenitor cells into NK cells by culturing hematopoietic progenitor cells in 3D suspension cultures. In some embodiments, the present disclosure provides a culture medium for amplifying NK cells in suspension. In some embodiments, the differentiation and / or expansion culture medium described herein comprises a serum-free basal culture medium having at least one exogenous factor driving differentiation and / or amplification. 3D suspension culture volume

[0103] In some embodiments, the cell populations of the present disclosure are cultured in 3D suspension cultures. In some embodiments, the volume of the 3D suspension culture is at least 0.01 ml, at least 0.1 ml, at least 1 ml, at least 10 ml, at least 100 ml, at least 1,000 ml, at least 10,000 ml, at least 100,000 ml, or at least 1,000,000 ml, including all intervening values ​​therebetween.

[0104] In some embodiments, the volume of the 3D suspension culture is about 0.01 mL to about 0.1 mL, about 0.1 mL to about 1 mL, about 1 mL to about 10 mL, about 10 mL to about 100 mL, about 100 mL to about 1,000 mL, about 1,000 mL to about 10,000 mL, about 10,000 mL to about 100,000 mL, or about 100,000 mL to about 1,000,000 mL, including all intervals therebetween. In some embodiments, the volume of the 3D suspension culture is about 1 mL to about 1,000,000 mL. In some embodiments, the volume of the 3D suspension culture is about 100 mL to about 1,000 mL. In some embodiments, the volume of the 3D suspension culture is about 200 mL to about 2,000 mL. In some embodiments, the volume of the 3D suspension culture is about 500 mL to about 2,000 mL. In some embodiments, the volume of the 3D suspension culture is about 1,000 mL to about 1,000,000 mL.

[0105] In some embodiments, the 3D suspension culture volume is contained in a container. In some embodiments, the container is a flask, a multi-layer flask, a bottle, a dish or a bioreactor.

[0106] In some embodiments, the bioreactor is a hollow fiber bioreactor, a packed bed bioreactor, a stirred tank bioreactor, a rocking motion bioreactor, a stirred tank bioreactor, and / or a wave bioreactor.

[0107] In some embodiments, the methods described herein are performed in a bioreactor. In some embodiments, the 3D culture suspension system is a bioreactor. In some embodiments, the 3D culture suspension system is a bioreactor of about 100 mL to about 1,000 mL. In some embodiments, the 3D culture suspension system is a bioreactor of about 200 mL to about 2,000 mL. In some embodiments, the 3D culture suspension system is a bioreactor of about 500 mL to about 2,000 mL. 3D suspension culture agitation

[0108] In some embodiments, the 3D suspension culture is stirred. In some embodiments, the stirring is measured at a rate of revolutions per minute (RPM). In some embodiments, the 3D suspension culture is stirred at a rate of at least 1RPM, at least 5RPM, at least 10RPM, at least 15RPM, at least 20RPM, at least 25RPM, at least 30RPM, at least 35RPM, at least 40RPM, at least 45RPM, at least 50RPM, at least 55RPM, at least 60RPM, at least 65RPM, at least 70RPM, at least 75RPM, at least 80RPM, at least 85RPM, at least 90RPM, at least 95RPM, at least 100RPM, at least 105RPM, at least 110RPM, at least 120RPM, at least 130RPM, at least 140RPM, at least 150RPM, at least 160RPM, at least 170RPM, at least 180RPM, at least 190RPM, at least 200RPM, at least 210RPM, at least 220RPM, at least 230RPM, at least 240RPM, at least 250RPM, at least 260RPM, at least 270RPM, at least 280RPM, at least 290RPM, at least 300RPM, at least 350RPM, at least 350RPM, at least 0RPM, at least 140RPM, at least 150RPM, at least 160RPM, at least 170RPM, at least 180RPM, at least 190RPM, at least 200RPM, at least 250RPM, at least 300RPM, at least 350RPM, at least 400RPM, at least 450RPM, at least 500RPM, at least 550RPM, at least 600RPM, at least 650RPM, at least 700RPM, at least 750RPM, at least 800RPM, at least 850RPM, at least 900RPM, at least 950RPM or at least 1,000RPM, including all intervals therebetween.

[0109] In some embodiments, the 3D suspension culture is agitated at a rate of about 1 RPM to about 5 RPM, about 5 RPM to about 10 RPM, about 10 RPM to about 15 RPM, about 15 RPM to about 20 RPM, about 20 RPM to about 25 RPM, about 25 RPM to about 30 RPM, about 30 RPM to about 35 RPM, about 35 RPM to about 40 RPM, about 40 RPM to about 45 RPM, about 45 RPM to about 50 RPM, about 50 RPM to about 55 RPM, about 55 RPM to about 60 RPM. RPM, about 60RPM to about 65RPM, about 65RPM to about 70RPM, about 70RPM to about 75RPM, about 75RPM to about 80RPM, about 80RPM to about 85RPM, about 85RPM to about 90RPM, about 90RPM to about 95RPM, about 95RPM to about 100RPM, about 100RPM to about 105RPM, about 105RPM to about 115RPM, about 110RPM to about 120RPM, about 120RPM to about 130RPM, about 130RPM The present invention relates to a motor vehicle that is powered by a motor vehicle of the present invention and has a motor vehicle speed of 150 to about 160 RPM, about 160 to about 170 RPM, about 170 to about 180 RPM, about 180 to about 190 RPM, about 190 to about 200 RPM, about 200 to about 250 RPM, about 250 to about 300 RPM, about 300 to about 350 RPM, about 350 to about 400 RPM, about 400 to about 450 RPM, about 4 50RPM to about 500RPM, about 500RPM to about 550RPM, about 550RPM to about 600RPM, about 600RPM to about 650RPM, about 650RPM to about 700RPM, about 700RPM to about 750RPM, about 750RPM to about 800RPM, about 800RPM to about 850RPM, about 850RPM to about 900RPM, about 900RPM to about 950RPM, or about 950RPM to about 1,000RPM, including all intervals therebetween.

[0110] In some embodiments, the 3D suspension culture is stirred at a rate of about 1 RPM to about 1,000 RPM. In some embodiments, the 3D suspension culture is stirred at a rate of about 10 RPM to about 500 RPM. In some embodiments, the 3D suspension culture is stirred at a rate of about 50 RPM to about 100 RPM.

[0111] In some embodiments, the 3D suspension culture is stirred at a rate of about 70 RPM. In some embodiments, the 3D suspension culture is stirred at a rate of 70 RPM. 3D suspension medium replacement

[0112] In some embodiments, the culture medium of the 3D suspension culture is replaced at least once during the methods described herein. In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the culture medium volume of the 3D suspension culture is replaced, including all interval values ​​in between.

[0113] In some embodiments, about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 70% to 75%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, or about 95% to 100% of the medium volume of the 3D suspension culture is replaced, including all interval values ​​in between.

[0114] In some embodiments, medium replacement occurs on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day 44, day 45, day 46, day 47, day 48, day 49, day 50, day 51, day 52 Day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day 44, day 45, day 46, day 47, day 48, day 49 or day 50.

[0115] In some embodiments, the culture medium is replaced at least once a week and not on other days. For example, in some embodiments, the culture medium is replaced on the 1st day of the week and not on the 2nd to 7th day. In some embodiments, the culture medium is replaced on the 2nd day of the week and not on the 1st and 3rd to 7th day. In some embodiments, the culture medium is replaced on the 3rd day of the week and not on the 1st to 2nd and 4th to 7th day. In some embodiments, the culture medium is replaced on the 4th day of the week and not on the 1st to 3rd and 5th to 7th day. In some embodiments, the culture medium is replaced on the 5th day of the week and not on the 1st to 4th and 6th to 7th day. In some embodiments, the culture medium is replaced on the 6th day of the week and not on the 1st to 5th and 7th day. In some embodiments, the culture medium is replaced on the 7th day of the week and not on the 1st to 6th day.

[0116] In some embodiments, the culture medium is replaced at least twice a week. In some embodiments, the culture medium is replaced on the 1st and 2nd day of the week but not on the 3rd to 7th day. In some embodiments, the culture medium is replaced on the 1st and 3rd day of the week but not on the 2nd and 4th to 7th day. In some embodiments, the culture medium is replaced on the 1st and 4th day of the week but not on the 2nd to 3rd and 5th to 7th day. In some embodiments, the culture medium is replaced on the 1st and 5th day of the week but not on the 2nd to 4th and 6th to 7th day. In some embodiments, the culture medium is replaced on the 1st and 6th day of the week but not on the 2nd to 5th and 6th to 7th day. In some embodiments, the culture medium is replaced on the 1st and 7th day of the week but not on the 2nd to 6th day. In some embodiments, the culture medium is replaced on the 2nd and 3rd day of the week but not on the 2nd and 3rd to 7th day. In some embodiments, the culture medium is replaced on the 2nd and 4th day of the week but not on the 1st, 3rd and 5th to 7th day. In some embodiments, the culture medium is replaced on the 2nd and 5th day of the week but not on the 1st, 3rd, 5th, 6th and 7th day. In some embodiments, the culture medium is replaced on days 2 and 6 of the week but not on days 1, 3, 4, 5, and 7. In some embodiments, the culture medium is replaced on days 2 and 7 of the week but not on days 1 and 3 to 6. In some embodiments, the culture medium is replaced on days 3 and 4 of the week but not on days 1, 2, and 5 to 7. In some embodiments, the culture medium is replaced on days 3 and 5 of the week but not on days 1, 2, 4, and 6 to 7. In some embodiments, the culture medium is replaced on days 3 and 6 of the week but not on days 1, 2, 4, 5, and 7. In some embodiments, the culture medium is replaced on days 3 and 7 of the week but not on days 1, 2, 4, 5, and 7. In some embodiments, the culture medium is replaced on days 4 and 5 of the week but not on days 1 to 3 and 6 to 7. In some embodiments, the culture medium is replaced on days 4 and 6 of the week but not on days 1 to 3, 5, and 7. In some embodiments, the culture medium is replaced on days 4 and 7 of the week but not on days 1 to 3, 5, and 6. In some embodiments, the culture medium is changed on days 5 and 6 of the week but not on days 1 to 4 and 7. In some embodiments, the culture medium is changed on days 5 and 7 of the week but not on days 1 to 4 and 6. In some embodiments, the culture medium is changed on days 6 and 7 of the week but not on days 1 to 5.

[0117] In some embodiments, the culture medium is replaced at least three times a week. In some embodiments, the culture medium is replaced on the 1st to 3rd day but not on the 4th to 7th day. In some embodiments, the culture medium is replaced on the 1st, 2nd and 4th day of the week but not on the 3rd and 5th to 7th day. In some embodiments, the culture medium is replaced on the 1st, 2nd and 5th day of the week but not on the 3rd, 4th, 6th and 7th day. In some embodiments, the culture medium is replaced on the 1st, 2nd and 6th day of the week but not on the 3rd, 4th, 5th and 7th day. In some embodiments, the culture medium is replaced on the 1st, 2nd and 7th day of the week but not on the 3rd to 6th day. In some embodiments, the culture medium is replaced on the 1st, 3rd and 4th day but not on the 2nd and 5th to 7th day. In some embodiments, the culture medium is replaced on the 1st, 3rd and 5th day of the week but not on the 4th, 5th, 6th and 7th day. In some embodiments, the culture medium is replaced on the 1st, 3rd and 6th day of the week but not on the 2nd to 5th and 7th day. In some embodiments, the culture medium is replaced on the 1st, 3rd and 7th day of the week but not on the 2nd and 4th to 6th day. In some embodiments, the culture medium is replaced on days 1, 3, and 7 of the week but not on days 2 and 4 to 6. In some embodiments, the culture medium is replaced on days 1, 4, and 5 but not on days 2, 3, 6, and 7. In some embodiments, the culture medium is replaced on days 1, 4, and 6 of the week but not on days 2 to 4 and 7. In some embodiments, the culture medium is replaced on days 1, 4, and 7 of the week but not on days 2, 3, 5, and 6. In some embodiments, the culture medium is replaced on days 1, 5, and 6 of the week but not on days 2 to 4 and 7. In some embodiments, the culture medium is replaced on days 1, 5, and 7 of the week but not on days 2 and 4 to 6. In some embodiments, the culture medium is replaced on days 2, 3, and 4 but not on days 1 and 5 to 7. In some embodiments, the culture medium is replaced on days 2, 3, and 5 of the week but not on days 1, 4, 6, and 7. In some embodiments, the culture medium is replaced on days 2, 3, and 6 of the week but not on days 1, 4, 5, and 7. In some embodiments, the culture medium is replaced on days 2, 3, and 7 of the week but not on days 1, 4 to 6. In some embodiments, the culture medium is replaced on days 2, 4, and 5 but not on days 1, 3, 6, and 7. In some embodiments, the culture medium is replaced on days 2, 4, and 6 of the week but not on days 1, 3, 5, and 7. In some embodiments, the culture medium is replaced on days 2, 4, and 7 of the week but not on days 1, 3, 5, and 6. In some embodiments, the culture medium is replaced on days 2, 5, and 6 of the week but not on days 1, 3, 4, and 7. In some embodiments, the culture medium is replaced on days 2, 5, and 7 of the week but not on days 1, 3, 4, and 6. In some embodiments, the culture medium is replaced on days 2, 6, and 7 of the week but not on days 1, 3, 4, and 5.In some embodiments, the culture medium is replaced on days 3, 4, and 5 of the week but not on days 1, 2, 6, and 7. In some embodiments, the culture medium is replaced on days 3, 4, and 6 of the week but not on days 1, 2, 5, and 7. In some embodiments, the culture medium is replaced on days 3, 4, and 7 of the week but not on days 1, 2, 5, and 6. In some embodiments, the culture medium is replaced on days 3, 5, and 6 of the week but not on days 1, 2, 4, and 7. In some embodiments, the culture medium is replaced on days 3, 5, and 7 of the week but not on days 1, 2, 4, and 6. In some embodiments, the culture medium is replaced on days 3, 6, and 7 of the week but not on days 1, 2, 4, and 5. In some embodiments, the culture medium is replaced on days 4, 5, and 6 of the week but not on days 1 to 3 and 7. In some embodiments, the culture medium is replaced on days 4, 5, and 7 of the week but not on days 1 to 3 and 6. In some embodiments, the culture medium is replaced on days 4, 6, and 7 of the week but not on days 1 to 3 and 5. In some embodiments, the culture medium is changed on days 5, 6, and 7 but not on days 1-4 of the week.

[0118] In some embodiments, the culture medium is replaced at least four times a week. In some embodiments, the culture medium is not replaced on the 1st to 3rd day but on the 4th to 7th day. In some embodiments, the culture medium is not replaced on the 1st, 2nd and 4th day of the week but on the 3rd and 5th to 7th day. In some embodiments, the culture medium is not replaced on the 1st, 2nd and 5th day of the week but on the 3rd, 4th, 6th and 7th day. In some embodiments, the culture medium is not replaced on the 1st, 2nd and 6th day of the week but on the 3rd, 4th, 5th and 7th day. In some embodiments, the culture medium is not replaced on the 1st, 2nd and 7th day of the week but on the 3rd to 6th day. In some embodiments, the culture medium is not replaced on the 1st, 3rd and 4th day but on the 2nd and 5th to 7th day. In some embodiments, the culture medium is not replaced on the 1st, 3rd and 5th day of the week but on the 4th, 5th, 6th and 7th day. In some embodiments, the culture medium is not replaced on the 1st, 3rd and 6th day of the week but on the 2nd to 5th and 7th day. In some embodiments, the culture medium is not replaced on days 1, 3, and 7 of the week but is replaced on days 2 and 4 to 6. In some embodiments, the culture medium is not replaced on days 1, 3, and 7 of the week but is replaced on days 2 and 4 to 6. In some embodiments, the culture medium is not replaced on days 1, 4, and 5 but is replaced on days 2, 3, 6, and 7. In some embodiments, the culture medium is not replaced on days 1, 4, and 6 of the week but is replaced on days 2 to 4 and 7. In some embodiments, the culture medium is not replaced on days 1, 4, and 7 of the week but is replaced on days 2, 3, 5, and 6. In some embodiments, the culture medium is not replaced on days 1, 5, and 6 of the week but is replaced on days 2 to 4 and 7. In some embodiments, the culture medium is not replaced on days 1, 5, and 7 of the week but is replaced on days 2 and 4 to 6. In some embodiments, the culture medium is not replaced on days 2, 3, and 4 but is replaced on days 1 and 5 to 7. In some embodiments, the culture medium is not replaced on days 2, 3, and 5 of the week but is replaced on days 1, 4, 6, and 7. In some embodiments, the culture medium is not replaced on the 2nd, 3rd and 6th day of the week but is replaced on the 1st, 4th, 5th and 7th day. In some embodiments, the culture medium is not replaced on the 2nd, 3rd and 7th day of the week but is replaced on the 1st, 4th to 6th day. In some embodiments, the culture medium is not replaced on the 2nd, 4th and 5th day but is replaced on the 1st, 3rd, 6th and 7th day. In some embodiments, the culture medium is not replaced on the 2nd, 4th and 6th day of the week but is replaced on the 1st, 3rd, 5th and 7th day. In some embodiments, the culture medium is not replaced on the 2nd, 4th and 7th day of the week but is replaced on the 1st, 3rd, 5th and 6th day. In some embodiments, the culture medium is not replaced on the 2nd, 5th and 6th day of the week but is replaced on the 1st, 3rd, 4th and 7th day. In some embodiments, the culture medium is not replaced on the 2nd, 5th and 6th day of the week but is replaced on the 1st, 3rd, 4th and 7th day. In some embodiments, the culture medium is not replaced on the 2nd, 5th and 7th day of the week but is replaced on the 1st, 3rd, 4th and 6th day.In some embodiments, the culture medium is not replaced on the 2nd, 6th and 7th day of the week but is replaced on the 1st, 3rd, 4th and 5th day. In some embodiments, the culture medium is not replaced on the 3rd, 4th and 5th day of the week but is replaced on the 1st, 2nd, 6th and 7th day. In some embodiments, the culture medium is not replaced on the 3rd, 4th and 6th day of the week but is replaced on the 1st, 2nd, 5th and 7th day. In some embodiments, the culture medium is not replaced on the 3rd, 4th and 7th day of the week but is replaced on the 1st, 2nd, 5th and 6th day. In some embodiments, the culture medium is not replaced on the 3rd, 5th and 6th day of the week but is replaced on the 1st, 2nd, 4th and 7th day. In some embodiments, the culture medium is not replaced on the 3rd, 5th and 7th day of the week but is replaced on the 1st, 2nd, 4th and 6th day. In some embodiments, the culture medium is not replaced on the 3rd, 5th and 7th day of the week but is replaced on the 1st, 2nd, 4th and 6th day. In some embodiments, the culture medium is not replaced on the 3rd, 6th and 7th day of the week but is replaced on the 1st, 2nd, 4th and 5th day. In some embodiments, the culture medium is not replaced on the 4th, 5th and 6th day of the week but is replaced on the 1st to 3rd and 7th day. In some embodiments, the culture medium is not changed on days 4, 5, and 7 of the week but is changed on days 1 to 3 and 6. In some embodiments, the culture medium is not changed on days 4, 6, and 7 of the week but is changed on days 1 to 3 and 5. In some embodiments, the culture medium is not changed on days 5, 6, and 7 of the week but is changed on days 1 to 4.

[0119] In some embodiments, the culture medium is replaced at least five times a week. In some embodiments, the culture medium is not replaced on the 1st and 2nd day of the week but on the 3rd to 7th day. In some embodiments, the culture medium is not replaced on the 1st and 3rd day of the week but on the 2nd and 4th to 7th day. In some embodiments, the culture medium is not replaced on the 1st and 4th day of the week but on the 2nd to 3rd and 5th to 7th day. In some embodiments, the culture medium is not replaced on the 1st and 5th day of the week but on the 2nd to 4th and 6th to 7th day. In some embodiments, the culture medium is not replaced on the 1st and 6th day of the week but on the 2nd to 5th and 6th to 7th day. In some embodiments, the culture medium is not replaced on the 1st and 7th day of the week but on the 2nd to 6th day. In some embodiments, the culture medium is not replaced on the 2nd and 3rd day of the week but on the 2nd and 3rd to 7th day. In some embodiments, the culture medium is not replaced on the 2nd and 4th day of the week but on the 1st, 3rd and 5th to 7th day. In some embodiments, the culture medium is not replaced on the 2nd and 5th day of the week but on the 1st, 3rd, 5th, 6th and 7th day. In some embodiments, the culture medium is not replaced on the 2nd and 6th day of the week but is replaced on the 1st, 3rd, 4th, 5th and 7th day. In some embodiments, the culture medium is not replaced on the 2nd and 7th day of the week but is replaced on the 1st and 3 to 6th day. In some embodiments, the culture medium is not replaced on the 3rd and 4th day of the week but is replaced on the 1st, 2nd and 5 to 7th day. In some embodiments, the culture medium is not replaced on the 3rd and 5th day of the week but is replaced on the 1st, 2nd, 4th and 6 to 7th day. In some embodiments, the culture medium is not replaced on the 3rd and 6th day of the week but is replaced on the 1st, 2nd, 4th, 5th and 7th day. In some embodiments, the culture medium is not replaced on the 3rd and 7th day of the week but is replaced on the 1st, 2nd, 4th, 5th and 7th day. In some embodiments, the culture medium is not replaced on the 4th and 5th day of the week but is replaced on the 1st to 3rd and 6 to 7th day. In some embodiments, the culture medium is not replaced on the 4th and 6th day of the week but is replaced on the 1st to 3rd, 5th and 7th day. In some embodiments, the culture medium is not changed on days 4 and 7 of the week but is changed on days 1 to 3, 5, and 6. In some embodiments, the culture medium is not changed on days 5 and 6 of the week but is changed on days 1 to 4 and 7. In some embodiments, the culture medium is not changed on days 5 and 7 of the week but is changed on days 1 to 4 and 6. In some embodiments, the culture medium is not changed on days 6 and 7 of the week but is changed on days 1 to 5.

[0120] In some embodiments, the culture medium is replaced at least six times a week and is replaced on other days. For example, the culture medium is not replaced on the 1st day of the week but on the 2nd to 7th day. In some embodiments, the culture medium is not replaced on the 1st day of the week but on the 2nd to 7th day. In some embodiments, the culture medium is not replaced on the 2nd day of the week but on the 1st and 3rd to 7th day. In some embodiments, the culture medium is not replaced on the 3rd day of the week but on the 1st to 2nd and 4th to 7th day. In some embodiments, the culture medium is not replaced on the 4th day of the week but on the 1st to 3rd and 5th to 7th day. In some embodiments, the culture medium is not replaced on the 5th day of the week but on the 1st to 4th and 6th to 7th day. In some embodiments, the culture medium is not replaced on the 6th day of the week but on the 1st to 5th and 7th day. In some embodiments, the culture medium is not replaced on the 7th day of the week but on the 1st to 6th day.

[0121] In some embodiments, the culture medium is changed every day of the week. 3D suspension culture seeding density

[0122] In some embodiments, the cell populations of the present disclosure are seeded in 3D suspension culture at a density of at least 1×10 3 cells, at least 1×10 4 cells, at least 1×10 5 cells, at least 1×10 6 cells, at least 1×10 7 cells, at least 1×10 8 cells or at least 1×10 9 cells.

[0123] In some embodiments, the cell populations of the present disclosure are seeded in 3D suspension culture at a density of about 1×10 3 cells to 1×10 4 cells, about 1×10 4 cells to 1×10 5 cells, about 1×10 5 cells to 1×10 6 cells, about 1×10 6 cells to 1×10 7 cells, about 1×10 7 cells to 1×10 8 cells or about 1×10 8 cells to 1×10 9 cells. Differentiation and Expansion Media

[0124] In some embodiments, the disclosure provides a culture medium for differentiating stem cells into hematopoietic progenitor cells. In some embodiments, the disclosure provides a culture medium for differentiating hematopoietic progenitor cells into NK cells. In some embodiments, the invention provides a culture medium for amplifying NK cells. In some embodiments, the differentiation and / or expansion culture medium described herein comprises a serum-free basal culture medium having at least one exogenous factor driving differentiation and / or amplification. Xeno-free culture medium

[0125] In some embodiments, the differentiation and / or expansion medium described herein is a medium with well-defined components. As used herein, a "well-defined medium" refers to a growth medium suitable for in vitro culture of human or animal cells, wherein all chemical components are known. In some embodiments, the differentiation and / or expansion medium includes a basal medium. In some embodiments, the basal medium includes Iscove's modified Dulbecco's medium, serum albumin, human insulin, human transferrin, and 2-mercaptoethanol. In some embodiments, the basal medium includes human serum albumin. In some embodiments, the basal medium does not include raw materials of animal origin.

[0126] In some embodiments, the basal medium is selected from StemSpan SFEMII medium (STEMCELL Technologies; serum-free), Stemline II (Sigma-Aldrich; completely 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 (STEMCELL Technologies; serum-free and animal component-free) or Hematopoietic Progenitor Cell Expansion Medium XF (PromoCell; serum-free and xeno-free medium). In some embodiments, the basal medium is StemSpan SFEMII medium. In some embodiments, the basal medium is Stemline II medium. In some embodiments, the basal medium is StemDiff APEL 2 medium. In some embodiments, the hematopoietic progenitor cell differentiation medium and the NK cell differentiation medium have the same basal medium. In some embodiments, the hematopoietic progenitor cell differentiation medium and the NK cell differentiation medium have different basal mediums. Exogenous factors

[0127] In some embodiments, differentiation and / or expansion culture medium described herein include exogenous factors. In some embodiments, the method disclosed herein includes contacting different cell populations with various exogenous factors in a culture medium without xenogeneics, to drive cell differentiation into, for example, hematopoietic progenitor cells and / or NK cells. In some embodiments, the exogenous factors include, but are not limited to, cytokines, such as interleukins, fibroblast growth factor (FGF), stem cell factor (SCF), phosphatidylinositol 3-kinase (PI3K) inhibitors, FMS-like tyrosine kinase 3 ligands (FLT3L), bone morphogenetic protein (BMP) pathway activators, pyrimidine and indole derivatives, and aryl hydrocarbon receptor antagonists.

[0128] In some embodiments, the exogenous factors suitable for differentiation and / or expansion culture medium are cytokines. Cytokines include interferons, interleukins and growth factors, which are small proteins that play an important role in cell signaling. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is selected from IL-2, IL-7, IL-12, IL-15, IL-18 and any combination thereof. In some embodiments, the cytokine is a growth factor. In some embodiments, the growth factor is selected from fibroblast growth factor, vascular endothelial growth factor and any combination thereof.

[0129] In some embodiments, the exogenous factor is interleukin 2 (IL-2). IL-2 is a secretory cytokine produced by activated CD4+ and CD8+ T lymphocytes, which is important for the proliferation of T lymphocytes and B lymphocytes. IL-2 is a member of the interleukin 2 (IL2) cytokine subfamily, which includes IL-4, IL-7, IL-9, IL-15, IL-21, erythropoietin and thrombopoietin.

[0130] In some embodiments, the exogenous factor is interleukin 7 (IL-7). IL-7 is a member of the interleukin 2 (IL2) cytokine subfamily. Lymphoid differentiation and activation are primarily dependent on IL-7 signaling.

[0131] 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. After a brief stimulation with IL-12, NK cells can acquire memory-like properties.

[0132] In some embodiments, the exogenous factor is interleukin 15 (IL-15). IL-15 is a member of the interleukin 2 (IL2) cytokine subfamily. IL-15 regulates NK cell activation and proliferation.

[0133] In some embodiments, the exogenous factor is interleukin 18 (IL-18). IL-18 is a proinflammatory cytokine of the IL-1 family that is constitutively found as a precursor in the cytoplasm of a variety of immune cells. IL-18 has been shown to potently activate NK cells.

[0134] In some embodiments, the exogenous factor is low-density lipoprotein (LDL). LDL induces an increase in NK cell proliferation and cytotoxic activity.

[0135] In some embodiments, the exogenous factor is fibroblast growth factor (FGF). The FGF family member is a cell signaling protein 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.

[0136] In some embodiments, the exogenous factor is FMS-like tyrosine kinase 3 ligand (FLT3L). FLT3L is an essential growth factor for NK cells and has been shown to play an important role in the expansion of early hematopoietic progenitor cells and the generation of mature peripheral NK cells.

[0137] In some embodiments, the exogenous factor is stem cell factor (SCF).SCF plays an important role in the survival of stem cells and the self-renewal and maintenance of stem cells.

[0138] 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 cystine knot growth factors. The VEGF family includes five family members. In some embodiments, the exogenous factor is VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. In some embodiments, the exogenous factor is VEGF-165. VEGF165 is a 38.2kDa disulfide-bonded homodimeric protein consisting of two polypeptide chains of 165 amino acids.

[0139] In some embodiments, the exogenous factor is an aryl hydrocarbon inhibitor. The aryl hydrocarbon receptor is a transcription factor that regulates gene expression. The aryl hydrocarbon receptor plays a role in regulating immunity, stem cell maintenance and cell differentiation. Antagonism of the aryl hydrocarbon receptor has been shown to promote the renewal and expansion of stem cells. In some embodiments, the exogenous factor is an aryl hydrocarbon receptor antagonist selected from PD98059, StemRegenin1 (SR1), GNF351, BAY 2416964, CH-223191, perillaldehyde, PDM-11 and BAY-218. In some embodiments, the exogenous factor is SR1.

[0140] In some embodiments, the exogenous factor is an inhibitor of phosphatidylinositol 3-kinase (PI3K). PI3K comprises a family of lipid and serine / threonine kinases that catalyze the transfer of phosphate to the D-3' position of inositol lipids to generate phosphoinositide-3-phosphate (PIP), phosphoinositide-3,4-bisphosphate (PIP2), and phosphoinositide-3,4,5-triphosphate (PIP3), which in turn serve as second messengers in signaling cascades by docking proteins containing Pleckstrin-homology, FYVE, Phox, and other phospholipid binding domains into a variety of signaling complexes typically located at the plasma membrane.

[0141] PI3K inhibitors include, but are not limited to, Idelalisib, Copanlisib, Duvelisib, Alpelisib, Umbralisib, Buplisib, Copanlisib, Dactolisib, Duvelisib, Idelalisib, Leniolisib, Parsaclisib, Paxalisib, Taselisib, Zandelisib ib, Inavolisib, Apitolisib, Bimiralisib, Eganelisib, Fimepinostat, Gedatolisib, Linperlisib, Nemiralisib, Pictilisib, Pilaralisib, Samotolisib, Seletalisib, Serabelisib, Sonolisib, Tenalisib, Voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, Acalisib, Omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, Hibiscone C, IC87114, LY294002, and PI-103.In some embodiments, the exogenous factor is Idelalisib, Copanlisib, Duvelisib, Alpelisib, Umbralisib, Bupallisib, Copanlisib, Dactolisib, Duvelisib, Idelalisib, Leniolisib, Parsaclisib, Paxalisib, Taselisib, Zandelisib ib, Inavolisib, Apitolisib, Bimiralisib, Eganelisib, Fimepinostat, Gedatolisib, Linperlisib, Nemiralisib, Pictilisib, Pilaralisib, Samotolisib, Seletalisib, Serabelisib, Sonolisib, Tenalisib, Voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, Acalisib, Omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, Hibiscone C, IC87114, LY294002, PI-103, or any combination thereof. In some embodiments, the exogenous factor is LY294002.

[0142] In some embodiments, the exogenous factor is an activator of the BMP pathway. Bone morphogenetic protein (BMP) is produced in the form of a large precursor molecule, which is proteolytically hydrolyzed into a mature peptide after translation. BMP exerts its effect through a specific transmembrane receptor located on the surface of the target cell. BMP receptors are serine threonine kinases, which are similar to TGF-β receptors and are divided into two subgroups: type I and type II receptors. BMP can only strongly bind to the heterotetrameric complex of these receptors. The formation of this complex is necessary for BMP signal transduction. In the target cell, the BMP signal is transmitted to the nucleus by a specific signaling molecule called Smad, which is also responsible for inhibiting the BMP signal.

[0143] BMP is a multifunctional cytokine and a member of the transforming growth factor-β superfamily. BMP receptors mediate BMP signaling by activating Smad. BMP ligands bind to BMP receptors BMPRI and BMPRII. Phosphorylated BMPRII activates BMPRI. Phosphorylated BMPRI then phosphorylates receptor-activated Smad protein (R-Smad), which binds to co-mediator-Smad (Co-Smad) and enters the nucleus, where they regulate gene expression. BMP pathway activators include those disclosed in WO2014011540, WO 2014062138, and WO 2005117994, which are incorporated herein by reference. BMP pathway activators include, but are not limited to, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, and BMP-4. In some embodiments, the BMP pathway activator is BMP-4. In some embodiments, the exogenous factor is BMP-4.

[0144] In some embodiments, the exogenous factor is a ROCK inhibitor. Rho-associated kinase (ROCK) is a serine / threonine kinase that serves the downstream effectors of Rho kinase (three subtypes exist, namely RhoA, RhoB and RhoC). ROCK inhibitors include, but are not limited to, polynucleotides, polypeptides and small molecules. ROCK inhibitors contemplated herein can reduce ROCK expression and / or ROCK activity. Exemplary examples of ROCK inhibitors encompassed herein include, but are not limited to, anti-ROCK antibodies, dominant negative ROCK variants, siRNA, shRNA, miRNA and antisense nucleic acids targeting ROCK.

[0145] Exemplary ROCK inhibitors contemplated herein include, but are not limited to, Thiazovivin, Y27632, Fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxy-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridinyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridinyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridinyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, as well as the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety. In some embodiments, the ROCK inhibitor is Thiazovivin, Y27632, or Pyrintegrin. In some embodiments, the ROCK inhibitor is Y27632. Mesoderm / embryoid body differentiation medium

[0146] In some embodiments, the present disclosure provides a differentiation medium for producing mesoderm and / or embryoid bodies from stem cells. In some embodiments, mesodermal cells are produced by iPSC or hESC. When stem cells begin to differentiate, three different germ layers are formed: ectoderm, mesoderm and endoderm. Immune cells, such as NK cells, are differentiated from mesodermal cells. Embryoid bodies are three-dimensional aggregates that can be differentiated into cells of three germ layers. In some embodiments, the mesodermal cells are produced by embryoid bodies. In some embodiments, the mesodermal cells produced by the compositions and methods of the present disclosure are further differentiated into hematopoietic progenitor cells. In some embodiments, the mesodermal cells produced by the compositions and methods of the present disclosure are further differentiated into NK cells.

[0147] In some embodiments, a stem cell population (e.g., iPSC or hESC) 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 includes a BMP pathway activator and FGF. In some embodiments, the exogenous factor includes a BMP pathway activator and VEGF. In some embodiments, the exogenous factor includes a BMP pathway activator and a ROCK inhibitor. In some embodiments, the exogenous factor includes FGF and VEGF. In some embodiments, the exogenous factor includes FGF and a ROCK inhibitor. In some embodiments, the exogenous factor includes VEGF and a ROCK inhibitor. In some embodiments, the exogenous factors include BMP pathway activators, FGF and VEGF. In some embodiments, the exogenous factors include BMP pathway activators, FGF and ROCK inhibitors. In some embodiments, the exogenous factors include FGF, VEGF and ROCK inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF and ROCK inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF and ROCK inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and ROCK inhibitors.

[0148] 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

[0149] In some embodiments, BMP4, FGF2, VEGF and / or ROCK inhibitor are used in the mesoderm formation step. For example, the mesoderm formation step may include contacting the cell population with BMP4 and FGF2; contacting with BMP4, FGF2 and ROCK inhibitor; contacting with BMP4 and VEGF; contacting with BMP4, VEGF and ROCK inhibitor; contacting with FGF2 and VEGF; contacting with FGF2, VEGF and ROCK inhibitor; contacting with BMP4, FGF2 and VEGF; contacting with BMP4, FGF2, VEGF and ROCK inhibitor; or contacting with any one of BMP4, FGF2, VEGF and ROCK inhibitor alone without using the others.

[0150] In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 31 ng / ml, about 32 ng / ml, about 33 ng / ml, about 34 ng / ml, about 35 ng / ml, about 36 ng / ml, about 37 ng / ml, about 38 ng / ml, about 39 ng / ml, about 40 ng / ml, about 41 ng / ml, about 42 ng / ml In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1-50 ng / ml, about 2-30 ng / ml, about 2-40 ng / ml, about 2-50 ng / ml, about 2-6 ng / ml, about 2-7 ng / ml, about 2-8 ng / ml, about 2-9 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, about 100 ng / ml, or any range derivable therein.

[0151] In some embodiments, the BMP pathway activator is BMP4. In some embodiments, BMP4 is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, BMP4 is present in the differentiation medium at about 1-50 ng / ml.

[0152] In some embodiments, FGF2 is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml. l, about 10ng / ml, about 11ng / ml, about 12ng / ml, about 13ng / ml, about 14ng / ml, about 15ng / ml, about 16ng / ml, about 17ng / ml, about 18ng / ml, about 19ng / ml, about 20ng / ml, about 21ng / ml, about 22ng / ml, about 23ng / ml, about 24ng / ml, about 25ng / ml, about 26ng / ml, about 27ng / ml , about 28ng / ml, about 29ng / ml, about 30ng / ml, about 35ng / ml, about 40ng / ml, about 45ng / ml, about 50ng / ml, about 55ng / ml, about 60ng / ml, about 65ng / ml, about 70ng / ml, about 75ng / ml, about 80ng / ml, about 85ng / ml, about 90ng / ml, about 95ng / ml, about 100ng / ml, about 110ng / ml In some embodiments, FGF2 is present in the differentiation medium at about 1-100 ng / ml.

[0153] In some embodiments, VEGF is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, VEGF is present in the differentiation medium at about 5-100 ng / ml, or any range derivable therein. In some embodiments, VEGF is present in the differentiation medium at about 5-100 ng / ml.

[0154] In some embodiments, the ROCK inhibitor 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, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM In some embodiments, the ROCK inhibitor is present in the differentiation medium at about 0.1-20 μM.

[0155] In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, Y27632 is present at a concentration of about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM In some embodiments, Y27632 is present in the differentiation medium at about 0.1-100 μM, about 10 μM, about 15 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about 100 μM, or any range derivable therein. In some embodiments, Y27632 is present in the differentiation medium at about 0.1-100 μM.

[0156] In some embodiments, the mesodermal differentiation medium comprises a BMP pathway activator, FGF and VEGF. In some embodiments, the mesodermal differentiation medium comprises a BMP pathway activator, FGF, VEGF and a ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises a xeno-free basal medium with clear components, a BMP pathway activator, FGF and VEGF. In some embodiments, the mesodermal differentiation medium comprises a xeno-free basal medium with clear components, a BMP pathway activator, FGF, VEGF and a ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises BMP4, FGF2 and VEGF-165. In some embodiments, the mesodermal differentiation medium comprises BMP4, FGF, VEGF-165 and a ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises BMP4, FGF, VEGF-165 and a ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises BMP4, FGF, VEGF-165 and Y27632.

[0157] In some embodiments, the mesodermal differentiation medium comprises 1-50ng / mL BMP4, 1-150ng / mL FGF2, and 1-100ng / mL VEGF-165. In some embodiments, the mesodermal differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM ROCK inhibitor. In some embodiments, the mesodermal differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM Y27632. Hematopoietic Progenitor Cell Differentiation Medium

[0158] In some embodiments, the present disclosure provides differentiation medium for generating HP cells from mesodermal cells and embryoid body cells. In some embodiments, the mesodermal cells and embryoid body cells generated by the compositions and methods of the present disclosure are further differentiated into hematopoietic progenitor cells.

[0159] In some embodiments, the HP cell population is cultured with at least one exogenous factor to form differentiated NK cells. In some embodiments, the exogenous factor is a BMP pathway activator. In some embodiments, the exogenous factor is FGF. In some embodiments, the exogenous factor is VEGF. In some embodiments, the exogenous factor is SCF. In some embodiments, the exogenous factor is TPO. In some embodiments, the exogenous factor is LDL. In some embodiments, the exogenous factor is a PI3K inhibitor. In some embodiments, the exogenous factor is a pyrimidoindole derivative. In some embodiments, the exogenous factor is an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor is a TGF-β receptor inhibitor. In some embodiments, the exogenous factor is selected from BMP pathway activators, FGF, VEGF, SCF, TPO, LDL, PI3K inhibitors, and any combination thereof. In some embodiments, the exogenous factor is selected from BMP pathway activators, FGF, VEGF, SCF, TPO, LDL, and any combination thereof. In some embodiments, the exogenous factor is selected from the group consisting of a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, a pyrimidoindole derivative, an aryl hydrocarbon receptor antagonist, a TGF-β receptor inhibitor, and any combination thereof.

[0160] In some embodiments, the exogenous factors include BMP pathway activators and FGF. In some embodiments, the exogenous factors include BMP pathway activators and VEGF. In some embodiments, the exogenous factors include BMP pathway activators and SCF. In some embodiments, the exogenous factors include BMP pathway activators and TPO. In some embodiments, the exogenous factors include BMP pathway activators and LDL. In some embodiments, the exogenous factors include BMP pathway activators and PI3K inhibitors. In some embodiments, the exogenous factors include FGF and VEGF. In some embodiments, the exogenous factors include FGF and SCF. In some embodiments, the exogenous factors include FGF and TPO. In some embodiments, the exogenous factors include FGF and LDL. In some embodiments, the exogenous factors include FGF and PI3K inhibitors. In some embodiments, the exogenous factors include VEGF and SCF. In some embodiments, the exogenous factors include VEGF and TPO. In some embodiments, the exogenous factors include VEGF and LDL. In some embodiments, the exogenous factors include VEGF and a PI3K inhibitor. In some embodiments, the exogenous factors include SCF and TPO. In some embodiments, the exogenous factors include SCF and LDL. In some embodiments, the exogenous factors include SCF and a PI3K inhibitor. In some embodiments, the exogenous factors include TPO and LDL. In some embodiments, the exogenous factors include TPO and a PI3K inhibitor. In some embodiments, the exogenous factors include LDL and a PI3K inhibitor.

[0161] In some embodiments, the exogenous factors include BMP pathway activators, FGF and VEGF. In some embodiments, the exogenous factors include BMP pathway activators, FGF and SCF. In some embodiments, the exogenous factors include BMP pathway activators, FGF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, FGF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and SCF. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF and SCF. In some embodiments, the exogenous factors include FGF, VEGF and TPO. In some embodiments, the exogenous factors include FGF, VEGF and LDL. In some embodiments, the exogenous factors include FGF, VEGF and LDL. In some embodiments, the exogenous factors include FGF, SCF and TPO. In some embodiments, the exogenous factors include FGF, SCF and LDL. In some embodiments, the exogenous factors include FGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, TPO and LDL. In some embodiments, the exogenous factors include FGF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, LDL and PI3K inhibitors. 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 PI3K inhibitors. In some embodiments, the exogenous factors include VEGF, TPO and LDL.In some embodiments, the exogenous factors include VEGF, TPO, and a PI3K inhibitor.

[0162] In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and SCF. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF and TPO. In some embodiments, the exogenous factors include FGF, VEGF, SCF and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, TPO and LDL.In some embodiments, the exogenous factors include FGF, VEGF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, TPO, LDL and PI3K inhibitors.

[0163] In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF and TPO. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL and PI3K inhibitors.In some embodiments, the exogenous factors include FGF, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include VEGF, SCF, TPO, LDL and PI3K inhibitors.

[0164] In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF, TPO and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, FGF, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include BMP pathway activators, VEGF, SCF, TPO, LDL and PI3K inhibitors. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO, LDL and PI3K inhibitors.

[0165] In some embodiments, the exogenous factors include BMP pathway activators, FGF, VEGF, SCF, TPO, LDL, and PI3K inhibitors.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF and TPO. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, TPO and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, 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, SCF, LDL and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO and LDL. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO and LY294002.In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, SCF, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include VEGF-165, SCF, TPO, LDL, and LY294002.

[0170] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, TPO and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, TPO and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, TPO, LDL and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, TPO, LDL and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO, LDL, and LY294002.

[0171] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, TPO, LDL, and LY294002.

[0172] In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 31 ng / ml, about 32 ng / ml, about 33 ng / ml, about 34 ng / ml, about 35 ng / ml, about 36 ng / ml, about 37 ng / ml, about 38 ng / ml, about 39 ng / ml, about 40 ng / ml, about 41 ng / ml, about 42 ng / ml In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1-50 ng / ml, about 2-30 ng / ml, about 2-40 ng / ml, about 2-50 ng / ml, about 2-6 ng / ml, about 2-7 ng / ml, about 2-8 ng / ml, about 2-9 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, about 100 ng / ml, or any range derivable therein.

[0173] 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 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, BMP4 is present in the differentiation medium at about 1-50 ng / ml.

[0174] In some embodiments, FGF2 is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, FGF2 is present in the differentiation medium at about 1-50 ng / ml.

[0175] In some embodiments, VEGF is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, VEGF is present in the differentiation medium at about 1-100 ng / ml, or any range derivable therein. In some embodiments, VEGF is present in the differentiation medium at about 1-100 ng / ml.

[0176] In some embodiments, SCF is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, SCF is present in the differentiation medium at about 1-100 ng / ml, or any range derivable therein. In some embodiments, SCF is present in the differentiation medium at about 1-100 ng / ml.

[0177] In some embodiments, TPO is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, the TPO is present in the differentiation medium at about 1-100 ng / ml, or any range derivable therein. In some embodiments, the TPO is present in the differentiation medium at about 1-100 ng / ml.

[0178] In some embodiments, LDL is present in the differentiation medium at a concentration of about 0.1-500 μg / ml, about 1-250 μg / ml, about 1-150 μg / ml, about 5-100 μg / ml, about or about 0.1 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 μg / ml, about In some embodiments, LDL is present in the differentiation medium at about 1-50 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, about 100 μg / ml, or any range derivable therein. In some embodiments, LDL is present in the differentiation medium at about 1-50 μg / ml.

[0179] In some embodiments, the PI3K inhibitor 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, 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 In some embodiments, the PI3K inhibitor is present in the differentiation medium at about 0.1-100 μM.

[0180] 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, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 6 In some embodiments, LY294002 is present in the differentiation medium at about 0.1-100 μM, about 10 μM, about 15 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about 100 μM, or any range derivable therein. In some embodiments, LY294002 is present in the differentiation medium at about 0.1-100 μM.

[0181] In some embodiments, the pyrimidoindole 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, 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. In some embodiments, the pyrimidoindole derivative is present in the differentiation medium at about 0.1-10 μM.

[0182] In some embodiments, the pyrimidoindole 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, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM In some embodiments, UM729 is present in the differentiation medium at about 0.1-10 μM, about 10 μM, about 15 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about 100 μM, or any range derivable therein. In some embodiments, UM729 is present in the differentiation medium at about 0.1-10 μM.

[0183] 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, 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. In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at about 0.1-10 μM.

[0184] In some embodiments, the aryl hydrocarbon receptor antagonist is StemRegenin 1 (SR1). In some embodiments, SR1 is present at a concentration of about 0.1-500 μM, about 1-250 μM, about 1-150 μM, about 5-100 μM, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM In some embodiments, SR1 is present in the differentiation medium at about 0.1-10 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about 100 μM, or any range derivable therein. In some embodiments, SR1 is present in the differentiation medium at about 0.1-10 μM.

[0185] In some embodiments, the TGF-β receptor inhibitor 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, 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. In some embodiments, the TGF-β receptor inhibitor is present in the differentiation medium at about 0.1-20 μM.

[0186] 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, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μ In some embodiments, GW788388 is present in the differentiation medium at about 0.1-20 μM.

[0187] 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, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μ In some embodiments, SB431542 is present in the differentiation medium at about 0.1-20 μM.

[0188] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, and VEGF. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor. In some embodiments, the HP differentiation medium comprises BMP4, FGF2, and VEGF-165. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and a ROCK inhibitor. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and a ROCK inhibitor.

[0189] In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 5-50ng / mL FGF2, and 1-100ng / mL VEGF-165. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 1-20μM ROCK inhibitor. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 1-20μM Y27632.

[0190] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and a pyrimidoindole derivative. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a pyrimidoindole derivative, and an aryl hydrocarbon receptor antagonist.

[0191] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, SCF, TPO and LDL. In some embodiments, the HP differentiation medium comprises BMP4, FGF2, VEGF-165, SCF, TPO and LDL. In some embodiments, the HP differentiation medium comprises 50ng / mL BMP4, 5-50ng / mL FGF2, 1-100ng / mL VEGF-165, 1-100ng / mL SCF, 1-100ng / mL TPO and 1-50g / ml LDL.

[0192] In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and pyrimidoindole derivatives. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, a pyrimidoindole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, a pyrimidoindole 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.

[0193] In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, and 0.1-10 μM UM729. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, and 0.1-10 μM SR1. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM UM729, and 0.1-10 μM SR1.

[0194] 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 pyrimidoindole derivative, and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a pyrimidoindole derivative, and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a 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 pyrimidoindole derivative, a aryl hydrocarbon receptor antagonist, and a TGF-β receptor inhibitor.

[0195] 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.

[0196] In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM GW788388. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, 0.1-10μM UM729, and 0.1-20μM GW788388. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM SR1, and 0.1-20 μM GW788388. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM UM729, 0.1-10 μM SR1, and 0.1-20 μM GW788388.

[0197] 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.

[0198] In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, and 0.1-20μM SB431542. In some embodiments, the HP differentiation medium comprises 1-50ng / mL BMP4, 1-50ng / mL FGF, 1-100ng / mL VEGF-165, 0.1-10μM UM729, and 0.1-20μM SB431542. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM SR1, and 0.1-20 μM SB431542. In some embodiments, the HP differentiation medium comprises 1-50 ng / mL BMP4, 1-50 ng / mL FGF, 1-100 ng / mL VEGF-165, 0.1-10 μM UM729, 0.1-10 μM SR1, and 0.1-20 μM SB431542. NK cell differentiation medium

[0199] In some embodiments, the present disclosure provides a differentiation medium for producing NK cells from HP cells. In some embodiments, NK cells are produced from HP cells. In some embodiments, HP cells produced by the compositions and methods of the present disclosure are further differentiated into NK cells.

[0200] In some embodiments, the HP cell colony is cultured with at least one exogenous factor to form differentiated NK cells. In some embodiments, the exogenous factor is stem cell factor (SCF). In some embodiments, the exogenous factor is IL-7. In some embodiments, the exogenous factor is IL-15. In some embodiments, the exogenous factor is IL-12. In some embodiments, the exogenous factor is FLT3L. In some embodiments, the exogenous factor is a pyrimidoindole 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, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists.

[0201] In some embodiments, the exogenous factors include SCF and IL-7. In some embodiments, the exogenous factors include SCF and IL-15. In some embodiments, the exogenous factors include SCF and IL-12. In some embodiments, the exogenous factors include SCF and FLT3L. In some embodiments, the exogenous factors include SCF and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7 and IL-15. In some embodiments, the exogenous factors include IL-7 and IL-12. In some embodiments, the exogenous factors include IL-7 and FLT3L. In some embodiments, the exogenous factors include IL-7 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-15 and IL-12. In some embodiments, the exogenous factors include IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-12 and FLT3L. In some embodiments, the exogenous factors include IL-12 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-12 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists.

[0202] 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 pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12 and IL-15. In some embodiments, the exogenous factors include SCF, IL-12 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-12 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-12 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-7, IL-12 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-12, IL-15 and pyrimidoindole derivatives.In some embodiments, the exogenous factors include IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors include IL-12, FLT3L, and a pyrimidoindole derivative. In some embodiments, the exogenous factors include IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist.

[0203] 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 pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and aryl hydrocarbon receptor antagonists.In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-15, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists.

[0204] 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 pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, and pyrimidoindole derivatives.In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, pyrimidoindole derivatives, and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, pyrimidoindole derivatives, and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L, pyrimidoindole derivatives, and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L, pyrimidoindole derivatives, and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-12, IL-15, FLT3L, pyrimidoindole derivatives, and aryl hydrocarbon receptor antagonists.

[0205] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L, pyrimidoindole derivatives and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L, pyrimidoindole derivatives, and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, pyrimidoindole derivatives, and aryl hydrocarbon receptor antagonists.

[0206] 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 pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-7 and aryl hydrocarbon receptor antagonists. In some embodiments, the exogenous factors include SCF, IL-12 and IL-15. In some embodiments, the exogenous factors include SCF, IL-12 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-12 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-12 and SR1. In some embodiments, the exogenous factors include SCF, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include SCF, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, pyrimidoindole derivatives and SR1. In some embodiments, the exogenous factors include IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-7, IL-12 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-12 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-15 and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, IL-15 and SR1. In some embodiments, the exogenous factors include IL-7, FLT3L and pyrimidoindole derivatives. In some embodiments, the exogenous factors include IL-7, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, UM729 and SR1. In some embodiments, the exogenous factors include IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-12, IL-15 and UM729. In some embodiments, the exogenous factors include IL-12, IL-15 and SR1.In some embodiments, the exogenous factors include IL-12, FLT3L and UM729. In some embodiments, the exogenous factors include IL-12, FLT3L and SR1.

[0207] In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and UM729. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-12, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and UM729. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L and UM729. In some embodiments, the exogenous factors include IL-7, IL-12, FLT3L and SR1.In some embodiments, the exogenous factors include IL-7, IL-12, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, IL-15, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, FLT3L, UM729 and SR1.

[0208] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-15, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-7, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-12, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include SCF, IL-15, FLT3L, UM729 and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, 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, 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.

[0209] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, FLT3L and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, 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.

[0210] In some embodiments, SCF is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, SCF is present in the differentiation medium at about 1-50 ng / ml, or any range derivable therein.

[0211] In some embodiments, IL-7 is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, the IL-7 is present in the differentiation medium at about 1-50 ng / ml, about 2-30 ng / ml, about 2-40 ng / ml, about 2-50 ng / ml, about 2-6 ng / ml, about 2-7 ng / ml, about 2-8 ng / ml, about 2-9 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, about 100 ng / ml or any range derivable therein. In some embodiments, the IL-7 is present in the differentiation medium at about 1-50 ng / ml.

[0212] In some embodiments, IL-12 is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, In some embodiments, the IL-12 is present in the differentiation medium at about 1-100 ng / ml, or any range derivable therein. In some embodiments, the IL-12 is present in the differentiation medium at about 1-100 ng / ml.

[0213] In some embodiments, IL-15 is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, In some embodiments, the IL-15 is present in the differentiation medium at about 1-100 ng / ml, or any range derivable therein. In some embodiments, the IL-15 is present in the differentiation medium at about 1-100 ng / ml.

[0214] In some embodiments, FLT3L is present in the differentiation medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, In some embodiments, FLT3L is present in the differentiation medium at about 1-100 ng / ml, or any range derivable therein.

[0215] In some embodiments, the pyrimidoindole 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, 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. In some embodiments, the pyrimidoindole derivative is present in the differentiation medium at about 0.1-10 μM.

[0216] In some embodiments, the pyrimidoindole 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, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM In some embodiments, UM729 is present in the differentiation medium at about 0.1-10 μM, about 10 μM, about 15 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about 100 μM, or any range derivable therein. In some embodiments, UM729 is present in the differentiation medium at about 0.1-10 μM.

[0217] 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, 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. In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at about 0.1-10 μM.

[0218] In some embodiments, the aryl hydrocarbon receptor antagonist is StemRegenin 1 (SR1). In some embodiments, SR1 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, about or about 0.1 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about In some embodiments, the StemRegenin 1 (SR1) is present in the differentiation medium at about 0.1-10 μM.

[0219] 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 pyrimidoindole derivatives. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L and an aryl hydrocarbon receptor antagonist. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, pyrimidoindole derivatives and an aryl hydrocarbon receptor antagonist.

[0220] 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.

[0221] In some embodiments, the NK cell differentiation medium comprises 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, and 1-100 ng / mL FLT3L. In some embodiments, the NK cell differentiation medium comprises 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, and 0.1-10 μM pyrimidoindole derivatives. In some embodiments, the NK cell differentiation medium comprises 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, and 0.1-10 μM aryl hydrocarbon receptor antagonist. In some embodiments, the NK cell differentiation medium comprises 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimidoindole derivatives, and 0.1-10 μM aryl hydrocarbon receptor antagonist.

[0222] In some embodiments, the NK cell differentiation medium comprises 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L and SR1. In some embodiments, the NK cell differentiation medium comprises 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L and 0.1-10 μM UM729. In some embodiments, the NK cell differentiation medium comprises 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 1-10 μM SR1, and 0.1-10 μM UM729.

[0223] In some embodiments, the NK cell differentiation medium comprises a defined xeno-free basal medium, 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, and 0.1-10 μM SR1. In some embodiments, the NK cell differentiation medium comprises a defined xeno-free basal medium, 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, and 0.1-10 μM UM729. In some embodiments, the NK cell differentiation medium comprises a defined xeno-free basal medium, 1-50 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM SR1, and 0.1-10 μM UM729. NK Cell Expansion Medium

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

[0225] In some embodiments, the differentiated NK cell colony is cultured with at least one exogenous factor to form mature NK cells. 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 an activated bead. In some embodiments, the exogenous factor is selected from IL-2, IL-7, IL-12, IL-15, IL-18 and an activated bead.

[0226] In some embodiments, the exogenous factors include IL-2 and IL-7. In some embodiments, the exogenous factors include IL-2 and IL-12. In some embodiments, the exogenous factors include IL-2 and IL-15. In some embodiments, the exogenous factors include IL-2 and IL-18. In some embodiments, the exogenous factors include IL-2 and activation beads. In some embodiments, the exogenous factors include IL-7 and IL-12. In some embodiments, the exogenous factors include IL-7 and IL-15. In some embodiments, the exogenous factors include IL-7 and IL-18. In some embodiments, the exogenous factors include IL-7 and activation beads. In some embodiments, the exogenous factors include IL-12 and IL-15. In some embodiments, the exogenous factors include IL-12 and IL-18. In some embodiments, the exogenous factors include IL-12 and activation beads. In some embodiments, the exogenous factors include IL-15 and IL-18. In some embodiments, the exogenous factors include IL-15 and activation beads. In some embodiments, the exogenous factors include IL-18 and activation beads.

[0227] In some embodiments, the exogenous factors include IL-2, IL-7 and IL-12. In some embodiments, the exogenous factors include IL-2, IL-7 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-7 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-12 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-7, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-7, IL-12 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-7, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-15, IL-18 and activation beads.

[0228] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-2, IL-15, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-7, IL-15, IL-18 and activation beads. In some embodiments, the exogenous factors include IL-12, IL-15, IL-18 and activation beads.

[0229] 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. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18 and activated beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18 and activated beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, IL-18 and activated beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, IL-18 and activated beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, IL-18 and activated beads.

[0230] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads.

[0231] In some embodiments, the exogenous factors include IL-2 and IL-7. In some embodiments, the exogenous factors include IL-2 and IL-12. In some embodiments, the exogenous factors include IL-2 and IL-15. In some embodiments, the exogenous factors include IL-2 and IL-18. In some embodiments, the exogenous factors include IL-2 and activation beads 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 activation 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 activation 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 activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-18 and activation beads coated with anti-CD2 / anti-NKp46.

[0232] In some embodiments, the exogenous factors include IL-2, IL-7 and IL-12. In some embodiments, the exogenous factors include IL-2, IL-7 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-7 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7 and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-12 and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-12 and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-15, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-18, and activation 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 activation 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 activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12, IL-15, and IL-18. In some embodiments, the exogenous factors include IL-12, IL-15, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

[0233] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and IL-15. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-7, IL-18 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-15, IL-18, and activation 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 activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-12, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-7, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

[0234] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15 and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, IL-18 and activated beads coated with anti-CD2 / anti-NKp46. In some embodiments, the exogenous factors include IL-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 activation beads coated with anti-CD2 / anti-NKp46.

[0235] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

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

[0237] In some embodiments, IL-7 is present in the expansion medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about In some embodiments, the IL-7 is present in the expansion medium at about 1-50 ng / mL.

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

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

[0240] In some embodiments, IL-18 is present in the expansion medium at a concentration of about 0.1-500 ng / ml, about 1-250 ng / ml, about 1-150 ng / ml, about 5-100 ng / ml, about or about 0.1 ng / ml, about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, In some embodiments, the IL-18 is present in the expansion medium at about 1-100 ng / mL, or any range derivable therein. In some embodiments, the IL-18 is present in the expansion medium at about 1-100 ng / mL.

[0241] In some embodiments, activation beads are present in the expansion medium.

[0242] In some embodiments, the activation beads coated with anti-CD2 / anti-NKp46 are present in the expansion medium in a ratio based on the number of differentiated NK cells. For example, there is one activation bead per NK cell, or the ratio of activation beads to NK cells is 1:1.

[0243] In some embodiments, the ratio of activated beads: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.

[0244] In some embodiments, the ratio of activated beads:NK cells is about 1:1 to 2:1, about 2:1 to 3:1, about 3:1 to 4:1, about 4:1 to 5:1, about 5:1 to 6:1, about 6:1 to 7:1, about 7:1 to 8:1, about 8:1 to 9:1, about 9:1 to 10:1, about 10:1 to 15:1, about 15:1 to 20:1, about 20:1 to 25:1, about 25:1 to 30:1, about 30:1 to 35:1, about 35:1 to 40:1, about 40:1 to 45:1, or about 45:1 to 50:1.

[0245] In some embodiments, the ratio of NK cells: 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.

[0246] In some embodiments, the ratio of NK cells: activation beads is about 1:1 to 2:1, about 2:1 to 3:1, about 3:1 to 4:1, about 4:1 to 5:1, about 5:1 to 6:1, about 6:1 to 7:1, about 7:1 to 8:1, about 8:1 to 9:1, about 9:1 to 10:1, about 10:1 to 15:1, about 15:1 to 20:1, about 20:1 to 25:1, about 25:1 to 30:1, about 30:1 to 35:1, about 35:1 to 40:1, about 40:1 to 45:1, or about 45:1 to 50:1.

[0247] In some embodiments, the NK cell expansion medium comprises IL-2, IL-7, IL-12, IL-15, IL-18 and activation beads. In some embodiments, the NK cell expansion medium comprises a defined xeno-free basal medium comprising IL-2, IL-7, IL-12, IL-15, IL-18 and activation beads.

[0248] In some embodiments, the NK cell expansion medium comprises IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46. In some embodiments, the NK cell expansion medium comprises a defined xeno-free basal medium comprising IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads coated with anti-CD2 / anti-NKp46.

[0249] In some embodiments, the NK cell expansion medium comprises 1-50 ng / mL IL-2, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL IL-18, and activation beads at a cell: bead ratio of 1: 1. In some embodiments, the NK cell expansion medium comprises a defined xeno-free basal medium comprising 1-50 ng / mL IL-2, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL IL-18, and activation beads at a cell: bead ratio of 1: 1.

[0250] In some embodiments, the NK cell expansion medium comprises 1-50ng / mL IL-2, 1-50ng / mL IL-7, 1-100ng / mL IL-12, 1-100ng / mL IL-15, 1-100ng / mL IL-18, and activation beads coated with anti-CD2 / anti-NKp46 at a cell: bead ratio of 1: 1. In some embodiments, the NK cell expansion medium comprises a defined xeno-free basal medium, comprising 1-50ng / mL IL-2, 1-50ng / mL IL-7, 1-100ng / mL IL-12, 1-100ng / mL IL-15, 1-100ng / mL IL-18, and activation beads coated with anti-CD2 / anti-NKp46 at a cell: bead ratio of 1: 1. Differentiation method

[0251] In some aspects, the present disclosure provides a method for producing hematopoietic progenitor cells by stem cells in a 3D culture system. In some aspects, the present disclosure provides a method for producing NK cells by stem cells in a 3D culture system. In some aspects, the present disclosure provides a method for producing NK cells by hematopoietic progenitor cells in a 3D culture system. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, and differentiating the hematopoietic progenitor cells into NK cells.

[0252] In some aspects, the present disclosure provides a method for producing common lymphoid progenitor cells (CLP) by stem cells in a 3D culture system. In some embodiments, the method includes differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, and differentiating the hematopoietic progenitor cells into CLP. CLP refers to a cell that is a precursor of a lymphoid cell. CLP is a cell that can be transformed into a hematopoietic cell type. In some embodiments, CLP is CD45+CD7+CD5+ / lo CD3-CD56-. In some embodiments, CLP is CD45+CD5+ / lo CD7+. In some aspects, the present disclosure provides a method for producing NK cells by CLP in a 3D culture system. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, differentiating the hematopoietic progenitor cells into CLP, and differentiating the CLP into NK cells.

[0253] In some aspects, the present disclosure provides a method for generating pRENK cell progenitors (preNKP) from stem cells in a 3D culture system. In some embodiments, the method comprises differentiating stem cells into hematopoietic progenitors, differentiating the hematopoietic progenitors into CLPs, and differentiating the CLPs into PreNKPs in a 3D culture system. PreNKPs are intermediate cells between CLPs and NKPs. In some embodiments, PreNKPs are Lin- / CD244+ / C-Kit 低 / IL-7Ra+ / FLT3- / CD122-. In some aspects, the present disclosure provides a method for producing NK cells from preNKP. In some embodiments, the method for producing NK cells comprises differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into preNKPs, and differentiating the preNKPs into NK cells.

[0254] In some aspects, the present disclosure provides a method for producing NK cell precursors (NKP) by stem cells in a 3D culture system. In some embodiments, the method includes differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into PreNKPs, and differentiating the PreNKPs into NKPs. NKP is the last cell before the final NK lineage is finalized. In some embodiments, NKP is Lin- / NK1.1-DX5- / IL-7Ra+ / CD122+ / NKG2D+. In some aspects, the present disclosure provides a method for producing NK cells by NKP in a 3D culture system. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into preNKPs, differentiating the preNKPs into NKPs, and differentiating the NKPs into NK cells.

[0255] In some aspects, the present disclosure provides a method for producing immature NK (iNK) cells from stem cells in a 3D culture system. In some embodiments, the method includes differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into PreNKPs, differentiating the PreNKPs into NKPs, and differentiating the NKPs into iNK cells. In some aspects, the present disclosure provides a method for producing NK cells from iNK cells in a 3D culture system. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into preNKPs, differentiating the preNKPs into NKPs, differentiating the NKPs into iNK cells, and differentiating the iNK cells into NK cells.

[0256] In some aspects, the present disclosure provides a method for producing mature NK (mNK) cells from stem cells in a 3D culture system. In some aspects, the present disclosure provides a method for producing mature NK cells from immature NK cells in a 3D culture system. In some embodiments, the method for producing NK cells includes differentiating stem cells into hematopoietic progenitor cells in a 3D culture system, differentiating the hematopoietic progenitor cells into CLPs, differentiating the CLPs into preNKPs, differentiating the preNKPs into NKPs, differentiating the NKPs into iNK cells, and differentiating the iNK cells into mNK cells.

[0257] In some embodiments, the methods provided herein are xeno-free. In some embodiments, the methods provided herein do not contain animal-derived raw materials. Expression markers

[0258] By for example flow cytometry detection markers, such as CD56, CD94, CD117, NKG2D, DNAM-1 and NKp46, the differentiation of source cells to NK cells can be assessed. Differentiation can also be assessed by the morphological characteristics of NK cells, such as large size, high protein synthesis activity in abundant endoplasmic reticulum (ER) and / or preformed particles. The maturation of NK cells can be assessed by detecting one or more functionally related markers, such as CD94, CD161, NKp44, DNAM-1, 2B4, NKp46, CD94, KIR and NKG2 family (such as NKG2D) of activated receptors. The maturation of NK cells can also be assessed by detecting specific markers at different developmental stages. For example, in one embodiment, preNKP cells are CD34+, CD45RA+, CD10+, CD117- and / or CD161-. In another embodiment, immature NK cells are CD34-, CD117+, CD161+, NKp46- and / or CD94 / NKG2A-. In another embodiment, CD56bright NK cells are CD117+, NKp46+, CD94 / NKG2A+, CD16- and / or KIR+ / -. In another embodiment, CD56dim NK cells are CD117-, NKp46+, CD94 / NKG2A+ / -, CD16+ and / or KIR+. In a specific embodiment, the maturation of NK cells (e.g., TSNK cells) is determined by the percentage of NK cells (e.g., TSNK cells) of CD161-, CD94+ and / or NKp46+. In a more specific embodiment, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65% or 70% of mature NK cells (e.g., TSNK cells) are NKp46+. In another more specific embodiment, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of mature NK cells (e.g., TSNK cells) are CD94+. In another more specific embodiment, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of mature NK cells (e.g., TSNK cells) are CD161-.

[0259] In some embodiments, by detecting, for example, the expression level of CD3, CD7 or CD127, CD10, CD14, CD15, CD16, CD33, CD34, CD56, CD94, CD117, CD161, NKp44, NKp46, NKG2D, DNAM-1, 2B4 or TO-PRO-3, using, for example, antibodies against one or more of these cell markers, to assess the differentiation of source cells into NK cells. Such antibodies can be conjugated with a detectable marker, for example, as a fluorescent marker, for example, FITC, R-PE, PerCP, PerCP-Cy5.5, APC, APC-Cy7 or APC-H7.

[0260] In some embodiments, the frequency of cell populations with a desired expression pattern is higher in 3D culture compared to 2D culture. In some embodiments, the 3D culture system can produce at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more hematopoietic progenitor cells (CD34+, CD45+, and CD43+) compared to 2D culture. In some embodiments, the 3D culture system can produce at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% more preNKP cells (CD34+, CD45RA+, CD10+, CD117- and / or CD161-) compared to 2D culture. In some embodiments, the 3D culture system produces at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% more immature NK cells (CD34-, CD117+, CD161+, NKp46- and / or CD94 / NKG2A-) compared to 2D culture. In some embodiments, the 3D culture system produces at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% more CD56bright NK cells (CD117+, NKp46+, CD94 / NKG2A+, CD16- and / or KIR+ / -) compared to 2D culture. In some embodiments, the 3D culture system produces at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% more CD56dim NK cells (CD117-, NKp46+, CD94 / NKG2A+ / -, CD16+ and / or KIR+) compared to 2D culture. In some embodiments, the 3D culture system produces at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% more mature NK cells (CD161-, CD94+ and / or NKp46+) compared to 2D culture. Source cells

[0261] In some embodiments, NK cells are generated from source cells.Any progenitor cell known in the art can be used as a source cell in the methods of the present disclosure.

[0262] In some embodiments, the source cell is hESC. In some embodiments, the source cell is iPSC. NK cells derived from iPSC may also be referred to as iPSC-derived NK cells.

[0263] In immunotherapy, the source cells are allogeneic or autologous, meaning from a donor or from a subject, respectively. In some embodiments, the source cells are allogeneic. In some embodiments, the source cells are autologous.

[0264] In some embodiments, the source cell is a peripheral blood cell. As used herein, the term "peripheral blood cell" is used to refer to cells derived from circulating blood and comprising hematopoietic stem cells capable of proliferation, selective differentiation and maturation. Therefore, peripheral blood NK cells may also be referred to as differentiated blood-derived NK cells (bdNK).

[0265] In some embodiments, the source cells include hematopoietic stem cells characterized as CD34+ and / or CD45+.

[0266] In some embodiments, the source cells include common lymphoid progenitor cells characterized as CD45+CD7+CD56-.

[0267] In some embodiments, NK cells can be produced by induced pluripotent stem cells (iPSC). iPSC is a type of pluripotent stem cells derived from adult cells, which are genetically reprogrammed to an embryonic stem cell-like state by forcibly expressing genes and factors important for maintaining the defining characteristics of embryonic stem cells. iPSC can be produced from tissues with somatic cells, including but not limited to skin, dental tissue, peripheral blood and urine. In order to produce iPSC, somatic cells can be reprogrammed by the following method, including but not limited to transient expression of reprogramming factors, virus-free methods, adenovirus, plasmid, minicircle vector (minicircle vector), episomal vector, Sendai virus, synthetic mRNA, self-replicating RNA, retrovirus, slow virus, PhiC31 integrase, excisable transposon, gene editing or recombinant protein based on CRISPR. A method for producing iPSC is disclosed in US9315779B2, US10370452B2, US11319555B2 and US20210015859A1, and these documents are incorporated herein by reference as a whole. Mesoderm / embryoid body formation

[0268] In some embodiments, the methods described herein include generating mesodermal cells from iPSC and / or hESC. When stem cells begin to differentiate, three different germ layers are formed: ectoderm, mesoderm, and endoderm. Immune cells, such as NK cells, are differentiated from mesodermal cells. In some embodiments, the mesodermal cells generated by the methods disclosed herein are further differentiated into NK cells.

[0269] The mesoderm forming step may comprise contacting the iPSC or hESC cell population with one or more factors in a defined expansion medium for a specific period of time. In some embodiments, the mesoderm cells are formed from embryoid bodies.

[0270] In some embodiments, stem cells are contacted with differentiation medium as described herein for a period of time to produce mesodermal cells and / or embryoid bodies. In some embodiments, the iPSC or hESC cell colony is contacted with the differentiation medium in a 3D culture system for a period of time sufficient to produce mesodermal cells and / or embryoid bodies. In some embodiments, the period of time sufficient to produce mesodermal cells from stem cells is at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

[0271] In some embodiments, the duration of the mesoderm formation step is about 12 hours to 24 hours, about 24 hours to 48 hours, about 48 hours to 72 hours, about 72 hours to 96 hours, or about 96 hours to 120 hours.

[0272] In some embodiments, cells derived from source cells are placed in a container to induce cell aggregation and cluster formation. In some embodiments, the container is a plate with holes or microwells, such as a 96-well plate and / or an Aggrewell plate. TM Plates (microplates; STEMCELL Technologies Inc., Vancouver, Canada). In some embodiments, Aggrewell TM Plates are prepared in plates with micropores to form cell aggregates of uniform size and shape. In some embodiments, at least 1 cell, at least 10 cells, at least 100 cells, at least 1,000 cells, at least 10,000 cells, or at least 50,000 cells are seeded in each well. In some embodiments, about 1 cell to 10 cells, about 10 cells to 100 cells, about 100 cells to 1,000 cells, about 1,000 cells to 10,000 cells, or about 10,000 cells to 50,000 cells are seeded in each well. Differentiation into hematopoietic progenitor cells

[0273] In some aspects, the present disclosure provides a method for producing NK cells from hematopoietic progenitor cells. In some embodiments, the method for producing NK cells includes differentiating the hematopoietic progenitor cells into NK cells. In some embodiments, the methods provided herein are xeno-free.

[0274] One aspect of the present invention is that the method for producing NK cells may include a hematopoietic progenitor cell differentiation step. The hematopoietic progenitor cell differentiation step may include contacting a specific time period with an embryoid body cell colony and one or more factors in a well-defined differentiation medium, thereby inducing the formation of hematopoietic progenitor cells in the cell colony. Hematopoietic progenitor cells are then defined by a combination of expression markers.

[0275] In some embodiments, mesoderm and / or embryoid somatic cells are contacted with differentiation medium as described herein for a period of time to produce hematopoietic progenitor cells. In some embodiments, the mesoderm and / or embryoid somatic cells are contacted with the differentiation medium in a 3D culture system to be enough to produce a time period of hematopoietic progenitor cells. In some embodiments, the time period sufficient to produce hematopoietic progenitor cells by mesoderm and / or embryoid somatic 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.

[0276] In some embodiments, the duration of the step of differentiating into hematopoietic progenitor cells is about 1 day to 2 days, about 2 days to 3 days, about 3 days to 4 days, about 4 days to 5 days, about 5 days to 6 days, about 6 days to 7 days, about 7 days to 8 days, about 8 days to 9 days, about 9 days to 10 days, about 10 days to 11 days, about 11 days to 12 days, about 12 days to 13 days, about 13 days to 14 days, about 14 days to 15 days, about 15 days to 16 days, about 16 days to 17 days, about 17 days to 18 days, about 18 days to 19 days, or about 19 days to 20 days.

[0277] In some embodiments, the hematopoietic progenitor cells express CD34, CD43 and CD45. In some embodiments, the methods of the present disclosure increase the percentage of CD34+CD43+CD45+ triple positive cells.

[0278] In some embodiments, the methods of the present disclosure generate a cell population of CD34+CD43+CD45+ triple positive 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%.

[0279] In some embodiments, the methods of the present disclosure produce a cell population of CD34+CD43+CD45+ triple positive cells from iPSCs with a purity of about 40% to 50%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90%, or about 90% to 100%. Differentiation into NK cells

[0280] In some aspects, the disclosure provides methods for producing NK cells from hematopoietic progenitor cells. In some embodiments, the method for producing NK cells comprises differentiating hematopoietic progenitor cells as described. In some embodiments, the methods provided herein are xeno-free.

[0281] One aspect of the present disclosure is that the method for producing NK cells may include an NK differentiation step. The NK differentiation step may include contacting a HP cell population with one or more factors in a differentiation medium with clear components for a specific time period, thereby inducing the formation of NK cells in the cell population. In some embodiments, the HP cell population is contacted with the differentiation medium in a 3D suspension culture for a time period sufficient to form NK cells. NK cells are then defined by a combination of expression markers.

[0282] In some embodiments, hematopoietic progenitor cells are contacted with differentiation medium as described herein for a period of time to produce NK cells. In some embodiments, the time period sufficient to produce NK cells from hematopoietic progenitor cells is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days or at least 40 days.

[0283] In some embodiments, the duration of the NK differentiation step is about 1 day to 2 days, about 2 days to 3 days, about 3 days to 4 days, about 4 days to 5 days, about 5 days to 6 days, about 6 days to 7 days, about 7 days to 8 days, about 8 days to 9 days, about 9 days to 10 days, about 10 days to 11 days, about 11 days to 12 days, about 12 days to 13 days, about 13 days to 14 days, about 14 days to 15 days, about 15 days to 16 days, about 16 days to 17 days, about 17 days to 18 days, about 18 days to 19 days, or about 19 days to 20 days, about From about 20 days to 21 days, from about 21 days to 22 days, from about 22 days to 23 days, from about 23 days to 24 days, from about 24 days to 25 days, from about 25 days to 26 days, from about 26 days to 27 days, from about 27 days to 28 days, from about 28 days to 29 days, from about 29 days to 30 days, from about 30 days to 31 days, from about 31 days to 32 days, from about 32 days to 33 days, from about 33 days to 34 days, from about 34 days to 35 days, from about 35 days to 36 days, from about 36 days to 37 days, from about 37 days to 38 days, from about 38 days to 39 days, or from about 39 days to 40 days.

[0284] In some embodiments, the differentiated NK cells comprise the markers CD34, CD43, CD45, and LFA1. In some embodiments, the methods of the present disclosure increase the percentage of CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0285] In some embodiments, the methods of the present disclosure produce a NK cell population of CD34+CD43+CD45+LFA1+ quadruple positive 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% from hematopoietic progenitor cells.

[0286] In some embodiments, the methods of the present disclosure produce a NK cell population of CD34+CD43+CD45+LFA1+ quadruple positive cells with a purity of about 40% to 50%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90%, or about 90% to 100% from hematopoietic progenitor cells. NK cell maturation

[0287] In some aspects, the present disclosure provides a method for producing mature NK cells from differentiated NK cells. In some embodiments, the method for producing mature NK cells includes differentiating the NK cells. In some embodiments, the method provided herein is xeno-free.

[0288] One aspect of the present disclosure is that the method for producing NK cells may include an NK maturation step. The NK cell maturation step may include contacting a differentiated NK cell population with one or more factors in a component-defined expansion medium for a specific time period, thereby inducing NK cell maturation in the cell population. In some embodiments, the method includes contacting a differentiated NK cell population with one or more factors in a 3D culture system for a time period sufficient to induce NK cell maturation. Mature NK cells are then defined by a combination of expression markers.

[0289] In some embodiments, the differentiated NK cells are contacted with a maturation medium as described herein for a period of time to produce mature NK cells. In some embodiments, the time period sufficient to produce mature NK cells from hematopoietic progenitor cells is at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 168 hours, at least 192 hours, at least 216 hours, or at least 240 hours.

[0290] In some embodiments, the duration of the maturation step is about 12 hours to 24 hours, about 24 hours to 48 hours, about 48 hours to 72 hours, about 72 hours to 96 hours, about 96 hours to 120 hours, about 120 hours to 144 hours, about 144 hours to 168 hours, about 168 hours to 192 hours, about 192 hours to 216 hours, or about 216 hours to 240 hours.

[0291] In some embodiments, the mature NK cells comprise the markers CD34, CD43, CD45, and LFA1. In some embodiments, the methods of the present disclosure increase the percentage of CD34+CD43+CD45+LFA1+ quadruple positive cells. In some embodiments, the methods increase the expression of activation markers. In some embodiments, the activation markers include NKp46, NKG2D, LFA1, and / or CD16. In some embodiments, the methods reduce the expression of inhibitory markers. In some embodiments, the inhibitory markers include CD161 and CD73.

[0292] In some embodiments, the methods of the present disclosure produce a mature NK cell population of CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- 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% from differentiated NK cells.

[0293] In some embodiments, the methods of the present disclosure produce a mature NK cell population of CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells with a purity of about 40% to 50%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90% or about 90% to 100% from differentiated NK cells.

[0294] In some embodiments, the maturation step reduces the CD56- cell population.

[0295] In some embodiments, the methods of the present disclosure produce CD56- 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 a mature NK cell population.

[0296] In some embodiments, the methods of the present disclosure produce a mature NK cell population with a CD56- cell purity of about 40% to 50%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90%, or about 90% to 100% from differentiated NK cells. Exemplary Differentiation Methods

[0297] In some embodiments, the method for differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising BMP4, FGF2 and VEGF in a 3D culture system. In some embodiments, the method for differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising 1-50ng / mL BMP4, 1-150ng / mL FGF2 and 5-100ng / mL VEGF in a 3D culture system. In some embodiments, the method for differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising BMP4, FGF2 and VEGF in a 3D culture system for 12 to 120 hours. In some embodiments, the method for differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising 1-50ng / mL BMP4, 1-150ng / mL FGF2 and 5-100ng / mL VEGF in a 3D culture system for 12 to 120 hours.

[0298] In some embodiments, the method for differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising BMP4, FGF2, VEGF and ROCK inhibitors in a 3D culture system. In some embodiments, the method for differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising 1-50ng / mL BMP4, 1-150ng / mL FGF2, 5-100ng / mL VEGF and 0.1-20μM ROCK inhibitors in a 3D culture system. In some embodiments, the method for differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising BMP4, FGF2, VEGF and ROCK inhibitors in a 3D culture system for 12-120 hours. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM ROCK inhibitor in a 3D culture system for 12 to 120 hours.

[0299] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a serum-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 in 3D culture. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising 1-50ng / mL BMP4, 1-150ng / mL FGF2, 5-100ng / mL VEGF and 0.1-20μM Y27632 in a 3D culture system. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells includes contacting a stem cell population with a xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 in a 3D culture system for 12-120 hours. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises contacting a stem cell population with a xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM Y27632 in a 3D culture system for 12 to 120 hours.

[0300] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2 and VEGF in a 3D culture system to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in a 3D culture system to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, and 5-100 ng / mL VEGF in a 3D culture system to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 μg / ml LDL, and about 1-100 ng / mL TPO in a 3D culture system to produce hematopoietic progenitor cells. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2 and VEGF in a 3D culture system for 12-120 hours to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in 3D culture for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2 and 5-100 ng / mL VEGF in a 3D culture system for 12-120 hours to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL and about 1-100 ng / mL TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells.

[0301] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and a ROCK inhibitor in a 3D culture system to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in a 3D culture system to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM ROCK inhibitor in a 3D culture system to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising -50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, and about 1-100 ng / mL TPO in a 3D culture system to produce hematopoietic progenitor cells. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and a ROCK inhibitor in a 3D culture system for 12-120 hours to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM ROCK inhibitor in a 3D culture system for 12-120 hours to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 μg / ml LDL, and about 1-100 ng / mL TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells.

[0302] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 in a 3D culture system to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in a 3D culture system to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM Y27632 in a 3D culture system to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising -50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 ug / mL LDL, and about 1-100 ng / mL TPO in a 3D culture system to produce hematopoietic progenitor cells. In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 in a 3D culture system for 12-120 hours to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells. In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, and 0.1-20 μM Y27632 in a 3D culture system for 12-120 hours to produce a mesodermal cell population, and (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising -50 ng / mL BMP4, 1-150 ng / mL FGF2, 5-100 ng / mL VEGF, about 1-100 ng / mL SCF, about 1-50 μg / ml LDL, and about 1-100 ng / mL TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells.

[0303] In some embodiments, the hematopoietic progenitor cell formation step is followed by a NK cell differentiation step. In some embodiments, the method of HP differentiation into NK comprises contacting the HP cell population with a xeno-free NK differentiation medium in a 3D culture system. In some embodiments, the method of HP differentiation into NK comprises contacting the HP cell population with a xeno-free NK differentiation medium in a 3D culture system for 15-25 days.

[0304] In some embodiments, the method of differentiating stem cells into NK cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2 and VEGF to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and a PI3K inhibitor to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system to produce NK cells.

[0305] In some embodiments, the method of differentiating stem cells into NK cells comprises (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, and 5-100 ng / mL VEGF to produce a population of mesodermal cells, (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 μg / ml LDL, about 100 ng / mL TPO, and 5-100 μM of a PI3K inhibitor to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free NK differentiation medium in a 3D culture system to produce NK cells.

[0306] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2 and VEGF in a 3D culture system for 12-120 hours to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO and a PI3K inhibitor in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free NK differentiation medium in a 3D culture system for 15-25 days to produce NK cells.

[0307] In some embodiments, the method of differentiating stem cells into NK cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and a ROCK inhibitor in a 3D culture system to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in a 3D culture system to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system to produce NK cells.

[0308] In some embodiments, a method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM ROCK inhibitor in a 3D culture system to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 μg / ml LDL, and about 100 ng / mL TPO in a 3D culture system to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system to produce NK cells.

[0309] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor in a 3D culture system for 12-120 hours to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system for 15-25 days to produce NK cells.

[0310] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF and 1-20 μM ROCK inhibitor in a 3D culture system for 12-120 hours to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 μg / ml LDL and about 100 ng / mL TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system for 15-25 days to produce NK cells.

[0311] In some embodiments, the method of differentiating stem cells into NK cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 in a 3D culture system to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in a 3D culture system to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system to produce NK cells.

[0312] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF and 1-20 μM Y27632 in a 3D culture system to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 μg / ml LDL and about 100 ng / mL TPO in a 3D culture system to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system to produce NK cells.

[0313] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF and Y27632 in a 3D culture system for 12-120 hours to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL and TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system for 15-25 days to produce NK cells.

[0314] In some embodiments, the method of differentiating stem cells into hematopoietic progenitor cells comprises (i) contacting a stem cell population with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF and 1-20 μM Y27632 in a 3D culture system for 12-120 hours to produce a mesodermal cell population, (ii) contacting the mesodermal cell population with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 μg / ml LDL and about 100 ng / mL TPO in a 3D culture system for 2-20 days to produce hematopoietic progenitor cells, and (iii) contacting the hematopoietic progenitor cell population with a third xeno-free NK differentiation medium in a 3D culture system for 15-25 days to produce NK cells.

[0315] In some or any of the foregoing embodiments, the NK differentiation medium comprises SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimidoindole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the pyrimidoindole derivative is UM729. In some embodiments, the aryl hydrocarbon receptor is SR1. In some embodiments, the NK differentiation medium comprises SCF, IL-7, IL-15, IL-12, FLT3L, UM729, and SR1.

[0316] In some or any of the foregoing embodiments, the NK differentiation medium comprises 5-50ng / mL SCF, 5-50ng / mL IL-7, 5-100ng / mL IL-15, 5-100ng / mL IL-12, 5-100ng / mL FLT3L, 1-10μM pyrimidoindole derivatives, and 1-10μM aryl hydrocarbon receptor antagonists. In some embodiments, the NK differentiation medium comprises 5-50ng / mL SCF, 5-50ng / mL IL-7, 5-100ng / mL IL-15, 5-100ng / mL IL-12, 5-100ng / mL FLT3L, 1-10μM UM729, and 1-10μM SR1.

[0317] In some or any of the foregoing embodiments, the 3D culture system is agitated at about 50 RPM to about 100 RPM.In some or any of the foregoing embodiments, the 3D culture system is a bioreactor.

[0318] In some embodiments, the stem cells and cells differentiated therefrom comprise gene editing. In some embodiments, the gene editing is gene knockout. In some embodiments, the gene editing is FKBP12 gene knockout. In some embodiments, the gene editing is B2M gene knockout.

[0319] In some embodiments, the stem cell comprises a knock-in gene. In some embodiments, the knock-in gene encodes an exogenous receptor. In some embodiments, the exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the exogenous receptor is a rapamycin-activated cytokine receptor (RACR). In some embodiments, the stem cell and the cells differentiated therefrom comprise gene knockout and gene knock-in. In some embodiments, the gene knock-in is located at the gene knockout. Characteristics of NK cells

[0320] In some embodiments, NK cells produced by the 3D suspension culture methods of the present disclosure have improved or enhanced properties compared to NK cells produced by 2D culture methods.

[0321] In some embodiments, the NK cells produced by the 3D suspension culture methods of the present disclosure have enhanced expansion. In some embodiments, the NK cells have at least 50-fold expansion, at least 100-fold expansion, at least 150-fold expansion, at least 200-fold expansion, at least 250-fold expansion, at least 300-fold expansion, at least 350-fold expansion, at least 400-fold expansion, at least 450-fold expansion, at least 500-fold expansion, at least 1,000-fold expansion, at least 10,000-fold expansion, at least 100,000-fold expansion, or at least 1,000,000-fold expansion compared to NK cells produced by 2D culture methods.

[0322] In some embodiments, the NK cells have about 50-fold to 100-fold expansion, about 100-fold to 150-fold expansion, about 150-fold to 200-fold expansion, about 200-fold to 250-fold expansion, about 250-fold to 300-fold expansion, about 300-fold to 350-fold expansion, about 350-fold to 400-fold expansion, about 400-fold to 450-fold expansion, about 450-fold to 500-fold expansion, about 500-fold to 1,000-fold expansion, about 1,000-fold to 10,000-fold expansion, about 10,000-fold to 100,000-fold expansion, or about 100,000-fold to 1,000,000-fold expansion compared to NK cells produced by other 2D culture methods.

[0323] In some embodiments, the differentiated NK cells produced by the 3D suspension culture method of the present disclosure reduce more tumor cell growth than the differentiated NK cells produced by the 2D culture method. In some embodiments, the differentiated NK cells produced by the 3D suspension culture method reduce at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of tumor cell growth more than the differentiated NK cells produced by the 2D culture method.

[0324] In some embodiments, differentiated NK cells produced by a 3D suspension culture method reduce tumor cell growth by about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90%, or about 90% to 100% more than differentiated NK cells produced by a 2D culture method.

[0325] In some embodiments, mature NK cells produced by the 3D suspension culture method of the present disclosure reduce more tumor cell growth compared to the 2D culture method. In some embodiments, mature NK cells produced by the 3D suspension culture method reduce tumor cell growth by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% more than mature NK cells produced by the 2D culture method.

[0326] In some embodiments, mature NK cells produced by a 3D suspension culture method reduce tumor cell growth by about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90%, or about 90% to 100% more than mature NK cells produced by a 2D culture method.

[0327] In some embodiments, the methods of the present disclosure produce differentiated NK cell populations. In some embodiments, the 3D suspension culture methods of the present disclosure produce differentiated NK cell populations of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% more CD34+CD43+CD45+LFA1+ quadruple positive cells compared to the 2D culture methods.

[0328] In some embodiments, the 3D suspension culture method of the present disclosure produces about 40% to 50%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90%, or about 90% to 100% more differentiated NK cell populations of CD34+CD43+CD45+LFA1+ quadruple positive cells compared to the 2D culture method.

[0329] In some embodiments, the 3D suspension culture method of the present disclosure produces a mature NK cell population. In some embodiments, the method of the present disclosure produces at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% more CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells of mature NK cell population compared to the 2D culture method.

[0330] In some embodiments, compared to the 2D culture method, the 3D suspension culture method of the present disclosure produces about 40% to 50%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90% or about 90% to 100% more mature NK cell populations of CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells. Gene Editing

[0331] Genome editing generally refers to the process of editing or changing the nucleotide sequence of a genome, preferably in an accurate, desired and / or predetermined manner. Examples of the compositions, systems and methods of genome editing described herein use site-directed nucleases to shear (cut) or cut (cleave) DNA at the precise target position in the genome, thereby producing double-strand breaks (DSBs) in DNA. Such breaks can be repaired by endogenous DNA repair pathways, such as homology-directed repair (HDR) and / or non-homologous end joining (NHEJ) repair (see, e.g., Cox et al., (2015) Nature Medicine 21 (2): 121-31).

[0332] In some embodiments, the cells described herein (e.g., stem cells, HP, NK) are genetically modified. In some embodiments, the modification involves knocking out one or more endogenous genes and / or knocking in exogenous target genes using DNA targeting proteins and nucleases or RNA-guided nucleases. In some embodiments, the target gene is knocked into a specific target locus. In some embodiments, the target gene is a synthetic cytokine receptor complex. In some embodiments, the target gene is a chimeric antigen receptor (CAR). In some embodiments, RACR is knocked into the target locus.

[0333] In some embodiments, the transformation comprises contacting the cell with a DNA targeting protein and a nuclease or an RNA guided nuclease. In some embodiments, the DNA targeting protein and the nuclease or the RNA guided nuclease comprises a zinc finger protein (ZFP), a clustered regularly interspaced short palindromic nucleic acid (CRISPR) or a TAL effector nuclease (TALEN). In some embodiments, Crispr-CAS9 is used. In some embodiments, Crispr-MAD7 is used.

[0334] Rejection of cell therapy agents (e.g., CAR T cells) is at least due to the mismatch of human leukocyte antigens (HLA) between donors and recipients. A recently determined solution is to destroy the expression of genes involved in the rejection, such as T cell receptor alpha constant region (TRAC), beta-2-microglobulin (B2M) and signal regulatory protein alpha (SIRPA). Therefore, in some embodiments, cells described herein (e.g., iPSC, CIL) are genetically engineered to knock out B2M loci, TRAC loci and / or SIRPA loci. In some embodiments, cells described herein are genetically engineered to knock out B2M loci. In some embodiments, cells described herein are genetically engineered to knock out TRAC loci. In some embodiments, cells described herein are genetically engineered to knock out SIPRA loci.

[0335] In some embodiments, the cells described herein are genetically engineered to have rapamycin resistance. In some embodiments, the cells are genetically engineered to disrupt genes associated with rapamycin recognition. In some embodiments, the cells are genetically engineered to disrupt the mTOR gene. In some embodiments, the cells are genetically engineered to disrupt the FKBP12 gene. In some embodiments, the cells are genetically engineered to knock out the FKB12 ​​gene, thereby inducing rapamycin resistance.

[0336] In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in an endogenous gene. In some embodiments, the synthetic cytokine receptor is engineered into the gene so that expression of the endogenous gene is not disrupted. In some embodiments, the synthetic cytokine receptor is engineered into a safe harbor locus.

[0337] In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB).

[0338] In some embodiments, the target gene inserted into the endogenous locus is a synthetic cytokine receptor complex. In some embodiments, an endogenous promoter of a specific locus is used. In some embodiments, additional promoters may be included so that two or more promoters drive the expression of an exogenous target gene.

[0339] In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor complex in a disrupted gene. For example, in some embodiments, the cells include a disrupted B2M gene and a nucleotide sequence encoding a synthetic cytokine receptor in the disrupted B2M gene.

[0340] In some embodiments, the cells described herein (eg, iPSCs, CILs) comprise (i) a disrupted B2M locus, and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex under the control of an endogenous B2M promoter and an EEF1A promoter.

[0341] In some embodiments, the cells described herein (e.g., iPSC, HP, NK) comprise (i) a disrupted B2M locus, and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex inserted into the endogenous B2M gene and under the control of the endogenous B2M promoter and the EEF1A promoter. Genome Editing System

[0342] In some embodiments, the system for editing cells described herein comprises a site-directed nuclease, such as a CRISPR / Cas system and an optional gRNA. In some embodiments, the system comprises an engineered nuclease. In some embodiments, the system comprises a site-directed nuclease. In some embodiments, the site-directed nuclease comprises a CRISPR / Cas nuclease system. In some embodiments, the Cas nuclease is Cas9. In some embodiments, the nuclease is Mad7. In some embodiments, the guide RNA comprising the CRISPR / Cas system is an sgRNA. CRISPR / Cas Core Acid enzyme system

[0343] The naturally occurring CRISPR / Cas system is a genetic defense system that provides a form of acquired immunity in prokaryotes. CRISPR is a clustered regularly interspaced short palindromic repeat (CRISPR) C lustered R egularly I Interspaced S hort P alindromic R CRISPR is an abbreviation for epeats, a family of DNA sequences found in the genomes of bacteria and archaea that contain DNA fragments (spacer DNA) that are similar to foreign DNA previously exposed to the cell (e.g., by a virus that infects or attacks prokaryotes). These DNA fragments are used by prokaryotes to detect and destroy similar foreign DNA that is reintroduced (e.g., from a similar virus in a subsequent attack). Transcription of the CRISPR locus results in the formation of RNA molecules containing the spacer sequence, which bind to and target the Cas (CRISPR-associated) protein that can recognize and cut foreign exogenous DNA. Many types and categories of CRISPR / Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37: 67-78).

[0344] Engineered versions of the CRISPR / Cas system have been developed in a variety of forms to mutate or edit the genomic DNA of cells from other species. The general method of using the CRISPR / Cas system involves heterologous expression or introduction of a site-directed nuclease (e.g., Cas nuclease) in combination with a guide RNA (gRNA) into a cell, resulting in a DNA cleavage event (e.g., formation of a single-strand or double-strand break (SSB or DSB)) at a precise, targetable location in the cellular genomic DNA backbone. The way in which the DNA cleavage event is repaired by the cell provides an opportunity to edit the genome by adding, removing, or modifying (substituting) DNA nucleotides or sequences (e.g., genes). i. Guide RNA (gRNA)

[0345] The engineered CRISPR / Cas system comprises at least two components: 1) guide RNA (gRNA) molecules and 2) Cas nucleases, which interact to form gRNA / Cas nuclease complexes. gRNA at least comprises a user-defined targeting domain called "spacer", which comprises a nucleotide sequence and CRISPR repeats. In the engineered CRISPR / Cas system, by generating a gRNA comprising a spacer, the gRNA / Cas nuclease complex is targeted to a specific target sequence of interest in a target nucleic acid (e.g., a genomic DNA molecule), and the spacer has a nucleotide sequence that can bind to the specific target sequence in a complementary manner (see Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011)). Therefore, the spacer provides the targeting function of the gRNA / Cas nuclease complex.

[0346] In naturally occurring type II CRISPR / Cas systems, "gRNA" consists of two RNA chains: 1) CRISPR RNA (crRNA) containing spacers and CRISPR repeats, and 2) trans-activating CRISPR RNA (tracrRNA). In type II CRISPR / Cas systems, a portion of crRNA containing CRISPR repeats and a portion of tracrRNA hybridize to form a crRNA:tracrRNA duplex, which interacts with a Cas nuclease (e.g., Cas9). As used herein, the term "split gRNA" or "modular gRNA" refers to a gRNA molecule comprising two RNA chains, wherein the first RNA chain integrates crRNA function and / or structure, the second RNA chain integrates tracrRNA function and / or structure, and wherein the first and second RNA chains partially hybridize.

[0347] Therefore, in some embodiments, the gRNA comprises two RNA molecules. In some embodiments, the gRNA comprises CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In some embodiments, the gRNA is a split gRNA. In some embodiments, the gRNA is a modular gRNA. In some embodiments, the split gRNA comprises a first strand, the first strand comprising a spacer and a first complementary region from 5' to 3'; and a second strand, the second strand comprising a second complementary region from 5' to 3'; and optionally a tail domain.

[0348] In some embodiments, the crRNA includes a spacer comprising a nucleotide sequence complementary to and hybridized with a sequence complementary to a target sequence on a target nucleic acid (e.g., a genomic DNA molecule). In some embodiments, the crRNA includes a region complementary to and hybridized with a portion of the tracrRNA.

[0349] In some embodiments, the tracrRNA may include all or part of a wild-type tracrRNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the tracrRNA may include a truncated or modified variant of a wild-type tracrRNA. The length of the tracrRNA may depend on the CRISPR / Cas system used. In some embodiments, the tracrRNA may include a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 nucleotides. In certain embodiments, the length of the tracrRNA is at least 26 nucleotides. In additional embodiments, the length of the tracrRNA is at least 40 nucleotides. In some embodiments, the tracrRNA may include certain secondary structures, such as one or more hairpins or stem-loop structures, or one or more bulge structures. Single guide RNA (sgRNA)

[0350] The engineered CRISPR / Cas nuclease system is usually combined into a single RNA molecule by adding a joint between crRNA and tracrRNA, referred to herein as "single guide RNA" (sgRNA). Not bound by theory, similar to double-stranded crRNA and tracrRNA, sgRNA will form a complex with Cas nuclease (e.g., Cas9), guide Cas nuclease to the target sequence and activate Cas nuclease to cut target nucleic acid (e.g., genomic DNA). Therefore, in some embodiments, the gRNA may include operably connected crRNA and tracrRNA. In some embodiments, the sgRNA may include a crRNA covalently linked to tracrRNA. In some embodiments, the crRNA and the tracrRNA are covalently linked via a joint. In some embodiments, the sgRNA may include a stem-loop structure by base pairing between the crRNA and the tracrRNA. In some embodiments, the sgRNA includes a spacer, a first complementary region, a connecting domain, a second complementary region, and an optional tail domain from 5' to 3'.

[0351] The sgRNA can be unmodified or modified. For example, the modified sgRNA can include one or more 2'-O-methyl phosphorothioate nucleotides.

[0352] As an illustration, the guide RNA or other smaller RNA used in the CRISPR / Cas system can be easily synthesized by chemical means, as described herein and in the art. Although the chemical synthesis procedure is constantly expanding, as the polynucleotide length increases significantly over 100 or so nucleotides, it is often more challenging to purify these RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE). A method for producing RNA of greater length is to produce two or more molecules connected together. Longer RNAs, such as RNA encoding Cas9 endonucleases, are more easily produced enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic production of RNA, such as enhancing stability, reducing the possibility or degree of innate immune response and / or enhancing other properties, as described in the art. Spacer

[0353] In some embodiments, the gRNA comprises a spacer sequence. The spacer sequence is a sequence that defines the target site of a target nucleic acid (e.g., DNA). The target nucleic acid is a double-stranded molecule: one strand comprises a target sequence adjacent to a PAM sequence, referred to as a "PAM strand," and the second strand is referred to as a "non-PAM strand," which is complementary to the PAM strand and the target sequence. Both the gRNA spacer and the target sequence are complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is complementary to the non-PAM strand of the target nucleic acid. Therefore, in some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is identical to the PAM strand. The gRNA spacer sequence hybridizes with the complementary strand (e.g., the non-PAM strand of the target nucleic acid / target site). In some embodiments, the spacer is sufficiently complementary to the complementary strand (e.g., non-PAM strand) of the target sequence to target the Cas nuclease to the target nucleic acid. In some embodiments, the spacer is at least 80%, 85%, 90%, or 95% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer is 100% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 1, 2, 3, 4, 5, 6 or more nucleotides that are not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 1 nucleotide that is not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 2 nucleotides that are not complementary to the non-PAM strand of the target nucleic acid.

[0354] In some embodiments, the 5' terminal nucleotide of the gRNA includes the 5' terminal nucleotide of the spacer. In some embodiments, the spacer is located at the 5' end of the crRNA. In some embodiments, the spacer is located at the 5' end of the sgRNA. In some embodiments, the length of the spacer is about 15-50, about 20-45, about 25-40, or about 30-35 nucleotides. In some embodiments, the length of the spacer is about 19-22 nucleotides. In some embodiments, the length of the spacer is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the spacer is 19 nucleotides. In some embodiments, the length of the spacer is 20 nucleotides, and in some embodiments, the length of the spacer is 21 nucleotides.

[0355] In some embodiments, the nucleotide sequence of the spacer is designed or selected using a computer program. The computer program may use variables such as predicted melting temperatures, secondary structure formation, predicted annealing temperatures, sequence identity, genomic background, chromatin accessibility, %GC, frequency of genomic occurrence (e.g., identical or similar but varying at one or more points due to mismatches, insertions or deletions), methylation status, and / or the presence of SNPs.

[0356] In some embodiments, the spacer comprises at least one or more modified nucleotides, such as those described herein. The invention provides gRNA molecules comprising spacers, which may comprise the nucleobase uracil (U), and any DNA encoding a gRNA comprising a spacer will comprise the nucleobase thymine (T) at the corresponding position, the spacer comprising the nucleobase uracil (U). ii. Method for preparing gRNA

[0357] Methods for preparing gRNA are known to those skilled in the art and include, but are not limited to, in vitro transcription (IVT), synthesis and / or chemical synthesis methods, or combinations thereof. Enzymatic (IVT), solid phase, liquid phase, combinatorial synthesis methods, small area synthesis, and ligation methods are used. In one embodiment, gRNA is prepared using IVT enzymatic synthesis methods. Methods for preparing polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062. Therefore, the present invention also includes polynucleotides, such as DNA, constructs, and vectors, for in vitro transcription of the gRNA described herein.

[0358] In some embodiments, a non-naturally modified nucleobase is introduced into a polynucleotide, such as a gRNA, during or after synthesis. In certain embodiments, the modification is on a nucleoside linkage, a purine or pyrimidine base, or a sugar. In some embodiments, the modification is introduced at the end of a polynucleotide; using chemical synthesis or using a polymerase. Examples of modified nucleic acids and their synthesis are disclosed in PCT application PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0359] In some embodiments, enzymatic or chemical ligation methods are used to conjugate polynucleotides or regions thereof to different functional moieties (e.g., targeting or delivery agents, fluorescent markers, liquids, nanoparticles, etc.) Conjugates of polynucleotides and engineered polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0360] In some embodiments, the disclosure provides nucleic acids encoding gRNA described herein, such as vectors. In some embodiments, the nucleic acid is a DNA molecule. In other embodiments, the nucleic acid is an RNA molecule. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding crRNA. In some embodiments, the nucleotide sequence encoding crRNA comprises a spacer flanking all or part of a repetitive sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding tracrRNA. In some embodiments, the crRNA and the tracrRNA are encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracrRNA are encoded by a single nucleic acid. In some embodiments, the crRNA and the tracrRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the tracrRNA are encoded by the same strand of a single nucleic acid.

[0361] In some embodiments, the gRNA provided by the present disclosure is chemically synthesized by any method described in the art (see, e.g., WO / 2005 / 01248). Although chemical synthesis procedures are constantly expanding, as the length of polynucleotides increases significantly beyond 100 or so nucleotides, purification of these RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging. A method for producing RNA of greater length is to produce two or more molecules connected together.

[0362] In some embodiments, more than one guide RNA can be used with the CRISPR / Cas nuclease system. Each guide RNA can contain different targeting sequences so that the CRISPR / Cas system cuts more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability within the Cas9 RNP complex. When more than one guide RNA is used, each guide RNA can be encoded on the same or different vectors. The promoter used to drive the expression of more than one guide RNA is the same or different.

[0363] The guide RNA can target any sequence of interest through the targeting sequence (e.g., spacer sequence) of the crRNA. In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule are 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1-6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 5 or 6 mismatches.

[0364] The length of the targeting sequence may depend on the CRISPR-Cas system and components used. For example, different Cas9 proteins from different bacterial species have different optimal targeting sequence lengths. Therefore, the length of the targeting sequence can be 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, 35, 40, 45, 50 or more than 50 nucleotides. In some embodiments, the length of the targeting sequence can be 18-24 nucleotides. In some embodiments, the length of the targeting sequence can be 19-21 nucleotides. In some embodiments, the length of the targeting sequence can be 20 nucleotides.

[0365] In some embodiments of the present disclosure, the CRISPR / Cas nuclease system includes at least one guide RNA. In some embodiments, the guide RNA and the Cas protein can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex. The guide RNA can guide the Cas protein to a target sequence on a target nucleic acid molecule (e.g., a genomic DNA molecule), wherein the Cas protein cuts the target nucleic acid. In some embodiments, the CRISPR / Cas complex is a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex is a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex is a Cas9 / guide RNA complex. In some embodiments, the CRISPR / Cas complex is an engineered 2-type V-type CRISPR system. In some embodiments, the endonuclease is Mad7. iii. Cas nuclease

[0366] In some embodiments, the present disclosure provides compositions and systems (e.g., engineered CRISPR / Cas systems) comprising a site-directed nuclease, wherein the site-directed nuclease is a Cas nuclease. The Cas nuclease may include at least one domain that interacts with a guide RNA (gRNA). In addition, the Cas nuclease is directed to a target sequence by a guide RNA. The guide RNA interacts with the Cas nuclease and the target sequence so that once the target sequence is directed, the Cas nuclease is able to cut the target sequence. In some embodiments, the guide RNA provides specificity for cutting the target sequence, and the Cas nuclease is universal and paired with different guide RNAs to cut different target sequences.

[0367] In some embodiments, the CRISPR / Cas system includes components derived from type I, type II, or type III systems. The latest classification scheme for CRISPR / Cas loci defines class 1 and class 2 CRISPR / Cas systems, with types I to V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR / Cas systems have a single protein effector. Type II, type V, and type VI Cas proteins are single protein, RNA-guided endonucleases, referred to herein as "class 2 Cas nucleases". Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0368] In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is from a type 2 CRISPR / Cas system (a single protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein). Proteins of the Cas9 and Cpf1 families are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing appropriate guide RNAs, as further described herein.

[0369] Type II CRISPR / Cas system components are from Type IIA, Type IIB, or Type IIC systems. Cas9 and its orthologs are contemplated. Non-limiting exemplary species from which the Cas9 nuclease or other components may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.), Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis), Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldariu, Bacillus pseudomycoide, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivariu, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium), Polaromonas naphthalenivorans, Polaromonas sp.), Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus), Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacterracemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes), Oscillatoria sp.), Petrotoga mobilis, Thermosiphoafricanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, or Acaryochloris marina. In some embodiments, the Cas9 protein is from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein is from Streptococcus thermophilus (StCas9). In some embodiments, the Cas9 protein is from Neisseria meningitidis (NMCAS9). In some embodiments, the Cas9 protein is from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 protein is from Campylobacter jejuni (CjCas9).

[0370] In some embodiments, the Cas nuclease may include more than one nuclease domain. For example, the Cas9 nuclease may include at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces DSB in the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to contain no functional HNH-like nuclease domain. In some embodiments in which only one nuclease domain is functional, the Cas9 nuclease is a nickase capable of introducing a single-strand break ("nick") into a target sequence. In some embodiments, the conservative amino acids in the Cas9 nuclease domain are substituted to reduce or change the nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid replacement in a RuvC-like nuclease domain. Exemplary amino acid replacements in the RuvC-like nuclease domain include D10A (based on Streptococcus pyogenes (S.pyogenes) Cas9 nuclease). In some embodiments, the nickase comprises an amino acid replacement in a HNH-like nuclease domain. Exemplary amino acid replacements in the HNH-like nuclease domain include E762A, H840A, N863A, H983A and D986A (based on Streptococcus pyogenes (S.pyogenes) Cas9 nuclease). In some embodiments, the nuclease system described herein includes a nickase and a pair of guide RNAs, which are complementary to the sense strand and antisense strand of the target sequence, respectively. The guide RNA guides the nickase targeting and introduces DSB by producing a nick (i.e., double nick) on the opposite strand of the target sequence. Using a chimeric Cas9 nuclease, a domain or region of the protein is replaced by a portion of a different protein. For example, the Cas9 nuclease domain is replaced with a domain from a different nuclease, such as Fok1. The Cas9 nuclease is an engineered nuclease.

[0371] In some embodiments, the Cas nuclease is from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of a cascade complex of a type I CRISPR / Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type VI CRISPR / Cas system.

[0372] In some embodiments, the Cas nuclease is a Mad endonuclease. The CRISPR / Mad system is closely related to type V (Cpf1-like) of the 2-family of Cas enzymes. In some embodiments, the CRISPR-Mad system employs Eubacterium rectale Mad7 endonuclease or a variant thereof. The Mad7-crRNA complex cuts the target DNA by identifying PAM5'-YTTN. Engineered nucleases

[0373] In some embodiments, the cells described herein are genetically engineered with a site-directed nuclease, wherein the site-directed nuclease is an engineered nuclease. Exemplary engineered nucleases are meganucleases (e.g., homing endonucleases), ZFNs, TALENs, and megaTALs.

[0374] Naturally occurring meganucleases can recognize and cut double-stranded DNA sequences of about 12 to 40 base pairs and are generally divided into five families. In some embodiments, the meganuclease is selected from the LAGLIDADG family, the GIY-YIG family, the HNH family, the His-Cys box family, and the PD-(D / E)XK family. In some embodiments, the DNA binding domain of the meganuclease is engineered to recognize and bind to sequences other than its homologous target sequence. In some embodiments, the DNA binding domain of the meganuclease is fused to a heterologous nuclease domain. In some embodiments, the meganuclease, such as a homing endonuclease, is fused to a TAL module to produce a hybrid protein, such as a "meganuclease". By recognizing the target sequence of the DNA binding domain of the meganuclease and the TAL module, the meganuclease has improved DNA targeting specificity.

[0375] ZFN is a fusion protein comprising a zinc finger DNA binding domain ("zinc finger" or "ZF") and a nuclease domain. Each naturally occurring ZF can bind to three consecutive base pairs (DNA triplet), and the ZF repeats are combined to recognize the DNA target sequence and provide sufficient affinity. Therefore, the engineered ZF repeats are combined to recognize longer DNA sequences, such as 9, 12, 15 or 18 base pairs (bp), etc. In some embodiments, the ZFN comprises a ZF fused to a nuclease domain from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain comprises a dimerization domain, such as when the nuclease is dimerized and active, and a pair of ZFNs comprising ZF repeats and nuclease domains are designed to target a target sequence comprising two half target sequences recognized by each ZF repeat on opposite strands of the DNA molecule, with a sequence connected to each other (sometimes referred to as a spacer in the literature). For example, the length of the interconnected sequence is 5 to 7 bp. When both ZFNs of the pair are bound, the nuclease domain can dimerize and introduce a DSB within the interconnected sequence. In some embodiments, the dimerization domain of the nuclease domain comprises a knob-into-hole motif that promotes dimerization. For example, the ZFN comprises a knob-into-hole motif in the dimerization domain of FokI.

[0376] The DNA binding domain of TALEN typically contains a variable number of 34 or 35 amino acid repeats ("modules" or "TAL modules"), each module binding a single DNA base pair, A, T, G or C. The adjacent residues at positions 12 and 13 of each module ("repeat-variable diresidues" or RVDs) specify the single DNA base pair that the module binds. Although the module for recognizing G may also have an affinity for A, TALEN benefits from a simple recognition code - there is one module for each of the 4 bases - which greatly simplifies the customization of the DNA binding domain that recognizes a specific target sequence. In some embodiments, TALEN may include a nuclease domain from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain can dimerize to be active, and a pair of TALENs are designed for targeting a target sequence, which includes two half-target sequences recognized by each DNA binding domain on opposite strands of the DNA molecule, with an interconnected sequence between the two half-target sequences. For example, each half target sequence is in the range of 10 to 20 bp, and the length of the interconnected sequence is 12 to 19 bp. When the two TALENs of the pair are bound, the nuclease domain can dimerize and introduce a DSB within the interconnected sequence. In some embodiments, the dimerization domain of the nuclease domain can include a knob-in-hole motif that promotes dimerization. For example, the TALEN can include a knob-in-hole motif in the dimerization domain of FokI. Target

[0377] In some embodiments, the site-directed nuclease described herein directs and cuts (e.g., introduces DSB) a target nucleic acid molecule. In some embodiments, the target nucleic acid molecule is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1α (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin β (ACTB). In some embodiments, the target nucleic acid molecule is a bloodline gene. In some embodiments, the bloodline gene is a C-type protein tyrosine phosphatase receptor (PTPRC), IL2RG, or IL2RB. In some embodiments, the target nucleic acid is a gene associated with rapamycin response. In some embodiments, the target nucleic acid is FKBP12. In some embodiments, the target nucleic acid is B2M, TRAC, or SIRPA.

[0378] The target nucleic acid molecule is any DNA molecule endogenous or exogenous to the cell. As used herein, the term "endogenous sequence" refers to a sequence inherent to the cell. In some embodiments, the target nucleic acid molecule is a genomic DNA (gDNA) molecule or a chromosome from a cell or in a cell. In some embodiments, the target sequence of the target nucleic acid molecule is a genomic sequence from a cell or in a cell. In some embodiments, the target sequence may be located in a non-coding sequence between a coding sequence of a gene, an intron sequence of a gene, a transcription control sequence of a gene, a translation control sequence of a gene, or a gene. In some embodiments, the gene may be a protein coding gene. In other embodiments, the gene may be a non-coding RNA gene. In some embodiments, the target sequence may include all or part of a disease-related gene.

[0379] In some embodiments, the target sequence may be located at a non-gene functional site in the genome that controls aspects of chromatin organization, such as a scaffold site or a locus control region. In some embodiments, the target sequence may be a gene safe harbor site, i.e., a locus that promotes safe genetic modification.

[0380] In some embodiments, the target sequence may be adjacent to a protospacer adjacent motif (PAM), which is a short sequence recognized by the CRISPR / Cas complex. In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4 nucleotides of the 3' end of the target sequence. In some embodiments, the target sequence may include the PAM. The length and sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from a consensus sequence or a specific PAM sequence of a specific Cas nuclease or Cas ortholog, including Ran et al., (2015) Nature, 520:186-191 (2015) Figure 1Those disclosed in, which are incorporated herein by reference. In some embodiments, the PAM can be 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG (SpCas9 WT, SpCas9 nickase, dimer dCas9-Fok1, SpCas9-HF1, SpCas9 K855A, ESpCas9 (1.0), ESpCas9 (1.1)), NGAN or NGNG (SpCas9 VQR variant), NGAG (SpCas9 EQR variant), NGCG (SpCas9 VRER variant), NAAG (SpCas9 QQR1 variant), NNGRRT or NNGRRN (SaCas9), NNNRRT (KKH (SaCas9), NNNNRYAC (CjCas9), NNAGAAW (St1Cas9), NAACA (TdCas9), NGGNG (St3Cas9), NG (FnCas9), NAAAAN (TdCas9), NNAAAAW (StCas9), NNNNACA (CjCas9), GNNNCNNA (PmCas9), and NNNNGATT (NmCas9) (see, e.g., Cong et al., (2013) Science 339:819-823; Kleinstiver et al., (2015) Nat Biotechnol 33:1293-1298; Kleinstiver et al., (2015) Nature 523:481-485; Kleinstiver et al., (2016) Nature 529:490-495; Tsai et al., (2014) Nat Biotechnol 32:569-576; Slaymaker et al., (2016) Science 351:84-88; Anders et al., (2016) Mol Cell 61:895-902; Kim et al., (2017) Nat Comm 8:14500; Fonfara et al., (2013) Nucleic Acids Res 42:2577-2590; Garneau et al., (2010) Nature 468:67-71; Magadan et al., (2012) PLoS ONE7:e40913; Esvelt et al., (2013) Nat Methods 10(11):1116-1121 (wherein N is defined as any nucleotide, W is defined as A or T, R is defined as purine (A) or (G), and Y is defined as pyrimidine (C) or (T)). In some embodiments, the PAM sequence is NGG. In some embodiments, the PAM sequence is NGAN.In some embodiments, the PAM sequence is NGNG. In some embodiments, the PAM is NNGRRT. In some embodiments, the PAM sequence is NGGNG. In some embodiments, the PAM sequence can be NNAAAAW. Ribonucleoprotein

[0381] In some embodiments, the site-directed polypeptide (e.g., Cas nuclease) and genome targeting nucleic acid (e.g., gRNA or sgRNA) can each be administered separately to a cell or subject. In some embodiments, the site-directed polypeptide can be pre-complexed with one or more guide RNAs or one or more sgRNAs. Such pre-complexed substances are referred to as ribonucleoprotein particles (RNPs). In some embodiments, the nuclease system comprises a ribonucleoprotein (RNP). In some embodiments, the nuclease system comprises a Cas9 RNP, which comprises a purified Cas9 protein complexed with a gRNA. In some embodiments, the nuclease system comprises a Mad7 RNP, which comprises a purified Mad7 protein complexed with a gRNA. Cas9 and Mad7 proteins can be expressed and purified by any means known in the art. Ribonucleoproteins are assembled in vitro and can be delivered directly to cells using standard electroporation or transfection techniques known in the art. Engineered stem cells

[0382] In some embodiments, the present disclosure provides engineered stem cells that transiently or stably express a synthetic cytokine receptor complex. In some embodiments, the present disclosure provides engineered stem cells that stably express a synthetic cytokine receptor complex.

[0383] In some embodiments, the engineered stem cell comprises a genome comprising a nucleotide sequence encoding a synthetic cytokine receptor complex. In some embodiments, the genome further comprises a disrupted B2M, TRAC and / or SIRPA locus. In some embodiments, the genome further comprises a disrupted FKBP12 locus.

[0384] In some embodiments, the engineered stem cell comprises a genome comprising (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted B2M locus, and (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cell comprises a genome comprising (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted TRAC locus, and (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cell comprises a genome comprising (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted SIRPA locus, and (iii) a disrupted FKBP12 locus. In some embodiments, the engineered stem cell comprises a genome comprising (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted B2M locus, (iii) a disrupted TRAC locus, and (iv) a disrupted FKBP12 locus. In some embodiments, the engineered stem cell comprises a genome comprising (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted B2M locus, (iii) a disrupted SIRPA locus, and (iv) a disrupted FKBP12 locus. In some embodiments, the engineered stem cell comprises a genome comprising (i) a nucleotide sequence encoding a synthetic cytokine receptor complex, (ii) a disrupted SIRPA locus, (iii) a disrupted TRAC locus, and (iv) a disrupted FKBP12 locus. Engineered cells

[0385] In some embodiments, the cell colonies described herein are genetically engineered. In some embodiments, the source cells are genetically engineered. In some embodiments, the mesoderm cells are genetically engineered. In some embodiments, the embryoid somatic cells are genetically engineered. In some embodiments, the hematopoietic progenitor cells are genetically engineered. In some embodiments, the differentiated NK cells are genetically engineered. In some embodiments, the mature NK cells are genetically engineered. In some embodiments, genetic engineering reduces the expression of endogenous genes. In some embodiments, genetic engineering increases the expression of endogenous genes.

[0386] In some embodiments, the genetically engineered cell comprises introducing foreign DNA into the cell. In some embodiments, the foreign DNA is a gene. In some embodiments, the foreign DNA changes the expression of an endogenous gene.

[0387] In some embodiments, genetic engineering includes introducing RNA into the cell, such as interfering RNA (RNAi), double-stranded RNA (dsRNA), small interfering RNA (siRNA), and / or micro RNA (miRNA).

[0388] In some embodiments, genetic engineering involves introducing DNA into cells, such as a plasmid or a bacterial artificial chromosome (BAC).

[0389] In some embodiments, genetic engineering includes the introduction of: (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 includes a zinc finger protein (ZFP), a TAL protein, or a clustered regularly spaced short palindromic nucleic acid (CRISPR) specific to a gene. In some embodiments, the destruction includes the introduction of a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas9 combination that specifically binds, recognizes, or hybridizes to the gene. In some embodiments, the introduction is performed by introducing a nucleic acid into the cell, the nucleic acid comprising a sequence encoding a DNA binding protein, a DNA binding nucleotide, and / or a complex comprising a DNA binding protein or a DNA binding nucleotide. In some embodiments, the nucleic acid is a viral vector.

[0390] In some embodiments, the genetically engineered cells described herein include a chimeric antigen receptor (CAR). In some embodiments, genetically engineered stem cells include CAR. In some embodiments, genetically engineered hematopoietic progenitor cells include CAR. In some embodiments, genetically engineered NK cells include CAR.

[0391] In some embodiments, the genetically engineered cells described herein comprise a rapamycin-activated cytokine receptor (RACR). In some embodiments, genetically engineered stem cells comprise RACR. In some embodiments, genetically engineered hematopoietic progenitor cells comprise RACR. In some embodiments, genetically engineered NK cells comprise RACR.

[0392] In some embodiments, the genetically engineered cells described herein comprise CAR and RACR. In some embodiments, genetically engineered stem cells comprise CAR and RACR. In some embodiments, genetically engineered hematopoietic progenitor cells comprise CAR and RACR. In some embodiments, genetically engineered NK cells comprise CAR and RACR.

[0393] In some embodiments, the genetically engineered NK cells may comprise an inactivating mutation. In some embodiments, the inactivating mutation is a nonsense mutation. In some embodiments, the nonsense mutation is a premature stop codon. In some embodiments, the inactivating mutation is a missense mutation. Synthetic cytokine receptor complex

[0394] In some embodiments, the cells described herein are genetically engineered to express a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor comprises a synthetic γ chain and a synthetic β chain, each comprising a dimerization domain. The dimerization domain controllably dimerizes in the presence of a non-physiological ligand, thereby activating the signaling of the synthetic cytokine receptor.

[0395] The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an intracellular domain. In some embodiments, the intracellular domain is 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- or C-terminal to the IL-2G intracellular domain.

[0396] The synthetic β chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain. In some embodiments, the intracellular domain is selected from the intracellular domain of interleukin-2 receptor subunit β (IL-2RB), the intracellular domain of interleukin-7 receptor subunit β (IL-7RB) or the intracellular domain of interleukin-21 receptor subunit β (IL-21RB). In some embodiments, the intracellular domain comprises interleukin-2 / interleukin-15 receptor subunit β (IL-2 / 15RB). In some embodiments, the intracellular domain comprises an interleukin-15 receptor α subunit. The synthetic γ chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an intracellular domain of interleukin-2 receptor subunit γ (IL-2RG). The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2RB or IL-7RB intracellular domain).

[0397] The non-physiological ligand can activate the synthetic cytokine receptor in the cytotoxic innate lymphoid cells to induce the expansion and / or activation of the engineered cytotoxic innate lymphoid cells. In a preferred embodiment, the non-physiological ligand is rapamycin or a rapamycin analog (rapalog), and such a synthetic cytokine receptor is called a rapamycin-activated cytokine receptor (RACR).

[0398] In some embodiments, the non-physiological ligand activates the synthetic cytokine receptor in the NK cell to induce the expansion of the NK cell. In some embodiments, the activation of the synthetic cytokine receptor causes the number of NK cells to increase by at least about 10 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, or at least about 4000 times compared to uninduced cells.

[0399] In some embodiments, the NK cells increase by about 10 times to about 100 times, about 50 times to about 200 times, about 100 times to about 300 times, about 200 times to about 400 times, about 300 times to about 500 times, about 400 times to about 1000 times, about 500 times to about 1500 times, about 1000 times to about 2000 times, about 1500 times to about 2500 times, about 2000 times to about 3000 times, about 2500 times to about 3500 times, about 3000 times to about 4000 times, or any value between these ranges. Intracellular domain

[0400] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain. In some embodiments, the IL2Rg domain comprises the sequence shown in SEQ ID NO: 1. In some embodiments, the IL2Rg common gamma chain intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 1.

[0401] In some embodiments, the sequence of the IL2RG common gamma chain intracellular domain is as shown in SEQ ID NO: 1: ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET.

[0402] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain and a second transmembrane receptor protein comprising an IL-2RB intracellular domain and a second dimerization domain.

[0403] In some embodiments, the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL2RB) intracellular domain. In some embodiments, the IL-2 receptor subunit beta is referred to as an IL-2 / IL-15 receptor beta subunit. In some embodiments, the IL2RB intracellular domain comprises the sequence shown in SEQ ID NO: 2. In some embodiments, the IL2RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 2.

[0404] In some embodiments, the sequence of the IL2RB intracellular domain is as shown in SEQ ID NO: 2: NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0405] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain and a second transmembrane receptor protein comprising an IL-7RB intracellular domain and a second dimerization domain.

[0406] In some embodiments, the synthetic beta chain comprises an interleukin-7 receptor subunit beta (IL7RB) intracellular domain. In some embodiments, the IL7RB intracellular domain comprises the sequence shown in SEQ ID NO: 3. In some embodiments, the IL7RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 3.

[0407] In some embodiments, the sequence of the IL7RB intracellular domain is as shown in SEQ ID NO:3: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0408] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain and a first dimerization domain and a second transmembrane receptor protein comprising an IL-21RB intracellular domain and a second dimerization domain.

[0409] In some embodiments, the synthetic β chain comprises an interleukin-21 receptor subunit β (IL21RB) intracellular domain. In some embodiments, the IL21RB intracellular domain comprises the sequence shown in SEQ ID NO: 4. In some embodiments, the IL21RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 4.

[0410] In some embodiments, the sequence of the IL21RB intracellular domain is as shown in SEQ ID NO:4: SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVLDAEGPCTWPCSCE DDGYPALDLDAGLEEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS Dimerization domain

[0411] The dimerization domain may be a heterodimerization domain, including but not limited to FK506-binding protein (FKBP) and FKBP12-rapamycin binding (FRB) domains of 12 kD in size, which are known in the art to dimerize in the presence of rapamycin or a rapamycin analog. The FRB domain may comprise a polypeptide sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 6 or SEQ ID NO: 7. The FKBP domain may comprise a polypeptide sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 5.

[0412] In some embodiments, the sequence of an exemplary FKBP domain is shown in SEQ ID NO:5: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0413] In some embodiments, the sequence of an exemplary FRB domain is shown in SEQ ID NO:6: ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK

[0414] In some embodiments, the sequence of the variant FRB domain (FRB mutant domain) is as shown in SEQ ID NO:7: ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK

[0415] Alternatively, the first dimerization domain and the second dimerization domain may be a 12 kD FK506 binding protein (FKBP) and a calcineurin domain, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0416] In some embodiments, the dimerization domain is a homodimerization domain selected from: i) FK506 binding protein (FKBP) with a size of 12 kD; ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); The corresponding non-physiological ligands are i) FK1012, AP1510, AP1903 or AP20187; ii) Cyclosporine A (CsA); or iii) coumermycin or its analogs.

[0417] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a cyclophilin domain.

[0418] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0419] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0420] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1). Transmembrane domain

[0421] The transmembrane region is a sequence of a synthetic cytokine receptor that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0422] In some embodiments, the sequence of the transmembrane (TM) domain is as shown in SEQ ID NO:8: VVISVGSMGLIISLLCVYFWL

[0423] In some embodiments, the sequence of the TM domain is as shown in SEQ ID NO:9: VAVAGCVFLLISVLLLSGL

[0424] In some embodiments, the sequence of the TM domain is as shown in SEQ ID NO: 10: PILLTISILSFFSVALLVILACVLW

[0425] In some embodiments, the sequence of the TM domain is as shown in SEQ ID NO: 11: GWNPHLLLLLLLVIVFIPAFW

[0426] In some embodiments, the sequence of the CD8a signal sequence is as shown in SEQ ID NO: 12: MALPVTALLLPLALLLHAARP Non-physiological ligands

[0427] In various embodiments of the compositions and methods of the present disclosure, the system comprises a non-physiological ligand. Exemplary small molecules that can be used as ligands include, but are not limited to, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazine-2-yl)oxy]benzoic acid (aMPOB), folic acid, rhodamine, acetazolamide, and CA9 ligand.

[0428] In some embodiments, the synthetic cytokine receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0429] In some embodiments, the non-physiological ligand is a rapamycin analog.

[0430] In some embodiments, the non-physiological ligand is rapamycin.

[0431] In some embodiments, the non-physiological ligand is AP21967.

[0432] In some embodiments, the non-physiological ligand is FK506.

[0433] In some embodiments, the non-physiological ligand is FK1012. In some embodiments, the non-physiological ligand is AP1510. In some embodiments, the non-physiological ligand is AP1903. In some embodiments, the non-physiological ligand is AP20187. In some embodiments, the non-physiological ligand is cyclosporine-A (CsA). In some embodiments, the non-physiological ligand is coumermycin.

[0434] In some embodiments, the synthetic cytokine receptor complex is activated by folic acid, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, rapamycin analog (Rapalog) (analog of rapamycin), CD28 ligand, poly(his) tag, Strep tag, FLAG tag, VS tag, Myc tag, HA tag, NE tag, biotin, digoxigenin, dinitrophenol or its derivatives.

[0435] In some embodiments, the non-physiological ligand may be an inorganic or organic compound less than 1000 Daltons.

[0436] In some embodiments, the ligand can be rapamycin or a rapamycin analog (Rapalog). In some embodiments, the rapamycin analog comprises a variant of rapamycin with one or more of the following modifications relative to rapamycin: demethylation, elimination or substitution of a methoxy group at C7, C42 and / or C29; elimination, derivatization or substitution of a hydroxyl group at C13, C43 and / or C28; reduction, elimination or derivatization of a ketone at C14, C24 and / or C30; replacement of a 6-piperidinylcarboxylic acid ring with a 5-membered prolyl ring; and optional substitution on a cyclohexyl ring or replacement of a cyclohexyl ring with a substituted cyclopentyl ring.

[0437] Thus, in some embodiments, the rapamycin analog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, Temsirolimus (CCI-779), C20-methallyl rapamycin, C16-(S)-3-methylindorapamycin, C16-(S)-3-methylindorapamycin (C16-iRap), AP21967 (A / C heterodimer, Takara ), sodium mycophenolic acid, benidipine hydrochloride, rapamine, AP23573 (Ridaforolimus), AP1903 (Rimiducid), or their metabolites, derivatives and / or combinations thereof.

[0438] In some embodiments, the ligand comprises FK1012 (a semisynthetic dimer of FK506), tacrolimus (FK506), FKCsA (a complex of FK506 and cyclosporine), rapamycin, coumermycin, gibberellin, HaXS dimerizer (a chemical dimerizer for Halo tags and SNAP tags), TMP-H tag (trimethoprim halogenase protein dimerizer), or ABT-737 or a functional derivative thereof.

[0439] 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 an amount within a range defined by any two of the above amounts.

[0440] 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.

[0441] 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.

[0442] In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 100 nM.

[0443] In some embodiments, the non-physiological ligand is present or provided at 1 nM. In some embodiments, the non-physiological ligand is present or provided at 10 nM. In some embodiments, the non-physiological ligand is present or provided at 100 nM. In some embodiments, the non-physiological ligand is present or provided at 1000 nM. Cytoplasmic FRB

[0444] The FRB domain is a domain of approximately 100 amino acids derived from the mTOR protein kinase. It 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 transduced cells and promotes sustained activation of transduced cells, giving the cells a proliferative advantage over native cells.

[0445] In some embodiments, the synthetic cytokine receptor complex comprises a cytoplasmic polypeptide bound to a ligand or a complex comprising the ligand.

[0446] In some embodiments, the cytoplasmic polypeptide comprises a FRB domain. In some embodiments, the cytoplasmic polypeptide comprises a FRB domain and the ligand is rapamycin. The cytoplasmic FRB domain may comprise a polypeptide sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 6 or SEQ ID NO: 7. The FRB domain may be a naked FRB domain consisting essentially of a polypeptide having a polypeptide sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 6 or SEQ ID NO: 7. Advantageously, the cytoplasmic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapamycin analogs). Chimeric Antigen Receptor

[0447] In some embodiments, the cells described herein are genetically engineered to express a chimeric antigen receptor (CAR).

[0448] In some embodiments, the present disclosure encompasses a CAR system for treating a cancer subject.In some embodiments, the NK cells of the present disclosure comprise a CAR sequence (CAR-NK cells).

[0449] In some embodiments, by transfecting a cell colony with an expression vector encoding a CAR construct, NK cells are engineered to express a CAR construct. Illustrative examples of cell colonies that can be transfected include HSC, blood progenitor cells, common lymphoid progenitor cells, or NK cells. Suitable methods for preparing a transduced NK cell colony expressing a selected CAR construct are well known to those skilled in the art, and include retroviruses, slow viruses (virus-mediated CAR gene delivery systems), Sleeping Beauty, and PiggyBac (including transposon / transposase systems of non-viral mediated CAR gene delivery systems), to name a few examples. In some embodiments, any transduction method encompassed by the present disclosure can be used to produce NK cells expressing CAR. CAR targeted agents

[0450] Typically, CAR is produced by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding a transmembrane and intracellular domain, and transducing cells with the polynucleotide and with the corresponding VH or VL (if necessary). Many variants of CAR are well known in the art, and the present disclosure encompasses the use of any known variants. In addition, VL / VH pairs and scFvs of countless haptens are known in the art, or can generally be produced by conventional methods. Therefore, the present disclosure encompasses the use of any known hapten binding domain.

[0451] Various methods for targeting CAR and cells expressing CAR have been described in the art, including, for example, in US2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, fluorescein or fluorescein isothiocyanate (FITC) moieties can be conjugated to agents that bind to desired target cells (e.g., cancer cells), so that CAR-NK cells expressing anti-fluorescein / FITC chimeric antigen receptors can be selectively targeted to target cells labeled by conjugates. In different variants, other haptens recognized by CAR can be used to replace fluorescein / FITC. CAR can be produced using various scFv sequences known in the art or scFv sequences produced by conventional and conventional methods. Further exemplary scFv sequences of fluorescein / FITC and other haptens are provided, for example, in WO2021 / 076788, the disclosure of which is incorporated herein by reference.

[0452] In some embodiments, the CAR system of the present disclosure uses a CAR that targets a portion that is not produced or expressed by the cells of the subject to be treated. Therefore, the CAR system allows NK cells to focus on targeting target cells, such as cancer cells. By administering small conjugate molecules together with NK cells expressing CAR, NK cell responses can only target cells that express tumor receptors, thereby reducing off-target toxicity, and due to the rapid removal of small conjugate molecules, the activation of NK cells can be more easily controlled. As an additional advantage, NK cells expressing CAR can be used as "universal" cytotoxic cells to target a variety of tumors without the need to prepare separate CAR constructs. The targeting portion recognized by CAR can also remain constant. All that needs to be changed is the ligand portion of the small conjugate molecule to allow the system to target cancer cells of different characteristics (identity).

[0453] In one embodiment, the present disclosure provides examples of such conjugate molecule / CAR systems.

[0454] In some embodiments, the CAR system of the present disclosure utilizes a conjugate molecule as a bridge between cells expressing CAR and targeted cancer cells. The conjugate molecule is a conjugate comprising a hapten and a cell targeting portion, such as any suitable tumor cell specific ligand. Exemplary haptens that can be recognized and bound by CAR include small molecular weight organic molecules, such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxigenin, and fluorescein and its derivatives, including FITC (fluorescein isothiocyanate), NHS-fluorescein and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, desmin, Centyrin and DARPin. Suitable cell targeting portions that can be used as haptens of CAR themselves include desmin (see Kolmar H. et al., The FEBS Journal. 2008. 275 (11): 26684-90), Centyrin and DARPin (see Reichert, JMMAbs 2009. 1 (3): 190-209).

[0455] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), which is a ligand bound by PSMA-positive human prostate cancer cells with nanomolar affinity (Kd=14nM; see Kularatne, SA 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 a targeting moiety, and the DUPA derivative is described in WO 2015 / 057852, which is incorporated herein by reference.

[0456] In some embodiments, the cell targeting moiety is a CCK2R ligand, which is a ligand bound by CCK2R-positive cancer cells (e.g., thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal cancer, and colon cancer; see Wayua. C. et al., Molecular Pharmaceutics. 2013. Epub).

[0457] In some embodiments, the cell targeting moiety is folate, folic acid, or an analog thereof, which is a ligand bound by folate receptors on cancer cells, including ovarian, cervical, endometrial, lung, kidney, brain, breast, colon, and head and neck cancers; see Sega, EI et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

[0458] In some embodiments, the cell targeting moiety is a NK-1R ligand. For example, receptors for NK-1R ligands are found in colon cancer and pancreatic cancer. In some embodiments, the NK-1R ligand can be synthesized according to the methods disclosed in International Patent Application PCT / US2015 / 044229, which is incorporated herein by reference.

[0459] 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 of 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 (neuromediator K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0460] In some embodiments, the cell targeting moiety is a CAIX ligand. For example, a receptor for a CAIX ligand found in renal cancer, ovarian cancer, vulvar cancer, and breast cancer. A CAIX ligand may also be referred to herein as CA9.

[0461] In some embodiments, the cell targeting moiety is a ligand for gamma glutamyl transpeptidase, which is overexpressed in, for example, ovarian cancer, colon cancer, liver cancer, astrocytoma, melanoma, and leukemia.

[0462] In some embodiments, the cell targeting moiety is a CCK2R ligand. A receptor for CCK2R ligand found in cancers such as thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal cancer, and colon cancer.

[0463] In one embodiment, the cell targeting moiety may have a mass of less than about 10,000 Daltons, less than about 9,000 Daltons, less than about 8,000 Daltons, less than about 7,000 Daltons, less than about 6,000 Daltons, less than about 5,000 Daltons, less than about 4,500 Daltons, less than about 4,000 Daltons, less than about 3,500 Daltons, less than about 3,000 Daltons, less than about 2,500 Daltons, less than about 2,000 Daltons, less than about 1,500 Daltons, less than about 1,000 Daltons, or less than about 500 Daltons. In another embodiment, the small molecule ligand may have a mass of about 1 to about 10,000 Daltons, about 1 to about 9,000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7,000 Daltons, about 1 to about 6,000 Daltons, about 1 to about 5,000 Daltons, about 1 to about 4,500 Daltons, about 1 to about 4,000 Daltons, about 1 to about 3,500 Daltons, about 1 to about 3,000 Daltons, about 1 to about 2,500 Daltons, about 1 to about 2,000 Daltons, about 1 to about 1,500 Daltons, about 1 to about 1,000 Daltons, or about 1 to about 500 Daltons.

[0464] In an exemplary embodiment, the connection in the conjugate described herein can be a direct connection (e.g., a reaction between an isothiocyanate group of FITC and a free amine group of a small molecule ligand), or the connection can be carried out through an intermediate linker. In one embodiment, if present, the intermediate linker can be any biocompatible linker known in the art, such as a divalent linker. In an exemplary embodiment, the divalent linker can include about 1 to about 30 carbon atoms. In another exemplary embodiment, the divalent linker can include about 2 to about 20 carbon atoms. In other embodiments, a lower molecular weight divalent linker (i.e., those with an approximate molecular weight of about 30 to about 300 Da) is used. In another embodiment, suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 atoms or more.

[0465] In some embodiments, the hapten and cell targeting moiety can be directly conjugated by reaction between, for example, the isothiocyanate group of FITC and the free amine group of a small ligand (e.g., folate, DUPA, and CCK2R ligand). However, it may be helpful to use a linker domain to connect the two molecules because it can provide flexibility and stability. Examples of suitable linker domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) carbohydrates; 5) non-natural peptidoglycans; 6) polyvinylpyrrolidone; 7) Pluronic F-127. Suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 atoms or more.

[0466] In some embodiments, the linker may be a bivalent linker, which may comprise one or more spacers.

[0467] An exemplary conjugate of the present disclosure is FITC-folic acid

[0468] An exemplary conjugate of the present disclosure is FITC-CA9

[0469] 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.

[0470] Although the affinity with which a ligand binds to a cancer cell receptor can vary, and in some cases low affinity binding may be preferred (e.g., about 1 μM), the binding affinity of a ligand to a cancer cell receptor is typically at least about 100 μM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0471] Examples of conjugates and methods for their preparation are provided in U.S. patent applications US2017 / 0290900, US2019 / 0091308, and US2020 / 0023009, all of which are incorporated herein by reference. CAR constructs

[0472] In some embodiments, the binding portion of the CAR can be, for example, a single chain fragment variable region (scFv), Fab, Fv, Fc or (Fab')2 fragment of an antibody, etc. It is not within the scope of the present invention to use an unaltered (ie, full-size) antibody, such as IgG, IgM, IgA, IgD or IgE, in or as a CAR.

[0473] In some embodiments, the costimulatory domain is used to enhance t...

Claims

1. A method for generating a CD34+ / CD43+ / CD45+ cell population, the method comprising: (i) culturing the progenitor cell population in a two-dimensional (2D) culture system for a period of time sufficient to form progenitor cell aggregates; (ii) passage the progenitor cell aggregates from the 2D culture system to a three-dimensional (3D) suspension culture system; (iii) contacting the progenitor cell aggregates with a differentiation medium in the 3D suspension culture system for a period of time sufficient to generate the CD34+ / CD43+ / CD45+ cell population.

2. A method for differentiating a stem cell population into a hematopoietic progenitor cell population, the method comprising: (i) culturing the stem cell population in a 2D culture system for a period of time sufficient to form stem cell aggregates; (ii) passage the stem cell aggregates from the 2D culture system to a 3D suspension culture system; (iii) contacting the stem cell aggregates in the 3D suspension culture system with a differentiation medium for a period of time sufficient to differentiate the stem cell population into the hematopoietic progenitor cell population, wherein the differentiation medium comprises a bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).

3. The method according to claim 1 or 2, wherein the 3D suspension culture has a volume between 50-50,000 ml.

4. The method according to any one of claims 1 to 3, wherein the 3D suspension culture is agitated.

5. The method of claim 3, wherein the 3D suspension culture is stirred at a rate of 10 revolutions per minute (RPM) to 100 RPM.

6. The method according to claim 4 or 5, wherein the 3D suspension culture is stirred at a rate of 70 RPM.

7. The method according to any one of claims 2 to 6, wherein the hematopoietic progenitor cell population comprises CD34+ / CD43+ / CD45+ cells.

8. The method according to any one of claims 2 to 7, wherein the BMP pathway activator is BMP4.

9. The method according to any one of claims 2 to 8, wherein the FGF is FGF2.

10. The method of any one of claims 2 to 9, wherein the VEGF is VEGF-165.

11. The method according to any one of claims 2 to 10, wherein the differentiation medium comprises a Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor.

12. The method of claim 11, wherein the ROCK inhibitor is Y27632.

13. The method according to any one of claims 2 to 12, wherein the differentiation medium comprises stem cell factor (SCF).

14. The method according to any one of claims 2 to 13, wherein the differentiation medium comprises thrombopoietin (TPO).

15. The method according to any one of claims 2 to 14, wherein the differentiation medium comprises low-density lipoprotein (LDL).

16. The method according to any one of claims 2 to 15, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor.

17. The method of any one of claims 2 to 16, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF, SCF, TPO and the LDL.

18. The method of any one of claims 2 to 17, wherein (iii) comprises contacting the population of stem cell aggregates with the differentiation medium for 1-5 days, wherein the differentiation medium comprises the BMP pathway activator, the FGF, the VEGF and optionally the ROCK inhibitor.

19. The method according to any one of claims 2 to 18, wherein (iii) comprises (a) contacting the stem cell aggregates with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF and the ROCK inhibitor for 1-5 days to produce embryoid bodies or mesodermal cells, and (b) contacting the embryoid bodies or mesodermal cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO and the LDL for 1-15 days.

20. The method of claim 19, wherein the differentiation medium comprises 1-50 ng / mL BMP, 1-50 ng / mL FGF, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO and 1-50 μg / mL LDL or any combination thereof.

21. The method of any one of claims 1 to 20, wherein the progenitor cell or stem cell is an induced pluripotent stem cell (iPSC).

22. The method of any one of claims 1 to 21, wherein the progenitor cell or stem cell is a human embryonic stem cell (hESC).

23. The method according to any one of claims 2 to 22, wherein the differentiation medium is serum-free.

24. The method of any one of claims 2 to 23, wherein the method is xeno-free.

25. A method for producing a NK cell population, the method comprising: (a) culturing a stem cell population in a 2D culture system for a period of time sufficient to form stem cell aggregates; (b) subculturing the stem cell aggregates from the 2D culture system to a 3D suspension culture system; (c) contacting the stem cell aggregates in the 3D suspension culture system with a first culture medium comprising a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor for a period of time sufficient to generate embryoid bodies; (d) contacting the embryoid bodies with a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL for a period of time sufficient to generate a population of hematopoietic progenitor cells; (e) contacting the population of hematopoietic progenitor cells with a second differentiation medium for a period of time sufficient to produce the population of NK cells.

26. The method of claim 25, wherein the second differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

27. The method of any one of claims 25 to 26, wherein the culture medium comprises 1-100 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimido-[4,5-b]-indole derivative, 0.1-10 μM AhR antagonist, and any combination thereof.

28. The method of claim 26 or 27, wherein the pyrimido-[4,5-b]-indole derivative is UM729 and the AhR inhibitor is SR1.

29. The method of any one of claims 25 to 28, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, and the ROCK inhibitor is Y27632.

30. The method of any one of claims 25 to 29, wherein each culture medium in steps (b)-(e) is serum-free.

31. The method of any one of claims 25 to 30, wherein the method is xeno-free.

32. The method of any one of claims 25 to 31, wherein the first culture medium, the first differentiation medium, and the second differentiation medium each comprise the same basal medium.

33. The method of any one of claims 25 to 31, wherein the first culture medium, the first differentiation medium, and the second differentiation medium each comprise a different basal culture medium.

34. The method of any one of claims 25 to 31, wherein the first differentiation medium and the second differentiation medium each comprise the same basal medium, and the first medium comprises a basal medium different from the first and second differentiation medium.

35. The method of any one of claims 25 to 31, wherein the first differentiation medium and the second differentiation medium each comprise a basal medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

36. The method of any one of claims 25 to 35, wherein the time period of step (b) is 2-8 days, the time period of step (c) is 1-5 days, the time period of step (d) is 3-15 days, and the time period of step (e) is 11-25 days.

37. The method of any one of claims 25 to 36, wherein steps (a)-(e) occur within 40-50 days.

38. The method of any one of claims 25 to 37, wherein the stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

39. The method of any one of claims 25 to 38, wherein the population of hematopoietic progenitor cells comprises about 50% to about 100% CD34+ / CD43+ / CD45+ cells.

40. The method of any one of claims 25 to 38, wherein the NK cell population comprises about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

41. The method of any one of claims 25 to 40, comprising expanding the NK cell population, wherein the NK cell population is expanded about 1,000 to about 10,000 fold.

42. The method of any one of claims 2 to 41, wherein the stem cell population is genetically engineered or edited.

43. The method of any one of claims 25 to 42, wherein the NK cell population is genetically engineered or edited.

44. A cell population comprising hematopoietic progenitor cells produced by the method of any one of claims 2 to 24.

45. The cell population of claim 44, wherein the hematopoietic progenitor cells are CD34+ / CD43+ / CD45+.

46. ​​The cell population of claim 44 or 45, comprising 30-50% hematopoietic progenitor cells.

47. A cell colony comprising NK cells produced by the method according to any one of claims 25 to 43.

48. The cell population of claim 47, wherein the NK cells are CD45+ / CD56+ / LFA1+.

49. The cell population of claim 47 or 48, comprising 60-100% NK cells.

50. A pharmaceutical composition comprising the cell population according to any one of claims 44 to 49.

51. A method for producing a hematopoietic progenitor cell population, the method comprising: (a) genetically engineering a stem cell population to express synthetic cytokine receptors for non-physiological ligands, Wherein the cytokine receptors include: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain; (b) culturing the stem cell population in a 2D culture system for a period of time sufficient to form stem cell aggregates; (c) passage the stem cell aggregates from the 2D culture system to a 3D suspension culture system; and (d) contacting the stem cell aggregates in the 3D suspension culture system with a differentiation medium for a period of time sufficient to generate hematopoietic progenitor cells.

52. A method for producing a NK cell population, the method comprising: (a) genetically engineering a stem cell population to express synthetic cytokine receptors for non-physiological ligands, Wherein the cytokine receptors include: A synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain; (b) culturing the stem cell population in a 2D culture system for a period of time sufficient to form stem cell aggregates; (c) passage the stem cell aggregates from the 2D culture system to a 3D suspension culture system; (d) contacting the stem cell aggregates in the 3D suspension culture system with a first differentiation medium for a period of time sufficient to generate hematopoietic progenitor cells; and (e) contacting the population of hematopoietic progenitor cells with a second differentiation medium for a period of time sufficient to produce the population of NK cells.

53. The method of claim 51 or 52, wherein the intracellular domain of the synthetic β chain polypeptide is selected from the intracellular domain of interleukin-2 receptor subunit β (IL-2RB), the intracellular domain of interleukin-7 receptor subunit β (IL-7RB) and / or the intracellular domain of interleukin-21 receptor subunit β (IL-21RB).

54. The method of any one of claims 51 to 53, wherein the nucleotide sequence is inserted by homology directed repair (HDR).

55. The method of any one of claims 51 to 54, wherein the vector comprises a nucleic acid comprising, from 5' to 3', (a) a nucleotide sequence homologous to a region located upstream of a target site, (b) a nucleotide sequence encoding a synthetic cytokine receptor for a non-physiological ligand, and (c) a nucleotide sequence homologous to a region located downstream, wherein a double-strand break occurs at the target site in an endogenous gene, and the nucleic acid is exchanged with the homologous nucleotide sequence of the endogenous gene.

56. The method of any one of claims 51 to 55, wherein the nucleotide sequence is inserted by non-homologous end joining (NHEJ).

57. The method of any one of claims 51 to 56, wherein the cell is engineered with an RNA-guided endonuclease.

58. The method of claim 57, wherein the RNA-guided endonuclease is selected from the group consisting of Cas endonuclease, Mad endonuclease, and Cpf1 endonuclease.

59. The method of claim 58, wherein the RNA-guided endonuclease is Cas9 or Mad7.

60. The method of any one of claims 51 to 59, wherein the method comprises disrupting a target gene and inserting the nucleotide sequence into the disrupted target gene, wherein disrupting the target gene comprises contacting the stem cell population with (i) a gRNA targeted to a target site in the target gene and (ii) an RNA-guided endonuclease.

61. The method of claim 60, wherein the target gene is selected from the group consisting of B2M, TRAC and SIRPA.

62. The method of any one of claims 51 to 61, comprising engineering the stem cell population to be resistant to rapamycin.

63. The method of claim 62, wherein engineering the stem cell population to be resistant to rapamycin comprises knocking out the FKBP12 gene.

64. The method of any one of claims 51 and 53 to 63, wherein the differentiation medium comprises a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor.

65. The method of claim 64, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, and the ROCK inhibitor is Y27632.

66. The method of any one of claims 64 to 65, wherein the differentiation medium comprises SCF, TPO and LDL.

67. The method of any one of claims 51 and 53 to 63, wherein (d) comprises contacting the population of stem cell aggregates with the differentiation medium for 1-5 days, wherein the differentiation medium comprises a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor.

68. The method of any one of claims 51 and 53 to 63, wherein (d) comprises (i) contacting stem cell aggregates with a differentiation medium comprising a BMP pathway activator, FGF, VEGF and a ROCK inhibitor for 1-5 days to produce embryoid bodies or mesodermal cells, and (ii) contacting the embryoid bodies or mesodermal cells with a differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO and the LDL inhibitor for 1-15 days.

69. The method of claim 68, wherein the differentiation medium comprises 1-50 ng / mL BMP, 1-50 ng / mL FGF, 5-100 ng / mL VEGF, 0.1-20 μM ROCK inhibitor, 1-200 ng / mL SCF, 1-100 ng / mL TPO and 1-50 μg / mL LDL or any combination thereof.

70. The method of any one of claims 52 to 63, wherein the first differentiation medium comprises a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor.

71. The method of any one of claims 52 to 63, wherein (d) comprises contacting the population of stem cell aggregates with the first differentiation medium for 1-5 days, wherein the first differentiation medium comprises a BMP pathway activator, FGF, VEGF, and optionally a ROCK inhibitor.

72. The method of any one of claims 52 to 63, wherein (d) comprises (i) contacting stem cell aggregates with a culture medium comprising a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor for 1-5 days to produce embryoid bodies or mesodermal cells, and (ii) contacting the embryoid bodies or mesodermal cells with a first differentiation culture medium comprising the BMP pathway activator, the FGF, the VEGF, SCF, TPO, and the LDL for 1-15 days.

73. The method of any one of claims 67 to 72, wherein the BMP pathway activator is BMP4, the FGF is FGF2, the VEGF is VEGF-165, and the ROCK inhibitor is Y27632.

74. The method of any one of claims 52 to 73, wherein the second differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-[4,5-b]-indole derivative, and an AhR inhibitor.

75. The method of any one of claims 52 to 73, wherein the second differentiation medium comprises 1-100 ng / mL SCF, 1-50 ng / mL IL-7, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL FLT3L, 0.1-10 μM pyrimido-[4,5-b]-indole derivatives, 0.1-10 μM AhR antagonists, and any combination thereof.

76. The method of claim 74 or 75, wherein the pyrimido-[4,5-b]-indole derivative is UM729 and the AhR inhibitor is SR1.

77. The method of any one of claims 52 to 67, wherein the first differentiation medium and the second differentiation medium are serum-free.

78. The method of any one of claims 51 to 77, wherein the method is xeno-free.

79. The method of any one of claims 52 to 78, wherein the first differentiation medium and the second differentiation medium each comprise the same basal medium.

80. The method of any one of claims 52 to 78, wherein the first differentiation medium and the second differentiation medium each comprise a different basal medium.

81. The method of any one of claims 52 to 78, wherein the first differentiation medium and the second differentiation medium each comprise a basal medium comprising Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

82. The method of any one of claims 52 to 81, wherein the time period for step (b) is 2-8 days, the time period for step (c) is 1-5 days, the time period for step (d) is 3-15 days, and the time period for step (e) is 11-25 days.

83. The method of any one of claims 52 to 82, wherein steps (a)-(e) occur within 40-50 days.

84. The method of any one of claims 52 to 83, wherein the stem cell is an induced pluripotent stem cell (iPSC) or a human embryonic stem cell (hESC).

85. The method of any one of claims 51 to 84, wherein the population of hematopoietic progenitor cells comprises about 50% to about 100% CD34+ / CD43+ / CD45+ cells.

86. The method of any one of claims 52 to 85, wherein the NK cell population comprises about 60% to about 100% CD43+ / CD45+ / CD56+ / LFA1+ cells.

87. The method of any one of claims 52 to 78, comprising expanding the NK cell population, wherein the NK cell population is expanded from about 1,000 to about 10,000 fold.

88. The method of any one of claims 51 to 87, wherein the stem cell population is genetically engineered or edited.

89. The method of any one of claims 52 to 88, wherein the NK cell population is genetically engineered or edited.

90. The method of any one of claims 51 to 89, wherein the stem cell is an iPSC.

91. A cell population produced by the method of any one of claims 51 to 90.

92. A pharmaceutical composition comprising the cell population of claim 83.

93. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the cell population of any one of claims 44 to 49 and 91 or the pharmaceutical composition of claim 50 or 84.

94. A kit comprising a cell population according to any one of claims 44 to 49 and 83 and instructions for administering the cell population to a subject in need thereof.

95. The kit of claim 94, wherein the subject has cancer.

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