Stem cell differentiation in suspension culture

Abstract

Xeno-free methods and compositions are provided for generating hematopoietic progenitors and natural killer (NK) cells in 3D suspension culture. TIFF2025526380000021.tif83159

Description

[Technical Field]

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

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

[0003] NK cells for use in immunotherapy can be obtained from primary sources such as peripheral blood or umbilical cord blood. Artificial sources of NK cells include pluripotent stem cells, including induced pluripotent stem cells (iPSCs), which are cells derived from somatic cells (commonly fibroblasts or peripheral blood mononuclear cells (PBMCs)) that have been induced to allow for unlimited proliferation and differentiation into other cell types when subjected to appropriate differentiation conditions, and human embryonic stem cells (hESCs). NK cells can be derived from iPSCs by sequentially differentiating them into hematopoietic progenitor cells (HPCs), also known as hematopoietic stem cells (HSCs).

[0004] After primary NK cells or iPSC-NK cells are obtained, they can be expanded ex vivo before being administered to patients. Methods for differentiating iPSCs into NK cells often involve the use of feeder cells or serum-containing media. To provide cells suitable for in vivo administration, there remains a need for compositions and methods related to generating NK cells without the use of animal-derived materials such as xenogeneic factors, e.g., fetal bovine serum (FBS) and / or feeder cells, and enabling scale-up production. Summary of the Invention

[0005] overview The present disclosure is based at least in part on the discovery of a method for differentiating stem cells into hematopoietic progenitors and NK cells in suspension.As provided herein, hematopoietic progenitors and NK cells generated in a three-dimensional suspension culture system exhibit higher purity and increased frequency of desired cell markers compared to two-dimensional culture systems for differentiation.Without wishing to be bound by theory, the suspension culture method described herein is suitable for large-scale production of hematopoietic progenitors and / or NK cells.Furthermore, 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 aspects, the present disclosure provides: (i) culturing a population of progenitor cells 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 in the 3D suspension culture system with a differentiation medium for a period of time sufficient to generate a population of CD34+ / CD43+ / CD45+ cells. The present invention provides a method for generating a population of CD34+ / CD43+ / CD45+ cells, comprising:

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

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

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

[0010] In some embodiments, the population of hematopoietic progenitors 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, 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 a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor. In some embodiments, the differentiation medium comprises a BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL.

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

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

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

[0017] In some embodiments, the progenitor or stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the progenitor or stem cells are human embryonic stem cells (hESCs).

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

[0019] In some aspects, the present disclosure provides: (a) culturing a population of stem cells in a 2D culture system for a period of time sufficient to form stem cell aggregates; (b) passaging 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 medium comprising a BMP pathway activator, FGF, VEGF, and optionally an inhibitor of ROCK 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 progenitors; (e) contacting the population of hematopoietic precursors with a second differentiation medium for a period of time sufficient to generate a population of NK cells. The present invention provides a method for generating a population of NK cells, comprising:

[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 to 100 ng / mL SCF, 1 to 50 ng / mL IL-7, 1 to 100 ng / mL IL-12, 1 to 100 ng / mL IL-15, 1 to 100 ng / mL FLT3L, 0.1 to 10 uM pyrimido-[4,5-b]-indole derivative, 0.1 to 10 uM AhR antagonist, or 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 inhibitor of ROCK is Y27632.

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

[0025] In some embodiments, the first culture medium, 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 different basal media. In some embodiments, the first differentiation medium and the second differentiation medium each comprise the same basal medium, and the first culture medium comprises a different basal medium from the first and second differentiation media. 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.

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

[0027] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

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

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

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

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

[0032] In some aspects, the present disclosure provides pharmaceutical compositions comprising the cell populations of the present disclosure.

[0033] In some aspects, the present disclosure provides: (a) genetically engineering a population of stem cells to express a synthetic cytokine receptor for a non-physiological ligand, Cytokine receptors are 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. Including, process, (b) culturing the population of stem cells in the 2D culture system for a period of time sufficient to form stem cell aggregates; (c) passaging 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 progenitors. The present invention provides a method for generating a population of hematopoietic progenitors, comprising:

[0034] In some aspects, the present disclosure provides: (a) genetically engineering a population of stem cells to express a synthetic cytokine receptor for a non-physiological ligand, Cytokine receptors are 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. Including, process, (b) culturing the population of stem cells in the 2D culture system for a period of time sufficient to form stem cell aggregates; (c) passaging 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 progenitors; and (e) contacting the population of hematopoietic precursors with a second differentiation medium for a period of time sufficient to generate a population of NK cells. The present invention provides a method for generating a population of NK cells, comprising:

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

[0036] In some embodiments, the nucleotide sequence is inserted via 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 the 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, such that a double-stranded break occurs at the target site in the endogenous gene and the nucleic acid is exchanged for the homologous nucleotide sequence of the endogenous gene.

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

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

[0040] In some embodiments, the method includes disrupting a target gene and inserting a nucleotide sequence into the disrupted target gene, wherein disrupting the target gene includes contacting a population of stem cells with (i) a gRNA that targets a target site within 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, an FGF, a 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 to 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 to 5 days to generate embryoid bodies or mesodermal cells, and (ii) contacting the embryoid bodies or mesodermal cells with a differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL for 1 to 15 days.

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

[0047] In some embodiments, the first differentiation medium comprises a BMP pathway activator, an FGF, a 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 to 5 days, the first differentiation medium comprising 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 to 5 days to generate embryoid bodies or mesodermal cells, and (ii) contacting the embryoid bodies or mesodermal cells with a first differentiation medium comprising a BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL for 1 to 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 to 100 ng / mL SCF, 1 to 50 ng / mL IL-7, 1 to 100 ng / mL IL-12, 1 to 100 ng / mL IL-15, 1 to 100 ng / mL FLT3L, 0.1 to 10 uM pyrimido-[4,5-b]-indole derivative, 0.1 to 10 uM AhR antagonist, or 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 method is 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 different basal media. In some embodiments, the first differentiation medium and the second differentiation medium each comprise a basal medium containing Iscove's modified Dulbecco's medium, bovine serum albumin, recombinant human insulin, human transferrin, and 2-mercaptoethanol.

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

[0058] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

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

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

[0061] In some embodiments, the method includes expanding the 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 population of stem cells is genetically engineered or edited, hi some embodiments, the population of NK cells is genetically engineered or edited.

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

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

[0065] In some embodiments, the method comprises administering to the subject an effective amount of the population of cells or pharmaceutical composition.

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

[0067] In some embodiments, the subject has cancer. [Brief explanation of the drawings]

[0068] [Figure 1] FIG. 1 provides a schematic diagram illustrating an exemplary method ("Method 1") for differentiating stem cells into hematopoietic progenitors and NK cells in suspension culture. [Figure 2] FIG. 2 provides a schematic illustrating an exemplary method for differentiating stem cells into hematopoietic progenitors in suspension culture ("Method 2"). [Figure 3A]Figures 3A-3E show the characterization of hematopoietic progenitors (HPs) differentiated from stem cells based on the protocol provided in Figures 1 and 2. HPs were characterized on day 15 unless otherwise indicated. Figure 3A shows 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, ranging from 60-80% of all cells triple-positive for CD34 / CD43 / CD45. Figure 3C is a graph showing the average expansion of HPs compared to iPSCs seeded on day 0. Figure 3D is a graph showing the expansion of HPs generated using the indicated method at day 12 compared to a standard 2D differentiation protocol. Figure 3E provides a representative brightfield microscopy image of EBs prior to HP harvest. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4A] Figures 4A-4E show the characterization of natural killer (NK) cells differentiated from stem cells using Method 1. NK cells were characterized on day 40. Figure 4A shows flow cytometry analysis performed by gating cells to quantify the percentage of CD45+CD56+LFA1+ cells. Figure 4B is a graph showing NK purity of total cells positive for CD34 / CD45 / CD56 / LFA1. Figure 4C is a graph showing NK expansion compared to iPSCs seeded on day 0. Figure 4D is a graph showing NK expansion generated using Method 1 compared to a standard 2D differentiation protocol. Figure 4E provides a representative brightfield microscopy image of NK cells. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E]See legend to Figure 4A. [Figure 5] Figures 5A and 5B show D40-differentiated iNK cells incubated with breast adenocarcinoma MDA-MB231 cells at various T:E ratios. NK cells and MDA-MB231 cells were incubated in the absence (unstimulated, Figure 5A) or presence (stimulated, Figure 5B) of the cytokines IL-2 and IL-15. iNK cells reduced MDA proliferation in a dose-responsive manner. [Figure 6] FIG. 6 provides a schematic diagram illustrating an exemplary experimental design for differentiating genetically engineered stem cells into hematopoietic progenitors and NK cells in suspension culture. [Figure 7] Figure 7 is a graph showing the ratio of hematopoietic progenitor cells (HP) to either parental wild-type iPSCs or engineered FKBP12 knockout iPSCs encoding RACR (B2M-EF1a-RACR and FKBP12 KO cells) after 14 days in 3D suspension culture. Cells were transferred into 3D suspension culture using either gentle cell dissociation reagent (GCDR) or EDTA. DETAILED DESCRIPTION OF THE INVENTION

[0069] Detailed Description In some aspects, the present disclosure provides compositions and methods for generating hematopoietic progenitors, common lymphoid progenitors, pre-NK progenitors, NK progenitors, 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.

[0070] definition All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0071] Unless the context indicates otherwise, the various features described herein may be used in any combination with any feature or combination of features described herein, and each feature may be excluded or omitted from a combination.

[0072] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context indicates otherwise. The use of "and / or" refers to all possible combinations of one or more of the listed items.

[0073] As used herein, "subject" refers to a recipient of an 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.

[0074] As used herein, "treat," "treating," or "treatment" refers to any type of action or administration that benefits a subject with a disease or disorder, including improvement of the patient's condition (i.e., improvement, reduction, or amelioration of one or more symptoms, and a partial or complete response to treatment).

[0075] The term "effective amount" refers to an amount effective to bring about a desired biochemical, cellular, or physiological response. The term "therapeutically effective amount" refers to a therapeutic amount, dose, or administration regimen effective to elicit a desired therapeutic effect.

[0076] As used herein, a "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently linked via an ester bond between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar moiety of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

[0077] As used herein, "polypeptide" refers to a polymer of amino acid residues linked together by peptide bonds that forms part of (or the whole of) a protein.

[0078] It will be understood by those skilled in the art that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. It will further be understood that those skilled in the art may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide will be expressed.

[0079] Nucleic acids can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides that contain synthetic or modified nucleotides. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for the uses described herein, polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or life span of the polynucleotide of interest.

[0080] The term "variant" refers to a polynucleotide or polypeptide that has 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 the reference polynucleotide or polypeptide.

[0081] The term "inactivating mutation" refers to a mutation in a genomic sequence that disrupts the function of a gene. Inactivating mutations can be in any sequence region (e.g., coding or non-coding) that contributes to gene expression. Examples include, but are not limited to, cis-acting elements (enhancers) or sequences that are subject to transcription (e.g., mRNA transcript sequences). Inactivating mutations include mutations that render a gene or its encoded protein nonfunctional or that reduce the function of a gene or its encoded protein.

[0082] As used herein, the term "sequence identity" or "identity" in relation to polynucleotide or polypeptide sequences refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences match at each position in the alignment over the entire length of the reference sequence. "Percent identity" is the number of matched positions in the optimal alignment divided by the sum of the length of the reference sequence plus the length of any gaps in the reference sequence in the alignment. The optimal alignment is the alignment that results in the highest percent identity. Aligning sequences to determine percent identity can be achieved by several well-known methods, including using mathematical algorithms such as those in the BLAST suite or Clustal Omega sequence analysis program. Unless otherwise specified, the term "sequence identity" in the claims refers to the sequence identity calculated by BLAST version 2.12.0 using default parameters. Also, unless otherwise specified, the alignment is the alignment of all or part of the polynucleotide or polypeptide sequence of interest over the entire length of the reference sequence.

[0083] As used herein, the term "engineered" refers to cells that have been stably transduced with a heterologous polynucleotide or that have been subjected to gene editing to introduce, delete, or modify a polynucleotide within the cell, or cells that have been transiently transduced with a polynucleotide to cause a stable phenotypic change in the cell.

[0084] As used herein, the term "stem cell" is used to refer to a cell with an undifferentiated phenotype that can differentiate into, for example, hematopoietic progenitors and / or NK cells.

[0085] As used herein, the term "pluripotency" means that stem cells can form substantially all of the differentiated cell types of an organism, at least during culture.For example, embryonic stem cells are a type of pluripotent stem cells that can form cells from each of three germ layers, namely, ectoderm, mesoderm and endoderm.

[0086] As used herein, the term " induced pluripotent stem cell " and " iPSC " refer to the cell derived from somatic cells, which is reprogrammed into pluripotent state and can proliferate, selectively differentiate and mature.iPSC is the stem cell that is produced from differentiated adult cells, newborn cells or fetal cells, that is, reprogrammed, which can be induced or changed into the cell that can differentiate into the tissues of all three germ layers or cortical layers, namely, mesoderm, endoderm and ectoderm.The iPSC produced does not refer to the cell that is found in nature.

[0087] As used herein, the terms "hematopoietic stem cell," "hematopoietic precursor," or "hematopoietic progenitor cell" refer to stem cells that can give rise to both mature myeloid and lymphoid cell types, including natural killer cells, T cells, and B cells. Hematopoietic stem cells are typically characterized by CD34+.

[0088] The term "progenitor" refers to a cell that has partially differentiated into a desired cell type. Progenitor cells retain some degree of pluripotency and can differentiate into multiple cell types.

[0089] As used herein, "differentiate" or "differentiated" refers to the process and conditions under which an undifferentiated or immature (e.g., unspecialized) cell acquires the characteristics of a mature (specialized) cell, thereby acquiring a specific morphology and function. Stem cells (unspecialized) are often exposed to various conditions (e.g., growth factors and morphogenetic factors) to induce a specific lineage commitment or differentiation of the stem cell.

[0090] As used herein, "expanding" or "expansion" refers to increasing the number and / or purity of a cell type within a cell population by mitosis of cells with limited proliferative capacity, e.g., NK cells.

[0091] As used herein, "activity," "activate," or "activation" refers to the stimulation of activating receptors on cytotoxic innate lymphoid cells, resulting in cell division, cytokine secretion (e.g., IFNγ and / or TNFα), and / or release of cytolytic granules to regulate or support the immune response.

[0092] As used herein, "xeno-free" refers to compositions and methods that lack animal-derived materials (e.g., fetal bovine serum). In some embodiments, the xeno-free methods described herein do not include feeder cells.

[0093] As used herein, "2D culture" refers to growing a cell culture on a flat surface, such as the bottom of a Petri dish or flask, with the cells in contact with the flat surface.

[0094] As used herein, "3D suspension culture" refers to an artificially created environment that allows cells to grow in all three dimensions or interact with their surroundings in all three dimensions.3D suspension culture allows cells to grow in all directions in vitro, similar to growing in vivo.These three-dimensional cultures are usually grown in bioreactors, small capsules that allow cells to grow into spheroids, or 3D cell colonies.

[0095] The term "bioreactor" refers to any manufactured device or system that supports a biologically active environment. In some aspects, a bioreactor is a vessel in which the process of growing an organism takes place.

[0096] 3D suspension culture In some embodiments, the present disclosure provides a method for differentiating stem cells into hematopoietic progenitors in 3D suspension culture. In some embodiments, the method includes subculturing cells from 2D culture to 3D suspension culture. In some embodiments, the present disclosure provides a method for differentiating hematopoietic progenitors into NK cells by culturing the hematopoietic progenitors in 3D suspension culture. In some embodiments, the present disclosure provides a medium for expanding NK cells in suspension. In some embodiments, the differentiation medium and / or expansion medium described herein comprises a serum-free basal medium containing at least one exogenous factor that promotes differentiation and / or expansion.

[0097] 3D suspension culture volume In some embodiments, the cell populations of the present disclosure are cultured in a 3D suspension culture, 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, inclusive of all intervening values.

[0098] 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, inclusive of all intervening values. 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.

[0099] In some embodiments, the volume of the 3D suspension culture is contained in a vessel, hi some embodiments, the vessel is a flask, a multi-layer flask, a bottle, a dish, or a bioreactor.

[0100] 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.

[0101] In some embodiments, the methods described herein are carried out in a bioreactor. In some embodiments, the 3D culture suspension is a bioreactor. In some embodiments, the 3D culture suspension is a bioreactor of about 100 mL to about 1,000 mL. In some embodiments, the 3D culture suspension is a bioreactor of about 200 mL to about 2,000 mL. In some embodiments, the 3D culture suspension is a bioreactor of about 500 mL to about 2,000 mL.

[0102] Agitation of 3D suspension cultures In some embodiments, the 3D suspension culture is agitated. In some embodiments, the agitation is measured at a rate of revolutions per minute (RPM). In some embodiments, the 3D suspension culture is agitated at a rate of at least 1 RPM, at least 5 RPM, at least 10 RPM, at least 15 RPM, at least 20 RPM, at least 25 RPM, at least 30 RPM, at least 35 RPM, at least 40 RPM, at least 45 RPM, at least 50 RPM, at least 55 RPM, at least 60 RPM, at least 65 RPM, at least 70 RPM, at least 75 RPM, at least 80 RPM, at least 85 RPM, at least 90 RPM, at least 95 RPM, at least 100 RPM, at least 105 RPM, at least 110 RPM, at least 120 RPM, inclusive of all intervening values. The mixture is stirred at a speed of at least 130 RPM, at least 140 RPM, at least 150 RPM, at least 160 RPM, at least 170 RPM, at least 180 RPM, at least 190 RPM, at least 200 RPM, at least 250 RPM, at least 300 RPM, at least 350 RPM, at least 400 RPM, at least 450 RPM, at least 500 RPM, at least 550 RPM, at least 600 RPM, at least 650 RPM, at least 700 RPM, at least 750 RPM, at least 800 RPM, at least 850 RPM, at least 900 RPM, at least 950 RPM or at least 1,000 RPM.

[0103] In some embodiments, the 3D suspension cultures are rotated at a speed ranging from about 1 RPM to about 5 RPM, from about 5 RPM to about 10 RPM, from about 10 RPM to about 15 RPM, from about 15 RPM to about 20 RPM, from about 20 RPM to about 25 RPM, from about 25 RPM to about 30 RPM, from about 30 RPM to about 35 RPM, from about 35 RPM to about 40 RPM, from about 40 RPM to about 45 RPM, from about 45 RPM to about 50 RPM, from about 50 RPM to about 55 RPM, and from about 55 RPM to about 60 RPM, inclusive of all intervening speeds. RPM ~ about 60RPM, about 60RPM - about 65RPM, about 65RPM - about 70RPM, about 70RPM - about 75RPM, about 75RPM - about 80RPM, about 80RPM - about 85RPM, about 85RPM - about 90RPM, Approximately 90RPM to approximately 95RPM, approximately 95RPM to approximately 100RPM, approximately 100RPM to approximately 105RPM, approximately 105RPM to approximately 115RPM, approximately 110RPM to approximately 120RPM, approximately 120RPM to approximately 130RPM, approximately 1 30RPM to about 140RPM, about 140RPM to about 150RPM, about 150RPM to about 160RPM, about 160RPM to about 170RPM, about 170RPM to about 180RPM, about 180RPM to about 190RPM, about 190RPM to about 200RPM, about 200RPM to about 250RPM, about 250RPM to about 300RPM, about 300RPM to about 350RPM, about 350RPM to about 400RPM, about 400RPM to about 450RPM, The mixture is stirred at a speed of about 450 RPM to about 500 RPM, about 500 RPM to about 550 RPM, about 550 RPM to about 600 RPM, about 600 RPM to about 650 RPM, about 650 RPM to about 700 RPM, about 700 RPM to about 750 RPM, about 750 RPM to about 800 RPM, about 800 RPM to about 850 RPM, about 850 RPM to about 900 RPM, about 900 RPM to about 950 RPM, or about 950 RPM to about 1,000 RPM.

[0104] In some embodiments, the 3D suspension culture is agitated at a rate of about 1 RPM to about 1,000 RPM. In some embodiments, the 3D suspension culture is agitated at a rate of about 10 RPM to about 500 RPM. In some embodiments, the 3D suspension culture is agitated at a rate of about 50 RPM to about 100 RPM.

[0105] In some embodiments, the 3D suspension culture is agitated at a rate of about 70 RPM. In some embodiments, the 3D suspension culture is agitated at a rate of 70 RPM.

[0106] 3D suspension culture medium replacement In some embodiments, the medium of the 3D suspension culture is changed 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 medium volume of the 3D suspension culture is changed, including all intervening values.

[0107] In some embodiments, about 1%-5%, about 5%-10%, about 10%-15%, about 15%-20%, about 20%-25%, about 25%-30%, about 30%-35%, about 35%-40%, about 40%-45%, about 45%-50%, about 50%-55%, about 55%-60%, about 60%-65%, about 65%-70%, about 70%-75%, about 75%-80%, about 80%-85%, about 85%-90%, about 90%-95%, or about 95%-100% of the medium volume of the 3D suspension culture is exchanged, including all intervening values.

[0108] In some embodiments, a medium change occurs on day 1, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of differentiation.

[0109] In some embodiments, the medium is changed at least once a week and not changed on other days. For example, in some embodiments, the medium is changed on day 1 but not on days 2-7 of that week. In some embodiments, the medium is changed on day 2 but not on days 1 and 3-7 of that week. In some embodiments, the medium is changed on day 3 but not on days 1-2 and 4-7 of that week. In some embodiments, the medium is changed on day 4 but not on days 1-3 and 5-7 of that week. In some embodiments, the medium is changed on day 5 but not on days 1-4 and 6-7 of that week. In some embodiments, the medium is changed on day 6 but not on days 1-5 and 7 of that week. In some embodiments, the medium is changed on day 7 but not on days 1-6 of that week.

[0110] In some embodiments, the medium is changed at least twice a week. In some embodiments, the medium is changed on days 1 and 2, but not on days 3-7 of that week. In some embodiments, the medium is changed on days 1 and 3, but not on days 2 and 4-7 of that week. In some embodiments, the medium is changed on days 1 and 4, but not on days 2-3 and 5-7 of that week. In some embodiments, the medium is changed on days 1 and 5, but not on days 2-4 and 6-7 of that week. In some embodiments, the medium is changed on days 1 and 6, but not on days 2-5 and 6-7 of that week. In some embodiments, the medium is changed on days 1 and 7, but not on days 2-6 of that week. In some embodiments, the medium is changed on days 2 and 3, but not on days 2 and 3-7 of that week. In some embodiments, the medium is changed on days 2 and 4, but not on days 1, 3, and days 5-7 of that week. In some embodiments, the medium is changed on days 2 and 5, but not on days 1, 3, 5, 6, and 7 of that week. In some embodiments, the medium is changed on days 2 and 6, but not on days 1, 3, 4, 5, and 7 of that week. In some embodiments, the medium is changed on days 2 and 7, but not on days 1, 3-6 of that week. In some embodiments, the medium is changed on days 3 and 4, but not on days 1, 2, and days 5-7 of that week. In some embodiments, the medium is changed on days 3 and 5, but not on days 1, 2, 4, and days 6-7 of that week. In some embodiments, the medium is changed on days 3 and 6, but not on days 1, 2, 4, 5, and 7 of that week. In some embodiments, the medium is changed on days 3 and 7, but not on days 1, 2, 4, 5, and 7 of that week. In some embodiments, the medium is changed on days 4 and 5, but not on days 1-3 and 6-7 of that week. In some embodiments, the medium is changed on days 4 and 6, but not on days 1-3, 5, and 7 of that week. In some embodiments, the medium is changed on days 4 and 7, but not on days 1-3, 5, and 6 of that week.In some embodiments, the medium is changed on days 5 and 6, but not on days 1-4 and 7 of that week. In some embodiments, the medium is changed on days 5 and 7, but not on days 1-4 and 6 of that week. In some embodiments, the medium is changed on days 6 and 7, but not on days 1-5 of that week.

[0111] In some embodiments, the medium is changed at least three times per week. In some embodiments, the medium is changed on days 1-3, but not on days 4-7. In some embodiments, the medium is changed on days 1, 2, and 4, but not on days 3 and 5-7 of that week. In some embodiments, the medium is changed on days 1, 2, and 5, but not on days 3, 4, 6, and 7 of that week. In some embodiments, the medium is changed on days 1, 2, and 6, but not on days 3, 4, 5, and 7 of that week. In some embodiments, the medium is changed on days 1, 2, and 7, but not on days 3-6 of that week. In some embodiments, the medium is changed on days 1, 3, and 4, but not on day 2 and days 5-7. In some embodiments, the medium is changed on days 1, 3, and 5, but not on days 4, 5, 6, and 7 of that week. In some embodiments, the medium is changed on days 1, 3, and 6, but not on days 2-5 and 7 of that week. In some embodiments, the medium is changed on days 1, 3, and 7, but not on days 2 and 4-6 of that week. In some embodiments, the medium is changed on days 1, 3, and 7, but not on days 2 and 4-6 of that week. In some embodiments, the medium is changed on days 1, 4, and 5, but not on days 2, 3, 6, and 7. In some embodiments, the medium is changed on days 1, 4, and 6, but not on days 2-4 and 7 of that week. In some embodiments, the medium is changed on days 1, 4, and 7, but not on days 2, 3, 5, and 6 of that week. In some embodiments, the medium is changed on days 1, 5, and 6, but not on days 2-4 and 7 of that week. In some embodiments, the medium is changed on days 1, 5, and 7, but not on days 2 and 4-6 of that week. In some embodiments, the medium is changed on days 2, 3, and 4, but not on days 1 and 5-7 of that week. In some embodiments, the medium is changed on days 2, 3, and 5, but not on days 1, 4, 6, and 7 of that week. In some embodiments, the medium is changed on days 2, 3, and 6, but not on days 1, 4, 5, and 7 of that week. In some embodiments, the medium is changed on days 2, 3, and 7, but not on days 1, 4-6 of that week.In some embodiments, the medium is changed on days 2, 4, and 5, but not on days 1, 3, 6, and 7. In some embodiments, the medium is changed on days 2, 4, and 6, but not on days 1, 3, 5, and 7 of that week. In some embodiments, the medium is changed on days 2, 4, and 7, but not on days 1, 3, 5, and 6 of that week. In some embodiments, the medium is changed on days 2, 5, and 6, but not on days 1, 3, 4, and 7 of that week. In some embodiments, the medium is changed on days 2, 5, and 7, but not on days 1, 3, 4, and 6 of that week. In some embodiments, the medium is changed on days 2, 6, and 7, but not on days 1, 3, 4, and 5 of that week. In some embodiments, the medium is changed on days 3, 4, and 5, but not on days 1, 2, 6, and 7 of that week. In some embodiments, the medium is changed on days 3, 4, and 6, but not on days 1, 2, 5, and 7 of that week. In some embodiments, the medium is changed on days 3, 4, and 7, but not on days 1, 2, 5, and 6 of that week. In some embodiments, the medium is changed on days 3, 5, and 6, but not on days 1, 2, 4, and 7 of that week. In some embodiments, the medium is changed on days 3, 5, and 7, but not on days 1, 2, 4, and 6 of that week. In some embodiments, the medium is changed on days 3, 6, and 7, but not on days 1, 2, 4, and 5 of that week. In some embodiments, the medium is changed on days 4, 5, and 6, but not on days 1-3 and 7 of that week. In some embodiments, the medium is changed on days 4, 5, and 7, but not on days 1-3 and 6 of that week. In some embodiments, the medium is changed on days 4, 6, and 7, but not on days 1-3 and 5 of the week. In some embodiments, the medium is changed on days 5, 6, and 7, but not on days 1-4 of the week.

[0112] In some embodiments, the medium is changed at least four times per week. In some embodiments, the medium is not changed on days 1-3, but is changed on days 4-7. In some embodiments, the medium is not changed on days 1, 2, and 4, but is changed on days 3 and 5-7 of that week. In some embodiments, the medium is not changed on days 1, 2, and 5, but is changed on days 3, 4, 6, and 7 of that week. In some embodiments, the medium is not changed on days 1, 2, and 6, but is changed on days 3, 4, 5, and 7 of that week. In some embodiments, the medium is not changed on days 1, 2, and 7, but is changed on days 3-6 of that week. In some embodiments, the medium is not changed on days 1, 3, and 4, but is changed on day 2 and 5-7. In some embodiments, the medium is not changed on days 1, 3, and 5, but is changed on days 4, 5, 6, and 7 of that week. In some embodiments, the medium is not changed on days 1, 3, and 6, but is changed on days 2-5 and 7 of that week. In some embodiments, the medium is not changed on days 1, 3, and 7, but is changed on days 2 and 4-6 of that week. In some embodiments, the medium is not changed on days 1, 3, and 7, but is changed on days 2 and 4-6 of that week. In some embodiments, the medium is not changed on days 1, 4, and 5, but is changed on days 2, 3, 6, and 7. In some embodiments, the medium is not changed on days 1, 4, and 6, but is changed on days 2-4 and 7 of that week. In some embodiments, the medium is not changed on days 1, 4, and 7, but is changed on days 2, 3, 5, and 6 of that week. In some embodiments, the medium is not changed on days 1, 5, and 6, but is changed on days 2-4 and 7 of that week. In some embodiments, the medium is not changed on days 1, 5, and 7, but is changed on days 2 and 4-6 of the week. In some embodiments, the medium is not changed on days 2, 3, and 4, but is changed on days 1 and 5-7 of the week. In some embodiments, the medium is not changed on days 2, 3, and 5, but is changed on days 1, 4, 6, and 7 of the week. In some embodiments, the medium is not changed on days 2, 3, and 6, but is changed on days 1, 4, 5, and 7 of the week. In some embodiments, the medium is not changed on days 2, 3, and 7, but is changed on days 1, 4-6 of the week.In some embodiments, the medium is not changed on days 2, 4, and 5, but is changed on days 1, 3, 6, and 7. In some embodiments, the medium is not changed on days 2, 4, and 6, but is changed on days 1, 3, 5, and 7 of the week. In some embodiments, the medium is not changed on days 2, 4, and 7, but is changed on days 1, 3, 5, and 6 of the week. In some embodiments, the medium is not changed on days 2, 5, and 6, but is changed on days 1, 3, 4, and 7 of the week. In some embodiments, the medium is not changed on days 2, 5, and 7, but is changed on days 1, 3, 4, and 6 of the week. In some embodiments, the medium is not changed on days 2, 6, and 7, but is changed on days 1, 3, 4, and 5 of the week. In some embodiments, the medium is not changed on days 3, 4, and 5, but is changed on days 1, 2, 6, and 7 of the week. In some embodiments, the medium is not changed on days 3, 4, and 6, but is changed on days 1, 2, 5, and 7 of the week. In some embodiments, the medium is not changed on days 3, 4, and 7, but is changed on days 1, 2, 5, and 6 of the week. In some embodiments, the medium is not changed on days 3, 5, and 6, but is changed on days 1, 2, 4, and 7 of the week. In some embodiments, the medium is not changed on days 3, 5, and 7, but is changed on days 1, 2, 4, and 6 of the week. In some embodiments, the medium is not changed on days 3, 6, and 7, but is changed on days 1, 2, 4, and 5 of the week. In some embodiments, the medium is not changed on days 4, 5, and 6, but is changed on days 1-3 and 7 of the week. In some embodiments, the medium is not changed on days 4, 5, and 7, but is changed on days 1-3 and 6 of the week. In some embodiments, the medium is changed on days 1-3 and 5 of the week, but not on days 4, 6, and 7. In some embodiments, the medium is changed on days 1-4 of the week, but not on days 5, 6, and 7.

[0113] In some embodiments, the medium is changed at least five times per week. In some embodiments, the medium is not changed on days 1 and 2, but is changed on days 3-7 of that week. In some embodiments, the medium is not changed on days 1 and 3, but is changed on days 2 and 4-7 of that week. In some embodiments, the medium is not changed on days 1 and 4, but is changed on days 2-3 and 5-7 of that week. In some embodiments, the medium is not changed on days 1 and 5, but is changed on days 2-4 and 6-7 of that week. In some embodiments, the medium is not changed on days 1 and 6, but is changed on days 2-5 and 6-7 of that week. In some embodiments, the medium is not changed on days 1 and 7, but is changed on days 2-6 of that week. In some embodiments, the medium is not changed on days 2 and 3, but is changed on days 2 and 3-7 of that week. In some embodiments, the medium is not changed on days 2 and 4, but is changed on days 1, 3, and 5-7 of that week. In some embodiments, the medium is not changed on days 2 and 5, but is changed on days 1, 3, 5, 6, and 7 of that week. In some embodiments, the medium is not changed on days 2 and 6, but is changed on days 1, 3, 4, 5, and 7 of that week. In some embodiments, the medium is not changed on days 2 and 7, but is changed on days 1 and 3-6 of that week. In some embodiments, the medium is not changed on days 3 and 4, but is changed on days 1, 2, and 5-7 of that week. In some embodiments, the medium is not changed on days 3 and 5, but is changed on days 1, 2, 4, and 6-7 of that week. In some embodiments, the medium is not changed on days 3 and 6, but is changed on days 1, 2, 4, 5, and 7 of that week. In some embodiments, the medium is not changed on days 3 and 7, but is changed on days 1, 2, 4, 5, and 7 of the week. In some embodiments, the medium is not changed on days 4 and 5, but is changed on days 1-3 and 6-7 of the week. In some embodiments, the medium is not changed on days 4 and 6, but is changed on days 1-3, 5, and 7 of the week. In some embodiments, the medium is not changed on days 4 and 7, but is changed on days 1-3, 5, and 6 of the week.In some embodiments, the medium is not changed on days 5 and 6, but is changed on days 1-4 and 7 of the week. In some embodiments, the medium is not changed on days 5 and 7, but is changed on days 1-4 and 6 of the week. In some embodiments, the medium is not changed on days 6 and 7, but is changed on days 1-5 of the week.

[0114] In some embodiments, the medium is changed at least 6 times per week, with changes on other days. For example, the medium is not changed on day 1, but is changed on days 2-7 of that week. In some embodiments, the medium is not changed on day 1, but is changed on days 2-7 of that week. In some embodiments, the medium is not changed on day 2, but is changed on days 1 and 3-7 of that week. In some embodiments, the medium is not changed on day 3, but is changed on days 1-2 and 4-7 of that week. In some embodiments, the medium is not changed on day 4, but is changed on days 1-3 and 5-7 of that week. In some embodiments, the medium is not changed on day 5, but is changed on days 1-4 and 6-7 of that week. In some embodiments, the medium is not changed on day 6, but is changed on days 1-5 and 7 of that week. In some embodiments, the medium is not changed on day 7, but is changed on days 1-6 of that week.

[0115] In some embodiments, the medium is changed every other day of the week.

[0116] Seeding density of 3D suspension cultures In some embodiments, the cell populations of the present disclosure comprise at least 1 x 10 3 cells, at least 1 x 10 4 cells, at least 1 x 10 5 cells, at least 1 x 10 6 cells, at least 1 x 10 7 cells, at least 1 x 10 8 cells or at least 1 x 10 9 Cells are seeded in 3D suspension culture at a density of 1000 x g.

[0117] In some embodiments, the cell population of the present disclosure comprises about 1 x 103 cells ~1×10 4 cells, approximately 1 x 10 4 cells ~1×10 5 cells, approximately 1 x 10 5 cells 1×10 6 cells, approximately 1 x 10 6 cells ~1×10 7 cells, approximately 1 x 10 7 cells ~1×10 8 cells, or approximately 1 x 10 8 cells 1×10 9 Cells are seeded in 3D suspension culture at a density of 1000 x g.

[0118] Differentiation and Expansion Growth Medium In some embodiments, the present disclosure provides a medium for differentiating stem cells into hematopoietic progenitors. In some embodiments, the present disclosure provides a medium for differentiating hematopoietic progenitors into NK cells. In some embodiments, the present disclosure provides a medium for expanding NK cells. In some embodiments, the differentiation medium and / or expansion medium described herein comprises a serum-free basal medium containing at least one exogenous factor that promotes differentiation and / or expansion.

[0119] xeno-free medium In some embodiments, the differentiation medium and / or expansion medium described herein is a defined medium. As used herein, "defined medium" refers to a growth medium suitable for in vitro culture of human or animal cells, all chemical components of which are known. In some embodiments, the differentiation medium and / or expansion medium comprises a basal medium. In some embodiments, the basal medium comprises Iscove's modified Dulbecco's medium, serum albumin, human insulin, human transferrin, and 2-mercaptoethanol. In some embodiments, the basal medium comprises human serum albumin. In some embodiments, the basal medium does not contain animal-derived ingredients.

[0120] In some embodiments, the basal medium is selected from StemSpan SFEM II medium (STEMCELL Technologies; serum-free), Stemline II (Sigma-Aldrich; defined, serum-free, and animal component-free, GMP manufactured), CTS NK Xpander medium (Gibco; serum-free and animal component-free medium), STEMdiff Hematopoietic-EB Basal Medium (STEMCELL Technologies; serum-free), STEMdiff APEL 2 Medium (STEM CELL Technologies; serum-free and animal component-free), or Hematopoietic Progenitor Expansion Medium XF (PromoCell; serum-free and xeno-free medium). In some embodiments, the basal medium is StemSpan SFEM II medium. In some embodiments, the basal medium is Stemline II medium. In some embodiments, the basal medium is STEMdiff APEL 2 medium. In some embodiments, the hematopoietic progenitor differentiation medium and the NK cell differentiation medium have the same basal medium. In some embodiments, the hematopoietic progenitor differentiation medium and the NK cell differentiation medium have different basal media.

[0121] extrinsic factors In some embodiments, the differentiation medium and / or expansion medium described herein comprises exogenous factors. In some embodiments, the methods of the present disclosure comprise contacting different cell populations with various exogenous factors in a xeno-free medium to promote the differentiation of cells into, for example, hematopoietic progenitors and / or NK cells. In some embodiments, the exogenous factors include, but are not limited to, cytokines, such as interleukins, fibroblast growth factors (FGFs), stem cell factors (SCFs), phosphatidylinositol 3-kinase (PI3K) inhibitors, FMS-like tyrosine kinase 3 ligands (FLT3Ls), bone morphogenetic protein (BMP) pathway activators, pyrimido-indole derivatives, and aryl hydrocarbon receptor antagonists.

[0122] In some embodiments, the exogenous factor suitable for use in the differentiation medium and / or expansion medium is a cytokine. 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.

[0123] In some embodiments, the exogenous factor is interleukin 2 (IL-2). IL-2 is a secreted cytokine produced by activated CD4+ and CD8+ T lymphocytes and is important for the proliferation of T and B lymphocytes. IL-2 is a member of the interleukin 2 (IL2) cytokine subfamily, which includes IL-4, IL-7, IL-9, IL-15, IL-21, erythropoietin, and thrombopoietin.

[0124] In some embodiments, the exogenous factor is interleukin 7 (IL-7). IL-7 is a member of the interleukin 2 (IL2) cytokine subfamily. Lymphocyte differentiation and activation are critically dependent on IL-7 signaling.

[0125] In some embodiments, the exogenous factor is interleukin 12 (IL-12). IL-12 is a cytokine with a wide range of biological activities that acts on T cells and natural killer cells. NK cells can acquire memory-like properties after brief stimulation with IL-12.

[0126] In some embodiments, the exogenous factor is interleukin 15 (IL-15). IL-15 is a member of the interleukin 2 (IL2) cytokine subfamily. IL-15 regulates the activation and proliferation of NK cells.

[0127] In some embodiments, the exogenous factor is interleukin 18 (IL-18). IL-18 is a pro-inflammatory cytokine of the IL-1 family that is constitutively found as a precursor in the cytoplasm of various immune cells. IL-18 has been shown to potently activate NK cells.

[0128] In some embodiments, the exogenous factor is low-density lipoprotein (LDL), which induces increased proliferation and cytotoxic activity of NK cells.

[0129] In some embodiments, the exogenous factor is a fibroblast growth factor (FGF). FGF family members are cell signaling proteins produced by macrophages. The FGF family includes 23 members. In some embodiments, the exogenous factor is FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23. In some embodiments, the exogenous factor is FGF2.

[0130] 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 a key role in the expansion of early hematopoietic precursors and the generation of mature peripheral NK cells.

[0131] In some embodiments, the exogenous factor is stem cell factor (SCF), which plays an important role in stem cell survival, as well as stem cell self-renewal and maintenance.

[0132] In some embodiments, the exogenous factor is vascular endothelial growth factor (VEGF). The VEGF family is a subfamily of growth factors, the platelet-derived growth factor family of cystine-knot growth factors. The VEGF family includes five family members. In some embodiments, the exogenous factor is VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D. In some embodiments, the exogenous factor is VEGF-165. VEGF165 is a 38.2 kDa disulfide-linked homodimeric protein consisting of two 165 amino acid polypeptide chains.

[0133] 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 stem cell renewal and expansion. In some embodiments, the exogenous factor is an aryl hydrocarbon receptor antagonist selected from PD98059, StemRegenin 1 (SR1), GNF351, BAY 2416964, CH-223191, perillaldehyde, PDM-11, and BAY-218. In some embodiments, the exogenous factor is SR1.

[0134] 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 produce phosphoinositol-3-phosphate (PIP), phosphoinositol-3,4-bisphosphate (PIP2), and phosphoinositol-3,4,5-triphosphate (PIP3). Phosphoinositol-3-phosphate (PIP), phosphoinositol-3,4-bisphosphate (PIP2), and phosphoinositol-3,4,5-triphosphate (PIP3) act as second messengers in signal transduction cascades by docking proteins containing pleckstrin homology domains, FYVE domains, Phox domains, and other phospholipid-binding domains to various signaling complexes, often at the plasma membrane.

[0135] PI3K inhibitors include, but are not limited to, idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, duvelisib, idelalisib, leniolisib, palsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linpelisib, nemiralisib, pictilisib, pilaralisib, samotricisib, seletalisib, selavelisib, sonolicisib, tenalisib, voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, Acalisib, Omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, Hibiscone C, IC87114, LY294002, and PI-103. In some embodiments, the exogenous agent is selected from the group consisting of idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, duvelisib, idelalisib, leniolisib, palsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linpelisib, nemiralisib, pictilisib, pilalalisib, samotricisib, seretalisib, selavelisib, sonolicisib, tenalisib, voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, acalisib, omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, Hibiscon C, IC87114, LY294002, PI-103, or any combination thereof. In some embodiments, the exogenous agent is LY294002.

[0136] In some embodiments, the exogenous factor is an activator of the BMP pathway. Bone morphogenetic proteins (BMPs) are produced as large precursor molecules that are proteolytically processed into mature peptides after translation. BMPs act through specific transmembrane receptors located on the cell surface of target cells. BMP receptors are serine-threonine kinases similar to TGF-β receptors and are divided into two subgroups: type I receptors and type II receptors. BMPs can only tightly bind to the heterotetrameric complex of these receptors. This complex formation is essential for BMP signal transduction. Inside target cells, BMP signals are transmitted to the nucleus via specific signaling molecules called Smads, which are also involved in the suppression of BMP signals.

[0137] BMP is a multifunctional cytokine that is a member of the transforming growth factor beta superfamily. BMP receptors mediate BMP signaling by activating Smads. BMP ligands bind to the BMP receptors BMPRI and BMPRII. Phosphorylated BMPRII activates BMPRI. Phosphorylated BMPRI then phosphorylates receptor-activated Smad proteins (R-Smads), which associate with common mediator Smads (co-Smads) and enter the nucleus, where they regulate gene expression. BMP pathway activators include the agents disclosed in International Publication Nos. 2014011540, 2014062138, and 2005117994, which are incorporated herein by reference. BMP pathway activators include, 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.

[0138] In some embodiments, the exogenous factor is a ROCK inhibitor.Rho-associated kinase (ROCK) is a serine / threonine kinase that serves as a downstream effector of Rho kinase (there are three isoforms thereof: RhoA, RhoB, and RhoC).ROCK inhibitors include, but are not limited to, polynucleotides, polypeptides, and small molecules.The ROCK inhibitors contemplated herein can reduce ROCK expression and / or ROCK activity.The illustrative examples of ROCK inhibitors contemplated herein include, but are not limited to, anti-ROCK antibodies, dominant-negative ROCK mutants, siRNA, shRNA, miRNA, and antisense nucleic acids that target ROCK.

[0139] 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, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, and the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated by reference in its entirety. In some embodiments, the ROCK inhibitor is thiazovivin, Y27632, or pyrintegrin. In some embodiments, the ROCK inhibitor is Y27632.

[0140] Mesoderm / embryoid body differentiation medium In some embodiments, the present disclosure provides a differentiation medium for generating mesoderm and / or embryoid bodies from stem cells. In some embodiments, mesoderm cells are generated from iPSCs or hESCs. As stem cells begin to differentiate, three different germ layers are formed: ectoderm, mesoderm, and endoderm. Immune cells, such as NK cells, differentiate from mesoderm cells. Embryoid bodies are three-dimensional aggregates that can differentiate into cells of all three germ layers. In some embodiments, mesoderm cells are produced from embryoid bodies. In some embodiments, mesoderm cells produced by the compositions and methods of the present disclosure are further differentiated into hematopoietic precursors. In some embodiments, mesoderm cells produced by the compositions and methods of the present disclosure are further differentiated into NK cells.

[0141] In some embodiments, a population of stem cells (e.g., iPSCs or hESCs) is cultured with at least one exogenous factor to form mesoderm and / or embryoid body cells. In some embodiments, the exogenous factor is a bone morphogenetic protein (BMP) activator. In some embodiments, the exogenous factor is FGF. In some embodiments, the exogenous factor is VEGF. In some embodiments, the exogenous factor is a ROCK inhibitor. In some embodiments, the exogenous factor is selected from a BMP pathway activator, FGF, VEGF, a ROCK inhibitor, and any combination thereof. In some embodiments, the exogenous factor comprises a BMP pathway activator and an FGF. In some embodiments, the exogenous factor comprises a BMP pathway activator and a VEGF. In some embodiments, the exogenous factor comprises a BMP pathway activator and a ROCK inhibitor. In some embodiments, the exogenous factor comprises an FGF and a VEGF. In some embodiments, the exogenous factor comprises an FGF and a ROCK inhibitor. In some embodiments, the exogenous factor comprises a VEGF and a ROCK inhibitor. In some embodiments, the exogenous factor comprises a BMP pathway activator, an FGF, and a VEGF. In some embodiments, the exogenous factor comprises a BMP pathway activator, an FGF, and a ROCK inhibitor. In some embodiments, the exogenous factor comprises an FGF, a VEGF, and a ROCK inhibitor. In some embodiments, the exogenous factor comprises a BMP pathway activator, an FGF, and a ROCK inhibitor. In some embodiments, the exogenous factor comprises a BMP pathway activator, an FGF, and a ROCK inhibitor. In some embodiments, the exogenous factor comprises a BMP pathway activator, an FGF, and a ROCK inhibitor.

[0142] 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 comprise BMP4, FGF2, and Y27632. In some embodiments, the exogenous factors comprise BMP4, FGF2, VEGF-165, and Y27632.

[0143] In some embodiments, BMP4, FGF2, VEGF and / or ROCK inhibitor are used in the mesoderm formation step.For example, the mesoderm formation step can include contacting a cell population with BMP4 and FGF2; with BMP4, FGF2 and ROCK inhibitor; with BMP4 and VEGF; with BMP4, VEGF and ROCK inhibitor; with FGF2 and VEGF; with FGF2, VEGF and ROCK inhibitor; BMP4, FGF2 and VEGF; BMP4, FGF2, VEGF and ROCK inhibitor; or individually with any one of BMP4, FGF2, VEGF and ROCK inhibitor.

[0144] In some embodiments, bone morphogenetic protein (BMP) activators are present in an amount of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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 2 In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at a concentration of about 0 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1 to 50 ng / ml.

[0145] In some embodiments, the BMP pathway activator is BMP4. In some embodiments, BMP4 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, BMP4 is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, BMP4 is present in the differentiation medium at about 1-50 ng / ml.

[0146] In some embodiments, FGF2 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about 12 ng / ml. g / 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 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, approximately 80ng / ml, approximately 85ng / ml, approximately 90ng / ml, approximately 95ng / ml, approximately 100ng / ml, approximately 110ng / ml, approximately 120ng / ml, approximately 130ng / ml In some embodiments, FGF2 is present in the differentiation medium at a concentration of about 140 ng / ml, about 150 ng / ml, about 160 ng / ml, about 170 ng / ml, about 180 ng / ml, about 190 ng / ml, about 200 ng / ml, about 250 ng / ml, about 300 ng / ml, about 350 ng / ml, about 400 ng / ml, about 450 ng / ml, or about 500 ng / ml, or any range derivable therein. In some embodiments, FGF2 is present in the differentiation medium at about 1-100 ng / ml.

[0147] In some embodiments, VEGF is at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, VEGF is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, VEGF is present in the differentiation medium at about 5-100 ng / ml.

[0148] In some embodiments, the ROCK inhibitor is at about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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, In some embodiments, the ROCK inhibitor is present in the differentiation medium at a concentration of about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derivable therein. In some embodiments, the ROCK inhibitor is present in the differentiation medium at about 0.1-20 μM.

[0149] In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, Y27632 is present in an amount of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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, or about 22 μM. In some embodiments, Y27632 is present in the differentiation medium at a concentration of about 0.1 to 100 μM.

[0150] In some embodiments, the mesoderm differentiation medium comprises a BMP pathway activator, FGF, and VEGF. In some embodiments, the mesoderm differentiation medium comprises a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor. In some embodiments, the mesoderm differentiation medium comprises a defined xeno-free basal medium, a BMP pathway activator, FGF, and VEGF. In some embodiments, the mesoderm differentiation medium comprises a defined xeno-free basal medium, a BMP pathway activator, FGF, VEGF, and a ROCK inhibitor. In some embodiments, the mesoderm differentiation medium comprises BMP4, FGF2, and VEGF-165. In some embodiments, the mesoderm differentiation medium comprises BMP4, FGF, VEGF-165, and a ROCK inhibitor. In some embodiments, the mesoderm differentiation medium comprises BMP4, FGF, VEGF-165, and Y27632.

[0151] In some embodiments, the mesoderm differentiation medium comprises 1 to 150 ng / mL BMP4, 1 to 50 ng / mL FGF2, and 1 to 100 ng / mL VEGF-165. In some embodiments, the mesoderm differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 0.1 to 20 μM ROCK inhibitor. In some embodiments, the mesoderm differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 0.1 to 20 μM Y27632.

[0152] Hematopoietic progenitor differentiation medium In some embodiments, the present disclosure provides differentiation media for generating HP cells from mesoderm cells and embryoid body cells. In some embodiments, the mesoderm cells and embryoid body cells produced by the compositions and methods of the present disclosure are further differentiated into hematopoietic progenitors.

[0153] In some embodiments, a population of HP cells 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 pyrimido-indole derivative. In some embodiments, the exogenous factor is an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor is a TGF-β receptor inhibitor. In some embodiments, the exogenous factor is selected from a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, and any combination thereof. In some embodiments, the exogenous factor is selected from a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, and any combination thereof. In some embodiments, the exogenous factor is selected from a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, a PI3K inhibitor, a pyrimido-indole derivative, an aryl hydrocarbon receptor antagonist, a TGF-β receptor inhibitor, and any combination thereof.

[0154] In some embodiments, the exogenous factor comprises a BMP pathway activator and FGF. In some embodiments, the exogenous factor comprises a BMP pathway activator and VEGF. In some embodiments, the exogenous factor comprises a BMP pathway activator and SCF. In some embodiments, the exogenous factor comprises a BMP pathway activator and TPO. In some embodiments, the exogenous factor comprises a BMP pathway activator and LDL. In some embodiments, the exogenous factor comprises a BMP pathway activator and a PI3K inhibitor. In some embodiments, the exogenous factor comprises FGF and VEGF. In some embodiments, the exogenous factor comprises FGF and SCF. In some embodiments, the exogenous factor comprises FGF and TPO. In some embodiments, the exogenous factor comprises FGF and LDL. In some embodiments, the exogenous factor comprises an FGF and a PI3K inhibitor. In some embodiments, the exogenous factor comprises VEGF and SCF. In some embodiments, the exogenous factor comprises VEGF and TPO. In some embodiments, the exogenous factor comprises VEGF and LDL. In some embodiments, the exogenous factor comprises VEGF and a PI3K inhibitor. In some embodiments, the exogenous factor comprises SCF and TPO. In some embodiments, the exogenous factor comprises SCF and LDL. In some embodiments, the exogenous factor comprises SCF and a PI3K inhibitor. In some embodiments, the exogenous factor comprises TPO and LDL. In some embodiments, the exogenous factor comprises TPO and a PI3K inhibitor. In some embodiments, the exogenous factor comprises LDL and a PI3K inhibitor.

[0155] In some embodiments, the exogenous factor comprises a BMP pathway activator, FGF, and VEGF. In some embodiments, the exogenous factor comprises a BMP pathway activator, FGF, and SCF. In some embodiments, the exogenous factor comprises a BMP pathway activator, FGF, and TPO. In some embodiments, the exogenous factor comprises a BMP pathway activator, FGF, and LDL. In some embodiments, the exogenous factor comprises a BMP pathway activator, FGF, and a PI3K inhibitor. In some embodiments, the exogenous factor comprises a BMP pathway activator, VEGF, and SCF. In some embodiments, the exogenous factor comprises a BMP pathway activator, VEGF, and TPO. In some embodiments, the exogenous factor comprises a BMP pathway activator, VEGF, and LDL. In some embodiments, the exogenous factor comprises a BMP pathway activator, VEGF, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, SCF, and TPO. In some embodiments, the exogenous factors comprise a BMP pathway activator, SCF, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, TPO, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise FGF, VEGF, and SCF. In some embodiments, the exogenous factors comprise FGF, VEGF, and TPO. In some embodiments, the exogenous factors comprise 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 a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, TPO, and LDL. In some embodiments, the exogenous factors include FGF, TPO, and a PI3K inhibitor.In some embodiments, the exogenous factors include FGF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include VEGF, SCF, and TPO. In some embodiments, the exogenous factors include VEGF, SCF, and LDL. In some embodiments, the exogenous factors include VEGF, SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include VEGF, TPO, and LDL. In some embodiments, the exogenous factors include VEGF, TPO, and a PI3K inhibitor.

[0156] In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, and SCF. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, and TPO. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, and TPO. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, TPO, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, FGF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, FGF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, SCF, and TPO. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, SCF, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, TPO, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, TPO, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, SCF, TPO, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, TPO, LDL, and a PI3K inhibitor.In some embodiments, the exogenous factors include FGF, VEGF, SCF, and TPO. In some embodiments, the exogenous factors include FGF, VEGF, SCF, and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, TPO, and LDL. In some embodiments, the exogenous factors include FGF, VEGF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors include FGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors include FGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, SCF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, TPO, LDL, and a PI3K inhibitor.

[0157] In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, and TPO. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, TPO, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, FGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, FGF, SCF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, FGF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors comprise a BMP pathway activator, VEGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, VEGF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, SCF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL, and a PI3K inhibitor.In some embodiments, the exogenous factors include FGF, VEGF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, SCF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, SCF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include VEGF, SCF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include VEGF, SCF, TPO, LDL, and a PI3K inhibitor.

[0158] In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, TPO, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, FGF, SCF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include a BMP pathway activator, VEGF, SCF, TPO, LDL, and a PI3K inhibitor. In some embodiments, the exogenous factors include FGF, VEGF, SCF, TPO, LDL, and a PI3K inhibitor.

[0159] In some embodiments, the exogenous factors include a BMP pathway activator, FGF, VEGF, SCF, TPO, LDL, and a PI3K inhibitor.

[0160] 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 factor comprises VEGF-165 and SCF. In some embodiments, the exogenous factor comprises VEGF-165 and TPO. In some embodiments, the exogenous factor comprises VEGF-165 and LDL. In some embodiments, the exogenous factor comprises VEGF-165 and LY294002. In some embodiments, the exogenous factor comprises SCF and TPO. In some embodiments, the exogenous factor comprises SCF and LDL. In some embodiments, the exogenous factor comprises SCF and LY294002. In some embodiments, the exogenous factor comprises TPO and LDL. In some embodiments, the exogenous factor comprises TPO and LY294002. In some embodiments, the exogenous factor comprises LDL and LY294002.

[0161] 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.

[0162] 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.

[0163] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, and TPO. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, TPO, and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, TPO, and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, LDL, and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, SCF, TPO, and LDL. In some embodiments, the exogenous factors include BMP4, FGF2, SCF, TPO, and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, SCF, LDL, and LY294002. In some embodiments, the exogenous factors include BMP4, FGF2, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, TPO, and LDL. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, TPO, and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, SCF, LDL, and LY294002. In some embodiments, the exogenous factors include BMP4, VEGF-165, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include BMP4, SCF, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO, and LDL. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO, and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO, and LY294002.In some embodiments, the exogenous factors include FGF2, VEGF-165, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO, and LDL. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, TPO, and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, SCF, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, VEGF-165, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include FGF2, SCF, TPO, LDL, and LY294002. In some embodiments, the exogenous factors include VEGF-165, SCF, TPO, LDL, and LY294002.

[0164] 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, TPO, 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.

[0165] In some embodiments, the exogenous factors include BMP4, FGF2, VEGF-165, SCF, TPO, LDL, and LY294002.

[0166] In some embodiments, bone morphogenetic protein (BMP) activators are present in an amount of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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 2 In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at a concentration of about 0 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, the bone morphogenetic protein (BMP) activator is present in the differentiation medium at about 1 to 50 ng / ml.

[0167] In some embodiments, the bone morphogenetic protein (BMP) activator is BMP4. In some embodiments, BMP4 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, BMP4 is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, BMP4 is present in the differentiation medium at about 1-50 ng / ml.

[0168] In some embodiments, FGF2 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, FGF2 is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, FGF2 is present in the differentiation medium at about 1-50 ng / ml.

[0169] In some embodiments, VEGF is at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, VEGF is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, VEGF is present in the differentiation medium at about 1-100 ng / ml.

[0170] In some embodiments, SCF is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about In some embodiments, SCF is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, SCF is present in the differentiation medium at about 1-100 ng / ml.

[0171] In some embodiments, TPO is administered at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about In some embodiments, TPO is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, TPO is present in the differentiation medium at about 1 to 100 ng / ml.

[0172] In some embodiments, LDL is about 0.1 to 500 μg / ml, about 1 to 250 μg / ml, about 1 to 150 μg / ml, about 5 to 100 μg / ml, about or about 0.1 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 μg / ml, about 19 μg / ml, about 20 μg / ml, about In some embodiments, LDL is present in the differentiation medium at a concentration of 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 or 100 μg / ml, or any range derivable therein. In some embodiments, LDL is present in the differentiation medium at about 1-50 μg / ml.

[0173] In some embodiments, the PI3K inhibitor is at about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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, In some embodiments, the PI3K inhibitor is present in the differentiation medium at a concentration of about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derivable therein. In some embodiments, the PI3K inhibitor is present in the differentiation medium at about 0.1 to 100 μM.

[0174] In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, LY294002 is present at a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 100 μM, 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 2 In some embodiments, LY294002 is present in the differentiation medium at a concentration of about 1 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 or 100 μM, or any range derivable therein. In some embodiments, LY294002 is present in the differentiation medium at about 0.1-100 μM.

[0175] In some embodiments, the Pyrimido-indole Derivatives have a saturation of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 62 μM, about 63 μM, about 64 μM, about 65 μM, about 66 μM, about 67 μM, about 68 μM, about 69 μM, about 70 μM, about 71 μM, about 72 μM, about 73 μM In some embodiments, the pyrimido-indole derivative is present in the differentiation medium at a concentration of about 0.1 to 10 μM.

[0176] In some embodiments, the pyrimido-indole derivative is UM729. In some embodiments, UM729 has 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, or about 22 μM. In some embodiments, UM729 is present in the differentiation medium at a concentration of about 0.1-10 μM.

[0177] In some embodiments, the aryl hydrocarbon receptor antagonist has a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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 The aryl hydrocarbon receptor antagonist is present in the differentiation medium at a concentration of about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derivable therein. In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at about 0.1 to 10 μM.

[0178] In some embodiments, the aryl hydrocarbon receptor antagonist is StemRegenin 1 (SR1). In some embodiments, SR1 has a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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, or 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 or 100 μM, about or 100 μM, or any range derivable therein. In some embodiments, SR1 is present in the differentiation medium at about 0.1-10 μM.

[0179] In some embodiments, the TGF-β receptor inhibitor is about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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 In some embodiments, the TGF-β receptor inhibitor is present in the differentiation medium at a concentration of about 0.1 to 20 μM.

[0180] In some embodiments, the TGF-β receptor inhibitor is GW788388. In some embodiments, GW788388 is present in an amount of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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, In some embodiments, GW788388 is present in the differentiation medium at a concentration of about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derivable therein. In some embodiments, GW788388 is present in the differentiation medium at about 0.1-20 μM.

[0181] In some embodiments, the TGF-β receptor inhibitor is SB431542. In some embodiments, GW788388 is present in an amount of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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, In some embodiments, SB431542 is present in the differentiation medium at a concentration of about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derivable therein. In some embodiments, SB431542 is present in the differentiation medium at about 0.1 to 20 μM.

[0182] 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 Y27632.

[0183] In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 5 to 50 ng / mL FGF2, and 1 to 100 ng / mL VEGF-165. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 1 to 20 μM ROCK inhibitor. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 1 to 20 μM Y27632.

[0184] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and a pyrimido-indole derivative. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0185] 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 50 ng / mL BMP4, 5-50 ng / mL FGF2, 1-100 ng / mL VEGF-165, 1-100 ng / mL SCF, 1-100 ng / mL TPO, and 1-50 μg / mL LDL.

[0186] In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and a pyrimido-indole derivative. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and SR1. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, and UM729. In some embodiments, the HP differentiation medium comprises BMP4, FGF, VEGF-165, UM729, and SR1.

[0187] In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 0.1 to 10 μM UM729. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 0.1 to 10 μM SR1. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, 0.1 to 10 μM UM729, and 0.1 to 10 μM SR1.

[0188] In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a pyrimido-indole derivative, and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, an aryl hydrocarbon receptor antagonist, and a TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises a BMP pathway activator, FGF, VEGF, a pyrimido-indole derivative, an aryl hydrocarbon receptor antagonist, and a TGF-β receptor inhibitor.

[0189] 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.

[0190] In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 0.1 to 20 μM TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 0.1 to 20 μM GW788388. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, 0.1 to 10 μM UM729, and 0.1 to 20 μM GW788388. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, 0.1 to 10 μM SR1, and 0.1 to 20 μM GW788388. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, 0.1 to 10 μM UM729, 0.1 to 10 μM SR1, and 0.1 to 20 μM GW788388.

[0191] 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.

[0192] In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 0.1 to 20 μM TGF-β receptor inhibitor. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, and 0.1 to 20 μM SB431542. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, 0.1 to 10 μM UM729, and 0.1 to 20 μM SB431542. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, 0.1 to 10 μM SR1, and 0.1 to 20 μM SB431542. In some embodiments, the HP differentiation medium comprises 1 to 50 ng / mL BMP4, 1 to 50 ng / mL FGF, 1 to 100 ng / mL VEGF-165, 0.1 to 10 μM UM729, 0.1 to 10 μM SR1, and 0.1 to 20 μM SB431542.

[0193] NK cell differentiation medium In some embodiments, the present disclosure provides a differentiation medium for generating NK cells from HP cells. In some embodiments, NK cells are generated from HP cells. In some embodiments, HP cells produced by the compositions and methods of the present disclosure are further differentiated into NK cells.

[0194] In some embodiments, a population of HP cells 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 pyrimido-indole derivative. In some embodiments, the exogenous factor is an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor is selected from SCF, IL-7, IL-15, IL-12, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0195] In some embodiments, the exogenous factors comprise SCF and IL-7. In some embodiments, the exogenous factors comprise SCF and IL-15. In some embodiments, the exogenous factors comprise SCF and IL-12. In some embodiments, the exogenous factors comprise SCF and FLT3L. In some embodiments, the exogenous factors comprise SCF and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7 and IL-15. In some embodiments, the exogenous factors comprise IL-7 and IL-12. In some embodiments, the exogenous factors comprise IL-7 and FLT3L. In some embodiments, the exogenous factors comprise IL-7 and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7 and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-15 and IL-12. In some embodiments, the exogenous factor comprises IL-15 and FLT3L. In some embodiments, the exogenous factor comprises IL-15 and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises IL-15 and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises IL-12 and FLT3L. In some embodiments, the exogenous factor comprises IL-12 and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises IL-12 and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factor comprises FLT3L and a pyrimido-indole derivative. In some embodiments, the exogenous factor comprises FLT3L and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous agent comprises a pyrimido-indole derivative and an aryl hydrocarbon receptor antagonist.

[0196] In some embodiments, the exogenous factors comprise SCF, IL-7, and IL-12. In some embodiments, the exogenous factors comprise SCF, IL-7, and IL-15. In some embodiments, the exogenous factors comprise SCF, IL-7, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-7, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, and IL-15. In some embodiments, the exogenous factors comprise SCF, IL-12, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-12, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-12, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, and IL-15. In some embodiments, the exogenous factors comprise IL-7, IL-12, and FLT3L. In some embodiments, the exogenous factors comprise IL-7, IL-12, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-12, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise IL-7, IL-15, and a pyrimido-indole derivative.In some embodiments, the exogenous factors comprise IL-7, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise IL-12, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-12, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist.

[0197] In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, and IL-15. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist.In some embodiments, the exogenous factors comprise SCF, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0198] In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-12, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0199] In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0200] In some embodiments, the exogenous factors comprise SCF, IL-7, and IL-12. In some embodiments, the exogenous factors comprise SCF, IL-7, and IL-15. In some embodiments, the exogenous factors comprise SCF, IL-7, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-7, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-7, and an aryl hydrocarbon receptor antagonist. In some embodiments, the exogenous factors comprise SCF, IL-12, and IL-15. In some embodiments, the exogenous factors comprise SCF, IL-12, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-12, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-12, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, IL-15, and SR1. In some embodiments, the exogenous factors comprise SCF, FLT3L, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise SCF, FLT3L, and SR1. In some embodiments, the exogenous factors comprise SCF, a pyrimido-indole derivative, and SR1. In some embodiments, the exogenous factors comprise IL-7, IL-12, and IL-15. In some embodiments, the exogenous factors comprise IL-7, IL-12, and FLT3L. In some embodiments, the exogenous factors comprise IL-7, IL-12, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-12, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise IL-7, IL-15, and a pyrimido-indole derivative. In some embodiments, the exogenous factors comprise IL-7, IL-15, and SR1. In some embodiments, the exogenous factors comprise IL-7, FLT3L, and a pyrimido-indole derivative.In some embodiments, the exogenous factors comprise IL-7, FLT3L, and SR1. In some embodiments, the exogenous factors comprise IL-7, UM729, and SR1. In some embodiments, the exogenous factors comprise IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise IL-12, IL-15, and UM729. In some embodiments, the exogenous factors comprise IL-12, IL-15, and SR1. In some embodiments, the exogenous factors comprise IL-12, FLT3L, and UM729. In some embodiments, the exogenous factors comprise IL-12, FLT3L, and SR1.

[0201] In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, and IL-15. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, and UM729. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, and UM729. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, FLT3L, and UM729. In some embodiments, the exogenous factors comprise SCF, IL-7, FLT3L, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, UM729, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, and UM729. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-12, FLT3L, and UM729. In some embodiments, the exogenous factors comprise SCF, IL-12, FLT3L, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-12, UM729, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-15, FLT3L, and UM729. In some embodiments, the exogenous factors comprise SCF, IL-15, FLT3L, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-15, FLT3L, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-15, FLT3L, and SR1. In some embodiments, the exogenous factors comprise SCF, FLT3L, UM729, and SR1. In some embodiments, the exogenous factors comprise 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 comprise IL-7, IL-12, IL-15, and SR1. In some embodiments, the exogenous factors comprise IL-7, IL-12, FLT3L, and UM729. In some embodiments, the exogenous factors comprise IL-7, IL-12, FLT3L, and SR1. In some embodiments, the exogenous factors comprise IL-7, IL-12, UM729, and SR1. In some embodiments, the exogenous factors comprise IL-7, IL-15, FLT3L, and UM729. In some embodiments, the exogenous factors comprise IL-7, IL-15, FLT3L, and SR1. In some embodiments, the exogenous factors comprise IL-7, IL-15, FLT3L, and SR1. In some embodiments, the exogenous factors include IL-7, FLT3L, UM729, and SR1.

[0202] In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, and FLT3L. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, IL-15, and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, and UM729. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, FLT3L, and SR1. In some embodiments, the exogenous factors include SCF, IL-7, IL-12, UM729, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, FLT3L, and UM729. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, FLT3L, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-15, UM729, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, FLT3L, UM729, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-12, IL-15, FLT3L, and UM729. In some embodiments, the exogenous factors comprise 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, FLT3L, UM729, and SR1.In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, FLT3L, and UM729. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, FLT3L, and SR1. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, UM729, and SR1. In some embodiments, the exogenous factors comprise IL-7, IL-12, FLT3L, UM729, and SR1. In some embodiments, the exogenous factors comprise IL-7, IL-15, FLT3L, UM729, and SR1. In some embodiments, the exogenous factors comprise IL-12, IL-15, FLT3L, UM729, and SR1.

[0203] In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, FLT3L, and UM729. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, FLT3L, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, IL-15, UM729, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, FLT3L, UM729, and SR1. In some embodiments, the exogenous factors comprise SCF, IL-7, IL-12, FLT3L, UM729, and SR1. In some embodiments, the exogenous factors include SCF, IL-12, IL-15, FLT3L, UM729, and SR1. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, FLT3L, UM729, and SR1.

[0204] In some embodiments, SCF is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about In some embodiments, SCF is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, SCF is present in the differentiation medium at about 1-50 ng / ml.

[0205] In some embodiments, IL-7 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, IL-7 is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, IL-7 is present in the differentiation medium at about 1-50 ng / ml.

[0206] In some embodiments, IL-12 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, IL-12 is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, IL-12 is present in the differentiation medium at about 1-100 ng / ml.

[0207] In some embodiments, IL-15 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, IL-15 is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, IL-15 is present in the differentiation medium at about 1-100 ng / ml.

[0208] In some embodiments, FLT3L is at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, In some embodiments, FLT3L is present in the differentiation medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, FLT3L is present in the differentiation medium at about 1-100 ng / ml.

[0209] In some embodiments, the Pyrimido-indole Derivatives have a saturation of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 62 μM, about 63 μM, about 64 μM, about 65 μM, about 66 μM, about 67 μM, about 68 μM, about 69 μM, about 70 μM, about 71 μM, about 72 μM, about 73 μM In some embodiments, the pyrimido-indole derivative is present in the differentiation medium at a concentration of about 0.1 to 10 μM.

[0210] In some embodiments, the pyrimido-indole derivative is UM729. In some embodiments, UM729 has 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, or about 22 μM. In some embodiments, UM729 is present in the differentiation medium at a concentration of about 0.1-10 μM.

[0211] In some embodiments, the aryl hydrocarbon receptor antagonist has a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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 The aryl hydrocarbon receptor antagonist is present in the differentiation medium at a concentration of about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, or any range derivable therein. In some embodiments, the aryl hydrocarbon receptor antagonist is present in the differentiation medium at about 0.1 to 10 μM.

[0212] In some embodiments, the aryl hydrocarbon receptor antagonist is StemRegenin 1 (SR1). In some embodiments, SR1 has a concentration of about 0.1 to 500 μM, about 1 to 250 μM, about 1 to 150 μM, about 5 to 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, or 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 or 100 μM, about or 100 μM, or any range derivable therein. In some embodiments, StemRegenin 1 (SR1) is present in the differentiation medium at about 0.1 to 10 μM.

[0213] In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, and FLT3L. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, and a pyrimido-indole derivative. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, and an aryl hydrocarbon receptor antagonist. In some embodiments, the NK cell differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist.

[0214] 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.

[0215] 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 of a pyrimido-indole derivative. 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 of an 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 of a pyrimido-indole derivative, and 0.1-10 μM of an aryl hydrocarbon receptor antagonist.

[0216] In some embodiments, the NK cell differentiation medium comprises 1 to 50 ng / ml SCF, 1 to 50 ng / ml IL-7, 1 to 100 ng / ml IL-12, 1 to 100 ng / ml IL-15, 1 to 100 ng / ml FLT3L, and SR 1. In some embodiments, the NK cell differentiation medium comprises 1 to 50 ng / ml SCF, 1 to 50 ng / ml IL-7, 1 to 100 ng / ml IL-12, 1 to 100 ng / ml IL-15, 1 to 100 ng / ml FLT3L, and 0.1 to 10 μM UM729. In some embodiments, the NK cell differentiation medium comprises 1 to 50 ng / ml SCF, 1 to 50 ng / ml IL-7, 1 to 100 ng / ml IL-12, 1 to 100 ng / ml IL-15, 1 to 100 ng / ml FLT3L, 1 to 10 μM SR1, and 0.1 to 10 μM UM729.

[0217] 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 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.

[0218] NK cell expansion medium In some embodiments, the present disclosure provides expansion media for generating mature NK cells from differentiated NK cells. In some embodiments, the differentiated NK cells are generated from HP cells. In some embodiments, the differentiated NK cells produced by the compositions and methods of the present disclosure are further expanded into mature NK cells.

[0219] In some embodiments, the population of differentiated NK cells 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 activation bead. In some embodiments, the exogenous factor is selected from IL-2, IL-7, IL-12, IL-15, IL-18, and an activation bead.

[0220] In some embodiments, the exogenous factors comprise IL-2 and IL-7. In some embodiments, the exogenous factors comprise IL-2 and IL-12. In some embodiments, the exogenous factors comprise IL-2 and IL-15. In some embodiments, the exogenous factors comprise IL-2 and IL-18. In some embodiments, the exogenous factors comprise IL-2 and activation beads. In some embodiments, the exogenous factors comprise IL-7 and IL-12. In some embodiments, the exogenous factors comprise IL-7 and IL-15. In some embodiments, the exogenous factors comprise IL-7 and IL-18. In some embodiments, the exogenous factors comprise IL-7 and activation beads. In some embodiments, the exogenous factors comprise IL-12 and IL-15. In some embodiments, the exogenous factors comprise IL-12 and IL-18. In some embodiments, the exogenous factors comprise IL-12 and activation beads. In some embodiments, the exogenous factors comprise IL-15 and IL-18. In some embodiments, the exogenous factors comprise IL-15 and activation beads. In some embodiments, the exogenous factors comprise IL-18 and activation beads.

[0221] 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 comprise IL-2, IL-18, and activation beads. In some embodiments, the exogenous factors comprise IL-7, IL-12, and IL-15. In some embodiments, the exogenous factors comprise IL-7, IL-12, and IL-18. In some embodiments, the exogenous factors comprise IL-7, IL-12, and activation beads. In some embodiments, the exogenous factors comprise IL-7, IL-15, and IL-18. In some embodiments, the exogenous factors comprise IL-7, IL-15, and activation beads. In some embodiments, the exogenous factors comprise IL-7, IL-18, and activation beads. In some embodiments, the exogenous factors comprise IL-12, IL-15, and IL-18. In some embodiments, the exogenous factors comprise IL-12, IL-15, and IL-18. In some embodiments, the exogenous factors comprise 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.

[0222] 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 comprise IL-2, IL-12, IL-15, and activated beads. In some embodiments, the exogenous factors comprise IL-2, IL-12, IL-18, and activated beads. In some embodiments, the exogenous factors comprise IL-2, IL-15, IL-18, and activated beads. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, and IL-18. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-15, and activated beads. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-18, and activated beads. In some embodiments, the exogenous factors comprise IL-7, IL-12, IL-18, and activated beads. In some embodiments, the exogenous factors include IL-12, IL-15, IL-18, and activation beads.

[0223] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15, and IL-18. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15, and activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18, and activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15, IL-18, and activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, IL-18, and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, IL-18, and activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, IL-18, and activation beads.

[0224] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads.

[0225] 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 anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-15 and IL-18. In some embodiments, the exogenous factors include IL-15 and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-18 and anti-CD2 / anti-NKp46 coated activation beads.

[0226] 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 anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated activation beads. 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 anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-2, IL-18, and anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors comprise IL-7, IL-18, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors comprise IL-12, IL-15, and IL-18. In some embodiments, the exogenous factors comprise IL-12, IL-15, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors comprise IL-12, IL-18, and anti-CD2 / anti-NKp46 coated activation beads.In some embodiments, the exogenous factors include IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads.

[0227] 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 anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-18, and anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-18, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-2, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated 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 anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-18, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-7, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-12, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads.

[0228] 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 anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-18, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-2, IL-7, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-2, IL-12, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the exogenous factors include IL-7, IL-12, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads.

[0229] In some embodiments, the exogenous factors include IL-2, IL-7, IL-12, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads.

[0230] In some embodiments, IL-2 is administered at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about In some embodiments, IL-2 is present in the expansion medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about or 100 ng / ml, or any range derivable therein. In some embodiments, IL-2 is present in the expansion medium at about 1-50 ng / ml.

[0231] In some embodiments, IL-7 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about In some embodiments, IL-7 is present in the expansion medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, IL-7 is present in the expansion medium at about 1-50 ng / ml.

[0232] In some embodiments, IL-12 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about In some embodiments, IL-12 is present in the expansion medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, IL-12 is present in the expansion medium at about 1 to 100 ng / ml.

[0233] In some embodiments, IL-15 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about In some embodiments, IL-15 is present in the expansion medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about or 100 ng / ml, or any range derivable therein. In some embodiments, IL-15 is present in the expansion medium at about 1 to 100 ng / ml.

[0234] In some embodiments, IL-18 is present at a concentration of about 0.1 to 500 ng / ml, about 1 to 250 ng / ml, about 1 to 150 ng / ml, about 5 to 100 ng / ml, 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, or about In some embodiments, IL-18 is present in the expansion medium at a concentration of about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, or any range derivable therein. In some embodiments, IL-18 is present in the expansion medium at about 1 to 100 ng / ml.

[0235] In some embodiments, activation beads are present in the expansion growth medium.

[0236] In some embodiments, the anti-CD2 / anti-NKp46 coated activation beads are present in the expansion medium in a ratio based on the number of differentiated NK cells, e.g., one activation bead for each NK cell, i.e., a 1:1 ratio of activation beads to NK cells.

[0237] In some embodiments, the activating bead:NK cell ratio 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.

[0238] In some embodiments, the activating bead:NK cell ratio 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.

[0239] In some embodiments, the NK cell:activating bead ratio 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.

[0240] In some embodiments, the NK cell:activation bead ratio 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.

[0241] 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 and comprises IL-2, IL-7, IL-12, IL-15, IL-18, and activation beads.

[0242] In some embodiments, the NK cell expansion medium comprises IL-2, IL-7, IL-12, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads. In some embodiments, the NK cell expansion medium comprises defined xeno-free basal medium and comprises IL-2, IL-7, IL-12, IL-15, IL-18, and anti-CD2 / anti-NKp46 coated activation beads.

[0243] 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 defined xeno-free basal medium and 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.

[0244] 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 anti-CD2 / anti-NKp46 coated activation beads at a cell:bead ratio of 1:1. In some embodiments, the NK cell expansion medium comprises defined xeno-free basal medium and 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 anti-CD2 / anti-NKp46 coated activation beads at a cell:bead ratio of 1:1.

[0245] Differentiation method In some aspects, the present disclosure provides methods for generating hematopoietic progenitors from stem cells in a 3D culture system. In some aspects, the present disclosure provides methods for generating NK cells from stem cells in a 3D culture system. In some aspects, the present disclosure provides methods for generating NK cells from hematopoietic progenitors in a 3D culture system. In some embodiments, the method for generating NK cells comprises differentiating stem cells into hematopoietic progenitors and differentiating the hematopoietic progenitors into NK cells in a 3D culture system.

[0246] In some aspects, the present disclosure provides methods for generating common lymphoid progenitors (CLPs) from stem cells in a 3D culture system. In some embodiments, the methods include differentiating the stem cells into hematopoietic progenitors and differentiating the hematopoietic progenitors into CLPs in the 3D culture system. CLPs refer to cells that are precursors of lymphoid cells. CLPs are cells capable of hematopoietic transformation into hematopoietic cell types. In some embodiments, CLPs are CD45+CD7+CD5+ / lo CD3-CD56-. In some embodiments, CLPs are CD45+CD5+ / lo CD7+. In some aspects, the present disclosure provides methods for generating NK cells from CLPs in a 3D culture system. In some embodiments, the method for generating NK cells includes differentiating the stem cells into hematopoietic progenitors, differentiating the hematopoietic progenitors into CLPs, and differentiating the CLPs into NK cells in the 3D culture system.

[0247] In some aspects, the present disclosure provides a method for generating pre-NK cell precursors (pre-NKPs) from stem cells in a 3D culture system. In some embodiments, the method includes differentiating the stem cells into hematopoietic precursors, differentiating the hematopoietic precursors into CLPs, and differentiating the CLPs into pre-NKPs in a 3D culture system. Pre-NKPs are intermediate cells between CLPs and NKPs. In some embodiments, pre-NKPs are Lin- / CD244+ / c-Kit low In some aspects, the present disclosure provides a method for generating NK cells from pre-NKPs, wherein the pre-NKPs are IL-7Ra+ / FLT3- / CD122-. In some embodiments, the method for generating NK cells includes differentiating stem cells into hematopoietic progenitors, differentiating the hematopoietic progenitors into CLPs, differentiating the CLPs into pre-NKPs, and differentiating the pre-NKPs into NK cells in a 3D culture system.

[0248] In some aspects, the present disclosure provides a method for generating NK cell precursors (NKPs) from stem cells in a 3D culture system. In some embodiments, the method comprises differentiating stem cells into hematopoietic precursors, differentiating the hematopoietic precursors into CLPs, differentiating the CLPs into pre-NKPs, and differentiating the pre-NKPs into NKPs in a 3D culture system. NKPs are the last cells before final NK lineage commitment. In some embodiments, the NKPs are Lin- / NK1.1-DX5- / IL-7Ra+ / CD122+ / NKG2D+. In some aspects, the present disclosure provides a method for generating NK cells from NKPs in a 3D culture system. In some embodiments, the method for generating NK cells comprises differentiating stem cells into hematopoietic precursors, differentiating the hematopoietic precursors into CLPs, differentiating the CLPs into pre-NKPs, differentiating the pre-NKPs into NKPs, and differentiating the NKPs into NK cells in a 3D culture system.

[0249] In some aspects, the present disclosure provides a method for generating immature NK (iNK) cells from stem cells in a 3D culture system. In some embodiments, the method comprises differentiating the stem cells into hematopoietic precursors, differentiating the hematopoietic precursors into CLPs, differentiating the CLPs into pre-NKPs, differentiating the pre-NKPs into NKPs, and differentiating the NKPs into iNK cells in the 3D culture system. In some aspects, the present disclosure provides a method for generating NK cells from iNK cells in a 3D culture system. In some embodiments, the method for generating NK cells comprises differentiating the stem cells into hematopoietic precursors, differentiating the hematopoietic precursors into CLPs, differentiating the CLPs into pre-NKPs, differentiating the pre-NKPs into NKPs, differentiating the NKPs into iNK cells, and differentiating the iNK cells into NK cells in the 3D culture system.

[0250] In some aspects, the present disclosure provides methods for generating mature NK (mNK) cells from stem cells in a 3D culture system. In some aspects, the present disclosure provides methods for generating mature NK cells from immature NK cells in a 3D culture system. In some embodiments, the method for generating NK cells includes differentiating stem cells into hematopoietic precursors, differentiating the hematopoietic precursors 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 in a 3D culture system.

[0251] In some aspects, the methods provided herein are xeno-free. In some aspects, the methods provided herein are free of animal-derived materials.

[0252] Expression markers The differentiation of source cells into NK cells can be assessed, for example, by flow cytometry, by detecting markers such as CD56, CD94, CD117, NKG2D, DNAM-1, and NKp46. Differentiation can also be assessed by NK cell morphological characteristics, such as large size, high protein synthesis activity within the abundant endoplasmic reticulum (ER), and / or preformed granules. NK cell maturation can be assessed by detecting one or more functionally related markers, such as CD94, CD161, NKp44, DNAM-1, 2B4, NKp46, CD94, KIR, and the NKG2 family of activating receptors (e.g., NKG2D). NK cell maturation can also be assessed by detecting specific markers during various developmental stages. For example, in one embodiment, pre-NKP 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 proportion of NK cells (e.g., TSNK cells) that are CD161-, CD94+, and / or NKp46+. In a more specific embodiment, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the mature NK cells (e.g., TSNK cells) are NKp46+. In another more specific embodiment, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the mature NK cells (e.g., TSNK cells) are CD94+.In another more specific embodiment, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the mature NK cells (e.g., TSNK cells) are CD161-.

[0253] In some embodiments, differentiation of source cells into NK cells is assessed by detecting expression levels of, e.g., CD3, CD7, or CD127, CD10, CD14, CD15, CD16, CD33, CD34, CD56, CD94, CD117, CD161, NKp44, NKp46, NKG2D, DNAM-1, 2B4, or TO-PRO-3, e.g., using antibodies to one or more of these cell markers. Such antibodies can be conjugated to a detectable label, e.g., as a fluorescent label, e.g., FITC, R-PE, PerCP, PerCP-Cy5.5, APC, APC-Cy7, or APC-H7.

[0254] In some embodiments, the frequency of cell populations with desired expression patterns is higher in 3D cultures compared to 2D cultures. In some embodiments, 3D culture systems 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 cultures. In some embodiments, the 3D culture system may produce at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more pre-NKP 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.

[0255] source cells In some aspects, the NK cells are generated from source cells. Any progenitor cells known in the art can be used as source cells in the methods of the present disclosure.

[0256] In some embodiments, the source cells are hESCs. In some embodiments, the source cells are iPSCs. NK cells derived from iPSCs may alternatively be referred to as iPSC-derived NK cells.

[0257] In immunotherapy, the source cells are allogeneic or autologous, meaning that they come from a donor or the subject, respectively. In some embodiments, the source cells are allogeneic. In some embodiments, the source cells are autologous.

[0258] In some embodiments, the source cells are peripheral blood cells. As used herein, the term "peripheral blood cells" refers to cells derived from circulating blood, including hematopoietic stem cells capable of proliferation, selective differentiation, and maturation. Thus, peripheral blood NK cells may alternatively be referred to as differentiated blood-derived NK cells (bdNK).

[0259] In some embodiments, the source cells include hematopoietic stem cells characterized as being CD34+ and / or CD45+.

[0260] In some embodiments, the source cells include common lymphoid progenitor cells characterized as CD45+CD7+CD56-.

[0261] In some embodiments, NK cells can be generated from induced pluripotent stem cells (iPSCs). iPSCs are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed into an embryonic stem cell-like state by the forced expression of genes and factors critical for maintaining the distinct characteristics of embryonic stem cells. iPSCs can be generated from tissues containing somatic cells, including, but not limited to, skin, dental tissue, peripheral blood, and urine. To generate iPSCs, somatic cells can be reprogrammed by methods including, but not limited to, transient expression of reprogramming factors, virus-free methods, adenoviruses, plasmids, minicircle vectors, episomal vectors, Sendai virus, synthetic mRNA, self-replicating RNA, retroviruses, lentiviruses, PhiC31 integrase, excisable transposons, CRISPR-based gene editing, or recombinant proteins. Methods for generating iPSCs are disclosed in U.S. Patent No. 9,315,779, U.S. Patent No. 10,370,452, U.S. Patent No. 11,319,555 and U.S. Patent No. 20210015859, which are incorporated by reference in their entireties.

[0262] Mesoderm / embryoid body formation In some embodiments, the methods described herein comprise generating mesodermal cells from iPSCs and / or hESCs. When stem cells begin to differentiate, three different germ layers are formed: ectoderm, mesoderm, and endoderm. Immune cells such as NK cells differentiate from mesodermal cells. In some embodiments, the mesodermal cells produced by the methods of the present disclosure are further differentiated into NK cells.

[0263] The mesoderm formation step can include contacting a population of iPSCs or hESCs with one or more factors for a specified period of time in a defined expansion growth medium. In some embodiments, mesoderm cells are formed from embryoid bodies.

[0264] In some embodiments, stem cells are contacted with differentiation medium as described herein for a period of time to generate mesodermal cells and / or embryoid bodies.In some embodiments, iPSC or hESC cell populations are contacted with differentiation medium in 3D culture system for a period of time sufficient to generate mesodermal cells and / or embryoid bodies.In some embodiments, the period of time sufficient to generate 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.

[0265] In some embodiments, the mesoderm formation step has a duration of 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.

[0266] In some embodiments, cells derived from source cells are placed in a container to induce the cells to aggregate and form clusters. In some embodiments, the container is a plate with wells or microwells, for example, a 96-well plate and / or an Aggrewell™ plate (microwell plate; STEMCELL Technologies Inc., Vancouver, Canada). In some embodiments, cell clusters are prepared in a plate with microwells, for example, using an Aggrewell™ plate, to form aggregates of cells with uniform size and shape. In some embodiments, at least 1 cell, at least 10 cells, at least 100 cells, at least 1,000 cells, at least 10,000 cells, or at least 50,000 cells are seeded into each well. In some embodiments, 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 into each well.

[0267] Differentiation into hematopoietic precursors In some aspects, the present disclosure provides methods for generating NK cells from hematopoietic progenitor cells. In some embodiments, the methods for generating NK cells include differentiating hematopoietic progenitors into NK cells. In some embodiments, the methods provided herein are xeno-free.

[0268] One aspect of the present disclosure is that the method for producing NK cells can include a hematopoietic progenitor differentiation step. The hematopoietic progenitor differentiation step can include contacting an embryoid body cell population with one or more factors in a defined differentiation medium for a specified period of time, thereby inducing the formation of hematopoietic progenitors within the cell population. Hematopoietic progenitors are then defined by expressing a combination of markers.

[0269] In some embodiments, mesoderm and / or embryoid body cells are contacted with the differentiation medium described herein for a period of time to generate hematopoietic progenitor cells.In some embodiments, mesoderm and / or embryoid body cells are contacted with the differentiation medium in a 3D culture system for a period of time sufficient to generate hematopoietic progenitor cells.In some embodiments, the period of time sufficient to generate hematopoietic progenitor cells from mesoderm and / or embryoid body cells is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days or at least 20 days.

[0270] In some embodiments, the differentiation process into hematopoietic progenitors has a duration of about 1 to 2 days, about 2 to 3 days, about 3 to 4 days, about 4 to 5 days, about 5 to 6 days, about 6 to 7 days, about 7 to 8 days, about 8 to 9 days, about 9 to 10 days, about 10 to 11 days, about 11 to 12 days, about 12 to 13 days, about 13 to 14 days, about 14 to 15 days, about 15 to 16 days, about 16 to 17 days, about 17 to 18 days, about 18 to 19 days, or about 19 to 20 days.

[0271] In some embodiments, 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.

[0272] In some embodiments, the methods of the present disclosure generate populations of cells from iPSCs with a purity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% CD34+CD43+CD45+ triple positive cells.

[0273] In some embodiments, the methods of the present disclosure generate populations of cells from iPSCs with a purity of about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100% CD34+CD43+CD45+ triple-positive cells.

[0274] NK cell differentiation In some aspects, the present disclosure provides methods for generating NK cells from hematopoietic progenitor cells. In some embodiments, the methods for generating NK cells include differentiating the hematopoietic progenitor cells. In some embodiments, the methods provided herein are xeno-free.

[0275] One aspect of the present disclosure is that the method for producing NK cells can include an NK differentiation step. The NK differentiation step can include contacting an HP cell population with one or more factors in a defined differentiation medium for a specified period of time, thereby inducing the formation of NK cells within the cell population. In some embodiments, the HP cell population is contacted with a differentiation medium in a 3D suspension culture for a period of time sufficient to form NK cells. NK cells are then defined by expressing a combination of markers.

[0276] In some embodiments, hematopoietic progenitor cells are contacted with a differentiation medium described herein for a period of time to generate NK cells. In some embodiments, the period of time sufficient to generate NK cells from hematopoietic progenitor cells is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days, or at least 40 days.

[0277] In some embodiments, the NK differentiation step lasts for about 1 to 2 days, about 2 to 3 days, about 3 to 4 days, about 4 to 5 days, about 5 to 6 days, about 6 to 7 days, about 7 to 8 days, about 8 to 9 days, about 9 to 10 days, about 10 to 11 days, about 11 to 12 days, about 12 to 13 days, about 13 to 14 days, about 14 to 15 days, about 15 to 16 days, about 16 to 17 days, about 17 to 18 days, about 18 to 19 days, or about 19 to 20 days, or about 20 to 21 days. , about 21 to 22 days, about 22 to 23 days, about 23 to 24 days, about 24 to 25 days, about 25 to 26 days, about 26 to 27 days, about 27 to 28 days, about 28 to 29 days, about 29 to 30 days, about 30 to 31 days, about 31 to 32 days, about 32 to 33 days, about 33 to 34 days, about 34 to 35 days, about 35 to 36 days, about 36 to 37 days, about 37 to 38 days, about 38 to 39 days, or about 39 to 40 days.

[0278] In some embodiments, the differentiated NK cells comprise the markers CD34, CD43, CD45, and LFA 1. In some embodiments, the methods of the disclosure increase the percentage of CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0279] In some embodiments, the methods of the present disclosure generate a population of NK cells from hematopoietic progenitor cells with a purity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0280] In some embodiments, the methods of the present disclosure generate populations of NK cells from hematopoietic progenitor cells with a purity of about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100% CD34+CD43+CD45+LFA1+ quadruple positive cells.

[0281] NK cell maturation In some aspects, the present disclosure provides methods for generating mature NK cells from differentiated NK cells. In some embodiments, the methods for generating mature NK cells include differentiating the NK cells. In some embodiments, the methods provided herein are xeno-free.

[0282] One aspect of the present disclosure is that the method for producing NK cells can include an NK maturation step. The NK maturation step can include contacting a differentiated NK cell population with one or more factors in a limited expansion growth medium for a specified period of time, thereby inducing NK cell maturation within 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 period of time sufficient to induce NK cell maturation. Mature NK cells are then defined by expressing a combination of markers.

[0283] In some embodiments, the differentiated NK cells are contacted with a maturation medium described herein for a period of time to generate mature NK cells. In some embodiments, the period of time sufficient to generate mature NK cells from hematopoietic progenitor cells is at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 168 hours, at least 192 hours, at least 216 hours, or at least 240 hours.

[0284] In some embodiments, the maturation step has a duration of 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.

[0285] In some embodiments, 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 expression of activation markers. In some embodiments, activation markers include NKp46, NKG2D, LFA1, and / or CD16. In some embodiments, the methods decrease expression of inhibitory markers. In some embodiments, inhibitory markers include CD161 and CD73.

[0286] In some embodiments, the methods of the disclosure generate a population of mature NK cells from differentiated NK cells with a purity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells.

[0287] In some embodiments, the methods of the present disclosure generate a population of mature NK cells from differentiated NK cells with a purity of about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100% CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells.

[0288] In some embodiments, the maturation process reduces the population of CD56- cells.

[0289] In some embodiments, the methods of the present disclosure generate a population of mature NK cells from differentiated NK cells with a purity of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% CD56- cells.

[0290] In some embodiments, the methods of the present disclosure generate populations of mature NK cells from differentiated NK cells with a purity of about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100% CD56- cells.

[0291] Exemplary Differentiation Methods In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system with a xeno-free differentiation medium comprising BMP4, FGF2, and VEGF. In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system with a xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, and 5-100 ng / mL VEGF. In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system with a xeno-free differentiation medium comprising BMP4, FGF2, and VEGF for 12-120 hours. In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system for 12 to 120 hours with a xeno-free differentiation medium comprising 1 to 50 ng / mL BMP4, 1 to 150 ng / mL FGF2, and 5 to 100 ng / mL VEGF.

[0292] In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system with a xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor. In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system 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 some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system with a xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor for 12-120 hours. In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system for 12 to 120 hours with a xeno-free differentiation medium comprising 1 to 50 ng / mL BMP4, 1 to 150 ng / mL FGF2, 5 to 100 ng / mL VEGF, and 0.1 to 20 μM ROCK inhibitor.

[0293] In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture with a serum-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632. In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system 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 some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system with a xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 for 12-120 hours. In some embodiments, a method for differentiating stem cells into hematopoietic progenitors comprises contacting a population of stem cells in a 3D culture system for 12 to 120 hours with a xeno-free differentiation medium comprising 1 to 50 ng / mL BMP4, 1 to 150 ng / mL FGF2, 5 to 100 ng / mL VEGF, and 0.1 to 20 μM Y27632.

[0294] In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells in the 3D culture system with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO to generate hematopoietic progenitors. In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, and 5-100 ng / mL VEGF to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells in the 3D culture system 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 to generate hematopoietic progenitors. In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF for 12 to 120 hours to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells in the 3D culture with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO for 2 to 20 days to generate hematopoietic progenitors.In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising 1-50 ng / mL BMP4, 1-150 ng / mL FGF2, and 5-100 ng / mL VEGF for 12-120 hours to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells in a 3D culture system 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 for 2-20 days to generate hematopoietic progenitors.

[0295] In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells in the 3D culture system with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO to generate hematopoietic progenitors. In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells in a 3D culture system 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 to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells in the 3D culture system with a second xeno-free differentiation medium comprising about 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 to generate hematopoietic progenitors. In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor for 12 to 120 hours to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells in a 3D culture system with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO for 2 to 20 days to generate hematopoietic progenitors.In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells in a 3D culture system 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 for 12-120 hours to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells in the 3D culture system 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 for 2-20 days to generate hematopoietic progenitors.

[0296] In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 to generate a population of mesodermal cells, and (ii) contacting the population of mesodermal cells in the 3D culture system with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO to generate hematopoietic progenitors. In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells in a 3D culture system 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 to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells in the 3D culture system with a second xeno-free differentiation medium comprising about 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 to generate hematopoietic progenitors. In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 in a 3D culture system for 12 to 120 hours to generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO in a 3D culture system for 2 to 20 days to generate hematopoietic progenitors.In some embodiments, a method of differentiating stem cells into hematopoietic progenitors comprises: (i) contacting a population of stem cells 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 generate a population of mesodermal cells; and (ii) contacting the population of mesodermal cells 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 generate hematopoietic progenitors.

[0297] In some embodiments, the hematopoietic progenitor cell formation step is followed by an NK cell differentiation step. In some embodiments, the method for differentiating HP into NK comprises contacting a population of HP cells with a xeno-free NK differentiation medium in a 3D culture system. In some embodiments, the method for differentiating HP into NK comprises contacting a population of HP cells with a xeno-free NK differentiation medium in a 3D culture system for 15 to 25 days.

[0298] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor to generate hematopoietic progenitors; and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free NK differentiation medium in a 3D culture system to generate NK cells.

[0299] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, and 5-100 ng / mL VEGF to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, about 100 ng / mL SCF, about 50 μg / mL LDL, about 100 ng / mL TPO, and 5-100 μM PI3K inhibitor to generate hematopoietic progenitors; and (iii) contacting the population of hematopoietic progenitor cells in a 3D culture system with a third xeno-free NK differentiation medium to generate NK cells.

[0300] In some embodiments, a method of differentiating stem cells into hematopoietic progenitors comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, and VEGF in a 3D culture system for 12 to 120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, TPO, and a PI3K inhibitor in a 3D culture system for 2 to 20 days to generate hematopoietic progenitors; and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free NK differentiation medium in a 3D culture system for 15 to 25 days to generate NK cells.

[0301] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells in the 3D culture system with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic progenitor cells in the 3D culture system with a third xeno-free NK differentiation medium to generate NK cells.

[0302] In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM ROCK inhibitor to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells in the 3D culture system 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 to generate hematopoietic progenitors; and (iii) contacting the population of hematopoietic progenitor cells in the 3D culture system with a third xeno-free NK differentiation medium to generate NK cells.

[0303] In some embodiments, a method of differentiating stem cells into hematopoietic progenitors comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and a ROCK inhibitor in a 3D culture system for 12 to 120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO in a 3D culture system for 2 to 20 days to generate hematopoietic progenitors; and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free NK differentiation medium in a 3D culture system for 15 to 25 days to generate NK cells.

[0304] In some embodiments, a method for differentiating stem cells into hematopoietic progenitors includes the steps of: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM ROCK inhibitor in a 3D culture system for 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM ROCK inhibitor in a 3D culture system for 2-20 days to generate a population of mesodermal cells; (iii) contacting the population of hematopoietic progenitor cells with a second xeno-free differentiation medium comprising about 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 to generate hematopoietic progenitors; and (iv) contacting the population of hematopoietic progenitor cells with a third xeno-free NK differentiation medium in the 3D culture system for 15-25 days to generate NK cells.

[0305] In some embodiments, a method of differentiating stem cells into NK cells comprises: (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells in the 3D culture system with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO to generate hematopoietic precursors; and (iii) contacting the population of hematopoietic progenitor cells in the 3D culture system with a third xeno-free NK differentiation medium to generate NK cells.

[0306] In some embodiments, a method of differentiating stem cells into hematopoietic progenitors includes: (i) contacting a population of stem cells in a 3D culture system with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM Y27632 to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells in the 3D culture system 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 to generate hematopoietic progenitors; and (iii) contacting the population of hematopoietic progenitor cells in the 3D culture system with a third xeno-free NK differentiation medium to generate NK cells.

[0307] In some embodiments, a method of differentiating stem cells into hematopoietic progenitors comprises: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising BMP4, FGF2, VEGF, and Y27632 in a 3D culture system for 12 to 120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a second xeno-free differentiation medium comprising BMP4, FGF2, VEGF, SCF, LDL, and TPO in a 3D culture system for 2 to 20 days to generate hematopoietic progenitors; and (iii) contacting the population of hematopoietic progenitor cells with a third xeno-free NK differentiation medium in a 3D culture system for 15 to 25 days to generate NK cells.

[0308] In some embodiments, a method for differentiating stem cells into hematopoietic progenitors includes the steps of: (i) contacting a population of stem cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM Y27632 in a 3D culture system for 12-120 hours to generate a population of mesodermal cells; (ii) contacting the population of mesodermal cells with a first xeno-free differentiation medium comprising 5-50 ng / mL BMP4, 5-50 ng / mL FGF2, 5-100 ng / mL VEGF, and 1-20 μM Y27632 in a 3D culture system for 2-20 days to generate a population of mesodermal cells; (iii) contacting the population of hematopoietic progenitor cells with a second xeno-free differentiation medium comprising about 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 to generate hematopoietic progenitors; and (iv) contacting the population of hematopoietic progenitor cells with a third xeno-free NK differentiation medium in the 3D culture system for 15-25 days to generate NK cells.

[0309] In some or any of the foregoing embodiments, the NK differentiation medium comprises SCF, IL-7, IL-15, IL-12, FLT3L, a pyrimido-indole derivative, and an aryl hydrocarbon receptor antagonist. In some embodiments, the pyrimido-indole 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.

[0310] In some or any of the foregoing embodiments, the NK differentiation medium comprises 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μM of a pyrimido-indole derivative, and 1-10 μM of an aryl hydrocarbon receptor antagonist. In some embodiments, the NK differentiation medium comprises 5-50 ng / mL SCF, 5-50 ng / mL IL-7, 5-100 ng / mL IL-15, 5-100 ng / mL IL-12, 5-100 ng / mL FLT3L, 1-10 μM UM729, and 1-10 μM SR1.

[0311] 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.

[0312] 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.

[0313] In some embodiments, the stem cells comprise 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 cells and cells differentiated therefrom comprise a gene knockout and a gene knockin. In some embodiments, the gene knockin is located in the gene knockout.

[0314] Characteristics of NK cells In some embodiments, NK cells produced by the disclosed 3D suspension culture methods have improved or enhanced properties compared to NK cells produced by 2D culture methods.

[0315] In some embodiments, NK cells produced by the 3D suspension culture methods of the present disclosure have enhanced expansion, ie, NK cells have at least a 50-fold expansion, at least a 100-fold expansion, at least a 150-fold expansion, at least a 200-fold expansion, at least a 250-fold expansion, at least a 300-fold expansion, at least a 350-fold expansion, at least a 400-fold expansion, at least a 450-fold expansion, at least a 500-fold expansion, at least a 1,000-fold expansion, at least a 10,000-fold expansion, at least a 100,000-fold expansion, or at least a 1,000,000-fold expansion compared to NK cells produced by 2D culture methods.

[0316] In some embodiments, the NK cells have an expansion / expansion factor of about 50 to 100, an expansion / expansion factor of about 100 to 150, an expansion / expansion factor of about 150 to 200, an expansion / expansion factor of about 200 to 250, an expansion / expansion factor of about 250 to 300, an expansion / expansion factor of about 300 to 350, an expansion / expansion factor of about 350 to 400, an expansion / expansion factor of about 400 to 450, an expansion / expansion factor of about 450 to 500, an expansion / expansion factor of about 500 to 1,000, an expansion / expansion factor of about 1,000 to 10,000, an expansion / expansion factor of about 10,000 to 100,000, or an expansion / expansion factor of about 100,000 to 1,000,000.

[0317] In some embodiments, differentiated NK cells produced by the disclosed 3D suspension culture methods reduce tumor cell proliferation more than differentiated NK cells produced by 2D culture methods, hi some embodiments, differentiated NK cells produced by 3D suspension culture methods reduce tumor cell proliferation by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% more than differentiated NK cells produced by 2D culture methods.

[0318] In some embodiments, differentiated NK cells produced by 3D suspension culture methods reduce tumor cell proliferation by about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100% more than differentiated NK cells produced by 2D culture methods.

[0319] In some embodiments, mature NK cells produced by the disclosed 3D suspension culture methods reduce tumor cell proliferation more than 2D culture methods, ie, 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 2D culture methods.

[0320] In some embodiments, mature NK cells produced by 3D suspension culture methods reduce tumor cell proliferation by about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100% more than mature NK cells produced by 2D culture methods.

[0321] In some embodiments, the disclosed methods produce differentiated NK cell populations. In some embodiments, the disclosed 3D suspension culture methods produce differentiated NK cell populations that have 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 2D culture methods.

[0322] In some embodiments, the 3D suspension culture methods of the present disclosure produce differentiated NK cell populations that are about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100% more CD34+CD43+CD45+LFA1+ quadruple positive cells compared to 2D culture methods.

[0323] In some embodiments, the disclosed 3D suspension culture methods produce mature NK cell populations that have 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 compared to 2D culture methods.

[0324] In some embodiments, the 3D suspension culture methods of the present disclosure produce mature NK cell populations that are about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100% more CD34+CD43+CD45+LFA1+NKp46+NKG2D+LFA1+CD161-CD73- cells compared to 2D culture methods.

[0325] Gene editing Genome editing generally refers to the process of editing or changing the nucleotide sequence of genome, preferably in a precise, desired, and / or predetermined manner.The example of genome editing composition, system and method described herein uses site-specific nuclease to cut DNA at precise target position in genome, thereby creating double-strand break (DSB) in DNA.This break can be repaired by endogenous DNA repair pathways, such as homology-directed repair (HDR) and / or non-homologous end joining (NHEJ) repair (see, for example, Cox et al., (2015) Nature Medicine 21(2):121-31).

[0326] In some embodiments, the cells described herein (e.g., stem cells, HP, NK) are genetically modified. In some embodiments, the modification involves using DNA targeting proteins and nucleases or RNA-guided nucleases to knock out one or more endogenous genes, and / or knock in an exogenous gene of interest. In some embodiments, the gene of interest is knocked in to a specific locus of interest. In some embodiments, the gene of interest is a synthetic cytokine receptor complex. In some embodiments, the gene of interest is a chimeric antigen receptor (CAR). In some embodiments, a RACR is knocked in to a locus of interest.

[0327] In some embodiments, the modification comprises contacting the cell with a DNA targeting protein and nuclease or an RNA-guided nuclease. In some embodiments, the DNA targeting protein and 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.

[0328] Rejection of cellular therapeutics (e.g., CAR T cells) is at least due to human leukocyte antigen (HLA) mismatches between donors and recipients. One recently identified solution is to disrupt the expression of genes involved in this rejection, such as T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M), and signal regulatory protein alpha (SIRPA). Thus, in some embodiments, the cells described herein (e.g., iPSCs, CILs) are genetically engineered to knock out the B2M locus, the TRAC locus, and / or the SIRPA locus. In some embodiments, the cells described herein are genetically engineered to knock out the B2M locus. In some embodiments, the cells described herein are genetically engineered to knock out the TRAC locus. In some embodiments, the cells described herein are genetically engineered to knock out the SIPRA locus.

[0329] In some embodiments, the cells described herein are genetically engineered to be rapamycin resistant. In some embodiments, the cells are genetically engineered to disrupt the gene 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 to induce rapamycin resistance.

[0330] In some aspects, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in an endogenous gene. In some aspects, the synthetic cytokine receptor is engineered into the gene such that expression of the endogenous gene is not disrupted. In some aspects, the synthetic cytokine receptor is engineered into a safe harbor locus.

[0331] In some embodiments, the cell described herein has been 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).

[0332] In some embodiments, the gene of interest inserted into the endogenous locus is a synthetic cytokine receptor complex. In some embodiments, the endogenous promoter of a particular locus is used. In some embodiments, additional promoters can be included so that two or more promoters drive the expression of the exogenous gene of interest.

[0333] 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 within the disrupted B2M gene.

[0334] In some aspects, the cells (e.g., iPSCs, CILs) described herein comprise (i) a disrupted B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex under the control of the endogenous B2M promoter and the EEF1A promoter.

[0335] In some aspects, the cells described herein (e.g., iPSCs, HPs, NKs) 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 EEF1A promoter.

[0336] Systems for genome editing In some embodiments, the system for editing cells described herein comprises a site-specific nuclease, such as a CRISPR / Cas system, and optionally a gRNA. In some embodiments, the system comprises an engineered nuclease. In some embodiments, the system comprises a site-specific nuclease. In some embodiments, the site-specific 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.

[0337] CRISPR / Cas nuclease system The naturally occurring CRISPR / Cas system is a genetic defense system that provides a form of adaptive immunity in prokaryotes. CRISPRs are a set of clustered regularly interspaced short palindromic repeats (CRISPRs). C Lustered R regularly I Interspaced S hort P alindromic R CRISPR / Cas is an abbreviation for CRISPR / Cas systems, a family of DNA sequences found in bacterial and archaeal genomes that contain fragments of DNA (spacer DNA) similar to foreign DNA previously exposed to cells, for example, by viruses that infect or attack the prokaryote. These fragments of DNA are used by prokaryotes to detect and destroy similar foreign DNA upon reintroduction from a similar virus during a subsequent attack, for example. Transcription of the CRISPR locus results in the formation of an RNA molecule containing a spacer sequence that associates with and targets Cas (CRISPR-associated) proteins, which can recognize and cleave foreign exogenous DNA. Numerous types and classes of CRISPR / Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

[0338] Engineered versions of the CRISPR / Cas system have been developed in many formats to mutate or edit the genomic DNA of cells from other species. A common approach using the CRISPR / Cas system involves heterologously expressing or introducing a site-specific nuclease (e.g., a Cas nuclease) in combination with a guide RNA (gRNA) into cells, resulting in a DNA cleavage event (e.g., forming a single-strand or double-strand break (SSB or DSB)) in the backbone of the cell's genomic DNA at a precise, targetable location. The manner in which DNA cleavage events are repaired by cells provides an opportunity to edit the genome by adding, removing, or modifying (substituting) DNA nucleotides or sequences (e.g., genes).

[0339] i. Guide RNA (gRNA) An engineered CRISPR / Cas system includes at least two components: 1) a guide RNA (gRNA) molecule and 2) a Cas nuclease that interacts to form a gRNA / Cas nuclease complex. The gRNA contains at least a user-defined targeting domain called a "spacer," which includes a nucleotide sequence and a CRISPR repeat sequence. In an engineered CRISPR / Cas system, the gRNA / Cas nuclease complex targets a specific target sequence of interest within a target nucleic acid (e.g., a genomic DNA molecule) by generating a gRNA containing a spacer with 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)). Thus, the spacer provides the targeting function of the gRNA / Cas nuclease complex.

[0340] In naturally occurring Type II CRISPR / Cas systems, the "gRNA" is composed of two RNA strands: 1) a CRISPR RNA (crRNA) containing a spacer and CRISPR repeats, and 2) a trans-activating CRISPR RNA (tracrRNA). In Type II CRISPR / Cas systems, a portion of the crRNA containing the CRISPR repeats and a portion of the tracrRNA hybridize to form a crRNA:tracrRNA duplex that 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 strands, where the first RNA strand incorporates crRNA function and / or structure and the second RNA strand incorporates tracrRNA function and / or structure, and the first and second RNA strands are partially hybridized.

[0341] Thus, in some embodiments, the gRNA comprises two RNA molecules. In some embodiments, the gRNA comprises a CRISPR RNA (crRNA) and a 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 comprising, from 5' to 3', a spacer and a first complementary region, and a second strand comprising, from 5' to 3', a second complementary region, and optionally a tail domain.

[0342] In some embodiments, the crRNA comprises a spacer comprising a nucleotide sequence that is complementary to and hybridizes with a sequence complementary to a target sequence on a target nucleic acid (e.g., a genomic DNA molecule). In some embodiments, the crRNA comprises a region that is complementary to and hybridizes with a portion of the tracrRNA.

[0343] In some embodiments, the tracrRNA may comprise all or part of the wild-type tracrRNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the tracrRNA may comprise a truncated or modified variant of the wild-type tracr RNA. The length of the tracr RNA may depend on the CRISPR / Cas system used. In some embodiments, the tracrRNA may comprise 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 length. In certain embodiments, the tracrRNA is at least 26 nucleotides in length. In additional embodiments, the tracrRNA is at least 40 nucleotides in length. In some embodiments, the tracrRNA may comprise a specific secondary structure, such as, for example, one or more hairpin or stem-loop structures, or one or more bulges.

[0344] Single guide RNA (sgRNA) Engineered CRISPR / Cas nuclease systems often combine a crRNA and a tracrRNA into a single RNA molecule, referred to herein as a "single guide RNA" (sgRNA), by adding a linker between these components. Without being bound by theory, the sgRNA, like the duplexed crRNA and tracrRNA, forms a complex with a Cas nuclease (e.g., Cas9), guides the Cas nuclease to the target sequence, and activates the Cas nuclease to cleave the target nucleic acid (e.g., genomic DNA). Thus, in some embodiments, a gRNA may comprise an operably linked crRNA and tracrRNA. In some embodiments, a sgRNA may comprise a crRNA covalently linked to a tracrRNA. In some embodiments, the crRNA and tracrRNA are covalently linked via a linker. In some embodiments, a sgRNA may comprise a stem-loop structure via base pairing between the crRNA and tracrRNA. In some embodiments, the sgRNA comprises, from 5' to 3', a spacer, a first region of complementarity, a linking domain, a second region of complementarity, and optionally a tail domain.

[0345] The sgRNA can be unmodified or modified. For example, a modified sgRNA can include one or more 2'-O-methyl phosphorothioate nucleotides.

[0346] For example, guide RNAs used in CRISPR / Cas systems, or other even smaller RNAs, can be easily synthesized by chemical means, as exemplified herein and described in the art. Although chemical synthesis procedures are constantly expanding, the purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as the length of polynucleotides increases significantly beyond about 100 nucleotides. One approach used to generate even longer RNAs is to generate two or more molecules that are ligated together. Much longer RNAs, such as those encoding Cas9 endonuclease, are more easily generated enzymatically. During or after chemical synthesis and / or enzymatic generation of RNA, various types of RNA modifications can be introduced, such as modifications that improve stability, reduce the likelihood or severity of innate immune responses, and / or enhance other attributes, as described in the art.

[0347] Spacer In some embodiments, the gRNA comprises a spacer sequence. The spacer sequence is a sequence that defines a target site of a target nucleic acid (e.g., DNA). The target nucleic acid is a double-stranded molecule, with one strand comprising a target sequence adjacent to a PAM sequence, referred to as the "PAM strand," and the second strand, referred to as the "non-PAM strand," being 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. Thus, 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 to 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 of the target sequence (e.g., the non-PAM strand) 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 one nucleotide that is not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises two nucleotides that are not complementary to the non-PAM strand of the target nucleic acid.

[0348] In some embodiments, the 5'-most nucleotide of the gRNA comprises the 5'-most 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 spacer is about 15-50, about 20-45, about 25-40, or about 30-35 nucleotides in length. In some embodiments, the spacer is about 19-22 nucleotides in length. In some embodiments, the spacer is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the spacer is 19 nucleotides in length. In some embodiments, the spacer is 20 nucleotides in length, and in some embodiments, the spacer is 21 nucleotides in length.

[0349] In some embodiments, the nucleotide sequence of the spacer is designed or selected using a computer program. The computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic frequency (e.g., of identical or similar sequences that differ in one or more spots due to mismatches, insertions or deletions), methylation status, and / or the presence of SNPs.

[0350] In some embodiments, the spacer comprises at least one or more modified nucleotides, such as those described herein. The present disclosure provides gRNA molecules comprising a spacer that may comprise the nucleobase uracil (U), and any DNA encoding a gRNA comprising a spacer comprising the nucleobase uracil (U) will comprise the nucleobase thymine (T) at the corresponding position.

[0351] ii. gRNA production method Methods for producing gRNA are known to those skilled in the art, including, but not limited to, in vitro transcription (IVT), synthetic and / or chemical synthesis methods, or a combination thereof. Enzyme (IVT) synthesis methods, solid-phase synthesis methods, liquid-phase synthesis methods, combined synthesis methods, small-region synthesis and ligation methods are utilized. In one embodiment, gRNA is produced using IVT enzymatic synthesis methods. Methods for producing polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062. Therefore, the present disclosure also includes polynucleotides, such as DNA, constructs, and vectors, used to in vitro transcribe the gRNA described herein.

[0352] In some embodiments, non-natural modified nucleobases are introduced into polynucleotides, such as gRNAs, during or after synthesis. In certain embodiments, the modifications are on internucleoside linkages, purine or pyrimidine bases, or sugars. In some embodiments, the modifications are introduced into the end of a polynucleotide by chemical synthesis or using a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in PCT Application No. PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0353] In some embodiments, enzymatic or chemical ligation methods are used to conjugate polynucleotides or regions thereof with various functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0354] In some embodiments, the present disclosure provides a nucleic acid, e.g., a vector, encoding a gRNA described herein. 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 a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a spacer flanking all or part of a repeat sequence derived from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a tracrRNA. In some embodiments, the crRNA and tracrRNA are encoded by two separate nucleic acids. In other embodiments, the crRNA and tracrRNA are encoded by a single nucleic acid. In some embodiments, the crRNA and tracrRNA are encoded by opposing strands of a single nucleic acid. In other embodiments, the crRNA and tracrRNA are encoded by the same strand of a single nucleic acid.

[0355] In some embodiments, the gRNA provided by the present disclosure is chemically synthesized by any means described in the art (see, for example, International Publication No. 2005 / 01248). Although chemical synthesis procedures are constantly expanding, the purification of such RNA by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as the length of polynucleotides increases significantly beyond about 100 nucleotides. One approach used to generate even longer RNA is to generate two or more molecules that are ligated together.

[0356] In some embodiments, multiple guide RNAs can be used with CRISPR / Cas nuclease system.Each guide RNA can contain different targeting sequences, so that CRISPR / Cas system can cleave multiple target nucleic acids.In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability in Cas9 RNP complex.When multiple guide RNAs are used, each guide RNA can be coded on the same or different vectors.The promoters used to promote the expression of multiple guide RNAs can be the same or different.

[0357] The guide RNA can target any sequence of interest via 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 to 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.

[0358] 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. Thus, the targeting sequence may comprise 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 length. In some embodiments, the targeting sequence may comprise 18-24 nucleotides in length. In some embodiments, the targeting sequence may comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence may comprise 20 nucleotides in length.

[0359] 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), e.g., 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), and the Cas protein cleaves 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 class 2 type V CRISPR system. In some embodiments, the endonuclease is Mad7.

[0360] iii. Cas nuclease In some embodiments, the present disclosure provides compositions and systems (e.g., engineered CRISPR / Cas systems) comprising a site-specific nuclease, wherein the site-specific nuclease is a Cas nuclease. The Cas nuclease may include at least one domain that interacts with a guide RNA (gRNA). Furthermore, the Cas nuclease is guided to a target sequence by the guide RNA. The guide RNA interacts with the Cas nuclease and the target sequence such that the Cas nuclease can cleave the target sequence after being guided to the target sequence. In some embodiments, the guide RNA provides specificity for cleaving the target sequence, and the Cas nuclease is universal and can be paired with various guide RNAs to cleave various target sequences.

[0361] In some embodiments, the CRISPR / Cas system comprises components from type I, type II, or type III systems. Updated classification schemes for CRISPR / Cas loci define 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, V, and 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.

[0362] In some embodiments, the Cas nuclease is derived from a type II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is derived from a class 2 CRISPR / Cas system (a single-protein Cas nuclease such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 family of proteins are enzymes with DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as further described herein.

[0363] Type II CRISPR / Cas system components are derived from Type IIA, Type IIB, or Type IIC systems, and include Cas9 and its orthologs. Non-limiting exemplary species from which Cas9 nuclease or other components may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus species, Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacteria, Neisseria meningitidis, Campylobacter jejuni, and the like. jejuni, Pasteurella multocida, Fibrobacter succinogenes, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Strepto- sporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius acidocaldarius, Bacillus pseudomycoidespseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacteria, Polaromonas naphthalenivorans, Polaromonas species, Crocosphaera watsonii, Cyanothece spp., Microcystis aeruginosa, Synechococcus spp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosumvinosum, Marinobacter spp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Tedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc spp., Arthrospira maxima, Arthrospira platensis platensis, Arthrospira spp., Lyngbya spp., Microcoleus chthonoplastes, Oscillatoria spp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria or Acaryochloris marina. In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein is derived from Streptococcus thermophilus (StCas9). In some embodiments, the Cas9 protein is derived from Neisseria meningitidis (NeisseriaIn some embodiments, the Cas9 protein is derived from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 protein is derived from Campylobacter jejuni (CjCas9).

[0364] In some embodiments, a Cas nuclease may contain multiple nuclease domains. For example, a Cas9 nuclease may contain 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 a DSB into 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 so that it does not contain a functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified so that it does not contain a functional HNH-like nuclease domain. In some embodiments, where only one of the nuclease domains is functional, the Cas9 nuclease is a nickase capable of introducing a single-strand break ("nick") into a target sequence. In some embodiments, conserved amino acids within the Cas9 nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid substitution in the RuvC-like nuclease domain. Exemplary amino acid substitutions within the RuvC-like nuclease domain include D10A (based on S. pyogenes Cas9 nuclease). In some embodiments, the nickase comprises an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions within the HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on S. pyogenes Cas9 nuclease). In some embodiments, the nuclease systems described herein comprise a nickase and a pair of guide RNAs complementary to the sense and antisense strands of a target sequence, respectively, where the guide RNAs guide the nickase to target and introduce DSBs by generating nicks on opposite strands of the target sequence (i.e., double nicking).The chimeric Cas9 nuclease is used, in which one domain or region of the protein is replaced by a part of a different protein.For example, the Cas9 nuclease domain is replaced by the domain from a different nuclease, such as Fok1.Cas9 nuclease is a modified nuclease.

[0365] In some embodiments, the Cas nuclease is derived 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.

[0366] In some embodiments, the Cas nuclease is a Mad endonuclease. The CRISPR / Mad system is closely related to type V (Cpf1-like) of the class 2 family of Cas enzymes. In some embodiments, the CRISPR-Mad system uses the Eubacterium rectale Mad7 endonuclease or a variant thereof. The Mad7-crRNA complex cleaves the target DNA by identifying the PAM 5'-YTTN.

[0367] Engineered nucleases In some embodiments, the cells described herein are genetically engineered using site-specific nucleases, and the site-specific nucleases are engineered nucleases. Exemplary engineered nucleases are meganucleases (e.g., homing endonucleases), ZFNs, TALENs, and megaTALs.

[0368] Naturally occurring meganucleases can recognize and cleave double-stranded DNA sequences of approximately 12 to 40 base pairs and are generally classified 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 a meganuclease is engineered to recognize and bind to a sequence other than its cognate target sequence. In some embodiments, the DNA-binding domain of a meganuclease is fused to a heterologous nuclease domain. In some embodiments, meganucleases, such as homing endonucleases, are fused to TAL modules to create hybrid proteins, such as "megaTAL" proteins. MegaTAL proteins have improved DNA targeting specificity by recognizing the target sequence of the meganuclease DNA-binding domain and the target sequence of the TAL module.

[0369] ZFNs are fusion proteins containing 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 ZF repeats are combined to recognize DNA target sequences and provide sufficient affinity. Thus, engineered ZF repeats are combined to recognize even longer DNA sequences, such as 9 bp, 12 bp, 15 bp, or 18 bp. In some embodiments, ZFNs contain a ZF fused to a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain includes a dimerization domain and a pair of ZFNs containing ZF repeats, such as when the nuclease becomes active upon dimerization. The nuclease domain is designed to target a target sequence that includes two halves of a target sequence recognized by each ZF repeat on opposite strands of a DNA molecule, with an interconnecting sequence (sometimes referred to in the literature as a spacer) between them. For example, the interconnecting sequence is 5-7 bp long. Upon binding of both ZFNs of the pair, the nuclease domains dimerize and can introduce a DSB within the interconnecting sequence. In some embodiments, the dimerization domain of the nuclease domain includes a knob-into-hole motif to promote dimerization. For example, the ZFN includes a knob-into-hole motif in the dimerization domain of FokI.

[0370] The DNA-binding domain of a TALEN typically contains a variable number of 34 or 35 amino acid repeats ("modules" or "TAL modules"), each of which binds to 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 RVD) specify the single DNA base pair to which the module binds. While the module used to recognize G may also have affinity for A, TALENs benefit from a simple recognition code (one module for each of the four bases), which greatly simplifies the customization of DNA-binding domains that recognize specific target sequences. In some embodiments, a TALEN may contain a nuclease domain derived from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain can dimerize to become active, and a pair of TALENs is designed to target a target sequence that contains two half target sequences recognized by each DNA-binding domain on opposite strands of a DNA molecule, with an interconnected sequence between them. For example, the target sequence of each half is within a 10-20 bp range, and the interconnecting sequence is 12-19 bp long. Upon binding of both TALENs of a pair, the nuclease domains can dimerize and introduce a DSB within the interconnecting sequence. In some embodiments, the dimerization domain of the nuclease domain can contain a knob-into-hole motif to promote dimerization. For example, a TALEN can contain a knob-into-hole motif in the dimerization domain of FokI.

[0371] target site In some embodiments, the site-specific nucleases described herein are directed to and cleave a target nucleic acid molecule (e.g., introduce a DSB). In some embodiments, the target nucleic acid molecule is 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). In some embodiments, the target nucleic acid molecule is a blood lineage gene. In some embodiments, the blood lineage gene is protein tyrosine phosphatase receptor type C (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.

[0372] A target nucleic acid molecule is any DNA molecule that is endogenous or exogenous to a cell. As used herein, the term "endogenous sequence" refers to a sequence that is natural to a cell. In some embodiments, the target nucleic acid molecule is a genomic DNA (gDNA) molecule or chromosome derived from or within a cell. In some embodiments, the target sequence of the target nucleic acid molecule is a genomic sequence derived from or within a cell. In some embodiments, the target sequence may be located in the coding sequence of a gene, the intron sequence of a gene, the transcriptional control sequence of a gene, the translational control sequence of a gene, or the non-coding sequence between genes. 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.

[0373] In some embodiments, the target sequence may be located at a non-gene functional site within the genome that controls aspects of chromatin organization, e.g., a scaffold site or locus control region. In some embodiments, the target sequence may be a genetic safe harbor site, i.e., a locus that facilitates safe genetic modification.

[0374] In some embodiments, the target sequence may be adjacent to a protospacer adjacent motif (PAM), 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 at the 3' end of the target sequence. In some embodiments, the target sequence may include a PAM. The length and sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from consensus sequences or specific PAM sequences for specific Cas nucleases or Cas orthologs, including those disclosed in Figure 1 of Ran et al., (2015) Nature, 520:186-191 (2015), which is incorporated herein by reference. In some embodiments, the PAM may 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, dimeric dCas9-Fok1, SpCas9-HF1, SpCas9 K855A, eSpCas9(1.0), eSpCas9(1.1)), NGAN or NGNG (SpCas9 VQR mutant), NGAG (SpCas9 EQR mutant), NGCG (SpCas9 VRER mutant), NAAG (SpCas9 QQR1 mutant), NNGRRT or NNGRRN (SaCas9), NNNRRT (KKH), SaCas9), NNNNRYAC(CjCas9), NNAGAAW(St1Cas9), NAAAAC(TdCas9), NGGNG(St3Cas9), NG(FnCas9), NAAAAN(TdCas9), NNAAAAW(StCas9), NNNNACA(CjCas9), GNNNCNNA(PmCas9), and NNNNGATT(NmCas9) (e.g., Cong et al. 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. al.,(2013)Nucleic Acids Res 42:2577-2590;Garneau et al.,(2010)Nature 468:67-71;Magadan et al.,(2012)PLoS ONE 7:e40913;Esvelt et al.,(2013)Nat Methods 10(11):1116-1121 (wherein N is defined as any nucleotide, W is defined as either A or T, R is defined as a purine (A) or (G), and Y is defined as a 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.

[0375] Ribonucleoproteins In some embodiments, the site-specific 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-specific polypeptide can be pre-complexed with one or more guide RNAs or one or more sgRNAs. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs). In some embodiments, the nuclease system comprises a ribonucleoprotein (RNP). In some embodiments, the nuclease system comprises a Cas9 RNP comprising purified Cas9 protein complexed with a gRNA. In some embodiments, the nuclease system comprises a Mad7 RNP comprising purified Mad7 protein complexed with a gRNA. The Cas9 and Mad7 proteins can be expressed and purified by any means known in the art. The ribonucleoproteins can be assembled in vitro and delivered directly to cells using standard electroporation or transfection techniques known in the art.

[0376] Engineered stem cells In some aspects, the present disclosure provides engineered stem cells that transiently or stably express a synthetic cytokine receptor complex. In some aspects, the present disclosure provides engineered stem cells that stably express a synthetic cytokine receptor complex.

[0377] 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 locus, a TRAC locus, and / or a SIRPA locus. In some embodiments, the genome further comprises a disrupted FKBP12 locus.

[0378] In some embodiments, the engineered stem cells comprise 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 cells comprise 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 cells comprise 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 cells comprise 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.

[0379] Engineered cells In some embodiments, the cell populations described herein are genetically engineered. In some embodiments, source cells are genetically engineered. In some embodiments, mesodermal cells are genetically engineered. In some embodiments, embryoid body cells are genetically engineered. In some embodiments, hematopoietic progenitor cells are genetically engineered. In some embodiments, differentiated NK cells are genetically engineered. In some embodiments, mature NK cells are genetically engineered. In some embodiments, the genetic engineering decreases expression of an endogenous gene. In some embodiments, the genetic engineering increases expression of an endogenous gene.

[0380] In some embodiments, genetically engineering a cell comprises introducing foreign DNA into the cell. In some embodiments, the foreign DNA is a gene. In some embodiments, the foreign DNA alters expression of an endogenous gene.

[0381] In some embodiments, the genetic manipulation comprises introducing RNA into the cell, such as interfering RNA (RNAi), double-stranded RNA (dsrna), small interfering RNA (siRNA) and / or microRNA (miRNA).

[0382] In some embodiments, the genetic manipulation involves introducing DNA, such as a plasmid or bacterial artificial chromosome (BAC), into the cell.

[0383] In some embodiments, the genetic manipulation involves introducing (a) a fusion protein comprising a DNA-targeting protein and a nuclease, or (b) an RNA-guided nuclease. For example, in some embodiments, the DNA-targeting protein or RNA-guided nuclease comprises a gene-specific zinc finger protein (ZFP), a TAL protein, or a clustered regularly interspaced short palindromic nucleic acid (CRISPR). In some embodiments, the disruption involves introducing a combination of a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or CRISPR-Cas9 that specifically binds to, specifically recognizes, or specifically hybridizes to the gene. In some embodiments, the introduction is carried out by introducing into the cell a nucleic acid comprising a sequence encoding a DNA-binding protein, a DNA-binding nucleotide, and / or a complex comprising the DNA-binding protein or the DNA-binding nucleotide. In some embodiments, the nucleic acid is a viral vector.

[0384] In some embodiments, the genetically engineered cells described herein comprise a chimeric antigen receptor (CAR). In some embodiments, the genetically engineered stem cells comprise a CAR. In some embodiments, the genetically engineered hematopoietic progenitors comprise a CAR. In some embodiments, the genetically engineered NK cells comprise a CAR.

[0385] In some embodiments, the genetically engineered cells described herein comprise a rapamycin-activated cytokine receptor (RACR). In some embodiments, the genetically engineered stem cells comprise a RACR. In some embodiments, the genetically engineered hematopoietic progenitors comprise a RACR. In some embodiments, the genetically engineered NK cells comprise a RACR.

[0386] In some embodiments, the genetically engineered cells described herein comprise a CAR and a RACR. In some embodiments, the genetically engineered stem cells comprise a CAR and a RACR. In some embodiments, the genetically engineered hematopoietic progenitors comprise a CAR and a RACR. In some embodiments, the genetically engineered NK cells comprise a CAR and a RACR.

[0387] 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.

[0388] Synthetic cytokine receptor complexes In some embodiments, the cells described herein are genetically engineered to express a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor comprises a synthetic gamma chain and a synthetic beta chain, each of which comprises a dimerization domain. The dimerization domains controllably dimerize in the presence of a non-physiological ligand, thereby activating signaling of the synthetic cytokine receptor.

[0389] 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 the interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain), and N-terminal or C-terminal to the IL-2G intracellular domain.

[0390] The synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain. In some embodiments, the intracellular domain is selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. In some embodiments, the intracellular domain comprises an interleukin-2 / interleukin-15 receptor subunit beta (IL-2 / 15RB). In some embodiments, the intracellular domain comprises an interleukin-15 receptor alpha subunit. The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2RB or IL-7RB intracellular domain).

[0391] The non-physiological ligand can activate a synthetic cytokine receptor in the cytotoxic innate lymphoid cells, inducing the expansion and / or activation of the engineered cytotoxic innate lymphoid cells. In a preferred embodiment, the non-physiological ligand is rapamycin or a rapalog, such a synthetic cytokine receptor referred to as the rapamycin-activated cytokine receptor (RACR).

[0392] In some embodiments, the non-physiological ligand activates a synthetic cytokine receptor in the NK cell, inducing NK cell expansion. In some embodiments, activation of the synthetic cytokine receptor results in at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, at least about 2500-fold, at least about 3000-fold, at least about 3500-fold, or at least about 4000-fold greater numbers of NK cells compared to uninduced cells.

[0393] In some embodiments, NK cells are increased by about 10-fold to about 100-fold, about 50-fold to about 200-fold, about 100-fold to about 300-fold, about 200-fold to about 400-fold, about 300-fold to about 500-fold, about 400-fold to about 1000-fold, about 500-fold to about 1500-fold, about 1000-fold to about 2000-fold, about 1500-fold to about 2500-fold, about 2000-fold to about 3000-fold, about 2500-fold to about 3500-fold, about 3000-fold to about 4000-fold, or any value between these ranges.

[0394] Intracellular domain 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 set forth 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 to SEQ ID NO: 1.

[0395] In some embodiments, the sequence of the IL2RG common gamma chain intracellular domain is set forth in SEQ ID NO:1. TIFF2025526380000002.tif11159

[0396] 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.

[0397] In some embodiments, the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL2RB) intracellular domain. In some embodiments, IL-2 receptor subunit beta refers to the IL-2 / IL-15 receptor beta subunit (IL-2 / 15RB). In some embodiments, the IL2RB intracellular domain comprises the sequence set forth in SEQ ID NO:2. In some embodiments, the IL2RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:2.

[0398] In some embodiments, the sequence of the IL2RB intracellular domain is set forth in SEQ ID NO:2. TIFF2025526380000003.tif33159

[0399] 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.

[0400] 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 set forth in SEQ ID NO:3. In some embodiments, the IL7RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:3.

[0401] In some embodiments, the sequence of the IL7RB intracellular domain is set forth in SEQ ID NO:3. TIFF2025526380000004.tif26159

[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-21RB intracellular domain and a second dimerization domain.

[0403] In some embodiments, the synthetic beta chain comprises an interleukin-21 receptor subunit beta (IL21RB) intracellular domain. In some embodiments, the IL21RB intracellular domain comprises the sequence set forth in SEQ ID NO:4. In some embodiments, the IL21RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity to SEQ ID NO:4.

[0404] In some embodiments, the sequence of the IL21RB intracellular domain is set forth in SEQ ID NO:4. TIFF2025526380000005.tif33159

[0405] Dimerization domain The dimerization domain may be a heterodimerization domain, including, but not limited to, the 12 kD FK506-binding protein (FKBP), which is known in the art to dimerize in the presence of rapamycin or a rapalog, and the FKBP12-rapamycin binding (FRB) domain. The FRB domain may comprise a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6 or SEQ ID NO:7. The FKBP domain may comprise a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:5.

[0406] In some embodiments, the sequence of an exemplary FKBP domain is set forth in SEQ ID NO:5. TIFF2025526380000006.tif12157

[0407] In some embodiments, the sequence of an exemplary FRB domain is set forth in SEQ ID NO:6. TIFF2025526380000007.tif12158

[0408] In some embodiments, the sequence of the mutant FRB domain (FRB variant domain) is set forth in SEQ ID NO:7. TIFF2025526380000008.tif12158

[0409] Alternatively, the first and second dimerization domains may be FK506-binding protein (FKBP) and calcineurin domains, which are 12 kD in size and known in the art to dimerize in the presence of FK506 or an analog thereof.

[0410] In some embodiments, the dimerization domain is: i) FK506-binding protein (FKBP) of size 12 kD; ii) cyclophilin A (CypA); or iii) gyrase B (CyrB); a homodimerization domain selected from The corresponding non-physiological ligands are: i) FK1012, AP1510, AP1903 or AP20187; ii) cyclosporine-A (CsA); or iii) coumermycin or its analogues. is.

[0411] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a cyclophilin domain.

[0412] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0413] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0414] 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).

[0415] Transmembrane domain The transmembrane domain is the sequence of the 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.

[0416] In some embodiments, the sequence of the transmembrane (TM) domain is shown as SEQ ID NO:8. TIFF2025526380000009.tif3128

[0417] In some embodiments, the sequence of the TM domain is set forth as SEQ ID NO:9. TIFF2025526380000010.tif3128

[0418] In some embodiments, the sequence of the TM domain is set forth as SEQ ID NO:10. TIFF2025526380000011.tif3128

[0419] In some embodiments, the sequence of the TM domain is shown as SEQ ID NO:11. TIFF2025526380000012.tif4128

[0420] In some embodiments, the sequence of the CD8a signal sequence is shown as SEQ ID NO:12. TIFF2025526380000013.tif3128

[0421] Non-physiological ligands In various embodiments of the compositions and methods of the present disclosure, the system includes a non-physiological ligand. Exemplary small molecules useful as ligands include, but are not limited to, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid (aMPOB), folate, rhodamine, acetazolamide, and CA9 ligand.

[0422] In some embodiments, the synthetic cytokine receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0423] In some embodiments, the non-physiological ligand is a rapalog.

[0424] In some embodiments, the non-physiological ligand is rapamycin.

[0425] In some embodiments, the non-physiological ligand is AP21967.

[0426] In some embodiments, the non-physiological ligand is FK506.

[0427] 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 cyclosporin-A (CsA). In some embodiments, the non-physiological ligand is coumermycin.

[0428] In some embodiments, synthetic cytokine receptor complexes activated by folate, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, a rapalog (rapamycin analog), CD28 ligand, poly(his) tag, Strep-tag, FLAG-tag, VS-tag, Myc-tag, HA-tag, NE-tag, biotin, digoxigenin, dinitrophenol, or derivatives thereof.

[0429] In some embodiments, the non-physiological ligand can be an inorganic or organic compound less than 1000 daltons.

[0430] In some embodiments, the ligand can be rapamycin or a rapamycin analog (rapalog). In some embodiments, rapalogs include variants of rapamycin that have one or more of the following modifications to rapamycin: demethylation, removal, or replacement of the methoxy at C7, C42, and / or C29; removal, derivatization, or replacement of the hydroxy at C13, C43, and / or C28; reduction, removal, or derivatization of the ketone at C14, C24, and / or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative substitutions on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring.

[0431] Thus, in some embodiments, the rapalog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, temsirolimus (CCI-779), C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-(S)-3-methylindolerapamycin (C16-iRap), AP21967 (A / C Heterodimerizer, Takara Bio®), mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573 (ridaforolimus), AP1903 (rimiduside), or metabolites, derivatives and / or combinations thereof.

[0432] In some embodiments, the ligand comprises FK1012 (a semi-synthetic dimer of FK506), tacrolimus (FK506), FKCsA (a conjugate of FK506 and cyclosporine), rapamycin, coumermycin, gibberellin, HaXS dimerizer (a chemical dimerizer of HaloTag and SNAP-tag), TMP-HTag (a trimethoprim haloenzyme protein dimerizer), or ABT-737 or a functional derivative thereof.

[0433] In some embodiments, the non-physiological ligand is administered in an amount between 0 nM and 1000 nM, e.g., 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 0.0nM, 70.0nM, 75.0nM, 80.0nM, 90.0nM, 95.0nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM or 1000nM, or an amount within a range defined by any two of the above amounts.

[0434] 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.

[0435] 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.

[0436] In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 100 nM.

[0437] 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.

[0438] Cytosolic FRB The FRB domain is a domain of about 100 amino acids derived from mTOR protein kinase.The FRB domain can be expressed in the cytosol as a freely diffusible soluble protein.Advantageously, the FRB domain reduces the inhibitory effect of rapamycin on mTOR in transduced cells, promotes the consistent activation of transduced cells, and gives cells a growth advantage over natural cells.

[0439] In some embodiments, the synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds to a ligand, or a complex that includes the ligand.

[0440] In some embodiments, the cytosolic polypeptide comprises an FRB domain. In some embodiments, the cytosolic polypeptide comprises an FRB domain and the ligand is rapamycin. The cytosolic FRB domain can comprise a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6 or SEQ ID NO:7. The FRB domain can be a naked FRB domain consisting essentially of a polypeptide having a polypeptide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6 or SEQ ID NO:7. Advantageously, the cytosolic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalogs).

[0441] Chimeric Antigen Receptor In some embodiments, the cells described herein are genetically engineered to express a chimeric antigen receptor (CAR).

[0442] In some embodiments, the present disclosure contemplates a CAR system for use in treating a subject with cancer. In some embodiments, the NK cells of the present disclosure comprise a CAR sequence (CAR-NK cells).

[0443] In some embodiments, NK cells are engineered to express a CAR construct by transfecting the cell population with an expression vector encoding the CAR construct. Illustrative examples of cell populations that can be transfected include HSCs, blood progenitor cells, common lymphoid progenitor cells, or NK cells. Suitable means for preparing a transduced population of NK cells expressing a selected CAR construct are well known to those skilled in the art and include, to name a few, retroviruses, lentiviruses (viral-mediated CAR gene delivery systems), Sleeping Beauty, and PiggyBac (transposon / transposase systems, including non-viral-mediated CAR gene delivery systems). In some embodiments, any of the transduction methods contemplated in this disclosure may be used to generate CAR-expressing NK cells.

[0444] Targeting agents for CAR Conventionally, CAR is produced by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding a transmembrane domain and an intracellular domain, and then transducing the polynucleotide and corresponding VH or VL into cells as needed.Many variations of CAR are known in the art, and the present disclosure contemplates the use of any of the known variations.In addition, VL / VH pairs and scFvs for numerous haptens are known in the art or can be routinely produced by conventional methods.Therefore, the present disclosure contemplates the use of any known hapten binding domain.

[0445] Various methods for targeting CARs and CAR-expressing cells have been described in the art, including, for example, U.S. Patent No. 2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, fluorescein or fluorescein isothiocyanate (FITC) moieties can be conjugated to an agent that binds to desired target cells (such as cancer cells), thereby allowing CAR-NK cells expressing anti-fluorescein / FITC chimeric antigen receptors to selectively target target cells labeled by the conjugate. Alternatively, other haptens recognized by CARs may be used instead of fluorescein / FITC. CARs can be produced using various scFv sequences known in the art or scFv sequences produced by conventional, routine methods. Further exemplary scFv sequences for fluorescein / FITC and other haptens are provided, for example, in International Publication No. WO 2021 / 076788, the disclosure of which is incorporated herein by reference.

[0446] In some embodiments, the CAR system of the present disclosure utilizes a CAR that targets a moiety that is not produced or expressed by the cells of the subject being treated. Therefore, this CAR system allows for the focused targeting of NK cells to target cells, such as cancer cells. By administering a small conjugate molecule together with the CAR-expressing NK cells, the NK cell response can target only cells that express the tumor receptor, thereby reducing off-target toxicity, and NK cell activation can be more easily controlled due to the rapid clearance of the small conjugate molecule. As an additional advantage, CAR-expressing NK cells can be used as "universal" cytotoxic cells to target a wide variety of tumors without the need to prepare separate CAR constructs. The targeting moiety recognized by the CAR can also remain constant. Only the ligand portion of the small conjugate molecule needs to be changed to allow the system to target different identities of cancer cells.

[0447] In one aspect, the present disclosure provides an illustration of this conjugate molecule / CAR system.

[0448] In some embodiments, the CAR system of the present disclosure utilizes a conjugate molecule as a bridge between CAR-expressing cells and target cancer cells. The conjugate molecule is a conjugate comprising a hapten and a cell targeting moiety, such as any suitable tumor cell-specific ligand. Exemplary haptens that can be recognized and bound by CAR include fluorescein and its derivatives, including FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, knottins, centyrin, and DARPins, as well as low molecular weight organic molecules such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin, and digoxigenin. Suitable cell-targeting moieties that may themselves act as haptens for CAR include knottins (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centrins, and DARPins (see Reichert, JMM Abs 2009. 1(3):190-209).

[0449] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), a ligand bound by PSMA-positive human prostate cancer cells with nanomolar affinity (KD=14 nM; see Kularatne, SA et al., Mol Pharm. 2009.6(3):780-9). In one embodiment, the DUPA derivative can be a small molecule ligand linked to the targeting moiety, and DUPA derivatives are described in WO 2015 / 057852, which is incorporated herein by reference.

[0450] In some embodiments, the cell targeting moiety is a CCK2R ligand that is a ligand bound by CCK2R-positive cancer cells (e.g., thyroid, lung, pancreatic, ovarian, brain, stomach, gastrointestinal stromal, and colon cancers; see Wayua, C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0451] In some embodiments, the cell targeting moiety is folate, folic acid, or an analog thereof, a ligand bound by folate receptors on cancer cells, including ovarian, cervical, endometrial, lung, renal, brain, breast, colon, and head and neck cancers (see Sega, EI et al., Cancer Metastasis Rev. 2008.27(4):655-64).

[0452] In some embodiments, the cell targeting moiety is an NK-1R ligand. Receptors for NK-1R ligands are found, for example, on colon and pancreatic cancers. In some embodiments, NK-1R ligands can be synthesized according to the methods disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0453] In some embodiments, the cell targeting moiety can be a peptide ligand, for example, the ligand can be a peptide ligand that is an endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide belonging to the tachykinin family that targets tachykinin receptors. Such regulatory peptides include substance P (SP), neurokinin A (substance K), an...

Claims

1. A method for differentiating a population of stem cells into a population of hematopoietic precursors, (i) A step of culturing the population of stem cells in a two-dimensional (2D) culture system for a period of time sufficient to form stem cell aggregates. (ii) A step of subculturing the stem cell aggregates from the 2D culture system to the 3D suspension culture system, (iii) Contacting the stem cell aggregates in the 3D suspension culture system with a differentiation medium containing bone morphogenetic protein (BMP) pathway activator, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) for a period of time sufficient to differentiate the population of stem cells into the population of hematopoietic precursors. The method, including the method described above.

2. The method according to claim 1, wherein the population of hematopoietic precursors comprises CD34+ / CD43+ / CD45+ cells.

3. A method for generating a population of NK cells, (a) A step of culturing a population of stem cells in a 2D culture system for a period of time sufficient to form stem cell aggregates. (b) A step of subculturing the stem cell aggregates from the 2D culture system to the 3D suspension culture system, (c) A step of contacting the stem cell aggregates in the 3D suspension culture system with a first medium containing a BMP pathway activator, FGF, VEGF, and optionally an inhibitor of ROCK for a period of time sufficient to generate embryoid bodies. (d) A step of contacting the embryoid body with a first differentiation medium containing BMP pathway activator, FGF, VEGF, SCF, TPO, and LDL for a period of time sufficient to generate a population of hematopoietic precursors. (e) The step of contacting the population of hematopoietic precursors with a second differentiation medium for a period of time sufficient to generate the population of NK cells. The method, including the method described above.

4. The method according to any one of claims 1 to 3, wherein the 3D suspension culture is seeded at a density of at least 1 × 10³ cells.

5. The method according to any one of claims 1 to 3, wherein the 3D suspension culture has a volume of at least 1 mL.

6. The 3D suspension culture is Seeded at a density of at least 1 × 10³ cells, Having a volume of at least 1 mL, The method according to any one of claims 1 to 3.

7. The method according to claim 1 or 2, wherein the 3D suspension culture is a flask, multilayer flask, bottle, dish or bioreactor, and optionally wherein the 3D suspension culture is a bioreactor, the bioreactor is a hollow fiber bioreactor, a packed bed bioreactor, a stirred tank bioreactor, an oscillating bioreactor, and / or a wave bioreactor.

8. The method according to claim 1 or 2, wherein the 3D suspension culture is a non-tissue culture treated 6-well plate.

9. The method according to claim 1 or 2, wherein the 3D suspension culture is stirred.

10. The method according to claim 9, wherein the 3D suspension culture is stirred at a speed of 10 revolutions per minute (RPM) to 100 RPM.

11. The method according to claim 1 or 2, wherein the BMP pathway activator is BMP4, the FGF is FGF2, and / or the VEGF is VEGF-165.

12. The method according to claim 1 or 2, wherein the differentiation medium comprises one or more of the following: a Rho-related coiled-coil forming protein serine / threonine kinase (ROCK) inhibitor, stem cell factor (SCF), thrombopoietin (TPO), low-density lipoprotein (LDL), phosphatidylinositol 3-kinase (PI3K) inhibitor, aryl hydrocarbon (AhR) inhibitor / antagonist, and pyrimido-indole derivatives.

13. The PI3K inhibitor is idelalisib, copanlisib, duvelisib, alpelisib, umbralicib, buparlicib, dactricib, reniolisib, palsacricib, paxalisib, taselicib, zandelisib, inavolisib, apitricib, vimiralisib, eganerisib, fimepinostat, gedatricib, linperisib, nemiralisib, pictilisib, piraralisib, samotricib, ceretalisib, ceravelicib, sonolicib, tenalisib, boxtalisib, AMG The method according to claim 12, selected from 319, AZD8186, GSK2636771, SF1126, acalicib, omiparicib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, Hibiscon C, IC87114, LY294002, and PI-103.

14. The differentiation medium is The BMP pathway activator, the FGF, the VEGF, and the ROCK inhibitor; or The BMP pathway activator, the FGF, the VEGF, SCF, SR1, and ROCK inhibitor including, The method according to claim 1 or 2.

15. (iii) The method according to claim 1 or 2, wherein the population of stem cell aggregates is brought into contact with the differentiation medium for 1 to 5 days, the differentiation medium comprising the BMP pathway activator, the FGF, the VEGF, and optionally a ROCK inhibitor.

16. The method according to claim 1 or 2, wherein the stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

17. The method according to claim 1 or 2, wherein the differentiation medium is serum-free and / or the method is heterogeneous.

18. The method according to claim 3, wherein the second differentiation medium comprises IL-15, SCF, and a pyrimido-indole derivative.

19. The method according to claim 3, wherein the second differentiation medium comprises SCF, IL-7, IL-12, IL-15, FLT3L, a pyrimide-[4,5-b]-indole derivative, and an AhR inhibitor.

20. The method according to claim 3, wherein the second differentiation medium comprises 1 to 100 ng / mL of SCF, 1 to 50 ng / mL of IL-7, 1 to 100 ng / mL of IL-12, 1 to 100 ng / mL of IL-15, 1 to 100 ng / mL of FLT3L, 0.1 to 10 μM of a pyrimido-[4,5-b]-indole derivative, 0.1 to 10 μM of an AhR antagonist, and any combination thereof.

21. The method according to claim 3, wherein the stem cells are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

22. The population of hematopoietic precursors comprises approximately 50% to approximately 100% CD34+ / CD43+ / CD45+ cells, or the population of NK cells comprises approximately 60% to approximately 100% CD43+ / CD45+ / CD56+ / LFA1+ cells. The method according to claim 3.

23. A population of cells comprising a hematopoietic precursor produced by the method described in claim 1.

24. A population of cells comprising NK cells produced by the method described in claim 3.

25. A pharmaceutical composition comprising a population of cells according to claim 23 or 24.

26. Use of an effective amount of a population of cells according to claim 23 or 24 for the manufacture of a drug for the treatment of cancer in a subject.

27. ​​A population of cells according to claim 23 or 24 for use in a method of treating cancer in a subject, comprising the step of administering an effective amount of the population of cells to the subject.

28. Use of an effective amount of the pharmaceutical composition according to claim 25 for the manufacture of a drug for the treatment of cancer in a subject.

29. The pharmaceutical composition according to claim 25, for use in a method of treating cancer in a subject, comprising the step of administering an effective amount of the pharmaceutical composition to the subject.

30. A kit comprising a population of cells according to claim 23 or 24, and instructions for administering the population of cells to a subject in need thereof.

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