Method of differentiation of pluripotent stem cells to natural killer cells

Abstract

The present invention discloses a method for substantially expanding the population of natural killer (NK) cells through the use of modified cellular culture conditions. By employing a targeted induction cocktail, including a WNT signaling pathway activator and bone morphogenetic protein (BMP), this technique facilitates the differentiation of pluripotent stem cells (PSCs) into NK cells. The core innovation lies in optimizing culture conditions, which significantly enhances the proliferation of NK cells. This approach streamlines the production of NK cells for therapeutic uses, emphasizing the expansion phase within the culture environment.

Description

PATENT ATTORNEY DOCKET NO.: N2041-03301 METHOD OF DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO NATURAL KILLER CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 561,673, filed March 5, 2024, the disclosure of the prior application is considered to be part of and is herein incorporated by reference in the disclosure of this application in its entirety. FIELD

[0002] The present invention relates generally to natural killer cells (NK cells) and more specifically to methods of generating NK cells from pluripotent stem cells (PSCs). BACKGROUND

[0003] Natural killer cells, also known as NK cells or large granular lymphocytes (LGL), are a type of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of known innate lymphoid cells (ILC) and represent 5–20% of all circulating lymphocytes in humans. The role of NK cells is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells and other intracellular pathogens acting around 3 days after infection and respond to tumor formation. Typically, immune cells detect the major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release and causing the death of the infected cell by lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named “natural killers” because of the notion that they do not require activation to kill cells that are missing “self” markers of MHC class 1. This role is especially important because harmful cells that are missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocyte cells.

[0004] In addition to natural killer cells being effectors of innate immunity, both activating and inhibitory NK cell receptors play important functional roles, including self-tolerance and the sustaining of NK cell activity. NK cells also play a role in the adaptive immune response; numerous experiments have demonstrated their ability to readily adjust to the immediate environment and formulate antigen-specific immunological memory, fundamental for responding to secondary infections with the same antigen. The role of NK cells in both the innate and adaptive immune responses is becoming increasingly important in research using NK cell activity as a potential cancer therapy.PATENT ATTORNEY DOCKET NO.: N2041-03301 SUMMARY

[0005] The present disclosure relates to the surprising discovery that a cocktail for endothelial induction, containing both bone morphogenic protein (BMP) and a WNT signaling activator, generates endothelial-like precursor cells. These cells possess a blend of characteristics from both endothelial and hematopoietic precursors, enabling their differentiation into terminally differentiated hematopoietic cells, such as natural killer (NK) cells. Moreover, this approach facilitates a significant expansion of the NK cell population through modifications to the cellular culture conditions. The present disclosure features methods to address the unmet need in the art to develop methods for PSC differentiation that are efficient and yield NK cell cultures in a short period of time using convenient culture conditions.

[0006] In some aspects, the present disclosure provides a method of producing natural killer (NK) cells comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and / or a bone morphogenetic protein (BMP); b) contacting the culture from step a) with a vascular endothelial growth factor (VEGF) thereby generating a population of CD34+precursor cells; c) incubating the population of CD34+precursor cells in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of cytokines, including, as non-limiting examples, interleukin-7 (IL-7), NK activating cytokines, e.g., interleukin-15 (IL-15) or other NK activating cytokines featured herein, Stem cell factor (also known as SCF, KIT-ligand, KL, or steel factor), and FMS-like tyrosine kinase 3 ligand (FLT3L) or one or more other cytokines featured herein, thereby creating a population of CD34+ / CD45+suspension cells; and d) incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, IL-15, SCF, and / or FLT3L, thereby producing NK cells. In some embodiments, the culture medium comprises at least one or more of: a WNT signaling pathway activator, a BMP, a VEGF. In some embodiments, the culture medium is supplemented with one or more cytokines and / or growth factors, including any of the cytokines featured above and disclosed herein. In some embodiments, the culture medium is supplemented with one or more NK activating cytokines, including any of the NK activating cytokines featured above or herein.

[0007] In one aspect, the present disclosure provides a method of producing natural killer NK cells comprising: a) contacting a culture of PSCs with a WNT signaling pathway activator and / or a BMP, wherein the PSCs are grown for about 1-7 days; b) contacting the culture of PSCs with a VEGF for about 1-7 days following step a) thereby generating a population of CD34+precursor cells at least about 80% enriched for CD34+; c) incubating the population of CD34+precursor cells for about 7-28 days in a culture medium lacking any serum-derived agent while optionallyPATENT ATTORNEY DOCKET NO.: N2041-03301 supplementing the medium with one or more of cytokines, including, as non-limiting examples, IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L, thereby creating a population of CD34+ / CD45+suspension cells; and d) incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7-35 days, thereby producing NK cells.

[0008] As featured and disclosed herein activators of the WNT signaling pathway include, as non- limiting examples, CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)- 2pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile); WNT family ligands; RSPO co- agonists; lithium chloride; TDZD8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione); BIO- Acetoxime ((2’Z,3’E)-6-Bromoindirubin-3′-acetoxime); A1070722 (1-(7-Methoxyquinolin- 4-yl)-3-[6 (trifluoromethyl)pyridin-2-yl]urea); HLY78 (4-Ethyl-5,6-Dihydro-5-methyl- [1,3]dioxolo[4,5-j]phenanthridine); CID 11210285 hydrochloride (2-Amino-4-(3,4- (methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero) arylpyrimidines; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, activation of the WNT signaling pathway may be achieved through repression of a WNT signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway inhibitor. In some embodiments, the WNT signaling pathway activator is a GSK3 inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0009] In some embodiments, the WNT signaling pathway activator is at a concentration ranging between about 1 µM to 10 µM. In some embodiments, the WNT signaling pathway activator is added to the PSC culture at a concentration that ranges from 0.5 to 1.0 µM, from 1.0 to 2.0 µM, from 2.0 to 3.0 µM, from 3.0 to 4.0 µM, from 4.0 to 5.0 µM, from 5.0 to 6.0 µM, from 6.0 to 7.0 µM, from 7.0 to 8.0 µM, from 8.0 to 9.0 µM, from 9.0 to 10 µM, or ranging between any two concentrations referred to above or herein. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator is CHIR99021 at a concentration ranging from between about 5µM to about 10 µM. In some embodiments, CHIR99021 is at a concentration of about 8 µM.

[0010] As featured and disclosed herein, a bone morphogenetic protein includes, as non-limiting examples, a BMP family ligand which activates the BMP pathway, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP 15. In some embodiments, activation of the BMP pathway may be achieved through repression of a BMPPATENT ATTORNEY DOCKET NO.: N2041-03301 pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the BMP pathway, or an antibody or small molecule directed to a BMP pathway inhibitor. In some embodiments, BMP is BMP4.

[0011] In some embodiments, the BMP, which is selected from any of the BMPs featured herein, is at a concentration ranging from between about 5 ng / ml to 50 ng / ml. In some embodiments, the BMP is at a concentration that ranges from 1 to 2 ng / ml, from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 15 ng / ml, from 15 to 20 ng / ml, from 20 to 25 ng / ml, from 25 to 30 ng / ml, from 30 to 35 ng / ml, from 35 to 40 ng / ml, from 40 to 45 ng / ml, from 45 to 50 ng / ml, from 50 to 55 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, BMP4 is at a concentration of about 25 ng / ml.

[0012] As featured and disclosed herein, a vascular endothelial growth factor includes, as non- limiting examples, a VEGF family ligand which activates the VEGF pathway, e.g., VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placenta growth factor (PIGF). In some embodiments, the VEGF is VEGFA.

[0013] In some embodiments, the VEGF, which is selected from any of the VEGFs featured herein, is at a concentration ranging from between about 50 ng / ml to 500 ng / ml. In some embodiments, the VEGF is at a concentration that ranges from 25 to 50 ng / ml, from 50 to 75 ng / ml, from 75 to 100 ng / ml, from 100 to 125 ng / ml, from 125 to 150 ng / ml, from 150 to 175 ng / ml, from 175 to 200 ng / ml, from 200 to 225 ng / ml, from 225 to 250 ng / ml, from 250 to 275 ng / ml, from 275 to 300 ng / ml, from 300 to 325 ng / ml, from 325 to 350 ng / ml, from 350 to 375 ng / ml, from 375 to 400 ng / ml, from 400 to 425 ng / ml, from 425 to 450 ng / ml, from 450 to 475 ng / ml, from 475 to 500 ng / ml, from 500 to 525 ng / ml, from 500 to 550 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, VEGFA is at a concentration of about 200 ng / ml.

[0014] In some embodiments, contacting the culture of PSCs comprises one or more agents selected from about 1-10 µM WNT signaling pathway activator, about 5-50 ng / ml BMP, and about 50-500 ng / ml VEGF.

[0015] In some embodiments, contacting the culture of PSCs comprises about 8 µM CHIR99021, about 25 ng / ml BMP4 and / or about 200 ng / ml VEGFA.

[0016] As featured and disclosed herein, growth factors are signaling molecules that regulate fundamental cellular processes such as differentiation by binding to cell surface receptors. Cytokines are a subtype of growth factors that regulate cellular processes including, as non- limiting examples, proliferation, differentiation, and communication, primarily in hematopoieticPATENT ATTORNEY DOCKET NO.: N2041-03301 and immune cell types. In some embodiments, the culture medium is supplemented with one or more growth factors. In some embodiments, the growth factors are cytokines. In some embodiments, the cytokines are selected from: SCF, FLT3L, and interleukins including, as non- limiting examples, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-17, and IL-18.

[0017] As featured and disclosed herein, cytokines involved in NK activation, or “NK activating cytokines,” include, as non-limiting examples, IL-12, IL-15, IL-18, IL-2, and CCL5. These NK activating cytokines alert NK cells to the presence of viral pathogens and recruit the NK cells to the affected area. In some embodiments, the culture medium is supplemented with one or more NK activating cytokines, including, as a non-limiting example, IL-15.

[0018] In some embodiments, the culture medium for step c), incubating the culture of CD34+precursor cells, and step d), incubating the culture of CD34+ / CD45+suspension cells, comprises about 4–40 ng / ml of a cytokine, for example, IL-7 or any cytokine featured above or herein. In some embodiments, the IL-7 is at a concentration ranging from about 4 to about 40 ng / ml. In some embodiments, the IL-7 is at a concentration that ranges from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 15 ng / ml, from 15 to 20 ng / ml, from 20 to 25 ng / ml, from 25 to 30 ng / ml, from 30 to 35 ng / ml, from 35 to 40 ng / ml, from 40 to 45 ng / ml, from 45 to 50 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, IL-7 is at a concentration of about 20 ng / ml.

[0019] In some embodiments, the culture medium for step c), incubating the culture of CD34+precursor cells, and step d) incubating the culture of CD34+ / CD45+suspension cells, comprises about 2 to 20 ng / ml of an NK activating cytokine, for example, IL-15 or any other NK activating cytokine featured herein. In some embodiments, the NK activating cytokine, for example, IL-15 or any other NK activating cytokine featured herein is at a concentration ranging from about 2 to about 20 ng / ml. In some embodiments, the NK activating cytokine, for example, IL-15 or any other NK activating cytokine featured herein is at a concentration that ranges from 1 to 2 ng / ml, from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 11 ng / ml, from 11 to 12 ng / ml, from 12 to 13 ng / ml, from 13 to 14 ng / ml, from 14 to 15 ng / ml, from 15 to 16 ng / ml, from 16 to 17 ng / ml, from 17 to 18 ng / ml, from 18 to 19 ng / ml, from 19 to 20 ng / ml, from 20 to 21 ng / ml, from 21 to 22 ng / ml, from 22 to 23 ng / ml, from 23 to 24 ng / ml, from 24 to 25 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, IL- 15 is at a concentration of about 10 ng / ml.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0020] In some embodiments, the culture medium for step c), incubating the culture of CD34+precursor cells, and step d) incubating the culture of CD34+ / CD45+suspension cells, comprises about 4–40 ng / ml SCF. In some embodiments, the SCF is at a concentration ranging from about 4 to about 40 ng / ml. In some embodiments, the SCF is at a concentration that ranges from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 15 ng / ml, from 15 to 20 ng / ml, from 20 to 25 ng / ml, from 25 to 30 ng / ml, from 30 to 35 ng / ml, from 35 to 40 ng / ml, from 40 to 45 ng / ml, from 45 to 50 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, SCF is at a concentration of about 20 ng / ml.

[0021] In some embodiments, the culture medium for step c), incubating the culture of CD34+precursor cells, and step d) incubating the culture of CD34+ / CD45+suspension cells, comprises about 1–20 ng / ml FLT3L. In some embodiments, the FLT3L is at a concentration ranging from about 1 to about 20 ng / ml. In some embodiments, the FLT3L is at a concentration that ranges from 0.1 to 0.5, from 0.5 to 1, from 1 to 2 ng / ml, from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 11 ng / ml, from 11 to 12 ng / ml, from 12 to 13 ng / ml, from 13 to 14 ng / ml, from 14 to 15 ng / ml, from 15 to 16 ng / ml, from 16 to 17 ng / ml, from 17 to 18 ng / ml, from 18 to 19 ng / ml, from 19 to 20 ng / ml, from 20 to 21 ng / ml, from 21 to 22 ng / ml, from 22 to 23 ng / ml, from 23 to 24 ng / ml, from 24 to 25 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, FLT3L is at a concentration of about 10 ng / ml.

[0022] In some embodiments, the culture medium in step c) incubating the culture of CD34+precursor cells or in step d) incubating the culture of CD34+ / CD45+suspension cells is supplemented with one or more cytokines, including, as non-limiting examples, IL-7, an NK activating cytokine, such as IL-15 or any other NK activating cytokine featured herein, SCF, and / or FLT3L.

[0023] In some embodiments, the culture medium for step c), incubating the culture of CD34+precursor cells, and step d) incubating the culture of CD34+ / CD45+suspension cells, comprises one or more cytokines. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is an NK activating cytokine, such as IL-15 or any other NK activating cytokine featured herein.

[0024] In some embodiments, the culture medium for step c) incubating the population of CD34+precursor cells and step d) incubating the population of CD34+ / CD45+suspension cells comprises about 4–40 ng / ml IL-7, about 2–20 ng / ml IL-15, about 4–40 ng / ml SCF, and / or about 1–20 ng / ml FLT3L.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0025] In some embodiments, the culture medium for step c) incubating the population of CD34+precursor cells and step d) incubating the population of CD34+ / CD45+suspension cells comprises about 20 ng / ml IL-7, about 10 ng / ml IL-15, about 20 ng / ml SCF, and / or about 10 ng / ml FLT3L.

[0026] As featured and disclosed herein, serum-derived agents include, as non-limiting examples, serum, blood platelet lysate, plasma, albumin derived from blood, and any component or derivative of blood plasma obtained from blood, such as by blood serum fractionation. In some embodiments, the serum-derived agent is selected from serum, blood platelet lysate, or albumin derived from blood. In some embodiments, the serum-derived agent is serum. In some embodiments, the serum-derived agent is blood platelet lysate. In some embodiments, the serum- derived agent is albumin. In some embodiments, the serum-derived agent is plasma. In some embodiments albumin or other proteins present in and / or derived from serum may include or exclude recombinant versions of albumin or other proteins.

[0027] In some embodiments of the incubation in step d), the population of CD34+ / CD45+suspension cells are incubated in in a culture medium comprising at least one serum-derived agent and cytokines. In some embodiments of the incubation in step d), the population of CD34+ / CD45+suspension cells are incubated in in a culture medium comprising at least one serum-derived agent and cytokines including at least one NK activating cytokine, such as IL-15 or any other NK activating cytokine featured herein. In some embodiments of the incubation in step d), the population of CD34+ / CD45+suspension cells are incubated in in a culture medium comprising at least one serum-derived agent and cytokines, such as IL-7, an NK activating cytokine such as IL- 15 or any other NK activating cytokine featured herein, SCF, and FLT3L. In some embodiments of the incubation in step d), the population of CD34+ / CD45+suspension cells are incubated in in a culture medium comprising serum and IL-7, IL-15, SCF, and FLT3L.

[0028] In some embodiments, the cells of step c) incubating the population of CD34+precursor cells and d) incubating the population of CD34+ / CD45+suspension cells are transiently CD34+.

[0029] In some embodiments, the contacting in step a), that is, the culture of PSCs are grown in the presence of a WNT signaling pathway activator and / or a bone morphogenetic protein BMP for about 1-7 days. In some embodiments, the contacting in step a) is for more than 7 days. In some embodiments, the contacting in step a) is for up to 1, 2, 3, 4, 5, 6, or 7, or more than 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step a) is for at least 1 day, 2 days, 3 days 4 days, 5 days, 6 days, or 7 days. In some embodiments, the contacting in step a) is for 1 day. In some embodiments, the contacting in step a) is for 2 days. In some embodiments, the contacting in step a) is for 3 days. In some embodiments, the contacting in step a) is for 4 days. In somePATENT ATTORNEY DOCKET NO.: N2041-03301 embodiments, the contacting in step a) is for 5 days. In some embodiments, the contacting in step a) is for 6 days. In some embodiments, the contacting in step a) is for 7 days. In some embodiments, the contacting in step a) is for more than 7 days. In some embodiments, the contacting in step a) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0030] In some embodiments, the contacting in step b) follows contacting the culture of PSCs from step a) with a VEGF for about 1-7 days, thereby generating a population of CD34+precursor cells. In some embodiments, the contacting in step b) is for more than 7 days. In some embodiments, the contacting in step b) is for up to 1, 2, 3, 4, 5, 6, or 7, or more than 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step b) is for at least 1 day, 2 days, 3 days 4 days, 5 days, 6 days, 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step b) is for 1 day. In some embodiments, the contacting in step b) is for 2 days. In some embodiments, the contacting in step b) is for 3 days. In some embodiments, the contacting in step b) is for 4 days. In some embodiments, the contacting in step b) is for 5 days. In some embodiments, the contacting in step b) is for 6 days. In some embodiments, the contacting in step b) is for 7 days. In some embodiments, the contacting in step b) is for more than 7 days. In some embodiments, the contacting in step b) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0031] In some embodiments, the contacting in step b), that is, contacting the culture of PSCs from step a) with a VEGF, results in generating a population of CD34+precursor cells at least about 80% enriched for CD34+. In some embodiments, the population of CD34+precursor cells at least about 60, 65, 70, 75, 80, 85, 90, 95%, or any percentage ranging between any two percentages referred to above or herein enriched for CD34+. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 60% enriched for CD34+. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 65% enriched for CD34+. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 70% enriched for CD34+. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 75% enriched for CD34+. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 80% enriched for CD34+. In some embodiments, the contacting in step b) generatesPATENT ATTORNEY DOCKET NO.: N2041-03301 a population of CD34+precursor cells at least 85% enriched for CD34+. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 90% enriched for CD34+. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 95% enriched for CD34+.

[0032] In some embodiments, the incubating in step c), that is, incubating the population of CD34+precursor cells from step b) for about 7-28 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L, thereby creating a population of CD34+ / CD45+suspension cells. In some embodiments, the incubating in step c) is for up to 7 days, 8 days, 9 days 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days 24 days, 25 days, 26 days, 27 days, or 28 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the incubating in step c) is for more than 7 days. In some embodiments, the incubating in step c) is for at least 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, or 28, or more than 28 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the incubating in step c) is for 7 days. In some embodiments, the incubating in step c) is for 8 days. In some embodiments, the incubating in step c) is for 9 days. In some embodiments, the incubating in step c) is for 10 days. In some embodiments, the incubating in step c) is for 11 days. In some embodiments, the incubating in step c) is for 12 days. In some embodiments, the incubating in step c) is for 13 days. In some embodiments, the incubating in step c) is for 14 days. In some embodiments, the incubating in step c) is for 15 days. In some embodiments, the incubating in step c) is for 16 days. In some embodiments, the incubating in step c) is for 17 days. In some embodiments, the incubating in step c) is for 18 days. In some embodiments, the incubating in step c) is for 19 days. In some embodiments, the incubating in step c) is for 20 days. In some embodiments, the incubating in step c) is for 21 days. In some embodiments, the incubating in step c) is for 22 days. In some embodiments, the incubating in step c) is for 23 days. In some embodiments, the incubating in step c) is for 24 days. In some embodiments, the incubating in step c) is for 25 days. In some embodiments, the incubating in step c) is for 26 days. In some embodiments, the incubating in step c) is for 27 days. In some embodiments, the incubating in step c) is for 28 days. In some embodiments, the incubating in step c) is for more than 28 days. In some embodiments, the incubating in step c) is for 7 to 8 days, 7 to 9 days, 7 to 10 days, 7 to 11 days, 7 to 12 days, 7 to 13 days, 7 to 14 days, 7 to 15 days, 7 to 16 days, 7 to 17 days, 7 to 18 days, 7 to 19 days, 7 to 20 days, 7 to 21 days, 7 toPATENT ATTORNEY DOCKET NO.: N2041-03301 22 days, 7 to 23 days, 7 to 24 days, 7 to 25 days, 7 to 26 days, 7 to 27 days, 7 to 28 days, or more than 28 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0033] In some embodiments, the incubation of population of CD34+precursor cells in c) is in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L. In some embodiments, the incubation of population of CD34+precursor cells in c) is in a culture medium lacking any serum-derived agent while optionally supplementing the medium with at least one of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L. In some embodiments, the incubation of population of CD34+precursor cells in c) is in a culture medium lacking any serum-derived agent while optionally supplementing the medium with a combination of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0034] In some embodiments, the incubating in step d), that is, incubating the population of CD34+ / CD45+suspension cells from c) in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7-35 days, thereby producing NK cells. In some embodiments, the incubating in step d) is for up to 7 days, 8 days, 9 days 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the incubating in step d) is for more than 7 days. In some embodiments, the incubating in step d) is for at least 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, or 35, or more than 35 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the incubating in step d) is for 7 days. In some embodiments, the incubating in step d) is for 8 days. In some embodiments, the incubating in step d) is for 9 days. In some embodiments, the incubating in step d) is for 10 days. In some embodiments, the incubating in step d) is for 11 days. In some embodiments, the incubating in step d) is for 12 days. In some embodiments, the incubating in step d) is for 13 days. In some embodiments, the incubating in step d) is for 14 days. In some embodiments, the incubating in step d) is for 15 days. In some embodiments, the incubating in step d) is for 16 days. In some embodiments, the incubating in step d) is for 17 days. In some embodiments, the incubating inPATENT ATTORNEY DOCKET NO.: N2041-03301 step d) is for 18 days. In some embodiments, the incubating in step d) is for 19 days. In some embodiments, the incubating in step d) is for 20 days. In some embodiments, the incubating in step d) is for 21 days. In some embodiments, the incubating in step d) is for 22 days. In some embodiments, the incubating in step d) is for 23 days. In some embodiments, the incubating in step d) is for 24 days. In some embodiments, the incubating in step d) is for 25 days. In some embodiments, the incubating in step d) is for 26 days. In some embodiments, the incubating in step d) is for 27 days. In some embodiments, the incubating in step d) is for 28 days. In some embodiments, the incubating in step d) is for 29 days. In some embodiments, the incubating in step d) is for 30 days. In some embodiments, the incubating in step d) is for 31 days. In some embodiments, the incubating in step d) is for 32 days. In some embodiments, the incubating in step d) is for 33 days. In some embodiments, the incubating in step d) is for 34 days. In some embodiments, the incubating in step d) is for 35 days. In some embodiments, the incubating in step d) is for more than 35 days. In some embodiments, the incubating in step d) is for 7 to 8 days, 7 to 9 days, 7 to 10 days, 7 to 11 days, 7 to 12 days, 7 to 13 days, 7 to 14 days, 7 to 15 days, 7 to 16 days, 7 to 17 days, 7 to 18 days, 7 to 19 days, 7 to 20 days, 7 to 21 days, 7 to 22 days, 7 to 23 days, 7 to 24 days, 7 to 25 days, 7 to 26 days, 7 to 27 days, 7 to 28 days, 7 to 29 days, 7 to 29 days, 7 to 30 days, 7 to 31 days, 7 to 32 days, 7 to 33 days, 7 to 34 days, 7 to 35 days, or more than 35 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0035] In some embodiments, contacting the culture of PSCs of step a) with a WNT signaling pathway activator and a BMP is for about 2-5 days. In some embodiments, contacting the culture of PSCs of step a) with a WNT signaling pathway activator and a BMP signaling pathway activator is for about 3 to 5 days. In some embodiments, contacting the culture of PSCs of step a) with a WNT signaling pathway activator and a BMP signaling pathway activator is for about 4 to 5 days. In some embodiments, contacting the culture of PSCs of step a) a WNT signaling pathway activator and a BMP signaling pathway activator is for about 2, 3, 4, 5 days, or for any length of time ranging between any two of the recited number of days.

[0036] In some embodiments, contacting the PSCs with a VEGF is for about 2-5 days. In some embodiments, contacting the PSCs with a VEGF is for about 3 to 5 days. In some embodiments, contacting the PSCs with a VEGF is for about 4 to 5 days. In some embodiments, contacting the PSCs with a VEGF is for about 2, 3, 4, 5 days, or for any length of time ranging between any two of the recited number of days.

[0037] In some embodiments, the culture of PSCs is an adherent layer of cells.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0038] In some embodiments, the layer of cells is grown in a two-dimensional culture system or on microcarriers.

[0039] In some embodiments, the PSCs are cultured on a coated surface comprising a laminin coating.

[0040] In some embodiments, the method further comprising collecting the NK cells in suspension in a cell culture medium.

[0041] In some embodiments, the PSCs are human PSCs (hPSCs).

[0042] In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stems cells (hESCs).

[0043] In some embodiments, the CD34+precursor cell is a CD34+endothelial-like precursor cell.

[0044] In some embodiments, the NK cells are at least about 60, 65, 70, 75, 80, 85, 90, or 95% enriched. In some embodiments, the NK cells are at least 60% enriched. In some embodiments, the NK cells are at least 65% enriched. In some embodiments, the NK cells are at least 70% enriched. In some embodiments, the NK cells are at least 75% enriched. In some embodiments, the NK cells are at least 80% enriched. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 85% enriched for CD34+. In some embodiments, the NK cells are at least 90% enriched. In some embodiments, the NK cells are at least 95% enriched.

[0045] In some embodiments, the NK cells are at least about 80% enriched.

[0046] In some embodiments, the NK cells are CD56+, 2B4+, NKp30+, NKp44+, NKp46+, NKG2D+, and / or CD16-.

[0047] In some embodiments, the NK cells are CD56brightor CD56dim.

[0048] In some embodiments, the NK cells are cytotoxic NK cells.

[0049] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7– 35 days enriches the number of cells by at least 50-fold to 300-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, FLT3L and SCF for about 7-14 days in a culture medium lacking any serum-derived agent. In some embodiments, the number of cells are enriched by at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300-fold, or any fold enrichment ranging between any two fold enrichment numbers referred to above or herein.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0050] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7- 35 days enriches the number of cells by at least 100-fold compared to incubating the population of CD34+precursor cells for about 7-28 days in a culture medium lacking any serum-derived agent. This enrichment occurs while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0051] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7- 35 days enriches the number of cells by at least 200-fold compared to incubating the population of CD34+precursor cells for about 7-28 days in a culture medium lacking any serum-derived agent. This enrichment occurs while optionally supplementing the medium with one or more of more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0052] In another aspect, the present disclosure provides a method of producing natural killer (NK) cells from pluripotent stem cells (PSCs) comprising: a) generating CD34+hemogenic endothelium (HE) cells by: (i) contacting a culture of PSCs with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), wherein the PSCs grown for about 3 days; and (ii) subsequently contacting the cells of (i) with a vascular endothelial growth factor (VEGF) for about 4 days; thereby generating a population of cells comprising at least 80% CD34+HE cells; b) culturing the CD34+HE cells of step a) in a medium lacking any serum-derived agents for about 7-28 days while optionally adding one or more of IL-7, IL-15, SCF and / or FLT3L, thereby generating a transient population of suspension cells comprising at least 80% CD34+ / CD45+hematopoietic precursor cells (HPCs); c) contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7-14 days in a culture medium lacking any serum-derived agent; and d) transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine, such as IL-15 or any other NK activating cytokine disclosed herein, FLT3L and / or SCF for about 7-35 days, thereby inducing NK cell differentiation from PSCs.

[0053] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 50-fold to 300-fold compared to the number of cells inPATENT ATTORNEY DOCKET NO.: N2041-03301 contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7-14 days in a culture medium lacking any serum-derived agent. In some embodiments, the number of cells are enriched by at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300-fold, or any fold enrichment ranging between any two fold enrichment numbers referred to above or herein.

[0054] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 100-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7- 14 days in a culture medium lacking any serum-derived agent.

[0055] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 200-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7- 14 days in a culture medium lacking any serum-derived agent.

[0056] In some embodiments, the NK cells are produced in a feeder-independent manner.

[0057] In some embodiments, the present disclosure provides a method of producing hemogenic endothelium (HE) cells by: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and / or a bone morphogenetic protein (BMP), wherein the PSCs are grown for about 1-7 days; and b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF) for about 1-7 days following step a) thereby generating a population of CD34+hemogenic endothelium (HE) cells.

[0058] In some embodiments, the contacting in step a), that is, growing the culture of PSCs in the presence of a WNT signaling pathway activator and / or a bone morphogenetic protein BMP, is for about 1-7 days. In some embodiments, the contacting in step a) is for more than 7 days. In some embodiments, the contacting in step a) is for up to 1, 2, 3, 4, 5, 6, or 7, or more than 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step a) is for at least 1 day, 2 days, 3 days 4 days, 5 days, 6 days, or 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step a) is for 1 day. In some embodiments, the contacting in step a) is for 2 days. In some embodiments, the contacting in step a) is for 3 days. In some embodiments, the contacting in step a) is for 4 days. In some embodiments, the contacting in step a) is for 5 days. In some embodiments, the contacting in step a) is for 6 days. In some embodiments, the contacting in step a) is for 7 days. In somePATENT ATTORNEY DOCKET NO.: N2041-03301 embodiments, the contacting in step a) is for more than 7 days. In some embodiments, the contacting in step a) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0059] In some embodiments, contacting the culture with VEGF in step b) optionally further comprises contacting the culture with forskolin or SB431542 to encourage endothelial differentiation or hematopoietic differentiation, respectively. In some aspects, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 µM, or more than 20 µM of either forskolin or SB431542, or any concentration of either forskolin or SB431542 ranging between any two of the recited concentrations referred to above or herein is added to the culture medium during the contacting step b). In some embodiments, about 10 µM of forskolin or SB431542 is optionally contacted with the culture in the contacting step b).

[0060] In some embodiments, the contacting in step b) follows contacting the culture of PSCs from step a) with a VEGF for about 1-7 days, thereby generating a population of CD34+hemogenic endothelium cells. In some embodiments, the contacting in step b) is for more than 7 days. In some embodiments, the contacting in step b) is for up to 1, 2, 3, 4, 5, 6, or 7, or more than 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step b) is for at least 1 day, 2 days, 3 days 4 days, 5 days, 6 days, or 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step b) is for 1 day. In some embodiments, the contacting in step b) is for 2 days. In some embodiments, the contacting in step b) is for 3 days. In some embodiments, the contacting in step b) is for 4 days. In some embodiments, the contacting in step b) is for 5 days. In some embodiments, the contacting in step b) is for 6 days. In some embodiments, the contacting in step b) is for 7 days. In some embodiments, the contacting in step b) is for more than 7 days. In some embodiments, the contacting in step b) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein. INCORPORATION BY REFERENCE

[0061] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.PATENT ATTORNEY DOCKET NO.: N2041-03301 BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0063] FIG. 1 illustrates a screening of the indicated basic media to support NK cell differentiation and proliferation from iPSC-HPCs. All media were supplemented with SCF, FLT3L, IL-7, and IL-15. Cells remained in the original differentiation wells throughout (media switch on d14).

[0064] FIG. 2 illustrates an end-point expression analysis by flow cytometry. Low NKp44 and CD38 levels in the defined medium may indicate a more naive NK phenotype.

[0065] FIG. 3A shows that defined APEL2 medium with factors does not support longer-term NK cell expansion after transfer of HPCs to independent culture wells, regardless of cell density (n=3 per data point).

[0066] FIG. 3B shows that the NK cell population collapse timing is related to the timing of the endothelial to hematopoietic transitions (EHT).

[0067] FIG. 4 illustrates a differentiation time course analysis in chemically defined medium using three independent GMP iPSC lines (flow cytometry data). Note the critical transition point from precursors to NK cells around day 28.

[0068] FIG. 5A shows that the media switch at the transition point between precursors and NK cells enables cell expansion upon differentiation (n=6 similar conditions). Note the logarithmic scale. Right panel: Flow cytometric analysis at the end of the time course showing 100% 2B4+CD56+, 96% NKp30+NKp46+, and 97% NKp44+NKp46+of the cells that had the media switch.

[0069] FIG. 5B schematically illustrates a NK cell expansion protocol with indicated media changes and added signaling molecules. B = BMP signaling stimulation; W = WNT signaling stimulation; V = VEGFA; S = SCF; F = FLT3L.

[0070] FIG. 6 illustrates an RNA-seq data analysis highlighting NK cell-specific cluster with selected marker genes and enrichment terms.

[0071] FIG. 7 shows the results of killing assays using NK cells derived from 3 iPSC lines.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0072] FIG. 8 illustrates linking the differentiation paradigms of the present study to context- dependent strategies for upscaling. Ball sizes are to reflect cell numbers. DETAILED DESCRIPTION OF THE INVENTION

[0073] In one embodiment, the present disclosure features methods and compositions relating to the surprising finding that employing a cocktail for endothelial induction, comprising both bone morphogenic protein (BMP) and a WNT signaling activator, leads to the generation of endothelial- like precursor cells. These cells exhibit combined characteristics of both endothelial and hematopoietic precursors, capable of differentiating into terminally differentiated hematopoietic cells, including natural killer (NK) cells. Furthermore, a significant expansion of the NK cell population is achieved by modifying the cellular culture conditions.

[0074] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0076] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only to the appended claims.

[0077] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” include one or more methods and / or steps of the type described herein, which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0078] As used herein, the terms “about” and “substantially” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, givenPATENT ATTORNEY DOCKET NO.: N2041-03301 the context in which it is used, “about” and “substantially” will mean up to plus or minus 10% of the particular term.

[0079] By “contacting” it is meant that the cells are cultured with one or more agents of interest, added to the defined basal or supplemented medium. That is, the cells are cultured in their regular culture basal or supplemented medium, in which a desired concentration of one or more agents of interest is added. For example, the cells are cultured with one or more of: a WNT signaling pathway activator, a BMP signaling pathway activator, and a VEGF signaling pathway activator. In some embodiments, the cells are cultured with one or more cytokines. As another example, by “contacting,” it is meant that the HSCs are cultured with one or more agents of interest, added to the culture medium lacking serum-derived agents.

[0080] A “signaling pathway activator,” as used herein, refers to any molecule that is capable of activating, enhancing, or inducing a signaling pathway of interest. A signaling pathway is a series of chemical reactions in which a group of molecules in a cell work together to control a cell function, such as cell differentiation. A cell receives signals from its environment when a molecule, such as a hormone or growth factor, binds to a specific protein receptor on or in the cell. After the first molecule in the pathway receives a signal, it activates another molecule. This process is repeated through the entire signaling pathway until the last molecule is activated and cell function is carried out. Abnormal activation of signaling pathways or inhibition of a signaling pathway may lead to diseases or, in the case of pluripotent cells, to an alteration of the pluripotent state and therefore to differentiation. The term “molecule” includes, as non-limiting examples, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines. Non-limiting examples of polynucleotides include short interfering nucleic acid (siNA), antisense, enzymatic nucleic acid molecules, 2',5'-oligoadenylate, triplex forming oligonucleotides, aptamers, and decoys. Biologically active molecules include antibodies (e.g., monoclonal, chimeric, humanized, etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys, and analogs thereof, and small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), antagomirs, and short hairpin RNA (shRNA) molecules.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0081] Natural killer cells, also known as NK cells or large granular lymphocytes (LGL), are a type of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of known innate lymphoid cells (ILC) and represent 5–20% of all circulating lymphocytes in humans. They have different functions, including cytolytic granule-mediated cell apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and cytokine-induced NK and cytotoxic T lymphocyte (CTL) activation.

[0082] NK cells are cytotoxic; small granules in their cytoplasm contain proteins such as perforin and proteases known as granzymes. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell, creating an aqueous channel through which the granzymes and associated molecules can enter, inducing either apoptosis or osmotic cell lysis. The distinction between apoptosis and cell lysis is important in immunology: lysing a virus-infected cell could potentially release the virions, whereas apoptosis leads to the destruction of the virus inside. α-defensins, antimicrobial molecules, are also secreted by NK cells and directly kill bacteria by disrupting their cell walls in a manner analogous to that of neutrophils.

[0083] Infected cells are routinely opsonized with antibodies for detection by immune cells. Antibodies that bind to antigens can be recognized by FcγRIII (CD16) receptors expressed on NK cells, resulting in NK activation, the release of cytolytic granules, and consequent cell apoptosis. This is a major killing mechanism of some monoclonal antibodies like rituximab (Rituxan), ofatumumab (Arzerra), and others.

[0084] The release of certain cytokines in response to viral infection play a crucial roles in NK cell activation. These stress molecules serve to signal to the NK cell the presence of viral pathogens in the affected area. For example, cytokines involved in NK activation, or “NK activating cytokines” include, as non-limiting examples, IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections, while the adaptive immune response generates antigen-specific cytotoxic T cells that can clear the infection. NK cells work to control viral infections by secreting IFNγ and TNFα. IFNγ activates macrophages for phagocytosis and lysis, and TNFα acts to promote direct NK tumor cell killing. Patients deficient in NK cells prove to be highly susceptible to the early phases of herpes virus infection.

[0085] Tumor-infiltrating NK cells have been reported to play a critical role in promoting drug- induced cell death in human triple-negative breast cancer. Since NK cells recognize target cells when they express non-self HLA antigens (but not self), autologous (patients' own) NK cell infusions have not shown any antitumor effects. Instead, investigators are working on using allogeneic cells from peripheral blood, which requires that all T cells be removed before infusionPATENT ATTORNEY DOCKET NO.: N2041-03301 into the patients to remove the risk of graft versus host disease, which can be fatal. This can be achieved using an immunomagnetic column (CliniMACS). In addition, because of the limited number of NK cells in the blood (only 10% of lymphocytes are NK cells), their number needs to be expanded in culture. This can take a few weeks, and the yield is donor-dependent.

[0086] The methods described herein detail the cultivation of human pluripotent stem cells under specific cellular culture conditions that lead to the generation of a population of CD34+hematopoietic precursor cells. These cells are then selectively stimulated to differentiate into NK cells, with the subsequent alteration of culture conditions facilitating a substantial expansion of the NK cell population.

[0087] In some embodiments, the disclosure pertains to deriving NK cells by contacting pluripotent stem cells (PSCs) with a natural killer cell induction composition. As detailed herein, the composition of natural killer cell induction may vary and generally includes effective amounts of one or more of the following: a WNT signaling pathway activator, a BMP signaling pathway activator, and a VEGF signaling pathway activator.

[0088] In some embodiments, the present disclosure provides a method of producing natural killer (NK) cells comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and / or a bone morphogenetic protein (BMP); b) contacting the culture from step a) with a vascular endothelial growth factor (VEGF) thereby generating a population of CD34+precursor cells; c) incubating the population of CD34+precursor cells in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of cytokines, including, as non-limiting examples, interleukin-7 (IL-7), an NK activating cytokine such as interleukin-15 (IL-15) or any other NK activating cytokine featured herein, stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (FLT3L), thereby creating a population of CD34+ / CD45+suspension cells; and d) incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as interleukin-15 (IL-15) or any other NK activating cytokine featured herein, SCF, and / or FLT3L, thereby producing NK cells. In some aspects, the culture medium comprises at least one or more of: a WNT signaling pathway activator, a BMP, a VEGF. In some aspects, the culture medium is supplemented with one or more cytokines and / or growth factors, including any of the cytokines featured above and disclosed herein.

[0089] In one aspect, the present disclosure provides a method of producing natural killer (NK) cells comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and / or a bone morphogenetic protein (BMP), wherein the PSCs are grown for about 1-7 days; b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF)PATENT ATTORNEY DOCKET NO.: N2041-03301 for about 1-7 days following step a) thereby generating a population of CD34+precursor cells at least about 80% enriched for CD34+; c) incubating the population of CD34+precursor cells for about 7-28 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of interleukin-7 (IL-7), an NK activating cytokine such as interleukin-15 (IL-15) or any other NK activating cytokine featured herein, stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (FLT3L), thereby creating a population of CD34+ / CD45+suspension cells; and d) incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7-35 days, thereby producing NK cells.

[0090] The WNT signaling pathways are a group of signal transduction pathways that begin with proteins that pass signals into a cell through cell surface receptors. WNT signaling pathways use either nearby paracrine (cell-cell) communication or autocrine (same-cell) communication. Three WNT signaling pathways have been characterized: the canonical WNT pathway, the noncanonical planar cell polarity pathway, and the noncanonical WNT / calcium pathway. All three pathways are activated by the binding of a WNT-protein ligand to a Frizzled family receptor, which passes the biological signal to the Dishevelled protein inside the cell. The canonical WNT pathway leads to regulation of gene transcription and is thought to be negatively regulated in part by the SPATS1 gene. The noncanonical planar cell polarity pathway regulates the cytoskeleton, which is responsible for the shape of the cell. The noncanonical WNT / calcium pathway regulates calcium inside the cell. WNT signaling was first identified for its role in carcinogenesis, then for its function in embryonic development. The embryonic processes controlled by WNT signaling include body axis patterning, cell fate specification, cell proliferation, and cell migration. These processes are necessary for the proper formation of important tissues, including bone, heart, and muscle. Its role in embryonic development was discovered when genetic mutations in WNT pathway proteins produced abnormal fruit fly embryos. Later research found that the genes responsible for these abnormalities also influenced breast cancer development in mice. WNT signaling also controls tissue regeneration in adult bone marrow, skin, and intestine.

[0091] In some embodiments, an inducing agent useful for the induction of cardiomyocytes may include an activator of the WNT signaling pathway. As featured and disclosed herein activators of the WNT signaling pathway include, as non-limiting examples, CHIR99021 (6-[[2-[[4-(2,4- Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2pyrimidinyl]amino]ethyl]amino]-3- pyridinecarbonitrile); WNT family ligands; RSPO co-agonists; lithium chloride; TDZD8 (4- Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione); BIO-Acetoxime ((2’Z,3’E)-6-BromoindirubinPATENT ATTORNEY DOCKET NO.: N2041-03301 -3’-acetoxime); A1070722 (1-(7-Methoxyquinolin-4-yl)-3-[6(trifluoromethyl)pyridin-2-yl]urea); HLY78 (4-Ethyl-5,6-Dihydro-5-methyl-[1,3]dioxolo[4,5-j]phenanthridine); CID 11210285 hydrochloride (2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero)arylpyrimidines; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, activation of the WNT signaling pathway may be achieved through repression of a WNT signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway inhibitor. In some embodiments, contacting the adherent culture of PSCs includes a culture medium comprising about 1 to 10 µM WNT signaling pathway activator.

[0092] In some aspects, the WNT signaling pathway activator is added to the PSC culture at a concentration that ranges from about 1 µM to 10 µM. For example, the PSC are grown in a culture medium that includes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 µM or more. In some embodiments, the WNT signaling pathway activator is added to the PSC culture at a concentration that ranges from 0.5 to 1.0 µM, from 1.0 to 2.0 µM, from 2.0 to 3.0 µM, from 3.0 to 4.0 µM, from 4.0 to 5.0 µM, from 5.0 to 6.0 µM, from 6.0 to 7.0 µM, from 7.0 to 8.0 µM, from 8.0 to 9.0 µM, from 9.0 to 10 µM, or ranging between any two concentrations referred to above or herein. In some embodiments, the culture medium includes about 8 µM WNT signaling pathway activator. In some embodiments, the WNT signaling pathway activator is a GSK3 inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the WNT signaling pathway activator comprises CHIR99021.

[0093] The transforming growth factor beta (TGF-β) superfamily includes TGF-β proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), Activins, Inhibins, Nodal, Lefty, and Mülllerian inhibiting substance (MIS). Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines and metabologens. Originally discovered for their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. The important functioning of BMP signals in physiology is emphasized by the multitude of roles for dysregulated BMP signaling in pathological processes. BMPs interact with specific receptors on the cell surface, referred to as bone morphogenetic protein receptors (BMPRs). Signal transduction through BMPRs results in the mobilization of members of the SMAD family of proteins. The signaling pathways involving BMPs, BMPRs, and SMADs are important in the development of the heart, central nervous system, and cartilage, as well as postnatal bone development. They have an important role duringPATENT ATTORNEY DOCKET NO.: N2041-03301 embryonic development in embryonic patterning and early skeletal formation. As such, disruption of BMP signaling can affect the body plan of the developing embryo. For example, BMP4 and its inhibitors, noggin and chordin, help regulate the polarity of the embryo (i.e., back-to-front patterning). Specifically, BMP4 and its inhibitors play a major role in neurulation and the development of the neural plate. BMP4 signals ectoderm cells to develop into skin cells, but the secretion of inhibitors by the underlying mesoderm blocks the action of BMP4 to allow the ectoderm to continue on its normal course of neural cell development.

[0094] In some embodiments, an inducing agent useful for the induction of natural killer cells may include an activator the BMP signaling pathway. Activators and inhibitors of the BMP signaling pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like, that activate or inhibit at least one component of the BMP signaling pathway, resulting in a corresponding activation or inhibition of cellular BMP signaling. As featured and disclosed herein, a bone morphogenetic protein includes, as non-limiting examples, a BMP family ligand, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP 15. In some embodiments, contacting the adherent culture of PSCs includes a culture medium comprising about 10 to 100 ng / ml BMP.

[0095] In some aspects, the BMP is added to the PSC culture at a concentration that ranges from about 5 ng / ml to about 50 ng / ml. For example, the PSC are grown in a culture medium that includes about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 ng / ml or more. In some embodiments, the BMP is at a concentration that ranges from 1 to 2 ng / ml, from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 15 ng / ml, from 15 to 20 ng / ml, from 20 to 25 ng / ml, from 25 to 30 ng / ml, from 30 to 35 ng / ml, from 35 to 40 ng / ml, from 40 to 45 ng / ml, from 45 to 50 ng / ml, from 50 to 55 ng / ml, or ranging between any two concentrations referred to above or herein. In some embodiments, the culture medium includes about 25 ng / mL BMP. In some embodiments, BMP is BMP4. In some embodiments, BMP4 is at a concentration of about 25 ng / ml.

[0096] As further described in the examples, BMP4 can be prepared by resuspension in various solutions. For example, dry BMP4 can be resuspended with PBS / 0.01% HSA or in citric acid (as recommended by the manufacturer). PBS / 0.01% HSA may reduce the biological activity of BMP4, as compared to its activity when prepared in citric acid. One of the experts in the art would easily recognize that a concentration of 5–50 ng / ml of BMP4 could be significantly lowered if the BMP4 is resuspended in citric acid and therefore has greater biological activity.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0097] In some embodiments, an inducing agent useful for the induction of NK cells may include an activator of the vascular endothelial growth factor (VEGF) signaling pathway. As featured and disclosed herein, a vascular endothelial growth factor includes, as non-limiting examples, a VEGF ligand, e.g., VEGF (VEGFA), VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placenta growth factor (PIGF). In some instances, an activator or inhibitor of the VEGF signaling pathway may also include activators or inhibitors of related signal transduction pathways, including, as a non- limiting example, the Ras / MAPK signal transduction pathway. Activators and inhibitors of the FGF signaling pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the VEGF signaling pathway, resulting in a corresponding activation or inhibition in cellular VEGF signaling. In some embodiments, contacting the adherent culture of PSCs includes a culture medium comprising about 50 to 500 ng / ml VEGF.

[0098] In some aspects, the VEGF is added to the PSC culture at a concentration that ranges from about 50 ng / ml to about 500 ng / ml. In some embodiments, the VEGF is at a concentration that ranges from 25 to 50 ng / ml, from 50 to 75 ng / ml, from 75 to 100 ng / ml, from 100 to 125 ng / ml, from 125 to 150 ng / ml, from 150 to 175 ng / ml, from 175 to 200 ng / ml, from 200 to 225 ng / ml, from 225 to 250 ng / ml, from 250 to 275 ng / ml, from 275 to 300 ng / ml, from 300 to 325 ng / ml, from 325 to 350 ng / ml, from 350 to 375 ng / ml, from 375 to 400 ng / ml, from 400 to 425 ng / ml, from 425 to 450 ng / ml, from 450 to 475 ng / ml, from 475 to 500 ng / ml, from 500 to 525 ng / ml, from 500 to 550 ng / ml, or ranging between any two concentrations referred to above or herein. For example, the PSC are grown in a culture medium that includes about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 ng / ml or more. In some embodiments, the culture medium includes about 200 ng / mL VEGF. In some embodiments, the VEGF is VEGFA. In some embodiments, VEGFA is at a concentration of about 200 ng / ml.

[0099] In some embodiments, contacting the adherent culture of PSCs includes one or more agents selected from about 1 to 10 µM WNT signaling pathway activator, about 10-100 ng / ml BMP, and about 50–500 ng / ml VEGF.

[0100] In some embodiments, contacting the adherent culture of PSCs includes about 8 µM CHIR99021, about 25 ng / ml BMP4, and / or about 200 ng / ml VEGFA.

[0101] As featured and disclosed herein, growth factors are signaling molecules that regulate fundamental cellular processes such as differentiation by binding to cell surface receptors to initiate signal transduction. Cytokines are a subtype of growth factors regulating cellular processes including, as non-limiting examples, proliferation, differentiation, and communication, primarilyPATENT ATTORNEY DOCKET NO.: N2041-03301 in hematopoietic and immune cell types. In some aspects, the culture medium is supplemented with one or more growth factors. In some aspects, the growth factors are cytokines selected from: Stem cell factor (also known as SCF, KIT-ligand, KL, or steel factor), FMS-like tyrosine kinase 3 ligand (FLT3L), and interleukins including, as non-limiting examples, Interleukin 7 (IL-7) and Interleukin-15 (IL-15). In some embodiments, the cytokine is an interleukin, including, as non- limiting examples: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-17, and IL-18.

[0102] As featured and disclosed herein, cytokines involved in NK activation, or “NK activating cytokines,” include, as non-limiting examples, IL2, IL-12, IL-15, IL-18, and CCL5. These NK activating cytokines alert NK cells to the presence of viral pathogens and recruit the NK cells to the affected area. In some embodiments, the culture medium is supplemented with one or more NK activating cytokines, including, as a non-limiting example, IL-15.

[0103] IL-7 is a cytokine and hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus. It is also produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells, but is not produced by normal lymphocytes. IL-7 stimulates the differentiation of multipotent (pluripotent) hematopoietic stem cells into lymphoid progenitor cells. It also stimulates the proliferation of all cells in the lymphoid lineage (B cells, T cells, and NK cells). It is important for proliferation during certain stages of B-cell maturation, T and NK cell survival, development, and homeostasis.

[0104] In some aspects, the IL-7 is added to the PSC culture at a concentration that ranges from about 4 ng / mL to 40 ng / ml. In some embodiments, the IL-7 is at a concentration ranging from about 4 to about 40 ng / ml. In some embodiments, the IL-7 is at a concentration that ranges from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 15 ng / ml, from 15 to 20 ng / ml, from 20 to 25 ng / ml, from 25 to 30 ng / ml, from 30 to 35 ng / ml, from 35 to 40 ng / ml, from 40 to 45 ng / ml, from 45 to 50 ng / ml, or ranging between any two concentrations referred to above or herein. For example, the PSC are grown in a culture medium that includes about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 µM or more. In some embodiments, the culture medium includes about 20 ng / mL IL- 7.

[0105] IL-15 is a cytokine with structural similarity to Interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following infection by a virus(es). This cytokine induces the proliferation of natural killer cells, i.e., cells of the innate immune system, whose principal role is to kill virallyPATENT ATTORNEY DOCKET NO.: N2041-03301 infected cells. IL-15 regulates the activation and proliferation of T and natural killer (NK) cells. IL-15 exemplifies NK activating cytokines, which includes, as non-limiting examples, IL-2, IL- 12, IL-15, and CCL5. Survival signals that maintain memory T cells in the absence of antigen are provided by IL-15. This cytokine is also implicated in NK cell development. In rodent lymphocytes, IL-15 prevents apoptosis by inducing BCL2L1 / BCL-x (BCL-xL), an inhibitor of the apoptosis pathway. In humans with celiac disease, IL-15 similarly suppresses apoptosis in T lymphocytes by inducing Bcl-2 and / or Bcl-xL.

[0106] In some aspects, the NK activating cytokine, such as interleukin-15 (IL-15) or any other NK activating cytokine featured herein, is added to the PSC culture at a concentration that ranges from about 2 ng / mL to 20 ng / ml. In some embodiments, the NK activating cytokine, such as interleukin-15 (IL-15) or any other NK activating cytokine featured herein is at a concentration ranging from about 2 to about 20 ng / ml. In some embodiments, the NK activating cytokine, such as interleukin-15 (IL-15) or any other NK activating cytokine featured herein is at a concentration that ranges from 1 to 2 ng / ml, from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 11 ng / ml, from 11 to 12 ng / ml, from 12 to 13 ng / ml, from 13 to 14 ng / ml, from 14 to 15 ng / ml, from 15 to 16 ng / ml, from 16 to 17 ng / ml, from 17 to 18 ng / ml, from 18 to 19 ng / ml, from 19 to 20 ng / ml, from 20 to 21 ng / ml, from 21 to 22 ng / ml, from 22 to 23 ng / ml, from 23 to 24 ng / ml, from 24 to 25 ng / ml, or ranging between any two concentrations referred to above or herein. For example, the PSC are grown in a culture medium that includes about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 ng / ml or more of the NK activating cytokine, such as interleukin-15 (IL-15) or any other NK activating cytokine featured herein. In some embodiments, the culture medium includes about 10 ng / mL IL-15.

[0107] SCF is a cytokine that binds to the c-KIT receptor (c-KIT / CD117). SCF exists both as a transmembrane protein and a soluble protein and plays an important role in hematopoiesis, spermatogenesis, and melanogenesis. SCF plays an important role in hematopoiesis during embryonic development. Sites where hematopoiesis takes place, such as the fetal liver and bone marrow, all express SCF. SCF may serve as guidance cues that direct hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which a stem cell resides), and it plays an important role in HSC maintenance. SCF plays a role in the regulation of HSCs in the stem cell niche in the bone marrow. SCF has been shown to increase the survival of HSCs in vitro and contribute to the self-renewal and maintenance of HSCs in vivo. HSCs at all stages of development express the same levels of the c-KIT. The stromal cells that surround HSCs are a component of the stem cell niche, and they release a number of ligands, including SCF. In the bone marrow,PATENT ATTORNEY DOCKET NO.: N2041-03301 HSCs and hematopoietic progenitor cells are adjacent to stromal cells, such as fibroblasts and osteoblasts. These HSCs remain in the niche by adhering to ECM proteins and to the stromal cells themselves. SCF has been shown to increase adhesion and thus may play a large role in ensuring that HSCs remain in the niche. SCF may be used along with other cytokines to culture HSCs and hematopoietic progenitors. The expansion of these cells ex vivo would allow advances in bone marrow transplantation, in which HSCs are transferred to a patient to re-establish blood formation. One of the problems with injecting SCF for therapeutic purposes is that SCF activates mast cells. The injection of SCF has been shown to cause allergic-like symptoms and the proliferation of mast cells and melanocytes.

[0108] In some aspects, the SCF is added to the PSC culture at a concentration that ranges from about 4 ng / ml to 40 ng / ml. In some embodiments, the SCF is at a concentration that ranges from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 15 ng / ml, from 15 to 20 ng / ml, from 20 to 25 ng / ml, from 25 to 30 ng / ml, from 30 to 35 ng / ml, from 35 to 40 ng / ml, from 40 to 45 ng / ml, from 45 to 50 ng / ml, or ranging between any two concentrations referred to above or herein. For example, the PSC are grown in a culture medium that includes about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 µM or more. In some embodiments, the culture medium includes about 20 ng / ml of SCF.

[0109] FLT3L is an endogenous small molecule that functions as a cytokine and growth factor that increases the number of immune cells (lymphocytes (B cells and T cells)) by activating the hematopoietic progenitors. It acts by binding to and activating FLT3 (CD135), which is found on what (in mice) are called multipotent progenitor (MPP) and common lymphoid progenitor (CLP) cells. It also induces the mobilization of the hematopoietic progenitors and stem cells in vivo, which may help the system kill cancer cells. FLT3L is crucial for steady-state plasmacytoid dendritic cell (pDC) and classical dendritic cell (cDC) development. A lack of FLT3L results in low levels of dendritic cells.

[0110] In some aspects, the FLT3L is added to the PSC culture at a concentration that ranges from about 2 ng / mL to 20 ng / mL. In some embodiments, the FLT3L is at a concentration ranging from about 1 to about 20 ng / ml. In some embodiments, the FLT3L is at a concentration that ranges from 0.1 to 0.5, from 0.5 to 1, from 1 to 2 ng / ml, from 2 to 3 ng / ml, from 3 to 4 ng / ml, from 4 to 5 ng / ml, from 5 to 6 ng / ml, from 6 to 7 ng / ml, from 7 to 8 ng / ml, from 8 to 9 ng / ml, from 9 to 10 ng / ml, from 10 to 11 ng / ml, from 11 to 12 ng / ml, from 12 to 13 ng / ml, from 13 to 14 ng / ml, from 14 to 15 ng / ml, from 15 to 16 ng / ml, from 16 to 17 ng / ml, from 17 to 18 ng / ml, from 18 to 19 ng / ml, from 19 to 20 ng / ml, from 20 to 21 ng / ml, from 21 to 22 ng / ml, from 22 to 23 ng / ml, fromPATENT ATTORNEY DOCKET NO.: N2041-03301 23 to 24 ng / ml, from 24 to 25 ng / ml, or ranging between any two concentrations referred to above or herein. For example, the PSC are grown in a culture medium that includes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ng / ml or more. In some embodiments, the culture medium includes about 10 ng / mL FLT3L.

[0111] For NK cell differentiation, the culture medium of HSCs may be switched to an “undefined medium.” As used herein, the term “undefined basal medium” or “undefined medium” is typically meant to refer to media formulations containing serum-derived agents such as human platelet lysate (hPL) or, alternatively, blood serum. Such media containing serum or serum-derived agents may be commercially available or prepared “in-house” by users. As used herein, the “undefined medium” is referred to as a “serum-derived agent containing medium.” As detailed above, such a serum-derived agent containing medium can be “basal,” “supplemented,” or “complete.”

[0112] In some embodiments, serum-derived agents include, as non-limiting examples, serum, blood platelet lysate, plasma, albumin derived from blood, and any component or derivative of blood plasma obtained from blood, such as by blood serum fractionation. In some embodiments, the serum-derived agent is selected from serum, blood platelet lysate, or albumin derived from blood. In some embodiments, the serum-derived agent is serum. In some embodiments, the serum- derived agent is blood platelet lysate. In some embodiments, the serum-derived agent is albumin. In some embodiments, the serum-derived agent is plasma.

[0113] In the methods described herein, pluripotent stem cells are differentiated into CD34+ hematopoietic precursor cells, which are then differentiated into NK cells. The methods described herein generate CD34+hematopoietic precursor cells that may or may not be different from hematopoietic stem cells, which are CD34+ / CD45+. Such an intermediate population of CD34+ / CD45+hematopoietic precursor cells emerges from CD34+ / CD45- hemogenic endothelial- like cells and vanishes thereafter to generate fully differentiated NK cells, characterized among others by being CD56+NK cells.

[0114] In some aspects, the CD34+precursor cells are contacted with about 4 to 40 ng / mL IL-7, about 2 to 20 ng / mL IL-15, about 4 to 40 ng / mL SCF, and / or about 1 to 20 ng / mL FLT3L. In some embodiments, the CD34+ / CD45+suspension cells are contacted with about 4 to 40 ng / mL IL-7, about 2 to 20 ng / mL IL-15, about 4 to 40 ng / mL SCF, and / or about 1 to 20 ng / mL FLT3L.

[0115] In some embodiments, the culture medium for incubating the culture of the culture of CD34+precursor cells and incubating the culture of CD34+ / CD45+suspension cells comprises a cytokine. In some embodiments, the cytokine is an interleukin, including, as non-limiting examples: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-17, and IL-18. In some embodiments, the cytokine is a NK activating cytokine, such asPATENT ATTORNEY DOCKET NO.: N2041-03301 IL-15 or any NK activating cytokine featured above or herein. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-15.

[0116] In some embodiments, the CD34+precursor cells are contacted with about 20 ng / ml IL-7, about 10 ng / ml IL-15, about 20 ng / ml SCF, and / or about 10 ng / ml FLT3L. In some embodiments, the CD34+ / CD45+suspension cells are contacted with about 20 ng / ml IL-7, about 10 ng / ml IL- 15, about 20 ng / ml SCF, and / or about 10 ng / ml FLT3L.

[0117] In some aspects, the contacting in step a), that is, growing the culture of PSCs in the presence of a WNT signaling pathway activator and / or a bone morphogenetic protein BMP is for about 1-7 days. In some embodiments, the contacting in step a) is for more than 7 days. In some embodiments, the contacting in step a) is for up to 1, 2, 3, 4, 5, 6, or 7, or more than 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step a) is for at least 1 day, 2 days, 3 days 4 days, 5 days, 6 days, or 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step a) is for 1 day. In some embodiments, the contacting in step a) is for 2 days. In some embodiments, the contacting in step a) is for 3 days. In some embodiments, the contacting in step a) is for 4 days. In some embodiments, the contacting in step a) is for 5 days. In some embodiments, the contacting in step a) is for 6 days. In some embodiments, the contacting in step a) is for 7 days. In some embodiments, the contacting in step a) is for more than 7 days. In some embodiments, the contacting in step a) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0118] In some aspects, the contacting in step b) follows contacting the culture of PSCs from step a) with a VEGF for about 1-7 days, thereby generating a population of CD34+precursor cells. In some embodiments, the contacting in step b) is for more than 7 days. In some embodiments, the contacting in step b) is for up to 1, 2, 3, 4, 5, 6, or 7, or more than 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step b) is for at least 1 day, 2 days, 3 days 4 days, 5 days, 6 days, or 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step b) is for 1 day. In some embodiments, the contacting in step b) is for 2 days. In some embodiments, the contacting in step b) is for 3 days. In some embodiments, the contacting in step b) is for 4 days. In some embodiments, the contacting in step b) is for 5 days. In some embodiments, the contacting in step b) is for 6 days. In some embodiments, the contacting in step b) is for 7 days. In some embodiments, the contacting in stepPATENT ATTORNEY DOCKET NO.: N2041-03301 b) is for more than 7 days. In some embodiments, the contacting in step b) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0119] In some aspects, the incubation of population of CD34+precursor cells in step c) is in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more cytokines. In some embodiments, one or more of the cytokines is a NK activating cytokine such as IL-15 or any of the NK activating cytokines featured above or herein. In some aspects, the incubation of population of CD34+precursor cells in step c) is in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L. In some embodiments, the population of CD34+precursor cells in step c) are incubated in a culture medium lacking any serum-derived agent while optionally supplementing the medium with at least one of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L. In some embodiments, the population of CD34+precursor cells in step c) are incubated in a culture medium lacking any serum-derived agent while optionally supplementing the medium with a combination of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0120] In some aspects, the incubating in step c) is for up to 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, or 28, or more than 28 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the incubating in step c) is for 7 days. In some embodiments, the incubating in step c) is for 8 days. In some embodiments, the incubating in step c) is for 9 days. In some embodiments, the incubating in step c) is for 10 days. In some embodiments, the incubating in step c) is for 11 days. In some embodiments, the incubating in step c) is for 12 days. In some embodiments, the incubating in step c) is for 13 days. In some embodiments, the incubating in step c) is for 14 days. In some embodiments, the incubating in step c) is for 15 days. In some embodiments, the incubating in step c) is for 16 days. In some embodiments, the incubating in step c) is for 17 days. In some embodiments, the incubating in step c) is for 18 days. In some embodiments, the incubating in step c) is for 19 days. In some embodiments, the incubating in step c) is for 20 days. In some embodiments, the incubating in step c) is for 21 days. In some embodiments, the incubating in step c) is for 22 days. In some embodiments, the incubating in step c) is for 23 days. In some embodiments, the incubating in step c) is for 24 days. In some embodiments, the incubating in step c) is for 25 days. In some embodiments, the incubating inPATENT ATTORNEY DOCKET NO.: N2041-03301 step c) is for 26 days. In some embodiments, the incubating in step c) is for 27 days. In some embodiments, the incubating in step c) is for 28 days. In some embodiments, the incubating in step c) is for more than 28 days. In some embodiments, the incubating in step c) is for 7 to 8 days, 7 to 9 days, 7 to 10 days, 7 to 11 days, 7 to 12 days, 7 to 13 days, 7 to 14 days, 7 to 15 days, 7 to 16 days, 7 to 17 days, 7 to 18 days, 7 to 19 days, 7 to 20 days, 7 to 21 days, 7 to 22 days, 7 to 23 days, 7 to 24 days, 7 to 25 days, 7 to 26 days, 7 to 27 days, 7 to 28 days, or more than 28 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0121] n some embodiments, the population of CD34+ / CD45+suspension cells in step d) are incubated in in a culture medium comprising at least one serum-derived agent and one or more cytokines, including NK activating cytokines such as IL-15 or any NK activating cytokine featured above or herein. In some embodiments, the population of CD34+ / CD45+suspension cells in step d) are incubated in in a culture medium comprising at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0122] In some aspects, the incubating in step d), that is, incubating the population of CD34+ / CD45+suspension cells from step c) in a culture medium containing at least one serum- derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7-35 days, thereby producing NK cells. In some embodiments, the incubating in step d) is for at least 7 days, 8 days, 9 days 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the incubating in step d) is for more than 7 days. In some embodiments, the incubating in step d) is for at least 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, or 35, or more than 35 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the incubating in step d) is for 7 days. In some embodiments, the incubating in step d) is for 8 days. In some embodiments, the incubating in step d) is for 9 days. In some embodiments, the incubating in step d) is for 10 days. In some embodiments, the incubating in step d) is for 11 days. In some embodiments, the incubating in step d) is for 12 days. In some embodiments, the incubating in step d) is for 13 days. In some embodiments, the incubating in step d) is for 14 days. In some embodiments, the incubating in step d) is for 15 days. In some embodiments, the incubating in step d) is for 16 days. In somePATENT ATTORNEY DOCKET NO.: N2041-03301 embodiments, the incubating in step d) is for 17 days. In some embodiments, the incubating in step d) is for 18 days. In some embodiments, the incubating in step d) is for 19 days. In some embodiments, the incubating in step d) is for 20 days. In some embodiments, the incubating in step d) is for 21 days. In some embodiments, the incubating in step d) is for 22 days. In some embodiments, the incubating in step d) is for 23 days. In some embodiments, the incubating in step d) is for 24 days. In some embodiments, the incubating in step d) is for 25 days. In some embodiments, the incubating in step d) is for 26 days. In some embodiments, the incubating in step d) is for 27 days. In some embodiments, the incubating in step d) is for 28 days. In some embodiments, the incubating in step d) is for 29 days. In some embodiments, the incubating in step d) is for 30 days. In some embodiments, the incubating in step d) is for 31 days. In some embodiments, the incubating in step d) is for 32 days. In some embodiments, the incubating in step d) is for 33 days. In some embodiments, the incubating in step d) is for 34 days. In some embodiments, the incubating in step d) is for 35 days. In some embodiments, the incubating in step d) is for more than 35 days. In some embodiments, the incubating in step d) is for 7 to 8 days, 7 to 9 days, 7 to 10 days, 7 to 11 days, 7 to 12 days, 7 to 13 days, 7 to 14 days, 7 to 15 days, 7 to 16 days, 7 to 17 days, 7 to 18 days, 7 to 19 days, 7 to 20 days, 7 to 21 days, 7 to 22 days, 7 to 23 days, 7 to 24 days, 7 to 25 days, 7 to 26 days, 7 to 27 days, 7 to 28 days, 7 to 29 days, 7 to 29 days, 7 to 30 days, 7 to 31 days, 7 to 32 days, 7 to 33 days, 7 to 34 days, 7 to 35 days, or more than 35 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0123] In some embodiments, contacting the adherent culture of PSCs with a WNT signaling pathway activator and a BMP takes about 2–5 days. In some embodiments, contacting the culture of PSCs of step a) with a WNT signaling pathway activator and a BMP signaling pathway activator is for about 3 to 5 days. In some embodiments, contacting the culture of PSCs of step a) with a WNT signaling pathway activator and a BMP signaling pathway activator is for about 4 to 5 days. In some embodiments, contacting the culture of PSCs of step a) a WNT signaling pathway activator and a BMP signaling pathway activator is for about 2, 3, 4, 5 days, or for any length of time ranging between any two of the recited number of days.

[0124] In some embodiments, the PSCs are subsequently contacted with a VEGF for about 2–5 days. In some embodiments, contacting the PSCs with a VEGF is for about 3 to 5 days. In some embodiments, contacting the PSCs with a VEGF is for about 4 to 5 days. In some embodiments, contacting the PSCs with a VEGF is for about 2, 3, 4, 5 days, or for any length of time ranging between any two of the recited number of days.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0125] In some embodiments, contacting the population of CD34+precursor cells with IL-7, IL- 15, SCF, and / or FLT3L is for about 5–10 days, and contacting the population of CD34+ / CD45+suspension cells with IL-7, IL-15, FLT3L, and / or SCF is for at least about 7–21 days. In some embodiments, contacting the population of CD34+precursor cells with IL-7, IL-15, SCF, and / or FLT3L is for about 4 to 5 days, 5 to 6 days, 5 to 7 days, 5 to 8 days, 5 to 9 days, 5 to 10 days, or more than 10 days, or for any length of time ranging between any two of the recited number of days referred to above or herein, and contacting the population of CD34+ / CD45+suspension cells with IL-7, IL-15, FLT3L, and / or SCF is for at least about 6 to 7 days, 7 to 8 days, 7 to 9 days, 7 to 10 days, 7 to 11 days, 7 to 12 days, 7 to 13 days, 7 to 14 days, 7 to 15 days, 7 to 16 days, 7 to 17 days, 7 to 18 days, 7 to 19 days, 7 to 20 days, 7 to 21 days, or more than 21 days, or any length of time ranging between any of the two recited number of days referred to above or herein.

[0126] In some embodiments, contacting the population of CD34+precursor cells with IL-7, IL- 15, SCF, and / or FLT3L is for about 5–10 days, followed by contacting the population of CD34+ / CD45+suspension cells with IL-7, IL-15, FLT3L, and / or SCF for at least about 7–21 additional days. In some embodiments, contacting the population of CD34+precursor cells with IL-7, IL-15, SCF, and / or FLT3L is for about 4 to 5 days, 5 to 6 days, 5 to 7 days, 5 to 8 days, 5 to 9 days, 5 to 10 days, or more than 10 days, or for any length of time ranging between any two of the recited number of days referred to above or herein, and contacting the population of CD34+ / CD45+suspension cells with IL-7, IL-15, FLT3L, and / or SCF is for at least about 6 to 7 additional days, 7 to 8 additional days, 7 to 9 additional days, 7 to 10 additional days, 7 to 11 additional days, 7 to 12 additional days, 7 to 13 additional days, 7 to 14 additional days, 7 to 15 additional days, 7 to 16 additional days, 7 to 17 additional days, 7 to 18 additional days, 7 to 19 additional days, 7 to 20 additional days, 7 to 21 additional days, or more than 21 additional days, or any length of time ranging between any of the two recited number of days referred to above or herein.

[0127] In some embodiments, the culture of PSCs is an adherent layer of cells. In some embodiments, the layer of cells is grown in a two-dimensional culture system or on microcarriers.

[0128] In some embodiments, the PSCs are cultures on scaffolds composed of microcarriers, which are beads or particles. The beads may be microscopic or macroscopic and may further be dimensioned so as to permit penetration into tissues or compacted to form a particular geometry.

[0129] In some embodiments, the framework for the cell cultures comprises particles that, in combination with the cells, form a three-dimensional tissue. The cells attach to the particles and to each other to form a three-dimensional tissue. Beads, or microcarriers, are typically considered a two-dimensional system or scaffold.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0130] As used herein, a “microcarrier” refers to a particle having a size of nanometers to micrometers, where the particles may be any shape or geometry, being irregular, non-spherical, spherical, or ellipsoid. The size of the microcarriers suitable for the purposes herein can be of any size suitable for the particular application.

[0131] In some embodiments, the size of microcarriers suitable for the three-dimensional tissues may be those administrable by injection. In some embodiments, the microcarriers have a particle size range of at least about 1 μm, at least about 10 μm, at least about 25 μm, at least about 50 μm, at least about 100 μm, at least about 200 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, at least about 700 μm, at least about 800 μm, at least about 900 μm, at least about 1000 μm, or any range of sizes between any two of the sizes referred to above or herein.

[0132] In some embodiments, the microcarriers are made of biodegradable materials. In some embodiments, microcarriers comprising two or more layers of different biodegradable polymers may be used. In some embodiments, at least an outer first layer has biodegradable properties for forming the three-dimensional tissues in culture, while at least a biodegradable inner second layer, with properties different from the first layer, is made to erode when administered into a tissue or organ.

[0133] In some embodiments, the microcarriers are porous microcarriers. Porous microcarriers refer to microcarriers having interstices through which molecules may diffuse in or out of the microparticle. In other embodiments, the microcarriers are non-porous microcarriers. A nonporous microparticle refers to a microparticle in which molecules of a select size do not diffuse in or out of the microparticle.

[0134] Microcarriers for use in the compositions are biocompatible and have low or no toxicity to cells. The microcarriers may comprise various polymers, natural or synthetic, charged (i.e., anionic or cationic) or uncharged, biodegradable, or nonbiodegradable. The polymers may be homopolymers, random copolymers, block copolymers, graft copolymers, or branched polymers.

[0135] In some embodiments, the microcarriers comprise non-biodegradable microcarriers. Non- biodegradable microcapsules and microcarriers include, but are not limited to, those made of polysulfones, poly (acrylonitrile-co-vinyl chloride), ethylene-vinyl acetate, and hydroxyethyl methacrylate-methyl-methacrylate copolymers. These are useful to provide tissue bulking properties or in embodiments where the microcarriers are eliminated by the body.

[0136] In some embodiments, the microcarriers comprise degradable scaffolds. These include microcarriers made from naturally occurring polymers, non-limiting examples of which include,PATENT ATTORNEY DOCKET NO.: N2041-03301 among others, fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginate, or poly- amino acids such as poly-lysine.

[0137] In some embodiments, the degradable microcarriers are made of synthetic polymers, non- limiting examples of which include, among others, polylactide (PLA), polyglycolide (PGA), poly (lactide-co-glycolide) (PLGA), poly (caprolactone), polydioxanone trimethylene carbonate, polyhybroxyalkonates (e.g., poly (hydroxybutyrate), poly (ethyl glutamate), poly DTH iminocarbony (bisphenol A iminocarbonate), poly (ortho ester), and polycyanoacrylates.

[0138] In some embodiments, the microcarriers comprise hydrogels, which are typically hydrophilic polymer networks filled with water. Hydrogels have the advantage of selectively triggering polymer swelling. Depending on the composition of the polymer network, swelling of the microparticle may be triggered by a variety of stimuli, including pH, ionic strength, thermal, electrical, ultrasound, and enzyme activities. Non-limiting examples of polymers useful in hydrogel compositions include, among others, those formed from polymers of poly (lactide-co- glycolide); poly (N-isopropylacrylamide); poly (methacrylic acid-g-polyethylene glycol); polyacrylic acid and poly (oxypropylene-co-oxyethylene) glycol; and natural compounds including, as non-limiting examples, chrondroitan sulfate, chitosan, gelatin, fibrinogen, or mixtures of synthetic and natural polymers, for example, chitosan-poly (ethylene oxide). The polymers may be crosslinked reversibly or irreversibly to form gels adaptable for forming three- dimensional tissues.

[0139] In some embodiments, the microcarriers or beads for use in the present disclosure are composed wholly or partly of dextran.

[0140] In some embodiments, the PSCs are cultured on a coated surface, including a laminin coating.

[0141] In some embodiments, the CD34+precursor cell is a CD34+endothelial-like precursor cell.

[0142] In some embodiments, the NK cells are further collected in suspension in a cell culture medium.

[0143] The methods described herein allow for the differentiation of NK cells that grow in suspension from CD34+endothelial-like precursor cells that are adherent. Therefore, during the course of differentiation, intermediate and transient CD34+and CD45+cells emerge in suspension from the CD34+endothelial-like precursor cells and are differentiated into NK cells. After at least four weeks in culture, the methods described herein yield at least 80% pure or enriched NK cells in suspension. Since the cells are in suspension, they can easily be aspirated and collected in the culture medium.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0144] In some embodiments, the NK cells are at least about 60, 65, 70, 75, 80, 85, 90, or 95% enriched. In some embodiments, the NK cells are at least 60% enriched. In some embodiments, the NK cells are at least 65% enriched. In some embodiments, the NK cells are at least 70% enriched. In some embodiments, the NK cells are at least 75% enriched. In some embodiments, the NK cells are at least 80% enriched. In some embodiments, the contacting in step b) generates a population of CD34+precursor cells at least 85% enriched for CD34+. In some embodiments, the NK cells are at least 90% enriched. In some embodiments, the NK cells are at least 95% enriched. In some embodiments, the NK cells are at least about 80% enriched.

[0145] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7– 35 days enriches the number of cells by at least 50-fold to 300-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7-14 days in a culture medium lacking any serum-derived agent. In some embodiments, the number of cells are enriched by at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300-fold, or any fold enrichment ranging between any two percentages referred to above or herein.

[0146] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7– 35 days enriches the number of cells by at least 50-fold compared to the number of cells in incubating the population of CD34+precursor cells for about 7–14 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0147] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7– 35 days enriches the number of cells by at least 100-fold compared to the number of cells in incubating the population of CD34+precursor cells for about 7–14 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0148] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7– 35 days enriches the number of cells by at least 150-fold compared to the number of cells in incubating the population of CD34+precursor cells for about 7–14 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0149] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7– 35 days enriches the number of cells by at least 200-fold compared to the number of cells in incubating the population of CD34+precursor cells for about 7–14 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0150] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7- 35 days enriches the number of cells by at least 250-fold compared to the number of cells in incubating the population of CD34+precursor cells for about 7-28 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0151] In some embodiments, incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L for about 7- 35 days enriches the number of cells by at least 300-fold compared to the number of cells in incubating the population of CD34+precursor cells for about 7-28 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0152] In another aspect, the present disclosure provides a method of producing natural killer (NK) cells from pluripotent stem cells (PSCs) comprising: a) generating CD34+hemogenicPATENT ATTORNEY DOCKET NO.: N2041-03301 endothelium (HE) cells by: (i) contacting a culture of PSCs with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), wherein the PSCs grown for about 3 days; and (ii) subsequently contacting the cells of (i) with a vascular endothelial growth factor (VEGF) for about 4 days; thereby generating a population of cells comprising at least 80% CD34+HE cells; b) culturing the CD34+HE cells of step a) in a medium lacking any serum-derived agents for about 7-28 days while optionally adding one or more of IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, SCF and / or FLT3L, thereby generating a transient population of suspension cells comprising at least 80% CD34+ / CD45+hematopoietic precursor cells (HPCs); c) contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7-14 days in a culture medium lacking any serum-derived agent; and d) transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days, thereby inducing NK cell differentiation from PSCs.

[0153] In some embodiments, differentiated NK cells are CD56+, NKp30+, NKp44+, NKp46+, NKG2D+, NKG2A+, KIR2D+and / or CD16+.

[0154] NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3−). NK cells (belonging to the group of innate lymphoid cells) are one of the three kinds of cells differentiated from the common lymphoid progenitor, the other two being B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation. NK cells differ from natural killer T cells (NKTs) phenotypically, by origin and by respective effector functions; often, NKT cell activity promotes NK cell activity by secreting interferon gamma. In contrast to NKT cells, NK cells do not express T-cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD16 (FcγRIII) and CD57 in humans. The NKp46 cell surface marker constitutes another NK cell marker of preference being expressed in both humans, several strains of mice and in three common monkey species.

[0155] NK cells can be classified as CD56brightor CD56dim. CD56brightNK cells are similar to T helper cells in exerting their influence by releasing cytokines. CD56brightNK cells constitute the majority of NK cells, being found in bone marrow, secondary lymphoid tissue, liver, and skin. CD56dimNK cells are primarily found in the peripheral blood and are characterized by their cell killing ability. CD56dimNK cells are always CD16 positive (CD16 is the key mediator of antibody- dependent cellular cytotoxicity (ADCC). CD56brightcan transition into CD56dimby acquiring CD16.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0156] In some embodiments, the differentiated NK cells are CD56brightor CD56dim.

[0157] In some embodiments, the differentiated NK cells are cytotoxic NK cells.

[0158] In some aspects, the NK cells are produced in a feeder-independent manner.

[0159] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, an NK activating cytokine such as IL-15 or any other NK activating cytokine featured herein, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 50-fold to 300-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7- 14 days in a culture medium lacking any serum-derived agent. In some embodiments, the number of cells are enriched by at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300-fold, or any fold enrichment ranging between any two percentages referred to above or herein.

[0160] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 50-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7-14 days in a culture medium lacking any serum-derived agent.

[0161] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 100-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7- 14 days in a culture medium lacking any serum-derived agent.

[0162] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 150-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7- 14 days in a culture medium lacking any serum-derived agent.

[0163] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 200-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7- 14 days in a culture medium lacking any serum-derived agent.

[0164] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 daysPATENT ATTORNEY DOCKET NO.: N2041-03301 enriches the number of cells by at least 250-fold compared to the number of cells in contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7- 14 days in a culture medium lacking any serum-derived agent.

[0165] In some embodiments, transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days enriches the number of cells by at least 300-fold compared to the number of cells in contacting the CD34 CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7-14 days in a culture medium lacking any serum-derived agent.

[0166] Stem cells are undifferentiated cells that have the ability to self-renew indefinitely and to remain in said undifferentiated state. As opposed to embryonic stem cells, which can only be isolated from the inner mass of a blastocyst, there are three known accessible sources of adult stem cells: the bone marrow, which requires the drilling of a bone; the adipose tissue, which is accessible by liposuction; and the blood, from which the cells can be extracted among other cells. The term “pluripotent stem cells,” as used herein, refers to cells that are capable of generating all the cell types of an organism, i.e., cells derived from any of the three germ layers. On the other hand, multipotent stem cells can differentiate into several cell types, but only those of a closely related family of cells, generally the cell types of the organ from which they originate. Most adult stem cells are multipotent, but small amounts of pluripotent adult stem cells can be retrieved from the umbilical cord or other tissues. The sources of cells used for cell therapy include stem cells such as embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs).

[0167] In some embodiments, the PSCs used in the methods described herein are human (hPSCs), and in some embodiments, the human PSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

[0168] By “generating” or “producing” CD34+hematopoietic precursor cells, it is meant that the present methods provide physical and chemical culture conditions that have been optimized to induce the differentiation of PSCs into CD34+hematopoietic precursor cells. The differentiation method described herein yields a CD34+hematopoietic precursor cell population that is enriched for CD34+hematopoietic precursor cells. For example, greater than 80%, greater than 85%, greater than 90%, greater than 95%, 96%, 97%, 98%, or 99% CD34+hematopoietic precursor cells are obtained in short time and using convenient culture conditions.

[0169] Physical culture conditions include but are not limited to the culture environment of the cell (e.g., adherent versus suspension culture, or in two-dimensional versus in three-dimensional culture systems), the pH of the culture medium, the gas concentration in the incubator (e.g., CO2concentration, O2 concentration), and the temperature.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0170] There are two basic systems for growing cells in culture, as monolayers on an artificial substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture). The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading (i.e., tissue-culture treated). However, many cell lines can also be adapted for suspension culture.

[0171] In some embodiments, the culture of PSCs is an adherent layer of cells. In some embodiments, the layer of cells is grown in a two-dimensional culture system or on microcarriers.

[0172] In addition to the treatment of the tissue-culture surface, cells can require to be grown on coated surfaces, to enhance or improve their adhesion and / or spreading (i.e., using a coating). “Coating” as an additional surface treatment stands for all additional modifications made to increase cell adhesion in addition to the standard plasma or corona treatment which is performed on all cell culture plastic by manufacturer. Usually, coating is done with proteins or peptides. Various proteins can be used to coat tissue-culture treated dishes, including poly-L-Lysine, poly- D-Lysine, poly-Ornithine, gelatin, collagen I, IV, fibronectin, laminin, vitronectin, osteopontin, fibronectin domains, Matrigel ™ (several components of the extracellular matrix with bound growth factors etc.), collagen gels, alginate gels, and lactate gels.

[0173] In some embodiments, the PSCs are cultured on a coated surface including a laminin coating.

[0174] Physical culture conditions include the gas concentration in the incubator. Incubation of cell cultures is typically performed in normal atmosphere with 15-22% oxygen and 5% CO2 for expansion and seeding. In some embodiments, the PSCs are grown in a humidified atmosphere including about 5% CO2concentration, and normoxic conditions (non-hypoxic O2concentration). While hypoxic culture conditions are thought to support stem cell performance in general, in the present methods, the PSCs are cultured under conditions that are not hypoxic conditions. As used herein, “normoxic” conditions refer to culture conditions including atmospheric O2concentration (e.g., about 15-25% O2concentration). As used herein, hypoxic conditions are characterized by a lower oxygen concentration as compared to the oxygen concentration of ambient air (approximately 15%-25% oxygen).

[0175] Chemical culture conditions include but are not limited to the agents or molecules that are added to the culture medium to achieve the desired effects sought after (i.e., differentiation of PSCs into CD34+hematopoietic precursor cells). The terms “agent” and “molecule” are used interchangeably and include, but are not limited to, small molecules (including small molecules that do not have optimal cell-permeability), lipids, nucleosides, nucleotides, nucleic acids,PATENT ATTORNEY DOCKET NO.: N2041-03301 polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines.

[0176] In the methods described herein, pluripotent stem cells are differentiated into CD34+hematopoietic precursor. As used herein, “CD34+hematopoietic precursor” or “CD34+hematopoietic progenitor” refers to transient cells that express some of hematopoietic stem cells (HSCs) markers, and display some of their characteristics, without being presenting all the HSCs markers and characteristics. For example, HSCs are usually characterized as CD34+ / CD45+non- adherent cells. After about 7 days of culture under the conditions described herein, the CD34+hematopoietic precursor cells are CD34+but they remain CD144+and CD45-, they are also still adherent cells.

[0177] Unless otherwise specifically describes, pluripotent stem cells are maintained in a stem cell medium, suitable for the culture and propagation of pluripotent stem cells. Stem cell basal media are well known in the art; non-limiting examples of such suitable media include but are not limited to StemPro34 ™. For HSC differentiation, the pluripotent stem cells are switched to a “defined medium”. As used herein, the term “basal medium” generally refers to a base medium, without any additives added by the used (for example, a basal medium refers to a medium as commercially available). Those generally include water, nutrients, salts and amino acids, but no additive or supplements. A basal medium can be supplemented with general additives to obtain a “supplemented basal media”. Non-limiting examples of supplements include but are not limited to insulin or ascorbate for example. A basal medium can also be completed with specific signaling molecules such as those identified by a user as necessary to achieve a particular goal with the cell culture, such as for example driving the differentiation of a cell type of interest into a target cell type. Such complete basal medium can be referred to as a “final”, “complete” or “cell-specific” medium.

[0178] In the context of the present disclosure, the cell culture media are additionally referred to based on their use. For example, the terms “defined basal medium” and “defined medium” are meant to refer to ready-to-use medium formulations for the generation of CD34+ / CD45+hematopoietic precursor cells that only contain specific components in quantifiable amounts, e.g., they do not contain serum-derived agents (blood serum or components directly isolated from it or other animal or tissue-derived products isolated from organisms or cells). However, by definition herein, recombinant serum albumin is considered essential even in the defined medium, and it is not considered “serum-derived.” Such ready-to-use formulations can be found in commercially available products or in “in-house” compositions developed by users. Without wishing to be limited to any specific formulation, it is provided that a general formulation for a defined mediumPATENT ATTORNEY DOCKET NO.: N2041-03301 can include a basal medium including, as non-limiting examples, DMEM, DMEM / F12, IMDM, or mixture thereof, and supplements comprising one of more of the following without being limited to: insulin, insulin combined with transferrin and selenium, serum albumin, human recombinant serum albumin, polyvinylalcohol (PVA), lipids or fatty acids, glutamine, alanyl- glutamine (Glutamax), amino acids in general, antioxidants such as ascorbic acid, ascorbic acid- 2-phosphate, or thiol compounds, and inorganic salts (for the supplemented version). Non-limiting examples of commercially available defined basal media include, but are not limited to: (1) APEL ™ including 1X Iscove’s modified Dulbecco’s medium (IMDM), 1X Ham’s F-12 nutrient mixture, Albucult (rh Albumin) (5 mg / ml), Polyvinylalcohol (PVA), Linoleic acid (100 ng / ml), Linolenic acid (100 ng / ml), SyntheChol (synthetic cholesterol) (2.2 mg / ml), a-Monothioglycerol (a-MTG) (3.9 ml per100ml), rh Insulin-transferrin-selenium-ethanolamine solution (rhITS-Eth), protein-free hybridoma mixture II (PFHMII) (5%), ascorbic acid 2 phosphate (50µg / ml), GlutamaxI (L-alanyl-L-glutamine) (2 mM) and penicillin / streptomycin (50 U Pen G / 50 mg streptomycin sulfate); (2) APELII ™ or APEL2 ™ including 1X Iscove’s modified Dulbecco’s medium (IMDM), 1X Ham’s F-12 nutrient mixture, Albucult (rh Albumin) (5 mg / ml), Polyvinylalcohol (PVA), Linoleic acid (100 ng / ml), Linolenic acid (100 ng / ml), SyntheChol (synthetic cholesterol) (2.2 mg / ml), a-Monothioglycerol (a-MTG) (3.9 ml per100ml), rh Insulin- transferrin-selenium-ethanolamine solution (rhITS-Eth), ascorbic acid 2 phosphate (50µg / ml), GlutamaxI (L-alanyl-L-glutamine) (2 mM) and penicillin / streptomycin (50 U Pen G / 50 mg streptomycin sulfate); (3) APALII including 1X Iscove’s modified Dulbecco’s medium (IMDM), Polyvinylalcohol (PVA, 0.1%), Albucult (rh Albumin) (0.2%), ascorbic acid 2 phosphate (250 uM), lipids (1%), rh Insulin-transferrin-selenium-ethanolamine solution (rhITS-Eth, 0.1%); (4) ESSENTIAL 6 ™ or E6 ™ including DMEM / F12, insulin, transferrin, sodium selenium, ascorbic acid-2-phosphate, and NaHCO3; and (5) ESSENTIAL 8 ™ or E8 ™ including DMEM / F12, L- ascorbic acid-2-phosphate magnesium (64 mg / l), sodium selenium (14 µg / l), insulin (19.4 mg / l), NaHCO3 (543 mg / l) and transferrin (10.7 mg / l).

[0179] Such basal or supplemented defined media used for HE generation can then be completed with small molecules of interest as needed.

[0180] By “contacting,” it is meant that the cells are cultured with one or more agents of interest, added to the culture medium lacking serum-derived agents. That is, the cells are cultured in their regular culture basal or supplemented medium, in which a desired concentration of one or more agents of interest is added. For example, the cells are cultured with a WNT signaling pathway activator, a BMP, and / or a VEGF.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0181] A “signaling pathway activator,” as used herein, refers to any molecule that is capable of activating, enhancing, or inducing a signaling pathway of interest. A signaling pathway is a series of chemical reactions in which a group of molecules in a cell work together to control a cell function, such as cell differentiation. A cell receives signals from its environment when a molecule, such as a hormone or growth factor, binds to a specific protein receptor on or in the cell. After the first molecule in the pathway receives a signal, it activates another molecule. This process is repeated through the entire signaling pathway until the last molecule is activated and cell function is carried out. Abnormal activation of signaling pathways or inhibition of a signaling pathway may lead to diseases or, in the case of pluripotent cells, to an alteration of the pluripotent state and therefore to differentiation. The term “molecule” includes, but is not limited to, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines. Non-limiting examples of polynucleotides include short interfering nucleic acid (siNA), antisense, enzymatic nucleic acid molecules, 2',5'-oligoadenylate, triplex- forming oligonucleotides, aptamers, and decoys. Biologically active molecules include antibodies (e.g., monoclonal, chimeric, humanized, etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex-forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys, and analogs thereof, and small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), antagomirs, and short hairpin RNA (shRNA) molecules.

[0182] The WNT signaling pathways are a group of signal transduction pathways that begin with proteins that pass signals into a cell through cell surface receptors. WNT signaling pathways use either nearby cell-cell communication (paracrine) or same-cell communication (autocrine). Three WNT signaling pathways have been characterized: the canonical WNT pathway, the noncanonical planar cell polarity pathway, and the noncanonical WNT / calcium pathway. All three pathways are activated by the binding of a WNT-protein ligand to a Frizzled family receptor, which passes the biological signal to the disheveled protein inside the cell. The canonical WNT pathway leads to regulation of gene transcription and is thought to be negatively regulated in part by the SPATS1 gene. The noncanonical planar cell polarity pathway regulates the cytoskeleton, which is responsible for the shape of the cell. The noncanonical WNT / calcium pathway regulates calcium inside the cell. WNT signaling was first identified for its role in carcinogenesis, then for itsPATENT ATTORNEY DOCKET NO.: N2041-03301 function in embryonic development. The embryonic processes it controls include body axis patterning, cell fate specification, cell proliferation, and cell migration. These processes are necessary for the proper formation of important tissues, including bone, heart, and muscle. Its role in embryonic development was discovered when genetic mutations in WNT pathway proteins produced abnormal fruit fly embryos. Later research found that the genes responsible for these abnormalities also influenced breast cancer development in mice. WNT signaling also controls tissue regeneration in adult bone marrow, skin, and intestine.

[0183] Activators of the WNT signaling pathway include, as non-limiting examples, CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)- 2pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile); WNT family ligands; RSPO co- agonists; lithium chloride; TDZD8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione); BIO- Acetoxime ((2’Z,3’E)-6-Bromoindirubin-3′-acetoxime); A1070722 (1-(7-Methoxyquinolin- 4-yl)-3-[6 (trifluoromethyl)pyridin-2-yl]urea); HLY78 (4-Ethyl-5,6-Dihydro-5-methyl- [1,3]dioxolo[4,5-j]phenanthridine); CID 11210285 hydrochloride (2-Amino-4-(3,4- (methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero) arylpyrimidines; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, activation of the WNT signaling pathway may be achieved through repression of a WNT signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway inhibitor. In some embodiments, the WNT signaling pathway activator is a GSK3 inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0184] The transforming growth factor beta (TGF-β) superfamily includes TGF-β proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), activins, inhibitors, nodal, lefty, and Mülllerian inhibiting substances (MIS). Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines and metabologens. Originally discovered for their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. The important functioning of BMP signals in physiology is emphasized by the multitude of roles for dysregulated BMP signaling in pathological processes.

[0185] BMPs interact with specific receptors on the cell surface, referred to as bone morphogenetic protein receptors (BMPRs). Signal transduction through BMPRs results in the mobilization of members of the SMAD family of proteins. The signaling pathways involving BMPs, BMPRs, and SMADs are important in the development of the heart, central nervousPATENT ATTORNEY DOCKET NO.: N2041-03301 system, and cartilage, as well as postnatal bone development. They have an important role during embryonic development in embryonic patterning and early skeletal formation. As such, disruption of BMP signaling can affect the body plan of the developing embryo. As featured and disclosed herein, a bone morphogenetic protein includes, as non-limiting examples, a BMP family ligand, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP 15.

[0186] For example, BMP4 and its inhibitors, noggin and chordin, help regulate the polarity of the embryo (i.e., back-to-front patterning). Specifically, BMP-4 and its inhibitors play a major role in neurulation and the development of the neural plate. BMP-4 signals ectoderm cells to develop into skin cells, but the secretion of inhibitors by the underlying mesoderm blocks the action of BMP-4 to allow the ectoderm to continue its normal course of neural cell development.

[0187] In some embodiments, the BMP is BMP4.

[0188] Vascular endothelial growth factor (VEGF), originally known as vascular permeability factor (VPF), is a signal protein produced by many cells that stimulates the formation of blood vessels. To be specific, VEGF is a sub-family of growth factors in the platelet-derived growth factor family of cystine-knot growth factors. They are important signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate, such as in hypoxic conditions. The serum concentration of VEGF is high in bronchial asthma and diabetes mellitus. VEGF's normal function is to create new blood vessels during embryonic development, new blood vessels after injury, muscle following exercise, and new vessels (collateral circulation) to bypass blocked vessels. A vascular endothelial growth factor includes, as non-limiting examples, a VEGF family ligand, e.g., VEGF (VEGFA), VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placenta growth factor (PIGF). In some embodiments, the VEGF is VEGF-A.

[0189] In some embodiments, the chemical culture conditions of the presently described methods include a mixture of agents, including a WNT signaling pathway activator, a BMP, and / or a VEGF.

[0190] For example, the mixture of agents includes a WNT signaling pathway activator and a BMP. In an additional example, the mixture of agents includes a WNT signaling pathway activator, a BMP, and VEGF alone. In another example, the mixture of agents includes a WNT signaling pathway activator alone, a BMP alone, or a VEGF alone.

[0191] In some embodiments, the PSCs are grown on a substrate in the presence of a WNT signaling pathway activator and a bone morphogenetic protein (BMP) for about 1–8 days. ForPATENT ATTORNEY DOCKET NO.: N2041-03301 example, the cells are grown for about 1, 2, 3, 4, 5, 6, 7, 8, or more days in the presence of a WNT signaling pathway activator and a BMP.

[0192] In some embodiments, following the initial culture in the presence of a WNT signaling pathway activator and a BMP, the PSCs are grown on a substrate in the presence of a VEGF for about 1–8 days. In some embodiments, the PSCs are grown on a substrate in the presence of VEGF for about 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein. For example, following the initial culture, the cells are grown for about 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein, in the presence of VEGF.

[0193] In some embodiments, following the initial culture in the presence of a WNT signaling pathway activator and a BMP, the PSCs are grown on a substrate in the presence of VEGF, a WNT signaling activator, and a BMP for about 1–8 days. In some embodiments, the PSCs are grown on a substrate in the presence of VEGF, a WNT signaling activator, and a BMP for about 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein. For example, following the initial culture, the cells are grown for about 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein, in the presence of a VEGF, a WNT signaling activator, and a BMP.

[0194] In some embodiments, the culture of PSCs is contacted with a WNT signaling pathway activator and a BMP for about 3 days, and then with a VEGF for about 4 additional days.

[0195] In some embodiments, the culture of PSCs is contacted with a WNT signaling pathway activator and a BMP for about 2 days, 3 days, or more than 3 days, or any length of time ranging between any of the two recited number of days referred to above or herein, and then with a VEGF for about 3 additional days, 4 additional days, or more than 4 additional days, or any length of time ranging between any of the two recited number of days referred to above or herein.

[0196] In some embodiments, contacting the adherent culture of PSCs with a WNT signaling pathway activator, a BMP, and / or a VEGF generates CD34+hemogenic endothelium (HE).

[0197] Hemogenic endothelium (HE) is composed of a special subset of endothelial cells scattered within blood vessels that can differentiate into hematopoietic cells. The development of hematopoietic cells in the embryo proceeds sequentially from the mesoderm through the hemangioblast to the hemogenic endothelium and hematopoietic progenitors. Hemangioblasts are multipotent precursor cells that can differentiate into both hematopoietic and endothelial cells.PATENT ATTORNEY DOCKET NO.: N2041-03301 Hemangioblasts are the progenitors that form the blood islands and are capable of differentiating into endothelial progenitor cells (EPCs) and blood cells. Hemangioblasts have been first extracted from embryonic cultures and manipulated by cytokines to differentiate along either the hematopoietic or endothelial routes.

[0198] In some aspects, the present disclosure provides a method of producing hemogenic endothelium (HE) cells by: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and / or a bone morphogenetic protein (BMP), wherein the PSCs are grown for about 1-7 days; and b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF) for about 1-7 days following step a) thereby generating a population of CD34+HE cells. In some aspects, the contacting in step a), that is, growing the culture of PSCs in the presence of a WNT signaling pathway activator and / or a bone morphogenetic protein BMP is for about 1- 7 days.

[0199] In some embodiments, the contacting in step a) is for more than 7 days. In some embodiments, the contacting in step a) is for up to 1, 2, 3, 4, 5, 6, or 7, or more than 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step a) is for at least 1 day, 2 days, 3 days 4 days, 5 days, 6 days, or 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step a) is for 1 day. In some embodiments, the contacting in step a) is for 2 days. In some embodiments, the contacting in step a) is for 3 days. In some embodiments, the contacting in step a) is for 4 days. In some embodiments, the contacting in step a) is for 5 days. In some embodiments, the contacting in step a) is for 6 days. In some embodiments, the contacting in step a) is for 7 days. In some embodiments, the contacting in step a) is for more than 7 days. In some embodiments, the contacting in step a) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0200] In some aspects, contacting the culture with VEGF in step b) optionally further comprises contacting the culture with forskolin or SB431542 to encourage endothelial differentiation or hematopoietic differentiation, respectively. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 µM, or more than 20 µM of either forskolin or SB431542, or any concentration of either forskolin or SB431542 ranging between any two of the recited concentrations referred to above or herein is added to the culture medium during the contacting step b). In some embodiments, about 10 µM of forskolin or SB431542 is optionally contacted with the culture in the contacting step b).PATENT ATTORNEY DOCKET NO.: N2041-03301

[0201] In some aspects, the contacting in step b) follows contacting the culture of PSCs from step a) with a VEGF for about 1-7 days, thereby generating a population of CD34+hemogenic endothelium cells. In some embodiments, the contacting in step b) is for more than 7 days. In some embodiments, the contacting in step b) is for up to 1, 2, 3, 4, 5, 6, or 7, or more than 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step b) is for at least 1 day, 2 days, 3 days 4 days, 5 days, 6 days, or 7 days, or for any length of time ranging between any two of the number of days referred to above or herein. In some embodiments, the contacting in step b) is for 1 day. In some embodiments, the contacting in step b) is for 2 days. In some embodiments, the contacting in step b) is for 3 days. In some embodiments, the contacting in step b) is for 4 days. In some embodiments, the contacting in step b) is for 5 days. In some embodiments, the contacting in step b) is for 6 days. In some embodiments, the contacting in step b) is for 7 days. In some embodiments, the contacting in step b) is for more than 7 days. In some embodiments, the contacting in step b) is for 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, or more than 8 days, or for any length of time ranging between any two of the recited number of days referred to above or herein.

[0202] Presented below are examples discussing methods of inducing the differentiation of PSCs into natural killer (NK) cells and substantially expanding the population of NK cells contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. EXAMPLES

[0203] The invention is further illustrated by the following non-limiting examples: EXAMPLE 1 Design of a Natural Killer Cell Differentiation Protocol

[0204] In a stepwise iterative developmental process, a new combination of factors promoting NK cell differentiation at high efficiency has been identified using a simple and GMP-compatible workflow.

[0205] The NK cell differentiation and expansion protocol described herein is divided into three steps. The first stage focuses on the production of CD34+ / CD45- hemogenic endothelium (HE) cells from human pluripotent stem cells (hPSCs) utilizing an activator of the WNT signaling pathway, bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF).PATENT ATTORNEY DOCKET NO.: N2041-03301 Subsequently, the process advances to the second stage, wherein the CD34+ / CD45- hemogenic endothelium cells are cultured in a serum-free culture medium, with the optional addition of interleukin-7 (IL-7), an NK activating cytokine, such as interleukin-15 (IL-15) or any other NK activating cytokine featured herein, stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (FLT3L) to form CD34+ / CD45+hematopoietic precursor cells (HPCs). The final stage entails differentiating these precursor cells into NK cells within a culture medium that includes at least one serum-derived component, including, as non-limiting examples, serum itself, blood platelet lysate, or serum-purified albumin, or any component or derivative of blood plasma obtained from blood, such as by blood serum fractionation, in addition to the continuous application of differentiation-inducing factors such as IL-7, an NK activating cytokine such as IL- 15 or any other NK activating cytokine featured herein, SCF, and FLT3L.

[0206] This approach highlights a significant leap forward in NK cell manufacturing, offering a feeder-free, regulatory-compliant method that sidesteps the safety concerns associated with traditional feeder cell techniques. By facilitating the expansion of NK cells to volumes suitable for clinical application, this protocol presents a pivotal development in the therapeutic deployment of NK cells, streamlining production while ensuring safety and efficacy. EXAMPLE 2 Materials and Methods of Natural Killer Cells Differentiation

[0207] Table 1: Materials for Cell CulturePATENT ATTORNEY DOCKET NO.: N2041-03301PATENT ATTORNEY DOCKET NO.: N2041-03301PATENT ATTORNEY DOCKET NO.: N2041-03301

[0208] Table 2: Antibodies used for Flow Cytometry (all to Store at 2-8°C or, alternatively, at - 20°C after addition of 80% (v / v) glycerol to 10% final):PATENT ATTORNEY DOCKET NO.: N2041-03301

[0209] Methods:

[0210] Maintenance of hiPSCs (R26 line):

[0211] hiPSCs were split on Monday mornings and Thursday afternoons at 200,000-250,000 (up to 6 wells) per 6-well, respectively. Cells for experiments were replated on Thursdays using the same cells as for hiPSC maintenance. Differentiation was initiated on Fridays.

[0212] The following steps (1)-(41) provide an exemplary protocol starting from preparing splitting hiPSCs on a Monday:

[0213] Monday: (1) Coat 2-46-wells for hiPSC maintenance with 3 µl iMatrix-511 per well in 2 ml XF medium with 10 µM Y-27632 (1:1000 Y-27632: 1µl Y per 1 ml medium) for at least 1 hour at 37°C. (2) Prewarm Accutase and approximately 10 ml of XF medium. Transfer required volume of Accutase (1 ml per well to be harvested) to separate tube. Add 1:1000 Y-27632 to both solutions and mix. (3) Maintenance wells of hiPSCs should be 50-100% confluent and overall undifferentiated. Vigorously agitate plate with cells to collect all dead cells in supernatant. Completely soak off medium and wash with 2 ml PBS. Replace by 1 ml prewarmed Accutase containing Y-27632 and transfer to incubator for 10 min. Most cells should come off by gently agitating the plate. If this is not the case, prolong digestion for 2 more min and so forth, until cells lift off virtually by themselves. (4) Add 3 ml of XF medium containing Y-27632 to 15 ml tube. Flush cells off from each well by pipetting up and down 3-4 times. Transfer cells from all wells to 15 ml tube containing 3 ml XF medium + Y. Centrifuge at 300 g for 3 min. Supernatant should be clear and cells should form a compact pellet. (5) Soak off supernatant and resuspend in 2 ml of XF medium + Y per harvested 6-well by 3 times pipetting up and down with a 1 ml pipette. Immediately and gently transfer 10 µl to a counting chamber and quantify cell titer. (6) Plate out 200,000 cells per well into each precoated 6-well. Transfer to incubator and agitate plate slowly describing and infinity symbol inside the incubator.

[0214] Tuesday: (7) Feed cells with 2.5 ml prewarmed XF medium per well.

[0215] Wednesday: (8) Feed cells with 3 ml prewarmed XF medium per well.

[0216] Thursday:PATENT ATTORNEY DOCKET NO.: N2041-03301 (9) Confirm that cells are subconfluent and fully undifferentiated. In the afternoon, coat wells with 6 µl iMatrix-511 and split cells as above except that only 1-2 wells are used, and cells plated at 200,000 cells per well. The remaining cells in suspension are to be plated onto 6-wells precoated with 6 µl iMatrix-511 at 450,000 cells (50,000 cells per cm2) per each well for the experiment. The number of wells may vary depending on experimental design. (10) For a 12-well differentiation, coat the wells with 3 µl iMatrix-511 in 1ml PBS for 1h in the incubator. Plate 200,000 cells per well in 1ml medium including 10µM ROCK inhibitor.

[0217] Friday: (11) In the afternoon, feed maintenance wells of hiPSCs with 6 ml XF medium per well over the weekend.

[0218] Differentiation of hiPSCs (R26 line) into endothelial progenitor cells (EPCs):

[0219] Friday: (12) Confirm even distribution of hiPSCs of the wells to be used for differentiation. Cells should be flat and form loose colonies at around 70-90% confluence. (13) Prepare and / or pre-warm slightly more than 6 ml MEI differentiation medium per differentiation well. Thaw required aliquots of CHIR99021 and BMP4 at RT for several (2-5) min, then mix by flicking the tubes. Optionally do the same for additional factors to be tested. (14) Prepare differentiation media for the different conditions in the best way, depending on the experimental design. For example, if all wells shall receive the same amount of CHIR but different concentrations of BMP4, prepare a master mix of MEI differentiation medium with CHIR. Then distribute into individual 15 (or 50) ml tubes and add required amounts of other factor(s) as appropriate. Alternatively, depending on the design of the experiment, the additional factors can directly be added to the wells following replacement of the consumed maintenance by differentiation medium. (15) Replace old XF medium by 6 ml fresh MEI medium following vigorous agitation of the differentiation plate(s). Place back into incubator over the weekend. (16) 12-well version: Use 3ml of MEI differentiation media per well.

[0220] Monday: (17) Prepare and / or pre-warm slightly more than 3.5 ml MEI differentiation medium per differentiation well. Thaw required aliquots of VEGFA and SB431542 at RT for several (2-5) min, then mix by flicking the tubes. Optionally do the same for additional factors to be tested. Replace old MEI medium by fresh MEI medium following vigorous agitation of the differentiation plate(s) (18) 12-well version: Use 1ml per well.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0221] Tuesday: (19) Prepare and / or pre-warm slightly more than 3.5 ml MEI differentiation medium (same as on Monday) per differentiation well. Thaw required aliquots of VEGFA and SB431542 at RT for several (2-5) min, then mix by flicking the tubes. Optionally do the same for additional factors to be tested. Replace old MEI medium by fresh MEI medium following vigorous agitation of the differentiation plate(s). (20) 12-well version: Use 1ml per well.

[0222] Wednesday: (21) Prepare and / or pre-warm slightly more than 3.5 ml MEI differentiation medium (same as on Monday) per differentiation well. Thaw required aliquots of VEGFA and SB431542 at RT for several (2-5) min, then mix by flicking the tubes. Optionally do the same for additional factors to be tested. Replace old MEI medium by fresh MEI medium following vigorous agitation of the differentiation plate(s). (22) 12-well version: Use 1ml per well.

[0223] Thursday: (23) Prepare and / or pre-warm slightly more than 3.5 ml MEI differentiation medium (same as on Monday) per differentiation well. Thaw required aliquots of VEGFA and SB431542 at RT for several (2-5) min, then mix by flicking the tubes. Optionally do the same for additional factors to be tested. Replace old MEI medium by fresh MEI medium following vigorous agitation of the differentiation plate(s). (24) 12-well version: Use 1ml per well.

[0224] Differentiation of hemogenic endothelium into NK cells: (25) On Friday (day 7) aspirate the old MEI medium and very gently wash each well once with 1ml PBS. Replace with prewarmed NK differentiation medium + IL-3 at a volume of 1ml per well in a 12 well plate. Add the media very slowly, expect 0-30% of the clusters to detach. Between days 10 and 14, the first suspension cells should appear. Ideal cultures will have d14 yields of 1.5 million cells per well, while the minimum yield at this point should be 0.5 million cells. (26) NK expansion version: Aspirate old media and replace with 1ml fully defined media (such as APEL2) without washing and without adding factors. On Monday (day 10) do a full media change with the same media, and optionally SCF, with or without IL-7 for increased endothelial to hematopoietic transitions (EHT) yields. (27) On Friday (day 14) perform a ‘spin change’ by pooling the suspension cells (very gently using a p1000 pipette, dripping the old media over the adhesive layer with as little pressure as possible before transferring the suspension cells to a collection tube) then centrifuging at 300g forPATENT ATTORNEY DOCKET NO.: N2041-03301 8 mins and resuspending the pellet in new NK dif. Medium (without IL-3). Do not allow the adherent cells to dry out and add half the final volume of fresh media (0.5ml) immediately after collecting the suspension cells. Ideally use 15ml tubes for centrifugation. Resuspend the cells in half the final plate media volume (6ml total), then evenly distribute the cells into the wells (0.5ml each). (28) NK expansion version: Aspirate the old media and any suspension cells, replace with 1ml of the same media formulation from day 10. (29) On day 17 (Monday), repeat the spin change procedure however now resuspend in 2ml per well. From day 20 onwards, perform half media changes (HMC) twice a week every 3-4 days. For example, as early as possible on Mondays, and as late as possible on Thursdays to achieve an ideal media change interval average of 3.5 days. For HMC, carefully tilt the plate towards you approximately 30 degrees, then remove 0.9ml from the surface of each well with a p1000 without disturbing the layer of suspension cells at the bottom. Gently drip in 1ml fresh NK differentiation medium, avoiding disturbing the sedimented cells. (30) NK expansion version: On day 17, transfer 200K cells to a new well (12-well) and add 1ml of fully defined media (such as APEL2) containing the 4 factors (4F) SCF, FLT3L, IL-7 and IL-15 at 20, 10, 20 and 10ng / ml, respectively. (31) For cell number tracking during HMCs, measure the exact media volume after discarding 0.9ml by taking up 0.8ml with a p1000 pipette, and then taking up the remaining volume of the well by slowly turning the pipette until air is sucked in and record this value. Gently mix this volume, resuspending all suspension cells, then take 10µl for counting (Haemocytometer or diluting in 90 µl NK basal medium and then using the NC-200). Multiply the per milliliter cell count by the measured media volume for an accurate cell count for that well. Be aware that this disturbs the adhesive layer, decreasing NK cell production compared to undisturbed cells. (32) On day 21 (NK expansion only) add 1ml fresh media with the same formulation as day 17 (4F). (33) On days 24 and 26 (NK expansion only), perform HMCs with the same medium. (34) On day 28 (NK expansion only) transfer half of the cells into a new well and add 1ml of NK dif. Medium with all 4 factors at the usual concentrations (NK dif. 4F). Expect the 200K day 17 cells to have expanded to 3-4 million NK progenitor cells by this point. Day 28 is the last possible harvest point before the NK progenitor population collapse without exposure to undefined media such as those containing hPL. (35) On day 31 (NK expansion only), split the cells 1 in 10, or targeting >500K per well in 1ml NK dif. 4F media. Perform HMC on days 33 and 35, with another 1 in 10 split on day 38, againPATENT ATTORNEY DOCKET NO.: N2041-03301 targeting >500K per well in 1ml NK dif. 4F. Repeat this until the NK cells stop expanding, with an expected duration of >3 weeks resulting in >1000-fold expansion. (36) Do not isolate the suspension cells from the adherent layer until they are ready for expansion (day 28-35, >90% CD56 positive). Cell numbers should slowly decrease from 4E6 on day 40 to 2.5E6 by day 70.

[0225] Flow cytometry: (37) For performing flow cytometry 1ml of culture media with cells are spun down at 400g for 3 mins, then resuspended in 1 ml PBS and again pelleted at 400g for 3 mins. (38) Distribute 0.5 million cells per staining group in 98µl PBS in 1.5 ml tubes. (39) Add 2µl of respective antibody for staining, tap to mix, and incubate for 20 min at RT in the dark. (40) Washing steps: Add 400 µl of PBS to each tube then centrifuge at 400 g for 2 mins. Discard the supernatant. (41) Add 300 µl of PBS to each tube and resuspend. Perform flow cytometry. After the detection is completed, save the file on G-drive, and analyze the flow cytometry result with analysis software.

[0226] NK killing assay:

[0227] Target K562 cells were resuspended in 2.5 µM Cell Trace Violet (CTV, Thermofisher, C34564) in PBS at 1 million cells / ml for 20 minutes at 37°C. The staining reaction was stopped by adding a 5-fold volume of hPL-based medium (DF12 with 15% hPL - PL BioScience - 1x Glutamax, and 250 µM 2-phospho-L-ascorbate, without additional cytokines) for 5 minutes at 37°C. These cells were then resuspended in fresh hPL-based medium at 0.5 million cells / ml. NK cells were also resuspended at 0.5 million cells / ml in the same medium. These two groups were mixed at 1:5, 1:1 and 5:1 ratios of NK:K562 in a final volume of 225 µl in a round-bottom 96- well plate. A 100% K562 control condition was included. The cells were briefly spun down and then incubated at 37°C. After 3.5 hr, 30 µl of 20 µM CellEvent Caspase-3 / 7 Green (Thermofisher, C10423) in PBS was mixed into each well, and the cells again spun down and incubated at 37°C. 5 minutes before the end, 30 µl of 10 µM SYTOX 7AAD (Thermofisher, S10274) in PBS was mixed into each well. After 4 hours total, the plate was analyzed by flow cytometry using a Miltenyi MACS Quant10. Gating strategy (established on K562 control cells): P1: all events in the forward / side scatter plot minus the CTV-negative debris. P2: Doublet exclusion. P3: CTV- positive cells. P4 / 5: Caspase-3 / 7 green on one axis, 7AAD on the other, measuring caspase single- positive cells and caspase / 7AAD double-positive cells relative to the K562 control group which can be up to 5% positive.PATENT ATTORNEY DOCKET NO.: N2041-03301

[0228] Abbreviations and standard reagents:EXAMPLE 3 Generation and Characterization of Natural Killer Cells Differentiated from Human Pluripotent Stem Cells

[0229] Differentiation into another cell type of high relevance in immuno-oncology, specifically natural killer (NK) cells, was thoroughly investigated. An established set of four signaling factors served as cues to explore which basic medium would optimally support NK differentiation. Screening various commercial and custom basic media, all supplemented with identical signaling molecules, highlighted significant differences. A formulation based on the chemically defined APEL medium was notably superior, demonstrating enhanced cell counts and NK differentiation efficiencies, as evidenced by CD56 expression (FIG. 1). Another medium, DF12 supplemented with human platelet lysate (hPL) and L-ascorbate, facilitated NK cell induction but was less effective in promoting sustained cell expansion (blue curve in FIG. 1).

[0230] Moreover, these media consistently enabled NK cell induction across different independent iPSC lines utilizing the specified HPC platform (FIG. 2). With experiments conducted in original wells containing residual adherent cells, an effort was made to eliminate potential indirect effects and to assess upscaling strategies by transferring d17 HPCs to new vessels. Interestingly, cell numbers initially increased over approximately 10 days in APEL2PATENT ATTORNEY DOCKET NO.: N2041-03301 medium with added factors, before a decline was observed, although many live cells maintained an NK cell fate (FIG. 3A). The timing of the declination of the cell numbers was related to the timing of endothelial-to-hematopoietic transitions (EHT) (FIG.3B). This suggests that neither the defined APEL2-based medium nor the undefined alternatives alone were sufficient for significant expansion following NK cell differentiation.

[0231] An intriguing observation was made regarding the dynamics of HPC loss and NK cell identity gain, where culture collapse in APEL2-based medium around four weeks coincided with a marked HPC-to-NK transition (FIG.4). It appeared that while the defined medium could support progenitor cell expansion, it was less effective post-transition to early NK cell stages. The study then explored whether switching to undefined hPL medium could enhance proliferation and yield purely differentiated NK cells, as indicated by CD56 expression and the presence of functional markers (2B4, NKp30, NKp44, NKp46; FIG. 5A and FIG. 5B). This switch resulted in a significant increase in cell proliferation and yielded highly pure NK cells.

[0232] Flow cytometry analysis indicated that concluding the differentiation process with the undefined, yet GMP-compatible, hPL-based medium could produce more mature cells, as shown by higher CD38 levels and the activation marker NKp44 (FIG.4). RNA-seq analysis of iPSC-NK cells derived in hPL-based medium confirmed the robust expression of genes essential for cytotoxicity (FIG. 6). These cells demonstrated significant cytotoxic activity against K562 leukemia cells, achieving a 90% cytotoxicity at a 5:1 effector-to-target cell ratio within just four hours (FIG. 7).

[0233] It was noted that iPSC-MSCs from certain iPSC lines exhibited remarkable proliferation capacity, suggesting the feasibility of intermediate banking to preserve expansion potential downstream (FIG. 8, second panel). In contrast, iPSC-derived cardiomyocytes showed limited expansion capacity, indicating the necessity for upscaling at the iPSC stage (FIG. 8, third panel). For immune cells, upscaling at a precursor stage might be beneficial to avoid exhaustion of the final differentiated product (FIG. 8, fourth panel). To the best of the current understanding, this strategy for expanding iPSC-NK cells upon differentiation represents a novel approach.

[0234] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and thatPATENT ATTORNEY DOCKET NO.: N2041-03301 methods and structures within the scope of these claims and their equivalents be covered accordingly.

Claims

PATENT ATTORNEY DOCKET NO.: N2041-03301 CLAIMS What is claimed is:

1. A method of producing natural killer (NK) cells comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and / or a bone morphogenetic protein (BMP), wherein the PSCs are grown for about 1- 7 days; b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF) for about 1-7 days following step a) thereby generating a population of CD34+precursor cells at least about 80% enriched for CD34+; c) incubating the population of CD34+precursor cells for about 7-28 days in a culture medium lacking any serum-derived agent while optionally supplementing the medium with one or more of interleukin-7 (IL-7), NK activating cytokine optionally IL-15, stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L), thereby creating a population of CD34+ / CD45+suspension cells; and d) incubating the population of CD34+ / CD45+suspension cells in a culture medium containing at least one serum-derived agent and IL-7, NK activating cytokine optionally IL-15, SCF, and FLT3L for about 7-35 days, thereby producing NK cells.

2. The method of claim 1, wherein the serum-derived agent is selected from serum, blood platelet lysate, albumin derived from blood, or a component or derivative of blood plasma obtained from blood.

3. The method of claim 1, wherein the cells of step c) and / or step d) are transiently CD34+.

4. The method of claim 1, wherein culture medium of step c) and / or step d) comprises about 4-40 ng / ml IL-7, about 2-20 ng / ml IL-15, about 4-40 ng / ml SCF and / or about 1-20 ng / ml FLT3L.

5. The method of claim 4, wherein the culture medium comprises about 20 ng / ml IL-7, about 10 ng / ml IL-15, about 20 ng / ml SCF and / or about 10 ng / ml FLT3L.

6. The method of claim 1, wherein contacting the culture of PSCs comprises one or more agents selected from about 1-10 µM WNT signaling pathway activator, about 5-50 ng / ml BMP, about 50-500 ng / ml VEGF.

7. The method of claim 6, wherein the WNT signaling pathway activator is a GSK3 inhibitor.PATENT ATTORNEY DOCKET NO.: N2041-03301 8. The method of claim 7, wherein the GSK3 inhibitor is CHIR99021.

9. The method of claim 6, wherein the BMP is BMP4.

10. The method of claim 6, wherein the VEGF is VEGF-A.

11. The method of claim 6, wherein contacting the culture of PSCs comprises about 8 µM CHIR99021, about 25 ng / ml BMP4 and / or about 200 ng / ml VEGFA.

12. The method of claim 1, wherein contacting the culture of PSCs of step a) with a WNT signaling pathway activator and a BMP is for about 2-5 days.

13. The method of claim 1, wherein subsequently contacting the PSCs with a VEGF is for about 2-5 days.

14. The method of claim 1, wherein the NK cells are produced in a culture selected from: an adherent layer of cells, a two-dimensional culture system, on microcarriers, on a coated surface comprising a laminin coating, a three-dimensional culture, and a suspension culture.

15. The method of claim 1, further comprising collecting the NK cells in suspension in a cell culture medium.

16. The method of claim 1, wherein the PSCs are human PSCs (hPSCs).

17. The method of claim 16, wherein the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stems cells (hESCs).

18. The method of claim 1, wherein the CD34+precursor cell is a CD34+endothelial-like precursor cell.

19. The method of claim 1, wherein the NK cells are at least about 80% enriched.

20. The method of claim 1, wherein the NK cells are CD56+, 2B4+, NKp30+, NKp44+, NKp46+, NKG2D+, and / or CD16-.

21. The method of claim 1, wherein the NK cells are CD56brightor CD56dim.

22. The method of claim 1, wherein the NK cells are cytotoxic NK cells.

23. The method of claim 1, wherein d) enriches the number cells by at least 100-fold compared to the number of cells in c).

24. The method of claim 1, wherein d) enriches the number cells by at least 200-fold compared to the number of cells in c).PATENT ATTORNEY DOCKET NO.: N2041-03301 25. A method of producing natural killer (NK) cells from pluripotent stem cells (PSCs) comprising: a) generating CD34+hemogenic endothelium (HE) cells by: (i) contacting a culture of PSCs with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), wherein the PSCs grown for about 3 days; and (ii) subsequently contacting the cells of (i) with a vascular endothelial growth factor (VEGF) for about 4 days; thereby generating a population of cells comprising at least 80% CD34+HE cells; b) culturing the CD34+HE cells of step a) in a medium lacking any serum-derived agents for about 7-28 days while optionally adding one or more of IL-7, IL-15, SCF and / or FLT3L, thereby generating a transient population of suspension cells comprising at least 80% CD34+ / CD45+hematopoietic precursor cells (HPCs); c) contacting the CD34+ / CD45+HPCs of step b) with one or more of IL-7, IL-15, FLT3L and SCF for about 7-14 days in a culture medium lacking any serum-derived agent; and d) transferring the CD34+ / CD45+HPCs into a culture medium containing at least one serum-derived agent and IL-7, IL-15, FLT3L and SCF for about 7-35 days, thereby inducing NK cell differentiation from PSCs.

26. The method of claim 25, wherein the NK cells are CD56+, 2B4+, NKp30+, NKp44+, NKp46+, NKG2D+, and / or CD16-.

27. The method of claim 25, wherein the NK cells are CD56brightor CD56dim.

28. The method of claim 25, wherein the NK cells are cytotoxic NK cells.

29. The method of claim 25, wherein d) enriches the number cells by at least 100-fold compared to the number of cells in c).

30. The method of claim 25, wherein d) enriches the number cells by at least 200-fold compared to the number of cells in c).

31. The method of claim 1 or 25, wherein the NK cells are produced in a feeder-independent manner.

32. A method of producing hemogenic endothelium (HE) cells comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and / or a bone morphogenetic protein (BMP), wherein the PSCs are grown for about 1- 7 days; andPATENT ATTORNEY DOCKET NO.: N2041-03301 b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF) for about 1-7 days following step a) thereby generating a population of CD34+hemogenic endothelium cells.

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