Methods for differentiating pluripotent stem cells into natural killer cells

Abstract

This invention discloses a method for the large-scale expansion of natural killer (NK) cell populations using modified cell culture conditions. By employing a targeted induction cocktail mixture comprising a WNT signaling pathway activator and bone morphogenetic protein (BMP), this technique promotes the differentiation of pluripotent stem cells (PSCs) into NK cells. The core innovation lies in optimizing culture conditions, which significantly enhances NK cell proliferation. This method simplifies the production of NK cells for therapeutic purposes, focusing on the expansion phase within the culture environment.

Description

[0001] Citations of relevant applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 561,673, filed March 5, 2024, pursuant to 35 USC § 119(e), the disclosure of which is considered part of the disclosure of this application and is incorporated herein by reference in its entirety. Technical Field

[0003] This invention generally relates to natural killer cells (NK cells), and more particularly to a method for generating NK cells from pluripotent stem cells (PSCs). Background Technology

[0004] Natural killer cells, also known as NK cells or large granular lymphocytes (LGLs), are a type of cytotoxic lymphocyte that is crucial to the innate immune system. They belong to the rapidly expanding family of known innate lymphoid cells (ILCs) and comprise 5–20% of all circulating lymphocytes in humans. NK cells play a role similar to cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virally infected cells and other intracellular pathogens, exerting their effects approximately 3 days after infection, and responding to tumor formation. Normally, immune cells detect the major histocompatibility complex (MHC) presented on the surface of infected cells, triggering the release of cytokines and leading to the death of infected cells through lysis or apoptosis. However, NK cells are unique because they possess the ability to recognize and kill stressed cells in the absence of antibodies and MHC, enabling a more rapid immune response. They are named "natural killer" because they are thought to kill cells lacking the MHC class I "self" marker without requiring activation. This role is particularly important because harmful cells lacking the MHC I marker cannot be detected and destroyed by other immune cells, such as T lymphocytes.

[0005] Besides natural killer cells serving as effectors of innate immunity, both activating and inhibitory NK cell receptors play important functional roles, including self-tolerance and maintaining NK cell activity. NK cells also play a role in adaptive immune responses; numerous experiments have demonstrated their ability to rapidly adapt to the immediate environment and form antigen-specific immune memories, which are crucial for responding to secondary infections with the same antigens. The role of NK cells in innate and adaptive immune responses is becoming increasingly important in research utilizing NK cell activity as a potential cancer therapy. Summary of the Invention

[0006] This disclosure relates to a surprising discovery that a cocktail mixture containing both bone morphogenetic protein (BMP) and a WNT signaling activator for endothelial induction can generate endothelial-like precursor cells. These cells possess characteristics of both endothelial precursors and hematopoietic precursors, enabling them to differentiate into terminally differentiated hematopoietic cells, such as natural killer (NK) cells. Furthermore, this method promotes a significant expansion of the NK cell population by modifying cell culture conditions. This disclosure provides a method for addressing an unmet need in the art, namely, a method for developing efficient and convenient culture conditions to generate NK cell cultures for PSC differentiation in a short time.

[0007] In some aspects, this disclosure provides a method for producing natural killer (NK) cells, comprising: a) contacting a pluripotent stem cell (PSC) culture with a WNT signaling pathway activator and / or bone morphogenetic protein (BMP); b) contacting the culture from step a) with vascular endothelial growth factor (VEGF) to generate CD34. + Precursor cell population; c) CD34 + The precursor cell population was incubated in a medium lacking any serum-derived reagents, while optionally supplementing the medium with one or more cytokines. As a non-limiting example, these cytokines included interleukin-7 (IL-7), NK-activating cytokines (such as interleukin-15 (IL-15) or other NK-activating cytokines described herein), stem cell factors (also known as SCF, KIT-ligand, KL, or steel factor), and FMS-like tyrosine kinase 3 ligand (FLT3L), or one or more other cytokines described herein, thereby producing CD34. + / CD45 + Suspension cell population; and d) CD34 + / CD45 + Suspension cell populations are incubated in a medium containing at least one serum-derived reagent and IL-7, IL-15, SCF, and / or FLT3L to produce NK cells. In some embodiments, the medium contains at least one or more of the following: WNT signaling pathway activator, BMP, and VEGF. In some embodiments, the medium is supplemented with one or more cytokines and / or growth factors, including any cytokines provided above and disclosed herein. In some embodiments, the medium is supplemented with one or more NK-activating cytokines, including any NK-activating cytokines provided above or herein.

[0008] In one aspect, this disclosure provides a method for producing natural killer (NK) cells, comprising: a) contacting a PSC culture with a WNT signaling pathway activator and / or BMP, wherein the PSC growth lasts for approximately 1-7 days; b) after step a), contacting the PSC culture with VEGF for approximately 1-7 days, thereby generating at least approximately 80% enriched CD34. + CD34 + Precursor cell population; c) CD34 + The precursor cell population was incubated in a medium lacking any serum-derived reagents for approximately 7–28 days, while optionally supplementing the medium with one or more cytokines. As a non-limiting example, these cytokines included IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, thereby producing CD34. + / CD45 + Suspension cell population; and d) CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L to produce NK cells.

[0009] As provided and disclosed herein, activators of the WNT signaling pathway, as non-limiting examples, include: CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarboxynitrile); WNT family ligands; RSPO co-agonists; lithium chloride; TDZD8 (4-benzyl-2-methyl- 1,2,4-Thiadiazolidine-3,5-dione); BIO-acetone oxime ((2'Z,3'E)-6-bromoindorubin-3'-acetone oxime); A1070722 (1-(7-methoxyquinoline-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea); HLY78 (4-ethyl-5,6-dihydro-5-methyl-[1,3]dioxolane[4,5-j]phenanthridine); CID 11210285 hydrochloride (2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero)arylpyrimidine; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, activation of the WNT signaling pathway can be achieved by inhibiting a WNT signaling pathway inhibitor. As non-limiting examples, this includes using an inhibitory nucleic acid targeting a WNT signaling pathway inhibitor, or an antibody or small molecule targeting 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.

[0010] In some embodiments, the concentration of the WNT signaling pathway activator is in the range of about 1 µM to 10 µM. In some embodiments, the WNT signaling pathway activator is added to PSC cultures at concentrations in the range of 0.5 to 1.0 µM, 1.0 to 2.0 µM, 2.0 to 3.0 µM, 3.0 to 4.0 µM, 4.0 to 5.0 µM, 5.0 to 6.0 µM, 6.0 to 7.0 µM, 7.0 to 8.0 µM, 8.0 to 9.0 µM, 9.0 to 10 µM, or at concentrations between the above or any two concentrations mentioned herein. In some embodiments, the WNT signaling pathway activator includes CHIR99021. In some embodiments, the WNT signaling pathway activator is CHIR99021 at a concentration in the range of about 5 µM to about 10 µM. In some embodiments, the concentration of CHIR99021 is about 8 µM.

[0011] As provided and disclosed herein, bone morphogenetic proteins include, as non-limiting examples, BMP family ligands that activate the BMP pathway, such as BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP15. In some embodiments, activation of the BMP pathway can be achieved by inhibiting BMP pathway inhibitors, including, as non-limiting examples, the use of inhibitory nucleic acids targeting BMP pathway inhibitors, or antibodies or small molecules targeting BMP pathway inhibitors. In some embodiments, the BMP is BMP4.

[0012] In some embodiments, the concentration of any BMP selected herein is in the range of about 5 ng / ml to 50 ng / ml. In some embodiments, the concentration of BMP is in the range of 1 to 2 ng / ml, 2 to 3 ng / ml, 3 to 4 ng / ml, 4 to 5 ng / ml, 5 to 6 ng / ml, 6 to 7 ng / ml, 7 to 8 ng / ml, 8 to 9 ng / ml, 9 to 10 ng / ml, 10 to 15 ng / ml, 15 to 20 ng / ml, 20 to 25 ng / ml, 25 to 30 ng / ml, 30 to 35 ng / ml, 35 to 40 ng / ml, 40 to 45 ng / ml, 45 to 50 ng / ml, 50 to 55 ng / ml, or a range between the above or any two of the concentrations mentioned herein. In some embodiments, the concentration of BMP4 is about 25 ng / ml.

[0013] As provided and disclosed herein, by way of non-limiting example, vascular endothelial growth factor includes VEGF family ligands that activate the VEGF pathway, such as VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placental growth factor (PIGF). In some embodiments, VEGF is VEGFA.

[0014] In some embodiments, the concentration of any VEGF selected from those provided herein is in the range of about 50 ng / ml to 500 ng / ml. In some embodiments, the concentration of VEGF is 25 to 50 ng / ml, 50 to 75 ng / ml, 75 to 100 ng / ml, 100 to 125 ng / ml, 125 to 150 ng / ml, 150 to 175 ng / ml, 175 to 200 ng / ml, 200 to 225 ng / ml, 225 to 250 ng / ml, 250 to 275 ng / ml, 275 to 300 ng / ml, 300 to 325 ng / ml, 325 to 350 ng / ml, 350 to 375 ng / ml, 375 to 400 ng / ml, 400 to 425 ng / ml, 425 to 450 ng / ml, 450 to 475 ng / ml, 475 to 500 ng / ml, 500 to 525 ng / ml. Concentrations in the range of 500 to 550 ng / ml, or between the above or any two of the concentrations mentioned herein. In some embodiments, the concentration of VEGFA is about 200 ng / ml.

[0015] In some embodiments, the PSC culture is contacted with one or more reagents selected from the following: about 1-10 µM WNT signaling pathway activator, about 5-50 ng / ml BMP, and about 50-500 ng / ml VEGF.

[0016] In some implementations, the PSC culture is contacted with approximately 8 µM CHIR99021, approximately 25 ng / ml BMP4, and / or approximately 200 ng / ml VEGFA.

[0017] As provided 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 primarily regulate cellular processes in hematopoietic and immune cell types, including, as unrestricted examples, proliferation, differentiation, and communication. In some embodiments, the culture medium is supplemented with one or more growth factors. In some embodiments, the growth factor is a cytokine. In some embodiments, the cytokine is selected from SCF, FLT3L, and interleukins, including, as unrestricted 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.

[0018] As provided and disclosed herein, cytokines involved in NK activation, or "NK-activating cytokines," include IL-12, IL-15, IL-18, IL-2, and CCL5 as non-limiting examples. These NK-activating cytokines alert NK cells to the presence of viral pathogens and recruit NK cells to affected areas. In some embodiments, the culture medium is supplemented with one or more NK-activating cytokines, including IL-15 as a non-limiting example.

[0019] In some implementations, CD34 is used for step c) incubation. + Precursor cell culture and step d) incubation of CD34 + / CD45 + The culture medium for suspension cell cultures contains approximately 4–40 ng / ml of a cytokine, such as IL-7 or any cytokine provided above or herein. In some embodiments, the concentration of IL-7 is in the range of approximately 4 to approximately 40 ng / ml. In some embodiments, the concentration of IL-7 is in the range of 2–3 ng / ml, 3–4 ng / ml, 4–5 ng / ml, 5–6 ng / ml, 6–7 ng / ml, 7–8 ng / ml, 8–9 ng / ml, 9–10 ng / ml, 10–15 ng / ml, 15–20 ng / ml, 20–25 ng / ml, 25–30 ng / ml, 30–35 ng / ml, 35–40 ng / ml, 40–45 ng / ml, 45–50 ng / ml, or a range between any two of the above or mentioned herein. In some implementations, the concentration of IL-7 is approximately 20 ng / ml.

[0020] In some implementations, CD34 is used for step c) incubation. + Precursor cell culture and step d) incubation of CD34 + / CD45 +The culture medium for the suspension cell culture contains approximately 2 to 20 ng / ml of NK-activating cytokines, such as IL-15 or any other NK-activating cytokines provided herein. In some embodiments, the concentration of the NK-activating cytokines, such as IL-15 or any other NK-activating cytokines provided herein, is in the range of approximately 2 to approximately 20 ng / ml. In some embodiments, NK-activating cytokines, such as IL-15 or any other NK-activating cytokines provided herein, are used at concentrations of 1 to 2 ng / ml, 2 to 3 ng / ml, 3 to 4 ng / ml, 4 to 5 ng / ml, 5 to 6 ng / ml, 6 to 7 ng / ml, 7 to 8 ng / ml, 8 to 9 ng / ml, 9 to 10 ng / ml, 10 to 11 ng / ml, 11 to 12 ng / ml, 12 to 13 ng / ml, 13 to 14 ng / ml, 14 to 15 ng / ml, 15 to 16 ng / ml, 16 to 17 ng / ml, 17 to 18 ng / ml, 18 to 19 ng / ml, 19 to 20 ng / ml, 20 to 21 ng / ml, 21 to 22 ng / ml, 22 to 23 ng / ml, 23 to 24 ng / ml, 24 to 25 ng / ml. Concentrations in the range of ng / ml, or concentrations in the range between the above or any two concentrations mentioned herein. In some embodiments, the concentration of IL-15 is about 10 ng / ml.

[0021] In some implementations, CD34 is used for step c) incubation. + Precursor cell culture and step d) incubation of CD34 + / CD45 + The culture medium for suspension cell cultures contains approximately 4–40 ng / ml of SCF. In some embodiments, the concentration of SCF is in the range of approximately 4 to approximately 40 ng / ml. In some embodiments, the concentration of SCF is in the range of 2–3 ng / ml, 3–4 ng / ml, 4–5 ng / ml, 5–6 ng / ml, 6–7 ng / ml, 7–8 ng / ml, 8–9 ng / ml, 9–10 ng / ml, 10–15 ng / ml, 15–20 ng / ml, 20–25 ng / ml, 25–30 ng / ml, 30–35 ng / ml, 35–40 ng / ml, 40–45 ng / ml, 45–50 ng / ml, or a range between the above or any two of the concentrations mentioned herein. In some embodiments, the concentration of SCF is approximately 20 ng / ml.

[0022] In some implementations, CD34 is used for step c) incubation. +Precursor cell culture and step d) incubation of CD34 + / CD45 + The culture medium for suspension cell cultures contains approximately 1–20 ng / ml FLT3L. In some embodiments, the concentration of FLT3L is in the range of approximately 1 to approximately 20 ng / ml. In some embodiments, the concentration of FLT3L is 0.1 to 0.5, 0.5 to 1, 1 to 2 ng / ml, 2 to 3 ng / ml, 3 to 4 ng / ml, 4 to 5 ng / ml, 5 to 6 ng / ml, 6 to 7 ng / ml, 7 to 8 ng / ml, 8 to 9 ng / ml, 9 to 10 ng / ml, 10 to 11 ng / ml, 11 to 12 ng / ml, 12 to 13 ng / ml, 13 to 14 ng / ml, 14 to 15 ng / ml, 15 to 16 ng / ml, 16 to 17 ng / ml, 17 to 18 ng / ml, 18 to 19 ng / ml, 19 to 20 ng / ml, 20 to 21 ng / ml, 21 to 22 ng / ml, 22 to 23 ng / ml, 23 to 24 ng / ml, 24 to 25 ng / ml. Concentrations in the range of ng / ml, or concentrations in the range between the above or any two concentrations mentioned herein. In some embodiments, the concentration of FLT3L is about 10 ng / ml.

[0023] In some implementations, step c) involves incubating CD34. + Incubate CD34 in precursor cell culture or step d) + / CD45 + The culture medium in the suspension cell culture is supplemented with one or more cytokines, including IL-7 as a non-limiting example, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF and / or FLT3L.

[0024] In some implementations, CD34 is used for step c) incubation. + Precursor cell culture and step d) incubation of CD34 + / CD45 + The culture medium for the suspension cell culture contains 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 provided herein.

[0025] In some implementations, CD34 is used for step c) incubation. + Precursor cell population and step d) incubation of CD34 + / CD45 +The culture medium for the suspension cell population contains approximately 4–40 ng / ml IL-7, approximately 2–20 ng / ml IL-15, approximately 4–40 ng / ml SCF and / or approximately 1–20 ng / ml FLT3L.

[0026] In some implementations, CD34 is used for step c) incubation. + Precursor cell population and step d) incubation of CD34 + / CD45 + The culture medium for the suspension cell population contains approximately 20 ng / ml IL-7, approximately 10 ng / ml IL-15, approximately 20 ng / ml SCF and / or approximately 10 ng / ml FLT3L.

[0027] As provided and disclosed herein, as a non-limiting example, serum-derived reagents include: serum, platelet lysate, plasma, blood-derived albumin, and any component or derivative of blood-derived plasma (such as those separated by serum fractionation). In some embodiments, the serum-derived reagent is selected from serum, platelet lysate, or blood-derived albumin. In some embodiments, the serum-derived reagent is serum. In some embodiments, the serum-derived reagent is platelet lysate. In some embodiments, the serum-derived reagent is albumin. In some embodiments, the serum-derived reagent is plasma. In some embodiments, albumin or other proteins present in and / or derived from serum may include or exclude recombinant forms of albumin or other proteins.

[0028] In some embodiments of the incubation process in step d), CD34 is... + / CD45 + Suspension cell populations are incubated in a culture medium containing at least one serum-derived reagent and cytokines. In some embodiments of the incubation step d), CD34... + / CD45 + Suspension cell populations were incubated in a medium containing at least one serum-derived reagent and cytokines, including at least one NK-activating cytokine, such as IL-15 or any other NK-activating cytokine provided herein. In some embodiments of the incubation in step d), CD34... + / CD45 + Suspension cell populations were incubated in a medium containing at least one serum-derived reagent and cytokines such as IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L. In some embodiments of the incubation in step d), CD34... + / CD45 +Suspension cell populations were incubated in a medium containing serum as well as IL-7, IL-15, SCF and FLT3L.

[0029] In some implementations, step c) involves incubating CD34. + Precursor cell population and step d) incubation of CD34 + / CD45 + The cells in the suspension cell population are transient CD34 cells. + .

[0030] In some embodiments, the contact in step a), i.e., the growth of PSC cultures in the presence of WNT signaling pathway activators and / or bone morphogenetic protein BMP, lasts for approximately 1-7 days. In some embodiments, the contact in step a) lasts for more than 7 days. In some embodiments, the contact in step a) lasts for up to 1, 2, 3, 4, 5, 6, or 7 days, or more than 7 days, or any length of time between the above or any two of the numbers mentioned herein. In some embodiments, the contact in step a) lasts for at least 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, the contact in step a) lasts for 1 day. In some embodiments, the contact in step a) lasts for 2 days. In some embodiments, the contact in step a) lasts for 3 days. In some embodiments, the contact in step a) lasts for 4 days. In some embodiments, the contact in step a) lasts for 5 days. In some embodiments, the contact in step a) lasts for 6 days. In some embodiments, the contact in step a) lasts for 7 days. In some embodiments, the contact in step a) lasts for more than 7 days. In some implementations, the contact in step a) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0031] In some implementations, the contact in step b) follows step a), where the PSC culture from step a) is contacted with VEGF for approximately 1-7 days, thereby generating CD34. +Precursor cell population. In some embodiments, the contact in step b) lasts for more than 7 days. In some embodiments, the contact in step b) lasts for up to 1, 2, 3, 4, 5, 6, or 7 days, or more than 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step b) lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step b) lasts for 1 day. In some embodiments, the contact in step b) lasts for 2 days. In some embodiments, the contact in step b) lasts for 3 days. In some embodiments, the contact in step b) lasts for 4 days. In some embodiments, the contact in step b) lasts for 5 days. In some embodiments, the contact in step b) lasts for 6 days. In some embodiments, the contact in step b) lasts for 7 days. In some embodiments, the contact in step b) lasts for more than 7 days. In some implementations, the contact in step b) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0032] In some implementations, the contact in step b), i.e., contacting the PSC culture from step a) with VEGF, results in the formation of at least about 80% enrichment of CD34. + CD34 + Precursor cell population. In some implementations, CD34 + The precursor cell population has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any percentage between the above or any two percentages mentioned herein, of CD34. + Enrichment. In some implementations, the contact in step b) generates at least 60% enrichment of CD34. + CD34 + Precursor cell population. In some embodiments, the contact in step b) generates at least 65% enrichment of CD34. + CD34 + Precursor cell population. In some embodiments, the contact in step b) generates at least 70% enrichment of CD34. + CD34 + Precursor cell population. In some embodiments, the contact in step b) generates at least 75% enrichment of CD34. + CD34 + Precursor cell population. In some embodiments, the contact in step b) generates at least 80% enrichment of CD34. + CD34+ Precursor cell population. In some embodiments, the contact in step b) generates at least 85% enrichment of CD34. + CD34 + Precursor cell population. In some embodiments, the contact in step b) generates at least 90% enrichment of CD34. + CD34 + Precursor cell population. In some embodiments, the contact in step b) generates at least 95% enrichment of CD34. + CD34 + Precursor cell population.

[0033] In some implementations, the incubation in step c) is, i.e., the incubation of CD34 from step b). + The precursor cell population was incubated in a medium lacking any serum-derived reagents for approximately 7–28 days, with optional supplementation of this medium with one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L, thereby generating CD34. + / CD45 +Suspended cell population. In some embodiments, the incubation in step c) lasts for up to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the incubation in step c) lasts for more than 7 days. In some embodiments, the incubation in step c) lasts 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 days, or more than 28 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the incubation in step c) lasts for 7 days. In some embodiments, the incubation in step c) lasts for 8 days. In some embodiments, the incubation in step c) lasts for 9 days. In some embodiments, the incubation in step c) lasts for 10 days. In some embodiments, the incubation in step c) lasts for 11 days. In some embodiments, the incubation in step c) lasts for 12 days. In some embodiments, the incubation in step c) lasts for 13 days. In some embodiments, the incubation in step c) lasts for 14 days. In some embodiments, the incubation in step c) lasts for 15 days. In some embodiments, the incubation in step c) lasts for 16 days. In some embodiments, the incubation in step c) lasts for 17 days. In some embodiments, the incubation in step c) lasts for 18 days. In some embodiments, the incubation in step c) lasts for 19 days. In some embodiments, the incubation in step c) lasts for 20 days. In some embodiments, the incubation in step c) lasts for 21 days. In some embodiments, the incubation in step c) lasts for 22 days. In some embodiments, the incubation in step c) lasts for 23 days. In some embodiments, the incubation in step c) lasts for 24 days. In some embodiments, the incubation in step c) lasts for 25 days. In some embodiments, the incubation in step c) lasts for 26 days. In some embodiments, the incubation in step c) lasts for 27 days. In some embodiments, the incubation in step c) lasts for 28 days. In some embodiments, the incubation in step c) lasts for more than 28 days.In some implementations, the incubation in step c) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0034] In some implementations, CD34 in c) + The precursor cell population is incubated in a culture medium lacking any serum-derived reagents, optionally supplemented with one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L. In some embodiments, c) CD34 + The precursor cell population is incubated in a culture medium lacking any serum-derived reagents, optionally supplemented with at least one of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L. In some embodiments, c) CD34 + The precursor cell population was incubated in a medium lacking any serum-derived reagents, with the medium optionally supplemented with a combination of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L.

[0035] In some implementations, the incubation in step d) is, i.e., incubating CD34 from c) + / CD45 +Suspension cell populations are incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent and IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L to produce NK cells. In some embodiments, the incubation in step d) lasts for up to 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 days, or any length of time between the above or any two mentioned herein. In some embodiments, the incubation in step d) lasts for more than 7 days. In some embodiments, the incubation in step d) lasts 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 days, or more than 35 days, or any length of time between the above or any two of the numbers mentioned herein. In some embodiments, the incubation in step d) lasts for 7 days. In some embodiments, the incubation in step d) lasts for 8 days. In some embodiments, the incubation in step d) lasts for 9 days. In some embodiments, the incubation in step d) lasts for 10 days. In some embodiments, the incubation in step d) lasts for 11 days. In some embodiments, the incubation in step d) lasts for 12 days. In some embodiments, the incubation in step d) lasts for 13 days. In some embodiments, the incubation in step d) lasts for 14 days. In some embodiments, the incubation in step d) lasts for 15 days. In some embodiments, the incubation in step d) lasts for 16 days. In some embodiments, the incubation in step d) lasts for 17 days. In some embodiments, the incubation in step d) lasts for 18 days. In some embodiments, the incubation in step d) lasts for 19 days. In some embodiments, the incubation in step d) lasts for 20 days. In some embodiments, the incubation in step d) lasts for 21 days. In some embodiments, the incubation in step d) lasts for 22 days. In some embodiments, the incubation in step d) lasts for 23 days. In some embodiments, the incubation in step d) lasts for 24 days. In some embodiments, the incubation in step d) lasts for 25 days. In some embodiments, the incubation in step d) lasts for 26 days. In some embodiments, the incubation in step d) lasts for 27 days. In some embodiments, the incubation in step d) lasts for 28 days. In some embodiments, the incubation in step d) lasts for 29 days. In some implementations, the incubation in step d) lasts for 30 days.In some embodiments, the incubation in step d) lasts for 31 days. In some embodiments, the incubation in step d) lasts for 32 days. In some embodiments, the incubation in step d) lasts for 33 days. In some embodiments, the incubation in step d) lasts for 34 days. In some embodiments, the incubation in step d) lasts for 35 days. In some embodiments, the incubation in step d) lasts for more than 35 days. In some implementations, the incubation in step d) lasts 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 any length of time between the above or any two of the days listed herein.

[0036] In some embodiments, the PSC culture of step a) is contacted with the WNT signaling pathway activator and BMP for approximately 2-5 days. In some embodiments, the PSC culture of step a) is contacted with the WNT signaling pathway activator and BMP signaling pathway activator for approximately 3 to 5 days. In some embodiments, the PSC culture of step a) is contacted with the WNT signaling pathway activator and BMP signaling pathway activator for approximately 4 to 5 days. In some embodiments, the PSC culture of step a) is contacted with the WNT signaling pathway activator and BMP signaling pathway activator for approximately 2, 3, 4, or 5 days, or for any duration between any two of the listed days.

[0037] In some embodiments, the PSC is exposed to VEGF for approximately 2-5 days. In some embodiments, the PSC is exposed to VEGF for approximately 3 to 5 days. In some embodiments, the PSC is exposed to VEGF for approximately 4 to 5 days. In some embodiments, the PSC is exposed to VEGF for approximately 2, 3, 4, or 5 days, or for any length of time between any two of the listed number of days.

[0038] In some implementations, the PSC culture is an adherent cell layer.

[0039] In some implementations, the cell layer is grown in a two-dimensional culture system or on a microcarrier.

[0040] In some implementations, PSCs are cultured on a coated surface containing a laminin coating.

[0041] In some embodiments, the method further includes collecting suspended NK cells in a cell culture medium.

[0042] In some implementations, the PSC is a human PSC (hPSC).

[0043] In some implementations, the hPSC is a human induced pluripotent stem cell (hiPSC) or a human embryonic stem cell (hESC).

[0044] In some implementations, CD34 + The precursor cells are CD34 + Endothelial-like precursor cells.

[0045] In some embodiments, NK cells are enriched at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, NK cells are enriched at least 60%. In some embodiments, NK cells are enriched at least 65%. In some embodiments, NK cells are enriched at least 70%. In some embodiments, NK cells are enriched at least 75%. In some embodiments, NK cells are enriched at least 80%. In some embodiments, the contact in step b) generates at least 85% enrichment of CD34. + CD34 + Precursor cell population. In some embodiments, NK cells are enriched at least 90%. In some embodiments, NK cells are enriched at least 95%.

[0046] In some implementations, NK cells are enriched at least about 80%.

[0047] In some implementations, NK cells are CD56. + 2B4 + NKp30 + NKp44 + NKp46 + NKG2D + and / or CD16 - .

[0048] In some embodiments, the NK cells are CD56. 高表达 (CD56) bright CD56 明亮 ) or CD56 低表达 (CD56) dim CD56 暗淡 ).

[0049] In some embodiments, the NK cells are cytotoxic NK cells.

[0050] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, to reduce cell numbers compared to those in step b) where CD34 was increased. + / CD45 + HPC cells are enriched at least 50-fold to 300-fold when exposed to one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines described herein, FLT3L, and SCF in a culture medium lacking any serum-derived reagent for approximately 7–14 days. In some embodiments, the cell number enrichment is 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, or 300-fold, or any enrichment fold between the above or any two mentioned herein.

[0051] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, resulting in a cell number compared to CD34. + The precursor cell population was enriched at least 100-fold for approximately 7–28 days in a medium lacking any serum-derived reagents. This enrichment occurred with the optional supplementation of the medium with one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines described herein, SCF, and FLT3L.

[0052] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, resulting in a cell number compared to CD34. +The precursor cell population was enriched at least 200-fold after incubation in a medium lacking any serum-derived reagents for approximately 7–28 days. This enrichment occurred with the optional supplementation of the medium with one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines described herein, SCF, and FLT3L.

[0053] On the other hand, this disclosure provides a method for producing natural killer (NK) cells from pluripotent stem cells (PSCs), comprising: a) generating CD34 cells by... + Hematopoietic endothelial (HE) cells: (i) PSC cultures were contacted with WNT signaling pathway activators and bone morphogenetic protein (BMP), wherein PSC growth was sustained for approximately 3 days; and (ii) the cells from (i) were subsequently contacted with vascular endothelial growth factor (VEGF) for approximately 4 days; thereby generating cells containing at least 80% CD34. + a) A population of HE cells; b) CD34 cells from step a) cultured in a medium lacking any serum-derived reagents. + HE cells are maintained for approximately 7–28 days, with optional addition of one or more of IL-7, IL-15, SCF, and / or FLT3L, thereby generating cells containing at least 80% CD34. + / CD45 + a transient suspension of hematopoietic progenitor cells (HPCs); c) CD34 in step b) + / CD45 + HPC was exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days; and d) CD34 was... + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent and IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines disclosed herein), FLT3L, and / or SCF for approximately 7–35 days, thereby inducing differentiation of PSC cells into NK cells.

[0054] In some implementations, CD34 + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent and IL-7, IL-15, FLT3L, and SCF and remained there for approximately 7–35 days, allowing the cell number to increase compared to that in step b) when CD34 was introduced. + / CD45 +HPC cells enriched at least 50-fold to 300-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagent for approximately 7–14 days. In some embodiments, the cell number enrichment is 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, or 300-fold, or any enrichment fold between the above or any two mentioned herein.

[0055] In some implementations, CD34 + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent and IL-7, IL-15, FLT3L, and SCF and remained there for approximately 7–35 days, allowing the cell number to increase compared to that in step b) when CD34 was introduced. + / CD45 + HPC cells enriched at least 100-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days.

[0056] In some implementations, CD34 + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent and IL-7, IL-15, FLT3L, and SCF and remained there for approximately 7–35 days, allowing the cell number to increase compared to that in step b) when CD34 was introduced. + / CD45 + HPC cells enriched at least 200-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days.

[0057] In some implementations, NK cells are produced in a feeder-independent manner.

[0058] In some embodiments, this disclosure provides a method for generating hematopoietic endothelial (HE) cells by: a) contacting a pluripotent stem cell (PSC) culture with a WNT signaling pathway activator and / or bone morphogenetic protein (BMP), wherein PSC growth lasts for approximately 1-7 days; and b) after step a), contacting the PSC culture with vascular endothelial growth factor (VEGF) for approximately 1-7 days to generate CD34. +Hematopoietic endothelial (HE) cell population.

[0059] In some embodiments, the contact in step a) i.e., the growth of PSC cultures in the presence of WNT signaling pathway activators and / or bone morphogenetic protein BMP, lasts for approximately 1-7 days. In some embodiments, the contact in step a) lasts for more than 7 days. In some embodiments, the contact in step a) lasts for up to 1, 2, 3, 4, 5, 6, or 7 days, or more than 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step a) lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step a) lasts for 1 day. In some embodiments, the contact in step a) lasts for 2 days. In some embodiments, the contact in step a) lasts for 3 days. In some embodiments, the contact in step a) lasts for 4 days. In some embodiments, the contact in step a) lasts for 5 days. In some embodiments, the contact in step a) lasts for 6 days. In some embodiments, the contact in step a) lasts for 7 days. In some implementations, the contact in step a) lasts for more than 7 days. In some implementations, the contact in step a) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0060] In some embodiments, contacting the culture with VEGF in step b) optionally further includes contacting the culture with forskolin or SB431542 to promote endothelial differentiation or hematopoietic differentiation, respectively. In some aspects, during contact step b), any one of forskolin or SB431542, or at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 µM, or greater than 20 µM, or any concentration of forskolin or SB431542 between the concentrations listed above or herein, is added to the culture medium. In some embodiments, during contact step b), about 10 µM of forskolin or SB431542 is optionally contacted with the culture.

[0061] In some implementations, the contact in step b) occurs after the PSC culture from step a) has been in contact with VEGF for approximately 1-7 days, thereby generating CD34. +Hematopoietic endothelial cell population. In some embodiments, the contact in step b) lasts for more than 7 days. In some embodiments, the contact in step b) lasts for up to 1, 2, 3, 4, 5, 6, or 7 days, or more than 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step b) lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step b) lasts for 1 day. In some embodiments, the contact in step b) lasts for 2 days. In some embodiments, the contact in step b) lasts for 3 days. In some embodiments, the contact in step b) lasts for 4 days. In some embodiments, the contact in step b) lasts for 5 days. In some embodiments, the contact in step b) lasts for 6 days. In some embodiments, the contact in step b) lasts for 7 days. In some embodiments, the contact in step b) lasts for more than 7 days. In some implementations, the contact in step b) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0062] By incorporating citations

[0063] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0064] The patent or application document contains at least one color drawing. A copy of the patent or application disclosure with the color drawing will be provided by the official authority upon request and payment of the necessary fees. Novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments (in which the principles of the invention are utilized) and the accompanying drawings, in which: Figure 1 The basal culture media shown are used to support NK cell differentiation and proliferation from iPSC-HPC. All media were supplemented with SCF, FLT3L, IL-7, and IL-15. Cells were always retained in the original differentiation wells (medium was changed on day 14).

[0065] Figure 2 Endpoint expression analysis performed by flow cytometry is shown. Lower NKp44 and CD38 levels in the defined culture medium indicate a more naive NK phenotype.

[0066] Figure 3A The results showed that regardless of cell density, the factor-bound APEL2 medium did not support long-term NK cell expansion after HPC was transferred to individual culture wells (n = 3 per data point).

[0067] Figure 3B The time of NK cell population collapse was shown to be correlated with the time of endothelial-to-hematopoietic transition (EHT).

[0068] Figure 4 Differentiation time progression analysis (flow cytometry data) using three independent GMP iPSC cell lines in chemically defined media is shown. Note the critical transition point from precursor cells to NK cells around day 28.

[0069] Figure 5A The change of culture medium at the transformation point between precursor cells and NK cells shows that the cells were able to expand after differentiation (n = 6 similar conditions). Note the logarithmic coordinates. Right figure: Flow cytometry analysis at the end of the time course shows that 100% of the cells that underwent culture medium change were 2B4. + CD56 + 96% are NKp30 + NKp46 + And 97% are NKp44 + NKp46 + .

[0070] Figure 5B The schematic diagram illustrates the NK cell expansion protocol, indicating the culture medium change and the addition of signaling molecules. B = BMP signaling stimulation; W = WNT signaling stimulation; V = VEGFA; S = SCF; F = FLT3L.

[0071] Figure 6 The RNA-seq data analysis is shown, highlighting NK cell-specific clusters as well as selected marker genes and enrichment terms.

[0072] Figure 7 The results of a killing assay using NK cells derived from three iPSC cell lines are shown.

[0073] Figure 8 This illustrates the connection between the differentiation paradigm of this study and strategies for amplifying context-dependent factors. The size of the sphere reflects the number of cells. Detailed Implementation

[0074] In one embodiment, this disclosure provides a method and composition involving a surprising discovery: using a cocktail mixture for endothelial induction comprising both bone morphogenetic protein (BMP) and a WNT signaling activator, resulting in the generation of endothelial-like precursor cells. These cells exhibit combined characteristics of both endothelial precursors and hematopoietic precursors and are capable of differentiating into terminally differentiated hematopoietic cells, including natural killer (NK) cells. Furthermore, a significant expansion of the NK cell population was achieved through modified cell culture conditions.

[0075] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this invention, it should be understood that modifications and variations are covered within the spirit and scope of this disclosure. Preferred methods and materials are now described.

[0077] Before describing the compositions and methods of the present invention, it should be understood that the invention is not limited to the specific compositions, methods, and experimental conditions described, as such compositions, methods, and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0078] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, references to “the method” include one or more methods and / or steps of the type described herein, as will become apparent to those skilled in the art upon reading this disclosure and the like.

[0079] As used herein, the terms “about” and “substantially” will be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. If the use of a term is unclear to those skilled in the art based on the context in which it is used, “about” and “substantially” will be interpreted as being within plus or minus 10% of that particular term.

[0080] "Contact" means culturing cells with one or more reagents of interest added to a defined basal or supplemental culture medium. That is, culturing cells in their standard basal or supplemental culture medium with the desired concentration of one or more reagents of interest added. For example, culturing cells with one or more of WNT signaling pathway activators, BMP signaling pathway activators, and VEGF signaling pathway activators. In some embodiments, cells are cultured with one or more cytokines. As another example, "contact" means culturing HSCs with one or more reagents of interest added to a culture medium lacking serum-derived reagents.

[0081] As used herein, a “signaling pathway activator” refers to any molecule 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 cellular functions, such as cell differentiation. A cell receives a signal from its environment when a molecule (such as a hormone or growth factor) binds to a specific protein receptor on or within the cell. After the first molecule in the pathway receives the signal, it activates another molecule. This process is repeated throughout the signaling pathway until the last molecule is activated and the cellular function is performed. Aberrant activation or inhibition of a signaling pathway can lead to disease, or, in the case of pluripotent cells (multifunctional cells), to an alteration of pluripotency and thus differentiation. The term “molecule” includes, as non-limiting examples, small molecules (including those that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers (such as proteins, peptides, hormones, carbohydrates, or polyamines). Non-limiting examples of polynucleotides include short interfering nucleic acids (siNA), antisense nucleic acids (aitisense), enzymatic nucleic acid molecules, 2',5'-oligoadenic acid, triple-stranded oligonucleotides, aptamers, and decoys. Bioactive molecules include antibodies (e.g., monoclonal antibodies, chimeric antibodies, humanized antibodies, etc.), cholesterol, hormones, antiviral agents, peptides, proteins, chemotherapeutic agents, small molecules, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triple-stranded oligonucleotides, 2,5-A chimeras, isoforms, aptamers, decoys and their analogues, as well as small nucleic acid molecules such as short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antagomir (anti-miRNA oligonucleotide), and short hairpin RNA (shRNA) molecules.

[0082] Natural killer cells, also known as NK cells or large granular lymphocytes (LGLs), are a type of cytotoxic lymphocyte that is crucial to the innate immune system. They belong to the rapidly expanding known innate lymphocyte (ILC) family and account for 5-20% of all circulating lymphocytes in humans. They have different functions, including cytolytic granule-mediated apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and cytokine-induced activation of NK and cytotoxic T lymphocytes (CTLs).

[0083] NK cells are cytotoxic; their cytoplasm contains small particles containing proteins (such as perforin) and proteases called granzymes. When released near a target cell, perforin creates pores in the target cell's cell membrane, generating aqueous channels through which granzymes and related molecules can enter, inducing apoptosis or osmotic cell lysis. The distinction between apoptosis and cell lysis is crucial in immunology: lysis of virus-infected cells may release virions, while apoptosis results in the destruction of the internal virus. Alpha-defensins (antimicrobial molecules) are also secreted by NK cells and directly kill bacteria by disrupting their cell walls in a manner similar to neutrophils.

[0084] Infected cells are typically opsonized with antibodies for use in immune cell detection. Antibodies that bind to antigens can be recognized by the FcγRIII (CD16) receptor expressed on NK cells, leading to NK cell activation, cytolysis, granule release, and subsequent apoptosis. This is the primary killing mechanism of some monoclonal antibodies, such as rituximab (Rituxan) and oflamb (Arzerra).

[0085] Certain cytokines released in response to viral infection play a crucial role in NK cell activation. These stress molecules signal to NK cells the presence of viral pathogens in the affected area. Cytokines involved in NK activation, or “NK-activating cytokines,” include, as unrestricted examples, IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells are activated in response to interferon or macrophage-derived cytokines. These are used to control viral infection, while the adaptive immune response generates antigen-specific cytotoxic T cells that can clear the infection. NK cells control viral infection by secreting IFNγ and TNFα. IFNγ activates macrophages for phagocytosis and lysis, and TNFα promotes direct NK tumor cell killing. Patients with NK cell deficiencies have been shown to be highly susceptible to the early stages of herpesvirus infection.

[0086] Tumor-infiltrating NK cells have been reported to play a crucial 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 HLA antigens), autologous (patient-associated) NK cell infusion has not shown any anti-tumor effect. Instead, researchers are working to use allogeneic cells from peripheral blood, which requires the removal of all T cells before infusion into the patient to eliminate the risk of graft-versus-host disease, which can be fatal. This can be achieved using immunomagnetic columns (CliniMACS). Furthermore, due to the limited number of NK cells in the blood (only 10% of lymphocytes are NK cells), their numbers need to be expanded in cultures. This can take several weeks, and the yield depends on the donor.

[0087] The method described in this article details the culture of human pluripotent stem cells under specific cell culture conditions, resulting in the generation of CD34. + Hematopoietic progenitor cell population. These cells are then selectively stimulated to differentiate into NK cells, and subsequently, the culture conditions are altered to promote a significant expansion of the NK cell population.

[0088] In some embodiments, this disclosure relates to obtaining NK cells by contacting pluripotent stem cells (PSCs) with a natural killer cell inducing composition. As described in detail herein, the natural killer cell inducing composition 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.

[0089] In some embodiments, this disclosure provides a method for generating natural killer (NK) cells, comprising: a) contacting a pluripotent stem cell (PSC) culture with a WNT signaling pathway activator and / or bone morphogenetic protein (BMP); b) contacting the culture from step a) with vascular endothelial growth factor (VEGF) to generate CD34. + Precursor cell population; c) CD34 + The precursor cell population was incubated in a medium lacking any serum-derived reagents, while optionally supplementing the medium with one or more cytokines, including, as a non-restrictive example, interleukin-7 (IL-7), NK-activating cytokines (such as interleukin-15 (IL-15) or any other NK-activating cytokines provided herein), stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (FLT3L), thereby producing CD34. + / CD45 + Suspension cell population; and d) CD34 + / CD45 +Suspension cell populations are incubated in a medium containing at least one serum-derived reagent and IL-7, NK-activating cytokines (such as interleukin-15 (IL-15) or any other NK-activating cytokines disclosed herein), SCF, and / or FLT3L to produce NK cells. In some aspects, the medium contains at least one or more of WNT signaling pathway activators, BMP, and VEGF. In some aspects, the medium is supplemented with one or more cytokines and / or growth factors, including any cytokines described above and disclosed herein.

[0090] In one aspect, this disclosure provides a method for producing natural killer (NK) cells, comprising: a) contacting a pluripotent stem cell (PSC) culture with a WNT signaling pathway activator and / or bone morphogenetic protein (BMP), wherein the PSC growth lasts for approximately 1-7 days; b) after step a), contacting the PSC culture with vascular endothelial growth factor (VEGF) for approximately 1-7 days, thereby generating at least approximately 80% enriched CD34. + CD34 + Precursor cell population; c) CD34 + The precursor cell population was incubated in a medium lacking any serum-derived reagents for approximately 7–28 days, with optional supplementation of one or more of interleukin-7 (IL-7), NK-activating cytokines (such as interleukin-15 (IL-15) or any other NK-activating cytokines provided herein), stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (FLT3L) to produce CD34. + / CD45 + Suspension cell population; and d) CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L to produce NK cells.

[0091] The WNT signaling pathway is a set of signal transduction pathways that begin by transmitting signals to proteins within the cell via cell surface receptors. The WNT signaling pathway utilizes either neighboring paracrine (cell-to-cell) communication or autocrine (same-cell) communication. Three WNT signaling pathways have been characterized: the classical WNT pathway, the non-classical planar cell polarity pathway, and the non-classical WNT / calcium pathway. All three pathways are activated by the binding of WNT-protein ligands to Frizzled family receptors, which transmit biological signals to dishevelled proteins within the cell. The classical WNT pathway leads to the regulation of gene transcription and is thought to be partially negatively regulated by the SPATS1 gene. The non-classical planar cell polarity pathway regulates the cytoskeleton, which is responsible for cell shape. The non-classical WNT / calcium pathway regulates intracellular calcium. WNT signaling was initially recognized for its role in oncogenesis and subsequently for its function in embryonic development. Embryonic processes controlled by WNT signaling include body axis pattern formation, cell fate determination, cell proliferation, and cell migration. These processes are essential for the proper formation of vital tissues, including bone, heart, and muscle. Its role in embryonic development was discovered when gene mutations in WNT pathway proteins resulted in abnormal fruit fly embryos. Later studies found that these abnormal genes also affected the development of breast cancer in mice. WNT signaling also controls tissue regeneration in adult bone marrow, skin, and intestines.

[0092] In some embodiments, the inducer that can be used to induce cardiomyocytes may include an activator of the WNT signaling pathway. As described and disclosed herein, as a non-limiting example, activators of the WNT signaling pathway include: CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarboxynitrile); WNT family ligands; RSPO co-agonists; lithium chloride; TDZD8 (4-benzyl-2-methyl- 1,2,4-Thiadiazolidine-3,5-dione); BIO-acetone oxime ((2'Z,3'E)-6-bromoindorubin-3'-acetone oxime); A1070722 (1-(7-methoxyquinoline-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea); HLY78 (4-ethyl-5,6-dihydro-5-methyl-[1,3]dioxolane[4,5-j]phenanthridine); CID 11210285 hydrochloride (2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero)arylpyrimidine; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, activation of the WNT signaling pathway can be achieved by inhibiting a WNT signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting a WNT signaling pathway inhibitor, or an antibody or small molecule targeting a WNT signaling pathway inhibitor. In some embodiments, adherent cultures of PSCs are contacted with a culture medium containing approximately 1 to 10 µM of a WNT signaling pathway activator.

[0093] In some aspects, a WNT signaling pathway activator is added to PSC cultures at concentrations ranging from about 1 µM to 10 µM. For example, PSCs are cultured in media containing 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 PSC cultures at concentrations ranging from 0.5 to 1.0 µM, 1.0 to 2.0 µM, 2.0 to 3.0 µM, 3.0 to 4.0 µM, 4.0 to 5.0 µM, 5.0 to 6.0 µM, 6.0 to 7.0 µM, 7.0 to 8.0 µM, 8.0 to 9.0 µM, 9.0 to 10 µM, or at concentrations between the above or any two mentioned herein. In some embodiments, the culture medium contains about 8 µM of 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.

[0094] The transforming growth factor β (TGF-β) superfamily includes TGF-β protein, bone morphogenetic proteins (BMPs), growth differentiation factor (GDF), glial-derived neurotrophic factor (GDNF), activin, inhibin, Nodal, Lefty, and Müllerian inhibitory substance (MIS). Bone morphogenetic proteins (BMPs) are a group of growth factors, also known as cytokines and metabolic regulators. Initially discovered for their ability to induce bone and cartilage formation, BMPs are now considered to constitute a key set of morphogenetic signals that coordinate tissue structure throughout the body. The important physiological functions of BMP signaling are highlighted by the numerous roles of dysregulated BMP signaling in pathological processes. BMPs interact with specific receptors on cell surfaces called bone morphogenetic protein receptors (BMPRs). Signal transduction via BMPRs leads to the mobilization of members of the SMAD protein family. Signaling pathways involving BMPs, BMPRs, and SMADs are important in the development of the heart, central nervous system, and cartilage, as well as in postnatal skeletal development. They play a crucial role in embryonic pattern formation and early skeletal formation during embryonic development. Therefore, disruption of BMP signaling can affect the body plan (body construction, body shape pattern) of a developing embryo. For example, BMP4 and its inhibitors, noggin and chordin, help regulate embryonic polarity (i.e., back-to-front pattern formation). In particular, BMP4 and its inhibitors play a major role in neurogenesis and neural plate development. BMP4 signals ectoderm cells to develop into skin cells, but inhibitors secreted by the underlying mesoderm block the action of BMP4, allowing the ectoderm to continue its normal neural cell development process.

[0095] In some embodiments, the inducer used to induce natural killer cells may include an activator of 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, etc., which activate or inhibit at least one component of the BMP signaling pathway, resulting in the corresponding activation or inhibition of cellular BMP signaling. As provided and disclosed herein, as a non-limiting example, bone morphogenetic proteins include BMP family ligands such as BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP15. In some embodiments, adherent cultures of PSCs are contacted with a culture medium containing approximately 10 to 100 ng / ml BMP.

[0096] In some respects, BMP is added to PSC cultures at concentrations ranging from about 5 ng / ml to about 50 ng / ml. For example, PSCs are cultured in media containing about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 ng / ml or more. In some embodiments, the concentration of BMP is in the range of 1 to 2 ng / ml, 2 to 3 ng / ml, 3 to 4 ng / ml, 4 to 5 ng / ml, 5 to 6 ng / ml, 6 to 7 ng / ml, 7 to 8 ng / ml, 8 to 9 ng / ml, 9 to 10 ng / ml, 10 to 15 ng / ml, 15 to 20 ng / ml, 20 to 25 ng / ml, 25 to 30 ng / ml, 30 to 35 ng / ml, 35 to 40 ng / ml, 40 to 45 ng / ml, 45 to 50 ng / ml, 50 to 55 ng / ml, or a range between the above or any two concentrations mentioned herein. In some embodiments, the culture medium contains about 25 ng / mL of BMP. In some embodiments, BMP is BMP4. In some embodiments, the concentration of BMP4 is about 25 ng / ml.

[0097] As further described in the examples, BMP4 can be prepared by resuspending it in various solutions. For example, dried BMP4 can be resuspended in PBS / 0.01% HSA, or in citric acid (as recommended by the manufacturer). The bioactivity of BMP4 may be reduced by PBS / 0.01% HSA compared to its activity when prepared in citric acid. One skilled in the art will readily recognize that if BMP4 is resuspended in citric acid and thus exhibits higher bioactivity, the concentration of BMP4 can be significantly reduced from 5 to 50 ng / ml.

[0098] In some embodiments, inducers that can be used to induce NK cells may include activators of the vascular endothelial growth factor (VEGF) signaling pathway. As provided and disclosed herein, vascular endothelial growth factors include VEGF ligands such as VEGF (VEGFA), VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placental growth factor (PIGF). In some cases, activators or inhibitors of the VEGF signaling pathway may also include activators or inhibitors of related signal transduction pathways, including the Ras / MAPK signal transduction pathway as a non-limiting example. 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, etc., which activate or inhibit at least one component of the VEGF signaling pathway, resulting in the corresponding activation or inhibition of cellular VEGF signaling. In some embodiments, adherent cultures of PSCs are contacted with a culture medium containing approximately 50 to 500 ng / ml VEGF.

[0099] In some respects, VEGF is added to PSC cultures at concentrations ranging from about 50 ng / ml to about 500 ng / ml. In some embodiments, the concentration of VEGF is 25 to 50 ng / ml, 50 to 75 ng / ml, 75 to 100 ng / ml, 100 to 125 ng / ml, 125 to 150 ng / ml, 150 to 175 ng / ml, 175 to 200 ng / ml, 200 to 225 ng / ml, 225 to 250 ng / ml, 250 to 275 ng / ml, 275 to 300 ng / ml, 300 to 325 ng / ml, 325 to 350 ng / ml, 350 to 375 ng / ml, 375 to 400 ng / ml, 400 to 425 ng / ml, 425 to 450 ng / ml, 450 to 475 ng / ml, 475 to 500 ng / ml, 500 to 525 ng / ml. Concentrations in the range of 500 to 550 ng / ml, or between any two of the above or mentioned herein. For example, PSCs are cultured in a medium containing 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 medium contains about 200 ng / mL VEGF. In some embodiments, VEGF is VEGFA. In some embodiments, the concentration of VEGFA is about 200 ng / ml.

[0100] In some embodiments, the adherent culture of PSC is contacted with one or more reagents selected from about 1 to 10 µM WNT signaling pathway activator, about 10-100 ng / ml BMP and about 50-500 ng / ml VEGF.

[0101] In some implementations, the PSC culture is contacted with approximately 8 µM CHIR99021, approximately 25 ng / ml BMP4, and / or approximately 200 ng / ml VEGFA.

[0102] As provided 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 that primarily regulate cellular processes, including (as unrestricted examples) proliferation, differentiation, and communication, in hematopoietic and immune cell types. In some respects, culture media are supplemented with one or more growth factors. In some respects, growth factors are selected from cytokines such as stem cell factor (also known as SCF, KIT-ligand, KL, or steel factor), FMS-like tyrosine kinase 3 ligand (FLT3L), and interleukins, including (as unrestricted examples) interleukin-7 (IL-7) and interleukin-15 (IL-15). In some embodiments, the cytokines are interleukins, including 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 as non-limiting examples.

[0103] As provided and disclosed herein, cytokines involved in NK activation, or "NK-activating cytokines," include IL-2, IL-12, IL-15, IL-18, and CCL5 as non-limiting examples. These NK-activating cytokines alert NK cells to the presence of viral pathogens and recruit NK cells to affected areas. In some embodiments, the culture medium is supplemented with one or more NK-activating cytokines, including IL-15 as a non-limiting example.

[0104] 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 not by normal lymphocytes. IL-7 stimulates the differentiation of multipotent 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 the proliferation of B cells during certain stages of B cell maturation, as well as for the survival, development, and homeostasis of T and NK cells.

[0105] In some aspects, IL-7 is added to PSC cultures at a concentration ranging from about 4 ng / mL to 40 ng / mL. In some embodiments, the concentration of IL-7 is in the range of about 4 to about 40 ng / mL. In some embodiments, the concentration of IL-7 is in the range of 2 to 3 ng / mL, 3 to 4 ng / mL, 4 to 5 ng / mL, 5 to 6 ng / mL, 6 to 7 ng / mL, 7 to 8 ng / mL, 8 to 9 ng / mL, 9 to 10 ng / mL, 10 to 15 ng / mL, 15 to 20 ng / mL, 20 to 25 ng / mL, 25 to 30 ng / mL, 30 to 35 ng / mL, 35 to 40 ng / mL, 40 to 45 ng / mL, 45 to 50 ng / mL, or a range between the above or any two of the concentrations mentioned herein. For example, PSCs are grown in media containing approximately 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 µM or more. In some embodiments, the media contains approximately 20 ng / mL IL-7.

[0106] IL-15 is a cytokine structurally similar to interleukin-2 (IL-2). Similar to IL-2, IL-15 binds to and signals through a complex consisting of the IL-2 / IL-15 receptor β chain (CD122) and a common γ chain (γ-C, CD132). Following viral infection, monocytes (and some other cells) secrete IL-15. This cytokine induces the proliferation of natural killer cells (cells of the innate immune system whose primary function is to kill virus-infected cells). IL-15 regulates the activation and proliferation of T cells and natural killer (NK) cells. IL-15 is an example of NK-activating cytokines, which include IL-2, IL-12, IL-15, and CCL5 as unrestricted examples. IL-15 provides the signaling necessary to maintain the survival of memory T cells in the absence of antigens. This cytokine is also involved 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 people with celiac disease, IL-15 similarly inhibits T lymphocyte apoptosis by inducing Bcl-2 and / or Bcl-xL.

[0107] In some embodiments, NK-activating cytokines (such as interleukin-15 (IL-15) or any other NK-activating cytokines provided herein) are added to PSC cultures at concentrations ranging from about 2 ng / mL to 20 ng / mL. In some embodiments, the concentration of NK-activating cytokines (such as interleukin-15 (IL-15) or any other NK-activating cytokines provided herein) is in the range of about 2 to about 20 ng / mL. In some embodiments, the concentrations of NK-activating cytokines (such as interleukin-15 (IL-15) or any other NK-activating cytokines provided herein) are 1 to 2 ng / ml, 2 to 3 ng / ml, 3 to 4 ng / ml, 4 to 5 ng / ml, 5 to 6 ng / ml, 6 to 7 ng / ml, 7 to 8 ng / ml, 8 to 9 ng / ml, 9 to 10 ng / ml, 10 to 11 ng / ml, 11 to 12 ng / ml, 12 to 13 ng / ml, 13 to 14 ng / ml, 14 to 15 ng / ml, 15 to 16 ng / ml, 16 to 17 ng / ml, 17 to 18 ng / ml, 18 to 19 ng / ml, 19 to 20 ng / ml, 20 to 21 ng / ml, 21 to 22 ng / ml, 22 to 23 ng / ml, 23 to 24 ng / ml. Concentrations in the range of 24 to 25 ng / ml, or between any two of the above or mentioned herein. For example, PSCs are grown in a medium containing about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 ng / ml or more of NK-activating cytokines (such as interleukin-15 (IL-15) or any other NK-activating cytokines provided herein). In some embodiments, the medium contains about 10 ng / mL of IL-15.

[0108] SCF is a cytokine that binds to the c-KIT receptor (c-KIT / CD117). SCF exists as both a transmembrane and soluble protein and plays important roles in hematopoiesis, spermatogenesis, and melanin production. SCF plays a crucial role in hematopoiesis during embryonic development. SCF is expressed in sites of hematopoiesis, such as the fetal liver and bone marrow. SCF acts as a guidance cue to guide hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which stem cells reside), and it plays an important role in HSC maintenance. SCF plays a role in the regulation of HSCs in the stem cell niche within the bone marrow. SCF has been shown to increase HSC survival in vitro and promote HSC self-renewal and maintenance in vivo. HSCs at all developmental stages express the same levels of c-KIT. Stromal cells surrounding HSCs are components of the stem cell niche, and they release various ligands, including SCF. In the bone marrow, HSCs and hematopoietic progenitor cells are adjacent to stromal cells, such as fibroblasts and osteoblasts. These HSCs are held in nests by adhering to ECM proteins and the stromal cells themselves. SCF has been shown to increase adhesion and therefore play a significant role in ensuring HSCs remain in nests. SCF can be used in conjunction with other cytokines to culture HSCs and hematopoietic progenitor cells. In vitro expansion of these cells will advance bone marrow transplantation, in which HSCs are transferred into a patient to re-establish hematopoiesis. One problem with injecting SCF for therapeutic purposes is that SCF activates mast cells. Injection of SCF has been shown to cause allergic-like symptoms as well as proliferation of mast cells and melanocytes.

[0109] In some aspects, SCF is added to PSC cultures at concentrations ranging from about 4 ng / ml to 40 ng / ml. In some embodiments, the concentration of SCF is in the range of 2 to 3 ng / ml, 3 to 4 ng / ml, 4 to 5 ng / ml, 5 to 6 ng / ml, 6 to 7 ng / ml, 7 to 8 ng / ml, 8 to 9 ng / ml, 9 to 10 ng / ml, 10 to 15 ng / ml, 15 to 20 ng / ml, 20 to 25 ng / ml, 25 to 30 ng / ml, 30 to 35 ng / ml, 35 to 40 ng / ml, 40 to 45 ng / ml, 45 to 50 ng / ml, or a range between the above or any two of the concentrations mentioned herein. For example, PSCs are cultured in media containing about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 µM or more. In some implementations, the culture medium contains approximately 20 ng / ml of SCF.

[0110] FLT3L is an endogenous small molecule that functions as a cytokine and growth factor, increasing the number of immune cells (lymphocytes (B cells and T cells)) by activating hematopoietic progenitor cells. It works by binding to and activating FLT3 (CD135), which is present on cells (in mice) called multipotent progenitors (MPPs) and common lymphoprogenitors (CLPs). It also induces the mobilization of hematopoietic progenitor cells and stem cells in vivo, which may contribute to the systemic killing of cancer cells. FLT3L is essential for the development of homeostatic plasmacytoid dendritic cells (pDCs) and classical dendritic cells (cDCs). A deficiency of FLT3L leads to low levels of dendritic cells.

[0111] In some embodiments, FLT3L is added to PSC cultures at a concentration ranging from about 2 ng / mL to 20 ng / mL. In some embodiments, the concentration of FLT3L is in the range of about 1 to about 20 ng / mL. In some embodiments, the concentration of FLT3L is 0.1 to 0.5, 0.5 to 1, 1 to 2 ng / ml, 2 to 3 ng / ml, 3 to 4 ng / ml, 4 to 5 ng / ml, 5 to 6 ng / ml, 6 to 7 ng / ml, 7 to 8 ng / ml, 8 to 9 ng / ml, 9 to 10 ng / ml, 10 to 11 ng / ml, 11 to 12 ng / ml, 12 to 13 ng / ml, 13 to 14 ng / ml, 14 to 15 ng / ml, 15 to 16 ng / ml, 16 to 17 ng / ml, 17 to 18 ng / ml, 18 to 19 ng / ml, 19 to 20 ng / ml, 20 to 21 ng / ml, 21 to 22 ng / ml, 22 to 23 ng / ml, 23 to 24 ng / ml, 24 to 25 ng / ml. Concentrations in the range of ng / ml, or concentrations between any two of the concentrations mentioned above or herein. For example, PSCs are cultured in a medium containing 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 medium contains about 10 ng / mL FLT3L.

[0112] For NK cell differentiation, the medium for HSCs can be replaced with an “undefined medium.” As used herein, the terms “undefined basal medium” or “undefined medium” generally refer to a medium formulation containing serum-derived reagents, such as human platelet lysate (hPL) or optional blood serum. Such mediums containing serum or serum-derived reagents are commercially available or can be prepared by the user “in-house.” As used herein, “undefined medium” is referred to as “medium containing serum-derived reagents.” As detailed above, such mediums containing serum-derived reagents can be “basal,” “supplementary,” or “complete.”

[0113] In some embodiments, the serum-derived reagent includes, as non-limiting examples, serum, platelet lysate, plasma, blood-derived albumin, and any component or derivative of blood-derived plasma (e.g., obtained by serum fractionation). In some embodiments, the serum-derived reagent is selected from serum, platelet lysate, or blood-derived albumin. In some embodiments, the serum-derived reagent is serum. In some embodiments, the serum-derived reagent is platelet lysate. In some embodiments, the serum-derived reagent is albumin. In some embodiments, the serum-derived reagent is plasma.

[0114] In the method described herein, pluripotent stem cells differentiate into CD34. + Hematopoietic progenitor cells then differentiate into NK cells. The method described in this article generates CD34. + Hematopoietic progenitor cells, which can interact with hematopoietic stem cells (which are CD34 cells). + / CD45 + (Different or the same) This CD34 + / CD45 + Intermediate cell population of hematopoietic progenitor cells from CD34 + / CD45 - It appears in hematopoietic endothelial-like cells and subsequently disappears to generate fully differentiated NK cells, characterized by CD56. + NK cells.

[0115] In some respects, CD34 + Precursor cells are contacted with approximately 4 to 40 ng / mL IL-7, approximately 2 to 20 ng / mL IL-15, approximately 4 to 40 ng / mL SCF, and / or approximately 1 to 20 ng / mL FLT3L. In some embodiments, CD34 is... + / CD45 + Suspended cells were exposed to approximately 4 to 40 ng / mL IL-7, approximately 2 to 20 ng / mL IL-15, approximately 4 to 40 ng / mL SCF, and / or approximately 1 to 20 ng / mL FLT3L.

[0116] In some implementations, CD34 is used for incubation. + Precursor cell culture and incubation of CD34 + / CD45 + The culture medium for the suspension cell culture contains cytokines. In some embodiments, the cytokines are 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. In some embodiments, the cytokines are NK-activating cytokines, such as IL-15 or any NK-activating cytokines provided above or herein. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-15.

[0117] In some implementations, CD34 is made + Precursor cells are contacted with approximately 20 ng / ml IL-7, approximately 10 ng / ml IL-15, approximately 20 ng / ml SCF, and / or approximately 10 ng / ml FLT3L. In some embodiments, CD34 is... + / CD45 + Suspended cells were exposed to approximately 20 ng / ml IL-7, approximately 10 ng / ml IL-15, approximately 20 ng / ml SCF, and / or approximately 10 ng / ml FLT3L.

[0118] In some aspects, the contact in step a) i.e., the growth of PSC cultures in the presence of WNT signaling pathway activators and / or bone morphogenetic protein BMP, lasts for approximately 1-7 days. In some embodiments, the contact in step a) lasts for more than 7 days. In some embodiments, the contact in step a) lasts for at most 1, 2, 3, 4, 5, 6, or 7 days, or more than 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step a) lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step a) lasts for 1 day. In some embodiments, the contact in step a) lasts for 2 days. In some embodiments, the contact in step a) lasts for 3 days. In some embodiments, the contact in step a) lasts for 4 days. In some embodiments, the contact in step a) lasts for 5 days. In some embodiments, the contact in step a) lasts for 6 days. In some embodiments, the contact in step a) lasts for 7 days. In some implementations, the contact in step a) lasts for more than 7 days. In some implementations, the contact in step a) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0119] In some respects, the contact in step b) occurs after the PSC culture from step a) has been in contact with VEGF for approximately 1–7 days, thereby generating CD34. +Precursor cell population. In some embodiments, the contact in step b) lasts for more than 7 days. In some embodiments, the contact in step b) lasts for up to 1, 2, 3, 4, 5, 6, or 7 days, or more than 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step b) lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step b) lasts for 1 day. In some embodiments, the contact in step b) lasts for 2 days. In some embodiments, the contact in step b) lasts for 3 days. In some embodiments, the contact in step b) lasts for 4 days. In some embodiments, the contact in step b) lasts for 5 days. In some embodiments, the contact in step b) lasts for 6 days. In some embodiments, the contact in step b) lasts for 7 days. In some embodiments, the contact in step b) lasts for more than 7 days. In some implementations, the contact in step b) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0120] In some respects, CD34 in step c) + The incubation of the precursor cell population is carried out in a culture medium lacking any serum-derived reagents, while optionally supplementing the medium with one or more cytokines. In some embodiments, the one or more cytokines are NK-activating cytokines, such as IL-15 or any NK-activating cytokines provided above or herein. In some aspects, CD34 in step c) + The precursor cell population is incubated in a culture medium lacking any serum-derived reagents, optionally supplemented with one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L. In some embodiments, CD34 in step c) + The precursor cell population is incubated in a medium lacking any serum-derived reagents, while optionally supplementing the medium with at least one of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L. In some embodiments, CD34 in step c) + The precursor cell population was incubated in a medium lacking any serum-derived reagents, with the medium optionally supplemented with a combination of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L.

[0121] In some aspects, the incubation in step c) lasts 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 days, or longer than 28 days, or any length of time between the above or any two mentioned herein. In some embodiments, the incubation in step c) lasts for 7 days. In some embodiments, the incubation in step c) lasts for 8 days. In some embodiments, the incubation in step c) lasts for 9 days. In some embodiments, the incubation in step c) lasts for 10 days. In some embodiments, the incubation in step c) lasts for 11 days. In some embodiments, the incubation in step c) lasts for 12 days. In some embodiments, the incubation in step c) lasts for 13 days. In some embodiments, the incubation in step c) lasts for 14 days. In some embodiments, the incubation in step c) lasts for 15 days. In some embodiments, the incubation in step c) lasts for 16 days. In some embodiments, the incubation in step c) lasts for 17 days. In some embodiments, the incubation in step c) lasts for 18 days. In some embodiments, the incubation in step c) lasts for 19 days. In some embodiments, the incubation in step c) lasts for 20 days. In some embodiments, the incubation in step c) lasts for 21 days. In some embodiments, the incubation in step c) lasts for 22 days. In some embodiments, the incubation in step c) lasts for 23 days. In some embodiments, the incubation in step c) lasts for 24 days. In some embodiments, the incubation in step c) lasts for 25 days. In some embodiments, the incubation in step c) lasts for 26 days. In some embodiments, the incubation in step c) lasts for 27 days. In some embodiments, the incubation in step c) lasts for 28 days. In some embodiments, the incubation in step c) lasts for more than 28 days. In some implementations, the incubation in step c) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0122] In some implementations, CD34 in step d) + / CD45 +Suspension cell populations are incubated in a culture medium containing at least one serum-derived reagent and one or more cytokines, including NK-activating cytokines such as IL-15 or any NK-activating cytokines described above or herein. In some embodiments, CD34 from step d) is... + / CD45 + Suspension cell populations were incubated in a medium containing at least one serum-derived reagent as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L.

[0123] In some respects, the incubation in step d), i.e., the incubation of CD34 from step c), + / CD45 +Suspension cell populations are incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent and IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L to generate NK cells. In some embodiments, the incubation in step d) lasts for at least 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 days, or any length of time between the above or any two mentioned herein. In some embodiments, the incubation in step d) lasts for more than 7 days. In some embodiments, the incubation in step d) lasts 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 days, or more than 35 days, or any length of time between the above or any two of the numbers mentioned herein. In some embodiments, the incubation in step d) lasts for 7 days. In some embodiments, the incubation in step d) lasts for 8 days. In some embodiments, the incubation in step d) lasts for 9 days. In some embodiments, the incubation in step d) lasts for 10 days. In some embodiments, the incubation in step d) lasts for 11 days. In some embodiments, the incubation in step d) lasts for 12 days. In some embodiments, the incubation in step d) lasts for 13 days. In some embodiments, the incubation in step d) lasts for 14 days. In some embodiments, the incubation in step d) lasts for 15 days. In some embodiments, the incubation in step d) lasts for 16 days. In some embodiments, the incubation in step d) lasts for 17 days. In some embodiments, the incubation in step d) lasts for 18 days. In some embodiments, the incubation in step d) lasts for 19 days. In some embodiments, the incubation in step d) lasts for 20 days. In some embodiments, the incubation in step d) lasts for 21 days. In some embodiments, the incubation in step d) lasts for 22 days. In some embodiments, the incubation in step d) lasts for 23 days. In some embodiments, the incubation in step d) lasts for 24 days. In some embodiments, the incubation in step d) lasts for 25 days. In some embodiments, the incubation in step d) lasts for 26 days. In some embodiments, the incubation in step d) lasts for 27 days. In some embodiments, the incubation in step d) lasts for 28 days. In some embodiments, the incubation in step d) lasts for 29 days. In some implementations, the incubation in step d) lasts for 30 days.In some embodiments, the incubation in step d) lasts for 31 days. In some embodiments, the incubation in step d) lasts for 32 days. In some embodiments, the incubation in step d) lasts for 33 days. In some embodiments, the incubation in step d) lasts for 34 days. In some embodiments, the incubation in step d) lasts for 35 days. In some embodiments, the incubation in step d) lasts for more than 35 days. In some implementations, the incubation in step d) lasts 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 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 any length of time between the above or any two of the numbers listed herein.

[0124] In some embodiments, the adherent culture of PSCs is contacted with the WNT signaling pathway activator and BMP for approximately 2-5 days. In some embodiments, the PSC culture from step a) is contacted with the WNT signaling pathway activator and BMP signaling pathway activator for approximately 3 to 5 days. In some embodiments, the PSC culture from step a) is contacted with the WNT signaling pathway activator and BMP signaling pathway activator for approximately 4 to 5 days. In some embodiments, the PSC culture from step a) is contacted with the WNT signaling pathway activator and BMP signaling pathway activator for approximately 2, 3, 4, or 5 days, or for any duration between any two of the listed days.

[0125] In some embodiments, the PSC is then exposed to VEGF for approximately 2-5 days. In some embodiments, the PSC is exposed to VEGF for approximately 3 to 5 days. In some embodiments, the PSC is exposed to VEGF for approximately 4 to 5 days. In some embodiments, the PSC is exposed to VEGF for approximately 2, 3, 4, or 5 days, or for any length of time between any two of the listed number of days.

[0126] In some implementations, CD34 is made + Precursor cell populations are exposed to IL-7, IL-15, SCF, and / or FLT3L for approximately 5–10 days, and CD34 is activated. + / CD45 + Suspended cell populations are exposed to IL-7, IL-15, FLT3L, and / or SCF for at least approximately 7–21 days. In some embodiments, CD34 is... +Precursor cell populations are exposed to IL-7, IL-15, SCF, and / or FLT3L for approximately 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 any duration between the above or any two of the days listed herein, and CD34 + / CD45 + Suspended cell populations were exposed to IL-7, IL-15, FLT3L and / or SCF for at least approximately 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 between the above or any two of the numbers listed herein.

[0127] In some implementations, CD34 is made + Precursor cell populations are exposed to IL-7, IL-15, SCF, and / or FLT3L for approximately 5–10 days, subsequently leading to CD34 activation. + / CD45 + The suspended cell population is exposed to IL-7, IL-15, FLT3L, and / or SCF for at least approximately 7–21 additional days. In some embodiments, CD34 is made... + Precursor cell populations are exposed to IL-7, IL-15, SCF, and / or FLT3L for approximately 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 any duration between the above or any two of the days listed herein, and CD34 + / CD45 + The suspension cell population was exposed to IL-7, IL-15, FLT3L and / or SCF 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 between the above or any two of the days listed herein.

[0128] In some embodiments, the PSC culture is an adherent cell layer. In some embodiments, the cell layer is grown in a two-dimensional culture system or on microcarriers.

[0129] In some implementations, PSCs are cultured on a scaffold composed of microcarriers, which are beads or particles. The beads can be microscopic or macroscopic and can be further sized to allow penetration into tissues or compacted to form a specific geometry.

[0130] In some implementations, the framework for cell culture comprises particles that combine with cells to form a three-dimensional tissue. Cells attach to the particles and to each other to form a three-dimensional tissue. Beads or microcarriers are generally considered to be two-dimensional systems or scaffolds.

[0131] As used herein, “microcarrier” refers to particles having a size ranging from nanometer to micrometer, wherein the particles can be of any shape or geometry, including irregular, non-spherical, spherical, or ellipsoidal. The size of the microcarrier suitable for the purposes of this document can be any size appropriate for a particular application.

[0132] In some embodiments, the size of the microcarriers suitable for three-dimensional tissues can be an injectable size. 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 size range between the above or any two sizes mentioned herein.

[0133] 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 the outer first layer has biodegradable properties for forming three-dimensional tissues in cultures, while at least the inner second layer, which is biodegradable and has different properties from the first layer, is designed to erode upon application to tissues or organs.

[0134] In some embodiments, the microcarrier is a porous microcarrier. A porous microcarrier is a microcarrier with gaps through which molecules can diffuse into or out of the microparticle. In other embodiments, the microcarrier is a non-porous microcarrier. Non-porous microparticles are microparticles of a selected size through which molecules do not diffuse into or out of the microparticle.

[0135] Microcarriers used in the composition are biocompatible and have low or no cellular toxicity. Microcarriers can comprise a variety of polymers, natural or synthetic, charged (i.e., anionic or cationic) or uncharged, biodegradable or non-biodegradable. The polymers can be homopolymers, random copolymers, block copolymers, graft copolymers, or branched polymers.

[0136] In some embodiments, the microcarriers comprise non-biodegradable microcarriers. Non-biodegradable microcapsules and microcarriers include, but are not limited to, those made of polysulfone, poly(acrylonitrile-co-vinyl chloride), ethylene-vinyl acetate, and hydroxyethyl methacrylate-methyl methacrylate copolymers. These can be used to provide tissue-filling properties or in embodiments where the microcarriers are eliminated by the body.

[0137] In some embodiments, the microcarriers comprise a biodegradable scaffold. These include microcarriers made from naturally occurring polymers, among which non-limiting examples include: fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginate, or polyamino acids (such as polylysine).

[0138] In some embodiments, the biodegradable microcarriers are made of synthetic polymers, among which non-limiting examples include: polylactide (PLA), polyglycolic acid (PGA), poly(lactide-co-glycolic acid) (PLGA), poly(caprolactone), polydioxanone trimethylene carbonate, polyhydroxyalkanoates (e.g., poly(hydroxybutyrate), poly(ethyl glutamate), poly(DTH iminocarbonate), poly(orthoester), and polycyanoacrylate).

[0139] In some embodiments, the microcarriers comprise hydrogels, which are typically water-filled hydrophilic polymer networks. Hydrogels have the advantage of selectively triggering polymer swelling. Depending on the composition of the polymer network, the swelling of the microparticles can be triggered by a variety of stimuli, including pH, ionic strength, heat, electricity, ultrasound, and enzyme activity. Non-limiting examples of polymers that can be used in hydrogel compositions include those formed from poly(lactide-co-glycolic acid); poly(N-isopropylacrylamide); poly(methacrylic acid-grafted polyethylene glycol); polyacrylic acid and poly(oxypropylene-co-ethylene glycol); and natural compounds, including chondroitin sulfate, chitosan, gelatin, fibrinogen, or synthetic mixtures with natural polymers, such as chitosan-poly(ethylene oxide), as non-limiting examples. The polymers are reversibly or irreversibly crosslinked to form gels suitable for forming three-dimensional tissues.

[0140] In some embodiments, the microcarriers or beads used in this disclosure are composed wholly or partially of dextran.

[0141] In some implementations, PSCs are cultured on a coated surface, including a laminin coating.

[0142] In some implementations, CD34 + The precursor cells are CD34 +Endothelial-like precursor cells.

[0143] In some embodiments, NK cells are further collected in a suspension state in a cell culture medium.

[0144] The method described in this article allows for the extraction of CD34 from wall-mounted CD34. + Endothelial-like precursor cells differentiate into NK cells that grow in suspension. Therefore, during differentiation, intermediate and transient CD34... + and CD45 + Cells from CD34 + Endothelial-like precursor cells emerge in suspension and differentiate into NK cells. After at least four weeks of culture, the method described herein produces NK cells in suspension with a purity or suspension density of at least 80%. Because the cells are in suspension, they can be easily aspirated and collected from the culture medium.

[0145] In some embodiments, NK cells are enriched at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, NK cells are enriched at least 60%. In some embodiments, NK cells are enriched at least 65%. In some embodiments, NK cells are enriched at least 70%. In some embodiments, NK cells are enriched at least 75%. In some embodiments, NK cells are enriched at least 80%. In some embodiments, the contact in step b) generates at least 85% enrichment of CD34. + CD34 + Precursor cell population. In some embodiments, NK cells are enriched at least 90%. In some embodiments, NK cells are enriched at least 95%. In some embodiments, NK cells are enriched at least about 80%.

[0146] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, to achieve a cell number relative to that of CD34 in step b). + / CD45 +HPC cells enriched at least 50-fold to 300-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagent for approximately 7–14 days. In some embodiments, the cell number enrichment is 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, or 300-fold, or any enrichment factor between the above or any two percentages mentioned herein.

[0147] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, resulting in a cell number compared to CD34. + The precursor cell population was enriched at least 50-fold when incubated for approximately 7–14 days in a medium lacking any serum-derived reagents (with optional supplementation of one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L).

[0148] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, resulting in a cell number compared to CD34. + The precursor cell population was enriched at least 100-fold when incubated for approximately 7–14 days in a medium lacking any serum-derived reagents (with optional supplementation of one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L).

[0149] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, resulting in a cell number compared to CD34.+ The precursor cell population was enriched at least 150-fold when incubated for approximately 7–14 days in a medium lacking any serum-derived reagents (optionally supplemented with one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L).

[0150] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, resulting in a cell number compared to CD34. + The precursor cell population was enriched by at least 200-fold when incubated for approximately 7–14 days in a medium lacking any serum-derived reagents (with optional supplementation of the medium with one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L).

[0151] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, resulting in a cell number compared to CD34. + The precursor cell population was enriched at least 250-fold when incubated for approximately 7–28 days in a medium lacking any serum-derived reagents (with optional supplementation of one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L).

[0152] In some implementations, CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing at least one serum-derived reagent, as well as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L, resulting in a cell number compared to CD34. +The precursor cell population was enriched by at least 300-fold when incubated in a medium lacking any serum-derived reagents (optionally supplemented with one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and FLT3L) for approximately 7–28 days.

[0153] On the other hand, this disclosure provides a method for generating natural killer (NK) cells from pluripotent stem cells (PSCs), comprising: a) generating CD34 cells by... + Hematopoietic endothelial (HE) cells: (i) PSC cultures were contacted with WNT signaling pathway activators and bone morphogenetic protein (BMP), wherein PSC growth was sustained for approximately 3 days; and (ii) the cells from (i) were subsequently contacted with vascular endothelial growth factor (VEGF) for approximately 4 days; thereby generating cells containing at least 80% CD34. + a) A population of HE cells; b) CD34 cells from step a) cultured in a medium lacking any serum-derived reagents. + HE cells were sustained for approximately 7–28 days, with optional addition of one or more of IL-7, NK-activating cytokines such as IL-15 or any other NK-activating cytokines provided herein, SCF, and / or FLT3L, thereby generating cells containing at least 80% CD34. + / CD45 + a transient suspension of hematopoietic progenitor cells (HPCs); c) CD34 in step b) + / CD45 + HPC was exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days; and d) CD34 was... + / CD45 + HPC cells were transferred to a culture medium containing at least one serum-derived reagent as well as IL-7, IL-15, FLT3L and SCF for approximately 7–35 days, thereby inducing differentiation of PSC cells into NK cells.

[0154] In some implementations, the differentiated NK cells are CD56. + NKp30 + NKp44 + NKp46 + NKG2D + NKG2A + KIR2D + and / or CD16 + .

[0155] NK cells can be activated by the presence of CD56 and the absence of CD3 (CD56). + CD3 - NK cells (belonging to the innate lymphocyte group) are one of three cell types that differentiate from common lymphoprogenitor cells, the other two being B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus before entering circulation. NK cells differ phenotypedly, in origin, and in their respective effector functions from natural killer T cells (NKT); typically, NKT cell activity is enhanced by the secretion of interferon-γ. Compared to NKT cells, NK cells do not express T cell antigen receptors (TCRs) or the pan-T marker CD3 or surface immunoglobulin (Ig) B cell receptors, but they typically express the surface markers CD16 (FcγRIII) and CD57 in humans. The NKp46 cell surface marker constitutes another preferred NK cell marker, expressed in humans, several mouse strains, and three common monkey species.

[0156] NK cells can be divided into CD56 高表达 or CD56 低表达 CD56 高表达 NK cells are similar to T helper cells, exerting their influence by releasing cytokines. CD56 高表达 NK cells constitute the majority of NK cells and are found in the bone marrow, secondary lymphoid tissues, liver, and skin. CD56 低表达 NK cells are mainly found in peripheral blood and are characterized by their cytotoxic ability. CD56 低表达 NK cells are always CD16 positive (CD16 is a key mediator of antibody-dependent cell-mediated cytotoxicity (ADCC)). CD56 高表达 You can convert CD16 to CD56. 低表达 .

[0157] In some implementations, the differentiated NK cells are CD56. 高表达 or CD56 低表达 .

[0158] In some implementations, the differentiated NK cells are cytotoxic NK cells.

[0159] In some respects, NK cells are produced in a feeder-free manner.

[0160] In some implementations, CD34 + / CD45 +HPC cells were transferred to a medium containing at least one serum-derived reagent and IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), FLT3L, and SCF for approximately 7–35 days, resulting in a cell count relative to that of CD34 cells in step b). + / CD45 + HPC cells enriched at least 50-fold to 300-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagent for approximately 7–14 days. In some embodiments, the cell number enrichment is 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, or 300-fold, or any enrichment factor between the above or any two percentages mentioned herein.

[0161] In some implementations, CD34 + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent, as well as IL-7, IL-15, FLT3L, and SCF, and remained there for approximately 7–35 days, resulting in a cell count relative to that of CD34 cells obtained in step b). + / CD45 + HPC cells enriched at least 50-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days.

[0162] In some implementations, CD34 + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent, as well as IL-7, IL-15, FLT3L, and SCF, and remained there for approximately 7–35 days, resulting in a cell count relative to that of CD34 cells obtained in step b). + / CD45 + HPC cells enriched at least 100-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days.

[0163] In some implementations, CD34 + / CD45 +HPC cells were transferred to a medium containing at least one serum-derived reagent, as well as IL-7, IL-15, FLT3L, and SCF, and remained there for approximately 7–35 days, resulting in a cell count relative to that of CD34 cells obtained in step b). + / CD45 + HPC cells enriched at least 150-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days.

[0164] In some implementations, CD34 + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent, as well as IL-7, IL-15, FLT3L, and SCF, and remained there for approximately 7–35 days, resulting in a cell count relative to that of CD34 cells obtained in step b). + / CD45 + HPC cells enriched at least 200-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days.

[0165] In some implementations, CD34 + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent, as well as IL-7, IL-15, FLT3L, and SCF, and remained there for approximately 7–35 days, resulting in a cell count relative to that of CD34 cells obtained in step b). + / CD45 + HPC cells enriched at least 250-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days.

[0166] In some implementations, CD34 + / CD45 + HPC cells were transferred to a medium containing at least one serum-derived reagent, as well as IL-7, IL-15, FLT3L, and SCF, and remained there for approximately 7–35 days, resulting in a cell count relative to that of CD34 cells obtained in step b). + / CD45 + HPC cells enriched at least 300-fold when exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days.

[0167] Stem cells are undifferentiated cells capable of indefinite self-renewal and maintaining the undifferentiated state described above. Unlike embryonic stem cells, which can only be isolated from the inner cell mass of the blastocyst, there are three known sources of adult stem cells: bone marrow (requiring bone drilling); adipose tissue (obtained through liposuction); and blood (from which cells can be extracted from other cells). As used herein, the term "pluripotent stem cell" refers to a cell capable of generating all cell types of an organism (i.e., cells derived from any of the three germ layers). On the other hand, pluripotent stem cells can differentiate into several cell types, but only into cell types of closely related cell families, typically the cell types of their organ of origin. Most adult stem cells are pluripotent, but small amounts of pluripotent adult stem cells can be retrieved from the umbilical cord or other tissues. Cell sources used for cell therapy include stem cells such as embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs).

[0168] In some embodiments, the PSC used in the methods described herein is a human PSC (hPSC), and in some embodiments, the human PSC is an induced pluripotent stem cell (hiPSC) or a human embryonic stem cell (hESC).

[0169] "Generate" or "Produce" CD34 + Hematopoietic progenitor cells refer to the method of this invention that provides cells optimized to induce PSC differentiation into CD34. + Physical and chemical culture conditions for hematopoietic progenitor cells. The differentiation method described in this article produces CD34-enriched cells. + CD34 of hematopoietic progenitor cells + Hematopoietic progenitor cell populations. For example, obtaining CD34+ cells with greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% purity within a short time using convenient culture conditions. + Hematopoietic precursor cells.

[0170] Physical culture conditions include, but are not limited to, the cell culture environment (e.g., adherent culture versus suspension culture, or in a two-dimensional culture system versus in a three-dimensional culture system), the pH of the culture medium, the gas concentration in the incubator (e.g., CO2 concentration, O2 concentration), and the temperature.

[0171] There are two basic systems for growing cells in cultures: as a monolayer on an artificial substrate (i.e., adherent culture) or in free suspension in a culture medium (suspension culture). Most cells derived from vertebrates, with the exception of hematopoietic cell lines and a few other cell types, are adherent-dependent and must be cultured on a suitable substrate that has been specially treated to allow cell adhesion and spreading (i.e., tissue culture treatment). However, many cell lines can also be adapted for suspension culture.

[0172] In some embodiments, the PSC culture is an adherent cell layer. In some embodiments, the cell layer is grown in a two-dimensional culture system or on microcarriers.

[0173] In addition to surface treatments for tissue culture, cells may also need 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, represents all additional modifications made to increase cell adhesion beyond the standard plasma or corona treatment performed by the manufacturer on all cell culture plastics. Typically, coating is performed using proteins or peptides. A variety of proteins can be used to coat culture dishes treated with tissue culture, including poly-L-lysine, poly-D-lysine, polyornithine, gelatin, type I collagen, type IV collagen, fibronectin, laminin, hydrin, osteopontin, fibronectin domains, and Matrigel. TM (Contains several components of the extracellular matrix that bind growth factors, etc.), collagen gel, alginate gel, and lactate gel.

[0174] In some embodiments, PSCs are cultured on a coated surface including a laminin coating.

[0175] Physical culture conditions include the gas concentration in the incubator. Cell cultures are typically incubated in a normal atmosphere (atmosphere) with 15-22% oxygen and 5% CO2 for expansion and seeding. In some embodiments, PSCs are grown in a humidified atmosphere including approximately 5% CO2 concentration and normoxic conditions (non-hypoxic O2 concentration). While hypoxic culture conditions are generally considered to support stem cell performance, in this method, PSCs are cultured under non-hypoxic conditions. As used herein, "normoxic" conditions refer to culture conditions including atmospheric O2 concentration (e.g., approximately 15-25% O2 concentration). As used herein, hypoxic conditions are characterized by a lower oxygen concentration compared to the oxygen concentration of ambient air (approximately 15%-25% oxygen).

[0176] Chemical culture conditions include, but are not limited to, additions to the culture medium to achieve the desired effect (i.e., differentiation of PSCs into CD34). + Reagents or molecules (hematopoietic progenitor cells). The terms “reagent” and “molecule” are used interchangeably and include, but are not limited to, small molecules (including those that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers such as proteins, peptides, hormones, carbohydrates, or polyamines.

[0177] In the method described herein, pluripotent stem cells differentiate into CD34. +Hematopoietic precursor. As used in this article, "CD34" + "Hematopoietic precursor" or "CD34" + "Hematopoietic progenitor cells" refer to transient cells that express some of the markers of hematopoietic stem cells (HSCs) and exhibit some of their characteristics, but not all of them. For example, HSCs are often characterized by CD34. + / CD45 + Non-adherent cells. After culturing for approximately 7 days under the conditions described herein, CD34... + Hematopoietic precursor cells are CD34 + But they are still CD144 + and CD45 - They are still adherent cells.

[0178] Unless otherwise specified, pluripotent stem cells are maintained in stem cell culture media suitable for the culture and proliferation 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. TM For HSC differentiation, pluripotent stem cells are replaced with a “defined culture medium.” As used herein, the term “basal medium” generally refers to a basal medium that does not contain any additives added by the user (e.g., basal medium refers to a commercially available medium). These typically include water, nutrients, salts, and amino acids, but do not contain additives or supplements. Basal media may be supplemented with generic additives to obtain “supplemented basal media.” Non-limiting examples of supplements include, but are not limited to, insulin or ascorbic acid. Basal media may also include specific signaling molecules, such as those identified by the user as necessary to achieve specific goals with the cell culture (e.g., driving a target cell type to differentiate into a target cell type). Such complete basal media may be referred to as “final,” “complete,” or “cell-specific” media.

[0179] In the context of this disclosure, cell culture medium is additionally referred to based on its intended use. For example, the terms "defined basal medium" and "defined medium" refer to media used for the production of CD34. + / CD45 +Ready-to-use culture medium formulations for hematopoietic progenitor cells contain only quantifiable amounts of specific components, such as serum-derived reagents (serum or components directly isolated therefrom, or products of other animal or tissue origin isolated from organisms or cells). However, recombinant serum albumin is considered essential even in the defined culture medium, and it is not considered "serum-derived." Such ready-to-use formulations are available in commercially available products or in user-developed "in-house" compositions. Not wishing to be limited to any particular formulation, a general formulation of a defined culture medium may include a basal medium (including, as a non-limiting example, DMEM, DMEM / F12, IMDM, or mixtures thereof) and a supplement containing one or more of, but not limited to, insulin, a combination of insulin with transferrin and selenium, serum albumin, recombinant human serum albumin, polyvinyl alcohol (PVA), lipids or fatty acids, glutamine, alanyl-glutamine (Glutamax), common amino acids, antioxidants (such as ascorbic acid, ascorbic acid-2-phosphate, or thiols), and inorganic salts (for supplemental versions). Non-limiting examples of commercially available defined basal media include, but are not limited to: (1) APEL TM The mixture included 1X Iscove modified Dulbecco medium (IMDM), 1X Ham's F-12 nutrient mixture, Albucult (recombinant albumin) (5 mg / ml), polyvinyl alcohol (PVA), linoleic acid (100 ng / ml), linolenic acid (100 ng / ml), SyntheChol (synthetic cholesterol) (2.2 mg / ml), α-monothioglycerol (α-MTG) (3.9 ml / 100 ml), recombinant human insulin-transferrin-selenoethanolamine solution (rhITS-Eth), protein-free hybridoma mixture II (PFHMII) (5%), ascorbic acid-2-phosphate (50 µg / ml), Glutamax I (L-alanyl-L-glutamine) (2 mM) and penicillin / streptomycin (50 U penicillin G / 50 mg streptomycin sulfate); (2) APELII TM Or APEL2 TMThe following are included: (1) 1X Iscove modified Dulbecco medium (IMDM), 1X Ham's F-12 nutrient mixture, Albucult (recombinant human albumin) (5 mg / ml), polyvinyl alcohol (PVA), linoleic acid (100 ng / ml), linolenic acid (100 ng / ml), SyntheChol (synthetic cholesterol) (2.2 mg / ml), α-monothioglycerol (α-MTG) (3.9 ml / 100 ml), recombinant human insulin-transferrin-selenoethanolamine solution (rhITS-Eth), ascorbic acid 2-phosphate (50 µg / ml), GlutamaxI (L-alanyl-L-glutamine) (2 mM) and penicillin / streptomycin (50 U penicillin G / 50 mg streptomycin sulfate); (3) APALII, including 1X Iscove modified Dulbecco medium (IMDM), polyvinyl alcohol (PVA, 0.1%), Albucult (recombinant human albumin) (0.2%), ascorbic acid-2-phosphate (250 µM), lipids (1%), recombinant human insulin-transferrin-selenoethanolamine solution (rhITS-Eth, 0.1%); (4) ESSENTIAL 6 TM Or E6 TM This includes DMEM / F12, insulin, transferrin, sodium selenite, ascorbic acid-2-phosphate, and NaHCO3; and (5) ESSENTIAL 8 TM Or E8 TM The formula includes DMEM / F12, magnesium L-ascorbic acid-2-phosphate (64 mg / l), sodium selenite (14 µg / l), insulin (19.4 mg / l), NaHCO3 (543 mg / l) and transferrin (10.7 mg / l).

[0180] Such a basic or supplement-defined culture medium for HE generation can then include small molecules of interest as needed.

[0181] "Contact" refers to culturing cells with one or more reagents of interest added to a culture medium lacking serum-derived reagents. That is, culturing cells in their standard basal or supplemental culture medium with the desired concentration of one or more reagents of interest added. For example, culturing cells with WNT signaling pathway activators, BMP, and / or VEGF.

[0182] As used herein, a “signaling pathway activator” refers to any molecule 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 cellular functions, such as cell differentiation. A cell receives a signal from its environment when a molecule (such as a hormone or growth factor) binds to a specific protein receptor on or within the cell. After the first molecule in the pathway receives the signal, it activates another molecule. This process repeats throughout the signaling pathway until the last molecule is activated and the cellular function is performed. Aberrant activation or inhibition of a signaling pathway can lead to disease, or, in the case of pluripotent cells, to an alteration of pluripotency and thus differentiation. The term “molecule” includes, but is not limited to, small molecules (including those that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers (such as proteins, peptides, hormones, carbohydrates, or polyamines). Non-limiting examples of polynucleotides include short interfering nucleic acids (siNA), antisense nucleic acids, enzymatic nucleic acid molecules, 2',5'-oligoadenic acid, triplet-forming oligonucleotides, aptamers, and decoy molecules. Bioactive molecules include antibodies (e.g., monoclonal, chimeric, humanized antibodies, etc.), cholesterol, hormones, antiviral agents, peptides, proteins, chemotherapeutic agents, small molecules, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplet-forming oligonucleotides, 2,5-A chimeras, isoforms, aptamers, decoy molecules and their analogues, as well as small nucleic acid molecules such as short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antagomir RNA, and short hairpin RNA (shRNA) molecules.

[0183] The WNT signaling pathway is a set of signal transduction pathways that begin by transmitting signals to proteins within the cell via cell surface receptors. The WNT signaling pathway utilizes either neighbor-cell communication (paracrine) or intracellular communication (autocrine). Three WNT signaling pathways have been characterized: the classical WNT pathway, the non-classical planar cell polarity pathway, and the non-classical WNT / calcium pathway. All three pathways are activated by the binding of WNT-protein ligands to Frizzled family receptors, which transmit biological signals to disheveled proteins within the cell. The classical WNT pathway leads to the regulation of gene transcription and is thought to be partially negatively regulated by the SPATS1 gene. The non-classical planar cell polarity pathway regulates the cytoskeleton, which is responsible for cell shape. The non-classical WNT / calcium pathway regulates intracellular calcium. WNT signaling was initially recognized for its role in oncogenesis and subsequently for its function in embryonic development. Embryonic processes it controls include body axis patterning, cell fate determination, cell proliferation, and cell migration. These processes are essential for the proper formation of vital tissues, including bone, heart, and muscle. The role of WNT pathway proteins in embryonic development was discovered when mutations in these genes resulted in abnormal fruit fly embryos. Subsequent studies found that genes responsible for these abnormalities also influence the development of breast cancer in mice. WNT signaling also controls tissue regeneration in adult bone marrow, skin, and intestines.

[0184] As non-limiting examples, activators of the WNT signaling pathway include: CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarboxylonitrile); WNT family ligands; RSPO co-agonists; lithium chloride; TDZD8 (4-benzyl-2-methyl-1,2,4-dichlorophenyl) -Thiadiazolidine-3,5-dione); BIO-acetone oxime ((2'Z,3'E)-6-bromoindorubin-3'-acetone oxime); A1070722 (1-(7-methoxyquinoline-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea); HLY78 (4-ethyl-5,6-dihydro-5-methyl-[1,3]dioxolane[4,5-j]phenanthridine); CID 11210285 hydrochloride (2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero)arylpyrimidine; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, activation of the WNT signaling pathway can be achieved by inhibiting a WNT signaling pathway inhibitor. As non-limiting examples, this includes using an inhibitory nucleic acid targeting a WNT signaling pathway inhibitor, or an antibody or small molecule targeting 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.

[0185] The transforming growth factor β (TGF-β) superfamily includes TGF-β protein, bone morphogenetic proteins (BMPs), growth differentiation factor (GDF), glial-derived neurotrophic factor (GDNF), activin, inhibin, Nodal, Lefty, and Müllerian inhibitory substances (MIS). Bone morphogenetic proteins (BMPs) are a group of growth factors, also known as cytokines and metabolic regulators. Initially discovered for their ability to induce bone and cartilage formation, BMPs are now considered to constitute a key set of morphogenetic signals that coordinate the structure of tissues throughout the body. The important physiological functions of BMP signaling are highlighted by the numerous roles of dysregulated BMP signaling in pathological processes.

[0186] BMPs interact with specific receptors on the cell surface called bone morphogenetic protein receptors (BMPRs). Signal transduction via BMPRs leads to the mobilization of members of the SMAD protein family. Signaling pathways involving BMPs, BMPRs, and SMADs are important in the development of the heart, central nervous system, and cartilage, as well as in postnatal skeletal development. They play a crucial role in embryonic pattern formation and early skeletal formation during embryonic development. Therefore, disruption of BMP signaling can affect the body blueprint of the developing embryo. As described and disclosed herein, bone morphogenetic proteins include, as non-limiting examples, BMP family ligands such as BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP15.

[0187] For example, BMP-4 and its inhibitors, head proteins and notochordin, help regulate embryonic polarity (i.e., dorsal-to-ventral pattern formation). In particular, BMP-4 and its inhibitors play a major role in neurogenesis and neural plate development. BMP-4 signals ectoderm cells to develop into skin cells, but inhibitors secreted by the underlying mesoderm block the action of BMP-4, allowing the ectoderm to continue its normal neural cell development process.

[0188] In some implementations, BMP is BMP4.

[0189] Vascular endothelial growth factor (VEGF), originally called vascular permeability factor (VPF), is a signaling protein produced by various cells that stimulates angiogenesis. Specifically, VEGF belongs to the platelet-derived growth factor subfamily of cystine knot growth factor. They are important signaling proteins involved in both angiogenesis (de novo formation of the embryonic circulatory system) and angiogenesis (growth of blood vessels from a pre-existing vascular system). It is part of the system that restores oxygen supply to tissues when blood circulation is insufficient (such as under hypoxic conditions). Serum concentrations of VEGF are higher in bronchial asthma and diabetes. The normal functions of VEGF are to generate new blood vessels during embryonic development, to generate new blood vessels after injury, to generate muscle after exercise, and to generate new blood vessels (collateral circulation) to bypass obstructed vessels. Vascular endothelial growth factor includes, as non-limiting examples, VEGF family ligands such as VEGF (VEGFA), VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placental growth factor (PIGF). In some embodiments, VEGF is VEGF-A.

[0190] In some embodiments, the chemical culture conditions of the method of the present invention include a reagent mixture comprising a WNT signaling pathway activator, BMP, and / or VEGF.

[0191] For example, the reagent mixture includes a WNT signaling pathway activator and BMP. In another example, the reagent mixture includes a WNT signaling pathway activator, BMP, and VEGF alone. In yet another example, the reagent mixture includes a WNT signaling pathway activator alone, BMP alone, or VEGF alone.

[0192] In some implementations, PSCs are grown on a substrate in the presence of WNT signaling pathway activators and bone morphogenetic protein (BMP) for approximately 1–8 days. For example, cells are grown in the presence of WNT signaling pathway activators and BMP for approximately 1, 2, 3, 4, 5, 6, 7, 8 days or more.

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

[0194] In some embodiments, after initial culture in the presence of a WNT signaling pathway activator and BMP, PSCs are grown on a substrate in the presence of VEGF, WNT signaling activator, and BMP for approximately 1–8 days. In some embodiments, PSCs are grown on a substrate in the presence of VEGF, WNT signaling activator, and BMP for approximately 1–2 days, 1–3 days, 1–4 days, 1–5 days, 1–6 days, 1–7 days, 1–8 days, or more than 8 days, or for any length of time between any two of the above or any two listed herein. For example, after initial culture, cells are grown in the presence of VEGF, WNT signaling activator, and BMP for approximately 1, 2, 3, 4, 5, 6, 7, 8 days, or more than 8 days, or for any length of time between any two of the above or any two listed herein.

[0195] In some implementations, PSC cultures are contacted with WNT signaling pathway activators and BMP for approximately 3 days, and then with VEGF for approximately 4 additional days.

[0196] In some implementations, the PSC culture is contacted with the WNT signaling pathway activator and BMP for about 2 days, 3 days or more, or any length of time between any two of the above or any two of the numbers listed herein, and then contacted with VEGF for about 3 additional days, 4 additional days or more, or any length of time between any two of the above or any two of the numbers listed herein.

[0197] In some embodiments, adherent cultures of PSCs are contacted with WNT signaling pathway activators, BMP, and / or VEGF to generate CD34. + Hematopoietic endothelium (HE).

[0198] Hematopoietic endothelium (HE) is composed of a specialized subset of endothelial cells scattered throughout blood vessels that can differentiate into hematopoietic cells. In the embryonic stage, hematopoietic cell development proceeds sequentially from the mesoderm through angiogenesis cells to hematopoietic endothelium and hematopoietic progenitor cells. Angiogenesis cells are pluripotent precursor cells that can differentiate into both hematopoietic cells and endothelial cells. Angiogenesis cells are progenitor cells that form blood islands and can differentiate into endothelial progenitor cells (EPCs) and blood cells. Angiogenesis cells are initially extracted from embryonic cultures and are manipulated by cytokines to differentiate along either the hematopoietic or endothelial pathway.

[0199] In some aspects, this disclosure provides a method for producing hematopoietic endothelial (HE) cells by: a) contacting a pluripotent stem cell (PSC) culture with a WNT signaling pathway activator and / or bone morphogenetic protein (BMP), wherein PSC growth lasts for approximately 1-7 days; and b) after step a) contacting the PSC culture with vascular endothelial growth factor (VEGF) for approximately 1-7 days, thereby generating CD34. + HE cell population. In some respects, the contact in step a), i.e., growing PSC cultures in the presence of WNT signaling pathway activators and / or bone morphogenetic protein BMP, lasts for approximately 1–7 days.

[0200] In some embodiments, the contact in step a) lasts for more than 7 days. In some embodiments, the contact in step a) lasts for as long as 1, 2, 3, 4, 5, 6, or 7 days, or more than 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step a) lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step a) lasts for 1 day. In some embodiments, the contact in step a) lasts for 2 days. In some embodiments, the contact in step a) lasts for 3 days. In some embodiments, the contact in step a) lasts for 4 days. In some embodiments, the contact in step a) lasts for 5 days. In some embodiments, the contact in step a) lasts for 6 days. In some embodiments, the contact in step a) lasts for 7 days. In some embodiments, the contact in step a) lasts for more than 7 days. In some implementations, the contact in step a) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0201] In some aspects, contacting the culture with VEGF in step b) optionally further includes contacting the culture with laurin or SB431542 to promote endothelial differentiation or hematopoietic differentiation, respectively. In some embodiments, any one of laurin or SB431542 at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 µM, or greater than 20 µM, or any concentration of laurin or SB431542 between the concentrations listed above or herein, is added to the culture medium during contacting step b). In some embodiments, in contacting step b), about 10 µM of laurin or SB431542 is optionally contacted with the culture.

[0202] In some respects, the contact in step b) occurs approximately 1–7 days after step a) when the PSC culture is contacted with VEGF, thereby generating CD34. +Hematopoietic endothelial cell population. In some embodiments, the contact in step b) lasts for more than 7 days. In some embodiments, the contact in step b) lasts for up to 1, 2, 3, 4, 5, 6, or 7 days, or more than 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step b) lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, or any length of time between any two of the above or mentioned herein. In some embodiments, the contact in step b) lasts for 1 day. In some embodiments, the contact in step b) lasts for 2 days. In some embodiments, the contact in step b) lasts for 3 days. In some embodiments, the contact in step b) lasts for 4 days. In some embodiments, the contact in step b) lasts for 5 days. In some embodiments, the contact in step b) lasts for 6 days. In some embodiments, the contact in step b) lasts for 7 days. In some embodiments, the contact in step b) lasts for more than 7 days. In some implementations, the contact in step b) lasts 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 any length of time between the above or any two of the numbers listed herein.

[0203] The following presents examples of methods for inducing PSC differentiation into natural killer (NK) cells and for significantly expanding them into NK cell populations contemplated for the applications discussed. These examples are provided to further illustrate implementations of the invention but are not intended to limit the scope of the invention. While they are typical of what may be used, other procedures, methods, or techniques known to those skilled in the art may be used alternatively.

[0204] Example

[0205] The present invention is further illustrated by the following non-limiting embodiments: Example 1

[0206] Design of a natural killer cell differentiation protocol

[0207] In a step-by-step iterative development process, a new combination of factors that promotes NK cell differentiation with high efficiency was identified using a simple and GMP-compliant workflow.

[0208] The NK cell differentiation and expansion protocol described in this article consists of three steps. The first stage focuses on generating CD34 from human pluripotent stem cells (hPSCs) using activators of the WNT signaling pathway, bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF). + / CD45 - Hematopoietic endothelial (HE) cells. Subsequently, the process progresses to the second stage, in which CD34... + / CD45 - Hematopoietic endothelial cells were cultured in serum-free medium, optionally supplemented with interleukin-7 (IL-7), NK-activating cytokines (such as interleukin-15 (IL-15) or any other NK-activating cytokines provided herein), stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (FLT3L) to form CD34. + / CD45 + Hematopoietic progenitor cells (HPCs). The final stage requires the differentiation of these progenitor cells into NK cells in a culture medium containing at least one serum-derived component (including, as a non-limiting example, serum itself, platelet lysate or serum-purified albumin, or any component or derivative of blood-derived plasma, such as obtained by serum fractionation), in addition to the continuous application of differentiation-inducing factors, such as IL-7, NK-activating cytokines (such as IL-15 or any other NK-activating cytokines provided herein), SCF, and FLT3L.

[0209] This method represents a significant leap forward in NK cell manufacturing, providing a feeder-free, regulatory-compliant approach that circumvents the safety concerns associated with traditional feeder cell techniques. By accelerating the expansion of NK cells to clinically suitable volumes, this approach represents a key advancement in the therapeutic deployment of NK cells, simplifying production while ensuring safety and efficacy.

[0210] Example 2

[0211] Materials and methods for natural killer cell differentiation

[0212] Table 1: Materials used for cell culture

[0213] Table 2: Antibodies for flow cytometry (all stored at 2–8°C, or optionally at -20°C after adding 80% (v / v) glycerol to a final concentration of 10%):

[0214] method: Maintenance of hiPSC (R26 line): HiPSCs were passaged at 200,000–250,000 cells per 6 wells (maximum 6 wells) on Monday morning and Thursday afternoon, respectively. Cells used for experiments were replastered on Thursday using the same cells as the hiPSCs for maintenance culture. Differentiation was initiated on Friday.

[0215] The following steps (1)-(41) provide an exemplary scheme for preparing a hiPSC for succession starting from Monday: on Monday: (1) Coat 2-4 6-well plates with 3 µl iMatrix-511 / well in 2 ml XF medium containing 10 µM Y-27632 (1:1000 Y-27632:1 µl Y / 1 ml medium) for maintenance culture of hiPSC, and continue coating at 37°C for at least 1 hour.

[0216] (2) Preheat Accutase and approximately 10 ml of XF medium. Transfer the required volume of Accutase (1 ml per well to be harvested) to separate tubes. Add Y-27632 at a ratio of 1:1000 to both solutions and mix.

[0217] (3) The maintenance wells of hiPSCs should be 50-100% confluent and undifferentiated. Vigorously shake the plate containing cells to collect all dead cells in the supernatant. Completely aspirate the culture medium and wash with 2 ml of PBS. Replace with 1 ml of pre-warmed Accutase containing Y-27632 and incubate for 10 min. Most cells should detach by gently shaking the plate. If this is not the case, extend the digestion for another 2 min, and so on, until the cells almost detach spontaneously.

[0218] (4) Add 3 ml of XF medium containing Y-27632 to a 15 ml tube. Rinse the cells from each well by pipetting up and down 3-4 times. Transfer the cells from all wells to a 15 ml tube containing 3 ml of XF medium + Y. Centrifuge at 300 g for 3 min. The supernatant should be clear and the cells should form a tight pellet.

[0219] (5) Aspirate the supernatant and resuspend each harvested 6-well plate in 2 ml of XF medium + Y medium, and pipette up and down 3 times with a 1 ml pipette. Immediately and gently transfer 10 µl to a counting chamber and determine the cell concentration.

[0220] (6) Spread 200,000 cells per well into each pre-coated 6-well plate. Transfer to an incubator and gently shake the plate at an infinite number of ticks inside the incubator.

[0221] Tuesday: (7) Replace the cells with pre-warmed XF medium (feed, replenish, feed), 2.5 ml per well.

[0222] Wednesday: (8) Change the medium for the cells with pre-warmed XF medium, 3 ml per well.

[0223] Thursday: (9) Confirm that the cells are sub-sinking and completely undifferentiated. In the afternoon, coat the well plates with 6 µl of iMatrix-511 and passage the cells as described above, except that only 1-2 wells are used and the cells are seeded at 200,000 cells per well. The remaining cells in the suspension will be seeded at 450,000 cells per well (50,000 cells / cm²). 2 The solution is applied to 6-well plates pre-coated with 6 µl iMatrix-511 for experiments. The number of wells can be varied depending on the experimental design.

[0224] (10) For differentiation of 12-well plates, the wells were coated with 3 µl iMatrix-511 in 1 ml PBS and incubated for 1 h. The cells were then seeded at 200,000 per well in 1 ml of medium containing 10 µM ROCK inhibitor.

[0225] Friday: (11) In the afternoon, the maintenance culture wells of hiPSC were changed with XF medium, 6 ml per well, until the end of the weekend.

[0226] Differentiation of hiPSCs (R26 lineage) into endothelial progenitor cells (EPCs): Friday: (12) Confirm that hiPSCs are evenly distributed in the wells used for differentiation. Cells should be flattened and form loose clonal populations at approximately 70-90% confluence.

[0227] (13) Prepare and / or preheat slightly more than 6 ml of MEI differentiation medium per differentiation well. Thaw the desired amount of CHIR99021 and BMP4 at room temperature for (2–5) min, then mix by gently flicking the tube. Optionally perform the same procedure for additional factors to be tested.

[0228] (14) Prepare differentiation media under different conditions in the optimal manner according to the experimental design. For example, if all wells are to receive the same amount of CHIR but different concentrations of BMP4, prepare a master mixture of MEI differentiation media and CHIR. Then aliquot into individual 15 (or 50) ml tubes and add appropriate amounts of other factors as needed. Alternatively, depending on the experimental design, additional factors can be added directly to the wells after replacing the consumed maintenance medium with differentiation media.

[0229] (15) After vigorously shaking the differentiation plate, replace the old XF medium with 6 ml of fresh MEI medium. Return it to the incubator for the weekend.

[0230] (16) 12-well version: Use 3 ml MEI differentiation medium per well.

[0231] on Monday: (17) Prepare and / or preheat slightly more than 3.5 ml of MEI differentiation medium per differentiation well. Thaw the required aliquots of VEGFA and SB431542 at room temperature for (2–5) min, then mix by gently flicking the tube. Optionally perform the same procedure for any additional factors to be tested. After vigorous shaking of the differentiation plate, replace the old MEI medium with fresh MEI medium.

[0232] (18) 12-well version: Use 1 ml per well.

[0233] Tuesday: (19) Prepare and / or pre-warm slightly more than 3.5 ml of MEI differentiation medium (same as Monday) per differentiation well. Thaw the required aliquots of VEGFA and SB431542 at room temperature for (2–5) min, then mix by gently flicking the tube. Optionally perform the same procedure for any additional factors to be tested. After vigorous shaking of the differentiation plate, replace the old MEI medium with fresh MEI medium.

[0234] (20) 12-well version: use 1 ml per well.

[0235] Wednesday: (21) Prepare and / or pre-warm slightly more than 3.5 ml of MEI differentiation medium (same as Monday) per differentiation well. Thaw the required aliquots of VEGFA and SB431542 at room temperature for (2–5) min, then mix by gently flicking the tube. Optionally perform the same procedure for any additional factors to be tested. After vigorous shaking of the differentiation plate, replace the old MEI medium with fresh MEI medium.

[0236] (22) 12-well version: use 1 ml per well.

[0237] Thursday: (23) Prepare and / or pre-warm slightly more than 3.5 ml of MEI differentiation medium (same as Monday) per differentiation well. Thaw the required aliquots of VEGFA and SB431542 at room temperature for (2–5) min, then mix by gently flicking the tube. Optionally perform the same procedure for any additional factors to be tested. After vigorous shaking of the differentiation plate, replace the old MEI medium with fresh MEI medium.

[0238] (24) 12-well version: Use 1 ml per well.

[0239] Differentiation of hematopoietic endothelial cells into NK cells: (25) On Friday (Day 7), aspirate the old MEI medium and wash each well very gently once with 1 ml PBS. Replace with pre-warmed NK differentiation medium + IL-3, 1 ml per well in a 12-well plate. Add the medium very slowly, expecting 0-30% cell clumps to detach. The first suspension cells should appear between Day 10 and Day 14. Ideally, the culture yield will be 1.5 million cells per well on Day 14, while the minimum yield at this point should be 500,000 cells.

[0240] (26) NK amplification version: Aspirate the old medium and replace it with 1 ml of fully defined medium (such as APEL2) without washing and adding factors. On Monday (day 10), perform a complete medium change with the same medium and optionally use SCF, with or without IL-7, to increase endothelial-to-hematopoietic transformation (EHT) yield.

[0241] (27) On Friday (Day 14), perform a 'centrifugation change' by collecting the suspension cells (using a p1000 pipette, very gently, with the least pressure, drop the old medium onto the adherent layer, then transfer the suspension cells to a collection tube), centrifuging at 300 g for 8 min, and resuspending the pellet in fresh NK differentiation medium (without IL-3). Do not allow the adherent cells to dry out, and immediately add half the final volume of fresh medium (0.5 ml) after collecting the suspension cells. Ideally, use 15 ml tubes for centrifugation. Resuspend the cells in half the final plate medium volume (6 ml total), then distribute the cells evenly into the wells (0.5 ml per well).

[0242] (28) NK amplification version: Aspirate the old culture medium and any suspended cells, and replace with 1 ml of the same culture medium formulation as on day 10.

[0243] (29) On day 17 (Monday), repeat the centrifugation and medium change procedure, but now resuspend each well in 2 ml. From day 20 onwards, perform half-volume medium changes (HMC) twice a week, every 3–4 days. For example, as early as possible on Monday and as late as possible on Thursday to achieve an ideal average medium change interval of 3.5 days. For HMC, carefully tilt the plate about 30 degrees toward yourself, and then use p1000 to aspirate 0.9 ml from the surface of each well without disturbing the suspended cell layer at the bottom. Gently drop in 1 ml of fresh NK differentiation medium, avoiding disturbing the settled cells.

[0244] (30) NK amplification version: On day 17, 200K cells were transferred to new wells (12-well plates) and 1 ml of fully defined medium (such as APEL2) containing four factors (4F) SCF, FLT3L, IL-7 and IL-15 (20, 10, 20 and 10 ng / ml, respectively) was added.

[0245] (31) For cell count tracking during HMC, measure the exact culture medium volume after discarding 0.9 ml: aspirate 0.8 ml with a p1000 pipette, then slowly rotate the pipette to aspirate the remaining volume in the well until air is aspirated, and record the value. Gently mix this volume to resuspend all suspended cells, then take 10 µl for counting (hemocytometer or diluted in 90 µl of NK basal medium, then using NC-200). Multiply the cell count per ml by the measured culture medium volume to obtain the accurate cell count for that well. Note that this will interfere with the adherent layer and reduce NK cell yield compared to undisturbed cells.

[0246] (32) On day 21 (NK amplification only), add 1 ml of fresh medium with the same formulation as day 17 (4F).

[0247] (33) On day 24 and day 26 (NK amplification only), HMC was performed using the same medium.

[0248] (34) On day 28 (NK expansion only), half of the cells were transferred to new wells and 1 ml of NK differentiation medium (NK dif. 4F) containing all four factors (at normal concentrations) was added. It was expected that the 200K cells from day 17 had expanded to 3-4 million NK progenitor cells by this point. Day 28 was the last possible harvest point before the NK progenitor cell population collapsed without exposure to undefined media (such as those containing hPL).

[0249] (35) On day 31 (NK expansion only), passage cells 1:10, or aim for >500K per well in 1 ml NK dif. 4F medium. Perform HMC on days 33 and 35, and passage again 1:10 on day 38, again aiming for >500K per well in 1 ml NK dif. 4F. Repeat this process until NK cell expansion stops, expected to last >3 weeks, achieving >1000-fold expansion.

[0250] (36) Do not separate the suspension cells from the adherent layer until they are ready for expansion (days 28–35, >90% CD56 positive). The cell count should be slowly reduced from 4E6 on day 40 to 2.5E6 on day 70.

[0251] Flow cytometry: (37) For flow cytometry, 1 ml of culture medium containing cells is centrifuged at 400 g for 3 min, then resuspended in 1 ml of PBS and centrifuged again at 400 g for 3 min.

[0252] (38) In a 1.5 ml tube, 500,000 cells from each staining group were dispensed into 98 µl of PBS.

[0253] (39) Add 2 µl of the corresponding antibody for staining, gently pat to mix, and incubate at room temperature in the dark for 20 min.

[0254] (40) Washing procedure: Add 400 µl PBS to each tube and centrifuge at 400 g for 2 min. Discard the supernatant.

[0255] (41) Add 300 µl of PBS to each tube and resuspend. Perform flow cytometry. After the analysis is complete, save the file to G-drive and analyze the flow cytometry results using analysis software.

[0256] NK lethality test: K562 cells were resuspended in PBS containing 2.5 µM CellTrace-Violet (CTV, Thermo Fisher Scientific, C34564) at 1 million cells / ml and incubated at 37°C for 20 minutes. Staining was terminated by adding 5 volumes of hPL-based medium (DF12 containing 15% hPL (PL BioScience), 1x Glutamax, and 250 µM 2-phosphate-L-ascorbic acid, without additional cytokines) and incubating at 37°C for 5 minutes. These cells were then resuspended in fresh hPL-based medium at 500,000 cells / ml. NK cells were also resuspended in the same medium at 500,000 cells / ml. Both groups were mixed in 96-well round-bottom plates at NK:K562 ratios of 1:5, 1:1, and 5:1, to a final volume of 225 µl. A control containing 100% K562 was used. Cells were briefly centrifuged and then incubated at 37°C. After 3.5 hours, 30 µl of PBS containing 20 µM CellEvent Caspase-3 / 7 Green (Thermo Fisher Scientific, C10423) was added to each well, and the cells were centrifuged again and incubated at 37°C. Five minutes before the end of the incubation period, 30 µl of PBS containing 10 µM SYTOX 7AAD (Thermo Fisher Scientific, S10274) was added to each well. After a total of 4 hours, the plate was analyzed by flow cytometry using a Miltenyi MACS Quant10. Gating strategy (based on K562 control cells): P1: All events in the forward / lateral scatter plot minus CTV-negative fragments. P2: Exclusion of diploids. P3: CTV-positive cells. P4 / 5: One axis is Caspase-3 / 7 green, and the other axis is 7AAD. Caspase single positive cells and Caspase / 7AAD double positive cells are measured relative to the K562 control group (which can be up to 5% positive).

[0257] Abbreviations and Standard Reagents:

[0258] Example 3

[0259] Generation and characterization of natural killer cells differentiated from human pluripotent stem cells

[0260] Differentiation into another cell type highly relevant to immuno-oncology, particularly natural killer (NK) cells, has been thoroughly investigated. A set of four established signaling factors were used as clues to explore which basal medium would optimally support NK differentiation. Screening of various commercial and custom basal media, all supplemented with the same signaling molecules, revealed significant differences. The formulation based on chemically defined APEL medium was significantly superior to the others, exhibiting increased cell counts and NK differentiation efficiency, as demonstrated by CD56 expression. Figure 1 Another medium, DF12 supplemented with human platelet lysate (hPL) and L-ascorbic acid, promoted NK cell induction but was less effective in promoting sustained cell proliferation. Figure 1 (The blue curve in the image).

[0261] Furthermore, these culture media consistently achieve NK cell induction in different independent iPSC lines using a designated HPC platform. Figure 2 Since the experiments were conducted in original wells containing residual adherent cells, efforts were made to eliminate potential indirect effects and evaluate the scale-up strategy by transferring the HPCs on day 17 to new containers. Interestingly, in APEL2 medium supplemented with the factor, cell numbers increased in the initial 10 days and then decreased, although various viable cells maintained NK cell fate (…). Figure 3A The time of cell count decline is related to the time of endothelial-to-hematopoietic transition (EHT). Figure 3B This indicates that neither the defined APEL2-based culture medium nor the undefined alternative is sufficient on its own to achieve significant expansion after NK cell differentiation.

[0262] An interesting observation was obtained regarding the kinetics of HPC loss and NK cell identity acquisition, where culture collapse at approximately four weeks in APEL2-based medium coincided with significant HPC-NK conversion. Figure 4 This indicates that while the defined culture medium supports progenitor cell proliferation, its effectiveness is diminished after transformation into early NK cells. The study then explored whether switching to undefined hPL medium could enhance proliferation and generate pure, differentiated NK cells, as indicated by CD56 expression and the presence of functional markers (2B4, NKp30, NKp44, NKp46). Figure 5A and Figure 5B This replacement resulted in a significant increase in cell proliferation and the production of highly purified NK cells.

[0263] Flow cytometry analysis showed that terminating the differentiation process with an undefined but GMP-compliant hPL-based medium yielded more mature cells, as indicated by higher CD38 levels and the activation marker NKp44. Figure 4RNA-seq analysis of iPSC-NK cells derived in hPL-based media confirmed robust expression of genes essential for cytotoxicity. Figure 6 These cells exhibited significant cytotoxic activity against K562 leukemia cells, achieving 90% cytotoxicity within just four hours at an effector cell to target cell ratio of 5:1. Figure 7 ).

[0264] The significant proliferation capacity of iPSC-MSCs from certain iPSC lines demonstrates the feasibility of intermediate banking (intermediate cell bank, intermediate cryopreservation) to preserve downstream expansion potential. Figure 8 (Second subfigure). In contrast, cardiomyocytes derived from iPSCs showed limited proliferative capacity, highlighting the necessity of amplification at the iPSC stage ( Figure 8 (Third sub-figure). For immune cells, scaling up at the precursor stage may be beneficial to avoid depletion of the final differentiation product. Figure 8 (Figure 4). Based on current understanding, this strategy of expanding iPSC-NK cells after differentiation represents a novel approach.

[0265] While preferred embodiments of the 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. Many variations, modifications, 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 can be used to practice the invention. The scope of the invention is intended to be defined by the appended claims, and thereby covers the methods and structures within the scope of those claims and their equivalents.

Claims

1. A method for producing natural killer (NK) cells, comprising: a) Contact pluripotent stem cell (PSC) cultures with WNT signaling pathway activators and / or bone morphogenetic protein (BMP), wherein PSC growth lasts for approximately 1–7 days; b) Following step a), the PSC culture is exposed to vascular endothelial growth factor (VEGF) for approximately 1–7 days to generate at least approximately 80% enriched CD34. + CD34 + Precursor cell population; c) The CD34 + The precursor cell population was incubated in a medium lacking any serum-derived reagents for approximately 7–28 days, while optionally supplementing the medium with one or more of the following: interleukin-7 (IL-7); NK-activating cytokines, optionally IL-15; stem cell factor (SCF); and FMS-like tyrosine kinase 3 ligand (FLT3L), thereby producing CD34. + / CD45 + Suspended cell populations; and d) The CD34 + / CD45 + Suspension cell populations were incubated for approximately 7–35 days in a medium containing: at least one serum-derived reagent; IL-7; NK-activating cytokine, optionally IL-15; SCF and FLT3L. This leads to the production of NK cells.

2. The method according to claim 1, wherein, The serum-derived reagent is selected from serum, platelet lysate, albumin derived from blood, or plasma components or plasma derivatives obtained from blood.

3. The method according to claim 1, wherein, The cells in step c) and / or step d) are transient CD34 cells. + .

4. The method according to claim 1, wherein, The culture medium in step c) and / or step d) contains approximately 4-40 ng / ml IL-7, approximately 2-20 ng / ml IL-15, approximately 4-40 ng / ml SCF and / or approximately 1-20 ng / ml FLT3L.

5. The method according to claim 4, wherein, The culture medium contains approximately 20 ng / ml IL-7, approximately 10 ng / ml IL-15, approximately 20 ng / ml SCF and / or approximately 10 ng / ml FLT3L.

6. The method according to claim 1, wherein, Expose PSC cultures to one or more of the following reagents: approximately 1–10 µM WNT signaling pathway activator, approximately 5–50 ng / ml BMP, approximately 50–500 ng / ml VEGF.

7. The method according to claim 6, wherein, The WNT signaling pathway activator is a GSK3 inhibitor.

8. The method according to claim 7, wherein, The GSK3 inhibitor is CHIR99021.

9. The method according to claim 6, wherein, The BMP is BMP4.

10. The method according to claim 6, wherein, The VEGF mentioned is VEGF-A.

11. The method according to claim 6, wherein, The PSC culture was contacted with approximately 8 µM CHIR99021, approximately 25 ng / ml BMP4 and / or approximately 200 ng / ml VEGFA.

12. The method according to claim 1, wherein, The PSC culture from step a) is kept in contact with the WNT signaling pathway activator and BMP for approximately 2-5 days.

13. The method according to claim 1, wherein, The PSC was then kept in contact with VEGF for approximately 2-5 days.

14. The method according to claim 1, wherein, The NK cells are produced in cultures selected from: adherent cell layers, two-dimensional culture systems, microcarriers, coated surfaces including laminin coatings, three-dimensional cultures, and suspension cultures.

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

16. The method according to claim 1, wherein, The PSC is a human PSC (hPSC).

17. The method according to claim 16, wherein, hPSCs are either human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

18. The method according to claim 1, wherein, CD34 + The precursor cells are CD34 + Endothelial-like precursor cells.

19. The method according to claim 1, wherein, The NK cells were enriched at least approximately 80%.

20. The method according to claim 1, wherein, The NK cells were CD56. + 2B4 + NKp30 + NKp44 + NKp46 + NKG2D + and / or CD16 - .

21. The method according to claim 1, wherein, The NK cells were CD56. 高表达 or CD56 低表达 .

22. The method according to claim 1, wherein, The NK cells mentioned are cytotoxic NK cells.

23. The method according to claim 1, wherein, d) enriched the number of cells by at least 100 times compared to the number of cells in c).

24. The method according to claim 1, wherein, d) enriched the number of cells by at least 200 times compared to the number of cells in c).

25. A method for producing natural killer (NK) cells from pluripotent stem cells (PSCs), comprising: a) Generate CD34 using the following method + Hematopoietic endothelial (HE) cells: (i) Contacting PSC cultures with WNT signaling pathway activators and bone morphogenetic protein (BMP), wherein the PSC growth lasts for approximately 3 days; and (ii) Subsequently, the cells from (i) were exposed to vascular endothelial growth factor (VEGF) for approximately 4 days; This generates CD34 containing at least 80% + HE cell population; b) Take the CD34 from step a) + HE cells were cultured in a medium lacking any serum-derived reagents for approximately 7–28 days, with optional addition of one or more of IL-7, IL-15, SCF, and / or FLT3L, to generate cells containing at least 80% CD34. + / CD45 + Transient suspension of hematopoietic progenitor cells (HPC); c) Make CD34 in step b) + / CD45 + HPC was exposed to one or more of IL-7, IL-15, FLT3L, and SCF in a culture medium lacking any serum-derived reagents for approximately 7–14 days; and d) The CD34 + / CD45 + HPC cells were transferred to a culture medium containing at least one serum-derived reagent, as well as IL-7, IL-15, FLT3L, and SCF, and remained there for approximately 7–35 days. This induces the differentiation of PSCs into NK cells.

26. The method according to claim 25, wherein, The NK cells were CD56. + 2B4 + NKp30 + NKp44 + NKp46 + NKG2D + and / or CD16 - .

27. The method according to claim 25, wherein, The NK cells were CD56. 高表达 or CD56 低表达 .

28. The method according to claim 25, wherein, The NK cells mentioned are cytotoxic NK cells.

29. The method according to claim 25, wherein, d) enriched the number of cells by at least 100 times compared to the number of cells in c).

30. The method according to claim 25, wherein, d) enriched the number of cells by at least 200 times compared to the number of cells in c).

31. The method according to claim 1 or 25, wherein, The NK cells are produced in a feeder-free manner.

32. A method for producing hematopoietic endothelial (HE) cells, comprising: a) Contacting pluripotent stem cell (PSC) cultures with WNT signaling pathway activators and / or bone morphogenetic proteins (BMPs), wherein PSC growth lasts approximately 1–7 days; and b) Following step a), the PSC culture is exposed to vascular endothelial growth factor (VEGF) for approximately 1–7 days to generate CD34. + Hematopoietic endothelial cell population.

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