Method for differentiating pluripotent stem cells into hematopoietic progenitor cells and stem cells

Abstract

The present invention relates to CD34 + A method for producing a population of hematopoietic progenitor cells is provided. + Hematopoietic progenitor cells are used in methods for producing natural killer (NK) cells, methods for inducing differentiation of NK cells from pluripotent stem cells (PSCs), and methods for generating terminally differentiated hematopoietic cells from PSCs. Differentiation of immune cells, such as NK cells, from PSCs involves the use of a hemogenic endothelial induction cocktail containing a WNT signaling pathway activator, bone morphogenetic protein, and / or vascular endothelial growth factor. Methods for producing hematopoietic stem cells from pluripotent stem cells are also provided.

Description

[Technical Field]

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 406,185, filed September 13, 2022, and U.S. Provisional Patent Application No. 63 / 449,506, filed March 2, 2023. The disclosures of the prior applications are considered part of the disclosure of this application in their entireties and are incorporated herein by reference.

[0002] The present invention relates generally to hematopoietic cells, and more particularly to methods for generating hematopoietic progenitor and stem cells from pluripotent stem cells (PSCs). [Background technology]

[0003] Pluripotent stem cells are capable of self-renewal and can give rise to all three major cell groups that make up the human body: ectoderm (cells of the skin and nervous system), endoderm (including cells of the gastrointestinal tract and respiratory tract, endocrine gland cells, liver cells, and pancreatic cells), and mesoderm (bone, cartilage, most circulatory system cells, muscle cells, connective tissue cells, and others). Pluripotent stem cells can be induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Because they can reproduce indefinitely and give rise to every cell type in the body, they represent a potential source for the development of therapeutic cells. Among many other things, PSCs can be differentiated into hematopoietic progenitor cells, which have the potential to generate any cell type from the hematopoietic cell lineage.

[0004] Hematopoietic stem cells (HSCs) are stem cells that give rise to other blood cells through hematopoiesis. In adults, hematopoiesis occurs in the red bone marrow, located in the core of most bones. Red bone marrow is derived from the mesoderm. During hematopoiesis, all mature blood cells are produced. Production demand (on average, humans produce over 500 billion blood cells daily) must be balanced with the need to regulate the number of each blood cell type in circulation. Hematopoietic stem cells give rise to different types of blood cells: myeloid cells and lymphoid cells. Both the myeloid and lymphoid lineages are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes, as well as platelets. Lymphoid cells include T cells, B cells, natural killer cells, and innate lymphoid cells. Hematopoietic tissues contain cells with long-term and short-term regenerative potential, as well as committed multipotent, oligopotent, and unipotent progenitors. Hematopoietic stem cells make up 1:10,000 of the cells in bone marrow tissue. In clinical settings, they are used for HSC transplantation in the treatment of cancer and other immune system disorders.

[0005] Natural killer cells, also known as NK cells or large granular lymphocytes (LGLs), belong to a rapidly expanding family of known innate lymphoid cells (ILCs) and represent 5–20% of all circulating lymphocytes in humans. They are a type of cytotoxic lymphocyte important to the innate immune system. NK cells' role is similar to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide a rapid response against virus-infected cells and other intracellular pathogens, acting approximately three days after infection, and respond to tumorigenesis. Typically, immune cells detect major histocompatibility complexes (MHC) displayed on the surface of infected cells, triggering cytokine release and causing the infected cell's death by lysis or apoptosis. However, NK cells are unique because they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, enabling a much more rapid immune response. They are called "natural killer cells" because they do not require activation to kill cells lacking the MHC class I "self" marker. This role is particularly important because harmful cells that lack the MHC I marker cannot be detected and destroyed by other immune cells, such as T lymphocytes.

[0006] In addition to natural killer cells being effectors of innate immunity, both activating and inhibitory NK cell receptors play important functional roles, including self-tolerance and maintenance of NK cell activity. NK cells also play a role in adaptive immune responses, and numerous experiments have demonstrated their ability to readily adapt to the immediate environment and form antigen-specific immune memory, which is the basis for responding to secondary infections with the same antigen. The role of NK cells in both innate and adaptive immune responses has become increasingly important in research into the use of NK cell activity as a potential cancer therapy. Summary of the Invention [Problem to be solved by the invention]

[0007] There remains an unmet need in the art to develop methods of PSC differentiation that yield efficient and pure cell cultures in short periods of time using simple culture conditions. [Means for solving the problem]

[0008] (Summary of the Invention) The present invention is based on the seminal discovery that the combination of an endothelial-inducing cocktail containing bone morphogenetic proteins (BMPs) and WNT signaling activators generates endothelial-like progenitor cells that exhibit hybrid characteristics of endothelial and hematopoietic progenitor cells, which can further give rise to terminally differentiated hematopoietic cells such as NK cells. The combination of factors can be used to generate hematopoietic stem cells.

[0009] In one embodiment, the present invention relates to a method for detecting CD34 + 1. A method of producing a population of hematopoietic progenitor cells, comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) and allowing the PSCs to grow on a substrate for about 1-8 days; and b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF), alone or in combination with the WNT signaling pathway activator and / or BMP, for about 1-8 days after step a), wherein the cells produced after step b) are CD34+ in the entire population of cells. + cells, thereby enriched by at least about 80% for CD34 + The present invention provides a method for producing a population of progenitor cells.

[0010] In one embodiment, CD34+ hemogenic endothelium (HE) is generated by contacting adherent cultures of PSCs with a WNT signaling pathway activator, BMP, and / or VEGF. + The progenitor cell population is exposed to a cocktail of drugs to induce CD34 +In some embodiments, the hematopoietic lineage cells are natural killer (NK) cells or other immune cells. In one embodiment, the culture of PSCs is contacted with a WNT signaling pathway activator and a BMP for about 3 days, and with VEGF for an additional about 4 days. In some embodiments, the cells treated for about 1 week are CD34 + , K.D.R. + , CD31 + and CD45 - In another embodiment, CD34 + The cells also express CD144 + It is also.

[0011] In another embodiment, the present invention provides a method for producing natural killer (NK) cells, comprising: a) contacting a culture of PSCs with a WNT signaling pathway activator and / or a BMP and allowing the PSCs to grow on a substrate for about 1-8 days; b) contacting the culture of PSCs with VEGF, alone or in combination with a WNT signaling pathway activator and / or a BMP, for about 1-8 days after step a), thereby producing CD34 + generating a population of progenitor cells, and after step b) determining whether the cells generated are CD34 + c) at least about 80% enriched for CD34 cells; + The method includes contacting a population of progenitor cells with one or more of interleukin 7 (IL-7), IL-15, SCF, and FMS-like tyrosine kinase 3 ligand (FLT3L), thereby producing NK cells.

[0012] In one embodiment, c) CD34 + Contacting a population of progenitor cells includes (i) CD34 + (ii) contacting the progenitor cells with IL-7, IL-15, SCF, and FLT3L for about 5 to 10 days; and + In another embodiment, the progenitor cells are transiently transfected with CD34. +and CD45 + In one embodiment, the contacting of adherent cultures of PSCs comprises one or more agents selected from about 1-10 μM of a WNT signaling pathway activator, about 5-50 ng / ml of BMP, and about 50-500 ng / ml of VEGF. In some embodiments, the WNT signaling pathway activator is a GSK3 inhibitor. In various embodiments, the GSK3 inhibitor is CHIR99021. In other embodiments, the BMP is BMP4. In some embodiments, the VEGF is VEGF-A. In another embodiment, the contacting of adherent cultures of PSCs comprises about 8 μM of CHIR99021, about 25 ng / ml of BMP4, and / or about 200 ng / ml of VEGFA. In one embodiment, CD34 + The progenitor cells are contacted with about 4-40 ng / ml of IL-7, about 2-20 ng / ml of IL-15, about 4-40 ng / ml of SCF, and / or about 1-20 ng / ml of FLT3L. + The progenitor cells are contacted with about 20 ng / ml IL-7, about 10 ng / ml IL-15, about 20 ng / ml SCF, and / or about 10 ng / ml FLT3L. In one embodiment, the contacting of the adherent culture of PSCs in a) with a WNT signaling pathway activator and BMP is for about 2 to 5 days. In another embodiment, the PSCs are subsequently contacted with VEGF for about 2 to 5 days. In one embodiment, CD34 + Contacting the population of progenitor cells with IL-7, IL-15, SCF and / or FLT3L for approximately 5-10 days followed by CD34 +Contacting the population of progenitor cells with IL-7, IL-15, FLT3L and / or SCF is for at least about 7-21 days. In another embodiment, the culture of PSCs is an adherent layer of cells. In some embodiments, the layer of cells is grown in a two-dimensional culture system or on microcarriers. In other embodiments, the PSCs are cultured on a coated surface comprising a laminin coating. In one embodiment, NK cells are further collected from the suspension in cell culture medium. In another embodiment, the PSCs are human PSCs (hPSCs). In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs). In various embodiments, CD34 + Progenitor cells express CD34 + In one embodiment, the NK cells are enriched by at least about 80%.

[0013] In an additional embodiment, the present invention provides a method for inducing differentiation of PSCs into NK cells, comprising: a) (i) contacting an adherent culture of PSCs with a WNT signaling pathway activator and a BMP for about 3 days; and (ii) contacting an adherent culture of PSCs of i) with VEGF for about 4 days, thereby increasing the differentiation rate of at least 80% of CD34 + By generating a population of cells containing hemogenic endothelial (HE) cells, CD34 + b) generating HE cells; and + The HE cells are contacted with one or more of IL-7, IL-15, SCF and / or FLT3L for about 7 days, thereby reducing at least 80% of the CD34 + / CD45 + b) generating a transient population of cells containing hematopoietic stem cells (HSCs), and c) subsequently CD34 + / CD45 + The method includes contacting HSCs with one or more of IL-7, IL-15, FLT3L, and SCF for at least about 7 to 21 days, thereby inducing differentiation of PSCs into NK cells.

[0014] In one embodiment, the differentiated NK cells are CD56 + , NKp30 +, NKp44 + , NKp46 + , NKG2D + , NKG2A + , KIR2D + and / or CD16 + In another embodiment, the differentiated NK cells are CD56 bright or CD56 dim In various embodiments, the differentiated NK cells are cytotoxic NK cells.

[0015] In a further embodiment, the present invention provides a kit comprising: a) an HE induction cocktail comprising a WNT signaling pathway activator, BMP and / or VEGF; b) an NK induction cocktail comprising IL-7, IL-15, SCF and / or FLT3L; and c) instructions for inducing differentiation of pluripotent stem cells (PSCs) into NK cells.

[0016] In one embodiment, the kit further comprises a laminin-coated surface.

[0017] In one embodiment, the present invention provides a method for generating terminally differentiated hematopoietic cells from PSCs, comprising: a) (i) contacting an adherent culture of PSCs with a WNT signaling pathway activator and a BMP, and allowing the PSCs to grow on a substrate for about 2-5 days; and (ii) contacting the adherent culture of PSCs with VEGF for about 2-5 days after step (i), wherein the cells produced after step (ii) are CD34+ / CD36+ cells throughout the population of cells. + cells, thereby enriched by at least about 80% for CD34 + By generating progenitor cells, CD34 + generating hematopoietic progenitor cells, and b) CD34 + The progenitor cells are contacted with a mixture of drugs to induce CD34 + The present invention provides a method for inducing differentiation of progenitor cells into terminally differentiated hematopoietic cells, thereby generating terminally differentiated hematopoietic cells.

[0018] In one embodiment, the terminally differentiated hematopoietic cell is an immune cell or an erythrocyte. In various embodiments, the immune cell is selected from the group consisting of a macrophage, a T cell, and a NK cell.

[0019] In another embodiment, the present invention provides a method for detecting CD34 + 1. A method of producing a population of hematopoietic stem cells, comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) and allowing the PSCs to grow on a substrate for about 1-8 days; and b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF), alone or in combination with the WNT signaling pathway activator and / or BMP, for about 1-8 days after step a), wherein the cells produced after step b) are at least about 80% enriched for CD34+ hematopoietic stem cells in the overall population of cells, thereby increasing the CD34 + The present invention provides a method for producing a population of hematopoietic stem cells.

[0020] In one embodiment, CD34+ hemogenic endothelium (HE) is generated by contacting adherent cultures of PSCs with a WNT signaling pathway activator, BMP, and / or VEGF. In another embodiment, the PSCs are human PSCs (hPSCs). In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

[0021] In a further embodiment, the present invention provides a method for generating hematopoietic stem cells from pluripotent stem cells (PSCs), comprising: a) contacting an adherent culture of induced pluripotent stem cells (iPSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), and allowing the PSCs to grow on a substrate for about 2-5 days; and b) contacting the adherent culture of PSCs with vascular endothelial growth factor (VEGF) for about 2-5 days after step (a), wherein the cells produced after step (b) are CD34+ in the entire population of cells. + cells, thereby enriched by at least about 80% for CD34 + The present invention provides a method for producing progenitor cells, thereby producing hematopoietic stem cells.

[0022] In one embodiment, the iPSCs are human iPSCs (hiPSCs). [Brief explanation of the drawings]

[0023] [Figure 1] 1 is an exemplary diagram of the method of the present invention. [Figure 2A] 2A and 2B are exemplary schematic diagrams of the methods of the invention. Figure 2A illustrates a method of the invention. Figure 2B illustrates a method of the invention in which cells are cultured in stem cell medium for the first 7 days of induction. Figure 2C illustrates a method of the invention in which cells are cultured in stem cell medium, defined serum-free and animal component-free PSC differentiation medium, or hPL-containing medium. [Figure 2B] 2A and 2B are exemplary schematic diagrams of the methods of the invention. Figure 2A illustrates a method of the invention. Figure 2B illustrates a method of the invention in which cells are cultured in stem cell medium for the first 7 days of induction. Figure 2C illustrates a method of the invention in which cells are cultured in stem cell medium, defined serum-free and animal component-free PSC differentiation medium, or hPL-containing medium. [Figure 2C] 2A and 2B are exemplary schematic diagrams of the methods of the invention. Figure 2A illustrates a method of the invention. Figure 2B illustrates a method of the invention in which cells are cultured in stem cell medium for the first 7 days of induction. Figure 2C illustrates a method of the invention in which cells are cultured in stem cell medium, defined serum-free and animal component-free PSC differentiation medium, or hPL-containing medium. [Figure 3A]Figure 3 illustrates cell characterization and survival in different methods of the present invention. Figure 3A is a line graph illustrating the expression of HSC markers (CD34), leukocyte markers (CD45), and endothelial markers (CD144) by pluripotent stem cells over the course of an induction protocol. Figure 3B is a line graph illustrating the expression of endothelial and hematopoietic markers in adherent cells and cells in suspension over the course of an induction protocol. Figure 3C is a line graph illustrating the variation in cell number in suspension based on the stem cell medium, defined medium, or hPL-containing medium used for cell differentiation. Figure 3D is a line graph illustrating cell viability in different media (stem cell basal medium, defined medium, or hPL-containing medium) used for cell differentiation. Figure 3E is a line graph illustrating the difference in cell number in suspension based on the date of cell transfer. [Figure 3B] Figure 3 illustrates cell characterization and survival in different methods of the present invention. Figure 3A is a line graph illustrating the expression of HSC markers (CD34), leukocyte markers (CD45), and endothelial markers (CD144) by pluripotent stem cells over the course of an induction protocol. Figure 3B is a line graph illustrating the expression of endothelial and hematopoietic markers in adherent cells and cells in suspension over the course of an induction protocol. Figure 3C is a line graph illustrating the variation in cell number in suspension based on the stem cell medium, defined medium, or hPL-containing medium used for cell differentiation. Figure 3D is a line graph illustrating cell viability in different media (stem cell basal medium, defined medium, or hPL-containing medium) used for cell differentiation. Figure 3E is a line graph illustrating the difference in cell number in suspension based on the date of cell transfer. [Figure 3C]Figure 3 illustrates cell characterization and survival in different methods of the present invention. Figure 3A is a line graph illustrating the expression of HSC markers (CD34), leukocyte markers (CD45), and endothelial markers (CD144) by pluripotent stem cells over the course of an induction protocol. Figure 3B is a line graph illustrating the expression of endothelial and hematopoietic markers in adherent cells and cells in suspension over the course of an induction protocol. Figure 3C is a line graph illustrating the variation in cell number in suspension based on the stem cell medium, defined medium, or hPL-containing medium used for cell differentiation. Figure 3D is a line graph illustrating cell viability in different media (stem cell basal medium, defined medium, or hPL-containing medium) used for cell differentiation. Figure 3E is a line graph illustrating the difference in cell number in suspension based on the date of cell transfer. [Figure 3D] Figure 3 illustrates cell characterization and survival in different methods of the present invention. Figure 3A is a line graph illustrating the expression of HSC markers (CD34), leukocyte markers (CD45), and endothelial markers (CD144) by pluripotent stem cells over the course of an induction protocol. Figure 3B is a line graph illustrating the expression of endothelial and hematopoietic markers in adherent cells and cells in suspension over the course of an induction protocol. Figure 3C is a line graph illustrating the variation in cell number in suspension based on the stem cell medium, defined medium, or hPL-containing medium used for cell differentiation. Figure 3D is a line graph illustrating cell viability in different media (stem cell basal medium, defined medium, or hPL-containing medium) used for cell differentiation. Figure 3E is a line graph illustrating the difference in cell number in suspension based on the date of cell transfer. [Figure 3E]Figure 3 illustrates cell characterization and survival in different methods of the present invention. Figure 3A is a line graph illustrating the expression of HSC markers (CD34), leukocyte markers (CD45), and endothelial markers (CD144) by pluripotent stem cells over the course of an induction protocol. Figure 3B is a line graph illustrating the expression of endothelial and hematopoietic markers in adherent cells and cells in suspension over the course of an induction protocol. Figure 3C is a line graph illustrating the variation in cell number in suspension based on the stem cell medium, defined medium, or hPL-containing medium used for cell differentiation. Figure 3D is a line graph illustrating cell viability in different media (stem cell basal medium, defined medium, or hPL-containing medium) used for cell differentiation. Figure 3E is a line graph illustrating the difference in cell number in suspension based on the date of cell transfer. [Figure 4A] Photographs illustrating bright light imaging of cells during the course of the induction protocol. Figure 4A shows cells at day 0. Figure 4B shows cells at day 3. Figure 4C shows cells at day 7. Figure 4D shows cells at day 10. Figure 4E shows cells at day 14. Figure 4F shows cells at day 21. [Figure 4B] Photographs illustrating bright light imaging of cells during the course of the induction protocol. Figure 4A shows cells at day 0. Figure 4B shows cells at day 3. Figure 4C shows cells at day 7. Figure 4D shows cells at day 10. Figure 4E shows cells at day 14. Figure 4F shows cells at day 21. [Figure 4C] Photographs illustrating bright light imaging of cells during the course of the induction protocol. Figure 4A shows cells at day 0. Figure 4B shows cells at day 3. Figure 4C shows cells at day 7. Figure 4D shows cells at day 10. Figure 4E shows cells at day 14. Figure 4F shows cells at day 21. [Figure 4D]Photographs illustrating bright light imaging of cells during the course of the induction protocol. Figure 4A shows cells at day 0. Figure 4B shows cells at day 3. Figure 4C shows cells at day 7. Figure 4D shows cells at day 10. Figure 4E shows cells at day 14. Figure 4F shows cells at day 21. [Figure 4E] Photographs illustrating bright light imaging of cells during the course of the induction protocol. Figure 4A shows cells at day 0. Figure 4B shows cells at day 3. Figure 4C shows cells at day 7. Figure 4D shows cells at day 10. Figure 4E shows cells at day 14. Figure 4F shows cells at day 21. [Figure 4F] Photographs illustrating bright light imaging of cells during the course of the induction protocol. Figure 4A shows cells at day 0. Figure 4B shows cells at day 3. Figure 4C shows cells at day 7. Figure 4D shows cells at day 10. Figure 4E shows cells at day 14. Figure 4F shows cells at day 21. [Figure 5] 1 is a photograph illustrating colonies produced by cells isolated on day 14 using a clonogenic assay kit. [Figure 6A] Figure 6A is a bar graph illustrating the percentage of colony forming cells (CFCs) in control conditions (including all inducers), in the absence of IL15 (-IL15), and with SCF alone. Figure 6A is a bar graph illustrating the percentage of CFCs on day 14. Figure 6B is a bar graph illustrating the percentage of CFCs on day 21. Figure 6C is a bar graph illustrating the percentage of CFCs on day 28. [Figure 6B] Figure 6A is a bar graph illustrating the percentage of colony forming cells (CFCs) in control conditions (including all inducers), in the absence of IL15 (-IL15), and with SCF alone. Figure 6A is a bar graph illustrating the percentage of CFCs on day 14. Figure 6B is a bar graph illustrating the percentage of CFCs on day 21. Figure 6C is a bar graph illustrating the percentage of CFCs on day 28. [Figure 6C] Figure 6A is a bar graph illustrating the percentage of colony forming cells (CFCs) in control conditions (including all inducers), in the absence of IL15 (-IL15), and with SCF alone. Figure 6A is a bar graph illustrating the percentage of CFCs on day 14. Figure 6B is a bar graph illustrating the percentage of CFCs on day 21. Figure 6C is a bar graph illustrating the percentage of CFCs on day 28. [Figure 7] In Figure 6, photographs illustrating colony size under the conditions described are shown on day 21 (top row) and day 28 (bottom row). [Figure 8] Photographs illustrating cells after 14 days in culture under standard differentiation conditions. Round cells are those budding off from adherent cells to become HSCs, i.e., suspension cells migrating to the supernatant, which resembles hematopoiesis in vivo. [Figure 9A] Figure 9A is a graph illustrating the percentage of CD56+ cells. Figure 9B is a graph illustrating the expression of CD16 and CD56 proteins in differentiated cells. Figure 9C is a graph illustrating the expression of CD3 protein in differentiated cells. [Figure 9B] Figure 9A is a graph illustrating the percentage of CD56+ cells. Figure 9B is a graph illustrating the expression of CD16 and CD56 proteins in differentiated cells. Figure 9C is a graph illustrating the expression of CD3 protein in differentiated cells. [Figure 9C] Figure 9A is a graph illustrating the percentage of CD56+ cells. Figure 9B is a graph illustrating the expression of CD16 and CD56 proteins in differentiated cells. Figure 9C is a graph illustrating the expression of CD3 protein in differentiated cells. [Figure 10A]Figure 10 illustrates CD56 expression in cells over time. Figure 10A is a line graph illustrating the percentage of CD56 expression over time. Figure 10B is a line graph illustrating the difference in CD56 expression by cells based on the medium used for cell differentiation (stem cell medium, defined medium, or hPL-containing medium). [Figure 10B] Figure 10 illustrates CD56 expression in cells over time. Figure 10A is a line graph illustrating the percentage of CD56 expression over time. Figure 10B is a line graph illustrating the difference in CD56 expression by cells based on the medium used for cell differentiation (stem cell medium, defined medium, or hPL-containing medium). [Figure 11] 1 is a graph illustrating the total number of cells over time in a suspension containing NK cells. [Figure 12A] Figure 12A is a bar graph illustrating the percentage of caspase 3 / 7+ or dead K562 cells after 4 hours of incubation with NK cells. Figure 12B is a bar graph illustrating the percentage of cells after 4 hours of incubation with NK cells obtained after 35 days of differentiation. Figure 12A is a bar graph illustrating the percentage of caspase 3 / 7+ or dead K562 cells after 4 hours of incubation with NK cells obtained after 56 days of differentiation. [Figure 12B] Figure 12A is a bar graph illustrating the percentage of caspase 3 / 7+ or dead K562 cells after 4 hours of incubation with NK cells. Figure 12B is a bar graph illustrating the percentage of cells after 4 hours of incubation with NK cells obtained after 35 days of differentiation. Figure 12A is a bar graph illustrating the percentage of caspase 3 / 7+ or dead K562 cells after 4 hours of incubation with NK cells obtained after 56 days of differentiation. [Figure 13A]Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 13B] Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 13C]Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 13D] Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 13E]Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 13F] Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 13G]Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 13H] Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 13I]Figure 13A is a graph illustrating protein expression in differentiated NK cells as measured by flow cytometry. Figure 13A is a graph confirming that the differentiated cells are NK cells, as indicated by CD56 expression. Figure 13B is a graph illustrating CD56 and CD16 protein expression. Figure 13C is a graph illustrating NKp30 protein expression. Figure 13D is a graph illustrating NKp44 protein expression. Figure 13E is a graph illustrating NKp46 protein expression. Figure 13F is a graph illustrating NKG2D protein expression. Figure 13G is a graph illustrating NKG2A protein expression. Figure 13H is a graph illustrating NKG2C protein expression. Figure 13I is a graph illustrating KIR3D and KIR2D protein expression. [Figure 14A]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14B]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14C]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14D]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14E]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14F]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14G]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14H]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14I]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 14J]Figure 14A is a graph illustrating the difference in protein expression in CD56bright and CD56dim NK cells. Figure 14A is a graph illustrating the percentage of NK cells expressing CD56bright (top) and CD56dim (bottom). Figure 14B is a graph illustrating CD56 and CD16 expression in CD56bright (top) and CD56dim (bottom) NK cells from cells gated as shown in Figure 14A (arrows). Figure 14C is a graph illustrating side and forward scatter analysis in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14D is a graph illustrating CD56 expression in CD56bright (top) and CD56dim (bottom) NK cells from the isolated population illustrated in Figure 14C (right arrow). Figure 14E is a graph illustrating NKp46 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14F is a graph illustrating NKp30 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14G is a graph illustrating percNKp44 expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14H is a graph illustrating NKG2D expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14I is a graph illustrating NKG2A expression in CD56bright (top) and CD56dim (bottom) NK cells. Figure 14J is a graph illustrating NKG2C expression in CD56bright (top) and CD56dim (bottom) NK cells. [Figure 15A]

[0033] Figure 15 illustrates the use of the methods of the invention to generate T cells. Figure 15A shows an exemplary schematic diagram of the methods of the invention to generate T cells. Figure 15B shows a graph illustrating the flow cytometry profile of T cells. [Figure 15B]

[0033] Figure 15 illustrates the use of the methods of the invention to generate T cells. Figure 15A shows an exemplary schematic diagram of the methods of the invention to generate T cells. Figure 15B shows a graph illustrating the flow cytometry profile of T cells. [Figure 16A]16A illustrates the use of the methods of the invention to generate macrophages. Figure 16A shows an exemplary schematic diagram of the methods of the invention to generate macrophages. Figure 16B shows a graph illustrating the flow cytometry profile of macrophages. [Figure 16B] 16A illustrates the use of the methods of the invention to generate macrophages. Figure 16A shows an exemplary schematic diagram of the methods of the invention to generate macrophages. Figure 16B shows a graph illustrating the flow cytometry profile of macrophages. [Figure 17] 1 is an exemplary schematic diagram of a method of the present invention for generating hemogenic endothelium (HE). [Figure 18A] Figure 18 illustrates the number of cells harvested from HE and the number of colonies obtained from HE after 14 and 17 days. Figure 18A is a graph illustrating the number of HSC cells obtained from HE after 14 and 17 days. Figure 18B is a graph illustrating the number of HSC colonies obtained from HE after 14 and 17 days. [Figure 18B] Figure 18 illustrates the number of cells harvested from HE and the number of colonies obtained from HE after 14 and 17 days. Figure 18A is a graph illustrating the number of HSC cells obtained from HE after 14 and 17 days. Figure 18B is a graph illustrating the number of HSC colonies obtained from HE after 14 and 17 days. [Figure 19A] Figures 19A and 19B illustrate cytometric analysis of HSC cells harvested on days 14 and 17. Figure 19A illustrates cytometric analysis of CD34, CD90, CD38, and CD45 of HSC cells harvested on day 14. Figure 19B illustrates cytometric analysis of CD34, CD90, CD38, and CD45 of HSC cells harvested on day 17. [Figure 19B] Figures 19A and 19B illustrate cytometric analysis of HSC cells harvested on days 14 and 17. Figure 19A illustrates cytometric analysis of CD34, CD90, CD38, and CD45 of HSC cells harvested on day 14. Figure 19B illustrates cytometric analysis of CD34, CD90, CD38, and CD45 of HSC cells harvested on day 17. [Figure 20]1 is an exemplary schematic diagram of the method of the present invention for generating hemogenic endothelium (HE) and its HSCs in various cell culture media. [Figure 21] 1 is a graph illustrating the number of NK cells after cryopreservation of HSCs. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is based on the seminal discovery that the combination of an endothelial-inducing cocktail of bone morphogenetic proteins (BMPs) and WNT signaling activators generates endothelial-like progenitor cells that exhibit hybrid characteristics of endothelial and hematopoietic progenitor cells, which further give rise to terminally differentiated hematopoietic cells such as NK cells. The combination of factors can be used to generate hematopoietic stem cells.

[0025] Before the compositions and methods of the present invention are described, it is to be understood that the invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0026] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps, of the type described herein that will become apparent to those skilled in the art upon reading this disclosure and so forth.

[0027] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0028] 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, it is understood that modifications and variations are encompassed within the spirit and scope of the present disclosure. Preferred methods and materials are described herein.

[0029] In one embodiment, the present invention relates to a method for detecting CD34 + 1. A method of producing a population of hematopoietic progenitor cells, comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) and allowing the PSCs to grow on a substrate for about 1-8 days; and b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF), alone or in combination with the WNT signaling pathway activator and / or BMP, for about 1-8 days after step a), wherein the cells produced after step b) are CD34+ in the entire population of cells. + cells, thereby enriched by at least about 80% for CD34 + The present invention provides a method for producing a population of progenitor cells.

[0030] The methods described herein involve the use of CD34 + Cell culture conditions are described in which human pluripotent stem cells are expanded, resulting in the generation of a population of hematopoietic progenitor cells.

[0031] Stem cells are undifferentiated cells that have the ability to self-renew indefinitely and remain in an undifferentiated state. In contrast to embryonic stem cells, which can only be isolated from the inner mass of a blastocyst, there are three known sources of adult stem cells: bone marrow, which requires bone puncture; adipose tissue, which can be obtained by lipofection; and blood, from which cells can be extracted among other cells. The term "pluripotent stem cells," as used herein, refers to cells capable of generating all cell types of an organism, i.e., cells derived from any of the three germ layers. Multipotent stem cells, on the other hand, can differentiate into several cell types but are a type of closely related cell family and generally can differentiate only into the cell type of the organ from which they originate. While most adult stem cells are multipotent, a small number of pluripotent adult stem cells can be recovered from the umbilical cord or other tissues. Sources of cells used in cell therapy include stem cells such as embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs).

[0032] In some embodiments, the PSCs used in the methods described herein are human (hPSCs), and in some cases, the human PSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

[0033] CD34 + "Generating" or "producing" hematopoietic progenitor cells means that the methods of the present invention produce CD34 hematopoietic progenitor cells from PSCs. + The present invention provides physical and chemical culture conditions that are optimized to induce differentiation into hematopoietic progenitor cells. + Hematopoietic progenitor cells are enriched in CD34 + yielding a hematopoietic progenitor cell population, e.g., greater than 80%, greater than 85%, greater than 90%, 95%, 96%, 97%, or greater than 99% CD34 + Hematopoietic progenitor cells are obtained in a short period of time using simple culture conditions.

[0034] Physical culture conditions include, but are not limited to, the cell culture environment (e.g., adherent culture vs. suspension culture, or two-dimensional culture system vs. 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.

[0035] There are two basic systems for growing cells in culture: as a monolayer on an artificial substrate (i.e., adherent culture) or suspended in culture medium (suspension culture). The majority of cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and must be cultured on a suitable substrate that is specifically treated (i.e., tissue culture-treated) to allow cell attachment and spreading. However, many cell lines can also be adapted to suspension culture.

[0036] In another embodiment, the culture of PSCs is an adherent layer of cells. In some embodiments, the layer of cells is grown in a two-dimensional culture system or on microcarriers.

[0037] In addition to treating tissue culture surfaces, it may be necessary to grow cells on coated surfaces (i.e., using a coating) to enhance or improve their adhesion and / or spreading. "Coating" as an additional surface treatment refers to any additional modifications made to increase cell adhesion in addition to the standard plasma or corona treatment performed by manufacturers on all cell culture plastics. Coatings are typically made with proteins or peptides. Various proteins can be used to coat tissue culture-treated dishes, including poly-L-lysine, poly-D-lysine, poly-ornithine, gelatin, collagen I, collagen IV, fibronectin, laminin, vitronectin, osteopontin, fibronectin domains, Matrigel™ (several components of the extracellular matrix bound to growth factors, etc.), collagen gel, alginate gel, and lactate gel.

[0038] In other embodiments, the PSCs are cultured on a coated surface comprising a laminin coating.

[0039] Physical culture conditions include gas concentrations in the incubator. Cell culture incubation is typically performed under standard atmosphere with 15-22% oxygen and 5% CO2 for expansion and seeding. In various embodiments, PSCs are grown in a humidified environment containing approximately 5% CO2 and normoxic conditions (non-hypoxic O2 concentrations). While hypoxic culture conditions are generally considered to support stem cell performance, in the methods of the present invention, PSCs are cultured under conditions that are not hypoxic. As used herein, "normoxic" conditions refer to culture conditions that include atmospheric O2 concentrations (e.g., approximately 15-25% O2 concentrations). As used herein, hypoxic conditions are characterized by a low oxygen concentration compared to the oxygen concentration of ambient air (approximately 15%-25% oxygen).

[0040] Chemical culture conditions include those compounds that are added to the culture medium to achieve the desired effect being sought (i.e., PSC to CD34 + These include, but are not limited to, agents or molecules that achieve differentiation into hematopoietic progenitor cells. The terms "agent" and "molecule" are used interchangeably and include, but are not limited to, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers and other polymers, such as proteins, peptides, hormones, carbohydrates, or polyamines.

[0041] In the methods described herein, the pluripotent stem cells are CD34 + As used herein, "CD34 + Hematopoietic precursors” or “CD34 + "Hematopoietic progenitor" refers to a transient cell that expresses some of the hematopoietic stem cell (HSC) markers and displays some of these characteristics, but not all of the HSC markers and characteristics. For example, HSCs express CD34 + / CD45 + After approximately 7 days of culture under the conditions described herein, CD34 + Hematopoietic progenitor cells express CD34 +However, these are CD23 + and CD45 + These remain adherent cells.

[0042] Unless otherwise specified, pluripotent stem cells are maintained in a stem cell medium suitable for the culture and propagation of pluripotent stem cells. Stem cell basal media are well known in the art, and non-limiting examples of such suitable media include, but are not limited to, StemPro34™. For HSC differentiation, pluripotent stem cells are transferred to a "defined medium." As used herein, the term "basal medium" generally refers to a basal medium that does not have any additives added by the user (e.g., basal medium refers to commercially available media). These generally contain water, nutrients, salts, and amino acids, but do not contain additives or supplements. Basal medium can be supplemented with common additives to obtain a "supplemented basal medium." Non-limiting examples of supplements include, but are not limited to, insulin or ascorbic acid. Basal media can also be completed with specific signaling molecules, such as those identified by the user as necessary to achieve a specific goal using cell culture, such as driving differentiation of a desired cell type into a target cell type. Such complete basal media are sometimes referred to as "final," "complete," or "cell-specific" media.

[0043] In the context of the present invention, cell culture media are additionally referred to based on their use (e.g., to regenerate HE or induce differentiation of cells). For example, the terms "defined basal medium" and "defined medium" are intended to refer to ready-to-use medium formulations for the production of HE that contain only quantifiable amounts of specific components, e.g., do not contain serum or components isolated directly from serum or other animal- or tissue-derived products isolated from organisms or cells. Such ready-to-use formulations can be found in commercially available products or "in-house" compositions developed by the user. Without wishing to be limited to any particular formulation, it is provided that a typical formulation of a defined medium may include a basal medium such as DMEM, DEME / F12, IMDM, or mixtures thereof, and supplements including, but not limited to, one or more of the following: insulin (optionally combined with transferrin and selenium), serum albumin (preferably human recombinant), polyvinyl alcohol (PVA), lipids / fatty acids, glutamine / alanyl glutamine (Glutamax) / common amino acids, antioxidants such as ascorbic acid / ascorbic acid-2-phosphate or thiol compounds, and inorganic salts (supplemented). Non-limiting examples of commercially available defined basal media include, but are not limited to: APEL™ containing 1x Iscove's Modified Dulbecco's Medium (IMDM), 1x Ham's F-12 Nutrient Mix, Albucult (rh 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 per 100 ml), rh insulin-transferrin-selenium-ethanolamine solution (rhITS-Eth), protein-free hybridoma mixture II (PFHMII) (5%), ascorbic acid diphosphate (50 μg / ml), Glutamax I (L-alanyl-L-glutamine) (2 mM), and penicillin / streptomycin (50 U Pen G / 50 mg streptomycin sulfate); APELII™ or APEL2™ containing 1x Iscove's Modified Dulbecco's Medium (IMDM), 1x Ham's F-12 Nutrient Mix, Albucult (rhAlbumin) (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 per 100 ml), rhInsulin-Transferrin-Selenium-Ethanolamine Solution (rhITS-Eth), ascorbic acid diphosphate (50 μg / ml), GlutamaxI (L-alanyl-L-glutamine) (2 mM), and penicillin / streptomycin (50 U Pen G / 50 mg Streptomycin sulfate); · 1× Iscove's Modified Dulbecco's Medium (IMDM), containing polyvinyl alcohol (PVA, 0.1%), Albumult (rhAlbumin) (0.2%), ascorbic acid diphosphate (250uM), lipids (1%), rhInsulin-Transferrin-Selenium-Ethanolamine Solution (rhITS-Eth, 0.1%), APELII; ESSENTIAL6™ or E6™ containing DMEM / F12, insulin, transferrin, sodium selenium, ascorbic acid-2-phosphate, and NaHCO3; and ESSENTIAL8™ or E8™ containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate (64 mg / l), sodium selenium (14 μg / l), insulin (19.4 mg / l), NaHCO3 (543 mg / l) and transferrin (10.7 mg / l).

[0044] Such basal or supplemented defined media used for HE production can then be supplemented with small cells of interest as needed.

[0045] By "contacting" is meant that the cells are cultured with one or more agents of interest that are added to a defined basal or supplemented medium (or a basal or supplemented medium containing hPL, as described in more detail below). That is, the cells are cultured in a normal culture basal or supplemented medium to which a desired concentration of one or more agents of interest has been added. For example, the cells are cultured with a WNT signaling pathway activator, a BMP, and / or a VEGF.

[0046] As used herein, "pathway signaling activator" refers to any molecule that can activate, enhance, or induce a signaling pathway of interest. A signaling pathway is a series of chemical reactions in which a group of molecules in a cell act together to control a cellular function, 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 entire signaling pathway until the final molecule is activated and the cellular function is carried out. Abnormal activation of a signaling pathway or inhibition of a signaling pathway can result in disease or, in the case of pluripotent cells, can result in a change in the pluripotent state and therefore in differentiation. The term "molecule" includes, but is not limited to, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers and other polymers, such as proteins, peptides, hormones, carbohydrates, or polyamines. Non-limiting examples of polynucleotides include small interfering nucleic acids (siNA), antisense, enzymatic nucleic acid molecules, 2',5'-oligoadenylate, triplex-forming oligonucleotides, aptamers, and decoys. Biologically active molecules include antibodies (e.g., monoclonal, chimeric, humanized, etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutic agents, small molecules, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex-forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys and analogs thereof, small nucleic acid molecules such as small interfering nucleic acids (siNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antagomir, and short hairpin RNA (shRNA) molecules.

[0047] The Wnt signaling pathway is a group of signaling pathways that begin with proteins that transmit signals intracellularly via cell surface receptors. Wnt signaling pathways utilize either paracrine (communication between nearby cells) or autocrine (communication within the same cell). Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt / calcium pathway. All three pathways are activated by binding of Wnt protein ligands to Frizzled family receptors, which transmit biological signals to the Dishevelled protein within the cell. The canonical Wnt pathway regulates gene transcription and is thought to be negatively regulated, in part, by the SPATS1 gene. The non-canonical planar cell polarity pathway regulates the cytoskeleton, which is involved in cell shape. The non-canonical Wnt / calcium pathway regulates intracellular calcium. Wnt signaling was first identified for its role in carcinogenesis and then for its function in embryonic development. Embryonic processes regulated by Wnt signaling include axial patterning, cell fate specification, cell proliferation, and cell migration. These processes are necessary for the correct formation of important tissues, including bone, heart, and muscle. Its role in embryonic development was discovered when genetic mutations in Wnt pathway proteins resulted in abnormal Drosophila embryos. Subsequent studies found that the genes responsible for these abnormalities also affect breast cancer development in mice. Wnt signaling also controls tissue regeneration in adult bone marrow, skin, and intestine.

[0048] In some embodiments, the WNT signaling pathway activator is a GSK3 inhibitor. In various embodiments, the GSK3 inhibitor is CHIR99021.

[0049] The transforming growth factor beta (TGF beta) superfamily includes TGF beta proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), activins, inhibins, Nodal, Lefty, and Muellerian inhibitors (MISs). Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines and metabologens. Initially discovered for their ability to induce bone and cartilage formation, BMPs are now considered to constitute a group of central morphogenetic signals that orchestrate tissue architecture throughout the body. The important function of BMP signals in physiology is highlighted by the frequent role of dysregulated BMP signaling in pathological processes.

[0050] BMPs interact with specific cell surface receptors called bone morphogenetic protein receptors (BMPRs). Signaling through BMPRs results in the recruitment of members of the SMAD family of proteins. Signaling pathways involving BMPs, BMPRs, and SMADs are important in the development of the heart, central nervous system, and cartilage, as well as postnatal bone development. They play important roles in embryonic patterning and early skeletal formation during embryogenesis. Thus, disruption of BMP signaling can affect the structure of the developing embryo. For example, BMP4 and its inhibitors, noggin and chordin, help regulate embryonic polarity (i.e., dorsal-to-anterior patterning). Specifically, BMP-4 and its inhibitors play key roles in neurulation and neural plate development. BMP-4 signals ectodermal cells to generate skin cells, but secretion of inhibitors by the underlying mesoderm blocks the action of BMP-4, allowing the ectoderm to continue the normal course of neurogenesis.

[0051] In one embodiment, the BMP is BMP4.

[0052] Vascular endothelial growth factor (VEGF), originally known as vascular permeability factor (VPF), is a signaling protein produced by many cells that stimulates the formation of blood vessels. Specifically, VEGF is a subfamily of growth factors, the platelet-derived growth factor family of cystine-knot growth factors. They are key signaling proteins involved in both angiogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). They are part of a system that restores oxygen supply to tissues when blood circulation is inadequate, such as in hypoxic conditions. Serum concentrations of VEGF are elevated in bronchial asthma and diabetes mellitus. The normal function of VEGF is to generate new blood vessels during embryonic development, generate new blood vessels after injury, generate muscle after exercise, and generate new blood vessels that bypass blocked blood vessels (collateral circulation).

[0053] In some embodiments, the VEGF is VEGF-A.

[0054] In various embodiments, the chemical culture conditions of the described methods include a cocktail of agents including a WNT signaling pathway activator, a BMP, and / or a VEGF.

[0055] For example, the mixture of drugs includes a WNT signaling pathway activator and a BMP. In a further example, the mixture of drugs includes only a WNT signaling pathway activator, a BMP, and a VEGF. In another example, the mixture of drugs includes only a WNT signaling pathway activator, only a BMP, or only a BMP.

[0056] In one embodiment, the PSCs are grown on a substrate in the presence of a WNT signaling pathway activator and a bone morphogenetic protein (BMP) for about 1-8 days. For example, the cells are grown in the presence of a WNT signaling pathway activator and a BMP for about 1, 2, 3, 4, 5, 6, 7, 8, or more days.

[0057] In another embodiment, after initial culture in the presence of a WNT signaling pathway activator and a BMP, the PSCs are expanded on a substrate in the presence of VEGF for about 1-8 days, e.g., after initial culture, the cells are expanded in the presence of VEGF for about 1, 2, 3, 4, 5, 6, 7, 8, or more days.

[0058] In yet another embodiment, after initial culture in the presence of a WNT signaling pathway activator and a BMP, the PSCs are expanded on a substrate in the presence of VEGF, a WNT signaling pathway activator, and a BMP for about 1-8 days. For example, after initial culture, the cells are expanded in the presence of a VEGF, a WNT signaling pathway activator, and a BMP for about 1, 2, 3, 4, 5, 6, 7, 8, or more days.

[0059] In one embodiment, a culture of PSCs is contacted with a WNT signaling pathway activator and a BMP for about 3 days, and then with VEGF for about an additional 4 days.

[0060] In one embodiment, CD34+ hemogenic endothelium (HE) is generated by contacting adherent cultures of PSCs with a WNT signaling pathway activator, BMP and / or VEGF.

[0061] Hemopoietic endothelium (HE) consists of a specialized subset of endothelial cells scattered within blood vessels that can differentiate into hematopoietic cells. Embryonic hematopoietic cell development proceeds sequentially from mesoderm to hemangioblasts to hemogenic endothelium and hematopoietic precursors. Hemangioblasts are multipotent progenitor cells that can differentiate into both hematopoietic and endothelial cells. Hemangioblasts are the progenitors that form blood islands. Hemangioblasts are initially extracted from embryonic cultures and manipulated with cytokines to differentiate along either the hematopoietic or endothelial pathway.

[0062] In an exemplary embodiment, CD34 + Contacting the population of progenitor cells further includes contacting the cells with a transforming growth factor beta (TGFβ) / SMAD2 / SMAD3 pathway signaling inhibitor.

[0063] As used herein, a "pathway signaling inhibitor" refers to any molecule capable of inhibiting a signaling pathway of interest. A signaling pathway is a series of chemical reactions in which a group of molecules in a cell act together to control a cellular function, 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 in the cell. After the first molecule in the pathway receives the signal, this molecule activates another molecule. This process is repeated throughout the signaling pathway until the last molecule is activated and the cellular function is carried out. Abnormal activation of a signaling pathway or inhibition of a signaling pathway can result in disease or, in the case of pluripotent cells, can result in an alteration in the pluripotent state and therefore alteration in differentiation.

[0064] As used herein, a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor refers to any molecule that can inhibit TGFβ / SMAD2 / SMAD3. Signaling pathway inhibition is the opposite of signaling pathway upregulation. In this process, small molecules called "signaling inhibitors" or "pathway signaling inhibitors" block the communication between different molecules in the pathway, interrupting the molecular signaling cascade. TGFβ / SMAD2 / SMAD3 pathway signaling inhibitors include, for example, any molecules that inhibit TGFβ receptor type I (or ALK5) and the related ALK4 and ALK7. Non-limiting examples of TGFβ / SMAD2 / SMAD3 pathway signaling inhibitors include SB431542, LY3200882, TP0427736 HCl, RepSox, SB525334, GW788388, BIBF-0775, SD-208, galunisertib, vactosertib, A-83-01, LY2109761, SB505124, LY364947, and LDN-212854.

[0065] In some embodiments, contacting the cell with a TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor comprises contacting the cell with about 1-25 μM of the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor.

[0066] In one embodiment, the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is SB431542.

[0067] In some embodiments, the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor is added to the culture at a concentration ranging from about 5 μM to 20 μM. For example, cells are grown in a culture medium containing about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μM, or more. In one embodiment, the mixture contains about 10 μM of the TGFβ / SMAD2 / SMAD3 pathway signaling inhibitor. In another embodiment, the mixture contains about 5-20 μM of SB431542. In some embodiments, the mixture contains about 8-15 μM of SB431542. In other embodiments, the mixture contains about 10 μM of SB431542.

[0068] In another embodiment, the method comprises administering to a subject a subject a CD34 + The progenitor cell population is exposed to a cocktail of drugs to induce CD34 + It further includes inducing differentiation of the progenitor cells into terminally differentiated cells of the hematopoietic lineage.

[0069] "Terminally differentiated cells of the hematopoietic lineage" refer to any terminally differentiated cell that can emerge from one of three blood cell lineages, including the embryonic, lymphoid, and myeloid lineages. Erythroid cells are oxygen-carrying red blood cells. Lymphoid cells are the cornerstone of the adaptive immune system and are derived from a common lymphoid precursor. The lymphoid lineage is primarily composed of T and B cells and natural killer cells (i.e., white blood cells). Myeloid cells, including granulocytes, megakaryocytes, and macrophages, are derived from a common myeloid precursor and are involved in diverse roles such as innate immunity, adaptive immunity, and blood clotting.

[0070] In some embodiments, the cells of the hematopoietic lineage are natural killer (NK) cells or other immune cells.

[0071] In some embodiments, the cells treated for about 1 week are CD34 + , K.D.R. + , CD31 + and CD45 - In another embodiment, CD34 + The cells also express CD144 + It is also.

[0072] In another embodiment, the present invention provides a method for producing natural killer (NK) cells, comprising: a) contacting a culture of PSCs with a WNT signaling pathway activator and / or a BMP and allowing the PSCs to grow on a substrate for about 1-8 days; b) contacting the culture of PSCs with VEGF, alone or in combination with a WNT signaling pathway activator and / or a BMP, for about 1-8 days after step a), thereby increasing the CD34 + generating a population of progenitor cells, and after step b) determining whether the cells generated are CD34 + c) at least about 80% enriched for CD34 cells; + The method includes contacting a population of progenitor cells with one or more of interleukin 3 (IL-3), IL-7, IL-15, SCF, and FMS-like tyrosine kinase 3 ligand (FLT3L), thereby producing NK cells.

[0073] In one embodiment, the present invention provides a method for producing natural killer (NK) cells, comprising: a) contacting a culture of PSCs with a WNT signaling pathway activator and / or a BMP and allowing the PSCs to grow on a substrate for about 1-8 days; b) contacting the culture of PSCs with VEGF, alone or in combination with a WNT signaling pathway activator and / or a BMP, for about 1-8 days after step a), thereby increasing the CD34 + generating a population of progenitor cells, and after step b) determining whether the cells generated are CD34 +c) at least about 80% enriched for CD34 cells; + The method includes contacting a population of progenitor cells with one or more of IL-7, IL-15, SCF, and FMS-like tyrosine kinase 3 ligand (FLT3L), thereby producing NK cells.

[0074] Natural killer cells, also known as NK cells or large granular lymphocytes (LGLs), belong to a rapidly expanding family of known innate lymphoid cells (ILCs) and are a type of cytotoxic lymphocyte important to the innate immune system, representing 5-20% of all circulating lymphocytes in humans. They have distinct functions, including cytolytic granule-mediated cell apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and cytokine-induced NK and cytotoxic T lymphocyte (CTL) activation.

[0075] NK cells are cytotoxic, and their small cytoplasmic granules contain proteins such as perforin and proteases known as granzymes. By releasing perforin in close proximity to a cell selected for killing, it creates pores in the target cell's plasma membrane, creating aqueous channels through which granzymes and related molecules can enter, inducing either apoptosis or osmotic cytolysis. The distinction between apoptosis and cytolysis is important in immunology; lysing virus-infected cells can potentially release virions, while apoptosis results in the destruction of the virus itself. Alpha-defensins, antimicrobial molecules, are also secreted by NK cells and directly kill bacteria by disrupting their cell walls in a manner similar to that of neutrophils.

[0076] Infected cells are routinely opsonized with antibodies for detection by immune cells. Antibody binding to antigens is recognized by the FcγRIII (CD16) receptor expressed on NK cells, resulting in NK activation and the release of cytolytic granules, which can result in cell apoptosis. This is the primary killing mechanism of some monoclonal antibodies, such as rituximab (Rituxan) and ofatumumab (Azzera).

[0077] Cytokines play an important role in NK cell activation. These are stress molecules released from cells upon viral infection and thus serve to signal NK cells about the presence of viral pathogens in the infected area. Cytokines involved in NK activation include IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells are activated in response to interferon or macrophage-derived cytokines. These help contain viral infections, while the adaptive immune response generates antigen-specific cytotoxic T cells that can clear the infection. NK cells work to control viral infections by secreting IFNγ and TNFα. IFNγ activates macrophages for phagocytosis and lysis, and TNFα acts to promote direct NK tumor cell killing. Patients with NK cell deficiencies have been shown to be highly susceptible to early-stage herpesvirus infections.

[0078] Tumor-infiltrating NK cells have been reported to play an important role in promoting drug-induced cell death in human triple-negative breast cancer. Because NK cells recognize target cells when they express non-self (rather than self) HLA antigens, autologous (patient-specific) NK cell infusions have not shown any antitumor effects. Instead, researchers have studied syngeneic cells derived from peripheral blood, which require depletion of all T cells before infusion into the patient to eliminate the risk of potentially fatal graft-versus-host disease. This can be achieved using an immunomagnetic column (CliniMACS). Additionally, due to the limited number of NK cells in blood (only 10% of lymphocytes are NK cells), these numbers must be expanded in culture. This can take several weeks, and the yield is donor-dependent.

[0079] Interleukin-3 (IL-3), a protein encoded by the IL3 gene in humans, is also known as colony-stimulating factor, multi-CSF, mast cell growth factor, MULTI-CSF, MCGF, MGC79398, or MGC79399. IL-3 is produced as a monomer by activated T cells, monocytes / macrophages, and stromal cells. The primary function of the IL-3 cytokine is to regulate the concentrations of various blood cell types. It induces proliferation and differentiation of both early pluripotent stem cells and committed progenitors. It also has many other specific effects, such as platelet regeneration and potentially aids in early antibody isotype switching. IL-3 can stimulate the differentiation of immature myelomonocytic cells, leading to their transformation into macrophage and granulocyte populations. IL-3 signaling can affect a wide range of cell lineages, which is why it is independently named "multi-CSF." Interleukin-3 stimulates the differentiation of multipotent hematopoietic stem cells into myeloid progenitors, or, with the addition of IL-7, into lymphoid progenitors. In addition, IL-3 stimulates the proliferation of all cells of the myeloid lineage (granulocytes, monocytes, and dendritic cells) together with other cytokines, such as erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6.

[0080] IL-7 is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus. IL-7 is also produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells, but not by normal lymphocytes. IL-7 stimulates the differentiation of multipotent (pluripotent) hematopoietic stem cells into lymphoid progenitor cells (in contrast to myeloid progenitor cells, the differentiation of which is stimulated by IL-3). IL-7 also stimulates the proliferation of all cells of the lymphoid lineage (B cells, T cells, and NK cells). IL-7 is important for proliferation at certain stages of B cell maturation and for the survival, development, and homeostasis of T and NK cells.

[0081] Interleukin-15 (IL-15) is a cytokine structurally similar to interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and common gamma chain (gamma C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) after viral infection. This cytokine induces the proliferation of natural killer cells, i.e., cells of the innate immune system whose primary role is to kill virus-infected cells. IL-15 regulates the activation and proliferation of T cells and natural killer (NK) cells. IL-15 provides a survival signal that maintains memory T cells in the absence of antigen. This cytokine is also involved in the development of NK cells. In rodent lymphocytes, IL-15 prevents apoptosis by inducing BCL2L1 / BCL-x(L), an inhibitor of the apoptosis pathway. In humans with celiac disease, IL-15 similarly suppresses T lymphocyte apoptosis by inducing Bcl-2 and / or Bcl-xL.

[0082] Stem cell factor (SCF, also known as KIT ligand, KL, or steel factor) is a cytokine that binds to the c-KIT receptor (CD117). SCF exists as both a transmembrane and a soluble protein and plays an important role in hematopoiesis, spermatogenesis, and melanogenesis.

[0083] SCF plays an important role in hematopoiesis during embryonic development. Sites where hematopoiesis occurs, such as the fetal liver and bone marrow, all express SCF. SCF may serve as a guidance cue to direct hematopoietic stem cells (HSCs) to the stem cell niche (the microenvironment where stem cells reside) and plays an important role in HSC maintenance. SCF plays a role in regulating HSCs in the stem cell niche in the bone marrow. SCF has been shown to increase HSC survival in vitro and contribute to HSC self-renewal and maintenance in vivo. HSCs at all stages of development express the same receptor as SCF (c-KIT) at levels similar to SCF. Stromal cells surrounding HSCs are components of the stem cell niche and release numerous ligands, including SCF. In the bone marrow, HSCs and hematopoietic progenitor cells are adjacent to stromal cells such as fibroblasts and osteoblasts. These HSCs remain in the niche by adhering to ECM proteins and the stromal cells themselves. SCF has been shown to increase adhesion and may therefore play a major role in ensuring HSC retention in the niche. SCF can be used with other cytokines to cultivate HSCs and hematopoietic precursors. Ex vivo expansion of these cells allows for advances in bone marrow transplantation, where HSCs are transferred to patients to reestablish blood formation. One problem with therapeutically injecting SCF is that it activates mast cells. Injection of SCF has been shown to cause allergy-like symptoms and proliferation of mast cells and melanocytes.

[0084] FLT3L-like tyrosine kinase 3 ligand (FLT3L) is an endogenous small molecule that acts as a cytokine and growth factor to increase the number of immune cells (lymphocytes (B cells and T cells)) by activating hematopoietic progenitors. It acts by binding to and activating FLT3 (CD135), which is found on what are called multipotent progenitor (MPP) and common lymphoid progenitor (CLP) cells (in mice). It also stimulates the in vivo mobilization of hematopoietic progenitors and stem cells, which can help the system kill cancer cells. FLT3L is important for the development of steady-state plasmacytoid dendritic cells (pDC) and classical dendritic cells (cDC). Lack of FLT3L results in low levels of dendritic cells.

[0085] In one embodiment, CD34 + Contacting a population of progenitor cells includes (i) CD34 + (ii) contacting the progenitor cells with IL-3, IL-7, IL-15, SCF, and FLT3L for about 5 to 10 days; and + The method includes contacting the progenitor cells with IL-7, IL-15, FLT3L, and SCF for at least about 7 to 21 days. For example, CD34 + The population of progenitor cells is cultured with IL-3, IL-7, IL-15, SCF and FLT3L for about 5, 6, 7, 8, 9, 10 or more days, followed by culture with IL-7, IL-15, FLT3L and SCF (i.e., in the absence of IL-3) for at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days.

[0086] It has been found that contacting the cells with IL-3 is optional. Thus, in another embodiment, CD34 + Contacting a population of progenitor cells includes (i) CD34 + (ii) contacting the progenitor cells with IL-7, IL-15, SCF, and FLT3L for about 5 to 10 days; and +The method includes contacting the progenitor cells with IL-7, IL-15, FLT3L, and SCF for at least about 7 to 21 days. For example, CD34 + The population of progenitor cells is cultured with IL-7, IL-15, SCF and FLT3L for about 5, 6, 7, 8, 9, 10 or more days, followed by culture with IL-7, IL-15, FLT3L and SCF for at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more additional days.

[0087] For NK cell differentiation, HSCs may be switched to an "undefined medium." As used herein, the terms "undefined basal medium" or "undefined medium" are intended to refer to a medium formulation that typically contains human platelet lysate (hPL) or, alternatively, serum as a supplement. Such media containing plasma or serum-derived components may be commercially available or may be prepared "in-house" by the user. As used herein, an "undefined medium" is referred to as an "hPL-containing medium." As detailed above, such hPL-containing medium may be "basal," "supplemented," or "complete."

[0088] By "contacting" is meant that the HSCs are cultured with one or more agents of interest that are added to the hPL-containing medium.

[0089] In the methods described herein, the pluripotent stem cells are CD34 + The method described herein differentiates hematopoietic stem cells (HSCs) into hematopoietic progenitors, which in turn differentiate into NK cells. + Generates hematopoietic progenitor cells, which are CD34 + / CD45 + CD34 + / CD45 + A transient intermediate population of cells is CD34 + They emerge from hematopoietic progenitor cells and subsequently disappear to generate fully differentiated NK cells, which express, among other things, CD56 + In another embodiment, the cells are transiently transfected with CD34 +and CD45 + is.

[0090] In one embodiment, the contacting of the adherent culture of PSCs comprises one or more agents selected from about 1-10 μM of a WNT signaling pathway activator, about 10-100 ng / ml of BMP, and about 50-500 ng / ml of VEGF.

[0091] The WNT signaling pathway activator is added to the PSC culture at a concentration ranging from about 1 μM to 10 μM. For example, PSCs are grown in a culture medium containing about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM or more. In one embodiment, the mixture contains about 8 μM of the WNT signaling pathway activator.

[0092] BMPs are added to PSC cultures at concentrations ranging from about 5 ng / ml to 50 ng / ml. For example, PSCs are grown in culture medium containing about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / ml or more. In one embodiment, the mixture contains about 25 ng / ml of BMP.

[0093] As further described in the Examples, BMP4 can be prepared by resuspending it in various solutions. For example, anhydrous BMP4 can be resuspended in PBS / 0.01% HAS or in citric acid (as recommended by the manufacturer). PBS / 0.01% HAS may reduce the biological activity of BMP4 compared to the activity when prepared in citric acid. Those skilled in the art will readily recognize that when BMP4 is resuspended in citric acid, the concentration of BMP4 can be significantly reduced from 5 to 50 ng / ml, thus resulting in greater biological activity.

[0094] VEGF is added to PSC cultures at a concentration ranging from about 50 ng / ml to 500 ng / ml. For example, PSCs are grown in culture 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 one embodiment, the mixture contains about 200 ng / ml of VEGF.

[0095] In another embodiment, the contacting the adherent culture of PSCs comprises about 8 μM CHIR99021, about 25 ng / ml BMP4 and / or about 200 ng / ml VEGFA.

[0096] In one embodiment, CD34 + The progenitor cells are contacted with about 1 to 10 ng / ml of IL-3, about 4 to 40 ng / ml of IL-7, about 2 to 20 ng / ml of IL-15, about 4 to 40 ng / ml of SCF, and / or about 1 to 20 ng / ml of FLT3L. + The precursor cells are contacted with about 4 to 40 ng / ml of IL-7, about 2 to 20 ng / ml of IL-15, about 4 to 40 ng / ml of SCF, and / or about 1 to 20 ng / ml of FLT3L.

[0097] IL-3 is added to PSC cultures at a concentration ranging from about 1 ng / ml to 10 ng / ml. For example, PSCs are grown in culture medium containing about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ng / ml or more. In one embodiment, the mixture contains about 5 ng / ml of IL-3.

[0098] IL-7 is added to PSC cultures at a concentration ranging from about 4 ng / ml to 40 ng / ml. For example, PSCs are grown in culture medium containing about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 μM, or more. In one embodiment, the mixture contains about 20 ng / ml of IL-7.

[0099] IL-15 is added to PSC cultures at a concentration ranging from about 2 ng / ml to 20 ng / ml. For example, PSCs are grown in culture medium containing about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 ng / ml or more. In one embodiment, the mixture contains about 10 ng / ml of IL-15.

[0100] SCF is added to PSC cultures at a concentration ranging from about 4 ng / ml to 40 ng / ml. For example, PSCs are grown in culture medium containing about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 μM, or more. In one embodiment, the mixture contains about 20 ng / ml of SCF.

[0101] FLT3L is added to PSC cultures at a concentration ranging from about 2 ng / ml to 20 ng / ml. For example, PSCs are grown in a culture 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 one embodiment, the mixture contains about 10 ng / ml of FLT3L.

[0102] In various embodiments, CD34 + The progenitor cells are contacted with about 5 ng / ml IL-3, about 20 ng / ml IL-7, about 10 ng / ml IL-15, about 20 ng / ml SCF, and / or about 10 ng / ml FLT3L. + The progenitor cells are contacted with about 20 ng / ml IL-7, about 10 ng / ml IL-15, about 20 ng / ml SCF and / or about 10 ng / ml FLT3L.

[0103] In one embodiment, contacting the adherent culture of PSCs in a) with the WNT signaling pathway activator and BMP is for about 2 to 5 days.

[0104] In another embodiment, the PSCs are then contacted with VEGF for about 2 to 5 days.

[0105] In one embodiment, CD34 +Contacting the population of progenitor cells with IL-3, IL-7, IL-15, SCF and / or FLT3L for approximately 5-10 days induces CD34 + The contacting of the population of progenitor cells with IL-7, IL-15, FLT3L and / or SCF is for at least about 7 to 21 days. + Contacting the population of progenitor cells with IL-7, IL-15, SCF and / or FLT3L for approximately 5-10 days followed by CD34 + Contacting the population of progenitor cells with IL-7, IL-15, FLT3L and / or SCF is for at least about an additional 7 to 21 days.

[0106] In another embodiment, the culture of PSCs is an adherent layer of cells. In some embodiments, the layer of cells is grown in a two-dimensional culture system or on microcarriers.

[0107] In some embodiments, PSCs are cultured on a scaffold composed of microcarriers, which are beads or particles. The beads can be microscopic or macroscopic and can be sized to allow penetration into tissue or compressed to form a specific shape. In one embodiment, the framework of the cell culture comprises particles that combine with the cells to form a three-dimensional tissue. The cells bind to the particles and each other to form the three-dimensional tissue. The beads or microcarriers are typically considered two-dimensional systems or scaffolds.

[0108] As used herein, "microcarrier" refers to particles having a size ranging from nanometers to micrometers, which may be of any shape or form, including irregular, non-spherical, spherical, or ellipsoidal. Microcarrier sizes suitable for purposes herein may be any suitable size for a particular application. In some embodiments, microcarriers suitable for three-dimensional tissues may be sizes that can be administered by injection. In some embodiments, 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, or at least about 1000 μm.

[0109] In some embodiments, the microcarriers are made from biodegradable materials. In some embodiments, microcarriers comprising two or more layers of different biodegradable polymers can be used. In some embodiments, at least a first outer layer has biodegradable properties to form a three-dimensional tissue in culture, while at least a second inner biodegradable layer has properties different from the first layer and is made to erode when administered to a tissue or organ.

[0110] In some aspects, the microcarrier is a porous microcarrier. A porous microcarrier refers to a microcarrier that has interstices through which molecules can diffuse into or out of the microparticle. In other embodiments, the microcarrier is a non-porous microcarrier. A non-porous microparticle refers to a microparticle that does not allow molecules of a selected size to diffuse into or out of the microparticle.

[0111] The microcarriers used in the present compositions are biocompatible and have low or no toxicity to cells. Microcarriers can include a variety of polymers, natural or synthetic, charged (i.e., anionic or cationic) or uncharged, biodegradable or non-biodegradable. Polymers can be homopolymers, random polymers, block copolymers, graft copolymers, and branched polymers.

[0112] In some embodiments, the microcarrier comprises a non-biodegradable microcarrier. Non-biodegradable microcapsules and microcarriers include, but are not limited to, those made from polysulfone, poly(acrylonitrile-co-vinyl chloride), ethylene vinyl acetate, and hydroxyethyl methacrylate-methyl methacrylate copolymers. These are useful for providing tissue bulking properties or in embodiments where the microcarrier is eliminated from the body.

[0113] In some embodiments, the microcarriers comprise degradable scaffolds. These include microcarriers made from naturally occurring polymers, non-limiting examples of which include, among others, fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginate, or polyamino acids such as polylysine. In other embodiments, the degradable microcarriers are made from synthetic polymers, non-limiting examples of which include, among others, polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly(caprolactone), polydioxanone trimethylene carbonate, polyhydroxyalkonates (e.g., poly(hydroxybutyrate)), poly(ethyl glutamate), poly(DTH iminocarbony(bisphenol A iminocarbonate)), poly(orthoesters), and polycyanoacrylates.

[0114] 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, microparticle swelling can be triggered by various stimuli, including pH, ionic strength, heat, electricity, ultrasound, and enzymatic activity. Non-limiting examples of polymers useful in hydrogel compositions include those formed from polymers of poly(lactide-co-glycolide), poly(N-isopropylacrylamide), poly(methacrylic acid-g-polyethylene glycol), polyacrylic acid, and poly(oxypropylene-co-oxyethylene) glycol, among others, as well as natural compounds such as chondroitin sulfate, chitosan, gelatin, fibrinogen, or mixtures of synthetic and natural polymers, e.g., chitosan-poly(ethylene oxide). Polymers can be reversibly or irreversibly crosslinked to form gels adaptable for the formation of three-dimensional tissues.

[0115] In an exemplary embodiment, the microcarriers or beads used in the present invention are composed entirely or partially of dextran.

[0116] In other embodiments, the PSCs are cultured on a coated surface comprising a laminin coating.

[0117] In various embodiments, CD34 + Progenitor cells express CD34 + They are endothelial-like progenitor cells.

[0118] In one embodiment, the NK cells are further collected from the suspension in the cell culture medium.

[0119] The methods described herein involve the use of CD34, which is an adhesive + It allows differentiation of endothelial-like progenitor cells into NK cells grown in suspension. Thus, during the differentiation process, intermediate and transient CD34 + CD45 + The cells are CD34 +Endothelial-like progenitor cells emerge in suspension and differentiate into NK cells. After at least one week of culture, the methods described herein produce at least 80% pure or enriched NK cells in suspension. Because the cells are in suspension, they can be easily aspirated and collected in culture medium.

[0120] In one embodiment, the NK cells are enriched by at least about 80%.

[0121] In an additional embodiment, the present invention provides a method for inducing differentiation of PSCs into NK cells, comprising: a) (i) contacting an adherent culture of PSCs with a WNT signaling pathway activator and a BMP for about 3 days; and (ii) contacting an adherent culture of PSCs of i) with VEGF for about 4 days, thereby increasing the differentiation rate of at least 80% of CD34 + By generating a population of cells containing hemogenic endothelial (HE) cells, CD34 + b) generating HE cells; and + The HE cells are contacted with one or more of IL-3, IL-7, IL-15, SCF and / or FLT3L for about 7 days, thereby reducing at least 80% of the CD34 + / CD45 + b) generating a transient population of cells containing hematopoietic stem cells (HSCs), and c) subsequently CD34 + / CD45 + The method includes contacting HSCs with one or more of IL-7, IL-15, FLT3L, and SCF for at least about 7 to 21 days, thereby inducing differentiation of PSCs into NK cells.

[0122] In a further embodiment, the present invention provides a method for inducing differentiation of PSCs into NK cells, comprising: a) (i) contacting an adherent culture of PSCs with a WNT signaling pathway activator and a BMP for about 3 days; and (ii) contacting an adherent culture of PSCs of i) with VEGF for about 4 days, thereby increasing the differentiation rate of at least 80% of CD34 + By generating a population of cells containing hemogenic endothelial (HE) cells, CD34 + b) generating HE cells; and+ The HE cells are contacted with one or more of IL-7, IL-15, SCF and / or FLT3L for about 7 days, thereby reducing at least 80% of the CD34 + / CD45 + b) generating a transient population of cells containing hematopoietic stem cells (HSCs), and c) subsequently CD34 + / CD45 + The method includes contacting HSCs with one or more of IL-7, IL-15, FLT3L, and SCF for at least about 7 to 21 days, thereby inducing differentiation of PSCs into NK cells.

[0123] In one embodiment, the differentiated NK cells are CD56 + , NKp30 + , NKp44 + , NKp46 + , NKG2D + , NKG2A + , KIR2D + and / or CD16 + is.

[0124] NK cells are characterized by the presence of CD56 and CD3 (CD56 + , CD3 +NK cells (belonging to the group of innate lymphoid cells) are one of three cell types differentiated from a common lymphoid precursor; the other two are B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, from where they enter the circulation. NK cells differ from natural killer T cells (NKT) in morphology, origin, and corresponding effector functions; NKT cell activity often promotes NK cell activity by secreting interferon gamma. In contrast to NKT cells, NK cells do not express the T cell antigen receptor (TCR) or pan-T marker CD3 or surface immunoglobulin (Ig) B cell receptor, but typically express the surface markers CD16 (FcγRIII) and CD57 in humans. The NKp46 cell surface marker constitutes another NK cell marker with preferential expression in both humans, some strains of mice, and three common monkey species.

[0125] NK cells express CD56 bright or CD56 dim CD56 bright NK cells are similar to T helper cells in that they exert their effects by releasing cytokines. bright NK cells constitute the majority of NK cells found in the bone marrow and secondarily in lymphoid tissues, liver, and skin. dim NK cells are found primarily in peripheral blood and are characterized by their cell-killing ability. dim NK cells are always CD16 positive (CD16 is a major mediator of antibody-dependent cellular cytotoxicity (ADCC)). bright By obtaining CD16, CD56 dim It can transition to.

[0126] In one embodiment, the differentiated NK cells are CD56 bright or CD56 dim is.

[0127] In various embodiments, the differentiated NK cells are cytotoxic NK cells.

[0128] In a further embodiment, the present invention provides a kit comprising: a) an HE induction cocktail comprising a WNT signaling pathway activator, BMP and / or VEGF; b) an NK induction cocktail comprising IL-3, IL-7, IL-15, SCF and / or FLT3L; and c) instructions for inducing differentiation of pluripotent stem cells (PSCs) into NK cells.

[0129] In one embodiment, the present invention provides a kit comprising: a) an HE induction cocktail comprising a WNT signaling pathway activator, BMP and / or VEGF; b) an NK induction cocktail comprising IL-7, IL-15, SCF and / or FLT3L; and c) instructions for inducing differentiation of pluripotent stem cells (PSCs) into NK cells.

[0130] In one embodiment, the kit further comprises a laminin-coated surface.

[0131] In one embodiment, the present invention provides a method for generating terminally differentiated hematopoietic cells from PSCs, comprising: a) (i) contacting an adherent culture of PSCs with a WNT signaling pathway activator and a BMP, and allowing the PSCs to grow on a substrate for about 2-5 days; and (ii) contacting the adherent culture of PSCs with VEGF for about 2-5 days after step (i), wherein the cells produced after step (ii) are CD34+ / CD36+ cells throughout the population of cells. + cells, thereby enriched by at least about 80% for CD34 + By generating progenitor cells, CD34 + generating hematopoietic progenitor cells, and b) CD34 + The progenitor cells are contacted with a mixture of drugs to induce CD34 + The present invention provides a method for inducing differentiation of progenitor cells into terminally differentiated hematopoietic cells, thereby generating terminally differentiated hematopoietic cells.

[0132] In one embodiment, the terminally differentiated hematopoietic cell is an immune cell or an erythrocyte. In various embodiments, the immune cell is selected from the group consisting of a macrophage, a T cell, and a natural killer (NK) cell.

[0133] In another embodiment, the invention provides a method of producing a population of CD34+ hematopoietic stem cells, the method comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) and allowing the PSCs to grow on a substrate for about 1-8 days; and b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF), alone or in combination with the WNT signaling pathway activator and / or a BMP, for about 1-8 days after step a), wherein the cells produced after step b) are at least about 80% enriched for CD34+ hematopoietic stem cells in the overall population of cells, thereby producing a population of CD34+ hematopoietic stem cells.

[0134] In one embodiment, CD34+ hemogenic endothelium (HE) is generated by contacting adherent cultures of PSCs with a WNT signaling pathway activator, BMP, and / or VEGF. In another embodiment, the PSCs are human PSCs (hPSCs). In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

[0135] In a further embodiment, the present invention provides a method of generating hematopoietic stem cells from pluripotent stem cells (PSCs), the method comprising: a) contacting an adherent culture of induced pluripotent stem cells (iPSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), and allowing the PSCs to grow on a substrate for about 2-5 days; and b) contacting the adherent culture of PSCs with vascular endothelial growth factor (VEGF) for about 2-5 days after step (a), wherein the cells produced after step (b) are at least about 80% enriched for CD34+ cells in the overall population of cells, thereby generating CD34+ progenitor cells, thereby generating hematopoietic stem cells.

[0136] In one embodiment, the iPSCs are human iPSCs (hiPSCs).

[0137] The CD34 from PSC contemplated for the application under discussion + Examples are presented below that discuss methods of inducing differentiation into hematopoietic progenitor cells. The following examples are provided to further illustrate embodiments of the invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methods, or techniques known to those of skill in the art may alternatively be used. [Example]

[0138] [Example 1] Design of natural killer cell differentiation protocol Novel combinations of factors that promote NK cell differentiation with high efficiency in a stepwise iterative developmental process have been identified using a simple GMP-compatible workflow (see Figures 1 and 2).

[0139] The NK cell differentiation protocol described herein is divided into two steps.

[0140] The first step aims to differentiate endothelial-like cells from iPSCs using mesodermal inducers such as BMP and WNT followed by VEGF treatment. The present invention is based on the discovery that endothelial-like progenitor cells obtained after a differentiation protocol using BMP and WNT followed by VEGF treatment exhibit biplicative characteristics of endothelial and hematopoietic progenitor cells, i.e., hemogenic endothelial (HE) cells, which are endothelial-like cells that also give rise to cells of the hematopoietic lineage, first as hematopoietic stem cells (marked by CD34 and CD45), then as precursors of myeloid and lymphoid lineages, and then as terminally differentiated cells such as erythrocytes, macrophages, T cells, and NK cells. Interestingly, HE cells indeed co-express endothelial and hematopoietic markers, such as CD31, CD144, and CD34, respectively.

[0141] The protocol described herein generates HE cells in a defined and controlled manner using BMP and / or WNT stimulation of adherent iPSCs followed by VEGF treatment in combination / followed by treatment of the cells with one or more of IL3, IL7, IL15, SCF and / or FLT3L to promote further hematopoietic differentiation and NK cell induction.

[0142] HE cells are the common precursor of all hematopoietic cells, including all of the cell types mentioned above. Therefore, step 1 can be combined with known inductive production procedures for other cell types, such as T cells. Thus, the overall approach can be universal. An optimized procedure generates >90% pure HE cells, followed by >90% pure HSCs as the primary intermediate, and then >90% pure NK cells.

[0143] Because the HE cell layer continuously generates new HSCs and these suspension cells (which bud from HE precursors and migrate into the supernatant) have a propensity to proliferate, the protocol also produces a yield of NK cells.

[0144] [Example 2] Materials and Methods

[0145] [Table 1] TIFF2025530564000003.tif249169TIFF2025530564000004.tif140169

[0146] [Table 2]

[0147] method: Maintenance of hiPSD (R26 lineage): hiPSCs were split on Monday morning and then split into 200,000–250,000 cells per 6 wells (up to 6 wells) on Thursday afternoon. Cells were replated for experiments on Thursday using the same cells as for hiPSC maintenance. Differentiation was initiated on Friday.

[0148] Monday: For hiPSC maintenance, coat two to four of the six wells with 3 μl of iMatrix-511 per well in 2 ml of XF medium with 10 μM Y-27632 (1:1000 Y-27632: 1 μl Y per ml medium) for at least 1 hour at 37°C.

[0149] Preheat Accutase and approximately 10 ml of XF medium. Transfer the required volume of Accutase (1 ml per well) to a separate tube. Add 1:1000 Y-27632 to both solutions and mix. hiPSC maintenance wells should be 50-100% confluent and generally undifferentiated.

[0150] Vigorously agitate the plate with cells to collect all dead cells in the supernatant. Absorb the medium completely and wash with 2 ml of PBS. Replace with 1 ml of pre-warmed Accutase containing Y-27632 and transfer to the incubator for 10 minutes. Gentle agitation of the plate should cause most of the cells to detach. If this is not the case, extend the digestion for another 2 minutes or so until the cells have effectively detached on their own.

[0151] Add 3 ml of XF medium containing Y-27632 to a 15 ml tube. Pipette up and down 3-4 times to flush the cells from each well. Transfer the cells from all wells to a 15 ml tube containing 3 ml of XF medium + Y. Centrifuge at 300 g for 3 minutes. The supernatant should be clear and the cells should form a dense pellet.

[0152] The supernatant is absorbed and resuspended in 2 ml of XF medium + Y per 6 wells collected by pipetting up and down 3 times with a 1 ml pipette. Immediately afterwards, gently transfer 10 μl to a counting chamber to quantify the cell titer.

[0153] Seed 200,000 cells per well into each of the 6 pre-coated wells. Transfer to the incubator and gently agitate the plate, as shown in the inscription and infinity symbol inside the incubator.

[0154] Tuesday: Feed the cells with 2.5 ml of pre-warmed XF medium per well.

[0155] Wednesday: · Feed the cells with 3 ml of pre-warmed XF medium per well.

[0156] Thursday: Ensure cells are subconfluent and completely undifferentiated. That afternoon, coat wells with 6 μl of iMatrix-511 and split cells as above, except use only 1-2 wells, seeding cells at 200,000 cells per well. Transfer remaining cells in the supernatant to 6 wells pre-coated with 6 μl of iMatrix-511, seeding cells at 450,000 cells per well (1 cm) for the experiment. 2 The cells are seeded at 50,000 cells per well (50,000 cells per well). The number of wells can vary depending on the experimental design.

[0157] For 12-well differentiation, coat wells with 3 μl of iMatrix-511 in 1 ml of PBS for 1 minute in the incubator. Seed 200,000 cells per well in 1 ml medium containing 10 μM ROCK inhibitor.

[0158] Friday: That afternoon, feed the hiPSC maintenance wells with 6 ml of XF medium per well over the weekend.

[0159] Differentiation of hiPSC (R26 line) into hemogenic endothelium Friday: Ensure that the wells used for differentiation have an even distribution of hiPSCs. The cells should be flat and form loose colonies at approximately 70-90% confluence.

[0160] Prepare and / or pre-warm slightly more than 6 ml of StemPro 34 medium per differentiation well. Thaw the required aliquots of CHIR99021 and BMP4 at RT for a few minutes (2-5 min), then mix by flicking the tube. Optionally, do the same for any additional factors to be tested.

[0161] Depending on the experimental design, prepare differentiation medium for different conditions in an optimal manner. For example, if all wells receive the same amount of CHIR but different concentrations of BMP4, prepare a master mix of stem cell medium with CHIR. Then, distribute it into individual 15 (or 50) ml tubes and add the required amount of other factors, if appropriate. Alternatively, depending on the experimental design, additional factors can be added directly to the wells after replacing the spent maintenance medium with differentiation medium.

[0162] After vigorously agitating the differentiation plate, replace the old XF medium with 6 ml of fresh stem cell medium. Place back in the incubator over the weekend.

[0163] 12-well format: Use 3 ml of stem cell differentiation medium per well.

[0164] Monday: Prepare and / or preheat slightly more than 3.5 ml of stem cell differentiation medium per differentiation well. Thaw the required aliquots of VEGFA and SB431542 at RT for a few minutes (2-5 minutes), then mix by flicking the tube. Optionally, do the same for any additional factors to be tested. After vigorously agitating the differentiation plate, replace the old stem cell medium with fresh stem cell medium.

[0165] 12-well type: Use 1m per well.

[0166] Tuesday: Prepare and / or pre-warm slightly more than 3.5 ml of stem cell differentiation medium per differentiation well (same as on Monday). Thaw the required aliquots of VEGFA and SB431542 at RT for a few minutes (2-5 minutes), then mix by flicking the tube. Optionally, do the same for any additional factors to be tested. After vigorously agitating the differentiation plate, replace the old stem cell medium with fresh stem cell medium.

[0167] 12-well type: Use 1m per well.

[0168] Wednesday: Prepare and / or pre-warm slightly more than 3.5 ml of stem cell differentiation medium per differentiation well (same as on Monday). Thaw the required aliquots of VEGFA and SB431542 at RT for a few minutes (2-5 minutes), then mix by flicking the tube. Optionally, do the same for any additional factors to be tested. After vigorously agitating the differentiation plate, replace the old stem cell medium with fresh stem cell medium.

[0169] 12-well type: Use 1m per well.

[0170] Thursday: Prepare and / or pre-warm slightly more than 3.5 ml of stem cell differentiation medium per differentiation well (same as on Monday). Thaw the required aliquots of VEGFA and SB431542 at RT for a few minutes (2-5 minutes), then mix by flicking the tube. Optionally, do the same for any additional factors to be tested. After vigorously agitating the differentiation plate, replace the old stem cell medium with fresh stem cell medium.

[0171] 12-well type: Use 1m per well.

[0172] Differentiation of hemogenic endothelium into NK cells On Friday (day 7), aspirate the old stem cell medium and wash each well very gently once with 1 ml of PBS. Replace with prewarmed hPL-containing medium + IL3 at a volume of 1 ml per well of a 12-well plate. Add medium very slowly, allowing 0-30% of the clusters to detach. The first suspension cells should appear between days 10 and 14. An ideal culture will have a d14 yield of 1.5 million cells per well, with a minimum yield of 0.5 million cells at this point.

[0173] On Friday (day 14), pool the suspended cells (using a p1000 pipette very gently, drop the old medium onto the adherent layer with as little pressure as possible before transferring the suspended cells to a collection tube), then perform a "spin exchange" by centrifuging at 300g for 8 minutes and resuspending the pellet in new hPL-containing medium (without IL3). Do not allow the adherent cells to dry, and immediately after collecting the suspended cells, add half the final volume of fresh medium (0.5 ml). 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 each).

[0174] On day 17 (Monday), repeat the spin-exchange procedure, but now resuspend the cells in 2 ml per well. From day 20 onward, perform half-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, achieving an average interval of 3.5 days. For the HMC, carefully tilt the plate toward you at approximately 30 degrees, then remove 0.9 ml from the surface of each well with a p1000 tip without disturbing the suspended cell layer at the bottom. Gently add dropwise to 1 ml of fresh hPL-containing medium, avoiding disturbing the settled cells.

[0175] To track cell counts during HMC, measure the exact medium volume after discarding 0.9 ml by removing 0.8 ml with a p1000, then gently rotating the pipette until air is aspirated to remove the remaining volume in the well; record this value. Gently mix this volume to resuspend all suspended cells, then remove 10 μl for counting (using a hemocytometer or diluting with 90 μl of PBS followed by an NC-200). For an accurate cell count in that well, multiply the measured medium volume by the number of cells per million. Note that this will disturb the adherent layer and reduce NK cell production compared to undisturbed cells.

[0176] Do not isolate suspension cells from the adherent layer until ready for expansion (day 28-35, >90% CD56 positivity). Cell numbers should slowly decrease by 4E6 at day 40 to 2.5E6 at day 70.

[0177] Flow cytometry To perform flow cytometry, 1 ml of cell culture medium is spun down at 400 g for 3 minutes, then resuspended in 1 ml of PBS and pelleted again at 400 g for 3 minutes.

[0178] Distribute 500,000 cells per staining group into 98 μl of PBS in a 1.5 ml tube.

[0179] Add 2 μl of the corresponding staining antibody, mix by tapping gently, and incubate for 20 min at RT in the dark.

[0180] Washing step: Add 400 μl of PBS to each tube, then centrifuge at 400 g for 2 minutes. Discard the supernatant.

[0181] Add 300 μl of PBS to each tube and resuspend. Perform flow cytometry. After detection is complete, analyze the flow cytometry results using analysis software.

[0182] NK killing assay: Determine the ratio of effector to target cells to be tested. Keep the total number of combined cells at approximately 1 million + / - 30% respectively. Plan to have a minimum of 200K target cells in the population, so that after processing, at least 30K are detected by the p3 gate (Cell trace violet positive) during FACS analysis.

[0183] Calculate the total number of target cells + 10% reserve across all test ratios, including the "target cells only" spontaneous cell death control group. Resuspend these cells in 1 ml of PBS. Retain a final negative control group of 1E6 pure target cells that are not stained to measure autofluorescence levels but otherwise undergo the same processing steps as the other groups.

[0184] Add 0.5 μl of cell trace violet (final concentration 2.5 μM) and incubate at 37°C for 15 minutes.

[0185] 5 ml of hPL-containing medium is added and incubated for an additional 10 minutes to stop the staining reaction.

[0186] During these last two steps (25 min), prepare 1.5 ml tubes with the required number of NK cells for each test ratio at a concentration of 1E6 NK cells per ml in hPL-containing medium.

[0187] After stopping the cell trace violet staining of the target cells, pellet them at 300g for 5 minutes and resuspend them in hPL-containing medium at 1E6 / ml (e.g., 3E6 planned cells + 300K reserve cells resuspended in 3.3 ml of hPL-containing medium).

[0188] Distribute the target cells into the 1.5 ml tube of prepared NK cells at the calculated ratio.

[0189] Pulse spin down the cells in a microfuge. Gently tap to loosen any pellet that may have formed.

[0190] Open the lid and place the tube in an incubator at 37°C and 5% CO2 for 3.5 hours.

[0191] Add 1 μl of cell even caspase 3 / 7 green reagent to each tube (2 μM final), invert and tap to mix, pulse precipitate again, and place cells in the incubator for a further 25 minutes.

[0192] Discard 600 μl from the top of each tube, being careful not to disturb the cells that have settled to the bottom, for a final volume of 400 μl per tube. Add 0.5 μl of Sytox 7-AAD Live / Dead stain to each tube, mix, and let sit for 5 minutes. While adding the Syntox, use a p10 pipette to remove any large cell clumps that may clog the FACS machine.

[0193] After a 5 minute incubation, briefly vortex the cells and withdraw all 400 μl prior to FACS analysis.

[0194] FACT Settings: Take a small sample of stained target cells and observe the baseline value at a slow speed while viewing the blue, green, and infrared channels. When viewing the green versus red scatter plot, adjust the detector sensitivity so that the negative control cells are primarily in the bottom left corner. Ensure that the blue channel is not overexposed (cell trace violet is very bright). When viewing the SSC versus FSC plot, gate all cells away from the strip in the bottom left corner (including dead cells in the upper left region). Exclude doublets in P2. Use the P3 gate to select only purple cells (compared to the unstained control). For target cells only, view the P3 gate using red versus green scatter, with the quadrant set to have approximately 97% of the cells in the lower left quadrant. 3% of the cells in this group should be spontaneously apoptotic (upper left quadrant) or dead (right half). Use these gates for all other effector / target ratios, and set the stop gate to 30K events in P3.

[0195] [Example 3] Generation and characterization of natural killer cells differentiated from human pluripotent stem cells A novel method for differentiating NK cells from iPSCs has been developed that allows for higher yields, greater purity, and increased simplicity compared to the current gold standard of spin embryoid body (EB) formation. This is achieved by replacing the laborious and unclear EB phase with directed differentiation of iPSCs into hemogenic endothelial cells, which are further differentiated into hematopoietic stem / progenitor cells, recapitulating the natural transition of cell types during embryonic development. The protocol was developed with GMP-compliant processes in mind and allows for stock NK cells derived from HLA-homozygous iPSC banks. This platform significantly reduces the barrier to entry for cancer research groups, enabling them to transfer their self-developed CAR receptors from the laboratory to the clinic, widening the access to potent immunotherapy for patients.

[0196] The objectives of this study were to develop a novel method to produce hematopoietic stem / progenitor cells from iPSCs by using precise directed differentiation instead of spontaneous differentiation and further differentiate these intermediate cell types into NK cells with high purity / yield, to characterize NK cells using widely recognized NK cell surface markers and demonstrate their activity by killing assays, and to establish a GMP-compatible iPSC-based platform with various common NK-related gene editing ready for insertion of tumor-specific CAR receptors.

[0197] iPSCs were maintained by weekly passage of 10,000 iPSCs in Laminin-511 coated 6-well plates. Cells were expanded in Miltenyi iPSC brew XF, and cells reached 70-90% confluence on day 7 of expansion. NK differentiation - iPSCs were seeded into a 12-well format one day before initiating degradation. From day 0 to day 7, iPSCs were initially differentiated into hemogenic endothelium, expressing CD34 / CD144. + From day 7 to day 14, hPL-containing medium with IL3 is applied (as described by Miller & McCullar). From day 14 onwards, the same medium is used without IL3, with half of the medium replaced twice weekly (see Figures 2A-2C, 3A-3E, and 4A-4F).

[0198] Quiescence of iPSC-derived hematopoietic stem / progenitor cells was performed. The importance of all factors during the first week of NK cell differentiation was assessed using a clonogenic assay. 3000 cells were mixed with 2 ml of complete methylcellulose medium (EPO, IL3, GM-CSF, and SCF) and distributed into two wells of a 6-well plate (1 ml = 1500 cells per well). After 7 days of incubation (37°C, 5% CO2), colonies (defined as clusters of >20 cells) were quantified. Factor withdrawal during the first week of NK differentiation (d7-d14) had little effect on colony-forming cell (CFC) yield (see Figures 6A-6C), but at later time points, these conditions increased CFC yield at the expense of target cell differentiation. The overwhelming majority of d14 colonies were of the granulocyte / macrophage lineage, with rare BFU-E (burst-forming unit-erythroid) colonies (see Figure 5). Colony-forming cells from different differentiation conditions gave rise to small colonies over time: ctrl condition represents complete differentiation medium with all factors, -IL-15 represents medium without IL-15, and SCF represents medium with SCF only (all had IL-3 at week 1) (see Figures 7 and 8).

[0199] The purity and long-term culture stability of functional NK cells were assessed. Flow cytometry was performed using a Miltenyi MACS Quant10 flow cytometer. Cells were stained with Miltenyi 1:50 REAfinity FACS antibody, washed, and analyzed immediately. Gating: P1: lymphocyte region, P2: doublet exclusion, P3: CD56 + Cells (NK marker panel only). In each case, 30,000 cells were measured in the final gate. An isotype control antibody was used as a negative control (see Figures 9A-9C).

[0200] As shown in Figures 10A-10B, highly pure NK cells were obtained after 2-3 weeks of HSPC derivation, as exemplified by the percent CD56 expression over time. As shown in Figure 10B, the basal medium used for cell differentiation significantly reduced the CD56 expression obtained during the course of the differentiation protocol. + affected the percentage of cells.

[0201] As shown in Figure 11, NK cells persisted in the culture medium and were harvested without a significant decrease in cell numbers obtained over 56 days. Following the exemplary protocol described in Figure 2C, for example, the HE cell layer continuously generated new HSCs, and these suspension cells (which budded from HE precursors and migrated into the supernatant) were easily harvested from the cell culture supernatant. As evidenced in Figures 3B and 3E, initially, the cells in suspension were not all NK cells (as indicated by the absence of the CD56 marker), but the proportion of NK cells increased over time, achieving enrichment of at least 80% and up to 98% NK cells. As detailed in Figure 3E, the timing of cell transfer affected cell reconstitution and harvested cell numbers.

[0202] Killing assays were performed at different time points during the differentiation protocol. K562 target cells (line: ACC10) were stained with cell trace violet. Cells were mixed with NK cells at the indicated ratios and incubated for 4 hours (37°C, 5% CO). 30 minutes prior to the end, cell event caspase 3 / 7 green was added. Five minutes prior to the end, Syntox 7AAD live / dead stain was added. Cells were analyzed by flow cytometry. Gating: P1: cell fragment exclusion, P2: doublet exclusion, P3: cell trace violet-positive cells (K562 target cells). Stop gate: 30,000 events in the P3 gate (see Figures 12A and 12B). NK cells generated by this protocol demonstrated robust killing of target K562 cells after 35 days. This killing ability remained unchanged when measured after 56 days of culture.

[0203] The cells were further characterized by flow cytometry to assess the expression of different cell surface markers, including CD56, CD16, NKp30, NKp44, NKp46, NKG2D, NKG2A, NKG2C, KIR2D, and KIR3D (see Figures 13A-13I). NK cells generated by this method were strongly positive for natural cytotoxicity receptors, including NKp30, NKp44, and NKp46, and the majority were positive for NKG2D. Only approximately 5% of NK cells were positive for CD16, suggesting a low capacity for antibody-directed cytotoxicity. In addition, inhibitory receptors of the KIR family were only detected in a small percentage of cells, potentially enhancing the cytotoxicity of these NK cells.

[0204] The cells were further analyzed to confirm that the differentiation protocol was CD56 bight and CD56 dim The generation of NK cells was assessed (see Figures 14A-14I). dim NK cells can be differentiated without feeder cells using a completely defined medium. These cells display a distinct surface marker profile; only a few are CD16- or NKp44-positive, while the majority are NKG2D-positive NK cells. The arrows on the right indicate the cells gated in the next graph; the percentages shown subsequently refer to the total cells in the previous gate. In Figure 14C, the lymphocyte region (p1) is gated, as shown in Figure 14D, and then CD56-positive cells (p3) are gated; only these are shown in all subsequent histograms. The P2 gate, not shown, filters out approximately 10% of the cells that are doublets. In Figures 14E–14J, the thin line on the right side of each histogram indicates the readings of cells treated with an isotype control antibody (used as a negative control).

[0205] CD56 bight and CD56 dimTo induce NK cell generation, the hPL-containing medium (DMEM F12 + 15% hPL) was replaced with defined serum-free and animal component-free PSC differentiation medium from day 7 onwards. This provides the experimental design and results, including cell count and viability results, as illustrated in Figures 2C and 3C-3C, respectively. This experiment also tested using stem cell medium (StemPro34™) throughout the entire differentiation (rather than just days 1-7), but this did not work, as can be seen from the suspension cell count results (see Figure 3D).

[0206] A protocol was successfully developed to differentiate iPSCs into HSPCs using precisely defined conditions, yielding large numbers of CD34+ cells. These cells, when analyzed by clonogenic assays, were able to produce primarily CFU-GM colonies, with a minority of BFU-E colonies. These HSPC intermediates could be further differentiated into NK cells upon exposure to a cocktail of growth factors. After initial optimization of differentiation conditions, 99% NK cell purity was achieved, with a total yield of approximately 50 million NK-differentiated cells per plate after 35 days of culture in a 12-well format. After further optimization, the same yield and purity were achieved after only 28 days (data not shown).

[0207] [Example 4] Generation of T cells from isolated iPSC-derived HSC-like cells As illustrated in Figure 15A, the methods of the present invention can be used to generate HE and HSC cells as described above, which are then differentiated into T cells using a combination of IL-7, SCF, and FLT3L.

[0208] As shown in Figure 15B (left flow cytometry panel), HSC-like hematopoietic progenitors marked with CD34 and CD43 can be readily obtained from iPSC-independent cells. As illustrated in the right panel of Figure 15B, using stimulation of Notch signaling, HSCs can be further differentiated into T cell progenitors and definitive T cells marked by CD5 and CD7, and CD4 and CD8, respectively. Although iPSC-line-dependent effects are possible, these data support the notion that iPSC-derived HSCs are pluripotent and can give rise to major lymphoid cell types, including T cells.

[0209] [Example 5] Generation of monocytes / macrophages from isolated iPSC-derived HSC-like cells Based on the protocol illustrated in Figure 16A, which relies on the generation of HPC intermediate cells and their transfer to new culture vessels, monocytes / primary macrophages were obtained using M-CSF stimulation. This procedure yielded essentially pure monocytes / macrophages marked with CD14, CD16, CD163, CD86, and MHC class II (illustrated in Figure 16B).

[0210] Differentiation was also achieved by substantial cell expansion. The data highlight the pluripotency of iPSC-derived HSCs, as they can be readily differentiated along the myeloid lineage.

[0211] [Example 6] HSC Generation and Differentiation Platform Using the protocol described in the Examples above, an HSC production platform was developed. As illustrated in Figure 17 (top left), HE clusters were visible in a typical culture on day 7, and their formation was complete. Small white dots were visible on the well surface of the culture vessel. By day 14 of differentiation (see Figure 17, top center), when endothelial-hematopoietic transition was underway, HSCs were visible as a white halo of dispersed cells around the HE clusters.

[0212] Clonogenic assays were performed at this time point to demonstrate the potency of the multilineage HSCs. Of note, flow cytometry data analysis demonstrated the identity of CD34+, CD90+, CD38- HSCs (data not shown).

[0213] As illustrated in Figure 17, bottom left panel, endothelial-hematopoietic transition (EHT) occurred between days 7 and 17 when hPL-containing medium was used. On day 7 (Figure 17, top left), hemogenic endothelium (HE) formed clusters on the endothelial monolayer. On day 14 (Figure 17, top center), EHT of the clusters progressed, and many suspended cells were visible. When these suspended cells were removed and the medium was replaced, HE clusters were still clearly visible (Figure 17, top right). On day 17 (Figure 17, bottom center), most HE clusters were completely dispersed in the supernatant in the form of HSC suspension cultures. When these cells were removed and the medium was replaced, a cluster-free endothelial monolayer was revealed (Figure 17, bottom right).

[0214] As illustrated in Figure 18A, when generating HSCs using hPL-containing medium, approximately half of the EHT was performed by day 14, and the remaining half was completed by day 17. Typically, from a single well of a 12-well plate, a yield of 1-3 million HSCs was expected by day 14. After an additional 4 days of culture, clonogenic assays of day 14 cells showed that the generated HSCs were able to form white blood cell colonies. The clonogenic assay medium used was methylcellulose containing IL3, IL6, EPO, and SCF.

[0215] As shown in Figure 18B, counting the number of colonies resulting from inoculation of 1000 HSCs on days 14 or 17 revealed that approximately 35% of the cells were able to give rise to colonies (defined as >20 cells). Analysis of the colonies classified them as predominantly granulocyte / macrophage (GM-CFU) colony-forming units. A small number of red "burst-forming unit-erythroid" (BFU-E) colonies were also present.

[0216] As shown in Figure 19A, surface marker analysis on day 14 revealed a population of CD34+, CD90+, CD38- HSCs by flow cytometry. Surface marker analysis on day 17 revealed a small population of CD34+, CD90+, CD38- HSCs with a clear tendency toward differentiation (CD38+) visualized by flow cytometry (Figure 19B).

[0217] [Example 7] Production of HSCs in various cell culture media Between days 7 and 17, hemogenic endothelial clusters undergo endothelial-hematopoietic transition (EHT), during which adherent cells disperse into the supernatant, forming a suspension cell culture of HSCs. This was first demonstrated using a medium containing human platelet lysate (hPL). Therefore, we investigated whether the medium had this effect and demonstrated that by using a completely defined serum-free and animal component-free PSC differentiation medium, different types of HSCs could be generated with distinct differentiation potentials and surface marker profiles. HSCs derived from four different basal media were characterized with (4F) or without any additional factors (0F) and for their subsequent ability to differentiate into NK cells. All suspension cells were isolated on day 14 and transferred to new culture vessels for continuous differentiation. This was repeated on day 17 using the suspension cells resulting from day 14. Finally, cells reconstituted in the initial wells after day 17 were also evaluated. On day 38, suspension cells from all three groups were analyzed for NK identity. The protocol is illustrated schematically in Figure 20, where arrows indicate the analysis points. Cells with a surface marker profile of CD34+ / CD90+ / CD38- were considered HSCs.

[0218] By day 14, all conditions (hPL-containing medium, defined medium 1, defined medium 2, and defined medium 3) were able to generate CD34+ / CD90+ / CD38- HSCs. Self-made defined medium 2 consisted of AscP, VA, albumin (human), lipids (chemically defined), ITS-X, and IMD. Cytokines added to the basal medium included 5 ng / ml IL3, 20 ng / ml TPO, 20 ng / ml SCF, and 10 ng / ml FLT3L. The number of CD90+ / CD38- cells, representing the calculated total HSCs, in a single plate of a 12-well plate at the time of analysis was as follows: hPL-containing medium (no cytokines): 2,300 cells hPL-containing medium (all cytokines): 3,600 cells Defined Medium 1 (no cytokines): 2,500 cells Defined Medium 1 (all cytokines): 3,400 cells Defined Medium 2 (no cytokines): 3,000 cells Defined Medium 2 (all cytokines): 5,500 cells Defined Medium 3 (no cytokines): 10,700 cells Defined Medium 3 (all cytokines): 41,500 cells.

[0219] All conditions were able to generate CD34+ / CD90+ / CD38- HSCs on day 17. The number of CD90+ / CD38- cells, representing the calculated total HSCs, in a single plate of a 12-well plate at the time of analysis was as follows: hPL-containing medium (no cytokines): 7,800 cells hPL-containing medium (all cytokines): 12,000 cells Defined Medium 1 (no cytokines): 8,300 cells Defined Medium 1 (all cytokines): 39,000 cells Defined Medium 2 (no cytokines): 81,000 cells Defined Medium 2 (all cytokines): 214,000 cells Defined Medium 3 (no cytokines): 93,000 cells Defined Medium 3 (all cytokines): 75,000 cells.

[0220] By day 38 (reconstituted on day 17), all conditions were able to generate CD56+ NK cells. The number of NK cells, representing the calculated total HSCs, in a single plate of a 12-well plate at the time of analysis was as follows: hPL-containing medium (no cytokines): 1.1M cells hPL-containing medium (all cytokines): 0.3M cells Defined Medium 1 (no cytokines): 0.6M cells Defined Medium 1 (all cytokines): 2.6M cells Defined Medium 2 (no cytokines): 1.7M cells Defined Medium 2 (all cytokines): 2.7M cells Defined Medium 3 (no cytokines): 0.2M cells Defined Medium 3 (all cytokines): 0.3M cells.

[0221] On day 38 (transferred on day 14), only the DF12 / hPL-based medium was able to generate D56+ NK cells. The number of NK cells, representing the calculated total HSCs, in a single plate of a 12-well plate at the time of analysis was as follows: hPL-containing medium (no cytokines): 0.5M cells hPL-containing medium (all cytokines): 0.7M cells Defined Medium 1 (no cytokines): 0M cells Defined Medium 1 (all cytokines): 0M cells Defined Medium 2 (no cytokines): 0M cells Defined Medium 2 (all cytokines): 0M cells Defined Medium 3 (no cytokines): 0M cells Defined Medium 3 (all cytokines): 0M cells.

[0222] On day 38 (transferred on day 17), Nutri T-based medium alone failed to produce D56+ NK cells. The number of NK cells, representing the calculated total HSCs, in a single plate of a 12-well plate at the time of analysis was as follows: hPL-containing medium (no cytokines): 0.5M cells hPL-containing medium (all cytokines): 0.3M cells Defined Medium 1 (no cytokines): 0M cells Defined Medium 1 (all cytokines): 0M cells Defined Medium 2 (no cytokines): 0.4M cells Defined Medium 2 (all cytokines): 1.2M cells Defined Medium 3 (no cytokines): 0.3M cells Defined Medium 3 (all cytokines): 0.3M cells.

[0223] [Example 8] Cryopreservation of HSCs HSCs were cryopreserved on days 14, 17 and 21 and thawed in hPL-containing medium + / - IL15 (days 0-10, then all + IL15).

[0224] It was demonstrated that HSCs generated using hPL-containing medium could be cryopreserved, later thawed in culture, and differentiated into NK cells. During the first 10 days of culture, half of the wells were supplemented with IL15, while the other half were not. Three other factors (SCF, FLT3L, and IL7) were supplemented to both conditions at their defined concentrations. As illustrated in Figure 21, cell numbers from the various conditions showed that only cryopreserved cells expanded significantly upon differentiation on days 14 and 17.

[0225] Flow cytometry data of differentiation cultures on days 7, 14, 21 and 28 post-thaw were analyzed.

[0226] On day 7, numerous CD15-positive cells were observed, suggesting the initial production of granulocytes. A small population of CD14-positive cells was also present, representing monocytes. The lack of a distinct CD56-positive population indicates that NK cells are not present at this time point (data not shown).

[0227] Detection of CD15-positive cells on day 14 suggested the presence of granulocytes. A population of CD14-positive cells indicated the presence of monocytes. A distinct CD56-positive population was visible, indicating that primary NK cells had differentiated in all groups at this time point (data not shown).

[0228] By day 21, the largest population in most groups was CD14 / CD15 double positive. A distinct CD56 positive population was visible, indicating that NK cells had differentiated in all groups at this time point (data not shown).

[0229] Day 28. The majority of cells were CD56 positive in all groups, confirming that cryopreserved HSCs could be thawed into NK differentiation medium (data not shown).

[0230] [Table 3]

[0231] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. CD34 ＋ 1. A method for producing a population of hematopoietic progenitor cells, comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) and allowing the PSCs to grow on a substrate for about 1-8 days; and b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF), alone or in combination with a WNT signaling pathway activator and / or a BMP, for about 1-8 days after step a); the cells produced after step b) are enriched for CD34+ cells in the overall population of cells by at least about 80%; As a result, CD34 ＋ To generate a population of progenitor cells A method comprising:

2. Contacting adherent cultures of PSCs with WNT signaling pathway activators, BMPs, and / or VEGF induces CD34 ＋ The method of claim 1, wherein hemogenic endothelium (HE) is produced.

3. CD34 ＋ The population of progenitor cells is contacted with the drug mixture to induce CD34 ＋ The method of claim 2, further comprising inducing differentiation of the progenitor cells into terminally differentiated cells of the hematopoietic lineage.

4. 4. The method of claim 3, wherein the cells of the hematopoietic lineage are natural killer (NK) cells or other immune cells.

5. 10. The method of claim 1, wherein the culture of PSCs is contacted with the WNT signaling pathway activator and the BMP for about 3 days and with VEGF for an additional about 4 days.

6. After about a week of treatment, the cells were found to be CD34 ＋ , K.D.R. ＋ , CD31 ＋ and CD45 － The method according to claim 1 or 5, wherein

7. CD34 ＋ The cells also express CD144 ＋ The method of claim 1 or 5, wherein

8. 1. A method for producing natural killer (NK) cells, comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and / or a bone morphogenetic protein (BMP) and allowing the PSCs to grow on a substrate for about 1-8 days; b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF), alone or in combination with a WNT signaling pathway activator and / or BMP, for about 1-8 days after step a), thereby increasing the CD34 ＋ generating a population of progenitor cells, wherein the cells produced after step b) are CD34 ＋ be at least about 80% enriched for cells; and c) CD34 ＋ contacting the population of progenitor cells with one or more of interleukin 7 (IL-7), IL-15, SCF, and FMS-like tyrosine kinase 3 ligand (FLT3L); thereby producing NK cells A method comprising:

9. c) CD34 ＋ contacting the population of progenitor cells (i) CD34 ＋ contacting the progenitor cells with IL-7, IL-15, SCF, and FLT3L for about 5-10 days; and (ii) CD34 ＋ contacting the progenitor cells with IL-7, IL-15, FLT3L and SCF for at least about 7-21 days; The method of claim 8, comprising:

10. The cells are transiently transfected with CD34 ＋ and CD45 ＋ The method of claim 9, wherein

11. 9. The method of claim 8, wherein contacting the adherent culture of PSCs comprises one or more agents selected from about 1-10 μM of a WNT signaling pathway activator, about 5-50 ng / ml of a BMP, and about 50-500 ng / ml of VEGF.

12. 12. The method of claim 11, wherein the WNT signaling pathway activator is a GSK3 inhibitor.

13. 13. The method of claim 12, wherein the GSK3 inhibitor is CHIR99021.

14. The method of claim 11 , wherein the BMP is BMP4.

15. The method of claim 11, wherein the VEGF is VEGF-A.

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

17. 9. The method of claim 8, wherein c) comprises about 4 to 40 ng / ml of IL-7, about 2 to 20 ng / ml of IL-15, about 4 to 40 ng / ml of SCF, and / or about 1 to 20 ng / ml of FLT3L.

18. 18. The method of claim 17, wherein c) comprises about 20 ng / ml IL-7, about 10 ng / ml IL-15, about 20 ng / ml SCF and / or about 10 ng / ml FLT3L.

19. 9. The method of claim 8, wherein contacting the adherent culture of PSCs in a) with the WNT signaling pathway activator and the BMP is for about 2 to 5 days.

20. 9. The method of claim 8, wherein the subsequent contacting of the PSCs with VEGF is for about 2 to 5 days.

21. CD34 ＋ Contacting the population of progenitor cells with IL-7, IL-15, SCF and / or FLT3L for about 5-10 days followed by CD34 ＋ 12. The method of claim 11, wherein contacting the population of progenitor cells with IL-7, IL-15, FLT3L and / or SCF is for at least about 7-21 days.

22. The method of claim 8 , wherein the culture of PSCs is an adherent layer of cells.

23. 23. The method of claim 22, wherein the layer of cells is grown in a two-dimensional culture system or on microcarriers.

24. 23. The method of claim 22, wherein the PSCs are cultured on a coated surface comprising a laminin coating.

25. 9. The method of claim 8, further comprising collecting the NK cells from the suspension in the cell culture medium.

26. 9. The method of claim 8, wherein the PSCs are human PSCs (hPSCs).

27. 27. The method of claim 26, wherein the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

28. CD34 ＋ Progenitor cells are CD34 ＋ The method of claim 8, wherein the cells are endothelial-like progenitor cells.

29. 9. The method of claim 8, wherein the NK cells are enriched by at least about 80%.

30. 1. A method for inducing differentiation of pluripotent stem cells (PSCs) into natural killer (NK) cells, comprising: a) (i) contacting adherent cultures of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) for about 3 days; and (ii) contacting the adherent culture of PSCs of i) with vascular endothelial growth factor (VEGF) for about 4 days; Thereby, at least 80% of CD34 ＋ By generating a population of cells comprising hemogenic endothelial (HE) cells, CD34 ＋ generating HE cells; b) CD34 of a) ＋ The HE cells are contacted with one or more of IL-7, IL-15, SCF and / or FLT3L for about 7 days, thereby reducing at least 80% of the CD34 ＋ / CD45 ＋ generating a transient population of cells comprising hematopoietic stem cells (HSCs); and c) Subsequently, CD34 of b) ＋ / CD45 ＋ contacting the HSCs with one or more of IL-7, IL-15, FLT3L, and SCF for at least about 7-21 days; This induces differentiation of PSCs into NK cells. A method comprising:

31. Differentiated NK cells express CD56 ＋ , NKp30 ＋ , NKp44 ＋ , NKp46 ＋ , NKG2D ＋ , NKG2A+, KIR2D ＋ and / or CD16 ＋ 31. The method of claim 30, wherein:

32. Differentiated NK cells express CD56 ｂｒｉｇｈｔ or CD56 ｄｉｍ 31. The method of claim 30, wherein:

33. The method of claim 30, wherein the differentiated NK cells are cytotoxic NK cells.

34. a) a hemogenic endothelial (HE)-inducing cocktail containing a WNT signaling pathway activator, bone morphogenetic protein (BMP) and / or vascular endothelial growth factor (VEGF); b) a natural killer (NK)-inducing cocktail comprising interleukin-7 (IL-7), IL-15, SCF, and / or FLT3L; and c) Instructions for inducing differentiation of pluripotent stem cells (PSCs) into NK cells Includes a kit.

35. 35. The kit of claim 34, further comprising a laminin-coated surface.

36. 1. A method for generating terminally differentiated hematopoietic cells from pluripotent stem cells (PSCs), comprising: a) (i) contacting an adherent culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) and allowing the PSCs to grow on the substrate for about 2-5 days; and (ii) contacting adherent cultures of PSCs with vascular endothelial growth factor (VEGF) for about 2-5 days after step (i), and the cells produced after step (ii) exhibiting CD34 activity throughout the cell population; ＋ cells, thereby enriching for CD34 ＋ By generating progenitor cells, CD34 ＋ generating hematopoietic progenitor cells; and b) CD34 of a) ＋ The progenitor cells are contacted with the mixture of drugs to induce CD34 ＋ Induce differentiation of progenitor cells into terminally differentiated hematopoietic cells, thereby generating terminally differentiated hematopoietic cells. A method comprising:

37. 37. The method of claim 36, wherein the terminally differentiated hematopoietic cell is an immune cell or an erythrocyte.

38. 38. The method of claim 37, wherein the immune cells are selected from the group consisting of macrophages, T cells, and natural killer (NK) cells.

39. CD34 ＋ 1. A method for producing a population of hematopoietic stem cells, comprising: a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) and allowing the PSCs to grow on a substrate for about 1-8 days; and b) contacting the culture of PSCs with vascular endothelial growth factor (VEGF), alone or in combination with a WNT signaling pathway activator and / or a BMP, for about 1-8 days after step a); the cells produced after step b) are enriched for CD34+ hematopoietic stem cells in the overall population of cells by at least about 80%; As a result, CD34 ＋ To generate a population of hematopoietic stem cells A method comprising:

40. 40. The method of claim 39, wherein CD34+ hemogenic endothelium (HE) is generated by contacting adherent cultures of PSCs with a WNT signaling pathway activator, BMP and / or VEGF.

41. 40. The method of claim 39, wherein the PSCs are human PSCs (hPSCs).

42. 42. The method of claim 41, wherein the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

43. 1. A method for generating hematopoietic stem cells from pluripotent stem cells (PSCs), comprising: a) contacting adherent cultures of induced pluripotent stem cells (iPSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), and allowing the iPSCs to grow on the substrate for about 2-5 days; and b) contacting adherent cultures of PSCs with vascular endothelial growth factor (VEGF) for about 2-5 days after step (a), and determining that the cells produced after step (b) express CD34 throughout the population of cells; ＋ cells, thereby enriching for CD34 ＋ Generate progenitor cells, thereby generating hematopoietic stem cells A method comprising:

44. 44. The method of claim 43, wherein the iPSCs are human iPSCs (hiPSCs).

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