Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts

Abstract

To provide methods of generating natural killer (NK) cells and dendritic cells (DCs).SOLUTION: The methods utilize human hemangioblasts as intermediate cells to generate natural killer (NK) cells and dendritic cells (DCs). In various embodiments, the methods do not require the use of stromal feeder layers. In various embodiments, the hemangioblasts can be differentiated from human embryonic stem cells (hESCs). In other embodiments, the hemangioblasts can be differentiated from induced pluripotent (iPS) cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the generation of natural killer (NK) cells and dendritic cells (DC) from hemangioblasts. [Background technology]

[0002] All publications in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and explicitly indicated to be incorporated herein by reference. The following description contains information that may be useful in understanding the present invention. It is not an admission that any information provided herein is prior art or relevant to the claimed invention, nor is any publication explicitly or implicitly referenced prior art.

[0003] Studies with human and mouse embryonic stem cells (ESCs) have shown that common precursors of both vascular cell lineages (endothelial and smooth muscle cells) and hematopoietic cell lineages, called hemangioblasts, can be produced from ESC-derived embryoid bodies in culture. Our group has developed a facile strategy to efficiently and reproducibly generate hemangioblasts from multiple hESC lineages under serum- and stroma-free conditions, which is important for the application of hemangioblast production in regenerative medicine. Previous studies have shown that hESC-derived hemangioblasts can effectively differentiate into erythroid and myeloid lineages, but their ability to produce lymphoid lineage cells, including those with immunotherapeutic potential, remains relatively unknown.

[0004] Natural killer (NK) cells arise from the lymphoid lineage, are part of the innate immune system, and have been found to detect and kill certain types of tumor cells, and can therefore be used in anti-cancer therapy. Dendritic cells (DCs) often arise from the myeloid lineage (from monocytes), are part of the adaptive immune system, and can enhance antigen-specific immune responses due to their ability to present antigens, and can be used to stimulate both naive and memory T cells (e.g., DC-based vaccine therapy). Summary of the Invention [Problem to be solved by the invention]

[0005] Given their immunotherapeutic potential, there is a need in the art for methods to generate natural killer (NK) cells and dendritic cells (DCs). [Means for solving the problem]

[0006] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods that are intended to be exemplary and illustrative, not limiting in scope.

[0007] Various embodiments of the present invention provide a method of generating natural killer (NK) cells comprising providing hemangioblasts; culturing the hemangioblasts on methylcellulose and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, SCF, and FL; harvesting the cultured cells; and culturing the harvested cells in a liquid medium comprising human serum and a second cytokine mixture comprising IL7, IL15, SCF, and FL to generate NK cells.

[0008] In various embodiments, the methylcellulose may be H4236 methylcellulose, while in other embodiments, the methylcellulose may be H4536 methylcellulose.

[0009] In various embodiments, the concentration of IL2 may be about 5-10 ng / ml, IL3 may be about 1-10 ng / ml, IL6 may be about 1-10 ng / ml, IL7 may be about 5-20 ng / ml, IL15 may be about 5-10 ng / ml, SCF may be about 10-50 ng / ml, and FL may be about 10-50 ng / ml.

[0010] In various embodiments, the hemangioblasts may be cultured for about 6-8 days. In various embodiments, the harvested cells may be cultured for about 14-21 days. In various embodiments, the method may further include changing the medium once a week to refresh the second cytokine mixture.

[0011] In various embodiments, the hemangioblasts can be differentiated from human embryonic stem cells (hESCs). In other embodiments, the hemangioblasts can be differentiated from induced pluripotent (iPS) cells.

[0012] In various embodiments, the NK cells may be immature NK cells and may be CD56+ and CD16-. In other embodiments, the NK cells may be mature NK cells and may be CD56- and CD16+, or may be CD56lo and CD16+.

[0013] Various embodiments of the present invention provide methods of generating natural killer (NK) cells, comprising providing hemangioblasts; culturing the hemangioblasts in a liquid medium comprising human serum and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, and SCF; harvesting the cultured cells; and culturing the harvested cells in a liquid medium comprising human serum and a second cytokine mixture comprising IL7, IL15, SCF, and FL to generate NK cells.

[0014] In various embodiments, the concentration of IL2 may be about 5-10 ng / ml, IL3 may be about 1-10 ng / ml, IL6 may be about 1-10 ng / ml, IL7 may be about 5-20 ng / ml, IL15 may be about 5-10 ng / ml, SCF may be about 10-50 ng / ml, and FL may be about 10-50 ng / ml.

[0015] In various embodiments, the hemangioblasts may be cultured for about 6 to 8 days. In various embodiments, the harvested cells may be cultured for about 14 to 21 days.

[0016] In various embodiments, the method may further comprise changing the medium once a week to refresh the second cytokine mixture.

[0017] In various embodiments, the hemangioblasts can be differentiated from human embryonic stem cells (hESCs). In other embodiments, the hemangioblasts can be differentiated from induced pluripotent (iPS) cells.

[0018] In various embodiments, the NK cells may be immature NK cells and may be CD56+ and CD16-. In other embodiments, the NK cells may be mature NK cells and may be CD56- and CD16+, or may be CD56lo and CD16+.

[0019] Various embodiments of the invention provide natural killer (NK) cells generated by any of the methods of the invention. Other embodiments of the invention provide pharma- ceutically acceptable compositions comprising an amount of NK cells generated by any of the methods of the invention.

[0020] Various embodiments of the present invention provide a method of generating dendritic cells (DCs), comprising providing hemangioblasts; culturing the hemangioblasts in a liquid medium containing human serum, SCF, FL, IL3, and GM-CSF; adding IL4 to the liquid medium; and further culturing the hemangioblasts to generate DCs.

[0021] In various embodiments, the hemangioblasts may be cultured for about 7 to 11 days. In various embodiments, the hemangioblasts after addition of IL4 may be cultured for about 8 to 10 days.

[0022] In various embodiments, the method may further include adding a cytokine mixture comprising IL1b, TNFα, and IL6 to induce maturation of DCs. In various embodiments, the cytokine mixture may be added for about 48 hours. In various embodiments, the cytokine mixture may further comprise a cytokine selected from the group consisting of PGE2, IFNα2b, poly I:C, IFNγ, and combinations thereof.

[0023] In various embodiments, the method may further comprise adding LPS, IFNγ, and / or S-28463 to stimulate IL12p70 production from DCs and / or HLA-DR expression from DCs.

[0024] In various embodiments, the concentration of SCF may be about 20-100 ng / ml, FL may be about 10-50 ng / ml, IL3 may be about 5-50 ng / ml, GM-CSF may be about 50-100 ng / ml, and IL4 may be about 50-100 ng / ml. In various embodiments, the concentration of IL1b may be about 10 ng / ml, TNFγ may be about 10 ng / ml, and IL6 may be about 150 ng / ml. In various embodiments, the concentration of PGE2 may be about 1 μg / ml, IFNα2b may be about 3000 units / ml, poly I:C may be about 20 μg / ml, and IFNγ may be about 20 ng / ml.

[0025] In various embodiments, the DCs may be mature DCs and may express CD83, hi other embodiments, the DCs may be mature DCs and have increased expression of CD209, HLA DR, and / or CD11c.

[0026] Various embodiments of the invention provide dendritic cell (DC) cells generated by any of the methods of the invention. Other embodiments of the invention provide pharma- ceutically acceptable compositions comprising an amount of DCs generated by any of the methods of the invention.

[0027] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

[0028] Representative embodiments are illustrated in the referenced drawings: It is intended that the embodiments and drawings disclosed herein are to be regarded as illustrative and not restrictive. For example, the present invention provides the following: (Item 1) 1. A method for generating natural killer (NK) cells, comprising: Providing hemangioblast cells; culturing the hemangioblasts on methylcellulose and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, SCF, and FL; harvesting the cultured cells; and Culturing the collected cells in a liquid medium containing human serum and a second cytokine mixture containing IL7, IL15, SCF, and FL to generate NK cells. (Item 2) The method according to claim 1, wherein the methylcellulose is H4236 methylcellulose. The method according to claim 1, wherein the methylcellulose is H4536 methylcellulose. 2. The method according to item 1, wherein the concentrations of IL2 are about 5 to 10 ng / ml, IL3 are about 1 to 10 ng / ml, IL6 are about 1 to 10 ng / ml, IL7 are about 5 to 20 ng / ml, IL15 are about 5 to 10 ng / ml, SCF are about 10 to 50 ng / ml, and FL are about 10 to 50 ng / ml. (Item 5) 2. The method according to item 1, wherein the hemangioblasts are cultured for about 6 to 8 days. (Item 6) 2. The method according to item 1, wherein the recovered cells are cultured for about 14 to 21 days. (Item 7) 2. The method of claim 1, further comprising changing the medium once a week to refresh the second cytokine mixture. (Item 8) 9. The method of claim 1, wherein the hemangioblasts are differentiated from human embryonic stem cells (hESCs). 2. The method of claim 1, wherein the hemangioblasts are differentiated from induced pluripotent (iPS) cells. (Item 10) 2. The method of claim 1, wherein the NK cells are immature NK cells and are CD56+ and CD16-. (Item 11) The method of claim 1, wherein the NK cells are mature NK cells and are CD56- and CD16+, or are CD56lo and CD16+. (Item 12) A natural killer (NK) cell generated by the method according to any one of items 1 to 11. (Item 13) 13. A pharma- ceutically acceptable composition comprising an amount of the NK cell according to item 12. (Item 14) 1. A method for generating natural killer (NK) cells, comprising: Providing hemangioblast cells; culturing the hemangioblasts in a liquid medium comprising human serum and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, and SCF; harvesting the cultured cells; and Culturing the collected cells in a liquid medium containing human serum and a second cytokine mixture containing IL7, IL15, SCF, and FL to generate the NKs. (Item 15) Item 15. The method according to item 14, wherein the concentrations of IL2 are about 5 to 10 ng / ml, IL3 are about 1 to 10 ng / ml, IL6 are about 1 to 10 ng / ml, IL7 are about 5 to 20 ng / ml, IL15 are about 5 to 10 ng / ml, SCF are about 10 to 50 ng / ml, and FL are about 10 to 50 ng / ml. (Item 16) 15. The method according to item 14, wherein the hemangioblasts are cultured for about 6 to 8 days. (Item 17) Item 15. The method according to item 14, wherein the harvested cells are cultured for about 14 to 21 days. (Item 18) 15. The method of claim 14, further comprising changing the medium once a week to refresh the second cytokine mixture. (Item 19) 15. The method of claim 14, wherein the hemangioblasts are differentiated from human embryonic stem cells (hESCs). (Item 20) 21. The method of claim 14, wherein the hemangioblasts are differentiated from induced pluripotent (iPS) cells. 15. The method of claim 14, wherein the NK cells are immature NK cells and are CD56+ and CD16-. (Item 22) The method of claim 14, wherein the NK cells are mature NK cells and are CD56- and CD16+ or are CD56lo and CD16+. (Item 23) A natural killer (NK) cell produced by the method according to any one of items 14 to 22. (Item 24) 25. A pharma- ceutically acceptable composition comprising an amount of the NK cell according to item 24. (Item 25) 1. A method for generating dendritic cells (DCs), comprising: Providing hemangioblast cells; Culturing the hemangioblasts in a liquid medium containing human serum, SCF, FL, IL3, and GM-CSF; adding IL4 to the liquid medium; and further culturing said hemangioblasts to generate said DCs. (Item 26) 26. The method of claim 25, wherein the hemangioblasts are cultured for about 7 to 11 days. (Item 27) 26. The method according to item 25, wherein the hemangioblasts are cultured for about 8 to 10 days after the addition of IL4. (Item 28) 26. The method of claim 25, further comprising adding a cytokine mixture comprising IL1b, TNFα, and IL6 to induce maturation of the DCs. (Item 29) 29. The method of claim 28, wherein the cytokine mixture is added for about 48 hours. (Item 30) 29. The method of claim 28, wherein the cytokine mixture further comprises a cytokine selected from the group consisting of PGE2, IFNα2b, Poly I:C, IFNγ, and combinations thereof. (Item 31) 26. The method of claim 25, further comprising adding LPS, IFNγ, and / or S-28463 to stimulate IL12p70 production from the DCs and / or HLA-DR expression from the DCs. (Item 32) 26. The method according to item 25, wherein the concentrations of SCF are about 20 to 100 ng / ml, FL are about 10 to 50 ng / ml, IL3 are about 5 to 50 ng / ml, GM-CSF are about 50 to 100 ng / ml, and IL4 are about 50 to 100 ng / ml. (Item 33) 29. The method of claim 28, wherein the concentrations of IL1b is about 10 ng / ml, TNFγ is about 10 ng / ml, and IL6 is about 150 ng / ml. (Item 34) 31. The method according to item 30, wherein the concentrations of PGE2 are about 1 μg / ml, IFNα2b are about 3000 units / ml, poly I:C are about 20 μg / ml, and IFNγ are about 20 ng / ml. (Item 35) The method of item 25, wherein the DC is a mature DC and expresses CD83. (Item 36) 26. The method of claim 25, wherein the DC is a mature DC and has increased expression of CD209, HLA DR, and / or CD11c. (Item 37) A dendritic cell (DC) cell produced by the method according to any one of Items 25 to 36. (Item 38) A pharma- ceutically acceptable composition comprising an amount of DC cells according to item 37. [Brief description of the drawings]

[0029] [Figure 1-1] FIG. 1 shows that hemangioblasts are bipotent progenitor cells that can give rise to both hematopoietic (A-C) and vascular (D-F) lineages according to various embodiments of the present invention. [Figure 1-2] FIG. 1 shows that hemangioblasts are bipotent progenitor cells that can give rise to both hematopoietic (A-C) and vascular (D-F) lineages according to various embodiments of the present invention. [Figure 2-1] Figure 1 shows that cells with immunotherapeutic capabilities, such as dendritic cells, can be differentiated from hemangioblasts according to various embodiments of the present invention. A. Dendritic cell surface marker expression at day 28 of differentiation. B. Bar graph showing that DC surface marker expression can be increased by contact with maturation cytokines for 48 hours. C. Histogram showing that DCs can take up and process DQ-ovalbumin antigen in a 30 minute assay. D. Wright-Giemsa staining of DCs, 20x. E. Wright-Giemsa staining of DCs, 100x. [Figure 2-2]Figure 1 shows that cells with immunotherapeutic capabilities, such as dendritic cells, can be differentiated from hemangioblasts according to various embodiments of the present invention. A. Dendritic cell surface marker expression at day 28 of differentiation. B. Bar graph showing that DC surface marker expression can be increased by contact with maturation cytokines for 48 hours. C. Histogram showing that DCs can take up and process DQ-ovalbumin antigen in a 30 minute assay. D. Wright-Giemsa staining of DCs, 20x. E. Wright-Giemsa staining of DCs, 100x. [Diagram 3] Figure 1 shows that upon contact with lymphoid inducing cytokines, hESC-derived hemangioblasts can give rise to CD56low / CD16+ natural killer cells in a feeder-free culture system according to various embodiments of the present invention. A. During methylcellulose culture, a subset of hemangioblasts acquires the common leukocyte antigen, CD45. B. Transfer of hemangioblasts to liquid culture (containing human serum and a cytokine mixture) allows acquisition of the NK cell marker, CD56. C. Emergence of CD56low / CD16+ NK cells after a total of 28-40 days of differentiation. [Figure 4] FIG. 1 shows that intracellular flow cytometry, as well as the classical 51Cr release assay, can be used to assess NK-mediated cytotoxic activity according to various embodiments of the present invention. [Diagram 5] FIG. 1 shows that hemangioblast-derived NK effector cells are able to induce apoptosis of K562 erythroleukemia target cells after a standard 4-hour co-culture according to an embodiment of the present invention. [Figure 6-1] FIG. 1 illustrates a process for generating NK cells according to an embodiment of the present invention. [Figure 6-2] FIG. 1 illustrates a process for generating NK cells according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] All references cited herein are incorporated by reference as if fully set forth herein. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., J. Wiley & Sons (New York, NY 2001);March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5 th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), will provide one of skill with a general guide to many of the terms used in this application.

[0031] One skilled in the art will identify many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined as follows.

[0032] As used throughout this specification and the claims which follow, the meanings of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Also, as used herein, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise.

[0033] The term "embryonic stem cell" (ES cell) is used herein as it is used in the art. This term includes cells obtained from the inner cell mass of human blastocysts or morulae, including those that have been serially passaged as cell lines. ES cells may be obtained from fertilization of an egg cell with a sperm, and also using DNA, nuclear transfer, parthenogenesis, or by means of generating ES cells homozygous in the HLA region. ES cells are also cells obtained from zygotes, blastomeres, or blastocyst stage mammalian embryos that have developed by fusion of a sperm with an egg cell, nuclear transfer, parthenogenesis, androgenetics, or by reprogramming chromatin and subsequent integration of the reprogrammed chromatin into the cell membrane to generate cells. Embryonic stem cells, regardless of their source or the particular method used to generate them, can be identified based on (i) their ability to differentiate into cells of all three germ layers, (ii) their expression of at least Oct4 and alkaline phosphatase, and (iii) their ability to generate teratomas when transplanted into immunodeficient animals.

[0034] As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are obtained. Pluripotent stem cells are functionally defined as stem cells that: (a) have the ability to induce teratomas when transplanted into immunodeficient (SCID) mice; (b) have the ability to differentiate into cell types of all three germ layers (e.g., can differentiate into ectodermal, mesodermal, and endodermal cell types); and (c) express one or more markers of embryonic stem cells (e.g., express Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). Representative pluripotent stem cells can be generated, for example, using methods known in the art. Exemplary pluripotent stem cells include embryonic stem cells obtained from the ICM of a blastocyst stage embryo, as well as from one or more blastomeres of a cleavage or morula stage embryo (which may be done without destroying the remainder of the embryo, if desired). Such embryonic stem cells may be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgeny. Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells) generated by reprogramming somatic cells by expressing a combination of factors (hereinafter referred to as reprogramming factors). iPS cells may be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that may be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that may be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4, Sox2, Nanog, and Lin28. In other embodiments, somatic cells are reprogrammed by expression of at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors.Induced pluripotent stem cells can be produced by expressing a combination of reprogramming factors in somatic cells. In certain embodiments, at least two reprogramming factors are expressed in somatic cells for successful reprogramming of somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells for successful reprogramming of somatic cells. In other embodiments, at least four reprogramming factors are expressed in somatic cells for successful reprogramming of somatic cells. Induced pluripotent stem cells can be produced by protein transduction of reprogramming factors into somatic cells. In certain embodiments, at least two reprogramming proteins are transduced into somatic cells for successful reprogramming of somatic cells. In other embodiments, at least three reprogramming proteins are transduced into somatic cells for successful reprogramming of somatic cells. In other embodiments, at least four reprogramming proteins are transduced into somatic cells for successful reprogramming of somatic cells.

[0035] In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells. Induced pluripotent stem cells are functionally defined and include cells that have been reprogrammed using any of a variety of methods (integrative vectors, non-integrative vectors, chemical means, etc.).

[0036] Pluripotent stem cells may be from any species. Embryonic stem cells have been successfully obtained, for example, in mice, several species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one skilled in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including, but not limited to, humans, non-human primates, rodents (mouse, rat), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), felines (domestic and wild felines, such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), and the like. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.

[0037] Similarly, iPS cells may be from any species. These iPS cells have been successfully generated using mouse and human cells. iPS cells have been successfully generated using embryonic, fetal (fetus), neonatal (pup), and adult tissues. Thus, iPS cells can be easily generated using donor cells from any species. Thus, iPS cells can be generated from any species, including, but not limited to, humans, non-human primates, rodents (mouse, rat), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), felines (domestic and wild felines, such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), and the like. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.

[0038] Induced pluripotent stem cells may be generated using as a starting point virtually any somatic cell at any stage of cellular development. For example, the cells may be from embryonic, fetal (pup), neonatal (pup), juvenile, or adult donors. Exemplary somatic cells that may be used include fibroblasts, such as skin fibroblasts obtained by skin sample or biopsy, synovial cells from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. Skin and cheek provide suitable cell sources that are readily available and easily accessible, but virtually any cell may be used. In certain embodiments, the somatic cells are not fibroblasts.

[0039] The terms "hemangioblast" and "hemangio-colony forming cell" are used interchangeably throughout this application. These cells have numerous structural and functional characteristics. Among the characteristics of these cells is the ability to engraft into bone marrow when administered to a host. These cells can be described based on numerous structural and functional properties, including but not limited to, expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers. Hemangio-colony forming cells have the ability to differentiate to give rise to at least hematopoietic or endothelial cell types. Preferably, hemangio-colony forming cells are bipotent and have the ability to differentiate to give rise to at least hematopoietic and endothelial cell types. Thus, the hemangio-colony forming cells of the present invention are at least unipotent, and preferably bipotent. However, in addition, hemangio-colony forming cells may have a higher degree of developmental potential and, in certain embodiments, may differentiate to give rise to cell types of other lineages. In certain embodiments, the hemangio-colony forming cells have the capacity to differentiate to give rise to cardiac cells (eg, cardiomyocytes) and / or other mesodermal derivatives such as smooth muscle cells.

[0040] The terms "non-engrafting hemangioblasts" or "non-engrafting hemangio cells" are used throughout this application to refer to a population of cells that share some of the characteristics of hemangio-colony forming cells. However, non-engrafting hemangio cells can be distinguished in that they do not engraft into bone marrow when administered to an immunocompromised host. Despite this difference, non-engrafting hemangio cells may share one or more (2, 3, 4, 5, 6, 7, 8, 9, 10) of the functional or structural features and characteristics of hemangio-colony forming cells. For example, in certain embodiments, non-engrafting hemangio cells are loosely adherent to each other. In other embodiments, non-engrafting hemangio cells do not express one or more (2, 3, 4) of the following proteins: CD34, KDR, CD133, CD31. Without wishing to be bound by theory, non-engrafting hemangio cells may provide a unique stem cell population that is somewhat more specialized than hemangio-colony forming cells, but still has the capacity to generate a range of hematopoietic cell types.

[0041] The present inventors have developed an in vitro culture system for producing cells with immunotherapeutic potential from human ESCs. This strategy differs from the prior art in that it involves using hESC-derived hemangioblasts as an intermediate cell source. Using feeder-free culture conditions, the present inventors were able to direct the differentiation of hemangioblasts into both natural killer (NK) and dendritic cells (DC). NK cells arise from the lymphoid lineage, are part of the innate immune system, and have been found to detect and kill certain types of tumor cells, and therefore can be used in anti-cancer therapy. DCs often arise from the myeloid lineage (from monocytes), are part of the adaptive immune system, and can be used to enhance antigen-specific immune responses due to their ability to present antigens and stimulate both naive and memory T cells (e.g., DC-based vaccine therapy).

[0042] Interactions between various activating and inhibitory signals control three main functions of NK cells: cytokine release, natural cytotoxicity, and antibody-dependent cellular cytotoxicity. Using hemangioblasts generated from both H7 and HuES-3 hESC lines, we found that mature CD56low / -, CD16+ NK cells could be produced and that their production did not require the use of a stromal feeder layer. The differentiation procedure involves an initial 4-day culture to generate embryoid bodies, followed by 10-14 days of culture in methylcellulose supplemented with a set of cytokines and growth factors to produce and expand the hemangioblast population. An additional 14-21 days in liquid culture with the addition of human serum and a cytokine mixture allows for the differentiation of NK cells, as assessed by flow cytometry. A non-radioactive cytotoxicity assay similar to the 51Cr release assay indicates that these hemangioblast-derived NK cells harbor natural cytotoxic functions, as they can effectively induce apoptosis of target K562 erythroblastic leukemia cells after a standard 4-hour co-culture. The use of hemangioblasts as an intermediate cell source may enhance the capacity and / or efficiency of hESCs to differentiate in vitro and, importantly, enable the development of feeder-free systems to produce cells with immunotherapeutic potential.

[0043] Embodiments of the present invention provide methods of generating lymphoid lineage cells. In various embodiments, the present invention provides methods of generating natural killer (NK) cells.

[0044] In various embodiments, a method for generating NK cells includes providing hemangioblasts; plating and culturing the hemangioblasts on methylcellulose and IL2, IL3, IL6, IL7, IL15, SCF, and FL; harvesting the cells; and re-plating / culturing the harvested cells in liquid medium comprising human serum, IL7, IL15, SCF, and FL.

[0045] In various embodiments, the liquid medium is changed once a week. In various embodiments, the methylcellulose is H4236 methylcellulose. In other embodiments, the methylcellulose is H4536 methylcellulose. In various embodiments, the concentrations of cytokines are IL2 (5-10 ng / ml), IL3 (1-10 ng / ml), IL6 (1-10 ng / ml), IL7 (5-20 ng / ml), IL15 (5-10 ng / ml), SCF (10-50 ng / ml), and FL (10-50 ng / ml). In various embodiments, harvesting of cells is performed after culturing the cells for 6-8 days. In various embodiments, the cells are re-seeded and then cultured for an additional 14-21 days. In various embodiments, the liquid medium is αMEM or DMEM:F12.

[0046] In another embodiment, a method for generating natural killer (NK) cells includes providing hemangioblasts; culturing the hemangioblasts in a liquid medium comprising IL2, IL3, IL6, IL7, IL15, SCF, and human serum; harvesting the cells; and culturing the harvested cells in a liquid medium comprising human serum, IL7, IL15, SCF, and FL.

[0047] In various embodiments, the liquid medium is changed once a week. As before, in various embodiments, the concentrations of cytokines are IL2 (5-10 ng / ml), IL3 (1-10 ng / ml), IL6 (1-10 ng / ml), IL7 (5-20 ng / ml), IL15 (5-10 ng / ml), SCF (10-50 ng / ml), and FL (10-50 ng / ml). In various embodiments, harvesting of cells is performed after culturing the cells for 6-8 days. In various embodiments, the cells are re-seeded and then cultured for an additional 14-21 days. In various embodiments, the liquid medium is αMEM or DMEM:F12.

[0048] Various embodiments of the invention provide natural killer cells generated by the methods of the invention. In various embodiments, the NK cells are provided in a pharma- ceutically acceptable composition comprising an amount of the NK cells generated by the methods of the invention.

[0049] Another embodiment of the invention provides a method of generating dendritic cells (DCs). In one embodiment, the method of generating DCs includes providing hemangioblasts; seeding and culturing the hemangioblasts in a liquid medium containing human serum, SCF, FL, IL3, and GM-CSF; adding IL4 to the liquid medium; and further culturing the cells.

[0050] In other embodiments, the method further comprises adding a cytokine mixture comprising IL1b, TNFα, and IL6 to induce maturation of DCs. In a further embodiment, the cytokine mixture further comprises a cytokine selected from the group consisting of PGE2, IFNα2b, Poly I:C, IFNγ, and combinations thereof. In other embodiments, the method further comprises adding LPS, IFNγ, and / or S-28463 to stimulate IL12p70 production from DCs and / or HLA-DR expression from DCs.

[0051] In various embodiments, the liquid medium is changed every 6-7 days. In various embodiments, the liquid medium is αMEM or DMEM:F12. In various embodiments, the cells are cultured for 7-11 days. In various embodiments, IL4 is added after the cells have been cultured for 7-11 days. In various embodiments, the cells are cultured for an additional 8-10 days after the addition of IL4.

[0052] In various embodiments, the concentrations of human serum, SCF, FL, IL3, and GM-CSF are human serum (10-20%), SCF (20-100 ng / ml), FL (10-50 ng / ml), IL3 (5-50 ng / ml), and GM-CSF (50-100 ng / ml). In another embodiment, the concentration of IL4 is 50-100 ng / ml. In various embodiments, the concentrations of IL1b, TNFα, and IL6 are IL1b (10 ng / ml), TNFα (10 ng / ml), and IL6 (150 ng / ml). In various embodiments, the concentrations of PGE2, IFNα2b, poly I:C, and IFNγ are PGE2 (1 μg / ml), IFNα2b (3000 units / ml), poly I:C (20 μ / ml), and IFNγ (20 ng / ml).

[0053] Because the efficiency of generating dendritic cells from hemangioblasts is relatively good, future studies with DCs may include optimizing conditions for new functional assays and developing various new functional assays. For example, modification of the components that make up the DC maturation mixture may improve the IL12p70 secretion assay. Although we currently use a mixture of six different cytokines for maturation, the addition of LPS and / or IFNγ may be necessary to stimulate IL12p70 production from blast-derived DCs. Similarly, a synthetic compound, S-28463, can support an increase in both IL12p70 production and HLA-DR expression.

[0054] Various embodiments of the invention provide dendritic cells generated by the methods of the invention. In various embodiments, the DCs are provided in a pharma- ceutically acceptable composition comprising an amount of DCs generated by the methods of the invention.

[0055] In various embodiments of the present invention, hemangioblasts can be obtained by a method including: providing hESCs; culturing the hESCs in a medium containing cytokines to generate embryoid bodies (EBs); disaggregating the EBs; filtering the individual cells; seeding the individual cells into methylcellulose containing TPO, VEGF, FL, and bFGF; and recovering the blast-like cells from the methylcellulose.

[0056] In various embodiments, hESCs are first cultured for 4 days before disaggregating the EBs. In various embodiments, the cytokines for generating embryoid bodies include VEGF and BMP4. In various embodiments, VEGF and BMP4 are used throughout EB formation. In another embodiment, the cytokines for generating embryoid bodies further include bFGF. In one embodiment, bFGF is added after the first 2 days of hESC culture. In various embodiments, disaggregating the EBs includes disaggregating the EBs with trypsin, followed by inactivating the trypsin with serum-containing medium. In one embodiment, the trypsin is 0.05%. In various embodiments, filtering the individual cells includes filtering the individual cells through a 40 μM cell strainer. In various embodiments, the methylcellulose is H4436 or H4536 methylcellulose. In various embodiments, the concentrations of cytokines are TPO (50 μg / ml), VEGF (50 μg / ml), FL (50 μg / ml), and bFGF (20-50 μg / ml). In various embodiments, the blast-like cells are harvested from the methylcellulose on days 6-10.

[0057] In certain embodiments, such as the preparation of hemangioblasts for use in generating NK cells, the methylcellulose may contain additional cytokines. These additional cytokines are selected from the group consisting of IL2, IL7, IL15, and combinations thereof. In various embodiments, the concentrations of these cytokines are IL2 (1-10 μg / ml), IL7 (1-20 μg / ml), and IL15 (1-10 μg / ml). In various embodiments, the methylcellulose cultures are seeded at a concentration of 50,000-150,000 cells / ml.

[0058] In certain embodiments, the hemangioblasts are generated in erythropoietin-free methylcellulose. In one embodiment, the erythropoietin-free methylcellulose is H4536 methylcellulose.

[0059] In alternative embodiments, pluripotent stem cells (including iPS cells and human iPS cells) are used in place of hESCs. In other embodiments, the hemangioblasts may be non-engrafting hemangioblasts.

[0060] International applications PCT / US09 / 43050 and PCT / US09 / 43043, both filed May 6, 2009, are incorporated herein by reference as if fully set forth in their entireties, and provide additional guidance in generating hemangioblasts and non-engrafting hemangioblasts.

[0061] In various embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of the natural kill cells or dendritic cells of the present invention together with a pharma- ceutically acceptable excipient. By "pharma-ceutically acceptable excipient" is meant generally an excipient that is safe, non-toxic, and useful for preparing a desired pharmaceutical composition, including excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

[0062] In various embodiments, pharmaceutical compositions according to the present invention can be formulated for delivery by any route of administration, which can refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, transmucosal, transdermal, or parenteral.

[0063] "Parenteral" refers to routes of administration generally involving injection, including intraorbital, infusion, intraarterial, intravesical, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, intrathecal, subcapsular, subcutaneous, transmucosal, or transtracheal. For parenteral routes, the compositions may be in the form of a solution or suspension for infusion or injection, or as a lyophilized powder.

[0064] The pharmaceutical composition according to the present invention may also contain any pharma- ceutically acceptable carrier. As used herein, "pharma-ceutically acceptable carrier" refers to a pharma-ceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharma-ceutically acceptable" in that it must be compatible with the other components of the formulation. This also means that each component of the carrier must be suitable for use in contact with any tissue or organ with which it may come into contact, and must not pose a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that unduly outweighs its therapeutic benefits. EXAMPLES

[0065] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Where specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0066] Example 1 early differentiation The initial differentiation procedure for both cell types is the same and involves culturing hESCs in Stemline II (Sigma) supplemented with cytokines for 4 days to generate embryoid bodies (EBs). The cytokines, VEGF and BMP4, are used throughout the EB culture, with bFGF added after the first 2 days. After a total of 4 days, the resulting EBs are disaggregated with 0.05% trypsin, which is then inactivated with serum-containing medium. Individual cells are then filtered through a 40 μM cell strainer, counted, and plated onto H4436 or H4536 methylcellulose (Stem Cell Technologies) containing additional cytokines such as TPO (50 μg / ml), VEGF (50 μg / ml), FL (50 μg / ml), and bFGF (20-50 μg / ml). For NK differentiation, the cytokines IL2 (1-10 μg / ml), IL7 (1-20 μg / ml), and / or IL15 (1-10 μg / ml) can also be added at this stage. To generate and expand the hemangioblast population, methylcellulose cultures are seeded at a concentration of 50,000-150,000 cells per ml. Blast-like cells are harvested from the methylcellulose on days 6-10 and further differentiated by one of the procedures described below.

[0067] Example 2 NK differentiation Blast cells may be replated in H4236 methylcellulose supplemented with IL2 (5-10 ng / ml), IL3 (1-10 ng / ml), IL6 (1-10 ng / ml), IL7 (5-20 ng / ml), IL15 (5-10 ng / ml), SCF (10-50 ng / ml), and FL (10-50 ng / ml) or in liquid culture containing the same cytokines and 10-20% human serum. After 6-8 days of culture, cells are harvested and replated in liquid medium (αMEM or DMEM:F12) supplemented with 10-20% human serum and the cytokines IL7 (5-20 ng / ml), IL15 (5-10 ng / ml), SCF (10-50 ng / ml), and FL (10-50 ng / ml) for an additional 14-21 days. Weekly medium changes are used to refresh the cytokine mixture.

[0068] Flow cytometry is used intermittently throughout the differentiation procedure to assess the immunophenotype of the cells and the acquisition of NK cell surface markers, including CD34, CD45, CD56, CD16, CD94, NKG2D, CD3, CD7, CD4, CD8a, and CD45RA. Tests examining the function of hemangioblast-derived NK cells include: (1) natural cytotoxicity assays using K562 erythroleukemia target cells, (2) IFNγ production in response to IL12 / IL18 or phorbol myristate acetate treatment, (3) intracellular flow cytometry for the presence of perforin and granzyme B enzymes, and (4) antibody-dependent cellular cytotoxicity assays using Raji cells and anti-CD20 antibodies.

[0069] So far, we have been able to generate NK cells from both H7 and HuES3 hESCs. 51A non-radioactive cytotoxicity assay similar to the Cr release assay indicates that our hemangioblast-derived NK cells harbor natural cytotoxic function as they can effectively induce apoptosis of target K562 erythroblastic leukemia cells after a standard 4-h co-culture.

[0070] Example 3 DC differentiation Blast cells are seeded in liquid medium (αMEM or DMEM:F12) supplemented with 10-20% human serum and the cytokines SCF (20-100 ng / ml), FL (10-50 ng / ml), IL3 (5-50 ng / ml), and GM-CSF (50-100 ng / ml). After 7-11 days of culture, IL4 (50-100 ng / ml) is also added to the cultures and the cells are expanded for another 8-10 days. Medium changes are performed every 6-7 days. To induce DC maturation, an additional cytokine mixture (10 ng / ml IL1b, 10 ng / ml TNFγ, 150 ng / ml IL6) can be added to the cultures for 48 hours.

[0071] Flow cytometry is used intermittently throughout the differentiation procedure to assess the immunophenotype of the cells and the acquisition of DC surface markers. Cell surface markers include CD11c, CD209 (DC-SIGN), HLA ABC (MHC class I), HLA DR (MHC class II), CD1a, and CD14. Maturation is assessed by acquisition of the T cell costimulatory receptor (CD83), and expression of CD209, HLA DR, and CD11c may also increase upon maturation. In some assays, additional cytokines such as 1 μg / ml PGE2, 3000 units / ml IFNα2b, 20 μg / ml poly I:C, or IFNγ 20 ng / ml can be added to the maturation mixture to enhance DC functional responses. Assays addressing the functionality of hemangioblast-derived DCs include: (1) antigen uptake by DQ-ovalbumin (BD Biosciences) treatment, (2) transwell migration in response to the chemoattractant, MIP-3b, (3) allogeneic mixed lymphocyte reaction assays to determine the ability of DCs to increase proliferation of HLA-mismatched T cells, (4) IL12-p70 secretion upon DC stimulation, and (5) antigen presentation assays to determine whether antigen-loaded DCs can induce IFNγ production in peripheral blood mononuclear cells previously primed with antigen.

[0072] We were able to generate DC cells from both HuES3 and MA01 hESCs. In response to a 48-hour maturation cytokine cocktail, we observed upregulation of CD83, HLA-DR, CD11c, and CD209. These DCs are found to be able to take up and proteolyze DQ-ovalbumin in a 30-minute assay.

[0073] Example 4 Altering hemangioblast growth conditions The NK and DC differentiation procedures described above were performed using hemangioblasts grown in H4436 methylcellulose. However, H4536, an erythropoietin-free methylcellulose from Stem Cell Technologies, can also be used to efficiently generate hemangioblasts. These "EPO-minus" hemangioblasts are very similar to the original "EPO-plus" blasts, and they are capable of differentiating into a variety of hematopoietic and vascular cell types. Preliminary results suggest that the use of H4536 may offer significant advantages over H4436 methylcellulose for differentiating hemangioblasts into various hematopoietic lineages, including NK cells and DCs. The absence of EPO in the blast growth medium was found to reduce the percentage of cells expressing the erythroid marker CD235a and increase the percentage of cells expressing CD34, CD45, and CD41a. Because of this difference in cell surface marker expression, "EPO-minus" growth conditions can enhance differentiation into myeloid and / or lymphoid lineages.

[0074] Example 5 Generation of MKs from iPS cells Using the OP9 co-culture system, the present inventors demonstrate that MKs can be generated from iPS cells. Thousands of CD41a+MKs were generated from hundreds of thousands of iPS cells.

[0075] Example 6 NK cell differentiation from human ESCs The differentiation procedure was carried out as follows: H7 ESCs were differentiated into embryoid bodies (EBs) for 4 days. EBs were harvested and transferred to cytokine-rich methylcellulose for 10-15 days to generate and expand hemangioblasts. Hemangioblasts were harvested and placed in feeder-free liquid medium supplemented with 10-20% human AB serum along with a panel of cytokines for an additional 14-17 days with half-medium changes every 3-4 days. Immunophenotyping (using flow cytometry): Immature NK cells were CD56bright, CD16lo, KIRlo, CD117+, CD94-, NKG2D+. Using a modification of the above procedure, 20-30% of the viable cells after 32 days of differentiation were CD56+CD16-. Mature NK cells were CD56dim, CD16hi, KIRhi, CD117lo / -, CD94+, NKG2D+. Using the above procedure, 20% of the viable cells after 31 days of differentiation were CD56-CD16+, and 5% of them were CD56loCD16+.

[0076] Functional assays: Natural cytotoxicity: Mature NK cells can induce apoptosis of target cells such as human K562 erythroleukemia cells, MCF7 cells, U87 cells, PC3 cells, and NTERA2 cells. The efficiency of cytotoxicity is evaluated using the "3FC" assay. This assay is similar to the 51Cr release assay but does not require radioactivity. See Derby et al., Immunol. Letters 78: 35-39 (2001). The heterogeneous population of mature NK cells described above (item B2-a) was found to induce apoptosis in 65-70% of K562 cells in a standard 4-hour experiment.

[0077] Antibody-dependent cell-mediated cytotoxicity (ADCC): FcyRIII (CD16) on the surface of NK cells binds to the fc region of anti-CD20 antibody bound to target cells, inducing ADCC. Raji cells (derived from Burkitt's lymphoma) are preincubated with anti-CD20 antibody and used as targets in the ADCC assay. (Tsirigotis et al., J of Steroid Biochem and Mol Bio 108: 267-271 (2008)).

[0078] IFNγ cytokine production: Immature NK cells produce large amounts of IFNγ in response to overnight treatment with ionomycin or IL12 plus PMA (phorbol myristate acetate) plus IL18. IFNγ secretion is blocked with brefeldin a and cells are stained for cell surface markers and IFNγ using intracellular flow cytometry. See Woll et al. J. of Immunol. 175: 5095-5103 (2005).

[0079] In vivo immunotherapeutic potential of NK cells using a xenograft mouse model: Bioluminescent (luciferase-containing) K562 cells are injected into tumor-bearing NOD / SCID mice followed by a bolus of NK cells and daily IP injections of IL2 and IL15. Bioluminescence imaging is used to monitor in vivo NK immunotherapeutic potential over time. See Woll et al. Blood 113 (24): 6094-6101 (2009).

[0080] Example 7 By using hemangioblasts as bone marrow repopulating cells or by differentiating them into dendritic cells, natural killer cells, T cells, and / or mesenchymal stem cells (MSCs), we can generate large-scale, effective cell-based therapies to fight cancer, HIV, and / or automimmune diseases. We were able to achieve differentiation of dendritic cells (DCs) from both hESCs and iPS cells with 40-55% efficiency. In addition to two new functional assays, side-by-side comparisons with DCs derived from human bone marrow confirmed that hESC-derived DCs now share many similar characteristics with human BM-derived DCs and also identified areas requiring further optimization. In the case of natural killer cell differentiation, side-by-side comparisons with human bone marrow-derived NK cells confirmed that ex vivo NK cell differentiation was less efficient, even when using a bone marrow cell source. We found that blast-derived NK cells exhibited natural cytotoxicity capabilities. Antibody-dependent cellular cytotoxicity assays are performed. For T cell differentiation, we have successfully generated a human Delta ligand-expressing OP9 stromal cell line to stimulate Notch signaling and have used this stromal cell line to stimulate T cell differentiation of hemangioblasts.

[0081] Hemangioblast-derived dendritic cells (40-55% efficiency) The cell surface markers CD11c, 45, 209 show similar expression in blast-derived DCs and human bone marrow-derived DCs, whereas HLA-DR is expressed at much lower levels in blast DCs than in human BM DCs.

[0082] Functional assays (previously reported for antigen uptake and migration assays). IL12-p70 secretion: IL12p70 is secreted by mature DCs to induce Th1-directed responses from CD4+ T cells. Human BM-derived DCs can produce more than 500 pg / ml of IL12p70, whereas blast-derived DCs did not produce detectable IL12p70 upon maturation. Mixed lymphocyte reaction (MLR) assay: The MLR assay determines the ability of DCs to stimulate proliferation of allogeneic T cells. Cord blood mononuclear cells (CBMCs, which contain T cells) were used as responders, fluorescently labeled, and their proliferation was measured after co-culture with immature or mature blast-derived DCs for 4-5 days. Preliminary results show that responder cells proliferate in response to mature (m)DCs.

[0083] Hemangioblast-derived natural killer cells: Cell surface markers CD45, CD7, CD94, CD56, CD16, and NKG2D were assessed on blast-derived and human bone marrow-derived NK cells.

[0084] NK Differentiation Efficiency: Recombinant human Sox7 protein was investigated for its ability to increase the pool of CD34+ cells starting from precursors of NK differentiation. Results suggest that rhSox7 does not dramatically affect the % of CD34+ cells. Murine AFT024 was investigated as a stromal feeder layer that could provide important cell-cell contacts and secreted factors for NK cell differentiation. AFT024-stroma co-culture did not enhance either human BM or blast NK differentiation as shown by cell surface marker expression.

[0085] Hemangioblast-derived T cells: Notch signaling is important for T cell differentiation. Therefore, the cDNA of human delta-like ligand 1 (hDLL1) was cloned into a retroviral vector based on MSCV-ires-GFP, and the resulting viral supernatant was used to infect OP9 stromal cells. Upon virus integration, OP9 cells would express hDLL1 on their cell surface and become gfp positive. FACS-based sorting was used to purify the most gfp positive cells from a heterogeneous pool of infected OP9 cells. These OP9-hDL1S ("S" in sorting) were expanded and characterized. Q-RT-PCR, immunofluorescence, and flow cytometry all confirm the expression of hDLL1 protein in these cells.

[0086] Example 8 Both umbilical cord blood and peripheral blood mononuclear cells are used as responders. We use human bone marrow-derived DCs as positive control effectors.

[0087] E4BP4 has been shown to be important for NK lineage development (see Gascoyne et al. Nature Immunology 10(10): 1118-1125, 2009) and may provide the transcriptional program required for greater efficiency in vitro.

[0088] NK cell differentiation. We clone E4BP4 cDNA into a retroviral vector to overexpress it in hemangioblasts and evaluate its ability to increase NK differentiation. RT-PCR is used to monitor the expression of various KIR receptor isoforms, as well as the enzymes perforin and granzyme B, which are important for NK cell function. For functional assays, we have shown that blast-derived NK cells exhibit natural cytotoxicity. Thus, we evaluate the antibody-dependent cellular cytotoxicity (ADCC) ability of blast-derived NK cells. The reagents required for the ADCC assay include Burkitt's lymphoma-derived Raji cells and anti-CD20 antibodies. Co-culture of CD20-expressing Raji cells with NK cells should induce a specific ADCC response, which will be monitored by flow cytometry means.

[0089] NK References: 1. Woll, P, et al. J. of Immunology. 175: 5095-5103 (2005). 2. Woll, P et al. Blood113(24): 6094-101 (2009). 3. Bordoni et al. Hepatology 39: 1508-1516 (2004). 4. Tabatoabaei-Zavareh et al. PLOS One Issue 2, e232 (2007). 5. McCullar, V et al. Exp. Hematology 36(5): 598-608 (2008). 6. Freud, AG et al. Immunity 22:295-304 (2005). 7. Yu, H et al. Blood 92 (10): 3647-3657 (1998).

[0090] DC literature: 1. Su et al. Clinical Cancer Research 14(19): 6207-6217 (2008). 2. Tseng et al. Regenerative Medicine 4(4): 513-526 (2009). 3. Bandi et al. AIDS Research and Therapy 5:1 (2008). (open-access) 4. Slukvin, II et al. J of Immunology 176: 2924-2932 (2006).

[0091] Various embodiments of the present invention are described in the detailed description. Although these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations of the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be included within the scope of this description as well. Unless otherwise noted, it is the intention of the inventors that the words and phrases in this specification and claims are to be given their ordinary and customary meanings to those skilled in the relevant technical field(s).

[0092] The foregoing description of various embodiments of the present invention known to the applicant at the time of filing this application is presented and is intended to be illustrative and explanatory. This description is not intended to be exhaustive or to limit the invention to the precise form disclosed, as numerous modifications and variations are possible in light of the above teachings. The described embodiments serve to illustrate the principles of the present invention and its practical application, and to enable others skilled in the art to use the present invention in various embodiments with various modifications as appropriate for the particular use contemplated. Therefore, it is not intended that the present invention be limited to the specific embodiments disclosed for carrying out the invention.

[0093] While specific embodiments of the present invention have been shown and described, it will be apparent to one skilled in the art that, based on the teachings herein, changes and modifications can be made without departing from the present invention and its broader aspects, and therefore the appended claims are intended to encompass within their scope all such changes and modifications that are within the true spirit and scope of the present invention. It will be generally understood by those skilled in the art that the terms used in this specification are generally intended to be "open-ended" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.).

Claims

1. Providing hemangioblasts; culturing the hemangioblasts on methylcellulose and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, stem cell factor (SCF), and fms-related tyrosine kinase 3 ligand (FL); harvesting the cultured cells; and Culturing the harvested cells in a liquid medium containing human serum and a second cytokine mixture containing IL7, IL15, stem cell factor (SCF), and fms-related tyrosine kinase 3 ligand (FL) to generate natural killer (NK) cells. A natural killer (NK) cell produced by a method comprising the steps of:

2. The natural killer (NK) cell of claim 1, wherein the NK cell is an immature NK cell and is CD56+ and CD16-.

3. The natural killer (NK) cell of claim 1, wherein the NK cell is a mature NK cell and is CD56- and CD16+, or is CD56lo and CD16+.

4. A pharma- ceutically acceptable composition comprising an amount of natural killer (NK) cells according to any one of claims 1 to 3.

5. Providing hemangioblasts; culturing the hemangioblasts in a liquid medium comprising human serum and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, and stem cell factor (SCF); harvesting the cultured cells; and Culturing the harvested cells in a liquid medium containing human serum and a second cytokine mixture containing IL7, IL15, stem cell factor (SCF), and fms-related tyrosine kinase 3 ligand (FL) to generate natural killer (NK) cells. A natural killer (NK) cell produced by a method comprising the steps of:

6. The natural killer (NK) cell of claim 5, wherein the NK cell is an immature NK cell and is CD56+ and CD16-.

7. The natural killer (NK) cell of claim 5, wherein the NK cell is a mature NK cell and is CD56- and CD16+, or is CD56lo and CD16+.

8. A pharma- ceutically acceptable composition comprising an amount of the NK cells according to any one of claims 5 to 7.

9. Providing hemangioblasts; culturing the hemangioblasts in a liquid medium containing human serum, stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (FL), IL3, and GM-CSF; adding IL4 to the liquid medium; and Further culturing the hemangioblasts to generate dendritic cells (DCs). A dendritic cell (DC) generated by a method comprising:

10. The dendritic cell (DC) of claim 9, wherein the DC is a mature DC and expresses CD83.

11. The dendritic cell (DC) of claim 9, wherein the DC is a mature DC and has increased expression of CD209, HLA DR, and / or CD11c.

12. A pharma- ceutically acceptable composition comprising an amount of dendritic cells (DCs) according to any one of claims 9 to 11.

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