Hemangio-colony forming cells

Abstract

To provide a method of generating and expanding human hemangioblasts or hemangio-colony forming cells in vitro.SOLUTION: The present invention provides methods for generating human hemangioblasts or hemangio-colony forming lineage cells (i.e., hematopoietic and endothelial cells) that may be used in therapeutic applications. The methods of the invention provide further utility in that they enable the generation of large numbers of human hemangioblasts or hemangio-colony forming cells as well as hematopoietic and endothelial cells, and cells differentiated therefrom, that may be used at commercial scale.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to methods for generating and expanding human hemangio-colony forming cells, and more specifically, to methods for generating and expanding human hemangio-colony forming cells in vitro using a two-step process comprising a first step of culturing human embryo-derived stem cells in a serum-free medium in the presence of one or more growth factors in an amount sufficient to induce differentiation of the embryonic stem cells into embryoid bodies, and a second step of culturing embryoid bodies in a serum-free medium in the presence of one or more growth factors in an amount sufficient to expand the human hemangio-colony forming cells in the medium containing the embryoid bodies. The present invention also relates to human hemangio-colony forming cell preparations. Furthermore, the present invention relates to methods for differentiating human hemangio-colony forming units along hematopoietic or endothelial lineages, as well as methods for producing partially or fully differentiated cell types from hemangio-colony forming cells. Hemangio-colony forming cells and cells differentiated therefrom have a variety of uses in vitro and in vivo. The ability to generate large quantities of expanded human hemangio-colony forming cells in vitro provides, among other things, the possibility of derivation of numerous human hemangio-colony forming derivative cell types, e.g., human hematopoietic stem cells or endothelial cells, differentiated hematopoietic cells, e.g., red blood cells and platelets, that are useful in a variety of therapeutic applications. Also, the expanded numbers of human hemangio-colony forming cells induced by the present invention may be utilized in novel therapeutic strategies in the treatment of hematopoietic and endothelial cell disorders or in blood banks. [Background technology]

[0002] Hematopoietic stem cells (HSCs) are capable of self-renewal and can give rise to all blood cell lineages. These cells, present in the bone marrow, are the basis of the adult hematopoietic system. Transplantation of these cells is currently the most common cell-based therapy applied in clinical practice.

[0003] HSC transplantation is used to treat patients with acute or chronic leukemia, aplastic anemia, and various immune deficiency syndromes, as well as various malignant non-hematological tumors and autoimmune diseases. In the case of patients with malignant hematological tumors, transplantation of HSCs can save them from treatment-induced hypoplasia that can occur after high-dose chemotherapy and / or radiation therapy.

[0004] Although the clinical utility of HSC transplantation is widespread, the three major sources of HSC (human bone marrow, mobilized peripheral blood, and umbilical cord blood) are limited by the lack of a matched donor for two-thirds of patients requiring somatic HSC transplantation. For example, for any particular patient, there is only a 25% chance that a sibling will be an identical human leukocyte antigen (HLA) match.

[0005] HLA are proteins on the cell surface that help the immune system identify cells as autologous (belonging to the body) or non-autologous (foreign or derived from outside the body). These HLA proteins are encoded by a cluster of genes that form a region located on human chromosome 6 known as the major histocompatibility complex, or MHC, and it is known that proteins encoded by the MHC locus play an important role in graft rejection. Thus, HLA proteins are also called MHC proteins. Although matching the MHC molecules of the graft with those of the recipient can significantly improve the success of clinical transplants, rejection cannot be prevented even in cases where the transplant is between siblings with the same HLA. Such rejection can be triggered by differences between the autocompatibility antigens. These polymorphic antigens are generally "non-autologous" peptides bound to the MHC molecules on the transplanted tissue cells, and without the use of immunosuppressive drugs, differences between the autocompatibility antigens often cause the immune system of the transplant recipient to reject the transplant even if the MHC antigens are matched.

[0006] There are three types of HLA types I and II. A person (usually a sibling) who is a match for HLA types I and II is called a related donor. Matching of HLA types I, B, and C, and HLA types II, DRB1 and DQB1, between the recipient and donor is associated with increased survival (Mori et al., 2002 Blood (99): 4200-6). For patients without a matched related donor, a matched HLA type is sought through donor banks. However, the number of patients who need cell or tissue transplants, such as HSC transplants, far exceeds the available supply of cells and tissues suitable for transplantation. Under these circumstances, it is not surprising that a good match between the recipient's HMC proteins and the MHC proteins of the transplant is not possible. Thus, many transplant recipients must wait until an MHC-matched transplant is available, or accept a non-MHC-matched transplant and endure high doses of immunosuppressive drugs and risk rejection. Therefore, the ability to generate and manipulate HSCs and / or induce tolerance to transplants in recipients would be of considerable advantage in the treatment and management of human disease.

[0007] Based on avian embryo studies and subsequent work in frogs and mammals, it has been established that the developmental programs of blood and endothelium are closely linked. For example, endothelial and hematopoietic cells appear simultaneously and in close proximity in blood islands of the yolk sac. The blood islands of the yolk sac are derived from aggregates of mesodermal cells that colonize the yolk sac. The centers of these aggregates develop into embryonic hematopoietic cells, while the peripheral population differentiates into endothelial cells that form the blood vessels that surround the internal blood cells. These observations support the idea that endothelial and hematopoietic cells share a common ancestor.

[0008] Additionally, both endothelial and hematopoietic lineages in lionfish and mouse embryos share expression of certain genes, such as Flk1, Flt1, Tie1, Tie2, CD34, Sc1, and Runx1 (Fina et al., 1990 Blood(75):2417-2426; Millauer et al., 1993 Cell(72):835-846; Yamaguchi et al., 1993 Development(118):489-498; Anagnostou et al., 1994 PNAS USA(91):3974-3978; Kallianpur et al., 1994 Blood(83):1200-12081; Young et al., 1995 Blood(85):96-105, Asahara et al., 1997 Science(275):964-967; Kabrun et al., 1997 Development(124):2039-2048). Similarly, certain gene mutations affect both endothelial and hematopoietic cell development (Shalaby et al., 1995 Mature(376):62-66; Robb et al., 1995 PNAS USA(92):7075-7079; Shivdasani et al., 1995 Nature(373):432-434; Stainier et al., 1995 Development(121):3141-3150; Bollerot et al., 2005 APMIS(113):790-803). Furthermore, deletion of Flk1 or Flt1, the receptors for vascular endothelial growth factor (VEGF), disrupts hematopoietic and endothelial development in mouse embryos.

[0009] The in vitro generation of mouse hemangioblasts from mouse embryonic stem cells has been reported (Choi et al., 1998 Development 125:727-732). Human progenitor cells capable of developing into both hematopoietic and endothelial cells have also been derived from human ES cells (Wang et al., 2004 Immunity (21):31-41 and Wang et al., J Exp Med (201):1603-1614), but at best only small amounts are derived from many cells. Also, there are no methods or conditions for the in vitro expansion of hemangioblast progenitor cells.

[0010] Thus, a need remains for methods of generating and expanding large numbers of human hemangioblasts and hemangioblast derivative cell types, i.e., hematopoietic cells and endothelial cells, and any solutions / mixtures containing such quantities of hemangioblasts or derivative cell types. Such methods would increase the availability of cells for transplantation and be useful in a variety of other therapeutic applications, such as inducing immunological tolerance.

[0011] There is also a significant need for available blood for transfusions. The Red Cross and other blood supply companies say there is almost always a shortage of blood. This is especially true for patients with specific blood types, Rh+ patients, or after accidents or disasters resulting in mass casualties. Also, during times of war, troops need emergency blood available for use in treating combat-related trauma. The present invention provides differentiated hematopoietic cells for use in blood banks and transfusions. The cells and methods of the present invention may be a safe and reliable advancement over methods that have traditionally relied on blood donations, and may help prevent critical shortages of available blood. Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention overcomes the above-disclosed problems by providing a method for generating and expanding human hemangioblasts or hemangio-colony forming cells in vitro. The novel methods disclosed herein allow for the proliferation of human hemangioblasts or hemangio-colony forming cells to produce cells for a variety of therapeutic applications. The present invention also provides a method for generating human hemangioblasts or hemangio-colony forming lineage cells (i.e., hematopoietic cells and endothelial cells) for therapeutic applications. The methods of the present invention are useful in that they can generate large numbers of human hemangioblasts or hemangio-colony forming cells that can be used on a commercial scale, as well as hematopoietic and endothelial cells and cells differentiated therefrom. [Means for solving the problem]

[0013] The invention provides a method for generating and expanding human hemangio-colony forming cells in vitro, comprising the steps of: (a) culturing a cell culture comprising human embryo-derived cells in serum-free medium in the presence of one or more growth factors in an amount sufficient to induce differentiation of human embryo-derived cells into embryoid bodies; and (b) adding one or more growth factors to said culture comprising embryoid bodies and continuing to culture said culture in serum-free medium, wherein said growth factors are present in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body culture, wherein said stem cells, embryoid bodies and hemangio-colony forming cells are cultured in serum-free medium throughout steps (a) and (b).

[0014] The present invention also provides a method for generating and expanding human hemangio-colony forming cells in vitro, the method comprising the steps of: (a) culturing a cell culture comprising human embryo-derived cells in a serum-free medium in the presence of one or more growth factors in an amount sufficient to induce differentiation of human embryo-derived cells into embryoid bodies; (b) adding one or more growth factors to the culture comprising embryoid bodies and continuing to culture the culture in serum-free medium, wherein the growth factors are present in an amount sufficient to expand human hemangio-colony forming cells in the embryoid body culture; (c) disaggregating the embryoid bodies into single cells; and (d) adding one or more growth factors to the culture comprising the single cells and continuing to culture the culture in serum-free medium, wherein the growth factors are present in an amount sufficient to expand human hemangio-colony forming cells in the culture comprising the single cells, wherein the stem cells, embryoid bodies and hemangio-colony forming cells are cultured in serum-free medium throughout steps (a) through (d).

[0015] In certain embodiments of the invention, the embryo-derived cells are embryonic stem cells.

[0016] In certain embodiments of the methods of generating and expanding human hemangio-colony forming cells of the present invention, the growth factor is a protein comprising a homeobox protein or a functional variant or active fragment thereof. In certain embodiments, the homeobox protein is a protein comprising HOXB4 or a functional variant or active fragment thereof. It is also contemplated that the HOXB4 is a mammalian HOXB4, including mouse and human HOXB4. The HOXB4 protein is a full-length HOXB4 protein. In additional embodiments, the HOXB4 comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4.

[0017] In certain embodiments of the methods of generating and expanding human hemangio-colony forming cells of the invention, the growth factor comprising HOXB4 is a fusion protein comprising HOXB4 and a protein transduction domain (PTD). In additional embodiments, the PTD and HOXB4 are joined through a linker. In additional embodiments, the PTD is a TAT protein, functional variant or active fragment thereof (including a TAT polypeptide). In certain embodiments, the TAT protein comprises the amino acid sequence of SEQ ID NO: 14. In additional embodiments, the PTD comprises one or more copies of a TAT polypeptide having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the PTD is SEQ ID NO: 15.

[0018] In certain embodiments of the methods of generating and expanding human hemangio-colony forming cells of the invention, the growth factor is selected from the group consisting of vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), stem cell factor (SCF), Flt-3L (FL), thrombopoietin (TPO) and erythropoietin (EPO). In additional embodiments, vascular endothelial growth factor (VEGF) or bone morphogenetic protein (BMP), or both, are added to the culturing step (a) within 0 to 48 hours of culturing the cell culture comprising the hES cells. In yet additional embodiments, stem cell factor (SCF), Flt-3L (FL) or thrombopoietin (TOP), or any combination thereof, is added to the cell culture comprising the hES cells within 48 to 72 hours after initiation of the culturing step (a).

[0019] In certain embodiments of the methods of generating and expanding human hemangio-colony forming cells of the invention, a growth factor comprising a HOXB4 protein or a functional variant or active fragment (or domain) thereof is added to the above step, wherein the protein is added multiple times, for example, once per day or once every other day.

[0020] In certain embodiments of the methods of generating and expanding human hemangio-colony forming cells of the invention, the growth factor comprising a homeobox protein is added to step (b) within 48-72 hours after initiation of step (a).In certain embodiments of the methods of the invention, the HOXB4 protein or a functional variant or active fragment thereof is added to step (b) within 48-72 hours after initiation of step (a).

[0021] In certain embodiments, the methods of generating and expanding human hemangio-colony forming cells of the invention further comprise the step of adding erythropoietin (EPO) to step (b). In certain embodiments, the methods of the invention described in paragraph 0016 further comprise the step of adding erythropoietin (EPO) to steps (b) and (d).

[0022] In certain embodiments, the methods of generating and expanding human hemangio-colony forming cells of the invention further comprise step(s) of purifying and / or isolating the human hemangio-colony forming cells. Hemangio-colony forming cells may be purified by utilizing immunoaffinity column chromatography with an anti-CD71 antibody. Hemangio-colony forming cells may be separated by size and / or morphology.

[0023] In certain embodiments of the methods of generating and expanding human hemangio-colony forming cells of the invention, the cell cultures comprising human embryo-derived stem cells are derived from a library of human embryonic stem cells, the library of human embryonic stem cells comprising stem cells that are hemizygous or homozygous for one or more MHC alleles present in the human population, each member of the library of stem cells being hemizygous or homozygous for the other set of MHC alleles relative to the remaining members of the library. In additional embodiments, the library of human embryonic stem cells comprises stem cells that are hemizygous or homozygous for every MHC allele present in the human population. These methods generate a library of human hemangio-colony forming cells that are hemizygous or homozygous for one or more MHC alleles present in the human population, each member of the library of stem cells being hemizygous or homozygous for the other set of MHC alleles relative to the remaining members of the library. In additional embodiments, these methods generate libraries of human hemangio-colony forming cells that are hemizygous or homozygous for every MHC allele present in a human population. Thus, the invention also provides libraries of human hemangio-colony forming cells produced by such methods. This library can be used as follows:

[0024] The present invention provides a method of treating a patient in need of treatment comprising administering to said patient human hematopoietic stem cells or human endothelial cells, the method comprising the steps of: (a) selecting said patient; (b) identifying MHC proteins expressed on the surface of said patient's cells; (c) providing a library of human hemangio-colony forming cells as described in paragraph 0024 above; (d) selecting human hemangio-colony forming cells from the library that match the MHC proteins of said patient on said patient's cells; (e) differentiating said human hemangio-colony forming cells identified in step (d) into human hematopoietic stem cells, endothelial cells, or both, as appropriate; and (f) administering said hematopoietic stem cells, endothelial cells, or both from step (e) to said patient. The method can be performed at a regional center, e.g., a hospital or medical center, or any other suitable facility.

[0025] In certain embodiments, the methods of generating and expanding human hemangio-colony forming cells of the invention further comprise growing said human hemangio-colony forming cells under conditions suitable for inducing differentiation of said human hemangio-colony forming cells into human hematopoietic stem cells.

[0026] In certain embodiments, the methods of generating and expanding human hemangio-colony forming cells of the invention further comprise growing said human hemangio-colony forming cells under conditions suitable for inducing differentiation of said human hemangio-colony forming cells into human endothelial cells. In additional embodiments, the conditions suitable for inducing differentiation of said human hemangio-colony forming cells into human endothelial cells comprise growing said human hemangio-colony forming cells on a fibrinogen-coated culture plate.

[0027] In certain embodiments of the invention, the methods of generating and expanding human hemangio-colony forming cells include producing at least 10,000 human hemangio-colony forming cells, at least 50,000 human hemangio-colony forming cells, at least 100,000 human hemangio-colony forming cells, at least 500,000 human hemangio-colony forming cells, at least 1×10 6 ≥ 2 x 10 human hemangio-colony forming cells 6≥ 3 x 10 human hemangio-colony forming cells 6 ≥ 4 x 10 human hemangio-colony forming cells or 4 x 10 6 These methods produce cell solutions that can contain between 10,000 and 4 million human hemangio-colony forming cells.

[0028] Thus, the present invention also provides a method for the production of a cell line comprising the steps of: 10,000 or more human hemangio-colony forming cells; 50,000 or more human hemangio-colony forming cells; 100,000 or more human hemangio-colony forming cells; 500,000 or more human hemangio-colony forming cells; 1×10 6 ≥ 2 x 10 human hemangio-colony forming cells 6 ≥ 3 x 10 human hemangio-colony forming cells 6 ≥ 4 x 10 human hemangio-colony forming cells or 4 x 10 6 Provided are solutions of human hemangio-colony forming cells (which can be grown in serum-free medium) comprising one or more human hemangio-colony forming cells. These solutions are injectable into a subject. These solutions can be serum-free. Of these solutions, the human hemangio-colony forming cells are capable of differentiating into at least hematopoietic cells and endothelial cells, but have greater developmental potential to differentiate into other cell types. The invention also provides the use of these solutions for the manufacture of a medicament for treating a disease treatable by administration of hemangio-colony forming cells, hematopoietic cells or endothelial cells.

[0029] The invention also provides a method for producing a solution of human hemangio-colony forming cells suitable for injection into a patient comprising the steps of isolating said cell solution as described in the previous paragraph and placing said cells in a solution suitable for injection into a patient.The invention also provides a method for producing a solution of human hemangio-colony forming cells suitable for freezing comprising the steps of isolating said cell solution as described in the previous paragraph and placing said cells in a solution suitable for freezing.

[0030] The invention also provides a method of administering human hematopoietic stem cells to a patient in need thereof, comprising the steps of: (a) selecting a patient in need thereof; (b) providing human hemangio-colony forming cells generated and expanded by the methods of the invention; (c) differentiating the human hemangio-colony forming cells into human hematopoietic stem cells; and (d) administering some or all of the human hematopoietic stem cells to the patient.

[0031] The invention also provides a method of administering human hematopoietic stem cells to a patient in need thereof, comprising the steps of: (a) selecting a patient in need thereof; (b) providing human hemangio-colony forming cells in an amount of 10,000 or more cells; (c) differentiating the human hemangio-colony forming cells into human hematopoietic stem cells; and (d) administering some or all of the human hematopoietic stem cells to the patient.

[0032] The invention also provides a method of administering human endothelial cells to a patient in need thereof, comprising the steps of: (a) selecting a patient in need thereof; (b) providing human hemangio-colony forming cells generated and expanded by the methods of the invention; (c) differentiating the human hemangio-colony forming cells into human endothelial cells; and (d) administering some or all of the human endothelial cells to the patient.

[0033] The invention also provides a method of administering human endothelial cells to a patient in need thereof, the method comprising the steps of: (a) selecting a patient in need thereof; (b) providing human hemangio-colony forming cells in an amount of 10,000 or more cells; (c) differentiating the human hemangio-colony forming cells into human endothelial cells; and (d) administering some or all of the human endothelial cells to the patient.

[0034] In certain embodiments, said human hemangio-colony forming cells are present in an amount between 10,000 and 4 million cells.

[0035] The invention also provides a method of treating an endothelial cell disorder in a patient in need of such treatment, comprising the steps of: (a) selecting a patient in need of such treatment; (b) providing human hemangio-colony forming cells generated and expanded by the methods described above; (c) differentiating the human hemangio-colony forming cells into human endothelial cells; and (d) administering some or all of the human endothelial cells to the patient.

[0036] The invention also provides a method of treating an endothelial cell disorder in a patient in need of such treatment, comprising the steps of: (a) selecting a patient in need of such treatment; (b) providing human hemangio-colony forming cells in an amount of at least 10,000 cells; (c) differentiating the human hemangio-colony forming cells into human endothelial cells; and (d) administering some or all of the human endothelial cells to the patient.

[0037] In certain embodiments, said human hemangio-colony forming cells are present in an amount between 10,000 and 4 million cells.

[0038] Endothelial cell disorders that may be treated by these methods include myocardium infarction, stroke, atherosclerosis, and ischemia. Ischemia can originate from the brain, limbs, heart, lungs, skin, and eyes.

[0039] The present invention provides a method for producing human hematopoietic stem cells in vitro, comprising the steps of: The present invention provides a method comprising the steps of: (a) providing human hemangio-colony forming cells generated and expanded by the methods of the invention; and (b) growing said human hemangio-colony forming cells under conditions suitable for inducing differentiation of said human hemangio-colony forming cells into human hematopoietic stem cells.

[0040] The invention provides a method for producing human endothelial cells in vitro, the method comprising the steps of: (a) providing human hemangio-colony forming cells generated and expanded by the methods of the invention; and (b) growing the human hemangio-colony forming cells under conditions suitable for inducing differentiation of the human hemangio-colony forming cells into human endothelial cells.

[0041] The present invention also provides a method for expanding hemangioblast cells, comprising growing mammalian hemangioblast cells in serum-free medium in the presence of a protein comprising a homeobox protein (e.g., HOXB4), or a functional equivalent or active fragment thereof, in an amount sufficient to support the expansion of said hemangioblast cells. The expanded hemangioblast cells may be enriched, purified, or isolated from cord blood, peripheral blood, or bone marrow. The hemangioblast cells may be human hemangioblast cells. The method may produce hemangioblast cells that are enriched, purified, or isolated from 10,000 to 4×10 6 A solution containing one or more hemangio-colony forming cells may be produced. The HOXB4 protein used in this method may be any protein suitable for use in the methods of generating and expanding human hemangio-colony forming cells of the invention.

[0042] The invention also provides a method of inducing immune tolerance using human hemangio-colony forming cells generated and expanded or expanded by the methods of the invention. The tolerized human hemangio-colony forming cells are selected to share tissue synthesis markers with the allograft and may be administered to a human recipient prior to or simultaneously with an allograft or cell therapy that regenerates cellular functions required by the patient. The immune tolerance generated then reduces the risk of acute or chronic rejection of the allograft or cell therapy.

[0043] The large numbers of hemangio-colony forming cells generated by the methods disclosed herein allow for tolerance protocols in which toxic pretreatments may be omitted. Donor human hemangio-colony forming cells may be administered to a human recipient prior to transplantation of a graft or prior to transplantation of organs, tissues, or cells that match the donor hemangio-colony forming cells. The human hemangio-colony forming cells and graft are obtained from the same donor. The graft may be derived from differentiated donor human hemangio-colony forming cells. The human hemangio-colony forming cells and graft are obtained separately from another donor that matches the donor. The human hemangio-colony forming cells may be administered to a human recipient to induce tolerance in the recipient in need of tolerance.

[0044] In certain embodiments, the present invention relates to a method of inducing tolerance in a human recipient to a donor allograft, the method comprising the steps of: (a) administering to the recipient an agent that inhibits T cell co-stimulation; (b) introducing into the recipient human hemangio-colony forming cells that are generated and expanded or expanded by the methods described herein; and (c) transplanting the allograft into the recipient. The human hemangio-colony forming cells (donor cells) cause the recipient to tolerate the allograft. In certain embodiments, the agent that inhibits T cell co-stimulation is an agent that inhibits or blocks CD40 ligand-CD40 co-stimulatory interactions. The method may further comprise administering an agent that inhibits CD28-B7 interactions. The method may or may not include thymic irradiation and / or T cell depletion or inactivation. The above-mentioned methods may also result in mixed chimerism in the absence of hematopoietic space generated by total body irradiation.

[0045] The present invention also provides a method of enhancing tolerance in a human recipient to a donor allograft, the method comprising: (a) generating thymic space in the recipient; (b) reducing or inactivating donor-reactive T cells in the recipient; (c) introducing donor human hemangio-colony forming cells of the donor or donor-matching donor human hemangio-colony forming cells into the recipient; and (d) transplanting the allograft into the recipient, wherein the donor hemangio-colony forming cells induce tolerance to the allograft. In certain embodiments, the method does not include hematopoietic space generating irradiation. The method can include generating thymic space by treating the recipient with one or more therapies selected from thymic irradiation (fractionable), steroids, corticosteroids, brequinar, or immunosuppressants or drugs.

[0046] The present invention also provides methods for producing cells suitable for use in blood transfusions. For example, the present invention provides methods for producing red blood cells that can be used in blood transfusions. As such, the present invention provides a solution useful in alleviating the chronic shortage of blood utilizing donated blood.

[0047] In certain embodiments, the invention provides methods for generating differentiated hematopoietic cells in vitro from human hemangio-colony forming cells, the method comprising the steps of: (a) providing human hemangio-colony forming cells; and (b) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells.

[0048] In certain embodiments, the invention provides a method of transfusion utilizing hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, the method comprising the steps of: (a) providing human hemangio-colony forming cells; (b) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells; and (c) transfusing the differentiated hematopoietic cells.

[0049] In certain embodiments, said hemangio-colony forming cells are recovered from frozen cultures.

[0050] In certain embodiments, the invention provides a method of transfusion utilizing hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, the method comprising the steps of: (a) culturing a cell culture comprising human embryo-derived cells in the presence of one or more growth factors in an amount sufficient to induce differentiation of said human embryo-derived cells into embryoid bodies; (b) adding one or more growth factors to said culture comprising embryoid bodies; (c) differentiating said hemangio-colony forming cells into differentiated hematopoietic cells; and (d) transfusing said differentiated hematopoietic cells, wherein said growth factors are present in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body culture.

[0051] In certain embodiments, the embryo-derived cells, embryoid bodies and hemangio-colony forming cells are grown in serum-free medium during step (a) of the method.

[0052] In certain embodiments, the embryo-derived cells are embryonic stem cells.

[0053] In certain embodiments, the invention provides a method of transfusion utilizing hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, the method comprising the steps of: (a) culturing a cell culture comprising human embryo-derived cells in the presence of one or more growth factors in an amount sufficient to induce differentiation of said human embryo-derived cells into embryoid bodies; (b) adding one or more growth factors to said culture comprising embryoid bodies; (c) differentiating said hemangio-colony forming cells into differentiated hematopoietic cells; and (d) transfusing said differentiated hematopoietic cells, wherein said growth factors are present in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body culture.

[0054] In certain embodiments, the stem cells, embryoid bodies and hemangio-colony forming cells are grown in serum-free medium during steps (a) and (b) of the method.

[0055] In certain embodiments of the invention, said embryo-derived cells are embryonic stem cells.

[0056] In certain embodiments, said hemangio-colony forming cells are recovered from frozen cultures.

[0057] In certain embodiments, the invention provides a method of transfusion utilizing hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, the method comprising the steps of: (a) culturing a cell culture comprising human embryo-derived cells in the presence of one or more growth factors in an amount sufficient to induce differentiation of said human embryo-derived cells into embryoid bodies; (b) adding one or more growth factors to said culture comprising embryoid bodies, wherein said growth factors are present in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body culture; (c) disaggregating said embryoid bodies into single cells; (d) adding one or more growth factors to said culture comprising said single cells, wherein said growth factors are present in an amount sufficient to expand human hemangio-colony forming cells in said culture comprising said single cells; (e) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells; and (f) transfusing said differentiated hematopoietic cells.

[0058] In certain embodiments, the stem cells, embryoid bodies and hemangio-colony forming cells are grown in serum-free medium throughout steps (a)-(d) of the method.

[0059] In certain embodiments of the invention, the embryo-derived cells are embryonic stem cells.

[0060] In certain embodiments, the growth factor is a protein comprising a homeobox protein, or a functional variant or active fragment thereof, hi certain embodiments, the homeobox protein comprises a HOXB4 protein, or a functional or active variant thereof.

[0061] In certain embodiments, the differentiated hematopoietic cells are produced as a single cell type. In certain embodiments, the single cell type is selected from red blood cells, platelets, or phagocytes. In certain embodiments, the phagocytes are selected from granulocytes: neutrophils, basophils, eosinophils, lymphocytes, or monocytes. In certain embodiments, the red blood cells express hemoglobin F. In certain embodiments, the single cell types are mixed to appropriately equalize the ratio of differentiated cell types found in blood, where the ratio of differentiated cell types found in blood is 96% red blood cells, 1% platelets, and 3% phagocytes.

[0062] In certain embodiments, multiple differentiated hematopoietic cell types are produced in the same step. In certain embodiments, the multiple differentiated hematopoietic cell types are selected from red blood cells, platelets, or phagocytes. In certain embodiments, the phagocytes are selected from granulocytes, neutrophils, basophils, eosinophils, lymphocytes, or monocytes. In certain embodiments, the red blood cells express hemoglobin F. In certain embodiments, the multiple differentiated hematopoietic cell types are produced in proportions approximately equal to the proportion of differentiated hematopoietic cell types found in blood, where the proportion of differentiated cell types found in blood is 96% red blood cells, 1% platelets, and 3% phagocytes.

[0063] In certain embodiments, plasma is added to the differentiated hematopoietic cells prior to transfusion.

[0064] In certain embodiments, the hemangio-colony forming cells are matched with the patient to ensure that differentiated hematopoietic cells of the patient's own blood type are produced. In certain embodiments, the hemangio-colony forming cells are negative for antigenic factors A, B, Rh, or any combination thereof. In certain embodiments, the differentiated hematopoietic cells are red blood cells, wherein the step of differentiating the red blood cells includes erythropoietin (EPO).

[0065] The invention also provides human hemangio-colony forming cells, which are differentiated to produce at least hematopoietic or endothelial cell types, where the human hemangio-colony forming cells are loosely adherent to other human hemangio-colony forming cells.

[0066] In certain embodiments, the present invention provides human hemangio-colony forming cells, which are differentiated to generate at least hematopoietic or endothelial cell types, where the human hemangio-colony forming cells are loosely adherent to other human hemangio-colony forming cells.

[0067] In certain embodiments, the invention provides human hemangio-colony forming cells, which are differentiated to produce at least hematopoietic or endothelial cell types, where the human hemangio-colony forming cells are loosely adherent to other human hemangio-colony forming cells, and where the human hemangio-colony forming cells do not express CD34 protein.

[0068] In certain embodiments, the present invention provides human hemangio-colony forming cells, which are differentiated to generate at least hematopoietic or endothelial cell types, wherein the human hemangio-colony forming cells do not express any of the following proteins: CD23, CD31, KDR, and CD133.

[0069] In certain embodiments, the invention provides cell cultures comprising human hemangio-colony forming cells, which cells are differentiated to generate at least hematopoietic or endothelial cell types, wherein the human hemangio-colony forming cells are loosely adherent to each other.

[0070] In certain embodiments, the invention provides cell cultures comprising human hemangio-colony forming cells, which cells are differentiated to generate at least hematopoietic or endothelial cell types, wherein the human hemangio-colony forming cells are loosely adherent to each other.

[0071] In certain embodiments, the invention provides cell cultures comprising a substantially purified population of human hemangio-colony forming cells, which cells are differentiated to produce at least hematopoietic or endothelial cell types, where the human hemangio-colony forming cells are adherent to each other, and where the human hemangio-colony forming cells do not express CD34 protein.

[0072] In certain embodiments, the invention provides cell cultures comprising human hemangio-colony forming cells differentiated from embryo-derived cells, wherein the human hemangio-colony forming cells are loosely adherent to each other.

[0073] In certain embodiments, the cell culture comprises 1×10 6 or more human hemangio-colony forming cells. In certain embodiments, the cell culture comprises 5×10 6 or more human hemangio-colony forming cells.

[0074] In certain embodiments, the embryo-derived cell is an embryonic stem cell line. In certain embodiments, the embryo-derived cell is selected from an embryo, a blastomere, a blastocyt, or an inner cell mass.

[0075] In certain embodiments, the present invention provides a pharmaceutical preparation comprising human hemangio-colony forming cells, which cells are differentiated to produce at least hematopoietic or endothelial cell types, wherein the human hemangio-colony forming cells are loosely adherent to each other.

[0076] In certain embodiments, the present invention provides a pharmaceutical preparation comprising human hemangio-colony forming cells, which cells are differentiated to produce at least hematopoietic or endothelial cell types, wherein the human hemangio-colony forming cells are loosely adherent to each other.

[0077] In certain embodiments, the present invention provides a pharmaceutical formulation comprising human hemangio-colony forming cells, which cells are differentiated to produce at least a hematopoietic or endothelial cell type, wherein the human hemangio-colony forming cells do not express any of the following proteins: CD34, CD31, KDR, and CD133.

[0078] In certain embodiments, the pharmaceutical formulation comprises 1×10 6 In certain embodiments, the pharmaceutical formulation comprises 5×10 or more human hemangio-colony forming cells. 6 or more human hemangio-colony forming cells.

[0079] In certain embodiments, the pharmaceutical preparation comprising human hemangio-colony forming cells is differentiated from an embryo-derived cell. In certain embodiments, the embryo-derived cell is an embryonic stem cell. In certain embodiments, the embryo-derived cell is selected from an embryo, a blastomere, a blastula, or an inner cell mass.

[0080] In certain embodiments, the present invention provides cryopreserved preparations of any of the aforementioned human hemangio-colony forming cells.

[0081] In a specific embodiment, the present invention provides a method for producing a 1×10 6 Provided are cryopreserved preparations of the above human hemangio-colony forming cells, wherein the human hemangio-colony forming cells do not express any of the following proteins: CD34, CD31, KDR, and CD133.

[0082] In certain embodiments, the cryopreserved formulation contains 5×10 6 or more human hemangio-colony forming cells.

[0083] In certain embodiments, cryopreserved preparations comprising human hemangio-colony forming cells do not express CD34 protein. In certain embodiments, cryopreserved preparations comprising human hemangio-colony forming cells do not express CD34, CD31, CD133 and KDR proteins. In certain embodiments, cryopreserved preparations comprising human hemangio-colony forming cells do not express LM02 and GATA2 proteins.

[0084] In certain embodiments, the invention provides cultures comprising hematopoietic cells differentiated from human hemangio-colony forming cells as described above.

[0085] In certain embodiments, the invention provides cultures comprising endothelial cells differentiated from human hemangio-colony forming cells as described above.

[0086] In certain embodiments, the invention provides cultures comprising smooth muscle cells differentiated from human hemangio-colony forming cells as described above.

[0087] In certain embodiments, the invention provides cultures comprising cardiomyocytes differentiated from human hemangio-colony forming cells as described above.

[0088] In a particular embodiment, the present invention provides the use of human hemangio-colony forming cells as described above in the manufacture of a medicament for treating a condition in a patient in need of said human hemangio-colony forming cells.

[0089] In certain embodiments, the invention provides the use of such cell cultures in the manufacture of a medicament for treating a condition in a patient in need thereof.

[0090] In certain embodiments, the present invention provides the use of such a pharmaceutical formulation in the manufacture of a medicament for treating a condition in a patient in need thereof.

[0091] For example, the present invention provides the following: (Item 1) A method for generating and expanding human hemangio-colony forming cells in vitro, comprising: (a) culturing a cell culture comprising human embryo-derived cells in serum-free medium in the presence of at least one growth factor in an amount sufficient to induce differentiation of the human embryo-derived cells into embryoid bodies; (b) continuing to culture said culture in serum-free medium by adding at least one growth factor to said culture comprising embryoid bodies, said growth factor being added in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body culture; The method wherein said embryo-derived cells, embryoid bodies and hemangio-colony forming cells are cultured in serum-free medium throughout steps (a) and (b) of the method. 2. A method for generating and expanding human hemangio-colony forming cells in vitro, comprising: (a) culturing a cell culture comprising human embryo-derived cells in serum-free medium in the presence of at least one growth factor in an amount sufficient to induce differentiation of the human embryo-derived cells into embryoid bodies; (b) continuing to culture said culture in serum-free medium by adding at least one growth factor to said culture containing embryoid bodies, said growth factor being added in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body culture; (c) disaggregating the embryoid bodies into single cells; (d) continuing to culture said culture in serum-free medium by adding at least one growth factor to said culture comprising said single cell, said growth factor being added in an amount sufficient to cause the expansion of human hemangio-colony forming cells in said culture comprising said single cell; The embryo-derived cells, embryoid bodies and hemangio-colony forming cells are cultured in serum-free medium throughout steps (a)-(d) of the method. (Item 3) The method according to item 1 or 2, wherein the embryo-derived cell is an embryonic stem cell. (Item 4) The method according to item 1 or 2, wherein the growth factor is a protein containing a homeobox protein or a functional or active variant thereof. (Item 5) The method according to Item 4, wherein the homeobox protein comprises HOXB4 protein or a functional or active variant thereof. (Item 6) The method according to any one of items 1 to 5, wherein the growth factor is selected from the group consisting of vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), stem cell factor (SCF), Flt-3L (FL), thrombopoietin (TPO), and erythropoietin (EPO). (Item 7) The method according to item 6, wherein the vascular endothelial growth factor (VEGF) or bone morphogenetic protein (BMP) or both are added to the step (a) within 0 to 48 hours of cell culture. (Item 8) The method according to item 6, wherein the stem cell factor (SCF), Flt-3L (FL), or thrombopoietin (TPO), or any combination thereof, is added to the culture within 48 to 72 hours from the start of step (a). (Item 9) The method according to item 4 or 5, wherein the growth factor is added in step (b) within 48 to 72 hours from the start of step (a). (Item 10) The method according to item 5, wherein the growth factor is added to the culture step (b) within 48 to 72 hours from the start of step (a). (Item 11) The method according to Item 1, further comprising the step of adding erythropoietin (EPO) to step (b). (Item 12) The method according to Item 2, further comprising the step of adding erythropoietin (EPO) to steps (b) and (d). (Item 13) The method according to any one of items 1 to 3, further comprising a step of purifying the human hemangio-colony forming cells from the culture. (Item 14) The method according to Item 13, wherein the step of purifying the hemangio-colony forming cells comprises using immunoaffinity column chromatography using an anti-CD71 antibody. (Item 15) The method according to any one of items 1 to 3, further comprising a step of isolating the human hemangio-colony forming cells from the culture. (Item 16) The method according to Item 15, wherein the step of separating the hemangio-colony forming cells comprises separating the hemangio-colony forming cells by size and / or morphology. (Item 17) The method according to item 4 or 5, wherein the growth factor is a fusion protein comprising HOXB4 and a protein transduction domain (PTD). (Item 18) The method according to Item 17, wherein the PTD and the HOXB4 are joined by a linker. (Item 19) The method according to item 17, wherein the protein transduction domain (PTD) is a TAT protein, a functional variant or an active fragment thereof. (Item 20) The method according to Item 5, wherein the HOXB4 is mammalian HOXB4. (Item 21) The method according to Item 20, wherein the mammalian HOXB4 is mouse or human HOXB4. (Item 22) The method according to Item 21, wherein the mammalian HOXB4 is human HOXB4. (Item 23) The method according to Item 5, wherein the HOXB4 comprises a full-length protein. (Item 24) The method according to Item 22, wherein the HOXB4 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. (Item 25) The method described in Item 19, wherein the TAT protein comprises the amino acid sequence of SEQ ID NO:14. (Item 26) The method described in Item 17, wherein the PTD comprises one or more TAT polypeptide copies having the amino acid sequence of SEQ ID NO:14. (Item 27) The cell culture comprising human embryo-derived cells is derived from a library of human embryonic stem cells, the library of human embryonic stem cells comprising stem cells that are each hemizygous or homozygous for at least one MHC allele present in a human population, each member of the stem cell library being hemizygous or homozygous for a different set of MHC alleles relative to the remaining members of the library, and the method generates a library of hemangio-colony forming cells that are each hemizygous or homozygous for at least one MHC allele present in a human population, each member of the stem cell library being hemizygous or homozygous for a different set of MHC alleles relative to the remaining members of the library, the method comprising any one of items 1 to 3. (Item 28) The method of Item 27, wherein the library of human embryonic stem cells comprises stem cells that are hemizygous or homozygous for every MHC allele present in the human population, the method generating a library of hemangio-colony forming cells comprising hemangio-colony forming cells that are hemizygous or homozygous for every MHC allele present in the human population. (Item 29) The method according to any one of items 1 to 3, further comprising culturing the human hemangio-colony forming cells under conditions suitable for inducing differentiation of the human hemangio-colony forming cells into human hematopoietic stem cells. (Item 30) The method according to any one of items 1 to 3, further comprising culturing the human hemangio-colony forming cells under conditions suitable for inducing differentiation of the human hemangio-colony forming cells into human endothelial cells. (Item 31) The method according to Item 30, wherein the conditions include culturing the human hemangio-colony forming cells on a culture plate coated with fibronectin. (Item 32) The culture is at least 1 × 10 6 4. The method according to any one of items 1 to 3, comprising human hemangio-colony forming cells. (Item 33) The culture is at least 2 × 10 6 33. The method of paragraph 32, comprising human hemangio-colony forming cells. (Item 34) The culture is at least 3×10 6 34. The method of paragraph 33, comprising human hemangio-colony forming cells. (Item 35) The culture is at least 4×10 6 35. The method of paragraph 34, comprising human hemangio-colony forming cells. (Item 36) At least 1×10 6 A human hemangio-colony forming cell solution comprising human hemangio-colony forming cells. (Item 37) The solution according to Item 36, which can be injected into a subject. (Item 38) The solution according to Item 36, which is suitable for freezing. (Item 39) At least 2 × 10 6 37. The solution according to item 36, comprising human hemangio-colony forming cells. (Item 40) At least 3×10 6 40. The solution according to item 39, comprising human hemangio-colony forming cells. (Item 41) At least 4×10 6 41. The solution according to item 40 comprising human hemangio-colony forming cells. (Item 42) The solution described in Item 36, wherein the human hemangio-colony forming cells are capable of differentiating into at least hematopoietic cells or endothelial cells. (Item 43) Use of a solution according to any one of items 36 to 42 in the manufacture of a medicament for the treatment of a disease treatable by administration of hemangio-colony forming cells, hematopoietic cells or endothelial cells. 44. A method for administering human hemangio-colony forming cells to a patient in need thereof, comprising: (a) selecting a patient in need of said administration; (b) providing human hemangio-colony forming cells produced by the method according to any one of items 1 to 35; (c) administering some or all of the human hemangio-colony forming cells to the patient. 45. A method for administering human hemangio-colony forming cells to a patient in need thereof, comprising: (a) selecting a patient in need of said administration; (b) providing human hemangio-colony forming cells in an amount of at least 10,000 cells; (c) administering some or all of the human hemangio-colony forming cells to the patient. (Item 46) The method according to Item 45, wherein the human hemangio-colony forming cells are in an amount of 10,000 to 4,000,000 cells. (Item 47) A method for administering human hematopoietic stem cells to a patient in need thereof, comprising: (a) selecting a patient in need of said administration; (b) providing human hemangio-colony forming cells produced by the method according to any one of items 1 to 35; (c) differentiating the human hemangio-colony forming cells into human hematopoietic stem cells; (d) administering some or all of the human hematopoietic stem cells to the patient. (Item 48) A method for administering human hematopoietic stem cells to a patient in need thereof, comprising: (a) selecting a patient in need of said administration; (b) providing human hemangio-colony forming cells in an amount of at least 10,000 cells; (c) differentiating the human hemangio-colony forming cells into human hematopoietic stem cells; (d) administering some or all of the human hematopoietic stem cells to the patient. (Item 49) The method according to Item 48, wherein the human hemangio-colony forming cells are in an amount of 10,000 to 4,000,000 cells. (Item 50) A method for administering human endothelial cells to a patient in need thereof, comprising: (a) selecting a patient in need of said administration; (b) providing human hemangio-colony forming cells produced by the method according to any one of items 1 to 35; (c) differentiating the human hemangio-colony forming cells into human endothelial cells; and (d) administering some or all of the human endothelial cells to the patient. (Item 51) A method for administering human endothelial cells to a patient in need thereof, comprising: (a) selecting a patient in need of said administration; (b) providing human hemangio-colony forming cells in an amount of at least 10,000 cells; (c) differentiating the human hemangio-colony forming cells into human endothelial cells; and (d) administering some or all of the human endothelial cells to the patient. (Item 52) The method according to Item 51, wherein the human hemangio-colony forming cells are in an amount of 10,000 to 4,000,000 cells. (Item 53) A method for treating a hematopoietic stem cell disorder in a patient in need of treatment, comprising: (a) selecting a patient in need of treatment; (b) providing human hemangio-colony forming cells produced by the method according to any one of items 1 to 35; (c) differentiating the human hemangio-colony forming cells into human hematopoietic stem cells; (d) administering some or all of the human hematopoietic stem cells to the patient. (Item 54) A method for treating a hematopoietic stem cell disorder in a patient in need of treatment, comprising: (a) selecting a patient in need of treatment; (b) providing human hemangio-colony forming cells in an amount of at least 10,000 cells; (c) differentiating the human hemangio-colony forming cells into human hematopoietic stem cells; (d) administering some or all of the human hematopoietic stem cells to the patient. (Item 55) The method according to Item 54, wherein the human hemangio-colony forming cells are in an amount of 10,000 to 4,000,000 cells. (Item 56) A method for treating an endothelial cell disorder in a patient in need of treatment, comprising: (a) selecting a patient in need of treatment; (b) providing human hemangio-colony forming cells produced by the method according to any one of items 1 to 35; (c) differentiating the human hemangio-colony forming cells into human endothelial cells; (d) administering some or all of the human endothelial cells to the patient. (Item 57) A method for treating an endothelial cell disorder in a patient in need of treatment, comprising: (a) selecting a patient in need of treatment; (b) providing human hemangio-colony forming cells in an amount of at least 10,000 cells; (c) differentiating the human hemangio-colony forming cells into human endothelial cells; (d) administering some or all of the human endothelial cells to the patient. (Item 58) The method according to Item 57, wherein the human hemangio-colony forming cells are in an amount of 10,000 to 4,000,000 cells. (Item 59) The method according to item 57 or 58, wherein the endothelial cell disease is selected from the group consisting of myocardial infarction, stroke, atherosclerosis and ischemia. (Item 60) The method according to Item 59, wherein the ischemia occurs in the brain, limbs, heart, lungs, skin and eyes. (Item 61) A method for generating human hematopoietic stem cells in vitro, comprising: (a) providing human hemangio-colony forming cells produced by the method according to any one of items 1 to 34; (b) culturing said human hemangio-colony forming cells under conditions suitable for inducing differentiation of said human hemangio-colony forming cells into human hematopoietic stem cells. (Item 62) A method for generating human endothelial cells in vitro, comprising: (a) providing human hemangio-colony forming cells produced by the method according to any one of items 1 to 35; (b) culturing said human hemangio-colony forming cells under conditions suitable for inducing differentiation of said human hemangio-colony forming cells into human endothelial cells. (Item 63) A library of human hemangio-colony forming cells produced by the method described in Item 27 or 28. 64. A method of treating a human patient in need of treatment comprising administering to the patient human hematopoietic stem cells or human endothelial cells, (a) screening the patient; (b) identifying MHC proteins expressed on the cell surface of said patient; (c) providing a library of human hemangio-colony forming cells according to item 63; (d) selecting human hemangio-colony forming cells which match the patient's MHC proteins on the patient's cells; (e) differentiating the human hemangio-colony forming cells identified in step (d) into human hematopoietic stem cells, endothelial cells, or both, as appropriate; (f) administering some or all of the human hematopoietic stem cells, endothelial cells, or both obtained from step (e) to the patient. (Item 65) The method according to item 64, carried out at a regional center. 66. The method of claim 65, wherein the regional center is a hospital. (Item 67) A method for expanding mammalian hemangio-colony forming cells, comprising the step of culturing the hemangio-colony forming cells in serum-free medium in the presence of a protein containing a homeobox protein or a functional equivalent or active fragment thereof in an amount sufficient to support the growth of the hemangio-colony forming cells. (Item 68) The method of Item 67, wherein the hemangio-colony forming cells are enriched, purified or isolated from umbilical cord blood, peripheral blood or bone marrow. (Item 69) The method according to Item 67, wherein the hemangio-colony forming cells are human hemangio-colony forming cells. (Item 70) The hemangio-colony forming cells are at least 1×10 6 70. The method of paragraph 67, wherein the cells are cultured in vitro to yield hemangio-colony forming cells. (Item 71) The method according to Item 67, wherein the protein comprising a homeobox protein or a functional equivalent or active fragment thereof is a protein comprising a HOXB4 protein or a functional equivalent or active fragment thereof. (Item 72) The method according to Item 71, wherein the protein is a fusion protein comprising HOXB4 and a protein transduction domain (PTD). (Item 73) The method according to Item 72, wherein the protein transduction domain (PTD) is a TAT protein, a functional variant or an active fragment thereof. (Item 74) The method according to Item 71, wherein the HOXB4 is mammalian HOXB4. (Item 75) The method according to Item 74, wherein the mammalian HOXB4 is mouse or human HOXB4. (Item 76) The method according to Item 75, wherein the mammalian HOXB4 is human HOXB4. (Item 77) The method according to Item 71, wherein the HOXB4 comprises a full-length protein. (Item 78) The method described in Item 71, wherein the HOXB4 has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. (Item 79) The method described in Item 73, wherein the TAT protein comprises the amino acid sequence of SEQ ID NO:14. (Item 80) The method described in Item 72, wherein the PTD comprises one or more copies of a TAT polypeptide having the amino acid sequence of SEQ ID NO:14. (Item 81) The method described in items 4 or 5, wherein the growth factor is added to the culture of step (b) multiple times throughout step (b). (Item 82) The method described in Items 4 or 5, wherein the growth factor is added to the culture of steps (b) and (d) multiple times throughout steps (b) and (d). (Item 83) The method according to item 81 or 82, wherein the protein is added once a day. (Item 84) The method according to item 81 or 82, wherein the protein is added once every two days. (Item 85) An isolated hemangio-colony forming cell produced by the method described in Item 1. (Item 86) An isolated hemangio-colony forming cell produced by the method described in Item 2. (Item 87) A method for inducing tolerance in a human recipient to a donor allograft, comprising: (a) administering to the recipient an agent that inhibits T cell costimulation; (b) introducing into said recipient human hemangio-colony forming cells produced by the method of claim 1, said hemangio-colony forming cells being matched to the donor; (c) transplanting said allograft into said recipient, wherein said human hemangio-colony forming cells induce tolerance to said allograft in the recipient. (Item 88) The method described in Item 87, wherein the formulation that inhibits T cell costimulation is a formulation that inhibits CD40 ligand-CD40 interaction. (Item 89) The method described in Item 87, further comprising administering a formulation that inhibits CD28-B7 interaction. (Item 90) The method according to any one of Items 87 to 89, which does not include a thymic irradiation or T cell depletion or inactivation step. (Item 91) The method described in any one of Items 87 to 89, further comprising a step of administering thymic irradiation to the recipient. (Item 92) The method of any one of Items 87 to 89, further comprising a step of administering to the recipient a treatment to deplete or inactivate T cells. (Item 93) The method of any one of Items 87 to 89, further comprising a step of subjecting the recipient to a treatment to deplete or inactivate T cells and thymic irradiation. (Item 94) A method for inducing tolerance in a human recipient to a donor allograft, comprising: (a) creating an intrathymic space in the recipient; (b) depleting or inactivating donor-reactive T cells of the recipient; (c) introducing into said recipient human hemangio-colony forming cells produced by the method of clause 1, said hemangio-colony forming cells being matched to said donor; (d) transplanting the allograft into the recipient; The human hemangio-colony forming cells induce tolerance in the recipient to the allograft. (Item 95) The method described in Item 94, wherein the intrathymic space is formed by administering to the recipient at least one treatment selected from the group consisting of thymic irradiation, steroids, corticosteroids, brequinar, and immunosuppressant chemicals or drugs. (Item 96) The method according to Item 95, wherein the intrathymic space is formed by administering thymic irradiation to the recipient. (Item 97) The method described in Item 95, wherein the intrathymic space is formed by administering an immunosuppressive chemical or drug to the recipient. (Item 98) The method described in Item 95, wherein the intrathymic space is formed by administering cyclosporine to the recipient. (Item 99) The method described in Items 87 or 94, wherein hemangio-colony forming cells are administered multiple times to the recipient. (Item 100) The method according to item 87 or 94, wherein the resistance is induced in the absence of hematopoietic space formed by total body irradiation. (Item 101) The method described in Items 87 or 94, wherein the human hemangio-colony forming cells and the allograft are obtained from the same donor. (Item 102) The method described in Items 87 or 94, wherein the human hemangio-colony forming cells and the allograft are obtained from different donors. (Item 103) A human hemangio-colony forming cell capable of differentiating to form at least a hematopoietic cell type or an endothelial cell type, the human hemangio-colony forming cell having weak adhesiveness to other human hemangio-colony forming cells. (Item 104) A human hemangio-colony forming cell capable of differentiating to form at least hematopoietic cell types and endothelial cell types, the human hemangio-colony forming cell having weak adhesiveness to other human hemangio-colony forming cells. (Item 105) A human hemangio-colony forming cell capable of differentiating to form at least hematopoietic cell types and endothelial cell types, which has poor adhesion to other human hemangio-colony forming cells and does not express CD34 protein. (Item 106) A human hemangio-colony forming cell capable of differentiating to form at least hematopoietic cell types and endothelial cell types, which does not express any of the proteins CD34, CD31, KDR and CD133. (Item 107) A cell culture comprising human hemangio-colony forming cells capable of differentiating to form at least hematopoietic or endothelial cell types, wherein the human hemangio-colony forming cells are weakly adherent to one another. (Item 108) A cell culture comprising a substantially purified population of human hemangio-colony forming cells capable of differentiating to form at least hematopoietic cell types and endothelial cell types, wherein the hemangio-colony forming cells are weakly adherent to one another and do not express CD34 protein. (Item 109) A cell culture comprising human hemangio-colony forming cells differentiated from embryo-derived cells, wherein the hemangio-colony forming cells are weakly adherent to one another. (Item 110) A cell culture comprising human hemangio-colony forming cells capable of differentiating to form at least hematopoietic cell types and endothelial cell types, wherein the hemangio-colony forming cells are weakly adherent to one another. (Item 111) A pharmaceutical preparation comprising human hemangio-colony forming cells capable of differentiating to form at least hematopoietic or endothelial cell types, wherein the hemangio-colony forming cells are weakly adherent to one another. (Item 112) A pharmaceutical preparation comprising human hemangio-colony forming cells capable of differentiating to form at least hematopoietic cell types and endothelial cell types, wherein the hemangio-colony forming cells are weakly adherent to each other. (Item 113) A pharmaceutical preparation comprising human hemangio-colony forming cells capable of differentiating to form at least hematopoietic cell types and endothelial cell types, wherein the hemangio-colony forming cells do not express any of the proteins CD34, CD31, KDR and CD133. (Item 114) A cryopreserved preparation of hemangio-colony forming cells described in any one of Items 103 to 113. (Item 115) At least 1×10 6 1. A cryopreserved preparation comprising human hemangio-colony forming cells, wherein said hemangio-colony forming cells do not express any of the proteins CD34, CD31, KDR and CD133. (Item 116) The hemangio-colony forming cell described in Item 103, which does not express one or more proteins selected from CD34, CD31, KDR and CD133. (Item 117) The hemangio-colony forming cell described in Item 103, which does not express CD34 protein. (Item 118) A hemangio-colony forming cell described in Item 103 that does not express CD34, CD31, KDR and CD133. (Item 119) The hemangio-colony forming cell described in Item 103, which expresses LMO2 and GATA2 proteins. (Item 120) The hemangio-colony forming cell described in Item 116, which expresses LMO2 and GATA2 proteins. (Item 121) The hemangio-colony forming cell described in Item 117, which expresses LMO2 and GATA2 proteins. (Item 122) The hemangio-colony forming cell described in Item 118, which expresses LMO2 and GATA2 proteins. (Item 123) The hemangio-colony forming cell described in Item 104, which does not express one or more proteins selected from CD34, CD31, KDR and CD133. (Item 124) The hemangio-colony forming cell described in Item 104, which does not express CD34 protein. (Item 125) A hemangio-colony forming cell described in Item 104 that does not express CD34, CD31, KDR, and CD133. (Item 126) The hemangio-colony forming cell described in Item 104, which expresses LMO2 and GATA2 proteins. (Item 127) The hemangio-colony forming cell described in Item 123, which expresses LMO2 and GATA2 proteins. (Item 128) The hemangio-colony forming cell described in Item 124, which expresses LMO2 and GATA2 proteins. (Item 129) The hemangio-colony forming cell described in Item 125, which expresses LMO2 and GATA2 proteins. (Item 130) The hemangio-colony forming cell described in Item 105, which does not express one or more proteins selected from CD31, CD133 or KDR. (Item 131) A hemangio-colony forming cell described in Item 105 that does not express CD31, CD133 and KDR proteins. (Item 132) The hemangio-colony forming cell described in Item 105, which expresses LMO2 and GATA2 proteins. (Item 133) The hemangio-colony forming cell described in Item 130, which expresses LMO2 and GATA2 proteins. (Item 134) The hemangio-colony forming cell described in Item 131, which expresses LMO2 and GATA2 proteins. (Item 135) The hemangio-colony forming cell described in Item 106, which expresses LMO2 and GATA2 proteins. (Item 136) At least 1×10 6108. The cell culture of paragraph 107, comprising human hemangio-colony forming cells. (Item 137) At least 5×10 6 137. The cell culture of paragraph 136, comprising human hemangio-colony forming cells. (Item 138) At least 1×10 6 109. The cell culture of paragraph 108, comprising human hemangio-colony forming cells. (Item 139) At least 5×10 6 139. The cell culture of paragraph 138, comprising human hemangio-colony forming cells. (Item 140) The cell culture described in Item 109, wherein the embryo-derived cells are embryonic stem cell lines. (Item 141) The cell culture described in Item 109, wherein the embryo-derived cells are selected from embryos, blastomeres, blastulas or inner cell masses. (Item 142) At least 1×10 6 111. The cell culture of paragraph 110, comprising human hemangio-colony forming cells. (Item 143) At least 5×10 6 143. The cell culture of paragraph 142, comprising human hemangio-colony forming cells. (Item 144) The cell culture described in Item 107, wherein the hemangio-colony forming cells do not express one or more proteins selected from CD34, CD31, CD133 or KDR. (Item 145) The cell culture described in Item 107, wherein the hemangio-colony forming cells do not express CD34 protein. (Item 146) The cell culture described in Item 107, wherein the hemangio-colony forming cells do not express CD34, CD31, CD133 and KDR proteins. (Item 147) The cell culture described in Item 107, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 148) The cell culture described in Item 144, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 149) The cell culture described in Item 145, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 150) The cell culture described in Item 146, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 151) The cell culture described in Item 108, wherein the hemangio-colony forming cells do not express one or more proteins selected from CD31, CD133 or KDR. (Item 152) The cell culture described in Item 108, wherein the hemangio-colony forming cells do not express CD31, CD133 and KDR proteins. (Item 153) The cell culture described in Item 108, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 154) The cell culture described in Item 151, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 155) The cell culture described in Item 152, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 156) The cell culture described in Item 109, wherein the hemangio-colony forming cells do not express one or more proteins selected from CD34, CD31, CD133 or KDR. (Item 157) The cell culture described in Item 109, wherein the hemangio-colony forming cells do not express CD34 protein. (Item 158) The cell culture described in Item 109, wherein the hemangio-colony forming cells do not express CD34, CD31, CD133 and KDR proteins. (Item 159) The cell culture described in Item 109, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 160) The cell culture described in Item 156, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 161) The cell culture described in Item 157, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 162) The cell culture described in Item 158, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 163) The cell culture described in Item 110, wherein the hemangio-colony forming cells do not express one or more proteins selected from CD34, CD31, CD133 or KDR. (Item 164) The cell culture described in Item 110, wherein the hemangio-colony forming cells do not express CD34 protein. (Item 165) The cell culture described in Item 110, wherein the hemangio-colony forming cells do not express CD34, CD31, CD133 and KDR proteins. (Item 166) The cell culture described in Item 110, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 167) The cell culture described in Item 163, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 168) The cell culture described in Item 164, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 169) The cell culture described in Item 165, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 170) At least 1×10 6 12. The pharmaceutical formulation according to item 111, comprising human hemangio-colony forming cells. (Item 171) At least 5×10 6 171. The pharmaceutical formulation according to item 170, comprising human hemangio-colony forming cells. (Item 172) At least 1×10 6 13. The pharmaceutical formulation of item 112, comprising human hemangio-colony forming cells. (Item 173) At least 5×10 6 173. The pharmaceutical formulation according to item 172, comprising human hemangio-colony forming cells. (Item 174) At least 1×10 6 14. The pharmaceutical formulation of item 113, comprising human hemangio-colony forming cells. (Item 175) At least 5×10 6 175. The pharmaceutical formulation according to item 174, comprising human hemangio-colony forming cells. (Item 176) At least 5×10 616. The cryopreserved preparation of item 115, comprising human hemangio-colony forming cells. (Item 177) The cryopreserved preparation described in Item 115, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 178) The pharmaceutical preparation according to any one of Items 111 to 113, wherein the hemangio-colony forming cells are differentiated from embryo-derived cells. (Item 179) The pharmaceutical formulation described in Item 178, wherein the embryo-derived cells are embryonic stem cells. (Item 180) The pharmaceutical formulation described in Item 178, wherein the embryo-derived cells are selected from the group consisting of embryos, blastomeres, blastulas, and inner cell masses. (Item 181) The pharmaceutical preparation described in Item 111, wherein the hemangio-colony forming cells do not express one or more proteins selected from CD34, CD31, CD133 or KDR. (Item 182) The pharmaceutical preparation described in Item 111, wherein the hemangio-colony forming cells do not express CD34 protein. (Item 183) The pharmaceutical preparation described in Item 111, wherein the hemangio-colony forming cells do not express CD34, CD31, CD133 and KDR proteins. (Item 184) The pharmaceutical preparation described in Item 111, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 185) The pharmaceutical preparation described in Item 181, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 186) The pharmaceutical preparation described in Item 182, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 187) The pharmaceutical preparation described in Item 183, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 188) The pharmaceutical preparation described in Item 112, wherein the hemangio-colony forming cells do not express one or more proteins selected from CD34, CD31, CD133 or KDR. (Item 189) The pharmaceutical preparation described in Item 112, wherein the hemangio-colony forming cells do not express CD34 protein. (Item 190) The pharmaceutical preparation described in Item 112, wherein the hemangio-colony forming cells do not express CD34, CD31, CD133 and KDR proteins. (Item 191) The pharmaceutical preparation described in Item 112, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 192) The pharmaceutical preparation described in Item 188, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 193) The pharmaceutical preparation described in Item 189, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 194) The pharmaceutical preparation described in Item 190, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 195) The pharmaceutical preparation described in Item 113, wherein the hemangio-colony forming cells express LMO2 and GATA2 proteins. (Item 196) A culture comprising hematopoietic cells differentiated from hemangio-colony forming cells described in any one of Items 103 to 115. (Item 197) A culture comprising endothelial cells differentiated from hemangio-colony forming cells described in any one of Items 103 to 115. (Item 198) A culture comprising smooth muscle cells differentiated from hemangio-colony forming cells described in any one of Items 103 to 115. (Item 199) A culture comprising cardiomyocytes differentiated from hemangio-colony forming cells described in any one of Items 103 to 115. (Item 200) Use of the human hemangio-colony forming cells described in any one of Items 103 to 106 in the manufacture of a medicament for treating a disease in a patient in need of such treatment. (Item 201) Use of the cell culture described in any one of items 107 to 110 in the manufacture of a medicament for treating a disease in a patient in need of such treatment. (Item 202) Use of a pharmaceutical formulation according to any one of items 111 to 113 in the manufacture of a medicament for treating a disease in a patient in need of such treatment. 203. A method for generating differentiated hematopoietic cells from human hemangio-colony forming cells in vitro, comprising: (a) providing human hemangio-colony forming cells; (b) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells. (Item 204) A method for performing a blood transfusion using hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, comprising: (a) providing human hemangio-colony forming cells; (b) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells; (c) administering a transfusion to the differentiated hematopoietic cells. (Item 205) The method according to Items 203 or 204, wherein the hemangio-colony forming cells are recovered from a frozen culture. (Item 206) A method for performing a blood transfusion using hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, comprising: (a) culturing a cell culture comprising human embryo-derived cells in the presence of at least one growth factor in an amount sufficient to induce differentiation of the human embryo-derived cells into hemangio-colony forming cells; (b) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells; (c) administering a transfusion to the differentiated hematopoietic cells. (Item 207) The method according to Item 206, wherein the embryo-derived cells and hemangio-colony forming cells are cultured in serum-free medium throughout step (a) of the method. (Item 208) The method described in Item 206, wherein the embryo-derived cells are embryonic stem cells. (Item 209) A method for performing a blood transfusion using hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, comprising: (a) culturing a cell culture comprising human embryo-derived cells in the presence of at least one growth factor in an amount sufficient to induce differentiation of the human embryo-derived cells into embryoid bodies; (b) adding at least one growth factor to said cell culture containing embryoid bodies, said growth factor being added in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body culture; (c) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells; (d) administering a transfusion to the differentiated hematopoietic cells. 210. The method of claim 209, wherein the embryo-derived cells, embryoid bodies and hemangio-colony forming cells are cultured in serum-free medium throughout steps (a) and (b) of the method. (Item 211) The method described in Item 209, wherein the growth factor is a protein comprising a homeobox protein or a functional variant or active fragment thereof. (Item 212) The method described in Item 211, wherein the homeobox protein comprises HOXB4 protein or a functional or active variant thereof. (Item 213) The method described in Item 209, wherein the embryo-derived cells are embryonic stem cells. (Item 214) A method for performing a blood transfusion using hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, comprising: (a) culturing a cell culture comprising human embryo-derived cells in the presence of at least one growth factor in an amount sufficient to induce differentiation of the human embryo-derived cells into embryoid bodies; (b) adding at least one growth factor to said culture containing embryoid bodies, said growth factor being added in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body culture; (c) disaggregating the embryoid bodies into single cells; (d) adding at least one growth factor to the culture comprising said single cell, said growth factor being added in an amount sufficient to cause expansion of human hemangio-colony forming cells in the culture comprising said single cell; (e) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells; (f) performing a transfusion using the differentiated hematopoietic cells. (Item 215) The method according to Item 214, wherein the embryo-derived cells, embryoid bodies and hemangio-colony forming cells are cultured in serum-free medium throughout steps (a)-(d) of the method. (Item 216) The method described in Item 214, wherein the growth factor is a protein comprising a homeobox protein or a functional variant or active fragment thereof. (Item 217) The method described in Item 216, wherein the homeobox protein comprises HOXB4 protein or a functional or active variant thereof. (Item 218) The method described in Item 214, wherein the embryo-derived cells are embryonic stem cells. (Item 219) A method according to any one of Items 203 to 218, wherein the differentiated hematopoietic cells are generated into a single cell type. (Item 220) The method described in Item 219, wherein the single cell type is selected from red blood cells, platelets or phagocytes. (Item 221) The method described in Item 220, wherein the phagocytes are selected from granulocytes, neutrophils, basophils, eosinophils, lymphocytes or monocytes. (Item 222) The method described in Item 220, wherein the red blood cells express hemoglobin F. (Item 223) The method described in Item 219, wherein the single cell type is mixed in approximately the same proportions as the differentiated cell types found in blood, the proportions of differentiated cell types found in blood being 96% red blood cells, 1% platelets and 3% phagocytes. (Item 224) A method according to any one of Items 203 to 218, in which multiple differentiated hematopoietic cell types are produced in the same step. (Item 225) The method described in Item 224, wherein the multiple differentiated hematopoietic cell types are selected from red blood cells, platelets, or phagocytes. (Item 226) The method described in Item 225, wherein the phagocytes are selected from granulocytes, neutrophils, basophils, eosinophils, lymphocytes or monocytes. (Item 227) The method described in Item 225, wherein the red blood cells express hemoglobin F. (Item 228) The method described in Item 224, wherein the multiple differentiated hematopoietic cell types are generated in a ratio approximately equal to the ratio of differentiated hematopoietic cell types found in blood, the ratio of differentiated cell types found in blood being 96% red blood cells, 1% platelets and 3% phagocytes. (Item 229) A method according to any one of Items 203 to 218, further comprising adding plasma to the differentiated hematopoietic cells prior to transfusion. (Item 230) The method of any one of Items 203 to 218, wherein the hemangio-colony forming cells are matched to the patient such that differentiated hematopoietic cells of the patient's own blood type are produced. (Item 231) The method described in any one of Items 203 to 218, wherein the hemangio-colony forming cells are negative for antigenic factors A, B, Rh or any combination thereof. (Item 232) A method according to any one of Items 203 to 218, wherein the differentiated hematopoietic cells are red blood cells, and the step of differentiating the red blood cells includes erythropoietin (EPO). The present invention includes any combination of the above-described aspects and embodiments of the invention. [Brief description of the drawings]

[0092] [Figure 1] FIG. 1 illustrates hematopoietic CFUs induced from hemangioblasts generated from H1-GFP ES cells. [Diagram 2] 1 is a diagram illustrating the cell morphology of erythroid colony forming units (CFU-E) cultured in a serum-free (Stemline) culture medium. [Diagram 3] FIG. 1 is a diagram illustrating the cell morphology of multipotent colony forming units (CFU-GEMM / Mix) cultured in serum-free (Stemline) culture medium. [Figure 4] FIG. 1 is a diagram illustrating the cell morphology of pluripotent colony forming units (CFU-GEMM / Mix) cultured in serum-free (Stemline) culture medium. [Diagram 5]1 is a diagram illustrating the cell morphology of granulocyte colony forming units (CFU-G) and macrophage colony forming units (CFU-M) cultured in serum-free (Stemline) culture medium. [Figure 6] FIG. 1 is a diagram illustrating the cell morphology of granulocyte / macrophage colony forming units (CFU-GM) and megakaryocyte macrophage colony forming units (CFU-Mk) cultured in serum-free (Stemline) culture medium. [Figure 7] FIG. 1 illustrates the formation of tube-cord structures after replating hemangioblasts derived from (a) H9 ES cells and (b) ACT30 ES cells on Matrigel-based medium. [Figure 8] Figure 8 illustrates the tube-cord structure of hemangioblasts generated from H1-GFP ES cells cultured on fibronectin-coated plates in EGM-2 medium and then on Matrigel, as described in Example 2. Figure 9 also illustrates the uptake of Ac-LDL by cells when incubated with Alexa Fluor-labeled Ac-LDL, as described in Example 2. The top middle and top right panels show phase contrast pictures. [Figure 9] FIG. 1 illustrates the expression of von Willebrand factor (vWF) (light grey staining) in hemangioblasts generated from H1-GFP ES cells cultured on fibronectin-coated plates in EGM-2 medium, as described in Example 2. [Figure 10] FIG. 13 illustrates blood vessel formation in cross-sections of hematoxylin and eosin (H&E) stained Matrigel plugs injected into SCID mice, as described in Example 4. [Figure 11] FIG. 1 illustrates that blood vessels obtained from cross sections of Matrigel plugs are human hemangioblast-derived cells, as shown by positive staining for a human-specific nuclear antibody (light grey staining), as described in Example 4. [Figure 12] FIG. 1 illustrates the mRNA sequence of human HOXB4 (Accession Number: NM-024015.4; GI:85376187) (SEQ ID NO:2). [Figure 13] FIG. 1 illustrates the amino acid sequence of human HOXB4 (Accession Number: NP-076920.1; GI: 13273315) (SEQ ID NO: 1). [Figure 14] FIG. 1 illustrates the mRNA sequence of human HOXB4 (Accession Number: BC049204.1; GI:29351567) (SEQ ID NO:4). [Figure 15] 1 is a diagram illustrating the amino acid sequence of human HOXB4 (Accession Number: AAH49204.1; GI: 29351568) (SEQ ID NO: 3). [Figure 16]Figures illustrating phenotypic characterization of hemangioblasts (BL-CFC) derived from human ES cells as described in Examples 1 and 2: (a) hemangioblast colonies or blast colonies (BL-CFC or BC, ×400); (b) secondary EBs (×400); (c) blast cells (hES-BC cells) stained with Wright-Giemsa (×1000); (df) GATA-1 staining; (d) blast cells stained with GATA-1. (x600); (e) Blast cells stained with GATA-1 and DAPI (x600); (f) BM cells stained with GATA-1 and DAPI (x400); (gi) LMO2 staining: (g) Blast cells stained with LM02 (x600); (h) Blast cells stained with LMO2 and DAPI (x600); (i) K562 cells stained with LMO2 and DAPI (x1000); (jm) CD71 staining (light or light grey): (j) stained with CD71 Blast cells (×600); (k) Blast cells stained with CD71 and DAPI (×600); (m) BM cells stained with CD71 and DAPI (×1000); (np) CXCR-4 staining (light or light grey): (n) Blast cells stained with CXCR-4 (×600); (o) Blast cells stained with CXCR-4 and DAPI (×600); (p) BM cells stained with CXCR-4 and DAPI (×600); (qs) Epo-receptor staining (medium grey). :(q) Blast cells stained with Epo-receptor (x600);(r) Blast cells stained with Epo-receptor and DAPI (x600);(s) BM cells stained with Epo-receptor and DAPI (x600);(tv) Tpo-receptor staining (medium grey);(t) Blast cells stained with Tpo-receptor (x600);(u) Blast cells stained with Tpo-receptor and DAPI (x600);(v) BM cells stained with Tpo-receptor and DAPI (x1000). Note: in panels (d) and (n), hES-BC (angioblast) cells were double stained with GATA-1 and CXCR-4 antibodies but shown separately; in panels (q) and (t), hES-BC (angioblast) cells were additionally double stained with Epo-receptor and Tpo-receptor antibodies and shown separately. [Figure 17-1]FIG. 17a-u. Diagrams illustrating functional characterization of hemangioblasts (BL-CFC or blast cells) derived from human ES cells in vitro: (ad) hematopoietic CFUs derived from purified hemangioblasts: (a) CFU-erythroid (×100); (b) CFU-granulocyte (×100); (c) CFU-macrophage (×100); and (d) CFU-multilineage (mixture, ×100). );(eh) Wright-Giemsa staining of CFU-cells: (e) erythroid (×1000); (f) granulocyte (×1000); (g) macrophage (×400); and (h) mixed (×1000);(ik) Immunostaining of CFU-cells: (i) CFU-erythroid cells stained with CD235a (arrow, ×1000); (j) CFU-granulocyte cells stained with CD13 (arrow, ×1000);(k) CD CFU-hybrid cells stained with CD45 (arrow, ×1000); (mp and vy) FACS analysis of pooled CFU cells: (m) mouse IgG isotype control; (n) CD45; (o) CD13; and (p) CD235; (qu) purified hemangioblasts, or endothelial cells derived from blasts or hES-BC cells; (q) capillary-like structures formed on Matrigel after plating adherent cells derived from hemangioblasts (×100); (r) Ac-LDL uptake by hemangioblast-derived endothelial cells (gray) (×200); (s) expression of vWF in hemangioblast-derived endothelial cells whose nuclei are stained with DAPI (arrow) (×600); (t) PEC in hemangioblast-derived endothelial cells whose nuclei (circular feature in figure) are stained with DAPI. (u) Polarized localization of VE-cadherin (arrow) in hemangioblast-derived endothelial cells whose nuclei (circular features in the figure) are stained with DAPI (x200). See Example 2. [Figure 17-2]Figure 17v-cc. Diagrams illustrating functional characterization of hemangioblasts (BL-CFC or blast cells) derived from human ES cells in vitro: (v) mouse IgG isotype control; (w) CD45 and CD235a; (x) CD13 and CD45; and (y) CD13 and CD235a; (z-cc) purified hemangioblasts, or blast cells or endothelial cells derived from hES-BC cells: (z) Ac-LDL update (arrow head) and vWF expression (arrow, x600); (aa) Ac-LDL update (arrow head) and VE-cadherin expression (arrow head, x600); (bb) vWF expression (arrow) and CD31 expression (arrow head, x600); (cc) VE-cadherin expression (arrow) and CD31 expression (arrow head, x600). See Example 2. [Figure 18]Illustrative figures showing clonogenicity of blast colonies derived from hES cells: (ac) Clonogenicity of blast colonies: (a) and (b) Two types of blast colonies grown in a mixture of WA01-GFP and MAO1 EBs, demonstrating the clonal origin of the blast colonies: (a) Phase image (x100); (b) GFP image (x100); (c) Blast colony grown from a single cell (x400); (de) Growth of a single blast colony in liquid culture, where both hematopoietic and endothelial lineages are observed (x200): (d) x200; (e) x400; (fh) Endothelial cells derived from a single BC: (f) Capillary tubes formed on Matrigel after plating adherent cells derived from a single BC. Similar structures (×100); (g) Ac-LDL uptake by endothelial cells derived from a single BC, nuclei stained with DAPI (arrow) (×400); (h) vWF expression in single BC-derived endothelial cells, nuclei stained with DAPI (arrow) (×400); (im) hematopoietic CFUs derived from a single BC: (i) CFU-erythroid (×100); (j) CFU-granulocyte (×100); (k) CFU-macrophage (×100); and (m) CFU-multilineage (mixture, ×100). The gel represents the results of PCR analysis of GFP sequences in GFP+ and GFP- hES-BCs (angioblasts) derived from a plate mixture of WA01-GFP+ and MA01-GFP- cells. Lanes: WA01-GFP+, maternal WA01 / GFP+ hES cells; MA01-GFP-, maternal MA01 hES cells; H20, water negative control; BC-GFP+, GFP positive BC absorbed from cell-mixed plate; 1-10, GFP negative BC absorbed from cell-mixed plate. Myogenin gene was used as PCR reaction control. See Example 2. [Figure 19]Characterization of the tPTD-HOXB4 fusion protein is provided. (A) 6xHis-fused tPTD-HOXB4 recombinant protein was expressed in E. coli and purified using nickel ProBand resin. Two batches of desalted tPTD-HOXB4 protein (labeled (1) and (2)) were examined for purity and concentration using SDS-PAGE gels. (B) tPTD-HOXB4 protein was unstable in medium containing 5% FBS, but (C) remained intact in serum-free medium containing live ES cells (N=Stem Cell II medium alone; h=hours). [Figure 20-1] Figure 20a-b. Robust restoration of ischemic retinal vasculature after systemic injection of hES-derived hemangioblasts. Retinal ischemia was induced by applying hydrostatic pressure to the anterior chamber of the mouse eye for 2 hours. Seven days later, fluorescently labeled (GFP+) hemangioblasts were injected intravitreally or intravenously. One day later, the animals were euthanized. Eyes were enucleated and dissected, and retinas were flat-fixed and imaged via laser scanning confocal microscopy or sectioned for imaging. (a) Merged view of a representative control (uninjured) eye showing typical retinal vascular anatomy with no background green fluorescence, and where individual GFP+ (lower insert) and GFP- (upper insert) channels can also be seen; (b) Merged view of a treated eye from the same animal showing fluorescently labeled (GFP+) hemangioblast-derived cells (bright areas of the vasculature) introduced in the ischemic vasculature, as described in Example 5, where individual green (GFP+ hemangioblasts, lower insert) and unlabeled cells (upper insert) are also seen. See Example 5. [Figure 20-2]FIG. 20c-e. Robust restoration of ischemic retinal vasculature after systemic injection of hES-derived hemangioblasts. Retinal ischemia was induced by applying hydrostatic pressure to the anterior chamber of the mouse eye for 2 hours. After 7 days, fluorescently labeled (GFP+) hemangioblasts were injected intravitreally or intravenously. After 1 day, the animals were euthanized. The eyes were enucleated and dissected, and the retinas were flat-mounted and imaged via laser scanning confocal microscopy or sectioned for imaging. (c) Merged image of uninjured control group (no GFP fluorescence (light or light gray)), (d) and (e) merged images of ischemic eyes 2 days (d) and 7 days (e) after systemic hemangioblast injection (GFP+ cells appear light or light gray). See Example 5. [Figure 20-3] Figure 20f. Robust restoration of ischemic retinal vasculature after systemic injection of hES-derived hemangioblasts. Retinal ischemia was induced by applying hydrostatic pressure to the anterior chamber of the mouse eye for 2 hours. Seven days later, fluorescently labeled (GFP+) hemangioblasts were injected intravitreally or intravenously. One day later, the animals were euthanized. Eyes were enucleated and dissected, and retinas were flat-fixed and imaged via laser scanning confocal microscopy or sectioned for imaging. (f, X600, confocal), Fluorescent immunocytochemistry colocalizes hemangioblast (hES-BC)-derived endothelial cells to damaged vasculature in cross-section of mouse eyeball subjected to I / R injury; high magnification of a vessel lumen in the ganglion cell layer adjacent to the internal limiting membrane shows the lumen surrounded by peripheral endothelial cells (arrow, CD31) and mature endothelial cells (arrow, human nuclear antigen) derived from hemangioblast (hES-BC). Inset top and middle panels are the individual human nuclear antigen and CD31 channels used to generate the composite image. Bottom panels are low magnification views of such areas, with boxes indicating the areas represented in all panels. V=vitreous body; IPL=inner plexiform layer; RPE=retinal pigment epithelium layer; Ch=choroid. See Example 5. [Figure 21]Figures showing the introduction of hemangioblasts, or undifferentiated cells, or hES-BC cells into the retinal vasculature of diabetic rats. (a) and (b) are merged images of the retinal vasculature of a diabetic rat about 2 days after intravitreal hemangioblast injection, showing that both large and small vessels were introduced with hemangioblasts in large quantities (light or light gray areas), and (c) is a merged image of a control (nondiabetic) rat about 2 days after hemangioblast injection, showing that the hemangioblasts (or hemangioblast-derived cells) were not introduced into the vasculature but formed a sheet located on the top of the retina (soft gray layer). (d, X100), a section from a nondiabetic control rat about 2 days after intravitreal undifferentiated cell injection was negative for human nuclear antigen staining but clearly positive for the endothelium (CD31, arrow). (e, X100) and (f, X600, confocal) are rat eye sections from diabetic rats approximately 2 days after intravitreal hemangioblast injection and stained with CD31 and human nuclear antigen antibodies, demonstrating co-staining of CD31 and human nuclear antigen in cells lining the lumen of blood vessels in the ganglion cell layer of the retina, immediately dorsal to the internal limiting membrane separating the vitreous from the neural retina (arrows pointing to bright areas). See Example 6. [Figure 22]Figures showing endothelial differentiation in ischemic hind leg muscle and infarcted heart after hemangioblast injection. a, b, h, and i: Hemangioblast or hES-BC cell differentiation in infarcted heart. (a(200X)), infarcted myocardium sections from control mice immunostained with human-specific vWF antibody, which appears unstained with human vWF; (b, 200X) and (i, X600 confocal), infarcted myocardium sections 4 weeks after hemangioblast injection immunostained with human-specific vWF antibody (light grey or light areas in (b) and (i)); and (h) survival curves of sham-operated, medium control, and hemangioblast-treated mice. cg: Hemangioblast differentiation in ischemic hind leg muscle. (c, 50X), sections of hind leg muscle from control mice immunostained with human-specific vWF antibody, which appears not to stain with human vWF; (d, 50X) and (e, 600X, confocal), sections of ischemic hind leg muscle approximately 4 weeks after hemangioblast injection immunostained with human-specific vWF antibody (light grey); (f) Restoration of blood flow to the surgically induced ischemic leg. Hind leg blood flow was monitored continuously for 30 days after ligation in mice receiving 6x105 hemangioblasts and in mice receiving medium only. Blood flow was calculated as the flow velocity in the ischemic leg relative to the non-ischemic leg; (g) Laser Doppler blood flow images. Images of control (medium) and ischemic animals injected with BC cells (n=6 for each group). See Examples 7 and 8. [Diagram 23] Figure 1 shows GFP (lightest or brightest areas) and cTnI (medium grey) immunostaining of cardiac tissue sections from myocardial infarction (MI) mice. See Example 8. Arrows indicate double positive staining cells derived from injected hemangioblasts (hES-BCs). [Figure 24] This is a diagram showing the differentiation of hemangioblasts (hES-BC) into smooth muscle cells. When confirmed through PCR of RNA isolated from hemangioblasts (hES-BC), the hemangioblasts expressed smooth muscle-specific genes. Hemangioblasts (hES-BC) also differentiated into smooth muscle cells in vitro. Immunostaining for calponin and α-SMA showed that the differentiated cells expressed these two smooth muscle cell markers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0093] In order to provide a thorough understanding of the present invention, the following detailed description is set forth below. Unless otherwise defined, all 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. Methods and materials similar or equivalent to those described in the present invention can be used to describe or test the present invention, and suitable methods and materials are described below. The substances, materials, and examples are for illustrative purposes only and are not intended to be limiting.

[0094] All publications, patents, patent publications and applications, and other references mentioned herein are incorporated by reference in their entirety.

[0095] Throughout this specification, the word "comprise" or its variant forms, such as "comprises" or "comprising", is intended to include a stated integer or group of integers and not to exclude any other integer or group of integers.

[0096] In order to further clarify the present invention, the following terms and definitions are now provided.

[0097] The term "human embryonic stem cell" (hES cell) is used herein with the same meaning as in the art. This term includes cells derived from the inner cell mass of a human morula or blastocyst that can be serially passaged as a cell line. hES cells can be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, virgin fertilization, or through methods of generating hES cells using homozygotes for the HLA region. Human ES cells are also cells derived from mammalian embryos at the zygote, blastomere, or blastocyst stage produced through fusion of sperm and egg cell, nuclear transfer, virgin fertilization, or reprogramming of chromatin and subsequent transfer of the reprogrammed chromatin to the plasma membrane for cell production. Human embryonic stem cells of the present invention can include, but are not limited to, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells.

[0098] The term "protein transduction domain" ("PTD") is intended to mean any amino acid sequence that is translocated across a cell membrane into a cell or that, for example, enhances or confers the rate at which another molecule (e.g., a protein domain) to which the PTD is attached is translocated across a cell membrane into a cell. Protein transduction domains can be domains or sequences that occur naturally as part of a larger protein (e.g., the PTD of a viral protein, e.g., HIV TAT) or can be a synthetic or artificial amino acid sequence. overview The invention provides methods for the generation and expansion of human hemangio-colony forming cells from human embryo-derived cells, preparations and compositions comprising human hemangio-colony forming cells, methods for producing a variety of cell types partially or terminally differentiated from hemangio-colony forming cells, methods of therapeutically using hemangio-colony forming cells, and methods of therapeutically using a variety of cell types partially or terminally differentiated from hemangio-colony forming cells.

[0099] The terms "hemangioblast" and "hemangio-colony forming cell" are used interchangeably throughout this application. These cells may be described based on a variety of structural and functional characteristics, including, but not limited to, the expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers. Hemangio-colony forming cells are differentiated to generate at least hematopoietic cell types or endothelial cell types. Hemangio-colony forming cells are desirably bipotential, differentiated to generate at least hematopoietic cell types and endothelial cell types. Thus, the hemangio-colony forming cells of the present invention are at least unipotential, and preferably bipotential. However, in addition, hemangio-colony forming cells can have significant developmental potential and, in certain embodiments, are differentiated to generate cell types of other lineages. In one embodiment, the hemangio-colony forming cells are differentiated to produce other mesodermal derivatives, such as cardiac cells (eg, cardiomyocytes) and / or smooth muscle cells.

[0100] The invention also provides methods of expanding mammalian hemangio-colony forming cells obtained from any source including ES cells, blastocysts or blastomeres, umbilical cord blood from placenta or umbilical tissue, peripheral blood, bone marrow or other tissues, or obtained by other methods in the art. Human hemangio-colony forming cells may also be generated from human embryo-derived cells. Human embryo-derived cells may be a substantially homogeneous cell population, a heterogeneous cell population, or part or all of an embryonic tissue. As an example of embryo-derived cells that can be used in the methods of the invention, human hemangio-colony forming cells may be generated from human embryonic stem cells. Such embryonic stem cells include, for example, embryonic stem cells derived from or utilizing a blastocyst, a plated ICM, one or more blastomeres, or other portions of a preimplantation stage embryo or embryo-like structure, regardless of the mode of production, whether by fertilization, somatic cell nuclear transfer (SCNT), parthenogensis, androgenesis, or other sexual or asexual modes.

[0101] In certain embodiments, the hemangioblasts are further differentiated into hematopoietic cells, including, but not limited to, platelets and red blood cells. Such cells may be used in blood transfusions. The ability to generate large quantities of cells for transfusion would alleviate the chronic blood shortages experienced by blood banks and hospitals across the country. In certain embodiments, the methods of the present invention allow for the production of universal cells for transfusion. Specifically, O and Rh- type red blood cells can be readily generated to provide a universal blood source for transfusions.

[0102] The methods of the invention can easily expand hemangioblasts in large quantities in vitro for a variety of commercial and clinical applications. Expanding hemangioblasts in vitro means multiplying the hemangioblasts. The methods of the invention allow the expanded amount of human hematopoietic cells to reach commercially useful amounts, and the invention relates to large amounts of hemangioblasts and cell preparations comprising large amounts of human hemangioblasts (e.g., greater than 10,000, 100,000 or 500,000 cells). In certain embodiments, the cell preparations comprise 1×10 6In another embodiment, the cell preparation comprises 2×10 human hemangioblast cells or more. 6 In yet another embodiment, the cell line comprises 3×10 human hemangioblasts or more. 6 In yet another embodiment, the cell preparation comprises 4×10 human hemangioblast cells or more. 6 Contains more than cells.

[0103] The present invention relates to solutions, preparations and compositions comprising 10,000 to 4 million or more mammalian (e.g., human) hemangioblast cells. In such solutions, preparations and compositions, the number of hemangioblast cells can be any number within the range of 10,000 to 4 million or more. This number can be, for example, 20,000, 50,000, 100,000, 500,000, 1 million.

[0104] Similarly, the invention relates to preparations of progeny cells of human hemangioblasts (e.g., human hematopoietic cells, and human hematopoietic stem cells, including endothelial cells). The invention also relates to methods of producing, storing, and distributing hemangioblasts and / or hemangioblast lineage cells.

[0105] The present invention also provides methods and solutions suitable for transfusion into a human or animal patient. In specific embodiments, the present invention provides methods for producing red blood cells and / or platelets, and / or other hematopoietic cell types for transfusion. In one embodiment, the present invention is suitable for use in blood banks and hospitals to provide blood for transfusion in the treatment of vascular-related disorders or diseases, or following trauma. In one embodiment, the present invention provides red blood cells that are universal donor cells. In one embodiment, the red blood cells are functional and express hemoglobin F prior to transfusion.

[0106] The invention also provides human hemangio-colony forming cells, cell cultures comprising a substantially purified population of human hemangio-colony forming cells, pharmaceutical preparations comprising human hemangio-colony forming cells, and cryopreserved preparations of hemangio-colony forming cells. In one embodiment, the invention provides the use of the human hemangio-colony forming cells in the manufacture of a medicament for treating a condition in a patient in need of such treatment. In turn, the invention provides the use of the cell cultures in the manufacture of a medicament for treating a condition in a patient in need of such treatment. The invention also provides the use of the pharmaceutical preparations in the manufacture of a medicament for treating a condition in a patient in need of such treatment.

[0107] Hemangio-colony forming cells are identified and characterized based on these structural properties. Specifically, in one embodiment, the cells are unique in that they are loosely attached to one another (loosely attached to other hemangio-colony forming cells). Because the cells are loosely attached to one another, cultures or colonies of hemangio-colony forming cells can be disaggregated into single cells using mechanical disaggregation alone, without the need for enzymatic disaggregation methods. The cells are loosely attached to one another such that mechanical disaggregation alone, rather than enzymatic disaggregation or a combination of mechanical and enzymatic disaggregation, is sufficient to disaggregate the cultures or colonies without substantially damaging cell viability. In other words, mechanical disaggregation does not require the application of forces so great as to result in substantial cell damage or death, as compared to that observed following enzymatic disaggregation of cell aggregates.

[0108] Hemangio-colony forming cells may also be identified or characterized based on the expression or lack of expression (as assessed at the gene or protein level) of one or more markers. For example, in one embodiment, hemangio-colony forming cells may be identified or characterized based on the lack of expression of one or more of the following cell surface markers (e.g., the cells are characterized based on the lack of expression of one or more, two or more, three or more, or four or more of the following markers): CD34, KDR, CD133, or CD31. Alternatively, or alternatively, hemangio-colony forming cells may be identified or characterized based on the expression or lack of expression (as assessed at the gene or protein level) of one or more markers analyzed in Table 2.

[0109] The hemangio-colony forming cells of the present invention may be identified or characterized based on one or any combination of these structural or functional characteristics. It should be noted that the cells may be derived from any of a number of sources, e.g., embryonic, prenatal, or perinatal tissue, but regardless of source the term "hemangio-colony forming cells" is used to refer to cells that are capable of differentiating to produce at least hematopoietic and / or endothelial cell types and that have the structural or technical characteristics described above. In vitro differentiation of human embryonic stem cells to obtain embryoid bodies and hemangioblasts The present invention provides methods for generating and expanding human hemangioblasts derived from human embryonic stem cells or from human blastomeres or blastomeres. The hemangioblasts so generated may be purified and / or isolated.

[0110] Human hemangio-colony forming cells may also be formed from human embryo-derived cells. Human embryo-derived cells may be a substantially homogeneous cell population, a heterogeneous cell population, or part or all of an embryonic tissue. As an example of an embryo-derived cell that may be used in the methods of the present invention, human hemangio-colony forming cells may be generated from human embryonic stem cells. Such embryonic stem cells include, for example, embryonic stem cells derived from or using a blastula, a plated ICM, one or more blastomeres, or other parts of a pre-implantation stage embryo or embryo-like structure, regardless of generation by fertilization, somatic cell nuclear transfer (SCNT), unisexual reproduction, androgenesis, or other reproductive or non-reproductive means.

[0111] Additionally or alternatively, hemangio-colony forming cells can be generated from other embryo-derived cells. For example, hemangio-colony forming cells can be generated from or using plated embryos, ICMs, blastulas, trophoblast / trophoblast cells, one or more blastomeres, trophoblast stem cells, embryonic germ cells, or other parts of preimplantation stage embryos or embryo-like structures, regardless of generation by fertilization, somatic cell nuclear transfer (SCNT), unisexual reproduction, androgenesis, or other reproductive or non-reproductive means (without necessarily going through an embryonic stem cell derivation step). Similarly, hemangio-colony forming cells can be generated using cells or cell lines partially differentiated from embryo-derived cells. For example, where a human embryonic stem cell line is used to generate cells that are more developmentally earlier than the hemangio-colony forming cells in terms of developmental potential and adaptability, such embryo-derived cells can be used to generate hemangio-colony forming cells.

[0112] Additionally or alternatively, hemangio-colony forming cells can be generated from other fetal or perinatal sources, including, but not limited to, umbilical cord, umbilical cord blood, amniotic fluid, amniotic fluid stem cells, and placenta.

[0113] When hemangio-colony forming cells are generated from human embryonic tissue, an embryoid body formation step may be necessary. However, since the formation of embryoid bodies is at least partially conducive to recapitulating the three-dimensional interactions of germ layers occurring during early development, such a step is not necessarily required if the cells derived from the embryo already have organization or structure for substantially the same purpose as the formation of embryoid bodies. For example, when hemangio-colony forming cells are generated from plated blastula, a certain level of three-dimensional organization is already present in the blastula cells. Thus, an embryoid body formation step is not necessarily required to provide intercellular signals, inductive signals, or three-dimensional structure.

[0114] The methods and uses of the present invention may be used to generate hemangio-colony forming cells from embryo-derived cells. In certain embodiments, the embryo-derived cells are embryonic stem cells. In other particular embodiments, the embryo-derived cells are plated embryos, ICMs, blastulas, trophoblast / trophoblast cells, one or more blastomeres, trophoblast stem cells, or other parts of the early pre-implantation embryo. For any of the above, the embryo-derived cells may be derived from embryos generated by fertilization, somatic cell nuclear transfer (SCNT), parthenogenesis, androgenesis, or other reproductive or non-reproductive means.

[0115] Throughout this application, where the invention is disclosed in connection with generating hemangio-colony forming cells specifically from embryonic stem cells, the invention also includes generating hemangio-colony forming cells using cells generated from or using other embryo-derived cells for any of the same therapeutic uses.

[0116] In some aspects of the invention, human embryonic stem cells can be the starting material for this method. Embryonic stem cells can be cultured by any method known in the art, for example, in the presence or absence of nutrient-feeding cells.

[0117] Embryonic stem cells can form embryoid bodies ("EBs") in suspension in medium containing serum (Wang et al. 2005 J Exp Med(201):1603-1614; Wang et al. 2004 Immunity(21):31-41; Chadwick et al. 2003 Blood(102):906-915). However, the addition of serum poses several problems, including experimental variability, expense, potential for infectious agents, and limited supply. Additionally, in clinical and certain commercial applications, the use of serum requires compliance with additional U.S. and international regulations governing biological products.

[0118] The present invention provides methods for generating and expanding human hemangioblasts from embryonic stem cells in the absence of serum. Serum-free conditions are more advantageous for large-scale production under Good Manufacturing Practice (GMP) guidelines than conditions requiring serum. Serum-free conditions also extend the half-life of certain factors added to the medium (e.g., the half-life of growth factors, cytokines, and proteins including HOXB4 is increased in the medium in the absence of serum). In certain embodiments, serum-free medium is used throughout the methods of the present invention for generating and expanding human hemangioblasts.

[0119] In the first step of the method for generating and expanding human hemangioblasts, human stem cells are grown in serum-free medium and induced to differentiate into embryoid bodies. To induce embryoid body formation, embryonic stem cells are pelleted and supplemented with one or more morphogenic factors and cytokines, and then plated in serum-free medium (e.g., Stemline I or II medium (Sigma)) on low attachment (e.g., ultra-low attachment) culture plates. TM) in the medium. The morphogenetic factors and cytokines can include, but are not limited to, bone morphogenetic proteins (e.g., BMP2, BMP-4, BMP-7, but not BMP-3), and VEGF, SCF, and FL. The bone morphogenetic proteins and VEGF can be used alone or with other factors. The morphogenetic factors and cytokines can be added to the medium from 0 to 48 hours into cell culture. After incubation under these conditions, the plated ES cells can form EBs if incubated in the presence of early hematopoietic growth cytokines, including, but not limited to, thrombopoietin (TPO), Flt-3 ligand, and stem cell factor (SCF). In addition to TPO, Flt-3 ligand, and SCF, VEGF, BMP-4, and HoxB4 can also be added to the medium. In one embodiment, human ES cells are first grown in the presence of BMP-4 and VEGF165 (e.g., 25-100 ng / ml), and then grown in the presence of BMP-4, VEGF165, SCF, TPO, and FLT3 ligand (e.g., 10-50 ng / ml) and HoxB4 (e.g., 1.5-5 μg / ml of a triple protein transduction domain-HoxB4 fusion protein as disclosed herein). Additional factors may be added 48-72 hours after plating.

[0120] In the methods of the invention, human hemangioblasts are isolated from early embryoid bodies ("EBs"). Isolation of hemangioblasts from early EBs supports the proliferation of cells in vitro. For human cells, hemangioblasts are obtained from EBs grown for less than 10 days. In certain embodiments of the invention, hemangioblasts arise in human EBs grown for 2-6 days. According to one embodiment, hemangioblasts may be identified and isolated from human EBs grown for 4-6 days. In other embodiments, human EBs are grown for 2-5 days before isolating hemangioblasts. In certain embodiments, human EBs are grown for 3-4.5 days before isolating hemangioblasts.

[0121] In certain embodiments, primary EBs are washed and dissociated (e.g., by trypsin / EDTA or collagenase B). A selected number of cells (e.g., 2-5x10 5cells) are mixed with serum-free methylcellulose medium optimized for hemangioblast growth (e.g., BL-CFU medium, e.g., Stem Cell Technologies Catalogue H4436, or Hemangioblast Growth Medium (HGM), or any of the following MDM containing 1.0% methylcellulose, 1-2% bovine serum albumin, 0.1 mM 2-mercaptoethanol, 10 μg / ml rh-insulin, 200 μg / ml iron-bound human transferrin, 20 ng / ml rh-GM-CSF, 20 ng / ml rh-IL-3, 20 ng / ml rh-IL-6, 20 ng / ml rh-G-CSF) ("rh" stands for "recombinant human"). Such media may be supplemented with early stage cytokines (including but not limited to EPO, TPO, SCF, FL, FLt-3, VEGF, BMPs, such as BMP2, BMP4, and BMP7, but excluding BMP3) and HOXB4 (or additional homeobox proteins). In certain embodiments, erythropoietin (EPO) is added to the media. In additional embodiments, EPO, SCF, VEGF, BMP-4, and HoxB4 are added to the media. In additional embodiments, cells are grown in the presence of EPO, TPO, and FL. In certain embodiments where H9 is the starting human ES cell line, EPO, TPO, and FL are added to the media. In addition to EPO, TPO, and FL, media for cells derived from H9 or other ES cells may further include VEGF, BMP-4, and HoxB4.

[0122] Cells obtained by the method, including hemangioblasts (the cells can be in BL-CFU medium), are plated on ultra-low attachment culture plates and incubated in a CO2 incubator to grow hemangioblast colonies. Some cells may form secondary EBs. After 3-6 days, and in some cases after 3-4.5 days, hemangioblast colonies are observed. Hemangioblast colonies can be distinguished from other cells, such as secondary EBs, by their characteristic grape-like morphology and / or small size. Hemangioblasts can also be identified by expression of certain markers (e.g., expression of early hematopoietic cell markers and endothelial cell markers) and their ability to differentiate into at least hematopoietic and endothelial cells (see below, hemangioblast lineage cell induction). For example, even though hemangioblasts lack certain characteristics characteristic of mature endothelial or hematopoietic cells, these cells can be identified by the presence of certain markers (e.g., CD71+) and the absence of other markers (e.g., CD34-). Hemangioblasts may also express GATA-1 and GATA-2 proteins, CXCR-4, and TPO and EPO receptors. Hemangioblasts may also be characterized by low or absent expression of other markers (e.g., CD31, CD34, KDR, or other adhesion molecules). Hemangioblasts may also be characterized by expression of specific genes, such as genes associated with hemangioblast and early primitive erythroblast development, e.g., SCL, LMO2, FLT-I, embryonic fetal globin gene, NF-E2, GATA-1, EKLF, ICAM-4, glycophoriuns, and EPO receptor.

[0123] Thus, hemangioblasts can be separated by size (they are smaller than other cells) or purified with anti-CD71+ antibodies, for example, by immunoaffinity column chromatography.

[0124] Hemangioblasts may be separated by size and / or morphology by the following procedure: After 6-7 days of growth, the cell mixture contains EBs (which are round, clumps of many cells) and hemangioblasts (which are grape-shaped, smaller than EBs, and single cells). Thus, hemangioblasts may be separated based on morphology and size. Hemangioblasts may be picked out manually, for example, when the cell mixture is viewed under a microscope. The cells are then allowed to grow into colonies (each colony having 100-150 cells).

[0125] Human hemangioblast colonies induced as described above can be picked and replated on methylcellulose CFU-medium to form hematopoietic CFUs. In certain embodiments, the CFU-medium comprises Stem Cell Technologies H4436. In additional embodiments, the hemangioblasts are plated in Stemline II medium supplemented with cytokines and other factors. For example, individual BL-CFC colonies can be hand picked and transferred to fibronectin-coated plates containing Stemline II and recombinant human SCF (e.g., 20 ng / ml), TPO (e.g., 20 ng / ml), FL (e.g., 20 ng / ml), IL-3 (e.g., 20 ng / ml), VEGF (e.g., 20 ng / ml), G-CSF (e.g., 2Ong / ml), BMP-4 (e.g., 15 ng / ml), IL-6 (e.g., 10 ng / ml), IGF-1 (e.g., 10 ng / ml), endothelial cell growth supplement (ECGS, e.g., 100 μg / ml), and Epo (e.g., 3 U / ml). After one week of growth in the test tube, the cells can be carefully pipetted to remove non-adherent hematopoietic cells and used directly for hematopoietic CFU analysis. After removing non-adherent cells, the adherent colonies are transferred to EGM-2 endothelial cell medium (Cambrex). TM After one week of growth in medium containing 0.1% CO, expression of vWF can be examined. In vitro hemangioblast proliferation A particular aspect of the invention relates to the in vitro propagation of hemangioblasts, hi a particular embodiment, the hemangioblasts propagated by the methods of the invention are obtained from early embryoid bodies derived from human embryonic stem cells as described above.

[0126] In addition to deriving hemangioblasts from human embryonic stem cells (hES cells), the hemangioblasts to be expanded may also be isolated from other mammalian sources, such as mammalian embryos (Ogawa et al. 2001 Int Rev Immunol(20):21-44; U.S. Patent Publication 2004 / 0052771), umbilical cord blood from umbilical cord tissue and placenta (Pelosi, et al. 2002 Blood(100):3203-3208; Cogle et al. 2004 Blood(103):133-5), peripheral blood and bone marrow (Pelosi et al. 2002 Hematopoiesis(100):3203-3208). In certain embodiments, the non-human hemangioblasts to be expanded may be generated from non-human embryonic stem cells (such as mice and non-human primates). In certain embodiments, hemangioblasts are obtained from umbilical cord blood (UCB) or bone marrow by methods such as, for example, magnetic bead positive selection or purification techniques (e.g., MACS columns). Cells may be selected based on their CD71+ status and confirmed as CD34-. Isolated hemangioblasts may also be tested for potential generation of hematopoietic and endothelial cell lineages. In certain embodiments, hemangioblasts isolated or purified, and optionally enriched from embryos, umbilical cord blood, peripheral blood, bone marrow or other tissues, are greater than 95% pure.

[0127] Bone marrow-derived cells may be obtained from any stage of development of the donor individual, including prenatal (e.g., embryonic or fetal), infancy (e.g., birth to about 3 years in humans), childhood (e.g., about 3 to about 13 years in humans), adolescence (e.g., about 13 to about 18 years in humans), young adulthood (e.g., about 18 to about 35 years in humans), adulthood (e.g., about 35 to about 55 years in humans), or geriatric (e.g., about 55 years or older in humans).

[0128] Human bone marrow is obtained, for example, from the split sternum of a patient undergoing surgery. Hereinafter, bone marrow is defined as a volume of 0.1 to 1 mm 3After preservation in tissue chunks, the cells may be grown on mouse embryonic nutrient layers (e.g., mitomycin C-treated or irradiated nutrient layers). The bone marrow cells will attach to the plates over a culture period of 1-2 weeks, and hemangioblasts may be identified and isolated based on morphological characteristics and / or cell markers (see US 2004 / 0052771). The cells may then be grown and expanded in serum-free conditions according to methods disclosed herein.

[0129] Hemangioblasts can also be obtained by sorting bone marrow cells and cells derived from blood or other tissues. Sorting methods are widely known in the art and generally involve both positive selection (i.e., retention of cells based on a particular property) and negative selection (i.e., removal of cells based on a particular property). Enrichment and sorting methods for bone marrow-derived cells are best characterized for human and mouse cells.

[0130] Various enrichment and sorting methods for bone marrow-derived cells or other cells are known in the art. Positive selection methods, such as enrichment for cells expressing CD71, may be used. Negative selection methods to remove or reduce cells expressing CD3, CD10, CD11b, CD14, CD16, CD15, CD16, CD19, CD20, CD32, CD45, CD45R / B220, or Ly6G may also be used alone or in conjunction with positive selection techniques. In the case of bone marrow cells, if the donor bone marrow-derived cells are not autologous, negative selection may be performed on the cell preparation to reduce or remove differentiated T cells.

[0131] Generally, methods used to select / enrich bone marrow-derived cells, blood cells, or other cells utilize immunoaffinity techniques, although density centrifugation methods are also useful. Immunoaffinity techniques can take a variety of forms as known in the art, but generally utilize an antibody or antibody derivative in conjunction with some type of separation technique. The separation technique generally physically separates cells bound by the antibody from cells not bound by the antibody, although in some cases separation techniques that kill cells bound by the antibody may be used for negative selection.

[0132] Any suitable immunoaffinity technique may be used to select / enrich hemangioblasts derived from bone marrow-derived cells, blood cells or other cells, including fluorescence-activated cell sorting (FACS), panning, immunomagnetic separation, immunoaffinity chromatography, antibody-mediated complement fixation, immunotoxins, density gradient separation, etc. After treatment with the immunoaffinity process, the cells of interest (cells bound by the immunoaffinity reagent in the case of positive selection, or cells not bound by the immunoaffinity reagent in the case of negative selection) may be recovered and subjected to additional recovery immunoaffinity selection / enrichment.

[0133] Immunoaffinity selection / enrichment is typically performed by incubating a preparation of cells, including bone marrow-derived cells, with an antibody or an antibody-derived affinity reagent (e.g., an antibody specific for a given surface marker), followed by using the bound affinity reagent to select for or against the antibody-bound cells. The selection step typically involves physical separation, which can be achieved using antibodies bound (directly or indirectly) to a solid substrate (panning, immunoaffinity chromatography) or using a magnetic field to collect cells bound to magnetic particles through an affinity reagent (immunomagnetic separation), and directing droplets containing single cells into different containers depending on the presence or absence of bound affinity reagent (FACS). Alternatively, undesired cells can be removed from the bone marrow-derived cell preparation using affinity reagents that induce cytotoxic damage to cells bound by the affinity reagent. The cytotoxic damage can be activated by the affinity reagent (e.g., complement fixation) or localized to the target cells by the affinity reagent (e.g., immunotoxins such as ricin B chain).

[0134] Although the methods described herein relate to enriching cells from bone marrow-derived or blood cell preparations, one of skill in the art will be able to apply similar positive and negative selection techniques to cell preparations derived from other tissues.

[0135] Certain aspects of the invention relate to the in vitro propagation of hemangioblasts. In certain embodiments, the hemangioblasts propagated by the methods of the invention are obtained from early embryoid bodies derived from human embryonic stem cells as described above. In other embodiments, the hemangioblasts are isolated or enriched from human tissues (e.g., placenta or umbilical cord blood, peripheral blood, bone marrow, etc.).

[0136] In certain embodiments, the hemangioblasts are grown in the presence of a homeodomain protein (also referred to herein as a homeobox protein). In additional embodiments, the hemangioblasts are grown in the presence of HOXB4. In certain embodiments, HOXB4 is added to the hemangioblasts during the method of expansion of the hemangioblasts.

[0137] HOXB4 is a homeodomain transcription factor (also called HOX2F, HOX2, HOX-2.6, and in rats HOXA5) that is expressed intracellularly in the stem cell compartment of bone marrow and then downregulated during differentiation. Expression of the HOXB4 gene is associated with the retention of a primitive stem cell phenotype (Sauvageau et al. 1995 Genes Dev9:1753-1765; Buske et al. 2002 Blood 100:862-868; Thorsteinsdottir et al. 1999 Blood 94:2605-2612; Antonchuk et al. 2001 Exp Hematol 29:1125-1134).

[0138] HOXB4 used in the generation and proliferation of hemangioblasts in the methods of the present invention includes, but is not limited to, full-length HOXB4 (e.g., the HOXB4 polypeptide identified under public accession numbers GI:13273315, GI:29351568), as well as any functional variants and active fragments thereof. Wild-type HOXB4 protein may be encoded by the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or any other alternative allelic form of that protein. Such sequences may be accessed through publicly available databases such as Genbank. Alternatively, HOXB4 may be expressed ectopically in cells or provided in the culture medium. Ectopically expressed HOXB4 may be operably linked to an inducing promoter. HOXB4 provided in the culture medium may be secreted by other cell types (e.g., feeder layers) or added directly to the culture medium.

[0139] The present invention also relates to fusion proteins comprising HOXB4, including fusion proteins comprising full length HOXB4 or functional variants of HOXB4 or active fragments of HOXB4. In addition to HOXB4, the fusion proteins may also comprise any additional protein, protein domain or peptide. In certain embodiments, HOXB4 may be linked to a protein transduction domain (PTD) such that the protein is translocated from the medium into the cell and then to the nuclear compartment. The fusion proteins may or may not include one or more linker sequences located between the protein domains.

[0140] Functional variants of HOXB4 include mutational and allelic variants of HOXB4 and active fragments thereof. Functional variants of HOXB4 include any HOXB4 polypeptide and active fragments thereof that are capable of proliferating hemangioblasts by the methods of the present invention. HOXB4 functional variants also include HOXB4 polypeptides that have greater transcriptional activity compared to native HOXB4 proteins. HOXB4 variants include proteins in which one or more amino acid substitutions, additions and / or deletions are present relative to wild-type HOXB4. HOXB4 variants also include, but are not limited to, polypeptides that are 75% or more similar to the sequence provided in SEQ ID NO:1 or SEQ ID NO:3. Thus, HOXB4 variants include polypeptides that are 80%, 85%, 90%, 95% and 99% similar to the amino acid sequence provided in SEQ ID NO:1 or SEQ ID NO:3.

[0141] HOXB4 variants also include polypeptides encoded by nucleic acid sequences that are 80% or more identical to the nucleic acid sequence encoding its complement [e.g., a wild-type HOXB4 protein can be encoded by the nucleic acid sequence of SEQ ID NO:2 (GI:85376187) or SEQ ID NO:4 (GI:29351567)]. Thus, HOXB4 variants include HOXB4 polypeptides encoded by nucleic acid sequences that are 85%, 90%, 95% and 99% identical to the sequence provided in SEQ ID NO:2 or SEQ ID NO:4, or their complements.

[0142] Nucleic acid sequences encoding HOXB4 include, but are not limited to, any nucleic acid sequence that binds under stringent conditions with the nucleic acid sequence of SEQ ID NO: 2 or 4, or its complement or fragments. Similarly, nucleic acids that vary from the nucleic acids set forth in SEQ ID NO: 2 or 4 due to the degeneracy of the genetic code are also included within the scope of the present invention. HOXB4 variant polypeptides also include splice variants or other naturally occurring HOXB4 proteins or nucleic acid sequences.

[0143] Active fragments of HOXB4 include, but are not limited to, any fragment of the full-length HOXB4 polypeptide that can maintain hemangioblasts by the methods of the present invention. Thus, in one embodiment, the HOXB4 protein of the present invention is a HOXB4 protein lacking a portion of the N-terminus, for example, the N-terminal 31, 32, or 33 amino acids of full-length HOXB4. Any of the HOXB4 proteins can be fused with additional proteins or protein domains. For example, HOXB4 can be linked to a protein transduction domain (PTD).

[0144] Protein transduction domains, covalently or non-covalently linked to HOXB4, may enable HOXB4 to translocate across the cell membrane, allowing the protein to ultimately reach the nuclear compartment of the cell.

[0145] The PTD fused to the HOXB4 protein includes the PTD of the HIV transcriptional activator protein (TAT) (Tat47-57) (Schwarze and Dowdy 2000 Trends Pharmacol. Sci. 21:45-48; Krosl et al. 2003 Nature Medicine (9):1428-1432). In the HIV TAT protein, the amino acid sequence that confers membrane translocation activity corresponds to residues 47-57 (YGRKKRRQRRR, SEQ ID NO:5) (Ho et al., 2001, Cancer Research 61:473-477; Vives et al., 1997, J. Biol. Chem. 272:16010-16017). This sequence alone confers protein translocation activity. The TAT PTD also has the nine amino acid peptide sequence RKKRRQRRR (SEQ ID NO:6) (Park et al. MoI Cells 2002(30):202-8). The TAT PTD sequence can be any of the peptide sequences disclosed in Ho et al., 2001, Cancer Research 61:473-477, the disclosure of which is incorporated herein by reference, including YARKARRQARR (SEQ ID NO:7), YARAAARQARA (SEQ ID NO:8), YARAARRARARR (SEQ ID NO:9), and RARAARRAARA (SEQ ID NO:10).

[0146] Other proteins containing a PTD that can be fused to the HOXB4 protein of the present invention include the Herpes Simplex Virus 1 (HSV-I) DNA binding protein VP22 and the Drosophila Antennapedia (Antp) homeotic transcription factor (Schwarze et al. 2000 Trends Cell Biol. (10):290-295). For Antp, amino acids 43-58 (RQIKIWFQNRRMKWKK, SEQ ID NO:11) represent the protein transduction domain, and for HSV VP22 the PTD is represented by residues DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:12). Alternatively, an artificial peptide that confers transduction activity or HeptaARG (RRRRRRR, SEQ ID NO:13) can be used as the PTD of the present invention.

[0147] In additional embodiments, the PTD peptide can be a duplexed or multiplexed PTD peptide. In certain embodiments, the PTD is one or more of the TAT PTD peptide YARAAARQARA (SEQ ID NO: 14). In certain embodiments, the PTD is a multimer made up of three of the TAT PTD peptides YARAAARQARA (SEQ ID NO: 15). HOXB4 proteins fused or conjugated to multimeric PTDs, such as triplicate synthetic protein transduction domains (tPTDs), can exhibit reduced instability and increased stability in cells. Such HOXB4 constructs can also be stable in the presence of hES cells and in serum-free medium.

[0148] Techniques for preparing fusion genes encoding fusion proteins are known in the art. The ligation of various DNA fragments encoding substantially different polypeptide sequences can be performed by conventional techniques. In other embodiments, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that induce complementary extensions between two consecutive gene fragments that are subsequently annealed to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

[0149] In certain embodiments, a fusion gene encoding a purification leader sequence, such as a poly-(His) sequence, is attached to the N-terminus of a portion of a HOXB4 polypeptide or HOXB4-fusion protein to produce a Ni 2+ The fusion protein can be purified by affinity chromatography using a metal resin, and the purification leader sequence can then be removed by treatment with enterokinase to provide a purified HOXB4 polypeptide (see, e.g., Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).

[0150] In certain embodiments, the HOXB4 protein or a functional variant or active domain thereof is linked to the C-terminus or N-terminus of a second protein or protein domain (e.g., PDT) with or without an intervening linker sequence. The exact length and sequence of the linker and its orientation relative to the linked sequences can vary. The linker can contain, for example, 2, 10, 20, 30 or more amino acids and can be selected based on certain properties such as solubility, length, steric separation, etc. In certain embodiments, the linker can contain a functional sequence useful, for example, for purification, detection, or modification of the fusion protein. In certain embodiments, the linker comprises a polypeptide of two or more glycosines.

[0151] The protein domains and / or the linkers to which the domains are fused may be modified to alter the efficiency, stability and / or functional properties of HOXB4.

[0152] In certain embodiments, HOXB4 is ectopically expressed in the hemangioblast cells or provided in the culture medium. The ectopically expressed HOXB4 is operably linked to a regulatory sequence, which is recognized in the art and selected to direct expression of the HOXB4 polypeptide.

[0153] HOXB4 provided in the medium can be secreted by other cell types. Other cell types can be feeder layers, such as mouse stromal cell layers, that have been transduced to express secretable HOXB4. For example, HOXB4 can be fused or engineered to contain a hydrophobic sequence or a single peptide that promotes efflux and secretion of the protein. Alternatively, HOXB4 as a fusion protein covalently or non-covalently linked to a PTD can be added directly to the medium. Additionally, HOXB4 can be borne on a viral vector, such as a retroviral vector or an adenoviral vector. Such vectors can deliver hemangioblasts or other cells in the culture.

[0154] In certain embodiments, HOXB4 is added to the culture medium at selected times during the growth of the hemangioblasts, depending on the HOXB4 protein used. Because the hemangioblasts are grown in serum-free medium, HOXB4 is relatively stable. Thus, in certain embodiments, HOXB4 protein or fusion protein is added to the human hemangioblasts daily. In other embodiments, HOXB4 protein or fusion protein is added every other day, and in other embodiments, HOXB4 protein or fusion protein is added every two days. In one embodiment, the HOXB4 fusion protein, HOXB4-PTD, is added to the culture medium every two days.

[0155] In certain embodiments, the hemangioblasts may be grown in the presence of any other growth factors or proteins that are sufficient for the proliferation of such cells.

[0156] Hemangioblasts obtained from any source, including human or non-human ES cells, bone marrow, placenta or umbilical cord blood, peripheral blood, or other tissues, can be expanded by the method. Thus, in certain embodiments, a selected number of purified hemangioblasts or supplemented cells are mixed with a serum-free methylcellulose medium optimized for hemangioblast growth (e.g., BL-CFU medium, see Examples 1 and 2). This medium can be supplemented with early stage cytokines (including but not limited to EPO, TPO, FL, VGF, BMPs like BMP2, BMP4, and BMP7, but excluding BMP3) and HOXB4. In certain embodiments, erythropoietin (EPO) is added to the medium. In certain embodiments, EPO, TPO, and FL are added to the medium. The cells are then plated in ultra-low attachment culture plates and grown in a CO2 incubator. As discussed above, hemangioblast colonies exhibit a distinctive grape-like appearance and are relatively smaller than other cells, and thus can be distinguished from other cell types. Hemangioblasts may be tested for markers as well as their ability to further differentiate into hematopoietic or endothelial lineages. The hemangioblasts are then isolated and expanded in vitro. The medium used for expansion includes serum-free methylcellulose medium optimized for hemangioblast growth (e.g., BL-CFU) supplemented with early stage cytokines and HOXB4. Early stage cytokines include, but are not limited to, EPO, TPO, FL, VEGF, BMPs similar to BMP2, BMP4, and BMP7, but excluding BMP3. In certain embodiments, erythropoietin (EPO) is added to the medium. In additional embodiments, EPO, TPO, and FL are added to the medium.

[0157] Thus, the medium for hemangioblast growth includes VEGF, SCF, EPO, BMP-4, and HoxB4, and in certain embodiments, the medium may further include TPO and FL. Single cells produced from EBs cultured for approximately 3.5 days were collected, dissociated in 0.05% trypsin-0.53 mM EDTA (Invitrogen) for 2-5 min, and passed 3-5 times through a 22G needle to produce a single cell suspension. Cells were collected by centrifugation at 1,000 rpm for 5 min. The cell pellet was resuspended in 50-200 μl of Stemline I medium. To grow hemangioblasts, 2-5×10 5 Single cell suspensions derived from differentiation of hES cells were mixed with 2 ml of hemangioblast growth medium (HGM) containing 1.0% methylcellulose, 1-2% bovine serum albumin, 0.1 mM 2-mercaptoethanol, 10 μg / ml rh-insulin, 200 μg / ml iron-saturated human transferrin, 20 ng / ml rh-GM-CSF, 20 ng / ml rh-IL-3, 20 ng / ml rh-IL-6, 20 ng / ml rh-G-CSF, 3-6 units / ml rh-Epo, 50 ng / ml rh-SCF, 50 ng / ml rh-VEGF, 50 ng / ml rh-BMP-4, and 1.5 μg / ml tPTD-HoxB4 in Isocve's MDM with or without 50 ng / μl Tpo and FL. The cell mixture was plated onto ultra-low plates and incubated at 37° C. in 5% CO 2 for 4 to 6 days.

[0158] In certain circumstances, it may be desirable to obtain hemangioblasts from a patient or a patient relative and expand the hemangioblasts in vitro. Such circumstances include, for example, patients scheduled for chemotherapy or radiation therapy, or other circumstances in which autologous HSC transplantation (utilizing the patient's own stem cells) may be utilized. Accordingly, the present invention provides a method of treating a patient in need of cell-based therapy (e.g., a patient in need of hematopoietic reconstitution or treatment, or treatment of vascular injury, including ischemia, or vascular growth, see below) using the expanded hemangioblasts or hemangioblast lineage cells of the invention, where the hemangioblasts are obtained from the bone marrow, blood or other tissue of the patient or patient relative. Thus, in certain embodiments, a method of treating a patient in need of hemangioblasts (or hemangioblast lineage cells) may include the step of isolating the hemangioblasts from the patient or patient relative. The hemangioblasts isolated from the patient or patient relative may be expanded in vitro by the methods of the present invention and then administered to the patient. Alternatively, the expanded hemangioblasts may be further grown to generate hematopoietic or endothelial cells prior to treatment of the patient.

[0159] Human ES cells can also be obtained from such patients by any method known in the art, such as somatic cell nuclear transfer. The methods of the invention can then be used to generate hemangioblasts from the patient and propagate them from the patient's own ES cells. These hemangioblasts, or their lineage derivatives, can be administered to the patient or to the patient's relatives.

[0160] Using the methods of the present invention, human hemangioblasts can be expanded and then brought to commercial mass production for use in a variety of therapeutic and clinical applications, and hemangioblasts obtained by the methods disclosed herein can be further differentiated to generate hematopoietic or endothelial cell lineages for clinical use.

[0161] The hemangioblasts obtained from the methods of the present invention for generating and expanding human hemangioblasts from human ES cells have the potential to differentiate into at least endothelial or hematopoietic cells (i.e., they are at least bipotential). Other hemangioblasts may also be bipotential. However, other hemangioblasts may also be capable of differentiating into cells other than hematopoietic and endothelial cells (i.e., they are mixed or multipotential). Engineering MHC genes in human embryonic stem cells to obtain hemangioblasts with reduced complexity Human embryonic stem cells used as a starting point in the method of generating and expanding human hemangioblasts according to the invention can also be derived from a library of human embryonic stem cells, each of which is hemizygous or homozygous for one or more MHC alleles present in the human population. In certain embodiments, each member of the stem cell library is hemizygous or homozygous for a different set of MHC alleles compared to the remaining members of the library. In certain embodiments, the library of stem cells is hemizygous or homozygous for every MHC allele present in the human population. In the present invention, stem cells that are homozygous for one or more histocompatibility antigen genes include cells that are null for one or more (and in some embodiments, all) such genes. Null for a locus means that the gene is zero at that locus, i.e., both alleles of that gene have been deleted or inactivated. Stem cells null for every MHC gene can be generated by standard methods known in the art, such as gene targeting and / or loss of heterozygosity (LOH). See, for example, U.S. Patent Applications US 20040091936, US 20030217374 and US 20030232430, and U.S. Provisional Application No. 60 / 729,173, the disclosures of all of which are incorporated herein by reference.

[0162] The present invention thus relates to methods of obtaining hemangioblasts, including libraries of hemangioblasts with reduced MHC complexity. MHC complexity reduced hemangioblasts and hemangioblast lineage cells would increase the supply of cells available for therapeutic use as it eliminates the difficulties associated with patient matching. Such cells can be derived from stem cells engineered to be hemizygous or homozygous for the genes of the MHC complex.

[0163] Human ES cells can contain mutations in one of the sister chromosome alleles in the MHC complex of the cell. Various methods for generating gene mutations, such as gene targeting, can be used to modify the gene in the MHC complex. The modified allele in the MHC complex in the cell can then be engineered to be homozygous, so that the same allele is present on the sister chromosome. Methods such as loss of heterozygosity (LOH) can be used to engineer cells to have homozygous alleles in the MHC complex. For example, one or more genes in one set of MHC genes from a parental allele can be targeted to generate hemizygous cells. The other set of MHC genes can be removed by gene targeting or LOH to create a null cell line. This null cell line can be used again as an embryonic cell line to drop multiple HLA genes, or individual genes, to generate hemizygous or homozygous banks with an otherwise uniform genetic background.

[0164] In one embodiment, a library of ES cell lines, each member of the library being homozygous for one or more HLA genes, is used to derive hemangioblasts by the methods of the invention. In another embodiment, the invention provides a library of hemangioblasts (and / or hemangioblast lineage cells), where several ES cell lines are selected and differentiated into hemangioblasts. Such hemangioblasts and / or hemangioblast lineage cells can be used in patients in need of cell-based therapy.

[0165] Accordingly, certain embodiments of the invention relate to a method of administering reduced complexity human hemangioblasts, hematopoietic stem cells, or human endothelial cells derived from embryonic stem cells to a patient in need thereof. In certain embodiments, the method includes the steps of (a) identifying a patient in need of a treatment comprising administering human hemangioblasts, hematopoietic stem cells, or human endothelial cells, (b) identifying MHC proteins expressed on the cell surface of the patient, (c) providing a library of human hemangioblasts with reduced MHC complexity produced by the in vitro human hemangioblast generation and expansion method of the invention, (d) selecting human hemangioblast cells from the library that match the patient's MHC proteins to the cells, (e) selectively differentiating the human hemangioblast cells identified in step (d) into human hematopoietic stem cells, endothelial cells, or both, or cells further differentiated into one or both of these two lineages, as appropriate, and (f) administering any of the cells from steps (d) and / or (e) to the patient. The method can be performed at community centers, such as hospitals, clinics, doctors' offices, and other health care facilities, and the hemangioblasts selected as a match for the patient can be expanded prior to patient treatment if stored in low cell numbers. Human hemangio-colony forming cells / angioblasts In certain aspects, the present invention provides human hemangio-colony forming cells, which are a unique primitive cell type with a variety of therapeutic and other uses. Additionally, this cell type provides an important tool for studying the evolution of at least the hematopoietic and / or endothelial lineages. Thus, the present invention relates to compositions and various preparations (including pharmaceutical preparations) that comprise human hemangio-colony forming cells, as well as compositions and preparations (including pharmaceutical preparations) that comprise one or more cell types partially or periodically differentiated from hemangio-colony forming cells.

[0166] The terms "hemangioblast" and "hemangio-colony forming cells" will be used interchangeably throughout this application. These cells may be described based on a number of structural and functional characteristics, including, but not limited to, the expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers. Hemangio-colony forming cells are capable of differentiating to give rise to at least hematopoietic cell types or endothelial cell types. Hemangio-colony forming cells are preferably bipotential and capable of differentiating to give rise to at least hematopoietic cell types and endothelial cell types. Thus, the hemangio-colony forming cells of the present invention are at least unipotential, and preferably bipotential. Additionally, hemangio-colony forming cells may have an even greater level of evolutionary potential and, in certain embodiments, may differentiate to give rise to cell types of other lineages. In certain embodiments, the hemangio-colony forming cells may differentiate to give rise to other mesodermal derivatives, such as cardiac cells (e.g., cardiomyocytes) and / or smooth muscle cells.

[0167] Hemangio-colony forming cells may also be identified or characterized based on their structural properties. Specifically, and in certain embodiments, these cells are unique in that they are only loosely attached to one another (loosely attached to other hemangio-colony forming cells). Because these cells are only loosely attached to one another, cultures or colonies of hemangio-colony forming cells may be dissociated into single cells using only mechanical dissociation techniques and without the need for enzymatic dissociation techniques. Mechanical dissociation alone, rather than enzymatic dissociation or a combination of mechanical and enzymatic dissociation, is sufficient to disaggregate the cultures or colonies without substantially impairing the viability of the cells. That is, mechanical dissociation does not require as much force to cause substantial cell damage or death when compared to that observed following enzymatic dissociation of cell aggregates.

[0168] Hemangio-colony forming cells may also be identified or characterized based on the expression or lack of expression (assessed by gene or protein levels) of one or more markers. For example, in certain embodiments, hemangio-colony forming cells may be identified or characterized based on the lack of expression of one or more of the following cell surface markers: CD34, KDR, CD133, or CD31 (e.g., the cells may be characterized based on the lack of expression of one or more, two or more, three or more, or four or more of the markers). Additionally or alternatively, hemangio-colony forming cells may be identified or characterized based on expression of GATA2 and / or LMO2. Additionally or alternatively, hemangio-colony forming cells may be identified or characterized based on the expression or lack of expression (assessed by gene or protein levels) of one or more markers analyzed in Table 2.

[0169] The hemangio-colony forming cells of the present invention may be identified or characterized based on one or any combination of such structural or functional properties. Although these cells may be derived from any of a number of sources, e.g., embryonic, fetal or perinatal tissue, the term "hemangio-colony forming cells" applies to cells, regardless of source, that are capable of differentiating to give rise to at least hematopoietic and / or endothelial cell types and that possess one or more of the aforementioned structural or functional properties.

[0170] In the following, human hemangio-colony forming cells of the invention have one or more of the following structural characteristics: (a) are capable of differentiating to give rise to at least hematopoietic or endothelial cell types, (b) are capable of differentiating to give rise to at least hematopoietic and endothelial cell types, (c) are loosely adherent to each other (to other human hemangio-colony forming cells), (d) do not express CD34 protein, (e) do not express CD31 protein, (f) do not express KDR protein, (g) do not express CD133 protein, (h) express GATA2 protein, (i) express LMO2 protein. In certain embodiments, human hemangio-colony forming cells have two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more of the structural or functional characteristics described herein.

[0171] The present invention provides human hemangio-colony forming cells. Such cells are capable of differentiating to generate at least hematopoietic and / or endothelial cell types. In certain embodiments, the cells are characterized as being loosely adherent to other human hemangio-colony forming cells. Alternatively or additionally, the cells may also be described based on the expression or lack of expression of certain markers. For example, the cells may also be described based on the lack of expression of one or more of the following proteins: CD34, KDR, CD133, and CD31.

[0172] As mentioned above, one of the interesting properties of human hemangio-colony forming cells is that they are loosely attached to one another. Because these cells are only loosely attached to one another, cultures or colonies of hemangio-colony forming cells can be dissociated into single cells simply by mechanical dissociation techniques, without the need for enzymatic dissociation techniques. The cells are sufficiently loosely attached to one another that mechanical dissociation alone, rather than enzymatic dissociation or a combination thereof, is sufficient to disintegrate the cultures or colonies without substantially damaging the viability of the cells. That is, mechanical dissociation does not require so much force as to cause cell damage or death.

[0173] This property is not only useful for engineering the cells to distinguish them phenotypically from other cell types, but also has important therapeutic implications. 6 Bigger or even 1x10 7 Larger or more 1x1O 8 Large numbers of hemangio-colony forming cells (larger than 100 μg / ml) can be infused into humans or other animals with substantially less risk of causing blood clots or embolisms, or otherwise lodgement in the lungs. This is an important advance in cellular therapy. The ability to safely administer relatively large numbers of cells makes cellular therapy practical and feasible for the effective treatment of an increasing number of diseases and conditions.

[0174] The term "loosely attached" is described qualitatively above and refers to the behavior of human hemangio-colony forming cells relative to one another. Cultures or colonies of hemangio-colony forming cells can be dissociated into single cells simply utilizing mechanical dissociation techniques and without the need for enzymatic dissociation techniques. The cells are sufficiently loosely attached to one another that mechanical dissociation alone, rather than enzymatic dissociation or a combination thereof, is sufficient to disintegrate the culture or colony without substantially impairing the viability of the cells. That is, mechanical dissociation does not require so much force as to cause cell damage or death.

[0175] The terms may also be more quantitatively described. For example, and in certain embodiments, the term "loosely attached" is used to refer to cultures or colonies of hemangio-colony forming cells in which 50% or more of the cells in the culture can be dissociated into single cells using simply mechanical dissociation techniques and without the need for enzymatic dissociation techniques. In other embodiments, the terms refer to cultures in which 60%, 65%, 70% or more of the cells in the culture can be dissociated into single cells using simply mechanical dissociation techniques and without the need for enzymatic dissociation techniques. In yet other embodiments, the terms refer to cultures in which 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more or even 100% of the cells in the culture can be dissociated into single cells using simply mechanical dissociation techniques and without the need for enzymatic dissociation techniques.

[0176] The ability to dissociate the hemangio-colony forming cells using solely mechanical dissociation techniques, without the need for enzymatic dissociation techniques, can also be assessed based on the health and viability of the cells upon mechanical dissociation. That is, the cells are not loosely attached as defined herein if dissociation without enzymatic dissociation requires so much mechanical force that it would damage or kill a significant number of the cells. For example, and in certain embodiments, the term "loosely attached" refers to a culture of cells that can be dissociated into single cells using solely mechanical dissociation techniques, without the need for enzymatic dissociation techniques, without substantial damage to the health or viability of the cells, when compared to the same cells dissociated using enzymatic dissociation techniques. For example, the health or viability of the cells is reduced by less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or even less than 1% when compared to that observed when the same culture of cells is dissociated using enzymatic dissociation techniques.

[0177] Exemplary enzymatic dissociation techniques include, but are not limited to, treatment with trypsin, collagenase, or other enzymes that disrupt cell-cell or cell-matrix interactions. Exemplary mechanical dissociation techniques include, but are not limited to, one or more passes through a pipette.

[0178] Human hemangio-colony forming cells according to the present invention are structurally and functionally defined. Such cells may be generated from any of a number of sources, including embryonic, fetal, perinatal, and even adult tissues. By way of example, human hemangio-colony forming cells may be generated from human embryonic stem cells, other embryo-derived cells (blastocysts, blastomeres, ICM, embryos, trophoblast / trophoblast cells, trophoblast stem cells, primordial germ cells, embryonic germ cells, etc.), amniotic fluid, amniotic stem cells, placenta, placental stem cells, and umbilical cord.

[0179] The present invention provides human hemangio-colony forming cells, compositions comprising human hemangio-colony forming cells, and preparations (including pharmaceutical preparations) comprising human hemangio-colony forming cells. Particular features of such aspects of the invention are described in detail below. The present invention includes any combination of the following aspects and embodiments of the invention.

[0180] In one aspect, the invention provides human hemangio-colony forming cells, which are capable of differentiating to generate at least hematopoietic and / or endothelial cell types. In certain embodiments, the cells are loosely adherent to other human hemangio-colony forming cells. In certain embodiments, the cells do not express CD34 protein. In other particular embodiments, the cells do not express one or more of the following proteins (e.g., the cells do not express one or more, two or more, three or more, or four or more of the following proteins): CD34, CD31, CD133, KDR. In other particular embodiments, the cells express GATA2 and / or LMO2 protein. In other particular embodiments, the cells do or do not express one or more (e.g., at least one, two, three, four, five, six, seven, eight, nine, ten, etc.) of the markers set forth in Table 2. In other particular embodiments, the cells have an expression profile as set forth in Table 2.

[0181] In another aspect, the invention provides human hemangio-colony forming cells, which are capable of differentiating to generate at least hematopoietic and / or endothelial cell types, and which do not express any of the following proteins: CD34, CD31, KDR, and CD133. In particular embodiments, the cells are loosely adherent to other human hemangio-colony forming cells. In other embodiments, the cells express GATA2 and / or LMO2 proteins. In other particular embodiments, the cells express or do not express one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the markers set forth in Table 2. In other particular embodiments, the cells have an expression profile as set forth in Table 2.

[0182] In yet another aspect, the invention provides a cell culture comprising a substantially purified population of human hemangio-colony forming cells, the cells being capable of differentiating to produce at least hematopoietic and endothelial cell types, and the cells being loosely adherent to one another. In certain embodiments, the cells do not express CD34 protein. In other particular embodiments, the cells do not express one or more of the following proteins (e.g., the cells do not express one or more, two or more, three or more, or four or more of the following proteins): CD34, CD31, CD133, KDR. In other particular embodiments, the cells express GATA2 and / or LMO2. In other particular embodiments, the cells do or do not express one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) of the markers set forth in Table 2. In other particular embodiments, the cells have an expression profile as set forth in Table 2.

[0183] In yet another aspect, the invention provides cell cultures comprising human hemangio-colony forming cells differentiated from embryonic tissue. In certain embodiments, the hemangio-colony forming cells are loosely adherent to one another. In certain embodiments, the cells are capable of differentiating to produce at least hematopoietic and / or endothelial cell types and the cells are loosely adherent to one another. In certain embodiments, the cells do not express CD34 protein. In certain other embodiments, the cells do not express one or more of the following proteins (e.g., the cells do not express one or more, two or more, three or more, or four or more of the following proteins): CD34, CD31, CD133, KDR. In certain other embodiments, the cells express GATA2 and / or LMO2 protein. In certain other embodiments, the cells do or do not express one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) of the markers set forth in Table 2. In another particular embodiment, the cells have an expression profile as set forth in Table 2.

[0184] In yet another aspect, the invention provides cell cultures comprising human hemangio-colony forming cells, which are capable of differentiating to generate at least hematopoietic and / or endothelial cell types. In certain embodiments, the cells are loosely adherent to one another. In certain embodiments, the cells do not express CD34 protein. In other particular embodiments, the cells do not express one or more of the following proteins (e.g., the cells do not express one or more, two or more, three or more, or four or more of the following proteins): CD34, CD31, CD133, KDR. In other particular embodiments, the cells express GATA2 and / or LMO2 protein. In other particular embodiments, the cells express or do not express one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the markers set forth in Table 2. In other particular embodiments, the cells have an expression profile as set forth in Table 2.

[0185] In yet another aspect, the invention provides a pharmaceutical formulation comprising human hemangio-colony forming cells, which are capable of differentiating to generate at least hematopoietic and / or endothelial cell types. In certain embodiments, the hemangio-colony forming cells are loosely adherent to one another. In certain embodiments, the cells do not express CD34 protein. In other particular embodiments, the cells do not express one or more of the following proteins (e.g., the cells do not express one or more, two or more, three or more, or four or more of the following proteins): CD34, CD31, CD133, KDR. In other particular embodiments, the cells express GATA2 and / or LMO2 protein. In other particular embodiments, the cells express or do not express one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) of the markers set forth in Table 2. In other particular embodiments, the cells have an expression profile as set forth in Table 2. The pharmaceutical formulation may be prepared using any pharma- ceutically acceptable carrier or excipient.

[0186] In yet another aspect, the invention provides a pharmaceutical formulation comprising human hemangio-colony forming cells, the hemangio-colony forming cells do not express any of the following proteins: CD34, CD31, KDR, and CD133. In certain embodiments, the hemangio-colony forming cells are capable of differentiating to generate at least hematopoietic and / or endothelial cell types. In certain embodiments, the hemangio-colony forming cells are loosely adherent to one another. In other particular embodiments, the cells express GATA2 and / or LMO2 proteins. In other particular embodiments, the cells express or do not express one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the markers set forth in Table 2. In other particular embodiments, the cells have an expression profile as set forth in Table 2. The pharmaceutical formulation can be prepared using any pharma- ceutical acceptable carrier or excipient.

[0187] In certain embodiments of the above, the composition or pharmaceutical formulation comprises 1×10 5 In certain other embodiments of the above, the composition or pharmaceutical preparation comprises 1×10 6 That's it, 5×1O 6 That's it, 1×1O 7 or more, or 1×1O 7 Contains excess human hemangio-colony forming cells.

[0188] Additional cells, compositions, and preparations include cells that are partially or terminally differentiated from human hemangio-colony forming cells. For example, the invention includes compositions and preparations that include one or more hematopoietic and / or endothelial cell types differentiated from hemangio-colony forming cells. Exemplary hematopoietic cell types include hematopoietic stem cells, platelets, RBCs, lymphocytes, megakaryocytes, etc. As an additional example, the invention includes compositions and preparations that include one or more other cell types, e.g., one or more partially or terminally differentiated mesodermal cell types, differentiated from hemangio-colony forming cells.

[0189] In certain embodiments of the foregoing, the present invention provides cryopreserved preparations of human hemangiocolony cells or partially or terminally differentiated cells therefrom.

[0190] In certain of the foregoing embodiments, the present invention provides therapeutic uses of human hemangio-colony forming cells, or compositions or preparations of human hemangio-colony forming cells. Such cells and preparations can be used in the treatment of any condition or disease in the blood banking industry, not just those described throughout the specification. Furthermore, cells differentiated from human hemangio-colony forming cells, or compositions or preparations of human hemangio-colony forming cells, can be used therapeutically in the treatment of any condition or disease in the blood banking industry, not just those described throughout the specification.

[0191] The human hemangio-colony forming cells of the present invention can be used therapeutically. Additionally or alternatively, the human hemangio-colony forming cells can be used to study differentiation of endothelial and hematopoietic lineages or in diagnostic tests, for example, to identify factors that can be used to (i) maintain the human hemangio-colony forming cells or (ii) promote differentiation of the human hemangio-colony forming cells into one or more partially or terminally differentiated cell types. Furthermore, the human hemangio-colony forming cells can be used to generate one or more partially or terminally differentiated cell types for use in the laboratory or in vivo.

[0192] The human hemangio-colony forming cells of the present invention can be used in any of the methods or applications described herein, including, but not limited to, in the treatment of any of the diseases or conditions described herein. Cell preparations containing hemangioblasts grown in the laboratory In certain embodiments of the invention, mammalian (including human) hemangioblast cells are expanded to commercial quantities for use in a variety of therapeutic and clinical applications. In certain embodiments, the hemangioblasts are expanded to reach cell numbers on the order of 10,000 to 4 million (or more). Such cell numbers can be reached within 3 to 4 days of starting the initial preparation. Thus, the invention relates to preparations containing large amounts of hemangioblast cells, the preparations comprising at least 10,000, 50,000, 100,000, 500,000, one million, 2 million, 3 million, or 4 million cells.

[0193] The invention also relates to solutions, compositions, and preparations comprising large numbers of hemangioblasts, the solutions, compositions, and preparations comprising at least 10,000, 50,000, 100,000, 500,000, one million, 2 million, 3 million, or 4 million cells. The hemangioblasts can be human.

[0194] Another aspect of the invention relates to differentiating hemangioblasts obtained by the methods disclosed herein into hematopoietic or endothelial cell lineages, or both lineages, for subsequent use in clinical applications. Thus, the invention also relates to cell preparations comprising large amounts of hematopoietic or endothelial cells. The invention also relates to differentiating hemangioblasts obtained by the methods disclosed herein into other cell lineages besides hematopoietic and endothelial cells. Thus, the invention also relates to cell preparations comprising large amounts of other hemangioblast-differentiated cells.

[0195] Compositions and preparations comprising large numbers (e.g., tens of millions or millions) of hemangioblasts can be obtained by expanding hemangioblasts obtained as described herein. Accordingly, the present invention relates to compositions and preparations comprising large numbers of hemangioblasts obtained by expanding ES cells (e.g., human ES cells) or hemangioblasts obtained from umbilical cord blood, peripheral blood, or bone marrow blood. Further expansion methods are utilized, for example, for mouse, rat, bovine, or non-human primate hemangioblasts. The present invention also relates to compositions and preparations comprising large numbers of hemangioblasts from species other than humans. Hemangioblasts expanded by the methods of the present invention can be bi-potential; that is, the cells can be differentiated into endothelial cells or hematopoietic stem cells. In certain embodiments, human hemangioblasts generated and expanded from human ES cells are bi-potential. Hemangio-colony forming cells can be differentiated to provide at least hematopoietic cell types or endothelial cell types. Hemangio-colony forming cells are preferably bi-potential and can be differentiated to provide at least hematopoietic and endothelial cell types. In this function, the hemangio-colony forming cells of the present invention are at least uni-potential, preferably bi-potential. In addition, however, hemangio-colony forming cells can have a high degree of developmental potential and, in certain embodiments, can be differentiated to provide cell types of other lineages. In certain embodiments, hemangio-colony forming cells can be differentiated to provide other mesodermal differentiated bodies, for example, cardiac cells (e.g., cardiomyocytes) and / or smooth muscle cells. Mammalian hemangioblast cell marker As discussed above, hemangio-colony forming cells lack certain properties characteristic of adult endothelial or hematopoietic cells. However, such hemangio-colony forming cells or hemangioblasts may be identified by a variety of markers, such as, for example, CD71+, GATA-1 and GATA-2 proteins, CXCR-4, and receptors for TPO and EPO. In additional embodiments, hemangioblasts express LMO-2. Hemangioblasts may further be characterized by the absence or low expression of other markers. Thus, hemangioblasts may be CD34-CD31-, and KDR-. In additional embodiments, hemangioblasts may be CD34-, CD31-, KDR-, and CD133-.

[0196] Thus, in certain embodiments, hemangioblasts generated and expanded by the methods of the invention are characterized by the presence or absence of any one or more markers set forth in Table 2. For example, the hemangioblasts may test negative for expression of any one or more markers set forth in Table 2 designated with a "-" under "BL-CFC." Thus, in some embodiments, the hemangioblasts may be negative for CD34 expression. The cells may additionally or alternatively be negative for CD31, CD133, and / or KDR expression. In additional embodiments, the hemangioblasts may express any marker designated in Table 2 with a "+." For example, the cells may express one or more of the markers: LMO-2 and GATA-2. Marker expression may be assayed by any method, such as, for example, immunohistochemistry or immunoblotting to examine protein expression, or mRNA analysis to examine expression at the RNA level. Differentiation of hemangioblast lineage cells The methods and cell preparations of the present invention also relate to hemangioblast-differentiated cells. Human hemangioblasts generated and expanded according to the present invention, and mammalian hemangioblasts expanded according to the methods of the present invention, can be differentiated in the laboratory to yield not only hematopoietic cells (including hematopoietic stem cells (HSC)) or endothelial cells, but also cells further differentiated along these two lineages. Such cells can be subsequently used in the therapeutic and commercial applications described below.

[0197] In certain embodiments, the hematopoietic cells are derived by growing hemangioblasts in serum-free BL-CFC for 3-10 days. In other embodiments, single cell suspensions of hES-derived BL-CFC cells are grown for 10-14 days. It is most desirable to maintain serum-free conditions, as this facilitates cost reduction as well as compatibility with expansion production and regulatory guidelines. The hemangioblasts of the present invention can also be grown in serum-free Hem-culture (Bhatia et al. 1997 J Exp Med(186):619-624), which maintains human hematopoietic stem cells and contains BSA (e.g., 1% BSA), insulin (e.g., 5 μg / ml human insulin), transferrin medium or transferrin (e.g., 100 μg / ml human transferrin), L-glutamine, beta-mercaptoethanol (e.g., 10 -4 The hemangioblasts may also be grown in serum-free methylcellulose medium supplemented with a multilineage hematopoietic growth factor cocktail. Growth factors may include SCF (e.g., 300 ng / ml), granulocyte-colony-stimulating factor (G-CSF) (e.g., 50 ng / ml), Flt-3 (e.g., 300 ng / ml), IL-3 (e.g., 10 ng / ml), and IL-6 (e.g., 10 ng / ml). Other factors useful for obtaining hematopoietic cells from hemangioblasts include thrombopoietin (TPO) and VEGF (see, e.g., Wang et al. 2005 Ann NY Acad Sex (1044):29-40) and BMP-4. The hemangioblasts may also be grown in serum-free methylcellulose medium supplemented with a multilineage hematopoietic growth factor cocktail. Thus, hemangioblasts can be grown in methylcellulose in Iscove modified Dulbecco medium (IMDM) supplemented with early acting growth factors (e.g., c-kit ligand, flt3 ligand), multilineage growth factors (e.g., IL-3, granulocyte macrophage-CSF (GM-CSF)) and single lineage growth factors (e.g., G-CSF, M-CSF, EPO, TPO), VEGF and bFGF, and containing BSA, saturated human transferrin, human LDL. Alternatively, hemangioblasts can be grown in medium containing single lineage growth factors that support the growth of one type of hematopoietic cell (e.g., erythrocytes, macrophages or granulocytes).

[0198] In one embodiment, hemangioblast colonies are resuspended in Stemline I medium. The cells are then cultured in serum-free hematopoietic CFU medium (H4436, Stem Cell Technologies TM ) 1ml + tPTD-HoxB4 and 0.5% EX-CYTE (Serologicals Proteins Inc. TM ) 1.5 μg / ml. The cell mixture is then plated onto cell culture untreated plates and incubated at 37° C. for 10-14 days. Hematopoietic CFUs arising 10-14 days after initial plating can be characterized morphologically, e.g., by staining with Wright-Giemsa dye.

[0199] Hematopoietic cells can also be derived from hemangioblasts using other conditions known in the art (e.g., IMDM, 30% fetal calf serum (FCS), 1% bovine serum albumin (BSA), 10 -4 In another embodiment, basic fibroblast growth factor is used to increase the frequency of both BL-CFCs in EBs and promote hematopoietic differentiation (Faloon et al. 2000 Development(127):1931-1941). In another embodiment, growth factor hematopoietic (HAPO) is used to promote hemangioblast growth and hematopoietic differentiation (Liu et al. 2004 Blood(103):4449-4456). Hematopoietic differentiation can be assessed, for example, by CD45 status (CD45+) and CFU analysis.

[0200] To form hematopoietic cells, human hemangioblasts can be grown in CFU-medium for 3-10 days, or optionally for longer periods (e.g., 10-24 days). Human hemangioblasts of the invention can form colonies that form units containing only one of the following cell types (e.g., CFU-G, CFU-E, CFU-M, and CFU-GM) as well as CFUs that include granulocytes, erythrocytes, macrophages, and megakaryocytes (CFU-GEMM / mixed). In certain embodiments, single cell suspensions of hES-derived BL-CFC cells are grown for 10-14 days to induce hematopoietic cells such as erythrocytes, myeloid, macrophages, and multilineage hematopoietic cells.

[0201] Yet another aspect of the invention relates to human hemangioblasts obtained and expanded by the methods described herein, or endothelial cells derived from mammalian hemangioblasts expanded thereby, wherein the hemangioblasts can be grown under conditions favorable for epithelial maturation.

[0202] In certain embodiments of the invention, to obtain endothelial cells, hemangioblasts are first plated onto a fibronectin-coated surface and after 3-5 days (or 3-7 days in other embodiments), replated with a thick layer of Matrigel to support differentiation into endothelial cells. These conditions maintain the serum-free conditions established during hemangioblast development. Alternatively, hemangioblasts are grown in a known medium to support differentiation into endothelial cells. Such conditions may be, for example, 20% fetal bovine serum (FBS), 50 ng / ml endothelial cell growth supplement (i.e., pituitary extract), 10 IU / ml heparin, and 5 ng / ml human VEGF-A. 165(Terramani et al. 2000 In Vitro Cell Dev Biol Anim (36):125-132). Other conditions known in the art include medium supplemented with 25% FCS / horse serum, and in some embodiments, medium supplemented with heparin (e.g., 10 U / ml), insulin-like growth factor (IGFl) (e.g., 2 ng), and EC growth supplement (ECGS, e.g., 100 μg). Growth factors VEGF and EGF can also be used with HAPO to support endothelial differentiation (Liu et al. 2004). Hemangioblasts can also be seeded on collagen and fibronectin coated plates to promote differentiation into, for example, endothelial cells. The cells can be assayed for von Willebrand factor (vWF) and endothelial nitric oxide synthase (eNOS) and the ability to form endothelial networks in vitro.

[0203] Therefore, to form endothelial cells, hemangioblast colonies induced by the aforementioned method are picked and replated on fibronectin-coated culture plates optimized for the first step towards endothelial differentiation. The cells are cultured in EGM-2 or EGM-2MV complete medium (Cambrex). TM ) after 3-5 days, or in an alternative embodiment, 3-7 days, the cells are replated onto a surface that supports endothelial differentiation, such as a layer of Matrigel. After 16-24 hours of incubation, typical endothelial cell behavior is confirmed by the formation of branched tube-cords (see, e.g., FIG. 8). Endothelial specific assays, such as LDL-uptake, can also be used to confirm that the cells have endothelial characteristics.

[0204] In another aspect of the invention, the human hemangioblasts generated and expanded according to the invention and the mammalian hemangioblasts expanded by the methods of the invention can be differentiated in vitro to yield not only other cells, but also further differentiated cells from the cell lineages. Such further cell lineages can be derived from the hemangioblasts generated and expanded according to the invention and the mammalian hemangioblasts expanded by the methods of the invention because the hemangioblasts may have a greater degree of developmental potential following differentiation into hematopoietic and endothelial cells.

[0205] Clinical and Commercial Examples of Human Hemangioblasts and Hemangioblast Lineage Cells cell therapy While human hemangioblasts may differentiate in vivo into other hematopoietic or endothelial cells, they may be used in cell therapies where these two cell types are necessary or may enhance therapy. They may also be treated with any therapy or treatment that involves hemangioblast lineage cells (i.e., hematopoietic cells and / or endothelial cells). The following section describes methods of using the human hemangioblasts of the invention, generated and expanded by the methods of the invention, or expanded by the methods of the invention.

[0206] Certain embodiments of the invention contemplate treatments for increasing or treating hematopoietic cells and for increasing blood vessel growth and / or promoting vascular repair. Accordingly, in certain aspects, the invention relates to methods and compositions for treating a patient in need of hematopoietic cells or blood vessel growth or repair. The hemangioblasts can be administered to the subject's blood vessels by injection or via surgery. The patient or subject can be a human.

[0207] In certain embodiments of the invention, human hemangioblasts are used for transplantation, whereas in other cases, HSC transplantation may be used. Such transplantation may be used, for example, for hematopoietic reconstitution for the treatment of patients suffering from acute or chronic leukemia, aplastic anemia, and various immune deficiencies, as well as various non-hematological malignancies and autoimmune disorders, and to rescue patients from treatment-induced hypoplasia following high dose chemotherapy and / or radiation therapy. Such transplantation may be performed in vivo or ex vivo (e.g., bone marrow transplantation).

[0208] In another embodiment of the invention, the human hemangioblasts are used to treat patients in need of hematopoietic reconstitution or therapy, including those suffering from thalassemia, sickle cell anemia, aplastic anemia (also known as hypoplastic anemia), cytopenias, bone marrow hypoplasia, platelet deficiencies, hematopoietic malignancies such as leukemia, paroxysmal nocturnal hematuria (PNH), and ADA (e.g., deaminase (ADA)-deficient severe combined immune deficiency (SCID)).

[0209] Accordingly, a specific embodiment of the present invention relates to a method for treating a patient in need of hematopoietic reconstitution or hematopoietic therapy using the hemangioblasts of the present invention. Accordingly, the present invention relates to a method for treating a patient in need of hematopoietic reconstitution or therapy, comprising: selecting a patient in need thereof; generating and expanding or growing human hemangioblasts according to the methods of the present invention; and administering said human hemangioblasts to the patient. Accordingly, the method comprises differentiating the generated and expanded or grown human hemangioblasts into human hematopoietic cells and then administering the hematopoietic cells to the patient.

[0210] Alternative embodiments include methods for producing human hemangioblasts on a large scale and storing them in advance of a patient in need thereof. Thus, yet another embodiment of the present invention relates to a method for treating a patient in need of hematopoietic reconstitution or therapy, comprising selecting a patient in need thereof, prescribing human hemangioblasts already isolated and expanded by the above-described methods, and administering said human hemangioblasts to said patient. Similarly, said method may comprise differentiating said human hemangioblasts into human hematopoietic cells and administering said hematopoietic cells to the patient. In additional embodiments, hemangioblasts that are hemizygous or homozygous for one or more MHC alleles are grown, optionally grown in commercial quantities, and optionally stored by an enterprise. When a patient requires such cells or hemangioblast lineage cells, a clinician or hospital orders such cells from an enterprise.

[0211] Because the human hemangioblasts of the invention proliferate and differentiate into endothelial cells in an angiogenic microenvironment, the human hemangioblasts can be used in a therapeutic manner to provide new blood vessels at the site of injury in a patient or to induce repair of damaged blood vessels. Thus, in certain aspects, the invention relates to a method of promoting the growth of new blood vessels or repair of damaged vasculature. The human hemangioblasts of the invention can be used to treat endothelial injuries, such as those occurring after myocardial infarction, stroke, and ischemic brain, ischemic limb, and skin wounds, including ischemic limb, and wounds arising from diabetic animals or patients, and ischemic reperfusion injury in the retina. Other ischemic conditions that can be treated with the hemangioblasts of the invention include renal ischemia, pulmonary ischemia, and ischemic cardiomyopathy. The hemangioblasts can also be used to aid in the repair of damaged blood vessels following balloon angioplasty or the development of a cerebrovascular stent. The hemangioblasts can also be used following tissue transplantation, surgery, and radiation injury. Hemangioblasts can also be used to treat and / or prevent the progression of atherosclerosis as well as to repair endothelial cell damage that occurs in systemic sclerosis and Raylord phenomenon (RP) (Blann et al. 1993 J Rheumatol. (20): 1325-30).

[0212] The present invention thus provides a variety of methods related to providing vascular growth and healing in patients in need thereof. In one embodiment, the present invention provides a method of inducing new blood vessel formation in an ischemic tissue in a patient in need thereof, comprising administering to the patient an effective amount of a purified preparation of human hemangioblasts as described above, thereby forming new blood vessels in the ischemic tissue. Accordingly, a particular embodiment of the present invention provides a method of increasing angiogenesis in a patient in need thereof, comprising selecting a patient in need thereof, isolating human hemangioblasts as described above, and administering the hemangioblasts to the patient. In another embodiment, the present invention provides a method of treating damaged blood vessels in a patient in need thereof, comprising selecting a patient in need thereof, expanding or generating and expanding human hemangioblasts as described above, and administering the hemangioblasts to the patient. In addition to the above embodiments, hemangioblasts can be produced on a large scale and stored prior to selecting a patient in need of hemangioblasts. In additional embodiments, hemangioblasts that are hemizygous or homozygous for one or more MHC alleles are grown, optionally grown in commercial quantities, and optionally stored prior to selecting patients for hemangioblast therapy. Any of the above hemangioblasts or hemangioblast preparations can be administered directly (intravenously) into the circulatory system. In certain embodiments (e.g., when vascular restoration is required in the eye, such as for treatment of retinal ischemia / reperfusion injury), the hemangioblasts or hemangioblast preparations can be administered by intravitreal injection.

[0213] The hemangioblast or derivative cell solution or formulation can be administered by any route, and such administration can be determined on an individual basis, and the effective amount of the hemangioblast or derivative cell solution or formulation administered is an amount that is therapeutically effective and can be determined on an individual basis.

[0214] In additional embodiments, hemangioblast lineage cells find therapeutic use, e.g., in the treatment of the aforementioned indications. Thus, hemangioblasts generated and expanded or expanded by the methods described herein can first differentiate in vitro to produce hematopoietic and / or endothelial cells, and then produce cells that are further differentiated in the two lineages. Such cells can then be administered to a subject or patient, e.g., for the treatment of a hematopoietic condition, or for hematopoietic reconstitution, or for the treatment of ischemia or vascular injury.

[0215] HSCs derived from human hemangioblasts obtained by the methods disclosed herein grow to further expand HSCs and / or induce other hematopoietic lineage cell types. Certain aspects of the invention relate to the use of HSCs derived from hemangioblasts during transplantation. In additional embodiments, differentiated hematopoietic cells (e.g., granulocytes, erythrocytes, myeloid cells, megakaryocytes, platelets, macrophages, mast cells, and neutrophil leukocytes (Wiles and Keller 1991 Development(111):259) are used in a variety of therapies, such as transfusion therapy or the treatment of infection. Accordingly, other embodiments of the invention relate to methods of treating a patient in need of hematopoietic reconstitution or therapy using HSCs or hematopoietic lineage cells derived from the hemangioblasts of the invention.

[0216] In some aspects, the invention relates to a method of treating a patient in need of hematopoietic cells or a treatment comprising the steps of selecting a patient in need of treatment, expanding or isolating and expanding human hemangioblasts by the methods of the invention, differentiating the hemangioblasts into hematopoietic stem cells and / or mature hematopoietic cells, and administering the hematopoietic cells to the patient.

[0217] In another aspect of the invention, hemangioblasts are grown to give rise to endothelial cells according to the methods disclosed herein. The endothelium can then be used to induce repair of damaged blood vessels or provide new blood vessels at the site of injury in a patient. Thus, in some aspects, the invention relates to methods of promoting new blood vessel growth or repairing damaged vasculature, in which endothelial cells derived from hemangioblasts are used as therapeutic agents. The endothelial cells can be used to treat endothelial injuries, such as myocardial infarction and pulmonary ischemia, stroke and ischemic brain injury, limb ischemia and skin injuries including limb ischemia, and injuries occurring in patients, even in diabetic animals, intraretinal ischemic reperfusion injury, and renal ischemia. The endothelial cells can also be used to aid in repair of damaged blood vessels following placement of balloon angioplasty or endovascular stents as well as grafts, surgery, and subsequent radiation injury. The endothelial cells can also be used to repair endothelial cell damage occurring in systemic sclerosis and Raynaud's phenomenon, as well as to treat and / or prevent the progression of atherosclerosis.

[0218] The endothelial cells may be further differentiated and these cells may be suitably used to treat, for example, one or more of the "endothelial cell" diseases or conditions listed in the paragraph above.

[0219] Accordingly, one aspect of the present invention relates to a method of treating a patient with endothelial or vascular injury or in need of vascular growth or restoration, comprising the steps of selecting a patient in need of treatment, expanding or isolating and expanding human hemangioblasts by the methods of the present invention, differentiating the hemangioblasts into endothelial cells, and administering the endothelial cells to the patient. Blood Bank Another aspect of the invention provides methods for generating hematopoietic cells suitable for transplantation. Such cells and methods have many uses, but one particularly important use is in improving the compatibility of blood for transplantation. In some preferred embodiments, the invention provides red blood cells differentiated from hemangioblasts / hemangio-colony forming units. Such differentiated red blood cells can be used for transplantation.

[0220] Yet another aspect of the invention relates to a method for generating hematopoietic cells differentiated from hemangioblasts / hemangio-colony forming units for use in transfusions for individuals in need thereof. In some embodiments, the differentiated hematopoietic cells are transplanted to treat trauma, blood loss during surgery, blood disorders such as anemia, sickle cell anemia, or hemolytic disorders, or malignant disorders. In some embodiments, red blood cells are transplanted to treat trauma, blood loss during surgery, or blood disorders such as anemia, sickle cell anemia, or hemolytic disorders. In some embodiments, platelets are transplanted to treat congenital platelet disorders or malignant disorders. In some embodiments, a mixed population of red blood cells and platelets are transplanted.

[0221] It should be noted that many differentiated hematopoietic cell types, particularly red blood cells, generally exist as a mixed population in vivo. In particular, circulating red blood cells of various ages and levels of differentiation are found in vivo. Red blood cells also mature over time, expressing less fetal hemoglobin and more adult hemoglobin. The present invention contemplates transplantation of either a pure population of red blood cells, or a mixed population of red blood cells having various ages and levels of differentiation. In a specific embodiment, the present invention contemplates transplantation of red blood cells expressing fetal hemoglobin (hemoglobin F).

[0222] The present invention provides a method for generating differentiated hematopoietic cells from human hemangio-colony forming cells in vitro, said method comprising the steps of: (a) providing human hemangio-colony forming cells, and (b) differentiating said hemangio-colony forming cells into differentiated hematopoietic cells.

[0223] The present invention also provides a method for performing a blood transfusion using hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, said method comprising the steps of: (a) providing human hemangio-colony forming cells; (b) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells, and (c) performing a transfusion with said differentiated hematopoietic cells.

[0224] The present invention also provides a method for performing a blood transfusion using hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, said method comprising the steps of: (a) culturing a cell culture comprising human embryonic stem cells in a serum-free medium in the presence of one or more growth factors in an amount sufficient to induce differentiation of the embryonic stem cells into embryoid bodies; (b) adding one or more growth factors to said cultures containing embryoid bodies and continuing to culture said cultures in serum-free medium, wherein said growth factors are in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body cultures; (c) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells, and (d) performing a transfusion with said differentiated hematopoietic cells.

[0225] In some embodiments, the stem cells, embryoid bodies and hemangio-colony formation are grown throughout steps (a) and (b) of the method in serum-free medium.

[0226] The present invention also provides a method for performing a blood transfusion using hematopoietic cells differentiated in vitro from human hemangio-colony forming cells, said method comprising the steps of: (a) culturing a cell culture comprising human embryo-derived cells in a serum-free medium in the presence of one or more growth factors in an amount sufficient to induce differentiation of the embryo-derived cells into embryoid bodies; (b) adding one or more growth factors to said cultures containing embryoid bodies and continuing to culture said cultures in serum-free medium, wherein said growth factors are in an amount sufficient to expand human hemangio-colony forming cells in said embryoid body cultures; (c) disaggregating the embryoid bodies into single cells; (d) adding one or more growth factors to said culture comprising said single cell and continuing to culture said culture in serum-free medium, said growth factors being in an amount sufficient to expand human hemangio-colony forming cells in said culture comprising said single cell; (e) differentiating the hemangio-colony forming cells into differentiated hematopoietic cells, and (f) performing a transfusion with said differentiated hematopoietic cells.

[0227] In some embodiments, the embryo-derived cells, embryoid bodies, hemangio-colony forming cells and single cells are grown in serum-free medium throughout steps (a) through (d) of the method.

[0228] In some embodiments, the embryonic derived cells are embryonic stem cells.

[0229] In some embodiments, the growth factor is a protein comprising a homeobox protein, or a functional variant or active fragment thereof, hi some embodiments, the homeobox protein comprises a HOXB4 protein, or a functional variant or active fragment thereof.

[0230] In some embodiments, the differentiated hematopoietic cells are generated as single cell forms, such as red blood cells, platelets, and phagocytes. However, it should be noted that when single cell forms are generated, the cell forms may be heterogeneous in terms of the maturity or differentiation level of the particular cell type. For example, differentiated red blood cells may be heterogeneous in terms of maturity and cell age level. Without being bound by theory, such heterogeneity of red blood cells may be beneficial in order to mimic the way red blood cells are found in vivo.

[0231] In some embodiments, the single cell forms are mixed in the same proportions of differentiated cell forms found in blood. In some embodiments, multiple differentiated hematopoietic cell forms are generated in the same step. In some embodiments, the phagocytes are selected from granular leukocytes, neutrophils, basophils, eosinophils, lymphocytes, or monocytes. In some embodiments, the hematopoietic cell forms are generated in approximately the same proportions of differentiated hematopoietic cell forms found in blood (96% red blood cells, 1% platelets, and 3% phagocytes). In some embodiments, plasma is added to the differentiated hematopoietic stem cells prior to transplantation. In some embodiments, packed cells, e.g., packed red blood cells, are transplanted in the absence or substantial absence of plasma.

[0232] In some embodiments, the differentiated hematopoietic cells produced from the methods of the present application are functional. In some embodiments, the platelets produced from the methods of the present application are functional. In some embodiments, the phagocytes produced from the methods of the present application are functional. In some embodiments, the red blood cells produced from the methods of the present application are functional. In some embodiments, the red blood cells express hemoglobin F prior to transplantation. In some embodiments, the red blood cells transport oxygen. In some embodiments, the red blood cells have the same life span as naturally derived red blood cells. In some embodiments, the red blood cells have a life span that is 75% of the naturally derived red blood cell life span. In some embodiments, the red blood cells have a life span that is 50% of the naturally derived red blood cell life span. In some embodiments, the red blood cells have a life span that is 25% of the naturally derived red blood cell life span.

[0233] In some embodiments, the method of the present application achieves a yield of 1x10 per 100 mm dish. 6 Generate cells. In some examples, 2x10 cells per 100 mm dish. 6 In some cases, cells are produced at 3x10 per 100mm dish. 6 Cells are generated. In some examples, 4x10 cells per 100 mm dish 6 Cells are generated. In some examples, 5x10 cells per 100mm dish 6 Cells are generated. In some examples, 6x10 cells per 100mm dish 6 In some cases, 7x10 cells are produced per 100mm dish. 6 Cells are generated. In some examples, 8x10 cells per 100mm dish are 6 In some cases, 9x10 cells are produced per 100mm dish. 6 Cells are generated. In some embodiments, 1x10 cells are generated per 100 mm dish. 7 Cells are generated. In some examples, 5x10 cells per 100mm dish 7 Cells are generated. In some embodiments, 1x10 cells are generated per 100 mm dish. 8 Cells are generated.

[0234] In some embodiments, the differentiation step is performed using conditions known to those of skill in the art. In some embodiments, the differentiation step is performed using a method specific for differentiating cells into red blood cells (see WO 2005 / 118780, which is incorporated herein by reference). In some embodiments, the differentiation step is performed using a method specific for differentiating cells into platelets. In some embodiments, the differentiation step is performed using a method specific for differentiating cells into white blood cells.

[0235] Differentiation agents that may be used in accordance with the present invention include cytokines such as interferon-alpha A, interferon-alpha A / D, interferon-beta, interferon-gamma, interferon-gamma-inducible protein-10, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-1, interleukin-12, interleukin-13, interleukin-15, interleukin-17, keratinocyte growth factor, leptin, leukemia inhibitory factor, macrophage colony stimulating factor, and macrophage inflammatory protein-1 alpha.

[0236] The differentiation agents according to the invention may also be used to treat or inhibit the proliferation of growth factors, such as 6Ckine (recombinant), activin A, alpha A-interferon, alpha-interferon, amphiregulin, angiogenin, B-endothelial cell growth factor, betacellulin, B-interferon, brain-induced neurotrophic factor, C1O (recombinant), cardiotrophin-1, ciliary neurotrophic factor, cytokine-induced neutrophil chemotactic agent-1, endothelial cell growth supplement, eotaxin, epidermal growth factor, epithelial neutrophil activating peptide-78, erythropoietin. , estrogen receptor-alpha, estrogen receptor-B, fibroblast growth factor (acidic / basic, heparin stabilized, recombinant), FLT-3 / FLK-2 ligand (FLT-3 ligand), gamma-interferon, glial cell line-derived neurotrophic factor, Gly-His-Lys, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, GRO-alpha / MGSA, GRO-B, GRO-gamma, HCC-1, heparin-binding epidermal growth factor-like growth factor, hepatocyte growth factor, heregulin-alpha (EGF domain), insulin growth factor binding protein-1, insulin-like growth factor-binding protein-1 / IGF-1 complex, insulin-like growth factor, insulin-like growth factor II, 2.5S nerve growth factor (NGF), 7S-NGF, gluttony Cellular inflammatory protein-1B, macrophage inflammatory protein-2, macrophage inflammatory protein-3 alpha, macrophage inflammatory protein-3B, monocyte chemotactic protein-1, monocyte chemotactic protein-2, monocyte chemotactic protein-3, neurotrophic factor-3, neurotrophic factor-4, These include NGF-B (human or rat recombinant), oncostatin (human or mouse recombinant), pituitary extract, placenta growth factor, platelet-derived endothelial cell growth factor, platelet-derived growth factor, pleiotrophin, rantes, stem cell factor, stromal cell-derived factor, B / pre-B cell growth stimulating factor, thrombopoietin, transforming growth factor alpha, transforming growth factor-B1, transforming growth factor-B2, transforming growth factor-B3, transforming growth factor-B5, tumor necrosis factor (alpha and B), and vascular endothelial growth factor.

[0237] Differentiation agents according to the present invention include hormones and hormone antagonists, such as 17B-estradiol, adrenocorticotropic hormone, adrenomedullin, alpha-melanocyte-stimulating hormone, chorionic gonadotropin, corticosteroid binding globulin, corticosterone, dexamethasone, estriol, follicle-stimulating hormone, gastrin 1, glucagon, gonadotropins, hydrocortisone, insulin, insulin-type growth factor binding protein, L-3,3',5'-triiodothyronine, L-3,3',5-triiodothyronine, leptin, leutinizing hormone, L-thyroxine, melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary growth hormone, progesterone, prolactin, secretin, sex hormone globulin, thyrotropin, thyrotropin releasing factor, thyroxine binding globulin, and vasopressin.

[0238] Differentiation agents according to the present invention also include extracellular matrix components such as fibronectin, proteolytic hematopoietic colony forming cell hydrolyzed fragments of fibronectin, laminin, thrombospondin, aggrecan, and syndecans.

[0239] The differentiation agent according to the present invention may also be an antibody against various factors, such as an anti-low density lipoprotein receptor antibody, an anti-progesterone receptor, an intrabody, an anti-alpha interferon receptor chain 2 antibody, an anti-cc chemokine receptor 1 antibody, an anti-CD118 antibody, an anti-CD 119 antibody, anti-colony stimulating factor-1 antibody, anti-CSF-1 receptor / c-fin antibody, anti-epidermal growth factor (AB-3) antibody, anti-epidermal growth factor receptor antibody, anti-epidermal growth factor receptor, phospho-specific antibody, anti-epidermal growth factor (AB-1) antibody, anti-erythropoietin receptor antibody, anti-estrogen receptor antibody, anti-estrogen receptor, C-terminal antibody, anti-estrogen receptor-B antibody, anti-fibroblast growth factor receptor antibody, anti-fibroblast growth factor, basic antibody, anti-gamma interferon receptor chain antibody, anti-gamma interferon human recombinant antibody, anti-GFR alpha-1 C-terminal antibody, anti-GFR alpha-2 C-terminal antibody, anti-granulocyte colony-stimulating factor (AB-1) antibody, anti-granulocyte colony-stimulating factor receptor antibody, anti-insulin receptor antibody, anti-insulin anti-type growth factor-1 receptor antibody, anti-interleukin-6 human recombinant antibody, anti-interleukin-1 human recombinant antibody, anti-interleukin-2 human recombinant antibody, anti-leptin mouse recombinant antibody, anti-nerve growth factor recombinant antibody, anti-p60 chicken antibody, anti-parathyroid hormone type protein antibody, anti-platelet derived growth factor receptor antibody, anti-platelet derived growth factor receptor-B antibody, anti-platelet derived growth factor-alpha antibody, anti-progesterone receptor antibody, anti-retinoic acid receptor-alpha antibody, anti-thyroid hormone nuclear receptor antibody, anti-thyroid hormone nuclear receptor-alpha 1 / Bi antibody, anti-transferrin receptor / CD71 antibody, anti-transforming growth factor-alpha antibody, anti-transforming growth factor-B3 antibody, anti-tumor necrosis factor-alpha antibody, and anti-vascular endothelial growth factor antibody.

[0240] The invention also provides a library of differentiated hematopoietic cells capable of providing matched cells to potential patient recipients as described above. In a specific embodiment, the cells are stored in a frozen state. Thus, in one embodiment, the invention provides a method of carrying out a pharmaceutical business comprising providing a differentiated hematopoietic cell preparation homozygous for one or more compatible antigens, wherein the cells are selected from a cell bank comprising a library of human hemangio-colony forming cells that can be expanded by the methods disclosed herein, each hemangio-colony forming cell preparation being hemizygous or homozygous for one or more MHC alleles present in a human population, said hemangio-colony forming cell bank comprising cells that are hemizygous or homozygous, respectively, for a different set of MHC alleles relative to the remaining members of the cell bank. As described above, genetic targeting or loss of heterozygosity methods can be used to generate hemizygous or homozygous MHC allele stem cells that are used to derive hemangio-colony forming cells. In a specific embodiment, hemangio-colony forming cells of all blood types are included in the bank. In certain embodiments, the hemangio-colony forming cells are matched to the patient, thereby ensuring the production of differentiated hematopoietic cells of the patient's own blood type. In certain embodiments, the hemangio-colony forming cells are negative for antigenic factors A, B, Rh, or any combination thereof. In certain embodiments, the differentiated hematopoietic cells are universal donor cells. For example, hematopoietic cells that are type O Rh negative can be used universally for blood transfusions. In certain embodiments, the present invention provides methods for producing type O Rh negative red blood cells for universal transfusion.

[0241] In certain embodiments, red blood cells differentiated from hemangio-colony forming cells express fetal hemoglobin. Transfusion of red blood cells expressing fetal hemoglobin may be particularly useful in the treatment of sickle cell anemia. Thus, the present invention provides improved methods for the treatment of sickle cell anemia.

[0242] In one embodiment, after a particular hemangio-colony forming cell preparation is appropriately selected for a patient, it is then expanded and differentiated to reach a quantity suitable for treating the patient, obtaining differentiated hematopoietic cells, and administering the cells to the recipient. The method of conducting a pharmaceutical business may also include establishing a distribution system for distribution of the preparation for sale, or may include establishing a sales group for marketing the pharmaceutical preparation.

[0243] In any of the above, the hemangio-colony forming cells can be immediately differentiated or the hemangio-colony forming cells can be frozen for subsequent use. In certain embodiments, the invention provides methods for freezing and culturing hemangio-colony forming cells suitable for subsequent thawing and expansion, and differentiation into hematopoietic or endothelial lineages.

[0244] Human hemangio-colony forming cells can be used to generate a number of hematopoietic cell types that can be used for transfusion. For example, a number of homogeneous or heterogeneous populations of RBCs and / or platelets can be generated from human hemangio-colony forming cells. Hemangio-colony forming cells and hematopoietic cell types differentiated therefrom can be banked to provide a wide range of donated blood products and for use in transfusions and other therapies. Banking such products would help to alleviate the risk of dangerous storage of donated blood products. Furthermore, hemangio-colony forming cells and derived products can be genetically engineered in vivo to provide a universal donated blood product.

[0245] Thus, in certain embodiments, the present invention provides methods of conducting a blood banking business. Subject banking involves the derivation and storage (long or short term) of hemangio-colony forming cells and / or hematopoietic cell types (e.g., RBCs, platelets, lymphocytes, etc.) generated therefrom. The cells may be cryopreserved for long term storage or maintained in culture for relatively short term storage. The cells may be sorted and cross-matched in a manner very similar to the manner in which currently available blood products are sorted, and the cells may be stored on the basis of type. Additionally, and in certain embodiments, cells may be modified to specifically generate A-negative and / or B-negative and / or Rh-negative cells, thereby producing cells suitable for transfusion to any patient, either universally or nearly universally.

[0246] It should be noted that hemangio-colony forming cells and / or differentiated hematopoietic cell types may be generated using any of the methods of the present invention detailed throughout the specification.

[0247] In certain embodiments of blood banking practices, cells (hemangio-colony forming cells and / or differentiated hematopoietic cell types) are produced and stored at one or more central facilities. The cells may then be delivered, for example, to a hospital or treatment facility for use in treating patients. In other particular embodiments, the cells are maintained in cryopreservation and are specially thawed and prepared for transfusion based on the hospital or other treatment facility's regulations. Such regulations may be standing orders (e.g., to produce and provide a certain amount of a certain number of units of cells). In a specific embodiment, the method includes a system for billing hospitals or insurance companies for costs associated with the deposited products.

[0248] In certain embodiments of any of the above, the cells are allocated based on cell number, volume, or any unit acceptable to the user to quantify the dose administered to the patient and / or to compare the dose administered during a standard blood transfusion.

[0249] In certain embodiments, the cells are produced, stored, and administered as a mixed cell population, e.g., the preparation may include cells of altered developmental stages as well as distinct cell types, while in other embodiments, the cells are produced, stored, and / or administered as a substantially purified preparation of a single cell type.

[0250] In certain embodiments, the preparation of cells is screened for one or more infectious diseases. Screening can occur before or after generation or storage. For example, the preparation of cells can be screened to identify hepatitis, HIV, or other blood-borne infectious diseases that may be transmitted to the recipient of the product. Induction of tolerance in transplant recipients Human hemangioblasts generated and expanded by the methods of the invention, or expanded by the methods of the invention, can be used to induce immune tolerance. Immune tolerance refers to the suppression of an immune response in a graft recipient that would otherwise occur, for example, to the introduction of nonself MHC antigens (e.g., antigens shared with the graft and the tolerized hemangioblasts) to the recipient. Thus, tolerance refers to the suppression of immune responses induced by specific donor antigens, as opposed to the broad spectrum immune suppression that can be elicited using immunosuppressants. Tolerance can involve humoral responses, cellular responses, or both humoral and cellular responses. Tolerance can include the elimination and / or inactivation of pre-existing mature donor-reactive T cells, and the long-term (e.g., lifelong) elimination and / or inactivation of newly developed donor-reactive T cells.

[0251] The methods of generating and expanding human hemangioblasts described in the present invention provide several advantages in inducing tolerance. The methods of the present invention generate vast and hitherto inaccessible numbers of human hemangioblasts. Vast quantities of human hemangioblasts can be used to induce tolerance in transplant recipients using less toxic pre-conditioned protocols. Methods of the present invention are also provided for generating libraries of human hemangioblasts, each of which is hemizygous or homozygous for one or more MHC alleles present in the human population, with each member of the library of hemangioblasts being hemizygous or homozygous for a different set of MHC alleles relative to the other members in the library. Such libraries of human hemangioblasts can be used to select cells and select tolerant human hemangioblasts that can be matched to any available donor transplant.

[0252] Bone marrow transplantation and subsequent establishment of hematopoietic cells or mixed chimerism have previously been shown in both murine and human models to induce specific tolerance to new tissue types derived from hematopoietic stem cells. Hematopoietic cells or mixed chimerism refers to the production in the recipient of hematopoietic cells derived from both donor and recipient stem cells. Thus, when the recipient achieves hematopoietic cell chimerism, the recipient becomes tolerant to donor-specific antibodies. In many protocols for tolerance induction, tolerant donor cells are administered to the recipient and transplanted into the recipient's bone marrow. In order to create hematopoietic cell space in the recipient's bone marrow for the donor cells, some protocols require a step of creating hematopoietic cell space (e.g., by total body irradiation), which is typically toxic or harmful to the recipient. However, there is evidence from established models that when a very large number of donor tolerant cells are available, irradiation can be completely eliminated to achieve hematopoietic cell or mixed chimerism with the advantage of a less harmful pre-conditioned regimen. Thus, mixed chimerism can be achieved, for example, with the regulation of specific non-myeloablative recipients.

[0253] Thus, because the novel methods described herein can produce vast numbers of human hemangioblasts, the invention offers the advantage that regulatory protocols are less stringent or less deleterious immune tolerance is induced, e.g., the hematopoietic cell space formation step can be omitted if sufficient numbers of tolerant donor cells are used.

[0254] Thus, in certain embodiments of the invention, human hemangioblasts generated and expanded or expanded by the methods described herein can be used to induce immune tolerance. While not wishing to be bound by any theory based on the mechanism, human hemangioblasts can be received by and transplanted into the recipient's bone marrow to induce immune tolerance by producing mixed chimerism.

[0255] In particular embodiments, the donor human hemangioblasts are administered to a recipient patient (e.g., by intravenous injection) prior to transplanting a graft or transplanting an organ, tissue, or cells from the donor into the recipient patient. In particular embodiments, the human hemangioblasts are administered to induce tolerance in a patient (e.g., a graft or graft recipient) in need of induction of tolerance. Thus, in particular embodiments, a method of inducing tolerance in a human recipient patient includes the steps of: (a) selecting a patient in need of transplant or cell therapy; (b) administering to said patient human hemangioblasts derived from or matched to the donor, wherein said hemangioblasts are generated and expanded or propagated according to the present invention; and (c) transplanting the donor organ, tissue, or cell graft into the recipient patient, wherein said hemangioblasts induce tolerance to donor antigens. In particular embodiments, the patient is receiving an organ, tissue, or cell therapy, wherein said organ, tissue, or cells are obtained from a donor or a donor cell source. For example, hemangioblasts from a donor can be (1) expanded by the methods described above to generate large numbers of donor-tolerant cells, and (2) expanded and differentiated in vivo to obtain hematopoietic or endothelial cells or tissues that can then be transplanted into a recipient patient. In other embodiments, the organ, tissue, or cell therapy is not derived from the donor hemangioblasts, but is consistent with the donor hemangioblasts.

[0256] The term "matched" as used herein refers to how similar the HLA types are between the donor and recipient (e.g., graft). In one embodiment, the term "matched" refers to a match between the MHC I and / or MHC II alleles of the donor hemangioblasts and graft such that rejection does not occur. In another embodiment, the term "matched" refers to a match between the MHC I and / or MHC II alleles of the donor hemangioblasts and graft such that the donor graft is tolerized by the matching donor hemangioblasts. In yet another embodiment, the term "matched" refers to a match between the MHC I and / or MHC II alleles of the donor hemangioblasts and graft such that immunosuppression is not required.

[0257] The above-described methods for inducing tolerance to alloantigens or allografts may be used when there is a mismatch between the donor and recipient at the MHC locus or other locus sufficient to result in graft rejection. Thus, for example, in certain embodiments, mismatches may occur at one or more MHC loci or at one or more other loci that mediate recognition and rejection, such as minimal antigen loci. In some embodiments, for example, the recipient and donor may be mismatched in HLA alleles, resulting in one or more mismatched antigens. In the case of MHC type I and II loci, the donor and recipient may be, for example, matched at type I and mismatched at type II, mismatched at type I, mismatched at type II, matched at type I and matched at type II. Any combination of other loci that control recognition and rejection, such as minimal antigen loci, may be matched or mismatched. Mismatched at MHC I type means mismatched at one or more MHC type I loci, e.g., mismatched at one or more of HLA-A, HLA-B, or HLA-C. Mismatched at MHC II type means mismatched at one or more MHC type II loci, e.g., mismatched at one or more of DPA, DPB, DQA, DQB, DRA, or DRB. For example, the hemangioblast and graft may be matched at type II HLA-DRB1 and DQB1 alleles. The hemangioblast and graft may be matched at two or more type I HLA-A, B, or C, alleles (in addition to the matched DRB1 and DQB1 alleles).

[0258] In another embodiment, the tolerizing donor cells are cells derived from hemangioblasts generated and expanded or expanded by the above-described method. According to this embodiment, donor human hemangioblasts are differentiated in vitro to generate donor hematopoietic stem cells, which are then administered to a recipient patient to induce tolerance. In some of the above methods, donor hemangioblasts or hematopoietic stem cells derived therefrom are administered to the recipient to induce tolerance in the recipient (to the donor tolerizing cells) to prepare a matched transplant or graft.

[0259] In another embodiment, the method of inducing tolerance further comprises generating hematopoietic space (thereby promoting grafting of hemangioblasts or hematopoietic stem cells derived therefrom). In another embodiment, the method of inducing tolerance further comprises temporarily inhibiting rejection of donor hemangioblasts or hematopoietic stem cells derived therefrom, e.g., by removing and / or inactivating pre-existing donor-reactive T cells. To generate hematopoietic space, the method can include irradiation (e.g., total body, lymphoid, or selective thymic irradiation). To prevent rejection of donor cells, the method can further include administration of drugs or antibodies (e.g., cytostatic agents, antimetabolites, or anti-T cells or anti-CD8 or anti-CD4 antibodies) and / or other treatments that promote donor cell grafting and survival and formation of mixed chimerism (e.g., administration of stromal cells or growth factors, cytokines, etc. to the recipient, or administration of other agents that deplete or inactivate the recipient's natural antibodies). In certain embodiments, the radiation, antibodies, drugs and / or other agents administered are sufficient to inactivate thymocytes and / or T cells in the recipient to promote donor survival and / or generate hematopoietic space within the recipient. Such steps of generating hematopoietic space and / or temporarily inhibiting donor cell rejection can be performed, for example, prior to introducing donor hemangioblasts into the recipient. Alternatively, the patient can receive agents or methods to block, remove or inactivate T-cells simultaneously with administration of the donor tolerizing cells.

[0260] In certain embodiments, hematopoietic space creation is combined with immunosuppression. For example, the recipient is administered anti-T cell antibodies in combination with low-dose total body irradiation and / or thymic irradiation. In one embodiment, the recipient is administered anti-CD4 and anti-CD8 antibodies, followed by a mild, nonmyeloablative dose of total body irradiation (e.g., a dose that removes the recipient's bone marrow fraction so that the bone marrow is not irreparable) and selective thymic irradiation, or alternatively, an additional dose of a T cell inactivating antibody or a costimulatory blocking agent (e.g., CTLA4-Ig and / or anti-CD40L antibody). After irradiation, donor hemangioblasts or hematopoietic stem cells derived therefrom can be administered to the recipient (e.g., by intravenous injection). In this embodiment, total body irradiation to promote grafting of donor cells can be replaced by administering various donor human hemangioblasts or hematopoietic stem cells derived therefrom. Obtaining such various donor human cells can be performed by the methods described above.

[0261] In another embodiment, a treatment to deplete or inactivate recipient T cells can help promote survival of the administered donor tolerized human hemangioblasts and prevent graft inhibition. In another embodiment, the method can include clonal depletion of donor-reactive cells in the recipient patient. For example, the patient can be administered a mild dose of total body irradiation followed by T cell costimulatory blockade and administration of donor human hemangioblasts. Alternatively, the patient can be administered T cell costimulatory blockade and administration of various donor human hemangioblasts without irradiating the patient.

[0262] In other embodiments, tolerance may be achieved without myeloablative conditioning of the recipient. In one embodiment, the recipient may be fed donor human hemangioblasts with anti-CD40L to facilitate grafting of the donor hemangioblasts. For example, the recipient may be fed a variety of donor hemangioblasts with anti-CD40L monoclonal antibodies followed within days by a dose of CTLA4-Ig. Such a protocol removes donor-reactive T cells and blocks CD40-CD40L interactions. The novel methods described above for generating and expanding human hemangioblasts in vitro make such a mild tolerance protocol feasible.

[0263] After receptor modulation and / or depletion or blocking of donor-reactive T cells, donor-tolerized human hemangioblasts generated by the methods of the invention are administered to the recipient. Donor human hemangioblasts can be derived from hemangioblasts obtained from tissue or cell sources from the donor. Alternatively, donor human hemangioblasts can be obtained from a different non-donor source that is matched to the donor.

[0264] In certain embodiments, tolerance is induced by administering multiple doses of donor human hemangioblast cells (e.g., administration of 2, 3, 4 or more donor cells). Thus, tolerance can be induced by methods involving multiple administrations of donor tolerizing cells, where the multiple administrations are administered to the recipient within a period of a week or less.

[0265] In certain embodiments, the ability of the human hemangioblasts of the invention to induce immune tolerance can be assessed using different experimental model systems. For example, the ability to establish a human immune system in SCID mice has been used to study human immune responses in experimental models. It has been shown that a functional human immune system can be reconstituted in an immunodeficient recipient mouse using human fetal liver and thymus tissue. Similarly, the functional potency of the human hemangioblasts of the invention can be assessed using similar experimental model systems. For example, the ability of human hemangioblasts to replace human fetal liver in establishing a functional human immune system in a mouse can be assessed using the aforementioned experimental models. Also, in mice with a functional human immune system (e.g., when human fetal liver and thymus tissue are used to establish a human immune system in SCID mice to produce hu-SCID mice), human "donor" hemangioblasts (mismatched to the fetal liver and thymus tissue used to establish the hu-SCID mice) can be administered to the hu-SCID mice by any one of the methods described above to obtain mixed chimerism. When these animals are then transplanted with allografts matched for the donor hemangioblasts, tolerance to the donor antigens can be assessed.

[0266] In certain embodiments, the invention relates to cell preparations. An effective cell preparation includes two components: a first cell type that induces immune tolerance and a second cell type that regenerates a desired function. The two cell types can be produced by the methods of the invention and obtained from the same donor. For example, donor-derived human hemangioblasts can be used as tolerized donor cells. Donor-derived cells (e.g., embryonic stem cells, pluripotent stem cells or early progenitor cells, or hemangioblasts) can also be used to generate, for example, hematopoietic or endothelial cells (as described herein), neural cells, e.g., oligodendrocytes, hepatocytes, cardiac myoblasts or cardiac myoblast progenitor cells, or osteoblasts and their progenitors. Thus, donor human hemangioblasts can be used to induce tolerance in a recipient such that the recipient is tolerant to the donor hemangioblasts or cells or tissue derived from the donor embryonic or pluripotent stem cells.

[0267] In other embodiments, the two cellular components of the cell combinations of the invention can be obtained from different sources or donors, where the two sources or donors are identical. For example, hemangioblasts can be generated from an embryonic stem cell source, and the graft cells or tissue can be obtained from a source different from the embryonic stem cell source used to generate the human hemangioblasts. In such embodiments, the two sources are identical.

[0268] For any of the therapeutic purposes described herein, human hemangioblasts or hematopoietic cells derived therefrom for immune tolerance can be provided in the form of a pharmaceutical composition containing isotonic excipients manufactured under sufficiently sterile conditions for human administration. Hemangioblasts in gene therapy Another aspect of the invention relates to the use of hemangioblast cells, or hematopoietic or endothelial cells differentiated therefrom, or cells further differentiated from these cells, in gene therapy. The mammalian hemangioblast preparations of the invention can be used to deliver therapeutic genes to patients having diseases that can be treated by the gene product of the therapeutic gene. Hemangioblast cells are particularly useful for delivering therapeutic genes that are involved in or affect neovascularization (e.g., VEGF to induce the formation of collaterals in ischemic tissues), hematopoiesis (e.g., erythropoietin to induce erythropoiesis), vascular function (e.g., growth factors to induce proliferation of vascular smooth muscle to treat aneurysms), or blood cell function (e.g., clotting factors to reduce bleeding) or secrete hormones (e.g., growth hormone). Methods for gene therapy are described in the prior art. See, for example, U.S. Pat. No. 5,399,346 (Anderson et al.). Biocompatible capsules for the delivery of genetic material are described in PCT Publication WO 95 / 05452 (Baetge et al.). Methods for gene delivery to bone marrow-derived cells have also been reported (see U.S. Patent No. 6,410,015 (Gordon et al.)). Therapeutic genes can be any gene with clinical utility, including genes encoding gene products or proteins involved in disease prevention or treatment, or genes that have a cell regulatory effect involved in disease prevention or treatment. Such gene products can replace defective or missing gene products or proteins in a patient, or have a cell regulatory effect, allowing for the prevention or treatment of a disease or disorder in a patient.

[0269] Thus, the present invention further provides a method for delivering a therapeutic gene to a patient having a disease requiring gene therapy, the method comprising the steps of selecting a patient in need thereof, modifying a hemangioblast cell preparation such that the cells carry the therapeutic gene, and administering the modified preparation to the patient. Such preparations may be modified by techniques generally known in the art. Modification may include inserting into mammalian hemangioblast cells a DNA or RNA fragment encoding a gene product that enhances the therapeutic effect of the hemangioblasts. The gene is inserted in such a manner that the modified hemangioblasts produce the therapeutic gene product in the patient's body or have a desired therapeutic effect. In one embodiment, the hemangioblasts are prepared from a cell source obtained from the patient along with bone marrow. Any gene transfer procedure can be used to insert genes into hemangioblasts, such as naked DNA integration, direct injection of DNA, receptor-mediated DNA uptake, retroviral-mediated transfection, viral-mediated transfection, non-viral transfection, lipid-mediated transfection, electrotransfer, electrophoresis, calcium phosphate-mediated transfection, microinjection, or proteoliposomes, all of which can include gene therapy vectors. Other vectors that can be used include retroviral vectors, as well as vectors derived from DNA viruses and other RNA viruses. As is evident when RNA viruses are used, such viruses contain RNA encoding a therapeutic agent, such that hemangioblasts transfected with such RNA viruses provide DNA encoding a therapeutic gene product. Methods for introducing genes into cells are known in the art (see, e.g., Ausubel, id.).

[0270] According to another aspect of the invention, a purified preparation of human hemangioblasts modified to carry a therapeutic gene is provided in a container or commercial package, which further includes instructions for use of the preparation in a gene therapy method to prevent and / or treat a disease by delivery of a therapeutic gene. Thus, the invention further provides a commercial package (i.e., a kit) comprising a preparation of mammalian hemangioblasts of the invention, where such preparations have cells modified to carry a therapeutic gene, and instructions for treating a patient having a disease treatable by gene therapy. Other commercial applications and methods Certain aspects of the invention involve expanding human hemangioblasts to commercial quantities. In certain embodiments, human hemangioblasts are produced on a large scale, stored when needed, and supplied to hospitals, clinicians, or other health care institutions. Human hemangioblasts can be ordered and supplied at the appropriate time when a patient is prescribed, for example, ischemia or vascular injury, or when hematopoietic reconstitution is required. Thus, the invention relates to methods of generating and expanding human hemangioblasts to obtain cells on a commercial scale, cell preparations containing human hemangioblasts derived from said methods, and methods of providing (i.e., producing, storing when needed, and selling) human hemangioblasts to hospitals and clinics. Additionally, hemangioblast lineage cells can be produced in vitro, stored when needed, and sold to hospitals and clinicians.

[0271] Accordingly, certain aspects of the invention relate to methods of producing, storing and distributing hemangioblasts expanded by the methods described herein. After producing and expanding the human hemangioblasts in vitro, the human hemangioblasts may be harvested, purified and, if necessary, stored prior to treating a patient. Alternatively, if hemangioblast lineage cells are desired, the human hemangioblasts may be further differentiated in vitro prior to treating a patient. Thus, in certain embodiments, the invention provides methods of providing hemangioblasts to hospitals, health care centers and clinicians, where the hemangioblasts or hemangioblast lineage cells produced by the methods disclosed herein are stored and ordered at the request of the hospital, health care center or clinician and administered to a patient in need of hemangioblast or hemangioblast lineage therapy. In alternative embodiments, the hospital, health care center or clinician orders human hemangioblasts based on patient specific data, and the human hemangioblasts are produced according to the patient's condition and provided to the ordering hospital or clinician.

[0272] An additional aspect of the invention relates to libraries of hemangioblasts and / or hemangioblast lineage cells that can provide cells that match potential patient receptors. Thus, in one embodiment, the invention provides a method of conducting a pharmaceutical business comprising providing hemangioblast preparations homozygous for one or more tissue junction antigens, where the cells are selected from such cell banks that contain a human hemangioblast library that can be expanded by the methods disclosed herein, each hemangioblast preparation being hemizygous or homozygous for one or more MHC alleles present in the human population, and the hemangioblast bank being hemizygous or homozygous, respectively, for a different set of MHC alleles when compared to other members of the cell bank. As discussed above, gene targeting or loss of heterozygosity can be used to generate hemozygous or homozygous MHC allele stem cells that can be used to derive hemangioblasts. In one embodiment, after a particular hemangioblast preparation is selected to be appropriate for a patient, it is expanded to a quantity appropriate for treating the patient. Such methods may further include differentiating the hemangioblasts to obtain hematopoietic and / or endothelial cells prior to administering the cells to the recipient. The methods of conducting a pharmaceutical business may also include conducting a distribution system to distribute the formulation for sale, and may include establishing a sales group for marketing the pharmaceutical formulation.

[0273] Another aspect of the invention relates to the use of the human blood stem cells of the invention as research tools within the facilities of pharmaceutical, chemical, or biotechnology companies, hospitals, or academic or research institutions. For example, the human blood stem cells and blood stem cell derived cells (e.g., endothelial cells) can be used to screen and evaluate angiogenic and anti-angiogenic factors, or used for tissue engineering. Also, because the hemangioblasts obtained and expanded by the methods known herein have the dual potential to differentiate into hematopoietic and endothelial cells, they can be used for cell and molecular biology of hematopoiesis and angiogenesis. Furthermore, the human hemangioblasts can be used for the discovery of new markers of these cells, genes, growth factors, and differentiation factors that play a role in hematopoiesis and angiogenesis, or for the development of screening assays for potential toxic or protective agents and drug discovery.

[0274] In another embodiment of the invention, hemangioblast lineage cells (such as blood cells) can also be used commercially. Hematopoietic cells can be used to produce blood products such as hemoglobin and growth factors, which can be used for clinical and research applications.

[0275] The invention also includes methods for obtaining human ES cells from a patient and expanding human hemangioblasts derived from the ES cells, which can be stored and used to treat the patient from whom the ES were obtained, or a relative of the patient.

[0276] The above methods and applications relate to the treatment, pharmaceutical formulation and storage of hemangioblasts, and the present invention also relates to hemangioblast cell solutions suitable for such applications. Accordingly, the present invention relates to solutions of hemangioblast cells suitable for injection into a patient. Such solutions may include cells formulated in a physiologically acceptable fluid (e.g., normal saline, buffered saline, or balanced saline solution). The solutions may optionally include factors that promote in vivo cell differentiation. The solutions may be administered to a patient by intravascular administration (e.g., intravenous infusion) by techniques that are compatible with the methods used for bone marrow transplantation. In some embodiments, the cell solutions are administered into a peripheral vein, a superficial peripheral vein, or alternatively, into a central vein (e.g., via a central venous catheter). The number of cells in the solution may be approximately 10 2 More than or equal to about 10 9 In another embodiment, the number of cells in the solution is about 10 1 , 10 2 , 5X10 2 , 10 3 , 5X10 3 , 10 4 , 10 5 , 10 6 ,10 7 , or 10 8 Or about 5X10 2 , 10 3 , 5X10 3 , 10 4 , 10 5 , 10 6 ,10 7 , 10 8 , or 10 10 The upper and lower limits are non-independently selected, except that the lower limit is always less than the upper limit. Additionally, the cells can be administered in single or multiple doses.

[0277] The present invention will be further illustrated by reference to the following examples, which are merely illustrative and are not to be construed as limitations of the above-described invention. EXAMPLES

[0278] Example 1: Hemangioblasts derived from human ES cells exhibit both hematopoietic and endothelial potential Human embryonic stem cell lines H1 and H9 are cultured in mouse embryonic fibroblast (MEF) ES cell medium. Cultured human ES cells are separated, pelleted by centrifugation at 200 g, and resuspended in embryoid body (EB) formation medium. EB formation medium contains serum-free Stemline medium (Sigma®) supplemented with BMP-4 and VEGF. Cells are then plated onto ultra-low attachment culture plates and cultured in a CO2 incubator. After 24-48 hours, early hematopoietic cytokines (TPO, Flt-3, and SCF) are added and cultured for another 24-48 hours to form EBs.

[0279] Embryoid bodies (EBs) are cultured for 2-6 days or optionally 2-5 days, after which they are harvested, washed in PBS, and disaggregated into single cell suspension using trypsin / EDTA. EBs are counted and approximately 5-10X10 6 Cells / mL were cultured in BL-CFU medium (Stem Cell Technology TM ) and supplemented with cytokines and the PTD-HOXB4 fusion protein, the cells are then replated onto new ultra-low attachment culture plates to grow hemangioblast colonies.

[0280] EBs are monitored daily for the formation of hemangioblast colonies, which are observed at approximately day 3. Hemangioblasts are identified by their highly characteristic grape-shaped cell morphology.

[0281] A portion of the cells could further form EBs (secondary EBs). Secondary EBs do not have the grape-shaped morphology as can be seen in Figure 16b. Hemangioblasts are also smaller than secondary EBs.

[0282] These hemangioblast colonies are then selected, identified, and replated in methylcellulose CFU-medium to test their ability to form hematopoietic colony forming units (CFUs) and further differentiate.Similarly, these hemangioblast colonies are selected, identified, and replated on fibronectin-coated culture plates optimized for the first step towards endothelial differentiation.

[0283] To examine the generation of hematopoietic progenitors from the selected hemangioblast colonies, the growth of colony forming units (CFU) for granulocytes, erythrocytes, macrophages and megakaryocytes is measured after 3-10 days of growth.

[0284] To examine the development of endothelial cells from the isolated hemangioblast colonies, the ability of the hemangioblasts to form branched tube-cords when replated in a thick layer of Matrigel is measured.

[0285] The ability of isolated hemangioblast colonies to differentiate into endothelial cells may also be confirmed using other endothelial-specific assays, such as the presence of endothelial cell surface markers or LDL uptake. Example 2: Secondary characteristics of hemangioblasts or hESC-derived BC cells Human ES cell culture .

[0286] The hES cell lines used in this study were the H1, H7, and H9 cell lines previously described (NIH registered as WAO1, WA07, and WA09) and four lines derived from Advanced Cell Technology (MAO1, MA03, MA40, and MA09). Undifferentiated human ES cells were cultured on inactivated (mitomycin C-treated) mouse embryonic fibroblast (MEF) cells in complete hES medium until 80% confluent (Klimanskaya & McMahon, Approaches of derivation and maintenance of human ES cells: Detailed procedures and alternatives, in Handbook of Stem Cells. Volume 1: Embryonic Stem Cells, ed. Lanza, R. et al. (Elsevier / Academic Press, San Diego, 2004). Undifferentiated hES cells were then cultured for 2-5 min with 0.05% trypsin-0.53 m MEDTA (Invitrogen). TM ) and collect by centrifugation at 1,000 rpm for 5 minutes. EB formation To induce hemangioblast precursor (mesoderm) formation, hES cells (2 to 5×10 5 cells / ml) with BMP-4 and VEGF 165 (50ng / ml, R&D Systems TM ) supplemented with serum-free Stemline medium (e.g., Stemline I or II, Sigma-Aldrich) TM ) in ultra-low attachment plates (Corning TM) and cultured at 5% CO2. After 48 hours, the EB medium is supplemented and filled with a mixture of early hematopoietic / endothelial growth factors (cockatil). For example, half of the medium is removed after 48 hours and fresh medium is supplemented with BMP-4 and VEGF as well as SCF, TPO and FLT3 ligand (20ng / ml, R&D Systems) at the same final concentrations. Triple protein transduction domain (tPTD)-HoxB4 fusion protein (1.5μg / ml) is added to the culture medium for 48-72 hours to expand hemangioblasts and their precursors. Hemangioblast or blast proliferation After 3.5–5 days, EBs were cultured for 2–5 min with 0.05% trypsin-0.53 mM EDTA (Invitrogen TM ), and a single cell suspension is prepared by passing 3-5 times through a 22G needle and a 40μM strainer. Cells are collected by centrifugation at 1,000 rpm for 5 minutes and counted. The cell pellet is resuspended in 50-200 ml of serum-free Stemline medium. To expand the hemangioblasts, 2 to 5X10 5 Single cell suspensions obtained from EBs derived from differentiation of hES cells are mixed with 2 ml of BL-CFC / angioblast growth medium (BGM) containing Iscove's MDM, 1-2% bovine serum albumin, 0.1 mM 2-mercaptoethanol and 1.0% methylcellulose L in a mixture of growth factors. For example, 10 μg / ml rh-insulin, 200 μg / ml iron-saturated human transferrin, 20 ng / ml rh-GM-CSF, 20 ng / ml rh-IL-3, 20 ng / ml rh-IL-6, 20 ng / ml rh-G-CSF, 3 to 6 units / ml rh-EPO, 50 ng / ml rh-SCF, 50 ng / ml rh-FLt3 ligand, 50 ng / ml rh-VEGF, and 50 ng / ml rh-BMP-4 ("rh" stands for "human recombinant"), and 1.5 μg / ml of TPO and FL with or without PTD-HoxB4 fusion protein were added. The cell mixture was plated in ultra-low attachment plates (e.g., 2-5×10 cells / well in one well of a 6-well plate). 5The cells were plated in 1000 μl of ...

[0287] 0.35±0.01% and 0.52±0.06% of individual cells from WAO1 and MAO1 hES cells, respectively, developed into grape-shaped mother cell colonies (BCs). At day 3, BCs contained <10 cells and rapidly expanded to >100 cells over 4 to 6 days (Fig. 16a). Colonies were generally less dense than secondary EBs and more morphologically uniform (Fig. 16b). Wright-Giemsa staining and cytospin preparation of hES-derived mother cell colonies confirmed the morphological characteristics of immature blast cells (Fig. 16c). To extend this result to other hES cell lines (WA07[H7], WA09[H9], MAO1, MA03, MAO9, and MA40), supplementation with FL (50 ng / ml) and Tpo (50 ng / ml) is necessary for sustained growth of BC colonies (without FL and Tpo, small (10-20 cell) harvested hES-BCs die after 4-8 days). Discrepancies observed between other hESC lines may be due to intrinsic properties of this cell line, as previously observed by Bowles et al. (2006 Stem Cells 24:1359-1369). Epo (3-6 units / ml of human recombinant Epo) was also essential for growth and BC formation in all hES cells examined.

[0288] A time course (from days 2 to 6) of human BL-CFC formation in EBs was examined, and a narrow time period was found during which human ESC-derived EBs generated large numbers of BL-CFCs (or hES-BCs). On day 2, EBs generated hES-BCs at a low frequency (57.3±7.4 BC / 1x10 for WAO1 hES cells). 5 EB cells, mean ± SE, n = 3; MAO1 hES cells, 395 ± 10.4 BC / lx10 5EB cells, n=3), and hematopoietic (erythroid) precursors were generated on day 6. However, on day 3.5, EBs gave rise to numerous hemangioblasts or hES-BCs (347.4±11.1 BC / 1×10 for WAO1 hES cells) in all tested hESC lines, including those derived from both blastocysts (WAO1 and WA09) and single-dividing cells (MAO1 and MA09). 5 EB cells, n=3; MAO1 hES cells, 523.3±60.1 BC / lx10 5 EB cells, n=3). Although the efficiency varied depending on the hESC line and growth conditions (see Table 1), WAO1-GFP and MAO1 hES cells (12-13×10 6 One 6-well plate of cells was 22.18±3.51X10 5 (grown for 6-8 days, mean ± SE, n = 17), and 49.73 ± 7.23 × 10 6 (6-8 days of growth, n=9) and 396.4±91.63x10 6 (grown for 10-13 days, n=6) hemangioblasts or hES-BC cells were generated, and thus the cells can be readily grown under the well-defined and reproducible conditions described above.

[0289] [Table 1]

[0290] Cryopreservation of hemangioblasts Hemangioblasts (or blast cells or hES-BC cells) can be cryopreserved in serum-free medium. For cryopreservation of hemangioblasts, purified cells were divided into two equal parts: one half of the cells was directly plated for the two hematopoietic CFU assays and endothelial cell development, and the other half was resuspended in 8% DMSO in Stemline medium and stored in liquid nitrogen. The cells were then thawed and examined for hematopoietic and endothelial developmental potential. Once thawed, hemangioblasts were plated for hematopoietic and endothelial lineage development as described herein and compared to fresh, purified cells. For endothelial cell lineages, 78±2% (mean+SE, n=3) of cells were recovered after the freezing process, while 61±7% (n=3) of total hematopoietic CFUs and 46±4% (n=3) of CFU-erythrocytes remained when compared to fresh hES-BC cells. No loss of more primitive multipotential progenitors for CFU-mixing was observed following recovery of hES-BC cells upon storage in liquid nitrogen. Characteristics of hemangioblasts For chemical analysis of BL-CFC immune cells, purified BL-CFC were cytospun onto polylysine-treated glass slides and fixed with 4% paraformaldehyde. To examine the expression of maximal genes, primary antibodies were incubated overnight at 4°C, then secondary antibodies were labeled with fluorescent dyes, and finally examined under a fluorescent microscope. Normal human BM cells, K562 cells, and HUVECs were used as controls.

[0291] Immunocytochemical analysis revealed that hES cell-derived BL-CFC or BC expressed CD31, CD34, and KDR proteins but not other adhesion molecules, which are associated with various hemangioblasts. hES-BC expressed GATA-1 (Fig. 16d and 16e) and GATA-2 proteins. GATA-1 is a zinc finger transcription factor essential for both primitive (embryonic) and definitive (adult) hematopoiesis and is expressed in mouse hemangioblasts (Ferreira et al. 2005 MoI Cell Biol 25:1215-1227 and Yokomizo et al. 2007 EMBO J 26:184-196). GATA-2 is a zinc finger transcription factor that acts at multiple stages during hemangioblast development and differentiation (Lugus et al. 2007 Development 134:393-405). hES cell-derived BL-CFCs or BCs also expressed LMO2, a LIM-domain protein important for hemangioblast development (Gering et al. 2003 Development 130:6187-6199) (Fig. 16g and 16h) and CXCR-4 (Fig. 16n and 16o). CXCR-4 is a receptor for the chemokine SDF-I and is expressed on the surface of endothelial cells derived from hESCs, hematopoietic stem cells and mouse hemangioblasts. CXCR-4 plays an important role in the migration, retention and development of hematopoietic precursors in the bone marrow. The cells further express TPO and EPO receptors (Fig. 16t and 16u, and 16q and 16r, and Table 1) and react readily to specific antibodies for CD71, the transferrin receptor (Fig. 16j and 16k) (see Table 2 and Fig. 16d-v). The cells express little or no other adhesion molecules or CD31, CD34 and KDR (Table 2). The absence of CD34, CD31 and KDR expression, and the absence of CD133 expression, are unique profiles of surface marker expression.

[0292] For gene profiling analysis, total RNA was isolated from purified BL-CFC / hES-BC cells, 3.5-day-old EBs, and undifferentiated ES cells using the RNAeasy kit (Qiagen). The truncated antisense cRNA was used for hybridization to human U133.2 arrays (Affymetrix®, Inc) at the Core Genomics Facility at the University of Massachusetts (Worcester, MA). For expression profiling of β-cluster globin genes, individual CFU colonies were selected, RNA was isolated and amplified, and β-, γ-, and ε-globin gene expression was analyzed as previously described (Lu et al. 2004 Blood(103):4134-4141).

[0293] Affymetrix TM Molecular profiling by arrays shows a substantial increase in genes associated with hemangioblasts and early primitive erythroid cell development compared to early stage EBs (Table 2). FLT-I (a receptor for VEGF) as well as SCL and LMO2, two genes important in hemangioblast development (D'Souza et al. 2005 Blood(105):3862-3870; Park et al. 2004 Development(131):2749-2762; Gering et al. 2003 Development(130):6187-6199)) were readily detected in BL-CFCs / blastocyte colonies. Germline (ε-globin, 549-fold) and fatal (γ-globin, 817-fold) globin gene expression was dramatically increased in BL-CFCs / hES-BCs, and NF-E2 (12-fold), GATA-1 (6-fold), EKLF (7-fold), ICAM-4 (4-fold), glycophorin (14-fold), and Epo receptor (4-fold) message levels were also modestly increased.

[0294] [Table 2-1]

[0295] [Table 2-2]

[0296] Functional characterization of hemangioblasts Grape-like hemangioblast colonies were picked, manually isolated under a dissecting microscope, and resuspended in serum-free Stemline (Stemline I) medium, after which the cells were tested for lineage potential as described below. Clonality of hES cells To determine whether the hES-mother colonies were replicative and derived from a common bipotential progenitor cell, cell mixing experiments were performed as previously described in mouse studies (Choi et al. 1998 Development (125): 725-732; Kennedy et al. 1997 Nature (386): 488-493). Cells from EBs of WAO1-GFP and MAO1 hES cells were mixed and plated in BGM. hES-mother colonies were examined 4-6 hours later by phase and fluorescence microscopy. In a series of three experiments, 100% (77 out of 77) of the mother colonies were found to be GFP positive or GFP negative (no mixed BCs were observed, Figures 18a and 18b). To exclude the possibility that GFP-negative colonies contained cells with an inactive GFP gene, GFP-positive and GFP-negative colonies were examined for the presence of GFP sequences. 12 individual GFP-positive and 12 individual GFP-negative colonies were hand-picked under phase and fluorescence microscopy from the mixing experiments. Gene DNA was analyzed using a MicroDNA kit (Qiagen TM ) and performed a GFP-specific wjrR reaction. As an internal control for the PCR reaction, myogenin primers were included in every PCR reaction, which generates a 245 bp fragment. PCR products were resolved on a 2% agarose gel and visualized by ethidium bromide staining. Polymerase chain reaction (PCR) analysis confirmed the absence of GFP gene sequences in every negative colony (Figure 18, PCR analysis).

[0297] Dilution studies further confirmed the clonogenicity of primary and secondary BCs (Table 3, FIG. 18c). Limiting dilution studies revealed that different numbers (1.5×10 3 ~1.5X10 4 (100 cells / ml). After dilution of single EB-cell suspensions in 150 ml BGM (100 cells / ml), these were plated in fifteen ultra-low 96-well plates and incubated at 37°C for BC development. To confirm the clonal origin of the mother cell colonies, the plates were checked under masked conditions by two investigators 4 hours after plating. Wells with double or triple cell clumps were excluded from further investigation.

[0298] As reported by Kennedy et al. (1997 Nature 386:488-493) in mouse ES cell studies, human embryonic stem cell colony (hES-BC or BC) development is also cell density dependent with poor development at low cell numbers. Seven BCs developed from 15 96-well plates (Figure 18c). Additional limiting dilution studies were performed to determine whether the BCs contained cells with the potential to generate secondary BCs.

[0299] For secondary BC growth, primary mother cell colonies were hand-picked and dissociated into single cells. Single cells were then mixed with 20 ml BGM (200, 300, and 1,000 cells, respectively), serially diluted, and plated (0.1 ml / well) into six ultra-low 96-well plates. Wells with double or triple cell clumps were excluded from the study. Twenty-nine secondary BCs with the same morphological characteristics as the primary BCs were generated from each of the five 96-well plates. These experiments confirmed the clonogenicity of primary and secondary hES-BCs (Table 3). Bipotential capacity of hES-cell colonies To account for the bi-differentiation potential of clonally derived mother cells, two strategies were used. In the first method, individual hES-BC colonies were hand-picked and transferred to fibronectin-coated 48-well plates containing Stemline II with growth factors supporting the growth of hematopoietic and endothelial lineages for single BC expansion. Growth factors in liquid culture included recombinant human SCF (20 ng / ml), Tpo (20 ng / ml), FL (20 ng / ml), IL-3 (20 ng / ml), VEGF (20 ng / ml), G-CSF (2Ong / ml), BMP-4 (15 ng / ml), IL-6 (10 ng / ml), IGF-I (10 ng / ml), endothelial cell growth supplement factor (ECGS, 100 μg / ml), and Epo (3 U / ml). After 1 week of culture, non-adherent hematopoietic cells were removed by gentle pipetting and used directly for hematopoietic CFU analysis. After removing non-adherent cells, the adherent population was cultured in EGM-2 endothelial cell medium (Cambrex TM ) for more than one week, and then vWF expression was examined.

[0300] When individual BL-CFCs were transferred to liquid culture containing growth factors that support the growth of hematopoietic and endothelial lineages, both nonadherent and adherent cells gave rise to more than 60% (15 of 24) BL-CFCs (Figs. 18d and 18e, Table 2), and after replated in semisolid medium supplemented with various hematopoietic cytokines, 65% (16 of 24) of these formed hematopoietic colonies, including erythroid (Fig. 18i), myeloid (Figs. 18j and 18k), and multilineage (Fig. 18m) colonies. More than 95% (23 of 24) of individual BCs gave rise to adherent, stromal-like cells that took up Ac-LDL (Fig. 18g), expressed vWF (Fig. 18h), and formed capillary structures in Matrigel (Fig. 18f).

[0301] In the second method, individual BCs from primary and secondary mother colonies were picked and divided into two groups, one group tested for hematopoietic CFU and the other group tested for endothelial progenitor cell formation. For primary BCs, over 90% (22 out of 24) gave rise to hematopoietic and endothelial lineages, while 100% and 92% gave rise to hematopoietic and endothelial cells, respectively (Table 3). Similarly, seven individual BCs from limiting dilution experiments were also examined for their potential to differentiate into hematopoietic and endothelial lineages. Five BCs generated both hematopoietic and endothelial progeny cells (Table 3), showing lower efficiency compared to BCs from regular experiments (71% vs. 92%), which may be due to suboptimal developmental conditions. For 29 secondary BCs, over half (15 out of 29) gave rise to hematopoietic and endothelial lineages, while six (21%) and three (10%) gave rise to hematopoietic or endothelial cell lineages, respectively (Table 3). Even though primary BCs contain a heterogeneous population including precursors of hematopoietic and endothelial cells, secondary limiting dilution experiments clearly demonstrate the presence of hemangioblasts in primary BCs.

[0302] [Table 3]

[0303] Hematopoietic progenitor cell analysis For hematopoietic progenitor cell analysis, half of the cells were cultured in serum-free hematopoietic colony forming unit (CFU) medium (H4436, Stem Cell Technologies). TM )1ml, 0.5%EX-CYTE (Serologicals Proteins Inc. TM) and tPTD-HoxB4 at 1.5 μg / ml. Cells can also be mixed with serum-free hematopoietic colony forming cell assay medium containing Iscove's MDM (1.0% methylcellulose, 1-2% bovine serum albumin, 0.1 mM 2-mercaptoethanol, 10 μg / ml rh-insulin, 200 μg / ml iron-saturated human transferrin, 20 ng / ml rh-GM-CSF, 20 ng / ml rh-IL-3, 20 ng / ml rh-IL-6, 20 ng / ml rh-G-CSF, 3 units / ml rh-Epo, 50 ng / ml rh-SCF) and 1.5 to 5 μg / ml PTD-HoxB4 fusion protein. The cell mixtures were plated into untreated 12-well cell culture plates and cultured at 37° C. for 10-14 days.

[0304] Hematopoietic CFUs were examined under a microscope 10-14 days after initial plating. For morphological characterization, single hematopoietic CFUs were picked, washed twice with PBS, cytospun onto glass slides, stained with Wright-Giemsa dye, and cell morphology was examined under a microscope.

[0305] As can be seen in FIG. 1, illustrative of the hematopoietic potential of hemangioblasts, hematopoietic CFUs were observed from hemangioblasts generated from H1-GFP ES cells.

[0306] The morphologies of other hematopoietic CFUs observed are illustrated in Figures 2-6. The CFU for erythrocytes (CFU-E) was characterized by the morphology seen in Figure 2. The polyfunctional CFU (CFU-GEMM / Mix) was characterized by the morphology shown in Figures 3 and 4. The CFU for granulocytes (CFU-G) and the CFU for macrophages (CFU-M) were characterized by the morphology shown in Figure 5. The CFU for granulocytes / macrophages (CFU-GM) and the CFU for megakaryocyte macrophages (CFU-Mk) were characterized by the morphology shown in Figure 6.

[0307] As another illustration of the hematopoietic potential of hemangioblasts, when single cell suspensions were plated in serum-free methylcellulose medium containing various cytokines as mentioned above for 10-14 days, erythroid colonies (Fig. 17a), granulocytic (Fig. 17b), macrophage (Fig. 17c) and multilineage (Fig. 17d) hematopoietic cells were further observed. PACS analysis (Fig. 17v-y), Wright-Giemsa staining (Fig. 17e-g) and immunostaining (Fig. 17i-k) using antibodies against CD235a (erythroid), CD13 (myeloid) and CD45 (leukocyte) confirmed the identity of these hematopoietic lineages. Most CFUs (>50%) were of the erythroid core lineage and formed within a few days after plating (see e.g. Fig. 1).

[0308] The erythroid colonies resembled those of primitive erythroid cells with a bright red color, and Wright-Giemsa staining indicated that all erythrocytes were nucleated (Figure 17e-g). To further distinguish the stages of erythroid development, CFU colonies were analyzed for the expression patterns of β-cluster globin genes (Lu et al. 2004 Blood (103): 4134-4141). All erythroid and multilineage colonies expressed predominantly germinal ε-globin genes, and no β-globin gene message was detected (data not shown), suggesting a primitive yolk sac-like state.

[0309] For immunological characterization, CFU-E, CFU-G, and CFU-Mix cells cytospun onto glass slides were stained with anti-human CD235a, CD13, and CD45 antibodies. For FACS analysis shown in Figure 17m-p, CFU cells collected from one well were stained with mouse IgG1 isotype control, CD235a, CD13, and CD45 and analyzed on a Coulter flow cytometer. Wright-Giemsa staining, immunostaining, and FACS analysis confirmed the identity of these hematopoietic lineages (Figure 17e-p).

[0310] For the FACS analyses given in Figure 17v-y, a LSRII flow cytometer (BD Biosciences) was used. TMThree-color cytometric analysis was performed using standard procedures at BD Biosciences. Cells were collected from wells containing various types of hematopoietic CFU and isolated by diluting the methylcellulose with 5 volumes of IMDM medium. Aliquots of single cell suspensions were stained with isotype control or antigen-specific antibodies. The antibody combinations used were CD235a conjugated to fluorescein-isothiocyanate (FITC), CD45 conjugated to R-phycoerythrin (R-PE), and CD13 conjugated to allotropic cyanin (APC). Aliquots of cells were also stained with mouse IgG conjugated to APC, PE, and FITC as an isotype control (BD Biosciences). TM Samples were run on an LSRII flow cytometer and analyzed using FlowJo. TM Analysis was performed with version 6.3.2 software. Endothelial progenitor cell analysis For endothelial progenitor cell analysis, the remaining fraction of cells was incubated for 3-7 days in EGM-2 or EGM-2MV complete medium (Cambrex TM ) on fibronectin-coated plates (BD Bioscience TM ). The ability of isolated hemangioblast colonies to differentiate into endothelial cells was confirmed using tube formation on Matrigel, LDL uptake, and immunohistochemistry to detect the presence of endothelial cell surface markers. Tube formation on Matrigel Matrigel (BDBioscience TM) was added to each well of a 4-well tissue culture plate (0.2 ml) or a 96-well plate (0.05 ml) and allowed to solidify for 45-90 minutes at 37°C. After gel formation, 0.2-0.3 ml (e.g., 0.1 ml for a 96-well plate) of the cell suspension was placed on top of the Matrigel in EGM-2 or EGM-2MV medium containing 0.5-1.5X105 cells derived from the cultures and incubated at 37°C, 5% CO2. The formation of capillary-like (tube) structures was checked after 16-24 hours of incubation. Figure 7 shows representative photographs of tube-cord formation of hemangioblasts derived from H9 (a) and ACT30 (b) cells; after replated on Matrigel substrate medium, the hemangioblasts formed capillary-like structures and also gave rise to adherent cells that absorbed Ac-LDL. Figure 8(a) shows a representative photograph of tube-cord structures of hemangioblasts with endothelial potential derived from H1-GFP cells, and Figure 17q shows capillary-like structures formed on Matrigel after replating of adherent cells derived from hemangioblasts. Such cells can take up AC-LDL (data not shown). AC-LDL absorption Hemangioblasts grown on fibronectin-coated wells for 3–7 days were incubated with Alexa Fluor 647-labeled AC-LDL (Invitrogen TM ) at 10 μg / ml and incubated for 4-6 hours. Cells were then washed 3 times with 1XPBS and fixed with 4% paraformaldehyde for 30 minutes. AC-LDL absorption was visualized under a fluorescent microscope. For example, Figure 8b shows AC-LDL absorption of hemangioblasts derived from Hl-GFP cells. Figures 17z and 17aa show that incubation with Alexa Fluor594-labeled AC-LDL exhibits a punctuate staining pattern characteristic of endothelial cells. immunocytochemistry Hemangioblasts grown on fibronectin-coated wells for 3-7 days were washed three times with 1X PBS and fixed with 4% paraformaldehyde for 30 min. After permeabilization, cells were stained with primary anti-human vWF (Dako Biosciences) for expression of von Willebrand factor (vWF), PECAM-1 (CD31), VE-cadherin, KDR, and CD34. TM or Chemicon TM ), PECAM-1 and KDR (Cell Signaling Technologies TM ), VE-cadherin (R&D Systems TM ), and CD34 (Dako) antibodies at 4°C overnight, and then incubated with either Lodamin or FITC (Jackson Laboratory TM The expression of vWF protein was monitored by incubation with a goat anti-mouse IgG-Alexa Fluor 647 secondary antibody (Jackson Laboratory, NY, USA) for 30-60 min. TM ) for 30-60 min. After a final wash, the cells were checked under a fluorescent microscope. Figure 9 shows vWF expression in hemangioblasts derived from Hl-GFP ES cells. Figure 17s shows vWF expression (arrows) in hemangioblast-derived endothelial cells. Adherent cells in the vascular-like structures were positive for PECAM-1 (CD31), KDR and VE-cadherin (Figures 17t, 17u, and 17z-17cc).

[0311] Neovascularization was also observed when adherent cells or hand-picked BL-CFCs were mixed with Matrigel and implanted in vivo for 4-5 weeks. Histological examination of the Matrigel plugs confirmed the presence of microtubules that were immunoreactive with human-specific nuclear and vWF antibodies. See Figures 10 and 11. Example 3: Methods for producing and purifying representative HOXB4 proteins A representative HOXB4 protein is produced and purified, which is a TAT-HA-HOXB4 fusion protein.

[0312] The pTAT-HA-HoxB4 expression vector was generated by cloning a PCR fragment (sense primer 5'-TAC CTA CCC ATG GAC CAC TCG CCC-3' (SEQ ID NO:16) and antisense primer 5'-TCG TGG CTC CCG AAT TCG GGG GCA-3' (SEQ ID NO:17)) containing the HoxB4 ORF (open reading frame) with and without the N-terminal 32 amino acids into the NcoI and EcoRI sites of the pTAT-HA expression vector (amplified from Dr. S. F. Dowdy, University of California San Diego, LaJolla, CA), which contains the N-terminal TAT PTD. The generated pTAT-HA-HoxB4 plasmid was confirmed by sequence analysis and transformed into BL21(DE3)pLysS bacteria (Invitrogen). TM ) was transformed into

[0313] Bacterial cells growing in logarithmic phase were induced with 1 mM IPTG for 4 h at 30°C and harvested by centrifugation. Since the 6xHis-fused recombinant TAT-HoxB4 protein was found to be sequestered in inclusion bodies by the host bacteria, the cells were disrupted by sonication in a denaturing solution (6 M guanidinium, 20 mM NaPO4, and 0.5 M NaCl, pH 7.8), and then the 6xHis-fused recombinant TAT-HoxB4 protein was encapsulated in nickel resin (ProBond resin, Invitrogen). TM After several washes, the TAT-HoxB4 fusion protein was eluted with 20 mM NaPO4, pH 4.0, 0.5 M NaCl, 8 M urea, and 10 mM imidazole. The TAT-HoxB4 fusion protein was then desalted on a Sephadex G-25 column into IMDM medium, aliquoted, and immediately stored at -80°C.

[0314] The purity and concentration of the TAT-HoxB4 fusion protein were determined by SDS-PAGE gel electrophoresis and visualized by Coomassie blue staining. The purified TAT-HA-HoxB4 recombinant protein acts as a ≈37 KD protein. The concentration of the purified TAT-HA-HoxB4 protein was assessed by comparison with a BSA protein standard on the same gel.

[0315] Based on computer modeling, Ho et al. (2001 Cancer Res 61:474-477) demonstrated that the modified PTD possesses significantly improved protein delivery capability compared to the native TAT PTD both in vitro and in vivo. To prepare the modified PTD-HA-HoxB4 fusion protein, sense 5'-CGA TGG GGA TCC GGC TAC GCA CGC GCA GCT GCG CGC CAG GCT CGC GCC GGT GGA TCC ACC ATG-3' (SEQ ID NO: 18) and antisense 5'-CAT GGT GGA TCC ACC GGC GCG AGC CTG GCG CGC AGC TGC GCG TGC GTA GCC GGA TCC CCA TCG-3' (SEQ ID NO: 19) oligos were digested with BamHI and purified using a kit (Qiagen TM ) and purified. The TAT fragment in the pTAT-HA-HoxB4 plasmid was then replaced with the modified PTD fragment, and the triplicated PTD insert was selected based on restriction fragment size and confirmed by DNA sequence analysis. This plasmid is referred to as ptPTD-HA-HoxB4. The pTAT-HA-HoxB4 and ptPTD-HA-HoxB4 plasmids were transformed into BL21(DE3)pLysS bacteria (Invitrogen) and purified using the ELISA kit. TM) and induced HoxB4 fusion protein expression by adding 1 mM IPTG for 4 h at 30°C. The 6xHis-fused recombinant TAT-HoxB4 and tPTD-HoxB4 proteins were sequestered in inclusion bodies by the host bacteria, and the cells were disrupted by sonication in a denaturing solution (6 M guanidinium, 20 mM NaPO4, and 0.5 M NaCl, pH 7.8), after which the 6xHis-fused TAT-HoxB4 and tPTD-HoxB4 recombinant proteins were immobilized on nickel resin (ProBond resin, Invitrogen). TM After several washes, the TAT-HoxB4 and tPTD-HoxB4 fusion proteins were eluted with 20 mM NaPO4, pH 4.0, 0.5 M NaCl, 8 M urea + 10 mM imidazole. The TAT-HoxB4 and tPTD-HoxB4 fusion proteins were desalted on a Sephadex G-25 column into IMDM medium, aliquoted, and immediately stored at -80°C. The purity and concentration of the TAT-HoxB4 and tPTD-HoxB4 fusion proteins were determined by SDS-PAGE gel electrophoresis and visualized by Coomassie blue staining. The purified TAT-HoxB4 and tPTD-HoxB4 recombinant proteins act as ≈38 KD proteins. The concentrations of purified TAT-HoxB4 and tPTD-HoxB4 proteins were estimated by comparison with BSA protein standards on the same gel. For stability studies, TAT-HoxB4 and tPTDHoxB4 proteins were added to Stem line II medium or IMDM medium containing 5% FBS containing live hESCs in Ultra low 24-well plates and cultured at 37°C. Aliquots of medium were removed at various times, subjected to SDS-PAGE gel electrophoresis, and visualized by Coomassie blue staining.

[0316] In the literature [Beslu et al. (2002 Blood 1 00:22a)], the first 31 amino acids of the N-terminus of HoxB4 were omitted for its function, and the fact that the removal of the N-terminal 33 amino acids produced a more stable protein was recorded. The results confirmed that the full-length TAT-HoxB4 protein was unstable, forming a ≈30 KD protein after several melting cycles, while the N-terminally deleted TAT-HoxB4 and tPTD-HoxB4 proteins retained their original structure after repeated melting and overnight incubation at 37°C (Figure 19C). As shown in Figure 19A, tPTD-HoxB4 and TATHoxB4 were relatively pure proteins after multiple column chromatography under denaturing and desalting conditions. The purified tPTD-HoxB4 protein appeared as a single ≈38 KD band under native SDS-PAGE, with maximal solubility in PBS or IMDM medium at 250-300 μg / ml, as estimated by comparison with a BSA protein standard. Similar results were obtained with the TAT-HoxB4 fusion protein.

[0317] The literature [Krosl et al. (2003 Nat Medicine 9:1428-1432)] described that TAT-HoxB4 protein is highly unstable in FBS-containing environments and must be renewed every 2 hours to retain biological function. The results presented herein confirmed that tPTD-HoxB4 is unstable in medium containing 5% FBS, with a half-life of less than 1 hour (Figure 19B). FBS pretreatment at 70°C for 30 minutes eliminated most of the enzyme activity. To further examine its stability, tPTD-HoxB4 protein was added to serum-free stem line medium and cultured with hESCs at 37°C. As shown in Figure 19C, substantial fragments of tPTD-HoxB4 protein were detected even after 48 hours of culture in hESCs. Example 4: Matrigel plugs implanted in SCID mice generate endothelial cells Hemangioblast-derived endothelial cells or pure hemangioblasts derived from Hl-GFP human ES cells (1 × 10 6 Cells) and 700 μl of Matrigel (BD Biosciences TMThe cells were then resuspended in 4-week-old SCID mice (Jackson Laboratory TM ) was injected subcutaneously into the spinal region of the rat. Five weeks after injection, the Matrigel plugs were removed from the animals and frozen specimens were prepared from the plugs. Cross sections of the Matrigel plugs were fixed and stained with standard hematoxylin and eosin (H&E) staining as well as anti-human specific nuclear antibody (MAB1281, Chemicon). TM ) and human specific anti-vWF antibody followed by the corresponding fluorescent dye labeled secondary antibody (goat anti-mouse IgG-Texas Red secondary for MAB1281). Figure 10 demonstrates blood vessels in cross sections of H&E stained Matrigel plugs, and Figure 11 demonstrates that blood vessels in cross sections of Matrigel plugs are human hemangioblast derived cells positive for human specific nuclear antibody. Example 5: In vivo ocular ischemia / reperfusion studies Using two techniques, intravitreal injection and adoptive transfer, we directly examined the contribution of hES-derived hemangioblasts to the repair process in vivo. Ischemia / reperfusion (I / R) injury induced elevated intraocular pressure. I / R injury models cause damage to the retinal endothelium, including vaso-obliteration and the generation of acellular capillaries. Seven days after retinal I / R injury, mice were either systemically injected via retro-orbitalsinus (n=13) or intravitreally injected (n=4) with fluorescently labeled hemangioblasts harvested from day 6 hemangioblasts (also called "hES-BCs").

[0318] One day after injection, mice were euthanized, and retinas were removed and labeled with rhodamine-conjugated Ricinus communis agglutinin I. This agglutinin binds to a glycoprotein specifically expressed by endothelial cells and is therefore used to fluorescently label the external surface of blood vessels. Confocal microscopy demonstrated green fluorescent labeling of hemangioblast (hES-BC)-derived endothelial cells, which represented 33.5±10% of the retinal vasculature of the injured eye. No green fluorescence was observed in control eyes (FIGS. 20a and 20b, respectively), demonstrating the fact that hemangioblast (hES-BC) cells homed back to the acellular regions assimilated only to the injured area and caused reendothelialization of non-perfused acellular capillaries in the injured retina (FIGS. 21a and 21b). This recovery process was observed in all animals (n=17) in the same manner.

[0319] A second group of I / R injured animals (n=6) were handled as described above and then perfusion-fixed with 3-5 ml of TRITC-conjugated dextrin before sacrifice to visualize the vascular lumen of blood vessels and conclusively demonstrate that blood vessels restored by such cells were open and therefore functional. Red staining represented open blood vessels, green staining demonstrated hemangioblast (hES-BC) derived endothelial cells, and yellow staining indicated the locations where hemangioblast (hES-BC) cells had generated open vasculature. Figure 21d illustrates large blood vessels with yellow (red and green, appearing as light grey) fluorescence 2 days after hES-BC cell injection, indicating that the cells had integrated in the vessel wall and the vessels were perfused. Particularly notable in this animal were the numerous microvessels that were only observed in green, which appeared to represent new collateral compensatory neovasculature that had not yet matured sufficiently to generate open lumens and therefore remained dissected. Figure 20c illustrates the uninjured control eye of the same animal, clearly demonstrating that BC cells were not associated in any way with the uninjured vasculature. In retinal mounts from mice sacrificed 7 days after hES-BC cell injection, the number of green vessels (non-lumenized, non-perfused vessels) was lower compared to mice sacrificed 2 days after hES-BC cell injection. There were many more yellow vessels (appearing in light grey or white), representing vessels (green) derived from hES-BC cells perfused with TRITC-dextrin (red). This represented fully functional vessels, demonstrating that hemangioblast (hES-BC) cells had integrated into the vasculature of the injured eye, further expanding the area with perfused microvessels (Figure 2Oe).

[0320] Fluorescence immunohistochemistry shows the injured vasculature in cross sections of I / R injured mouse eyes, colocalizing hemangioblast (hES-BC) cell-derived endothelial cells, and a representative cross section of a ganglion cell layer blood vessel in the retina from an injured eye is shown (Figure 2Of). Colocalization of anti-CD31 and human nuclear antigen staining of blood vessels indicates that some of the endothelial cells in the vasculature were derived from hemangioblast (hES-BC) cells. Figure 20f shows a high-resolution photograph of a blood vessel lumen in the ganglion cell layer adjacent to the internal limiting membrane. The lumen is surrounded by endothelial cells (Figure 2Of, arrow, CD31) and mature endothelial cells derived from hemangioblast (hES-BC) cells (Figure 2Of, arrow, human nuclear antigen). method For the in vivo ischemia / reperfusion studies, C57BL6 / J mice (Jackson Laboratory) were 7 to 10 weeks old at the beginning of the study. Ischemia / reperfusion injury induced an increase in intraocular pressure (IOP). Mice were placed under inhalation anesthesia (isoflurane vapor) during induction of ischemia. The anterior chamber of the eye was cannulated with a 30-gauge needle attached to a saline infusion line, and the anterior chamber was subjected to hydrostatic pressure (80-90 mmHg, measured by TonoPen, Medtronic Solan, Jacksonville, Fla.) for 2 h. This induces retinal ischemia as confirmed by iris albinism and loss of red reflex. After 120 min, the needle was removed and IOP was normalized, which induces reperfusion injury. The contralateral eye served as a control.

[0321] Seven days after retinal ischemia-reperfusion injury, mice were injected systemically (n = 13) or intravitreally (n = 4) with fluorescently labeled hemangioblasts harvested from day 6 hES-BCs via retro-orbital sinus. Fluorescent labeling of hemangioblasts was performed using the dye PKH-67 (Sigma-Aldrich) according to the manufacturer's instructions. TM For systemic injections, each mouse received 4 × 10 5 For intravitreal injections, each mouse received 5 × 104 2 μl of hemangioblasts were administered. The contralateral control eyes of the intravitreal injection group were injected with an equivalent volume of sterile isotonic saline. One day later, mice were euthanized, and the eyes were removed and fixed in 4% paraformaldehyde for 1 hour. After washing, the eyes were opened with a circumferential limbic incision to remove the cornea and lens, followed by removal of the vitreous humor. The intact neural retina was then gently removed from the underlying choroid. The intact retina was placed in permeabilization buffer. The retinas were isolated and cultured overnight at 4°C in 1:1000 rhodamine-conjugated R. communis agglutinin I (Vector Laboratories) in 10 mM HEPES, 150 mM NaCl, and 0.1% Tween 20 (pH 7.5). After 24 hours, retinas were washed with 10 mM HEPES and 150 mM NaCl for 24 hours at 40°C and then mounted on cover slips for imaging using an MRC-1024 Confocal Laser Scanning Microscope (BioRad) at the Light Microscopy Facility at the University of Florida (Gainesville). Alternatively, retinas were imaged using a Zeiss laser scanning confocal microscope.

[0322] For microscopic examination using a Zeiss confocal laser microscope, the retina was placed on a glass coverslip and its curvature was flattened by a number 4 or 5 radial incision extending from the ciliary margin to within 1 mm of the optic disc. Simultaneous red and green fluorescent digital image capture of the prepared retina was performed with a Zeiss confocal laser microscope using a 1OX or 2OX objective. The z-section depth was held constant at 2 μm and the entire retinal vasculature (including superficial, intermediate, and deep vascular plexi) was scanned through the thickness of the neural retina, typically generating 25-35 z-section images per fluorescent channel. Image captures were made at random locations around the intermediate retina, as this is the region where most vascular damage is typically observed in this model. Three-dimensional z-stacks were imaged using ImageJ software (ImageJ 1.37c, Wayne Rasband, National Institutes of Health, USA, 2014) such that the three vascular plexi were seen in a single two-dimensional plane. http: / / rsb.info.nih.gov / ij / index.html ) was used to flatten it.

[0323] The second group of mice (n=6) with I / R injury to the right eye had approximately 2×10 5 PKH-67-labeled hES-BCs or hemangioblasts were administered in a left retro-orbital sinus injection in 100 μl. Mice were euthanized 2 and 7 days after injury (n=3 each) and perfused with 3-5 ml TRITC-conjugated dextrin (Sigma-Aldrich, St. Louis, MO, 5 mg / ml) in 4% buffered paraformaldehyde through the left ventricular hole. The eyes were then removed, dissected, and the retinas flat-mounted for digital image capture by confocal microscopy as previously described. The contralateral (uninjured) eyes served as the control group. Such a perfusion fixation method allows visualization of open vessels, since only functional vessels can be perfused. Example 6: In vivo restoration of diabetic models In vivo recovery was studied in spontaneously diabetic obese BBZDR / Wor rats with >3 months of type 2 diabetes that develop many features of preproliferative diabetic retinopathy, including acellular capillary and endothelial dysfunction, adventitial cell loss, and hematoretinal destruction (see Shi et al. 1998 Blood(92):362-367; Asahara et al. 1997 Science(275):964-967; Kalka et al. 2000 Circ. Res.(86):1198-1202; Murohara et al. 2000 J. Clin. Invest.(105):1527-1536; Urbich et al. 2004 Trends Cardiovasc. Med.(14):318-322).

[0324] The ability of BCs to participate in reendothelialization of damaged capillaries was examined using male obese type 2 diabetic BBZDR / Wor rats (Biomedical Research Models, Worcester, MA) with diabetes persisting for more than 3 months, as well as lean age-matched and sex-matched controls. Rats were immunosuppressed with intramuscular injections of cyclosporine (2 mg / kg / day) for 1 day prior to cell administration and continued for the duration of the study. hES-EBc were pelleted by centrifugation and 3 × 10 4 The cells were resuspended in sterile saline at a concentration of 100 / μl and 5 μl of this suspension was injected into each eye of six diabetic and control rats, respectively. Animals were euthanized on days 2 (n=3 diabetic, n=3 control) and 7 (n=3 diabetic, n=3 control) after hES-BC administration. Four of the six eyes from each treatment group were processed and retinal planum preparations were prepared as described in the mouse I / R study utilizing rhodamine-conjugated R. communis to visualize blood vessels. Rat retinas were further stained with single clone anti-GFP (Chemicon TM 1:250 in PBS) followed by FITC-conjugated goat anti-mouse IgG (Abeam, Temecula, CA). TMGFP-expressing BCs were visualized by reaction with 1:200 PBS (Citroenzyme Q100, Cambridge, MA; 1:200 in PBS). Digital image capture was performed with a focus microscope as described in the mouse I / R study.

[0325] The remaining two eyes from each treatment group were fixed, dehydrated in 2.5 M sucrose, and then inserted into OCT medium for cryosectioning. More than 50 sections (10 μm thick, keeping every 10th section) were collected and stained with monoclonal rat anti-CD31 (Abeam TM , 1:100 in PBS), rabbit anti-human vWF (Sigma-Aldrich TM After reacting with FITC-conjugated anti-rat IgG (Sigma-Aldrich, 1:320 in PBS) and AMCA-conjugated goat anti-rabbit IgG (Vector Laboratories, 1:200 in PBS) and 1:50 biotinylated anti-human nuclear antigen, the cells were incubated with 100 mM sodium dodecyl sulfate (NaCl) and 10 mM glycerol (NaCl) for 1 h. TM Sections were then incubated with 1:75 PBS (Vector Laboratories, Burlingame, VT) and finally with Texas Red-conjugated streptavidin (Vector Laboratories, 1:500 in PBS) (with appropriate washes between incubations). Sections were then incubated with Vectashield antifade medium (Vector Laboratories, TM ) and digital images were captured with a Zeiss Axioplan 2 fluorescence microscope coupled to a spot CCD camera.

[0326] As in the I / R mouse model, intravitreally administered hES-BC cells (angioblasts) were extensively integrated into areas with both wide and narrow vasculature (Figs. 21a and 21b). Fig. 20b also shows a large vessel (light grey vessel on the right side of Fig. 21b) that is almost entirely green and narrows very rapidly. Such "pinched-off" vessels are characteristic of preproliferative diabetic retinopathy, where microthrombi cause vascular degeneration and downstream ischemia. Extensively branching and continuous bovine vessels from such pinched wide vessels show a high degree of integration of green BC cells (illustrated in light grey in Fig. 21). In contrast, green hES-BC cells were not colocalized with residual vessels in the eye from nondiabetic control animals. Eyes from non-diabetic control animals confirmed that BC cells did not integrate into the intact vasculature but instead remained as sheets at the edge of the retina after intravitreal administration of hES-BC cells (Figure 21c, light grey area).

[0327] Immunohistochemical analysis confirmed colocalized staining wi...

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[Claim 1] The invention described in the specification.

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