Mesenchymal stromal cells and related uses

Abstract

The present invention relates generally to novel preparations of hemangioblast-derived mesenchymal stromal cells (MSCs) and methods of using such MSCs to treat disease conditions. The methods of the present invention produce substantial numbers of MSCs with a youthful phenotype that retain potency, which are useful in treating pathological conditions. For example, a pharmaceutical preparation suitable for use in a mammalian patient, comprising at least 10 6 Provided is a pharmaceutical preparation comprising mesenchymal stromal cells and a pharmaceutically acceptable carrier, wherein the mesenchymal stromal cells have the replicative capacity to undergo at least 10 population doublings in cell culture, with less than 25% of the cells undergoing cell death, senescence, or differentiation into non-MSC cells by the 10th doubling.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is filed under the heading "METHODS OF GENERATING MESENCHYMAL STROMAL CELLS This application claims priority to U.S. Provisional Patent Application No. 61 / 565,358, filed November 30, 2011, entitled "METHOD FOR USE IN HEMANGIOBLASTS" (Attorney Docket No. 75820.210001).

[0002] The present invention relates to the use of cell-based therapies to, for example, reduce the incidence of pathologies characterized by an inappropriate immune response in a subject, as well as to further affect the root cause of the pathology so that the abnormalities defining the pathology are reversed to normal. In particular, the present invention relates to mesenchymal stromal cells (MSCs) that retain a "youthful" cellular phenotype that confers high efficacy in reducing the incidence of pathologies in a subject. [Background technology]

[0003] Many pathologies, such as transplant rejection, inflammation, and autoimmune disorders, manifest clinically through unnecessary or excessive immune responses within the host. Immunosuppressive therapies have been developed to treat symptoms rather than the underlying causes of pathologies characterized by excessive immune responses. These therapies are effective in downregulating immune function, thus increasing the likelihood of serious adverse events, including cancer and opportunistic infections, as well as cataracts, hyperglycemia, contusions, and nephrotoxicity from drugs such as prednisone, cyclosporine, and tacrolimus.

[0004] Although therapies that do not suppress the entire immune system have been developed, there are still limitations associated with these regimens. Because these immunomodulatory treatments target narrow points within the immune system, they have different side effects, sometimes of reduced severity. Examples of such immunomodulatory therapies include the use of antibodies, such as anti-CD3 or anti-IL2R. While these immunomodulatory therapies are effective in inducing a state of increasing non-responsiveness, discontinuing these immunomodulatory therapies often leads to reversal of the undesirable pathology.

[0005] Mesenchymal stromal cells (MSCs) are multipotent stem cells with the ability to self-renew and differentiate into osteoblasts, chondrocytes, and adipocytes, among other mesenchymal cell lineages. In recent years, intensive research into the multilineage differentiation potential and immunomodulatory properties of human MSCs has demonstrated that these cells can be used to treat a variety of clinical conditions, including immunological disorders and degenerative diseases. Consequently, a steady increase in clinical trials using MSCs has been observed for a wide variety of conditions, including graft-versus-host disease (GVHD), myocardial infarction, and inflammation, as well as autoimmune diseases and disorders. Currently, clinical programs using MSCs rely on the isolation of these cells from adult sources and umbilical cord blood. The high cell numbers required for clinical use of MSCs (up to several million cells per kilogram of patient body weight) require reliable, reproducible, effective, and robust expansion protocols capable of generating large numbers of cells from those isolated from donor sources.

[0006] However, to achieve clinically meaningful cell numbers for cell therapy and tissue engineering applications, active ex vivo expansion of MSCs is essential. During in vivo aging, subsequent ex vivo cell passage of MSCs obtained from umbilical cord blood, fetal, and adult sources (e.g., bone marrow or adipose tissue) may cause replicative stress, chromosomal abnormalities, or other stochastic cell defects, leading to a progressive loss of the proliferative, clonogenic, and differentiation potential of the expanded MSCs, which may ultimately compromise the clinical safety and efficacy of MSCs in therapy. The use of aged MSCs should not be underestimated, as the cells have lost some of their differentiation potential and their secretory profile has also been altered. Senescence of MSCs in culture has been shown to induce cell growth arrest accompanied by telomere shortening, and whereas the propensity for differentiation into osteogenic lineages increases, a continuous decrease in adipogenic differentiation potential has been reported for bone marrow (BM) MSCs with increasing passages. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, several essential problems remain to be solved before MSCs can be used clinically. ESC-derived MSCs can be produced in sufficient quantities and in a highly controllable manner, thus alleviating the problems associated with donor-dependent sources. Long-term MSC engraftment is unnecessary, and there is no fundamental concern about major histocompatibility (MHC) mismatches [7, 8]. In the art, ESC-derived MSCs have been obtained through various methods, including coculture with murine OP9 cells or hand-picking techniques [9-13]. However, these methods are cumbersome and produce MSCs with low yields, variability, and lack of efficacy. Furthermore, it is desirable to maximize the efficacy of infused cells relative to CB-, BM-, or adipose-derived MSCs, both in terms of providing a cell product with a favorable therapeutic index, i.e., the ability to be used at reduced dosages (cell numbers), and / or the ability of MSCs to provide manageable therapies for inflammatory and autoimmune diseases for which CB-, BM-, or adipose-derived MSCs are not fully effective. [Means for solving the problem]

[0008] The present invention relates to mesenchymal stromal cells (MSCs) and methods for producing MSCs. The methods of the present invention produce significant numbers of high-quality mesenchymal stromal cells characterized by a youthful cellular phenotype that confers high potency. In one embodiment of the present invention, MSCs are derived from hemangioblasts. Preparations of the subject MSCs are useful in treating conditions involving unwanted immune responses, such as autoimmune diseases and disorders, and inflammatory diseases and disorders.

[0009] In one aspect, the present invention includes improved preparations of MSCs produced from hemangioblasts using improved methods for culturing hemangioblasts. In exemplary embodiments, the mesenchymal stromal cells of the present invention retain a high level of potency and do not aggregate or aggregate to a substantially lesser extent than mesenchymal stromal cells derived directly from embryonic stem cells (ESCs). Mesenchymal stromal cells produced by any one or more processes of the present invention can retain a higher level of potency and do not aggregate or aggregate to a substantially lesser extent than mesenchymal stromal cells derived from ESCs.

[0010] In one aspect, the present invention provides a pharmaceutical preparation comprising mesenchymal stromal cells, wherein the mesenchymal stromal cells are capable of undergoing at least 10 population doublings, e.g., at least 10 population doublings within about 22-27 days. In another aspect, the present invention provides a pharmaceutical preparation comprising mesenchymal stromal cells, wherein the mesenchymal stromal cells are capable of undergoing at least 15 population doublings, e.g., at least 15 population doublings within about 22-27 days. The pharmaceutical preparations of the present invention can be produced by in vitro differentiation of hemangioblasts. The mesenchymal stromal cells of the present invention can be primate cells, e.g., human cells. The mesenchymal stromal cells of the present invention can be capable of undergoing at least 15 population doublings. For example, the mesenchymal stromal cells of the present invention can be capable of undergoing at least 20, 25, 30, 35, 40, 45, 50, or more population doublings. The preparations of the present invention may have a % solubility in the blood of less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, or ... The preparations of the invention may contain less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, less than 0.0009%, less than 0.0008%, less than 0.0007%, less than 0.0006%, less than 0.0005%, less than 0.0004%, less than 0.0003%, less than 0.0002%, or less than 0.0001% pluripotent cells. Preferably, the preparations of the invention are free of pluripotent cells. Preparations of the invention may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% mesenchymal stromal cells.

[0011] In one embodiment, at least 50% of the mesenchymal stromal cells are positive for (i) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, (ii) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC, or (iii) any combination thereof. In another embodiment, at least 50% of said mesenchymal stromal cells are positive for (i) at least two of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, and (ii) all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC. In yet another embodiment, at least 50% of the mesenchymal stromal cells (i) are positive for all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC, and (ii) do not express or express low levels of at least one of CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and TLR3. Furthermore, at least 60%, 70%, 80%, or 90% of the mesenchymal stromal cells may be positive for (i) one or more of CD10, CD34, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, or (ii) one or more of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

[0012] In one embodiment, the pharmaceutical preparation of the present invention comprises an amount of mesenchymal stromal cells effective to treat or prevent an unwanted immune response in a subject in need thereof. The pharmaceutical preparation of the present invention may further comprise other cells, tissues, or organs for transplantation into a recipient in need thereof. Exemplary other cells or tissues include RPE cells, skin cells, corneal cells, pancreatic cells, liver cells, or cardiac cells or tissues containing any of the foregoing cells.

[0013] In another embodiment, the mesenchymal stromal cells of the present invention are not derived from bone marrow, and the potency of the preparation in an immunomodulatory assay exceeds the potency of a preparation of bone marrow-derived mesenchymal stromal cells. Potency can be tested by an immunomodulatory assay to determine the EC50 dose.

[0014] In one embodiment, a preparation of the invention retains about 50% to 100% of its proliferative potential after 10 population doublings.

[0015] In another embodiment, the mesenchymal stromal cells of the pharmaceutical preparation of the present invention are not derived directly from pluripotent cells, and said mesenchymal stromal cells (a) do not aggregate or aggregate in substantially lesser amounts than mesenchymal stromal cells derived directly from ESCs, (b) disperse more readily when dividing compared to mesenchymal stromal cells derived directly from ESCs, and (c) disperse in equal numbers. (d) when starting from ESCs, are more numerous than mesenchymal stromal cells derived directly from ESCs, and / or (e) acquire characteristic mesenchymal stromal cell surface markers earlier than mesenchymal stromal cells derived directly from ESCs.

[0016] The present invention further encompasses a method for producing mesenchymal stromal cells, comprising culturing hemangioblasts under conditions that result in mesenchymal stromal cells. The hemangioblasts may be cultured under feeder-free conditions. Furthermore, the hemangioblasts may be disposed on a matrix containing, for example, transforming growth factor β (TGF-β), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and / or platelet-derived growth factor (PDGF). The matrix may be selected from the group consisting of laminin, fibronectin, vitroctin, proteoglycan, entactin, collagen, collagen I, collagen IV, heparan sulfate, Matrigel (a soluble preparation obtained from Engelbreth-Holm-Swarm (EHS) murine sarcoma cells), human basement membrane extract, and any combination thereof. The matrix may also include a soluble preparation obtained from Engelbreth-Holm-Swarm murine sarcoma cells.

[0017] In one embodiment, the mesenchymal stromal cells of the present invention are mammalian cells. Preferably, the mesenchymal stromal cells of the present invention are human, canine, or equine cells.

[0018] In one aspect, the hemangioblasts can be cultured in a medium containing αMEM. In another aspect, the hemangioblasts can be cultured in a medium containing serum or a serum replacement. For example, the hemangioblasts can be cultured in a medium containing αMEM supplemented with 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% fetal bovine serum. In further exemplary embodiments, the medium can contain a higher percentage of fetal bovine serum, e.g., 20% or more, e.g., at least 25%, at least 30%, at least 35%, at least 40%, or even higher percentages of fetal bovine serum. The hemangioblasts can be cultured on the matrix for at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0019] In one embodiment, the hemangioblasts or hemangio-colony forming cells are differentiated from pluripotent cells, such as iPS cells or blastomeres. The pluripotent cells can be derived from one or more blastomeres without destroying a human embryo. Furthermore, hemangioblasts can be differentiated from pluripotent cells by a method comprising the steps of: (a) culturing the pluripotent cells to form cell clusters. In one embodiment, the pluripotent cells are cultured in the presence of vascular endothelial growth factor (VEGF) and / or bone morphogenetic protein 4 (BMP-4). VEGF and BMP-4 can be added to the pluripotent cell culture within 0 to 48 hours of initiating the cell culture, with the VEGF optionally being added at a concentration of 20 to 100 nM / mL and the BMP-4 optionally being added at a concentration of 15 to 100 ng / mL.

[0020] In one embodiment, hemangioblasts are differentiated from pluripotent cells by a method further comprising the step of (b) culturing the single cells in the presence of at least one growth factor in an amount sufficient to induce differentiation of the cluster of cells into hemangioblasts. The at least one growth factor added in step (b) can include one or more of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), Flt3L (FL), thrombopoietin (TPO), EPO, and / or tPTD-HOXB4. The at least one growth factor added in step (b) can be added to the culture within 36 to 60 hours of the initiation of step (a). Preferably, Preferably, one or more of the at least one growth factor added in step (b) can be added to the culture within 40 to 48 hours of the initiation of step (a). The at least one factor added in step (b) may include one or more of bFGF, VEGF, BMP-4, SCF, FL, and / or tPTD-HOXB4. When added in step (b), the concentrations of the growth factors can range approximately as follows: bFGF is about 20 to 25 ng / ml, VEGF is about 20 to 100 ng / ml, BMP-4 is about 15 to 100 ng / ml, SCF is about 20 to 50 ng / ml, FL is about 10 to 50 ng / ml, TPO is about 20 to 50 ng / ml, and tPTD-HOXB4 is about 1.5 to 5 U / ml.

[0021] In another embodiment, the method further comprises (c) optionally dissociating the clusters of cells into single cells. In another embodiment, the method further comprises (d) culturing the hemangioblasts in medium comprising at least one additional growth factor, wherein the at least one additional growth factor is in an amount sufficient to actively proliferate the hemangioblasts or hemangio-colony forming cells. The at least one additional growth factor in (d) can comprise one or more of insulin, transferrin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating growth factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), and / or tPTD-HOXB4. Exemplary concentrations in step (d) include about 10-100 μg / ml insulin, about 200-2,000 μg / ml transferrin, about 10-50 ng / ml GM-CSF, about 10-20 ng / ml IL-3, about 10-1,000 ng / ml IL-6, about 10-50 ng / ml G-CSF, about 3-50 U / ml EPO, about 20-200 ng / ml SCF, about 20-200 ng / ml VEGF, about 15-150 ng / ml BMP-4, and / or about 1.5-15 U / ml tPTD-HOXB4. Steps (a), (b), (c), and / or (d) can be performed in serum-free medium.

[0022] In one embodiment, the method of the invention produces at least 80 million, 85 million, 90 million, 95 million, 100 million, 125 million, or 150 million mesenchymal stromal cells. Hemangioblasts can be harvested at least 10, 11, 12, 13, 14, 15, 16, 17, or 18 days after inducing differentiation of the pluripotent cells. The mesenchymal stromal cells of the present invention can be produced within at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days from the start of inducing differentiation of the pluripotent cells. In another embodiment, the methods of the invention result in at least 80 million, 85 million, 90 million, 95 million, 100 million, 125 million, or 150 million mesenchymal stromal cells produced from about 200,000 hemangioblasts within about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of culture. The mesenchymal stromal cells of the present invention can be produced from hemangioblasts and / or hemangio-colony forming cells at a ratio of hemangioblasts to mesenchymal stromal cells of at least 1:200, 1:250, 1:300, 1:350, 1:400, 1:415, 1:425, 1:440, 1:450, 1:365, 1:475, 1:490, and 1:500. The cells can be human.

[0023] The present invention also contemplates mesenchymal stromal cells derived from hemangioblasts obtained by the methods described. In one embodiment, the present invention comprises mesenchymal stromal cells derived by in vitro differentiation of hemangioblasts, wherein at least 50% of said mesenchymal stromal cells are (i) CD10 , CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC, and (ii) do not express or express low levels of at least one of CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and TLR3. Alternatively, at least 50% of the mesenchymal stromal cells may (i) be positive for all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, or (ii) be positive for all of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC. At least 60%, 70%, 80%, or 90% of these mesenchymal stromal cells may (i) be positive for at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90, or (ii) be positive for at least one of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC. Preferably, the mesenchymal stromal cells of the present invention do not express or express low levels of at least one of CD31, CD34, CD45, CD133, FGFR2, CD271, Stro-1, CXCR4, and TLR3.

[0024] In another aspect, the invention encompasses a preparation of mesenchymal stromal cells as described herein, wherein the preparation has a concentration of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%. %, less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, less than 0.0009%, less than 0.0008%, less than 0.0007%, less than 0.0006%, less than 0.0005%, less than 0.0004%, less than 0.0003%, less than 0.0002%, or less than 0.0001% of pluripotent cells. Preferably, the preparations of the invention are free of pluripotent cells. The preparations of the invention can be substantially purified, and optionally comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% human mesenchymal stromal cells. Preparations of the invention can contain substantially identical levels of p53 and p21 protein, or 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater levels of p53 protein compared to p21 protein. The mesenchymal stromal cells of the invention can be allowed to undergo at least 5 population doublings in culture. Preferably, the mesenchymal stromal cells of the invention are allowed to undergo at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more population doublings.

[0025] In one aspect, the mesenchymal stromal cells of the present invention (a) do not aggregate or aggregate in a substantially smaller amount than mesenchymal stromal cells derived directly from ESCs, (b) disperse more readily when dividing compared to mesenchymal stromal cells derived directly from ESCs, (c) are more numerous than mesenchymal stromal cells derived directly from ESCs when starting with an equal number of ESCs, and / or (d) acquire characteristic mesenchymal stromal cell surface markers earlier than mesenchymal stromal cells derived directly from ESCs. The present invention contemplates pharmaceutical preparations comprising such mesenchymal stromal cells, which contain an amount of mesenchymal stromal cells effective for treating an unwanted immune response. The preparation can contain an amount of mesenchymal stromal cells effective for treating an unwanted immune response and can further include other cells or tissues for transplantation into a recipient in need thereof. Exemplary other cells include allogeneic or syngeneic pancreatic, neural, hepatic, RPE, or corneal cells or tissues containing any of the foregoing. The pharmaceutical preparation of the present invention is useful for treating multiple sclerosis, systemic sclerosis, hematological cancers, Myocardial infarction, organ transplant rejection, chronic allograft nephritis, cirrhosis, liver failure, heart failure, GvHD, tibial fractures, left ventricular dysfunction, leukemia, myelodysplastic syndrome, Crohn's disease, diabetes, chronic obstructive pulmonary disease, osteogenesis imperfecta, homozygous familial hypocholesterolemia, post-meniscectomy management, adult periodontitis, angiogenesis in patients with severe myocardial ischemia, spinal cord injury, bone dysplasia, severe ischemic lower limbs, diabetic foot disease, primary Sjögren's syndrome, osteoarthritis, cartilage defects, laminitis, multiple system atrophy, muscle atrophy The subject MSCs (including preparations or formulations thereof) can be used to treat respiratory conditions, particularly those with an inflammatory component or acute injury, such as adult respiratory distress syndrome, post-traumatic adult respiratory distress syndrome, transplant pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic obstructive bronchitis, bronchitis, allergic reactions, injury due to bacterial or viral pneumonia, asthma, exposure to irritants, and smoking. Furthermore, the subject MSCs (including formulations or preparations thereof) can be used to treat atopic dermatitis, allergic rhinitis, hearing loss (particularly autoimmune hearing loss or noise-induced hearing loss), and psoriasis.

[0026] The present invention encompasses kits comprising the mesenchymal stromal cells or mesenchymal stromal cell preparations described herein. The kits may include frozen or cryopreserved mesenchymal stromal cells or mesenchymal stromal cell preparations. The mesenchymal stromal cells or mesenchymal stromal cell preparations included in the kits may be encapsulated in a cell delivery vehicle.

[0027] Additionally, the present invention contemplates a method for treating a disease or disorder, comprising administering to a subject in need thereof an effective amount of mesenchymal stromal cells or mesenchymal stromal cell preparations described herein. This method further includes transplantation of other cells or tissues, such as retinal, RPE, corneal, neural, immune, bone marrow, liver, or pancreatic cells. Exemplary diseases or disorders that may be treated include multiple sclerosis, systemic sclerosis, hematologic cancers, myocardial infarction, organ transplant rejection, chronic allograft nephritis, cirrhosis, liver failure, heart failure, GvHD, tibia fracture, left ventricular dysfunction, leukemia, myelodysplastic syndrome, Crohn's disease, diabetes, chronic obstructive pulmonary disease, osteogenesis imperfecta, homozygous familial hypocholesterolemia, post-meniscectomy treatment, adult periodontitis, angiogenesis in patients with severe myocardial ischemia, spinal cord injury, bone dysplasia, severe limb ischemia, diabetic foot disease, and the like. These include, but are not limited to, primary Sjögren's syndrome, osteoarthritis, cartilage defects, laminitis, multiple system atrophy, amyotrophic lateral sclerosis, cardiac surgery, systemic lupus erythematosus, living donor kidney allotransplantation, non-malignant red blood cell disorders, burns, radiation burns, Parkinson's disease, microfractures, epidermal necrosis, severe coronary ischemia, idiopathic dilated cardiomyopathy, femoral head necrosis, lupus nephritis, bone void defects, ischemic stroke, post-stroke, acute radiation syndrome, pulmonary disease, arthritis, bone regeneration, uveitis, or a combination thereof. In one embodiment, the disease or disorder is uveitis. In another embodiment, the disease or disorder is an autoimmune disorder, such as multiple sclerosis, or an immune response to allogeneic cells.

[0028] The present invention further encompasses methods for treating bone loss or cartilage damage, comprising administering to a subject in need thereof an effective amount of mesenchymal stromal cells or a preparation of mesenchymal stromal cells described herein. The mesenchymal stromal cells of the present invention may be administered in combination with allogeneic or syngeneic transplanted cells or tissue, such as retinal pigment epithelial cells, retinal cells, or muscle cells.

[0029] The present invention includes methods of culturing hemangioblasts that produce preparations of MSCs that maintain potency despite an increasing number of cell divisions. The products exhibit improved therapeutic properties when administered to a mammalian host in need of such administration. [Brief explanation of the drawings]

[0030] [Figure 1] Figure 1 shows the production of FM-MA09-MSCs from pluripotent cells. This figure shows a microscopic view of the production of mesenchymal stromal cells from ESCs via hemangioblasts. [Figure 2] Figure 2 shows the phenotype of FM-MA09-MSCs derived from hemangioblasts derived from pluripotent cells. This figure shows the percentage of cells positive for MSC surface markers in the initial hemangioblast population (left side of the graph, hemangioblasts on days 7–11) and the percentage of cells positive for MSC surface markers after hemangioblast culture on Matrigel-coated plates (right side of the graph), as well as micrographs of mesenchymal stromal cells derived from hemangioblasts (right panel). [Figure 3] Figure 3 shows the phenotype of mesenchymal stromal cells derived from different culture methods. The figure shows the percentage of cells positive for MSC surface markers after culturing human embryonic stem cells (ESCs) on gelatin-coated plates (left panel), ESCs on Matrigel-coated plates (center panel), and hemangioblasts on Matrigel-coated plates (right panel). [Figure 4] Figure 4 shows the yield of mesenchymal stromal cells from pluripotent cells. The figure shows the yield of cells positive for MSC surface markers obtained from culturing ESCs on gelatin-coated plates (first column—no yield), ESCs on Matrigel-coated plates (second column), and hemangioblasts on Matrigel-coated plates (third column). [Figure 5] Figure 5 shows the acquisition of mesenchymal stromal cell markers. This figure depicts the time over which MSC surface markers are acquired using hemangioblasts (top line) and ESCs (bottom line). [Figure 6]Figure 6 shows the phenotype of mesenchymal stromal cells derived from different culture methods, showing the percentage of cells positive for MSC markers and negative for hematopoietic and endothelial cell markers after culturing ESCs on Matrigel-coated plates (left panel) and hemangioblasts on Matrigel-coated plates (right panel). [Figure 7] Figure 7 shows the differentiation potential of FM-MA09-MSCs. This figure illustrates the differentiation potential of mesenchymal stromal cells derived from hemangioblasts differentiated from MA09 ESCs to form adipocytes and osteocytes. [Figure 8] Figure 8 shows chondrogenic differentiation of MSCs. This figure shows chondrogenic differentiation of MA09 ESC hemangioblast-derived mesenchymal stromal cells by mRNA expression of aggrecan (chondroitin sulfate proteoglycan 1) and collagen IIa. [Figure 9] Figure 9 shows transient expression of CD309 by FM-MA09-MSCs. This figure shows the transient expression of the cell surface marker CD309. [Figure 10A] Figure 10A shows that T cell proliferation in response to mitogens is suppressed by FM-MA09-MSCs. This figure shows the suppression of hemangioblast-derived mesenchymal stromal cells on T cell proliferation induced by chemical stimulation (PMA / ionomycin). [Figure 10B] Figure 10B shows that T cell proliferation in response to antigen-presenting cells is suppressed by FM-MA09-MSCs. This figure shows hemangioblast-derived mesenchymal stromal cell suppression of T cell proliferation caused by exposure to dendritic cells. [Figure 11] Figure 11 shows that T cell proliferation in response to antigen-presenting cells is suppressed by FM-MA09-MSCs. Figure 11A shows that hemangioblast-derived mesenchymal stromal cells were able to increase the percentage of CD4 / CD25 double-positive Tregs induced in response to IL2 stimulation. Figure 11B shows that hemangioblast-derived mesenchymal stromal cells inhibit Th1 secretion of IFNγ. [Figure 12]Figure 12 shows that the pro-inflammatory cytokine IFNg stimulates changes in FM-MA09-MSC surface marker expression. This figure shows that interferon gamma can stimulate changes in MSC surface marker expression and enhance MSC immunosuppressive effects. [Figure 13] Figure 13 shows the increased potency and greater inhibitory effect of FM-MA09-MSCs compared to BM-MSCs. FM-MA09-MSCs exert a greater inhibitory effect on T cells than BM-MSCs (A). Increasing amounts of MSCs in coculture with PBMCs cause a dose-dependent decrease in T cell proliferation in response to PMA and ionomycin. Young (p4) FM-MA09-MSCs are the most potent of all cell types tested (B). FM-MA09-MSCs inhibit T cell proliferation in response to PMA to a greater extent than BM-MSCs do. A 5:1 ratio of PBMCs:MSCs was cocultured for 6 days (C). FM-MA09-MSCs inhibit T cell proliferation in response to increasing amounts of dendritic cells better than BM-MSCs do. In (A-C), the percentage of T cell proliferation was assessed by BrdU incorporation in the CD4+ and / or CD8+ cell populations. [Figure 14] Figure 14 shows that FM-MA09-MSCs enhance Treg induction: early-passage MSCs have a greater effect than late-passage MSCs. Non-adherent PBMCs (different donors) were cultured for 4 days with or without FM-MA09-MSCs + / - IL2. The percentage of CD4 / CD25 double-positive Tregs was assessed by flow cytometry. Young (p6) or old (p16-18) FM-MA09-MSCs were used. Black bars represent the average of six experiments. MSCs had a statistically significant effect on Treg induction overall (p=0.02). [Figure 15]Figure 15 shows enhanced Treg proliferation by FM-MA09-MSCs compared to BM-MSCs. FM-MA09-MSCs induce Treg proliferation better than BM-MSCs (A). The percentage increase in CD4 / CD25 double-positive Tregs is shown. The IL2 condition was set at 1, and the other groups are expressed as the induction rate above this level. MM is MA09-MSCs, BM is bone marrow MSCs, and "p" is the passage number (B). FM MA09-MSCs (MM) induce CD4 / CD25 / FoxP3 triple-positive Tregs better than BM-MSCs (C). The percentage of responding PBMCs that are CD4+ is consistent between the different treatment groups (D). The percentage of responding PBMCs that are CD25+ differs between the different treatment groups. FM-MA09-MSCs induce greater CD25 expression than BM-MSCs. This difference may explain the difference in Treg induction. [Figure 16] Figure 16 shows that FM-MA09-MSCs have greater proliferative potential than BM-MSCs. FM-MA09-MSCs have greater proliferative potential than BM-MSCs. Cumulative population doublings are plotted against days of culture. After initial plating of ESC-derived hemangioblasts or bone marrow-derived mononuclear cells, adherent cells were considered p0 MSCs. Serial MSC passages were replated at a density of 7000 cells / cm2 and harvested (every 3–5 days) when the cultures were approximately 70% confluent. [Figure 17] Figure 17 shows the process of FM-MA09-MSC production: Matrigel effect. Removing cells from Matrigel at early passage (i.e., p2) may temporarily slow MSC proliferation compared to maintaining them on Matrigel until p6. [Figure 18] Figure 18 shows that BM-MSCs and FM-MA09-MSCs undergo chondrogenesis. Safranin O staining (an indicator of cartilage matrix deposition) was performed on paraffin-embedded pellet mass cultures after 21 days. Images are at 40x magnification. [Figure 19]Figure 19 shows that under basal conditions, FM-MA09-MSCs secrete less PGE2 than BM-MSCs, yet the rate of increase upon stimulation with IFNγ or TNFα is greater (A). The amount of prostaglandin E2 secretion (pg / ml) is shown for BM-MSCs compared to FM-MA09-MSCs under basal or various stimulation conditions. The amount of PGE2 is normalized to cell number (B). The basal PGE2 value is set to 1 (black line), and PGE2 secretion under various stimulations is expressed as the rate of increase above the basal level. [Figure 20] Figure 20 shows that FM-MA09-MSCs maintain their phenotype over time. Flow cytometry analysis of different MSC populations is shown (A). Cell surface marker expression of FM-MA09-MSCs is maintained on three different substrates and compared to BM-MSCs (B). Cell surface marker expression of FM-MA09-MSCs is assessed over time (using serial subculture as shown). [Figure 21] Figure 21 shows that FM-MA09-MSCs express less Stro-1 and more CD10 than BM-MSCs. Flow cytometry analysis of different MSC populations is shown. Stro-1 expression is lower in FM-MA09-MSCs than in BM-MSCs at the indicated passage numbers. CD10 expression is higher in FM-MA09-MSCs than in BM-MSCs. Other markers are similar in both MSC populations. [Figure 22] Figure 22 shows that Stro-1 and CD10 expression in 10 different lots of early-passage FM-MA09-MSC consistently showed low Stro-1 expression and intermediate-range CD10 expression. Flow cytometry analysis of different MSC populations is shown. Ten different lots of FM-MA09-MSC were evaluated for Stro-1 and CD10 expression at the indicated passage numbers. Stro-1 expression was consistently low (average 5-10%) in the different lots of FM-MA09-MSC. CD10 expression was consistently in the intermediate range (average approximately 40%) in the different lots of FM-MA09-MSC. [Figure 23]Figure 23 shows that FM-MA09-MSCs maintain their size as they senesce in culture, while BM-MSCs increase in size with senescence. A forward scatter / side scatter dot plot (shown on the left) on a flow cytometer was used to obtain the size of MSCs. The percentage of cells in the "large" cells in the upper right quadrant was monitored and represented by a bar graph. [Figure 24] Figure 24 shows that CD10 and CD24 are upregulated in FM-MA09-MSCs compared to BM-MSCs. Gene expression analysis is shown for BM-MSCs and FM-MA09-MSCs in the basal state. Quantitative RT-PCR using Taqman probes was used to assess the expression of the indicated genes and normalized to two housekeeping genes. Averages of quadruplicate readings are shown with + / - standard deviation. [Figure 25] Figure 25 shows that Aire-1 and IL-11 are upregulated in FM-MA09-MSCs compared to BM-MSCs. Gene expression analysis is shown for BM-MSCs and FM-MA09-MSCs in the basal state. Quantitative RT-PCR using Taqman probes was used to assess the expression of the indicated genes and normalized to two housekeeping genes. The mean of quadruplicate readings is shown with + / - standard deviation. [Figure 26] Figure 26 shows that Ang-1 and CXCL1 are upregulated in FM-MA09-MSCs compared to BM-MSCs. Gene expression analysis is shown for BM-MSCs and FM-MA09-MSCs in the basal state. Quantitative RT-PCR using Taqman probes was used to assess the expression of the indicated genes and normalized to two housekeeping genes. Averages of quadruplicate readings are shown with + / - standard deviation. [Figure 27]Figure 27 shows that IL6 and VEGF are downregulated in FM-MA09-MSCs compared to BM-MSCs. Gene expression analysis is shown for BM-MSCs and FM-MA09-MSCs in the basal state. Quantitative RT-PCR using Taqman probes was used to assess the expression of the indicated genes and normalized to two housekeeping genes. Averages of quadruplicate readings are shown with + / - standard deviation. [Figure 28] Figure 28 shows that FM-MA09-MSCs and BM-MSCs exhibit increased indoleamine 2,3-dioxygenase (IDO) activity in response to IFNγ stimulation for 3 days. A comparison of MSCs stimulated with 50 ng / ml of IFNγ for 3 days was shown for their ability to convert tryptophan to kynurenine (a measure of IDO activity). For each MSC population, 1 million cells were lysed and used in the assay. [Figure 29] Figure 29 shows aging-associated changes in the expression of Aire-1 and prion protein (PrP) in FM-MA09-MSCs: two proteins involved in immunosuppression and proliferation, respectively. Western blot analysis of the expression of Aire-1 and PrP in whole cell lysates of FM-MA09-MSCs at different passage numbers (p) is shown. Actin expression is shown as a loading control. The difference between Aire-1 and PrP expression is expressed with reference to the actin loading control. [Figure 30] Figure 30 shows that FM-MA09-MSCs secrete less IL6 than BM-MSCs in the basal state. A cytokine array showing a positive control (four dots on the left) for normalization and IL6 in MSC culture supernatant (boxed) is displayed. BM-MSCs obtained from two different donors are compared with four different lots of FM-MA09-MSCs. [Figure 31]Figure 31 shows that FM-MA09-MSCs secrete less IL6 than BM-MSCs under basal and IFNγ-stimulated conditions. A cytokine array showing a positive control (four dots on the left) for normalization and IL6 in MSC culture supernatant (boxed) is displayed. Passage 7 BM-MSCs are compared to p7 FM-MA09-MSCs after 48 hours of + / - IFNγ treatment. [Figure 32] Figure 32 shows that FM-MA09-MSCs secrete less VEGF than BM-MSCs under basal and IFNγ-stimulated conditions. A cytokine array showing a positive control (four dots on the left) for normalization and VEGF (boxed) in MSC culture supernatants is displayed. Passage 7 BM-MSCs after 48 hours of + / - IFNγ treatment are compared to p7 FM-MA09-MSCs. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention relates to methods for producing mesenchymal stromal cells, preparations of mesenchymal stromal cells obtained by culturing hemangioblasts, methods for culturing hemangioblasts, and methods for treating disease conditions using mesenchymal stromal cells.

[0032] The methods of the present invention, in which hemangioblasts produce increased yields of mesenchymal stromal cells compared to prior processes, are more efficient than previous processes in producing substantially ESC-free mesenchymal stromal cells. The hemangioblast-derived mesenchymal stromal cells of the present invention retain a novel youthful phenotype defined by the expression or lack thereof of specific markers.

[0033] In certain embodiments, the MSC preparation of the present invention (at least 10 3 , 10 4 , 10 5 Or even 10 6The ESCs and / or human iPS cells (e.g., a culture comprising MSCs) may, on average, have a telomere length that is at least 30% of that of the ESCs and / or human iPS cells (or on average of a population of ESCs and / or human iPS cells), preferably at least 40, 50, 60, 70, 80 or even 90% of that of the ESCs and / or human iPS cells (or on average of a population of ESCs and / or human iPS cells). For example, the ESCs and / or human iPS cells (or population of ESCs and / or human iPS cells) may be the cells or cell population from which the MSCs were differentiated.

[0034] MSC preparations of the invention may, as a population, have an average terminal restriction fragment length (TRF) of greater than 4 kb, preferably greater than 5, 6, 7, 8, 9, 10, 11, 12, or even 13 kb, hi exemplary embodiments, MSCs of the invention may have an average TRF that is 10 kb or greater.

[0035] In certain embodiments, the MSC preparation of the present invention (at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 Or even 10 8 cultures with MSCs) can be obtained from other sources ( For example, the subject MSC preparations may have a replicative lifespan that exceeds that of MSC preparations derived from donated human tissue (e.g., cultures derived from human tissue for transplantation, such as fetal, infant, child, adolescent, or adult tissue). Replicative lifespan can be assessed by determining the number of population doublings or passages in culture before replicative senescence, i.e., 10, 20, 30, 40, or even 50% or more of the cells in culture become senescent before the next doubling or passage. For example, the subject MSC preparations may have a replicative lifespan that is at least 10 doublings greater than that of MSC preparations derived from donated human tissue (particularly those derived from adult bone marrow or adult adipose tissue), and preferably at least 20, 30, 40, 50, 60, 70, 80, 90, or even 100 population doublings. In certain embodiments, the MSC preparations of the invention may have a replicative lifespan that allows for at least 8 passages before 50% or more of the cells become senescent and / or differentiate into a non-MSC cell type (e.g., fibroblasts), more preferably at least 10, 12, 14, 16, 18, or even 20 passages before reaching this point. In certain embodiments, the MSC preparations of the invention may have a replicative lifespan that allows for at least 2-fold more doublings or passages, and more preferably at least 4-, 6-, 8-, or even 10-fold more doublings or passages compared to adult bone marrow-derived and / or adipocyte-derived MSC preparations (e.g., with an equivalent starting number of cells), before 50% or more of the cells become senescent and / or differentiate into a non-MSC cell type (e.g., fibroblasts).

[0036] In certain embodiments, the MSC preparation of the present invention (at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 Or even 10 8The subject MSC preparations (e.g., cultures comprising MSCs) have statistically significantly increased content and / or enzymatic activity of proteins involved in cell cycle regulation and senescence compared to passage 1 (P1), passage 2 (P2), passage 3 (P3), passage 4 (P4), and / or passage 5 (P5) MSC preparations, particularly bone marrow-derived MSCs and adipocyte-derived MSCs, derived from other sources (e.g., cultures derived from human tissue for transplantation, such as fetal, infant, pediatric, adolescent, or adult tissue). For example, the subject MSC preparations have a protease 26S subunit, non-ATPase regulatory subunit 11 (PSMD11) protein content that is less than 75%, and even more preferably less than 60, 50, 40, 30, 20, or even 10%, of the content in MSCs obtained from human tissue for transplantation (e.g., MSCs derived from adult bone marrow or adult adipose tissue).

[0037] In certain embodiments, the MSC preparation of the present invention (at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 Or even 10 8The cultures (e.g., cultures with MSCs) have statistically significantly increased content of proteins and / or enzymatic activity involved in cellular energy and / or lipid metabolism compared to passage 1 (P1), passage 2 (P2), passage 3 (P3), passage 4 (P4), and / or passage 5 (P5) MSC preparations derived from other sources (e.g., cultures derived from human tissue for transplantation, such as fetal, infant, child, adolescent, or adult tissue), particularly bone marrow-derived MSCs and adipocyte-derived MSCs. Illustratively, the subject MSC preparations have a protein content of one or more proteins involved in metabolic pathways related to ATP or NADHP synthesis, such as glycolysis (fructose bisphosphate aldolase A, ALDOA; aldo-ketoreductase family 1, member A1, AKR1A1); glyceraldehyde triphosphate, GAPDH, etc.), tricarboxylic acid cycle (TCA cycle) (isocitrate dehydrogenase 1, IDH1, etc.), pentose phosphate cycle (glucose hexaphosphate dehydrogenase, G6PD, etc.), and UDP-glucose biosynthesis in the glucuronic acid biosynthetic pathway (UDP-glucose 6-dehydrogenase, UGDH), that is less than 90%, and even more preferably less than 60, 50, 40, 30, 20, or even 10%, of the content in MSCs obtained from human tissue for transplantation (particularly MSCs derived from adult bone marrow or adult adipose tissue). By way of further example, the subject MSC preparations can be assayed for one or more proteins involved in lipid metabolism, such as enoyl-CoA hydratase, short chain, 1 (ECHS1) and / or acetyl-CoA acetyltransferase (ACAT2), to identify potential targets for transplantation. The MSCs have a protein content that is less than 90%, and even more preferably less than 60, 50, 40, 30, 20 or even 10%, of the content in MSCs obtained from adult tissue (particularly MSCs derived from adult bone marrow or adult adipose tissue).

[0038] In certain embodiments, the MSC preparation of the present invention (at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 Or even 10 8The cultures (e.g., cultures with MSCs) have a statistically significant increased content of proteins and / or enzymatic activity involved in cellular apoptosis compared to passage 1 (P1), passage 2 (P2), passage 3 (P3), passage 4 (P4) and / or passage 5 (P5) MSC preparations derived from other sources (e.g., cultures derived from human tissue for transplantation, such as fetal, infant, child, adolescent or adult tissue), particularly bone marrow-derived MSCs and adipocyte-derived MSCs. Illustratively, the subject MSC preparations have a protein content of one or more of the proteins annexin A1 (ANXA1), A2 (ANXA2), A5 (ANXA5), voltage-dependent anion-selective channel protein 1 (VDAC1), and / or glyceraldehyde triphosphate dehydrogenase (GAPDH) that is less than 90%, and even more preferably less than 60, 50, 40, 30, 20, or even 10%, of the content in MSCs obtained from human tissue for transplantation (particularly MSCs derived from adult bone marrow or adult adipose tissue).

[0039] Without being bound by theory, the statistically significant differences in the content and / or enzyme activity of proteins involved in cellular energy and / or lipid metabolism, and / or apoptosis exhibited by the hemangioblast-derived MSCs of the present invention are attributable, at least in part, to the homogeneous nature of the preparation. For example, the hemangioblast-derived MSCs of the present invention have homogeneous MHC gene expression, i.e., are fully MHC-matched, and differ from adult-derived MSC banks in which cells are derived from multiple different donors, i.e., are MHC-mismatched. Therapeutic doses of MSCs are approximately 2-8 million cells / kg (or approximately 130-500 million cells / dose).

[0040] Definition of Terms As used herein, "pluripotent cells" and "pluripotent stem cells" refer broadly to cells that, under appropriate conditions, maintain their undifferentiated state, exhibit a stable (preferably normal) karyotype, and have the potential to differentiate into all three germ layers (i.e., ectoderm, mesoderm, and endoderm), while also being capable of long-term or virtually indefinite proliferation in vitro. Typically, pluripotent cells (a) are capable of inducing teratomas when transplanted into immunodeficient (SCID) mice, (b) are capable of differentiating into all three germ layers (i.e., ectoderm, mesoderm, and endoderm), and (c) express at least one hES cell marker (e.g., Oct-4, alkaline phosphatase, SSEA 3 surface antigen, SSEA 4 surface antigen, NANOG, TRA 1 60, TRA 1 81, SOX2, REX1, etc.). Exemplary pluripotent cells can express Oct-4, alkaline phosphatase, SSEA 3 surface antigen, SSEA 4 surface antigen, TRA 1 60, and / or TRA 1 81. Further exemplary pluripotent cells include, but are not limited to, embryonic stem cells, induced pluripotent (iPS) cells, embryo-derived cells, pluripotent cells produced from embryonic germ (EG) cells (e.g., by culturing in the presence of FGF-2, LIF, and SCF), parthenogenetic ES cells, ES cells produced from cultured inner cell mass cells, ES cells produced from blastomeres, and ES cells produced by nuclear transfer (e.g., somatic cell nuclei transferred into recipient oocytes). Exemplary pluripotent cells can be produced without destruction of embryos. For example, induced pluripotent cells can be produced from cells obtained without embryo destruction. As a further example, pluripotent cells can be produced from biopsied blastomeres (which can be accomplished without harming the remainder of the embryo), and the remainder of the embryo can optionally be cryopreserved, cultured, and / or implanted into a suitable host. Pluripotent cells (from whatever source) can be differentiated into cells (e.g., MSCs, and hemangioblasts), Modifications can be made at the genetic level or in other ways to increase longevity, potency, homing, or to supply desired factors. By way of non-limiting example, pluripotent cells can be engineered to express Sirt 1 (thereby increasing lifespan), to express one or more telomerases, optionally under the control of an inducible or repressible promoter, to incorporate fluorescent labels, to incorporate iron oxide particles or other such reagents (which are used for tracking cells via in vivo imaging, MRI, etc., see Thu et al., Nat Med. 2012 Feb 26;18(3):463-7), to express bFGF, which can promote lifespan (see Go et al., J. Biochem. 142, 741-748 (2007)), to express CXCR4 for homing (see Shi et al., Haematologica. 2007 Jul;92(7):897-904), or to express recombinant TRAIL to induce caspase-mediated apoptosis in cancer cells, such as gliomas (see Sasportas et al., Proc Natl Acad Sci USA. 2009 Mar. 24;106(12):4822-7).

[0041] As used herein, "embryo" or "embryonic" refers broadly to a developing cell mass that has not been implanted into the endometrium of a maternal host. A "fetal cell" is a cell isolated from or contained within an embryo. It also includes blastomeres obtained early in the two-cell division stage and aggregated blastomeres.

[0042] "Embryonic stem cells" (ES cells or ESCs) encompass pluripotent cells produced from embryonic cells (such as cultured inner cell mass cells or cells obtained from cultured blastomeres) as well as induced pluripotent cells (described further below). Often, such cells are or have been serially passaged as cell lines. Embryonic stem cells can be used as pluripotent stem cells in the process of producing hemangioblasts described herein. For example, ES cells can be produced by methods known in the art, such as fertilization of egg cells with sperm or sperm DNA, nuclear transfer (including somatic cell nuclear transfer), or derivation from embryos produced by any method (including sexual or asexual means), such as parthenogenesis. As a further example, embryonic stem cells also include cells produced by somatic cell nuclear transfer, even if non-embryonic cells are used in the process. For example, ES cells may be derived from the ICM of a blastocyst-stage embryo as well as embryonic stem cells derived from one or more blastomeres. Such embryonic stem cells can be generated from embryonic material produced by reproductive or asexual means, including somatic cell nuclear transfer (SCNT), apolipogenesis, and androgeny. Furthermore, as discussed above (see "Pluripotent Cells"), ES cells may be genetically modified to increase longevity, potency, homing, or to provide desired factors in cells differentiated from such pluripotent cells (e.g., MSCs and hemangioblasts).

[0043] ES cells can be generated with homozygosity or hemizygosity in one or more HLA genes, for example, through genetic engineering, screening for spontaneous loss of heterozygosity, etc. ES cells may also be genetically modified to increase lifespan, potency, homing, or provide desired factors in cells differentiated from such pluripotent cells (e.g., MSCs and hemangioblasts). Regardless of their source or the particular method used to produce them, embryonic stem cells typically possess one or more of the following attributes: (i) the ability to differentiate into cells of all three germ layers, (ii) the expression of at least Oct-4 and alkaline phosphatase, and (iii) the ability to produce teratomas when transplanted into immunocompromised animals. Embryonic stem cells that can be used in embodiments of the present invention include, but are not limited to, human ES cells ("ESCs" or "hES cells"), such as MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Additional exemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. NIH Embryo See also Human Embryonic Stem Cell Registry. An exemplary human embryonic stem cell line that can be used is MA09 cells. The isolation and preparation of MA09 cells is described in Klimanskaya et al. (2006), "Human Embryonic Stem Cell Lines Derived from Human Embryonic Stem Cells." from Single Blastomeres," Nature 444:481-485. Human ES cells used according to exemplary embodiments of the present invention may be derived and maintained in accordance with GMP standards.

[0044] Exemplary hES cell markers include, but are not limited to, alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), repressed expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, surface antigen 30 (CD30), Crypto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand). Additional examples include embryonic stem cells capable of expressing Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, TRA 1 60, and / or TRA 1 81.

[0045] The ESCs may initially be co-cultured with murine embryonic feeder cells (MEFs). The MEF cells can be mitotically inactivated by exposure to mitomycin C prior to seeding the ESCs into the co-culture, thereby preventing the MEFs from proliferating in culture. Furthermore, ESC cell cultures can be examined microscopically, and colonies containing non-ESC cell morphology can be harvested and discarded, for example, using a stem cell cutting tool, by laser ablation, or other means. Typically, no additional MEF cells are used at the time of harvesting the ESCs for seeding to form embryoid bodies.

[0046] As used herein, "embryonic-derived cells" (EDCs) broadly refer to pluripotent morula-derived cells, including those from the inner cell mass, embryonic shield, or epidisc, blastocyst-derived cells, or other pluripotent stem cells of the early embryo, including primitive ectoderm, mesoderm, and endoderm and their derivatives. "EDCs" also include cell masses obtained from blastomeres and aggregated single blastomeres, and embryos obtained from different stages of development, but exclude human embryonic stem cells passaged as cell lines.

[0047] Exemplary ESC cell markers include, but are not limited to, alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), repressed expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN, surface antigen 30 (CD30), Crypto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand).

[0048] As used herein, "potency" broadly refers to the concentration, e.g., molar, of a reagent (such as hemangioblast-derived MSCs) that produces a defined effect. Potency can be defined in terms of the effective concentration (EC50), which does not include a measurement of the maximum effect, but instead includes effects at various locations along the concentration axis of a dose-response curve. Potency can also be determined from either a stepwise (EC50) or arithmetic dose-response curve (ED50, TD50, and LD50), although potency is preferably measured by EC50. The term "EC50" refers to the concentration of a drug, antibody, or toxicant that elicits a response midway between the baseline effect and the maximum effect after some specified exposure time. Thus, the EC50 of a graded dose-response curve represents the concentration of a compound at which 50% of its maximum effect is observed. The EC50 of a quantal dose-response curve represents the concentration of a compound at which 50% of a population responds after a specified exposure duration. This EC50 can be determined using animal studies in which a defined animal model exhibits a measurable physiological change in response to drug application; cell-based assays using specific cell lines that exhibit a measurable biological response upon drug addition; and / or enzymatic reactions in which the biological activity of a drug can be measured by the accumulation of a product following a drug-promoted chemical reaction. Preferably, an immunomodulatory assay is used to determine the EC50. Non-limiting examples of such immunomodulatory assays include intracellular cytokine, cytotoxicity, regulatory capacity, cell signaling capacity, proliferation capacity, apoptosis assessment, and other assays.

[0049] As used herein, "mesenchymal stem cells" (MSCs) refer to multipotent stem cells with the capacity for self-renewal and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes, among other mesenchymal cell lineages. In addition to these characteristics, MSCs may be identified by the expression of one or more markers further described herein. Such cells can be used to treat a variety of clinical conditions, including immunological disorders such as graft-versus-host disease (GVHD), myocardial infarction, and inflammation, as well as autoimmune diseases and disorders, and degenerative diseases. Unless the context dictates otherwise, MSCs can include cells obtained from adult sources and umbilical cord blood. MSCs (or the cells from which they are generated, such as multipotent cells) can be genetically modified or otherwise modified to increase longevity, potency, homing, or to provide desired factors into the MSCs or cells differentiated from such MSCs. By way of non-limiting example, the MSC cells can be engineered to express Sirt 1 (thereby increasing lifespan), to express one or more telomerases, optionally under the control of an inducible or repressible promoter, to incorporate a fluorescent label, to incorporate iron oxide particles or other such reagents (which are used for tracking cells via in vivo imaging, MRI, etc., see Thu et al., Nat Med. 2012 Feb 26;18(3):463-7), to express bFGF, which can promote lifespan (see Go et al., J. Biochem. 142, 741-748 (2007)), to express CXCR4 for homing (see Shi et al., Haematologica. 2007 Jul;92(7):897-904), or to express recombinant TRAIL to induce caspase-mediated apoptosis in cancer cells, such as gliomas (see Sasportas et al., Proc Natl Acad Sci USA. 2009 Mar. 24;106(12):4822-7).

[0050] As used herein, "therapy," "therapeutic," and "treating" refer to treating a disease, arresting or reducing the occurrence of a disease or its clinical symptoms, and / or alleviating the disease, resulting in the resolution of a disease or its clinical symptoms. Therapy includes preventing, inhibiting, treating, curing, improving, reducing, alleviating, and / or providing relief from a disease, signs of a disease, and / or symptoms of a disease. Therapy includes alleviating signs and / or symptoms in a patient with ongoing signs and / or symptoms of a disease (e.g., muscle weakness, multiple sclerosis). Therapy also encompasses "prevention" and "inhibition." Prevention includes preventing the occurrence of a disease after treating a patient for a disease, or reducing the onset or severity of a disease in a patient. The term "reduced," for purposes of therapy, broadly refers to a clinically significant decrease in signs and / or symptoms. Therapy includes treating the recurrence or recurrence of signs and / or symptoms (e.g., retinal degeneration, blindness). Therapy includes, but is not limited to, preventing the onset of signs and / or symptoms, as well as reducing existing signs and / or symptoms, and also reducing existing signs and / or symptoms. Therapy includes eliminating signs and / or symptoms. Therapy includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing relapse or recurrence of signs and / or symptoms (e.g., muscle weakness, multiple sclerosis).

[0051] To maintain regulatory compliance, an MSC bank must maintain an adequate supply of cells to provide a sufficient number of cells to treat at least several hundred to 10,000 patients, for example, and the MSC bank must have at least 50 billion MSCs. The present invention encompasses GMP-complaint and / or cryopreserved MSC banks. In one embodiment, the MSC preparation of the present invention comprises at least 10 10 In another embodiment, the MSC preparation of the present invention comprises at least 10 hemangioblast-derived MSCs. 11 , 10 12 , 10 13 , or 10 14An MSC preparation comprising hemangioblast-derived MSCs is provided.

[0052] As used herein, "normalizing a pathological condition" refers to restoring abnormal structure and / or function resulting from a disease to a more normal state. Normalization suggests that the progression of a pathological condition can be controlled and ameliorated by correcting abnormalities in the structure and / or function of tissues, organs, cell types, etc. resulting from a disease. For example, after treatment with the ESC-MSCs of the present invention, immune system abnormalities resulting from an autoimmune disease, such as MS, can be improved, corrected, and / or reversed.

[0053] induced pluripotent stem cells Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells), which are generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors ("reprogramming factors"). iPS cells can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. iPS cells can also be obtained from cell banks. Alternatively, iPS cells can be generated de novo (by processes known in the art) before they begin to differentiate into RPE cells or another cell type. iPS cell manufacturing can be the first step in the production of differentiated cells. iPS cells can be specifically generated using material from a particular patient or matched donor with the goal of generating histocompatible RPE cells. iPS cells can be generated from cells that are substantially non-immunogenic in the intended recipient, for example, from autologous cells or cells that are histocompatible with the intended recipient. Furthermore, as described above (see "Pluripotent Cells"), pluripotent cells, including iPS cells, may be genetically modified or otherwise modified to increase lifespan, potency, homing, or to provide factors desired in cells differentiated from such pluripotent cells (e.g., MSCs and hemangioblasts).

[0054] As another example, induced pluripotent stem cells may be generated by reprogramming somatic or other cells by contacting the cells with one or more reprogramming factors. For example, reprogramming factors may be expressed by a cell from an endogenous gene in response to factors, such as, for example, exogenous nucleic acids added to the cell, or from a small molecule, microRNA, etc., that promotes or induces expression of that gene (Suh and Blelloch, Development 138, 1653-1661 (2011); Miyosh et al., Cell Stem Cell (2011), doi:10.1016 / j.stem.2011.05.001; Sancho-Martinez et al., Journal of of Molecular Cell Biology (2011), 1-3; Anokye-Danso et al., Cell Stem Cell 8, 376-388, April 8, 2011; Orkin and Hochedlinger, Cell 145, 835-850, June 10, 2011 (each of which is incorporated herein by reference in its entirety). Reprogramming factors can be added to the culture medium, for example, to induce cell proliferation. Factors can be provided from exogenous sources and introduced into cells by methods known in the art, such as coupling to cell-penetrating peptides, proteins, or nucleic acid transfection agents, lipofection, electroporation, biolistic particle delivery systems (gene guns), microinjection, etc. iPS cells can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes called Oct3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4, Sox2, Nanog, and Lin28. In other embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors. In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent cells. iPS cells can typically be identified by the expression of the same markers as embryonic stem cells, although specific iPS cell lines may differ in their expression profiles.

[0055] Induced pluripotent stem cells can be produced by expressing one or more reprogramming factors in somatic cells or by inducing the expression of reprogramming factors. The somatic cells can be fibroblasts, such as skin fibroblasts, synovial fibroblasts, or lung fibroblasts, or non-fibroblast somatic cells. The somatic cells are reprogrammed by expressing at least one, two, three, four, or five reprogramming factors. The reprogramming factors can be selected from Oct3 / 4, Sox2, NANOG, Lin28, cMyc, and Klf4. Expression of the reprogramming factors can be induced by contacting the somatic cells with at least one agent, such as a small organic molecule agent, that induces expression of the reprogramming factors.

[0056] The somatic cells can also be reprogrammed using a combinatorial approach in which reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and expression of the reprogramming factors is induced (e.g., using small organic molecules). For example, the reprogramming factors can be expressed in somatic cells by infection with a viral vector, such as a retroviral or lentiviral vector. Furthermore, the reprogramming factors can be expressed in somatic cells using a non-integrating vector, such as an episomal plasmid. See, for example, Yu et al., Science. 2009 May 8;324(5928):797-801, which is incorporated herein by reference in its entirety. When the reprogramming factors are expressed using a non-integrating vector, the factors can be expressed using electroporation, transfection, or transformation of somatic cells with the vector. For example, in mouse cells, expression of four factors (Oct3 / 4, Sox2, cmyc, and Klf4) using an integrating viral vector is sufficient to reprogram somatic cells. In human cells, expression of four factors (Oct3 / 4, Sox2, NANOG, and Lin28) using an integrating vector is sufficient to reprogram somatic cells.

[0057] Once the reprogramming factors are expressed in the cells, the cells can be cultured. Over time, cells with ES characteristics will appear in the culture dish. The cells can be selected and subcultured, for example, based on ES morphology or on the expression of selectable or detectable markers. The cells can be cultured to produce a culture of cells that resemble ES cells (these are putative iPS cells). iPS cells can typically be identified by the expression of the same markers as other embryonic stem cells, although specific iPS cell lines may differ in their development. Exemplary iPS cells may express Oct-4, alkaline phosphatase, SSEA 3 surface antigen, SSEA 4 surface antigen, TRA 1 60, and / or TRA 1 81.

[0058] To confirm the pluripotency of iPS cells, the cells can be tested in one or more assays for pluripotency. For example, the cells can be tested for ES cell markers; the cells can be tested for their ability to produce teratomas when transplanted into SCID mice; or the cells can be evaluated for their ability to differentiate to produce cell types of all three germ layers. Once pluripotent iPS cells are obtained, they can be used to produce hemangioblasts and MSC cells.

[0059] hemangioblasts Hemangioblasts are multipotent and serve as a common precursor for both hematopoietic and endothelial cell lineages. During embryonic development, they are thought to arise as a transitional cell type that emerges during early mesoderm development and colonizes primitive blood islands (Choi et al., Development 125(4):725-732 (1998)). Once there, hemangioblasts can give rise to both primitive and definitive hematopoietic cells, HSCs, and endothelial cells (Mikkola et al., J. Hematother. Stem Cell Res 11(1):9-17 (2002)).

[0060] Hemangioblasts can be derived in vitro from both mouse ESCs (Kennedy et al., Nature (386):488-493 (1997); Perlingeiro et al., Stem Cells (21):272-280 (2003)) and human ESCs (References 14, 15; Yu et al., Blood 2010 116:4786-4794). Other studies claim to have isolated hemangioblasts from umbilical cord blood (Bordoni et al., Hepatology 45(5)1218-1228), from circulating CD34-lin-CD45-CD133 cells obtained from peripheral blood (Ciraci et al., Blood 118:2105-2115), and from mouse uterus (Sun et al., Blood 116(16):2932-2941 (2010)). Both mouse and human ESC-derived hemangioblasts have been obtained through the culture and differentiation of clusters of cells grown in liquid medium following expansion of the cells in semi-solid medium containing various cytokines and growth factors (Kennedy and Perlingeiro, References 14 and 15); see also U.S. Pat. No. 8,017,393, the entire contents of which are incorporated herein by reference. For purposes of this application, the term hemangioblast also encompasses hemangio-colony forming cells, as described in U.S. Pat. No. 8,017,393, which, in addition to differentiating into hematopoietic and endothelial cell lineages, can also become smooth muscle cells and are not CD34, CD31, KDR, or CD133 positive. Hemangioblasts useful in the methods described herein can be derived or obtained from any of these known methods. For example, embryoid bodies can be formed by culturing pluripotent cells under non-adherent conditions, e.g., on low-adhesion substrates or in "hanging drops." In these cultures, ES cells can form clumps or clusters of cells called embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 Feb;6(2):88-95, which is incorporated herein by reference in its entirety.Typically, embryoid bodies initially form as solid clumps or clusters of pluripotent cells; over time, some of the embryoid bodies contain fluid-filled cavities, the former referred to in this literature as "simple" EBs and the latter as "cystic" embryoid bodies. Cells in these EBs (both solid and cystic) can differentiate, producing a gradually increasing number of cells over time. Optionally, EBs can then be cultured as adherent cultures to form protrusions. Similarly, pluripotent cells that overgrow and form multilayered cell populations can also differentiate over time.

[0061] In one embodiment, hemangioblasts are generated by a process comprising: (a) culturing an ESC cell line for 2, 3, 4, 5, 6, or 7 days to form clusters of cells; and (b) inducing the clusters of cells to differentiate into hemangioblasts. In another embodiment, the clusters of cells in step (b) are cultured in a cytokine-rich, serum-free, methylcellulose-based medium (14, 15).

[0062] In one embodiment, hemangioblasts are produced by a process comprising: (a) culturing an ESC cell line selected from the group consisting of MA09, H7, H9, MA01, HuES3, and H1gfp for 2, 3, 4, 5, 6, or 7 days to allow the cells to form clusters; and (b) inducing differentiation into hemangioblasts by culturing in cytokine-rich, serum-free, methylcellulose-based medium.

[0063] In another embodiment, hemangioblasts are generated by inducing any of the pluripotent cells described herein. In other embodiments, hemangioblasts are generated by inducing differentiation of pluripotent cells selected from the group including blastocysts, plated ICMs, one or more blastomeres, or other parts of preimplantation stage embryos or germ layer structures (whether produced by germ cell proliferation, somatic cell nuclear transfer (SCNT), parthenogenesis, androgenetics, or sexual or asexual means), or ESCs derived through reprogramming (e.g., iPS cells). In yet other embodiments, hemangioblasts are generated from iPS cells, where the iPS cells are generated using exogenously added factors or proteins or other methods known in the art, such as microRNAs (see Zhou et al., Cell Stem Cell(4):1-4, 2009; Miyoshi et al., Cell Stem Cell(8):1-6, 2011; Danso et al., Cell Stem Cell(8):376-388, 2011).

[0064] In another aspect, the present disclosure provides preparations of mesenchymal stromal cells (MSCs) and methods for generating MSCs using hemangioblasts. The MSCs may differ from pre-existing MSCs in one or more aspects, as further described herein. In one embodiment, hemangioblasts are prepared by inducing pluripotent cells selected from the group including blastocysts, plated ICMs, one or more blastomeres, or other parts of pre-implantation stage embryos or germ layer structures (whether produced by germ cell regeneration, somatic cell nuclear transfer (SCNT), parthenogenesis, androgenetics, or by sexual or asexual means), or cells derived through reprogramming (e.g., iPS cells) in serum-free methylcellulose medium supplemented with penicillin / streptomycin (pen / strp), EX-CYTE® growth supplement (9.0-11.0 g / L cholesterol, and 13.0 g / L ATP). The hemangioblasts are harvested after at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days of culture in a medium supplemented with one or more ingredients selected from the group consisting of an aqueous concentrate (pH 7-8.4) containing ~18.0 g / L lipoproteins and fatty acids, Flt3-ligand (FL), vascular endothelial growth factor (VEGF), thrombopoietin (TPO), basic fibroblast growth factor (bFGF), stem cell-derived factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL3), and interleukin-6 (IL6). In a preferred embodiment of the present invention, the hemangioblasts are harvested after culturing for 6 to 14 days in a medium, for example, serum-free methylcellulose supplemented with the ingredients of the previous embodiment. In a preferred embodiment, the components are present in the medium at the following concentrations: Flt3-ligand (FL) at 50 ng / ml, vascular endothelial growth factor (VEGF) at 50 ng / ml, thrombopoietin (TPO) at 50 ng / ml, and basic fibroblast growth factor (bFGF) at 20 ng / ml, 50 ng / ml stem cell-derived factor (SCF), 20 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng / ml interleukin 3 (IL3), 20 ng / ml interleukin 6 (IL6), 50 ng / ml FL, 50 ng / ml VEGF, 50 ng / ml / ml TPO, and 30ng / ml bFGF.

[0065] In another embodiment, the clusters of cells substantially consisting of hemangioblasts are replated and cultured for at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 days to form a preparation of mesenchymal stem cells. In one embodiment, the mesenchymal stem cells are produced by a process comprising: (a) culturing the ESCs for 8-12 days; (b) harvesting the hemangioblasts that form the clusters of cells; (c) replating the hemangioblasts of step (b); and (d) culturing the hemangioblasts of step (c) for 14-30 days.

[0066] In one embodiment, hemangioblasts are harvested, replated, and cultured in liquid medium under feeder-free conditions, in which a feeder layer of cells, such as mouse embryonic fibroblasts, OP9 cells, or other cell types known to those of skill in the art, is included in the culture. In a preferred embodiment, the hemangioblasts are cultured on an extracellular matrix. In a further preferred embodiment, the hemangioblasts are cultured on an extracellular matrix comprising a soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, which gels at room temperature to form a reconstituted basement membrane (Matrigel). In yet another embodiment, the hemangioblasts are produced by a process comprising: (a) culturing the hemangioblasts on Matrigel for at least 7 days; and (b) transferring the hemangioblasts of step (a) to uncoated tissue culture plates and further culturing the hemangioblasts of step (b) for about 7 to 14 days. Hemangioblasts may be cultured on a substrate comprising one or more factors selected from the group consisting of transforming growth factor beta (TGF-β), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and / or platelet-derived growth factor (PDGF), human basement membrane extract (BME) (e.g., Cultrex BME, Trevigen) or EHS matrix, laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen (e.g., collagen I, collagen IV), and heparan sulfate. The matrix or matrix components may be mammalian, or more specifically, human, in origin. In one embodiment, hemangioblasts are cultured on Matrigel-coated plates in a serum-containing liquid medium, which may contain components selected from αMEM (Sigma-Aldrich) supplemented with 10-20% fetal bovine serum (αMEM + 20% FCS), αMEM supplemented with 10-20% heat-inactivated human AB serum, and IMDM supplemented with 10-20% heat-inactivated AB human serum.

[0067] Mesenchymal stromal cells generated by culturing hemangioblasts One embodiment of the present invention includes improved mesenchymal stromal cells, which can be generated from hemangioblasts using an improved process for culturing hemangioblasts.

[0068] The mesenchymal stromal cells of the invention are capable of maintaining a higher level of potency and are capable of not aggregating or are capable of aggregating substantially less than mesenchymal stromal cells derived directly from ESCs. In one embodiment of the invention, preparations of mesenchymal stromal cells produced by any one or more of the processes of the invention maintain a higher level of potency and are capable of not aggregating or are capable of aggregating substantially less than mesenchymal stromal cells derived directly from ESCs.

[0069] One embodiment of the present invention provides a process for culturing hemangioblasts to produce a preparation of mesenchymal stromal cells, wherein said mesenchymal stromal cells maintain a youthful phenotype. The pharmaceutical preparation of mesenchymal stromal cells of the present invention exhibits improved therapeutic properties when administered to a mammalian host in need of treatment.

[0070] One embodiment of the present invention provides a preparation of mesenchymal stromal cells produced by culturing human hemangioblasts. Another embodiment of the present invention provides a process for producing a preparation of mesenchymal stromal cells by culturing human hemangioblasts. In an embodiment of the process of the present invention, the human hemangioblasts are cultured in feeder-free conditions and then plated on a matrix. In yet another embodiment of the present invention, the matrix is ​​selected from the group consisting of transforming growth factor β (TGF-β), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, heparan sulfate, a soluble preparation obtained from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, Matrigel, and human basement membrane extract. In yet another embodiment, the matrix may be derived from mammalian or human sources.

[0071] In another embodiment, the hemangioblasts are cultured in medium containing serum or a serum replacement, such as αMEM supplemented with 20% fetal bovine serum, hi other embodiments, the hemangioblasts are cultured on the matrix for about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In yet another embodiment of the invention, the preparation of mesenchymal stromal cells is produced by a process comprising: (a) culturing hemangioblasts on Matrigel for about 7 days; and (b) transferring the hemangioblasts of step (a) from the Matrigel and growing the hemangioblasts on uncoated tissue culture dishes for an additional 9 to 100 days, i.e., about 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 days.

[0072] In one embodiment of the invention, the preparation of mesenchymal stromal cells is cultured in a medium containing serum or a serum replacement, such as αMEM supplemented with 20% fetal bovine serum. In other embodiments of the invention, the hemangioblasts are cultured on the matrix for about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0073] In one embodiment of the invention, the hemangioblasts are differentiated from ESCs. In another embodiment of the invention, the hemangioblasts of the previous embodiment are differentiated from ESCs, said ESCs being selected from the group comprising iPS, MA09, H7, H9, MA01, HuES3, H1gfp, inner cell mass cells, and blastomeres.

[0074] One embodiment of the present invention comprises a preparation of mesenchymal stromal cells produced by the process of differentiation of hemangioblasts from ESCs. In another embodiment of the present invention, the hemangioblasts of the previous embodiment are differentiated from ESCs, wherein the ESCs are selected from the group consisting of iPS, MA09, H7, H9, MA01, HuES3, H1gfp, inner cell mass cells, and blastomeres.

[0075] In one embodiment of the present invention, hemangioblasts are differentiated from ESCs by the steps of: (a) culturing ESCs in the presence of, for example, vascular endothelial growth factor (VEGF) and / or bone morphogenetic protein 4 (BMP-4) to form clusters of cells; (b) culturing the clusters of cells in the presence of at least one growth factor (e.g., basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), Flt 3L (FL), thrombopoietin (TPO), and / or tPTD-HOXB4) provided in an amount sufficient to induce differentiation of the clusters of cells into hemangioblasts; and (c) culturing the hemangioblasts in the presence of at least one additional growth factor (e.g., insulin, transferrin, granules, etc.). and culturing the cells in a medium comprising at least one additional growth factor (e.g., GM-CSF, interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), and tPTD-HOXB4, wherein the at least one additional growth factor is provided in an amount sufficient to expand the clusters of cells in the culture, and optionally, copper is added to any of steps (a) to (c).

[0076] In one embodiment of the present invention, the preparation of mesenchymal stromal cells is produced by culturing hemangioblasts, and the hemangioblasts are differentiated from ESCs by the steps of: (a) culturing ESCs in the presence of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 4 (BMP-4) to form clusters of cells within 0-48 hours of initiating the culturing; and (b) culturing the clusters of cells in the presence of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), Flt, provided in an amount sufficient to induce differentiation of the clusters of cells into hemangioblasts. (c) culturing the hemangioblasts in the presence of at least one growth factor selected from the group consisting of insulin, transferrin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony-stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), and tPTD-HOXB4, wherein the at least one additional growth factor is provided in an amount sufficient to expand clusters of human cells in the culture.

[0077] In another embodiment, the preparation of mesenchymal stromal cells is produced by a process comprising: (a) harvesting hemangioblasts at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after inducing ESCs to differentiate into said hemangioblasts; and (b) harvesting said mesenchymal stromal cells produced within about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days of inducing the hemangioblasts from step (a) to differentiate into said mesenchymal stromal cells.

[0078] In yet another embodiment, a preparation of at least 80 million, 85 million, 90 million, 100 million, 125 million, or 125 million mesenchymal stromal cells is generated from a culture of about 200,000 hemangioblasts within about 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, wherein said preparation of mesenchymal stromal cells has less than about 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% , less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, less than 0.0009%, less than 0.0008%, less than 0.0007%, less than 0.0006%, less than 0.0005%, less than 0.0004%, less than 0.0003%, less than 0.0002%, or less than 0.0001% human embryonic stem cells. In yet another embodiment, at least 80 million, 85 million, 90 million, 100 million, 125 million, or 150 million mesenchymal stromal cells are generated from about 200,000 hemangioblasts within about 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of culturing the hemangioblasts.

[0079] In one embodiment of the process of the invention, the preparation of mesenchymal stromal cells is substantially purified for human embryonic stem cells. In other embodiments of the process of the invention, the preparation of mesenchymal stromal cells is substantially purified for human embryonic stem cells such that the preparation comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% mesenchymal stromal cells.

[0080] In another embodiment of the invention, the preparations of mesenchymal stromal cells produced by any one or more of the processes of the invention do not form teratomas when introduced into a host.

[0081] In another embodiment of the invention, at least 50% of a preparation of mesenchymal stromal cells are positive for CD105 or CD73 within about 7-20 (e.g., 15) days of culture. In a preferred embodiment of the invention, at least 50% of a preparation of mesenchymal stromal cells produced by any one or more processes of the invention are positive for CD105 or CD73 after about 7-15 days of culture. In another embodiment of the invention, at least 80% of the mesenchymal stromal cells are positive for CD105 and CD73 within about 20 days of culture. In yet another embodiment of the invention, at least 80% of a preparation of mesenchymal stromal cells produced by any one or more processes of the invention are positive for CD105 and CD73 within about 20 days of culture.

[0082] In an exemplary embodiment, the present disclosure provides at least 10 6 The present invention provides a pharmaceutical preparation suitable for use in a mammalian patient, comprising mesenchymal stromal cells and a pharmaceutically acceptable carrier, wherein the mesenchymal stromal cells have the replicative capacity to undergo at least 10 population doublings, with less than 25% of the cells undergoing cell death and senescing or differentiating into non-MSCs by the 10th doubling.

[0083] In an exemplary embodiment, the present disclosure provides at least 10 6 The present invention provides a pharmaceutical preparation suitable for use in a mammalian patient, comprising mesenchymal stromal cells and a pharmaceutically acceptable carrier, wherein the mesenchymal stromal cells have the replicative capacity to undergo at least five passages in cell culture, with less than 25% of the cells undergoing cell death and senescing or differentiating into fibroblasts by the fifth passage.

[0084] In an exemplary embodiment, the present disclosure provides at least 10 6 A pharmaceutical preparation is provided that includes mesenchymal stromal cells and a pharmaceutically acceptable carrier, the mesenchymal stromal cells being differentiated from hemangioblasts.

[0085] In an exemplary embodiment, the present disclosure provides at least 10 8 The present invention provides a cryogenic cell bank comprising mesenchymal stromal cells, the mesenchymal stromal cells having the replicative capacity to undergo at least 10 population doublings in cell culture, with less than 25% of the cells undergoing cell death, senescence, or differentiation into fibroblasts by the 10th population doubling.

[0086] In an exemplary embodiment, the present disclosure provides at least 10 6 The present invention provides a purified cell preparation comprising mesenchymal stromal cells and less than 1% of any other cell type, wherein the mesenchymal stromal cells have the replicative capacity to undergo at least 10 population doublings in cell culture, with less than 25% of the cells undergoing cell death, senescence, or differentiation into non-MSC cells by the 10th population doubling.

[0087] The mesenchymal stromal cells of the present invention can be differentiated from a pluripotent stem cell source, such as an embryonic stem cell line or an induced pluripotent stem cell line. For example, all of the mesenchymal stromal cells in a preparation or bank can be differentiated from a common pluripotent stem cell source. Furthermore, the mesenchymal stromal cells can be differentiated from the pluripotent stem cell source, passaged in culture to expand the number of mesenchymal stromal cells, and isolated from the culture after fewer than 20 population doublings.

[0088] The mesenchymal stromal cells may be HLA genotypically identical. The mesenchymal stromal cells may be genomically identical.

[0089] At least 30% of the mesenchymal stromal cells may be positive for CD10. Additionally, at least 60% of the mesenchymal stromal cells may be positive for the markers CD73, CD90, CD105, CD13, CD29, CD44, and CD166, and HLA-ABC. In exemplary embodiments, at least 30% of the mesenchymal stromal cells may be positive for the markers CD31, CD34, CD45, CD133, FGFR2, CD271, Stro-1, CXCR4, and TLR3.

[0090] The mesenchymal stromal cells of the present invention can have a replication rate to undergo at least 10 population doublings in cell culture in less than 25 days. The mesenchymal stromal cells have an average terminal restriction fragment length (TRF) that can be greater than 8 kb. The mesenchymal stromal cells can have a statistically significant decrease in the content and / or enzyme activity of proteins involved in (i) cell cycle regulation and cellular senescence, (ii) cellular energy and / or lipid metabolism, and (iii) apoptosis compared to a mesenchymal stromal cell preparation derived from bone marrow that has undergone five population doublings. The mesenchymal stromal cells can have a statistically significant increase in the content and / or enzyme activity of proteins involved in cytoskeletal structure and associated cellular dynamics compared to bone marrow-derived mesenchymal stromal cells. The mesenchymal stromal cells cannot undergo a 75% or greater increase in cells with forward scatter values ​​greater than 5,000,000 as measured by flow cytometry over 10 population doublings in culture. The mesenchymal stromal cells are capable of expressing mRNA encoding interleukin-6 in a resting state at levels that may be less than 10% of the levels of IL-6 mRNA expressed in their resting state by mesenchymal stromal cells derived from bone marrow or adipose tissue.

[0091] The preparations of the invention may be suitable for administration to human patients. The preparations may be suitable for administration to non-human veterinary mammals.

[0092] In exemplary embodiments, the present disclosure provides pharmaceutical preparations comprising mesenchymal stromal cells, wherein said mesenchymal stromal cells are capable of undergoing at least 10 population doublings, wherein 10 population doublings occur within about 27 days, more preferably less than about 26 days, preferably less than 25 days, more preferably less than about 24 days, even more preferably less than about 23 days, even more preferably less than about 22 days, or less.

[0093] In an exemplary embodiment, the present disclosure provides a pharmaceutical preparation comprising mesenchymal stromal cells, wherein said mesenchymal stromal cells are capable of undergoing at least 15 population doublings.

[0094] The mesenchymal stromal cells may be allowed to undergo at least 20, 25, 30, 35, 40, 45, 50 or more population doublings.

[0095] In exemplary aspects, the present disclosure can provide a pharmaceutical preparation comprising mesenchymal stromal cells, wherein the mesenchymal stromal cells are capable of undergoing at least 15 population doublings, at least 20 population doublings, or at least 25 population doublings in culture.

[0096] The mesenchymal stromal cells of the present invention can be produced by in vitro differentiation of hemangioblasts. The mesenchymal stromal cells can be primate cells or other mammalian cells. The mesenchymal stromal cells can be human cells.

[0097] The population doubling occurs within about 35 days, more preferably within about 34 days, preferably within 33 days, more preferably within 32 days, even more preferably within 31 days, or even more preferably within about 30 days.

[0098] The preparations of the present invention may have less than about 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01% The total number of pluripotent cells in a culture may be less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, less than 0.0009%, less than 0.0008%, less than 0.0007%, less than 0.0006%, less than 0.0005%, less than 0.0004%, less than 0.0003%, less than 0.0002%, or less than 0.0001%.

[0099] The preparation of the present invention may not contain pluripotent cells.

[0100] Preparations of the invention may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% mesenchymal stromal cells.

[0101] At least 50% of the mesenchymal stromal cells may be positive for (i) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90; (ii) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC; (iii) CD105, CD73, and / or CD90; or (iv) any combination thereof. At least 50% of the mesenchymal stromal cells may be positive for (i) at least two of CD105, CD73, and / or CD90; (ii) at least two of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90; or (iii) all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC. At least 50% of the mesenchymal stromal cells may (i) be positive for all of CD105, CD73, and CD90; (ii) be positive for all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC; and / or (ii) be negative for CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and / or TLR3, or less than 5% or less than 10% of the cells may express CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and / or TLR3.At least 60%, 70%, 80%, or 90% of the mesenchymal stromal cells may be positive for (i) one or more of CD105, CD73, and CD90; (ii) one or more of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90; or (iii) one or more of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

[0102] The pharmaceutical preparations of the present invention can comprise an amount of mesenchymal stromal cells effective to treat an unwanted immune response in a subject in need thereof.

[0103] The pharmaceutical preparations of the present invention can contain other cells, tissues, or organs for transplantation into a recipient in need thereof. The other cells or tissues can be RPE cells, skin cells, corneal cells, pancreatic cells, liver cells, cardiac cells, or tissues containing any of the foregoing cells. The mesenchymal stromal cells are not derived from bone marrow, and the potency of this preparation in immunomodulatory assays can exceed the potency of preparations of bone marrow-derived mesenchymal stromal cells. Potency can be assayed by immunomodulatory assays to determine the EC50 dose. The preparation can retain approximately 50% to 100% of its differentiation potential after 10 population doublings.

[0104] The mesenchymal stromal cells cannot be derived directly from pluripotent cells, and the mesenchymal stromal cells (a) do not aggregate or aggregate in lesser amounts than mesenchymal stromal cells derived directly from pluripotent cells; (b) disperse more readily when dividing compared to mesenchymal stromal cells derived directly from pluripotent cells; (c) can outnumber mesenchymal stromal cells derived directly from pluripotent cells when starting with an equal number of pluripotent cells; and / or (d) acquire characteristic mesenchymal stromal cell surface markers earlier than mesenchymal stromal cells derived directly from pluripotent cells.

[0105] The mesenchymal stromal cells can be mammalian cells. The mesenchymal stromal cells can be human, canine, bovine, non-human primate, murine, feline, or equine cells.

[0106] In an exemplary embodiment, the present disclosure provides a method for generating mesenchymal stromal cells, the method comprising culturing hemangioblasts under conditions conducive to mesenchymal stem cells. The hemangioblasts can be cultured under feeder-free conditions. The hemangioblasts can be plated on a matrix. The matrix can comprise one or more of transforming growth factor beta (TGF-β), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and / or platelet-derived growth factor (PDGF). The matrix can be selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, heparan sulfate, Matrigel (a soluble preparation obtained from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), human basement membrane extract, and any combination thereof. The matrix can comprise a soluble preparation obtained from Engelbreth-Holm-Swarm mouse sarcoma cells.

[0107] The mesenchymal stromal cells can be mammalian cells. The mesenchymal stromal cells can be human, canine, bovine, non-human primate, murine, feline, or equine cells.

[0108] The hemangioblasts can be cultured in a medium containing αMEM. The hemangioblasts can be cultured in a medium containing serum or serum replacement. The hemangioblasts can be cultured in a medium containing αMEM supplemented with 0%, 0.1-0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% fetal bovine serum. The hemangioblasts can be cultured on the matrix for at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0109] The hemangioblasts are capable of differentiating from pluripotent cells.

[0110] The pluripotent cells can be iPS cells or blastomere-derived pluripotent cells, which can be derived from one or more blastomeres without destroying the human embryo.

[0111] The hemangioblasts can be differentiated from pluripotent cells by a method comprising the step of (a) culturing the pluripotent cells to form cell clusters. The pluripotent cells can be cultured in the presence of vascular endothelial growth factor (VEGF) and / or bone morphogenetic protein 4 (BMP-4). In step (a), the pluripotent cells can be cultured in the presence of vascular endothelial growth factor (VEGF) and / or bone morphogenetic protein 4 (BMP-4). The VEGF and BMP-4 can be added to the pluripotent cell culture within 0 to 48 hours of initiating the cell culture, and the VEGF can optionally be added at a concentration of 20 to 100 nM / mL, and the BMP-4 can optionally be added at a concentration of 15 to 100 ng / mL. The VEGF and BMP-4 can be added to the cell culture of step (a) within 0 to 48 hours of initiation of the cell culture, and the VEGF can optionally be added at a concentration of 20 to 100 nM / mL, and the BMP-4 can optionally be added at a concentration of 15 to 100 ng / mL. The hemangioblasts can be differentiated from pluripotent cells by a method further comprising the step of (b) culturing the clusters of cells in the presence of at least one growth factor in an amount sufficient to induce differentiation of the clusters of cells into hemangioblasts. The at least one growth factor added in step (b) can include one or more of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), Flt3L (FL), thrombopoietin (TPO), EPO, and / or tPTD-HOXB4.

[0112] The at least one growth factor added in step (b) may comprise one or more of about 20-25 ng / ml basic fibroblast growth factor (bFGF), about 20-100 ng / ml vascular endothelial growth factor (VEGF), about 15-100 ng / ml bone morphogenetic protein 4 (BMP-4), about 20-50 ng / ml stem cell factor (SCF), about 10-50 ng / ml Flt 3L (FL), about 20-50 ng / ml thrombopoietin (TPO), EPO, and / or 1.5-5 U / ml tPTD-HOXB4.

[0113] One or more of the at least one growth factor added in step (b) can be added to the culture within 36 to 60 hours, or within 40 to 48 hours, from the start of step (a).

[0114] The at least one growth factor added in step (b) can be added to the culture within 48 to 72 hours from the start of step (a).

[0115] The at least one growth factor added in step (b) may comprise one or more of bFGF, VEGF, BMP-4, SCF and / or FL.

[0116] The method of the invention may further comprise the step of (c) optionally dissociating said clusters of cells into single cells.

[0117] The method of the present invention may further comprise the step of (d) culturing the hemangioblasts in a medium containing at least one additional growth factor, wherein the at least one additional growth factor may be in an amount sufficient to proliferate the hemangioblasts.

[0118] In step (d), the at least one additional growth factor may include one or more of insulin, transferrin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP-4), and / or tPTD-HOXB4.

[0119] In step (d), the at least one additional growth factor may be about 10 to 100 μg / ml of insulin, about 200 to 2,000 μg / ml of transferrin, about 10 to 50 ng / ml of granulocyte-macrophage colony-stimulating factor (GM-CSF), about 10 to 20 ng / ml of interleukin-3 (IL-3), about 10 to 1,000 ng / ml of interleukin-6 (IL-6), about 10 to 50 The formulation may include one or more of: 100 ng / ml granulocyte colony-stimulating factor (G-CSF), 3-50 U / ml erythropoietin (EPO), about 20-200 ng / ml stem cell factor (SCF), about 20-200 ng / ml vascular endothelial growth factor (VEGF), about 15-150 ng / ml bone morphogenetic protein 4 (BMP-4), and / or about 1.5-15 U / ml tPTD-HOXB4.

[0120] The medium in steps (a), (b), (c) and / or (d) may be a serum-free medium.

[0121] The above method may further comprise the step of (e) mitotically inactivating the mesenchymal stromal cells.

[0122] It is possible to generate at least 80 million, 85 million, 90 million, 95 million, 100 million, 125 million, or 150 million mesenchymal stromal cells.

[0123] The hemangioblasts can be harvested at least 10, 11, 12, 13, 14, 15, 16, 17, or 18 days after inducing differentiation of the pluripotent cells.

[0124] The mesenchymal stromal cells may be generated within at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 days from initiating induction of differentiation of the pluripotent cells.

[0125] The present invention can result in at least 80 million, 85 million, 90 million, 95 million, 100 million, 125 million, or 150 million mesenchymal stromal cells generated from about 200,000 hemangioblasts within about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of culture.

[0126] The mesenchymal stromal cells of the present invention can be generated from hemangioblasts within about 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of culture as hemangioblasts at ratios of hemangioblasts to mesenchymal stromal cells of at least 1:200, 1:250, 1:300, 1:350, 1:400, 1:415, 1:425, 1:440, 1:450, 1:365, 1:475, 1:490, and 1:500.

[0127] The cells may be human cells.

[0128] In another aspect, the present disclosure provides mesenchymal stromal cells derived from hemangioblasts obtained by any of the methods described above.

[0129] In another aspect, the present disclosure provides mesenchymal stromal cells derived from in vitro differentiation of hemangioblasts.

[0130] At least 50% of the mesenchymal stromal cells are characterized by: (i) CD10, CD24, IL-11; AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC may all be positive, and (ii) CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and / or TLR3 may be negative, or less than 5% or less than 10% of the cells may express CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and / or TLR3.

[0131] At least 50% of the mesenchymal stromal cells may be positive for (i) all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90; or (ii) all of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

[0132] At least 60%, 70%, 80%, or 90% of the mesenchymal stromal cells may be positive for (i) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, and CD90; or (ii) at least one of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

[0133] The mesenchymal stromal cells may not express at least one of CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, or TLR3, or less than 5% or less than 10% of the cells may express at least one of CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, or TLR3.

[0134] In another aspect, the present disclosure provides a preparation of mesenchymal stromal cells as described above.

[0135] The preparation may comprise less than about 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03, less than 0.02%, less than 0.01%. The total number of pluripotent cells in a culture may be less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, less than 0.0009%, less than 0.0008%, less than 0.0007%, less than 0.0006%, less than 0.0005%, less than 0.0004%, less than 0.0003%, less than 0.0002%, or less than 0.0001%.

[0136] The preparation may be free of pluripotent cells.

[0137] The preparation can be substantially purified and optionally comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% human mesenchymal stromal cells.

[0138] The preparation may contain substantially similar levels of p53 and p21 protein, or may contain 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold greater levels of p53 protein compared to p21 protein.

[0139] The mesenchymal stromal cells or MSCs in this preparation are capable of undergoing at least 5 population doublings in culture, or are capable of undergoing at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more population doublings in culture. It is Noh.

[0140] The mesenchymal stromal cells (a) are unable to aggregate or are able to aggregate in lesser amounts than mesenchymal stromal cells derived directly from pluripotent cells; (b) are able to disperse more readily when dividing compared to mesenchymal stromal cells derived directly from pluripotent cells; (c) are able to disperse in greater numbers than mesenchymal stromal cells derived directly from pluripotent cells when starting with an equivalent number of pluripotent cells; and / or (d) acquire characteristic mesenchymal cell surface markers earlier than mesenchymal stromal cells derived directly from pluripotent cells.

[0141] In another aspect, the present disclosure provides a pharmaceutical preparation comprising any of the mesenchymal stromal cells or preparations of mesenchymal stromal cells described above.

[0142] The pharmaceutical preparation can include an amount of mesenchymal stromal cells effective to treat an unwanted immune response.

[0143] The pharmaceutical preparation can include an amount of mesenchymal stromal cells effective to treat an unwanted immune response, and may further include other cells or tissues for transplantation into a recipient in need thereof.

[0144] The other cells or tissues may be allogeneic or syngeneic pancreatic, neural, hepatic, RPE, corneal cells or tissue containing any of the foregoing.

[0145] The pharmaceutical preparations of the present invention may be used in treating autoimmune disorders or immune responses to allogeneic cells, or in treating multiple sclerosis, systemic sclerosis, hematological cancers, myocardial infarction, organ transplant rejection, chronic allograft nephritis, liver cirrhosis, liver failure, heart failure, GvHD, tibial fractures, left ventricular dysfunction, leukemia, myelodysplastic syndromes, Crohn's disease, diabetes, chronic obstructive pulmonary disease, osteogenesis imperfecta, homozygous familial hypocholesterolemia, post-meniscectomy treatment, adult periodontitis, angiogenesis in patients with severe myocardial ischemia, spinal cord injury, bone dysplasia , severe lower limb ischemia, diabetic foot disease, primary Sjogren's syndrome, osteoarthritis, cartilage defects, laminitis, multiple system atrophy, amyotrophic lateral sclerosis, cardiac surgery, systemic lupus erythematosus, living donor kidney allotransplant, non-malignant red blood cell disorders, burns, radiation burns, Parkinson's disease, microfractures, epidermal necrosis, severe coronary ischemia, idiopathic dilated cardiomyopathy, femoral head necrosis, lupus nephritis, bone void defects, ischemic stroke, post-stroke, acute radiation syndrome, pulmonary disease, arthritis, bone regeneration, uveitis, or a combination thereof.

[0146] In another aspect, the present disclosure provides a kit comprising any of the mesenchymal stromal cells or any of the preparations of mesenchymal stromal cells described above.

[0147] In another aspect, the present disclosure provides a kit comprising the mesenchymal stromal cells or preparations of mesenchymal stromal cells described above, wherein the cells or preparations of cells are capable of being frozen or cryopreserved.

[0148] In another aspect, the present disclosure provides a kit comprising the mesenchymal stromal cells or preparations of mesenchymal stromal cells described above, wherein the cells or preparations of cells can be encapsulated in a cell delivery vehicle.

[0149] In another aspect, the present disclosure provides a method for treating a disease or disorder, the method comprising administering to a subject in need thereof an effective amount of mesenchymal stromal cells or a preparation of mesenchymal stromal cells as described above.

[0150] The methods of the present invention can further include the generation of other cells or tissues. The cells or tissues can include retinal, RPE, corneal, neural, immune, bone marrow, liver, or pancreatic cells. The diseases or disorders include multiple sclerosis, systemic sclerosis, hematologic cancers, myocardial infarction, organ transplant rejection, chronic allograft nephritis, cirrhosis, liver failure, heart failure, GvHD, tibial fractures, left ventricular dysfunction, leukemia, myelodysplastic syndrome, Crohn's disease, diabetes, chronic obstructive pulmonary disease, osteogenesis imperfecta, homozygous familial hypocholesterolemia, post-meniscectomy treatment, adult periodontitis, angiogenesis in patients with severe myocardial ischemia, spinal cord injury, bone dysplasia, severe limb ischemia, diabetic The condition may be selected from foot disease, primary Sjogren's syndrome, osteoarthritis, cartilage defects, laminitis, multiple system atrophy, amyotrophic lateral sclerosis, cardiac surgery, systemic lupus erythematosus, living donor kidney allotransplant, non-malignant red blood cell disorders, burns, Parkinson's disease, microfractures, epidermal necrosis, severe coronary ischemia, idiopathic dilated cardiomyopathy, femoral head necrosis, lupus nephritis, bone void defects, ischemic stroke, post-stroke, acute radiation syndrome, pulmonary disease, arthritis, bone regeneration, or a combination thereof.

[0151] The disease or disorder may be uveitis. The disease or disorder may be an autoimmune disorder or an immune response to allogeneic cells. The autoimmune disorder may be multiple sclerosis.

[0152] In another aspect, the present disclosure provides a method of treating bone loss or cartilage damage, the method comprising administering to a subject in need thereof an effective amount of mesenchymal stromal cells or a preparation of mesenchymal stromal cells.

[0153] The mesenchymal stromal cells of the present invention may be administered in combination with allograft or syngeneic transplant cells or tissue, which may include retinal pigment epithelial cells, retinal cells, corneal cells, or muscle cells.

[0154] In another aspect, the present disclosure provides a pharmaceutical preparation comprising mitotically inactivated mesenchymal stromal cells, the mesenchymal stromal cells being capable of differentiating from hemangioblasts.

[0155] This medicine is 6 The composition may comprise mesenchymal stromal cells and a pharmaceutically acceptable carrier.

[0156] In another aspect, the present disclosure provides a pharmaceutical preparation comprising the mitotically inactivated mesenchymal stromal cells produced by the above method.

[0157] The preparation may be suitable for administration to a human subject.The preparation may be suitable for administration to a non-human veterinary mammal.

[0158] The pharmaceutical preparation may be free of pluripotent cells.

[0159] The pharmaceutical preparation can include an amount of mesenchymal stromal cells effective to treat an unwanted immune response in a subject in need thereof.

[0160] The pharmaceutical preparation can comprise an amount of mesenchymal stromal cells effective to treat a disease or condition selected from the group consisting of inflammatory respiratory conditions, respiratory conditions resulting from acute injury, adult respiratory distress syndrome, post-traumatic adult respiratory distress syndrome, transplant lung disease, chronic obstructive pulmonary disease, emphysema, chronic obstructive bronchitis, bronchitis, allergic reactions, injury resulting from bacterial pneumonia, injury resulting from viral pneumonia, asthma, exposure to irritants, smoking, atopic dermatitis, allergic rhinitis, hearing loss, autoimmune hearing loss, noise-induced hearing loss, psoriasis, and any combination thereof. .

[0161] Preparation of mesenchymal stromal cells In one embodiment of the present invention, a subject preparation of mesenchymal stromal cells (e.g., produced by culturing hemangioblasts) is provided, wherein the desired phenotype of the mesenchymal stromal cells is present earlier than mesenchymal stromal cells produced by ESC culture (see Figure 5). In another embodiment of the present invention, a subject preparation of mesenchymal stromal cells (e.g., produced by culturing hemangioblasts) is provided, wherein the desired phenotype of the mesenchymal stromal cells is present earlier than mesenchymal stromal cells produced by ESC culture, wherein the desired phenotype is defined by the expression of at least two markers selected from the group consisting of CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166, and HLA-abc.

[0162] Other embodiments of the invention include preparations of mesenchymal stromal cells, wherein the phenotype of the mesenchymal stromal cells is defined by expression of at least two markers selected from the group including CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166, and HLA-ABC. Still other embodiments of the invention include preparations of mesenchymal stromal cells, wherein the phenotype of the mesenchymal stromal cells is defined by expression of at least two markers selected from the group including CD9, CD13, CD29, CD44, CD73, CD90, and CD105, and wherein the mesenchymal stromal cells do not express CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E, and CD133.

[0163] In one embodiment of the present invention, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the subject mesenchymal stromal cells (e.g., generated by culturing hemangioblasts) display a phenotype defined by expression of CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166, and HLA-abc after about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days in culture. In one embodiment of the present invention, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the subject mesenchymal stromal cells (e.g., generated by culturing hemangioblasts) express CD9, CD13, CD29, or CD30 after about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days in culture. The cells exhibit a phenotype defined by the expression of at least two markers selected from the group comprising CD44, CD73, CD90, CD105, CD166, and HLA-abc, and the absence of expression of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E, CD133, and Stro-1. In the foregoing embodiment, the phenotype is further defined by markers selected from the group comprising AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1.

[0164] A preferred process of the present invention is provided, in which the number of mesenchymal stromal cells derived from hemangioblasts reaches about 2×10 within about 30 days of culturing the mesenchymal stromal cells. 5 Approximately 8 × 10 cells derived from hemangioblasts 7 , 8.5×10 7 , 9×10 7 , 9.5×10 7 , 1×10 8 , 1.25×10 8 , or 1.5 × 10 8In alternative embodiments of the present invention, mesenchymal stromal cells can be generated from hemangioblasts within about 30 days of culturing the mesenchymal stromal cells at ratios of hemangioblasts to mesenchymal stromal cells of about 1:200, 1:400, 1:415, 1:425, 1:440, 1:450, 1:465, 1:475, 1:490, and 1:500.

[0165] In preferred embodiments of the invention, the number of mesenchymal stromal cells obtained by culturing hemangioblasts is greater than the number of mesenchymal stromal cells obtained directly from ESCs. In other preferred embodiments of the invention, the number of mesenchymal stromal cells obtained by culturing hemangioblasts is at least 5, 10, 20, 22 times greater than the number of mesenchymal stromal cells obtained directly from ESCs (see Figure 4).

[0166] In another embodiment of the present invention, the subject mesenchymal stromal cell preparations do not form teratomas when introduced into a mammalian host.

[0167] One embodiment of the present invention provides a preparation of mesenchymal stromal cells produced by culturing hemangioblasts using any of the process embodiments of the present invention. One embodiment of the present invention includes a preparation of mesenchymal stromal cells produced by culturing hemangioblasts using any of the process embodiments of the present invention, wherein the phenotype of the preparation is defined by the presence of any or all of markers selected from the group including AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1. Another embodiment of the present invention includes a preparation of mesenchymal stromal cells produced by culturing hemangioblasts using any of the process embodiments of the present invention, wherein the phenotype of the preparation is defined by the presence of any or all of markers selected from the group including AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1, and wherein the preparation exhibits reduced expression of IL-6, Stro-1, and VEGF.

[0168] In one embodiment of the invention, a preparation of a subject mesenchymal stromal cell (e.g., produced by culturing hemangioblasts) is provided, said preparation comprising substantially similar levels of p53 and p21 protein, or the level of p53 relative to p21 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater. In one embodiment of the invention, a preparation of a subject mesenchymal stromal cell (e.g., produced by culturing hemangioblasts) is provided, said preparation comprising substantially similar levels of p53 and p21 protein, or the level of p53 relative to p21 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater. In one embodiment of the present invention, pharmaceutical preparations of the subject mesenchymal stromal cells (e.g., produced by culturing hemangioblasts) are provided, said pharmaceutical preparations containing substantially similar levels of p53 and p21 protein, or the level of p53 relative to p21 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater.

[0169] In one embodiment of the present invention, a preparation of the subject mesenchymal stromal cells (e.g., produced by culturing hemangioblasts) is provided, said preparation comprising substantially the same percentage of cells positive for p53 and p21 proteins, or the percentage of cells positive for p53 compared to p21 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater. In one embodiment of the present invention, a preparation of the subject mesenchymal stromal cells (e.g., produced by culturing hemangioblasts) is provided, said preparation comprising substantially the same percentage of cells positive for p53 and p21 proteins, or the percentage of cells positive for p53 compared to p21 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater. In one embodiment of the present invention, a pharmaceutical preparation of the subject mesenchymal stromal cells (e.g., produced by culturing hemangioblasts) is provided, said pharmaceutical preparation comprising substantially similar percentages of cells positive for p53 and p21 proteins, or the percentage of cells positive for p53 compared to p21 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater.

[0170] In one embodiment of the present invention, the subject mesenchymal stromal cells (e.g., hemangioblasts) are cultured. and (2) a preparation of S100A1, VIM, MYADM, PIM1, ANXA2, RAMP, MEG3, IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EML1, MYO1B, LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1, SLC35E1, ANXA2, HLA-E, CD59, BHLHB2, UCHL1, SUSP3, CREDBL2, OCRL, OSGIN2, SLEC3B, IDS, T GFBR2, TSPAN6, TM4SF1, MAP4, CAST, LHFPL2, PLEKHM1, SAMD4A, VAMP1, ADD1, FAM129A, HPDC1, KLF11, DRAM, TREM140, BHLHB3, MGC17330, TBC1D2, KIAA1 191, C5ORF32, C15ORF17, FAM791, CCDC104, PQLC3, EIF4E3, C7ORF41, DUSP18, SH3PX3, MYO5A, PRMT2, C8ORF61, SAMD9L, PGM2L1, HOM-TES-103, EPOR and TM EM112, or the group comprising S100A1, VIM, MYADM, PIM1, ANXA2, RAMP, MEG3, IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EMLI, MYO1B, LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1 and SLC35E1, or a substantially similar percentage of cells with background levels of senescence markers selected from the group consisting of S100A1, VIM, MYADM, PIM1, ANXA2, RAMP, MEG3, IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EMLI, MYO1B, LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1 and SLC35E1. NXA2, RAMP, MEG3, IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EML1, MYO1B, LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1, SLC 35E1, ANXA2, HLA-E, CD59, BHLHB2, UCHL1, SUSP3, CREDBL2, OCRL, OSGIN2, SLEC3B, IDS, TGFBR2, TSPAN6, TM4SF1, MAP4, CAST, LHFPL2, PLEKHM1, SAMD4A,from the group comprising VAMP1, ADD1, FAM129A, HPDC1, KLF11, DRAM, TREM140, BHLHB3, MGC17330, TBC1D2, KIAA1191, C5ORF32, C15ORF17, FAM791, CCDC104, PQLC3, EIF4E3, C7ORF41, DUSP18, SH3PX3, MYO5A, PRMT2, C8ORF61, SAMD9L, PGM2L1, HOM-TES-103, EPOR, TMEM112, or S100A1, The percentage of cells positive for a senescence marker selected from the group including VIM, MYADM, PIM1, ANXA2, RAMP, MEG3, IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EML1, MYO1B, LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1 and SLC35E1 is 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold greater than background. In one embodiment of the present invention, a preparation of the subject mesenchymal stromal cells (e.g., generated by culturing hemangioblasts) is provided, said preparation comprising: HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3, STMN1, CCT8, SFRS10, CBX3, CBX1, FLJ11021, DDX46, ACADM, KIAA0101, TYMS, BCAS2, CEP57, TDG, MAP2K6, CSRP2, GLMN, HMGN2, HNRPR, EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A, LOC 730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1, FKBP3, ARL5A, GMNN, ENY2, FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5, ZNF639, MRPL47, GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63, FAM33A, EPSTL1, CTR9, GAS5, ZNF711, MTO1 and CDP2, or HoxB3, HoxB7, MID1, SNAPC5,PPA, RG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3, STMN1, CCT8, SFRS10, CBX3, CBX1, FLJ11021, DDX46, ACADM, KIAA0101, TYMS, BCAS2, C EP57, TDG, MAP2K6, CSRP2, GLMN, HMGN2, HNRPR, EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A, LOC730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1, FKBP3 , ARL5A, GMNN, ENY2, FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5, ZNF639, MRPL47, GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63, FAM33A, EPSTL1, CTR9, GAS5, ZNF711, MTO1 and CDP2, or less than 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times background.

[0171] In one embodiment of the present invention, there is provided a preparation of the subject mesenchymal stromal cells (generated by culturing hemangioblasts), said preparation comprising: HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3, STMN1, CCT8, SFRS10, CBX3, CBX1, FLJ11021, DDX46, ACADM, KIAA0101, TYMS, BCAS2, CEP57, TDG, MAP2K6, CSRP2, GLMN, HMGN2, HNRPR, EIF3S from the group comprising: 1, PAPOLA, SFRS10, TCF3, H3F3A, LOC730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1, FKBP3, ARL5A, GMNN, ENY2, FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5, ZNF639, MRPL47, GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63, FAM33A, EPSTL1, CTR9, GAS5, ZNF711, MTO1 and CDP2, or HoxB3, Ho or a substantially similar percentage of cells with background levels of senescence markers selected from the group including HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, and SULT1A3, or HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3, STMN1, CCT8, SFRS10, CBX3, CBX1, FLJ11021, DDX46, ACADM, KIAA01 01, TYMS, BCAS2, CEP57, TDG, MAP2K6, CSRP2, GLMN, HMGN2, HNRPR, EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A, LOC730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1 , FKBP3, ARL5A, GMNN, ENY2, FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5, ZNF639, MRPL47, GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63,The percentage of cells positive for a senescence marker selected from the group including FAM33A, EPSTL1, CTR9, GAS5, ZNF711, MTO1, and CDP2, or selected from the group including HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, and SULT1A3 is less than 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the background level.

[0172] In another embodiment, hemangioblast-derived MSCs have a more youthful phenotype compared to adult-derived MSCs. In one embodiment, the subject MSCs are capable of undergoing at least 5, 10, 15, 20, 30, 35, 40, 45, 50, 55, 60, or more population doublings in culture. In contrast, adult-derived mesenchymal stromal cells typically undergo 2-3 population doublings in culture. In another embodiment, hemangioblast-derived MSCs have longer telomere lengths, greater telomere density, and / or greater telomere size compared to adult-derived MSCs. have immunosuppressive effects, fewer vacuoles, divide more rapidly, divide more easily in culture, have higher CD90 expression, are less lineage committed, or a combination thereof. In another embodiment, hemangioblast-derived MSCs have increased expression of transcripts that promote cell proliferation (i.e., have higher proliferative potential) and reduced expression of transcripts involved in terminal cell differentiation compared to adult-derived MSCs.

[0173] In another embodiment of the invention, a preparation of mesenchymal stromal cells is produced by any one or more of the processes of the invention, wherein said mesenchymal stromal cells are capable of undergoing at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more population doublings in culture.

[0174] In another embodiment of the invention, a subject preparation of mesenchymal stromal cells (produced by culturing hemangioblasts) is capable of undergoing at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more population doublings in culture. In another embodiment of the invention, a subject preparation of mesenchymal stromal cells is capable of undergoing at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more population doublings in culture, wherein after said population doublings, less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of the mesenchymal stromal cells have undergone replicative senescence. In other embodiments, the preparation is a pharmaceutical preparation.

[0175] In another embodiment of the invention, a preparation of mesenchymal stromal cells is provided, wherein said mesenchymal stromal cells have undergone at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 population doublings in culture.

[0176] In another embodiment of the present invention, there is provided a preparation of mesenchymal stromal cells, the mesenchymal stromal cells having undergone at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 population doublings in culture, and less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of the mesenchymal stromal cells have undergone replicative senescence, the mesenchymal stromal cells have a youthful phenotype and potency, and the preparation is a pharmaceutical preparation. The preparation can comprise a number of mesenchymal stromal cells effective for treating a disease, such as an immunological disorder, a degenerative disease, or other disease amenable to treatment using MSCs.

[0177] In another embodiment of the present invention, there is provided a subject preparation of mesenchymal stromal cells (produced by culturing hemangioblasts), wherein the mesenchymal stromal cells have undergone at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 population doublings in culture, and after such doublings, less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of the mesenchymal stromal cells have undergone replicative senescence, the mesenchymal stromal cells have a youthful phenotype and potency, and the preparation is a pharmaceutical preparation. The preparation can comprise a number of mesenchymal stromal cells effective for treating a disease, such as an immunological disorder, a degenerative disease, or other disease amenable to treatment using MSCs.

[0178] In another embodiment of the present invention, there is provided a subject preparation of mesenchymal stromal cells (produced by culturing hemangioblasts), wherein said mesenchymal stromal cells have undergone at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 population doublings in culture. In the previous embodiment, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, 5%, or less than 1% of said mesenchymal stromal cells have undergone replicative senescence, and said mesenchymal stromal cells have a youthful phenotype and potency, and said preparation The product is a pharmaceutical preparation, said pharmaceutical preparation comprising an effective number of mesenchymal stromal cells, and said pharmaceutical preparation is preserved.

[0179] In another embodiment, the invention provides a kit comprising a pharmaceutical preparation of mesenchymal stromal cells. In another embodiment, the invention provides a kit comprising a pharmaceutical preparation of mesenchymal stromal cells, said preparation being preserved. In another embodiment, the invention provides a kit comprising a pharmaceutical preparation of the subject mesenchymal stromal cells (produced by culturing hemangioblasts). In another embodiment, the invention provides a kit comprising a pharmaceutical preparation of the subject mesenchymal stromal cells (produced by culturing hemangioblasts), said preparation being preserved.

[0180] In another embodiment, the present invention provides a method of treating a condition, including but not limited to, an autoimmune disorder, uveitis, bone loss, or cartilage damage, by administering to a subject in need thereof an effective amount of hemangioblast-derived mesenchymal stromal cells.

[0181] The mesenchymal stromal cells of the present invention obtained by culturing hemangioblasts have improved properties compared to MSCs derived directly from ESCs. For example, ESC-derived MSCs aggregate in large numbers, are more difficult to disperse when dividing, and rarely produce the same number of MSCs starting from an equal number of ESCs, and take a longer time to acquire characteristic MSC cell surface markers compared to hemangioblast-derived MSCs. See Example 2 and Figures 3-6.

[0182] In one embodiment, the present invention provides a preparation of subject mesenchymal stromal cells (produced by culturing hemangioblasts), the preparation being effective in normalizing a pathological condition. In another embodiment of the present invention, a preparation of subject mesenchymal stromal cells (produced by culturing hemangioblasts), the preparation being effective in reducing excessive or unwanted immune responses. In another embodiment of the present invention, a preparation of subject mesenchymal stromal cells (produced by culturing hemangioblasts), the preparation being effective in alleviating autoimmune disorders. In another embodiment of the present invention, a normalization of a pathological condition is provided by administering to a host an effective amount of a subject mesenchymal stromal cell (produced by culturing hemangioblasts). Another embodiment of the present invention provides a normalization of a pathological condition, the normalization being characterized by an effect selected from the group consisting of cytokine release by the MSCs, stimulating an increase in the number of regulatory T cells, inhibiting a specific amount of IFNγ release from Th1 cells, and stimulating a specific amount of IL4 secretion from Th2 cells. In other embodiments, administration of a subject preparation of mesenchymal stromal cells (generated by culturing hemangioblasts) results in the release from said mesenchymal stromal cells of a cytokine selected from the group including transforming growth factor beta, indoleamine 2,3 dioxygenase, prostaglandin E2, hepatocyte growth factor, nitric oxide, interleukin 10, interleukin 6, macrophage colony-stimulating factor, and soluble human leukocyte antigen (HLA) G5.

[0183] In other embodiments of the present invention, administration of the subject mesenchymal stromal cell preparations (generated by culturing hemangioblasts) may be associated with the production of various proteins, including transforming growth factor beta, indoleamine 2,3-dioxygenase, prostaglandin E2, hepatocyte growth factor, nitric oxide, interleukin 10, interleukin 6, macrophage colony-stimulating factor, and soluble human leukocyte antigen (HLA) G5, interleukins 4, 8, 11, granulocyte-macrophage colony-stimulating factor, vascular endothelial growth factor, insulin-like growth factor 1, phosphatidylinositol-glycan biosynthesis class F protein, monocyte chemoattractant protein 1, stromal cell-derived factor 1, tumor necrosis factor 1, transforming growth factor beta, basic fibroblast growth factor, angiopoietin, erythro ... and chemokine ligands 1 and 2, interferon-γ-induced monokine, interferon-inducible protein 10, brain-derived neurotrophic factor, interleukin-1 receptor α, and chemokine ligands 1 and 2 are released from the mesenchymal stromal cells.

[0184] Pharmaceutical preparation of MSCs The MSCs of the present invention can be formulated with a pharmaceutically acceptable carrier. For example, the MSCs of the present invention can be administered alone or as a component of a pharmaceutical preparation, and the MSCs can be formulated for administration in any convenient manner for pharmaceutical use. One embodiment provides a pharmaceutical preparation of mesenchymal stromal cells combined with one or more pharmaceutically acceptable sterile, isotonic, aqueous or non-aqueous solutions selected from the group consisting of dispersions, suspensions, emulsions, sterile powders that are optionally reconstituted into sterile injectable solutions or dispersions immediately prior to use, antioxidants, buffers, bacteriostats, solutes or suspending agents, and thickening agents.

[0185] In one embodiment of the invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone about 5 to about 100 population doublings. In another embodiment of the invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone about 10 to about 80 population doublings. In another embodiment of the invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone about 25 to about 60 population doublings. In another embodiment of the invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone less than about 10 population doublings. In yet another embodiment of the invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone less than about 20 population doublings. In another embodiment of the invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone less than about 30 population doublings, and wherein the mesenchymal stromal cells have not undergone replicative senescence. In another embodiment of the present invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone fewer than about 30 population doublings, and wherein less than about 25% of the mesenchymal stromal cells have undergone replicative senescence. In another embodiment of the present invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone fewer than about 30 population doublings, and wherein less than about 10% of the mesenchymal stromal cells have undergone replicative senescence. In another embodiment of the present invention, a pharmaceutical preparation of mesenchymal stromal cells is provided, wherein the mesenchymal stromal cells have undergone fewer than about 30 population doublings, and wherein less than about 10% of the mesenchymal stromal cells have undergone replicative senescence, and wherein the mesenchymal stromal cells express a marker selected from the group including AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1.

[0186] The concentration of the pharmaceutical preparation of MSCs for injection can be any amount that is effective, e.g., an amount that is substantially free of ESCs. For example, the pharmaceutical preparation can include the numbers and types of MSCs described herein. In certain embodiments, the pharmaceutical preparation of MSCs contains about 1 x 10 MSCs for systemic administration to a host in need thereof. 6For local administration to a host containing or in need of the subject MSCs (e.g., generated by culturing hemangioblasts), about 1 x 10 4 The MSCs are obtained by culturing hemangioblasts.

[0187] Exemplary compositions of the present disclosure can be formulations suitable for use in treating human patients, such as pyrogen-free or essentially pyrogen-free and pathogen-free. When administered, pharmaceutical preparations for use in the present disclosure can be in a pyrogen-free, pathogen-free, physiologically acceptable form.

[0188] Preparations containing MSCs used in the methods described herein may be implanted as suspensions, gels, colloids, slurries, or mixtures. Also, at the time of injection, cryopreserved MSCs can be resuspended in commercially available balanced salt solutions to achieve the desired osmolality and concentration for administration by injection (i.e., bolus injection or intravenous injection).

[0189] One aspect of the present invention relates to a pharmaceutical preparation suitable for use in a mammalian patient, the pharmaceutical preparation comprising at least 10 6 , 10 7 , 10 8 Or even 10 9 Another embodiment of the present invention comprises at least 10 mesenchymal stromal cells and a pharmaceutically acceptable carrier. 6 , 10 7 , 10 8 Or even 10 9 A pharmaceutical preparation comprising a number of mesenchymal stromal cells and a pharmaceutically acceptable carrier, wherein the mesenchymal stromal cells are differentiated from hemangioblasts. 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13 Yet another aspect of the present invention provides a purified cell preparation that is free or substantially free of non-human cells and / or non-human animal products, the preparation comprising at least 10 cryogenic cell banks containing at least 10 mesenchymal stromal cells.6 , 10 7 , 10 8 Or even 10 9 mesenchymal stromal cells and less than 1% of any other cell type, more preferably less than 0.1%, less than 0.01% or even less than 0.001% of any other cell type. Specific preferred embodiments of the above preparations, compositions and banks include, but are not limited to, those listed in the following paragraphs.

[0190] In certain embodiments, the mesenchymal stromal cells have the replicative capacity to undergo at least 10 population doublings in culture, with less than 25, 20, 15, 10, or even 5% of the cells undergoing cell death, senescence, or differentiation into non-MSC cells (fibroblasts, adipocytes, and / or osteocytes) by the 10th doubling.

[0191] In certain embodiments, the mesenchymal stromal cells have the replicative capacity to undergo at least 15 population doublings in culture, with less than 25, 20, 15, 10, or even 5% of the cells undergoing cell death, senescence, or differentiation into non-MSC cells (fibroblasts, adipocytes, and / or osteocytes) by the 15th doubling.

[0192] In certain embodiments, the mesenchymal stromal cells have the replicative capacity to undergo at least 20 population doublings in culture, with less than 25, 20, 15, 10, or even 5% of the cells undergoing cell death, senescence, or differentiation into non-MSC cells (fibroblasts, adipocytes, and / or osteocytes) by the 20th doubling.

[0193] In certain embodiments, the mesenchymal stromal cells have the replicative capacity to undergo at least five passages in culture, with less than 25, 20, 15, 10, or even 5% of the cells undergoing cell death, senescence, or differentiation into non-MSC cells (fibroblasts, adipocytes, and / or osteocytes) by the fifth passage.

[0194] In certain embodiments, the mesenchymal stromal cells have the replicative capacity to undergo at least 10 passages in culture, with less than 25, 20, 15, 10, or even 5% of the cells undergoing cell death, senescence, or differentiation into non-MSC cells (fibroblasts, adipocytes, and / or osteocytes) by the 10th passage.

[0195] In certain embodiments, the mesenchymal stromal cells are differentiated from a pluripotent stem cell source, such as pluripotent stem cells expressing OCT-4, alkaline phosphatase, Sox2, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-80 (and such as an embryonic stem cell line or an induced pluripotent stem cell line), and more preferably from a general pluripotent stem cell source.

[0196] In certain embodiments, the mesenchymal stromal cells are HLA-genotypically identical.

[0197] In certain embodiments, the mesenchymal stromal cells are genomically identical.

[0198] In certain embodiments, at least 30%, 35%, 40%, 45% or even 50% of the mesenchymal stromal cells are CD10 positive.

[0199] In certain embodiments, at least 60%, 65%, 70%, 75%, 80%, 85% or even 90% of the mesenchymal stromal cells are positive for the markers CD73, CD90, CD105, CD13, CD29, CD44, CD166 and CD274 and HLA-ABC.

[0200] In certain embodiments, less than 30%, less than 25%, less than 20%, less than 15%, or even less than 10% of the mesenchymal stromal cells are positive for the markers CD31, CD34, CD45, CD133, FGFR2, CD271, Stro-1, CXCR4, and TLR3.

[0201] In certain embodiments, the mesenchymal stromal cells have a replication rate that undergoes at least 10 population doublings in cell culture in less than 25 days, less than 24 days, less than 23 days, less than 22 days, less than 21 days, or even less than 20 days.

[0202] In certain embodiments, the mesenchymal stromal cells have an average terminal restriction fragment length (TRF) greater than 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, or even 12 kb.

[0203] In certain embodiments, the mesenchymal stromal cells do not undergo a 75%, 70%, 65%, 60%, 55%, 50%, or even 45% or greater percent increase in cells having forward scatter values, as measured by flow cytometry, of greater than 5,000,000 over 10, 15, or even 20 population doublings in culture.

[0204] In certain embodiments, the mesenchymal stromal cells, in a resting state, express mRNA encoding interleukin-6 at a level that is less than 10%, less than 8%, less than 6%, less than 4%, or even less than 2% of the IL-6 mRNA level expressed by mesenchymal stromal cells derived from umbilical cord blood, bone marrow, or adipose tissue.

[0205] In certain embodiments, the mesenchymal stromal cells are at least 2, 4, 6, 8, 10, 20, 50, or even 100 times more potent than MSCs derived from cord blood, bone marrow, or adipose tissue.

[0206] In certain embodiments, when injected into a MOG35-55 EAE mouse model (such as C57BL / 6 mice immunized with MOG35-55 peptide), 1 million mesenchymal stromal cells will, on average, reduce a clinical score of 3.5 to less than 2.5, and even more preferably, reduce this clinical score to less than 2, 1.5, or even to less than 1.

[0207] In certain embodiments, the preparation is suitable for administration to a human patient and, more preferably, is pyrogen-free and / or free of non-human animal products.

[0208] In other embodiments, the preparation is suitable for administration to non-human veterinary animals, such as dogs, cats or horses.

[0209] Diseases and conditions treatable using MSCs derived from hemangioblast cultures MSCs have been shown to have therapeutic effects for a variety of diseases and conditions. In particular, MSCs migrate to the site of injury, exert immunosuppressive effects, and promote repair of damaged tissue. One embodiment of the present invention is provided, in which a pharmaceutical preparation of mesenchymal stromal cells reduces the onset of pathology. One embodiment of the present invention is provided, in which a pharmaceutical preparation of mesenchymal stromal cells reduces the onset of pathology. In another embodiment of the present invention, a pharmaceutical preparation of the subject MSCs (e.g., produced by culturing hemangioblasts) reduces the onset of a condition selected from wound healing, graft-versus-host disease (GvHD), disease, chronic eye disease, retinal degeneration, glaucoma, uveitis, acute myocardial infarction, chronic pain, hepatitis, and nephritis. In another embodiment of the present invention, a pharmaceutical preparation of mesenchymal stromal cells by culturing hemangioblasts reduces the onset of symptoms of equine laminitis. As another example, MSCs can be administered in combination with allogeneic transplant cells or tissue (e.g., including retinal pigment epithelial (RPE) cells, oligodendrocyte precursors, cells differentiated from ES cells, such as retina, cornea, muscle, such as skeletal muscle, smooth muscle, or cardiac muscle, or any combination thereof, or others), thereby reducing the likelihood of an immune response against the transplanted cells or tissue and potentially eliminating the need for other immunosuppression. The subject MSCs (produced by culturing hemangioblasts) described herein can be used in similar applications. One embodiment of the process of the invention is provided, wherein administering to a host a pharmaceutical preparation of the subject MSCs (generated by culturing hemangioblasts) reduces the need for future treatment. One embodiment of the process of the invention is provided, wherein administering to a host a pharmaceutical preparation of the subject MSCs (generated by culturing hemangioblasts) reduces the need for future treatment, said treatment suppressing immune function.

[0210] In one embodiment of the invention, a pharmaceutical preparation of the subject MSCs (produced by culturing hemangioblasts) is administered to a host for the treatment of a condition, including wound healing, multiple sclerosis, systemic sclerosis, hematologic cancer, myocardial infarction, tissue and organ transplantation, tissue and organ transplant rejection, chronic allograft nephritis, liver cirrhosis, liver failure, heart failure, GvHD, tibial fracture, left ventricular dysfunction, leukemia, myelodysplastic syndrome, Crohn's disease, diabetes mellitus type I or II, chronic obstructive pulmonary disease, pulmonary hypertension, chronic pain, osteogenesis imperfecta, homozygous familial hypocholesterolemia, post-meniscectomy treatment, adult periodontitis, vasculogenesis imperfecta in patients with severe myocardial ischemia. In some embodiments, the condition is selected from the list comprising: osteoarthritis, spinal cord injury, bone dysplasia, severe limb ischemia associated with diabetes mellitus, diabetic foot disease, primary Sjogren's syndrome, osteoarthritis, cartilage defects (e.g., articular cartilage defects), laminitis, multiple system atrophy, amyotrophic lateral sclerosis, cardiac surgery, resistant systemic lupus erythematosus, living donor kidney allograft, non-malignant red blood cell disorders, burns, radiation burns, Parkinson's disease, microfractures (e.g., in patients with knee articular cartilage defects), bullous epidermal necrosis, severe coronary ischemia, idiopathic dilated cardiomyopathy, femoral head necrosis, lupus nephritis, bone void defects, ischemic stroke, post-stroke, acute radiation syndrome, lung disease, arthritis, bone regeneration.

[0211] In other embodiments of the invention, pharmaceutical preparations of the subject MSCs (generated by culturing hemangioblasts) are administered to a host for the treatment of an autoimmune condition, including acute necrotizing hemorrhagic encephalomyelitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroiditis, and the like. cystic ulcer disease, autoimmune urticaria, axonal and neuronal neuropathy, concentric sclerosis (Balo's disease), Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas' disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multiple myelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, Crest's disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, Erythema nodosum, experimental allergic encephalomeningitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA) (see Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoprotein disease, inclusion body myositis, insulin-dependent diabetes mellitus (type 1), interstitial bladder Inflammation, juvenile arthritis, juvenile diabetes mellitus, Kawasaki disease, Lambert-Eaton syndrome, leukocyte-regulated vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucher-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatoid arthritis, PANDAS (pediatric autoimmune streptococcal psychiatric disorder), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria Vinuria, (PNH), Parry-Romberg syndrome (progressive facial hemiatrophy), Parsonage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polyglandular autoimmune syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, multisystem granulomatous disease, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, autoimmune azoospermia orchitis, stiff-body syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, cephalosal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, bullous dermatitis, leukoplakia, and Wegener's granulomatosis (currentlyGranulomatosis with polyangiitis (GPA) is a rare condition that can be treated with granulomatosis with polyangiitis (GPA).

[0212] Therapeutic regimens using MSCs derived from hemangioblast cultures The MSCs and pharmaceutical preparations comprising MSCs described herein can be used in cell-based therapies. In particular, the present invention provides methods for treating or preventing the diseases and conditions described herein, the methods comprising administering an effective amount of a pharmaceutical preparation comprising MSCs, the MSCs being derived from culturing hemangioblasts.

[0213] The MSCs of the present invention can be administered using therapeutic means known in the art, including, but not limited to, injection via intravenous, intramyocardial, transendocardial, intravitreal, or intramuscular routes or local implantation, depending on the particular condition being treated.

[0214] The mesenchymal stromal cells of the present invention can be administered via local implantation, utilizing a delivery device, which is biocompatible and biodegradable. The delivery devices of the present invention may be prepared from biocompatible fibers, biocompatible yarns, biocompatible foams, aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylenes, oxalates, polyamides, tyrosine-derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide-esters, polyoxaesters containing amino groups, poly(anhydrides), polyphosphazenes, biopolymers; homopolymers and copolymers of lactide, glycosides, ε-caprolactone, para-dioxanone, trimethylene carbonate; homopolymers and copolymers of lactide, glycosides, ε-caprolactone, para-dioxanone, trimethylene carbonate; fibrillar collagen, non-fibrillar collagen, collagen that has not been treated with pepsin, collagen combined with other polymers, growth factors, extracellular matrix proteins, biologically relevant peptide fragments, hepatocyte growth factor, platelet-derived growth factor, platelet-enriched plasma, insulin growth factor, growth differentiation factors, vascular endothelial growth factors, and the like. It may be manufactured using materials selected from the group including cell-derived growth factors, nicotinamide, glucagon-like peptides, tenascin-C, laminin, anti-rejection agents, analgesics, antioxidants, anti-apoptotic agents, anti-inflammatory agents and cytostatic agents.

[0215] The particular treatment regimen, route of administration, and adjunctive therapy may be adjusted based on the particular condition, the severity of the condition, and the patient's health status. Administration of a pharmaceutical preparation comprising MSCs may be effective to reduce the severity of symptoms of the condition and / or prevent further progression of symptoms of the condition.

[0216] Therapeutic regimens of the present invention can include administration of a single dose of MSCs. Alternatively, the therapeutic regimens described herein can include a course of treatment in which MSCs are administered multiple times over a period of time. Exemplary courses of treatment can include weekly, biweekly, monthly, quarterly, semi-annually, or annual treatment. Alternatively, treatment can require multiple administrations initially (e.g., once daily for the first week), with fewer and less frequent administrations thereafter.

[0217] In one embodiment, a preparation of mesenchymal stromal cells obtained by culturing hemangioblasts is administered to a patient one or more periodically throughout the patient's lifetime. In other embodiments of the present invention, a pharmaceutical preparation of the subject MSCs (e.g., produced by culturing hemangioblasts) is administered once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months, or once every 1-4 weeks. Alternatively, more frequent administration may be desirable for certain conditions or disorders. In one embodiment of the present invention, a pharmaceutical preparation of the subject MSCs (e.g., produced by culturing hemangioblasts) is administered once, multiple times, periodically throughout the patient's lifetime, or via a device, as appropriate for the particular patient and the patient's condition being treated. Similarly, treatment regimens that vary over time are contemplated. For example, more frequent treatments may be required at the beginning of treatment (e.g., daily or weekly treatments). Over time, as the patient's condition improves, fewer treatments, or even no further treatments, may be required.

[0218] According to the present invention, diseases or conditions can be treated or prevented by intravenous administration of the mesenchymal stem cells described herein. In some embodiments, the number of nucleotides is about 20 million, about 40 million, about 60 million, about 80 million, about 100 million, about 120 million, about 140 million, about 160 million, about 180 million, about 200 million, about 220 million, about 240 million, about 260 million, about 280 million, about 300 million, about 320 million, about 340 million, about 360 million, about 380 million, about 400 million, about 420 million, about 440 million, about 460 million, about 480 million, about 500 million, About 520 million pieces, about 540 million pieces, about 560 million pieces, about 580 million pieces, about 600 million pieces, about 620 million pieces, about 600 million pieces 40 million pieces, about 660 million pieces, about 680 million pieces, about 700 million pieces, about 720 million pieces, about 740 million pieces, about 706,0 In some embodiments, about 1 billion, about 2 billion, about 3 billion, about 4 billion, or about 5 billion cells or more are injected intravenously. In some embodiments, the number of cells ranges from about 20 million to about 4 billion cells, between about 40 million to about 1 billion cells, between about 60 million to about 750 million cells, between about 80 million to about 400 million cells, between about 100 million to about 350 million cells, and between about 175 million to about 250 million cells.

[0219] The methods described herein may further comprise the step of monitoring the effectiveness of the treatment or prevention using methods known in the art.

[0220] kit The present invention provides kits comprising any of the compositions described herein. The preparation of mesenchymal stromal cells may be encapsulated in a delivery device manufactured by methods known to those skilled in the art, including those described in U.S. Patent Application Publication No. 2002 / 0103542 and European Patent Application No. EP1454641, or stored according to methods known to those skilled in the art, including those described in U.S. Patent No. 8,198,085, PCT Application No. WO2004 / 098285, and U.S. Patent Application Publication No. 2012 / 0077181. In one embodiment of the present invention, the kit comprises at least about 8 x 10 7 pieces, 8.5×10 7 pieces, 9×10 7 pieces, 9.5×10 7 pieces, 1×10 8 pieces, 1.25×10 8 pieces, or 1.25 x 10 8 In another embodiment, the subject MSC preparation comprises about 8 x 10 hemangioblasts. 7 pieces, 8.5×10 7 pieces, 9×10 7 pieces, 9.5×10 7 pieces, 1×10 8 pieces, 1.25×10 8 pieces, or 1.25 x 10 8 Kits are provided containing a preparation of the subject MSCs (generated by culturing hemangioblasts), said preparation being a pharmaceutical preparation. 7 pieces, 8.5×10 7 pieces, 9×10 7 pieces, 9.5×10 7 pieces, 1×10 8 pieces, 1.25×10 8 pieces, or 1.25 x 10 8 A kit is provided comprising a pharmaceutical preparation of the subject MSCs (generated by culturing hemangioblasts), said pharmaceutical preparation being preserved. 7 pieces, 8.5×10 7 pieces, 9×10 7 pieces, 9.5×107 pieces, 1×10 8 pieces, 1.25×10 8 pieces, or 1.25 x 10 8 Kits are provided that include pharmaceutical preparations of the subject MSCs (generated by culturing hemangioblasts), said pharmaceutical preparations contained within a cell delivery vehicle.

[0221] Additionally, the kit may comprise cryopreserved MSCs or a preparation of cryopreserved MSCs, frozen MSCs or a preparation of frozen MSCs, thawed frozen MSCs or a preparation of thawed frozen MSCs.

[0222] Combinations of Various Embodiments and Concepts It is understood that the embodiments and concepts described herein can be used in combination. For example, the present invention provides a method of generating MSCs, the method comprising generating hemangioblasts from ESCs, culturing the hemangioblasts for at least 4 days, harvesting the hemangioblasts, replating the hemangioblasts on Matrigel-coated plates, and culturing the hemangioblasts described herein for at least 14 days, wherein the method generates at least 85 million MSCs, substantially free of ESCs. [Example]

[0223] The following examples are not intended to limit the invention in any way.

[0224] Example 1 - Generation of MSCs from hemangioblasts Hemangioblasts were generated from a clinical-stage single blastomere derived from the ESC line, MA09

[16] , as follows.

[0225] First, they were cultured in serum-free medium supplemented with a combination of morphogens and early hematopoietic cytokines (specifically, bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), thrombopoietin (Tpo), and fms-related tyrosine kinase 3 ligand (FL)). Early-stage cell clusters were generated from MA09 ESCs. Specifically, ESCs from one well of a 6-well tissue culture-treated plate were seeded into one well of a 6-well ultra-low attachment surface plate (Corning) in 3 mL of Stemline II medium (Sigma) supplemented with 50 ng / ml VEGF and 50 ng / ml BMP-4 (R&D) and incubated at 37°C with 5% CO2. Cell clusters formed over the first 24 hours. After 40–48 hours, half of the medium (1.5 mL) was replaced with fresh Stemline II medium supplemented with 50 ng / ml VEGF, 50 ng / ml BMP-4, and 20–22.5 ng / ml bFGF, and incubation continued for another 40–48 hours (i.e., 3.5–4 days in total).

[0226] Cell clusters were dissociated and single cells were plated in serum-free semi-solid blast colony growth medium (BGM). Specifically, cell clusters were dissociated with 0.05% trypsin-0.53 mM EDTA (Invitrogen) for 2-5 minutes. The cell suspension was pipetted up and down, and then DMEM + 10% FCS was added to inactivate the trypsin. The cells were then passed through a 40 μm strainer to obtain a single cell suspension. The cells were then counted and a concentration of 1-1.5 × 10 6 The cells were resuspended in Stemline II medium at 100 cells / ml.

[0227] Single cell suspension (0.3 ml, 3–4.5 × 10 5The cells were mixed with 2.7 ml of hemangioblast growth medium (H4536-based medium formulation described above) with brief vortexing and allowed to sit for 5 minutes. The cell mixture was then transferred to one well of a 6-well ultra-low attachment plate using a syringe (3 ml) fitted with an 18G needle and incubated at 37°C with 5% CO2.

[0228] Some of the cells developed into grape-like blast colonies (BCs). Specifically, BCs were visible on day 3 (typically containing fewer than 10 cells at the beginning of day 3), and after 4–6 days, grape-like hES-BCs were easily identified under a microscope (containing more than 100 cells per BC). The number of BCs present in the culture gradually increased over several days. After 6–7 days, BCs could be harvested using an open glass capillary.

[0229] Hemangioblasts could be harvested from cultures between days 7 and 12 and replated onto Matrigel-coated tissue culture plates in α-MEM + 20% FCS. Flow cytometry analysis showed that the expression levels of five cell surface markers typically found on MSCs were relatively low in the initial hemangioblast population (Figure 2, left panel, mean + / - standard deviation of four experiments). However, after 3 weeks of culture in MSC growth conditions, a homogenous population of adherent cells develops that stains >90% positive for these five characteristic MSC markers (Figure 2, right panel - days 22-23, mean + / - standard deviation of four experiments). The amount of time required for cells to differentiate in MSC culture conditions to acquire MSC surface markers may vary depending on the specific ESC line used, the day the hemangioblasts are harvested, and the number of hemangioblasts plated on Matrigel. In some experiments, the markers appear in 90% of the cells by 7-14 days, while in other experiments it may take 22-24 days for this many cells to acquire these MSC markers.

[0230] In relation to the above experiments, Figure 1 shows photomicrographs of the generation of FM-MA09-MSCs from pluripotent cells and the generation of mesenchymal stromal cells from ECS via hemangioblasts. In addition, Figure 2 includes the phenotype of FM-MA09-MSCs obtained from hemangioblasts derived from pluripotent cells generated as described above. This figure shows the percentage of cells positive for MSC surface markers in the initial hemangioblast population (left side of the graph, hemangioblasts from days 7 to 11) and the matrilineage. Figure 17 shows the percentage of cells positive for MSC surface markers after culturing hemangioblasts on gel-coated plates (right side of graph) and photomicrographs of mesenchymal stromal cells derived from hemangioblasts (right panel photographs). Furthermore, in relation to the above experiments, Figure 17 shows the process of FM-MA09-MSC generation and the effect of Matrigel, i.e., removing cells from Matrigel at early passage (i.e., p2) may temporarily slow MSC proliferation compared to those maintained on Matrigel until p6.

[0231] FIG. 18 shows that the resulting BM-MSCs and FM-MA09-MSCs undergo chondrogenesis.

[0232] Example 2 - Comparison of differentiation of ESCs and hemangioblast-derived MSCs This example describes a comparison of the differentiation of ESCs into MSCs by two methods: direct differentiation (in which ESCs were plated directly on gelatin or Matrigel) or the hemangioblast method (in which ESCs were first differentiated into hemangioblasts and then plated on Matrigel, as described in Example 1). Direct differentiation on gelatin generated MSC-like cells, but these cells lacked CD105 expression, suggesting incomplete selection of the MSC fate (Figure 3, left panel). When ESCs were plated directly on Matrigel, the resulting cells did not express CD105, as expected for MSCs (Figure 3, center panel). However, compared to MSCs produced by the hemangioblast method, directly differentiated MSC cells grew in clumps, were difficult to disperse when dividing, and rarely generated the same number of MSCs starting from an equal number of ESCs (Figure 4).

[0233] MSCs differentiated directly from ESCs also took longer to acquire characteristic MSC cell surface markers (Figure 5). Once MSCs were obtained, extensive immunophenotyping showed that MSCs obtained from both methods were positive for other markers typically found on MSCs, such as HLA-ABC, but negative for hemangioblast-associated markers, such as CD34 and CD45 (Figure 6). These results suggest that an intermediate hemangioblast stage allows for the robust production of homogenous MSCs from ESCs. Given these findings, additional studies on MSCs will be performed using hemangioblast-derived MSCs.

[0234] Additionally, experiments (described above) whose results are contained in Figures 3-6, 13, 15, 16, 19, and 21-27 compare the properties of ESC-MSCs or BM-MSCs versus hemangioblast-derived MSCs and reveal that these cells exhibit significant differences that may impact the therapeutic efficacy of these cells and compositions derived therefrom. In particular, Figure 3 shows the percentage of cells positive for MSC surface markers after culturing human embryonic stem cells (ESCs) on gelatin-coated plates (left panel), ESCs on Matrigel-coated plates (center panel), and hemangioblasts on Matrigel-coated plates (right panel). Furthermore, Figure 4 shows MSCs generated from pluripotent cells, Figure 5 illustrates the acquisition of mesenchymal stromal cell markers, and Figure 6 shows the phenotype of mesenchymal stromal cells derived from different culture methods, including the expression of MSC markers and the lack of expression of hematopoietic and endothelial cell markers. Furthermore, FM-MA09-MSCs were assayed to detect significant differences in potency and inhibitory effect (Figure 13), stimulation of Treg proliferation (Figure 15), proliferation capacity (Figure 16), PGE2 secretion (Figure 19), Stro-1 and CD10 expression (Figures 21-22), size maintenance during passage (Figure 23), CD10 and CD24 expression (Figure 24), Aire-1 and IL-11 expression (Figure 25), Ang-1 and CXCL1 expression (Figure 26), and IL6 and VEGF expression (Figure 27).

[0235] Example 3 - Hemangioblast-derived MSCs differentiate into other cell types MSCs, by definition, must be capable of giving rise to adipocytes, osteocytes, and chondrocytes. Using standard methods, Figure 7 shows the ability of hemangioblast-derived MSCs to differentiate into adipocytes and osteocytes, while Figure 8 shows their ability to differentiate toward chondrocytes via expression of chondrocyte-specific genes, and Figure 18 shows their ability to differentiate toward chondrocytes via Safranin O staining of pellet mass cultures.

[0236] Hemangioblast-derived MSCs are expected to differentiate into adipocytes, osteocytes, and chondrocytes. These differentiation pathways can be examined using methods previously reported in the art. See Karlsson et al., Stem Cell Research 3:39-50 (2009) (concerning the differentiation of hemangioblast-derived and directly ESC-derived MSCs into adipocytes and osteocytes). In particular, FM-MA09-MSCs exhibit differentiation potential, including the ability to differentiate into adipocytes and osteocytes (Figure 7). For differentiation into chondrocytes, a method from Gong et al., J. Cell. Physiol. 224:664-671 (2010) was adapted to study this process and subsequently examine the acquisition of chondrocyte-specific genes (e.g., aggrecan and collagen IIa) and glycosaminoglycan deposition through safranin O, alcian blue, and / or toluene blue staining. In particular, chondrogenic differentiation of MA09 ESC hemangioblast-derived mesenchymal stromal cells was detected by mRNA expression of aggrecan (chondroitin sulfate proteoglycan 1) and collagen IIa (Figure 8). Literature has reported that these three cell types (adipocytes, osteocytes, or chondrocytes derived from MSCs) express immunostimulatory HLA DR molecules (Le Blanc 2003; Gotherstrom 2004; Liu 2006). To confirm these reported observations, immunostaining and / or flow cytometry could be performed on these fully differentiated MSC cell types. This is important to ensure that differentiation of MSCs in an in vivo environment does not elicit an immune response from the host recipient. Among these three cell types, chondrogenic differentiation may be particularly important due to its potential for use in cartilage replacement therapy for sports injuries, aging-related joint pain, and osteoarthritis. For such therapies, MSCs do not necessarily need to be fully differentiated into chondrocytes to be used therapeutically.

[0237] Example 4 - Confirmation that MSCs derived from hemangioblasts are substantially free of ESCs MSCs must also lack the teratoma-forming propensity of ESCs. MSCs were confirmed to contain normal karyotypes by passage 12 (up to approximately 50 days in culture) (data not shown). To confirm that blast-derived MSCs do not contain traces of ESCs, teratoma formation assays were performed in NOD / SCID mice. 5 × 10 6 MSCs were injected subcutaneously into the left thigh muscle of three mice. CT2 ECs were used as a positive control, and the mice were monitored for 6 weeks to compare teratoma formation in MSC-injected mice versus ESCC-injected mice. No teratomas formed in the MSC-injected mice.

[0238] Example 5 - Reduction of EAE scores by hemangioblast-derived MSCs A pilot study was conducted to treat experimental autoimmune encephalomyelitis (EAE) in 6-8 week-old C57BL / 6 mice using hemangioblast-derived ESC-MSCs. EAE was initiated by injecting mice into the flank with 100 pL of an emulsion of 50 pg of MOG(35-55) peptide and 250 pg of M. tuberculosis in adjuvant oil (CFA) on day 0. The mice were then intraperitoneally injected with 500 ng of pertussis toxin. Six days later, the mice were intraperitoneally injected with either 1 million ESC-MSCs in PBS (n = 3) or vehicle (n = 4) as a control. Clinical scores were recorded for 29 days after immunization. A significant reduction in disease score was observed (data not shown). figure).

[0239] Example 6 - Confirmation of the efficacy of hemangioblast-derived ESC-MSCs in treating EAE and use of additional animal models of disease A. ESC-MSC testing in a mouse EAE model confirms their anti-EAE efficacy. To confirm the results obtained in Example 5, additional studies will be performed with a larger number of animals, varying cell doses, different administration protocols, and more controls. Clinical scores and mortality rates will be recorded. The degree of lymphocyte infiltration in the brain and spinal cord of mice will also be evaluated. The anti-EAE effect of MSCs is generally thought to involve immunosuppressive activity, such as suppression of Th17 cells, and is expected to reduce the degree of lymphocyte infiltration in the CNS.

[0240] B. ESC-MSCs are compared with mouse bone marrow (BM)-MSCs, human BM-MSCs, and human UCB-MSCs. Murine BM-MSCs were first used and thoroughly studied for EAE treatment [1]. Given their cross-species nature, ESC-MSCs can be directly compared with murine BM-MSCs for anti-EAE efficacy. Human UCB-MSCs have also been shown to have immunosuppressive activity

[19] . The anti-EAE activity of human UCB-MSCs and human BM-MSCs can also be compared with that of ESC-MSCs in an EAE mouse model. The age and passage of these various cell types may affect their anti-EAE behavior; therefore, we also intend to evaluate the causal role of age on the efficacy of MSCs in an EAE mouse model system.

[0241] C. Optimize the dose, route, and timing of ESC-MSC administration. Injection of ESC-MSCs can reduce EAE scores as recorded within 29 days after immunization. To test long-term prevention and cure of the disease, ESC-MSCs can be administered in various doses, routes, and times.

[0242] MSCs have been generated from H1gfp ESCs and confirmed to still express GFP at the MSC stage. EAE mice can be injected with these GFP+ ESC-MSCs, and their distribution can be tracked in vivo using the Xenogen In Vivo Imaging System. Through these approaches, various administration doses, routes, and timing of ESC-MSCs will be analyzed to provide information on the mechanism of action (i.e., pancreatic or endocrine effects) of MSCs for their anti-EAE activity, the longevity of MSCs in mice, and MSC biodistribution and excretion / elimination pathways.

[0243] Anti-EAE efficacy may be reflected by one or more of a reduction in clinical scores, increased survival rates, and / or regression of lymphocyte infiltration and demyelination of the CNS. Different ESC lines may have different inherent abilities to generate MSCs. Therefore, multiple ESC lines can be used in this study, and the acquisition of MSC markers can be monitored over time and compared for each ESC line. To further reduce variability between experiments using ESC-MSCs, large stocks of frozen ESC-MSCs can be produced in aliquots, and each aliquot can be used in multiple experiments.

[0244] D. Confirm the efficacy of hemangioblast-derived MSCs in other disease models. As mentioned above, MSCs may also have therapeutic activity against other types of autoimmune diseases, such as Crohn's disease, ulcerative colitis, and eye disorders (uveitis). Animal models for these diseases exist and are well known in the art (e.g., Pizarro et al., 2003; Duijvestein et al., 2011; Liang et al., 2012). (See, e.g., Copland et al., 2008; Copland et al., 2011). In vivo studies can be expanded to include evaluation of the therapeutic utility of MSCs in one or more of these animal model systems. Such models can allow applicants to examine the cytokine secretion profile of human MSCs by isolating and screening serum from injected animals for human cytokines. In particular, models of uveitis can be useful because local intravitreal injection can allow applicants to study the effects of MSCs in a non-systemic setting.

[0245] MSCs may also have significant therapeutic utility in treating osteoarthritic conditions, including articular cartilage loss and inflammation of affected joints (Noth et al., 2008). Models for studying osteoarthritis, cartilage loss, and joint inflammation are also well known in the art (see, e.g., Mobasheri et al., 2009). In some of these studies, human BM-MSCs are encapsulated within semi-solid scaffolds or microspheres and transplanted into affected joints of human subjects to determine whether MSCs have a local, non-systemic therapeutic effect on reducing inflammation and / or cartilage repair (Wakitani et al., 2002). Such methods will aid in determining the therapeutic utility of our ESC-angioblast-derived MSCs for treating degenerative joint conditions.

[0246] The expected lifespan of injected MSCs is very short [8], suggesting that long-term survival of transplanted cells is not required. Therefore, mitotically inactivated ESC-MSCs (e.g., irradiated or treated with mitomycin C) could also be tested for anti-EAE or other anti-disease effects in the animal models described above. If so, live ESC-MSCs may not be required, thus further reducing biosafety concerns from potentially residual ESC contaminants in the transplanted ESC-MSCs.

[0247] E. Results MSCs obtained from different donor-derived sources (mouse BM-MSCs, human BM-MSCs, and human UCB-MSCs) are expected to retain anti-EAE effects. However, these effects may vary between experiments because MSCs are obtained from donor-specific sources. In contrast, the ESC-MSCs of the present disclosure may have more consistent effects. Because many cell surface markers are used to characterize MSCs and not all MSCs express all markers, a subset of markers, such as CD73+ and CD45-, can be used to compare the efficacy of MSCs from different sources.

[0248] ESC-MSCs are expected to have therapeutic utility in animal models of Crohn's disease, ulcerative colitis, and uveitis, as these contain an autoimmune component and inflammatory response.

[0249] Mitotically inactivated MSCs (e.g., irradiated or mitomycin C-inactivated MSCs or ESC-MSCs) can at least partially retain immunosuppressive function because they still secrete cytokines and express cell surface markers associated with immunosuppressive function

[29] . However, these effects may be reduced due to their shortened lifespan in vivo. If so, the dose and frequency of administration of irradiated or otherwise mitotically inactivated cells could be increased to enhance immunosuppressive function. Mitotically inactivated MSCs and ESC-MSCs can at least partially retain immunosuppressive function because they still secrete cytokines and express cell surface markers associated with immunosuppressive function

[29] . However, these effects may be reduced due to their shortened lifespan in vivo. If so, the dose and frequency of administration of mitotically inactivated cells could be increased to enhance immunosuppressive function. , it is possible to increase it.

[0250] A second pilot study was conducted to treat EAE. Eight- to ten-week-old C57BL / 6 mice were immunized subcutaneously with the MOG35-55 peptide in complete Freund's adjuvant. This was combined with intraperitoneal injection of pertussis toxin. Six days later, 1 million live (or 2 million irradiated) hemangioblast-derived pluripotent / multipotent stromal cells were injected intraperitoneally per mouse. Disease severity was scored on a scale of 0 to 5 by monitoring the paw / trunk movements of the mice, as previously published. Results show a significant reduction in clinical scores with passage 4 hemangioblast-derived pluripotent / multipotent stromal cells and irradiated hemangioblast-derived pluripotent / multipotent stromal cells compared to vehicle controls (data not shown). Scoring for both pilot studies was performed according to the following protocol: a score of 1 indicated a limp tail, 2 indicated partial paralysis of the hind legs, 3 was complete paralysis of the hind legs, 4 was complete paralysis of the hind legs and partial paralysis of the front legs, and 5 was moribund.

[0251] In addition, the efficacy of the MSCs according to the invention and products derived therefrom for use in different therapies can be confirmed in other animal models, e.g. other transplant or autoimmune models, depending on the intended therapeutic indication.

[0252] Example 7 - Examination of the functional components of ESC-MSCs MSCs can be defined as plastic-adherent cells that express the following cell surface markers: CD105, CD73, CD29, CD90, CD166, CD44, CD13, and HLA-class I (ABC), while simultaneously being negative for CD34, CD45, CD14, CD19, CD11b, CD79a, and CD31 when cultured in non-induced conditions (e.g., in cytokine-free standard α-MEM + 20% FCS). Under these conditions, MSCs should express intracellular HLA-G and be negative for CD40 and HLA-class II (DR). Functionally, such cells should also be capable of differentiating into adipocytes, osteocytes, and chondrocytes, as assessed by standard in vivo culture assays. After 7 days of stimulation with interferon-γ (IFNγ), MSCs should express HLA-G on their cell surface, as well as CD40 and HLA-class II (DR) on their cell surface. Despite these requirements, MSCs derived from any source may contain some heterogeneity due to the pluripotency of ESCs, and MSC cultures derived from ESCs can contain cells of any lineage from the three germ layers. While the culture system described herein suggests that >90% of cells routinely exhibit the immunophenotypic and functional characteristics described above, some subpopulations of cells within an MSC culture may lack expression of one or more MSC cell surface markers and express one or more markers that should be present or absent. The extent of such subpopulations in the MSC cultures of the present invention is examined to determine the degree of heterogeneity. Multicolor flow cytometry (eight or more colors simultaneously) can be performed on a BD LSR II flow cytometer to determine the overlap between the markers described above. This can also help pinpoint the exact cell surface marker profile required for maximum immunosuppressive activity.

[0253] A. Characterizing the differentiation stage, subpopulations, and activation state of ESC-MSCs in relation to their immunosuppressive effects. A large time window exists (e.g., at least 14–28 days in MSC differentiation medium) for harvesting ESC-MSCs (see, e.g., Figure 1). Several studies have shown that MSCs tend to lose their immunosuppressive function, becoming older as they are serially passaged and senescent during prolonged culture. Thus, the cells were cultured for different time periods to determine whether a given number of days in MSC medium confers greater immunosuppressive activity. MSCs can be harvested at a specific time point in time. In fact, MSCs harvested at early time points (e.g., day 14 in MSC culture conditions) may contain precursor cells that have not yet fully acquired all of the characteristic MSC cell surface markers but retain very potent immunosuppressive effects. To define potentially useful MSC precursor populations, the expression of a wide range of cell surface markers is tracked throughout the MSC differentiation process, from days 7 to 28. At least 50% of the cultures will acquire the cell surface marker CD309 (also known as VEGFR2 and KDR) within 14 days in MSC culture conditions. CD309 is largely absent in the starting hemangioblast population (Figure 9, MA09 hemangioblasts harvested at the first time point, days 7 and 8), but it emerges within the first 2 weeks in MSC culture conditions and again declines to less than 5% of cells by day 28 (Figure 9, days 2, 3, and 4). This pattern has been observed not only in MA09 hemangioblast-derived MSCs, but also in those derived from MA01, H1gfp, and H7 ESCs. In these experiments, hemangioblasts were consistently negative for CD309, regardless of their harvest date (days 6–14). However, the percentage of MSCs that develop into MSCs that acquire CD309 expression may be reduced when developed from older hemangioblasts (e.g., d10 or d12 blastomeres). In a similar manner, it has been observed that the growth characteristics of hemangioblast-derived MSCs may differ depending on the harvest date of the hemangioblasts. MSCs developed from younger hemangioblasts (days 6 or 7) do not continue to proliferate as vigorously as MSCs developed from older hemangioblasts (days 8–12). The optimal number of days for hemangioblast harvest may be intermediate (8-10 days) to allow adequate acquisition of CD309 as a surrogate marker of MSC development while still maintaining a robust capacity for proliferation through 28 days and beyond. Studies are underway to optimize these aspects of MSC precursor development.

[0254] Except for CD105, CD90, and CD73, which have proven to be the most typical markers for MSCs (as described in the International Society for Cellular Therapy (Dominici et al., Cytotherapy 8(4):315-317 (2006)) as the minimal classification of MSCs), many other cell surface molecules not mentioned above, such as CD49a, CD54, CD80, CD86, CD271, VCAM, and ICAM, have also been proposed or used as MSC markers

[22] . Therefore, it is possible that ESC-MSCs may contain subpopulations that express various combinations of other markers during differentiation from hemangioblasts, and these may have diverse immunosuppressive activities. Subpopulations can be sorted based on one or more markers (alone or in combination) for analysis to compare their immunosuppressive activities using in vitro or in vivo methods (e.g., using FACS).

[0255] B. Optimizing differentiation and growth conditions to obtain large quantities of functional ESC-MSCs. Although preliminary experiments have shown that MSCs can be maintained in IMDM + 10% heat-inactivated human serum, the applicants have not yet tested these derivatives in this medium. Different culture conditions can be tested to determine whether alternative culture components (e.g., basal medium, serum source, serum replacement product, human serum platelet lysate) can enrich for the effective subpopulations described herein. Different animal-free basal media and defined culture systems (without FBS) for culturing ESCs and preparing MSCs will be evaluated. Specifically, StemPro® MSC SFM, available from Invitrogen, and the MSCM burette kit, available from Lonza, will be used to determine whether serum-free defined culture systems produce ESC-MSCs of the desired quality and quantity. Furthermore, various growth factors, such as FGF, PDGF, and TGFI3, as well as small chemicals that modulate signaling pathways or cell structure, can be used to enhance the quality and quantity of ESC-MSCs.

[0256] C. Results ESC-MSCs express the typical markers CD73 (ecto-5'-nucleotidase

[26] ), CD90, and CD105. Furthermore, Figure 20 shows that FM-MA09-MSCs produced according to the present invention maintain their phenotype over time (based on marker expression detected during flow cytometry analysis of different MSC populations over time and serial passages).

[0257] Example 8 - Mechanism of immunosuppression by ESC-MSC A. Examine how ESC-MSCs can suppress adaptive immune responses mediated by T cells. A common response of T cells within PBMCs is to proliferate when they are induced by mitogenic stimulants such as phytohemagglutinin (PHA) or phorbol myristate acetate (PMA) / ionomycin, or when they encounter antigen-presenting cells such as dendritic cells. This is best exemplified by the general proliferation of CD4+ and CD8+ T cells in a mixed leukocyte reaction (MLR) assay. Previous studies have suggested that MSCs can suppress T cell proliferation in MLR assays.

[0258] We examined the ability of the ESC-angioblast-derived MSCs of the present invention to inhibit T cell proliferation triggered by exposure to either chemical stimulation (PMA / ionomycin, Figures 10a and 13a) (PHA, Figure 13b) or APCs (dendritic cells, Figures 10b and 13c). We observed that MSCs attenuated the T cell proliferation response resulting from either chemical stimulation or coculture with APCs, and that this suppression occurred in a dose-dependent manner (Figure 10b, right graph). Furthermore, we found that mitotically inactivated MSCs (Figure 10b) were able to suppress T cell proliferation to the same extent as live MSCs, suggesting that mitotically inactivated MSCs may be useful in vivo for immunosuppression.

[0259] Various functional subsets of T cells exist, which perform specific roles involved in inducing pro-inflammatory responses, anti-inflammatory responses, or T cell anergy. Regulatory T cells (Tregs) are naturally occurring immunosuppressive T cells that, in normal settings, can be thought of as responsible for dampening hypersensitive autoreactive T cell responses. Although they normally represent a small fraction of the body's T cells, their distribution can be influenced by various environmental factors. MSCs have been shown to induce peripheral immune tolerance through the induction of Treg cells [33-35].

[0260] In a short-term, 5-day coculture assay, we observed that hemangioblast-derived MSCs were able to increase the percentage of CD4 / CD25 double-positive Tregs induced in response to IL2 stimulation, as in previous studies (Figures 11a, 14, and 15a). Coculture of mixed T cell populations derived from nonadherent peripheral blood mononuclear cells (PBMCs) with MSCs (at a ratio of 10 PBMCs:1 MSC) showed that Treg induction was nearly doubled when MSCs were included in the IL2-induced cultures. This degree of Treg induction is similar to that observed in the highly cited study by Aggarwal et al., published in Blood, 2005. The amount of FoxP3 induced within the CD4 / CD25 double-positive population was examined to confirm that these were true Tregs (Figure 15b). Intracellular flow cytometry was used to examine FoxP3 induction in the absence and presence of MSCs during IL2-induced T cell cultures. Both non-adherent PBMCs and purified CD4+ T cell populations can be used to test Treg induction in these assays. Without wishing to be bound by theory, ES-MSCs are more effective at inducing Tregs because they increase CD25 expression more effectively than BM-MSCs (Figure 15b).

[0261] Th1 and Th17 cells are thought to play important roles in MS and other autoimmune diseases. The differentiation and function of Th1 and Th17 CD4+ T cells were initially analyzed using in vitro assays; these can also be examined in EAE models or other animal models that can be used in the present invention. The effect of MSCs on Th1 differentiation in vitro has begun to be investigated. Culture conditions that promote Th1 specification from naive CD4+ T cells are known in the art (Aggarwal et al.). These culture conditions (containing anti-CD3, anti-CD28, and anti-CD4 antibodies along with human IL3 and IL12) were used to induce Th1 cells from naive, nonadherent PBMCs in the absence or presence of MSCs (10 PBMCs:1 MSC). After 48 hours of coculture, nonadherent cells were isolated, rinsed, and stimulated with PMA / ionomycin in new wells for 16 hours. After 16 hours of induction, supernatants were collected and analyzed for secretion of the Th1 cytokine IFNγ. As expected, PBMCs cultured with MSCs in Th1-inducing conditions for 48 hours were found to produce less IFNγ than those cultured without MSCs, suggesting that MSCs can suppress a key Th1 cell function, i.e., IFNγ secretion (Fig. 11b). Similar studies will be performed by differentiating Th17 cells in vitro and determining the effect of MSCs on the secretion of proinflammatory IL17 using an ELISA assay on the culture supernatant.

[0262] Th2 cells are known to secrete cytokines with anti-inflammatory effects, such as IL4. MSCs may be able to enhance Th2 differentiation and IL4 secretion. Similar to the above-described test for Th1 cells, Th2-inducing conditions will be used in a 48-hour culture system to stimulate Th2 differentiation from naive PBMCs containing T cells. The effect of MSC co-culture on IL4 secretion will be examined using an ELISA assay.

[0263] Recently, CD8 T cells have also been suggested to play a central role in the EAE model and the underlying mechanisms of MS

[30] . We will investigate whether ex vivo coculture with ESCs and MSCs can affect CD8 T cell function. To do this, non-adherent PBMCs or purified CD8 T cells will be exposed to the EAE-associated MBP110-118 peptide via the use of APCs. This will result in the emergence of antigen-specific CD8 T cell populations, which can be actively expanded using CD3 / CD28 Expander beads (Invitrogen). The presence of antigen-specific CD8 T cells can be confirmed by flow cytometry using a pentamer reagent specific for the MBP peptide (Proimmune). Restimulation with MBP110-118-loaded APCs will be performed to induce antigen-specific immune responses, including both active proliferation of antigen-specific CD8 T cells and secretion of IFNγ. Responses from T cells cultured in the absence or presence of MSCs will be compared to determine whether MSCs are able to suppress the induction of these cytotoxic EAE-associated antigen-specific T cells. Pentamer-specific flow cytometry (BrdU incorporation) and ELISA assays will be used for this purpose.

[0264] B. Determine whether inflammatory factors and intercellular adhesion molecules contribute to the immunosuppressive effects of ESC-MSCs. TGFβ, PGE2, IDO, nitric oxide (NO), and ICAM have been shown to be important for the immunosuppressive function of MSCs [7]. The secretion of these molecules and the expression of ICAM by ESC-MSCs will be examined using ELISA assays and flow cytometry.

[0265] The pro-inflammatory cytokine, IFNγ, is required for MSC activation

[23] , and various agonists of Toll-like receptors (TLRs), such as LPS and poly(I:C), have been shown to inhibit the proliferation of MS cells. It has been shown that IFNγ-activated MSCs can induce distinct subsets of C

[24] . For example, IFNγ-activated MSCs have recently been shown to have greater therapeutic efficacy than untreated MSCs in a mouse model of colitis (Duijvestein et al., 2011). This effect of IFNγ on MSC characteristics is beginning to be investigated. ESC-MSCs have been treated with IFNγ in vitro for up to 7 days, resulting in significant changes in the expression of cell surface markers. These results are consistent with previous observations (Gotherstrom et al., 2004; Rasmusson et al., 2006; Newman et al., 2009) and demonstrate that hemangioblast-derived ESC-MSCs function similarly to MSCs isolated from the soma. For example, in the quiescent state, MSCs do not express significant amounts of HLA G on their cell surface (<10%), but they do possess intracellular reservoirs of this unique class of immune tolerance HLA markers. Upon 7 days of IFNγ treatment, HLA G was readily detected on the cell surface (Figure 12) and could be induced to be secreted (although this remains to be tested). Furthermore, IFNγ treatment caused upregulation of CD40 and HLA DR expression on the cell surface (Figure 12). These changes are proposed to enhance their immunosuppressive effects. For example, we plan to use the in vitro coculture assay described above to examine whether pretreatment of MSCs with IFNγ can enhance their ability to induce Treg populations, suppress Th1 secretion of IFNγ, or enhance IL secretion from Th2 cells. IFNγ may also affect the ability of MSCs to inhibit general T cell proliferation in an MLR assay. The effects of TNFα, LPS, and / or poly I:C on these types of MSC immunosuppressive properties may also be examined.

[0266] C. Results As functional Tregs have been reported to upregulate the expression of transcription factors in response to inductive stimuli, the CD4 / CD25 double-positive population of Tregs induced from MSCs also expressed the transcription factor FoxP3 (Fig. ​(Fig.15b). 15b).

[0267] It is predicted that MSCs can inhibit to some extent the proinflammatory secretion of IL17 by Th17 cells, and that MSCs can significantly enhance IL4 secretion by anti-inflammatory Th2 cells. These observations, which have been made in previous studies, will help confirm the true functionality of hemangioblast-derived MSCs.

[0268] The ESC-MSCs should at least partially inhibit antigen-induced activation of CD8+ T cells. NK cell, macrophage, and dendritic cell function after ESC-MSC coculture can also be examined. The effects of ESC-MSCs on maturation, cytotoxicity, and / or specific cytokine production by these other types of immune cells will also be examined.

[0269] For example, the experiment in Figure 11A shows that hemangioblast-derived mesenchymal stromal cells increase the percentage of CD4 / CD25 double-positive Tregs induced in response to IL2 stimulation. Furthermore, the experiment in Figure 12 shows that the proinflammatory cytokine IFNg stimulates changes in FM-MA09-MSC surface markers, and interferon gamma stimulates changes in MSC surface marker expression, which can enhance the MSC immunosuppressive effect.

[0270] Furthermore, the experiment in Figure 14 demonstrates that FM-MA09-MSCs enhance Treg induction, particularly early-passage MSCs, which had a greater effect than late-passage MSCs. Non-adherent PBMCs (different donors) were cultured in the absence or presence of FM-MA09-MSCs, with or without IL2. The percentage of CD4 / CD25 double-positive Tregs was assessed by flow cytometry. Young (p6) or older (p16-18) FM-MA09-MSCs enhanced Treg induction. C was used. Black bars represent the mean value of six experiments. Overall, MSCs had a statistically significant effect on the induction of Tregs (p=0.02).

[0271] Example 9 - ESC-MSCs have increased potency and greater inhibitory effect than BM-MSCs To determine whether different MSC populations have different abilities to inhibit T cell proliferation, mixed lymphocyte reaction (MLR) assays were performed. The results suggest that ESC-MSCs are more potent than BM-MSCs in their ability to inhibit T cell proliferation in response to either mitogen stimulation ("one-way" MLR) (see Figures 13a and 13b) or antigen-presenting cells (dendritic cells, DCs, "two-way" MLR) (see Figure 13c).

[0272] This "one-way" MLR assay was performed as follows: Human PBMCs were purchased from AllCell. Upon thawing from frozen vials, PBMCs were plated for at least 1 hour or overnight in IMDM + 10% heat-inactivated human serum to selectively adhere mononuclear cells. Nonadherent cells (containing T cells) were used as a crude source of T cell responders. ESC-derived MSCs or BM-derived MSCs were used as inhibitors. These MSCs were either viable or mitotically arrested with mitomycin C. Nonadherent PBMCs and MSCs were mixed together at various ratios and cocultured for 5 days. On day 3, mitogens, phorbol-12-myristate 13-acetate (PMA) and ionomycin or phytohemagglutinin (PHA), were added to the culture to induce T cell proliferation. On day 4, bromodeoxyuridine (BrdU) was added. On day 5, T cell proliferation was assessed via flow cytometry staining with CD4, CD8, and BrdU-directed antibodies using a BrdU incorporation kit (B&D Biosystems). T cell proliferation was assessed as the percentage of CD4 and / or CD8 cells with BrdU incorporated into their DNA (i.e., BrdU) (shown in Figures 13a and 13b).

[0273] In the "two-way" MLR, ESC-derived MSCs or BM-derived MSCs were used as inhibitors, nonadherent peripheral blood mononuclear cells (PBMCs) were used as a crude source of T cell responders, and monocyte-derived dendritic cells (DCs) were used as stimulators. To induce DCs, plastic-adherent mononuclear cells were isolated from PBMCs. PBMCs were plated in IMDM + 10% heat-inactivated human serum (10% HuSer) for at least 1 hour or overnight to allow selective adherence of mononuclear cells. Nonadherent cells were removed, and adherent cells were cultured for 4 days in IMDM + 10% HuSer with SCF, FL, GM-CSF, IL3, and IL4. In this variation of the assay, no mitogen was added on day 3. BrdU alone was added 16–24 h before harvesting cells for flow cytometry as described above. In this assay, both MSCs and DCs were mitotically inactivated with mitomycin C (shown in Figure 13c).

[0274] Example 10 - Improved induction of Treg expression by early ESC-MSCs compared to BM-MSCs and late ESC-MSCs To determine whether the presence of MSCs could induce the active proliferation of regulatory T cells (Tregs) within the PBMC population, co-culture experiments were performed using PBMCs and MSCs. The results suggest that early ESC-MSCs induced the active proliferation of Tregs better than both BM-MSCs and late ESC-MSCs (see Figures 14 and 15).

[0275] Co-cultures were established using non-adherent PBMCs and different types of MSCs ("early" ESC-derived (approximately p5-6), "late" ESC-derived (approximately p12 or higher passage), and BM-derived) at a 10:1 ratio (PBMC:MSC). Co-cultures were incubated for 4 days in IMDM + 10% heat-inactivated human serum + 300 units / ml recombinant human IL2. The presence of Tregs was assessed using a FoxP3 intracellular flow cytometry staining kit (BioSource). The percentage of PBMCs staining positive for CD4, CD25, and FoxP3 was determined using the legend.

[0276] Example 11 - ESC-MSCs have great proliferation capacity. To determine whether the source of MSCs influences their proliferation capacity, we monitored the proliferation rates of different MSC populations over time. Results indicate that ESC-derived MSCs have greater proliferation capacity than BM-derived MSCs. Results also suggest that culturing ESC-MSCs on a substrate (such as Matrigel) for extended periods (up to six passages) helps maintain a higher proliferation rate than removing the cells from the substrate at earlier passages, such as p2 (Figures 16 and 17).

[0277] ESC-derived hemangioblasts were cultured at p0 in αMEM + 20% Hyclone FBS + l-glutamine + non-essential amino acids (=MSC growth medium) at 50,000 cells / cm on Matrigel-coated tissue culture plastic. 2 Bone marrow mononuclear cells were seeded at 50,000 cells / cm on defined tissue culture plastic in MSC growth medium as p0. 2 When cells reached approximately 50-60% confluence at p0 or 70-80% confluence at p1 or later (usually every 3-5 days), they were harvested using 0.05% trypsin-edta (Gibco). At harvest, cells were centrifuged, counted, and plated at 7000 cells / cm. 2 ESC-MSCs were detached from Matrigel and subsequently expanded on defined tissue culture plastic starting at p3 unless otherwise noted. Cumulative population doublings over time are plotted to demonstrate that the rate of proliferation of cells such as MSCs is maintained in culture.

[0278] Example 12 - ESC-MSCs undergo chondrogenic differentiation To determine the chondrogenic potential of different MSC populations, ESC-MSCs or BM-MSCs were seeded as pellet cultures and induced to differentiate into chondrocytes using differentiation medium (or maintained in defined MSC growth medium as a negative control). Results suggest that ESC-MSCs undergo chondrogenesis in a manner similar to that of BM-MSCs. Both ESC-MSCs and BM-MSC pellets demonstrate cartilage matrix (proteoglycan) deposition via Safranin O staining (see Figure 18).

[0279] To form chondrogenic pellet cultures, 2.5 x 10 5 ESC-MSC cells were centrifuged at 500 × g for 5 minutes in a 15 mL conical tube. The culture medium was aspirated, and 0.5 mL of chondrogenic medium (DMEM-HG (Life Technologies, Gaithersburg, MD) supplemented with 1 mM sodium pyruvate (Life Technologies), 0.1 mM ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO), 0.1 μM dexamethasone (Sigma-Aldrich), 1% ITS (Collaborative Biomedical Products, Bedford, MA), and 10 ng / mL TGF-β3 (Peprotech, Rocky Hill, NJ)) or control medium was added to the pellet. The medium was changed every 2–3 days, and the pellet culture was maintained for 21 days. At the end of 21 days, pellets were fixed in 4% paraformaldehyde and sent to MassHistology (Worcester, Mass.) for paraffin embedding, sectioning, and Safranin O staining using standard procedures.

[0280] Example 13 - Enhanced secretion of prostaglandin E2 (PGE2) under IFN-γ or TNF-α stimulation ESC-MSCs exert their immunomodulatory effects in part through the secretion of PGE2. Culture supernatants collected from FM ESC-MSCs and BM-MSCs show that BM-MSCs secrete higher levels of PGE2 than FM ESC-MSCs in the basal state. Experiments to determine PGE2 secretion under stimulatory conditions (with various concentrations of IFN-γ and / or TNF-α) show that FM-ESC-MSCs significantly increase their PGE2 secretion in response to stimulation (Figure 19). In fact, the induction rate of PGE2 secretion from basal to stimulated conditions is significantly greater for FM ESC-MSCs than for BM-MSCs. However, the actual crude amount of PGE2 secreted under stimulated conditions is similar for FM ESC-MSCs and BM-MSCs.

[0281] ESC-MSCs were cultured at 7.5 × 10 in a 6-well plate (BD Falcon, Franklin Lakes, NJ). 6 cells / cm 2 Cultures were maintained in medium for 24 hours and then stimulated with 10, 50, 100, or 200 ng / ml IFN-γ and / or 10, 25, or 50 ng / ml TNF-α (Peprotech). Supernatants were collected 3 days after induction and stored at -20°C. ESC-MSCs were harvested and counted, and PGE2 levels were normalized to cell number. PGE2 concentrations were measured using an ELISA kit (R&D PGE2 Parameter or Prostaglandin E2 Express EIA kit (Cayman Chemicals) was used according to the manufacturer's protocol.

[0282] Example 14 - ESC-MSC phenotype assessment The expression of various cell surface markers was evaluated in different MSC populations to determine their individual immunophenotypes. ESC-derived MSCs can differentiate on a variety of substrates. A panel of cell surface markers was examined to determine their expression profile on MSCs induced on three different matrices (Matrigel, fibronectin, or collagen I) compared with that on BM-MSCs. The results show similar patterns of expression for these markers regardless of the substrate used for their initial differentiation. Over 95% of the cells were positive for CD13, 29, 44, 73, 90, 105, 166, and HLA-ABC, whereas CD31, 34, 45, HLA-DR, FGFR2, and CD271 were negative (see Figure 20A). Stro-1 expression varied from approximately 5% in ESC-MSCs to approximately 30% in BM-MSCs.

[0283] MSCs slow down in proliferation and population doubling with increasing passage number. The aim of this experiment was to examine the surface marker expression of multiple different MSC markers in FM-ESC-MSCs from passages 3 to 17. Cells at all passages of FM-ESC-MSCs stained positive for CD90, CD73, CD105, HLA-ABC, CD166, CD13, and CD44. Cells were negative for CD34, CD45, TLR3, HLA-DR, CD106, CD133, and CD271 (see Figure 20B).

[0284] The same protocol was followed for each line / passage number. Cells were grown in T75 or T175 flasks in MSC medium. Cells were passaged every 3-4 days. Passaging consisted of washing the flask with PBS, harvesting the cells using cell dissociation medium TryPLE Express, and rinsing with MSC medium. Cells were counted for viability using trypan blue, and 50-100,000 viable cells were aliquoted per condition. The following antibodies were used: CD34-Fitc, CD34-PE, CD44-Fitc, CD73-PE, CD106-PE, CD45-APC (BD); HLA-DR-APC, CD90-Fitc, HLA-ABC-Fitc, CD133-APC, CD29 (ebioscience); CD166-PE, CD105-APC, CD13-PE, CD13-APC, CD271-Fitc, CD-Fitc, Stro-1-AF647, CD10 (Biolegend); and TLR3-Fitc (Santa Cruz Biotech). Propidium iodide was also added as a viability marker. Cells were incubated at room temperature for 30 minutes, centrifuged, passed through a 40 μm cell strainer, and analyzed using an Accuri C6 flow cytometer. For each cell type, cells were analyzed for MSCs. The population (FSC vs. SSC) was gated on PI negativity. Percent positivity was determined by gating on histogram plots and using an unstained cell population as a negative control. See Wagner W et al., Replicative Senescence of Mesenchymal Stem Cells: A Continuous and Organized Process. PLoS ONE (2008). 3(5):e2213. doi:10.1371 / journal.pone.0002213; and Musina et al., Comparison of Mesenchymal Stem Cells Obtained from Different Human Tissues. Cell Technologies in Biology and Medicine (2005) April, 1(2):504-509.

[0285] Furthermore, FM ESC-MSCs have higher levels of CD10 expression and lower levels of Stro-1 expression than FM ESC-MSCs and BM-MSCs (see Figure 21). This pattern of low Stro-1 expression (5-10% of cells) and intermediate CD10 expression (approximately 40% of cells) was confirmed in 10 different lots of FM-MA09-MSCs (see Figure 22). Flow cytometry was also used to assess cell size in different populations (see Figure 23). The results show that BM-MSCs increase in size as the cells are maintained in culture over time, whereas FM ES-derived MSCs maintain their size. Cell size was determined by forward scatter versus side scatter on a flow cytometry dot plot. Quadrant gates were used to divide the plot into four regions. The upper right quadrant contains large cells; that is, cells in this region have both a large forward scatter (cell volume) and a high side scatter (granularity).

[0286] ESC-MSCs were harvested as described above and washed in 1X DPBS (Life Technologies). 5 Cells were washed with flow buffer (3% FBS; Atlas Biologicals, Fort Collins, CO) and then incubated on ice for 45 minutes in 100 μL of flow buffer containing primary or isotype control antibodies. Cells were washed with 2 mL of flow buffer and incubated on ice in 100 μL of flow buffer containing secondary antibodies. Cells were finally washed, resuspended in flow buffer containing propidium iodide, and analyzed on an Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor, MI).

[0287] Example 15 - Gene expression analysis in ESC-MSCs The purpose of these studies was to determine the similarities or differences in mRNA expression between FM-ESC-MSCs and BM-MSCs. In the first set of experiments (basal experiments), the relative differences in mRNA expression between cells derived from FM-ESC-MSCs and BM-MSCs were compared by quantitative polymerase chain reaction (QPCR). Taqman probes (Life Technologies) for various genes were used to determine relative expression to the endogenous control, GAPDH, using the ΔΔCt method. From a list of 28 genes, the following genes were upregulated in the FM-ESC-MSC vs. BM-MSC basal experiments: AIRE, ANGPT1 (ANG-1), CXCL1, CD10, CD24, and IL11 (see Figures 24-26). IL6 and VEGF were downregulated in FM-ESC-MSCs vs. BM-MSCs (see Figure 27). The following genes did not differ significantly between MSC sources: ALCAM, FGF7, HGF, LGALS1, NT5E, and TNFSF1B (data not shown). The following genes were not detected in either MSC source: ANGPT2, CD31, CD34, CD45, HLA-G, IL2RA, IL3, and IL12B (data not shown). As a negative control, all MSCs were tested for expression of the hematopoietic progenitor markers CD34, CD41, and CD45. From these experiments, Applicants determined that FM-ESC-MSCs express several genes at higher or lower levels than equivalent BM-MSCs.

[0288] Additionally, we challenged MSCs to an environment mimicking an immune response by treating them with T cells and then adding the stimulatory agent phytohemagglutinin (PHA). ESC-MSCs were cultured for two days in the presence of T cells (unstimulated) or T cells plus PHA (stimulated) before adding 2.5 μg / ml PHA for an additional two days before harvesting RNA. Gene expression in unstimulated or stimulated ESC-MSCs is now compared to mRNA levels in unstimulated and stimulated BM-MSCs.

[0289] For the baseline experiments, FM-ESC-MSCs and BM-MSCs were cultured in 10-cm dishes at a starting density of approximately 500,000 cells for 4 days under the conditions described above. Additionally, the negative control for the baseline experiments was MA09 ESC-derived hematopoietic progenitor cells.

[0290] For stimulation experiments, FM-ESC-MSCs and BM-MSCs were cultured in 10-cm dishes at a starting density of approximately 500,000 cells for 3–4 days under the conditions described above. MSCs were then exposed to T cells for 2 days, followed by exposure to 2.5 μg / ml PHA. As controls, MSCs were grown in the presence of T cells without PHA, and T cells + PHA (without MSCs) were also grown separately. The medium was aspirated, washed twice in PBS, and aspirated dry. RNA was isolated using the RNAeasy kit (Qiagen) according to the manufacturer's instructions. RNA concentration and purity were confirmed using a Nanodrop 2000 (Thermo The data were analyzed using a BioRad Scientific. cDNA synthesis was performed using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Life Technologies) with 1 microgram of RNA as starting material. cDNA was diluted approximately 30-fold at 5 microliters per well. Diluted cDNA, 1 microliter of QPCR Taqman probe (Life Technologies), and 15 microliters of SSO Fast Mastermix (Biorad) were mixed in each well. QPCR was performed on a Biorad CFX 96. Data were analyzed using CFX Manager 2.1 (Biorad). Relative amounts of mRNA expression were determined using an endogenous control, GAPDH, and the ΔΔCt method.

[0291] Example 16 - Indoleamine 2,3-dioxygenase (IDO) enzyme activity in ESC-MSCs Indoleamine 2,3-dioxygenase (IDO) is an enzyme involved in the conversion of tryptophan to kynurenine. IFNγ-activated MSCs produce IDO, which may be partially responsible for their ability to suppress T cell proliferation because IDO interferes with T cell metabolism. In this study, we examined the IDO activity of BM-MSCs compared with ESC-MSCs. IDO expression was measured after stimulation with IFNγ or by co-culturing with T cells. The experiment showed that all MSC populations significantly increased IDO upon stimulation with IFNγ (Figure 28).

[0292] Cells are stimulated by either adding IFNγ (50 ng / ml) to the medium or by co-culturing with T cells for 3 days, and IDO expression is measured using a spectrophotometric assay. After stimulation, cells are harvested and 1-2×10 6 The cells were lysed. The lysate was collected and diluted 2x. The lysates were mixed 1:1 with IDO buffer (PBS containing 40 mM ascorbate, 20 μM methylene blue, 200 μg / ml catalase, and 800 μM L-tryptophan) and incubated at 37°C for 30 min. The reaction was stopped by adding 30% trichloroacetic acid and incubated at 52°C for 30 min. The lysates were centrifuged, and the supernatant was purified with Ehrlich's reagent (freshly prepared 0.8% p-dimethylaminobenzaldehyde in acetic acid). The solution was mixed 1:1 with tryptophan (hydride). After color development, the absorbance was read at 492 nm on a spectrophotometer. To assess the conversion of tryptophan to kynurenine, the OD values ​​were compared with standards of 0–1000 μM kynurenine.

[0293] See Meisel et al., Human bone marrow stromal cells inhibit allogeneic T-cell response by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood. (2004) June 15;103(12):4619-21.

[0294] See Braun, D. et al., A two-step induction of indoleamine 2,3-dioxygenase (IDO) activity during dendritic-cell maturation. Blood. (2005) October 1;106(7):2375-81.

[0295] Example 17 - Expression levels of Aire-1 and prion protein in ESC-MSCs The expression levels of Aire-1 and prion protein (PrP) were monitored using Western blot analysis to determine whether there were differences between different MSC populations (based on cell source, derivation method, or MSC passage number). Aire-1 helps induce the transcription of rare peripheral tissue-restricted antigens (PTAs), which are subsequently presented on MHC and moderate the response of neighboring T cells. Aire-1 also suppresses the expression of early T cell activator-1 (ETA-1), inhibiting T cell inflammatory responses. Prion protein (PrP) has been shown to enhance the proliferation and self-renewal of various stem cell populations (e.g., hematopoietic stem cells, neural stem cells), and its expression can be correlated with the growth characteristics of different MSC populations in culture. Results show an aging-associated decline in both proteins (after examining actin as a loading control for each sample). FM MA09-MSCs appear to maintain the expression of both Aire-1 and PrP over time (Figure 29).

[0296] Whole-cell lysates from MSCs were run on a 12% acrylamide SDS-PAGE gel according to standard protocols. Proteins were transferred to nitrocellulose membranes and blocked with 5% milk in PBS plus 0.05% Tween-20. The membranes were probed with antigens directed against Aire-1 (Santa Cruz Biotech) or prion protein (Abcam), followed by HRP-conjugated secondary antigens. Signals were developed using enhanced chemiluminescence reagents prior to analysis on a Biorad GelDoc Imaging System.

[0297] See Parekkadan et al., Molecular Therapy 20(1):178-186 (2011).

[0298] See Mohanty et al., Stem Cells 30:1134-1143 (2012).

[0299] Example 18 - ESC-MSC secretion of cytokines MSCs are known to secrete a variety of cytokines and growth factors both in the basal state and in response to various stimuli. More than 20 different secreted factors were analyzed using a cytokine array. The results indicate that there are several important differences between ESC-MSCs and BM-MSCs in terms of secreted factors in both the basal and stimulated states. BM-MSCs express higher levels of VEGF than ESC-MSCs in both the basal and IFNγ-stimulated states (see Figures 30-32).

[0300] Equal numbers of MSCs were initially plated, and supernatants were collected from the MSCs 3–4 days after plating. The CM was briefly centrifuged to remove cellular debris and frozen at −20°C. The CM was thawed and analyzed on RayBiotech (Norcross, GA) custom membrane arrays or various R&D Systems (Minneapolis, MN) off-the-shelf cytokine arrays according to the manufacturer's protocols.

[0301] Example 19 - Human ES cell culture for differentiation of MSCs The aim of this experiment was to evaluate different growth media used in hESC culture prior to differentiation into MSCs.

[0302] Human ES cells were generally cultured on irradiated or mitomycin C-treated mouse embryonic fibroblast (MEF) feeder cells in human cell growth medium (Knockout DMEM or DMEM / F12 (1:1) basal medium, 20% serum replacement, l-glutamine, non-essential amino acids, and 10 ng / ml bFGF). Passage was performed using 0.05% trypsin / EDTA. Alternatively, hESCs were cultured on MEF feeders in Primate Medium and passaged using dissociation solution (both purchased from ReproCELL). Results showed that Primate Medium consistently produced "better-looking" hESC colonies (less spontaneous differentiation, more rounded, firmer colonies) compared to cells grown on human ES cell growth medium containing Knockout DMEM.

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[0304] Each document cited herein (including U.S. patents, U.S. patent application publications, non-patent literature, etc.) is hereby incorporated by reference in its entirety.

Claims

1. 1. A pharmaceutical composition comprising a plurality of mesenchymal stromal cells (MSCs), wherein the MSCs are derived from hemangioblasts cultured in a medium containing serum or a serum replacement, under feeder-free conditions and / or on a matrix; and expression of CD10 is upregulated in the MSCs in the pharmaceutical composition compared to MSCs in bone marrow, and at least 30% of the MSCs in the pharmaceutical composition express CD10 on their surface; and The pharmaceutical composition, wherein expression of Stro-1 is downregulated in MSCs in the pharmaceutical composition compared to MSCs in bone marrow, and less than 30% of the MSCs in the pharmaceutical composition express Stro-1 on their surface.

2. The pharmaceutical composition of claim 1 , wherein the hemangioblasts are cultured in feeder-free conditions and on a matrix.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein at least 35%, 40%, 45% or 50% of the MSCs in the pharmaceutical composition express CD10 on their surface.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the MSCs in the pharmaceutical composition are positive for the markers CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274 and HLA-ABC.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein less than 25%, 20%, 15% or 10% of the MSCs in the pharmaceutical composition express Stro-1 on their surface.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein less than 30%, 25%, 20%, 15% or 10% of the MSCs in the pharmaceutical composition are positive for the markers CD31, CD34, CD45, CD133, FGFR2, CD271, CXCR4 and TLR3.

7. 7. The pharmaceutical composition according to any one of claims 1 to 6, wherein at least 40% of the MSCs in the pharmaceutical composition express CD10 on their surface and 5-10% of the MSCs in the pharmaceutical composition express Stro-1 on their surface.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the MSCs of the pharmaceutical composition have been passaged 4, 5 or 6 times.

9. 9. The pharmaceutical composition of claim 8, wherein the MSCs of the pharmaceutical composition have been passaged four times.

10. 10. The pharmaceutical composition of claim 9, wherein the MSCs of the pharmaceutical composition have been passaged five times.

11. The pharmaceutical composition of claim 10, wherein the MSCs of the pharmaceutical composition have been passaged six times.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the MSCs of the pharmaceutical composition are human cells.

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