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  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
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  • C12N2500/00—Specific components of cell culture medium
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  • C12N2500/10—Metals; Metal chelators
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/065—Modulators of histone acetylation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2303—Interleukin-3 (IL-3)
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2306—Interleukin-6 (IL-6)
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/70—Enzymes
  • C12N2501/72—Transferases [EC 2.]
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  • C12N2510/00—Genetically modified cells

Definitions

• expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).
• the term“inflammatory disease” refers to a disease or condition characterized by aberrant inflammation (e.g. an increased level of inflammation compared to a control such as a healthy person not suffering from a disease).
• inflammatory diseases include autoimmune diseases, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, ankylosing spondylitis, psoriasis, Sjogren’s syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, Behcet’s disease, Crohn’s disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis,
• carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
• exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid
• treatment includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms (e.g ., ocular pain, seeing halos around lights, red eye, very high intraocular pressure), fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.
• the length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient. In embodiments, the treating or treatment is not prophylactic treatment.
• Dosages may be varied depending upon the requirements of the patient and the compound being employed.
• the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
• the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects.
• Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
• administering means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
• Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
• HSCs can be used to treat diseases including, but not limited to: aplastic anemia, Fanconi anemia, Diamond-blackfan syndrome, Sickle cell disease, Thalassemia,
• Paroxysmal nocturnal hemoglobinuria Chediak-Higashi syndrome, Chronic granulomatous disease, Glanzmann thrombasthenia, Osteopetrosis, Lysosomal storage disorders, Gaucher disease, Niemann-Pick, Mucopolysaccharidosis, Glycoproteinoses, Immune deficiencies, Ataxia telangiectasia, DiGeorge syndrome, Severe combined immunodeficiency (SCID), Wiscott- Aldrich, Kostmann syndrome, Shwachman-Diamond syndrome, Leukemias, Acute myelogenous leukemia, Acute lymphoblastic leukemia, Hairy cell leukemia, Chronic lymphocytic leukemia,
• serum-free refers to medium which is free or substantially free of serum.“Substantially free of serum” as used herein refers to media which contains less than about 1% serum by weight, contains only trace amounts of serum, or contains undetectable amounts of serum.
• cell culture or“culture” is meant the maintenance of cells in an artificial, in vitro environment.
• HSCs hematopoietic stem cells
• ex vivo culture leads to expansion and differentiation of cells, at the expense of the most primitive pluripotent long-term stem cells (CD34 + CD90 + CD45RA ).
• CD34 + CD90 + CD45RA pluripotent long-term stem cells
• the medium described herein increases the numbers of both CD34 + cells and CD34 + CD90 + CD45RA cells during culture to levels not seen in other media systems.
• a supplement is provided and used with the medium for growth of HSCs.
• the supplement is provided as a stock solution, for example 2x, 5x, lOx, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 75x, 80x, 90x, lOOx stock solution (where“50x” indicates that the stock solution is a 50-fold solution, i.e., should be diluted 1:50 prior to use).
• the supplement is a chemically-defined medium.
• the supplement does not comprise protein.
• the supplement does not comprise serum.
• the supplement does not comprise an ingredient derived from an animal.
• the medium and/or supplement comprises an organic or inorganic salt selected from an aluminum salt, a barium salt, a cadmium salt, a copper salt, a magnesium salt, a manganese salt, a nickel salt, a potassium salt, a calcium salt, a silver salt, a tin salt, a zirconium salt, a sodium salt, or combinations thereof.
• the medium and/or supplement comprises an inorganic salt in a range of about 1 x 10 7 g/L to about 7.8 g/L, about 1 x 10 7 g/L to about 7.6 g/L, about 1 x 10 7 g/L to about 7.4 g/L, about 1 x 10 7 g/L to about 7.2 g/L, or about 1 x 10 7 g/L to about 6.9 g/L.
• the concentration may be any value or subrange within the recited ranges, including endpoints.
• the medium comprises an iron salt (e.g., FeS0 4 , FeCl 2 , FeCL, and/or Fe(N0 3 ) 3 ) in a range of about 5 x 10 6 g/L to about 1 x 10 3 g/L, about 5 x 10 5 g/L to about 5 x 10 4 g/L, or about 5 x 10 5 g/L to about 1 x 10 4 g/L.
• an iron salt e.g., FeS0 4 , FeCl 2 , FeCL, and/or Fe(N0 3 ) 3
• the medium and/or supplement comprises a potassium salt (e.g., KBr, KC1, and/or KI) in a range of about 1 x 10 3 g/L to about 10 g/L.
• the medium comprises a potassium salt (e.g., KBr, KC1, and/or KI) in a range of about 5 x 10 3 g/L to about 10 g/L, about 1 x 10 2 g/L to aboutlO g/L, about 5 x 10 2 g/L to about 10 g/L, or about 0.1 g/L to about 10 g/L.
• the medium and/or supplement comprises a zinc salt (ZnCl 2 and/or ZnS0 4 ) in a range of about 1 x 10 5 g/L to about 5 x 10 2 g/L.
• the medium comprises a zinc salt (ZnCl 2 and/or ZnS0 4 ) in a range of about 1 x 10 4 g/L to about 5 x 10 2 g/L, about 1 x 10 3 g/L to about 5 x 10 2 g/L, about 5 x 10 3 g/L to about 5 x 10 2 g/L, or about 1 x 10 2 g/L to about 5 x 10 2 g/L.
• the medium comprises a zinc salt (ZnCl 2 and/or ZnS0 4 ) in a range of about 1 x 10 5 g/L to about 5 x 1o -4 g/L, about 5 x 10 5 g/L to about 1 x 10 3 g/L, about 1 x l0 4 g/L to about 5 x 10 3 g/L, about 5 x 10 4 g/L to about 1 x 10 2 g/L, or about 1 x 10 3 g/L to about 5 x 10 2 g/L.
• the concentration may be any value or subrange within the recited ranges, including endpoints.
• the medium and/or supplement comprises a vitamin selected from para-aminobenzoic acid, vitamin B 12, biotin, choline (e.g., choline chloride), tocopherol (e.g., tocopherol acetate), folic acid, ascorbic acid, inositol, nicotinic acid, niacinamide, pantothenic acid, calcium pantothenate, pyridoxine, pyroxidone, riboflavin, thiamine, or combinations thereof.
• each of the previously mentioned vitamins may independently be excluded from a medium and/or supplement as described herein.
• the medium and/or supplement comprises the lipid (e.g., fatty acid) in a range of about 5 x 10 6 g/L to about 1 x 10 3 g/L, about 1 x 10 5 g/L to about 1 x 10 3 g/L, about 5 x 10 5 g/L to about 1 x 10 3 g/L, about 1 x 10 4 g/L to about 1 x 10 3 g/L, or about 5 x 10 4 g/L to about 1 x 10 3 g/L.
• the lipid e.g., fatty acid
• the medium and/or supplement comprises the energy source in a range between about 5 x 10 3 g/L and about 10 g/L, about 1 x 10 2 g/L and about 10 g/L, about 5 x 10 2 g/L and about 10 g/L, about 0.1 g/L and about 10 g/L, about 0.5 g/L and about 10 g/L, about 1 g/L and about 10 g/L, or about 5 g/L and about 10 g/L.
• the medium and/or supplement comprises the energy source in a range between about 1 x 10 3 g/L and about 5 g/L, about 1 x 10 3 g/L and about 1 g/L, about 1 x 10 3 g/L and about 0.5 g/L, about 1 x 10 3 g/L and about 0.1 g/L, about 1 x 10 3 g/L and about 5 x 10 2 g/L, or about 1 x 10 3 g/L and about 1 x 10 2 g/L.
• the concentration may be any value or subrange within the recited ranges, including endpoints.
• the medium and/or supplement comprises the amino acid or amino acid derivative in a range of about 1 x 10 4 g/L to about 5 g/L, about 1 x 10 4 g/L to about 1 g/L, about 1 x 10 4 g/L to about 0.01 g/L, about 1 x 10 4 g/L to about 0.05 g/L, about 1 x 10 4 g/L to about 0.01 g/L, about 1 x 10 4 g/L to about 5 x 10 3 g/L, or about 1 x 10 4 g/L to about 1 x 10 3 g/L.
• the medium and/or supplement comprises insulin in a range between about 0.005 g/L to about 0.1 g/L, about 0.005 g/L to about 0.05 g/L, or about 0.005 g/L to about 0.01 g/L.
• the concentration may be any value or subrange within the recited ranges, including endpoints.
• insulin may be excluded from a medium and/or supplement as described herein.
• the medium and/or supplement comprises transferrin in a range between about 0.01 g/L to about 0.5 g/L, about 0.05 g/L to about 0.5 g/L, about 0.1 g/L to about 0.5 g/L, about 1 g/L to about 5 g/L, about 5 g/L to about 10 g/L.
• the medium and/or supplement comprises transferrin in a range between about 0.005 g/L to about 0.1 g/L, about 0.005 g/L to about 0.05 g/L, or about 0.005 g/L to about 0.01 g/L.
• the concentration may be any value or subrange within the recited ranges, including endpoints.
• transferrin may be excluded from a medium and/or supplement as described herein.
• the antioxidative agent is selected from polyphenols, ascorbate, and carotenoids.
• the polyphenol is selected from those found naturally in fruits, wines, and teas.
• the ascorbate is selected from ascorbate or ascorbic acid.
• the carotenoid is selected from beta-carotene, alpha-carotene and lycopene.
• the antioxidative agent is selected from DL lipoic acid, DL tocopherol acetate, and ascorbic acid.
• the medium and/or supplement comprises an additional ingredient selected from an emulsifier, a surfactant, an antioxidant, a buffer, a poloxamer, a metal binding compound, or combinations thereof.
• the antioxidative agent is selected from polyphenols, ascorbate, and carotenoids.
• the polyphenol is selected from those found naturally in fruits, wines, and teas.
• the ascorbate is selected from ascorbate or ascorbic acid.
• the carotenoid is selected from beta-carotene, alpha-carotene and lycopene.
• the antioxidative agent is selected from DL lipoic Acid, DL tocopherol acetate, and ascorbic acid.
• the medium and/or supplement comprises an additional ingredient selected from hypoxanthine or salt thereof, thymidine, polysorbate, ethanolamine, putrescine, spermine, sperimidine, EDTA, 2-mercaptoethanol, B-glycerophosphate, and hydrocortisone.
• each additional ingredient may individually be excluded from a medium and/or supplement as described herein. Each additional ingredient may be present in the medium and/or supplement in any amount, for example between about 5 x 10 6 g/L and about 1 g/L.
• the medium and/or supplement comprises the additional ingredient in a range between about 5 x 10 6 g/L and about 1 x 10 5 g/L, about 1 x 10 5 g/L and about 5 x 10 5 g/L, about 5 x 10 5 g/L and about 1 x 10 4 g/L, about 1 x 10 4 g/L and about 5 x 10 4 g/L , about 5 x 1o -4 g/L and about 1 x 10 3 g/L, about 1 x 10 3 g/L and about 5 x 10 3 g/L , about 5 x 10 3 g/L and about 0.01 g/L, about 0.01 g/L and about 0.05 g/L, about 0.05 g/L and about 0.1 g/L, about 0. 1 g/L and about 0.5 g/L, or about 0.5 g/L and about 1 g/L.
• the concentration may be any value or subrange within the recited ranges,
• the medium and/or supplement comprises a histone
• HAT inhibitor refers to a substance capable of detectably decreasing the expression or activity of histone acetyltransferase.
• the inhibitor can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the inhibitor.
• expression or activity is 1.5-fold, 2-fold, 3-fold, 4- fold, 5-fold, lO-fold or lower than the expression or activity in the absence of the inhibitor.
• the HAT inhibitor is present at a concentration between about 1 x 10 4 g/L and about 5 x 10 4 g/L, about 5 x 10 4 g/L and about 0.001 g/L, about 0.001 g/L and about 0.005 g/L, about 0.005 g/L and about 0.01 g/L, about 0.01 g/L and about 0.05 g/L, about 0.05 g/L and about 0.01 g/L, about 0.01 g/L and about 0.05 g/L, about 0.05 g/L and about 0.1 g/L, or about 0.1 g/L and about 0.5 g/L.
• the concentration may be any value or subrange within the recited ranges, including endpoints.
• SCF is present in the medium and/or supplement at a concentration between about 0.0005 milligram per milliliter (mg/mL) and about 1 mg/mL. In embodiments, SCF is present in the medium and/or supplement at a concentration between about 0.0001 mg/mL and about 1 mg/mL, between about 0.005 mg/mL and about 1 mg/mL, about 0.01 mg/mL and about 1 mg/mL, about 0.05 mg/mL and about 1 mg/mL; about 0.1 mg/mL and about 1 mg/mL, or about 0.5 mg/mL and about 1 mg/mL.
• Flt3L is present at a concentration between about 0.0005 mg/mL and about 1 mg/mL. In embodiments, Flt3L is present in the medium and/or supplement at a
• Flt3L is present in the medium and/or supplement at a concentration between about 0.0005 mg/mL and about 0.5 mg/mL, about 0.0005 mg/mL and about 0.1 mg/mL, about 0.0005 mg/mL and about 0.05 mg/mL; about 0.0005 mg/mL and about 0.01 mg/mL, about 0.0005 mg/mL and about 0.005 mg/mL, or about 0.0005 mg/mL and about 0.001 mg/mL.
• TPO is present in the medium and/or supplement at a concentration between about 0.0005 mg/mL and about 0.005 mg/mL, about 0.005 mg/mL and about 0.05 mg/mL, about 0.05 mg/mL and about 0.5 mg/mL; about 0.5 mg/mL and about 1 mg/mL.
• G-CSF is present in the medium and/or supplement at a concentration between about 0.0001 mg/mL and about 1 mg/mL, between about 0.005 mg/mL and about 1 mg/mL, about 0.01 mg/mL and about 1 mg/mL, about 0.05 mg/mL and about 1 mg/mL; about 0.1 mg/mL and about 1 mg/mL, or about 0.5 mg/mL and about 1 mg/mL.
• G-CSF is present in the medium and/or supplement at a concentration between about 0.0005 mg/mL and about 0.5 mg/mL, about 0.0005 mg/mL and about 0.1 mg/mL, about 0.0005 mg/mL and about 0.05 mg/mL; about 0.0005 mg/mL and about 0.01 mg/mL, about 0.0005 mg/mL and about 0.005 mg/mL, or about 0.0005 mg/mL and about 0.001 mg/mL.
• G-CSF is present in the medium and/or supplement at a concentration between about 0.0005 mg/mL and about 0.005 mg/mL, about 0.005 mg/mL and about 0.05 mg/mL, about 0.05 mg/mL and about 0.5 mg/mL; about 0.5 mg/mL and about 1 mg/mL.
• GM-CSF is present in the medium and/or supplement at a concentration between about 0.00005 mg/mL and about 0.05 mg/mL, about 0.00005 mg/mL and about 0.01 mg/mL, about 0.00005 mg/mL and about 0.005 mg/mL; about 0.00005 mg/mL and about 0.001 mg/mL, about 0.00005 mg/mL and about 0.0005 mg/mL, or about 0.00005 mg/mL and about 0.0001 mg/mL.
• GM-CSF is present in the medium and/or supplement at a concentration between about 0.00005 mg/mL and about 0.0005 mg/mL, about 0.0005 mg/mL and about 0.005 mg/mL, about 0.005 mg/mL and about 0.05 mg/mL; about 0.05 mg/mL and about 0.1 mg/mL
• the HAT inhibitor is present in the medium or supplement at a concentration of between about 0.0001 g/L and about 0.01 g/L and the HD AC inhibitor is present in the medium or supplement at a concentration of between about 0.0005 g/L and about 0.1 g/L. In some embodiments, the HAT inhibitor is present in the medium and/or supplement at a concentration of between about 0.001 g/L and about 1 g/L and the HD AC inhibitor is present in the medium and/or supplement at a concentration of between about 1 g/L and about 5 g/L.
• the medium and/or supplement include a HAT inhibitor, a HD AC inhibitor and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt
• the HAT inhibitor is selected from 2,6-Bis[(3-bromo-4- hydroxyphenyl)methylene]cyclohexanone, MG149, C646, CPTH2, curcumin, A-485, anacardic acid, MB -3, and chalcones such as garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2- hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin
• the HD AC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin,
• panobinostat sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, sodium valproate (valproic acid; VP A), givinostat, MS-275, MGCD0103, and Scriptaid.
• the medium and/or supplement comprise a chalcone HAT inhibitor and a HD AC inhibitor selected from sodium butyrate, sodium phenylbutyrate, trichostatin A, and sodium valproate (VPA).
• the medium and/or supplement comprise a HAT inhibitor selected from garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2- hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin, and a HD AC inhibitor selected from sodium butyrate, sodium phenylbutyrate, trichostatin A, and sodium valproate (VPA).
• HAT inhibitor selected from garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2- hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin
• a HD AC inhibitor selected from sodium butyrate, sodium phenylbutyrate, trichostatin A, and sodium valproate (VPA).
• the medium and/or supplement include a HAT inhibitor, a HD AC inhibitor and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt
• the HAT inhibitor is selected from 2,6-Bis[(3-bromo-4- hydroxyphenyl)methylene]cyclohexanone, MG149, C646, CPTH2, curcumin, A-485, anacardic acid, MB -3, and chalcones such as garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2- hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin;
• the HD AC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydro
• the medium and/or supplement include a HAT inhibitor, a HD AC inhibitor and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt
• the HAT inhibitor is selected from 2,6-Bis[(3-bromo-4-hydroxyphenyl)methylene]cyclohexanone, MG149, C646, CPTH2, curcumin, A-485, anacardic acid, MB-3, and chalcones such as garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2-hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin;
• the HD AC inhibitor is selected from apicidin, belinostat, CI-994, CRA- 024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, tric
• the medium and/or supplement include a HAT inhibitor, a HD AC inhibitor and three or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt
• the HAT inhibitor is selected from 2,6-Bis[(3-bromo-4- hydroxyphenyl)methylene]cyclohexanone, MG149, C646, CPTH2, curcumin, A-485, anacardic acid, MB-3, and chalcones such as garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2- hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin;
• the HD AC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydrox
• the medium and/or supplement include a HAT inhibitor, a HD AC inhibitor and three or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt
• the HAT inhibitor is selected from 2,6-Bis[(3-bromo-4-hydroxyphenyl)methylene]cyclohexanone, MG149, C646, CPTH2, curcumin, A-485, anacardic acid, MB-3, and chalcones such as garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2-hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin;
• the HD AC inhibitor is selected from apicidin, belinostat, CI-994, CRA- 024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, tric
• the medium and/or supplement comprises a HAT inhibitor, a HD AC inhibitor, a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt
• the HAT inhibitor is selected from 2,6-Bis[(3-bromo-4-hydroxyphenyl)methylene]cyclohexanone, MG149, C646, CPTH2, curcumin, A-485, anacardic acid, MB-3, and chalcones such as garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2-hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin;
• the HD AC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A
• the medium and/or supplement comprises a HAT inhibitor, a HD AC inhibitor, a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt
• the HAT inhibitor is selected from 2,6-Bis[(3- bromo-4-hydroxyphenyl)methylene]cyclohexanone, MG149, C646, CPTH2, curcumin, A-485, anacardic acid, MB -3, and chalcones such as garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2-hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin;
• the HD AC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin
• the medium and/or supplement comprises a HAT inhibitor, a HD AC inhibitor and two or more of a lipid, an amino acid or amino acid derivative, an
• the HAT inhibitor is selected from 2,6-Bis[(3- bromo-4-hydroxyphenyl)methylene]cyclohexanone, MG149, C646, CPTH2, curcumin, A-485, anacardic acid, MB -3, and chalcones such as garcinol, isogarcinol, xanthohumol isoxanthohumol, 2-hydroxycalchone, 4-hydroxycalchone, yakuchinone A, and isoliquiritigenin; wherein the HD AC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, sodium valproate (valproic acid; VPA), givinostat, MS-275, MGCD0103, and Scriptaid; wherein the HD AC inhibitor is selected from apicidin,
• the medium and/or supplement comprises a HAT inhibitor, a HD AC inhibitor and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt
• the HAT inhibitor is selected from a chalcone such as garcinol, isogarcinol, xanthohumol isoxanthohumol, 2-hydroxycalchone, 4- hydroxycalchone, yakuchinone A, and isoliquiritigenin
• the HD AC inhibitor is selected from sodium butyrate, sodium phenylbutyrate, tricho statin A, and sodium valproate (VPA);
• antioxidative agent is selected from a DL lipoic acid, DL tocopheral acetate and ascorbic acid; and combinations thereof; and wherein the inorganic salt is selected from copper salt, a magnesium salt, a selenite salt, a potassium salt, a calcium salt, a zinc salt, an iron salt, a sodium salt, or
• the HD AC inhibitor and HAT inhibitor are at a weight ration of 1:1 to 1:30 HD AC inhibitor: HAT inhibitor.
• the medium and/or supplement comprise a chalcone HAT inhibitor and a HD AC inhibitor selected from sodium butyrate, sodium phenylbutyrate, trichostatin A, and sodium valproate (VPA).
• the medium and/or supplement comprise a HAT inhibitor selected from garcinol, isogarcinol, xanthohumol, isoxanthohumol, 2-hydroxycalchone, 4- hydroxycalchone, yakuchinone A, and isoliquiritigenin, and a HD AC inhibitor selected from sodium butyrate, sodium phenylbutyrate, trichostatin A, and sodium valproate (VPA).
• the present disclosure relates to a method of expanding stem cells, the method including growing stem cells in a growth medium including a basal medium, a histone acetyltransferase (HAT) inhibitor, a histone deacetylase (HD AC) inhibitor, and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt.
• a growth medium including a basal medium, a histone acetyltransferase (HAT) inhibitor, a histone deacetylase (HD AC) inhibitor, and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt.
• HAT histone acetyltransferase
• HD AC histone deacetylase
• the present disclosure relates to a method of expanding stem cells, the method including (i) adding a supplement including a histone acetyltransferase (HAT) inhibitor and a histone deacetylase (HD AC) inhibitor to a basal medium to form a growth medium, and (ii) growing stem cells in the growth medium, thereby expanding the stem cells.
• a supplement including a histone acetyltransferase (HAT) inhibitor and a histone deacetylase (HD AC) inhibitor
• HAT histone acetyltransferase
• HD AC histone deacetylase
• the present disclosure relates to a method of expanding primary cells from a subject, the method including growing the primary cells in a growth medium including a basal medium, a histone acetyltransferase (HAT) inhibitor and a histone deacetylase (HD AC) inhibitor, and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt.
• a growth medium including a basal medium, a histone acetyltransferase (HAT) inhibitor and a histone deacetylase (HD AC) inhibitor, and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt.
• HAT histone acetyltransferase
• HD AC histone deacetylase
• the present disclosure relates to a method of treating a subject in need of a therapy, the method including: (a) obtaining hematopoietic stem cells (HSCs); (b) expanding the HSCs in a growth medium including a basal medium, a histone acetyltransferase (HAT) inhibitor, and a histone deacetylase (HD AC) inhibitor, and at least two of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt; and (c) transferring the HSCs to the subject, thereby treating the subject.
• HSCs hematopoietic stem cells
• HAT histone acetyltransferase
• HD AC histone deacetylase
• HSCs Hematopoietic stem cells
• HD AC histone deacetylase
• the HSCs may be derived from the same subject that is to be treated and/or may be genetically modified prior to transferring the HSCs to the subject. Expanded HSCs may be used to treat a hematopoietic malignancy, cancer, an autoimmune disease, or a blood-based disease and/or after the subject has undergone chemotherapy.
• the subject may be human.
• the genome is edited using one or more genome editing reagents selected from a zinc-finger nuclease (ZFN), transcription activator- like effector nuclease (TALEN), meganuclease, and a clustered regularly interspaced short palindromic repeat (CRISPR) associated protein, and (c) optionally expanding the edited cell in HSC expansion media as described herein for a period of time (e.g., 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, or longer, or any amount of time in between).
• ZFN zinc-finger nuclease
• TALEN transcription activator- like effector nuclease
• CRISPR clustered regularly interspaced short palindromic repeat
• the present disclosure relates to a method of treating a subject in need of a therapy, the method including: (a) obtaining hematopoietic stem cells (HSCs) that were expanded using a medium as described herein; and (b) transferring the HSCs to the subject, thereby treating the subject.
• HSCs hematopoietic stem cells
• the present disclosure relates to a method of reprogramming a cell (e.g., a CD34+ cell, a PBMC) to an iPSC, the method including: (a) obtaining a cell to be re-programmed to an iPSC cell, (b) expanding the cell in HSC expansion media as described herein, (c) introducing the Yamanaka factors into the cell (see, for example Takahashi et al. (2007) Cell 131:861-872.), (d) culturing the cell from (c) on a matrix in HSC expansion media for a first period of time, and (e) replacing the HSC expansion media with iPSC media, thereby reprogramming the cell.
• a cell e.g., a CD34+ cell, a PBMC
• the method including: (a) obtaining a cell to be re-programmed to an iPSC cell, (b) expanding the cell in HSC expansion media as described herein, (c)
• (e) includes transitioning the cell from HSC expansion media to iPSC media.
• the HSC expansion media is completely replaced with iPSC media at once.
• the HSC expansion media is replaced with iPSC media in a stepwise fashion, until all of the HSC expansion media is replaced.
• the reprogramming factors are introduced to the expanded CD34+ cells using a CytoTuneTM iPS Reprogramming Kit (Thermo Fisher Scientific), such as CytoTuneTM -iPS 2.0 Sendai Reprogramming Kit or CTSTM CytoTuneTM -iPS 2.1 Sendai Reprogramming Kit.
• the present disclosure relates to a kit including a basal medium and a supplement, the medium and/or supplement including a histone acetyltransferase (HAT) inhibitor, a histone deacetylase (HD AC) inhibitor, and two or more of a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt.
• the medium and/or supplement including a HAT inhibitor, a HD AC inhibitor, a lipid, an amino acid or amino acid derivative, an antioxidative agent, and an inorganic salt.
• CD34 + CD90 + CD45RA cells in the expanded cell population.
• the CD34 + CD90 + CD45RA cell population is relevant in the context of HSC transplantation, since this population is believed to be more important in establishing long term engraftment.
• HAT inhibitor and HD AC inhibitor to competitor media did not result in a corresponding increase in the CD34 + CD90 + CD45RA subpopulation - indicating that the effect is not due solely to the presence of
• VPA/Garcinol but rather to the supplement more generally.
• the basal medium used in the following examples was manufactured as a IX formulation and stored at 4 °C, while the supplement was a 50X formulation stored at -20 °C.
• the thawed supplement was added to the basal medium, and specific growth factors were added to the complete media to promote HSC expansion.
• CD34 + cells from cord blood, bone marrow, or mobilized peripheral blood, as indicated below, were cultured in the presence of this media system for up to 14 days, in a 37 °C incubator. At Day 7 (or the indicated time point), cells were enumerated and assessed for phenotype by flow cytometry.
• the basal medium plus the supplement (at IX) and growth factors (SCF, Flt3L, TPO, IL-6 and IL-3) enable the expansion of HSCs. See, e.g., Fig. 1.
• SCF, Flt3L, TPO, IL-6 and IL-3 enable the expansion of HSCs. See, e.g., Fig. 1.
• HSC Medium as provided herein
• SCGM Stem Cell Growth Medium
• HSC expansion was initiated by culturing 5 x 10 3 CD34 + cells/mL in a 48 well plate for 7 days in a humidified 37 °C, 5% C0 2 incubator. At day 7, total nucleated cell (TNC) number and % viability were assessed using a Countess II cell counter (Thermo Fisher Scientific), and cell phenotype was performed by flow cytometry.
• TNC were washed in phosphate-buffered saline (PBS) and incubated for 20 minutes at room temperature with LIVE/DEAD® Fixable Yellow stain in PBS.
• Cells were washed in flow cytometry staining buffer, and incubated with a cocktail of monoclonal antibodies against human cell surface antigens: CD34 PE-Cy7, CD90 APC, CD45RA Pacific Blue, for 30 minutes at 4 °C.
• Cells were analyzed on a Attune NxT flow cytometer (Thermo Fisher Scientific), and FLOWJO® Software (version 7.6.5 Becton Dickinson).
• ALDH Aldehyde dehydrogenase
• CD34 + CD90 + CD45RA data points in the three donors were averaged and standard error calculated.
• HSC Medium used in this example is a xeno-free, serum-free medium specifically formulated to support expansion of hematopoietic stem cells (HSCs). Phenotypes targeted for expansion are total CD34 + cell population, with an emphasis on enrichment of the HSCs.
• CD34 + cells and the CD34 + CD90 + CD45RA subset are found in low proportions in mobilized peripheral blood, bone marrow, as well as cord blood. They are important for proper hematopoietic and immune function, and are studied for a variety of reasons including transplantation biology, stem cell biology, and hematopoietic development. HSC Medium enables superior expansion of not only the total CD34 + compartment, but also enriches for CD34 + CD90 + CD45RA stem cells, providing the researcher with more cells to work with (see figures 7-9).
• CFU colony forming unit
• CFC colony forming cell
• TNC total nucleated cells
• CFC colony forming cell
• Expanded HSC must maintain their ability to differentiate into various blood cell lineages. In vivo , this is assessed via engraftment of HSC into immunodeficient mice. As these experiments typically take months to complete, a surrogate assay to determine differentiation potential is the colony forming cell (CFC) assay.
• CFC colony forming cell
• cells are seeded into a semi- solid MethoCultTM medium that contains growth factors such as erythropoietin, GM-CSF, G-CSF, IL-3, IL-6, SCF, which support the growth of progenitor cells such as erythrocytes, granulocyte, and macrophage progenitors. The different colonies are observed after a 14 day culture period.
• CD34 + CD90 + CD45RA long term HSC Fig. 7E. Percentages of CD34+ and
• CD34 + CD90 + CD45RA shown are from the total live TNC population.
• Three human single donor purified CD34 + mPB were cultured in HSC Medium (HSC Basal and 50X Supplement) or three commercial media, all supplemented with growth factors (SCF, Flt3L, TPO, IL-3, and IL-6). Cells were cultured for 7 days, following which total nucleated cells (TNC) and % viability were determined using a Countess II, and phenotype assessed by flow cytometry as described below and in Fig. 9. Error bars denote standard deviation.
• HSC Medium demonstrates Consistent Lot-to-Lot Performance.
• CD34 + mPB expanded in three different lots of HSC Medium demonstrate equivalent levels of CD34 + cell expansion (Fig. 8A), TNC expansion (Fig. 8B), % viability (Fig. 8C), %
• CD34 + (Fig. 8D), and % CD34 + C D90 + C D45 R A long term HSC (Fig. 8E).
• Three human single donor purified CD34 + mPB were cultured in three different lots of HSC Medium (HSC Basal and 50X Supplement), all supplemented with growth factors. Cells were cultured for 7 days, following which total nucleated cells (TNC) and % viability were determined using a Countess II, and phenotype assessed as described in Fig. 9. Error bars denote standard deviation.
• Granulocyte/Erythroid/Monocyte/Megakaryocyte GEMM
• Erythroid E
• E Erythroid
• HSC Medium Expands Single donor human CD34 + mPB cells. All three human single donor CD34 + mPB that were expanded in HSC Medium demonstrated equivalent levels of expansion in CD34 + cells (Fig. 11 A), TNC (Fig. 11B), % viability (Fig. 11C), % CD34+ (Fig. 11D). Notably, the level of expansion of CD34 + CD90 + CD45RA long term HSC (Fig. 11E) varied amongst donors. Three human single donor purified CD34 + mPB were cultured in HSC Medium, supplemented with growth factors. Cells were cultured for 7 days, following which total nucleated cells (TNC) and % viability were determined using a Countess II, and phenotype assessed as described in Fig. 9. Error bars denote standard deviation within sample replicates.
• TNC total nucleated cells
• Fig. 12C highest ALDH expression on a per- cell basis (Geometric Mean Fluorescent Intensity), and (Fig. 12D) highest % of CD34 + cells staining positive for ALDH.
• Consistency in ALDH expression by expanded cells was observed between HSC Medium lots.
• Three human single donor purified CD34 + mPB were cultured in three lots of HSC Medium or three commercial media, all supplemented with growth factors. Cells were cultured for 7 days, following which total nucleated cells (TNC) were incubated in either DEAB or ALDEFLUORTM, according to manufacturer’s instructions. Cells were stained with antibodies to identify CD34 + cells, and analyzed for ALDH expression. Data displayed were pooled from the three single donor mPB cells. Error bars denote standard error.
• CD34+ cells Expanded in HSC Medium are capable of being genetically engineered using CRISPR/Cas9.
• lx 10 6 purified CD34+ cells from mobilized peripheral blood (mPB) from a single donors was individually cultured in HSC Medium described herein, supplemented with 100 ng/mL SCF, 100 ng/mL FLT-3L, 100 ng/mL TPO, 50 ng/mL IL-3, and 20 ng/mL IL-6 (all provided by Thermo Fisher Scientific). Cells were cultured for 2 days in a 37 °C incubator, 5% C02.
• GFP+ and GFP- -sorted cells were analyzed by flow cytometry for CD34 and CD90 using an Attune NxT flow cytometer (Thermo Fisher Scientific). The bars indicate the mean value of the 3 individual donors.
• Fig. 13B Transfected GFP+ cells exhibited a similar CD34, CD90 profile when compared to the un-transfected GFP- cells, demonstrating that expansion of CD34+ cells in HSC expansion media as described herein does not negatively impact the ability to genetically modify the cells.
• GFP+ genetically engineered cells displayed similar ability to differentiate compared to non-modified cells, using a colony forming assay (Fig. 13C).
• a xeno-free reprograming workflow is desirable.
• xeno-free workflows are desirable from a regulatory standpoint.
• the following experiment demonstrates that HSC’s can be reprogrammed to iPSCs using the xeno-free HSC expansion media as described herein.
• Single donor, cord-blood derived, CD34+ cells from 2 individuals were thawed and cultured in either OPTMIZERTM cell media (Thermo Fisher Scientific) containing SCF, IL-3, and GM-CSF; or STEMPROTM HSC Expansion media as described herein containing SCF, Flt3L, TPO, IL-3, and IL-6.
• Example 6 StemPro HSC Expansion Medium (Prototype) Enables Reprogramming of PBMC into iPSC with CTS CytoTune 2.1 and CytoTune 2.0.
• HSC HSC
• PBMCs CD34+ cells from three single donors were obtained, thawed and cultured in either STEMPROTM 34 cell media (Thermo Fisher Scientific) containing SCF, IL-3, and GM-CSF; or HSC Expansion media as described herein containing SCF, Flt3L, TPO, IL-3, and IL-6. During each day of culture, half the media was removed and replaced with fresh media containing cytokines.
• Cells were transduced with CTSTM CytoTuneTM 2.l-iPS Sendai Reprogramming Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol, at an MOI of 5-5-3 (KOS-LMyc-Klf4) in the presence of polybrene. Following transduction, the cells were split into two cultures; one culture was grown in normoxic conditions, the other culture was grown in hypoxic conditions, in the STEMPROTM 34 media or HSC Expansion media with growth factors as described above. Three days later, cells were plated onto recombinant human vitronectin (rhVTN-N) coated plates in STEMPROTM 34 media or HSC Expansion Media as described herein containing no cytokines.
• CTSTM CytoTuneTM 2.l-iPS Sendai Reprogramming Kit Thermo Fisher Scientific
• Example 7 Engraftment of CD34+ Cells Expanded in HSC Expansion Media.
• Human CD34+ Cells are obtained and expanded in HSC expansion media described herein for 7 days. Various concentrations of expanded cells, unexpanded cells (control) are transplanted into lethally irradiated immunodeficient mice. At different timepoints (2 months, 6 months), mice are euthanized and their bone marrow cells and spleen cells are harvested. Bone marrow and spleen cells are stained with antibodies against human CD34, CD33, CD45, Lineage, CD3 and CD19 cell surface markers and analyzed using flow cytometer.
• bone marrow cells of mice euthanized at 6 months as described above are transplanted into lethally irradiated immunodeficient mice (secondary transplant). At two months, the mice are euthanized and their bone marrow cells and spleen cells are harvested. Bone marrow and spleen cells are stained with antibodies against human CD34, CD33, CD45, Lineage, CD3 and CD19 cell surface markers and analyzed using flow cytometer. Presence of transplanted cells in bone marrow and spleen is observed, indicating self-renewal capacity of expanded cells.
• HSC medium The ability of HSC medium to promote expansion of primary cells derived from a human donor is evaluated. Different types of primary cells (e.g., macrophages, T cells) are expanded in HSC medium as described herein. It is expected that the HSC medium will increase expansion of some additional primary cell types, for example T cells.
• T cells Different types of primary cells
• Example 10 Treatment of Patients with HSCs Expanded in HSC Medium
• HSCs expanded as described herein to treat a patient having a disease treatable by HSCs is determined.
• HSCs are derived from an HSC source that is allogenic for the patient to be treated.
• HSCs are expanded ex vivo in HSC Medium. Expanded HSCs are administered to the patient in an amount effective to promote engraftment of the cells and treatment of the condition.

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