WO2019018256A1 - Utilisation de cellules souches somatiques pour augmenter le niveau de prmt - Google Patents

Utilisation de cellules souches somatiques pour augmenter le niveau de prmt Download PDF

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Publication number
WO2019018256A1
WO2019018256A1 PCT/US2018/042219 US2018042219W WO2019018256A1 WO 2019018256 A1 WO2019018256 A1 WO 2019018256A1 US 2018042219 W US2018042219 W US 2018042219W WO 2019018256 A1 WO2019018256 A1 WO 2019018256A1
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subject
micrometers
cells
stem cells
composition
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PCT/US2018/042219
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James Wang
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StemBios Technologies, Inc.
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Publication of WO2019018256A1 publication Critical patent/WO2019018256A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]

Definitions

  • Protein arginine methylation is a common posttranslational modification that
  • PRMTs Protein arginine methyltransferases
  • type I PRMTs catalyze the formation of asymmetric dimethylarginine
  • type II PRMTs catalyze the formation of symmetric dimethylarginine.
  • PRMT5 is the major type II enzyme identified thus far. It, together with its cofactor MEP50, o mediates the methylation of histones H2A and H4 at R3 and histone H3 at R8.
  • PRMT5 has been found in several transcriptional repressor complexes such as SIN3A/HDAC, MBD2/NURD, and N-CoR/SMRT. PRMT5 has been shown to repress globin gene expression through recruitment of DNMT3A, indicating a potential 5 crosstalk between histone arginine methylation and DNA methylation. The enzyme plays an important role in epigenetic regulation, which is linked to aging process in cells.
  • PRMT5 hematopoietic stem and progenitor cells
  • a method of increasing a PRMT level in a subject includes identifying a subject in need thereof; and administering to the subject a composition that contains small cells that are greater than 2 micrometers and less than 6 5 micrometers in size; wherein the small cells include somatic stem cells that are (i) pluripotent or multipotent; and (ii) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+), or CD34(+).
  • the identifying step includes detecting the level of a PRMT in a biological sample obtained from the subject. In some embodiments, the detected level is o lower than a control level.
  • the identified subject can have a condition associated with a PRMT dysfunction. For example, the subject can have a condition associated with aging or anemia.
  • the small cells in the composition administered to the subject can further include platelets.
  • 75% to 85% of the small cells can be platelets and 20% to 25% of the 5 small cells can be the somatic stem cells.
  • the composition contains 10 million to 500 million of the somatic stem cells.
  • the composition is prepared by a process that includes:
  • the composition is prepared.
  • 1.5 to 2.0 mg of the divalent cation chelating agent per millimeter of the blood sample can be mixed with the blood sample to obtain the mixture.
  • the divalent cation chelating agent is EDTA.
  • an action for increasing stem cell number is performed on the subject or donor subject.
  • the action can be administration of an effective amount of fucoidan or a granulocyte-colony stimulating factor.
  • the process for preparing the composition can further include, after collecting the o upper layer, adding a pharmaceutically acceptable excipient to the collected upper layer.
  • the process can further include, after collecting the upper layer, centrifuging the upper layer to obtain a cell pellet.
  • the pellet can be further washed and suspended in a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient can be one that is free of divalent ions.
  • the pharmaceutically acceptable 5 excipient is a saline solution.
  • the method further comprises, after the administering step, detecting a PRMT level in a biological sample obtained from the subject after the administering step.
  • compositions for increasing a PRMT level in a 0 subject contains small cells that are greater than 2 micrometers and less than 6 micrometers in size, wherein the small cells include somatic stem cells that are (i) pluripotent or multipotent; and (ii) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+), or CD34(+).
  • compositions containing certain small somatic stem cells can increase the level of a PRMT. Accordingly, described herein are methods and compositions for increasing a PRMT level in a subject and o for treating conditions associated with a defect in PRMT.
  • somatic stem cells There are various types of somatic stem cells, including totipotent stem cells, pluripotent stem cells, multipotent stem cells, and progenitor stem cells (also called unipotent stem cells).
  • Blastomere-like stem cells BLSCs
  • VSELs Very small embryonic -like stem cells
  • SB cells are pluripotent or multipotent somatic stem cells.
  • MSCs Mesenchymal stem cells
  • HSC hematopoietic stem cell
  • the size (Z) of a cell such as a stem cell, as used herein may refer to (1) the conventional definition of the size or representative length of a cell in the field of cell biology 0 or the field of stem cells, (2) the diameter of a cell especially when the cell is substantially spherical, (3) the length of the major axis of a cell especially when the cell is substantially ellipsoidal, (4) the width of a cell when the shape of the cell has an approximate shape of a square, (5) the length of a cell when the shape of the cell has an approximate shape of a rectangle, or (6) the greatest cross-sectional or transverse dimension of a cell,
  • the size (Z), 5 either the diameter, length, width, or greatest cross-sectional or transverse dimension can be determined or measured, for example, using an image of the cell obtained from an optical microscope or from an electron microscope (e.g., scanning electron microscope (SEM)), or using data (e.g., two-dimensional dot, contour or density plot) of the cell obtained from a flow
  • o microscope may be a two-dimensional (2D) cross section or three-dimensional (3D) structure of the cell.
  • the size (Z) of the cell may be obtained by measuring the greatest cross-sectional or transverse dimension of the cell in a 2D cross-sectional image obtained from an optical microscope or an electron microscope (e.g., SEM).
  • small cell e.g., small somatic stem cell
  • large cell refers to a cell having a size greater than 6 micrometers.
  • CD349(+) SB cells are pluripotent or multipotent somatic stem cells.
  • CD349(+) SB cells may also be CD9(+), Oct4(+), and Nanog(+), as well as CD133(-), CD90(-), CD34(-), and Sox2(-).
  • CD349(+) SB cells each have a size equal to or less than 4, 5 or 6 micrometers, such as between 0.1 and 6.0 micrometers, between 0.5 and 6.0 micrometers, between 1.0 and 6.0 micrometers, between 2.0 and 6.0 micrometers, between 0.1 and 5.0 micrometers, between 0.5 and 5.0 micrometers, between 1.0 and 5.0 micrometers, between 0.1 and 4.0 micrometers, between 0.5 and 4.0 micrometers, or between 1.0 and 4.0 micrometers.
  • 5 or 6 micrometers such as between 0.1 and 6.0 micrometers, between 0.5 and 6.0 micrometers, between 1.0 and 6.0 micrometers, between 2.0 and 6.0 micrometers, between 0.1 and 5.0 micrometers, between 0.5 and 5.0 micrometers, between 1.0 and 5.0 micrometers, between 0.1 and 4.0 micrometers, between 0.5 and 4.0 micrometers, or between 1.0 and 4.0 micrometers.
  • the size is greater than 2 micrometers and less than 6 micrometers.
  • Lgr5(+) SB cells are also pluripotent or multipotent somatic stem cells. They may also be Oct4(+) and Nanog(+), as well as CD133(-), CD66e(-), CD4(-), CD8(-), CD9(-), CDIO(-), CDll(-), CD16(-), CD17(-), CD18(-), CD19(-), CD20(-), CD21(-), CD31(-), CD42(-), CD63(-), CD34(-), Lin(-), CD38(-), CD90(-), CD45(-), CD349(-), and
  • the size of a Lgr5(+) SB cell can be equal to or less than 4, 5 or 6 micrometers, such as between 0.1 and 6.0 micrometers, between 0.5 and 6.0 micrometers, between 1.0 and 6.0 micrometers, between 2.0 and 6.0 micrometers, between 0.1 and 5.0 micrometers, between 0.5 and 5.0 micrometers, between 1.0 and 5.0 micrometers, between 0.1 and 4.0 micrometers, between 0.5 and 4.0 micrometers or between 1.0 and 4.0 micrometers.
  • a Lgr5(+) SB cell is greater than 2 micrometers and less than 6 micrometers in size.
  • Blastomere-like stem cells are CD66e(+) totipotent or pluripotent somatic stem cells. They can each have a size that is equal to or less than 4, 5 or 6 micrometers, such as between 0.1 and 6.0 micrometers, between 0.5 and 6.0 micrometers, between 1.0 and 6.0 micrometers, between 2.0 and 6.0 micrometers, between 0.1 and 5.0 micrometers, between 0.5 and 5.0 micrometers, between 1.0 and 5.0 micrometers, between 0.1 and 4.0 micrometers, between 0.5 and 4.0 micrometers or between 1.0 and 4.0 micrometers.
  • a BLSC can have a size that is greater than 2 micrometers and less than 6 micrometers.
  • VSELs are pluripotent somatic stem cells, which can be CD133(+) or CD34(+).
  • a VSEL can also be CD45(-) and Lin(-).
  • a VSEL can be CD133(+), CD45(-) and Lin(-), or CD34(+), CD45(-) and Lin(-).
  • the size of a VSEL can be equal to or less than 4, 5 or 6 micrometers, such as between 0.1 and 6.0 micrometers, between 0.5 and 6.0 micrometers, between 1.0 and 6.0 micrometers, between 2.0 and 6.0 micrometers, between 0.1 and 5.0 micrometers, between 0.5 and 5.0 micrometers, between 1.0 and 5.0 micrometers, between 0.1 and 4.0 micrometers, between 0.5 and 4.0 micrometers or between 1.0 and 4.0 micrometers.
  • a VSEL can be greater than 2 micrometers and less than 6 micrometers in size.
  • MSCs Mesenchymal stem cells
  • An MSC may express one or more of the cell surface markers CD 13, CD29, CD44, CD73, CD90 and CD105.
  • MSCs constitute a very heterogeneous population.
  • Some types of MSCs may be equal to or less than 4, 5 or 6 micrometers, such as between 0.1 and 6.0 micrometers, between 0.5 and 6.0 micrometers, between 1.0 and 6.0 micrometers, between 0.1 and 5.0 micrometers, between 0.5 and 5.0 micrometers, between 1.0 and 5.0 micrometers, between 0.1 and 4.0 micrometers, between 0.5 and 4.0 micrometers or between 1.0 and 4.0 micrometers, in size.
  • Other types of MSCs may be greater than 6, 7 or 10 micrometers in size.
  • HSCs Hematopoietic stem cells
  • They can be CD34(+), cKit(-), CD38(-), Lin(-) cells or CD150(+), CD244(-), and CD48(-) cells.
  • HSCs can be equal to or less than 4, 5 or 6 micrometers, such as between 0.1 and 6.0 micrometers, between 0.5 and 6.0 micrometers, between 1.0 and 6.0 micrometers, between 0.1 and 5.0 micrometers, between 0.5 and 5.0 micrometers, between 1.0 and 5.0 micrometers, between 0.1 and 4.0 micrometers, between 0.5 and 4.0 micrometers or between 1.0 and 4.0 micrometers in size.
  • Action (X) as used herein is an action that may be effective for increasing the number of one or more types of stem cells in vivo, e.g., in a human subject or non-human subject.
  • Actions (X) can include:
  • vitamins Vitamin A, B, B complex, B ⁇ , D, D3, E, etc.
  • macro and/or trace minerals e.g., calcium, sodium, potassium, fluorine, bromine, chromium, iodine, silicon, selenium, beryllium, lithium, cobalt, vanadium and/or nickel
  • trace minerals e.g., calcium, sodium, potassium, fluorine, bromine, chromium, iodine, silicon, selenium, beryllium, lithium, cobalt, vanadium and/or nickel
  • polysaccharides high molecular weight fucose-containing glycoproteins, seaweed (including green algae, blue-green algae, brown algae, and etc.), fucose, fucoidan (a major component of brown algae), oligo fucoidan, algae, brown algae containing fucoidan (for example, brown algae grown and produced in Okinawa, Japan), Japanese Mozuku, green algae, blue-green algae (or blue algae), brown algae (including mozuku, kelp, undaria, sargassum fusiforme, pinnatifida, and etc.), phytochemical (e.g., isoflavones or phytoestrogen), lycopene, epigallocatechin gallate (EGCG), green tea essence, gluconutrients (e.g., Xylose, Galactose, Glucose, Mannose N-acetylglucosamine, N-acetylgalaetosanmine, or N-acetylneuraminic acid),
  • Exercising such as walking, jogging, dancing, gymnastics, Yoga, aerobic exercise, and/or Taijiquan (Chinese shadow exercise);
  • Taking a certain nutrient for improving health of a certain organ in a body for example, taking lycopene to improve the health of prostate; 15. Taking a rehabilitation program to heal the injury, or to heal the wounds caused by surgery, or to cure a disease;
  • a medicinal liquor or called medicinal wine, medicated liquor or medicated wine
  • a medicinal liquor made from, e.g., immersing one Chinese medicine or multiple Chinese medicines in liquor or wine for a period of time, such as ginseng wine made from immersing ginseng in a high alcohol concentration rice wine for a month;
  • a specific disease e.g., a type of cancer, skin disease, kidney disease and/or so on
  • a specific disease e.g., a type of cancer, skin disease, or kidney disease
  • the lamp light or the light emitting diode (LED) light which may include a whole spectrum of visible lights, IR light, red light, green light, blue light, or UV light, or a combination of more than one of the above lights;
  • Hyperbaric oxygen therapy performed after injury or surgery for improving self-healing
  • G-CSF granulocyte-colony stimulating factor
  • a nutrient a nutrient product, a nutrient fluid, a nutrient drink, a nutrient liquid, or a nutrient food containing (1) varieties of amino acids (such as Arginine, Histidine, Lysine, Aspartic acid, Glutamic acid, Serine, Threonine, Asparagine, Glutamine, Cysteine, Valine, Proline, Glycine, Selenocysteine, Alanine, Isoleucine, Leucine, Phenylalanine, Methionine, Tyrosine, or Tryptophan), (2) balanced amino acids, or (3) 9 essential amino acids (i.e., Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan and Valine) for human bodies.
  • amino acids such as Arginine, Histidine, Lysine, Aspartic acid, Glutamic acid, Serine, Threonine, Asparagine, Glut
  • a medicinal liquor or called medicinal wine, medicated liquor, or medicated wine
  • a stem cell-containing composition (e.g., a stem cell-containing solution) can be prepared using an exemplary method described below.
  • An action (X), which may be one of the above-mentioned actions (X), is performed on a subject.
  • the subject for example, is a human (e.g., child, teenager, adult, or elderly) or a non-human animal.
  • a non-human animal include a primate (e.g., monkey or gorilla), dog, rodent (e.g., mouse or guinea pig), cat, horse, cow, cattle, sheep, pig, chicken, duck, goose, bird, and elephant.
  • the subject can ingest a stem cell-mobilization agent such as a fucoidan- containing compound.
  • the fucoidan-containing compound can be a brown algae supplement.
  • a pill of the brown algae supplement contains 80% of a mozuku powder, 15% of crystalline cellulose, 3% of sucrose fatty acid esters, and 2% of micro or fine silica (containing silicon dioxide).
  • the mozuku powder may be extracted from mozuku brown algae (one kind of seaweed) grown in the sea around and near Okinawa, Japan. The mozuku powder is then mixed with crystalline cellulose, sucrose fatty acid esters, and micro or fine silica (containing silicon dioxide) to form the pill of the brown algae supplement, which contains 0.1 grams of fucoidan.
  • the subject may ingest 20 or more pills (e.g., at least 30 pills) of the brown algae supplement or 2 grams or more (such as at least 3 grams) of fucoidan.
  • the subject may be injected with a granulocyte-colony stimulating factor (GCSF), i.e., a mobilization agent, or may be subjected to a course of GCSF injections.
  • GCSF granulocyte-colony stimulating factor
  • the subject waits for a period of time (e.g., a predetermined period of time), such as between 15 minutes and 60 minutes, between 20 minutes and 100 minutes, between 30 minutes and 4 hours, between 60 minutes and 90 minutes, between 0.5 hours and 3 hours, between 1 hour and 6 hours, between 1 hour and 12 hours, between 12 hours and 36 hours, or between 36 hours and 50 hours.
  • a period of time e.g., a predetermined period of time
  • a period of time e.g., a predetermined period of time
  • a period of time e.g., a predetermined period of time
  • somatic stem cells such as SB cells (i.e., CD349(+) and Lgr5(+) SB cells)
  • the peripheral blood of the subject thus becomes enriched with the one or more specific types of somatic stem cells.
  • the one or more specific types of somatic stem cells may be or may include one or more of the somatic stem cells described above.
  • the one or more specific types of somatic stem cells may be or may include somatic stem cells less than 6 micrometers in size, and more preferably greater than 2 micrometers in size, such as CD349(+) somatic stem cells and/or Lgr5(+) somatic stem cells.
  • Performing action (X) and waiting for a period are optional steps.
  • a blood sample can be obtained from a subject without first performing any action (X) on the subject.
  • a blood sample is obtained from the peripheral blood of the subject and placed into one or more containers (e.g., a bag, one or more syringes, or one or more tubes) containing a divalent cation chelating agent.
  • the blood sample is mixed with the divalent cation chelating agent in the container to form a mixture.
  • the divalent cation chelating agent e.g., an anticoagulant
  • EDTA ethylenediaminetetraacetic acid
  • K2 EDTA anticoagulant or K3 EDTA anticoagulant having a weight, e.g., greater than 70 mg, such as between 90 and 900 mg, between 120 and 450 mg, or between 150 and 400 mg.
  • the divalent cation chelating agent may be citrate having a weight, e.g., greater than 70 mg, such as between 90 and 900 mg, between 120 and 450 mg, or between 150 and 400 mg.
  • the blood sample contains a plurality of cells, including small cells less than 6 micrometers in size and large cells greater
  • the small cells for 5 example, contain platelets and small somatic stem cells less than 6 micrometers in size.
  • the small somatic stem cells contain the one or more specific types of somatic stem cells (i.e., SB cells, for example), BLSCs (i.e., CD66e(+) somatic stem cells), and VSELs (e.g., CD133(+) somatic stem cells and CD34(+) somatic stem cells).
  • the large cells for example, contain large somatic stem cells greater than 6 micrometers in size and lineage cells o such as red blood cells and white blood cells.
  • the blood sample may have a volume greater than or equal to 45 milliliters, such as between 60 and 500 milliliters, between 80 and 250 milliliters or between 100 and 200 milliliters.
  • the blood sample may be mixed with 1.5 mg or more, such as between 1.6 and 2.0 mg, of the divalent cation chelating agent (such as K2 EDTA, K3 EDTA, or citrate) per milliliter of the blood sample to form the 5 mixture in the container.
  • the divalent cation chelating agent such as K2 EDTA, K3 EDTA, or citrate
  • the process can include steps for stem cell activation and purification/isolation.
  • purification or “isolation” as used herein means substantial separation of small cells (e.g., cells greater than 2 micrometers and less than 6 micrometers in size) from large cells (e.g., cells greater o than 6 micrometers in size).
  • the mixture can be stored at a temperature between 2 degrees Celsius (°C) and 12°C, more preferably between 2 °C and 7 °C or at 4 °C, in a suitable facility (e.g., refrigerator or other device used to keep things cold) for a predetermined period of time.
  • the period of time can be between 3 hours and 72 hours, and more preferably between 3 hours and 6 hours, 5 between 6 hours and 72 hours, between 6 hours and 48 hours, between 16 hours and 72
  • the one or more specific types of somatic stem cells (e.g., SB cells) in the mixture may be activated by the divalent cation chelating agent (such as K2 EDTA, K3 EDTA, or 0 citrate), i.e., the cell cycle of the one or more specific types of somatic stem cells is activated from GO into Gl.
  • the divalent cation chelating agent such as K2 EDTA, K3 EDTA, or 0 citrate
  • the activation may relate to the ability of the divalent cation chelating agent to repress p53's function (presumably by chelating Zn 2+ ), thereby allowing the one or more specific types of somatic stem cells (e.g., SB cells) to exist from the GO quiescence stage into the Gl stage of the cell cycle.
  • chelating Zn 2+ by the divalent cation chelating agent may be a key step to activate the one or more specific types of somatic stem cells (e.g., SB cells). It is possible that the divalent cation chelating agent can chelate other divalent ions (e.g., Ca 2+ ), thereby activates the one or more specific types of somatic stem cells and forces them to proliferate and expand.
  • the upper layer may have a volume between 20 and 250 milliliters, between 40 and 125 milliliters, or between 50 and 100 milliliters.
  • the upper layer contains platelets, serum, and one or more specific types of small somatic stem cells (i.e., SB cells, for example), BLSCs (i.e., CD66e(+) somatic stem cells), and VSELs (e.g., CD133(+) somatic stem cells and CD34(+) somatic stem cells).
  • Most of the large cells containing lineage cells and the large somatic stem cells of the blood sample are in the lower layer.
  • the ratio of the volume of the supernatant to the volume of the blood sample may range from one third to one half.
  • substantially all of the upper layer may be collected or transferred into a liquid container, such as a bag, a syringe, or a glass bottle, to produce a stem cell-containing solution or stem cell mixture.
  • the upper layer e.g., a stem cell-containing solution
  • the number of small somatic stem cells in the stem cell-containing solution can be greater than or equal to 10 million (e.g., greater than or equal to 30 million, greater than or equal to 50 million, between 10 million and 500 million, between 25 million and 300 million, or between 30 million and 500 million).
  • the stem cell-containing solution may also contain the divalent cation chelating agent (e.g., EDTA) and/or growth factors.
  • the stem cell-containing solution barely includes or substantially excludes large cells (e.g., large somatic stem cells and lineage cells).
  • large cells can constitute less than 5% (e.g., less than 1%, 0.5%, or 0.01%) of the total number of cells in the stem cell-containing solution.
  • the number of red blood cells in the stem-cell containing solution e.g., the collected upper layer
  • the number of red blood cells per milliliter of the stem cell-containing solution is less than 10 3 .
  • the number of white blood cells per milliliter of the stem cell-containing solution can be less than 10 4 (e.g., less than 10 3 ).
  • the number of white blood cells per milliliter of the stem-cell containing solution is less than 10 2 .
  • the small cells can include platelets, Lgr5(+)cells, CD349(+) cells, CD133(+) cells, CD34(+), and CD66e(+) cells. Platelets can constitute 75% to 85% of the small cells in the stem cell-containing solution. Greater than 4% (e.g., greater than 5% or between 4.5% and 10%) of all of the small cells can be Lgr5+ somatic stem cells. CD349(+) somatic stem cells can constitute greater than 4% (e.g., greater than 5% or between 4.5% and 10%) of all of the small cells the stem cell-containing solution.
  • the small cells can be CD133(+) cells and CD34(+) cells combined.
  • Less than 6% (e.g., less than 5% or 4.5%) of the small cells can be CD66e(+) cells.
  • Any specific small cells can also be further isolated or depleted from the collected upper layer using flow cytometry or other conventional techniques (e.g. antibody-based techniques such as antibody-conjugated beads).
  • the collected upper layer can be used as is as a stem cell-containing solution (e.g., administered to a subject or stored) or further processed. For example, it can be further purified (e.g., filtered) or mixed with one or more additional components.
  • a suitable cell medium or solution free from Ca 2+ having a volume, e.g., greater than 400 milliliters, such as between 500 and 900 milliliters, can be added to the collected upper layer to make a stem cell-containing solution.
  • the suitable medium or solution free from Ca 2+ such as a NaCl- containing solution, may be further free from any divalent ions, including Mg 2+ .
  • the NaCl- containing solution for example, can be normal saline (e.g., a solution of 0.90% w/v of NaCl, about 300 mOsm/L or 9.0 gram per liter).
  • the stem cell-containing solution may be stored in a frozen storage temperature, e.g., equal to or less than -70°C or -80°C (e.g., between -75°C and -85°C) for an extended period of time (e.g., more than one week, one month, or one year).
  • a frozen storage temperature e.g., equal to or less than -70°C or -80°C (e.g., between -75°C and -85°C) for an extended period of time (e.g., more than one week, one month, or one year).
  • the frozen stem cell-containing solution can be quickly thawed and, optionally, mixed with the aforementioned suitable medium or solution free from Ca 2+ (e.g., 0.9% NaCl).
  • the stem cell-containing composition produced by the procedure described above can be used to increase the level of a PRMT in a subject.
  • the composition can also be used to treat a condition associated with a decreased level of a PRMT or a condition that can be treated by a PRMT agonist.
  • protein arginine methyltransferase or "PRMT” refers to an enzyme that catalyzes arginine methylation.
  • PRMT protein arginine methyltransferase
  • the mammalian genome encodes at least nine different PRMTs, i.e., PRMT1, PRMT2, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, PRMT8, and PRMT9.
  • PRMT level can refer to a PRMT protein level, mRNA level, cDNA level, or functional (e.g., enzymatic) level in any biological sample, e.g., blood sample, bone marrow sample, urine sample, or solid tissue sample, obtained from a subject.
  • a decreased PRMT level is a level that is lower than the level or range of levels found in healthy individuals or individuals without a condition associated with a decreased PRMT level.
  • a control level can be a level found in healthy individuals, in individuals without a condition associated with a decreased PRMT level, or in individuals before treatment with the stem cell-containing composition. Methods of measuring protein, mRNA, cDNA, and functional levels are well known in the art, e.g., ELISA, Western blotting, and real time PCR.
  • the level of a PRMT in a sample e.g., blood sample, bone marrow sample, urine sample, or solid tissue sample, obtained from the subject can be determined to monitor treatment efficacy.
  • a disease parameter or symptom in the subject can be evaluated before and/or after the administration.
  • Conditions that can be treated with the stem-cell containing composition include aging, anemia, other blood cell disorders, and T-cell related disorders.
  • the stem cell-containing composition described herein can be administered to a subject in need thereof via any route of administration, e.g., intravenous, intraarticular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, or subcutaneous route of administration.
  • the composition can contain between 10 million and 500 million small somatic stem cells. Autologous or allogeneic somatic stem cells can be used.
  • the composition can be administered to a subject, for example, every 1-14 days, every 2-4 weeks, every 1-6 months, or every 2-12 months, for a treatment period (e.g., 1-36 months or 2-10 years), or whenever needed.
  • a “subject” refers to a human or a non-human animal.
  • Treating” or “treatment” refers to administration of a compound or composition to a subject, who has a disorder, with the purpose to cure, alleviate, relieve, remedy, delay the onset of, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder.
  • An “effective amount” refers to an amount of the compound or composition that is capable of producing a medically desirable result in a treated subject.
  • the treatment method can be performed alone or in conjunction with other drugs or therapy.
  • peripheral blood samples 100 to 150 ml of peripheral blood samples were obtained from each of 91 subjects.
  • EDTA-coated tubes containing the blood samples were stored for 6 to 48 hours at 4°C until the blood separated into two distinct layers via gravity.
  • the top layer which contained SB cells, was collected and delivered autologously back to each of the subjects through intravenous injection.
  • the subjects received treatments every three to six months for a total of four treatments.

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  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant d'augmenter le niveau de la protéine arginine méthyltransférase (PRMT) chez un sujet, lequel procédé consiste à: identifier un sujet en ayant besoin; et administrer au sujet une composition qui contient de petites cellules dont la taille est supérieure à 2 micromètres et inférieure à 6 micromètres; les petites cellules comprenant des cellules souches somatiques qui sont (i) pluripotentes ou multipotentes ; et (i) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+), ou CD34(+).
PCT/US2018/042219 2017-07-17 2018-07-16 Utilisation de cellules souches somatiques pour augmenter le niveau de prmt WO2019018256A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055678A1 (en) * 2005-06-06 2010-03-04 Suomen Punainen Risti, Veripalvelu Method of profiling a cell population
US20120034194A1 (en) * 2010-08-04 2012-02-09 StemBios Technologies, Inc. Somatic Stem Cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055678A1 (en) * 2005-06-06 2010-03-04 Suomen Punainen Risti, Veripalvelu Method of profiling a cell population
US20120034194A1 (en) * 2010-08-04 2012-02-09 StemBios Technologies, Inc. Somatic Stem Cells

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BLANC ET AL.: "Regenerating muscle with arginine methylation", TRANSCRIPTION, vol. 8, no. 3, 27 May 2017 (2017-05-27), pages 175 - 178, XP055567542, Retrieved from the Internet <URL:doi:10.1080/21541264.2017.1291083> *
IRHIMEH ET AL.: "Fucoidan ingestion increases the expression of CXCR4 on human CD 34+ cells", EXP HEMATOL ., vol. 35, no. 6, June 2007 (2007-06-01), pages 989 - 994, XP025319723 *
LIU ET AL.: "Arginine methyltransferase PRMT5 is essential for sustaining normal adult hematopoiesis", J CLIN INVEST., vol. 125, no. 9, 1 September 2015 (2015-09-01), pages 3532 - 3544, XP055567547, Retrieved from the Internet <URL:doi:10.1172/JCI81749> *
STOPA ET AL.: "The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond", CELL MOL LIFE SCI, vol. 72, no. 11, June 2015 (2015-06-01), pages 2041 - 2059, XP035501545, Retrieved from the Internet <URL:doi:10.1007/s00018-015-1847-9> *

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