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WO2018119178A1 - Utilisation de cellules souches somatiques pour diminuer le taux de néprilysine - Google Patents

Utilisation de cellules souches somatiques pour diminuer le taux de néprilysine Download PDF

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Publication number
WO2018119178A1
WO2018119178A1 PCT/US2017/067795 US2017067795W WO2018119178A1 WO 2018119178 A1 WO2018119178 A1 WO 2018119178A1 US 2017067795 W US2017067795 W US 2017067795W WO 2018119178 A1 WO2018119178 A1 WO 2018119178A1
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subject
micrometers
cells
stem cells
upper layer
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James Wang
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Stembios Technologies Inc
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Stembios Technologies Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
    • G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
    • G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
    • G01N2333/96419—Metalloendopeptidases (3.4.24)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders

Definitions

  • NEP neutral endopeptidase
  • CD10 cluster of differentiation 10
  • Neprilysin is a zinc -dependent metalloprotease that cleaves peptides at the amino side of hydrophobic residues and inactivates several peptide hormones including glucagon, enkephalins, substance P, neurotensin, oxytocin, and bradykinin. It was shown that old NEP-deficient mice exhibited significantly improved ability to learn.
  • Dementia is a broad term that refers to an often gradual decrease in a person's mental abilities that is sufficient to interfere with daily functioning. Symptoms may include impairments in one or more of memory, reasoning, judgment, communication, focus, planning, and visual perception. There is currently no cure for dementia. Drags and non- drug treatments may help to manage the symptoms.
  • a method of decreasing the level of neprilysin 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 micrometers in size; wherein the small cells include somatic stem cells that are (i) pluripotent; and (i) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+), or CD34(+).
  • the identifying step includes detecting an elevated level of neprilysin, as compared to a control level, in a biological sample obtained from the subject.
  • the identified subject can have a condition associated with elevated neprilysin or condition that can be treated by decreasing neprilysin level.
  • the subject can have non- Alzheimer's dementia.
  • the subject has an age-related cognitive impairment.
  • 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 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 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 excipient is a saline solution.
  • the method further includes, after the administering step, detecting a level of neprilysin in a biological sample obtained from the subject after the administering step.
  • compositions for decreasing the level of neprilysin in a 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; and (i) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+), or CD34(+).
  • FIG. 1 is a graph showing the neprilysin (CD 10) levels in dementia patients treated with a stem cell composition.
  • compositions containing certain small somatic stem cells can decrease the level of neprilysin (CD 10) in subjects and improve the memory and other psychological symptoms of dementia (e.g., non- Alzheimer's dementia). Accordingly, described herein are methods and compositions for decreasing neprilysin level, or for treating conditions associated with elevated level of neprilysin.
  • 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 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), 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 cytometer.
  • An image of a cell obtained from an optical microscope or electron 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. Actions for Increasing Stem Cells
  • 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);
  • 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 l o 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;
  • 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 5 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 l o 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.
  • 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
  • somatic stem cells such as SB cells (i.e., CD349(+) and Lgr5(+) SB cells), to be mobilized into the subject's peripheral
  • 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
  • CD349(+) somatic stem cells and/or Lgr5(+) somatic stem cells are 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
  • the divalent cation chelating agent may be ethylenediaminetetraacetic acid (EDTA), such as K2 EDTA anticoagulant or K3 EDTA anticoagulant, having a weight, e.g., greater than 70 mg, such as 5 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 than 6 micrometers in size.
  • the small cells for o 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 cells5 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 o 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 greater5 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,0 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 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 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 5 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(+)5 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 o upper layer using flow cytometry or other conventional techniques (e.g. antibody-based
  • 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 cell5 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, o 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 decrease neprilysin level in a subject. For example, it can be used to treat a condition associated with elevated neprilysin level, or condition that can be treated with a neprilysin antagonist or by decreasing neprilysin level.
  • Neprilysin level can refer to a neprilysin protein level, mRNA level, cDNA, or
  • neprilysin level is a level that is higher than the level or range of levels found in healthy individuals or individuals without a condition associated with elevated neprilysin level.
  • 15 mRNA, cDNA, and enzymatic levels are well known in the art, e.g., ELISA, Western
  • the stem cell-containing composition Before the stem cell-containing composition is administered to a subject, whether the subject has an elevated level of neprilysin can be determined. Alternatively or in addition, after the composition is administered, the neprilysin level in the subject can be determined to
  • a disease parameter or symptom e.g., memory or learning abilities
  • a disease parameter or symptom e.g., memory or learning abilities
  • neprilysin sequences are known in the art, e.g., NCBI accession nos. NP_001341571 (human), NP_001341572 (human), NP_001276391 (mouse),
  • 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
  • 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.
  • Conditions that can be treated with the stem-cell containing composition include non- Alzheimer's dementia (e.g., vascular dementia), pain, psychological disorders, decreased cognitive ability (e.g., memory, learning, focus, or reasoning) associated with normal aging.
  • Dementia as used herein, is a general term for loss of memory and other mental abilities severe enough to interfere with daily life. The loss is more than what is expected for normal aging. Symptoms of dementia can vary greatly. A skilled practitioner would be able to determine whether a subject has dementia.
  • 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 four patients who had dementia.
  • 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.
  • the top layer which contained SB cells, were collected and delivered autologously back into the patients through intravenous injection.
  • neprilysin i.e., CD10

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Abstract

L'invention concerne un procédé permettant de diminuer le taux de néprilysine chez un sujet, lequel procédé consiste : à identifier un sujet en ayant besoin ; et à administrer au sujet une composition qui contient de petites cellules dont la taille est supérieure à 2 micromètres et inférieure à 6 micromètres ; lesdites petites cellules comprenant des cellules souches somatiques qui sont (i) pluripotentes ; et (i) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+) ou CD34(+).
PCT/US2017/067795 2016-12-23 2017-12-21 Utilisation de cellules souches somatiques pour diminuer le taux de néprilysine Ceased WO2018119178A1 (fr)

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Citations (6)

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Publication number Priority date Publication date Assignee Title
US20040076603A1 (en) * 2002-01-25 2004-04-22 Tony Peled Methods of expanding stem and progenitor cells and expanded cell populations obtained thereby
WO2004080448A2 (fr) * 2003-03-12 2004-09-23 Forschungsverbund Berlin E. V. Utilisation de molecules associees a la nep pour traiter des troubles comportementaux non immunogenes et non hypertensifs
US20070077201A1 (en) * 2004-09-29 2007-04-05 Reading Christopher L Stem cell expansion and uses
US20090156585A1 (en) * 2005-11-09 2009-06-18 Lili Feng Organic compounds
US20140161774A1 (en) * 2012-12-06 2014-06-12 StemBios Technologies, Inc. Lgr5+ SOMATIC STEM CELLS
US20160362472A1 (en) * 2015-04-08 2016-12-15 Hans Bitter Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)- expressing cell

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Publication number Priority date Publication date Assignee Title
US20040076603A1 (en) * 2002-01-25 2004-04-22 Tony Peled Methods of expanding stem and progenitor cells and expanded cell populations obtained thereby
WO2004080448A2 (fr) * 2003-03-12 2004-09-23 Forschungsverbund Berlin E. V. Utilisation de molecules associees a la nep pour traiter des troubles comportementaux non immunogenes et non hypertensifs
US20070077201A1 (en) * 2004-09-29 2007-04-05 Reading Christopher L Stem cell expansion and uses
US20090156585A1 (en) * 2005-11-09 2009-06-18 Lili Feng Organic compounds
US20140161774A1 (en) * 2012-12-06 2014-06-12 StemBios Technologies, Inc. Lgr5+ SOMATIC STEM CELLS
US20160362472A1 (en) * 2015-04-08 2016-12-15 Hans Bitter Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)- expressing cell

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