TW201907926A - Use of somatic stem cells for increasing prmt level - Google Patents

Abstract

A method of increasing a protein arginine methyltransferase (PRMT) level in a subject, comprising: 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 or multipotent; and (ii) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+), or CD34(+).

Description

Use of body stem cells to increase protein arginine methyltransferase (PRMT) levels

The present invention relates to the use of somatic stem cells for increasing the level of protein arginine methyltransferase (PRMT).

Protein arginine methylation is a common post-translational modification that regulates the function of proteins, including histones. Protein arginine methyltransferases (PRMTs) are classified based on the type of methyl arginine produced. For example, Type I PRMTs catalyze the formation of asymmetric dimethyl arginine, and Type II PRMTs catalyze the formation of symmetric dimethyl arginine. In humans, nine PRMTs have been identified. PRMT5 is the major type II enzyme identified to date. Together with its cofactor MEP50, it mediates the methylation of histone H2A at R3 and histone H3 at H4 and R8. Symmetric dimethylation of their histone residues typically produces repressive marks upon gene transcription. PRMT5 was found in several transcriptional repressor complexes, such as SIN3A/HDAC, MBD2/NURD, and N-CoR/SMRT. PRMT5 has been shown to inhibit globin gene expression by recruiting DNMT3A, indicating a potential crosstalk between histone arginine methylation and DNA methylation. This enzyme plays an important role in epigenetic regulation and is involved in the aging process in cells.

In addition, the function of PRMT5 in hematopoietic stem cells and precursor cells (HSPC) has been studied. The data show that PRMT5 plays a fundamental role in maintaining adult hematopoietic cells. Deletion of PRMT5 in mice results in a fatal, very severe aplastic anemia-like phenotype.

In one aspect, described herein is a method of increasing the PRMT level in a body. The method comprises identifying a subject in need thereof; and administering to the individual a composition comprising a plurality of small cells having a size greater than 2 microns and less than 6 microns; wherein the minicells comprise a plurality of individual stem cells, the stem cell lines (i) pluripotentl or multipotent; and (ii) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+ ), CD133 (+) or CD34 (+) or.

In some embodiments, the identifying step includes detecting a PRMT level in the biological sample taken from the individual. In some embodiments, the level detected is below a control level. The identified individual can have a disorder associated with a malfunction of PRMT. For example, the individual can have a condition associated with aging or anemia.

The small cells administered to the composition of the individual may further comprise platelets. For example, 75% to 85% of small cells may be platelets and 20% to 25% of small cells may be body stem cells. In some embodiments, the composition contains from 10 to 500 million individual stem cells.

In some embodiments, the composition is prepared by a process comprising: providing a mixture comprising a blood sample taken from the individual or a donor individual and a divalent cation chelating agent; The mixture is stored at a temperature between 2 ° C and 12 ° C for 3 to 72 hours, whereby the mixture is separated into an upper layer and a lower layer, wherein the upper layer comprises the minipopulations; and the collection The upper layer, thereby preparing the composition. 1.5 to 2.0 mg of the divalent cation chelating agent per ml of blood sample may be mixed with the blood sample to obtain the mixture. Preferably, the divalent cation chelating agent is EDTA.

Optionally, an action for increasing the number of stem cells is performed on the individual or donor individual prior to taking the blood sample to prepare the composition. The action can be administration of an effective amount of fucoidan or granulocyte-colony stimulating factor.

The process of preparing the composition further includes, after collecting the upper layer, adding a pharmaceutically acceptable excipient to the collected upper layer. Alternatively, the process may further comprise, after collecting the upper layer, centrifuging the upper layer to obtain a cell pellet. The precipitate can be further washed and suspended in a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient can be one that does not contain a divalent ion. In one embodiment, the pharmaceutically acceptable excipient is an aqueous salt solution.

In some embodiments, the method further comprises, after the administering step, detecting a PRMT level in the biological sample taken from the individual after the administering step.

In another aspect, a composition for increasing the PRMT level in a body is described herein. The composition comprises a plurality of small cells having a size greater than 2 microns and less than 6 microns, wherein the minicells comprise a plurality of individual stem cells, (i) pluripotency or versatility; and (ii) CD349 ( +), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+) or CD34(+).

The details of one or more embodiments are set forth in the drawings and the description below. Other features, objects, and advantages of the embodiments will be apparent from the description and appended claims.

Surprisingly, it has been found that compositions containing specific small body stem cells, such as Lgr5 (+) or CD349 (+) cells, can increase the level of PRMT. Accordingly, described herein are methods and compositions for increasing PRMT levels in a body and for treating disorders associated with PRMT deficiency. Somatic stem cell

There are many types of somatic stem cells, including totipotent stem cells, pluripotent stem cells, multipotent stem cells, and progenitor stem cells (also known as single energy). Unipotent stem cells). Blastomere-like stem cells (BLSCs) are totipotent or pluripotent stem cells. Very small embryonic-like stem cells (VSELs) are pluripotent stem cells. SB cells are pluripotent or multifunctional somatic stem cells. Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSC) are multifunctional somatic stem cells.

The size (Z) of a cell (such as a stem cell) as used herein may refer to: (1) a conventional definition of the size or representative length of a cell in the field of cell biology or stem cell, and (2) the diameter of a cell, especially when the cell is substantially When the upper is spherical, (3) the length of the long axis of the cell, especially when the cell is substantially elliptical, (4) the cell width when the cell shape has an approximately square shape, and (5) when the cell shape has an approximately rectangular shape The length of the cell or (6) the largest cross-sectional or lateral dimension of the cell. Determination of or size (Z) using cell images obtained by optical microscopy or electron microscopy (such as scanning electron microscopy (SEM)) or cell data obtained by flow cytometry (such as two-dimensional points, contours or density maps) , diameter, length, width or maximum cross section or lateral dimension. Scanning Electron Microscopy (SEM)). The cell image obtained from an optical microscope or an electron microscope may be a two-dimensional (2D) cross-section or a three-dimensional (3D) structure of the cell. As an example, the cell size (Z) can be obtained by measuring the cell's maximum cross-section or transverse dimension of a 2D cross-sectional image obtained by measuring an optical microscope or an electron microscope such as SEM.

"Small cells" (such as small body stem cells) refers to cells having a size less than 6 microns, such as between 2.0 and 6.0 microns. The term "large cell" refers to cells having a size greater than 6 microns.

CD349(+) SB cells are pluripotent or multifunctional somatic stem cells. CD349(+) SB cells can 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 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, Between 2.0 and 6.0 microns, between 0.1 and 5.0 microns, between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns Between or between 1.0 and 4.0 microns. Preferably, the size is greater than 2 microns and less than 6 microns.

Lgr5(+) SB cells are also pluripotent or multifunctional somatic stem cells. It can also be Oct4(+) and Nanog(+), as well as CD133(−), CD66e(−), CD4(−), CD8(−), CD9(−), CD10(−), CD11(−), CD16(−), CD17(−), CD18(−), CD19(−), CD20(−), CD21(−), CD31(−), CD42(−), CD63(−), CD34(−), Lin(−), CD38(−), CD90(−), CD45(−), CD349(−), and Sox2(−). The size of the Lgr5(+) SB cells may be equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between Between 2.0 and 6.0 microns, between 0.1 and 5.0 microns, between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns Between or between 1.0 and 4.0 microns. Preferably, the Lgr5(+) SB cells are greater than 2 microns in size and less than 6 microns in size.

Leukoid blastomeres (BLSCs) are CD66e (+) totipotent or pluripotent stem cells. They may each have a size equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 2.0 and Between 6.0 microns, between 0.1 and 5.0 microns, between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns or Between 1.0 and 4.0 microns. For example, a BLSC can have a size greater than 2 microns and less than 6 microns.

Minimal embryonic stem cells (VSELs) are pluripotent stem cells that can be CD133(+) or CD34(+). VSEL can also be CD45(−) and Lin(−). For example, VSEL can be CD133(+), CD45(−) and Lin(−) or CD34(+), CD45(−), and Lin(−). The size of the VSEL can be equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, and between 2.0 and 6.0 microns. Between 0.1 and 5.0 microns, between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns, or between 1.0 and Between 4.0 microns. The size of the VSEL can be greater than 2 microns and less than 6 microns.

Mesenchymal stem cells (MSCs) are multifunctional somatic stem cells. MSCs can exhibit one or more cell surface markers CD13, CD29, CD44, CD73, CD90 and CD105. MSCs constitute a very heterogeneous population. Some types of MSCs may be sized equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 0.1 and Between 5.0 microns, between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns, or between 1.0 and 4.0 microns. Other types of MSCs may be sized greater than 6, 7, or 10 microns.

Hematopoietic stem cells (HSCs) are multifunctional somatic stem cells. These may be CD34(+), cKit(−), CD38(−), Lin(−) cells or CD150(+), CD244(−) and CD48(−) cells. The size of the HSCs may be equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 0.1 and 5.0 microns. Between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns or between 1.0 and 4.0 microns. Increase the behavior of stem cells

Action (X) as used herein is an act that effectively increases the number of one or more stem cells in vivo (e.g., within a human or non-human individual). Action (X) may include: 1. taking a drug, such as a synthetic drug or a naturally derived compound; 2. taking herbal or Chinese herbal medicines, such as Cordyceps sinensis, ginseng, Lycium Chinense Mill, Ganoderma lucidum (Ganoderma lucidum), Taiwanese mushrooms (Taiwanofungus camphoratus), and/or Brazil mushrooms; 3. Taking nutrients or dietary supplements, such as nutritional pills or powders, including the following materials or elements: vitamins (vitamin A, B, B complex, B ₁₂ , D, D ₃ , E, etc.), large and/or trace minerals (such as calcium, sodium, potassium, fluorine, bromine, chromium, iodine, antimony, selenium, tellurium, lithium, cobalt, vanadium) And/or nickel), polysaccharides, high molecular weight trehalose glycoproteins, seaweeds (including green algae, blue-green algae, brown algae, etc.), trehalose, fucoidan (the main component of brown algae), oligo-fucoidan, Algae, brown algae containing fucoidan (such as brown algae produced and manufactured in Okinawa, Japan), Japanese Mozuku, green algae, blue-green algae (or cyanobacteria), brown algae (including brown algae, kelp, wakame, horsetail) Algae, kale, etc.) Physical compounds (such as isoflavones or phytoestrogens), lycopene, epigallocatechin gallate (EGCG), green tea extract, glucose nutrients (such as xylose, galactose, glucose, Mannose N-acetylglucosamine, N-acetonitrile chitosan or N-acetyl ceramide, fish oil, toona sinensis, and/or from plants, leaves, fruits, vegetables, fish, Nutrients extracted from seaweed or algae; 4. Implementing a vegetarian diet; 5. Taking or eating healthy or organic foods; 6. Taking alternative (non-traditional) medications; 7. Receiving alternative therapies or treatments, such as Gerson therapy (Gerson Therapy) or Bruuss cancer cure; 8. Acupuncture treatment; 9. Receiving massage, such as foot massage; 10. Exercise, such as walking, jogging, dancing, gymnastics, yoga, aerobic Exercise, and / or Tai Chi (China Shadow Movement); 11. Sleep (for the purpose of measuring sleep quality); 12. Meditation; 13. Implement a health improvement plan or disease designed by an individual, health professional or doctor Treatment plan; 14. Take certain nutrients to improve the health of specific organs in the body, such as taking lycopene to improve the health of the prostate; 15. Take rehabilitation programs to heal wounds or cure wounds caused by surgery or cure diseases; Take a liquid medicine (or medicated liquor, medicated liquid or medicated wine), such as immersing a Chinese medicine or a plurality of traditional Chinese medicines in wine for a period of time, such as immersing human cockroaches in rice wine of high alcohol concentration for one month. Adults who drink alcohol; 17. Take medications licensed by one or more government agencies or agencies, such as the US Food and Drug Administration (US FDA), for the treatment of specific diseases (such as specific cancers, skin diseases, kidney diseases, and/or Other diseases); 18. Taking or receiving treatments or therapies approved by the government to cure specific diseases (such as cancer, skin diseases or kidney diseases); 19. Conducting religious activities such as praying for peace or worshipping God; 20. Direct or indirect Exposure to sunlight or light (between 10 minutes before sunrise and 50 minutes after sunrise (with a large number of infrared (IR) lines); or around noon, for example, between 11:30 AM and 12:30 PM It Between (containing a large amount of ultraviolet (UV)); or in the afternoon, for example, between 50 minutes before sunset and 10 minutes after sunset (containing a large amount of infrared (IR) light); 21. Exposure to light or light-emitting diodes ( LED) light, which may include a complete spectrum of visible, IR, red, green, blue or UV light or a combination of more than one of the above; 22. Exercise or accept procedures, therapies for improving the body's self-healing , methods, devices, and/or systems, for example, methods or therapies (such as hyperbaric oxygen therapy) performed after an injury or surgery to improve self-healing; 23. drinking coffee, such as black coffee; 24. drinking tea, such as green tea , black tea or jasmine tea; 25. drinking red wine; 26. taking melatonin; 27. listening to music, such as the symphony of Mozart or Beethoven; 28. injecting substances containing fucoidan or oligofucose (such as Nutrients or supplements; 29. taking hormone supplements or receiving hormone injections; 30. Injecting particulate white blood cell community stimulating factor (G-CSF or GCSF), which is a glycoprotein; 31. receiving a GCSF injection course; and 32. ingesting camp Nutrients, nutrients, nutrient solutions, nutritious drinks, nutrient solutions or nutritious foods, which contain: (1) a variety of amino acids (such as arginine, histidine, lysine, aspartic acid, proline) , serine, threonine, aspartic acid, amidoxime, cysteine, valine, valine, glycine, selenocysteine, alanine, white Aminic acid, leucine, phenylalanine, methionine, tyrosine or tryptophan), (2) balanced amino acids or (3) 9 essential amino acids of the human body (ie, histamine) Acid, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. For example: (a) products produced or extracted from red bean, mung bean, black bean fermentation; (b) from fruit or fruit combinations (such as beets, apples, guava, kiwi, grapes, pineapple, red dragon fruit (fire dragon fruit) ), liquid, solution or beverage produced by fermentation of green papaya, tomato, and/or avocado; (c) liquid medicine (or called medicinal liquor, medicated liquid or medicated wine), such as one Chinese medicine or multiple Chinese medicines Soaked in wine for a period of time, such as a person who dipped in a high alcohol concentration of rice wine for one month. Composition containing stem cells

Compositions containing stem cells (such as solutions containing stem cells) can be prepared using the exemplary methods described below.

The action (X) performed on the individual may be one of the above actions (X). Individuals, for example, are humans (such as children, adolescents, adults, or the elderly) or non-human animals. Examples of non-human animals include primates (such as monkeys or orangutans), dogs, rodents (such as mice or guinea pigs), cats, horses, cows, oxen, sheep, pigs, chickens, ducks, geese, birds, and Elephant.

For example, an individual may ingest a stem cell activating agent, such as a compound containing fucoidan. The compound containing fucoidan may be a brown algae supplement. The pellet of the brown algae supplement contains 80% Japanese brown algae powder, 15% crystalline cellulose, 3% sucrose fatty acid ester, and 2% cerium oxide microparticles or fine granules (including cerium oxide). Japanese brown algae powder can be extracted from Japanese brown algae (a seaweed) grown around Okinawa, Japan, and nearby seas. Then, Japanese brown algae powder was mixed with crystalline cellulose, sucrose fatty acid esters, and cerium oxide microparticles or fine particles (containing cerium oxide) to form a pellet of brown algae supplement containing 0.1 g of fucoidan. An individual may consume 20 or more pills (such as at least 30 pills) of a brown algae supplement or 2 grams or more (such as at least 3 grams) of fucoidan. In another example, the individual may be injected with a particulate leukocyte stimulating factor (GCSF), ie, an activating agent or may be subjected to a GCSF injection regimen.

After performing action (X), the individual waits for a period of time (such as 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. Acting (X) and waiting for a period of time such that one or more specific types of somatic stem cells, such as SB cells (ie, CD349(+) and Lgr5(+) SB cells), activate from the bone marrow, such as an individual, to the individual Peripheral blood. Thus, the individual peripheral blood becomes enriched in the one or more specific types of somatic stem cells. One or more specific types of somatic stem cells, for example, can be or can include one or more of the above-described somatic stem cells. For example, the one or more particular types of somatic stem cells can be or can include somatic stem cells that are less than 6 microns in size, and more preferably, are larger than 2 microns in size, such as CD349(+) somatic stem cells and/or Lgr5 (+ Body stem cells.

Perform action (X) and wait for a period of time for any step. In other words, to make a composition containing stem cells, a blood sample can be taken from an individual without first having to perform any action (X) on the individual.

Immediately after the waiting step (if action (X) and waiting step), a blood sample is taken from the peripheral blood of the individual and placed in one or more containers containing a divalent cation chelating agent (such as a bag, a Or multiple syringes or one or more tubes). In the container, the blood sample is mixed with a divalent cation chelating agent to form a mixture. A divalent cation chelating agent, such as an anticoagulant, can be ethylenediaminetetraacetic acid (EDTA), such as a K2 EDTA anticoagulant or a K3 EDTA anticoagulant, having a weight, such as greater than 70 mg, such as Between 90 and 900 mg, between 120 and 450 mg or between 150 and 400 mg. Alternatively, the divalent cation chelating agent can be a citrate salt having a weight, such as greater than 70 mg, such as between 90 and 900 mg, between 120 and 450 mg, or between 150 and 400 mg. Blood samples contain a plurality of cells, including small cells smaller than 6 microns in size and large cells larger than 6 microns in size. Small cells, for example, contain platelets and small body stem cells that are less than 6 microns in size. For example, small body stem cells contain one or more specific types of somatic stem cells (ie, such as SB cells), BLSCs (ie, CD66e (+) somatic stem cells), and VSELs (such as CD133(+) somatic stem cells and CD34 (+) somatic stem cells). Large cells, for example, contain large body stem cells and lineage cells such as red blood cells and white blood cells that are larger than 6 microns in size. The blood sample can 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. In one example, the blood sample can be mixed in a container with a divalent cationic chelating agent (such as K2 EDTA, K3 EDTA, or citrate) greater than 1.5 mg per milliliter of blood sample (such as between 1.6 and 2.0 mg). To form a mixture.

Next, the mixture is treated to form a solution containing stem cells. The process can include the steps of activating and purifying/separating stem cells. As used herein, the terms "purified" or "isolated" are meant to substantially separate small cells (such as cell sizes greater than 2 microns and less than 6 microns) from large cells, such as cell sizes greater than 6 microns.

The mixture can be stored in a suitable facility (such as a refrigerator or other freezer) at a temperature between 2 ° C and 12 ° C, more preferably between 2 ° C and 7 ° C or 4 ° C. Equipment) for a predetermined period of time. The period of time may be between 3 hours and 72 hours, and more preferably between 3 hours and 6 hours, between 6 hours and 72 hours, between 6 hours and 48 hours, Between 16 hours and 72 hours, between 16 hours and 48 hours, between 36 hours and 60 hours, between 48 hours and 72 hours or about 48 hours. After the mixture is stored for a predetermined period of time, one or more specific types of somatic stem cells (such as SB cells) in the mixture may be activated with a divalent cation chelating agent (such as K2 EDTA, K3 EDTA or citrate), ie, one or The cell cycle of multiple specific types of somatic stem cells is activated from G0 to G1. This activation may be related to the ability of the divalent cation chelating agent to inhibit p53 function (assumed by chelation of Zn ²⁺ ) such that one or more specific types of somatic stem cells (such as SB cells) survive the G0 of the cell cycle. Period to G1. Since p53 protein requires Zn ²⁺ to properly entangle and form a functional protein, chelation of Zn ²⁺ with a divalent cation chelating agent can be a critical step in activating one or more specific types of somatic stem cells, such as SB cells. It is possible that the divalent cation chelating agent can sequester other divalent ions (such as Ca ²⁺ ) to activate the one or more specific types of somatic stem cells and force them to differentiate and expand.

When the mixture is stored, it is layered by gravity, including the upper and lower layers. The upper layer or supernatant 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 body stem cells (eg, also known as SB cells), BLSCs (ie, CD66e(+) somatic stem cells), and VSELs (such as CD133(+) somatic stem cells. With CD34(+) body stem cells). Most large cells containing lineage cells and large body stem cells in blood samples, such as large cells greater than 95%, 98%, or 99% in blood samples, are located in the lower layer. The ratio of the volume of the supernatant to the volume of the blood sample, for example, may range from one third to one half.

Substantially all of the upper layers can then be collected or transferred to a liquid container, such as a bag, syringe or glass bottle, to produce a solution containing stem cells or a mixture of stem cells. The upper layer, such as a solution containing stem cells, contains small cells including platelets and small body stem cells. The number of small body stem cells in the solution containing stem cells may be greater than or equal to 10 million (such as greater than or equal to 30 million, greater than or equal to 50 million, between 10 million and 50 million, Between 25 million and 300 million or between 30 and 500 million). The solution containing stem cells may also contain a divalent cation chelating agent such as EDTA and/or a growth factor.

Furthermore, solutions containing stem cells hardly include or substantially exclude large cells (such as large body stem cells and lineage cells). For example, large cells may constitute less than 5% of the total number of cells in a solution containing stem cells (such as 1%, 0.5%, or 0.01% or less). For example, the number of red blood cells in a solution containing stem cells, such as the collected upper layer, may be 10 ⁵ or less than 10 ⁴ per ml. Preferably, the number of red blood cells in the solution containing the stem cells is 10 ³ or less per ml. The number of white blood cells in the solution containing stem cells may be 10 ⁴ or less per ml (such as 10 ³ or less). Preferably, the number of white blood cells in the solution containing stem cells is 10 ² or less per ml.

In a solution containing stem cells, more than 95% (such as 99% or 99.99%) of all cells may be small cells. Small cells may include platelets, Lgr5(+) cells, CD349(+) cells, CD133(+) cells, CD34(+), and CD66e(+) cells. Platelets can constitute 75% to 85% of small cells in a solution containing stem cells. In all small cells, greater than 4% (such as greater than 5% or between 4.5% and 10%) may be Lgr5+ body stem cells. CD349(+) somatic stem cells can constitute more than 4% of all small cells in a solution containing stem cells (such as 5% or more or between 4.5% and 10%). In small cells, less than 2% (such as 1% or less) may be a combination of CD133(+) cells and CD34(+) cells. In small cells, there are 6% or less (such as 5% or 4.5% or less may be CD66e(+) cells.

Any particular small cell can also be further isolated or removed from the collected upper layer by flow cytometry or other conventional techniques such as antibody-based techniques such as antibody-bonded beads.

The collected upper layer can be used as it is as a solution containing stem cells (such as administration to an individual or stored) or further processed. For example, it can be further purified (such as filtration) or mixed with one or more other components. A suitable Ca ^{2+ -free} cell culture medium or solution (such as greater than 400 ml, such as between 500 and 900 ml) can be added to the collected upper layer to make a solution containing stem cells. Suitable Ca ^{2+ -free} cell culture media or solutions, such as NaCl-containing solutions, may be further free of any divalent ions, including Mg ²⁺ . The NaCl-containing solution, for example, may be a normal saline solution (such as a 0.90% w/v NaCl solution, about 300 mOsm/L or 9.0 g per liter).

The solution containing stem cells can be stored at a freezing storage temperature, such as -70 ° C or -80 ° C (eg between -75 ° C and -85 ° C) for a long period of time (such as a week, a Month or more). When ready for use, the frozen stem cell-containing solution can be quickly thawed and, optionally, mixed with a suitable Ca ^{2+ free} medium or solution (such as 0.9% NaCl) as described above. treatment method

The stem cell-containing composition produced by the above procedure can be used to increase the individual's PRMT level. The composition can also be used to treat conditions associated with a decrease in the level of PRMT or conditions that can be treated by a PRMT agonist.

The terms "protein arginine methyltransferase" or "PRMT" mean enzymes that catalyze the methylation of arginine. The mammalian genome encodes at least nine different PRMTs, namely, PRMT1, PRMT2, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, PRMT8, and PRMT9.

The PRMT level can refer to any biological sample taken from an individual, such as a blood sample, a bone marrow sample, a urine sample, or a solid tissue sample, the protein level of the PRMT, mRNA level, cDNA level, or functionality (such as enzyme). Level. A reduced PRMT level means that the level is lower than the level or level of the condition associated with a decrease in the PRMT level in a healthy individual or individual. The control level can be a level in a healthy individual, an individual without a PRMT level reduction, or an individual prior to treatment with a composition comprising stem cells. Methods for determining protein, mRNA, cDNA, and functional levels are well known in the art, such as ELISA, Western blotting, and real-time PCR.

Before and after administration of the composition to the individual, the level of the PRMT in the sample taken by the individual, such as a blood sample, a bone marrow sample, a urine sample, or a solid tissue sample, can be measured to monitor treatment efficacy. Optionally, the individual's disease parameters or symptoms can be assessed before and/or after administration.

Conditions that can be treated with compositions comprising stem cells include aging, anemia, other blood cell diseases, and T cell related diseases.

The stem cell-containing compositions described herein can be administered to a subject in need via any route of administration, such as intravenous, joint, conjunctival, intracranial, peritoneal, pleural, intramuscular, intrathecal or subcutaneous routes of administration. The composition may contain between 10 and 500 million small body stem cells. Autologous or allogeneic stem cells can be used. The composition may be, for example, every 1-14 days, every 2-4 weeks, every 1-6 months or every 2-12 months, a treatment period (such as 1-36 months or 2-10 years) or when needed Invest in individuals.

"Individual" means a human or non-human animal. "Treatment" or "treatment" means the administration of a compound or composition to an individual suffering from a disease for the purpose of curing, alleviating, alleviating, remedying, delaying the onset of the disease or ameliorating the disease, the symptoms of the disease, the disease state secondary to the disease or Predisposition. By "effective amount" is meant an amount of a compound or composition that produces a medically desired result in the individual being treated. Therapeutic methods can be performed alone or in combination with other drugs or therapies.

The following specific examples are for illustrative purposes only and are not intended to limit the remainder of the disclosure in any way. Without further elaboration, it is believed that one of ordinary skill in the art of the invention can Example

100 to 150 ml of peripheral blood samples were taken from each of the 91 individuals. The EDTA coated tube containing the blood sample was stored at 4 °C for 6 to 48 hours until the blood was separated into two different layers by gravity. The top layer was collected, which contained SB cells and was autologously delivered back to each body by intravenous injection. These individuals receive treatment every three to six months for a total of four treatments.

Blood samples are collected from the individuals before and after treatment. The performance level of PRMT5 in the sample was determined by RT-PCR. In 24 hours or 1 week after the first treatment, 67% of the individuals showed a significant increase in PRMT5 performance (i.e., at least 1.3 fold). See Table 1. In addition, these individual descriptions feel more energetic and sleep better. Table 1. Other embodiments

All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an identical, equivalent, or alternative feature. Therefore, unless expressly stated otherwise, each feature disclosed is only an example of a series of equivalent or similar features.

From the above description, the general features of the specific embodiments can be easily determined by those skilled in the art, and various changes can be made to the specific embodiments without departing from the spirit and scope thereof. And modified to make it suitable for a variety of uses and conditions. Therefore, other specific embodiments are also within the scope of the present patent application.

Claims (20)

A method of increasing the level of protein arginine methyltransferase (PRMT) in a body, comprising: identifying a subject in need thereof; and administering to the individual a plurality of sizes greater than 2 microns and less than 6 a composition of micron small cells; wherein the small cells comprise a plurality of individual stem cells, (i) pluripotentl or multipotent, and (ii) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+) or CD34(+). The method of claim 1, wherein the identifying step comprises detecting a PRMT level in a biological sample taken from the individual. The method of claim 2, wherein the PRMT is PRMT5. The method of claim 1, wherein the individual has a condition associated with abnormal PRMT function. The method of claim 4, wherein the condition is aging or anemia. The method of any one of claims 1 to 5, wherein the minicell further comprises platelets. The method of claim 6, wherein 75% to 85% of the small cells are platelets, and 20% to 25% of the small cells are the stem cells. The method of claim 7, wherein the composition comprises from 10 to 500 million such stem cells. The method of claim 8, wherein the composition is prepared by a process comprising: providing a mixture comprising a blood sample obtained from the individual or a donor individual and a divalent cation chelating agent The mixture is stored at a temperature between 2 ° C and 12 ° C for 3 to 72 hours, thereby separating the mixture into an upper layer and a lower layer, wherein the upper layer comprises the small populations; And collecting the upper layer, thereby preparing the composition. The method of claim 9, wherein the divalent cation chelating agent is EDTA. The method of claim 10, wherein 1.5 to 2.0 mg of a divalent cation chelating agent per ml of the blood sample is mixed with the blood sample to obtain the mixture. The method of claim 10, wherein an action for increasing the number of stem cells is performed on the individual or the donor individual prior to obtaining the blood sample. The method of claim 12, wherein the action is administering an effective amount of fucoidan or granulocyte-colony stimulating factor. The method of claim 10, wherein the process for preparing the composition further comprises, after collecting the upper layer, adding a pharmaceutically acceptable excipient to the collected upper layer. The method of claim 10, wherein the process for preparing the composition further comprises, after collecting the upper layer, centrifuging the upper layer to obtain a cell pellet. The method of claim 15, wherein the process for preparing the composition further comprises washing the precipitate and suspending the precipitate in a pharmaceutically acceptable excipient. The method of claim 15 or 16, wherein the pharmaceutically acceptable excipient does not contain divalent ions. The method of claim 17, wherein the pharmaceutically acceptable excipient is an aqueous saline solution. The method of any one of claims 1 to 18, wherein the composition is administered intravenously. The method of any one of claims 1 to 19, further comprising, after the administering step, detecting a PRMT level in the biological sample taken from the individual after the administering step.