TW201819623A - Composition and method for decreasing bilirubin level - Google Patents

Abstract

A method of decreasing a bilirubin level in a subject, comprising: identifying a subject in need thereof; and administering to the subject a composition that includes 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) plurripotent or totipotent; and (i) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+), or CD34(+).

Description

Composition and method for reducing bilirubin level

The present invention relates to compositions and methods for reducing bilirubin levels.

BACKGROUND OF THE INVENTION Bilirubin is a yellow pigment that is commonly found in the blood as well as in feces. When aged red blood cells break down in a normal and healthy procedure, bilirubin is produced in the body. After circulating in the blood, bilirubin will move to the liver. In the liver, bilirubin is discharged into the bile duct and stored in the gallbladder. The bilirubin is then released into the small intestine along with the bile to help digest the fat. Finally, bilirubin is excreted with the feces. A variety of conditions can lead to a high bilirubin level.

SUMMARY OF THE INVENTION In one aspect, described herein is a method of reducing bilirubin levels in a body. The method comprises: identifying a subject in need thereof; and administering to the individual (eg, intravenously) a composition comprising small cells having a size greater than 2 microns and less than 6 microns; wherein the minicells comprise a plurality of individuals Stem cells, which are: (i) versatile or pluripotent; and (ii) CD349(+), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+ ), CD133 (+) or CD34 (+).

In a specific embodiment, the identifying step in the method includes detecting a bilirubin level that is higher than a control level in the individual. In one embodiment, the identified individual does not have a liver condition or condition. The individual can have a gallbladder condition, a pancreatic condition, a viral infection, an autoimmune disease, a metabolic defect, or hemolytic anemia.

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

In one embodiment, the composition is prepared by a process, wherein the process comprises: providing a mixture comprising a blood sample obtained from the individual or a donor 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 contains the small populations; and collecting the The upper layer, thereby preparing the composition. 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. 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 obtaining the blood sample to prepare the composition. This action may be by administering an effective amount of fucoidan or a particulate white blood cell community stimulating factor.

The process for preparing the composition may further comprise, after collecting the upper layer, adding a pharmaceutically acceptable excipient to the collected upper layer. Alternatively, the process may further include, 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. The pharmaceutically acceptable excipient may be free of divalent ions. In a specific embodiment, the pharmaceutically acceptable excipient is a saline solution.

In a specific embodiment, the method further comprises, after the administering step, detecting a bilirubin level in a biological sample obtained from the individual after the administering step.

In another aspect, described herein is a composition for reducing the level of bilirubin in a body. The composition comprises 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) versatility or pluripotency; and (i) CD349 ( +), CD9(+), Oct4(+), Nanog(+), Lgr5(+), CD66e(+), CD133(+) or CD34(+).

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

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It has been unexpectedly discovered that a composition comprising specific small body stem cells (e.g., Lgr5 (+) or CD349 (+) cells) can reduce blood bilirubin levels in a patient. Accordingly, the methods described herein are used to treat hyperbilirubinemia as well as compositions. Somatic stem cell

There are many types of somatic stem cells, including totipotent stem cells, multifunctional stem cells, pluripotent stem cells, and precursor stem cells (also known as specific stem cells). Lecithin-like stem cells (BLSCs) are pluripotent or multifunctional somatic stem cells. Minimal embryonic stem cells (VSELs) are multifunctional somatic stem cells. SB cell line versatile or pluripotent stem cells. Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSC) are pluripotent stem cells.

The size (Z) of a cell such as a stem cell used herein may be (1) a conventional definition of the size or representative length of a cell in the field of cell biology or stem cell, (2) a cell Diameter, especially when the cell is substantially spherical, (3) the length of the major axis of a cell, especially when the cell is substantially ellipsoid, (4) the width of a cell, when the shape of the cell has an approximation a square shape, (5) the length of a cell, when the shape of the cell has a shape that approximates a rectangle, or (6) the maximum cross-section or lateral dimension of a cell. For example, an image of the cell obtained from an optical microscope or from an electron microscope (for example, a scanning electron microscope (SEM)) or data of the cell obtained from a first-class cytometer can be used (for example, two-dimensional Point, contour or density map) Determine or measure the dimension (Z), whether diameter, length, width, or maximum cross-section or lateral dimension. An image of a cell obtained from an optical or electron microscope may be a two-dimensional (2D) cross-section or a three-dimensional (3D) structure of the cell. As an example, the size (Z) of the cell can be obtained by measuring the maximum cross-sectional or lateral dimension of the cell in a 2D cross-sectional image obtained from an optical microscope or an electron microscope (e.g., SEM).

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

CD349(+) SB cell line versatile or pluripotent stem cells. CD349(+) SB cells can also be CD9(+), Oct4(+), and Nanog(+), as well as CD133(−), CD90(−), CD34(−), and Sox2(−). Each CD349(+) SB cell has 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, at 2.0 and 6.0. Between 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 between. Preferably, the size is greater than 2 microns and less than 6 microns.

Lgr5(+) SB cells are also multifunctional or pluripotent 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 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, at 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 Between microns. Preferably, a Lgr5(+) SB cell has a size greater than 2 microns and less than 6 microns.

Lecithin-like stem cells (BLSCs) are CD66e (+) pluripotent or multifunctional somatic stem cells. Each of them may 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, 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 multifunctional somatic stem cells that can be CD133(+) or CD34(+). A VSEL can also be CD45(−) and Lin(−). For example, a VSEL can be CD133(+), CD45(−), and Lin(−), or CD34(+), CD45(−), and Lin(−). A VSEL may be sized to be equal to or smaller 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, or between 1.0 and 4.0 microns. A VSEL may be sized greater than 2 microns and less than 6 microns.

Mesenchymal stem cells (MSCs) are pluripotent stem cells. One MSC can express one or more cell surface markers CD13, CD29, CD44, CD73, CD90, and CD105. MSCs constitute a very heterogeneous group. 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 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 pluripotent stem cells. It can 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, at 0.5. Between 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 action of stem cells

One of the actions (X) used herein is an action that effectively increases the number of one or more stem cells in a living body, for example, in a human or non-human individual. Action (X) may include: 1. taking a drug, such as a synthetic drug or a natural compound; 2. taking a herb or a Chinese herbal medicine such as Cordyceps, Ginseng, Wolfberry, Ganoderma Lucidum, Antrodia, and/or Brazilian Mushroom; A product or dietary supplement, such as a nutritional pill or a nutritional powder, which includes the following substances or elements: vitamins (vitamin A, B, B, B ₁₂ , D, D ₃ , E, etc.), macro and/or trace minerals Substances (eg, calcium, sodium, potassium, fluorine, bromine, chromium, iodine, antimony, selenium, tellurium, lithium, cobalt, vanadium and/or nickel), polysaccharides, high molecular weight trehalose-containing glycoproteins, seaweed ( Including green algae, blue-green algae, brown algae, etc.), trehalose, fucoidan (a major component of brown algae), oligofucose gum, algae, brown algae containing fucoidan (for example, grown and produced in Okinawa, Japan) Brown algae), Japanese water cloud, green algae, blue-green algae (or cyanobacteria), brown algae (including water clouds, kelp, wakame, sarcophagus, wakame, etc.), botanical compounds (eg, isoflavones or phytoestrogens) ), lycopene, epigallocatechin catechin gallate (E GCG), green tea, glycotrophic nutrients (eg, xylose, galactose, glucose, mannose N-acetylglucosamine, N-acetylgalactosamine, or N-acetamidine), fish oil, toon (toona sinensis), and/or nutrients extracted from plants, leaves, fruits, vegetables, fish, seaweed, or algae; 4. stimulating a vegetarian diet; 5. taking or eating healthy or organic foods; (non-traditional) medicine; 7. receiving an alternative therapy or treatment, such as Gerson therapy or Breuss cancer cure; 8. receiving acupuncture; 9. receiving a massage, such as a foot massage; 10. Exercise, such as walking, jogging, dancing, gymnastics, yoga, aerobics, and/or Tai Chi (Chinese shadow training); 11. Sleeping (for the purpose of measuring sleep quality); 12. Meditation; 13. Implementation by one person , a medical professional, or a medical improvement program designed by a medical doctor or a disease treatment plan; 14. taking a specific nutrient for improving the health of a particular organ in the body, for example, taking an improved prostate Healthy lycopene; 15. Participate in a rehabilitation program to heal an injured area, or heal a wound caused by surgery, or treat a disease; 16. Take a medicinal liquor (or medicinal liquor) Medicinal wine), medicated liquor or medicated wine, for example, by immersing a traditional Chinese medicine or a plurality of traditional Chinese medicines in a liquor or wine for a period of time, such as immersing ginseng in high Ginseng wine made in alcoholic alcoholic liquor for one month; 17. taken for treatment of a specific disease (eg, a cancer, approved by a government agency or agency such as the US Food and Drug Administration (US FDA) One or more drugs for skin diseases, kidney diseases and/or the like; 18. The use or acceptance of a drug approved by a government agency for the treatment of a particular disease (eg, a cancer, skin disease, or kidney disease) Treatment or therapy; 19. Participate in a religious activity, such as praying for peace or worshiping God; 20. Directly or indirectly exposed to sunlight or daylight (in the morning, for example, 10 minutes before sunrise) Between 50 minutes (including a large amount of infrared (IR) light); or near noon, for example between 11:30 am and 12:30 pm (including a large amount of ultraviolet (UV) light); or Afternoon, for example between 50 minutes before sunset and 10 minutes after sunset (including a large amount of infrared (IR) light)); 21. Exposure to light or light-emitting diode (LED) light, which may include the full spectrum of visible light, IR light, red light, green light, blue light or UV light, or a combination of more than one of the above; 22. Implementing or accepting plans, therapies, methods, instruments, and/or systems for improving the body's self-healing power, such as a method or therapy for improving self-healing force after injury or surgery (eg, hyperbaric oxygen therapy); 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 a substance containing fucoidan or oligofucose (for example, a nutrient or Supplement); 29. taking hormone supplements or accepting Injection of a granular white blood cell community stimulating factor (G-CSF or GCSF), which is a glycoprotein; 31. receiving a GCSF injection course; and 32. taking a nutritional product, a nutritional product, a nutrition a fluid, a nutritious beverage, a nutrient liquid, or a nutritious food comprising (1) a plurality of amino acids (such as arginine, histidine, lysine, aspartic acid, glutamic acid, serine , sulphonic acid, aspartic acid, glutamic acid, cysteine, valine, valine, glycine, selenocysteine, alanine, isoleucine, leucine , phenylalanine, methionine, tyrosine, or tryptophan), (2) balanced amino acids, or (3) 9 essential amino acids (ie, histidine, isoleucine) , leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. For example: (a) a product produced or extracted from the fermentation of red beans, mung beans, black beans; (b) a combination of a fruit or several fruits, such as beets, apples, guava, kiwi, grapes, pineapples, red dragons a liquid, fluid, or beverage produced by the fermentation of fruit (fire dragon fruit), green papaya, tomato, and/or avocado, etc.; (c) for example, soaking a Chinese medicine or a plurality of traditional Chinese medicines in liquor or wine (wine Medicinal liquor (also known as medicinal wine, medicated liquor, or medicated wine), such as immersing ginseng in high alcoholic rice wine for a period of time Ginseng wine made in the month; composition containing stem cells

A composition containing stem cells (e.g., a solution containing stem cells) can be prepared using an exemplary method described below.

An action (X) is performed on a body, which may be one of the above actions (X). The individual, for example, is a human (eg, a child, a teenager, an adult, or an elderly person) or a non-human animal. Examples of a non-human animal include a primate (eg, a monkey or gorilla), a dog, a rodent (eg, a mouse or guinea pig), a cat, a horse, a cow, a cow, a sheep, a pig, a chicken, a duck, Goose, bird, and elephant.

For example, the individual can ingest a stem cell-mobilization agent, such as a compound containing fucoidan. The compound containing fucoidan may be a brown algae supplement. A brown algae supplement pill contains 80% water cloud powder, 15% crystalline cellulose, 3% sucrose fatty acid ester, and 2% micro or fine powder (containing cerium oxide). The water cloud powder can be extracted from a water cloud brown algae (a seaweed) grown in the sea near Okinawa, Japan. The water cloud powder is then mixed with crystalline cellulose, sucrose fatty acid ester, and micro or fine mash (containing cerium oxide) to form the brown algae supplement pellet comprising 0.1 gram of fucoidan. The individual may ingest 20 or more (eg, at least 30) brown algae supplement pills or 2 grams or more (such as at least 3 grams) of fucoidan. In another example, the individual may ingest a particulate leukocyte community stimulating factor (GCSF), i.e., a mobilizer, or may receive a GCSF injection regimen.

After performing an action (X), the individual waits for a period of time (eg, for 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 at 36 hours Between 50 hours. Acting (X) and waiting for a period of time allows one or more specific types of somatic stem cells, such as SB cells (ie, CD349(+) and Lgr5(+) SB cells), to move from, for example, the individual's bone marrow to the individual Peripheral blood. The peripheral blood of the individual thus becomes rich in one or more specific types of somatic stem cells. The one or more specific types of somatic stem cells, for example, may be or may include one or more of the above-described somatic stem cells. For example, the one or more specific types of somatic stem cells may be or may include somatic stem cells having a size of less than 6 microns, and more preferably, body stem cells having a size greater than 2 microns, such as CD349(+) somatic stem cells and/or Lgr5. (+) body stem cells.

Perform action (X) and wait for a period of time to select an optional step. In other words, when preparing a composition containing stem cells, a blood sample can be obtained from one body without first performing any action (X) on the individual.

Immediately following the waiting step (if there is an action (X) and the waiting step), a blood sample is obtained from the peripheral blood of the individual and placed in one or more containers containing a divalent cation chelating agent. Medium (for example, a bag, one or more syringes, or one or more test tubes). The blood sample is mixed with the divalent cation chelating agent in the container to form a mixture. The divalent cation chelating agent, such as an anticoagulant, may be ethylenediaminetetraacetic acid (EDTA), such as a K2 EDTA anticoagulant or a K3 EDTA anticoagulant, having a weight, for example, 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, for example, greater than 70 mg, such as between 90 and 900 mg, between 120 and 450 mg, or at 150 and 400 mg. between. The blood sample contains a variety of cells, including small cells of size less than 6 microns and large cells of size greater than 6 microns. Such small cells, for example, comprise platelets and small body stem cells having a size of less than 6 microns. For example, the minisomal stem cells comprise the one or more specific types of somatic stem cells (ie, SB cells, eg,), BLSCs (ie, CD66e (+) somatic stem cells), and VSELs (eg, CD133(+) bodies). Stem cells and CD34(+) somatic stem cells). Such large cells, for example, comprise large body stem cells having a size greater than 6 microns and lineage cells such as red blood cells and white blood cells. 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 1.5 mg or more per milliliter of blood sample, such as between 1.6 and 2.0 mg, of the divalent cation chelating agent (such as K2 EDTA, K3 EDTA, or citrate) Mix to form the mixture in the container.

Next, the mixture is processed to form a solution containing stem cells. The process can include steps of stem cell activation and purification/separation. The term "purified" or "isolated" as used herein means the substantial separation of small cells (eg, cells having a size greater than 2 microns and less than 6 microns) from large cells (eg, cells having a size greater than 6 microns). .

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 (eg , a refrigerator or other device for keeping things cool) for a predetermined period of time. This 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, at 16 hours. Between 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 has been stored for a predetermined period of time, the one or more specific types of somatic stem cells (eg, SB cells) in the mixture may be by the divalent cation chelating agent (such as K2 EDTA, K3 EDTA, or The citrate is activated, that is, the cell cycle of the one or more specific types of somatic stem cells is activated from G0 into G1. The activation line is associated with the ability of the divalent cation chelating agent to inhibit the function of p53 (assumed by chelation of Zn ²⁺ ), thereby allowing the one or more specific types of somatic stem cells (eg, SB cells) to be from the cell cycle G0 leaves during the stationary period and enters the G1 phase. Since the p53 protein requires Zn ^{2+ to} properly fold and form a functional protein, chelation of Zn ²⁺ by the divalent cation chelating agent may activate the one or more specific types of somatic stem cells (eg, SB) A key step in the cell). The divalent cation chelating agent also has the potential to sequester other divalent ions (e.g., Ca2 ⁺ ) to activate the one or more specific types of somatic stem cells and promote their proliferation and expansion.

When the mixture is stored, it separates into different layers due to gravity, including an upper layer and a lower layer. The upper layer, or the supernatant, may have a volume between 20 and 250 milliliters, between 40 and 125 milliliters, or between 50 and 100 milliliters. The upper layer comprises platelets, serum, and one or more specific types of small body stem cells (i.e., SB cells, for example), BLSCs (i.e., CD66e (+) somatic stem cells), and VSELs (e.g., CD133 (+) bodies). Stem cells and CD34(+) somatic stem cells). The large cells of the blood sample containing the lineage cells and the large body stem cells, such as greater than 95%, 98% or 99% of the large blood 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 can be, for example, in the range of one third to one half.

Next, substantially all of the upper layer can be collected or transferred to a liquid container, such as a bag, a syringe, or a glass vial to produce a solution containing stem cells or a mixture of stem cells. The upper layer, for example, a solution containing stem cells, comprises 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 (eg, greater than or equal to 30 million, greater than or equal to 50 million, between 10 million and 500 million, at 2 thousand Between 5 million and 300 million, or between 30 million and 500 million). The solution containing stem cells may also comprise the divalent cation chelating agent (eg, EDTA) and/or growth factors.

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

More than 95% (eg, 99% or 99.99%) of all such cells in the solution containing stem cells can be small cells. Such minicells may include platelets, Lgr5(+) cells, CD349(+) cells, CD133(+) cells, CD34(+), and CD66e(+) cells. The platelets may constitute 75% to 85% of such small cells in the solution containing stem cells. More than 4% (eg, greater than 5% or between 4.5% and 10%) of all such small cells may be Lgr5+ somatic stem cells. The CD349(+) somatic stem cells may constitute greater than 4% (e.g., greater than 5% or between 4.5% and 10%) of all such minicells in the solution containing stem cells. Less than 2% (eg, less than 1% or 0.5%) of such minicells may be a combination of CD133(+) cells and CD34(+) cells. Less than 6% (eg, less than 5% or 4.5%) of such small cells can be CD66e(+) cells.

Flow cytometry or other conventional techniques (e.g., antibody-based techniques, such as antibody-conjugated beads) can also be used to further isolate or exhaust any particular small cells from the collected supernatant.

The collected upper layer can be used as a solution containing stem cells (e.g., administered to a body or stored) or further processed. For example, it can be further purified (eg, filtered) or mixed with one or more additional ingredients. A suitable cell culture medium or solution containing no Ca ²⁺ has a volume, for example, greater than 400 ml, such as between 500 and 900 ml, which can be added to the collected upper layer to produce a solution containing stem cells. The suitable medium or solution that does not contain Ca ²⁺ , such as a solution containing NaCl, may be further free of any divalent ions, including Mg ²⁺ . The NaCl-containing solution, for example, may be a physiological saline solution (for example, a solution of 0.90% w/v NaCl, about 300 mOsm/L or 9.0 g per liter).

The solution containing stem cells can be stored at a freezing storage temperature, for example, equal to or less than -70 ° C or -80 ° C (for example, between -75 ° C and -85 ° C) for a long period of time. Time (for example, greater than one week, one month, or one year). When ready for use, the frozen solution containing stem cells can be rapidly thawed and, optionally, mixed with a suitable medium or solution (e.g., 0.9% NaCl) that does not contain Ca2 ⁺ as described above. treatment method

The stem cell-containing solution or composition produced by the above method can be used to treat hyperbilirubinemia or to lower the level of bilirubin in one body.

The bilirubin attached to the sugar is either direct or bound bilirubin. Indirect or unbound bilirubin is an unattached bilirubin. All forms of bilirubin in the body are collectively referred to as total bilirubin. The term "bilirubin level" as used herein may mean an indirect bilirubin level, a direct bilirubin level, or a total bilirubin level. A high bilirubin level is higher than the level or level range found in healthy individuals or in individuals who do not have a condition associated with high bilirubin levels. For example, an adult's normal total bilirubin level can be less than 1.3 milligrams per minute (mg/dL). The normal level of direct bilirubin is usually between 0.1 and 0.4 mg/dL. In accordance with the specifications accepted in the art to which the present invention pertains, one skilled in the art can determine whether a body has a high bilirubin level.

Before the composition containing the stem cells is administered to a body, it is possible to determine whether the individual has a high bilirubin level. After the composition is administered, the level of bilirubin in the individual can also be determined to monitor therapeutic efficacy and make a therapeutic decision. Alternatively or additionally, other disease parameters or symptoms in the individual can be assessed before and/or after the administration.

The bilirubin level will increase for several reasons. For example, it occurs when more than normal red blood cells decompose, for example, when a body has fetal red blood cells or hemolytic anemia. Liver problems can also cause hyperbilirubinemia. For example, hepatitis, Gilbert disease, cirrhosis, and nonalcoholic fatty liver disease increase bilirubin levels. Bilirubin levels are affected by gallbladder or bile duct problems. For example, gallstones, gallbladder inflammation, gallbladder cancer, surgical removal of the gallbladder, shrinkage of the common bile duct, or pancreatitis can lead to hyperbilirubinemia. Specific viral infections (eg, Epstein-Barr virus infection), autoimmune diseases, metabolic defects, alcohol, and drugs (eg, acetaminophen, chlorpromazine, penicillin, female) Hormone steroids and assimilated steroids can also cause hyperbilirubinemia. The methods described herein can be used to reduce the level of hyperbilirubin in a subject in which the underlying condition or disorder of the individual has not been diagnosed.

The stem cell-containing composition described herein can be via any route of administration, for example, intravenous, intra-articular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, or subcutaneous The path is given to an individual in need. The composition may comprise between 10 million and 500 million small body stem cells. Autologous or allogeneic somatic stem cells can be used. The composition can be, for example, administered every 1-14 days, every 2-4 weeks, every 1-6 months, or every 2-12 months to a physical examination period (eg, 1-36) Months or 2-10 years), or when needed.

An "individual" refers to a human or a non-human animal. "Trating" or "treatment" refers to administering a compound or composition to an individual having a condition for the purpose of curing, alleviating, ameliorating, treating, delaying, or ameliorating the condition. The symptoms of the condition, the condition of the disease secondary to the condition, or the predisposition to develop the condition. By "effective amount" is meant an amount of the compound or composition that produces a medically desirable result in a treated individual. The method of treatment 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 disclosure in any way. Without further elaboration, it is believed that one of ordinary skill in the art of the invention can Example

Peripheral blood samples of 100 to 150 ml were obtained from patients A and B who had hyperbilirubinemia for more than several months. The samples were placed in EDTA coated tubes and incubated at 4 °C for 6 to 48 hours until the blood was separated into 2 distinct layers. The upper layer contains SB cells. The upper layer was collected from the patient's own sample and delivered back to the patient via intravenous injection. The plasma bilirubin level in each patient was found to be significantly reduced after treatment.

The plasma bilirubin level of patient A was 1 mg/dL before starting chemotherapy. About 3 months after the start of chemotherapy, the patient's bilirubin level reached 2.7 mg/dL. The chemotherapy was then discontinued for approximately 1 month until the bilirubin level dropped to 1.8 mg/dL. The patient then completed 8 chemotherapy sessions. About 5 months after the completion of chemotherapy, the patient's bilirubin level was 1.6 mg/dL. An SB cell composition was prepared and administered to the above patients. The patient received 2 treatments approximately 2 months apart. About 3 weeks after the second treatment, the patient's bilirubin level was reduced to 1.2 mg/dL.

The patient had a history of bilirubin levels below 1 mg/dL (eg, 0.5 to 0.7 mg/dL) before the patient's gallbladder was surgically removed. About one year after the surgery, the patient's bilirubin level was measured to be 1.5 mg/dL. One month later, the patient was treated with the above SB cell composition. The patient's bilirubin level was reduced to 1.2 mg/dL 2 weeks after the treatment. Other specific 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 (19)

A method of reducing a level of bilirubin in a body, comprising: identifying an individual 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 Where the small cells comprise a plurality of individual stem cells, which are: (i) versatility or pluripotency; and (ii) CD349(+), CD9(+), Oct4(+), Nanog(+) , Lgr5(+), CD66e(+), CD133(+) or CD34(+). The method of claim 1, wherein the identifying step comprises detecting a bilirubin level in the individual that is higher than a control level. The method of claim 2, wherein the individual does not have a liver condition. The method of claim 3, wherein the individual has a gallbladder condition, a pancreatic condition, a viral infection, an autoimmune disease, a metabolic defect, or hemolytic anemia. The method of claim 2, further comprising: detecting a bilirubin level in the individual after the administering step. The method of any one of claims 1 to 5, wherein the minicells further comprise 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 stem cells. The method of claim 7, wherein the composition comprises from 10 to 500 million of 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 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 contains the small populations; and the upper layer is collected, The composition was thus prepared. 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 cationic chelating agent per millimeter of blood sample is mixed with the blood sample to obtain the mixture. The method of claim 10, wherein the individual or the donor is subjected to an action for increasing the number of stem cells prior to obtaining the blood sample. The method of claim 12, wherein the action is administered an effective amount of fucoidan or a particulate leukocyte community 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 a saline solution. The method of any one of claims 1 to 18, wherein the composition is administered intravenously.