WO2017019850A1

WO2017019850A1 - Composition and method for inhibiting histone deacetylase

Info

Publication number: WO2017019850A1
Application number: PCT/US2016/044444
Authority: WO
Inventors: James Wang
Original assignee: StemBios Technologies, Inc.
Priority date: 2015-07-28
Filing date: 2016-07-28
Publication date: 2017-02-02
Also published as: HK1245144A1; EP3328400A1; CN107847525A; JP2018524360A; EP3328400A4; TW201707713A

Abstract

A method of inhibiting a histone deacetylase activity, comprising administering to a recipient subject in need thereof a composition that contains an effective amount of small somatic stem cells that are greater than 2 micrometers and less than 6 micrometers in size, wherein the small somatic stem cells include CD349+ somatic stem cells and Lgr5+ somatic stems cells.

Description

Composition and Method for Inhibiting Histone Deacetylase

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to US Provisional Application Nos. 62/197,742, 62/197,745, 62/197,753, 62/197,757, 62/197,760, 62/197,770, and 62/197,782, all filed on July 28, 2015. The contents of all prior applications are hereby incorporated herein by reference in their entirety.

BACKGROUND

Stem cells have the ability to self -renew to generate more stem cells and also to differentiate into other types of cells. They can thus be used to treat a variety of diseases.

Histone deacetylase has been shown as a drug target for disorders including cancers, immune disorders, and neurodegenerative disorders. A composition that can inhibit histone deacetylase activity has great potential to treat such disorders.

SUMMARY

In one aspect, described herein is a method of inhibiting a histone deacetylase activity. The method includes administering to a recipient subject in need thereof a composition that contains an effective amount of small somatic stem cells that are greater than 2 micrometers and less than 6 micrometers in size, wherein the small somatic stem cells include CD349(+) somatic stem cells and Lgr5(+) somatic stems cells. The method can further include a step of determining a histone deacetylase activity in the recipient subject before or after (or both) the administering step.

The recipient subject can have an autoimmune disorder, diabetes, cancer, neurodegenerative disorder, or virus infection. For example, the recipient subject can have dementia, Parkinson's disease, arthritis, ankylosing spondylitis, or diabetes.

In one embodiment, greater than 95% (e.g., greater than 99% or 99.99%) of all of the cells in the composition can be small cells. The small cells can include one or more types of cells including any of platelets, Lgr5(+) cells, CD349(+) cells, CD133(+) cells, CD34(+), and CD66e(+) cells. Platelets can constitute 75% to 85% of the small cells in the composition. Greater than 4% (e.g., greater than 5% or between 4.5% and 10%) of all of the small cells can be Lgr5+ somatic stem cells. CD349(+) somatic stem cells can constitute greater than 4% (e.g., greater than 5% or between 4.5% and 10%) of all of the small cells in the composition. Less than 2% (e.g., less than 1% or 0.5%) of 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.

Further, the composition can substantially exclude large cells that are greater than 6 micrometers in size. For example, 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 composition.

In one embodiment, the composition is an injectable solution that is administered intravenously. It can also contain a divalent cation chelating agent (e.g.,

ethylenediaminetetraacetic acid). The injectable solution can be prepared by a procedure including: providing a mixture that contains a blood sample and a divalent cation chelating agent; storing the mixture at a temperature between 2°C and 12°C for 3 to 72 hours, whereby the mixture separates into an upper layer and a lower layer, wherein the upper layer contains the small somatic stem cells; and collecting the upper layer, whereby the injectable solution is prepared. The blood sample used in the procedure can be obtained from the recipient subject or a donor subject.

Optionally, prior to obtaining the blood sample for preparing the injectable solution, an action for increasing stem cell number can be performed on the recipient subject or donor subject. For example, the action can be administration of fucoidan or a granulocyte-colony stimulating factor.

In another aspect, described herein is an injectable composition for use in decreasing a histone deacetylase activity in a subject. The composition contains an effective amount of small somatic stem cells that are greater than 2 micrometers and less than 6 micrometers in size, the small somatic stem cells including CD349+ somatic stem cells and Lgr5+ somatic stems cells.

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

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a flow chart illustrating a method for preparing a stem cell mixture according to an embodiment of the present disclosure.

Fig. 2 is a table showing the ingredient information of a brown algae fucoidan supplement (per pill).

Fig. 3 is a flow chart illustrating a method for stem cell activation, purification and concentration according to an embodiment of the present disclosure.

Fig. 4 is a graph showing stem cell data of six human subjects obtained before and 5 after oral ingestion of a brown algae fucoidan supplement.

Fig. 5A is a graph showing flow cytometry data obtained before a course of injections of granulocyte stimulating factor (GCSF or G-CSF).

Fig. 5B is a graph showing flow cytometry data obtained after a course of injections of granulocyte stimulating factor (GCSF or G-CSF).

o Fig. 6A is a flow cytometry dot plot showing forward scattering coefficient (FSC) versus side scattering coefficient (SSC).

Fig. 6B is a fluorescence histogram from flow cytometry analysis.

Fig. 6C is a FSC versus SSC flow cytometry dot plot.

Fig. 7A is a FSC versus SSC flow cytometry dot plot.

5 Fig. 7B is a CD61 fluorescence histogram from flow cytometry analysis.

Fig. 7C is a CD133 fluorescence histogram from flow cytometry analysis. Fig. 7D is a CD34 fluorescence histogram from flow cytometry analysis. Fig. 7E is a CD66e fluorescence histogram from flow cytometry analysis. Fig. 7F is a CD349 fluorescence histogram from flow cytometry analysis. o Fig. 7G is a Lgr5 fluorescence histogram from flow cytometry analysis.

Fig. 8 is a scatter plot of the data shown in Table 2.

DETAILED DESCRIPTION

It was unexpectedly discovered that a composition containing certain small somatic stem cells (e.g., Lgr5(+) and CD349(+) cells) inhibited histone deacetylase (HDAC) activity 5 in vivo. Accordingly, described herein are methods and compositions for inhibiting histone deacetylase.

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 0 stem cells). Blastomere-like stem cells (BLSCs) are totipotent or pluripotent somatic stem cells. Very small embryonic -like stem cells (VSELs) are pluripotent somatic stem cells. SB cells are pluripotent or multipotent somatic stem cells. Mesenchymal stem cells (MSCs) and hematopoietic stem cell (HSC) are multipotent somatic stem cells.

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. As an example, 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).

The term "small cell" (e.g., small somatic stem cell) refers to a cell having a size less than 6 micrometers (e.g., between 2.0 and 6.0 micrometers). The term "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.

Preferably, the size is greater than 2 micrometers and less than 6 micrometers.

Lgr5(+) SB cells are 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 Sox2(-). 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. Preferably, a Lgr5(+) SB cell is greater than 2 micrometers and less than 6 micrometers in size.

Blastomere-like stem cells (BLSCs) 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. For example, a BLSC can have a size that is greater than 2 micrometers and less than 6 micrometers.

Very small embryonic-like stem cells (VSELs) are pluripotent somatic stem cells, which 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(-). 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.

Mesenchymal stem cells (MSCs) are multipotent somatic 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. Hematopoietic stem cells (HSCs) are multipotent somatic 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 5 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

An action (X) as used herein is an action that may be effective for increasing the l o 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:

1. Taking drugs such as synthetic drugs or compounds derived from nature;

2. Taking herbs or Chinese herbal medicines, such as Cordyceps sinensis, ginseng, Lycium Chinense Mill, Ganoderma lucidum (lingzhi), Taiwanofungus camphoratus, and/or

15 Brazil mushroom;

3. Taking nutrients or dietary supplements, such as nutrition pills or powder, including the following materials or elements: vitamins (Vitamin A, B, B complex, Bi₂, 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),

20 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,

25 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), fish oil, China toona (toona sinensis), and/or nutrients extracted from plant, leaf, fruit, vegetable, fish, seaweed, or algae;

30 4. Practicing a vegetarian dietary;

5. Taking or eating healthy food or organic food; 6. Taking an alternative (non-traditional) medicine;

7. Being subjected to an alternative therapy or treatment such as the Gerson therapy or the Breuss cancer cure;

8. Being subjected to acupuncture;

9. Being subjected to massage such as foot massage;

10. Exercising such as walking, jogging, dancing, gymnastics, Yoga, aerobic exercise, and/or Taijiquan (Chinese shadow exercise);

11. Sleeping (for purpose of measuring the quality of sleep);

12. Meditating;

13. Practicing a health improvement program or a disease curing program designed by an individual, a health professional, or a medical doctor;

14. Taking a certain nutrient for improving health of a certain organ in a body, for example, taking lycopene to improve the health of prostate;

15. Taking a rehabilitation program to heal the injury, or to heal the wounds caused by surgery, or to cure a disease;

16. Taking a medicinal liquor (or called medicinal wine, medicated liquor or medicated wine) made from, e.g., immersing one Chinese medicine or multiple Chinese medicines in liquor or wine for a period of time, such as ginseng wine made from immersing ginseng in a high alcohol concentration rice wine for a month;

17. Taking one or more drugs approved by a government department or authority, such as U.S. food and drug administration (U.S. FDA), for curing a specific disease (e.g., a type of cancer, skin disease, kidney disease and/or so on);

18. Taking or being subjected to a treatment or therapy approved by a government department for curing a specific disease (e.g., a type of cancer, skin disease, or kidney disease);

19. Practicing a religious activity, such as praying for peace or worshiping God;

20. Being exposed directly or indirectly to sunshine or sunlight (in the morning between, for example, 10 minutes before sunrise and 50 minutes after sunrise (containing significant amount of infrared (IR) light); or around noon, for example, between 11:30 AM to 12:30 PM (containing significant amount of ultra-violet (UV) light); or in the afternoon, for example, between 50 minutes before sunset and 10 minutes after sunset (containing significant amount of infrared (IR) light)); 21. Being exposed to 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;

22. Exercising or being subjected to programs, therapies, methods, apparatus and/or 5 systems for improving body's self-healing, for example, a method or therapy (e.g.,

Hyperbaric oxygen therapy) performed after injury or surgery for improving self-healing;

23. Drinking coffee such as black coffee;

24. Drinking tea such green tea, black tea, or jasmine tea;

25. Drinking red wine;

0 26. Taking melatonin;

27. Listening to music such as Mozart's or Beethoven's symphony;

28. Injecting a substance (e.g., a nutrient or supplement) containing fucoidan or oligo fucoidan;

29. Taking hormone supplements or being subjected to a hormone injection;5 30. Injecting a granulocyte-colony stimulating factor (G-CSF or GCSF), which is a glycoprotein;

31. Being subjected to a course of GCSF injections; and

32. Taking 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, o 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. For examples: (a) Product produced or extracted 5 from the fermentation of red, green, black beans; (b) Liquid, fluid, or drink produced from fermentation of a fruit or a combination of fruits, such as sugar beet, apple, guava, kiwi, grape, pineapple, red pitaya (dragon fruit), green papaya, tomato, and/or avocado, etc.; (c) A medicinal liquor (or called medicinal wine, medicated liquor, or medicated wine) made from, e.g., immersing one Chinese medicine or multiple Chinese medicines in liquor or wine for a o period of time, such as ginseng wine made from immersing ginseng in a high alcohol

concentration rice wine for a month. Stem Cell-Containing Composition

A stem cell-containing composition (e.g., a stem cell-containing solution) can be prepared using an exemplary method illustrated by the flow chart shown in Fig. 1.

Referring to Fig. 1, in step 10, a subject takes or is subjected to an action (X), which may be one of the above-mentioned actions (X). The subject, for example, is a human (e.g., child, teenager, adult, or elderly) or a non-human animal. Examples of 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.

For example, in step 10, 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. Fig. 2 shows the ingredients of a brown algae supplement. A pill of the brown algae supplement contains 80% of a mozuku powder, 15% of crystalline cellulose, 3% of sucrose fatty acid esters, and 2% of micro or fine silica (containing silicon dioxide). The mozuku powder may be extracted from mozuku brown algae (one kind of seaweed) grown in the sea around and near Okinawa, Japan. The mozuku powder is then mixed with crystalline cellulose, sucrose fatty acid esters, and micro or fine silica (containing silicon dioxide) to form the pill of the brown algae supplement, which contains 0.1 grams of fucoidan. In step 10, the subject may ingest 20 or more pills (e.g., at least 30 pills) of the brown algae supplement or 2 grams or more (such as at least 3 grams) of fucoidan. In another example, in step 10, 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.

In step 11, after step 10 is performed, the subject waits for a period of time (e.g., a predetermined period of time), such as between 15 minutes and 60 minutes, between 20 minutes and 100 minutes, between 30 minutes and 4 hours, between 60 minutes and 90 minutes, between 0.5 hours and 3 hours, between 1 hour and 6 hours, between 1 hour and 12 hours, between 12 hours and 36 hours, or between 36 hours and 50 hours. Steps 10 and 11 allow one or more specific types of somatic stem cells, such as SB cells (i.e., CD349(+) and Lgr5(+) SB cells), to be mobilized into the subject's peripheral blood from, e.g., the subject's bone marrow. The peripheral blood of the subject thus becomes enriched with the one or more specific types of somatic stem cells. The one or more specific types of somatic stem cells, for example, may be or may include one or more of the somatic stem cells described above. For instance, the one or more specific types of somatic stem cells may be or may include somatic stem cells less than 6 micrometers in size, and more preferably greater than 2 micrometers in size, such as CD349(+) somatic stem cells and/or Lgr5(+) somatic stem cells.

Steps 10 and 11 are optional. In other words, to make a stem cell-containing composition, a blood sample can be obtained from a subject without first performing any action (X) on the subject.

In step 12, immediately after step 11 (if steps 10 and 11 are performed), a blood sample is extracted, drawn, taken, obtained, collected or derived 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, 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 between 90 and 900 mg, between 120 and 450 mg, or between 150 and 400 mg. Alternatively, 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 example, contain platelets and small somatic stem cells less than 6 micrometers in size. For instance, the small somatic stem cells contain the one or more specific types of somatic stem cells (i.e., SB cells, for example), BLSCs (i.e., CD66e(+) somatic stem cells), and VSELs

(e.g., CD133(+) somatic stem cells and CD34(+) somatic stem cells). The large cells, for example, contain large somatic stem cells greater than 6 micrometers in size and lineage cells 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. In an example, 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 mixture in the container.

Next, in step 13, the mixture is processed to form a stem-cell containing solution. The process can include steps for stem cell activation and purification/isolation, such as steps 21 and 22 illustrated in Fig. 3. The term "purification" or "isolation" as used herein means substantial separation of small cells (e.g., cells greater than 2 micrometers and less than 6 micrometers in size) from large cells (e.g., cells greater than 6 micrometers in size), e.g., the act of obtaining the small cells in the mixture or blood sample by removing the large cells in the mixture or blood sample.

Referring to Fig. 3, in step 21, the mixture formed in step 12 is 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, 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 around 48 hours. After the mixture has been stored for the predetermined period of time, the one or more specific types of somatic stem cells (e.g., SB cells) in the mixture are 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 activation relates to the ability of the divalent cation chelating agent to repress p53's function (presumably by chelating

Zn²⁺), 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. As the p53 protein requires Zn²⁺ to fold properly and form a functional protein, chelating Zn²⁺ by the divalent cation chelating agent would 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., Ca²⁺), thereby activates the one or more specific types of somatic stem cells and forces them to proliferate and expand.

In step 21, the mixture separates into multiple separate layers including an upper layer and a lower layer due to gravity. 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 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, such as greater than 95%, 98% or 99% of the large 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, for example, may range from one third to one half. Next, in step 22, 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, contains small cells, which include platelets and small somatic stem cells. 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.

In addition, the stem cell-containing solution barely includes or substantially excludes large cells (e.g., large somatic stem cells and lineage cells). For example, 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. For example, the number of red blood cells in the stem-cell containing solution (e.g., the collected upper layer) can be less than 10⁵ or 10⁴per milliliter. Preferably, the number of red blood cells per milliliter of the stem cell-containing solution is less than 10³. The number of white blood cells per milliliter of the stem cell-containing solution can be less than 10⁴ (e.g., less than 10³). Preferably, the number of white blood cells per milliliter of the stem-cell containing solution is less than 10².

Greater than 95% (e.g., 99% or 99.99%) of all of the cells in the stem cell-containing solution can be small cells. The small cells can include platelets, Lgr5(+)cells, CD349(+) cells, CD133(+) cells, CD34(+), and CD66e(+) cells. Platelets can constitute 75% to 85% of the small cells in the stem cell-containing solution. Greater than 4% (e.g., greater than 5% or between 4.5% and 10%) of all of the small cells can be Lgr5+ somatic stem cells. CD349(+) somatic stem cells can constitute greater than 4% (e.g., greater than 5% or between 4.5% and 10%) of all of the small cells the stem cell-containing solution. Less than 2% (e.g., less than

1% or 0.5%) of 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 from the collected upper layer using flow cytometry or other conventional techniques (e.g. antibody-based techniques such as antibody-conjugated beads).

The collected upper layer can be used as is as a stem cell-containing solution (e.g., administered to a subject or stored) or further processed. For example, it can be further purified (e.g., filtered) or mixed with one or more additional components. A suitable cell medium or solution free from Ca²⁺ 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 Ca²⁺, such as a NaCl- containing solution, may be further free from any divalent ions, including Mg²⁺. The NaCl- containing solution, for example, can be normal saline (e.g., a solution of 0.90% w/v of NaCl, about 300 mOsm/L or 9.0 gram per liter).

The stem cell-containing solution may be stored in a frozen storage temperature, e.g., equal to or less than -70°C or -80°C (e.g., between -75°C and -85°C) for an extended period of time (e.g., more than one week, one month, or one year). When ready for use, the frozen stem cell-containing solution can be quickly thawed and, optionally, mixed with the aforementioned suitable medium or solution free from Ca²⁺ (e.g., 0.9% NaCl).

Inhibiting Histone Deacetylase (HDAC) Activity

The stem cell-containing solution or composition produced by the procedure described above can be used to inhibit HDAC activity.

For example, the stem cell-containing composition can be administered (e.g., injected intravenously) to a subject to inhibit or decrease HDAC activity in the subject. The treatment can, for example, decrease HDAC activity in the subject by at least 20%, e.g., by at least 30%, 40%, 50%, 60%, 70% or more. Autologous or allogeneic somatic stem cells can be administered to the subject.

HDACs are key players in epigenetic regulation, which is involved in a number of biological processes and diseases. Epigenetic modifications are reversible. However, they may be imbalanced or aberrant in certain diseases. HDACs have been shown to be promising drug targets for a number of diseases including cancer, neurodegenerative disorders, immune disorders, diabetes, and cardiovascular disorders. See, e.g., Arguelles et al., Drug Discovery

Today, 21(3):499-509 (2015): Abel and Zukin, Curr. Opin. Pharmacol., 8(l):57-64 (2008); Irwin et al., Drug Development Research, 77: 109-123 (2016); and Huang, Journal of Cellular Physiology, 209:611-616 (2006). As the stem cell-containing solution is an HDAC inhibitor, it can be used to modulate these processes and treat the diseases.

For example, lineage reprogramming (also known as transdifferentiation) is a process where one mature somatic cell transforms into another mature somatic cell without undergoing an intermediate pluripotent state or progenitor cell type. The process involves the transition between different epigenetic states. Therefore, an epigenetic regulator plays a key role for lineage reprogramming. The stem cell-containing solution described herein can thus be administered to a subject to modulate lineage reprogramming in the subject. For example, the stem cell-containing solution can direct a first type of terminally differentiated cells to go back or transform into (i) somatic cells upstream of the first type of terminally differentiated cells, or (ii) a different type of terminally differentiated cells.

Accordingly, the stem cell-containing composition described herein can be used as an HDAC inhibitor for treating, for example, an immune disorder (e.g., an autoimmune disorder or inflammatory disorder), a neurodegenerative or neurological disorder, a viral infection, a cancer, or diabetes.

Autoimmune disorders or inflammatory disorders include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, ankylosing spondylitis, thrombocytopenic purpura, Hashimoto's thyroiditis, Graves' disease, Graver's disease, multiple sclerosis, inflammatory dermatoses, and inflammatory bowel diseases.

Neurodegenerative disorders include, for example, schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS).

A cancer to be treated can be, e.g., lymphoma (such as cutaneous T-cell lymphoma, peripheral T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or diffuse large B-cell lymphoma), lung cancer (such as non-small cell lung cancer or bronchial cancer), breast cancer, ovarian cancer, prostate cancer, colorectal cancer, multiple myeloma, liver cancer (such as hepatocellular carcinoma or intrahepatic cholangiocarcinoma), kidney cancer, gastric cancer, skin cancer (such as melanoma), thyroid cancer, esophageal cancer, brain cancer, pancreatic cancer, oral cancer, throat cancer (such as pharyngeal cancer or laryngeal cancer), cervical cancer, bone cancer, bladder cancer, leukemia, or carcinoma in situ.

A virus infection (or a disease caused by a virus infection) can be an HIV infection, acquired immunodeficiency syndrome (AIDS), herpesvirus infection (e.g., HCMV, HSV, or EBV infection), or HBV infection.

The stem cell-containing composition can be used to treated type 1 diabetes (or called diabetes mellitus type 1) or type 2 diabetes (or called diabetes mellitus type 2). A subject to be treated for one of the above-mentioned diseases or disorders can be identified by standard diagnosing techniques for that particular disease or disorder.

"Treating" refers to administration of a composition, agent or substance (e.g., a stem-cell containing solution) to a human subject, who is suffering from or is at risk for developing that disease or disorder, with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disease or disorder, the symptom of the disease or disorder, the disease state secondary to the disease or disorder, or the predisposition to the disease or disorder. An "effective amount" refers to an amount of the composition, agent or substance 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 therapies.

Either before or after administration (or both) of the stem cell-containing composition to a subject, HDAC activity in a sample (e.g., a peripheral blood sample or a sample of a tissue affected by the disease being treated) obtained from the subject can be assessed. For example, HDAC activity in a nuclear extract or HDAC proteins prepared from the sample can be assayed. A decrease in HDAC activity (e.g., by at least 20%, 30%, 40%, 50%, 60%, 70% or more) post administration indicates that the stem cell-containing solution is effective for inhibiting HDAC activity in the subject. A decrease in HDAC activity can also indicate that the treatment is effective for treating one of the disorders mentioned above. The level of HDAC activity in the subject after an administration of the stem cell-containing composition or during a course of administrations can also be used to make treatment decisions, e.g., to continue or discontinue with the treatment or to determine frequency of the treatment.

Techniques and reagents for assaying HDAC activity are known in the art. HDAC activity assay kits are also commercially available.

The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications cited herein are herein incorporated by reference in their entirety. EXAMPLES

Example 1 : Effects of stem cell mobilizing agents

Stem cell-containing solutions were prepared from peripheral blood samples obtained from six human subjects using the procedure described above. Also see Fig. 1 and Fig. 3.

Human subjects C, L, M, W, and Y each orally ingested 20 pills of the brown algae supplement described in Fig. 2. In other words, each of subjects C, L, M, W, and Y orally ingested at least 2 grams of fucoidan. Human subject P served as a control and did not ingest the brown algae supplement. Samples were drawn before and after the subjects ingested the pills.

As shown in Fig. 4, a significant increase in the number of SB cells (i.e., CD349(+)

SB cells and Lgr5(+) SB cells) was found at 1.5 hours after ingestion of the brown algae supplement in four subjects. No increase was found in the control subject. The data demonstrate that ingesting fucoidan, e.g., a brown algae supplement, can mobilize SB cells (e.g., CD349(+) SB cells and Lgr5(+) SB cells) into the peripheral blood (or the bloodstream) and enrich the peripheral blood with SB cells. In Fig. 4, 0 hr represents "before the ingestion of fucoidan" for human subjects C, L, M, W, and Y and also represents "at the beginning of a control test" for the human subject P; 1.5 hr represents "at 1.5 hours after the ingestion of fucoidan" for human subjects C, L, M, W, and Y and also represents "at 1.5 hours after the beginning of the control test" for human subject P; 24 hr represents "at 24 hours after the ingestion of fucoidan" for human subjects C, L, M, W, and Y and also represents "at 24 hours after the beginning of the control test" for human subject P.

The effect of GCSF on the mobilization of SB cells was also assessed. A human subject was injected a single dose of 5 micrograms/kg/day of GCSF for 5 consecutive days. A first peripheral blood sample was obtained from the subject before the first injection and a second peripheral blood sample was obtained from the subject 3.5 hours after the last injection. A significant increase in the number of Lgr5+ SB cells was found in the second sample as compared to the first sample.

Flow cytometry data of the two samples are shown in Figs. 5A and 5B. The black points in region Q5-LR in both figures represent Lgr5(+) cells. With respect to the first peripheral blood sample, as shown in Fig. 5 A, the number of Lgr5(+) cells in region Q5-LR is 1.6% of the total number of cells in regions Q5-UL, Q5-UR, Q5-LL, and Q5-LR. With respect to the second peripheral blood sample, as shown in Fig. 5B, the number of Lgr5(+) cells in region Q5-LR is 8% of the total number of cells in regions Q5-UL, Q5-UR, Q5-LL, and Q5-LR. Therefore, GCSF could mobilize SB cells (especially Lgr5(+) cells) into the peripheral blood (or the bloodstream) and enrich the peripheral blood with SB cells.

The above-described data demonstrated that ingesting a brown algae supplement or receiving a course of GCSF injections can mobilize SB cells into the peripheral blood of a subject.

Example 2: Cells in a stem cell-containing solution

A stem cell-containing solution was prepared from a peripheral blood sample obtained from a subject according to the method described above and illustrated in Fig. 1 and Fig. 3. The cell content of the stem cell-containing solution was analyzed.

The number of red blood cells per milliliter of the stem-cell containing solution was calculated based on the data shown in Figs. 6A and 6B. Fig 6A was obtained by analyzing 10 microliters (μΐ) of the stem-cell containing solution with a flow cytometer. Red blood cells (i.e., CD235a(+) cells) are shown in region R3 of Fig. 6A; the number of all cells in region R3 of Fig. 6A is 25000. Fig. 6B shows the distribution of CD235a fluorescence intensity in all cells in region R3 of Fig. 6A. In Fig. 6B, the fluorescence histogram is separated into region V3-L with low fluorescence intensity and region V3-R with high fluorescence intensity by vertical line 2a (i.e., reference). Region V3-R of Fig. 6B represents cells stained positive for CD235a, i.e., red blood cells; region V3-L of Fig. 6B represents cells stained negative for CD235a. The result of Fig. 6B indicates that the percentage of the number of red blood cells in region R3 of Fig. 6A to the number of all cells in region R3 of Fig. 6 A is 0.1%. By multiplying the number of all cells in region R3, i.e., 25000, by the percentage of the number of red blood cells in region R3 of Fig. 6A to the number of all cells in the region R3 of Fig. 6A, i.e., 0.1%, the number of red blood cells in 10 microliters of the stem-cell containing solution was found to be 25. The number of red blood cells per milliliter of the stem-cell containing solution was calculated from the number of red blood cells in 10 microliters of the stem-cell containing solution and found to be equal to 2500.

The number of white blood cells per milliliter of the stem-cell containing solution was calculated based on the flow cytometry data in Fig. 6C. The flow cytometry data in Fig. 6C was obtained by analyzing 50 microliters (μΐ) of the stem-cell containing solution. In Fig.

6C, region Rl is white-blood-cell (WBC) gating. Based on the flow cytometry data in Fig. 6C, the number of white blood cells in 50 microliters of the stem-cell containing solution was calculated and found to be equal to 30. Therefore, the number of white blood cells per milliliter of the stem-cell containing solution was calculated from the number of white blood cells in 50 microliters of the stem-cell containing solution and found to be equal to 600.

5 Fig. 7A shows a forward scattering (FSC) versus side scattering (SSC) flow

cytometry dot plot, which was obtained by analyzing 5.6 microliters (μΐ) of the stem-cell containing solution with a flow cytometer. In Fig. 7A, region R5 represents cells that are less than 6 micrometers and greater than 2 micrometers in size. In other words, all cells in region R5 are greater than 2 micrometers and less than 6 micrometers in size. Fig. 7B shows the o distribution of CD61 fluorescence intensity in all cells in region R5 of Fig. 7 A. In Fig. 7B, the fluorescence histogram is separated into region VI -L with low fluorescence intensity and region Vl-R with high fluorescence intensity by a vertical line (i.e., reference). Region Vl-R of Fig. 7B represents cells stained positive for CD61, i.e., platelets; region Vl-L of Fig. 7B represents cells stained negative for CD61. The result of Fig. 7B indicates that the number of5 platelets in region R5 80.5% all cells in region R5.

Fig. 7C shows the distribution of CD 133 fluorescence intensity in all cells region R5 of Fig. 7 A. In Fig. 7C, the fluorescence histogram is separated into region V3-L with low fluorescence intensity and region V3-R with high fluorescence intensity by a vertical line (i.e., reference). Region V3-R of Fig. 7C represents cells stained positive for CD133, i.e., VSELs; o region V3-L of Fig. 7C represents cells stained negative for CD133. The result of Fig. 7C indicates that the percentage of the number of CD133(+) cells in region R5 is 0.3% of the number of all cells in region R5. Fig. 7D shows the distribution of CD34 fluorescence intensity in all cells in region R5 of Fig. 7A. In Fig. 7D, the fluorescence histogram is separated into region Vl-L with low fluorescence intensity and region Vl-R with high

5 fluorescence intensity by a vertical line (i.e., reference). Region Vl-R of Fig. 7D represents cells stained positive for CD34, i.e., VSELs; region Vl-L of Fig. 7D represents cells stained negative for CD34. The result of Fig. 7D indicates that the number of CD34(+) cells in region R5 is 0.4% of the number of all cells in the region.

Fig. 7E shows the distribution of CD66e fluorescence intensity in all cells in region 0 R5 of Fig. 7A. In Fig. 7E, the fluorescence histogram is separated into region V2-L with low fluorescence intensity and region V2-R with high fluorescence intensity by a vertical line (i.e., reference). Region V2-R of Fig. 7E represents cells stained positive for CD66e, i.e., BLSCs; region V2-L of Fig. 7E represents cells stained negative for CD66e. The result of Fig. 7E indicates that the number of CD66e(+) cells in region R5 is 4% of all cells in the region.

Fig. 7F shows the distribution of CD349 fluorescence intensity in all cells in region R5 of Fig. 7 A. In Fig. 7F, the fluorescence histogram is separated into region V3-L with low fluorescence intensity and region V3-R with high fluorescence intensity by a vertical line (i.e., reference). Region V3-R of Fig. 7F represents cells stained positive for CD349, i.e., SB-1 cells; region V3-L of Fig. 7F represents cells stained negative for CD349. The result of Fig. 7F indicates that the number of CD349(+) cells in region R5 is 5.6% of the number of all cells in the region.

Fig. 7G shows the distribution of Lgr5 fluorescence intensity in all cells in region R5 of Fig. 7A. In Fig. 7G, the fluorescence histogram is separated into region V2-L with low fluorescence intensity and region V2-R with high fluorescence intensity by a vertical line (i.e., reference). Region V2-R of Fig. 7G represents cells stained positive for Lgr5, i.e., SB-2 cells; region V2-L of Fig. 7G represents cells stained negative for Lgr5. The result of Fig. 7G indicates that the number of Lgr5(+) cells in region R5 is 5.4% of all cells in the region.

Example 3: A stem cell-containing solution for inhibiting HDAC activity

Stem cell-containing solutions were prepared from peripheral blood samples obtained from ten human subjects. The subjects each ingested 20 pills of the brown algae supplement described in Fig. 2. A 150 milliliters peripheral blood sample was drawn from each subject 1.5 hours after ingestion. 20 milliliters of each blood sample (hereinafter the "20-milliliter blood sample") was analyzed by a flow cytometer to obtain the number of specific somatic stem cells in the sample. The specific somatic stem cells were small somatic stem cells greater than 2 micrometers and less than 6 micrometers in size including Lgr5(+) somatic stem cells, CD349(+) somatic stem cells, CD66e(+) somatic stem cells, CD133(+) somatic stem cells, and CD34(+) somatic stem cells. The rest of each peripheral blood sample was processed according to the procedure described above to obtain a stem cell- containing solution of about 65 milliliters. Each stem cell-containing solution contained the small somatic stem cells. The number of somatic stem cells in each stem cell-containing solution was calculated or estimated based on the number of somatic stem cells in the corresponding 20-milliliter blood sample. Each stem cell-containing solution was mixed with 500 milliliters of saline. The ten human subjects were each injected intravenously with his or her own stem cell containing- solution. Table 1 shows the number of somatic stem cells in each of the stem-cell containing solutions.

Table 1

HDAC activity in purified nucleated cells obtained from a peripheral blood sample of each of the ten human subjects was assayed. An average HDAC activity was calculated from two duplicates for each subject. As shown in Table 2 below, a significant decrease (at least 22%) in HDAC activity in each subject was found at 48 hours after intravenous injection of the stem cell-containing solution.

Fig. 8 is a scatter plot of the average HDAC activities shown in Table 2 with a linear regression line. As shown in Fig. 8, the coefficient of determination (i.e., R²) between an average HDAC activity before intravenous injection of the stem cell-containing solution and the corresponding percentage of decrease in the HDAC activity at 48 hours after the injection is as large as 0.7346, which suggests that the relationship between the two variables is very strong. The plot shows that the higher the HDAC activity before the injection, the higher the percentage of decrease in the HDAC activity at 48 hours after the injection.

The above-described data indicate that the stem cell-containing solution can significantly decrease HDAC activity in a human subject. Table 2

Example 4: A stem cell-containing solution for inhibiting HDAC activity in patients

Patients each with a neurodegenerative disorder, an autoimmune disorder, or diabetes 5 were treated with their own stem cell-containing solutions prepared using the procedure described above.

For each patient, HDAC activity and clinical assessment were obtained before and after the treatment. The results are shown in Tables 3, 4, and 5 below. The results demonstrate that, after administration of the stem cell-containing solution, there were a l o decrease in HDAC activity and a corresponding improvement in the patient's clinical

assessment.

Table 3

Patient number Disease Mobility/energy HDAC (RFU/min/ug)

Before After Before After

080-02 Dementia 2 3 151.35 48.325

0134-01 Parkinson's 6 8 258.5972 110.3889

0144-01 Parkinson's 4 6 273.3639 189.5944

0157-01 Parkinson's 3 4 282.3944 207.3667 Table 4

Table 5

5

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated l o otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the described embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments to adapt it to 15 various usages and conditions. Thus, other embodiments are also within the claims.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting a histone deacetylase activity, comprising administering to a recipient subject in need thereof a composition that contains an effective amount of small somatic stem cells that are greater than 2 micrometers and less than 6 micrometers in size, wherein the small somatic stem cells include CD349(+) somatic stem cells and Lgr5(+) somatic stems cells.

2. The method of claim 1, further comprising determining a histone deacetylase activity in the recipient subject before the administering step.

3. The method of claim 2, further comprising determining a histone deacetylase activity in the recipient subject after the administering step.

4. The method of claim 1, further comprising determining a histone deacetylase activity in the recipient subject before and after the administering step.

5. The method of claim 4, wherein the determining step is performed by measuring a histone deacetylase activity in nucleated cells in a peripheral blood sample obtained from the recipient subject.

6. The method of any of claims 1-5, wherein the composition is an injectable solution that is administered intravenously.

7. The method of claim 6, wherein the composition contains 10 million to 500 million of the small somatic stem cells.

8. The method of claim 7, wherein the composition contains a divalent cation chelating agent.

9. The method of claim 8, wherein the injectable solution is prepared by a procedure including:

providing a mixture that contains a blood sample and a divalent cation chelating agent;

storing the mixture at a temperature between 2°C and 12°C for 3 to 72 hours, whereby the mixture separates into an upper layer and a lower layer, wherein the upper layer contains the small somatic stem cells; and

collecting the upper layer, whereby the injectable solution is prepared.

10. The method of claim 9, wherein the blood sample is obtained from the recipient subject or a donor subject.

11. The method of claim 10, wherein, prior to obtaining the blood sample, an action for increasing stem cell number is performed on the recipient subject or donor subject.

12. The method of claim 11, wherein the action is administration of fucoidan or a granulocyte-colony stimulating factor.

13. The method of claim 9, wherein 1.5 to 2.0 mg of divalent cation chelating agent per millimeter of the blood sample is added the blood sample to obtain the mixture.

14. The method of claim 13, wherein the divalent cation chelating agent is ethylenediaminetetraacetic acid (EDTA).

15. The method of claim 9, wherein the procedure for preparing the injectable solution further includes, after collecting the upper layer, adding a pharmaceutically acceptable excipient to the collected upper layer.

16. The method of claim 15, wherein the pharmaceutically acceptable excipient is a saline solution.

17. The method of claim 6, wherein the small somatic stem cells include CD133(+) cells, CD34(+) cells, and CD66e(+) cells.

18. The method of claim 6, wherein the recipient subject has an autoimmune 5 disorder, diabetes, cancer, neurodegenerative disorder, or virus infection.

19. The method of claim 18, wherein the recipient subject has dementia, Parkinson's disease, arthritis, ankylosing spondylitis, or diabetes. l o

20. An injectable composition for use in decreasing a histone deacetylase activity in a subject, wherein the composition contains an effective amount of small somatic stem cells that are greater than 2 micrometers and less than 6 micrometers in size, the small somatic stem cells including CD349(+) somatic stem cells and Lgr5(+) somatic stems cells.

15 21. The injectable composition of claim 20, wherein the subject has an

autoimmune disorder, diabetes, cancer, neurodegenerative disorder, or virus infection

22. The injectable composition of claim 21, wherein the subject has dementia, Parkinson's disease, arthritis, ankylosing spondylitis, or diabetes.

20