阅读说明：本技术 成体多能干细胞的制备、扩增及应用 (Preparation, amplification and application of adult pluripotent stem cells ) 是由 李劭伟 胡敏 赫尔曼·彼得·洛伦兹 于 2018-04-03 设计创作，主要内容包括：存在于哺乳动物的外周血中的某些相对较小的细胞能够被活化以形成多能干细胞群。这些小细胞通常直径小于5μm,并且是CD45阳性的,在本文中称为CD45<Sup>+</Sup>细胞或休眠的微小细胞。因此,提供了含有来自血液样品的富集的休眠的微小细胞的细胞群和组合物、以及活化这些休眠的微小细胞的方法和组合物。分化后,活化的干细胞能够被用于各种治疗目的。(Certain relatively small cells present in the peripheral blood of mammals can be activated to form pluripotent stem cell populations. These minicells are typically less than 5 μm in diameter and are CD45 positive, referred to herein as CD45+ cells or resting minicells. Thus, cell populations and compositions containing enriched quiescent minicells from a blood sample, and methods and compositions for activating these quiescent minicells, are provided. After differentiation, the activated stem cells can be used for various therapeutic purposes.)

1. A composition comprising at least 1000 cells, wherein at least 50% of the cells are CD45+ cells that express CD45 and are less than 5 μm in diameter.

2. The composition of claim 1, wherein the CD45+ cells are further characterized as being CD34 positive and ABCG2 negative.

3. The composition of claim 1 or 2, wherein at least 75% of the cells are CD45+ cells.

4. The composition of claim 1 or 2, wherein at least 95% of the cells are CD45+ cells.

5. The composition of any one of claims 1-4, comprising at least 10,000 cells.

6. The composition of any one of claims 1-5, wherein less than 20% of the cells are red blood cells.

7. The composition of any one of claims 1-6, wherein the CD45+ cells are less than 3 μ ι η in diameter.

8. The composition of any one of claims 1-6, wherein the CD45+ cells are about 2 μ ι η to about 3 μ ι η in diameter.

9. The composition of any one of claims 1-8, wherein the CD45+ cells are further characterized as Lin negative.

10. The composition of any one of claims 1-9, wherein the CD45+ cells comprise small RNAs and ribosomal RNAs, wherein the ratio of small RNAs to ribosomal RNAs is at least about 20: 1. or at least about 15: 1. or at least about 10: 1. or at least about 5: 1.

11. The composition of any one of claims 1-10, wherein the CD45+ cells have a nucleoplasmic ratio (v/v) of at least about 9: 1. or at least about 8: 1. or at least about 7: 1. or at least about 6: 1. or at least about 5: 1.

12. The composition of any one of claims 1-11, wherein the CD45+ cells further express one or more markers selected from the group consisting of: CD44, CD150, Sca1, c-kit, Thy1.1(CD90.1), Oct4, SSEA1, Nanog, Vasa, CD133, and CD 105.

13. The composition of any one of claims 1-12, wherein the CD45+ cells do not express one or more markers selected from the group consisting of: CD41, Lin, E-cadherin, and CD 184.

14. The composition of any one of claims 1-13, wherein the CD45+ cells are capable of being activated by a medium comprising one or more factors, wherein the activated cells express ABCG 2.

15. A method of culturing cells, comprising: culturing a plurality of mammalian cells in a culture medium that is in contact with or has been conditioned by at least one selected from the group consisting of primary hepatocytes, human hepatoblastoma (HepG 2) cells, hepatocyte cell lines, and Mouse Embryonic Fibroblast (MEF) cell lines.

16. The method of claim 15, wherein the cells (a) are less than 5 μ ι η in diameter, and (b) express CD 45.

17. The method of claim 15, wherein the cell is further characterized as being CD34 positive and ABCG2 negative.

18. The method of claim 17, wherein the cell expresses ABCG2 after culture.

19. The method of claim 18, wherein the culturing is for at least 1 hour.

20. The method of claim 15, wherein the mammalian cells comprise at least one cell selected from the group consisting of: embryonic Stem (ES) cells, Hematopoietic Stem Cells (HSCs), Mesenchymal Stem Cells (MSCs), Endothelial Stem Cells (ESCs), mammary stem cells (mascs), Intestinal Stem Cells (ISCs), Neural Stem Cells (NSCs), adult Olfactory Stem Cells (OSCs), Neural Crest Stem Cells (NCSCs), Testicular Stem Cells (TSCs), and induced pluripotent stem cells (ipscs).

21. The method of any one of claims 15-20, wherein the hepatocyte cell line comprises AML12, HepaRG, or a combination thereof.

22. The method of any one of claims 15-21, wherein the culture medium is in contact with or has been conditioned by at least primary hepatocytes, HepG2 cells, a hepatocyte cell line, and a MEF cell line.

23. The method of any one of claims 15-21, wherein the culture medium is in contact with or has been conditioned by at least primary hepatocytes, HepG2 cells, and a hepatocyte cell line.

24. The method of any one of claims 15-21, wherein the culture medium is in contact with or has been conditioned by at least primary hepatocytes, HepG2 cells, and MEF cell lines.

25. The method of any one of claims 15-21, wherein the culture medium is in contact with or has been conditioned with at least primary hepatocytes, a hepatocyte cell line and a MEF cell line.

26. The method of any one of claims 15-21, wherein the culture medium is contacted with or has been conditioned with at least HepG2 cells, a hepatocyte cell line and a MEF cell line.

27. The method of any one of claims 15-21, wherein the culture medium is in contact with or has been conditioned by at least primary hepatocytes and HepG2 cells.

28. The method of any one of claims 15-21, wherein the culture medium is at least in contact with or has been conditioned by primary hepatocytes and a hepatocyte cell line.

29. The method of any one of claims 15-21, wherein the culture medium is in contact with or has been conditioned with at least primary hepatocytes and a MEF cell line.

30. The method of any one of claims 15-21, wherein the culture medium is in contact with or has been conditioned by at least HepG2 cells and a hepatocyte cell line.

31. The method of any one of claims 15-21, wherein the culture medium is contacted with or has been conditioned with at least HepG2 cells and MEF cell line.

32. The method of any one of claims 15-21, wherein the culture medium is in contact with or has been conditioned with at least a hepatocyte cell line and a MEF cell line.

33. The method of any one of claims 15-21, wherein the culture medium is at least in contact with or has been conditioned by primary hepatocytes.

34. The method of any one of claims 15-21, wherein the culture medium is at least in contact with or has been conditioned by HepG2 cells.

35. The method of any one of claims 15-21, wherein the culture medium is at least in contact with or has been conditioned with a hepatocyte cell line.

36. The method of any one of claims 15-21, wherein the culture medium is at least in contact with or has been conditioned with a MEF cell line.

37. The method of any one of claims 15-21, wherein the culture medium is contacted with or has been conditioned by at least two selected from the group consisting of primary hepatocytes, HepG2 cells, hepatocyte cell lines, and MEF cell lines, simultaneously or sequentially.

38. An isolated mammalian cell that expresses CD34 and ABCG2, but does not express CD 45.

39. The isolated mammalian cell of claim 38, further characterized as Oct4, Nanog and/or keratin epithelium positive, and Lin negative.

40. The isolated mammalian cell of claim 38 or 39, further expressing one or more markers selected from the group consisting of: CD117, CD29, CD44, CD73, CD90, CD105, Sca1, CD31, CD184, nestin and osteocalcin.

41. The isolated mammalian cell of claim 38 or 39, further expressing at least 2 markers selected from the group consisting of: CD117, CD29, CD44, CD73, CD90, CD105, Sca1, CD31, CD184, nestin and osteocalcin.

42. The isolated mammalian cell of claim 38 or 39, further expressing at least 3 markers selected from the group consisting of: CD117, CD29, CD44, CD73, CD90, CD105, Sca1, CD31, CD184, nestin and osteocalcin.

43. The isolated mammalian cell of claim 38 or 39, further expressing at least 4 markers selected from the group consisting of: CD117, CD29, CD44, CD73, CD90, CD105, Sca1, CD31, CD184, nestin and osteocalcin.

44. The isolated mammalian cell of any one of claims 38-43, wherein the cell does not express one or more markers selected from the group consisting of: CD3, CD4, CD8a, CD11b, CD13, CD140a, E-cadherin, and Lin.

45. The isolated mammalian cell of any one of claims 38-44, wherein the cell is Lin-.

46. The isolated mammalian cell of any one of claims 38-45, wherein the cell develops into a colony after being cultured in a medium comprising one or more factors for a period of time.

47. A population of cells of any one of claims 38-46.

48. A composition comprising the cell of any one of claims 38-46.

49. A stem cell derived from cells that (a) are less than 5 μ ι η in diameter, b) express CD45, and (c) do not express ABCG 2; wherein the stem cell expresses ABCG2 and is further characterized as (a) CD34+/CD45+, (b) CD34+/CD45-, (c) CD34-/CD45+, or (d) CD34-/CD 45-.

50. A composition comprising the stem cell of claim 49 in a pharmaceutically acceptable carrier or excipient.

51. A cell differentiated from the stem cell of claim 49.

52. A composition comprising the differentiated cells of claim 51 and a pharmaceutically acceptable carrier or excipient.

53. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the composition of claim 50 or 51.

54. The method of claim 53, wherein the disease or disorder is selected from: degenerative diseases, proliferative diseases, genetic diseases, injuries and organ failure.

Drawings

FIG. 1 shows an electron microscope image of dormant minute cells (dormant tiny cells) isolated from blood.

FIG. 2 is a graph showing nuclear staining for up to 7 days of resting minicells in vitro and activated stem cells derived therefrom, and a graph of GFP expression in cells at day 7.

Figure 3A is a graph showing ABCG2 expression in quiescent minicells and activated stem cells derived therefrom.

Figure 3B is a diagram showing a population of cells expressing CD34 and CD45 isolated and characterized as resting minicells.

FIG. 4 is a graph showing the ratio of sRNA to rRNA in quiescent minicells and activated stem cells derived therefrom, as compared to a hepatocyte cell line as a control.

FIG. 5 is a graph showing growth and expansion of quiescent minicells and stem cell colony formation in the activation/development system.

FIG. 6 is a diagram showing in vitro cell culture of purified CD34+/CD 45-stem cell populations from stem cell colonies.

Figure 7 is a diagram showing a purified stem cell population that expresses the CD34 marker but does not express CD 45.

FIG. 8 is a diagram showing that purified CD34+/CD 45-cell population is capable of differentiating and expressing neural cell specific markers.

FIG. 9 is a diagram showing that purified CD34+/CD 45-cell population is capable of expressing epidermal markers.

FIG. 10 is a graph showing that the purified CD34+/CD 45-cell population is capable of differentiating and expressing bone tissue specific staining.

FIG. 11 is a diagram showing that the purified CD34+/CD 45-cell population is capable of differentiating and expressing cardiomyocyte-specific markers.

FIG. 12 is a graph showing that the purified CD34+/CD 45-cell population is capable of differentiating and expressing hepatocyte and cholangiocyte epithelial-specific markers.

FIG. 13 is a graph showing faster rates of skin wound healing in wild type mice when treated with a purified CD34+/CD 45-cell population.

Figure 14 is a graph showing the growth of new functional skin in treated mice compared to scar tissue in control mice.

FIG. 15 is a graph showing faster rates of skin wound healing in diabetic mice when treated with purified CD34+/CD 45-cell populations.

Fig. 16 is a graph showing the growth of new skin after 16 days of treatment in diabetic mice compared to the barely closed wound in control mice.

FIG. 17 is a graph showing growth of bone tissue derived from a purified CD34+/CD 45-cell population.

FIG. 18 is a graph showing the repair of liver damage caused by CCl4 following transplantation of a purified CD34+/CD 45-cell population.

FIG. 19 is a diagram showing another cell culture of resting minicells isolated from blood co-cultured with an activation/development system.

FIG. 20 is a graph showing expression of hepatocyte-specific markers in stem cells co-cultured with an activation/development system.

FIG. 21 is a diagram showing treatment of liver injury using stem cells obtained from a co-culture system.

Fig. 22 is a graph showing expression of proliferation markers, growth factors, and cytokines of activated stem cells.

Detailed Description

Throughout this disclosure, various publications, patents, and published patent specifications are cited. The disclosures of these publications, patents and published patent specifications are incorporated by reference into this disclosure in their entirety.

Before the compositions and methods are described, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention, and is in no way intended to limit the scope of the present invention as set forth in the appended claims.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Russell et al (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the services Autosubel et al, eds. (2007) Current Protocols in Molecular Biology; the services Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al (1991) PCR 1: A Practical application (IRL Press at Oxford University Press); MacPherson et al (1995) PCR 2: A Practical access; Harlow and Lane Numbers (1999) Antibodies, A Laboratory Manual; Free et al (1982005) Culture of filtration Nucleic acids, 1984; filtration experiments, 1984; filtration Protocols, 1984; filtration experiments, 2; filtration Nucleic acids, 1984; filtration experiments, 2; filtration Nucleic acids, 1984; filtration Nucleic acids, 2; filtration Nucleic acids, 2, filtration Nucleic acids, 2; filtration Nucleic acids, 2, filtration Nucleic acids, 2; filtration Nucleic acids, 2, filtration Nucleic acids (1984) A Practical Guide to Molecular Cloning; Miller and calcium eds (1987) Gene Transfer Vectors for Mammarian Cells (Cold Spring Harbor Laboratory), Makrides ed. (2003) Gene Transfer and Expression In Mammarian Cells; Mayer and Walker eds (1987) biochemical Methods In Cells and Molecular Biology (Academic Press, London), Herzenberg et al (1996) We's Handbook of Experimental Immunology; Manipulating the Molecular organism Embryo: A Laboratory Manual, 3rd (carbohydrate) (Hartwig Laboratory), et al (carbohydrate), et al, Molecular analysis J (1987) PCR; experiment J, et al, Molecular analysis, et al (carbohydrate), Molecular analysis, et al, PCR, Molecular analysis, et al (1987) PCR, Molecular analysis, et al, Molecular analysis, PCR, et al, 3rd (carbohydrate), sample analysis, et al, carbohydrate, et al (1987), eds. (1988) Antibodies, A Laboratory Manual; Harlow and Lane, eds. (1999) Using Antibodies, A Laboratory Manual; Animal Cell Culture (R.I. Freshney, ed. (1987)); Zigova, Sanberg and Sanchez-Ramos, eds. (2002) Neural Stem Cells.

All numerical designations (e.g., pH, temperature, time, concentration, and molecular weight), including ranges, are approximations that vary ((+) or (-), as appropriate, in increments of 0.1 or 1. It is to be understood that all numerical designations are preceded by the term "about," although this is not always explicitly stated. The term "about" also includes, where appropriate, the precise value of "X" as well as small increments of "X", such as "X + 0.1 or 1" or "X-0.1 or 1". It is also to be understood that, although not always explicitly stated, the reagents described herein are exemplary only and equivalents thereof are known in the art.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the following terms have the following meanings.

As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of cells, including mixtures thereof.

As used herein, the term "comprising" or "containing" is intended to mean that the compositions and methods include the recited elements, but not exclude other elements. When used to define compositions and methods, "consisting essentially of … …" is meant to exclude other elements that have any significance to the combination for the purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics claimed. "consisting of … …" refers to trace elements and substantial process steps excluding other components. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

As used herein, the term "isolated" means separated from cellular and other components in which cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are generally related in nature. For example, an isolated polynucleotide is separated from 3 'and 5' contiguous nucleotides to which it is normally associated in its natural or native environment (e.g., on a chromosome). It will be apparent to those skilled in the art that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof need not be "isolated" to distinguish them from their naturally occurring counterparts. An isolated cell is a cell that is isolated from a tissue or cell of a dissimilar phenotype or genotype.

As used herein, "stem cells" define cells that are capable of dividing indefinitely in culture and producing specialized cells. Non-limiting examples of types of stem cells include somatic (adult) stem cells, embryonic stem cells, parthenogenetic stem cells (see Cibelli et al (2002) Science 295(5556): 819; U.S. patent publication Nos. 20100069251 and 20080299091), and/or induced pluripotent stem cells (iPS cells or iPSCs). Somatic stem cells are undifferentiated cells found in differentiated tissues that are capable of self-renewal (cloning) and (with certain limitations) differentiation to produce all the specialized cell types of the tissue from which they originate. Embryonic stem cells are primitive (undifferentiated) cells from embryos that have the potential to become a variety of specialized cell types. Non-limiting examples of embryonic stem cells include: HES2 (also known as ES 02) cell line available from ESI, Singapore and H1 or H9 (also known as WA 01) cell line available from WiCell, Madison, WI. Other cell lines are awaiting NIH review. See, e.g., grads. nih. gov/stem cells/registry/current. htm (last access time 3 months 13 days 2017). Pluripotent embryonic stem cells can be distinguished from other types of cells by the use of markers including, but not limited to: oct-4, alkaline phosphatase, CD30, TDGF-1, GCTM-2, Genesis, reproductive nuclear factor, SSEA1, SSEA3 and SSEA 4. Induced pluripotent stem cells (ipscs) are artificially derived stem cells (typically adult somatic cells) derived from non-pluripotent cells, which are generated by inducing the expression of one or more stem cell-specific genes. Specific genes for iPSC expression include, but are not limited to: the octamer transcription factor family, such as Oct-3/4; sox gene families such as Sox1, Sox2, Sox3, Sox15 and Sox 18; the Klf gene family, such as Klf1, Klf2, Klf4, and Klf 5; the Myc gene family, such as c-Myc and L-Myc; nanog gene families such as Octamer-4 (OCT 4), NANOG, and REX 1; or LIN 28. Examples of iPSCs are described in Takahashi et al, (2007) Cell (published online early on at 11.20.2007), Takahashi & Yamanaka (2006) Cell 126: 663-76, Okita et al, (2007) Nature 448: 260-262, Yu et al, (2007) Science (published online early on at 11.20.2007), and Nakagawa et al, (2007) Nat. Biotechnol (published online early on at 11.30.2007).

As used herein, the term "propagating" means growing or altering the phenotype of a cell or group of cells. The term "growth" or "expansion" refers to the proliferation of cells in the presence of a support medium, nutrients, growth factors, support cells, or any chemical or biological compound necessary to obtain the desired number of cells or cell types. In one embodiment, the growth/expansion of the cells results in regeneration of the tissue.

As used herein, the term "culturing" refers to the in vitro propagation of cells or organisms on or in various media. It is understood that progeny of a cell grown in culture may not be identical (i.e., morphological, genetic, or phenotypic) to the parent cell. "expansion" refers to any proliferation or division of cells.

As used herein and as set forth in more detail below, a "conditioned medium" is a medium that is cultured with mature cells that provide the medium with cellular factors, such as cytokines, growth factors, hormones, extracellular matrix, and some materials that will promote cell growth, development, and differentiation.

As used herein, the term "differentiation" describes the process by which non-specialized cells obtain the characteristics of specialized cells (e.g., skin, heart, liver, or muscle cells). By "directed differentiation" is meant the manipulation of stem cell culture conditions to induce differentiation into a particular cell type. "dedifferentiation" defines cells that return to a less fixed (committed) location in the cell lineage. As used herein, the term "differentiated" defines cells that exhibit a more fixed ("differentiated") location within a cell lineage.

As used herein, the "lineage" of a cell defines the inheritance of the cell, i.e., its precursors and progeny. The lineage of the cells places the cells within a genetic program of development and differentiation. As used herein, "cells that differentiate into a mesodermal (or ectodermal or endodermal) lineage" defines cells that become respectively of a particular mesodermal (or ectodermal or endodermal) lineage. Examples of cells that differentiate into mesodermal lineages or give rise to particular mesodermal cells include, but are not limited to, the following: adipogenic, smooth muscle-forming, chondrogenic, cardiogenic, dermogenic, hematopoietic, angiogenic, myogenic, nephrogenic, urogenic, osteogenic, pericardial-derived, or stromal. Examples of cells that differentiate into ectodermal lineages include, but are not limited to, epidermal cells, neuronal cells, and glial cells. Examples of cells that differentiate into endodermal lineages include, but are not limited to, cells that produce pancreas, liver, lung, stomach, intestine, and thyroid.

As used herein, the term "pluripotent stem cell" refers to a cell having the following properties: (i) capable of unlimited proliferation in vitro in an undifferentiated state; (ii) maintaining normal karyotype by long-term culture; and (iii) retains the potential to differentiate into derivatives of all three embryonic germ layers (endoderm, mesoderm and ectoderm) even after prolonged culture. Non-limiting examples of currently available pluripotent stem cells include embryonic stem cells and ipscs. As used herein, the term "embryonic-like" stem cells refers to cells derived from tissues, organs or blood that have the pluripotent characteristics of embryonic stem cells.

As used herein, the term "multi-lineage stem cell" or "pluripotent stem cell" refers to a stem cell that proliferates itself and at least two further differentiated progeny cells from different developmental lineages. Lineages may be able to be from the same germ layer (i.e., mesoderm, ectoderm, or endoderm), or from different germ layers. Examples of two progeny cells with different developmental lineages that differentiate from a multi-lineage stem cell are myoblasts and adipocytes (both derived from mesoderm, but producing different tissues). Another example is neurogenic cells (derived from ectoderm) and adipocytes (derived from mesoderm).

As used herein, the term "self-regenerable" means that the cell is capable of self-renewal in multiple passages without significant changes in cell properties. In one aspect, the number of passages is at least about 5, or at least 10, or alternatively at least about 15, 20, 30, 50 or 100.

As used herein, the term "substantially homologous" describes a population of cells in which greater than about 50%, or greater than about 60%, or greater than 70%, or greater than 75%, or greater than 80%, or greater than 85%, or greater than 90%, or greater than 95%, or greater than 99% of the cells have the same or similar phenotype. The phenotype can be determined by pre-selected cell surface markers or other markers.

As used herein, the term "purified population" of cells of interest refers to a population of cells that has been isolated from substantially all other cells present in their natural environment, and also refers to a population of cells when the proportion of cells of interest in a mixture of cells is greater than that found in their natural environment. For example, a purified cell population represents an enriched population of cells of interest, even though other cells and cell types are present in the enriched population. In some embodiments, a purified cell population represents at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of a mixed cell population, provided that the "purified" population has a greater percentage of cells of interest in the total cell population than in the population prior to purification.

As used herein, the term "population of cells" refers to a collection of more than one phenotypically and/or genotypically identical (clonal) or non-identical cells.

As used herein, the term "cell colony" or "colony" refers to a population of closely related cells formed as a result of cell growth. These terms are used regardless of the number of cells that make up the colony.

As used herein, the term "quiescent cells" is intended to encompass cells that are in a quiescent or quiescent state, which need to be activated before they are able to undergo growth or differentiation.

As used herein, the term "quiescent minicells" is intended to encompass cells in a quiescent or quiescent state, which need to be activated before they are able to undergo growth and/or differentiation. Dormant minicells are typically less than 5 μm in diameter.

As used herein, the term "activation" of a dormant cell refers to a measurable morphological, phenotypic, and/or functional change in the dormant state of the cell. This activation usually occurs simultaneously with the expression of specific markers of the activated cells. In one embodiment, activation occurs simultaneously with a change in cell growth and/or development.

As used herein, the term "activated stem cell" is intended to encompass a stem cell that is in an activated state and is capable of undergoing growth and/or differentiation under specific conditions.

As used herein, the term "composition" is intended to encompass a combination of an active agent and another carrier (e.g., a compound or composition, inert (e.g., a detectable agent or label) or active (e.g., an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc.)). The carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides and oligosaccharides; derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, including in the range of 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumin (e.g., Human Serum Albumin (HSA), recombinant human albumin (rHA)), gelatin, casein, and the like. Representative amino acid/antibody components (which can also serve as buffering agents) include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also included within the scope of the present invention, examples of which include, but are not limited to: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and inositol. In certain embodiments, the composition comprises a population or mixture of cells. In certain embodiments, the composition is formulated as a film, gel, patch, 3-D structure, or liquid solution.

As used herein, the term "pharmaceutically acceptable" means that the indicated material is not of a nature that would cause a reasonably prudent physician to avoid administration of the material to a patient, taking into account the disease or condition to be treated and the corresponding route of administration. For example, it is often desirable that such materials be substantially sterile.

As used herein, the term "pharmaceutically acceptable carrier (or culture medium)", which is used interchangeably with the term "biocompatible carrier (or culture medium)", means that the agents, cells, compounds, materials, compositions and/or dosages in such forms are not only compatible with the cells and other drugs being administered therapeutically, but are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers suitable for the present invention include liquids, semi-solids (e.g., gels), and solid materials (e.g., cell scaffolds and matrices, tube sheets, and other such materials known in the art and described in more detail herein). These semi-solid and solid materials can be designed to resist degradation in vivo (non-biodegradable), or they can be designed to degrade in vivo (biodegradable, bioerodible). The biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into body fluids (a water-soluble implant is one example), or may be broken down and eliminated by conversion to other materials or by natural pathways. For topical use, the pharmaceutically acceptable carrier is suitably formulated as a cream, ointment, pectin, gel, solution, suspension, or the like. These carriers are conventional in the art, for example, topical application of polyethylene glycol (PEG). These formulations may optionally comprise additional pharmaceutically acceptable ingredients, for example, diluents, stabilizers, and/or adjuvants.

As used herein, the term "solution" refers to solutions, suspensions, emulsions, drops, ointments, liquid washes, sprays, and liposomes, which are well known in the art. In some embodiments, the liquid solution contains an aqueous pH buffer that resists changes in pH when small amounts of acid or base are added.

As used herein, the term "pH buffer" refers to an aqueous buffer solution that resists changes in pH when small amounts of acid or base are added thereto. The pH buffered solution typically comprises a mixture of a weak acid and its conjugate base, and vice versa. For example, the pH buffered solution may comprise: phosphates such as sodium phosphate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate; boric acid and borates such as sodium borate and potassium borate; citric acid and citrates, e.g., sodium citrate and disodium citrate; acetates such as sodium acetate, potassium acetate; carbonates such as sodium carbonate and sodium bicarbonate, etc. The pH adjusting agent may include: for example, acids such as hydrochloric acid, lactic acid, citric acid, phosphoric acid, and acetic acid; and bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and the like. In some embodiments, the pH buffering agent is a Phosphate Buffered Saline (PBS) solution (i.e., containing sodium phosphate, sodium chloride, and in some formulations, potassium chloride and potassium phosphate).

As used herein, the term "formulation" or "preparation" refers to the process of combining different substances, including one or more pharmaceutically active ingredients, to produce a dosage form. In certain embodiments, two or more pharmaceutically active ingredients can be co-formulated into a single dosage form or combined dosage unit, or formulated separately and subsequently combined into a combined dosage unit. A sustained release formulation is a formulation designed to slowly release the therapeutic agent in vivo over an extended period of time, while an immediate release formulation is a formulation designed to rapidly release the therapeutic agent in vivo over a shortened period of time.

As used herein, the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, arresting, and/or stopping one or more clinical symptoms of a disease or disorder before, during, and/or after injury or intervention.

As used herein, the term "patient" or "subject" refers to an animal, including mammals, e.g., murine, canine, equine, bovine, simian, or human, that is treated with a pharmaceutical composition or according to the methods described herein.

As used herein, the term "delivery" refers to routes, methods, formulations, techniques and systems for transporting a pharmaceutical composition in vivo in order to safely achieve its desired therapeutic effect. The route of delivery can be any suitable route, including but not limited to intravascular, intravenous, intraarterial, intramuscular, cutaneous, subcutaneous, transdermal, intradermal, and intraepidermal routes. In some embodiments, an effective amount of the composition is formulated for application to or delivery into the skin of a patient. In some embodiments, an effective amount of the composition is formulated for delivery into the bloodstream of a patient.

As used herein, the term "effective amount" refers to a concentration or amount of a composition or agent (e.g., a composition, population of cells, or other agent described herein) that is effective to produce the desired result, including cell growth and/or differentiation in vitro or in vivo, or to treat a disease, disorder, or condition in a patient in need thereof. It will be appreciated that the number of cells to be administered will vary depending on the specifics of the condition to be treated, including but not limited to the size or total volume/surface area to be treated, and the proximity of the site of administration to the location of the area to be treated, as well as other factors familiar to medical biologists and/or treating physicians.

As used herein, the terms "useful life (or time)" and "useful conditions" refer to a period of time or other controlled conditions (e.g., temperature, humidity of an in vitro method) necessary or preferred for a pharmaceutical agent or composition to achieve its intended result (e.g., differentiation of cells into a predetermined cell type).

As used herein, the term "control" or "control group" refers to a surrogate object or sample used in an experiment for comparative purposes. Controls may be "positive" or "negative".

As used herein, the term "simultaneously" refers to simultaneous (i.e., combined) administration. In one embodiment, the administration is co-administration such that two or more pharmaceutically active ingredients (including any solid forms thereof) are delivered together at once.

As used herein, the term "sequentially" refers to separate (i.e., at different times) administration. In one embodiment, staggered administration allows two or more pharmaceutically active ingredients (including any solid forms thereof) to be delivered separately at different times.

As used herein, the term "target tissue" or "target organ" refers to the intended site of accumulation of stem cells as disclosed herein and/or differentiated cells derived from stem cells as disclosed herein after administration to a subject. For example, in some embodiments, the methods disclosed herein relate to a target tissue or target organ that has been damaged (e.g., by ischemia or other injury).

As used herein, the terms "autologous transfer," "autograft," and the like refer to treatment in which the cell donor is also the recipient of cell replacement therapy. The terms "allogenic transfer," "allograft," and the like refer to treatment in which the cell donor and the recipient of the cell replacement therapy belong to the same species but are not the same individual. Cell transfer in which the donor's cells are histocompatibly matched with the recipient is sometimes referred to as homologous transfer. The terms xenogeneic transfer, xenotransplantation, and the like refer to treatment in which the cell donor is a different species from the recipient of the cell replacement therapy.

As used herein, the term "ABCG 2" or "ATP-binding cassette (ABC) transporter G2" refers to a semi-transporter protein of the ABCG family that belongs to the ABCG/white subfamily and has the gene symbol ABCG 2. ABCG2 is also known as MXR (mitoxantrone resistance protein, Miyake et al, 1999.), BCRP (breast cancer resistance protein, Doyle et al, 1998), or ABCP (placenta-specific ABC transporter, Allikmets et al, 1998). GENBANK database discloses the amino acid and nucleic acid sequences of ABCG2 from humans (e.g., AAG 52982), mice (NM _ 011920.3), rats (BAC 76396.1), cats (XP _ 019684813.1), dogs (NP _ 001041486.1), pigs (NP _ 999175.1), cows (NP _ 001032555.2), and the like.

As used herein, the term "CD 34" refers to a cell surface marker found on certain hematopoietic stem cells and non-hematopoietic stem cells, and has the genetic symbol CD 34. The GENBANK ® database discloses the amino acid and nucleic acid sequences of CD34 from humans (e.g., AAB 25223), mice (NP-598415), rats (XP-223083), cats (NP-001009318), pigs (MP-999251), cows (NP-776434), and the like.

As used herein, the term "CD 45" refers to tyrosine phosphatase, also known as Leukocyte Common Antigen (LCA), and has the genetic symbol PTPRC. The gene corresponds to GENBANK accession numbers NP-002829 (human), NP-035340 (mouse), NP-612516 (rat), XP-002829 (dog), XP-599431 (cattle) and AAR16420 (pig). Amino acid sequences of other CD45 homologous chromosomes were also present in the GENBANK database, which included those from several fishes and several non-human primates.

As used herein, the term "lineage marker" or "Lin" refers to a molecule characteristic of a cell lineage, e.g., a cell surface marker, mRNA, or an internal protein. Lineage positive (Lin +) cells refer to a mixture of cells that express markers of the mature cell lineage. Lineage negative (Lin-) cells include stem and progenitor cells, which are not differentiated mature cells. In one aspect, "Lin" refers to a group of markers. The mouse lineage panel as used herein is capable of reacting with cells from the major hematopoietic cell lineage (e.g., T lymphocytes, B lymphocytes, monocytes/macrophages, granulocytes, NK cells, and erythrocytes). As used herein, the FITC anti-mouse lineage antibody cocktail was designed for flow cytometric identification of hematopoietic progenitor cells in mouse bone marrow. The components of the mixture comprise: anti-mouse CD3e, clone 145-2C 11; anti-mouse Ly-6G/Ly-6C, clone RB6-8C 5; anti-mouse CD11b, clone M1/70; anti-mouse CD45R/B220, clone RA3-6B 2; against mouse TER-119/erythrocytes, Ter-119 was cloned. The FITC anti-mouse lineage isotype control mixture contains an equivalent concentration of isotype matched negative control immunoglobulin.

As used herein, the term "keratin epithelial markers" refers to a group of markers including CK5, CK6, CK 8.

2. Isolation of quiescent minicells

The present inventors have surprisingly and unexpectedly found that certain relatively small cells from the peripheral blood of a mammalian subject can be activated into pluripotent stem cells. Pluripotency is demonstrated by the ability of cells to differentiate into all three germ layers. Furthermore, pluripotent stem cells can be rapidly and efficiently expanded in vitro using the novel activation and development systems developed herein. Furthermore, pluripotent stem cells derived from blood are not considered ethically and politically controversial, and do not require genetic manipulation. Another advantage of the present technology is that pluripotent stem cells are proven to be non-tumorigenic. Thus, the presently disclosed technology will pave the way for practical cell-based tissue repair and regeneration therapies, and will also provide new treatments for unmet medical needs in chronic and age-related diseases.

In one embodiment, the present disclosure provides a population of cells enriched for cells expressing CD45 and having a diameter of less than 5 μm. Such cells are referred to herein as "CD 45+ cells" or "resting minicells". In some embodiments, the composition or population of cells comprises at least 100 cells, 1000 cells, 10,000 cells, or 100,000 cells in total, and at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of them are quiescent minicells. In some embodiments, all of the dormant minicells are obtained from a blood sample of a mammalian subject. In some embodiments, the composition further comprises a relatively low percentage (e.g., less than about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%) of blood cells, e.g., red blood cells and white blood cells.

In some embodiments, the dormant minicells in the composition comprise intact and disrupted cells. In some embodiments, the ratio of the number of intact dormant minicells to disrupted dormant minicells in the cell population is less than 1:1, at least about 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1. 7: 1. 8: 1. 9: 1. 10: 1. 11: 1. 12: 1. 13: 1. 14: 1 or 15: 1.

In some embodiments, the dormant minicells are about 2 μm, or about 2.5 μm, or about 3 μm, or about 3.5 μm, or about 4 μm, or about 4.5 μm, or about 5 μm, or between about 2-3 μm, or between about 2-4 μm, or between about 2-5 μm, or less than 4 μm, or less than 3 μm in diameter. In some embodiments, the isolated resting minicells have a very high nuclear-to-cytoplasmic ratio (v/v, also referred to as the N: C ratio, or N/C). In some embodiments, the nuclear to cytoplasmic ratio of a resting minicell as disclosed herein can be at least 9: 1, or at least 8: 1. or at least 7: 1. or at least 6: 1. or at least 5: 1. when seeded in vitro in conventional cell culture media (e.g., α -MEM with 20% FBS), in some embodiments, resting minicells do not proliferate and become senescent within a few days. In some embodiments, the isolated dormant minicell population expresses markers CD34 and CD45, and does not express marker ABCG 2. In some embodiments, the resting minicells are further characterized as Lin-.

In some embodiments, the resting minicells can have a high ratio of small RNAs (srnas, including micrornas) to ribosomal RNAs (rrna). In some embodiments, the ratio of small RNA to ribosomal RNA is at least 22: 1, or at least 20: 1. or at least 18: 1. or at least 15: 1. or alternatively at least 12: 1. or at least 10: 1. or at least 9: 1. or at least 8: 1. or at least 7: 1. or at least 6: 1. or at least 5: 1. in certain aspects, low levels or minimal expression of mitochondrial markers (e.g., COX IV), Endoplasmic Reticulum (ER) markers (e.g., calnexin), and ribosomal markers (e.g., RPS 3) may also indicate that the isolated stem cell population is in a quiescent/quiescent state.

Dormant minicells can be analyzed using cell surface markers and intracellular markers (e.g., those shown in table 1 below).

TABLE 1

Antigens or markers	Exemplary GenBank accession numbers
		CD34	NM_001111059
CD45	NM_011210.4
		CD44	NM_009851
CD150	NM_013730.4
		CD90.1 (Thy1.1)	AY445633.1
CD105	NM_007932.2
		CD133	NM_008935.2
Sca1(Ly6a)	NM_010738
		c-kit (CD117)	NM_001122733.1
Oct4 (Oct3/4, Pou5f1)	BC068268.1
		SSEA1	NM_010242.3
Nanog	AY278951.1
		Vasa	NM_001145885

In some embodiments, the isolated quiescent minicells express one or more early stem cell markers (e.g., Oct4, Nanog, SSEA1, Vasa). In some embodiments, the quiescent minicells express one or more markers of the hematopoietic marker set (e.g., CD34, CD45, CD133, CD 150) and an MSC marker (e.g., CD44, CD105, Sca1, CD 90.1). In some embodiments, the isolated quiescent minicells express one or more markers identified in table 1. In one aspect, there are two markers, or three, or four, or five, identified in table 1 and added to the presence of all markers. In some embodiments, the quiescent minicells do not express E-cadherin, CD184, and/or CD 41.

In some embodiments, methods of isolating resting minicells as disclosed herein from a blood sample are provided. The dormant minicells can be isolated from peripheral blood by any method that allows for cell isolation. For example, the methods disclosed herein can include removing at least a portion of blood cells and platelets from a blood sample, and centrifuging the sample to obtain a pellet comprising resting minicells. In other aspects, the methods can include cell sorting and cell separation methods based on one or more identifying markers. For example, Fluorescence Activated Cell Sorting (FACS) or Magnetic Activated Cell Sorting (MACS) can be used to sort and separate dormant minicells. In some embodiments, other methods can be used to isolate resting minicells as disclosed herein. Examples of some of the isolation procedures are provided below in example 1.

In one aspect, a method of isolating resting minicells comprises: (1) removing at least a portion of the red blood cells from the blood sample; (2) removing at least a portion of the platelets from the sample; (3) centrifuging the sample at 400 Xg-3000 Xg; and (4) obtaining a pellet comprising the dormant minicells disclosed herein. In some aspects, the sample in step (3) may be centrifuged at about 400 xg, or about 500 xg, or about 800 xg, or about 1000 xg, or about 1200 xg, or about 1500 xg, or about 1800 xg, or about 2000 xg, or about 2200 xg, or about 2500 xg, or about 2800 xg, or about 3000 xg, or about 3500 xg, or about 4000 xg, or about 4500 xg, or about 5000 xg, or about 5500 xg, or about 6000 xg, or about 6500 xg, or about 7000 xg, or about 7500 xg, or about 8000 xg, or about 9000 xg, or about 10,000 xg, or more than 10,000 xg.

In some embodiments, methods are provided for isolating a particular enriched stem cell population based on a particular marker of the stem cells. For example, the method can be used to isolate a cell population enriched for CD34+/CD45+/ABCG 2-/Lin-cells (e.g., by FACS or MACS). In another example, the method may be used to isolate a cell population enriched for CD34+/ABCG 2-cells. In other embodiments, the method can be used to isolate a cell population enriched for cells having a particular combination of particular markers (e.g., markers for identifying quiescent minicells as disclosed herein).

In some embodiments, the dormant minicells can be isolated from other sources/tissues, so long as the tissue contains active dormant minicells as disclosed herein. In some embodiments, the dormant minicells can be isolated from a subject of any age. In some embodiments, the dormant minicells can be isolated from a subject at any time. In some embodiments, the dormant minicells may be isolated from animals such as, but not limited to, horses, dogs, pigs, cows, mice, apes, and humans.

3. Activation and development

To activate and culture isolated dormant minicell populations in vitro, an activation/development system has been established. In some embodiments, the activation/development system comprises one or more cell types/cell lines selected from the group consisting of primary hepatocytes, human hepatoblastoma (HepG 2) cells, hepatocyte cell lines, and Mouse Embryonic Fibroblast (MEF) cell lines. Non-limiting examples of hepatocyte cell lines include AML12 and HepaRG. In some embodiments, the activation/development system may include other types of hepatoblastoma cells and/or other types of embryonic fibroblast cell lines. In some embodiments, the activation/development system may include other types of cells and/or cell lines. In some aspects, the activation/development system may comprise at least one, or at least two, or at least three, or at least four of the above-described cells/cell lines.

In one aspect, the activation/development system comprises a cell mixture of at least primary hepatocytes, HepG2 cells, a hepatocyte cell line, and a MEF cell line. In another aspect, the activation/development system comprises a cell mixture of at least primary hepatocytes, HepG2 cells, and a hepatocyte cell line. In another aspect, the activation/development system comprises a cell mixture of at least primary hepatocytes, HepG2 cells, and MEF cell lines. In another aspect, the activation/development system comprises a cell mixture of at least primary hepatocytes, a hepatocyte cell line and a MEF cell line. In another aspect, the activation/development system comprises a cell mixture of at least HepG2 cells, a hepatocyte cell line and a MEF cell line. In another aspect, the activation/development system comprises a cell mixture of at least primary hepatocytes and HepG2 cells. In another aspect, the activation/development system comprises a cell mixture of at least primary hepatocytes and a hepatocyte cell line. In another aspect, the activation/development system comprises a cell mixture of at least primary hepatocytes and a MEF cell line. In another aspect, the activation/development system comprises a cell mixture of at least HepG2 cells and a hepatocyte cell line. In another aspect, the activation/development system comprises a cell mixture of at least HepG2 cells and MEF cell line. In another aspect, the activation/development system comprises a cell mixture of at least a hepatocyte cell line and a MEF cell line. In another aspect, the activation/development system comprises at least primary hepatocytes. In another aspect, the activation/development system comprises at least HepG2 cells. In another aspect, the activation/development system includes at least a hepatocyte cell line. In another aspect, the activation/development system comprises at least a MEF cell line.

In one embodiment, provided herein is a method of activating and culturing quiescent minicells in vitro. In some embodiments, the resting minicells can be co-cultured with a cell or mixture of cells disclosed herein using a Transwell plate. For example, cells or cell mixtures of the above cell/cell lines can be prepared and treated with mitomycin C to inactivate cell mitosis. The cell/cell mixture can then be seeded on the bottom of the cell culture plate in a co-culture medium. Isolated resting minicells can be seeded on a Transwell membrane to co-culture with the cell/cell mixture. The co-culture medium may include an α -MEM medium containing 5-50% FBS (fetal bovine serum). For example, a co-culture medium can include an α -MEM medium containing about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% FBS. In some embodiments, other media may be used in the methods disclosed herein. Optionally, other agents and factors may be added to the co-cultivation medium.

In another aspect, provided herein are methods of using conditioned media to activate and culture resting minicells. In some aspects, the culture medium may be treated with a mixture of the above-described cells or cells/cell lines prior to use in an activation/development system. For example, the cell/cell mixture described above can be suspended in cell culture media and then seeded into a cell culture dish/plate. The cell culture medium used to culture the cell/cell mixture may include DMEM containing 5-50% FBS. For example, the culture medium can comprise DMEM containing about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% FBS. In some embodiments, other media may be used in the methods disclosed herein. Optionally, other agents and factors may be added to the culture medium. Conditioned media can be collected from cell culture dishes/plates and the remaining cells in the media can be removed (e.g., by centrifugation and/or filtration) before the isolated resting minicells are cultured using the media. The collected conditioned medium may be mixed with the above co-culture medium for culturing the resting minicells. For example, the conditioned media disclosed herein can be about 5% (v/v), or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, based on the total volume of the media mixture. Or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%. Optionally, other agents and factors may be added to the medium mixture.

Embodiments of the present disclosure also provide a population of activated stem cells derived from the resting minicells disclosed herein. Activated stem cells can be obtained by culturing dormant minicells in the above-described activation/development system for an effective period of time. In some aspects, the culture time effective to activate the resting minicells may include, but is not limited to, at least 1 hour, or at least 2 hours, or at least 4 hours, or at least 12 hours, or at least 1 day, or at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 8 days, or at least 10 days, or at least 12 days, or at least 15 days, or at least 18 days, or at least 20 days. The cell culture medium may be changed every 1 day, or every 2 days, or every 3 days, or every 4 days, or every 5 or more days. In some embodiments, activated stem cells can be obtained by culturing the quiescent minicells in other culture systems, media, or conditions for an effective period of time. In some embodiments, activated stem cells are characterized as a highly heterogeneous mixture of cells, including various cell subpopulations characterized by different marker sets.

In some embodiments, other cell culture media and/or cell culture conditions may be used to activate and/or culture stem cells as disclosed herein. In some embodiments, the cell culture media and/or cell culture conditions used in the activation/development systems as disclosed herein may be used to culture other types of cells and/or other types of stem cells. For example, the cell culture medium and/or cell culture conditions used in the activation/development system may be used to culture one or more cells selected from the group consisting of: embryonic Stem (ES) cells, Hematopoietic Stem Cells (HSCs), Mesenchymal Stem Cells (MSCs), Endothelial Stem Cells (ESCs), mammary stem cells (mascs), Intestinal Stem Cells (ISCs), Neural Stem Cells (NSCs), adult Olfactory Stem Cells (OSCs), Neural Crest Stem Cells (NCSCs), and Testicular Stem Cells (TSCs), and induced pluripotent stem cells (ipscs).

In some embodiments, the activated stem cells express ABCG2, which is not expressed in resting minicells. In some aspects, nucleic acid staining (e.g., Hoechst 33342) gradually diffuses out of activated stem cells cultured in an activation/development system, whereas nucleic acid staining in quiescent minicells as disclosed herein does not diffuse in conventional media. In some embodiments, the high small RNA to ribosomal RNA ratio in resting minicells is significantly reduced upon stem cell activation. In some embodiments, the ratio of small RNA to ribosomal RNA of the activated stem cell can be less than 1: 4. or less than 1: 5. or less than 1: 6. or less than 1: 7. or less than 1: 8. or less than 1: 9. or less than 1: 10. In some embodiments, activated stem cells may have significantly increased COX IV, RPS3, and calnexin levels compared to quiescent minicells.

In some embodiments, also provided herein are activated stem cell populations of ABCG2+, which can be further characterized as several subpopulations of CD34+/CD45+, CD34+/CD45-, CD34-/CD45+, and/or CD34-/CD 45-. In one embodiment, at least about 50%, or at least about 55%, or at least about 60%, or at least about 66%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the population of activated stem cells is CD34+/CD 45-. In one aspect, less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5% of the activated stem cells are CD34+/CD45+, CD34-/CD45+, CD34-/CD45-, or any combination thereof.

In some embodiments, one or more subpopulations of activated stem cells as disclosed herein are capable of rapid expansion in an activation/development system in vitro. In some embodiments, the doubling time of a subpopulation of activated stem cells may vary. In some embodiments, the doubling time of one or more subpopulations of activated stem cells in the activation/development system may be more than 50 hours, or less than 45 hours, or less than 40 hours, or less than 35 hours, or less than 30 hours, or less than 25 hours, or between about 20 hours and about 50 hours, or between about 20 hours and about 40 hours, or between about 20 hours and about 30 hours. In some embodiments, one or more subpopulations of activated stem cells are capable of expansion in other cell culture systems and/or under other culture conditions in vitro.

Also provided herein are methods for purifying a subpopulation of activated stem cells derived from the resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of CD34+/CD45+ activated stem cells derived from resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of CD34+/CD 45-activated stem cells derived from resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of CD34-/CD45+ activated stem cells derived from resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of CD34-/CD 45-activated stem cells derived from resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of CD34+ activated stem cells derived from resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of CD 34-activated stem cells derived from resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of CD45+ activated stem cells derived from resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of CD 45-activated stem cells derived from resting minicells disclosed herein. In one aspect, a method is provided for purifying a subpopulation of activated stem cells having a set of markers derived from resting minicells disclosed herein.

In one embodiment, one or more cell colonies develop after a period of culture in the activation/development system (e.g., 10-20 days). In some aspects, resting minicells cultured in conventional media (e.g., α -MEM media containing 20% FBS) do not grow and expand, and do not develop into cell colonies.

In some embodiments, provided herein are methods for isolating cell colonies derived from the resting minicells disclosed herein. For example, cell colonies can be obtained using a cloning column. Alternatively, the cell colonies can be isolated by trypsinization and separation of the colonies from the cell culture plate/dish. It will be appreciated that other methods can be used to isolate cell colonies. At least 60%, or at least 70%, or at least 80%, or at least 95%, or at least 99% of the cells in the isolated stem cell colonies are determined (e.g., by FACS) to be CD34+/CD 45-. In one aspect, at least 90% of the stem cells in the isolated stem cell colony are CD34+/CD 45-.

Also provided herein are methods of purifying a population of activated stem cells from an isolated colony as disclosed herein. In one aspect, at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 99.9% of the cells in the isolated stem cell colony are CD34+/CD 45-. In some embodiments, in addition to CD34+/CD 45-cells, the cells in a colony may include subpopulations of cells that may be CD34+/CD45+, CD34-/CD45+, and/or CD34-/CD 45-. In one aspect, less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 2%, or less than about 1% of the cells in the isolated stem cell colony are CD34+/CD45+, CD34-/CD45+, CD34-/CD45-, or any combination thereof. In some embodiments, less than about 40%, alternatively less than about 30%, alternatively less than about 20%, alternatively less than about 10%, alternatively less than about 5%, alternatively less than about 2%, alternatively less than about 1%, alternatively less than about 0.1% of the cells in the purified cell population are not CD34+/CD 45-based on total number of cells. In some embodiments, methods such as MACS or FACS can be used to purify a particular population or enriched cell population from activated stem cells. In some embodiments, other methods can be used to purify a particular population or enriched cell population from activated stem cells.

In some embodiments, single cell dilution of activated stem cells can be performed and then cultured in an activation/development system as disclosed herein. After a period of time (e.g., about 10-20 days), one or more colonies derived from single cells may develop. Cell colonies derived from single cells had an embryoid body-like (EB-like) structure (fig. 6, D). In one aspect, substantially 100% of the cells in the single cell colony are CD34+/CD 45-. In one aspect, at least about 90%, alternatively at least about 95%, alternatively at least about 99%, alternatively at least about 99.9% of the cells in the single-cell colony are CD34+/CD 45-.

Also provided herein are methods for purifying a population of CD34+/CD 45-stem cells from isolated cell colonies or single cell colonies as disclosed herein. In one embodiment, the population of CD34+/CD 45-stem cells can be purified using, for example, MACS or FACS. The purification method is provided below in example 2. It will be appreciated that other methods can be used to purify the CD34+/CD 45-stem cell population from the cell colony. In some embodiments, the methods disclosed herein or other methods can be used to purify other cell populations from colonies.

Purified CD34+/CD 45-stem cells are about 20-30 μm in diameter, larger than resting minicells. For example, the diameter of purified CD34+/CD 45-stem cells can be less than 20 μm, or about 22 μm, or about 24 μm, or about 26 μm, or about 28 μm, or about 30 μm, or greater than 30 μm. Purified CD34+/CD 45-stem cells were further characterized by a relatively high nuclear to cytoplasmic ratio of about 5: 1. or about 6: 1. or about 7: 1. or about 8: 1. or about 9: 1. in one aspect, the nucleus of the purified CD34+/CD 45-stem cell comprises at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the total volume of the cell. Purified CD34+/CD 45-stem cells are capable of rapid (e.g., with a doubling time of about 26 hours) expansion in vitro in conventional cell culture media (e.g., α -MEM containing 20% FBS). In other aspects, the doubling time of purified CD34+/CD 45-stem cells can vary under other culture conditions. In some aspects, purified CD34+/CD 45-stem cells can be cultured in vitro as adherent cell cultures and/or suspension cell cultures. When purified CD34+/CD 45-stem cells were attached to cell culture dishes/plates and reached confluency, the cell arrangement was similar to that of epithelial cell cultures (fig. 6, a and B).

In one aspect, the purified CD34+/CD 45-stem cells were further analyzed by markers as set forth in Table 2 below. In some embodiments, the purified CD34+/CD 45-stem cells express one or more markers identified in table 2. In one aspect, there are two markers, or three, or four, or five, identified in table 2 and all markers are added to the presence. Other defined antigens are illustrated in table 2 and described below.

TABLE 2

Antigens or markers	Exemplary GenBank accession numbers
		CD34	NM_001111059
CD14	NM_009841.4
		CD19	NM_009844.2
CD117	NM_001122733.1
		CD29	NM_010578.2
CD44	NM_009851
		CD73	L12059.1
CD90.1 (Thy1.1)	AY445633.1
		CD31	NM_008816.3
CD105	NM_007932.2
		CD106	NM_011693.3
CD133	NM_008935.2
		CD184	NM_009911.3
Sca1(Ly6a)	NM_010738
		Oct4 (Oct3/4, Pou5f1)	BC068268.1
SSEA1	NM_010242.3
		Sox2	AB044284.1
Nanog	AY278951.1
		Vasa	NM_001145885
Keratin epithelium	(as described above)
		CK18	NM_010664.2
CK19	M28698.1
		Nestin	NM_016701.3
Osteocalcin (BGLAP)	NM_007541.3
		ABCG2	NM_011920.3

In some aspects, the purified CD34+/CD 45-stem cells can express one or more markers from the group: early markers (e.g., Oct4, Sox2, Nanog, Vasa, SSEA 1), hematopoietic markers (e.g., CD34, CD14, CD19, CD 117), MSC markers (e.g., CD44, CD73, thy1.1(CD90.1), CD105, CD106, Sca 1), epithelial markers (e.g., keratin epithelium, CK18, CK 19), neural stem cell markers (e.g., Nestin), osteoblast markers (e.g., osteocalcin), endothelial cell markers (e.g., CD 31), cell migration markers (e.g., CD 184), cellular integrin markers (e.g., CD 29), multiple types of stem cell (e.g., neuronal, HSC, endothelial) markers (e.g., CD 133).

In some embodiments, the purified population of CD34+/CD 45-stem cells expresses one or more of the following markers: oct4, Nanog, ABCG2, CD117, CD29, CD44, CD73, CD90.1, CD105, Sca1, CD31, CD184, keratin epithelium, nestin, and osteocalcin. In one aspect, there are two, or three, or four, or five, and up to all, of the markers Oct4, Nanog, ABCG2, CD117, CD29, CD44, CD73, CD90.1, CD105, Sca1, CD31, CD184, keratin epithelium, nestin, and osteocalcin. In one aspect, the purified CD34+/CD 45-stem cells express at least ABCG2, Oct4, Nanog, CD117, CD29, CD44, Sca1, CD31, CD184, CD133, keratin epithelium, nestin, and osteocalcin. In one aspect, the purified CD34+/CD 45-stem cells express at least ABCG2, Oct4, Sox2, CD19, CD29, CD90, CD31, CD184, CK18, nestin, and osteocalcin. In one aspect, the purified CD34+/CD 45-stem cells express at least ABCG2, SSEA1, CD14, CD29, CD105, CD31, CD184, CK19, nestin, and osteocalcin. In one aspect, the purified CD34+/CD 45-stem cells express at least ABCG2, Nanog, CD117, CD29, CD90, CD31, CD184, keratin epithelium, nestin, and osteocalcin. In one aspect, the purified CD34+/CD 45-stem cells express at least ABCG2, Nanog, CD117, CD90, CD31, CD133, nestin, and osteocalcin.

In some embodiments, the purified CD34+/CD 45-stem cells are further characterized as Lin-. In some embodiments, the purified CD34+/CD 45-stem cells are identified as negative in one or more of CD3, CD4, CD8a, CD11b, CD13, CD45, Lin, and CD140 a. In some embodiments, the purified CD34+/CD 45-stem cells do not express at least one, or at least two, or at least three, or at least four, or at least five, or up to all of CD3, CD4, CD8a, CD11b, CD13, CD45, Lin, and CD140 a.

Expression of early embryonic stage markers (e.g., Oct4, Sox2, Nanog, Vasa, SSEA 1) indicates that purified CD34+/CD 45-stem cells are in the early stage. Expression of keratin epithelial markers was also observed in purified CD34+/CD 45-stem cells. Keratin epithelial markers are known to be expressed in ectodermal cells at early embryonic stages, but not in adult blood cells.

4. Differentiation of activated stem cells

Purified populations of CD34+/CD 45-stem cells can also be identified by their pluripotency, e.g., the ability to differentiate into cell types from all three germ layers (ectoderm, mesoderm, and endoderm) using appropriate culture conditions and media. The differentiation state of the cells can be confirmed by identifying cell type-specific markers known to those skilled in the art.

The present disclosure provides methods for inducing the differentiation of a purified population of CD34+/CD 45-stem cells into an ectodermal lineage. Also provided are compositions or populations of differentiated cells in ectodermal lineage derived from purified CD34+/CD 45-stem cell populations. In one aspect, the purified CD34+/CD 45-stem cells are capable of differentiating into at least one cell type in the ectodermal lineage. For example, purified CD34+/CD 45-stem cells are capable of differentiating into neural and epithelial cells. On the other hand, purified CD34+/CD 45-stem cells are capable of differentiating into at least two, at least three, and increasing to all cell types in the ectodermal lineage. Non-limiting examples of cells that differentiate into ectodermal lineages include, but are not limited to, epidermal cells, neuronal cells, and glial cells.

In other aspects, methods of inducing a population of activated stem cells derived from quiescent minicells as disclosed herein to differentiate into an ectodermal lineage are also provided. Also provided are compositions or populations of cells differentiated in the ectodermal lineage from an activated stem cell population derived from a quiescent minicell as disclosed herein. In other aspects, methods are also provided for inducing differentiation of various subpopulations of an activated stem cell population derived from quiescent minicells as disclosed herein into ectodermal lineages. Also provided are compositions or populations of cells differentiated in the ectodermal lineage derived from various subpopulations of the activated stem cell population derived from the quiescent minicells as disclosed herein. In other aspects, methods of inducing activated stem cell subpopulations as disclosed herein to differentiate into ectodermal lineages are also provided. Also provided are compositions or populations of cells differentiated in the ectodermal lineage derived from a subpopulation of activated stem cells as disclosed herein.

The present disclosure provides methods for inducing the differentiation of a purified population of CD34+/CD 45-stem cells into a mesodermal lineage. Also provided are compositions or populations of differentiated cells in the mesodermal lineage derived from a purified population of CD34+/CD 45-stem cells. In one aspect, the purified CD34+/CD 45-stem cells are capable of differentiating into at least one cell type in the mesodermal lineage. For example, purified CD34+/CD 45-stem cells are capable of differentiating into cardiomyocytes and osteoblasts. On the other hand, purified CD34+/CD 45-stem cells are capable of differentiating into at least two, or at least three, or at least four, of the mesodermal lineages and increasing to all cell types. Non-limiting examples of cells that differentiate into mesodermal lineages include, but are not limited to, adipogenic, smooth myogenic, chondrogenic, cardiogenic, dermogenic, hematopoietic, angiogenic, myogenic, nephrogenic, urogenic, osteogenic, pericardial-derived, or stromal cells.

In other aspects, methods of inducing a population of activated stem cells derived from resting minicells as disclosed herein to differentiate into a mesodermal lineage are also provided. Also provided are compositions or populations of cells differentiated in the mesodermal lineage from an activated stem cell population derived from a quiescent minicell as disclosed herein. In other aspects, methods are also provided for inducing differentiation of various subpopulations of an activated stem cell population derived from quiescent minicells as disclosed herein into mesodermal lineages. Also provided are compositions or populations of cells differentiated in the mesodermal lineage derived from various subpopulations of the activated stem cell population derived from the quiescent minicells as disclosed herein. In other aspects, methods of inducing activated stem cell subpopulations as disclosed herein to differentiate into mesodermal lineages are also provided. Also provided are compositions or populations of cells differentiated in the mesodermal lineage derived from a subpopulation of activated stem cells as disclosed herein.

The present disclosure provides methods for inducing the differentiation of a purified population of CD34+/CD 45-stem cells into endodermal lineages. Also provided are compositions or populations of differentiated cells in the endodermal lineage derived from a purified population of CD34+/CD 45-stem cells. In one aspect, the purified CD34+/CD 45-stem cells are capable of differentiating into at least one cell type in the endodermal lineage. For example, purified CD34+/CD 45-stem cells are capable of differentiating into hepatocytes. In another aspect, purified CD34+/CD 45-stem cells are capable of differentiating into at least two, or at least three, or at least four, or at least five, and increasing to all cell types in the endodermal lineage. Non-limiting examples of cells that differentiate into endodermal lineages include, but are not limited to, cells in the pancreas, liver, lung, stomach, intestine, and thyroid.

In other aspects, methods of inducing a population of activated stem cells derived from resting minicells as disclosed herein to differentiate into endodermal lineages are also provided. Also provided are compositions or populations of cells differentiated in the endoderm lineage derived from an activated stem cell population of resting minicells as disclosed herein. In other aspects, methods are also provided for inducing differentiation of various subpopulations of an activated stem cell population derived from resting minicells as disclosed herein into endodermal lineages. Also provided are compositions or populations of cells differentiated in the endodermal lineage derived from various subpopulations of activated stem cell populations derived from resting minicells as disclosed herein. In other aspects, methods of inducing activated stem cell subpopulations as disclosed herein to differentiate into endodermal lineages are also provided. Also provided are compositions or populations of cells differentiated in the endodermal lineage derived from a subpopulation of activated stem cells as disclosed herein.

5. Application method

The present disclosure provides methods of using the activated stem cells disclosed herein to treat a disease in a subject in need thereof. In some embodiments, methods of treating a disease in a subject in need thereof using differentiated cells derived from activated stem cells disclosed herein are provided. Regenerative medicine includes therapies aimed at aiding in the repair, replacement or regeneration of damaged cells, tissues or organs. The methods disclosed herein may be used for cell-based therapies in reproductive medicine.

In some aspects, the methods and compositions disclosed herein can be used to treat a disease or disorder, for example, a degenerative disease, a proliferative disease, a genetic disease, an injury, and/or organ failure. Non-limiting examples of diseases or conditions include neurodegenerative diseases, neurological diseases (e.g., cognitive disorders and mood diseases), auditory diseases (e.g., deafness), osteoporosis, cardiovascular diseases, diabetes, metabolic disorders, respiratory diseases, drug-sensitive diseases, ocular diseases (e.g., macular degeneration), immunological diseases, hematological diseases, renal diseases, proliferative diseases, genetic diseases, trauma, stroke, organ failure, or loss of limbs. Other examples of diseases include neurodegenerative diseases, neurological diseases, ocular diseases, mood diseases, respiratory diseases, auditory diseases, cardiovascular diseases, immunological diseases, hematological diseases, metabolic diseases, renal diseases, proliferative diseases, genetic diseases, autoimmune diseases, drug-sensitive diseases, cognitive disorders, depression, deafness, osteoporosis, diabetes, macular degeneration, obesity, Alexander's disease, Alper's disease, alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, barton's disease, Canavan disease, Cockayne syndrome (Cockayne syndrome), corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington's disease, HIV, dementia-related dementia, cognitive disorders, and the like, Kennedy's disease, Krabbe's disease, Lewy body dementia, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, narcolepsy, neuroborreliosis (neuroborreliosis), Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, Primary lateral sclerosis, prion disease, Refsum's disease, Sandhofs disease, Schilder's disease, subacute comorbidities of spinal cord secondary to pernicious anemia, schizophrenia, spinocerebellar ataxia, Spinal Muscular Atrophy (SMA), Steele-Richardson-Olski disease, tabes dorsalis, acquired immunodeficiency, leukemia, lymphoma, hypersensitivity (hypersensitivity), severe combined immunodeficiency, acute diffuse hemolytic disease, Aidsura's disease, autoimmune disease, antiphospholipid profile, autoimmune disease, Addison's disease, Lipidism, neuroleptosis, neuroborreliosis, Parkinson's disease, neuroleptosis, Parkinson's disease, Parkinson Autoimmune hepatitis, bullous pemphigoid, celiac disease, dermatomyositis, type 1 diabetes, type 2 diabetes, Goodpasture's syndrome, Graves ' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, lupus erythematosus, myasthenia gravis, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, Sjogren's syndrome, temporal arthritis, vasculitis, Wegener's granulomatosis, aneurysm, angina pectoris, arrhythmia, atherosclerosis, cardiomyopathy, aortic calcified disease (CAVD), cerebrovascular accident (stroke), cerebrovascular disease, congenital heart disease, heart failure, myocarditis, coronary valvular disease, congestive heart disease, heart failure, coronary heart disease, and heart disease, Cardiomyopathy, diastolic dysfunction, endocarditis, hypertension, hypertrophic cardiomyopathy, mitral valve prolapse, myocardial infarction, venous thromboembolism, acid lipase disease, amyloidosis, Barth syndrome, biotin enzyme deficiency, carnitine palmitoyltransferase deficiency type II, Central pontine myelinolysis, muscular dystrophy, Farber's disease, glucose-6-phosphate dehydrogenase deficiency, gangliosidosis, trimethylaminouria, Lesch-Nyhan syndrome, lipid storage disorders, metabolic myopathy, methylmalonate uremia, mitochondrial myopathy, mucopolysaccharidosis, mucolipidosis, mucopolysaccharidosis, multiple CoA carboxylase deficiency, nonketohyperglycinemia, Pompe disease (Pompe disease), propionic acid disease, glycogen storage disease type I, urea circulatory metabolic disorder, hyperuricemia, hyperuricosuric disease, diabetes mellitus, cardiovascular disease, diabetes mellitus, Oxalate deposition, carcinoma, sarcoma, germ cell tumor, blastoma, prostate cancer, lung cancer, colorectal cancer, bladder cancer, skin melanoma, breast cancer, endometrial cancer, and ovarian cancer.

In one aspect, methods of treating a skin wound in a subject in need thereof using the activated stem cells disclosed herein or differentiated cells derived therefrom are provided. In one aspect, methods of treating liver injury in a subject in need thereof using the activated stem cells disclosed herein or differentiated cells derived therefrom are provided. In one aspect, methods of treating bone injury or disorder (e.g., arthritis, osteoporosis, etc.) in a subject in need thereof using the activated stem cells disclosed herein or differentiated cells derived therefrom are provided. In one aspect, methods of treating a neural injury or a neuronal degenerative disease in a subject in need thereof using the activated stem cells disclosed herein or differentiated cells derived therefrom are provided. In one aspect, methods of treating cardiac tissue damage or heart failure in a subject in need thereof using the activated stem cells disclosed herein or differentiated cells derived therefrom are provided. In one aspect, methods of treating a chronic disease (e.g., diabetes) in a subject in need thereof using the activated stem cells disclosed herein or differentiated cells derived therefrom are provided.

In one aspect, methods of autologous transfer of activated or differentiated cells disclosed herein are provided. In one aspect, methods of allo-transfer of activated or differentiated cells disclosed herein are provided. In one aspect, methods of homologous transfer of activated or differentiated cells disclosed herein are provided.

Examples