WO2025076394A1

WO2025076394A1 - Generation of stem cells with high plasticity

Info

Publication number: WO2025076394A1
Application number: PCT/US2024/050018
Authority: WO
Inventors: Shaowei Li
Original assignee: Apstem Therapeutics Inc
Current assignee: Apstem Therapeutics Inc
Priority date: 2023-10-06
Filing date: 2024-10-04
Publication date: 2025-04-10
Anticipated expiration: 2026-04-06

Abstract

A population of tiny, dormant cells is isolated from adult tissue, which upon activation and growth in vitro exhibits unique characteristics and is referred to herein as guide cells that are distinct from all known cell types. Guide cells can interact with somatic cells to generate a novel type of high-plasticity stem cells, referred to herein as guide integrated adult stem cells (giaSC). Accordingly, provided are cell populations and compositions with high-plasticity stem cells or guide cells, as well as methods of generating such cell populations and compositions. Also provided are methods of regulating somatic cells via interaction with guide cells or their cellular components. The compositions and methods can be used for various therapeutic purposes.

Description

GENERATION OF STEM CELLS WITH HIGH PLASTICITY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of United States Provisional Application Serial Number 63/588,596, filed October 6, 2023, the content of which is incorporated by reference in its entirely into the present disclosure.

BACKGROUND

[0002] Stem cell-based therapies hold tremendous promise for the treatment of serious diseases and injuries. The ability of stem cells to self-renew and their capacity for differentiation offers potential to repair, replace, or regenerate damaged cells, tissues, or organs and to maintain tissue homeostasis throughout the lifespan of a multicellular organism. Currently various types of stem cells are investigated in preclinical and clinical studies, including embryonic stem cells (ESCs), stem cells isolated from adult tissues (e.g., mesenchymal stem/stromal cells (MSCs), hematopoietic stem cells (HSCs), etc.), induced pluripotent stem cells (iPS cells or iPSCs), and a variety of specialized cells obtained by differentiation from the above cell sources among others.

[0003] The term “pluripotency” is used to describe the ability' of a cell to differentiate to derivatives of the three embryonic germ layers, while the teratoma assay has long been regarded as the “gold standard” for assessing pluripotency of human pluripotent stem cells. The only natural source of pluripotent stem cells is believed to exist at the very early embryonic stage (e.g., ESCs isolated from blastocyst), and the subsequent development and adult life are viewed as a continuum of decreasing potencies of differentiation. With respect to human ESCs, many problems have hindered their use in cell therapy, such as ethical issues, tumorigenicity (forming teratoma), and immune rejection. Although some of these problems, such as the ethical issues and immuno-rejection, may be avoided by iPSCs that are artificially generated by genetically reprogramming the patient’s own cells, the tumorigenicity and low- yield of iPSCs derivation is still unresolved. Current stem cell researchers believe that the adult body no longer reserves pluripotent stem cells, and naturally occurring adult stem cells are “tissue-specific” or “lineage-restricted.” Thus, current stem cells cannot fulfill the long desire in regenerative medicine for a source of stem cells that have high plasticity indicated by broad differentiation potential and capability to actively adapt to host environment without causing tumor formation. [0004] Therefore, there is a need for the generation and identification of adult stem cells with high plasticity, which can expand in vitro, have differentiation potential to cross germ layers, can actively adapt to adult tissue environment, and are non-tumorigenic. There is also a need for the establishment of a technology platform for producing, culturing, and preparing these high-plasticity stem cells in vitro for cell-based therapy for the treatment of tissue damages as well as degenerative diseases.

SUMMARY

[0005] The instant disclosure demonstrates that a population of tiny cells that have a diameter of less than 6 micrometers (pm) and are in a dormant state (referred to herein as "‘dormant guide cells.” or “d-GC”) can be isolated from adult tissues (e.g., blood). These dormant guide cells are activated and grow in vitro (hence becoming “activated/mature guide cells,” or “m- GC”), and exhibit unique characteristics and function. Further demonstrated in the instant disclosure is the interaction of activated/mature guide cells with somatic cells, leading to generation of a novel type of adult stem cells that have high plasticity. These high-plasticity stem cells (referred to herein as ‘‘guide integrated adult stem cells,” or ‘‘giaSC”) have been demonstrated to have the differentiation potential to cross germ layers. Large-scale genomic analysis shows that both guide cells and giaSC are distinct from all cell types as currently identified. The instant disclosure also shows transfer of cellular components, for example from activated/mature guide cells to somatic cells and/or between giaSC, which regulates somatic cells or adult stem cells. Further, giaSC can be expanded in vitro for banking and therapeutic uses. Moreover, giaSC are not considered ethically controversial, and the generation of giaSC does not require genetic manipulation. Yet another advantage of the present technology is that giaSC are demonstrated to be non-tumorigenic. Therefore, the presently disclosed technology platform will pave the way for practical cell-based therapy for the treatment of tissue damages as well as degenerative diseases.

[0006] The present disclosure, in one embodiment, provides a method of generating high- plasticity stem cells that comprises contacting somatic cells with guide cells and/or cellular components thereof, while the guide cells are characterized as CD49fYCD45⁺/CD90“, as disclosed herein. In some aspects, the high-plasticity stem cells have differentiation potential for more than one germ layer as disclosed herein. In some aspects, the high-plasticity stem cells actively adapt to host environment and regenerate and/or reconstitute tissue when transplanted in vivo. In some aspects, the high-plasticity stem cells are non-tumorigenic. In some embodiments, under culturing conditions such contacting allows establishment of cellcell interactions. In some aspects, cell-cell interactions allow transfer of the cellular components from the guide cells into the somatic cells. In some aspects, the transferred cellular components comprises RNA as disclosed herein. In some aspects, the cell-cell interaction is via tunneling nanotubes, gap junction, and/or exocytosis/endocytosis, as disclosed herein. In some aspects, the cell-cell interaction is established in a co-culture system, as disclosed herein. In some aspects, the guide cells and the somatic cells have a ratio of cell numbers that is at least about 1 : 1, as disclosed herein.

[0007] In some embodiments, the guide cells, as disclosed herein, is further characterized as CD44⁺. In some embodiments, the guide cells, as disclosed herein, is further characterized as SSEA4-. In some embodiments, the guide cells, as disclosed herein, is further characterized as SSEA3-. In some embodiments, the guide cells, as disclosed herein, is further characterized as CD324-. In some embodiments, the guide cells, as disclosed herein, is further characterized as CD73-. In some embodiments, the guide cells, as disclosed herein, is further characterized as CD105⁺. In some embodiments, the guide cells, as disclosed herein, is further characterized as CD52 . In some embodiments, the guide cells, as disclosed herein, is further characterized as positive in one or more markers selected from the group consisting of HLA-I, HLA-II, and HLA-E. In some embodiments, the guide cells, as disclosed herein, is further characterized as CD34^lo'^v/". In some embodiments, the guide cells, as disclosed herein, is further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, and CD56. In some embodiments, the guide cells, as disclosed herein, express very low level of POU5F1 gene (Oct4). In some embodiments, the guide cells, as disclosed herein, does not express one or more genes from the group of Lin28, Nanog, and Sox2. In some embodiments, the guide cells, as disclosed herein, comprises a plurality of RNA-rich granules in the cytoplasm. In some embodiments, the guide cells, as disclosed herein, comprises one or more pseudopodia to release the RNA- rich granules. In some embodiments, the somatic cells, as disclosed herein, are stem cells. In some embodiments, the somatic cells, as disclosed herein, are not stem cells. In some aspects, the somatic cells, as disclosed herein, are mesenchymal stromal cells. In some aspects, the somatic cells, as disclosed herein, have proliferative capacity, in some embodiments, the high-plasticity stem cells are characterized as CD49F/CD907CD34-, as disclosed herein. In some embodiments, the high-plasticity stem cell is further characterized as CD45^low. In some embodiments, the high-plasticity stem cell is further characterized as CD324^low. Also provided, in some embodiments, is a composition that comprises high- plasticity stem cells that are generated via the above methods, as disclosed herein.

[0008] Also provided, in another embodiment, is an isolated high-plasticity stem cell that is characterized as CD49ftyCD90⁺/CD34- and preferably has differentiation potential for more than one germ layer, as disclosed herein. In some embodiments, the high-plasticity stem cell is further characterized as CD45^low. In some embodiments, the high-plasticity stem cell is further characterized as CD324^low. In some embodiments, the high-plasticity stem cell is further characterized as SSEA4⁺. In some embodiments, the high-plasticity stem cell is further characterized as SSEA3-. In some embodiments, the high-plasticity stem cell is further characterized as CD73⁺. In some embodiments, the high-plasticity stem cell is further characterized as CD44⁺. In some embodiments, the high-plasticity⁷ stem cell is further characterized as CD105⁺. In some embodiments, the high-plasticity stem cell is further characterized as CD 146' . In some embodiments, the high-plasticity stem cell is further characterized as CD56⁺. In some embodiments, the high-plasticity stem cell is further characterized as CD52⁺. In some embodiments, the high-plasticity stem cell is further characterized as positive in HLA-I and/or HLA-E. In some embodiments, the high-plasticity stem cell is further characterized as negative in one or more markers selected from the group consisting of CD3, CD 19, CD20, and HLA-II. In some embodiments, the high-plasticity⁷ stem cell, as disclosed herein, expresses very low level of POU5F1 gene. In some embodiments, the high-plasticity stem cell does not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2. In some aspects, the high-plasticity⁷ stem cell as disclosed herein actively adapts to host environment and regenerate and/or reconstitute tissue when transplanted in vivo. In some embodiments, the high-plasticity⁷ stem cell as disclosed herein is non-tumorigenic.

[0009] In some embodiments, the high-plasticity stem cell expresses (a) one or more of endoderm markers, (b) one or more of mesoderm markers, and (c) one or more of ectoderm markers. In some embodiments, the high-plasticity stem cell expresses one or more of endoderm markers and one or more of mesoderm markers. In some embodiments, the high- plasticity stem cell expresses one or more of endoderm markers and one or more of ectoderm markers. In some embodiments, the high-plasticity stem cell expresses one or more of mesoderm markers and one or more of ectoderm markers. Non-limiting examples of the endoderm markers include AFP, CK7 (KRT7), albumin (ALB), CK.18 (KRT18), and CK.19 (KRT19). Non-limiting examples of the mesoderm markers include desmin (DES), osteocalcin (BGLAP), CD106 (VCAM-1), CD54 (ICAM-1), and CD146 (MCAM). Nonlimiting examples of the ectoderm markers include nestin (NES), notch-1, notch-2. MSI2, CD56 (NCAM1), and CD325 (N-cadherm or Cadhenn-2).

[0010] In some embodiments, the high-plasticity stem cell, as disclosed herein, has an approximately triangular cell body with one or more slender pseudopodia in adherent culture. In some embodiments, the high-plasticity stem cell, as disclosed herein, exchanged a plurality of cellular components, such as RNA, with an adjacent stem cell of the same kind. In some aspects, the high-plasticity stem cell, as disclosed herein, exchanged a plurality of cellular components, such as RNA, with an adjacent cell of a different kind. In some aspects, the exchange of cellular components is via tunneling nanotubes, gap junction, and/or exocytosis/endocytosis. In some embodiments, the high-plasticity stem cell, as disclosed herein, proliferates and forms colonies in culture. In some embodiments, also provided is a method of culturing the high-plasticity stem cell as disclosed herein. In some aspects, also provided is a method of differentiating the high-plasticity stem cell as disclosed herein. In some aspects, also provided is a cell differentiated from the high-plasticity stem cell as disclosed herein.

[0011] Also provided is a composition that comprises a population of the high-plasticity stem cells, as disclosed herein, in a pharmaceutically acceptable carrier or excipient. In some embodiments, also provided is a composition comprising a population of the differentiated cells, as disclosed herein, in a pharmaceutically acceptable carrier or excipient. In some embodiments, also provided is a composition comprising the cellular components isolated from the high-plasticity stem cell or the differentiated cell, as disclosed herein, in a pharmaceutically acceptable carrier or excipient. Also provided is a method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of the compositions as disclosed herein to the subject.

[0012] The present disclosure further provides, in another embodiment, a method of generating guide cells (<?.g., CD49fNCD45⁺/CD9CT) as disclosed herein, which comprises activating dormant guide cells that (a) have a diameter of less than 6 pm, (b) express CD49f and CD45, (c) do not express CD90, and (d) do not have detectable intracellular esterase activity and/or transcriptomic activity. In some aspects, the dormant guide cells, as disclosed herein, are isolated from peripheral blood. In some aspects, the dormant guide cells, as disclosed herein, are further characterized as CD44⁺. In some aspects, the dormant guide cells, as disclosed herein, are further characterized as SSEA4-. In some aspects, the dormant guide cells, as disclosed herein, are further characterized as SSEA3-. In some aspects, the dormant guide cells, as disclosed herein, are further characterized as CD324-. In some aspects, the dormant guide cells, as disclosed herein, are further characterized as CD73-. In some aspects, the dormant guide cells, as disclosed herein, are further characterized as positive in one or more markers selected from the group consisting of CD 105, HLA-I, and HLA-E. In some embodiments, the dormant guide cells express low level of CD34. In some aspects, the dormant guide cells, as disclosed herein, are characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, CD56, and HLA-1I. In some aspects, the dormant guide cells, as disclosed herein, express very low level of POU5F1 gene. In some aspects, the dormant guide cells, as disclosed herein, do not express one or more genes from the group of Lin28, Nanog, and Sox2. In some embodiments, the activating dormant guide cells comprises culturing the dormant guide cells in a hepatic environment as disclosed herein. In some aspects, the hepatic environment comprises a co-culture system with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines, as disclosed herein. In some aspects, the hepatic environment comprises a conditioned medium that is in contact with or has been conditioned with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines, as disclosed herein. In another aspect, the hepatic environment comprises a medium that is supplemented with at least one growth factor or cytokine that is released by primary hepatocytes or hepatic cell lines. In another aspect, the hepatic environment comprises a medium that is supplemented with one or more cytokines selected from the group consisting of CXCL1. CXCL2, CXCL3, CXCL5, CX3CL2, CCL2, IL6. IL8, IL15, albumin, ANXA1, CSF3, TNFSF10, PVR, and ULBP2. Also provided, in another embodiment, is a composition that comprises a population of guide cells (e.g., CD49fVCD45⁺/CD90“) generated by the above method as disclosed herein.

[0013] Also provided, in another embodiment, is a guide cell characterized with CD49f7CD45⁺/CD90“ that comprises RNA-rich granules in the cytoplasm and one or more pseudopodia, as disclosed herein. In some aspects, the RNA-rich granules can be released by the pseudopodia of the guide cell. In some aspects, also provided is a cell derived from the guide cell, as disclosed herein. In some embodiments, also provided is a method of culturing the guide cell as disclosed herein. In some aspects, also provided is a method of isolating cellular components from the guide cell as disclosed herein.

[0014] Also provided, in another embodiment, is a composition that comprises a population of the guide cells, as disclosed herein, in a pharmaceutically acceptable carrier or excipient. In some embodiments, also provided is a composition comprising cells derived from the guide cells, as disclosed herein, in a pharmaceutically acceptable carrier or excipient. In some embodiments, also provided is a composition comprising the cellular components isolated from the guide cells or the derived cells, as disclosed herein, in a pharmaceutically acceptable carrier or excipient. Also provided is a method of treating a disease or condition in a subject in need thereof, comprising administering an effective amount of the compositions as disclosed herein to the subject.

[0015] The present disclosure further provides, in one embodiment, a method of regulating a somatic cell that comprises contacting the somatic cell with a guide cell characterized with CD49f7CD45⁺/CD90“. During the contacting, in some aspects, cell-cell interaction is established which allows transfer of a plurality of cellular components from the guide cell into the somatic cell, as disclosed herein. In some aspects, the transferred cellular components comprise RNA, as disclosed herein. In some aspects, the cell-cell interaction is via tunneling nanotubes, gap junctions, and/or exocytosis/endocytosis, as disclosed herein. In some aspects, the somatic cell is regulated to change its plasticity, as disclosed herein. In some aspects, the somatic cell is regulated to acquire high plasticity, as disclosed herein. In some aspects, the acquired high plasticity comprises differentiation potential for more than one germ layer, as disclosed herein. In some aspects, the acquired high plasticity comprises active adaptation to host environment to regenerate and/or reconstitute tissue when transplanted in vivo. In some aspects, the somatic cell is changed to express (a) one or more of endoderm markers, (b) one or more of mesoderm markers, and (c) one or more of ectoderm markers. In some embodiments, the somatic cell is changed to express one or more of endoderm markers and one or more of mesoderm markers. In some embodiments, the somatic cell is changed to express one or more of endoderm markers and one or more of ectoderm markers. In some embodiments, the somatic cell is changed to express one or more of mesoderm markers and one or more of ectoderm markers. In some embodiments, the guide cell is derived from a dormant guide cell, as disclosed herein.

[0016] Also provided is a method of regulating a somatic cell that comprises transferring to the somatic cell a plurality of cellular components isolated from a guide cell (e.g., CD49E/CD45⁺/CD90“), as disclosed herein. In some aspects, the cellular components are isolated in exosomes and/or microvesicles as disclosed herein. In some aspects, the transferred cellular components include RNA. In some aspects, the cellular components are transferred via endocytosis of the somatic cell. In some embodiments, the cellular components are added to a cell culture of the somatic cell. In some embodiments, the cellular components are delivered to a vicinity of the somatic cell.

[0017] Further provided in some embodiments is a method of isolating a sub-population of dormant guide cell that has a diameter of less than 6 pm and does not have detectable intracellular esterase activity and/or transcriptomic activity, comprises (a) preparing adult tissue in a solution, (b) centrifuging the solution at 5,000xg-15,000xg and obtaining a cell pellet, and (c) enriching CD49U/CD45⁺/CD90“ cells from the cell pellet, as disclosed herein. Also provided is a composition of dormant guide cells isolated by the above method as disclosed herein. Embodiments also provide a composition that comprises at least 1000 cells, wherein at least 50% of the cells are dormant guide cells that (a) have a diameter of less than 6 pm, (b) express CD49f and CD45, (c) do not express CD90, and (d) do not have detectable intracellular esterase activity and/or transcriptomic activity.

BRIEF DESCRIPTION OF THE FIGURES

[0018] FIG. 1 shows a microscopic image of a heterogeneous population isolated from human unmobilized peripheral blood, which includes dormant guide cells with nuclear staining.

[0019] FIG. 2 shows activation of the dormant guide cells in vitro, with positive staining of a fluorescent dye indicating intracellular esterase activity and positive AO staining for RNA indicating transcriptomic activity after activation.

[0020] FIG. 3 shows the growth and maturation process in vitro and unique morphology of the activated/mature guide cells (m-GC) such as aggregation of RNA-rich granules and pseudopodia. [0021] FIG. 4 shows co-culture of the activated/mature guide cells with human umbilical cord-derived mesenchymal stomal cells (UC-MSCs), which leads to colony formation that include guide integrated adult stem cells (giaSC).

[0022] FIG. 5 shows the interaction between activated/mature guide cells and UC-MSCs, during which RNA-rich granules are transferred from activated/mature guide cells into UC- MSCs via tunneling nanotubes (TNTs).

[0023] FIG. 6 shows that giaSC isolated from the colonies collected from the co-culture can be further expanded to form colonies that stain positive for representative markers of three germ-layers (indicating high plasticity).

[0024] FIG. 7 shows flow cytometry results of exemplary markers in dormant guide cells, activated/mature guide cells, and giaSC.

[0025] FIG. 8 shows neural differentiation of giaSC in vitro.

[0026] FIG. 9 shows bone differentiation of giaSC in vitro.

[0027] FIG. 10 shows that cultured giaSC synthesize and release hepatic functional proteins, indicating hepatic or liver progenitor differentiation potential.

[0028] FIG. 11 shows that in vivo transplantation of giaSC into a full-thickness excisional wound model in FVB mouse leads to full skin regeneration and angiogenesis with presence of human cells in various types of mouse skin tissues.

[0029] FIG. 12 shows that co-culture of activated/mature guide cells with bone marrow- derived mesenchymal stromal cells (BM-MSC) leads to generation of giaSC that express representative markers of three germ layers.

[0030] FIG. 13 shows that co-culture of activated/mature guide cells with intestinal epithelial cells (lECs), which are not stem cells, leads to generation of giaSC that express representative markers of three germ layers.

[0031] FIG. 14 shows that systemic administration of giaSC into LPS-induced FVB mouse model leads to repair of small intestinal damage with presence of human cells in small intestinal tissues. DETAILED DESCRIPTION

[0032] Throughout this disclosure, various publications, patents and published patent specifications are referenced herein. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference in their entirety into the present disclosure.

[0033] Before the compositions and methods are described, it is to be understood that the disclosure is not limited to the particular methodologies, protocols, cell lines, assays, and reagents described, as these may var ⁷. It is also to be understood that the terminology used herein is intended to describe particular embodiments of the present disclosure, and is in no way intended to limit the scope of the present disclosure as set forth in the appended claims.

[0034] The practice of the present disclosure 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, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3^rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5^th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent NO. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);

Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^rd edition (Cold Spring Harbor Laboratory' Press (2002)); Current Protocols In Molecular Biology' (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzy mology’ (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)); Harlow and Lane, 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.

[0035] All numerical designations, e.g, pH. temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0. 1 or 1 where appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. The term “about” also includes the exact value “X” in addition to minor increments of “X” such as “X+ 0.1 or 1” or “X - 0.1 or 1”, where appropriate. It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplar}⁷ and that equivalents of such are known in the art.

1. Definitions

[0036] 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.

[0037] As used in the specification and claims, the singular forms “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.

[0038] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others.

“Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) claimed. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0039] As used herein, the term “stem cell” defines a cell with the ability to self-renew and differentiate (e.g., give rise to specialized cells). Non-limiting examples of types of stem cells include adult (somatic) stem cells, embryonic stem cells, parthenogenetic stem cells (see Cibelli et al. (2002) Science 295(5556):819; U.S. Patent Publ. Nos. 20100069251 and 20080299091), and/or induced pluripotent stem cells (iPS cells or iPSCs). An adult/somatic stem cell is an undifferentiated cell found in an adult or postnatal tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield specialized cell types of the tissue from which it originated. Non-limiting examples of adult stem cells include hematopoietic stem cells (HSCs), mesenchymal stem/stromal cells (MSCs), endothelial stem cells (ESCs), mammary stem cells (MaSCs), intestinal stem cells (ISCs), neural stem cells (NSCs), adult olfactory⁷ stem cells (OSCs), skin stem cells, retinal stem cells, and muscle stem cells. An embryonic stem cell is a primitive (undifferentiated) cell from the embryo that has the pluripotent potential to become a wide variety of specialized cell types. Non-limiting examples of embry onic stem cells include the HES2 (also known as ES02) cell line available from ESI, Singapore and the Hl or H9 (also known as WA01) cell line available from WiCell, Madison, WI. Additional lines are pending NIH review. See, for example, grants.nih.gov/stem_cells/registry/cunent.htm (last accessed March 13. 2017). Pluripotent embryonic stem cells can be distinguished from other types of cells by the use of markers including, but not limited to, Oct4, alkaline phosphatase, CD30, TDGF-1, GCTM-2, Genesis, Germ cell nuclear factor, SSEA1, SSEA3, and SSEA4. An induced pluripotent stem cell (iPSC) is an artificially derived stem cell from a non-pluripotent cell, typically a somatic cell, produced by inducing expression of one or more stem cell specific genes. An iPSC expresses specific genes including, but are not limited to, the family of octamer transcription factors, e.g., Oct-3/4; the family of Sox genes, e.g., Soxl, Sox2, Sox3, Sox 15 and Sox 18; the family of Klf genes, e.g., Klfl, Klf2, Klf4 and Klf5; the family of Myc genes, e.g., c-myc and L- myc; the family ofNanog genes, e.g., Octamer-4 (OCT4), NANOG and REXI; or LIN28. Examples of iPSCs are described in Takahashi et al. (2007) Cell advance online publication 20 November 2007; Takahashi & Yamanaka (2006) Cell 126:663-76; Okita et al. (2007) Nature 448:260-262; Yu et al. (2007) Science advance online publication 20 November 2007; and Nakagawa et al. (2007) Nat. Biotechnol. Advance online publication 30 November 2007.

[0040] As used herein, the term “somatic cell'’ is intended to refer to any of the body cells except the reproductive (germ) cells. In one aspect, somatic cells include adult stem cells. In another aspect, somatic cells include non-stem cells. In some embodiments, somatic cells include mesenchymal stem/stromal cells derived from a variety^ of tissues as disclosed herein. [0041] As used herein, the term '‘stem cell pl astici ty ' is intended to refer to the ability of stem cells to cross lineage boundaries to adopt the morphologic, antigenic, and/or functional characteristics of a different lineage outside their destined repertoire of differentiation.

[0042] As used herein and as set forth in more detail below, the term '‘high-plasticity stem cells” or “adult stem cells with high plasticity” is intended to refer to stem cells derived from adult or postnatal tissues that acquire high plasticity. In some aspects, the term “high plasticity” encompasses differentiation potential for more than one germ layer. In some aspects, the term “high plasticity” encompasses differentiation potential for all three germ layers (i.e., endoderm, mesoderm, and ectoderm). In some aspects, the term “high plasticity” encompasses capability' to actively adapt to host tissue environment to regenerate/reconstitute tissue without causing tumor formation. In some embodiments, the guide integrated adult stem cells (giaSC). as disclosed herein, is one type of high-plasticity stem cells.

[0043] As used herein, the term “dormant” or “quiescent,” as used herein, is intended to encompass cells that are in a state which are required to be activated before they can undergo growth or differentiation.

[0044] As used herein, the term “activation” or “activate” is intended to refer to a measurable morphological, phenotypic, and/or functional change in the dormant/ quiescent state of the cells. In some embodiments, such activation is concurrent with the expression of specific markers and/or cellular changes (e.g, in intracellular activity, growth and/or development, morphology, size, and/or cellular components). As used herein, the term “maturation” or “mature” is intended to describe cells that undergo growth and/or development and have acquired specific features and/or functions.

[0045] As used herein and as set forth in more detail below, the term “dormant guide cells” or “d-GC” is intended to refer to tiny cells that have a diameter of less than 6 micrometers (pm) and are in a dormant state. In one aspect, the dormant guide cells are isolated from adult peripheral blood. In another aspect, the dormant guide cells reside in other adult or postnatal tissues. In some embodiments, the dormant guide cells are precursors of activated/mature guide cells and are thus also named as “pre-guide cells” or “pre-GC.”

[0046] As used herein and as set forth in more detail below, the term “activated/mature guide cells” or “m-GC” is intended to refer to guide cells that exit dormancy and acquire specific cellular activity. In one aspect the dormant guide cells can undergo activation, grow th, and development under specific conditions in vitro to reach a mature state. In some aspects, the activated/mature guide cells have enlarged size compared to the dormant guide cells, and have unique morphology such as aggregated granules in the cytoplasm and one or more pseudopodia extending from cell surface. In some aspects, the activated/mature guide cells have RNA-rich granules in the cytoplasm and can release them via the pseudopodia and/or interact with other cells.

[0047] As used herein, the term "lineage” of a cell is defined as the developmental history of a differentiated cell as traced back to the cell from which it arises. In one aspect, the lineage defines the heredity of the cell (/.<?., its predecessors and progeny) and places the cell w ithin a hereditary' scheme of development and differentiation. As used herein, the term ‘‘germ layer” refers to a primary layer of cells that forms during embryonic development, and each germ layer eventually gives rise to certain tissue types in the body. As used herein, the term “three germ layers”, “three embryonic germ layers”, or “three primary' germ layers” refers to the endoderm (inner layer), the ectoderm (outer layer), and the mesoderm (middle layer). In one aspect, each germ layer gives rise to multiple types of tissues and comprises multiple lineages. As used herein, “a cell that differentiates into a mesodermal (or ectodermal or endodermal) lineage” defines a cell that becomes committed to a specific mesodermal (or ectodermal or endodermal) lineage, respectively. Examples of cells that differentiate into a mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic. leiomyogenic, chondrogenic, cardiogenic, dermatogenic, hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or stromal. Examples of cells that differentiate into ectodermal lineage include, but are not limited to epidermal cells, neurogenic cells, and neurogliagenic cells. Examples of cells that differentiate into endodermal lineage include, but are not limited to cells that give rise to the pancreas, liver, lung, stomach, intestine, and thyroid.

[0048] As used herein, the term “self-renewable” refers to a cell being able to self-renew' for over a number of passages without substantial changes of cell properties. In one aspect, the number of passages is at least about 2, or alternatively at least 5, or alternatively at least 10, or alternatively at least about 15, 20, 30, 50, or 100.

[0049] As used herein, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, cellular component, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated polynucleotide is separated from the 3’ and 5’ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. An isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. An isolated cellular component is a component that is separated from other cellular components of a cell.

[0050] As used herein, the term “propagate” or “grow” means to expand and/or alter the phenotype of a cell or population of cells. The term “expand” refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell ty pe. In one aspect, the propagation/growth/expansion of cells occurs in vitro. In another aspect, the propagation/growth/expansion of cells occurs in vivo. In one embodiment, the propagation/growth/expansion of cells results in the regeneration of tissue.

[0051] As used herein, the term “culturing” or “culture” refers to the in vitro propagation or grow th of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i. e. , morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.

[0052] As used herein and as set forth in more detail below, “conditioned medium” or “conditioned media” is medium which was cultured with a cell that provides cellular factors to the medium such as cytokines, growth factors, hormones, cellular components, exosomes, microvesicles, extracellular matrix, and some materials that would facilitate cell growth, development, and differentiation. In one embodiment, the conditioned medium was cultured with a somatic cell. In another embodiment, the conditioned medium was cultured with a cell line. In some embodiments, the conditioned medium was cultured with a stem cell.

[0053] As used herein, the term “differentiation” or “differentiate” describes the process whereby an unspecialized cell acquires the features of a specialized cell such as a brain (neuron), skin, heart, liver, bone, or muscle cell. As used herein, the term ‘’differentiated' defines a cell that takes on a more committed position within the lineage of a cell.

[0054] As used herein, the term “substantially homogeneous’⁷ describes a population of cells in which more than about 50%, or alternatively more than about 60%, or alternatively more than 70%, or alternatively more than 75%, or alternatively more than 80%, or alternatively more than 85%, or alternatively more than 90%, or alternatively more than 95%, or alternatively more than 99% of the cells are of the same or similar phenotype. Phenotype can be determined by a pre-selected cell surface marker or other marker.

[0055] As used herein, the term “purified population” of cells of interest refers to the cell population that has been isolated away from substantially all other cells that exist in their native environment, but also when the proportion of the cells of interest in a mixture of cells is greater than would be found in their native environment. For example, a purified population of cells represents an enriched population of the cells of interest, even if other cells and cell types are also present in the enriched population. In some embodiments, a purified population of cells 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 population of cells, with the proviso that the cells of interest comprise a greater percentage of the total cell population in the “purified” population than they did in the population prior to the purification.

[0056] As used herein, the term “population of cells” refers to a collection of more than one cell that is identical (clonal) or non-identical in phenotype and/or genotype.

[0057] As used herein, the term “cell colony” or “colony” refers to a grouping of closely associated cells formed as a result of cell grow th. These terms are used irrelevantly to the number of cells constituting the colony.

[0058] As used herein, the term “composition” is intended to encompass a combination of active agent and another carrier, e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which can also function in a buffering capacity, 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 intended within the scope of this disclosure, 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, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. In certain embodiments, a composition includes a population of cells or a mixture of cells. In certain embodiments, the composition is formulated as a film, gel, patch, 3-D structure, or liquid solution.

[0059] As used herein, the term '‘pharmaceutically acceptable” indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile.

[0060] As used herein, the term “pharmaceutically acceptable carrier”, “pharmaceutically acceptable excipient”, or “pharmaceutically acceptable medium” , which may be used interchangeably with the term '‘biologically compatible carrier (or excipient or medium)”, refers to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers suitable for use in the present disclosure include liquids, semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds and matrices, tubes sheets and other such materials as known in the art and described in greater detail herein). These semi-solid and solid materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable). A biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathw ays. For topical use, the pharmaceutically acceptable carrier is suitable for manufacture of creams, ointments, jellies, gels, solutions, suspensions, etc. Such carriers are conventional in the art, e.g.. for topical administration with polyethylene glycol (PEG). These formulations may optionally comprise additional pharmaceutically acceptable ingredients such as diluents, stabilizers, and/or adjuvants.

[0061] As used herein, the term '‘solution” refers to solutions, suspensions, emulsions, drops, ointments, liquid wash, sprays, and liposomes, which are well known in the art. In some embodiments, the liquid solution contains an aqueous pH buffering agent which resists changes in pH when small quantities of acid or base are added.

[0062] As used herein, the term “pH buffering agent” refers to an aqueous buffer solution which resists changes in pH when small quantities of acid or base are added to it. pH buffering solutions typically comprise a mixture of weak acid and its conjugate base, or vice versa. For example. pH buffering solutions may comprise phosphates such as sodium phosphate, sodium dihydrogen phosphate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate, 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 such as sodium citrate and disodium citrate; acetates such as sodium acetate and potassium acetate; carbonates such as sodium carbonate and sodium hydrogen carbonate, etc. pH adjusting agents can include, for example, acids such as hydrochloric acid, lactic acid, citric acid, phosphoric acid and acetic acid, and alkaline bases such as sodium hydroxide, potassium hydroxide, sodium carbonate and sodium hydrogen carbonate, etc. 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). [0063] As used herein, the term '‘formulated” or “formulation” refers to the process in which different substances, including one or more pharmaceutically active ingredients, are combined 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 which is designed to slowly release a therapeutic agent in the body over an extended period of time, w hereas an immediate release formulation is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time.

[0064] As used herein, the term “treating” or “treatment” refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and/or halting one or more clinical symptoms of a disease or disorder prior to, during, and/or after an injury or intervention.

[0065] As used herein, the term “patient” or “subject” refers to animals, including mammals, such as humans, murine, canine, equine, bovine, or simian, who are treated with the pharmaceutical compositions or in accordance with the methods described herein.

[0066] As used herein, the term “delivery”, “application”, or “administration” refers to routes, approaches, formulations, technologies, and systems for transporting a pharmaceutical composition in the body as needed 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, percutaneous, intradermal, and intraepidermal routes. In some embodiments, an effective amount of the composition is formulated for applying on the skin or delivery into the skin of a patient. In some embodiments, an effective amount of the composition is formulated for delivery into the blood stream of a patient. In some embodiments, an effective amount of the composition is formulated for deliver}' into a specific tissue or organ of a patient.

[0067] As used herein, the term '‘effective amount” refers to a concentration or amount of composition or a reagent, such as a composition as described herein, cell population or other agent, that is effective for producing an intended result, including cell growth and/or differentiation in vitro or in vivo, or for the treatment of 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 disorder to be treated, including but not limited to size or total volume/surface area to be treated, as well as proximity of the site of administration to the location of the region to be treated, among other factors familiar to the medicinal biologist and/or treating physician.

[0068] As used herein, the terms “effective period (or time)” and “effective conditions” refer to a period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods), necessary or preferred for an agent or composition to achieve its intended result, e.g., the differentiation of cells to a pre-determined cell type.

[0069] As used herein, the term “control” or “control group” refers to an alternative subject or sample used in an experiment for comparison purpose. A control can be “positive” or “negative”.

[0070] As used herein, the term “concurrently” refers to simultaneous (i.e., in conjunction) administration. In one embodiment, the administration is co-administration such that two or more pharmaceutically active ingredients, including any solid form thereof, are delivered together at one time.

[0071] As used herein, the term “sequentially” refers to separate (i.e., at different times) administration. In one embodiment, the administration is staggered such that two or more pharmaceutically active ingredients, including any solid form thereof, are delivered separately at different times.

[0072] As used herein, the term “target tissue” or “target organ” refers to an intended site for accumulation of the stem cells as disclosed herein and/or the differentiated cells derived from the stem cells as disclosed herein, following administration to a subject. For example, the methods as disclosed herein involve a target tissue or a target organ that has been damaged (e.g, by ischemia or other injury) in some embodiments.

[0073] As used herein, the terms “autologous transfer”, “autologous transplantation”, “autograft” and the like refer to treatments wherein the cell donor is also the recipient of the cell replacement therapy. The terms “allogeneic transfer”, “allogeneic transplantation”, “allograft” and the like refer to treatments wherein the cell donor is of the same species as the recipient of the cell replacement therapy, but is not the same individual. A cell transfer in which the donor’s cells have been histocompatibly matched with a recipient is sometimes referred to as a syngeneic transfer. The terms xenogeneic transfer, xenogeneic transplantation, xenograft and the like refer to treatments wherein the cell donor is of a different species than the recipient of the cell replacement therapy.

[0074] As used herein, the term '‘CD49f ’ refers to a member of the integrin alpha chain family of proteins, and having the gene symbol ITGA6. CD49f is also known as ITGA6 or ITGA6B (Integrin Subunit Alpha 6, Cariati et al., 2008), or VLA-6 (Hemler et al., 1989). The GENBANK® database discloses amino acid and nucleic acid sequences of CD49f from humans (e.g., NM_000210.4, NP_000201.2), mice (NP_032423.2). rats (NP_446177.2). dogs (XP_003640224.1), and others.

[0075] As used herein, the term “CD34” refers to a cell surface marker found on certain hematopoietic and non-hematopoietic stem cells, and having the gene symbol CD34. The GENBANK® database discloses amino acid and nucleic acid sequences of CD34 from humans (e.g., NM_001025109.2, NP_001020280.1), mice (NP_598415.1), rats (NP_001100672.1), pigs (NP_999251.1), dogs (NP_001003341.1), and others.

[0076] As used herein, the term '‘CD45” refers to a tyrosine phosphatase that has the gene symbol PTPRC. CD45 is also known as PTPRC (Protein ty rosine phosphatase, receptor ty pe C, Goff et al., 1999), or L-CA or LCA (leukocyte-common antigen, Pingel et al., 1989). This gene corresponds to GENBANK® Accession Nos. NM_002838.5 and NP_002829.3 (human), NP_035340.3 (mouse), NP_612516.2 (rat), XP_003130644.5 (pig), and others. The amino acid sequences of additional CD45 homologs are also present in the GENBANK® database, including those from several fish species and several non-human primates. Various isoforms of CD45 exist, e.g, CD45RA, CD45RB. CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45R0, and CD45R(ABC).

[0077] As used herein, the term “CD90” refers to cell surface glycoprotein and having the gene symbol THY1. CD90 is also known as Thyl or Thy-1 cell surface antigen. This gene corresponds to GENBANK® Accession Nos. NM_006288.5 and NP_006279.2 (human), NP_033408.1 (mouse), XP_017450965.1 (rat), NP_001274058.1 (dog), NP_001 139601 .1 (pig), and others.

[0078] As used herein, the term “CD73’^? refers to a cell surface enzyme that catalyzes the conversion of extracellular nucleotides to membrane-permeable nucleosides, and having the gene symbol NT5E. CD73 is also known as 5 ’-nucleotidase (5’-NT) or ecto-5’ -nucleotidase. This gene corresponds to GENBANK® Accession Nos. NM_002526.4 and NP_002517.1 (human), NP_035981.1 (mouse), NP_067587.2 (rat), XP_038531093.1 (dog), XP_001927130. 1 (pig), and others.

[0079] As used herein, the term “CD105” refers to a type I membrane glycoprotein and have the gene symbol ENG. CD 105 is also known as endoglin (ENG) and is a major glycoprotein of the vascular endothelium. This gene corresponds to GENBANK® Accession Nos. NM_001114753.3 and NP_001108225.1 (human), NP_001139820.1 (mouse), NP_001010968.1 (rat), XP 005625387.1 (dog), NP_999196. 1 (pig), and others.

[0080] As used herein, the term “CD146” refers to a membrane glycoprotein and gave the gene symbol MCAM. CD 146 is also known as melanoma cell adhesion molecule (MCAM) or cell surface glycoprotein MUC18. This gene corresponds to GENBANK® Accession Nos. NM_006500.3 and NP_006491.2 (human), NP_075548.2 (mouse), NP_076473.2 (rat), XP_022273915.1 (dog), XP_005667488.1 (pig), and others.

[0081] As used herein, the term '‘SSEA4” refers to a glycolipid epitope that is also known as stage Specific Embryo Antigen 4. As used herein, the term “SSEA3” refers to a glycosphingolipid that is also known as stage-specific embryonic antigen 3.

[0082] As used herein, the term '‘CD324,” “E-Cadherin,” or ‘'E-Cad” refers to a cell adhesion molecule and having the gene symbol CDH1. CD324 is also known as Cadherin-1, Epithelial Cadherin, or CDHE. The GENBANK® database discloses amino acid and nucleic acid sequences of CD324 from humans (e.g.. NM_004360.5 and NP_004351. 1), mice (NP_033994. 1), rats (NP_112624.1), pigs (NP_001156532.1), dogs (NP_001274054.1), and others.

[0083] As used herein, the term “CD44” refers to a cell surface adhesion receptor and having the gene symbol CD44. CD44 is also known as homing cell adhesion molecule (HCAM), phagocytic glycoprotein- 1 (Pgp-1), or ECM-IIL The GENBANK® database discloses amino acid and nucleic acid sequences of CD44 from humans (e.g., NM_000610.4 and

NP 00060E3), mice (NP_001034240.1), rats (XP_006234689.1), pigs (XP_020940943.1), dogs (NP_001183951.1), and others. [0084] As used herein, the term “CD56” refers to a homophilic binding glycoprotein and having the gene symbol NCAM. CD56 is also known as NCAM (neural cell adhesion molecule) or NCAM1 (neural cell adhesion molecule 1), and is expressed on the surface of neurons, glia and skeletal muscle. CD56 is the archetypal phenotypic marker of natural killer cells but can be expressed by many more immune cells, including alpha beta T cells, gamma delta T cells, dendritic cells, and monocytes. This gene corresponds to GENBANK® Accession Nos. NP_000606.3 (human), NP_001106675.1 (mouse), NC_051343.1 (rat), NP_001010950. 1 (dog), and others.

[0085] As used herein, the term '‘lineage markers” or “Lin” refers to characteristic molecules for cell lineages, e.g., cell surface markers, mRNAs, or internal proteins. In one aspect, “Lin” refers to a panel of markers. As used herein, the FITC anti-human lineage antibody cocktail is optimized for the detection of human peripheral blood T cells, B cells, NK cells, monocytes, and neutrophils. This cocktail is composed of CD3, CD14, CD16, CD19, CD20, and CD56. In another embodiment, the markers in such Lin panel is detected individually.

[0086] As used herein, the term “HLA” refers to human leukocyte antigens also known as the human version of the major histocompatibility complex (MHC) that play important roles in the immune responses. It is believed that HLA-I molecules are expressed on the surface of almost all nucleated cells, while HLA-II molecules are expressed only on B lymphocytes, antigen-presenting cells (e.g., monocytes, macrophages, and dendritic cells), and activated T lymphocytes. In one aspect. HLA-II as disclosed herein include human HLA-DR. DP. DQ. HLA-E is reported to inhibit natural killer (NK) cell-mediated lysis.

[0087] As used herein, the term “hepatic cell lines” refers to cell lines derived from liver. Non-limiting examples of hepatic cell lines include cell lines derived from hepatomas, immortalized hepatocytes, immortal hepatocytes isolated from transgenic animals, hepatocyte/hepatoma hybrid cells, genetically engineered hepatocytes. In one aspect, cell lines derived from hepatomas may include, but are not limited to, HepG2, HepG2.2. 15, HLE, HLF. HuH-7, Hep3B, PLC/PRF-5, SNU182, SNU354, SNU368, SNU387. SNU398.

SNU423, SNU449, SNU475, and HepaRG. In another aspect, hepatic cell lines may also be generated via immortalization of hepatocytes. In one embodiment, immortalized hepatocytes may include hepatocytes generated via transformation with virus genes or oncogenes (i.e., simian virus SV40 large T antigen, c-myc. cH-ras). or via transfection using recombinant plasmids. In another embodiment, immortal hepatocytes may be isolated from transgenic animals expressing viral transforming genes, oncogenes or growth factors. In yet another embodiment, hepatocyte/hepatoma hybrid cells may be generated that remain permanently growing while expressing adult hepatic enzymes. In another embodiment, hepatic cell lines also include genetically engineered hepatocytes expressing human drug-metabolizing enzymes.

2. Isolation of Dormant Guide Cells from Adult Tissue

[0088] The instant disclosure demonstrates that high-plasticity stem cells can be generated from cells isolated from adult tissues which have broad clinical applications. As shown in the examples, a population of tiny cells with a diameter of less than 6 microns and in a dormant state (referred to herein as “dormant guide cells.” or “d-GC”) can be isolated from adult tissues (e.g., blood), which upon activation and growth in vitro (hence becoming “activated/mature guide cells,” or “m-GC”) exhibits unique characteristics. One of such unique characteristics, as demonstrated in the examples, is that their interaction with somatic cells can lead to generation of a novel type of adult stem cells with high plasticity. These high-plasticity stem cells (referred to herein as “guide integrated adult stem cells,” or “giaSC”) have the unexpected ability to differentiate across germ layers and to regenerat e/reconstitute various tissues.

[0089] For instance, when differentiated in vitro, the giaSC can develop into neurons (ectoderm lineage), osteocytes (mesoderm lineage), and liver cells (endoderm lineage). In vivo testing show ed that topical transplantation of giaSC enhanced healing rate, new hair grow th, new blood vessel formation, angiogenesis, and full skin regeneration and reconstitution (Example 5).

[0090] These giaSC, as demonstrated in Example 4, are associated with markers that can be used to identify them. For instance, the giaSC can be positive with one or more of CD49f, CD90, SSEA4. CD73, CD44, CD105, CD146, CD52. and CD56. They can also express low- lev els of CD45 and/or CD324 (E-Cadherin). In addition, in some embodiments, the giaSC are negative in CD34, SSEA3, CD3, CD19, and/or CD20. Moreover, the giaSC do not express one or more genes from the group of Lin28, Nanog, and Sox2, and express very low⁷ level of POU5F1 gene. Furthermore, the giaSC can be identified as being positive in HLA-I, negative in HLA-II, and/or having low level of HLA-E. [0091] Also unexpectedly, activated/mature guide cells (m-GC) can interact with various different types of somatic cells to generate giaSC. The instant disclosure also shows transfer of cellular components, for example from m-GC to somatic cells and/or between giaSC, regulating somatic cells or adult stem cells. In Examples 3-6, the giaSC were prepared from the interaction between m-GC and human umbilical cord derived mesenchymal stromal cells (UC-MSCs). As demonstrated in Example 7 in addition, giaSC can be generated via interaction of m-GC with human bone marrow derived mesenchymal stromal cells (BM- MSCs), as well as with human intestinal epithelial cells (lECs). High plasticity was also confirmed for these giaSC, underscoring the universal capability of m-GC to interact with somatic cells and generate high-plasticity stem cells.

[0092] Large-scale genomic analysis (Example 4) shows that both guide cells (d-GC and m- GC) and giaSC are distinct from all cell types as currently identified. Further, giaSC can be expanded in vitro for banking and therapeutic uses. Moreover, giaSC are not considered ethically and politically controversial, and the generation process does not require genetic manipulation. Yet another advantage of the present technology is that giaSC are demonstrated to be non-tumorigenic (Example 6). Therefore, the presently disclosed technology platform will pave the way for practical cell-based therapy for the treatment of tissue damages as well as degenerative diseases.

[0093] In one embodiment, accordingly, this disclosure provides a composition or a cell population enriched with dormant guide cells that (a) have a diameter of less than 6 pm, (b) express CD49f and CD45, (c) do not express CD90, and (d) do not have detectable intracellular esterase activity and/or transcriptomic activity. In some embodiments, the composition or cell population includes a total of at least 100 cells, 1000 cells, 10,000 cells, 100,000 cells, or 1,000,000 cells, and at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of them are dormant guide cells. In some embodiments, the composition or cell population enriched with dormant guide cells are isolated from an adult tissue sample of a human subject. In some embodiments, the composition or cell population further includes cells other than the dormant guide cells, at a percentage of less than about 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%. In some embodiments, the composition or cell population enriched with dormant guide cells are isolated from a blood sample of a human subject. In some embodiments, the composition or cell population enriched with dormant guide cells are isolated from an unmobilized blood sample of a human subject (i. e., the human subject is not treated with mobilizing agents). In some embodiments, the composition further includes blood cells, such as red blood cells, white blood cells, and platelets. In some embodiment, the composition or cell population enriched with dormant guide cells includes blood cells at a percentage of less than about 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%.

[0094] In some embodiments, the composition includes both intact dormant guide cells and broken ones or cell debris. In some embodiments, the ratio of the number of intact dormant guide cells to the cell debris in the composition 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.

[0095] In some embodiments, the dormant guide cells have a diameter of about 1 pm, or alternatively about 1.5 pm, or alternatively about 2 pm, or alternatively about 2.5 pm, or alternatively about 3 pm, or alternatively about 3.5 pm, or alternatively about 4 pm, or alternatively about 4.5 pm, or alternatively about 5 pm, or alternatively about 5.5 pm, or alternatively about 6 pm, or alternatively between about 1-2 pm, or alternatively between about 1-3 pm, or alternatively between about 1-4 pm, or alternatively between about 1-5 pm, or alternatively between about 1-6 pm, or alternatively between about 2-3 pm, or alternatively between about 2-4 pm, or alternatively between about 2-5 pm, or alternatively between about 2-6 pm, or alternatively between about 3-4 pm, or alternatively between about 3-5 pm, or alternatively between about 3-6 pm. or alternatively between about 4-5 pm, or alternatively between about 4-6 pm, or alternatively betw een about 5-6 pm, or alternatively less than 6 pm, or alternatively less than 5 pm, or alternatively less than 4 pm, or alternatively less than 3 pm, or alternatively less than 2 pm.

[0096] In some embodiments, the isolated dormant guide cells have very high nucleuscytoplasm ratio (v/v, also referred to as N:C ratio, or N/C). In some embodiments, the nucleus-cytoplasm ratio of a dormant guide cell as disclosed herein may be at least 0.9, or alternatively at least 0.8. or alternatively at least 0.7, or alternatively at least 0.6.

[0097] In some embodiments, the isolated population that includes dormant guide cells is a heterogeneous cell mixture, including sub-populations of cells characterized by different sets of markers. In some embodiments, the isolated population of dormant guide cells includes a sub-population of CD49F cells. In some embodiments, the isolated population of dormant guide cells includes a sub-population of CD49E/CD90 cells. In some embodiments, the isolated population of dormant guide cells includes a sub-population of CD49f7CD45⁺ cells. In some embodiments, the isolated population of dormant guide cells includes a subpopulation of CD49E/CD457CD90- cells. In some embodiments, the sub-population of CD49f7CD45⁺/CD90“ cells is further characterized as CD44⁺. In some embodiments, the sub-population of CD49 /CD45⁺/CD90“ cells is further characterized as SSEA4-. In some embodiments, the sub-population of CD49f7CD45⁺/CD90“ cells is further characterized as SSEA3-. In some embodiments, the sub-population of CD49E/CD45VCD90- cells is further characterized as CD324-. In some embodiments, the sub-population of CD49f7CD45⁺/CD90“ cells is further characterized as CD73-. In some embodiments, the sub-population of CD4917CD45⁺/CD90- cells is further characterized as Lin-. In some embodiments, the sub-population of CD49E/CD45VCD90- cells is further characterized as CD146-. In some embodiments, the sub-population of CD49f7CD45⁺/CD90- cells expresses low level of CD34. In some embodiments, the sub-population of

CD49f /CD45⁺/CD90- cells expresses low level of CD105. In some embodiments, the subpopulation of CD49f7CD457CD90- cells expresses very low level of POU5F1 gene. In some embodiments, the sub-population of CD49f7CD45⁺/CD90- cells does not express Lin28. In some embodiments, the sub-population of CD49F/CD457CD90- cells does not express Nanog. In some embodiments, the sub-population of CD49f /CD457CD90- cells does not express Sox2. In some embodiments, the sub-population of CD49f7CD457CD90- cells is further characterized as HLA-I⁺. In some embodiments, the sub-population of CD49I7CD45⁺/CD90- cells is further characterized as HLA-II-. In some embodiments, the sub-population of CD49f7CD45⁺/CD90- cells expresses low level of HLA-E. In some embodiments, the sub-population of CD49I7CD457CD90- cells is further characterized as negative in CD3, CD19, CD20, and/or CD56. In some embodiments, the sub-population of CD49f /CD45⁺/CD90- cells is further characterized as positive in one or more markers from the group of CD44, CD 105, HLA-I. and HLA-E. In some embodiments, the sub-population of CD49r /CD457CD90- cells is further characterized as negative in one or more markers from the group of CD73. CD146, SSEA4, SSEA3, CD324, Lin, and HLA-II. In some embodiments, the sub-population of CD49f7CD45⁺/CD90 cells does not express one or more genes from the group of Lin28, Nanog, and Sox2.

[0098] In some embodiments, the dormant guide cells may be analyzed using the cell surface markers/antigen and intracellular markers such as those shown in Table 1, below. In one aspect, the cell surface markers can be analyzed by flow' cytometry or immunofluorescence staining, for example. In another aspect, the intracellular markers (such as Oct4, Lin28, Nanog, and Sox2) can be analyzed by RT-PCR, q-PCR, or RNA sequencing, for example.

Table 1

[0099] In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that expresses one or more early-stage stem cell markers (e.g., CD49f and/or ven,' low level of Oct4). In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that does not express one or more early-stage stem cell markers (e.g., SSEA4. SSEA3, Lin28, Nanog, and/or Sox2). In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that expresses one or more markers of the group of hematopoietic markers (e.g., CD45 and/or low level of CD34). In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that does not express one or more markers of the group of hematopoietic markers (e.g., Lin, and/or individual markers included in the Lin panel). In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that does not express one or more markers of the group of MSC markers (e.g., CD73, CD90, and/or CD146). In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that expresses low level of MSC marker CD 105. In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that expresses a cell surface adhesion molecule CD44. In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that does not express another cell surface adhesion molecule CD324 (E-Cadherin). In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that expresses HLA-I and does not express HLA-II. In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that expresses low level of HLA-E. In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that is positive in one or more markers from the group of CD49f, CD45, CD44. and CD 105. In some embodiments, the isolated population of dormant guide cells as disclosed herein includes a sub-population of cells that is negative in one or more markers from the group of CD90, CD73, CD146, SSEA4, SSEA3, Lin, CD324, Lin28, Nanog, and Sox2. In some embodiments, the isolated population of dormant guide cells as disclosed herein includes two or more sub-populations of cells that express one or more of the markers identified in Table 1. In some embodiment, the sub-populations of cells in the dormant guide cells as disclosed herein express various combinations of markers identified in Table 1.

[0100] In some embodiments, provided is a method of isolating dormant guide cells as disclosed herein from adult human tissue sample. Non-limiting examples of adult human tissue include human peripheral blood, umbilical cord blood, bone marrow, umbilical cord, placenta, adipose tissue, brain, blood vessels, skeletal muscle, skin, teeth, dental pulp, heart, liver, ovarian epithelium, testis, kidney, retina, hair follicles, intestine, lung, spleen, lymph node, thymus, pancreas, bone, ligament, and tendon. In some embodiments, provided is a method of isolating dormant guide cells as disclosed herein from human blood sample. Nonlimiting examples of human blood sample include human peripheral blood and umbilical cord blood. In some embodiments, the dormant guide cells may be isolated from other sources/tissues so long as the tissue contains viable dormant guide cells as disclosed herein. In some embodiments, the dormant guide cells may be isolated from a subject at any age. In some embodiments, the dormant guide cells may be isolated from a subject at any time. In some embodiments, the dormant guide cells can be isolated from animals such as, but not limited to, equine, canine, porcine, bovine, murine, simian, and human.

[0101] In some embodiments, the dormant guide cells can be isolated from the adult human tissue sample by any means that allows for isolation of cells. For example, to isolate the dormant guide cells from human blood sample, the methods as disclosed herein may include removing at least a portion of the red blood cells from the blood sample, and centrifuging the sample to obtain a cell pellet that includes the dormant guide cells. In another example, the methods as disclosed herein may include retrieving cell suspension from adult human tissue samples (e g., by digesting the tissue samples and removing undigested tissue) and centrifuging the cell suspension to obtain a cell pellet that includes the dormant guide cells. In other aspects, the methods may include cell sorting and cell isolation methods based on one or more identifying markers. For example, fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) may be used to sort and isolate the dormant guide cells or a sub-population of the dormant guide cells. In some embodiments, other methods can be used to isolate the dormant guide cells as disclosed herein. Examples of some isolation procedures are provided in Example 1, infra.

[0102] In one aspect, the method of isolation of dormant guide cells comprises (1) removing at least a portion of red blood cells from a blood sample; (2) centrifuging the sample at 5,000xg-15,000xg. and (3) obtaining a cell pellet that comprises the dormant guide cells as disclosed herein. In some aspects, the sample in step (2) may be centrifuged at about 5,000xg, or alternatively at about 5500xg, or alternatively at about 6,000xg, or alternatively at about 6500xg, or alternatively at about 7,000xg, or alternatively at about 7500xg, or alternatively at about 8,000xg, or alternatively at about 8,500xg, or alternatively at about 9,000xg, or alternatively at about 9,500xg or alternatively at about 10,000xg, or alternatively at about 10,500xg, or alternatively at about l EOOOxg, or alternatively at about l l,500xg, or alternatively at about 12,000xg, or alternatively at about 12,500xg, or alternatively at about 13,000xg, or alternatively at about 13,500xg, or alternatively at about 14,000xg, or alternatively at about 14.500xg, or alternatively at about 15,000xg, or alternatively between 6,000xg-14,000xg, or alternatively between 7,000xg-13,000xg, between 8,000xg-12,000xg, or alternatively between 9,000xg-12,000xg.

[0103] In some aspects, the method of isolation of dormant guide cells comprises (1) digesting a tissue sample and removing undigested tissue to obtain a cell suspension; (2) centrifuging the cell suspension at 5,000xg-15,000xg, and (3) obtaining a cell pellet that comprises the dormant guide cells as disclosed herein. In some aspects, the sample in step (2) may be centrifuged at about 5,000xg, or alternatively at about 5500xg, or alternatively at about 6,000xg, or alternatively at about 6500xg. or alternatively at about 7.000xg, or alternatively at about 7500xg, or alternatively at about 8,000xg, or alternatively at about 8,500xg, or alternatively at about 9,000xg, or alternatively at about 9,500xg, or alternatively at about 10,000xg, or alternatively at about 10,500xg, or alternatively at about l l,000xg, or alternatively at about 11.500xg, or alternatively at about 12,000xg, or alternatively at about 12.500xg, or alternatively at about 13.000xg, or alternatively at about 13.500xg, or alternatively at about 14,000xg, or alternatively at about 14,500xg, or alternatively at about 15,000xg, or alternatively between 6,000xg-14,000xg, or alternatively between 7,000xg- 13,000xg, between 8,000xg-12,000xg. or alternatively between 9,000xg-12,000xg.

[0104] In some embodiments, provided are methods of isolating a specific enriched population of dormant guide cells based on the specific marker(s) of the dormant guide cells. For example, the methods may be used to isolate a cell population enriched in CD49f cells (e.g, by FACS or MACS). In another example, the methods may be used to isolate a cell population enriched in CD49f7CD90“ cells. In another example, the methods may be used to isolate a cell population enriched in CD49E /CD45⁺ cells. In another example, the methods may be used to isolate a cell population enriched in CD49f7CD45⁺/CD90“ cells. In another example, the methods may be used to isolate a cell population enriched in

CD49f /CD34⁺/CD90“ cells. In another example, the methods may be used to isolate a cell population enriched in CD49f7CD45⁺/SSEA4“ cells. In another example, the methods may be used to isolate a cell population enriched in CD49F /CD9CF/SSEA4- cells. In another example, the methods may be used to isolate a cell population enriched in CD49E/CD45VCD73- cells. In another example, the methods may be used to isolate a cell population enriched in CD49f7CD45⁺/CD324“ cells. In another example, the methods may be used to isolate a cell population enriched in CD49E/Lin“ cells. In another example, the methods may be used to isolate a cell population enriched in CD49E/CD90-/Lin^_ cells. In another example, the methods may be used to isolate a cell population enriched in CD49f /CD45⁺/Lin“ cells. In another example, the methods may be used to isolate a cell population enriched in CD49f7CD34⁺/Lin“ cells. In another example, the methods may be used to isolate a cell population enriched in CD49L/CD44⁺/Lin cells. In another example, the methods may be used to isolate a cell population enriched in CD49F /CD44 CD90- cells. In another example, the methods may be used to isolate a cell population enriched in CD49f /CD44 /CD324- cells. In another example, the methods may be used to isolate a cell population enriched in CD49I'7CD45 /CD73 cells. In another example, the methods may be used to isolate a cell population enriched in CD49L/CD447CD73- cells. In another example, the methods may be used to isolate a cell population enriched in CD49f /CD73 /Lin- cells. In other embodiments, the methods may be used to isolate a cell population enriched in certain sub-populations of cells having certain sets of specific markers (e.g, various combinations of markers for identifying dormant guide cells as disclosed herein).

[0105] Provided in some embodiments are a method of isolating a sub-population of dormant guide cells that has a diameter of less than 6 pm, which comprises (a) preparing adult tissue in a solution, (b) centrifuging the solution at 5,000xg-15,000xg and obtaining a cell pellet, and (c) enriching CD49f7CD457CD90- cells from the cell pellet. In one aspect, the adult tissue is a blood sample. In one aspect, the adult tissue is a human blood sample, such as peripheral blood. In one aspect, the solution used to prepare the tissue sample include red blood cell lysis buffer or reagent. In one aspect, the centrifugate speed is more than 6,000xg, or alternatively more than 7,000xg, or alternatively more than 8,000xg, or alternatively more than 9,000xg, or alternatively more than 10,000xg, or alternatively more than 1 l.OOOxg, or alternatively more than 12.000xg, or alternatively more than 13,000xg. or alternatively more than 14,000xg. In one aspect, the centrifugate speed is between 6,000xg-14,000xg, or alternatively between 7,000xg-13,000xg, between 8,000xg-12,000xg, or alternatively between 9,000xg-12.000xg. Also provided in an embodiment is a composition that comprises at least 1000 cells, wherein at least 50% of the cells are dormant guide cells that (a) have a diameter of less than 6 pm, (b) express CD49f and CD45, and (c) do not express CD90. In one aspect, the dormant guide cells do not have detectable intracellular esterase activity (e.g, having negative staining for Calcein AM). In one aspect, the dormant guide cells do not have detectable transcriptomic activity (e.g, having negative AO staining for RNA). In one aspect, the composition comprises at least 60%, or alternatively at least 70%, or alternatively at least 80%, or alternatively at least 90% of dormant guide cells. [0106] When seeded in regular cell culture medium (e.g., DMEM with 10% FBS) in vitro, in some embodiments, the dormant guide cells do not proliferate and become senescent in a few days. In one aspect, these dormant guide cells require specific conditions to become activated and mature.

3. Activation and Maturation of Guide Cells

[0107] The instant disclosure further demonstrates that the isolated dormant guide cells can be activated and developed in vitro to reach a mature state (thus becoming "activated/mature guide cells” or “m-GC”) with unique characteristics and functions.

[0108] In one embodiment, provided herein is an activation system that includes compositions and/or systems that promote activation of the dormant guide cells. In some embodiments, the activation system further promotes development and maturation of the guide cells. In some embodiments, the activation system includes a hepatic environment. In one embodiment, the hepatic environment is established in a co-culture system with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. In another embodiment, the hepatic environment includes a medium that is in contact with or has been conditioned with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. In some embodiments, the activation system includes one or more growth factors, cytokines, hormones, cellular components, exosomes, microvesicles, proteins, and other materials. In some embodiments, any other cells, medium, growth factors, cytokines, hormones, cellular components, exosomes, microvesicles, proteins, and other materials that supports activation, development, growth, and/or maturation of the guide cells may be included in the activation system as disclosed herein.

[0109] In some embodiments, the primary hepatocytes can be isolated from animals such as, but not limited to, equine, canine, porcine, bovine, murine, simian, and human. Non-limiting examples of the hepatic cell lines include cell lines derived from hepatomas, immortalized hepatocytes, immortal hepatocytes isolated from transgenic animals, hepatocyte/hepatoma hybrid cells, and genetically engineered hepatocytes. Non-limiting examples of the cell lines derived from hepatomas include HepG2, HepG2.2. 15, HLE, HLF, HuH-7, Hep3B, PLC/PRF- 5, SNU182, SNU354, SNU368, SNU387, SNU398, SNU423, SNU449, SNU475, and HepaRG. In one embodiment, the immortalized hepatocytes include hepatocytes generated via transformation with virus genes or oncogenes (e.g., simian virus SV40 large T antigen, c- myc, cH-ra.s). and hepatocytes generated via transfection using recombinant plasmids. For example, the immortalized hepatocytes may include, but are not limited to THLE-2, THLE-3, L-02 (HL-7702), human hepatocyte lines (HHLs). In one embodiment, the immortal hepatocytes include hepatocytes isolated from transgenic animals expressing viral transforming genes, oncogenes or growth factors. In one embodiment, hepatic cell lines also include hepatocyte/hepatoma hybrid cells generated by fusing hepatocytes and hepatoma cells. In one embodiment, hepatic cell lines also include genetically engineered hepatocytes expressing human drug-metabolizing enzymes. In some aspects, the activation system may include at least one, or alternatively at least two, or alternatively at least three, or alternatively at least four of the above-mentioned cells/cell lines.

[0110] In one aspect, the activation system includes a cell mixture of at least primary' hepatocytes and one or more selected from the hepatic cell lines. In another aspect, the activation system includes at least one selected from the hepatic cell lines. In another aspect, the activation system includes a cell mixture of at least two selected from the hepatic cell lines. In another aspect, the activation system includes a cell mixture of at least three selected from the hepatic cell lines. In another aspect, the activation system includes a cell mixture of at least primary' hepatocytes and HepG2. In another aspect, the activation system includes a cell mixture of at least HepG2. In another aspect, the activation system includes a cell mixture of at least primary' hepatocytes, HepG2 and HepaRG. In another aspect, the activation system includes a cell mixture of at least HepG2 and HepaRG. In another aspect, the activation system includes a cell mixture of at least primary hepatocytes and at least one selected from THLE-2, THLE-3, L-02 (HL-7702), and HHLs. In another aspect, the activation system includes a cell mixture of at least one selected from THLE-2, THLE-3, L- 02 (HL-7702), and HHLs. In another aspect, the activation system includes a cell mixture of at least HepaRG and at least one selected from THLE-2, THLE-3, L-02 (HL-7702), and HHLs. In another aspect, the activation system includes a cell mixture of at least primary hepatocytes and HepaRG. In another aspect, the activation system includes a cell mixture of at least HepaRG. In another aspect, the activation system includes at least primary hepatocytes. In one embodiment, in addition to the activation system, provided is a composition or system that supports growth and/or development of the activated guide cells.

[OHl] In some embodiments, the activation system includes a conditioned medium that is in contact with or has been conditioned with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines, as disclosed herein. In one aspect, the activation system includes a conditioned medium that is in contact with or has been conditioned with the cells or cell mixtures as disclosed herein.

[0112] In some embodiments, the activation system may include one or more growth factors, cytokines, hormones, cellular components, exosomes, microvesicles, and proteins. In one aspect, the activation system includes one or more grow th factors and/or cy tokines released by at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. In some embodiments, the activation system includes one or more growth factors such as, but not limiting to, insulin-like growth factors (IGFs), fibroblast grow th factors (FGFs), hepatocyte grow th factor (HGF), bone morphogenetic proteins (BMPs), and transforming growth factors (TGFs). In some embodiments, the activation system includes one or more cytokines such as, but not limiting to, CXCL1, CXCL2. CXCL3, CXCL5, CX3CL2. CCL2, IL6, IL8, IL15, albumin, ANXA1, CSF3, TNFSF10, PVR, and ULBP2. In some embodiments, the activation system includes one or more cytokines from the group of CXCL1, CXCL2, CXCL3. and CXCL5. In some embodiments, the activation sy stem includes CCL2. In some embodiments, the activation system includes IL6. In some embodiments, the activation system includes IL8. In some embodiments, the activation system includes IL15. In some embodiments, the activation system includes albumin. In some embodiments, the activation system includes IL6. In some embodiments, the activation system includes one or more cytokines from the group of ANXA1, CSF3, TNFSF10, PVR, and ULBP2. In one aspect, the activation system includes serum. In another aspect, the activation system is serum-free.

[0113] In some embodiments, the cell culture medium and/or cell culture conditions used in the activation system 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 system may be used to culture one or more of cells selected from the group consisting of embryonic stem (ES) cells, hematopoietic stem cells (HSCs). mesenchymal stem/stromal 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). [0114] In one embodiment, provided herein is a method of promoting activation of the dormant guide cells in vitro. In some embodiment, also provided is a method of promoting maturation of the activated guide cells in vitro. In some embodiment, provided herein is a method of promoting activation and/or maturation of the dormant guide cells in the activation system as disclosed herein. In one aspect, the dormant guide cells are cultured in the activation system for an effective period of time. In some aspects, the culturing time effective for promoting activation and/or maturation of the guide cells may include, but not limited to, at least 1 hour, or alternatively at least 2 hours, or alternatively at least 4 hours, or alternatively at least 12 hours, or alternatively at least 1 day, or alternatively at least 2 days, or alternatively at least 3 days, or alternatively at least 4 days, or alternatively at least 5 days, or alternatively at least 8 days, or alternatively at least 10 days, or alternatively at least 12 days, or alternatively at least 15 days, or alternatively at least 18 days, or alternatively at least 20 days, or alternatively at least 25 days, or alternatively at least 30 days. In some aspects, the culturing time effective for promoting activation and/or maturation of the guide cells may include, but not limited to, between approximately 1 day and 30 days, or alternatively between approximately 5 days and 30 days, or alternatively between approximately 10 days and 30 days, or alternatively between approximately 15 days and 30 days, or alternatively between approximately 5 days and 25 days, or alternatively between approximately 10 days and 25 days, or alternatively between approximately 15 days and 25 days. The cell culture medium may be changed even' 1 day, or alternatively every 2 days, or alternatively every 3 days, or alternatively every 4 days, or alternatively every 5 or more days.

[0115] In one aspect, provided herein is a method of promoting activation and/or maturation of the dormant guide cells in a co-culture system with the cells or cell mixture as disclosed herein. In some embodiments, the co-culture system may be prepared using Transwell plates. For example, cells or a cell mixture of the above-mentioned cells/cell lines can be prepared and treated with Mitomycin C to mitotically inactivate the cells. Then the cells/cell mixture can be seeded on the bottom of cell culture plates in a co-culture medium. The isolated population of dormant guide cells can be seeded on the Transwell membranes to be cocultured with the cells/cell mixture. The co-culture medium may include DMEM medium with 5-50% of FBS (fetal bovine serum). For example, the co-culture medium may include DMEM medium with about 5%, or alternatively about 10%, or alternatively about 15%, or alternatively about 20%, or alternatively about 25%, or alternatively about 30%, or alternatively about 35%, or alternatively about 40%, or alternatively about 45%, or alternatively about 50% of FBS. In another embodiment, the co-culture medium may include medium with human serum or serum derived from other animals. In another embodiment, the co-culture medium may include medium without serum. In some embodiments, other culture medium may be used in the methods as disclosed herein. Optionally other reagents and factors may be added to the co-culture medium.

[0116] In another aspect, provided herein is a method of promoting activation and/or maturation of the dormant guide cells using conditioned medium. In one aspect, the conditioned media includes a medium that is in contact with or has been conditioned with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. For example, the above-mentioned cells/cell mixture can be suspended in cell culture medium and then seeded in a cell culture dish/plate. The cell culture medium for culturing the cells/cell mixture may include DMEM with 5-50% of FBS. For example, the medium may include DMEM with about 5%, or alternatively about 10%, or alternatively about 15%, or alternatively about 20%, or alternatively about 25%, or alternatively about 30%, or alternatively about 35%, or alternatively about 40%, or alternatively about 45%, or alternatively about 50% of FBS. In another embodiment, the co-culture medium may include medium with human serum or serum derived from other animals. In another embodiment, the cell culture medium for culturing the cells/cell mixture may include medium without serum. In some embodiments, other culture medium may be used in the methods as disclosed herein. Optionally other reagents and factors may be added to the medium. The conditioned medium can be collected from the cell culture dishes/plates, and the remaining cells in the collected conditioned medium can be removed (e g., via centrifugation and/or filtering) before use of the conditioned medium to culture the dormant guide cells. In one aspect, the collected conditioned medium may be directly used to culture the dormant guide cells as disclosed herein. In another aspect, the collected conditioned medium may be mixed with the above-mentioned co-culture medium, for culturing the dormant guide cells. For example, based on the total volume of the medium mixture, the conditioned medium as disclosed herein may be about 5% (v/v), or alternatively about 10%, or alternatively about 15%, or alternatively about 20%, or alternatively about 25%, or alternatively about 30%, or alternatively about 35%. or alternatively about 40%, or alternatively about 45%, or alternatively about 50%, or alternatively about 55%, or alternatively about 60%, or alternatively about 65%, or alternatively about 70%, or alternatively about 75%, or alternatively about 80%, or alternatively about 85%, or alternatively about 90%, or alternatively about 95%. Optionally other reagents and factors may be added to the mixture of medium. Examples of some activation and development procedures are provided in Example 2, infra.

[0117] In some embodiments, provided herein is a method of promoting activation and/or maturation of the dormant guide cells using one or more of growth factors, cytokines, hormones, cellular components, exosomes, microvesicles, and proteins. In one aspect, the methods include contacting the dormant guide cells with one or more growth factors and/or cytokines released by at least one selected from the group consisting of pnmary hepatocytes and hepatic cell lines, as disclosed herein. In one aspect, the methods include contacting the dormant guide cells with one or more grow th factors and/or cytokines of the activation system, as disclosed herein. In some embodiments, the methods of activation and/or maturation as disclosed herein may be applied separately or in combination thereof, and maybe applied concurrently or sequentially.

[0118] In some embodiments, provided are methods for detecting the activation of dormant guide cells by detecting cellular activity. In one example, the activated guide cells are stained positive for a fluorescent dye that detects intracellular esterase activity (e.g., Calcein AM, Calcein Red- AM, Calcein Red-Orange, AM), while dormant guide cells do not have detectable intracellular activity and thus show negative staining. In yet another example, acridine orange (AO) is a cell-permeant nucleic acid binding dye that emits green fluorescence when bound to DNA and red fluorescence when bound to RNA. Dormant guide cells mainly show green AO staining of the DNA in the nucleus and negative staining of the RNA, indicating undetectable transcriptomic activity. While the activated guide cells have RNA expression (transcriptomic activity) and thus show red signals for RNA in the cytoplasm and green or yellow nucleus for DNA. In some embodiments, other dyes or markers can be used to detect activation of dormant guide cells.

[0119] In some embodiments, provided are methods for detecting the maturation of activated guide cells by observing and/or measuring cellular components. In one example, the activated guide cells are cultured and developed in vitro (e.g., in the activation system or other condition/medium that facilitate maturation) and show changes in size and morphology. In some embodiments, the appearance of granules aggregated in the cytoplasm of guide cells indicates maturation. For example, the granules may be rich in RNA by AO staining. In some embodiments, guide cells at mature state also extend one or more pseudopodia from the cell surface. In some embodiments, other dyes or markers can be used to detect maturation of guide cells.

[0120] In some embodiments, the dormant guide cells as disclosed herein, upon activation, undergo a development and maturation process in vitro (thus becoming activated/mature guide cells or m-GC). In one aspect, the development and maturation process occurs in the activation system as disclosed herein. In one aspect, the development and maturation process takes more than 5 days, or alternatively more than 10 days, or alternatively more than 15 days, or alternatively more than 20 days, or alternatively more than 25 days, or alternatively more than 30 days, or alternatively between about 5 and about 30 days, or alternatively between about 10 and about 30 days, or alternatively between about 15 and about 30 days, or alternatively between about 5 and about 25 days, or alternatively between about 10 and about 25 days, or alternatively between about 15 and about 25 days. In some embodiments, the activated/mature guide cells have limited proliferation capacity. In one aspect, the activated/mature guide cells have low expression of cyclin DI (CCND1), MYC, and/or other proliferation-related genes.

[0121] In some embodiments, the size of guide cells enlarges during the development and maturation process. In one aspect, the size of an activated/mature guide cell is more than 10 pm, or alternatively more than 15 pm, or alternatively more than 20 pm, or alternatively more than 25 pm, or alternatively more than 30 pm, or alternatively more than 35 pm, or alternatively more than 40 pm, or alternatively between about 10 and about 40 pm, or alternatively between about 15 and about 40 pm, or alternatively between about 20 and about 40 pm, or alternatively between about 25 and about 40 pm, or alternatively between about 20 and about 35 pm, or alternatively between about 25 and about 35 pm.

[0122] In some embodiments, the morphology of guide cells changes during the development and maturation process. In one aspect, the activated/mature guide cells have aggregated granules in the cytoplasm. In another aspect, the aggregated granules are located in the center region of the activated/mature guide cells. In another aspect, the aggregated granules includes RNA (e.g., indicated by AO staining). In another aspect, the aggregated granules includes one or more of cellular components such as, but not limited to, polynucleotide, peptide, polypeptide, protein, chemicals, and lipids. In another aspect, the activated/mature guide cells have one or more pseudopodia extending from the cell surface. In another aspect, the activated/mature guide cells can release cellular components such as the granules as disclosed herein via the one or more pseudopodia. In some embodiments, the activated/mature guide cells die in a period of time after releasing cellular components (e.g., the RNA-rich granules). In one aspect, the activated/mature guide cells die in about 10 days, or alternatively in about 7 days, or alternatively in about 5 days, or alternatively in about 3 days after their cellular components are released.

[0123] In one embodiment, after culturing the dormant guide cells in the activation system for a period of time (e.g. , 5-20 days), one or more cell colonies are developed. The dormant guide cells cultured in regular medium (e.g., DMEM medium with 10% FBS) do not propagate and grow, and do not develop into cell colonies, in some aspects. In some embodiments, the cell colonies in the activation system includes high urity of activated/mature guide cells. In some embodiments, after a plurality of medium changes, the activated/mature guide cells constitute a highly enriched population. In one aspect, the percentage of activated/mature guide cells in the cell colonies and/or enriched population developed in the activation system is more than 50%, or alternatively more than 60%, or alternatively more than 70%, or alternatively more than 80%, or alternatively more than 90%, or alternatively more than 95%. In some embodiments, the cell colonies and/or enriched population in the activation system includes substantially homogeneous population of activated/mature guide cells. In some embodiments, provided herein are methods of isolating a cell colony of activated/mature guide cells from the activation system as disclosed herein. For example, a cell colony can be picked up using a cloning cylinder. Alternatively, a cell colony can be isolated by trypsinizing and detaching the colony from the cell culture plate/dish. It should be understood that other methods can also be employed to isolate the cell colonies.

[0124] Embodiments of the disclosure also provide a composition or population of activated/mature guide cells derived from the dormant guide cells as disclosed herein. The activated/mature guide cells can be obtained by culturing the dormant guide cells in the above-mentioned activation system for an effective period of time as disclosed herein. In some embodiments, the activated/mature guide cells may be obtained by culturing the dormant guide cells in other culture systems, culture medium, or conditions for an effective period of time. In some embodiments, also provided are compositions or populations of cells derived from the activated/mature guide cells as disclosed herein. In some aspects, the compositions as disclosed herein also includes a pharmaceutically acceptable carrier or excipient. In one aspect, the activated/mature guide cells can undergo modification, induction, manipulation and other processes to generate cells that have one or more features different from the activated/mature guide cells.

[0125] Also provided herein, in some embodiments, is a population of activated/mature guide cells, characterized as CD49E/CD45⁺/CD90-. In some embodiments, the population of activated/mature guide cells are generated by activating dormant guide cells as disclosed herein. Also provided herein, in some embodiments, is an isolated activated/mature guide cell, characterized with CD49E/CD45⁺/CD90- and comprises RNA-rich granules in the cytoplasm and one or more pseudopodia. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as CD44⁺. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as SSEA4-. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as SSEA3-. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as CD324-. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as CD73-. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as CD105⁺. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as CD52⁺. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as Lin-. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as CD146-. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as CD56-. In some embodiments, the activated/mature guide cell, as disclosed herein, is further characterized as HLA-I⁺. In some embodiments, the activated/mature guide cell, as disclosed herein, expresses low level of HLA-II. In some embodiments, the activated/mature guide cell, as disclosed herein, expresses low level of HLA-E. In some embodiments, the activated/mature guide cell, as disclosed herein, expresses very low level of CD34. In some embodiments, the activated/mature guide cell, as disclosed herein, expresses very low level of POU5F1 gene. In some embodiments, the activated/mature guide cell, as disclosed herein, does not express Lin28. In some embodiments, the activated/mature guide cell, as disclosed herein, does not express Nanog. In some embodiments, the activated/mature guide cell, as disclosed herein, does not express Sox2. In some embodiments, the activated/mature guide cell, as disclosed herein, does not express one or more markers selected from the group consisting of CD3, CD 19, and CD20. In some embodiments, provided is an activated/mature guide cell characterized as positive in one or more markers selected from the group consisting of CD49f, CD45, CD44, CD105, CD52, HLA-I, HLA-II, and HLA-E. In some embodiments, provided is an activated/mature guide cell characterized as negative in one or more markers selected from the group consisting of CD90, Lin, CD73, CD146, SSEA4, SSEA3, CD324, CD3, CD19, CD20, CD56, Lin28, Nanog, and Sox2. In some embodiments, the activated/mature guide cell, as disclosed herein, expresses genes and/or proteins associated with tunneling nanotube (TNT) formation and/or cell-cell interactions.

[0126] In some embodiments, provided herein are methods of isolating cellular components of the activated/mature guide cells, as disclosed herein. In one aspect, the isolated cellular components include RNA and/or RNA-rich granules. In another aspect, the isolated cellular components include microvesicles and/or exosomes. In another aspect, the isolated microvesicles and/or exosomes include RNA. In one embodiment, the cellular components as disclosed herein can be isolated from the culture medium of activated/mature guide cells, or can be isolated by digesting the activated/mature guide cells. It should be understood that other methods can also be employed to isolate the cellular components as disclosed herein. Embodiments of the disclosure also provide compositions including cellular components isolated from the activated/mature guide cells, as disclosed herein.

4. Generation of High-Plasticity Stem Cells (giaSC) via Interaction Between Guide Cells and Somatic Cells

[0127] The instant disclosure further demonstrates that the activated/mature guide cells can interact with somatic cells, leading to generation of a novel type of high-plasticity stem cells (also referred to herein as “guide integrated adult stem cell.” or “giaSC”).

[0128] In some embodiments, provided herein are methods of generating high-plasticity⁷ stem cells (e.g, giaSC) that comprises contacting somatic cells with guide cells and/or cellular components thereof, as disclosed herein. In one aspect, as a result of the contacting, cell-cell interaction is established that allows transfer of the cellular components (e.g., RNAs or RNA- rich granules) from the guide cells into the somatic cells. In some aspects, the cellular components are transferred via tunneling nanotubes (TNTs), gap junction (GJ), and/or exocytosis and endocytosis. In another aspect, the cellular components as disclosed herein are transferred into the somatic cells via other routes. [0129] In one embodiment, the cell-cell interaction is established in a co-culture system of the somatic cells and the activated/mature guide cells as disclosed herein. In one aspect, the activated/mature guide cells and the somatic cells have a ratio of cell numbers that is at least about 1: 1, or alternatively at least about 2: 1. or alternatively at least about 3: 1, or alternatively at least about 4: 1, or alternatively at least about 5: 1, or alternatively at least about 6: 1, or alternatively at least about 7:1, or alternatively at least about 8: 1, or alternatively at least about 9: 1 , or alternatively at least about 10: 1.

[0130] In another embodiment, the cellular components are isolated from the activated/mature guide cells and transferred to the somatic cells as disclosed herein. In one aspect, the isolated cellular components as disclosed herein are added to the culture medium of the somatic cells. In another aspect, the isolated cellular components as disclosed herein are delivered to a vicinity of the somatic cells. In one aspect, the isolated cellular components as disclosed herein are transferred into the somatic cells via endocytosis.

[0131] In some embodiments, the cell-cell interaction as disclosed herein is established in the activation system, as disclosed herein, for an effective period of time as disclosed herein. For example, the cell-cell interaction of somatic cells and activated/mature guide cells can be established in a hepatic environment as disclosed herein. In one aspect, the cell-cell interaction of somatic cells and activated/mature guide cells is established in a co-culture system with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines as disclosed herein. In another aspect, the cell-cell interaction of somatic cells and activated/mature guide cells is established in a conditioned medium that is in contact with or has been conditioned with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. In another aspect, the cell-cell interaction of somatic cells and activated/mature guide cells is established in a medium that includes one or more of the grow th factors and/or cytokines in the activation system as disclosed herein. In some aspects, the cell-cell interaction time effective for generating giaSC may include, but not limited to, at least 3 hours, or alternatively at least 6 hours, or alternatively at least 9 hours, or alternatively at least 12 hours, or alternatively at least 1 day, or alternatively at least 2 days, or alternatively at least 3 days, or alternatively at least 4 days, or alternatively at least 5 days. The cell culture medium may be changed every 1 day, or alternatively every 2 days, or alternatively every 3 or more days. Examples of some procedures to generate giaSC are provided in Example 3, infra. [0132] In one embodiment somatic cells used to generate giaSC include stem cells as disclosed herein. In one aspect, somatic cells used to generate giaSC include adult stem cells as disclosed herein. In one aspect, somatic cells used to generate giaSC include mesenchymal stem/stromal cells. In one aspect, the mesenchymal stem/stromal cells as disclosed herein can be derived from various types of adult tissue, such as umbilical cord, cord blood, bone marrow, adipose tissue, placenta, dental tissues, and other adult tissue. In another embodiment, somatic cells used to generate giaSC include non-stem cells. In another embodiment, somatic cells used to generate giaSC have proliferative capability.

[0133] Non-limiting example of somatic cells as disclosed herein include cells that make up all the internal organs (e.g., heart, liver, lungs, spleen, kidney, stomach, intestines), brain, skin, muscle, bones, blood, and connective tissue. In some embodiments, somatic cells that are used to generate giaSC are derived from one or more of organs such as brain, bones, bone marrow, colon, ears, eyes, heart, hair follicle, kidneys, joints, liver, lungs, lymph nodes, large intestine, mouth, mammary glands, nose, nerves, nasal cavity, ovaries, penis, pancreas, placenta, prostate, skin, spleen, stomach, spinal cord, small intestine, skeletal muscles, teeth, testes, tendons, tongue, thyroid, uterus, and veins. In some embodiments, somatic cells that are used to generate giaSC include over 220 types of somatic cells. In some embodiments, somatic cells that are used to generate giaSC are derived from one or more of organs such as arteries, appendix, adrenal glands, anus, brain, bones, bronchi, bladder, bone marrow, bulbourethral glands, colon, cervix, clitoris, capillaries, cerebellum, diaphragm, ears, eyes, fallopian tubes, genitals, gallbladder, heart, hair follicle, hypothalamus, interstitium, kidneys, joints, liver, lungs, larynx, ligaments, lymph nodes, large intestine, lymphatic vessel, mouth, mesentery, mammary glands, nose, nerves, nasal cavity⁷, ovaries, oesophagus/esophagus, penis, pancreas, pharynx, placenta, prostate, pineal gland, pituitary gland, parathyroid glands, rectum, skin, spleen, scrotum, stomach, spinal cord, small intestine, salivary glands, skeletal muscles, seminal vesicles, subcutaneous tissue, teeth, tonsils, testes, tendons, tongue, thyroid, trachea, thymus gland, ureters, urethra, uterus, vulva, veins, vagina, vas deferens, and vestigial organ. In one aspect, somatic cells that are used to generate giaSC are derived from heart. In another aspect, somatic cells that are used to generate giaSC are derived from liver. In another aspect, somatic cells that are used to generate giaSC are derived from lungs. In another aspect, somatic cells that are used to generate giaSC are derived from spleen. In another aspect, somatic cells that are used to generate giaSC are derived from kidney. In another aspect, somatic cells that are used to generate giaSC are derived from stomach. In another aspect, somatic cells that are used to generate giaSC are derived from intestines. In another aspect, somatic cells that are used to generate giaSC are derived from brain. In another aspect, somatic cells that are used to generate giaSC are derived from skin. In another aspect, somatic cells that are used to generate giaSC are derived from muscle. In another aspect, somatic cells that are used to generate giaSC are derived from bone marrow. In another aspect, somatic cells that are used to generate giaSC are derived from spinal cord. In another aspect, somatic cells that are used to generate giaSC are derived from pancreas. In another aspect, somatic cells that are used to generate giaSC are derived from blood. In another aspect, somatic cells that are used to generate giaSC are derived from umbilical cord. In another aspect, somatic cells that are used to generate giaSC are derived from placenta. In another aspect, somatic cells that are used to generate giaSC are derived from adipose tissue. In some embodiments, somatic cells used to generate giaSC includes a mixture of cells comprising any combination of the somatic cells as disclosed herein.

[0134] In some embodiments, provided herein is a composition or population of giaSC generated via interaction between somatic cells and activated/mature guide cells as disclosed herein. In one aspect, the giaSC may be obtained by co-culturing the somatic cells and activated/mature guide cells, as disclosed herein, for an effective period of time. In another aspect, the giaSC may be obtained by co-culturing the somatic cells and activated/mature guide cells in a hepatic environment, as disclosed herein, for an effective period of time. In one aspect, the giaSC may be obtained by culturing the somatic cells with cellular components isolated from the activated/mature guide cells, as disclosed herein, for an effective period of time. In another aspect, the giaSC may be obtained by culturing the somatic cells with cellular components isolated from the activated/mature guide cells in a hepatic environment, as disclosed herein, for an effective period of time. In another aspect, the giaSC may be obtained by culturing the somatic cells and activated/mature guide cells in other culture systems, culture medium, or conditions for an effective period of time. In some embodiments, also provided is a composition of cells differentiated from giaSC. In some aspects, the compositions as disclosed herein also include a pharmaceutically acceptable carrier or excipient.

[0135] Also provided herein, in some embodiments, is an isolated giaSC characterized as CD49f7CD90⁺/CD34“. In some embodiments, the giaSC, as disclosed herein, has differentiation potential for more than one germ layer. In some embodiments, the giaSC, as disclosed herein, is further characterized as CD45^low. In some embodiments, the giaSC, as disclosed herein, is further characterized as CD324^low. In some embodiments, the giaSC, as disclosed herein, is further characterized as SSEA4⁺. In some embodiments, the giaSC, as disclosed herein, is further characterized as SSEA3-. In some embodiments, the giaSC, as disclosed herein, is further characterized as CD73⁺. In some embodiments, the giaSC, as disclosed herein, is further characterized as CD44⁺. In some embodiments, the giaSC, as disclosed herein, is further characterized as CD105⁺. In some embodiments, the giaSC, as disclosed herein, is further characterized as CD146⁺. In some embodiments, the giaSC, as disclosed herein, is further characterized as CD56⁺. In some embodiments, the giaSC, as disclosed herein, is further characterized as CD52⁺. In some embodiments, the giaSC, as disclosed herein, is further characterized as HLA-I⁺. In some embodiments, the giaSC, as disclosed herein, is further characterized as HLA-ll . In some embodiments, the giaSC. as disclosed herein, further expresses low level of HLA-E. In some embodiments, the giaSC, as disclosed herein, further expresses very low level of POU5F1 gene. In some embodiments, the giaSC, as disclosed herein, does not express Lin28. In some embodiments, the giaSC, as disclosed herein, does not express Nanog. In some embodiments, the giaSC, as disclosed herein, does not express Sox2. In some embodiments, the giaSC, as disclosed herein, further expresses low level of CD45. In some embodiments, the giaSC, as disclosed herein, further expresses low level of CD324. In some embodiments, the giaSC. as disclosed herein, is further characterized as CD3-, CD19-, and/or CD20-. In some embodiments, provided is a giaSC characterized as positive in one or more markers from the group of CD49f, CD90, CD73, CD105. SSEA4, CD146, CD44, CD45, CD324, CD56, CD52, HLA-I, and HLA-E. In some embodiments, provided is a giaSC characterized as negative in one or more markers from the group of CD34, SSEA3, HLA-II, CD3, CD19, CD20, Lin28, Nanog, and Sox2. In some embodiments, the giaSC, as disclosed herein, expresses genes and/or proteins associated with tunneling nanotube (TNT) formation and/or cell-cell interactions. In some aspects, the acquired high plasticity comprises active adaptation to host environment to regenerate and/or reconstitute tissue when transplanted in vivo. In some embodiments, the giaSC, as disclosed herein, are non-tumorigenic.

[0136] In some embodiments. giaSC are characterized as CD49f7CD73⁺/CD34 . In some aspects, the giaSC, as disclosed herein, has differentiation potential for more than one germ layer. In some aspects, the giaSC, as disclosed herein, is further characterized as CD45^low. In some aspects, the giaSC, as disclosed herein, is further characterized as CD324^low In some aspects, the giaSC, as disclosed herein, is further characterized as SSEA4⁺. In some aspects, the giaSC, as disclosed herein, is further characterized as SSEA3-. In some aspects, the giaSC, as disclosed herein, is further characterized as CD90⁺. In some aspects, the giaSC, as disclosed herein, is further characterized as CD44⁺. In some aspects, the giaSC, as disclosed herein, is further characterized as CD105⁺. In some aspects, the giaSC, as disclosed herein, is further characterized as CD146⁺. In some aspects, the giaSC. as disclosed herein, is further characterized as CD56⁺. In some aspects, the giaSC, as disclosed herein, is further characterized as CD52⁺. In some aspects, the giaSC, as disclosed herein, is further characterized as HLA-I⁺. In some aspects, the giaSC, as disclosed herein, is further characterized as HLA-II-. In some aspects, the giaSC, as disclosed herein, further expresses low level of HLA-E. In some aspects, the giaSC, as disclosed herein, further expresses very low level of POU5F1 gene. In some aspects, the giaSC, as disclosed herein, does not express Lin28 gene. In some aspects, the giaSC, as disclosed herein, does not express Nanog gene. In some aspects, the giaSC, as disclosed herein, does not express Sox2 gene. In some aspects, the giaSC, as disclosed herein, further expresses low level of CD45. In some aspects, the giaSC, as disclosed herein, further expresses low level of CD324. In some aspects, the giaSC, as disclosed herein, is further characterized as CD3-, CD19-, and/or CD20“.

[0137] In some embodiments. giaSC are characterized as CD49f /SSEA4 /CD34 . In some embodiments, giaSC are characterized as CD49E/CD324^low/CD34-. In some embodiments, giaSC are characterized as CD49f7CD I46 /CD34 In some embodiments, giaSC are characterized as CD49E/CD45^low/CD34-. In some embodiments, giaSC are characterized as CD49f7CD105⁺/CD34“.

[0138] In some embodiments, the giaSC, as disclosed herein, expresses markers and/or genes that are not expressed by the guide cells (d-GC and/or m-GC). In some embodiments, the giaSC, as disclosed herein, does not express markers and/or genes that are expressed by the guide cells (d-GC and/or m-GC). In some embodiments, the giaSC, as disclosed herein, has expression level of one or more markers that is different from the expression level in the guide cells (d-GC and/or m-GC). In some embodiments, the giaSC, as disclosed herein, express markers and/or genes that are not expressed by the somatic cell that is used to generate the giaSC. In some embodiments, the giaSC, as disclosed herein, has expression level of one or more markers that is different from the expression level in the somatic cell that is used to generate the giaSC.

[0139] In some embodiments, the giaSC, as disclosed herein, has unique morphology. In one aspect, the giaSC has an approximately triangular cell body with one or more slender pseudopodia as an adherent cell in culture. In one aspect, the size of cell body of giaSC is more than 10 pm, or alternatively more than 15 pm, or alternatively more than 20 pm, or alternatively more than 25 pm, or alternatively more than 30 pm. or alternatively more than 35 pm, or alternatively more than 40 pm, or alternatively between about 10 and about 40 pm, or alternatively between about 15 and about 40 pm, or alternatively between about 20 and about 40 pm. or alternatively between about 25 and about 40 pm. or alternatively between about 20 and about 35 pm. or alternatively between about 25 and about 35 pm.

[0140] In one aspect, the giaSC, as disclosed herein, can interact with another giaSC and exchange a plurality of cellular components. In another aspect, the giaSC, as disclosed herein, can interact with different types of cells and exchange or transfer cellular components. In another aspect, the giaSC, as disclosed herein, can interact with other stem cells as disclosed herein. In another aspect, the giaSC, as disclosed herein, can interact with nonstem cells as disclosed herein. In some aspects, the exchanged or transferred cellular components include RNA. In some aspects, the giaSC, as disclosed herein, can interact with a cell of the same kind or a different kind via tunneling nanotubes, gap junction, and/or exocytosis/endocytosis. In another aspect, the giaSC, as disclosed herein, can interact with adjacent cellular and/or non-cellular components.

[0141] Embodiments of the disclosure provide compositions including cellular components isolated from the giaSC, as disclosed herein. In one aspect, the isolated cellular components include RNA and/or RNA-rich granules. In another aspect, the isolated cellular components include microvesicles and/or exosomes. In another aspect, the isolated microvesicles and/or exosomes include RNA. In some embodiments, provided herein are methods of isolating cellular components of the giaSC as disclosed herein. In one embodiment, the cellular components as disclosed herein can be isolated from the culture medium of the giaSC, or can be isolated by digesting the giaSC. It should be understood that other methods can also be employed to isolate the cellular components as disclosed herein. [0142] In some embodiments, the giaSC, as disclosed herein, expresses a plurality of markers of more than one germ layer. In one aspect, the giaSC, as disclosed herein, express a plurality⁷ of markers of all three germ layers. In some embodiment, the giaSC, as disclosed herein, express one or more of endoderm markers, one or more of mesoderm markers, and one or more of ectoderm markers. In another aspect, the giaSC, as disclosed herein, express one or more of endoderm markers and one or more of mesoderm markers. In some embodiment, the giaSC, as disclosed herein, express one or more of endoderm markers and one or more of ectoderm markers. In some embodiment, the giaSC, as disclosed herein, express one or more of mesoderm markers and one or more of ectoderm markers. Nonlimiting examples of ectoderm markers include nestin (NES), notch-1, notch-2, MSI2, CD56 (NCAM1), and CD325 (N-cadherin or Cadherin-2). Non-limiting examples of mesoderm markers include desmin (DES), osteocalcin (BGLAP), CD106 (VCAM-1), CD54 (ICAM-1), and CD146 (MCAM). Non-limiting examples of endoderm markers include AFP, CK7 (KRT7), albumin (ALB), CK18 (KRT18), and CK19 (KRT19). In some embodiments, the giaSC, as disclosed herein, do not cause tumor formation when transplanted in vivo. In contrast, ESCs and iPSCs are tumorigenic and can cause tumor (e.g., teratoma).

[0143] In some embodiments, the giaSC, as disclosed herein, can proliferate in vitro. In one aspect, the doubling time for the giaSC, as disclosed herein, is more than 60 hours, or alternatively less than 60 hours, or alternatively less than 50 hours, or alternatively less than 45 hours, or alternatively less than 40 hours, or alternatively less than 35 hours, or alternatively less than 30 hours, or alternatively between about 30 and about 60 hours, or alternatively between about 30 and about 50 hours, or alternatively between about 30 and about 45 hours, or alternatively between about 35 and about 60 hours, or alternatively between about 35 and about 50 hours, or alternatively between about 40 and about 50 hours. In some aspects, the giaSC, as disclosed herein, can expand for a plurality of passages, for example more than 5 passages, or alternatively more than 8 passages, or alternatively more than 10 passages, or alternatively more than 12 passages.

[0144] Embodiments of the disclosure provide compositions or systems that support the expansion of the giaSC as disclosed herein. In some embodiments, the giaSC can expand in the activation system as disclosed herein. In some embodiments, the giaSC can expand in the hepatic environment as disclosed herein. In one aspect, the compositions or systems for expanding the giaSC, as disclosed herein, include cell culture medium. In another aspect, the compositions or systems for expanding the giaSC. as disclosed herein, include serum. In another aspect, the compositions or systems for expanding the giaSC, as disclosed herein, are serum-free. In some embodiments, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more growth factors such as, but not limiting to, insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), hepatocyte grow th factor (HGF), bone morphogenetic proteins (BMPs), transforming growth factors (TGFs), tumor necrosis factors (e.g., TNFa, TNFP). In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more growth factors selected from the group of IGF1, IGF2, FGF2, HGF, and BMP5. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more growth factors selected from the group of IGF1, IGF2, FGF2, and HGF. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more growth factors selected from the group of IGF2, FGF2, HGF, and BMP5. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more growth factors selected from the group of IGF 1, FGF2, HGF, and BMP5. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more growth factors selected from the group of FGF2, HGF, and BMP5. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more growth factors selected from the group of IGF1, FGF2, and HGF. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more growth factors selected from the group of IGF1, FGF2, and BMP5. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include FGF2 and/or HGF. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include at least FGF2. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include at least HGF. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include at least IGF1. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include at least IGF2. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include at least BMP5. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more cytokines as disclosed herein. In some aspects, the compositions or systems for expanding the giaSC, as disclosed herein, include one or more of chemicals, growth factors, cytokines, hormones, cellular components, exosomes, microvesicles, extracellular matrix, and some materials that would facilitate the expansion of giaSC. In some embodiments, the giaSC can expand in other cell culture systems and/or under other culture conditions in vitro. Also provided, in some embodiments, are methods of culturing the giaSC, as disclosed herein, in the compositions or systems as disclosed herein. Also provided, in some embodiments, are methods of culturing the giaSC, as disclosed herein, in the activation system as disclosed herein. In some aspects, provided are methods of culturing the giaSC, as disclosed herein, in medium supplemented with one or more of the grow th factors as disclosed herein.

[0145] In some embodiments, the giaSC, as disclosed herein, can proliferate and form cell colonies. In one aspect, the giaSC within the cell colonies, as disclosed herein, can migrate outside of the colonies and further expand. In some embodiments, the giaSC colonies and/or cell culture, as disclosed herein, include high purity population of the giaSC, as disclosed herein. In one aspect, the percentage of giaSC in the cell colonies and/or culture is more than 50%, or alternatively more than 60%, or alternatively more than 70%, or alternatively more than 80%, or alternatively more than 90%, or alternatively more than 95%. In some embodiments, the giaSC colonies and/or culture includes substantially homogeneous population of the giaSC, as disclosed herein. In some embodiments, provided herein are methods of isolating a cell colony of the giaSC from the cell culture as disclosed herein. For example, a cell colony can be picked up using a cloning cylinder. Alternatively, a cell colony can be isolated by trypsinizing and detaching the colony from the cell culture plate/dish. It should be understood that other methods can also be employed to isolate a cell colony of the giaSC.

[0146] Embodiments of the disclosure also provide methods of regulating a somatic cell by contacting the somatic cell with the activated/mature guide cell, as disclosed herein. In some embodiments, during contacting cell-cell interaction is established which allows transfer of a plurality of cellular components from the activated/mature guide cell into the somatic cell, as disclosed herein. In one aspect, the transferred cellular components include exosomes and/or microvesicles. In one aspect, the transferred cellular components include RNA and/or RNA- rich granules. In some aspects, the cellular components include other materials. In one aspect, the cell-cell interaction is established in a co-culture system. In some aspects, the cell-cell interaction can be mediated by tunneling nanotubes, gap junction, and/or exocytosis/endocytosis. In some embodiments, further provided are methods of regulating a somatic cell by transferring to the somatic cell a plurality of cellular components isolated from the activated/mature guide cell, as disclosed herein. In one aspect, the cellular components of the activated/mature guide cell, as disclosed herein, are added to culture medium of the somatic cell. In one aspect, the cellular components, as disclosed herein, are delivered to a vicinity of the somatic cells. In some aspects, the cellular components as disclosed herein are transferred into the somatic cells via endocytosis. In some embodiment, the regulated somatic cell is derived from any adult tissue as disclosed herein. In some embodiment, the regulated somatic cell is a stem/stromal cell as disclosed herein. In some embodiment, the regulated somatic cell is not a stem cell as disclosed herein. In some embodiment, the regulated somatic cell has proliferative capability as disclosed herein.

5. Differentiation of High-Plasticity Stem Cells (giaSC)

[0147] The generated high-plasticity stem cells or giaSC can be identified by their high plasticity, e.g., the differentiation potential for more than one germ layers. In one aspect, the giaSC, as disclosed herein, has capacity to differentiate into cell types from all three germ layers (ectoderm, mesoderm, and endoderm) using appropriate culture conditions and medium. In another aspect, the giaSC, as disclosed herein, has capacity to differentiate into two of the three germ layers (e.g, ectoderm and mesoderm, or alternatively ectoderm and endoderm, or alternatively mesoderm and endoderm). Confirmation of the differentiation state of the cells can be performed by identification of cell type specific markers as known to those of skill in the art. Examples of some differentiation procedures are provided in Example 5, infra.

[0148] The present disclosure provides methods of inducing differentiation of the giaSC, as disclosed herein, into ectodermal lineage. Also provided are compositions or populations of differentiated cells in the ectodermal lineage, derived from the giaSC as disclosed herein. In one aspect, the giaSC, as disclosed herein, are capable of differentiation into at least one of the cell types in the ectodermal lineage. In another aspect, the giaSC, as disclosed herein, are capable of differentiation into at least two. at least three, and increasing up to all of the cell types in the ectodermal lineage. Non-limiting examples of cells that differentiate into ectodermal lineage include, but are not limited to epithelial cells, neurogenic cells, and neurogliagenic cells. Non-limiting examples of tissues derived from the ectoderm include some epithelial tissue (epidermis or outer layer of the skin, the lining for all hollow organs which have cavities open to a surface covered by epidermis), modified epidermal tissue (fingernails and toenails, hair, glands of the skin), all nerve tissue, salivary glands, and mucous glands of the nose and mouth.

[0149] The present disclosure also provides methods of inducing differentiation of the giaSC, as disclosed herein, into mesodermal lineage. Also provided are compositions or populations of differentiated cells in the mesodermal lineage, derived from the giaSC as disclosed herein. In one aspect, the giaSC, as disclosed herein, are capable of differentiation into at least one of the cell types in the mesodermal lineage. In another aspect, the giaSC, as disclosed herein, are capable of differentiation into at least two. or alternatively at least three, or alternatively at least four, and increasing up to all of the cell types in the mesodermal lineage. Nonlimiting examples of cells that differentiate into mesodermal lineage include, but are not limited to adipogenic, leiomyogenic, chondrogenic, cardiogenic, dermatogenic, hematopoetic, hemangiogenic. myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or stromal cells. Non-limiting examples of tissues derived from the mesoderm include muscles, fibrous tissue, bone, cartilage, fat or adipose tissue, blood and lymph vessels, and blood cells.

[0150] The present disclosure also provides methods of inducing differentiation of the giaSC, as disclosed herein, into endodermal lineage. Also provided are compositions or populations of differentiated cells in the endodermal lineage, derived from the giaSC as disclosed herein. In one aspect, the giaSC. as disclosed herein, are capable of differentiation into at least one of the cell types in the endodermal lineage. In another aspect, the giaSC, as disclosed herein, are capable of differentiation into at least two, or alternatively at least three, or alternatively at least four, or alternatively at least five, and increasing up to all of the cell types in the endodermal lineage. Non-limiting examples of cells that differentiate into endodermal lineage include, but are not limited to cells in the pancreas, liver, lung, stomach, intestine, and thyroid.

6. Method of Use

[0151] Regenerative medicine includes therapies designed to aid the repair, replacement, or regeneration of damaged cells, tissues, or organs, and to treat degenerative diseases. The methods and compositions as disclosed herein may be used in cell-based therapies in the regenerative medicine. [0152] The present disclosure provides methods of treating diseases in a subject in need thereof using the composition or population of the giaSC as disclosed herein. In some embodiments, provided are methods of treating diseases in a subject in need thereof using differentiated cells derived from the giaSC as disclosed herein. In some embodiments, provided are methods of treating diseases in a subject in need thereof using the cellular components isolated from the giaSC or the differentiated cells as disclosed herein.

[0153] Embodiments of the disclosure also provide methods of treating diseases in a subject in need thereof using the composition or population of the activated/mature guide cells as disclosed herein. In some embodiments, provided are methods of treating diseases in a subject in need thereof using cells derived from the activated/mature guide cells as disclosed herein. In some embodiments, provided are methods of treating diseases in a subject in need thereof using the cellular components isolated from the activated/mature guide cells and/or the derived cells as disclosed herein.

[0154] In some aspects, the methods and compositions disclosed herein may be used to treat diseases or conditions such as degenerative diseases, proliferative disorders, hereditary diseases, injuries, organ failures, and tissue damages. Non-limiting examples of the diseases or conditions include neurodegenerative disorders; neurological disorders such as cognitive impairment, and mood disorders; auditory disease such as deafness; osteoporosis; cardiovascular diseases; diabetes; metabolic disorders; respiratory’ diseases; drug sensitivity conditions; eye diseases such as macular degeneration; immunological disorders; hematological diseases; kidney diseases; proliferative disorders; genetic disorders, traumatic injury, stroke, organ failure, or loss of limb. Other examples of the diseases include a neurodegenerative disorder, a neurological disorder, an eye disease, a mood disorder, a respiratory disease, an auditory disease, a cardiovascular disease, an immunological disorder, a hematological disease, a metabolic disorder, a kidney disease, a proliferative disorder, a genetic disorder, an autoimmune disease, a drug sensitivity condition, a cognitive impairment, depression, deafness, osteoporosis, diabetes, macular degeneration, obesity, Alexander’s disease, Alper’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, ataxia telangiectasia. Batten disease, Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington’s disease, HIV-associated dementia, Kennedy’s disease, Krabbe’s disease, lewy body dementia, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, narcolepsy, neuroborreliosis. Parkinson’s disease, Pelizaeus-Merzbacher Disease, Pick’s disease, primary lateral sclerosis, a prion disease, Refsum’s disease, Sandhoffs disease, Schilder’s disease, subacute combined degeneration of spinal cord secondary to pernicious anaemia, schizophrenia, spinocerebellar ataxia, spinal muscular atrophy (SMA), Steele-Richardson-Olszewski disease, tabes dorsalis, acquired immune deficiency, leukemia, lymphoma, a hypersensitivity (allergy), severe combined immune deficiency, acute disseminated encephalomyelitis, addison’s disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, bullous pemphigoid, coeliac disease, dermatomy ositis, diabetes mellitus type 1, diabetes mellitus type 2. Goodpasture’s syndrome, Graves’ disease, Guillain- Barre syndrome, Hashimoto’s disease, idiopathic thrombocytopenic purpura, lupus erythematosus, myasthenia gravis, pemphigus, pernicious anaemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, Sjogren’s syndrome, temporal arthritis, vasculitis, Wegener’s granulomatosis, aneurysm, angina, arrhythmia, atherosclerosis, cardiomyopathy, calcific aortic valve disease (CAVD), cerebrovascular accident (stroke), cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease coronary, cardiomyopathy, diastolic dysfunction, endocarditis, hypertension, hypertrophic cardiomyopathy, mitral valve prolapse, myocardial infarction, venous thromboembolism, acid lipase disease, amyloidosis. Barth Syndrome, biotinidase deficiency, carnitine palmitoyl transferase deficiency type II, central pontine myelinolysis, muscular dystrophy, Farber’s Disease, glucose-6-phosphate dehydrogenase deficiency, gangliosidoses, trimethylaminuria, Lesch-Nyhan syndrome, lipid storage diseases, metabolic myopathies, methylmalonic aciduria, mitochondrial myopathies, mucopolysaccharidoses, mucolipidoses, mucolipidoses, mucopolysaccharidoses, multiple CoA carboxylase deficiency, nonketotic hyperglycinemia, Pompe disease, propionic acidemia, type I glycogen storage disease, urea cycle disorders, hyperoxaluria, oxalosis.

[0155] In one aspect, provided are methods of treating skin wounds in a subject in need thereof using the compositions or populations as disclosed herein. In another aspect, provided are methods of treating liver damages in a subject in need thereof the compositions or populations as disclosed herein. In another aspect, provided are methods of treating bone damages or conditions (e.g.. arthritis, osteoporosis, etc.) in a subject in need thereof using the compositions or populations as disclosed herein. In another aspect, provided are methods of treating brain injuries, stroke, damages of neurons and supporting cells, or neuron degenerative diseases in a subject in need thereof using the compositions or populations as disclosed herein. In another aspect, provided are methods of treating heart tissue damages or heart failure in a subject in need thereof using the compositions or populations as disclosed herein. In another aspect, provided are methods of treating chronic diseases such as diabetes in a subject in need thereof using the compositions or populations as disclosed herein.

[0156] In one aspect, provided are methods of autologous transfer of the compositions or populations as disclosed herein. In another aspect, provided are methods of allogeneic transfer of the compositions or populations as disclosed herein. In another aspect, provided are methods of transfer of the cellular components derived from the cell populations as disclosed herein. In another aspect, provided are methods of syngeneic transfer of the compositions or populations as disclosed herein.

[0157] Embodiment I provides a method of generating high-plasticity stem cells, comprising contacting somatic cells with guide cells and/or cellular components thereof, wherein the guide cells are characterized as CD49f /CD457CD90~. and wherein the high-plasticity stem cells preferably have differentiation potential for more than one germ layer. Embodiment 2 provides the method of embodiment 1 , wherein the contacting results in establishment of cellcell interaction that allows transfer of the cellular components from the guide cells into the somatic cells. Embodiment 3 provides the method of embodiment 1 or 2, wherein the cellular components comprise RNA. Embodiment 4 provides the method of embodiment 2 or 3, wherein the cell-cell interaction is via tunneling nanotubes. Embodiment 5 provides the method of embodiment 2 or 3, wherein the cell-cell interaction is via gap junction.

Embodiment 6 provides the method of embodiment 2 or 3, wherein the cell-cell interaction is via exocytosis and endocytosis. Embodiment 7 provides the method of any one of embodiments 1-6, wherein the contacting is established in a co-culture system. Embodiment 8 provides the method of any one of embodiments 1-7, wherein the guide cells and the somatic cells have a ratio of cell numbers that is at least about 1: 1.

[0158] Embodiment 9 provides the method of any one of embodiments 1-8, wherein the guide cells are further characterized as CD44⁺ and/or CD52⁺. Embodiment 10 provides the method of any one of embodiments 1 -9, wherein the guide cells are further characterized as SSEA4- and/or SSEA3-. Embodiment 11 provides the method of any one of embodiments 1-10, wherein the guide cells are further characterized as CD324- and/or CD73-. Embodiment 12 provides the method of any one of embodiments 1-11, wherein the guide cells are further characterized as positive in one or more markers selected from the group consisting of CD105, HLA-I, HLA-II, and HLA-E. Embodiment 13 provides the method of any one of embodiments 1-12, wherein the guide cells are further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19. CD20, and CD56. Embodiment 14 provides the method of any one of embodiments 1-13, wherein the guide cells do not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2. Embodiment 15 provides the method of any one of embodiments 1-14, wherein the guide cells comprise a plurality of RNA-rich granules in the cy toplasm. Embodiment 16 provides the method of any one of embodiments 1-15, wherein the guide cells comprise one or more pseudopodia.

[0159] Embodiment 17 provides the method of any one of embodiments 1-16, wherein the somatic cells comprise stem cells. Embodiment 18 provides the method of any one of embodiments 1-16, wherein the somatic cells comprise mesenchymal stromal cells. Embodiment 19 provides the method of any one of embodiments 1-16, wherein the somatic cells comprise non-stem cells. Embodiment 20 provides the method of embodiment 19, wherein the non-stem cells have proliferative capacity.

[0160] Embodiment 21 provides the method of any one of embodiments 1-20, wherein the high-plasticity stem cells are characterized as CD49E/CD90⁺/CD34“. An alternative embodiment provides the method of any one of embodiments 1-20, wherein the high- plasticity stem cells are characterized as CD49f /CD90 /CD73 /CD34~. An alternative embodiment provides the method of any one of embodiments 1-20. wherein the high- plasticity stem cells are characterized as CD49f7CD73⁺/CD34-. An alternative embodiment provides the method of any one of embodiments 1-20, wherein the high-plasticity stem cells are characterized as CD49E/CD73⁺/CD90-/CD34-. Embodiment 22 provides the method of any one of embodiments 1-21 and the alternative embodiments above, wherein the high- plasticity stem cells are further characterized as CD45^low and/or CD324^low. Embodiment 23 provides the method of any one of embodiments 1-22 and the alternative embodiments above, wherein the high-plasticity stem cells are further characterized as SSEA4⁺ and/or SSEA3-. Embodiment 24 provides the method of any one of embodiments 1-23 and the alternative embodiments above, wherein the high-plasticity stem cells are further characterized as CD 105¹ . Embodiment 25 provides the method of any one of embodiments 1-24 and the alternative embodiments above, wherein the high-plasticity stem cells are further characterized as positive in one or more markers selected from the group consisting of CD44, CD146, CD56, CD52, HLA-I, and HLA-E. Embodiment 26 provides the method of any one of embodiments 1-25 and the alternative embodiments above, wherein the high-plasticity stem cells are further characterized as negative in one or more markers selected from the group consisting of CD3, CD19, CD20, and HLA-II. Embodiment 27 provides the method of any one of embodiments 1 -26 and the alternative embodiments above, wherein the high- plasticity stem cells do not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2.

[0161] Embodiment 28 provides the method of any one of embodiments 1-27, wherein the high-plasticity stem cells express (a) one or more of endoderm markers, (b) one or more of mesoderm markers, and (c) one or more of ectoderm markers. Embodiment 29 provides the method of embodiment 28, wherein the endoderm markers comprise AFP, CK7 (KRT7). albumin (ALB), CK18 (KRT18), and CK19 (KRT19), wherein the mesoderm markers comprise desmin (DES), osteocalcin (BGLAP), CD106 (VCAM-1), CD54 (ICAM-1), and CD 146 (MCAM), and wherein the ectoderm markers comprise nestin (NES). notch- 1, notch- 2, MSI2, CD56 (NCAM1), and CD325 (N-cadherin or Cadherin-2). Embodiment 30 provides the method of any one of embodiments 1-29, wherein the high-plasticity stem cells have approximately triangular cell bodies with one or more slender pseudopodia.

Embodiment 31 provides the method of any one of embodiments 1-30, wherein the high- plasticity stem cells exchange a plurality of cellular components bet een each other and/or with cells of a different kind. Embodiment 32 provides the method of embodiment 31. wherein the plurality of cellular components comprise RNA. Embodiment 33 provides the method of embodiment 31 or 32, wherein the exchange of the plurality of cellular components is via tunneling nanotubes, gap junction, and/or exocytosis/endocytosis.

[0162] Embodiment 34 provides the method of any one of embodiments 1-33, wherein the guide cells are derived from dormant guide cells that (a) have a diameter of less than 6 pm, (b) express CD49f and CD45, (c) do not express CD90, and (d) do not have detectable intracellular esterase activity and/or transcriptomic activity. Embodiment 35 provides the method of embodiment 34, wherein the dormant guide cells are isolated from human blood. Embodiment 36 provides the method of embodiment 34 or 35, wherein the dormant guide cells are further characterized as CD44⁺. Embodiment 37 provides the method of any one of embodiments 34-36, wherein the dormant guide cells are further characterized as SSEA4- and/or SSEA3 . Embodiment 38 provides the method of any one of embodiments 34-37, wherein the dormant guide cells are further characterized as CD324- and/or CD73-. Embodiment 39 provides the method of any one of embodiments 34-38, wherein the dormant guide cells are further characterized as positive in one or more markers selected from the group consisting of CD34, CD 105, EILA-I, and HLA-E. Embodiment 40 provides the method of any one of embodiments 34-39, wherein the dormant guide cells are further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, CD56, and HLA-II. Embodiment 41 provides the method of any one of embodiments 34-40, wherein the dormant guide cells do not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2.

[0163] Embodiment 42 provides a composition comprising high-plasticity stem cells that are generated by the method of any one of embodiments 1-41. Embodiment 43 provides the composition of embodiment 42, further comprising a pharmaceutically acceptable carrier or excipient.

[0164] Embodiment 44 provides an isolated high-plasticity stem cell that is characterized as CD49f7CD90⁺/CD34“, and preferably has differentiation potential for more than one germ layer. Embodiment 45 provides the stem cell of embodiment 44, which is further characterized as CD45^low and/or CD324^low. Embodiment 46 provides the stem cell of embodiment 44 or 45, which is further characterized as SSEA4⁺ and/or SSEA3-.

Embodiment 47 provides the stem cell of any one of embodiments 44-46, which is further characterized as CD73⁺ and/or CD105⁺. Embodiment 48 provides the stem cell of any one of embodiments 44-47, which is further characterized as positive in one or more markers selected from the group consisting of CD44, CD 146, CD56, CD52. HLA-I, and HLA-E. Embodiment 49 provides the stem cell of any one of embodiments 44-48, which is further characterized as negative in one or more markers selected from the group consisting of CD3, CD19, CD20, and HLA-II. Embodiment 50 provides the stem cell of any one of embodiments 44-49, which does not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2. Embodiment 51 provides the stem cell of any one of embodiments 44-50, which has an approximately triangular cell body with one or more slender pseudopodia. Embodiment 52 provides the stem cell of any one of embodiments 44- 51, which exchanges a plurality of cellular components with an adjacent stem cell of the same kind or a different kind. Embodiment 53 provides the stem cell of embodiment 52, wherein the plurality of cellular components comprise RNA. Embodiment 54 provides the stem cell of embodiment 52 or 53, wherein the exchange of the plurality of cellular components is via tunneling nanotube, gap junction, and/or exocytosis/endocytosis. Embodiment 55 provides the stem cell of any one of embodiments 44-54, which proliferates and forms colonies in culture. Embodiment 56 provides the stem cell of any one of embodiments 44-55, further characterized by expression of one or more of endoderm markers, one or more of mesoderm markers, and one or more of ectoderm markers. Embodiment 57 provides the stem cell of any one of embodiments 44-55, further characterized by expression of one or more of endoderm markers and one or more of mesoderm markers. Embodiment 58 provides the stem cell of any one of embodiments 44-55, further characterized by expression of one or more of endoderm markers and one or more of ectoderm markers. Embodiment 59 provides the stem cell of any one of embodiments 44-55, further characterized by expression of one or more of mesoderm markers and one or more of ectoderm markers. Embodiment 60 provides the stem cell of any one of embodiments 56-58, wherein the endoderm markers comprise AFP, CK7 (KRT7), albumin (ALB), CK18 (KRT18), and CK19 (KRT19). Embodiment 61 provides the stem cell of any one of embodiments 56-57 and 59, wherein the mesoderm markers comprise desmin (DES), osteocalcin (BGLAP), CD 106 (VCAM-1), CD54 (ICAM- 1), and CD146 (MCAM). Embodiment 62 provides the stem cell of any one of embodiments 56 and 58-59, wherein the ectoderm markers comprise nestin (NES), notch-1, notch-2, MSI2, CD56 (NCAM1), and CD325 (N-cadherin or Cadherin-2). Embodiment 63 provides a composition comprising a population of high-plasticity stem cells of any one of embodiments 44-62 in a pharmaceutically acceptable carrier or excipient.

[0165] Embodiment 64 provides a composition comprising a plurality of cellular components isolated from the population of high-plasticity stem cells of any one of embodiments 44-62. Embodiment 65 provides the composition of embodiment 64. w herein the plurality of cellular components comprise microvesicles and/or exosomes. Embodiment 66 provides the composition of embodiment 64 or 65, w herein the plurality of cellular components comprise RNA. Embodiment 67 provides the composition of any one of embodiments 64-66, further comprising a pharmaceutically acceptable carrier or excipient.

[0166] Embodiment 68 provides a population of cells differentiated from a population of high-plasticity⁷ stem cells of any one of embodiments 44-62. Embodiment 69 provides a composition comprising the population of differentiated cells of embodiment 68 in a pharmaceutically acceptable carrier or excipient. Embodiment 70 provides a composition comprising a plurality of cellular components isolated from the population of differentiated cells of embodiment 68. Embodiment 71 provides the composition of embodiment 70, wherein the plurality of cellular components comprise microvesicles and/or exosomes. Embodiment 72 provides the composition of embodiment 70 or 71, wherein the plurality of cellular components comprise RNA. Embodiment 73 provides the composition of any one of embodiments 70-72, further comprising a pharmaceutically acceptable carrier or excipient.

[0167] Embodiment 74 provides a method of culturing a population of high-plasticity stem cells, comprising culturing the high-plasticity stem cell of any one of embodiments 44-62 in a medium that is supplemented with one or more grow th factors selected from the group consisting of IGF1, IGF2, FGF2, HGF, and BMP5. Embodiment 75 provides the method of embodiment 74, wherein the medium is supplemented with IGF1. Embodiment 76 provides a method of differentiating the high-plasticity stem cell of any one of embodiments 44-62 in a differentiation medium.

[0168] Embodiment 77 provides a method of treating a disease or condition in a human subject in need thereof, comprising administering an effective amount of the composition of any one of embodiments 43, 63, 67, 69, and 73 to the human subject. Embodiment 78 provides the method of embodiment 77, wherein the disease or condition is selected from the group consisting of degenerative diseases, proliferative disorders, hereditary diseases, injuries, tissue damages, and organ failures.

[0169] Embodiment 79 provides a method of generating guide cells characterized as CD49f7CD45⁺/CD90“, comprising activating dormant guide cells that (a) have a diameter of less than 6 pm, (b) express CD49f and CD45, (c) do not express CD90, and (d) do not have detectable intracellular esterase activity and/or transcriptomic activity. Embodiment 80 provides the method of embodiment 79, wherein the guide cells are further characterized as CD44⁺ and/or CD52⁺. Embodiment 81 provides the method of embodiment 79 or 80, wherein the guide cells are further characterized as SSEA4- and/or SSEA3-. Embodiment 82 provides the method of any one of embodiments 79-81, wherein the guide cells are further characterized as CD324- and/or CD73-. Embodiment 83 provides the method of any one of embodiments 79-82, wherein the guide cells are further characterized as positive in one or more markers selected from the group consisting of CD 105, HLA-I, HLA-II, and HLA-E. Embodiment 84 provides the method of any one of embodiments 79-83, wherein the guide cells are further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, and CD56. Embodiment 85 provides the method of any one of embodiments 79-84, wherein the guide cells do not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2. Embodiment 86 provides the method of any one of embodiments 79-85, wherein the guide cells comprise RNA-rich granules in the cytoplasm. Embodiment 87 provides the method of embodiment 86, wherein the guide cells comprise one or more pseudopodia to release the RNA-rich granules.

[0170] Embodiment 88 provides the method of any one of embodiments 79-87, wherein the dormant guide cells are isolated from human blood. Embodiment 89 provides the method of any one of embodiments 79-88, wherein the dormant guide cells are further characterized as CD44⁺. Embodiment 90 provides the method of any one of embodiments 79-89, wherein the dormant guide cells are further characterized as SSEA4- and/or SSEA3-. Embodiment 91 provides the method of any one of embodiments 79-90, wherein the dormant guide cells are further characterized as CD324- and/or CD73-. Embodiment 92 provides the method of any one of embodiments 79-91, wherein the dormant guide cells are further characterized as positive in one or more markers selected from the group consisting of CD34. CD105, HLA-I, and HLA-E. Embodiment 93 provides the method of any one of embodiments 79-92, wherein the dormant guide cells are further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, CD56, and HLA-II. Embodiment 94 provides the method of any one of embodiments 79-93, wherein the dormant guide cells do not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2.

[0171] Embodiment 95 provides the method of any one of embodiments 79-94, wherein the activating dormant guide cells comprises culturing the dormant guide cells in a hepatic environment. Embodiment 96 provides the method of embodiment 95, wherein the hepatic environment comprises a co-culture system with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. Embodiment 97 provides the method of embodiment 95, wherein the hepatic environment comprises a medium that is in contact with or has been conditioned with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. Embodiment 98 provides the method of embodiment 95, wherein the hepatic environment comprises a medium that is supplemented with at least one grow th factor or cytokine that is released by primary hepatocytes or hepatic cell lines. Embodiment 99 provides the method of embodiment 95, wherein the hepatic environment comprises a medium that is supplemented with one or more cytokines selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CX3CL2, CCL2, IL6, IL8, IL15, albumin, ANXA1, CSF3, TNFSF10, PVR, and ULBP2.

[0172] Embodiment 100 provides a composition comprising a population of the guide cells, w hich is generated by the method of any one of embodiments 79-99. Embodiment 101 provides the composition of embodiment 100, further comprising a pharmaceutically acceptable carrier or excipient. Embodiment 102 provides a composition comprising a plurality of cellular components isolated from a population of guide cells generated by the method of any one of embodiments 79-99. Embodiment 103 provides the composition of embodiment 102, wherein the plurality of cellular components comprise microvesicles and/or exosomes. Embodiment 104 provides the composition of embodiment 102 or 103, wherein the plurality' of cellular components comprise RNA. Embodiment 105 provides the composition of any one of embodiments 102-104. further comprising a pharmaceutically acceptable carrier or excipient.

[0173] Embodiment 106 provides an isolated guide cell characterized with CD49f7CD45⁺/CD90“, comprising RNA-rich granules in the cytoplasm and one or more pseudopodia. Embodiment 107 provides the guide cell of embodiment 106, wherein the RNA-rich granules can be released by the one or more pseudopodia. Embodiment 108 provides the guide cell of embodiment 106 or 107, which is further characterized as CD44⁺ and/or CD52⁺. Embodiment 109 provides the guide cell of any one of embodiments 106-108, which is further characterized as SSEA4- and/or SSEA3-. Embodiment 110 provides the guide cell of any one of embodiments 106-109, which is further characterized as CD324- and/or CD73-. Embodiment 111 provides the guide cell of any one of embodiments 106- 110, which is further characterized as positive in one or more markers selected from the group consisting of CD105, HLA-I, HLA-II, and HLA-E. Embodiment 112 provides the guide cell of any one of embodiments 106-111, w hich is further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, and CD56. Embodiment 113 provides the guide cell of any one of embodiments 106-112, which does not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2.

[0174] Embodiment 114 provides a composition comprising a population of the guide cells of any one of embodiments 106-113, and a pharmaceutically acceptable carrier or excipient. Embodiment 115 provides a composition comprising a plurality of cellular components isolated from a population of the guide cells of any one of embodiments 106-113. Embodiment 116 provides the composition of embodiment 115, wherein the plurality of cellular components comprises microvesicles and/or exosomes. Embodiment 117 provides the composition of embodiment 115 or 116, wherein the plurality of cellular components comprise RNA. Embodiment 118 provides the composition of any one of embodiments 115- 117, further comprising a pharmaceutically acceptable carrier or excipient.

[0175] Embodiment 119 provides a cell derived from the guide cell of any one of embodiments 106-113. Embodiment 120 provides a composition comprising a population of derived cells of embodiment 119 in a pharmaceutically acceptable carrier or excipient. Embodiment 121 provides a composition comprising a plurality of cellular components isolated from a population of derived cells of embodiment 119. Embodiment 122 provides the composition of embodiment 121, wherein the plurality of cellular components comprise microvesicles and/or exosomes. Embodiment 123 provides the composition of embodiment 121 or 122, wherein the plurality of cellular components comprise RNA. Embodiment 124 provides the composition of any one of embodiments 121-123, further comprising a pharmaceutically acceptable carrier or excipient.

[0176] Embodiment 125 provides a method of culturing the guide cell of any one of embodiments 106-113 in a hepatic environment. Embodiment 126 provides the method of embodiment 125, wherein the hepatic environment comprises a co-culture system with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. Embodiment 127 provides the method of embodiment 125, wherein the hepatic environment comprises a medium that is in contact with or has been conditioned with at least one selected from the group consisting of primary hepatocytes and hepatic cell lines. Embodiment 128 provides the method of embodiment 125, wherein the hepatic environment comprises a medium that is supplemented with at least one growth factor or cytokine that is released by primary hepatocytes or hepatic cell lines. Embodiment 129 provides the method of embodiment 125, wherein the hepatic environment comprises a medium that is supplemented with one or more cytokines selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CX3CL2, CCL2, IL6, IL8, IL15, albumin, ANXA1, CSF3, TNFSF10, PVR, and ULBP2.

[0177] Embodiment 130 provides a method of treating a disease or condition in a human subject in need thereof, comprising administering an effective amount of the composition of any one of embodiments 101, 105, 114, 118, 120, and 124 to the human subject. Embodiment 131 provides the method of embodiment 130, wherein the disease or condition is selected from the group consisting of degenerative diseases, proliferative disorders, hereditary diseases, injuries, tissue damages, and organ failures.

[0178] Embodiment 132 provides a method of regulating a somatic cell, comprising contacting the somatic cell with a guide cell characterized with CD49f7CD45⁺/CD90“ to establish cell-cell interaction which allow s transfer of a plurality of cellular components from the guide cell into the somatic cell. Embodiment 133 provides the method of embodiment 132, wherein the transferred plurality of cellular components comprise RNA. Embodiment 134 provides the method of embodiment 132 or 133, wherein the plurality of cellular components are transferred via tunneling nanotubes. Embodiment 135 provides the method of embodiment 132 or 133, wherein the plurality of cellular components are transferred via gap junction. Embodiment 136 provides the method of embodiment 132 or 133, wherein the plurality of cellular components are transferred via exocytosis and endocytosis. Embodiment 137 provides the method of any one of embodiments 132-136, wherein the cell-cell interaction is established in a co-culture system.

[0179] Embodiment 138 provides the method of any one of embodiments 132-137, wherein the guide cell is further characterized as CD44⁺ and/or CD52⁺. Embodiment 139 provides the method of any one of embodiments 132-138, wherein the guide cell is further characterized as SSEA4- and/or SSEA3-. Embodiment 140 provides the method of any one of embodiments 132-139, wherein the guide cell is further characterized as CD324- and/or CD73-. Embodiment 141 provides the method of any one of embodiments 132-140, wherein the guide cell is further characterized as positive in one or more markers selected from the group consisting of CD105, HLA-I, HLA-II, and HLA-E. Embodiment 142 provides the method of any one of embodiments 132-141, wherein the guide cell is further characterized as negative in one or more markers selected from the group consisting of Lin, CD 146, CD3, CD 19, CD20, and CD56. Embodiment 143 provides the method of any one of embodiments 132-142, wherein the guide cell does not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2. Embodiment 144 provides the method of any one of embodiments 132-143, wherein the guide cell comprises a plurality of RNA-rich granules in the cytoplasm. Embodiment 145 provides the method of embodiment 144, wherein the guide cell comprises one or more pseudopodia to release the RNA-rich granules.

[0180] Embodiment 146 provides the method of any one of embodiments 132-145, wherein the somatic cell is a stem cell. Embodiment 147 provides the method of any one of embodiments 132-145, wherein the somatic cell is a mesenchymal stromal cell. Embodiment 148 provides the method of any one of embodiments 132-145, wherein the somatic cell is not a stem cell. Embodiment 149 provides the method of embodiment 148, wherein the somatic cell has proliferative capacity. Embodiment 150 provides the method of any one of embodiments 132-149, wherein the somatic cell is regulated to acquire high plasticity. Embodiment 151 provides the method of embodiment 150, wherein the acquired high plasticity comprises differentiation potential for more than one germ layer.

[0181] Embodiment 152 provides the method of any one of embodiments 132-151, wherein the guide cell is derived from a dormant guide cell that (a) has a diameter of less than 6 pm, (b) expresses CD49f and CD45, (c) does not express CD90, and (d) does not have detectable intracellular esterase activity and/or transcriptomic activity. Embodiment 153 provides the method of embodiment 152. wherein the dormant guide cell is isolated from human blood. Embodiment 154 provides the method of embodiment 152 or 153, wherein the dormant guide cell is further characterized as CD44⁺. Embodiment 155 provides the method of any one of embodiments 152-154, wherein the dormant guide cell is further characterized as SSEA4- and/or SSEA3-. Embodiment 156 provides the method of any one of embodiments 152-155, wherein the dormant guide cell is further characterized as CD324- and/or CD73-.

Embodiment 157 provides the method of any one of embodiments 152-156, wherein the dormant guide cell is further characterized as positive in one or more markers selected from the group consisting of CD34, CD105, HLA-I, and HLA-E. Embodiment 158 provides the method of any one of embodiments 152-157, wherein the dormant guide cell is further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, CD56, and HLA-II. Embodiment 159 provides the method of any one of embodiments 152-158, wherein the dormant guide cell does not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2.

[0182] Embodiment 160 provides a method of regulating a somatic cell, comprising transferring to the somatic cell a plurality of cellular components isolated from a guide cell characterized with CD49E/CD45⁺/CD90“. Embodiment 161 provides the method of embodiment 160, wherein the plurality’ of cellular components comprise RNA. Embodiment 162 provides the method of embodiment 166 or 161, wherein the plurality of cellular components are isolated in exosomes and/or microvesicles. Embodiment 163 provides the method of embodiment any one of embodiments 160-162, wherein the plurality’ of cellular components are transferred via endocytosis of the somatic cell. Embodiment 164 provides the method of any one of embodiments 160-163, wherein the plurality of cellular components are added to a cell culture of the somatic cell. Embodiment 165 provides the method of any one of embodiments 160-164, wherein the plurality of cellular components are delivered to a vicinity of the somatic cell.

[0183] Embodiment 166 provides the method of any one of embodiments 160-165, wherein the guide cell is further characterized as CD44⁺ and/or CD52⁺. Embodiment 167 provides the method of any one of embodiments 160-166, wherein the guide cell is further characterized as SSEA4- and/or SSEA3-. Embodiment 168 provides the method of any one of embodiments 160-167, yvherein the guide cell is further characterized as CD324- and/or CD73 Embodiment 169 provides the method of any one of embodiments 160-168, wherein the guide cell is further characterized as positive in one or more markers selected from the group consisting of CD105, HLA-I, HLA-II, and HLA-E. Embodiment 170 provides the method of any one of embodiments 160-169, wherein the guide cell is further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, and CD56. Embodiment 171 provides the method of any one of embodiments 160-170, wherein the guide cell does not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2. Embodiment 172 provides the method of any one of embodiments 160-171, wherein the guide cell comprises a plurality of RNA-rich granules in the cytoplasm.

[0184] Embodiment 173 provides the method of any one of embodiments 160-172, yvherein the somatic cell is a stem cell. Embodiment 174 provides the method of any one of embodiments 160-172, wherein the somatic cell is a mesenchymal stromal cell. Embodiment 175 provides the method of any one of embodiments 160-172, wherein the somatic cell is not a stem cell. Embodiment 176 provides the method of embodiment 175, wherein the somatic cell has proliferative capacity. Embodiment 177 provides the method of any one of embodiments 160-176, wherein the somatic cell is regulated to acquire high plasticity. Embodiment 178 provides the method of embodiment 177, wherein the acquired high plasticity comprises differentiation potential for more than one germ layer.

[0185] Embodiment 179 provides a method of isolating a sub-population of dormant guide cells that has a diameter of less than 6 pm and does not have detectable intracellular esterase activity' and/or transcriptomic activity, comprising (a) preparing adult tissue in a solution, (b) centrifuging the solution at 5,000xg-15,000xg and obtaining a cell pellet, and (c) enriching CD49f7CD457CD90- cells from the cell pellet. Embodiment 180 provides the method of embodiment 179, wherein the adult tissue comprises human blood. Embodiment 181 provides the method of embodiment 180, wherein the solution comprises red blood cell lysis buffer. Embodiment 182 provides the method of any one of embodiments 179-181, wherein the solution is centrifuged at more than 8,000xg. Embodiment 183 provides the method of any one of embodiments 179-181, wherein the solution is centrifuged at more than 10,000xg.

[0186] Embodiment 184 provides the method of any one of embodiments 179-183, wherein the sub-population of dormant guide cells is further characterized as CD44⁺. Embodiment 185 provides the method of any one of embodiments 179-181, wherein the sub-population of dormant guide cells is further characterized as SSEA4- and/or SSEA3-. Embodiment 186 provides the method of any one of embodiments 179-182, wherein the sub-population of dormant guide cells is further characterized as CD324- and/or CD73-. Embodiment 187 provides the method of any one of embodiments 179-183, wherein the sub-population of dormant guide cells is further characterized as positive in one or more markers selected from the group consisting of CD34, CD 105, HLA-I, and HLA-E. Embodiment 188 provides the method of any one of embodiments 179-184, wherein the sub-population of dormant guide cells is further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19, CD20, CD56. and HLA-II. Embodiment 189 provides the method of any one of embodiments 179-185, wherein the sub-population of dormant guide cells does not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2. [0187] Embodiment 190 provides a composition of dormant guide cell isolated by method of any one of embodiments 179-189. Embodiment 191 provides a composition comprising at least 1000 cells, wherein at least 50% of the cells are dormant guide cells that (a) have a diameter of less than 6 pm, (b) express CD49f and CD45. (c) do not express CD90, and (d) do not have detectable intracellular esterase activity and/or transcnptomic activity. Embodiment 192 provides the composition of embodiment 191, wherein the dormant guide cells are isolated from human blood. Embodiment 193 provides the composition of embodiment 191 or 192, wherein the dormant guide cells are further characterized as CD44⁺. Embodiment 194 provides the composition of any one of embodiments 191-193. wherein the dormant guide cells are further characterized as SSEA4- and/or SSEA3-. Embodiment 195 provides the composition of any one of embodiments 191-194, wherein the dormant guide cells are further characterized as CD324 . Embodiment 196 provides the composition of any one of embodiments 191-195, wherein the dormant guide cells are further characterized as CD73-. Embodiment 197 provides the composition of any one of embodiments 191-196, wherein the dormant guide cells are further characterized as CD146-. Embodiment 198 provides the composition of any one of embodiments 191-197, wherein the dormant guide cells are further characterized as positive in one or more markers selected from the group consisting of CD34, CD105, HLA-I, and HLA-E. Embodiment 199 provides the composition of any one of embodiments 191-198, wherein the dormant guide cells are further characterized as negative in one or more markers selected from the group consisting of Lin, CD3. CD 19, CD20, CD56, and HLA-II. Embodiment 200 provides the composition of any one of embodiments 191-199, wherein the dormant guide cells do not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2.

EXAMPLES

Example 1. Isolation of Dormant Guide Cells from Adult Human Peripheral Blood

[0188] The following method was used to isolate dormant guide cells as disclosed herein from adult human peripheral blood. Human unmobilized peripheral blood was collected at the Stanford Blood Center, Stanford CA. All blood donors provided written informed consent, and 5-10 ml of peripheral blood was collected in Lithium-heparin tubes or ACD-A tubes (BD diagnostics) and kept at 4°C. [0189] The collected blood sample was mixed with red blood cells (RBC) lysis buffer for human (Alfa Aesar) at a ratio of 1 :9, and left to stand for 5 minutes at room temperature, to lyse the RBC. The suspension was then centrifuged at 9,000 xg for 40 minutes at 4°C. The supernatant was removed and the cell pellet was washed and resuspended in 5 ml of IxPBS for further analysis, or to be activated in an activation system as described below in Example 2.

[0190] The resuspended cell pellet was a heterogenous population that included tiny cells with nuclear staining (FIG. 1) and cell debris. The tiny cells’ diameter was below 6 pm. Some of the tiny cells’ diameter was around 3-5 pm. These tiny cells showed positive nuclear staining (Hoechst 33342) that occupied almost the entire cell with minimal cytoplasm, indicating a very high nucleus-cytoplasm (N/C) ratio (nearly 1) as measured by fluorescence microscopy. As demonstrated below, these freshly isolated tiny cells were dormant guide cells (or “d-GC”) indicated by negative staining of Calcein Red- AM (for intracellular esterase activity ) (FIG. 2A) and negative staining of Acridine Orange (AO) for RNAs (for transcriptomic activity) (FIG. 2B).

[0191] Further analysis was performed to study the marker profile, chromatin accessibility, and gene expression profile of the dormant guide cells. Flow cytometry was used to study exemplary antigen markers of the dormant guide cells C‘d-GC” in FIG. 7A). The flow cytometry results showed that the dormant guide cells was a heterogeneous population, including subpopulations. One sub-population of dormant guide cells was positive in CD49f and CD45, and negative in CD90. These dormant guide cells were also positive in CD44 and expressed a low level of CD34 and CD105. Further, they were negative in CD73, CD146, SSEA4, SSEA3, CD3, CD19, CD20, and CD56. Importantly, these dormant guide cells were also negative in CD324 (E-Cadherin) and Lin. Additional markers of this sub-population of dormant guide cells included very low level of POU5F1 gene, and lack of gene expression of Lin28, Nanog, and Sox2. Moreover, the sub-population of dormant guide cells was HLA- I 7HLA-IF and expressed low level of HLA-E. The HLA-II was evaluated by flow cytometry for anti-human HLA-DR, DP, DQ antibody (BioLegend).

[0192] In one experiment, sub-populations of dormant guide cells were further purified or enriched from the cell pellet. For example, the CD49f /CD457CD90 enriched cell fractions or sub-population were enriched by MACS following the protocol recommended by the manufacturer (Miltenyi Biotec Inc. San Diego, CA). In other examples, enriched cell fractions or sub-populations were obtained, via FACS or MACS, using antibodies targeting cell surface antigens of the cells. The enriched cell population or sub-population was then prepared for further analysis or activation.

Example 2. Activation and Maturation of Dormant Guide Cells In Vitro

[0193] The following methods were used to promote activation and maturation of the dormant guide cells obtained from adult human peripheral blood as described in Example 1 above. An activation system was prepared for activation of dormant guide cells and growth and maturation in vitro.

[0194] To promote activation and maturation of the dormant guide cells in culture, an activation system was prepared using a human hepatic cell line (HepaRG cells) to mimic a hepatic environment. The activation system was established in a co-culture system using Transwells. In another experiment, the activation system was established using conditioned medium from cell culture of hepatic cell line, as described below. In another experiment, grow th factors and cytokines w ere used in the activation system to mimic a hepatic environment.

[0195] For a co-culture system, HepaRG cells were treated with Mitomycin C for 2 hours to mitotically inactivate the cells, which were then inoculated on 6-well plates in DMEM with 10% FBS. About sixteen hours after inoculation, the cells adhered to the wells and were approximately 80% confluent. Then the isolated dormant guide cells w ere placed into the upper chamber of a Transwell (24-mm insert, Coming, Coming, New York), to be cocultured with the HepaRG cells in a co-culture medium of DMEM medium with 10% FBS. The isolated dormant guide cells w ere separated from the inactivated cells by the Transw-ell membrane (0.4 pm pore size). The same culture medium was used and changed every other day for both upper and lower chambers, for about 5 to 25 days.

[0196] In another experiment, conditioned medium was prepared that were used to activate and develop the isolated dormant guide cells in vitro. HepaRG cells were suspended in DMEM with 10% FBS and then seeded in a cell culture dish. The medium was changed every 2 days, and The medium was collected at 50%, 70%, and 90% cell confluency and pooled together. The pooled conditioned medium w as centrifuged at 3000xg to pellet the remaining cells in the medium. The conditioned medium was also filtered with the 0.2 pm filter unit. The pooled conditioned medium was stored at -20°C for short term storage or at - 80°C for long term storage. In one experiment, the conditioned medium was used directly to culture the isolated dormant guide cells. In another experiment, the conditioned medium was further supplemented with 5% serum to culture the isolated dormant guide cells. The isolated dormant guide cells were gently resuspended in the above-mentioned medium mixture, and then were seeded in culture dishes or plates. The medium was changed every other day. The cell culture was observed every day under microscope for cell grow th and formation of cell colonies for about 5 to 25 days.

[0197] To assess the activation status, a fluorescent dye (e.g, Calcein Red-Orange, AM, Calcein Red-AM, Calcein AM) was used to detect intracellular esterase activity⁷ (FIG.2A). The freshly isolated dormant guide cells showed positive nuclear staining (blue Hoechst 33342 staining) and negative staining of the red fluorescent dye (indicating lack of intracellular esterase activity), while after 1-hour culture in the activation system, more than 50% of the cells showed positive red staining, while overnight culture led to positive red staining in more than 90% (indicating activation with intracellular esterase activity) (FIG. 2A). In another experiment, acridine orange (AO) was used to detect RNA with red fluorescence that indicated transcriptomic activity of the cell (FIG. 2B). AO staining results showed that the freshly isolated guide cells were stained negative for RNA (indicating undetectable transcriptomic activity), while after overnight culture in the activation system, the guide cells were activated with abundant RNA red staining (indicating active transcription) (FIG. 2B). Both dormant and activated guide cells showed positive nuclear DNA green fluorescence using AO staining.

[0198] The activated guide cells (e.g., indicated by intracellular esterase activity and active transcription) were further cultured in the above-mentioned activation system for about 5-25 days for development and maturation. Cell growth, morphology changes, and colony formation yvere observed during culture (FIG. 3A). The dormant guide cells cultured in the regular medium (e.g., DMEM with 10% FBS) did not grow, but died. m-GC had enlarged size (about 25-35pm in diameter) and showed changes in morphology such as aggregated granules in the center region of the cells, indicating that the guide cells reached a mature state and hence referred to as “activated/mature guide cells” or “m-GC” (FIG. 3B). AO staining showed that the aggregated granules w ere rich in RNA (FIG. 3B). After continued culture and passaging, m-GCs showed pseudopodia extending from the cell surface. Release of the cellular contents such as the granules was also observed (FIG. 3C). After release of the cellular components, the m-GC died within a few days.

[0199] Further analysis was performed to study the marker profile, chromatin accessibility, gene expression profile of the activated/mature guide cells. Flow cytometry was used to study exemplary markers of the activated/mature guide cells (“m-GC” in FIG. 7B). The flow cytometry results showed that the activated/mature guide cells, derived from the dormant guide cells of Example 1, was a population with very high purity (>90%) that was positive in CD49f and CD45, and negative in CD90. The activated/mature guide cells were positive in CD44 and CD105, and expressed very low level of CD34. The activated/mature guide cells were negative in CD73, CD146, SSEA4, SSEA3, CD3, CD19, CD20, and CD56. Additional markers of these activated/mature guide cells include being negative in CD324 (E-Cadherin) and Lin. The activated/mature guide cells also expressed very low level of Oct4. and did not express Lin28, Nanog, and Sox2. Moreover, the activated/mature guide cells were HLA-I⁺ and expressed low level of HLA-II and HLA-E.

[0200] In one experiment, the colonies that include activated/mature guide cells in the activation system were isolated/collected, dissociated and re-seeded into fresh medium of the activation system for further culturing. Although cell grow th and colonies were observed during culture, the cell number did not significantly increase and the activated/mature guide cells showed limited proliferative capacity and low or minimal expression of proliferation- related genes such as MYC and CCND1 (Cychn DI).

Example 3. Generation of High-Plasticity Stem Cells (giaSC) via Interaction between Guide Cells and UC-MSCs In Vitro

[0201] The following method demonstrates that interaction of the activated/mature guide cells (m-GC) as described in Example 2 with umbilical cord derived mesenchymal stromal cells (UC-MSCs) generated high-plasticity stem cells, referred to herein as guide integrated adult stem cells (giaSC).

[0202] In one experiment, human umbilical cord tissue was collected during obstetrical delivery and kept at 4°C. UC-MSCs were isolated from human umbilical cord samples according to published protocol (N. Beeravolu, et.al., 2017). The m-GC as described in Example 2 above were labeled with Qtracker™ 625 red fluorescence dye (FIG. 4A). Human UC-MSCs were labeled with Calcein AM green fluorescence dye (FIG. 4B). The UC-MSCs were dissociated and added to adherent culture of m-GCs for co-culture (FIG. 4C) in the activation system, at a ratio of cell numbers that was 10: 1. In other experiments, the number of m-GC was more than that of UC-MSCs, for example, two times more, or three times more, or four times more. After co-culturing for a period of time (e.g., 12-72 hours), cell colonies emerged, which were surrounded by m-GC extending and transferring red cellular components into the colonies via tubular structures (TNTs) (FIG. 4D).

[0203] In another experiment, lower cell density was applied in the co-culture system, and tunneling nanotubes (TNTs) were formed between m-GC and UC-MSCs, with cellular components (red) transferred from m-GC (red) to UC-MSCs (green) (FIG. 5A). Acridine Orange (AO) staining showed that the cellular components transferred from m-GC to UC- MSCs included a lot of RNA-rich granules with red fluorescence (FIG. 5B).

[0204] The cell colonies in the co-culture system of m-GC and UC-MSCs were isolated, dissociated, and re-seeded in medium of the activation system. The cells derived from the colonies of the co-culture showed red granules received from m-GCs within green cytoplasm (FIG. 6A). These cells also demonstrated both self-renewal capacity (embryoid body -like cell colony formation and further expansion for multiple passages, FIG. 6B) and differentiation potential (expression of representative markers for three germ layer cells, FIG. 6F). Results showed that the colonies were stained positive for neural marker Nestin (example of ectoderm marker), muscle marker desmin and bone marker osteocalcin (example of mesoderm marker), as well as liver marker AFP (example of endoderm marker).

Therefore, the cells generated from the co-culture of m-GC and UC-MSCs were stem cells, hence termed “guide integrated adult stem cells'’ or “giaSC.”

[0205] In one experiment. giaSC were seeded in low density, and showed unique morphology of an approximately triangular cell body with slender and filiform pseudopodialike structure extending out from the cell body in adherent culture (FIG. 6C). Interaction via tunneling nanotubes (TNTs) was observed between neighboring giaSC, with transportation of RNA-rich granules (red) between each other (FIG. 6D). In one experiment, TNT structures between adherent giaSC were observed to be free-floating in the medium during cell culture. Thus, giaSC were different from both m-GC and UC-MSCs in cell morphology, growth pattern, and cell behaviors. Example 4. Identification and Expansion of giaSC In Vitro

[0206] The following methods were used to identify and expand giaSC in vitro as described in Example 3 above.

[0207] Further analysis was performed to study the marker profile, gene expression profile, and differentiation potential, which provide additional evidence of the distinctiveness and high-plasticity of giaSC compared to m-GC, UC-MSCs, as well as other cell types. Flow cytometry was used to study exemplary markers of the giaSC (FIG. 7C). Results showed that giaSC was a population of very high purity (e.g. , >90%) that was positive in CD49f and CD90, and negative in CD34. Further, the giaSC population expressed low level of CD45 and CD324, and was also positive in SSEA4, CD44, CD73, CD105, CD146, and CD56. The giaSC population was negative in SSEA3, CD3, CD19, and CD20. The giaSC population also expressed very low level of POU5F1 gene, and did not express Lin28, Nanog, and Sox2. Moreover, the giaSC population was positive in HLA-I and negative in HLA-II, and expressed low level of HLA-E. IF staining of m-GCs showed positive for CD45 and CD52. and negative for CD90 (FIG. 6E). UC-MSCs were stained positive for CD90 and negative for CD45 and CD52 (FIG. 6E). After integration of bioinformation from m-GCs into UC- MSCs, giaSCs were generated, showing positive for CD90 and CD52 and low level of CD45 (FIG. 6E). Large-scale genomic analysis showed that dormant guide cells (d-GC), activated/mature guide cells (m-GC), and giaSC were distinct in gene expression profile compared to other types of cells including stem cells (e.g., ESCs, iPSCs, MSCs, HSCs, and other stem cells) identified previously in public databases.

[0208] In one experiment, the giaSC population was further cultured and expanded for several passages with formation of cell colonies, from which proliferating cells crawled out and extended radially surrounding the colonies (FIG. 6B). Growth factors were used to culture the giaSC population, such as insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), bone morphogenetic proteins (BMPs), and transforming growth factors (TGFs). The a-MEM was supplemented with 10% serum and different combinations of IGF1, IGF2, FGF2, HGF, BMP5. In another experiment, the giaSC population was cultured in a-MEM supplemented with serum and IGF1. Example 5. Differentiation of giaSC In Vitro and Tissue Reconstitution In Vivo

[0209] The following methods were used to induce the differentiation of the giaSC population as described in Example 3 above.

[0210] In one experiment, a commercial neural differentiation induction media (NeuroCult™ NS-A Differentiation Kit, STEMCELL Technologies) was used to culture the giaSC, which gave rise to colonies that resemble neurospheres (FIG. 8A) and develop into typical neuron morphology (FIG. SB). Immunofluorescence (IF) staining results showed that these colonies were stained positive for Nestin (a marker of neuronal progenitor cells) and [33-tublin (one of the earliest markers of neuronal differentiation) (FIG. 8C). Further IF staining of neural- induced giaSCs showed positive expression for Nestin and [33-tublin, as well as neural stem cell marker Musashi RNA binding protein 2 (MSI2) and mature neuron marker NeuN (FIG. 8D). These results provided evidence of neuron differentiation of the giaSC as an example of ectoderm lineage. In control groups, MSCs and m-GCs in the neural induction medium failed to differentiate into neural cells.

[0211] In another example, the giaSC were cultured in a commercial bone differentiation media (MesenCult™ Osteogenic Differentiation Kit, STEMCELL Technologies) for 30 days and the cells showed positive staining with Alizarin Red dye that identifies calcium containing osteocytes and calcium deposits (FIG. 9A-B). These results provided evidence of bone differentiation of the giaSC as an example of mesoderm lineage.

[0212] In another experiment, when cultured in the activation system that mimics a hepatic environment, giaSC synthesized and released hepatic protein albumin (Alb) and hepatocyte growth factor (HGF) detected by ELISA assay (FIG. 10). These results showed that giaSC had differentiation potential into hepatic or liver progenitor cells as an example of endoderm plasticity.

[0213] An in vivo experiment was performed using a full-thickness wound model in the wildtype FVB mice. A circular 6 mm diameter full-thickness excisional wound was created using a biopsy punch in the dorsal skin of each FVB mouse. The human giaSC were mixed with a vehicle (human fibrin gel) and applied topically to the wound site and then covered by transparent film dressing Tegaderm for observation for 20 days. Human UC-MSCs were mixed with fibrin gel for topical application in a control group. Two other control groups were saline control and vehicle (fibrin-gel) control. The results showed that topical application of human giaSC/fibrin-gel enhanced healing rate, new hair growth, angiogenesis with new blood vessel formation (FIG. 11A), and full-layer skin tissue reconstitution and regeneration (FIG. 11B). By contrast, control groups (UC-MSCs/fibrin-gel, vehicle, saline) showed large scar area without new skin tissues such as blood vessels and hair follicles (FIG. 11A-B). The regenerated skin tissue at the wound site treated with human giaSC/fibrin-gel show ed presence of human cells derived from human giaSC as detected by human specific antibody (Ku80⁺ cells) in ectoderm representative tissues such as epidermis basal cells, hair follicles, and sebaceous glands, as well as in mesoderm representative tissues such as adipose tissue, blood vessels, and muscle tissue (FIG. 11C, indicated by arrows). Control groups treated with UC-MSCs, vehicle, and saline showed scar tissue at the wound site and negative staining of human Ku80. The engraftment of human cells derived from giaSC (Ku80 positive) in various skin tissues that originated from different germ layers indicated that giaSC had differentiation potential to cross germ layers in vivo and can actively adapt to the local skin environment to reconstitute and regenerate various tissues.

Example 6. Tumorigenicity Test of m-GC and giaSC In Vivo

[0214] The following method was used to evaluate the lumongenicity of m-GC and giaSC as described in Examples 2 and 3, respectively. In one experiment, a tumorigenicity test was performed for 4 months in immunodeficient nude mice with subcutaneous transplantation of human giaSC (IM cells, n=10), and the result showed no tumor formation in any of the tested mice. In another experiment, another tumorigenicity test was performed for 6 months in immunodeficient NOG mice with subcutaneous transplantation of the m-GC (IM cells, n=10) and the giaSC (IM cells. n=10), respectively, and the result showed no tumor formation in any of the transplanted mice.

[0215] In our experiments above, giaSC demonstrated in vitro differentiation into three germ layer cells and in vivo tissue repair and reconstitution without causing tumor, and thus present a significant advantage over cunent stem cells, especially ESCs and iPSCs that have tumorigenicity risks. Therefore, giaSC are referred to as high-plasticity stem cells that have both broad differentiation potential (e.g., across germ layers) and can actively adapt to host environment to regenerate/reconstitute various tissues without causing tumor. However, ESCs and iPSCs. albeit “pluripotent,” show very weak adaptability to the adult tissue environment and as a result develop into tumors. Thus, ESCs and iPSCs show low- plasticity. Once ESCs or iPSCs are differentiated into target cells (terminal differentiation) in vitro, the terminally differentiated cells lose the plasticity, especially the ability⁷ to actively adapt to the host environment.

Example 7. Generation of giaSC via Interaction between Guide Cells and Other Stem Cells and Non-Stem Cells In Vitro

[0216] The following methods were used to generate giaSC via interaction of the activated/mature guide cells (m-GC) as described in Example 2 above, with other stem cells (e.g, bone marrow derived mesenchymal stromal cells or BM-MSC) and non-stem cells (e.g., intestinal epithelial cells or lECs).

[0217] In one experiment, the m-GC as described in Example 2 above were labeled with Qtracker™ 625 red fluorescence dye (FIG. 12A). Human BM-MSCs were labeled with Calcein AM green fluorescence dye (FIG. 12B). The BM-MSCs were mixed and co-cultured with m-GC (FIG. 12C) in the activation system as described in Example 2 above. After a period of time (e.g., 12-72 hours), cell colonies emerged, which were surrounded by m-GC extending and transferring red cellular components into the colonies via TNTs (FIG. 12D). Cell-cell interaction (CCI) was observed between m-GC and BM-MSCs (FIG. 12D). RT- PCR analysis showed RNA expression of exemplary markers such as CD45, CD49f, CD73, CD90, CD105. and CD324 in the giaSC generated via interaction between m-GC and BM- MSC, which also include expression of ectoderm marker (e.g., nestin, notch-1, and notch-2), mesoderm markers (e.g, osteocalcin), and endoderm markers (e.g, CK18) (FIG. 12E).

These giaSC also express cyclin DI. c-Myc. HGF, VEGF, TGF-01, GDF15, CD106. and low level of EpCAM (FIG. 12E). The giaSC were further cultured in PSC Neural Induction Medium (Gibco) and were induced to acquire neurol cell morphology (FIG. 12F). In control groups, human BM-MSCs did not undergo neural differentiation in the neural induction medium. The results indicated that giaSC generated via interaction between m-GC and BM- MSC had differentiation potential for three germ layers.

[0218] In another experiment, the m-GC as described in Example 2 above were labeled with Qtracker™ 625 red fluorescence dye (FIG. 13A). Human intestinal epithelial cells (lECs) were labeled with Calcein AM green fluorescence dye (FIG. 13B). The lECs were mixed and co-cultured with m-GC (FIG. 13C) in the activation system as described in Example 2 above. After a period of time (e.g., 1-4 days), cell colonies emerged (FIG. 13F), and after further culture, cell-cell interaction (CCI) was observed between m-GC and lECs (FIG. 13D). Moreover, the m-GC were observed to release cellular components such as granules into the extracellular environment (indicated by arrow, FIG. 13D), and the granules were taken up into the cell colonies of giaSC (FIG. 13E). RT-PCR analysis showed RNA expression of exemplary markers such as CD49f, CD73, CD 105, and CD324 in the giaSC generated via interaction between m-GC and lECs, which also include expression of ectoderm marker (e.g., nestin, notch-1, and notch-2), mesoderm markers (e.g., osteocalcin), and endoderm markers (e.g, albumin, CK18, and CK19) (FIG. 13G). These giaSC also express cyclin DI, c-Myc, VEGF, TGF-(31, EpCAM, GDF15, and CD106 but does not express CD34 (FIG. 13G). These giaSCs did not showed expression of CD90, desmin, and HGF in the RT-PCR analysis. Immunofluorescence staining results (FIG. 13H) showed that these giaSC derived from lECs were stained positive for CD49f, CD45, SSEA4, and LGR5. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a bona fide biomarker for stem cells in multiple tissues and was first identified as a marker of intestinal stem cells. The results above indicated that giaSC were also generated via interaction between m-GC and lECs (a non-stem cell) and also have differentiation potential for three germ layers.

Example 8. Adaption and Differentiation of giaSCs In Vivo to Repair Small Intestinal Damage in FVB mice

[0219] The following methods were used to repair small intestinal damage in a lipopolysaccharide (LPS)-induced damage model by the giaSC population as described in Example 3 above.

[0220] LPS causes extensive small intestinal injury including damage to intestinal integrity and destruction and shortening of the villi. LPS-induced tissue damage model was established by intraperitoneal (i.p.) injections three times in wild-type FVB mice (10 mg/kg at DO, 5 mg/kg at Dll and 24). 3 days after the last LPS injection, surviving mice were randomly assigned to each group for treatment with human giaSCs (n=10), human UC-MSCs (n=10), and saline (n=8). Human giaSCs labeled using QTracker 625, a red fluorescent dye, were transplanted intravenously via retro-orbital injection (1 million cells per mouse). Control mice received human UC-MSCs labeled with QTracker 625 or saline. Of the giaSC- treated mic, 8 of 10 survived for 8 days; 5 of the treated with UC-MSCs and 4 of 8 treated with saline survived. All mice were sacrificed on day 8 after treatment, and 1 cm of ileum tissue, beginning at 1 cm proximal to the ileocecal junction, was removed for histological analysis. Because villus shortening is commonly utilized as a measure of small intestinal damage, we measured villus heights of the images post H&E staining. In order to investigate the engraftment of administered cells, a portion of the small intestine ileum tissue was embedded in OCT for frozen sectioning and the slides were immediately examined under fluorescent microscope for red signals of Qtracker™ 625. Rest of the ileum was further fixed for histological analysis by hematoxylin and eosin (H&E) and IHC using human Ku80 antibody. For undamaged control, human giaSCs (1 million cells per mouse) were injected (R.O.) into wild-ty pe FVB mice without LPS treatment (n=3). On day 8 after cell injection, the ileum tissue was fixed for histological analysis using anti-human Ku80 antibody.

[0221] Our results showed that villus heights were significantly greater in the small intestines of mice treated with giaSCs than in those of mice in the control groups (Fig. 14A), indicating protection against LPS-induced intestinal injury by giaSCs compared to that in control groups. In intestinal tissues of mice treated with giaSCs, red fluorescent signals from cells derived from the human giaSCs were detected in the crypts, at the bottom of the mucosal layer, and along the villi (Fig. 14B). The fluorescent signals coincided with locations of human Ku80⁺ cells detected by IHC (Fig. 14C-E). Many Ku80⁺ human giaSC-derived cells exhibited simple columnar epithelial morphology, originating from the crypts up both sides of the villi (Fig. 14C-D), indicating that human giaSCs had differentiated into epithelial cells that had replaced the mouse's own damaged cells. The upper parts of the villi were mainly composed of mouse simple columnar epithelial cells, with sporadic human Ku80⁺ cells (Fig. 14C) Human giaSC-derived cells were also observed in the smooth muscle layer of the small intestine and in the adipose tissue cells on the mesentery (Fig. 14E). Double immunofluorescence staining using antibodies specific for human and mouse ki67 antigens, respectively, revealed the presence of proliferating human cells derived from giaSCs and endogenous mouse cells in the small intestine villi treated with human giaSCs (Fig. 14H). The histological analysis of the giaSC-treated small intestines reveled less morphological damages than that in the control mice (Fig. 14C & 14F).

[0222] In mice treated with LPS and then with fluorescently labeled human UC-MSCs, no fluorescent signal was detected in the small intestine tissues (Fig. 14B), no Ku80⁺ cells were detected by IHC (Fig. 14F), and villus shortening and morphological damages were observed (Fig. 14A & 14F). In mice treated with LPS and then saline, small intestinal tissue was damaged and no Ku80⁺ cells were detected. The lack of Ku80⁺ cells confirmed the human Ku80-specificity of the antibody used. In wild-type (FVB) mice not treated with LPS but injected retro-orbitally with giaSCs, no human Ku80⁺ cells were detected in the healthy small intestinal tissues (Fig. 14G). This indicated that human giaSCs do not anchor in small intestinal tissues under normal physiological conditions. In summary, within 8 days post intravenous injection, human giaSCs had migrated to and anchored in the damaged small intestine of the mouse, and differentiated into small intestinal epithelial cells to participate in repair of the damaged tissue.

Claims

What is Claimed is:

1. A method of generating high-plasticity stem cells, comprising contacting somatic cells with guide cells and/or cellular components thereof, wherein the guide cells are characterized as CD49E/CD45⁺/CD90“, and wherein the high-plasticity stem cells preferably have differentiation potential for more than one germ layer.

2. The method of claim 1, wherein the contacting results in establishment of cellcell interaction that allows transfer of the cellular components from the guide cells into the somatic cells.

3. The method of claim 1 or 2, wherein the cellular components comprise RNA.

4. The method of claim 2 or 3, wherein the cell-cell interaction is via tunneling nanotubes.

5. The method of claim 2 or 3, wherein the cell-cell interaction is via gap junction.

6. The method of claim 2 or 3, wherein the cell-cell interaction is via exocytosis and endocytosis.

7. The method of any one of claims 1-6, wherein the contacting is established in a co-culture system.

8. The method of any one of claims 1-7, wherein the guide cells and the somatic cells have a ratio of cell numbers that is at least about 1: 1.

9. The method of any one of claims 1-8, wherein the guide cells are further characterized as CD44⁺ and/or CD52⁺.

10. The method of any one of claims 1-9, wherein the guide cells are further characterized as SSEA4- and/or SSEA3-.

11. The method of any one of claims 1-10, wherein the guide cells are further characterized as CD324- and/or CD73-.

12. The method of any one of claims 1-11, wherein the guide cells are further characterized as positive in one or more markers selected from the group consisting of CD 105, HLA-I, HLA-II. and HLA-E.

13. The method of any one of claims 1-12, wherein the guide cells are further characterized as negative in one or more markers selected from the group consisting of Lin, CD146, CD3, CD19. CD20, and CD56.

14. The method of any one of claims 1-13, wherein the guide cells do not express one or more genes selected from the group consisting of Lin28, Nanog, and Sox2.

15. The method of any one of claims 1-14, wherein the guide cells comprise a plurality of RNA-rich granules in the cytoplasm.

16. The method of any one of claims 1-15. wherein the guide cells comprise one or more pseudopodia.

17. The method of any one of claims 1-16, wherein the guide cells are derived from blood.

18. The method of claim 17, wherein the blood includes human peripheral blood.

19. The method of any one of claims 1-18. wherein the somatic cells comprise stem cells.

20. The method of any one of claims 1-18, wherein the somatic cells comprise mesenchymal stromal cells.

21 . The method of any one of claims 1-18, wherein the somatic cells comprise non-stem cells.

22. The method of claim 21, wherein the non-stem cells have proliferative capacity.

23. The method of any one of claims 1-22, wherein the high-plasticity stem cells are characterized as CD49E/CD90⁺/CD34“.

24. The method of claim 23, wherein the high-plasticity stem cells are further characterized as CD73⁺.

25. The method of any one of claims 1-22. wherein the high-plasticity stem cells are characterized as CD49 /CD73⁺/CD34“.

26. The method of claim 25, wherein the high-plasticity stem cells are characterized as CD90-.

27. The method of any one of claims 1-26, wherein the high-plasticity stem cells are further characterized as CD45^low and/or CD324^low.

28. The method of any one of claims 1-26. wherein the high-plasticity stem cells are further characterized as SSEA4⁺ and/or SSEA3-.

29. The method of any one of claims 1-28. wherein the high-plasticity stem cells are further characterized as CD105⁺.

30. The method of any one of claims 1-29, wherein the high-plasticity stem cells are further characterized as positive in one or more markers selected from the group consisting of CD44, CD 146. CD56, CD52, HLA-I, and HLA-E.

31. The method of any one of claims 1-30, wherein the high-plasticity’ stem cells are further characterized as negative in one or more markers selected from the group consisting of CD3. CD19, CD20, and HLA-II.

32. The method of any one of claims 1-31, wherein the high-plasticity stem cells do not express one or more genes selected from the group consisting of Lin28, Nanog, and

Sox2.

33. The method of any one of claims 1-32, wherein the high-plasticity stem cells express (a) one or more of endoderm markers, (b) one or more of mesoderm markers, and (c) one or more of ectoderm markers.

34. The method of claim 33, wherein the endoderm markers comprise AFP. CK7 (KRT7), albumin (ALB), CK18 (KRT18), and CK19 (KRT19), wherein the mesoderm markers comprise desmin (DES), osteocalcin (BGLAP), CD106 (VCAM-1), CD54 (ICAM- 1), and CD 146 (MCAM), and wherein the ectoderm markers comprise nestin (NES), notch- 1, notch-2, MSI2. CD56 (NCAM1), and CD325 (N-cadherin or Cadherin-2).

35. The method of any one of claims 1-34, wherein the high-plasticity stem cells have approximately triangular cell bodies with one or more slender pseudopodia.

36. The method of any one of claims 1-35. wherein the high-plasticity stem cells exchange a plurality of cellular components between each other and/or with cells of a different kind.

37. The method of claim 36, wherein the plurality of cellular components comprise RNA.

38. The method of claim 36 or 37, wherein the exchange of the plurality of cellular components is via tunneling nanotubes, gap junction, and/or exocytosis/endocytosis.

39. The method of any one of claims 1-38, wherein the guide cells are derived from dormant guide cells that (a) have a diameter of less than 6 pm, (b) express CD49f and CD45, (c) do not express CD90, and (d) do not have detectable intracellular esterase activity and/or transcriptomic activity.

40. The method of claim 39, wherein the dormant guide cells are isolated from human blood.

41. A composition comprising high-plasticity stem cells that are generated by the method of any one of claims 1 -40.