JP2025032034A - Rejuvenation agent and rejuvenation method - Google Patents

Abstract

[Problem] To provide a rejuvenating agent that restores cell function, inhibits senescence-associated secretory phenomenon (SASP), lengthens telomeres, and restores cell division ability.
The present invention relates to a rejuvenation agent for administration to a subject containing cells, the rejuvenation agent comprising microparticles derived from dental pulp-derived stem cells; a rejuvenation method;
[Selected Figure] Figure 1

Description

The present invention relates to a rejuvenation agent and a rejuvenation method.

There has been much research into the rejuvenation of cells and tissues, as well as the organisms that contain them. In previous studies, rejuvenation methods have been considered to involve at least one of the following: restoring cellular function, inhibiting senescence-associated secretory phenomena (SASP), lengthening telomeres, and restoring the ability to divide cells.

With regard to the restoration of cell function, Patent Document 1 describes a cell-free supernatant or a fraction of said cell-free supernatant that has previously supported the growth of a dedifferentiated plant cell suspension culture for use as a drug for skin treatment, the cell-free supernatant or fraction containing a peptide selected from peptide plant growth factors, plant transcription factors, epigenetic factors, and mixtures thereof, having a length of 4 to 300 amino acids, and the cell-free supernatant or fraction is free of cytoplasmic intracellular contents and membranes and/or cell walls resulting from cell lysis. It also describes the use of the cell-free supernatant or fraction as a repair and/or regenerator for animal skin cells by restoring aging in skin cells (i.e., fibroblasts).

Regarding the inhibition of SASP, Patent Document 2 describes a method for extending the life span of a subject, which comprises administering to the subject in need thereof therapeutically effective amounts of berberine, a vitamin A compound, and α-ketoglutaric acid (AKG). It also describes that SASP secreted by senescent cells contributes to the onset and progression of aging-related diseases.
Non-Patent Document 1 describes that treatment of old animals with small extracellular vesicles (sEVs) derived from adipose mesenchymal stem cells from young animals resulted in improvements in motor coordination, grip strength, fatigue resistance, fur regeneration, renal function, and the like, as well as a reduction in the aging markers SASP (IL-1β and IL-6).

Telomeres, in turn, are repeated DNA sequences that cap the ends of each chromosome and are associated with proteins that ensure and guarantee chromosomal integrity by protecting the ends of each chromosome from constant degradation during each cell cycle. Telomere shortening can result in cellular aging, and telomeres gradually shorten during the course of normal aging. Increasing the length of telomeres in cells leads to cell rejuvenation. In response to this, a method of rejuvenation is known in which cells are contacted with specific drugs to lengthen telomeres.
For example, Patent Document 3 describes a method for increasing telomere length in one or more human cells, comprising contacting one or more human cells with an agent that increases expression of Zscan4 in the human cells, wherein increased expression of Zscan4 induces telomere elongation in the one or more human cells compared to one or more corresponding human cells that have not been contacted with the agent.

Regarding restoring the ability of cells to divide, the ability of cells to divide generally decreases with age. In contrast, by restoring the ability of cells to divide, new healthy cells are generated.

JP 2018-512440 A JP 2023-027149 A JP 2023-099826 A

Sci. Adv. (2022) 8, 42, eabq2226

However, until now, few rejuvenation agents or methods have been known that can restore cell function, inhibit senescence-associated secretory phenomena (SASP), lengthen telomeres, and restore cell division ability.

The problem that the present invention aims to solve is to provide a rejuvenating agent that restores cell function, inhibits secretory phenomena associated with cellular aging, lengthens telomeres, and restores cell division ability.

The present inventors have found that a composition comprising a culture supernatant of dental pulp-derived stem cells, which contains microparticles derived from dental pulp-derived stem cells, restores cell function, inhibits secretory phenomena associated with cellular senescence, lengthens telomeres, and restores cell division ability.

Specifically, the present invention and its preferred configurations are as follows:

[1] A rejuvenating agent for administration to a subject comprising cells, comprising:
A rejuvenating agent comprising microparticles derived from dental pulp-derived stem cells.
[2] The rejuvenation agent described in [1], which is a composition comprising a culture supernatant of dental pulp-derived stem cells, wherein the culture supernatant contains microparticles.
[3] The rejuvenation agent according to [1], wherein the microparticles contain at least one of the rejuvenation-related miRNAs listed below.
Rejuvenation-related miRNA:
has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has- miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p and miR-93-5p.
[4] The rejuvenation agent described in [3], wherein the microparticles contain at least one of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p as rejuvenation-related miRNAs.
[5] The rejuvenation agent described in [3], wherein the microparticles contain rejuvenation-related miRNA at a higher concentration than the culture supernatant.
[6] The rejuvenating agent according to [1], wherein the composition does not contain a culture supernatant excluding microparticles.
[7] (2) The rejuvenating agent described in [1], which is an inhibitor of the expression of the following SASP factor.
SASP factors:
IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3 and MCP-1.
[8] The rejuvenation agent according to [1], wherein the cell is a somatic stem cell or a differentiated cell.
[9] The subject is a somatic stem cell or a differentiated cell,
The rejuvenating agent described in [1] can rejuvenate a subject that has been passaged n times more easily when administered with the rejuvenating agent than a subject that has been passaged n times without administration of the rejuvenating agent, where n is a natural number.
[10] (3) The rejuvenation agent described in [1], which suppresses shortening of the average telomere length at the ends of chromosomes possessed by a cell.
[11] (4) A rejuvenating agent as described in [1], which suppresses the number of positive cells stained with SA-β-gal when administered to a subject that has been passaged n times compared to the number of positive cells stained with SA-β-gal in a subject that has been passaged n times without administration of the rejuvenating agent.
[12] The rejuvenating agent described in [1], which has all of the following effects: recovery of cell function, inhibition of senescence-associated secretory phenomenon (SASP), lengthening of telomeres, and recovery of cell division ability.
[13] The rejuvenating agent according to [1], which satisfies all of the following (1) to (4):
(1) At least one of the following rejuvenation-related miRNAs is included:
Rejuvenation-related miRNA:
has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has- miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p and miR-93-5p;
(2) An inhibitor of the expression of the following SASP factor:
SASP factors:
IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3 and MCP-1;
(3) suppressing shortening of the average telomere length at the ends of chromosomes in cells;
(4) When a rejuvenating agent is administered to a subject that has been passaged n times, the number of positive cells stained with SA-β-gal contained in the subject is suppressed to be lower than the number of positive cells stained with SA-β-gal contained in a subject that has been passaged n times without administration of a rejuvenating agent.
[14] The rejuvenating agent described in [1], wherein the microparticles are exosomes.
[15] A rejuvenation method comprising administering an effective amount of the rejuvenation agent according to [1] to a subject containing cells.
[16] The rejuvenation method described in [15], wherein the amount of the rejuvenation agent administered is an amount that results in 500 or more microparticles per cell.

The present invention provides a rejuvenating agent that restores cell function, inhibits senescence-associated secretory phenomena (SASP), lengthens telomeres, and restores cell division ability.

FIG. 1 is a graph showing the read counts of 50 types of miRNAs with the highest read counts among the miRNAs expressed in exosomes of dental pulp-derived stem cells. FIG. 2 is a graph showing the expression of each SASP factor as a relative value (%), with Untreated being set at 100. FIG. 3 is a graph showing the average telomere length of each chromosome end of NHDF cells at passage number P5 and the average telomere length of each chromosome end of NHDF cells at passage number P12 when stimulated with the rejuvenating agent of Example 1 (culture supernatant of dental pulp-derived stem cells) or control (phosphate-buffered saline; PBS). FIG. 4 is a graph showing the percentage of SA-β-gal positive cells in aged ADMSCs (adipose-derived stem cells) at passage number P12 when stimulated with the rejuvenating agent of Example 1 (culture supernatant of dental pulp-derived stem cells) or control (phosphate buffered saline; PBS) for 72 hours (Day 3). FIG. 5 is a schematic diagram of Test Example 4 showing that the rejuvenating agent of the present invention (SHED-CM-EVs) has the effect of rejuvenating aged mesenchymal stem cells. FIG. 6 is a graph showing the relationship between the passage number of ADMSCs and the number of SA-β-gal positive cells at 0 hours of culture time.

The present invention will be described in detail below. The following explanation of the constituent elements may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "~" means a range that includes the numerical values written before and after "~" as the lower and upper limits.

[Rejuvenating agent]
The rejuvenating agent of the present invention is intended to be administered to a subject containing cells, and includes microparticles derived from dental pulp-derived stem cells.
The rejuvenating agent of the present invention has all of the following properties: recovery of cell function, inhibition of senescence-associated secretory phenomenon (SASP), lengthening of telomeres, and recovery of cell division ability.
Preferred embodiments of the rejuvenating agent of the present invention will be described below.

<Definition of rejuvenation>
In the present invention, rejuvenation refers to the restoration of cell function, the inhibition of secretory phenomena associated with cellular aging, the lengthening of telomeres, and the restoration of cell division ability.
Therefore, it is preferable that the rejuvenating agent of the present invention has all of the following effects: recovery of cell function, inhibition of senescence-associated secretory phenomenon (SASP), lengthening of telomeres, and recovery of cell division ability.

Specifically, the rejuvenating agent of the present invention preferably satisfies at least one of the following (1) to (4), more preferably satisfies at least two, particularly preferably satisfies at least three, and more particularly preferably satisfies all of them.
(1) is associated with the recovery of cell function, (2) is associated with the suppression of senescence-associated secretory phenomenon (SASP), (3) is associated with the lengthening of telomeres, and (4) is associated with the recovery of cell division ability.
(1) Contains at least one of the following rejuvenation-related miRNAs:
Rejuvenation-related miRNA:
has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has- miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p and miR-93-5p;
(2) An expression inhibitor of the following SASP factor:
SASP factors:
IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3 and MCP-1;
(3) suppressing shortening of the average telomere length at the ends of chromosomes in cells;
(4) When a rejuvenating agent is administered to a subject that has been passaged n times, the number of positive cells stained with SA-β-gal contained in the subject is suppressed to be lower than the number of positive cells stained with SA-β-gal contained in a subject that has been passaged n times without administration of a rejuvenating agent.

<Culture supernatant of dental pulp-derived stem cells>
The rejuvenating agent of the present invention is preferably a composition containing a culture supernatant of dental pulp-derived stem cells, and in this case, the culture supernatant preferably contains microparticles.

<Microparticles>
The rejuvenating agent of the present invention comprises microparticles derived from dental pulp-derived stem cells.
The microparticles are derived from dental pulp-derived stem cells, for example, by secretion, budding or dispersion from the mesenchymal stem cells of dental pulp-derived stem cells, and are exuded, released or shed into the cell culture medium.
The origin of the microparticles can be determined by a known method. For example, the microparticles can be determined to be derived from any stem cell, such as dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, or umbilical cord-derived stem cells, by the method described in J Stem Cell Res Ther (2018) 8:2. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticles.

(1) Rejuvenation-related miRNA
In the present invention, it is preferable that the microparticles contain at least one of the rejuvenation-related miRNAs described below.
Rejuvenation-related miRNA:
has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has- miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p and miR-93-5p.
In the present invention, miRNA (MicroRNAs) are RNA molecules having, for example, 21 to 25 bases (nucleotides). miRNA can regulate gene expression by degrading target gene (target) mRNA or suppressing it at the decoding stage.
In the present invention, miRNA may be, for example, a single-stranded (monomer) or a double-stranded (dimer). In addition, in the present invention, miRNA is preferably a mature miRNA cleaved by a ribonuclease such as Dicer.

In addition, the sequences of the miRNAs described herein, such as hsa-miR-199a-3p, are registered in a known database (e.g., miRBase database) in association with accession numbers, and a person skilled in the art can uniquely determine the sequence. For example, the accession number of hsa-miR-199a-3p is MIMAT0000232, and the sequence is registered in the miRBase database. Hereinafter, the accession numbers of each miRNA are omitted.
However, the miRNA in this specification also includes variants that differ from mature miRNAs such as hsa-miR-199a-3p by about 1 to 5 bases. In addition, each miRNA in this specification includes a polynucleotide consisting of a base sequence having identity to the base sequence of each miRNA (e.g., hsa-miR-199a-3p), or a polynucleotide consisting of a complementary base sequence thereof, which has the function of the miRNA in the present invention. "Identity" refers to the degree of identity when the sequences to be compared are appropriately aligned, and means the occurrence rate (%) of exact amino acid matches between the sequences. Alignment can be performed, for example, by using any algorithm such as BLAST. The identity is, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99%. A polynucleotide consisting of a base sequence having identity may have, for example, a point mutation, deletion and/or addition in the base sequence of miRNA. The number of bases such as point mutations is, for example, 1 to 5, 1 to 3, 1 to 2, or 1. In addition, a polynucleotide consisting of a complementary base sequence is, for example, a polynucleotide that hybridizes with a polynucleotide consisting of a base sequence of miRNA under stringent conditions, and includes a polynucleotide having the function of miRNA in the present invention. Examples of stringent conditions include, but are not limited to, the conditions described in [0028] of JP 2017-184642 A, the contents of which are incorporated herein by reference.

In the present invention, it is preferable that the microparticles contain rejuvenation-related miRNA (and TERT-related miRNA, described below) at a higher concentration than the culture supernatant of dental pulp-derived stem cells. Preferred embodiments of the rejuvenation-related miRNA contained in the microparticles are described below.

It is known that human miR-125a and miR-125b correspond to lin-4 in C. elegans, and that in C. elegans, lin-4 directly controls lifespan and loss of lin-4 function shortens lifespan (Human Genetics (2020) 139, 291-308). Therefore, it is speculated that human miR-125a-5p and has-miR-125b-5p are miRNAs related to lifespan.
miR-125b is known to be transferred from young fibroblasts to old fibroblasts via exosomes, promoting fibroblast migration and migration to combat aging (Journal of Nanobiotechnology (2020), 20, 144). Therefore, has-miR-125b-5p can be said to be a rejuvenation-related miRNA for anti-aging.

Int. J. Mol. Sci. (2020), 21(15):5281, Fig. 2, shows a schematic diagram of the role of microRNA regulation in human skin, including the involvement of miRNA in the development of skin components, the aging process, and cancer. This diagram shows that miR-199a and miR-199b are involved in hair development, miR-21 and miR-155 are involved in the development of Langerhans cells, and miR-155 is involved in photoaging of fibroblasts. Therefore, miR-155-5p, miR-199a-3p, miR-199b-5p, and miR-21-5p can be said to be rejuvenation-related miRNAs.
In addition, Fig. 3 of Int. J. Mol. Sci. (2020), 21(15):5281 provides a schematic diagram of the role of microRNAs in regulating important molecular signaling pathways in the skin aging process. The schematic diagram shows miRNAs (shown in red) that regulate cell signaling mechanisms and potentially promote aging, and miRNAs (shown in blue) that suppress aging, and describes that miR-93 is involved in the suppression of MMPs, P21, TGFβ, and E2F, miR-24 is involved in the suppression of P16, miR-155 is involved in the suppression of c-JUN, and miR-181a/b is involved in the suppression of SIRT1. Therefore, miR-155-5p, miR-181a-5p, miR-24-3p, and miR-93-5p can be said to be rejuvenation-related miRNAs.

Communications Biology (2021), 4, 427 describes that miR-199-3p, which circulates in the blood as a cell-free miRNA, is significantly reduced in the blood of aged mice compared to young mice. Furthermore, it has been shown that miR-199-3p has the function of promoting muscle differentiation and muscle regeneration, that administration of a miR-199 mimic that supplies miR-199-3p to aged mice delayed muscle fiber hypertrophy and muscle strength decline, and that systemic administration of a miR-199 mimic to mdx mice, known as an animal model of Duchenne muscular dystrophy (DMD), significantly improved the muscle strength of the mice. Therefore, cell-free miR-199a-3p and miR-199b-3p in the blood can be said to be rejuvenation-related miRNAs that have anti-aging effects such as hypertrophy of aged muscle fibers.
ACS Nano (2019) 13, 11273-11282 describes that in exosomes from three-dimensional spheroid-formed human fibroblasts (HDFs), hsa-miR-196a-5p and hsa-miR-744-5p were downregulated compared to exosomes from monolayer-cultured human fibroblasts (2D HDFs), whereas hsa-miR-133a-3p, hsa-miR-223-3p, hsa-5011-5p, hsa-miR-325, hsa-miR-199b-5p and hsa-miR-34a-5p were upregulated compared to both mesenchymal stem cell (MSC) and 2D HDF exosomes (Figure 2B). It has also been reported that UV rays induce enhanced oxidative stress in the skin, activating the TNF-α and NF-kB signaling pathways, resulting in collagen degradation and aging. It has also been reported that TIMP-1 and TGF-β, which are important for suppressing MMP and controlling matrix synthesis, were upregulated and TNF-α was downregulated in 3D HDF exosomes. It has also been reported that during the aging process, upregulation of MMP production and downregulation of collagen production lead to age-related skin disorders such as weakening of the dermal structure and reduced functional integrity, and that exosomes from HDFs formed into 3D spheroids were shown to restore the function of aged HDFs more than exosomes from mesenchymal stem cells (MSCs) or 2D HDFs. It has also been reported that exosomes from spheroid-formed HDFs may control dermal fibroblasts, induce efficient collagen biosynthesis, and improve skin inflammation caused by UVB irradiation, and that upregulation of miR-133a and miR-223 may contribute to this improvement. It has also been reported that exosomes from spheroid-formed HDFs suppress UVB-induced MMP1 expression, restore type I procollagen, and activate the TGF-β signal pathway, and that exosomes from spheroid-formed HDFs improve skin inflammation and aging through downregulation of TNF-α. Therefore, the above miRNAs, especially miR-223-3p, can be said to be rejuvenation-related miRNAs that improve age-related skin disorders.

In the present invention, it is preferable that the microparticles contain at least one of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p as rejuvenation-related miRNAs, more preferably at least two of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p, and particularly preferably all of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p.

The microparticles preferably contain at least one of the rejuvenation-related miRNAs, as a read count number obtained by analysis using IMOTA, of 10,000 or more, more preferably 30,000 or more, particularly preferably 50,000 or more, and more preferably 100,000 or more. The microparticles preferably contain has-miR-125a-5p and hsa-miR-199a-3p, as a Log2Ratio of the read count number obtained by analysis using IMOTA, of 30,000 or more, particularly preferably 50,000 or more, and more preferably 100,000 or more. The microparticles preferably contain hsa-miR-21-5p, more preferably 10,000 or more, and more preferably 30,000 or more.
The microparticles preferably contain at least one of the rejuvenation-related miRNAs, as a Log2Ratio of the number of read counts obtained by analysis using IMOTA, of 8.0 or more, more preferably 10.0 or more, and particularly preferably 12.0 or more. The microparticles preferably contain has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p, as a Log2Ratio of the number of read counts obtained by analysis using IMOTA, of 8.0 or more, more preferably 10.0 or more, and particularly preferably 12.0 or more.

The microparticles of the present invention preferably have expression levels of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p that are 1.1 times or more, more preferably 1.5 times or more, and particularly preferably 2 times or more, as compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells.

Here, the microparticles derived from the culture supernatant of dental pulp-derived stem cells contain about 2600 types of small RNA. Of these, about 1800 types are miRNA. Of these miRNAs, 180 to 200 types of miRNA are highly abundant. The miRNAs highly abundant in microparticles derived from dental pulp-derived stem cells are characterized by a high content of microRNAs related to the treatment of cranial nerve diseases and rejuvenation-related miRNAs, which was previously unknown and is a new finding discovered by the present inventor. This characteristic is significantly different from the types of miRNAs highly abundant in other mesenchymal stem cell microparticles. For example, the miRNAs highly abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to the treatment of cranial nerve diseases. In the context of the present invention, the rejuvenation-related miRNAs in microparticles derived from dental pulp-derived stem cells are much more abundant than the rejuvenation-related miRNAs in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells.

The microparticles preferably contain two or more types of rejuvenation-related miRNA, more preferably three or more types, even more preferably five or more types, particularly preferably eight or more types, and even more particularly preferably ten or more types.

(2) SASP Factor Expression Inhibitor The rejuvenating agent of the present invention is preferably an SASP factor expression inhibitor described below.
SASP factors:
IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3 and MCP-1.
The rejuvenating agent of the present invention can slow down the aging rate of cells (such as stem cells or differentiated cells, particularly tissue stem cells) and reduce SASP factors, which are aging factors released by aging cells (particularly aging stem cells).
Of these SASP factors, the rejuvenating agent of the present invention is more preferably an expression inhibitor of CCL5, CXCL10, IL-6, TNF-α and MCP-1.
In addition, the active ingredient as an expression inhibitor of SASP factors in the rejuvenating agent of the present invention is not particularly limited, but it is preferable that the microparticles contained in the rejuvenating agent of the present invention are expression inhibitors of these SASP factors.

IL-1β is a potent proinflammatory cytokine that lowers pain threshold, produces fever, and causes tissue injury, promotes T cell activation and antigen recognition, and also induces Th17 differentiation and maturation and is involved in the induction and secretion of prostaglandins. Transcription of IL-1β into mRNA is induced by microbial components, TNFα, IL-18, IL-1α, and IL-1β.
CCL5 (chemokine (CC motif) ligand 5) and CXCL10 (CXC motif chemokine 10) are chemokines that attract cytotoxic T cells that kill cancer cells.
PAI-1 (plasminogen activator inhibitor-1) is present in vascular endothelial cells, liver, platelets, fat cells, etc., and is released in large amounts into the blood due to vascular endothelial damage and platelet destruction. PAI-1 has the function of controlling fibrinolytic reactions by specifically and instantly inhibiting tissue plasminogen activator (t-PA), which helps the plasmin production reaction that dissolves formed thrombi.
IL-6 is a B cell differentiation factor; B cell stimulating factor-2; hepatocyte stimulating factor; hybridoma growth factor; and plasmacytoma growth factor. IL-6 is a representative inflammatory cytokine and is a multifunctional cytokine involved in the control of acute inflammatory responses, regulation of certain immune responses including B and T cell differentiation, bone metabolism, platelet production, epithelial proliferation, menstruation, neuronal differentiation, neuroprotection, aging, cancer, and inflammatory responses occurring in Alzheimer's disease. IL-6 is thought to play a role in the development of numerous diseases and disorders, including fatigue, cachexia, autoimmune diseases, skeletal diseases, cancer, heart disease, obesity, diabetes, asthma, Alzheimer's disease, and multiple sclerosis (see [0002] to [0005] of JP 2019-047787 A).
TNF (Tumor Necrosis Factor) is a representative inflammatory cytokine. TNFα, TNFβ and LTβ are known as TNF. Among them, TNFα mediates several important life functions including structural and functional organization of secondary lymphoid organs, apoptosis and antitumor activity, inhibition of viral replication, immunoregulation and inflammation. TNF also plays an important role in the pathogenesis of autoimmune diseases, acute phase reactions, septic shock, fever and cachexia (see [0004] in JP 2020-079306 A).
MMP-3 is a proteolytic enzyme produced by the surface cells of the synovial membrane of joints, and is closely related to the pathology of rheumatoid arthritis (RA) by dissolving cartilage, and is therefore considered a marker reflecting synovial proliferation. The produced MMP-3 accumulates in synovial fluid, and then migrates into the blood via blood vessels and lymphatic vessels, increasing serum MMP-3 levels, so that serum MMP-3 levels reflect the degree of synovial proliferation in RA.
MCP-1 (Monocyte chemoattractant protein-1) is a chemokine belonging to the CC chemokines, and has a strong chemotactic effect on monocytes and macrophages. In arteriosclerosis, MCP-1 promotes macrophage infiltration into lesions, and progresses the pathology through mechanisms such as foam cell formation and secretion of various cytokines. In addition to monocyte and macrophage migration, MCP-1 also directly promotes the proliferation of vascular smooth muscle cells and collagen production by fibroblasts, and is involved in the pathology of various cardiovascular diseases.

(3) Telomere lengthening/TERT-related miRNA
The rejuvenating agent of the present invention preferably inhibits the extension of the average telomere length at the ends of chromosomes possessed by cells, or inhibits the shortening of the average telomere length at the ends of chromosomes possessed by cells.
It is more preferable that the rejuvenating agent of the present invention suppresses shortening of the average telomere length at the ends of chromosomes possessed by cells.
Here, the ability of the rejuvenation agent of the present invention to lengthen telomeres is experimentally demonstrated in the Examples described below, and is also supported by the fact that the agent contains TERT-related miRNA.
Telomerase (TERT) confers replicative immortality to cancer cells, and its overexpression serves as a nearly universal marker of cancer. More than 50% of gene sequences encoding miRNAs are present in cancers and regions with high mutation rates, so miRNAs are involved in promoting cancer progression and silencing genes related to cancer progression. TERT-related miRNAs have been mainly studied as oncogenic miRNAs, which have the effect of activating TERT and extending telomeres.
Cancers (2020), 12(9), 2337 describes that miR-19b indirectly upregulates hTERT expression by inhibiting PITX1, an hTERT (human telomerase) suppressor gene. It also describes that miR-346 mediates the upregulation of hTERT through a competitive process with miR-138, which binds to the same region of the 3'UTR of hTERT mRNA as miR-346 but promotes the opposite effect on hTERT expression. In particular, it has been described that miR-21 affects telomerase activity through downregulation of PTEN, upregulating the expression of hTERT.
Open J Proteom Genom (2016), 1(1): 013-018 describes that miR-21 inhibits apoptosis and induces proliferation accompanied by negative regulation of hTERT expression via the phosphoinositide 3 kinase (PI3K) signaling pathway by binding to the 3'UTR of PTEN, inhibiting its translation. Regarding miR-19b, it has been described that PITX1 (Paired Like Homeodomain 1) mRNA is a direct target of miR-19b, and downregulation of PITX1 by miR-19b ultimately induces enhanced expression of hTERT mRNA. The middle sequence motif of miR-346 (nt 8-13, CCGCAU) binds to G-rich sequence binding factor 1 (GRSF-1) to form a bulge loop. This binding has been described to recruit hTERT mRNA to ribosomes for translation in an AG02-independent manner. It has been described that miR-155 acts as a key regulator in the development of breast cancer cells and is efficiently upregulated with decreased TRF1 protein levels; miR-155 mediates telomere elongation, increased telomere fragility, and chromosomal instability by decreasing the expression of TRF1 (telomeric repeat factor 1), which is part of the shelterin complex.
PLoS ONE (2016), 11(9): e0162077 describes that, when the expression levels and telomere length of miRNAs expressed in normal colonic mucosa of colon cancer patients were observed, it was confirmed that the higher the expression level of miRNA, the longer the telomere length. Many miRNAs have been described as miRNAs that increase telomere length, such as miR-134-5p, miR-5088 (miR-5088-3p, miR-5088-5p), and miR-887 (miR-887-3p, miR-887-5p).
PLoS ONE (2014), 9(4): e92088 describes combinations of miRNA and TERT-related proteins that increase target genes, including miR-181b and TERT, miR186 and TERF2IP, RAD50 and SIRT6, miR-96 and TERF2IP, and miR-15a and TATA box binding protein (TBP). For example, in a verification of TERF2IP and RAD50, it was described that only TERF2IP significantly decreased the amount of its transcript 60 minutes after exercise, and that the amount of miRNAs with binding potential (miR-186 and miR-96) increased accordingly. Based on this paper, miR-15a-5p, miR-181b-2-3p, miR-181b-3p, miR-181b-5p, miR-186-3p, miR-186-5p, and miR-96-5p can be said to be TERT-related miRNAs.
Almost all of these TERT-related miRNAs found by IMOTA are contained in dental pulp-derived microparticles. In particular, among the miRNAs that are ranked within the top 50 in terms of content in dental pulp-derived stem cell microparticles, there are six TERT-related miRNAs that target TERT: miR-16-5p, miR-143-3p, miR-181a-5p, miR-21-5p, miR-34a-5p, and miR-103a-3p.
The active ingredient in the rejuvenating agent of the present invention for telomere lengthening is not particularly limited, but it is preferable that the microparticles contained in the rejuvenating agent of the present invention are an active ingredient for telomere lengthening.
Although TERT-related miRNA promotes carcinogenesis, various miRNAs exist in the microparticles of dental pulp-derived stem cells and can regulate each other, so there is no problem of carcinogenicity (no need to conduct carcinogenicity experiments). TERT-related miRNAs are also present in the body and are thought to be necessary for maintaining some kind of homeostasis.

(4) Recovery of cell division ability The rejuvenating agent of the present invention is preferably capable of recovering cell division ability. Specifically, the rejuvenating agent of the present invention preferably suppresses the number of positive cells stained with SA-β-gal contained in a subject passaged n times when the rejuvenating agent is administered to the subject, compared to the number of positive cells stained with SA-β-gal contained in a subject passaged n times without administration of the rejuvenating agent.
The number of positive cells (proportion of positive cells) stained with SA-β-gal is an index representing the cell division ability of cells, and is expressed, for example, as a percentage. In general, the number of positive cells (proportion of positive cells) stained with SA-β-gal decreases with the number of passages.
n is an integer of 1 or more. There is no limitation on the value of n, but n may be, for example, 2 to 100, 4 to 20, 8 to 16, or 10 to 14. Regardless of the number of passages, administration of the rejuvenating agent of the present invention can make the cells younger than the number of passages.
The active ingredient in the rejuvenating agent of the present invention for restoring cell division ability is not particularly limited, but it is preferable that the microparticles contained in the rejuvenating agent of the present invention are an active ingredient for restoring cell division ability.

(Types of microparticles)
The microparticle is preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, and exovesicles, or microvesicles, and is more preferably an exosome.
The diameter of the microparticles is preferably from 10 to 1000 nm, more preferably from 30 to 500 nm, and particularly preferably from 50 to 150 nm.
Furthermore, it is desirable that the surface of the microparticles contains a molecule called tetraspanin, such as CD9, CD63, or CD81, and this may be CD9 alone, CD63 alone, or CD81 alone, or any combination of two or three of these.
Hereinafter, a preferred embodiment in which exosomes are used as the microparticles will be described, but the microparticles used in the present invention are not limited to exosomes.

Exosomes are preferably extracellular vesicles that are released from cells upon fusion of multivesicular bodies with the plasma membrane.
The surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells.
It is preferable that the exosome contains intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins.
Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another. Exosomes are easily traceable and can be targeted to specific regions.

(Fine particle content)
The content of microparticles in the culture supernatant of dental pulp-derived stem cells is not particularly limited. The culture supernatant of dental pulp-derived stem cells preferably contains 0.5×10 ⁸ or more microparticles, more preferably 1.0×10 ⁸ or more, particularly preferably 2.0×10 ⁸ or more, even more particularly preferably 2.5×10 ⁸ or more, and even more particularly preferably 1.0×10 ⁹ or more.
In addition, the concentration of microparticles in the culture supernatant of dental pulp-derived stem cells is not particularly limited. The culture supernatant of dental pulp-derived stem cells preferably contains microparticles at 1.0×10 ⁸ particles/mL or more, more preferably contains 2.0×10 ⁸ particles/mL or more, particularly preferably contains 4.0×10 ⁸ particles/mL or more, more particularly preferably contains 5.0×10 ⁸ particles/mL or more, and even more particularly preferably contains 2.0×10 ⁹ particles/mL or more.
A preferred embodiment of the microparticles of the present invention contains such a large amount or high concentration of microparticles, thereby making it possible to maintain a high amount of rejuvenation-related miRNA used in the rejuvenation agent.

<Other ingredients>
The composition containing the culture supernatant of dental pulp-derived stem cells may contain other components in addition to the microparticles, depending on the type of animal to which it is administered and the purpose, within the range that does not impair the effects of the present invention. Examples of other components include nutritional components, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, and antiseptics.
Examples of nutritional components include fatty acids and vitamins.
Examples of antibiotics include penicillin, streptomycin, and gentamicin.
Carriers can include materials known as pharma- ceutically acceptable carriers.
The composition containing the culture supernatant of dental pulp-derived stem cells may be the culture supernatant of dental pulp-derived stem cells itself, or may be a pharmaceutical composition further containing a pharma- ceutically acceptable carrier, excipient, etc. The purpose of the pharmaceutical composition is to facilitate the administration of microparticles to a subject.

Pharmaceutically acceptable carriers are preferably carriers (including diluents) that do not cause significant irritation to the subject and do not abrogate the biological activity and properties of the compound being administered. Examples of carriers are propylene glycol; saline; emulsions; buffer solutions; culture media, such as DMEM or RPMI; and cryopreservation media containing components that scavenge free radicals.

The composition containing the culture supernatant of dental pulp-derived stem cells may contain active ingredients of conventionally known rejuvenating agents. Those skilled in the art can appropriately modify the composition according to the intended use, the subject of administration, etc.

On the other hand, it is preferable that the composition containing the culture supernatant of dental pulp-derived stem cells and the rejuvenation agent of the present invention do not contain a specific substance. Below, the composition containing the culture supernatant of dental pulp-derived stem cells is explained, but the same applies to the rejuvenation agent of the present invention.
For example, a composition containing a culture supernatant of dental pulp-derived stem cells preferably does not contain dental pulp-derived stem cells.
In addition, the composition containing the culture supernatant of dental pulp-derived stem cells preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. Examples of other cytokines include those described in [0014] to [0020] of JP 2018-023343 A.
Furthermore, the composition containing the culture supernatant of dental pulp-derived stem cells preferably does not contain Siglec 9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec 9.
In addition, the composition containing the culture supernatant of dental pulp-derived stem cells preferably does not substantially contain serum (fetal bovine serum, human serum, sheep serum, etc.). In addition, the composition containing the culture supernatant of dental pulp-derived stem cells preferably does not substantially contain conventional serum substitutes such as Knockout serum replacement (KSR).
In a composition containing culture supernatant of dental pulp-derived stem cells, the content (solid content) of each of the other components described above is preferably 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

<Method for preparing culture supernatant of dental pulp-derived stem cells>
The culture supernatant of dental pulp-derived stem cells is not particularly limited.
The culture supernatant of dental pulp-derived stem cells is preferably substantially free of serum. For example, the culture supernatant of dental pulp-derived stem cells or the like preferably contains 1% by mass or less of serum, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

Dental pulp-derived stem cells may be derived from humans or non-human animals. Examples of non-human animals include animals (species) to which the microparticles of the present invention are administered, as described below, and mammals are preferred.

There is no particular limitation on the dental pulp-derived stem cells used in the culture supernatant. Stem cells from exfoliated deciduous teeth, deciduous tooth pulp stem cells obtained by other methods, and permanent tooth pulp stem cells (DPSCs) can be used. In addition to human deciduous tooth pulp stem cells and human permanent tooth pulp stem cells, dental pulp-derived stem cells derived from animals other than humans, such as porcine deciduous tooth pulp stem cells, can be used.
In addition to exosomes, dental pulp-derived stem cells can produce various cytokines such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, and chemokines. They can also produce many other physiologically active substances.
In the present invention, it is particularly preferable that the dental pulp-derived stem cells used in the culture supernatant of dental pulp-derived stem cells are dental pulp-derived stem cells that contain a large amount of protein, and it is preferable to use deciduous dental pulp stem cells. That is, in the present invention, it is preferable to use the culture supernatant of deciduous dental pulp stem cells.

The dental pulp-derived stem cells used in the present invention may be natural or genetically modified, so long as they can achieve the intended treatment.
In particular, in the present invention, immortalized stem cells derived from dental pulp can be used. By using immortalized stem cells capable of virtually infinite proliferation, the amount and composition of biological factors contained in the stem cell culture supernatant can be stabilized for a long period of time. There are no particular limitations on the immortalized stem cells derived from dental pulp. The immortalized stem cells are preferably non-cancerous immortalized stem cells. The immortalized stem cells derived from dental pulp can be prepared by adding the following low molecular weight compounds (inhibitors) alone or in combination to dental pulp-derived stem cells and culturing them.
The TGFβ receptor inhibitor is not particularly limited as long as it has an effect of inhibiting the function of the transforming growth factor (TGF) β receptor, and examples thereof include 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyridine, and the like. Examples of such compounds include pyrazole (A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all from Merck), and SB431542 (Sigma-Aldrich). A-83-01 is preferred.
The ROCK inhibitor is not particularly limited as long as it has an effect of inhibiting the function of Rho-binding kinase. Examples of the ROCK inhibitor include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (all Fujifilm Wako Pure Chemical Industries, Ltd.). Y-27632 is preferred.
The GSK3 inhibitor is not particularly limited as long as it inhibits GSK-3 (Glycogen synthase kinase 3), and examples thereof include A 1070722, BIO, and BIO-acetoxime (all from TOCRIS).
The MEK inhibitor is not particularly limited as long as it has an effect of inhibiting the function of MEK (MAP kinase-ERK kinase), and examples thereof include AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86-9766), SL327, U0126-EtOH (all manufactured by Selleck), PD98059, U0124, U0125 (all manufactured by Cosmo Bio Co., Ltd.), and the like.

When the rejuvenating agent of the present invention is used in regenerative medicine, due to the requirements of the Act on Safety of Regenerative Medicine, the culture supernatant of dental pulp-derived stem cells or these immortalized stem cells, and the composition containing microparticles derived therefrom, are in an embodiment that do not contain somatic stem cells other than dental pulp-derived stem cells. The culture supernatant of dental pulp-derived stem cells may contain mesenchymal stem cells or other somatic stem cells other than dental pulp-derived stem cells, but preferably does not contain them.
Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermal system, digestive system, bone marrow system, nervous system, etc. Examples of somatic stem cells from the dermal system include epithelial stem cells, hair follicle stem cells, etc. Examples of somatic stem cells from the digestive system include pancreatic (general) stem cells, hepatic stem cells, etc. Examples of somatic stem cells from the bone marrow system (other than mesenchymal stem cells) include hematopoietic stem cells, etc. Examples of somatic stem cells from the nervous system include neural stem cells, retinal stem cells, etc.
The culture supernatant of dental pulp-derived stem cells may contain stem cells other than somatic stem cells, but preferably does not contain them. Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).

The method for preparing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells is not particularly limited, and any conventional method can be used.
The culture supernatant of dental pulp-derived stem cells is a culture solution obtained by culturing dental pulp-derived stem cells. For example, the culture supernatant usable in the present invention can be obtained by separating and removing cellular components after culturing dental pulp-derived stem cells. Culture supernatants that have been appropriately subjected to various treatments (e.g., centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may also be used.

Dental pulp-derived stem cells for obtaining the culture supernatant of dental pulp-derived stem cells can be selected by standard methods, and can be selected based on cell size or morphology, or as adhesive cells. Dental pulp cells collected from shed deciduous or permanent teeth can be selected as adhesive cells or their subcultured cells. The culture supernatant of dental pulp-derived stem cells can be the culture supernatant obtained by culturing selected stem cells.

In addition, the "dental pulp-derived stem cell culture supernatant" is preferably a culture medium that does not contain the cells themselves obtained by culturing dental pulp-derived stem cells. In one embodiment, the dental pulp-derived stem cell culture supernatant used in the present invention preferably does not contain cells (regardless of the type of cell) as a whole. This characteristic clearly distinguishes the composition of this embodiment from various compositions that contain dental pulp-derived stem cells, as well as dental pulp-derived stem cells themselves. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed only of dental pulp-derived stem cell culture supernatant.
The culture supernatant of dental pulp-derived stem cells used in the present invention may contain both culture supernatants of stem cells derived from deciduous dental pulp and stem cells derived from adult dental pulp. The culture supernatant of dental pulp-derived stem cells used in the present invention preferably contains the culture supernatant of stem cells derived from deciduous dental pulp as an active ingredient, more preferably contains 50% by mass or more, and preferably contains 90% by mass or more. It is more particularly preferable that the culture supernatant of dental pulp-derived stem cells used in the present invention is a composition composed only of the culture supernatant of stem cells derived from deciduous dental pulp.

A basic medium or a basic medium to which serum or the like has been added can be used as the culture medium for dental pulp-derived stem cells to obtain the culture supernatant. In addition to Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM) (GIBCO, etc.), Ham's F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI1640 medium, etc. can be used as the basic medium. Examples of components that can be added to the medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various vitamins, and various minerals.
However, in order to prepare a serum-free "culture supernatant of dental pulp-derived stem cells", it is advisable to use a serum-free medium throughout the entire process or for the last or subsequent few subcultures. For example, a serum-free culture supernatant of dental pulp-derived stem cells can be prepared by culturing dental pulp-derived stem cells in a serum-free medium. A serum-free culture supernatant of dental pulp-derived stem cells can also be obtained by performing one or more subcultures and culturing the last or subsequent few subcultures in a serum-free medium. On the other hand, a serum-free culture supernatant of dental pulp-derived stem cells can also be obtained by removing serum from the collected culture supernatant using dialysis or solvent replacement using a column.

The conditions commonly used for culturing dental pulp-derived stem cells to obtain the culture supernatant can be applied as is. The method for preparing the culture supernatant of dental pulp-derived stem cells may be the same as the cell culture method described below, except that the steps of isolating and selecting stem cells are appropriately adjusted according to the type of stem cells. Isolation and selection of dental pulp-derived stem cells according to the type of stem cells can be appropriately performed by those skilled in the art.
In addition, special conditions may be applied to the culture of dental pulp-derived stem cells in order to produce a large amount of microparticles such as exosomes. Examples of the special conditions include low temperature conditions, low oxygen conditions, microgravity conditions, and co-culture with some kind of stimuli.

The culture supernatant of dental pulp-derived stem cells used in the present invention for preparing microparticles such as exosomes may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but it is preferable that it is substantially free of other components.
However, each type of additive used in preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.

(Preparation of Microparticles)
The microparticles can be prepared by purifying the microparticles from the culture supernatant of dental pulp-derived stem cells or the like.

The purification of the microparticles is preferably the separation of a fraction containing the microparticles from the culture supernatant of dental pulp-derived stem cells, and more preferably the isolation of the microparticles.
The microparticles can be isolated by separating them from non-associated components based on a property of the microparticle, for example, the microparticles can be isolated based on molecular weight, size, morphology, composition or biological activity.
The microparticles can be purified by separating a specific fraction (e.g., precipitate) containing a large amount of microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) in fractions other than the specified fraction may be removed. The removal of the solvent, dispersion medium, and unnecessary components from the microparticle composition does not have to be complete. Exemplary conditions for centrifugation include 100 to 20,000 g for 1 to 30 minutes.
Microparticles can be purified by filtering the culture supernatant of dental pulp-derived stem cells or the centrifuged product thereof. Unnecessary components can be removed by filtering. In addition, by using a filtering membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed simultaneously. The material and pore size of the filtering membrane used for filtering are not particularly limited. Filtration can be performed using a filtering membrane with an appropriate molecular weight or size cutoff by a known method. From the viewpoint of easy separation of exosomes, the pore size of the filtering membrane is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm.
In the present invention, the culture supernatant of dental pulp-derived stem cells, or its centrifuged or filtered product, can be separated using a further separation means such as column chromatography. For example, high performance liquid chromatography (HPLC) using various columns can be used. The column can be a size exclusion column or a binding column.
One or more properties or biological activities of the microparticles can be used to track the microparticles (or their activity) in each fraction at each processing step. For example, light scattering, refractive index, dynamic light scattering or UV-visible detectors can be used to track the microparticles. Or, specific enzyme activity, etc. can be used to track activity in each fraction.
As a method for purifying microparticles, the method described in paragraphs [0034] to [0064] of JP2019-524824A may be used, the contents of which are incorporated herein by reference.

The final form of the culture supernatant of dental pulp-derived stem cells is not particularly limited. For example, the culture supernatant of dental pulp-derived stem cells may be in the form of microparticles packed in a container together with a solvent or dispersion medium; in the form of microparticles gelled together with a gel and packed in a container; or in the form of microparticles frozen and/or dried to solidify and formulated or packed in a container. Examples of containers include tubes, centrifuge tubes, bags, etc. suitable for cryopreservation. The freezing temperature can be, for example, -20°C to -196°C.

The rejuvenation agent of the present invention has the advantages of being easy to mass-produce, being able to utilize the stem cell culture medium that was previously discarded as industrial waste, and being able to reduce the disposal cost of the stem cell culture medium, compared to compositions that can be used as conventional rejuvenation agents. In particular, when the culture supernatant of dental pulp-derived stem cells is a culture supernatant of human dental pulp-derived stem cells, the rejuvenation agent of the present invention has the advantage of being highly safe from an immunological standpoint and having few ethical issues when applied to humans. When the culture supernatant of dental pulp-derived stem cells is a culture supernatant of dental pulp-derived stem cells from the subject itself, the safety of applying the rejuvenation agent of the present invention to the subject will be higher and there will be fewer ethical issues.
The rejuvenating agent of the present invention is also used in restorative medicine. A composition containing microparticles derived from the culture supernatant of dental pulp-derived stem cells is preferably used in restorative medicine. Here, it is known that in regenerative medicine based on stem cell transplantation, stem cells are not the main players in regeneration, but the liquid components produced by stem cells repair organs together with the patient's own stem cells. The difficult problems associated with conventional stem cell transplantation, such as canceration, standardization, administration method, preservation, and culture method, are solved, and restorative medicine is possible using the culture supernatant of dental pulp-derived stem cells or a composition using microparticles derived therefrom. Compared to stem cell transplantation, when the rejuvenating agent of the present invention is used, tumor formation is less likely to occur because cells are not transplanted, and it can be said to be safer. In addition, the culture supernatant of dental pulp-derived stem cells used in the rejuvenating agent of the present invention has the advantage of being of a constant standardized quality. Since mass production and efficient administration methods can be selected, it can be used at low cost.

[Rejuvenation method]
The rejuvenation method of the present invention comprises administering an effective amount of the rejuvenation agent of the present invention to a subject containing cells.

There are no particular limitations on the step of administering the microparticles of the present invention to a subject.
The administration method can be sprayed or inhaled into the oral cavity, nasal cavity or airway, infusion, local administration, nasal drops, eye drops, sublingual administration, etc., and preferably is less invasive. The local administration method is preferably injection, rectal administration (including catheter, etc.), or urethral administration. In addition, electroporation is also preferred, which temporarily opens fine holes in the cell membrane by applying a voltage (electric pulse) to the skin surface, allowing the active ingredient to penetrate to the dermis layer, which cannot be reached by normal care. When administering locally, it can be intravenous administration, intraarterial administration, intraportal administration, intradermal administration, eye drops, sublingual administration, subcutaneous administration, transurethral administration, rectal administration, intramuscular administration, or intraperitoneal administration, and more preferably intraarterial administration, intravenous administration, subcutaneous administration, or intraperitoneal administration.
Also, various formulation techniques can be used to change the in vivo distribution of microparticles.Many methods of changing in vivo distribution are known to those skilled in the art.Examples of such methods include, for example, protection of exosomes in vesicles composed of substances such as proteins, lipids (e.g., liposomes), carbohydrates, or synthetic polymers.
The rejuvenating agent of the present invention administered to a subject may circulate within the subject's body and reach a desired tissue.
The number of administrations and the administration interval are not particularly limited. The number of administrations can be at least once a week, preferably at least five times, more preferably at least six times, and particularly preferably at least seven times. The administration interval is preferably one hour to one week, more preferably half a day to one week, and particularly preferably one day (once a day). However, this can be appropriately adjusted depending on the organism species to be administered and the symptoms of the subject to be administered.
The rejuvenating agent of the present invention is preferably used for administering the rejuvenating agent to a subject at least once a week during the effective therapeutic period. When the subject is a human, the more times the agent is administered per week, the more preferable, and the more preferable the agent is administered at least five times a week during the effective therapeutic period, and the more preferable the agent is administered every day.
In the present invention, the amount of the rejuvenating agent of the present invention administered is preferably an amount resulting in 500 or more microparticles per cell, more preferably an amount resulting in 800 or more microparticles per cell, and particularly preferably an amount resulting in 1000 or more microparticles per cell.

There is no particular limitation on the cells (cell types) to which the rejuvenating agent of the present invention is administered. The cells to which the rejuvenating agent of the present invention is administered are preferably somatic cells, more preferably somatic stem cells or differentiated cells. Examples of somatic stem cells include mesenchymal stem cells, neural stem cells, hematopoietic stem cells, vascular endothelial stem cells, hepatic stem cells, and epithelial stem cells, and mesenchymal stem cells are particularly preferred. Examples of mesenchymal stem cells include adipose-derived stem cells, umbilical cord-derived stem cells, dental pulp-derived stem cells, and bone marrow-derived stem cells.
The differentiated cells include cells other than stem cells, cells having functions, and the like, and specifically include fat cells.
There is no particular limitation on the animal (species) to which the rejuvenating agent of the present invention is administered. The animal to which the rejuvenating agent of the present invention is administered is preferably a mammal, a bird (chicken, quail, duck, etc.), or a fish (salmon, trout, tuna, bonito, etc.). The mammal may be a human or a non-human mammal, but is particularly preferably a human. The non-human mammal is more preferably a cow, a pig, a horse, a goat, a sheep, a monkey, a dog, a cat, a mouse, a rat, a guinea pig, or a hamster.
The subject to which the rejuvenating agent of the present invention is administered may be a cell itself. In this case, it is preferably a somatic cell, and more preferably a somatic stem cell or a differentiated cell. The preferred range of each of the somatic stem cell or the differentiated cell is the same as the preferred range of the somatic stem cell or the differentiated cell described in the subject cell (cell type) to which the rejuvenating agent of the present invention is administered.

The microparticles of the present invention may be used in combination with conventionally known rejuvenating agents.

The features of the present invention are explained in more detail below with reference to examples and comparative examples or reference examples. The materials, amounts used, ratios, processing contents, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

[Example 1]
<Preparation of culture supernatant of dental pulp-derived stem cells>
The culture supernatant of human deciduous dental pulp stem cells was prepared and separated according to the method described in Example 6 of Patent No. 6296622, except that DMEM medium was used instead of DMEM/HamF12 mixed medium. In the primary culture, fetal bovine serum (FBS) was added and cultured, and in the subculture, the primary culture medium was used to culture the supernatant of the subculture medium so that it did not contain FBS, and the culture supernatant of deciduous dental pulp stem cells was prepared. Note that DMEM is Dulbecco's modified Eagle's medium, and F12 is Ham's F12 medium.

<Exosome preparation>
Exosomes from dental pulp-derived stem cells were purified from the culture supernatant of the obtained dental pulp-derived stem cells using the following method.
The culture supernatant (100 mL) of deciduous dental pulp stem cells was filtered through a 0.22 micrometer pore size filter, and the solution was centrifuged at 100,000×g for 60 minutes at 4°C. The supernatant was decanted, and the exosome-enriched pellet was resuspended in phosphate-buffered saline (PBS). The resuspended sample was centrifuged at 100,000×g for 60 minutes. The pellet was again collected from the bottom of the centrifuge tube as the concentrated sample (approximately 100 μl). Protein concentration was determined by a micro BSA protein assay kit (Pierce, Rockford, IL). The composition containing exosomes (concentrated solution) was stored at -80°C.
A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used as the rejuvenating agent (microparticle composition sample) of Example 1.

The average particle size and concentration of the microparticles contained in the rejuvenating agent of Example 1 were evaluated.
The microparticles contained in the rejuvenating agent of Example 1 had an average particle size of 50 to 150 nm.
The rejuvenating agent of Example 1 was a high-concentration exosome solution of 1.0 × 10 ⁹ cells/ml or more, specifically, a high-concentration exosome solution of 2.0 × 10 ⁹ cells/ml.
In addition, the components of the obtained rejuvenation agent of Example 1 were analyzed by a known method. As a result, it was found that the rejuvenation agent of Example 1 does not contain stem cells derived from dental pulp, does not contain MCP-1, and does not contain Siglec 9. Therefore, it was found that the active ingredient of the rejuvenation agent of Example 1 is an active ingredient different from MCP-1 and Siglec 9, which are active ingredients of the culture supernatant of mesenchymal stem cells, and their analogues.

[Test Example 1]: (1) Restoration of cell function (search for microRNAs involved in rejuvenation)
Next-generation sequencing (NGS) analysis was performed on the small RNA contained in the rejuvenation agent of Example 1. The NGS analysis identified 1,787 miRNAs contained in the rejuvenation agent of Example 1 (exosomes of dental pulp-derived stem cells).
A search was conducted for microRNAs involved in rejuvenation among the miRNAs contained in the rejuvenation agent of Example 1. The rejuvenation-related miRNAs were extracted using IMOTA (Interactive Multi-Omics-Tissue Atlas). IMOTA is an interactive multi-omics atlas that allows you to investigate the interactions and expression levels of miRNA, mRNA, and proteins in each tissue and cell (Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770-D775, IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions). Here, we explored whether rejuvenation-related miRNAs were included.

The microparticles contained in the rejuvenation agent of the present invention contained the following miRNAs as rejuvenation-related miRNAs.
has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has- miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p and miR-93-5p.
Therefore, it was found that the rejuvenating agent of the present invention can restore the functions of cells.

The graph in Figure 1 shows the results of comparing the read counts of 50 types of miRNAs with the highest read counts among the miRNAs expressed in exosomes from dental pulp-derived stem cells. In Figure 1, the vertical axis is the read count. In addition, in Figure 1, the miRNAs enclosed in a box are rejuvenation-related miRNAs.

From Figure 1, it was found that the microparticles contained in the rejuvenation agent of the present invention contain a high concentration of rejuvenation-related miRNA. Therefore, the rejuvenation agent of the present invention can restore the function of cells.

[Test Example 2]: (2) Inhibition of Senescence-Associated Secretory Phenotype (SASP) Using Associated Secretary Phenotype (SASP) Antibody Sampler Kit #38461 from Cell Signaling Technology, SASP factors were quantitatively compared.
Specifically, the SASP factor included in this kit, IL-1β (D3U3E) Rabbit mAb #12703
CCL5/RANTES (R40) Antibody #2987
CXCL10 (D5L5L) Rabbit mAb #14969
PAI-1 (D9C4) Rabbit mAb #11907
IL-6 (D3K2N) Rabbit mAb #12153
TNF-α (D5G9) Rabbit mAb #6945
MMP-3 (D7F5B) Rabbit mAb #14351
MCP-1 Antibody (Carboxy-terminal Antigen) #39091
was used to quantitatively compare each SASP factor.
Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody, and colorimetric quantification was performed.

A culture supernatant of umbilical cord-derived stem cells was prepared in the same manner as in Example 1, except that umbilical cord-derived stem cells were used.
Exosomes from umbilical cord-derived stem cells were purified in the same manner as in Example 1, except that the culture supernatant of umbilical cord-derived stem cells was used (Comparative Example).

The rejuvenating agent of Example 1 (SHED-CM (culture supernatant of deciduous tooth dental pulp stem cells)-EVs) was purified twice by ultracentrifugation, thawed in PBS(-), and stored in a deep freezer at -80°C for approximately 8 months.
Exosomes from umbilical cord-derived stem cells (umbilical cord MSC-EVs) were purified twice by ultracentrifugation, thawed in PBS(-), and stored in a deep freezer at -80°C for approximately two months.
These EVs were treated at a ratio of 10,000 EVs/cell with aged adipose mesenchymal stem cells at passage number P8, with the number of SA-β-gal positive cells being 72%, and 72 hours later, SASP factors were treated according to the protocol of the above kit.
The results obtained as the average value of N=3 are shown in Fig. 2. Fig. 2 is a graph showing the expression of each SASP factor as a relative value (%), with Untreated being set at 100.

From Figure 2, it was found that the rejuvenating agent of the present invention can suppress senescence-associated secretory phenomenon (SASP). Specifically, it was found that it can suppress SASP factors such as IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3 and MCP-1. In particular, it was found that the rejuvenating agent of the present invention can significantly suppress CCL5, CXCL10, IL-6, TNF-α and MCP-1 compared to exosomes from umbilical cord-derived stem cells.

[Test Example 3]: (3) Telomere Lengthening Using commercially available normal human dermal fibroblasts (NHDF cells), the average telomere length of chromosome ends was compared depending on the passage number of NHDF cells.
Telomere length was measured using a telomere length qPCR kit from Cosmo Bio according to the protocol provided with the kit.
The average telomere length at each chromosome end was calculated according to the following formula 1.
Formula 1:
(Average telomere length at each chromosome end)
= (total telomere length of target sample per diploid cell)/92
First, in NHDF cells at passage number P5, the total telomere length of the target sample per diploid cell was 617 kB, and according to formula 1, the average telomere length of each chromosome end was 617/92=6.70 kb.
Next, NHDF cells at passage number P5 were plated into wells of a 24-well plate containing 1 mL of MSC growth medium. The cells in each well were stimulated with 20 μL of PBS (Control) or the rejuvenating agent of Example 1 resuspended in 20 μL of PBS (culture supernatant of dental pulp-derived stem cells containing exosomes, shown in FIG. 3 as SHED-CM-EVs) at a ratio of 1000 EV/cell. After that, the same operation was performed every time the passage was repeated, and in the NHDF cells at passage number P12, the average telomere length at each chromosome end was 439/92 = 6.02 Kb (SHED-CM-EVs).
On the other hand, in NHDF cells (control) that were passaged with the addition of PBS (control) to reach passage number P12, the total telomere length of the target sample per diploid cell was 439 kB, and the average telomere length of each chromosome end was 439/92 = 4.78 kb according to formula 1.
The results obtained are shown in FIG.
From the above, it was found that the rejuvenation agent of the present invention can suppress the shortening of telomere length due to cell division compared to the control, and can lengthen telomeres compared to control cells of the same passage number.

[Test Example 4]: (4) Recovery of cell division ability (reduction in the number of SA-β-gal positive cells in ADMSCs)
Approximately 4500 cells of commercially available passage P12 senescent ADMSCs (adipose derived stem cells) were plated into wells of a 24-well plate containing 1 mL of MSC growth medium.
The cells in each well were stimulated with 20 μL of PBS (control) or the rejuvenating agent of Example 1 (culture supernatant of dental pulp-derived stem cells containing exosomes (shown in FIG. 4 as SHED-CM-EVs) resuspended in 20 μL of PBS at a ratio of 1000 EV/cell. After incubation for 24 hours (Day 1), 48 hours (Day 2), or 72 hours (Day 3), the cells were fixed and stained to determine SA-β-gal activity. A reference example was also performed in which the cells in each well were stimulated with the rejuvenating agent of Example 1 at a ratio of 100 EV/cell.
Here, senescence-associated β-galactosidase (SA-β-gal) is specifically present in senescent cells. This hydrolase usually converts β-galactoside into monosaccharides, and accumulates in lysosomes in senescent cells. In Test Example 4, senescent ADMSCs containing SA-β-gal were stained blue using X-GAL as a substrate according to the protocol attached to the kit using Cosmo Bio's Cellular Senescence Detection Kit (SA-β-Gal Staining) (product number: CBA-230), and the number of SA-β-gal positive cells was quantified.
The number of SA-β-gal positive cells was quantified as the average value of N = 3 independent cultures, and the results are shown in Figure 4.

4, when the rejuvenating agent of Example 1 (culture supernatant of dental pulp-derived stem cells containing exosomes; SHED-CM-EVs) was added at a concentration of 1000EV/cell (1000 particles of exosomes per cell), not only was it possible to suppress the increase in the number of SA-β-gal positive cells, but it was also possible to significantly reduce the number of SA-β-gal positive cells by 22.7% (DAY 1), 29.9% (DAY 2), and 31.6% (DAY 3). In other words, it was found that the rejuvenating agent of Example 1 can restore cell division ability.
FIG. 5 is a schematic diagram of Test Example 4 showing that the rejuvenating agent of the present invention (SHED-CM-EVs) has the effect of rejuvenating aged mesenchymal stem cells.
Here, the relationship between the passage number of ADMSCs at 0 hours of culture time and the number of SA-β-gal positive cells was measured and determined by a separate test with N=3. The results are shown in FIG. 6. From FIG. 6, the number of SA-β-gal positive cells of ADMSCs at 0 hours of culture time was about 2% at passage number P2, about 9% at P4, about 11% at P6, about 15% at P8, about 28% at P10, and about 39% at P12. Therefore, it can be said that the rejuvenating agent of Example 1 was able to rejuvenate P12 ADMSCs to the equivalent of P10. In other words, the rejuvenating agent of the present invention was able to suppress the number of positive cells in SA-β-gal staining when the rejuvenating agent was administered to a subject that had been passaged n=12 times, compared to the number of positive cells in SA-β-gal staining contained in a subject that had been passaged n=12 times without administration of the rejuvenating agent.

From the above test examples 1 to 4, it was found that the rejuvenating agent of the present invention has all of the following effects: recovery of cell function, inhibition of secretory phenomena associated with cellular aging, lengthening of telomeres, and recovery of cell division ability.

Claims (16)

A rejuvenating agent for administration to a subject comprising cells, comprising:
A rejuvenating agent comprising microparticles derived from dental pulp-derived stem cells. A composition comprising a culture supernatant of dental pulp-derived stem cells,
The rejuvenating agent according to claim 1 , wherein the culture supernatant contains the microparticles. The rejuvenation agent according to claim 1, wherein the microparticles (1) contain at least one of the following rejuvenation-related miRNAs:
Rejuvenation-related miRNA:
has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has- miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p and miR-93-5p. The rejuvenation agent according to claim 3, wherein the microparticles contain at least one of has-miR-125a-5p, hsa-miR-199a-3p and hsa-miR-21-5p as the rejuvenation-related miRNA. The rejuvenation agent according to claim 3, wherein the microparticles contain the rejuvenation-related miRNA at a higher concentration than the culture supernatant. The rejuvenating agent according to claim 1, wherein the composition does not contain the culture supernatant excluding the microparticles. (2) The rejuvenating agent according to claim 1, which is an inhibitor of the expression of the following SASP factors:
SASP factors:
IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3 and MCP-1. The rejuvenation agent according to claim 1, wherein the cells are somatic stem cells or differentiated cells. The subject is a somatic stem cell or a differentiated cell,
The rejuvenating agent described in claim 1, wherein when the rejuvenating agent is administered to a subject that has been passaged n times, n being a natural number, the subject can be more rejuvenated than a subject that has been passaged n times without being administered the rejuvenating agent. (3) The rejuvenation agent according to claim 1, which inhibits shortening of the average telomere length at the ends of chromosomes possessed by the cells. (4) The rejuvenating agent according to claim 1, which suppresses the number of positive cells stained with SA-β-gal when administered to a subject that has been passaged n times, compared to the number of positive cells stained with SA-β-gal in a subject that has been passaged n times without administration of the rejuvenating agent. The rejuvenating agent according to claim 1, which has all of the following effects: recovery of cell function, inhibition of senescence-associated secretory phenomenon (SASP), lengthening of telomeres, and recovery of cell division ability. The rejuvenating agent according to claim 1, which satisfies all of the following (1) to (4).
(1) Contains at least one of the following rejuvenation-related miRNAs:
Rejuvenation-related miRNA:
has-miR-125a-5p, has-miR-125b-5p, has-miR-155-5p, has-miR-181a-5p, hsa-miR-199a-3p, has- miR-199b-3p, has-miR-199b-5p, hsa-miR-21-5p, has-miR-223-3p, has-miR-24-3p and miR-93-5p;
(2) An expression inhibitor of the following SASP factor:
SASP factors:
IL-1β, CCL5, CXCL10, PAI-1, IL-6, TNF-α, MMP-3 and MCP-1;
(3) suppressing shortening of the average telomere length at the ends of chromosomes in the cell;
(4) When the rejuvenating agent is administered to a subject that has been passaged n times, the number of positive cells stained with SA-β-gal contained in the subject is suppressed to be lower than the number of positive cells stained with SA-β-gal contained in the subject that has been passaged n times without administration of the rejuvenating agent. The rejuvenating agent according to claim 1, wherein the microparticles are exosomes. A method of rejuvenation comprising administering an effective amount of the rejuvenation agent of claim 1 to a subject containing cells. The rejuvenation method of claim 15, wherein the dosage of the rejuvenation agent is an amount resulting in 500 or more microparticles per cell.

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