Methods for treating senescence associated diseases

Abstract

To provide senolytic agents to eliminate senescent cells and methods for treating diseases and disorders associated with senescence.SOLUTION: Disclosed is a method for removing senescent cells from banked stem cells, the method comprising adding to the stem cells at least one senolytic agent. Also provided is a pharmaceutical composition comprising at least one senolytic agent for use in a method for treating a disease, disorder or condition associated with senescence in a subject, where the method comprises removing stem cells from the subject, culturing the cells with the pharmaceutical composition, and administering to the subject the cultured cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application is a PCT international patent application filed on August 14, 2020, and is a continuation of U.S. Provisional Patent Application No. 62 / 887,090, filed on August 15, 2019, entitled "USE OF SENOLYTIC AGENTS"; U.S. Provisional Patent Application No. 62 / 887,090, filed on August 23, 2019, entitled "CLEARING SENOLYTIC CELLS"; No. 62 / 890,893, entitled “FISETIN ATTENUATES SENESCENCE IN AGING, CULTURE-EXPANDED, AND BANKED HUMAN ADIPOSE-DERIVED STEM CELLS” filed on August 23, 2019; No. 62 / 890,910, entitled "AGED, CULTURE EXPANDED, AND BANKED HUMAN ADIPOSE DERIVED STEM CELLS," filed on January 9, 2020; No. 62 / 959,012, entitled "DETECTION AND EVALUATION OF SENESCENT CELLS," filed on March 4, 2020; and No. 62 / 985,242, entitled "SENESCENCE IN AGE-RELATED DISEASES AND CONDITIONS: IMPLICATIONS FOR ORTHOPEDICS, MUSCULOSKELETAL SYSTEM AND ORTHOBIOLOGICS," filed on March 4, 2020, the disclosures of each of which are incorporated by reference in their entireties herein.

[0002] Field of Disclosure The present disclosure relates to the use of senolytic agents to eliminate senescent cells. The present disclosure further relates to methods for treating aging-related diseases and disorders. [Background technology]

[0003] In cellular senescence, normally proliferating cells are in a permanent state of cell cycle arrest, where they no longer respond to growth stimuli and no longer divide. Cell cycle arrest of progenitor cells contributes to the loss of the ability to repair tissue. In addition, senescent cells produce proinflammatory and matrix degrading molecules, a response called senescence-associated secretory phenotype (SASP) (Non-Patent Document 1; Non-Patent Document 2). Cellular senescence is associated with the aging process and age-related diseases. As a result, targeting senescent cells has emerged as a therapeutic approach. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Childs et al., 2015 Nat Med 21(12):1424-1435 [Non-Patent Document 2] Paez-Ribes et al. 2019 EMBO Mol Med 11:1-19 Summary of the Invention [Means for solving the problem]

[0005] Brief Summary of the Invention In one aspect, the present invention provides a method for detecting senescent cells in a cell sample, the method comprising: staining the cells with C12FDG; and subjecting the cells to flow cytometry. In one embodiment, the cell sample comprises CD3+ T cells. In another embodiment, the cell sample comprises total peripheral blood mononuclear cells, bone marrow aspirate (BMA), whole blood, ex vivo cultured cells, or the like. The senescent cells are expanded stem cells, banked stem cells, freshly isolated stem cells, or endogenous stem cells. In yet another embodiment, the detected senescent cells are characterized according to the stage of senescence. In yet another embodiment, the characterization is based on the brightness of the signal. In a further embodiment, the stage of senescence is early stage (relatively low C12FDG positivity, "dim", low green fluorescence intensity on the flow cytometry plot), intermediate stage (relatively medium C12FDG positivity), or late stage (relatively high C12FDG positivity, "bright", high fluorescence intensity on the flow cytometry plot) as determined by normalized event gating by flow cytometry. In certain embodiments, late senescent cells are the target of senolytic or senescent therapeutic agents.

[0006] In another aspect, the invention provides a method for removing senescent cells from banked stem cells, the method comprising adding at least one senescent cell to the cells.

[0007] In yet another aspect, the invention provides a method of enriching banked stem cells, the method comprising adding at least one senolytic agent to the cells.

[0008] In certain embodiments of the method according to the invention, the banked stem cells are selected from the group consisting of ADSCs, ex vivo culture expanded stem cells, freshly isolated stem cells, endogenous stem cells, bone marrow aspirate concentrate (BMAC) stem cells, and whole blood stem cells. In additional embodiments, at least 25% of the senescent cells are removed, at least 50% of the senescent cells are removed, at least 75% of the senescent cells are removed, at least 80% of the senescent cells are removed, 85% of the senescent cells are removed, at least 90% of the senescent cells are removed, or at least 95% of the senescent cells are removed.

[0009] In yet another aspect, the present invention provides a method of treating a senescence-associated disease, disorder, or condition in a subject, the method comprising administering to the subject at least one senolytic agent.

[0010] In a further aspect, the present invention provides a method of treating a senescence-associated disease, disorder, or condition in a subject, the method comprising removing stem cells from the subject, culturing the cells with at least one senolytic agent, and administering the cultured cells to the subject.

[0011] In yet another aspect, the present invention provides a method of treating an age-related disease, disorder, or condition in a subject, the method comprising removing stem cells from the subject, culturing the cells with at least one senolytic agent, and administering the cultured cells to the subject.

[0012] In certain embodiments of the methods according to the invention, the disease, disorder, or condition is osteoarthritis.

[0013] In another aspect, the invention provides a method of improving a surgical outcome in a subject, the method comprising administering to the subject at least one senolytic agent, hi one embodiment, the senolytic agent is administered to a surgical site in the subject.

[0014] In yet another aspect, the invention provides a method of improving a surgical outcome in a subject, the method comprising removing stem cells from a subject, culturing the cells with at least one senolytic agent, and administering the cultured cells to the subject. In one embodiment, the cultured cells are administered to a surgical site in a patient.

[0015] In a particular embodiment of the method according to the invention, the surgical outcome is wound healing. In an additional embodiment of the method, the surgical site is a wound.

[0016] In another aspect, the present invention provides a method of removing senescent cells from an orthobiologic product, the method comprising adding at least one senolytic agent to the product. The orthobiologic product may be selected from the group consisting of, but not limited to, bone graft, autologous blood, platelet rich plasma (PRP), autologous conditioned serum, stem cells, bone marrow aspirate concentrate (BMAC), platelet rich plasma (PRP) PRP, alpha 2 macroglobulin (A2M), amniotic fluid, placental tissue, umbilical cord tissue, and hyaluronic acid.

[0017] In a particular embodiment of the method according to the invention, the at least one senolytic agent is fisetin.

[0018] In one aspect, the present invention provides a pharmaceutical composition comprising at least one senolytic agent for use in the treatment of an age-related disease, disorder, or condition.

[0019] In another aspect, the present invention provides pharmaceutical compositions comprising at least one senolytic agent for use in the treatment of a senescence-associated disease, disorder, or condition.

[0020] In yet another aspect, the present invention provides a pharmaceutical composition comprising at least one senolytic agent for use in improving surgical outcomes.

[0021] In a particular embodiment of the composition according to the invention, the at least one senolytic agent is Fisetin.

[0022] In one aspect, the invention provides a method of treating a subject having a senescence-associated condition or disease, the method comprising measuring senescence in a subject or a tissue sample from the subject, and administering a senolytic agent to the subject. In one embodiment, the subject or tissue sample from the subject comprises at least about 4% senescent cells. In another embodiment, the subject or tissue sample from the subject comprises at least about 4% to about 8% senescent cells. In another embodiment, the senolytic agent is fisetin.

[0023] In embodiments of the present disclosure that specify a selection of "at least one selected from the group consisting of ..." or simply "selected from the group consisting of," the use of the conjunction "and" between the last two items of the list following such language indicates that the items in the series are alternatives of one another, and that one (or more) of these items may be selected. It does not imply that each item is necessarily selected.

[0024] Other embodiments of the present invention will become apparent upon review of the detailed description that follows. [Brief description of the drawings]

[0025] [Figure 1-1] Figures 1A-1E show the effect of serial passaging on cellular senescence of ADSCs. Figure 1A graphically depicts the percentage of cells stained with C12FDG at each passage number (p3, p4, p6, and p8). Figures 1B-1E are representative histogram plots comparing unstained controls with C12FDG-stained ADSCs at passage 3 (Figure 1B), passage 4 (Figure 1C), passage 6 (Figure 1D), and passage 8 (Figure 1E). The R1 region indicates cells positive for C12FDG. One-way ANOVA results show that all successive passages are significantly different (p<.0001). [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Diagram 2] Figures 2A and 2B show the effect of serial passaging on cellular senescence in ADSCs. The bar graph in Figure 2A shows that the senescence-associated secretory phenotype (SASP) (factors IL-6, IL-8, and MCP-1) increase in concentration with passage number (p6 to p8). Figure 2B shows a plot of the % of cells positive for senescence-associated heterochromatin foci (SAHF H3K9 and H2AX) increasing with passage number (p4 to p18). Two-way ANOVA shows that SASP increases significantly (**=p<.01, ****=p<.0001). One-way ANOVA shows that SAHF increases significantly (**=p<.01). [Diagram 3] Figure 3 shows the percentage of viable cells in the form of a bar graph at various fisetin concentrations (50 μM, 25 μM, and 1 μM) for young female (YF), old female (OF), young male (YM), and old male (OM) subjects. Values ​​are relative to untreated control cells. One-way ANOVA shows no significant differences in the percentage of viable cells between treatments. [Figure 4-1] Figures 4A-4D show that fisetin treatment reduces β-galactosidase via C12FDG staining. The plots in Figure 4A show the percentage of cells positive for C12FDG at passages 6, 8, and 10 with or without fisetin treatment. The percentage of cells stained positive for C12FDG decreased with each passage with or without treatment. Figures 4B-4D show representative histogram plots comparing untreated controls to fisetin-treated ADSCs at passage 6 (Figure 4B), passage 8 (Figure 4C), and passage 10 (Figure 4D). The R1 region shows cells positive for C12FDG. One-way ANOVA statistics show significant differences for each treatment (*=p<0.05, **=p<0.01, ***=p<0.001) [Figure 4-2] Continued from Figure 4-1. [Diagram 5] Figures 5A-5C show that fisetin treatment reduces H3K9 and γ-H2AX senescence-associated heterochromatin foci (SAHF). The percentage reduction in cells stained positive for H3K9 and γ-H2AX in OF (older females) versus YF (younger females) treated or untreated with fisetin at passages 4 and 18 is summarized in Figure 5A. The results are also shown in the plots in Figure 5B (for passage 4) and Figure 5C (for passage 18). One-way ANOVA shows a significant reduction in SAHF (*=p<0.05, **=p<0.01, ****=p<0.0001). [Figure 6] 6 shows a bar graph depicting the concentration of SASP factors IL-6, IL-8, and MCP-1 in untreated cells versus cells treated with fisetin. One-way ANOVA statistics show significant differences for each treatment (*=p<0.05, **=p<0.01). [Figure 7]FIG. 7 shows a schematic of the protocol for T cell enrichment using Stem Cell Technologies Sepmatet™ tubes and RosetteSep™ Chemistry. [Figure 8] Figure 8 shows an example result of senescent cell detection using C12FDG staining and flow cytometry. The total number of cells in an event was obtained from the optimized set parameters and considered for further evaluation of cell subsets. Background cells were excluded / released. The subsets were classified as early senescent cells, mid-senescent cells, and late senescent cells. [Figure 9-1] Figures 9A, 9B, and 9C are flow cytometry plots (dot / density plots and histogram plots) showing populations of senescent cells. In Figure 9A, cell size is revealed to increase with the y-axis, while the intensity of senescence increases with the x-axis. Figure 9B shows cell populations corresponding to mid-senescence ("R2") and late-senescence ("R1"). The histogram plot in Figure 9C presents the data of Figure 9B in another way, further quantifying the percentage of mid- (24.6%) vs. late- (3.5%) senescent cells. [Figure 9-2] Continued from Figure 9-1. [Figure 10A] Figures 10A and 10B show flow cytometry dot / density plots: Figure 10A shows plots for a 25 year old male, a 55 year old male, and an 85 year old male. [Figure 10B] Figures 10A and 10B show flow cytometry dot / density plots, and Figure 10B shows the plots for a 41 year old female and a 77 year old female. [Figure 11A] Figure 11A shows the senescence profiling results of the samples of interest. Flow cytometry results are provided as dot plots, raw data, and average cell counts. Dim and bright senescent cells are visualized and quantified. [Figure 11B] FIG. 11B shows the abundance of dim versus bright senescent cells across study subsets in bar graph format. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Before the present disclosure is described, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, since such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.As used herein, the term "about" when used in relation to a specific recited numerical value means that the value may vary from the recited value by 1% or less.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0028] Although any methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety.

[0029] senescent cells Senescent cells include, but are not limited to, senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent chondrocytes, senescent mesenchymal cells, senescent macrophages, and senescent smooth muscle cells.

[0030] Senescent cells and senescent cell-associated molecules can be detected by techniques and procedures described in the art. For example, the presence of senescent cells in tissue can be analyzed by histochemical or immunohistochemical techniques to detect SA-β galactosidase (SA-βgal), a senescence marker (Dimri, et al. 1995 Proc. Natl Acad. Sci. USA 92: 9363-9367; Lee, et al. 2006 Aging Cell 5 (2): 187-195). The presence of senescent cell-associated polypeptide p16, specifically p16INK4a and p21Cip1, can be determined by immunochemical methods practiced in the art, such as immunoblotting analysis (Dimri, et al. 1996 Biol. Signals 5: 154-162). The expression of p16mRNA in cells can be measured by techniques practiced in the art, including quantitative PCR. The presence and levels of senescent cell-associated polypeptides, such as polypeptides of the SASP, commonly referred to as SASP factors or proteins, or the senescence messaging secretome (SMS), can be determined using automated high-throughput assays.

[0031] The presence of senescent cells is also associated with increased expression of growth factors, proteases, cytokines (e.g., inflammatory cytokines), chemokines, cell-associated metabolites, reactive oxygen species (e.g., H 2 O 2 ), as well as inflammation and / or other biological effects or effects that may promote or exacerbate the underlying disease of the subject. can be determined through detection of senescent cell-associated molecules, including other molecules that stimulate responses.

[0032] Senolytic agent Senolytic agents are agents that selectively target and induce apoptosis / death of senescent cells (Kirkland, et al. 2017 J Am Geriatr Soc 65(10):2297-2301; Zhu, et al. 2015 Aging Cell 14(4):644-658). Senolytic agents include, but are not limited to, flavonoids (quercetin, fisetin), tyrosine kinase inhibitors (e.g., dasatinib) + quercetin, alkaloids (piperongiumin), curcumin analogs, navitoclax, 17-DMAG, BCL-2 targeting agents (ABT-263, ABT-737), and combinations thereof. Specifically, these agents target the senescent cell anti-apoptotic pathway (SCAP), which is upregulated during aging. Senolytic agents may be included in the group of interventions known as "geroprotectors" or "senotherapy." Senolytic agents also include geroprotective nutrients such as, but not limited to, myricetin, N-acetylcysteine ​​(NAC), gamma tocotrienol, or epigallocatechin gallate (EGCG).

[0033] Aging markers / SASP Senescent cell markers include, but are not limited to, increased cell size, accumulation of lipofuscin, high expression of cell cycle regulators (e.g., p16INK4A), p21CIP1 and senescence-associated secretory phenotype (SASP) factors (e.g., but are not limited to, TNF-alpha, interleukin-6 (IL-6), multifunctional cytokine IL-1beta, chemokine CXCL10, RANTES / CCL5, and MCP-1, matrix metalloproteinase MMP3, and serine protease inhibitor PAI-1 (SASP: TNF-alpha, interleukin-6 (IL-6), multifunctional cytokine IL-1beta, chemokine CXCL10, RANTES / CCL5, and MCP-1, matrix metalloproteinase MMP3, and serine protease inhibitor PAI-1 (Tchkonia, et al. 2013 J Clin Invest 123,966-72; Sun,et al.2018 Trends Mol Med 24,871-885; Rao and Jackson 2016 Trends Cancer 2:676-687), increased senescence-associated β-galactosidase (SA-βgal) activity / accumulation of β-galactosidase, hemostatic factors (e.g., PAI-1), proteases, formation of senescence-associated heterochromatin foci (SAHF), and appearance of senescence-associated satellite swelling (SADS) and telomere-associated DNA damage foci (TAF) (Kirkland,et al.2017 J Am Geriatr Soc 2017 65(10):2297-2301; Coppe,et al.2010 ARP 5:99-118; Young and Narita 2009 EMBO Rep 10:228-30).

[0034] Senescence-associated secretory phenotype (SASP) refers to a phenotype that often occurs in senescent cells, displaying dramatic changes in their secretome (Coppe,et al.2010 ARP5:99-118; Young and Narita 2009 EMBO Rep 10:228-30). This entails the release of inflammatory cytokines, bradykinin, and chemokines, prostanoids, miRNAs, damage-associated molecular pattern proteins (DAMPs), tissue-damaging proteases (i.e., metalloproteases (MMPs)), factors that affect stem and progenitor cell function, hemostatic factors, and growth factors (Kirkland,et al.2017 J Am Geriatr Soc 2017 65(10):2297-2301). SASP factors include, but are not limited to, interleukins (IL-6, IL-8, IL-1β), monocyte chemotactic protein-1, and IL-1β. and plasminogen activator inhibitor-1.

[0035] Senescent cell-associated molecules include those described in the art as comprising senescence-associated secretory phenotype, senescence messaging secretome, and DNA damage secretion program (DDSP).As described in the art, these classifications of senescent cell-associated molecules include common molecules and are not intended to describe three separate groups of molecules.Senescent cell-associated molecules include specific expressed and secreted growth factors, proteases, cytokines, and other factors that may have strong autocrine and paracrine activity (e.g., Coppe, et al. 2006 J.Biol.Chem.281:29568-74; Coppe, et al. 2010 PLOS One 5:99-118; Krtolica, et al. 2001 PNAS USA98:12072-77; Parrinello, et al. 2005 J.Cell Sci.118:485-96). Extracellular matrix (ECM)-associated factors include inflammatory proteins and mediators of ECM remodeling that are strongly induced in senescent cells (see, e.g., Kuilman, et al. 2009 Nature Reviews 9:81-94). Other senescent cell-associated molecules include extracellular polypeptides (proteins) collectively described as DNA damage secretion program (DDSP) (see, e.g., Sun, et al. 2012 Nature 18:1359-1368). Senescent cell-associated proteins also include cell surface proteins (or receptors) expressed in senescent cells, including proteins that are present in low detectable amounts or are not present on the cell surface of non-senescent cells. Senescent cell-associated proteins also include cell surface markers whose expression is altered (lost or gained) during the senescence progression of a particular cell type (e.g., CD26, CD28), such as cluster of differentiation markers.

[0036] Senescent cell-associated molecules include secreted factors that may contribute to the proinflammatory phenotype of senescent cells (e.g., SASP). These factors include, but are not limited to, GM-CSF, GROα, GROαβγ, IGFBP-7, IL-1γ, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1α, MMP-1, MMP-10, MMP-3, amphiregulin, ENA-78, Eotaxin-3, GCP-2, GITR, HGF, ICAM-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, I L-10, MCP-4, MIF, MIP-3a, MMP-12, MMP-13, MMP-14, NAP2, Oncostatin M, Osteoproteogryn, PIGF, RANTES, sgp130, TIMP-2, TRAIL-R3, Acrp30, Angiogenin, Axl, bFGF, BLC, BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, I-309, IFN-γ, IGFBP-1, IGFBP-3, IL-1 R1, IL-11, IL-15, IL-2R-α, IL-6R, I-TAC, Leptin, LIF, MMP-2, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RII, thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1, TGF-β3, MIP-1-δ, IL-4, FGF-7, PDGF-BB, IL-16, BMP-4, MDC, MCP-4, IL-10, TIMP-1, Fit-3 ligand (Ligand), ICAM-1, Axl, CNTF, INFγ, EGF, BMP-6. Additional identified factors, including factors sometimes referred to in the art as senescence messaging secretome (SMS) factors, some of which are included in the list of SASP polypeptides, include, but are not limited to, IGF1, IGF2, and IGF2R, IGFBP3, IDFBP5, IGFBP7, PAl1, TGF-β, WNT2, IL-1α, IL-6, IL-8, and CXCR2-binding chemokines.Cell-associated molecules also include, but are not limited to, those molecules described in Sun, et al., Nature Medicine. Examples include the products of the genes MMP1, WNT16B, SFRP2, MMP12, SPINK1, MMP10, ENPP5, EREG, BMP6, ANGPTL4, CSGALNACT, CCL26, AREG, ANGPT1, CCK, THBD, CXCL14, NOV, GAL, NPPC, FAM150B, CST1, GDNF, MUCL1, NPTX2, TMEM155, EDN1, PSG9, ADAMTS3, CD24, PPBP, CXCL3, MMP3, CST2, PSG8, PCOLCE2, PSG7, TNFSF15, C17orf67, CALCA, FGFJ8, IL8, BMP2, MATN3, TFP1, SERPINI 1, TNFRSF25, and IL23A (Coppe, et al. 2010 ARP 5:99-118;Young and Narita 2009 EMBO Rep 10:228-30;Basisty,et al. 2020 PLOS Biology18(1):e300599). Senescent cell-associated proteins also include cell surface proteins (or receptors) expressed in senescent cells, including proteins that are present in low detectable amounts or that are absent from the cell surface of non-senescent cells.

[0037] SASP inhibitors SASP inhibitors are drugs that neutralize the senescence-associated secretory phenotype (SASP). SASP inhibitors are sometimes called antisenolytic drugs, which modulate the inflammatory secretome and complement senolytic drugs that target senescence and age-related diseases. Antisenolytic drugs selectively suppress the deleterious effects of senescence (Kang 2019 Mol Cells 42(12):821-827). SASP inhibitors include, but are not limited to, metformin, rapamycin, JAK1 / 2 inhibitors (e.g., ruxolitinib), and glucocorticoids. In certain embodiments, one or more SAP inhibitors may be used in combination with one or more senolytic drugs.

[0038] Methods for Treating or Ameliorating Age-Related Diseases and Conditions - Patent application The present disclosure includes methods for treating, ameliorating, or preventing (i.e., reducing the likelihood of occurrence) age-related diseases and conditions. Age-related diseases and conditions include conditions associated with senescence (Kirkland, et al. 2017 J Am Geriatr. Soc 65(10):2297-2301; Zhu,et al.2015 Aging Cell 14(4):644-658). The present disclosure also includes a method for treating, ameliorating, or preventing (i.e., reducing the likelihood of occurrence) at least one symptom or sign of an age-related disease or disorder in a subject. The method according to this aspect of the invention includes administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a senolytic agent. In a further embodiment, the composition further comprises at least one anti-aging agent. The age-related disease and condition is, in a further embodiment, a condition, disease, or disorder that is related to, associated with, or caused by cellular senescence. Senescent cells can be a nexus for multiple pathologies (Bhatia-Dey,et al.2016 Front Genet 7:13).

[0039] As used herein, the terms "treat", "treating" and the like refer to alleviating a symptom, eliminating the cause of a symptom, either temporarily or permanently, or preventing or delaying the appearance of symptoms of an age-related disease or condition in a subject. In certain embodiments, diseases or conditions associated with aging include, but are not limited to, cancer, tumorigenesis, metastasis, fibrosis, cardiovascular disease, myocardial infarction, atherosclerosis, cardiomyocyte hypertrophy, heart failure, peripheral vascular disease, premature aging of the hematopoietic system, obesity, obesity-induced metabolic syndrome, lipoatrophy, type 1 diabetes, type 2 diabetes, sarcopenia, inflammatory diseases, osteoarthritis, degenerative joint disease, kyphosis, osteoporosis, bone loss, herniated disc, rheumatoid arthritis, irritable bowel syndrome, inflammatory bowel disease, glaucoma, cataracts, lung disease, pulmonary insufficiency, idiopathic pulmonary fibrosis (IPF), sarcopenia, renal dysfunction / failure, tau-dependent pathologies, neuropathy, neurodegeneration, motor function disorders, cerebrovascular disease, emphysema. In a further embodiment, the age-related disease or condition is slow healing, including after injury (wound healing) or surgery (post-operative healing, response to physical therapy).

[0040] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal that exhibits one or more symptoms or signs of an age-related disease or condition and / or has been diagnosed with an age-related or condition. Throughout this disclosure, the terms "subject", "patient" and "subject in need thereof" are used interchangeably. The term "subject in need thereof" may also include a patient that exhibits (or has exhibited) one or more signs of an age-related disease or condition and / or exhibits aging biomarkers and / or SASP, for example, prior to treatment. The term "subject in need thereof" may also include a patient that is scheduled to undergo treatment and / or surgery, for example orthopedic surgery.

[0041] Pharmaceutical Compositions The present invention includes methods that include administering at least one senolytic agent to a subject, where the agent is contained within a pharmaceutical composition. The pharmaceutical compositions of the present invention may be formulated with suitable carriers, excipients, and other agents that provide suitable transport, delivery, tolerance, etc. A large number of suitable formulations can be found in formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures that include carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0042] A variety of delivery systems are known and may be used to administer the pharmaceutical compositions of the present invention, such as bioengineered scaffolds, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J.Biol.Chem.262:4429-4432). Methods of administration include, but are not limited to, intradermal, intraarticular, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. In a preferred embodiment, the compositions are administered orally.

[0043] In certain circumstances, the pharmaceutical composition may be delivered in a controlled release system. In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system may be placed close to the target of the composition, thus requiring only a fraction of the systemic dose (e.g., Goodson, 1984, Medical Applications of Controlled Release). (See Langer, 1990, Science 2, p. 115-138.) Other sustained release systems are described in This is discussed in a review by Ian Schneider, 49:1527-1533.

[0044] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending or emulsifying the drug in a sterile aqueous or oily medium that is conventionally used for injections. The aqueous medium for injection may be, for example, an isotonic solution containing saline, glucose and other auxiliary agents, etc., which may be used in combination with a suitable solubilizer such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adduct of hydrogenated castor oil)], etc. The oily medium may be, for example, sesame oil, soybean oil, etc., which may be used in combination with a solubilizer such as benzyl benzoate, benzyl alcohol, etc.

[0045] The pharmaceutical composition for oral or parenteral use is prepared in a dosage form suitable for the dosage of the active ingredient.Such dosage forms in a unit dosage include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0046] Dosing regimen The present invention includes methods comprising administering at least one senolytic agent to a subject at at least one dosing frequency. In additional embodiments, the methods of the present invention include administering at least one senolytic agent to a subject at two or more dosing frequencies. In still further embodiments, the dosing is such that a therapeutic response is achieved. A therapeutic response in this context constitutes a reduction in senescent cells. In certain embodiments, the dosing of the senolytic agent is chronic or acute, yet is essentially episodic (e.g., not taken daily) to minimize potential side effects from daily chronic treatment. In one embodiment, the senolytic agent is orally administered twice a month for one month (acute). In further embodiments, fisetin is orally administered twice a day in succession, followed by a routine 28-day break, over years or decades in a episodic regimen (chronic).

[0047] According to certain embodiments of the present invention, multiple doses of at least one senolytic agent may be administered to a subject over a defined time course. The method according to this aspect of the present invention includes sequentially administering multiple doses of the agent to the subject. As used herein, "sequential administration" means that each dose of the agent is administered to the subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods that include sequentially administering to a patient a single initial dose of the agent, followed by one or more secondary doses of the agent, and optionally then one or more tertiary doses of the agent.

[0048] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the temporal order of administration of at least one senolytic agent. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also called a "baseline dose"); a "secondary dose" is a dose administered after the first dose; and a "tertiary dose" is a dose administered after the second dose. The initial, secondary, and tertiary doses may all contain the same amount of agent, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of agent contained in the initial, secondary, and / or tertiary doses varies from each other during the course of treatment (e.g., optionally adjusted up or down).

[0049] According to certain embodiments, the methods of the invention include administering to the subject a second therapeutic agent in combination with at least one senolytic agent. The term "in combination" means that a second therapeutic agent is administered before, after, or simultaneously with the senolytic agent. The term "in combination with" also includes sequential or simultaneous administration of the senolytic agent and an additional therapeutic agent.

[0050] Dosage The amount of senolytic agent administered to a subject according to the method of the present invention is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to an amount of senolytic agent that results in one or more of the following: (a) a measurable reduction in senescent cells; and (b) an improvement in the symptoms of an age-related disease or condition. In one embodiment, the condition is wound healing. In another embodiment, the condition is osteoarthritis or a related orthopedic condition, such as osteoporosis.

[0051] The therapeutically effective amount of the senolytic agent may be about 0.05 mg to about 1000 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, or any amount of the senolytic agent therebetween. In a specific embodiment, 50 mg of the senolytic agent is administered. In additional embodiments, the senolytic agent is fisetin and is administered in vitro at about 1 μM to about 100 μM, hi yet another embodiment, the senolytic agent is fisetin and is administered to a human subject at about 1 mg / kg to about 100 mg / kg.

[0052] The amount of senolytic agent contained within an individual dose may be expressed in terms of milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, the senolytic agent may be administered to a patient at a dose of about 0.0001 to about 100 mg / kg of the patient's body weight.

[0053] Described herein are three approaches for using senescent cell elimination: i) elimination of senescent cells and stem cell banking and transplantation; ii) elimination of senescent cells in orthobiologic products; and iii) elimination of senescent cells in orthopedic surgery. Prong 1 is intended for the outside of the body, prong 2 is intended for the inside of the body, and prong 3 is intended for both the inside and outside of the body.

[0054] Senescent Cell Elimination and Stem Cell Banking and Transplantation It is desirable to store stem cells (called "stem cell banking") for future use, including being given to a recipient ("transplanted") for the treatment of a condition. This banking often involves expansion; however, the regenerative capacity of stem cells is reduced after ex vivo expansion (Monterras, et al. 2005 Science 309:2064-2067). When stem cells are banked, it is hypothesized that expanding them induces a senescent effect in the cells. As a result, banked and expanded cells injected into a subject usually have an increased proportion of senescent cells.

[0055] In one aspect, the disclosure provides a method for removing senescent cells from banked stem cells to minimize proliferation / accumulation of senescent cells upon (during) expansion of the banked cells. In additional embodiments, the banked cells are treated after expansion. In yet another embodiment, the banked cells are treated between expansion cycles. In a preferred embodiment, the cells are treated prior to freezing in liquid nitrogen. "Clearing" senescent cells means targeting and killing them. "Clearing" "Clearing" does not necessarily mean that all senescent cells are removed / killed. Rather, it means that the number of senescent cells in the subject after removal is significantly less than before. In some embodiments, 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%, or at least about 95% of the senescent cells are removed. In additional embodiments, the senescence therapeutic agent may reduce the senescent phenotype in stem cells in the above situations, not by removing, but rather by rejuvenating health and function. Such senomorphic agents may, for example, suppress markers of senescence or secretory phenotype of senescent cells without inducing cell death.

[0056] In one embodiment, a senolytic agent is used to remove senescent cells from the banked stem cells. In a further embodiment, previously banked stem cells are treated with a senolytic agent in vitro. This treatment results in the killing of senescent cells. The senescence-free (or senescence-reduced) stem cells are purified and enriched for reintroduction into the subject from whom the cells were originally taken for banking. In one embodiment, the senescence-free (or senescence-reduced) stem cells thus purified and enriched are reintroduced into a subject with eroded articular cartilage, for example to treat hip arthritis.

[0057] Aging is associated with the depletion of functional stem cells, mainly due to cellular senescence and increased proinflammatory signaling. To be considered as a potential treatment for musculoskeletal repair, adipose-derived stem cells (ADSCs) need to be culture-expanded before treatment. Similar to aging, long-term in vitro expansion of ADSCs results in significant accumulation of senescent cells. In this embodiment, senolytic drugs may be used to attenuate senescence and generate enriched stem cell populations.

[0058] In additional embodiments, senescent cells may be quantified in bone marrow aspirates, bone marrow concentrates, and / or peripheral blood using flow cytometry. Late senescent cells appear to be more abundant in bone marrow aspirates than in peripheral blood (data not shown).

[0059] Elimination of senescent cells in orthobiologic products The term "orthobiologics" or "orthobiologic products" as used herein refers to biological substances used to improve healing of bone, cartilage, tendons, and / or ligaments, for example, after injury or surgery. The products are considered biologics because they are made from substances found naturally in the body. Orthobiologics are advantageous in that they minimize the effects of degenerative diseases and allow for more rapid recovery from musculoskeletal injuries.

[0060] Orthobiologics typically include bone grafts, autologous blood, platelet-rich plasma (PPP), platelet-rich plasma (PRP) with derivatives with high or low white blood cell content (LR-PRP, LP-PRP), autologous conditioned serum, bone marrow aspirate concentrate (BMAC), and autologous stem cells. Orthobiologics may also include drugs such as anti-fibrotic agents, aging therapeutics, fat grafts such as microfragmented adipose tissue, nanofragmented adipose tissue, birth tissue-derived products, extracellular matrix (ECM) implants, supplements, alpha-2-macroglobulin (A2M), amniotic fluid, placental tissue, umbilical cord tissue, hyaluronic acid injections (or other mucus supplements), and stem cell injections.

[0061] In one embodiment, fisetin treatment may be added to orthobiologics in patients with moderate osteoarthritis of the knee and / or hip, reducing patient-reported pain and cartilage loss compared to patients not receiving orthobiologic senolytic therapy.

[0062] Elimination of senescent cells in orthopedic surgery Senescent cell burden has been shown to strongly correlate with age-related orthopedic conditions. Injection of senescent cells is sufficient to promote age-related conditions such as osteoarthritis, frailty, and reduced survival. Therefore, the development of therapies that selectively kill senescent cells is expected to delay the onset of the senescent phenotype, reduce the severity of age-related diseases, improve resilience, enhance survival, and extend lifespan (Xu, et al. 2018 Nat Med 24:1246-1256; Xu, et al. 2017 J Gerentol A Biol Sci Med Sci 72(6):780-785).

[0063] Moreover, targeting and eliminating senescent cells has been shown to alleviate age-related musculoskeletal decline. Thus, in one aspect, the present disclosure provides for treating a patient with a senolytic agent prior to harvesting stem cells to eliminate existing senescent cells in the body. The subject is administered, for example, fisetin or quercetin. Adipose or bone marrow or platelet-rich plasma cells are then harvested and expanded and / or concentrated for reinjection into the subject. As a result, the subject's surgical outcome is improved. For example, healing time is reduced, mobility is increased (e.g., joint mobility is improved as assessed by functional performance tests), joint cartilage is improved (e.g., assessed by MRI, T2 mapping, etc.), pain is reduced (e.g., assessed by PRO), scar tissue is reduced, and / or soft tissue healing is enhanced (i.e., following an ACLR procedure), and / or aging markers are reduced.

[0064] Improved surgical outcome refers to a positive change from baseline. In this context, the term "positive" refers to a change associated with better healing or other clinical outcomes such as improved pain scores or mobility. As used herein, the term "baseline" refers to the value of a subject's parameter before or during treatment according to the present invention.

[0065] To determine whether a surgical outcome / parameter is "improved," the parameter is quantified at baseline and at one or more time points after treatment. The difference between the value of the parameter at a particular time point after the start of treatment and the value of the parameter at baseline is used to ascertain whether there has been an "improvement." EXAMPLES

[0066] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers used (amounts, temperatures, etc.), but some experimental error and deviation must be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is temperature in degrees Celsius, and pressure is at or near atmospheric pressure.

[0067] Example 1 Stem cells accumulate senescence during ex vivo culture expansion Serial passaging (expansion) increases cellular senescence in ADSCs. Senescence is detected in ex vivo expanded cells. As reinjection of stem cells containing senescent cells is undesirable, cellular senescence was evaluated after each passage. To examine the effect of culture expansion of adipose-derived stem cells, cells at passage 2 were thawed and cultured until passage 8. Cells were maintained in T-75 or T-175 culture flasks using regular growth medium (DMEM:F-12 with 10% FBS and 1% penicillin / streptomycin). At 70–90% confluency, cells were passaged and reseeded in flasks at a 1:4 dilution. To test senescence at passages 3, 4, 6, and 8, cells were plated in triplicate in 12-well plates at a density of 40,000 cells / well. After attachment overnight, cells were incubated with 100 nM bafilomycin A1 at 37 °C and 5% CO. 2 The cells were treated with 33μM C12FDG for 1 hour. Then, 33μM C12FDG was added and incubated for 2 hours. The cells were then washed and harvested from the plate using TrypLE reagent. Cells positive for C12FDG substrate were quantified using an EasyCyte flow cytometer.

[0068] Senescence (% of senescent cells) increased with each passage, with a steep increase from passage 3 to passage 4. Figure 1A shows the effect of passaging on ADSCs. The 6% of senescence at passage 3 (P3) jumped to 30% at passage 4 (P4) and further increased to 45% at passage 6 (P6). Thus, cells passaged more than four times were no longer suitable for reinfusion. Rather, banked cells cultured up to passage 3 are optimal for reinfusion. Indeed, P1 to P4 are clinical grade cells. Figure 1B-E represent histogram plots of C12FDG staining. The R1 region shows cells positive for C12FDG.

[0069] Senescence-associated secretory phenotype (SASP) concentrations increase with serial passages. To determine the effect of passaging on senescence-associated secretory phenotype (SASP) and SAHF, passage 2 cells were thawed and cultured to the respective passage numbers tested. ADSCs were serially passaged 18 times and stained with antibodies against γ-H2AX and H3K9. These antibodies bind to senescence-associated heterochromatin foci (SAHF) and are indicative of gene silencing and DNA damage.

[0070] Specifically, cells were maintained in T-75 culture flasks using regular growth medium (DMEM:F-12 containing 10% FBS and 1% penicillin / streptomycin). At 70–90% confluency, cells were passaged and replated in flasks at a 1:4 dilution. At each tested passage number, cells were cultured at 5,000 cells / cm. 2 Cells were plated in chamber slides for SAHF staining at a density of 100x. Cells were seeded in triplicate for each group and incubated and allowed to adhere overnight. The next day, cells were fixed with cold 4% paraformaldehyde and stained with H2AX antibody using a 1:150 dilution and H3K9 antibody using a 1:250 dilution. DAPI was also used to stain nuclei. Five images per well and three wells per group were imaged using a Nikon Eclipse Ni-U microscope. Cells positive for H2AX and H3K9 antibodies were quantified using ImageJ software.

[0071] For SASP detection, the medium was collected after 24 h incubation with cells, centrifuged at 2000 × g for 10 min, transferred to a new microcentrifuge tube, and stored at −80 °C until testing. SASP was quantified using the Human Adipocyte Milliplex kit using an EMD Millipore Milliplex instrument.

[0072] Figures 2A and 2B show that with increasing passage number, senescence-associated secretory phenotypes (SASPs) and heterochromatin foci (SAHFs) also increased. IL-6, IL-8, and MCP-1 each increased in concentration from passage 6 to passage 8. Thus, it is shown herein that aging and culture expansion increase the level of senescence. Indeed, aged individuals and culture-expanded cells had a higher number of senescent cells compared to young individuals.

[0073] Example 2 Fisetin treatment is non-toxic to healthy stem cells Fisetin is a natural flavonoid found in many fruits and vegetables. Due to its hydroxyl group, fisetin is known as an antioxidant and reducing agent. It reduces the secretion of several pro-inflammatory factors, has anti-cancer activity, and blocks the mTOR and PI3K / AKT pathways, therefore fisetin is a potent therapeutic agent targeting senescent cells. Fisetin has the molecular formula C 15 H 10 O 6 , molecular weight 286.24 g / mol, CAS name / number: Fisetin, 2-(3,4-dihydroxyphenyl)-3,7-dihydroxychromen-4-one, 528-48-3, and chemical structure: [ka] has.

[0074] Because killing healthy, non-senescent cells when treated with fisetin would be harmful, it was necessary to determine the optimal fisetin dose. In an attempt to determine this concentration, healthy low-passage ADSCs were used to ensure that healthy cells were not dying.

[0075] Reagents included ADSC culture medium (CM) (DMEM-F12, Gibco #11320; 10% FBS, Gibco #16000044; 1% Penicillin Streptomycin, Gibco #15140122); 1x Ca and Mg Free DPBS (Gibco #14190250); TrypLE (Gibco #12605028); and Fisetin (Sigma #F4043).

[0076] adipose-derived stem cells We purchased banked ADSCs isolated from a 10-year-old male (YM), a 27-year-old female (YF), a 75-year-old male (OM), and a 79-year-old female (OF) and cultured them in regular growth medium (DMEM / F:12, 10% FBS, and 1% penicillin / streptomycin).

[0077] Fisetin Treatment Fisetin was added to the growth medium and allowed to act on the cells for 24 hours, after which the medium was replaced with regular growth medium for 24 hours before use in cytotoxicity, immunofluorescence, or flow cytometry.

[0078] Immunofluorescence Immunofluorescence results were generated to provide a proof of concept for detecting senescence by nuclear staining. γ-H2AX and H3K9 antibodies were used to stain for senescence-associated heterochromatin foci. Three replicate wells were given for each cell type, and each well was imaged six times for a total of 18 images per group. The number of cells positive for each marker was quantified using the Image J program. The collected data was processed using one-way ANOVA, and all but two treatment groups were found to be statistically significant in every group except for γH2AX in young female cells at passage 4 and old female cells at passage 18.

[0079] Flow cytometry Cells were incubated with 100 nM bafilomycin A1 at 37 °C and 5% CO 2 Then, 33 μM C12FDG was added and incubated for 2 hours. Thus, cells were stained with the fluorescent senescence-associated marker C12FDG, collected, and tested for senescence using flow cytometry after 50 μM fisetin treatment. Three replicates per treatment were used. Using InCyte software, unstained control cells were used. Fluorescence gating was determined by

[0080] Specifically, cells were thawed in a 37°C water bath. In a biosafety cabinet, cells (1 mL) were collected and added to 9 mL pre-warmed ADSC culture medium (CM, DMEM:F-12 with 10% FBS and 1% penicillin / streptomycin). Cells were centrifuged at 1000 x g for 5 min. Media was aspirated and cells were resuspended in 1 mL CM. 10 μL of cell suspension was pipetted into a microcentrifuge tube. 10 μL of cell suspension was mixed with 10 μL of trypan blue. 10 uL of cell suspension / trypan blue was pipetted onto a countess slide. Cell counts were performed using a Countess II The volume required to seed 300,000 cells / T-175 flask was calculated. 20ml of CM was added to the T-175 flask. The calculated cell suspension volume was added to the T-175 flask. The flask was placed in an incubator with 5% CO2 and 37°C. The cells were allowed to attach overnight. The flask was returned to the biosafety cabinet. The CM was aspirated and then washed by adding 10mL of 1xDPBS. The DPBS was aspirated. 20mL of pre-warmed CM was added and subsequently cultured in the incubator until 80% confluent, changing the medium every 2 days. At 70-90% confluence, the medium was aspirated and then washed with 10mL of 1xDPBS. 5mL of TrypLE was added. The flask was placed in the incubator and cell detachment was monitored under a microscope. Once detached, 5mL of CM was added to the flask. The cell suspension was collected in a 15 mL conical tube and centrifuged at 1000 xg for 5 minutes.

[0081] Media was aspirated and cells were resuspended in 1 mL CM. 10 μL of cell suspension was pipetted into a microcentrifuge tube. 10 μL of cell suspension was mixed with 10 μL of trypan blue. 10 uL of cell suspension / trypan blue was pipetted into a countess slide. Cell counts were recorded using a Countess II Automated Cell Counter. The volume required to seed 40,000 cells / well in a 12-well plate was calculated. 1 mL of CM was added per well followed by the calculated cell suspension volume (to each well). Flasks were placed in an incubator at 5% CO2 and 37°C. Cells were allowed to attach overnight. Flasks were returned to a biosafety cabinet. CM was aspirated and then washed by adding 1 mL of 1x DPBS per well. DPBS was aspirated. 1 mL of 50 uM Fisetin Supplemented Culture Media was added. The plates were cultured in the incubator for 24 hours. The plates were returned to the biosafety cabinet. The medium was aspirated and then washed by adding 1 mL of 1xDPBS per well. The DPBS was aspirated. 1 mL of pre-warmed CM was added. The plates were incubated until the cells were 80% confluent. The next step was to proceed with downstream use.

[0082] Thus, cells were cultured in a commercially available culture system using an inexpensive, commercially available growth media product, and a single dose treatment reduced the senescent cell population, allowing culture expansion without the accumulation of senescent cells.

[0083] cytotoxicity The toxicity of 50 μM, 25 μM, and 1 μM phycetin against low-passage (<p4) cells was measured using the CellTiter96® AQueous Cell Proliferation Assay. Cells were seeded at 40,000 cells / well in a 12-well plate. After adhering overnight, the cells were treated with 50 μM, 25 μM, and 1 μM phycetin for 24 hours. After treatment, the medium was changed back to normal growth medium for an additional 24 hours. Next, the cells were treated with the CellTiter 96 AQueous Cell Proliferation Assay according to the manufacturer's protocol. Values are based on untreated control cells.

[0084] Results The viability and proliferation of ADSCs were not impaired by phycetin exposure. Phycetin was found to be non-toxic to ADSCs at 1 - 50 μM (Figure 3). No significant difference in toxicity was observed between treatment concentrations. Treatment with 50 μM phycetin was not toxic to the health of low-passage ADSCs.

[0085] Example 3 Phycetin treatment results in a decrease in senescent stem cells Immunosuppressive agents such as rapamycin have previously been shown to reduce cellular senescence while improving myogenic and chondrogenic differentiation in muscle-derived stem cells from progeria mice. Therefore, the senescence-removing agent and antioxidant phycetin were investigated to reduce the senescence of ADSCs from senescent cells, cultured-expanded cells, and banked cells.

[0086] Adipose-derived stem cells (ADSCs) can be a promising tool for tissue regeneration and prevention of age-related musculoskeletal degeneration among other stem cells. However, these age-related conditions also affect the ADSC population in the form of cellular senescence. These senescent ADSCs may significantly lose their therapeutic potential when isolated from elderly individuals and / or cultured-expanded and banked. Therefore, there is a reason to eliminate these cells during culture expansion when a viable stem cell bank is generated for patients.

[0087] The use of senolytic drugs has been shown to reduce the effects of age-related diseases and improve lifespan. As a novel method to improve in vivo outcomes, senolytic agents have proven promising; however, their use for culture expansion and enrichment of banked human stem cells has not been demonstrated.

[0088] Based on the data from the previous examples, to determine the senescence potential of fisetin in vitro, a fisetin concentration of 50 μM was used to treat YF and OF cells. Senescent cells were quantified by immunofluorescence using γ-H2AX and H3K9. Similarly, YM and OM cells were treated with C for flow cytometry. 12 Quantification was performed using FDG. 12 FDG reacts with beta-galactosidase, a biomarker of aging, to produce a fluorogenic substrate that is detected and quantified by flow cytometry.

[0089] method Cells were seeded in triplicate in each group using the culture method and seeding density described above. After overnight attachment, the medium was changed. The untreated group used normal ADSC growth medium (DMEM:F-12 with 10% FBS and 1% penicillin / streptomycin) and the treated group was supplemented with 50 uM fisetin. After cells were treated for 24 hours, the medium was changed back to normal growth medium in all groups. After an additional 24 hours of culture in normal growth medium, the medium was collected for SASP quantification or the cells were processed for C12FDG or SAHF staining as described in the preceding examples.

[0090] Antibodies for H3K9 and H2AX (nuclear proteins involved in gene silencing in senescent cells) were used to quantitate the level of baseline senescence in stem cells from a 75-year-old male ("OM"). To determine the effect of fisetin on the elimination of senescent cells, ex vivo cultured stem cells from the same OM were treated with 50 μM fisetin. H3K9 and H2AX are indicative of essentially senescent cells.

[0091] result Herein, it is shown that fisetin, a natural flavonoid with proven senescent cell elimination, can reduce senescence accumulation in culture-expanded and banked ADSCs by single and intermittent doses during culture expansion. This is demonstrated herein by staining for senescence-associated β-galactosidase (C12FDG, Figure 4A-4D), senescence-associated heterochromatin foci (SAHFs) H3K9 and γH2AX (Figure 5A-5C), and senescence-associated secretory phenotype (Figure 6). These SAHFs are commonly found in senescent cells and indicate gene silencing and DNA damage, while β-galactosidase is standardly used as a biomarker of cellular senescence due to its overexpression in senescent and aged cells.

[0092] Thus, fisetin treatment reduced β-galactosidase, as assessed via C12FDG staining (Figure 4A-4D). Within the R1 region of Figure 4B-4D, cells positive for C12FDG are indicated by their respective peaks. Untreated cells appear to exhibit significantly larger peaks compared to treated cells. The left half of each scan shows untreated cells, with the "inner" peak representing senescent cells, showing a count of approximately 120. The right half of the scan shows cells treated with 50 μM fisetin, with the "inner" peak being senescent cells, showing a count of approximately 20. Thus, senescence was significantly reduced in fisetin-treated cells. Furthermore, fisetin treatment reduced H3K9 and γ-H2AX senescence-associated heterochromatin foci (SAHF) (Figure 5A-5C). The number of cells positive for H3K9 was significantly reduced in all groups (Figure 5A). γ-H2AX was decreased in low-passage OF cells, low-passage YF cells, and high-passage YF cells. All groups showed a decrease. Finally, fisetin treatment reduced the senescence-associated secretory phenotype (SASP) as shown in fisetin-treated cells cultured in serum-depleted culture medium for 24 hours, after which the culture medium was assayed for SASP using a Millipore Sigma Milliplex Instrument. Three replicates were used for each treatment. (Figure 6). The cytokines IL-6, IL-8, and MCP-1, known SASP factors, all showed a decrease in concentration after fisetin treatment, with the decrease in IL-6 and MCP-1 being particularly pronounced.

[0093] Example 4 Senescent cells are C 12 Detected using FDG staining and flow cytometry The ability to accurately detect senescent cells and their associated senescence-associated secretory phenotype (SASP) factors may facilitate understanding of individual patient responses to treatment and / or help determine intervention strategies in the clinic. Identification, quantification, and / or characterization of senescent / senescent cells may be used in each of the four prongs identified above that are contemplated for elimination of senescent cells. In one aspect, senescent cells are C 12Detected and / or quantified using FDG staining and flow cytometry.

[0094] The use of flow cytometry-assisted analysis of aged peripheral blood mononuclear cells is known for clinical diagnostic applications. Such analyses often rely on a specific subset of peripheral blood mononuclear cells (PBMCs), namely T cells, to reflect the age or senescence state of human whole blood over time. However, here we show that by selecting all T cells (CD3+), including both CD4 and CD8 subsets, rather than just one of the CD4 and CD8 subsets from PBMCs, we can provide a method to identify C. 12 We describe for the first time that it is possible to detect reproducible, age-correlated changes in senescent cell numbers using FDG staining.

[0095] C 12 FDG (5-dodecanoylaminofluorescein di-β-D-galactopyranoside) is a β-galactosidase substrate that has been covalently modified to contain a 12-carbon lipophilic moiety. Once inside the cell via the staining procedure, this substrate is cleaved by the β-galactosidase enzyme to generate a fluorescent product that is well retained by the cell, presumably by incorporating the lipophilic tail within the cell membrane. 12 The molecular formula of FDG is C 4 4 H 55 NO 16 , has a molecular weight of 853.9156 and a CAS name / number of dodecanamide, N-[3',6'-bis(β-D-galactopyranosyloxy)-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-5-yl]-138777-25-0.

[0096] Beta-galactosidase: [ka]

[0097] Senescence-associated beta-galactosidase (SA-β-gal or SABG) is a hydrolase that hypothesizes that catalyzes the hydrolysis of beta-galactosidase to monosaccharides only in senescent and aging cells. Senescence-associated beta-galactosidase is a biomarker of cellular senescence, due to its overexpression and accumulation, particularly in senescent and aging cells.

[0098] Whole blood was collected from subjects into a 50 ml syringe containing ACD-A and placed on a rocker. SepMate tubes (Stem Cell Technologies) were prepared using 15 ml of Lymphoprep in a sterile environment. 10 ml of whole blood was dispensed into a 50 ml conical tube. 500 μl (50 ul / ml) of RosetteSep (Stem Cell Technologies) was added to the 10 mL blood sample. For the other blood sample, total PBMCs were collected as described below without enrichment for T cells for comparison. The mixture was incubated for 10 minutes at room temperature (RT). An equal volume of dilution medium (DM-sterile PBS and 2% FBS) was added to the sample and mixed gently. The diluted sample was slowly added to the SepMate tube containing Lymphoprep density gradient medium. The mixture was centrifuged at 1200 x g for 10 minutes with the brake on. Enriched cells were collected by pouring the supernatant into a new tube. The enriched cells were washed by overlaying with DM and then centrifuged at 300×g for 10 min with low brake (setting 4). The supernatant was discarded and the cells were gently reconstituted in 10 ml of DM. The last two steps were repeated. The T cell enrichment protocol is shown diagrammatically in Figure 7. The pellet was reconstituted in 4 mL of C12 culture medium and distributed equally into two 15 ml conical tubes. One was filled with 30 μM C 12 One was treated with FDG and the other was left as an unlabeled control. The cap was loosened and the tube was incubated for 1 h, then incubated in 5% CO 2 Mix by inversion once every 30 min in a tissue culture incubator containing 0.5% CO. Cells were spun down and washed twice with 5 mL of cold PBS (300 × g with full brake) and then reconstituted in 1 mL PBS on ice protected from light.

[0099] C12FDG cells were analyzed via flow cytometry utilizing the Guava EasyCyte HT. Guava loading was according to the following example well allocation scheme (Table 1 below).

[0100] [Table 1]

[0101] Samples were acquired in triplicate when threshold and gain controls were set. Runs were saved as FCS files and gates were set to exclude the labeled control. Everything within the gate was C 12 - FDG was positive.

[0102] analysis Aged CD3 + To assess the percentage, number, and concentration (cells / ml) of T cells, an unstained control (i.e., C 12 The settings were adjusted using PBS alone (cells not treated with FDG). First, thresholds were set for FSC and SSC to catch cellular events. Next, the green channel gain control was set so that background events fell between 10 and 20 years. Finally, PBS alone (vehicle) ensured that no events were registered with the adjusted setting parameters.

[0103] Samples were run in triplicate and in every 5 wells run with water only. Typically, distinct "subpopulations" of senescent cells with varying degrees of intensity were found (Figure 8). These were 12These represent very "bright" cells that exhibit strong fluorescent signals due to high enzymatic degradation of FDG molecules. These cells are considered to be "late" senescent cells and may be the most physiologically destructive cells. In further embodiments, a cell sorter may be used to select (sort / accumulate) cell subsets classified as early stage senescent cells, intermediate stage senescent cells, and late stage senescent cells. In therapeutic settings (preclinical or clinical), sorted cells may be used to optimize the collection of healthy or non-senescent cells prior to autologous (autologous) or allogeneic (allogeneic) transplantation.

[0104] Advantages of the rapid assay include: i) C as a fluorescent stain; 12 ii) the use of FDG (as opposed to antibody staining immunophenotyping); ii) the use of benchtop flow cytometry; iii) due to the dynamics of the staining, the complete assay can be performed more quickly (in a few hours); iv) small sample volumes are required (only 2-5 ml of blood); v) the use of CD3+ T cell populations or total PBMC populations; vi) the ability to detect changes following senolytic treatment; vii) the ability to correlate senescent cell numbers with specific inflammatory factors and / or SASP in plasma; viii) the use on BMA and whole blood; and ix) sensitivity to potential stages of senescence.

[0105] Example 5 Fisetin treatment leads to reduction of senescent cells in 82-year-old man The senescent cells were treated with fisetin and then further characterized using flow cytometry (as described in Example 4 above). Peripheral blood CD3+ T cells of an 82-year-old male were evaluated for senescence and senescent cell populations before and after fisetin treatment (100 mg per day for 45 days). Before treatment, the subject measured approximately 29% senescent cells, and after treatment, the subject measured approximately 4% senescent cells (data not shown). Thus, the number of senescent cells was significantly reduced by treatment with the senolytic agent fisetin, as shown in Table 2 below.

[0106] [Table 2]

[0107] Example 6 Further characterization of senescent cells ("senescence profiling") Venipuncture (blood test) A qualified phlebotomist performed standard venipuncture from a vein in the antecubital (AC) region, forearm or hand using a 19-gauge butterfly needle. Approximately 35 mL of blood was drawn into one 30 cc syringe (with ACD-A) and one 5 cc (without ACD-A) syringe. Blood was used for CBC count (data not shown), multiplex immunoassay (data not shown), T cell expression assay (data not shown), and C12FDGFLOW assay using enriched T cells and total PBMC. For total PBMC and enriched T cells, SepMateTM tubes (Stem Cell Technologies) were used according to the manufacture protocol by LymphoprepTM (Stem Cell Technologies) gradient centrifugation. Specifically, in a separate tube, CD3+ T cells were enriched using RosetteSepTM commercial kit according to the manufacturer's instructions (Stem Cell Technologies #15021). A portion of the cells (enriched T cells and total PBMCs) were stained with 30 μM C12FDG in general culture medium supplemented with 10% FBS and analyzed by flow cytometry (Guava EasyCyte, Luminex). To assess the percentage, number, and concentration (cells / ml) of senescent CD3+ T cells and PBMCs, settings were first adjusted using unstained controls (i.e., cells not treated with C12FDG). First, thresholds were set for FSC and SSC to catch cellular events. Next, the green channel gain control was set so that background events fell between 10 and 20 years. Finally, PBS alone (vehicle) ensured no events were registered with the adjusted setting parameters. Samples were run in triplicate, with all 5 wells run with water alone.

[0108] Figure 9A shows control unlabeled cells. In the absence of labeling, no senescent cells are detectable. Staining of T cells revealed clear populations of high intensity bright cells and low intensity dim cells. This difference in intensity is due to the fluorescent marker C12FDG only fluorescing when hydrolyzed by β-galactosidase, an enzyme upregulated during senescence (leading to a range of signals due to the range of expression of the latter). The populations represent stages of senescence, with the brightest events being late senescence (high C12FDG signal) and presumably more pathological.

[0109] In one embodiment, the senescent cells may be further characterized. In another embodiment, the senescent cells may be characterized before and after treatment with a senolytic agent. Figures 9B and 9C show that the senescent cells are composed of a population of moderately senescent cells and highly senescent cells, with the moderately senescent cells corresponding to intermediate senescence and the highly senescent cells corresponding to late senescence.

[0110] The composition of senescent cells (e.g., mid- and late senescent cells) was compared between 25-year-old males, 55-year-old males, and 85-year-old males. The samples evaluated were peripheral blood samples. Figure 10A shows that 25-year-old males had a lower percentage of late senescent cells (0.6%) than 55-year-old males, and a lower percentage of late senescent cells (1.1%) than 85-year-old males (4.1%). Figure 10B shows that 41-year-old females had a lower total number of mid-+late senescent cells, a lower total number of late senescent cells, a lower percentage of mid-+late senescent cells, and a lower percentage of late senescent cells compared to 77-year-old females.

[0111] Senescence profiling was performed on 67 subjects, including male and female subjects of different age groups. Peripheral blood was collected from each subject and T cells were enriched in each sample for assessment of senescent cells. Total cells, dim cells, and bright cells (senescent cells) were measured using C12FDG cell staining and analysis on a flow cytometer device. Raw data obtained from the scatter plots were gated into two regions of interest: i) dim cells (events) and ii) bright cells (events). As described in the "Example of Interpretation of Results" below, each subject received the results of senescence profiling in a report in the format described above. The percentage of bright senescent cells versus the percentage of dim senescent cells was assessed. Each subject received the results of senescence profiling in a report in the format of Figure 11A. For subjects with multiple blood draws (longitudinal studies), the report reflects the change in senescent cells over time.

[0112] Analysis of stained blood cells (T cells) revealed distinct subpopulations of high intensity or "bright" cells and lower intensity "dim" cells. The fluorescent marker of interest (C12FDG, a senescence marker compound) only fluoresces when hydrolyzed by an enzyme (β-galactosidase) that is upregulated during senescence, resulting in a range of signals due to the range of β-galactosidase expression. This suggested that these populations represent stages of senescence, whereby the brightest events are later stages of senescence (high C12FDG signal) and therefore potentially more pathological. In one embodiment, these later or "bright" cells are indicative of a health condition, specifically, poor health or a higher risk of age-related and / or age-related diseases / disorders. This includes musculoskeletal conditions such as, but not limited to, osteoarthritis, osteoporosis, osteopenia, frailty risk, sarcopenia, tendinopathy, arthritis, and muscle weakness. This also includes other age-related comorbidities such as type 2 diabetes, metabolic syndrome, neurodegenerative diseases such as cognitive decline, and heart disease such as atherosclerosis. The data for the subject groups are summarized in Figure 11B. The percentage of bright senescent cells was significantly lower than the percentage of dim senescent cells (save for some outliers that are being further investigated).

[0113] Example 7 Removing senescent cells using a senolytic agent delayed osteoarthritis in progeria mice Hutchinson-Gilford Progeria Syndrome (HGPS) is an autosomal dominant disorder associated with premature aging (lamin A deficiency) and early death during childhood. Rapidly sclerotic skin, joint contractures, bone abnormalities, and growth retardation have been observed in affected subjects.

[0114] Zmpste24- / - (Z24- / -) mice are a reliable animal model of HGPS They are unable to produce lamin A, an essential component of the nuclear lamina. Premature senescence was observed in the Zmpste24- / - mouse model, and indeed an accumulation of senescent cells was observed in the mice.

[0115] We investigated the effect of senolytic drugs on osteoarthritis in Zmpste24− / − mice.

[0116] In vivo senolytic drug treatment Adult progeria Z24- / - mice were administered senolytic drugs by oral gavage (150 μl volume) starting at 2.5–3.0 months of age. D / Q (5 mg / kg in 10% PEG400 / 5.0% DMSO; 50 mg / kg) was administered as a single dose at 2.5 months or multiple doses at 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 months. Both single- and multiple-dose DQ mice were sacrificed at 5 months of age. Mice treated with FIS (100 mg / kg in 90% PEG400 / 10.0% ethanol) received a total of 4 weeks of drug treatment starting at 3.0 months of age and were sacrificed at 4.0 months of age. For 17-DMAG treatment (10 mg / kg, 1% DMSO-PBS), mice were treated three times a week starting at 2 months of age and then sacrificed at 4 months of age.

[0117] Histological evaluation of cartilage Knee joints from Z24- / - animals were decalcified using 10% disodium ethylenediaminetetraacetate (EDTA) plus 1% sodium hydroxide for 6 weeks. Whole knee joints were sectioned, dehydrated, and then paraffin-embedded to allow sagittally viewing of the articular center (at the level of the groove) and the articular edge (at the level of the condyle). To examine the articular cartilage, 5 μm paraffin sections were cut and stained with Alcian blue (to detect acidic glycosaminoglycan GAGs; mucopolysaccharides) and Safranin-O (to detect proteoglycan content). Images of the entire area of ​​the cartilage surface of each animal, both at the center (groove) and edge (condyle), were captured at ×200 magnification. Images were acquired using a Leica DMIRB microscope equipped with a Retiga digital camera and evaluated using Northern Eclipse software (v6.0; Empix Imaging).

[0118] Immunochemical staining of cartilage and primary chondrocytes Immunohistochemical staining of p16INK4a positive cells was used to assess the number of senescent chondrocytes in 5 μm paraffin sections from knee joints. Briefly, after deparaffinization, washing, and blocking with 5% donkey serum in PBS, sections were incubated overnight with rabbit anti-p16 antibody (Abcam, 1:100) in 5% donkey serum. Immunoreactivity was subsequently detected using goat anti-rabbit biotin (BA 1000, Vector Laboratories, Burlingame, CA, USA, 1:200 dilution), incubation with ABC reagent (PK 7200, Elite ABC kit, Vector Laboratories), and visualized with diaminobenzidine (DAB) staining (SK-4100, Vector Laboratories) according to the manufacturer's instructions.

[0119] Immunofluorescence was performed to detect abnormal cell nuclei in primary chondrocytes. Cultured cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). Cells were then incubated with Block Ace Solution (Dako Japan, Kyoto, Japan) and stained with an antibody against lamin A / C (1:50; Santa Cruz Biotechnology) as a sensitive marker of the nuclear lamina, followed by incubation with Alexa Fluor 594-conjugated rabbit anti-goat IgG1 (1:1,000; Molecular Probes, Grand Island, NY, USA) for 1 h. To visualize nuclei, nuclei were stained with DAPI solution (1:500; Sigma-Aldrich). h) for 5 min. Images were taken using a Leica equipped with a Retiga digital camera. Images were acquired on a DM IRB microscope and evaluated with Northern Eclipse software (v6.0; Empix Imaging).

[0120] Micro-CT analysis For fisetin treatment, mice were imaged using an eXplore Locus Utrapre-Clinical CT Scanner (GE Healthcare, London, ON) through the pre-Clinical CT Core Facility with the following acquisition settings: 140 kVp, 22 mA, 16 s rotation / exposure. Non-cardiac gated CT images were acquired at all time points. Simple backprojections were obtained from 0.154 μm image reconstructions and exported as DICOM images. Image analysis was performed using OsiriX software. Qualitative analysis of bone was performed on 2D and 3D reconstructions using OsiriX. MicroView imaging software was also used for quantitative analysis of bone mineral density.

[0121] Although no significant protective effect in bone was observed using D+Q, the use of the phytonutrient fisetin was effective. Z24- / - mice were treated with fisetin (100 mg / kg) weekly for 4 weeks starting at 3 months of age and then sacrificed at 4 months of age. We chose 4 months (instead of 5 months) as the sacrifice period to account for the more advanced pathology, which occurred at 5 months, a time point at which treatment may have a negligible effect, as seen in animals treated with D+Q. Z24- / - mice treated with FIS were found to have reduced transcription levels of the senescence marker p16INK4a as well as the SASP factors IL-6 and TGF-β (data not shown). In addition to the reduction of tissue senescence markers, weekly administration of FIS for several weeks starting at 3 months of age was able to significantly attenuate the loss of bone mineral density in Z24- / - mice, as evidenced by micro-CT analysis. Significantly higher scores were observed in Hounsfield unit (HU) intensity, bone mineral density (BMD), and specific bone surface (BS / BV) in fisetin-treated versus untreated Z24- / - mice. Thus, unlike D+Q, senolytic therapy with fisetin prevented age-related bone mineral density loss, suggesting the importance of senescence to bone loss during aging.

[0122] Example 9 Bone marrow aspirate concentrate containing senotherapeutic flavonoids to improve articular cartilage repair Microfracture (MFx), the most commonly used first-line treatment for cartilage damage, has been shown to have inferior long-term clinical outcomes, mainly due to the production of fibrocartilage repair tissue. Losartan administration was found to enhance microfracture for cartilage repair (Utsunomiya, et al. 2020 AJSM 48(4):974-984). Bone marrow aspirate concentrate (BMAC) is another biological product that has shown positive effects in bone and cartilage applications such as osteochondral defects, osteoarthritis, and bone nonunions. In addition, early clinical data suggests that BMAC may help stimulate a stronger hyaline cartilage repair tissue response through both chondrocyte differentiation and paracrine signaling of MSCs. Previous studies have reported that fisetin reduced cartilage destruction and subchondral bone plate thickness and alleviated synovitis in a mouse OA (osteoarthritis) model. However, the synergistic effect of adding BMAC and losartan to fisetin has not yet been investigated in an osteochondral model. The purpose of this example was to determine the effect of fisetin on cartilage repair in a rabbit model, as well as its synergistic effects with losartan and BMAC.

[0123] method Collection and processing of BMAC for autologous transplantation: Bone marrow aspirate (BMA) was collected from the intestine of each rabbit. Bone was collected by crest aspiration and processed using a benchtop centrifuge to prepare BMAC.

[0124] Surgical Procedure: Forty-eight skeletally mature New Zealand White rabbits were anesthetized to create a full-thickness osteochondral defect (OD) of 5 mm diameter in the patellar groove (depth: 2 mm). Microfracture surgery was performed within the defect by creating five holes, evenly spaced and 2 mm deep, in the subchondral bone using a micro-awl. After microfracture surgery, 0.5 ml of BMAC was injected intra-articularly in the left knee. Six and 12-week studies were performed using six rabbits in each group comparing the eight conditions listed below. At each endpoint, rabbits were euthanized for microCT, qPCR, and histological analysis.

[0125] result Preliminary data showed that the addition of BMAC or augmentation of fisetin and losartan to microfractures accelerated cartilage regeneration in a rabbit osteochondral defect model (data not shown). Therefore, the use of senescent therapeutic agents such as fisetin may eliminate senescent cells from orthobiologic BMAC, and this enrichment may activate stem cells in the BMAC and improve their regenerative capacity.

[0126] Example 10 Fisetin reduces early and late senescent cells Five patients, ranging in age from 56 to 77 years, were treated with 100 mg / day fisetin for 30 to 45 days. Notably, patients with a "high" baseline number of C12FDG+ T cells or "bright" cells showed a 10- to 20-fold decrease in "bright" C12FDG+ T cells (data not shown). At passage 19 (P19), pre-treated / untreated bright cells measured an average of 12.53%, while treated / post-treated bright cells measured an average of 1.15%. At passage 50 (P50), pre-treated / untreated bright cells measured an average of 19.80%, while treated / post-treated bright cells measured an average of 0.60%.

[0127] In intermediate senescent, or "early" stage senescent cell populations (i.e., those with more dim staining for C12FDG), the percentage of C12FDG+ cells was also reduced by 1-2 fold (data not shown). At passage 19 (P19), pretreated / untreated dim cells measured an average of 54.22%, while treated / posttreated dim cells measured an average of 26.70%. At passage 50 (P50), pretreated / untreated dim cells measured an average of 59.59%, while treated / posttreated dim cells measured an average of 27.55%.

[0128] Overall, the data demonstrated 1) the sensitivity of the methodology described herein for detecting changes in senescent cells using known senolytic drugs (senolytic drugs), and 2) the ability of orally administered fisetin to significantly reduce the number of "bright" or "advanced senescent" cells, cells believed to be the primary drivers of senescence-related pathology during aging. In some embodiments, patients with high baseline bright / late senescent cells respond particularly significantly to senotherapy treatment.

[0129] The present disclosure should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such variations are intended to be included within the scope of the appended claims.

Claims

1. 1. A method for detecting senescent cells in a cell sample, comprising staining the cells with C12FDG and subjecting the cells to flow cytometry.

2. The method of claim 1 , wherein the cell sample comprises CD3+ T cells.

3. 3. The method of claim 1 or 2, wherein the cell sample is total peripheral blood mononuclear cells, bone marrow aspirate (BMA), whole blood, ex vivo culture expanded stem cells, banked stem cells, freshly isolated stem cells, or endogenous stem cells.

4. The method according to any one of claims 1 to 3, wherein the detected senescent cells are characterized according to their stage of senescence.

5. The method of claim 4 , wherein the characterization is based on the brightness of the signal.

6. The method of claim 4 or 5, wherein the stage of senescence is early stage (relatively low C12FDG positivity), intermediate stage (relatively intermediate C12FDG positivity), or late stage (relatively high C12FDG positivity) as determined by normalized event gating by flow cytometry.

7. 1. A method for removing senescent cells from banked stem cells, the method comprising adding at least one senolytic agent to the cells.

8. 1. A method for enriching banked stem cells, the method comprising adding at least one senolytic agent to the cells.

9. 9. The method of claim 7 or 8, wherein the banked stem cells are selected from the group consisting of ADSCs, ex vivo culture expanded stem cells, freshly isolated stem cells, endogenous stem cells, bone marrow aspirate concentrate (BMAC) stem cells, and whole blood stem cells.

10. The method of any one of claims 7 to 9, wherein at least 25% of senescent cells are removed.

11. The method of any one of claims 7 to 10, wherein at least 50% of senescent cells are removed.

12. The method of any one of claims 7 to 11, wherein at least 75% of senescent cells are removed.

13. The method of any one of claims 7 to 12, wherein at least 80% of senescent cells are removed.

14. The method of any one of claims 7 to 13, wherein at least 85% of senescent cells are removed.

15. The method of any one of claims 7 to 14, wherein at least 90% of senescent cells are removed.

16. The method according to any one of claims 7 to 15, wherein at least 95% of senescent cells are eliminated. Law.

17. 1. A method of treating a senescence-associated disease, disorder, or condition in a subject, the method comprising administering to the subject at least one senolytic agent.

18. 1. A method for treating a disease, disorder, or condition associated with aging in a subject, the method comprising removing stem cells from the subject, culturing the cells with at least one senolytic agent, and administering the cultured cells to the subject.

19. 1. A method for treating an age-related disease, disorder, or condition in a subject, the method comprising removing stem cells from the subject, culturing the cells with at least one senolytic agent, and administering the cultured cells to the subject.

20. The method of any one of claims 17 to 19, wherein the disease, disorder, or condition is osteoarthritis.

21. 1. A method of improving a surgical outcome in a subject, the method comprising administering to the subject at least one senolytic agent.

22. 22. The method of claim 21, wherein the senolytic agent is administered to a surgical site in the subject.

23. 1. A method of improving a surgical outcome in a subject, the method comprising removing stem cells from a subject, culturing the cells with at least one senolytic agent, and administering the cells to the subject.

24. 24. The method of claim 23, wherein the cultured cells are administered to a surgical site in a patient.

25. The method of any one of claims 21 to 24, wherein the surgical outcome is wound healing.

26. 25. The method of claim 22 or 24, wherein the surgical site is a wound.

27. 1. A method for removing senescent cells from an orthobiologic product, the method comprising adding at least one senolytic agent to the product.

28. The method of any one of claims 7 to 27, wherein at least one senolytic agent is fisetin.

29. 1. A pharmaceutical composition comprising at least one senolytic agent for use in the treatment of an age-related disease, disorder, or condition.

30. A pharmaceutical composition comprising at least one senescent cell for use in the treatment of a disease, disorder, or condition associated with aging.

31. 1. A pharmaceutical composition comprising at least one senolytic agent for use in improving surgical outcomes.

32. The composition of any one of claims 29 to 31, wherein at least one senolytic agent is fisetin.

33. A method of treating a subject having an aging-related condition or disease, comprising administering to the subject or measuring senescence in a tissue sample derived from the subject, and administering a senolytic agent to the subject.

34. 34. The method of claim 33, wherein the subject or tissue sample from the subject comprises at least about 4% senescent cells.

35. 35. The method of claim 33 or 34, wherein the senolytic agent is fisetin.

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