WO2025069029A1 - Methods for rejuvenating human cells - Google Patents

Abstract

Described herein is a method using small molecules to rejuvenate human cells without induction of pluripotency or otherwise changing cellular identity of the cells following the described methods.

Description

METHODS FOR REJUVENATING HUMAN CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Benefit is claimed to US Provisional Patent Application No. 63/585,975, filed September 28, 2023; and to US Provisional Patent Application No. 63/570,325, filed March 27 , 2024. The contents of the foregoing patent applications are incorporated by reference herein in their entirety.

FIELD

[0002] Provided herein is a method using small molecules to rejuvenate human cells without induction of pluripotency or otherwise changing cellular identity of the end product.

BACKGROUND

[0003] Aging is a universal biological phenomenon experienced by nearly all living organisms. It is characterized by a progressive decline in physiological functions, ultimately leading to various severe illnesses and mortality. This degenerative process fundamentally originates from the accumulation of molecular damage from the moment of birth, one key aspect of which is the dysregulation of DNA methylation, a type of epigenetic alteration.

[0004] During embryonic development, the epigenetic architecture of DNA plays a critical role in guiding cell fate and identity. However, as organisms age, this precisely regulated landscape undergoes significant changes across all cells, a process closely correlated with chronological age. This correlation has facilitated the development of so-called "epigenetic clocks." These tools can estimate the chronological age of any mammalian tissue sample with a considerable degree of accuracy based on its methylation landscape, as derived from methodologies such as Illumina methylation arrays.

[0005] Various in vitro and in vivo experiments involving mammalian subjects have demonstrated that recognized longevity treatments, including but not limited to calorie restriction, and treatment with Rapamycin or Metformin, can decelerate the progression of the epigenetic clock. This slowdown corresponds to an extension of both lifespan and healthspan. Strikingly, these epigenetic clocks were found to be more accurate predictors of mortality than chronological age itself.

[0006] The process of reprogramming cells from older donors (whether animals or humans) into induced pluripotent stem cells (iPSCs), by applying Yamanaka factors (a transcription factor cocktail that consist of Oct4, Sox2, Klf4 and c-Myc, or "OSKM") over several weeks, has been demonstrated to reprogram various aspects of cellular physiology. This transformation includes a full reversal of the epigenetic clock, essentially resetting it to zero.

[0007] Recent studies have revealed the potential for short-term exposure to Yamanaka factors ("partial epigenetic reprogramming") to partially reverse the epigenetic clock without altering cell identity. This partial reversal has been associated with a broad rejuvenation of cellular physiology and could have important clinical implications.

[0008] However, using OSKM factors for clinical rejuvenation is not without risks. These factors have oncogenic potential and can efficiently induce pluripotency in a somatic transform a cell's identity into that of an iPSC, which carries an inherent risk of inducing teratoma formation in vivo. Therefore, it is imperative to find a safer approach to leverage the potential benefits of partial epigenetic reprogramming and rejuvenation so as to insure that treated cells do not form iPSCs.

SUMMARY

[0009] Described herein is a partial epigenetic reprogramming protocol using small molecules, that can rejuvenate human somatic cells such as fibroblasts and mesenchymal cells, for example by 5- 20, such as by 12-15 years, without forming iPSCs or changing the identity of the end product cells following the described method. The described methods provide significant improvements over previously described methods that require additional or alternative factors to achieve cellular regeneration or rejuvenation (see e.g., US 2021/0213069, US Patent No. 11,674,122, and US 2021/0205369).

[0010] The described methods involve (a) culturing human somatic cells in a first cell culture media supplemented with effective amounts of a DNA methyltransferase inhibitor, a DOTH inhibitor, an ALK5 inhibitor, a GSK3P inhibitor, retinoic acid receptor ligand, a ROCK1 inhibitor, a Hedgehog signaling activator, a coenzyme or cofactor, an inhibitor of VEGFR and PDGFR, an inhibitor of JAK1 and JAK2 protein kinases, and a Menin-MLL interaction inhibitor; (b) Culturing the human somatic cells from (a) in a second cell culture media supplemented with effective amounts of an ALK5 inhibitor, a GSK3P inhibitor, a retinoic acid receptor ligand, a ROCK1 inhibitor, a Hedgehog signaling activator, a coenzyme or cofactor, an inhibitor of VEGFR and PDGFR, an inhibitor of JAK1 and JAK2 protein kinases, a Menin- MLL interaction inhibitor, a DNA methyltransferase inhibitor, a Lysine-specific demethylase 1 inhibitor, a BMP signaling inhibitor, a fibroblast growth factor, an inhibitor of JNK-1 and JNK-2, a G9a and GLP inhibitor, a p38 MAPK inhibitor, and an inhibitor of the bromodomain-containing transcription factors CREBBP (CBP) and EP300; and (c) culturing the human somatic cells from (b) in a third cell culture media without supplementation, wherein the third culture media is media used in the art for culturing the subject somatic cell type.

[0011] In particular embodiments, the human somatic cells are cultured in the first cell culture medium for up to 15-30 days.

[0012] In other embodiments, the human somatic cells are cultured in the second cell culture medium for 7-13 days. [0013] In still other embodiments, the human somatic cells are cultured in the third cell culture medium from 2-25 days, such as 2-15 days.

[0014] In still other embodiments, the human somatic cells are cultured in the first cell culture medium for 23 days, the second culture medium for 8 days, and the third cell culture medium for 9 days.

[0015] In particular embodiments, the DNA methyltransferase inhibitor of the first cell culture media is DZNep.

[0016] In other embodiments, the DOTH inhibitor of the first cell culture media is selected from EPZ004777 and SGC0946.

[0017] In some embodiments, ALK5 inhibitor of the first and second cell culture media is selected from RepSox (E616452) and GW788388.

[0018] In still other embodiments, the GSK3P inhibitor of the first and second cell culture media is selected from CHIR99021 and Kenpaullone.

[0019] In particular embodiments, the retinoic acid receptor ligand of the first and second cell culture media is TTNPB.

[0020] In other particular embodiments, the ROCK1 inhibitor of the first and second cell culture media is selected from Y-27632 and Thiazovivin (Tzv).

[0021] In certain embodiments, the Hedgehog signaling activator of the first and second cell culture media is selected from Smoothened agonist (SAG), Hh-Agl.5, and Purmorphamine (Purmo).

[0022] In further particular embodiments, the coenzyme or cofactor of the first and second cell culture media is Nicotinamide.

[0023] In particular embodiments, the inhibitor of VEGFR and PDGFR of the first and second cell culture media is ABT869.

[0024] In some embodiments, the inhibitor of the JAK1 and JAK2 protein kinases of the first and second cell culture media is ruxolitinib.

[0025] In further particular embodiments, Menin-MLL interaction inhibitor of the first and second culture cell media is VTP50469.

[0026] In certain embodiments, the DNA methyltransferase inhibitor of the second cell culture media is 5-aza-dC.

[0027] In other embodiments, the Lysine-specific demethylase 1 inhibitor of the second cell culture media is Tranylcypromine.

[0028] In particular embodiments, the BMP signaling inhibitor of the second cell culture media is selected from Dorsomorphin and LDN193189.

[0029] In still further embodiments, the inhibitor of JNK-1 and JNK-2 of the second cell culture media is JNKIN8. [0030] In particular embodiments, the p38 MAPK inhibitor of the second cell culture media is selected from BIRB796 and PD169316.

[0031] In other embodiments, the inhibitor of the bromodomain-containing transcription factors CREBBP (CBP) and EP300 of the second cell culture media is SGC-CBP30.

[0032] In some embodiments, the first cell culture medium is supplemented with DZNep, EPZ004777, RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, and VTP50469.

[0033] In other embodiments, the second cell culture medium is supplemented with RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, VTP50469 + 5-aza-dC, Tranylcypromine, Dorsomorphin, bFGF, JNKIN8, UNC0224, BIRB796, and SGC-CBP30.

[0034] In still other embodiments, the first cell culture medium is supplemented with DZNep, EPZ004777, RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, and VTP50469 and the second cell culture medium is supplemented with RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, VTP50469 + 5-aza-dC, Tranylcypromine, Dorsomorphin, bFGF, JNKIN8, UNC0224, BIRB796, and SGC-CBP30.

[0035] In some embodiments, the human somatic cells are cultured in the third cell culture medium for sufficient time to return the cells to their original morphology, original replication rate, and original transcriptomic profile.

[0036] In certain embodiments, rejuvenating human somatic cells, without changing cell identity or inducing formation of pluripotent stem cells, is indicated by a reduction in cellular epigenetic age, and wherein maintaining cellular identity between the original and treated cells can be confirmed for example by determining confirming significant similarity between the transcriptomes of the rejuvenated and original cells.

[0037] In other embodiments, the described methods include a further step (d), which includes determining the epigenetic age of the rejuvenated human somatic cells.

[0038] The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

[0039] Figs. 1A and IB show rejuvenation of human fibroblast cells using the described methods. Cellular epigenetic age in the Normal Human Dermal Fibroblast (NHDF) 64F cells that came from a 64 years old [Female] (Fig. 1A) and NHDF 68F cells that came from a 68 years old [Female] (Fig. IB) under different culture conditions was measured at different stages of the described methods. Epigenetic age was measured by the Horvath Multitissue and Horvath skin & blood epigenetic clocks. These results show' marked epigenetic rejuvenation for both cell types after complete treatment. Left panels: culturing conditions and resultant epigenetic age by both methods. Right panels are graphical representations of the left panel results. Darker datapoints represent the multitissue clock; and the lighter datapoints represent the skin Sblood clock.

[0040] Fig. 1C are micrographs of NHDF 64F and NHDF 68F cultures at the indicated stages of the described rejuvenation methods for the indicated number of days. Sl= stage 1 and Sz- stage 2. Throughout the displayed process and in the final cultures, no IPSC cultures were observed. Normal fibroblast morphology was observed in the final cultures that went through the entire three stage method (Sl(2.3d)+ S2(8d) + S3 (12d)).

[0041] Fig. ID is a CellNet analysis chart of the RNAseq-derived transcriptome of non-treated (left column) and Rejuvenated (Sl(23d) S2(8d) S3(10d)) (right column) NHDF 63F cells. These results show that the treated rejuvenated cells still kept their fibroblastic identity.

[0042] Figs. 2A, 2B, and 2C show that the enriched media and minimal media that are not supplemented with the described small molecules do not induce cellular rejuvenation. Cellular epigenetic age in NHDF 63F cells (Fig. 2A), NHDF 68F cells (Fig. 2B), and NHDF 78F cells (Fig. 2C) under different culture conditions was determined, with particular focus on control conditions using enriched media and minimal media. Epigenetic age was measured by the Horvath multitissue and Horvath skin & blood clocks. Left panels: culturing conditions. Right panels show the determined epigenetic age following the indicated conditions. Darker datapoints represent the multitissue clock; and the lighter datapoints represent the skin & blood clock.

[0043] Figs. 3A and 3B show the results of culturing cells under culture conditions according to Schoenfeldt at al. These conditions are not sufficient to rejuvenate these cells in terms of epigenetic clock reversal. Cellular epigenetic age in NHDF 63F cells (Fig. 3A) and NHDF 68F cells (Fig. 3B) was assayed following culture conditions as noted and as detailed in Example 1. Epigenetic age was measured by the Horvath multitissue and Horvath skin & blood clocks. Top panels: culturing conditions. Bottom panels show the determined epigenetic age following the indicated conditions. Darker datapoints represent the multitissue clock; and the lighter datapoints represent the skin & blood clock.

[0044] Figs. 4A and 4B show rejuvenation of human fibroblast cells using the described methods and verified by RNAseq transcriptomic analysis (Fig. 4A) and the Horvath skin and blood clock (Fig. 4B). Epigenetic and transcriptomic ages of the treated cells was compared with non-treated (NT) cells that were cultured in 10% FBS supplemented RPMI without rejuvenating chemical supplementation (left-hand bars in both figures).

[0045] Figs. 5A and 5B show rejuvenation of two mesenchymal stem cells (MSC) lines using the described methods and verified by the Horvath skin and blood clock. The change in epigenetic clock in comparison to cells that were cultured in standard MSC medium is shown for MSC 63 Fcells (Fig. 5A) and MSC 62F cells (Fig. 5B). [0046] Fig. 6 shows rejuvenation of two human chondrocyte (HCH) cell types using the described methods, and verified by the Horvath multitissue and Horvath skin and blood clocks. The change in epigenetic clock in comparison to the same cell strains that were cultured in standard chondrocyte medium is shown for 66F and 82M cells.

DETAILED DESCRIPTION

[0047] Terms

[0048] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all molecular weight or molecular mass values are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0049] Analog, derivative or mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. A derivative is a biologically active molecule derived from the base structure. A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound. It is acknowledged that these terms may overlap in some circumstances. The currently described methods utilize small molecules in a multi-step process of cellular rejuvenation. It will be appreciated that analogs, derivatives, and mimetics of the small molecules specifically mentioned herein are encompassed by this disclosure.

[0050] Biological age: Health status of an organism or cell, as can be determined by relevant biomarkers. Correlates with the expected remaining lifespan of an organism, or division potential for mitotic cells in tissue culture. Biological age correlates with chronological age, but organisms with the same chronological age can have different biological ages.

[0051] Cell culture medium or media: synthetic set of culture conditions with the nutrients necessary to support the growth of a specific population of cells. Growth media generally include a carbon source, a nitrogen source and a buffer to maintain pH. In one embodiment, growth medium contains a minimal essential media, such as Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) medium. Additionally, the minimal essential media may be supplemented with additives such as horse, calf or fetal bovine serum. In the present disclosure, the cell culture media is supplemented with specific small molecules to affect the disclosed method of rejuvenation.

[0052] Chronological age: The age of an organism as a function of time, for example weeks or years.

[0053] Contacting: Placement in direct physical association. Includes both in solid and liquid form. Contacting can occur in vitro with isolated cells or in vivo by administering to a subject.

[0054] Differentiation refers to the process whereby unspecialized cells (e.g., stem cells or pluripotent stem cells) acquire specialized structural and/or functional features characteristic of more mature cells. Similarly, "differentiate" refers to this process. Typically, during differentiation, cellular structure alters, and tissue-specific proteins appear.

[0055] Effective amount of a compound: A quantity of compound sufficient to achieve a desired effect.

[0056] Epigenetic age: An estimate of chronological and biological age as determined by an algorithm that takes into account methylation levels of specific CpG sites of the human genome. Multiple algorithms of measuring this "epigenetic clock" have been developed, all of which can be used to determine epigenetic rejuvenation of cells following the subject methods. Particular methodology for measuring the epigenetic clock and its correlation to chronological and biological age have been described by Horvath (see Genome Biology. 14 (10): R115. doi:10.1186/gb-2013-14-10-rll5, 2013).

[0057] Expand: refers to a process by which the number of cells in a cell culture is increased due to cell division.

[0058] Inhibitor: A molecule or compound that decreases or prevents the action of another molecule or process, or in some instances that blocks the ability of a given chemical to bind to its receptor or other interacting molecule, thereby preventing a biological response, for example a second messenger cascade, kinase activity, methylation, or gene expression. Inhibitors are not limited to a specific type of compound. The inhibitors of the current disclosure are small molecules.

[0059] Isolated: A biological component, such as a cell or tissue that has been substantially separated or purified away from other biological components in the organism in which the component naturally occurs, i.e., other cells and tissues.

[0060] Rejuvenation (of a cell): Decreasing the biological age of a cell. Cellular rejuvenation can be determined by, among other indications, decreased epigenetic age of a cell, as measured by epigenetic clock algorithms such as the Horvath epigenetic clock. Rejuvenation of cells can also be determined by transcriptomic profiling of the cells prior to and following the rejuvenation process. While it is possible to decrease the epigenetic age of a cell to 0, prior methods that achieve this simultaneously also changed cellular identity to be iPSCs. In contrast, the methods described herein rejuvenate and decrease the epigenetic age of somatic cells by several years (e.g., 5-20 years) without changing the identity of the somatic cell at the completion of the method and without reducing the epigenetic age of the treated cell to 0. Accordingly, for example, after the described methods, a treated fibroblast that originally has an epigenetic age of 65 can be rejuvenated to be a fibroblast with an epigenetic age of 53.

[0061] Small Molecules: Chemical compounds having a molecular weight within a range of 50- 1,500 Daltons (see ebi.ac.uk/training/online/courses/metabolomics-introduction/what-is/small- molecules/).

[0062] Stem cell, Pluripotent Stem cell: A stem cell refers to a cell that can differentiate into more than one given cell type. A pluripotent stem cell naturally exists in the blastocyst embryo state, and can differentiate into all of the cells of the human body. An induced pluripotent stem cell (iPSC) has the properties of a pluripotent stem cell, but has been induced ex vivo from a differentiated cell by a process of cellular reprogramming. In contrast to differentiated cells that are derived from a subject of a specified age, such as differentiated somatic cells, the determined epigenetic age of an iPSC is at or near zero. For example, a fibroblast derived from a 60-year-old human may have an epigenetic age of around 60 according to the Horvath skin &blood clock, while iPSCs derived from the 60-year old's fibroblast will have an epigenetic age of 0.

[0063] In case of conflict, the present specification, including explanations of terms, will control. In addition, all the materials, methods, and examples are illustrative and not intended to be limiting.

[0064] Methods for Rejuvenating Somatic Cells with Small Molecules

[0065] Described herein is a method for rejuvenating isolated somatic cells in culture, but which does not change cell identity of the end-product cells or induce formation of pluripotent stem cells. The described method includes three stages of culturing the somatic cells in three distinct cell culture media, two of which are supplemented with small molecules that together rejuvenate somatic cells as evidenced by decreased cellular epigenetic age.

[0066] In the described method, in a first stage, isolated somatic cells are cultured for up to 15- 30 days in cell culture media supplemented with effective amounts of a DNA methyltransferase inhibitor, a DOTH inhibitor, a ALK5 inhibitor, a GSK3P inhibitor, retinoic acid receptor ligand, a ROCK1 inhibitor, a Hedgehog signaling activator, a coenzyme or cofactor, an inhibitor of VEGFR and PDGFR, an inhibitor of JAK1 and JAK2 protein kinases, and a Menin-MLL interaction inhibitor.

[0067] In a second stage, the cells from the first stage are then cultured for 7-13 days in cell culture media supplemented with effective amounts of a ALK5 inhibitor, a GSK3P inhibitor, a retinoic acid receptor ligand, a ROCK1 inhibitor, a Hedgehog signaling activator, a coenzyme or cofactor, an inhibitor of VEGFR and PDGFR, an inhibitor of JAK1 and JAK2 protein kinases, a Menin-MLL interaction inhibitor, a DNA methyltransferase inhibitor, a Lysine-specific demethylase 1 inhibitor, a BMP signaling inhibitor, fibroblast growth factor, an inhibitor of JNK-1 and JNK-2, a G9a and GLP inhibitor, a p38 MAPK inhibitor, and an inhibitor of the bromodomain-containing transcription factors CREBBP (CBP) and EP300.

[0068] In a third stage, the cells from the second stage are cultured for at least 2-10 days in standard cell culture media for supporting growth of the specific somatic cell type and allowing maintaining of the original cell identity.

[0069] Following the described three stage method, the epigenetic age of the cells will be reduced by about 5-20 years, for example by about 5, 10, 15, 20 years and all increments in between, as determined by standard measurements of the cellular epigenetic clock (e.g., the Horvath multitissue or skin&blood methods, see below). However, following the described method, the epigenetic age of the resultant cells will not indicate induction of pluripotency, and morphologically the cell type will not have changed at the end of the process, as can be seen for example by its transcriptomic pattern. For example, fibroblasts will remain fibroblasts, though with reduced epigenetic age.

[0070] Cell culture media for use in the described methods is standard, but in particular embodiments is tailored according to the particular somatic cell type being cultured. As indicated, the small molecule additives for use in the described methods are defined by their biological function. It will be appreciated therefore that for each of the additives, multiple variations are possible, all of which are encompassed by this disclosure.

[0071] Non-limiting examples of the DNA methyltransferase inhibitor for use in the described methods include 3-Deazaneplanocin A (DZNep); Azacitidine (5-aza-dC); (-) Neplanocin A (NepA; 5R- (6- amino-9H-purin-9-yl) -3- (hydroxymethyl) -3-cyclopentene-lS, 2 R-diol); Adenozine periodate oxidized ((Adox) [ (2S) -2- [ (1R) -1- (6-aminopurin-9-yl) -2-oxoethoxy] -3-hydroxypropanal); and 3- deazaadenosine (DZA) (1-p-D-ribofuranosyl-lH-imidazo [4, 5-c] pyridin-4-amine).

[0072] Non-limiting examples of the disruptor of telomeric silencing 1-like (DOTH) inhibitor for use in the described methods include EPZ004777 (7- [5-Deoxy-5-3-4-l, 1-dimethylethyl) phenyl] amino] carbonyl] amino] propyl] (1-methylethyl) amino] -p-D-ribofuranosyl] -7H-pyrrolo [2, 3-d] pyrimidineamine); SGC0946 (l-3-(2R, 3S, 4R, 5R) -5- (4-Amino-5-bromo-7H-pyrrolo [2, 3-d] pyrimidin-7-yl) -3, 4- dihydroxytetrahydrofuran-2-yl] methyl] (isopropyl) amino] propyl] -3- [4- (2, 2- dimethylethyl) phenyl] urea); and EPZ5676 ((2R, 3R, 4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- (lr, 3S) -3- (2- (5- (tert-butyl) -1H- benzo [d] imidazol-2-yl) ethyl) cyclobutyl) (isopropyl) amino) methyl) tetrahydrofuran-3, 4-diol).

[0073] Non-limiting examples of the ALK5 (transforming growth factor (TGF)-P) inhibitor for use in the described methods include RepSox (E616452; 2- (3- (6-Methylpyridin-2-yl) -lH-pyrazol-4-yl) -1, 5- naphthyridine); GW788388 (4- [4- [3- (2-Pyridinyl) -lH-pyrazol-4-yl] -2-pyridinyl] -N- (tetrahydro-2H- pyran-4-yl) -benzamide); SB 525334 (6- [2- (1, 1-Dimethylethyl) -5- (6-methyl-2-pyridinyl) -lH-imidazol-4- yl] quinoxaline); A 83-01 (3- (6-Methyl-2-pyridinyl) -N-phenyl-4- (4-quinolinyl) -lH-pyrazole-1- carbothioamide); SB 505124 (2- [4- (1, 3-Benzodioxol-5-yl) -2- (1, 1-dimethylethyl) -lH-imidazol-5-yl] -6- methyl-pyridine); and dorsomorphine.

[0074] Non-limiting examples of the glycogen synthesis kinase p (GSK3P) inhibitor for use in the described methods include CHIR99021 (6- 2- 4- (2, 4-Dichlorophenyl) -5- (5-methyl-lH-imidazol-2-yl) -2- pyrimidinyl] amino] ethyl] amino] -3-pyridinecarbonitrile]); Kenpaullone (9- Bromopaullone); BIO- acetoxime; SB-216763 (3- (2, 4-Dichlorophenyl) -4- (l-methyl-lH-indol-3-yl) -lH-pyrrole-2, 5-dione); CHIR99021 (trihydrochloride); GSK3 Inhibitor IX (( (2Z, 3E) -6'-bromo-3- (hydroxyimino) - [2, 3'- biindolinylidene] -2'-one); GSK 3 IX (6-Bromoindirubin-3'-oxime); GSK-3P Inhibitor XII (3- 6- (3- Aminophenyl) -7H-pyrrolo [2, 3-d] pyrimidin-4-yl] oxy] phenol); GSK-3 Inhibitor XVI (6- (2- (4- (2, 4- dichlorophenyl) -5- (4-methyl-lH-imidazol-2-yl) -pyrimidin-2-ylamino) ethyl-amino) -nicotinonitrile); SB- 415286 (3- [ (3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1 H-pyrrole-2, 5-dione); Bio ((2'Z, 3'E) -6-bromoindirubin-3'-oxime); and TD114-2 (6, 7, 9, 10, 12, 13, 15, 16, 18, 19-Decahydro-5, 29: 20, 25-dimetheno-26H-dibenzo [n, t] pyrrolo [3, 4-q] [1, 4, 7, 10, 13, 22] tetraoxadiazacyclote tracosine-26, 28 (27H) -dione).

[0075] Non-limiting examples of the retinoic acid receptor ligand for use in the described methods include TTNPB (4- [ (E) -2- (5, 6, 7, 8-Tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenyl) -1- propenyl] benzoic acid); Ch 55 (4- [ (IE) -3- [3, 5-bis (1, 1-Dimethylethyl) phenyl] -3-oxo-l-propenyl] benzoic acid); and AM580 (4- [ (5, 6, 7, 8-Tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenyl) carboxamido] benzoic acid],

[0076] Non-limiting examples of the Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) inhibitor for use in the described methods include Y-27632 ( [ (+) - (R) -trans-4- (1-aminoethyl) - N- (4-pyridyl) cyclohexanecarboxamide+++dihydrochloride) and Thiazovivin (Tzv).

[0077] Non-limiting examples of the Hedgehog signaling activator for use in the described methods include Smoothened agonist (SAG); Hh-Agl.5; and Purmorphamine (Purmo).

[0078] A non-limiting example of the cofactor or coenzyme for use in the described methods includes Nicotinamide.

[0079] Non-limiting examples of the inhibitor of vascular endothelial growth factor receptor (VEGFR) and platelet derived growth factor receptor (PDGFR) for use in the described methods include ABT 869 (Linifanib; N- [4- (3-amino-lH-indazol-4-yl) phenyl] -N'- (2-fluoro-5-methylphenyl) -urea); AG1296 (6, 7-Dimethoxy-3-phenylquinoxaline); and Valatanib.

[0080] A non-limiting example of the inhibitor of the Janus Kinase 1 (JAK1) and Janus Kinase 2

(JAK2) for use in the described methods includes ruxolitinib. [0081] Non-limiting examples of the Menin-MLL interaction inhibitor include VTP50469 (CAS #: 2169916-18-9), MI3454 (CAS #: 2134169-43-8), or WDR5-IN-4 (CAS #: 2407457-36-5).

[0082] A non-limiting example of the Lysine-specific demethylase 1 inhibitor includes Tranylcypromine.

[0083] Non-limiting examples of the BMP signaling inhibitor for use in the described methods include Dorsomorphin and LDN193189.

[0084] Non-limiting examples of the c-Jun N-terminal kinase-1 (JNK-1) and c-Jun N-terminal kinase-2 (JNK-2) inhibitor for use in the described methods includes JNKIN8; Sp600125; JNK-in-5; JNK-in- 7; and JNK-in-12.

[0085] A non-limiting example of the G9a and glucagon like peptide (GLP) inhibitor includes UNC0224.

[0086] Non-limiting examples of the p38 mitogen activated protein kinase (MAPK) inhibitor for use in the described methods include BIRB796; PD169316; AZD8330; and TAK-733.

[0087] A non-limiting example of the inhibitor of the bromodomain-containing transcription factors CREBBP (CBP) and EP300 for use in the described methods includes SGC-CBP30.

[0088] In particular embodiments of the described methods, the first cell culture medium is supplemented with effective amounts of DZNep, EPZ004777, RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, and VTP50469.

[0089] In other particular embodiments of the described methods, the second cell culture medium is supplemented with effective amounts of RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, VTP50469 + 5-aza-dC, Tranylcypromine, Dorsomorphin, bFGF, JNKIN8, UNC0224, BIRB796, and SGC-CBP30.

[0090] In a still further embodiment, the first cell culture medium is supplemented with effective amounts of DZNep, EPZ004777, RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, and VTP50469; and the second cell culture medium is supplemented with effective amounts of RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, VTP50469 + 5-aza-dC, Tranylcypromine, Dorsomorphin, bFGF, JNKIN8, UNC0224, BIRB796, and SGC-CBP30.

[0091] The described methods involve a sequence of cell culture stages, each with a defined culture medium, and each of which having a specific duration. In the first stage, in which the somatic cells are incubated in a first small molecule-supplemented cell culture medium, the cells are incubated for 15-30 days, such as 20-25 days or 21-23 days, and including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 days, and increments in between. In the second stage, in which the cells are incubated in a second small molecule-supplemented cell culture medium, the cells are incubated for 7- 13 days, including 7, 8, 9, 10, 11, 12, and 13 days, and increments in between. In the third stage, the cells are cultured in a third culture medium, which is a minimal media cell culture commonly used for the specific somatic cell type, for example FBS-supplemented RPML In this stage, the cells are incubated for at least 2-10 days, including at least 2, 3, 4, 5, 6, 7, 8, 9, and 10 days, and increments in between. In a particular embodiment of the described methods, the somatic cells are incubated in the first cell culture medium for 23 days, the second cell culture medium for 8 days, and the third cell culture medium for 9 days.

[0092] As noted, the described methods rejuvenate cells without changing cellular identity or inducing formation of pluripotent stem cells, such that by the completion of the described rejuvenation method, while the epigenetic clock of the cells has been decreased by several years (e.g., 5-20 years), cellular somatic identity is unchanged. Rejuvenation of cells and the retention of original cellular identity can be verified in several ways.

[0093] One method to determine cellular identity after processing the cells in the described methods is through observation of cellular morphology. For example, iPSC colonies possess a morphology that is distinct from that of normal human fibroblasts.

[0094] Similarly, somatic cells possess distinct biochemical identities which can be assayed to distinguish a somatic cell from an iPSC or from another type of somatic cell. In a particular embodiment, following the described method, a sample of the processed cells can be assayed for expressed mRNA. Subsets of expressed RNA or even the entire cellular transcriptome can be determined by methods known to the art, for example RNA-seq methodology to sequence the entire cellular transcriptome, and thereby distinguish one cell type from another by way of the expressed RNA. In another embodiment, cells can be assayed for expression of particular cell surface receptors that are specific to the particular somatic cell type, for example, by immunocytochemistry or immunofluorescence. Cells can also be sorted by standard methodologies such as FACS.

[0095] Additionally, it was observed that during the small molecule-supplemented stages of the described methods, the subject cells divide at a slower replication rate than that of the normal subject somatic cells. However, with the completion of stage three of the described methods, the rejuvenated cells will return to a typical replication rate for the particular cells. Accordingly, in particular embodiments, monitoring of cell division rate can, and determination of a return to a "normal" rate can further support acquisition of original cell identity.

[0096] In a further embodiment, rejuvenation of somatic cells is determined by measurement of telomere length. Telomere length is a well-known hallmark of cellular senescence and organismal aging, therefore the telomere length of chemically rejuvenated cells can be measured in comparison to original untreated cells to determine cellular rejuvenation. Telomere length can be measured in one non-limiting example by ddPCR using telomere specific primers.

[0097] Any isolated human somatic cell, derived from any tissue and any organ, can be the object of the described method. Somatic cells are all cells of a multicellular organism except for gametes. Particular non-limiting sources of human somatic cells that can be the object of the described methods include cells that are isolated from bone marrow, peripheral blood, umbilical cord blood, muscle, connective tissue, cartilage, organs such as but not limited to pancreas, liver, kidney, and skin. In particular embodiments, the somatic cells are skin derived cells such as fibroblasts, are of hematological origin including cells of the immune system, including T cells, B cells, and macrophages, adipose cells, epithelial cells, endothelial cells, mesenchymal-derived cells, parenchymal cells (for example, hepatocytes), neurological cells, and connective tissue cells. Among the foregoing cells are multipotent stem cells (for example, but not limited to, hematopoietic stem cells, mesenchymal stem cells, chondroblasts (e.g., chondrocytes), mammary stem cells, endothelial stem cells, intestinal stem cells, olfactory stem cells, neural stem cells, testicular cells, and neural crest stem cells) which while able to further differentiate into fully differentiated or unipotent cells, are not totipotent in the way of iPSCs.

[0098] The rejuvenation methods described herein reverse the epigenetic clock of somatic cells, such as by 5-20 years. This is in contrast to prior-described methods (see International Patent Publication Nos. WO2022213731A1 and WO2017091943A1) which reprogram somatic cells into induced pluripotent stem cells. It will be appreciated that induction of pluripotency (i.e., changing the identity of a somatic cell to an induced pluripotent stem cell), results in a complete resetting of the epigenetic clock to at or near zero. In contrast, the methods described herein decrease cellular epigenetic age, while maintaining cellular identity and without inducing pluripotent stem cells. In particular embodiments, epigenetic age is reduced by about 5-20 years, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or increments in between.

[0099] Somatic cells that have been rejuvenated by the described methods can be used in therapeutic and cosmetic autologous cell therapies. In such applications of the described methods, somatic cells are isolated from a subject to be treated, rejuvenated according to the described methods, expanded, and then used as needed for the particular therapeutic or cosmetic application. It will be appreciated that the downstream applications of the currently described methods are not limited in any way other than the ability to isolate a particular cell type, rejuvenate the cells according to the described method, and then use of the rejuvenated cells in the particular application.

[0100] The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

[0101] Example 1: Rejuvenation of Human Somatic Cells With Small Molecules

[0102] This example demonstrates the described method for rejuvenation of somatic cells with small molecules added to the cell culture media. Also described is the inability of a previously published method to decrease the epigenetic age of treated human somatic cells without changing cellular identity in the final product.

[0103] Fibroblast primary NHDF 63F, NHDF 64F, NHDF 78F, and NHDF 68F cells, obtained from dermal layer were used throughout the examples described herein. To test the described methods of cellular rejuvenation, the NHDF 63F, NHDF 64F, NHDF 78F, and NHDF 68F fibroblasts were cultured as follows.

[0104] In a first culturing stage, the fibroblasts that had been previously maintained in a minimal media were cultured in a first culture media (enriched media without small molecules) supplemented with 0.02pM DZNep (Cayman Chemical), 2 pM EPZ004777 (Cayman Chemical), 10 pM RepSox (Cayman Chemical), 10 pM CHIR99021 (Cayman Chemical), 2 pM TTNPB (Peprotech), 2 pM Y- 27632 (Adooq Bioscience), 0.5 pM SAG (Cayman Chemical), 1 mM Nicotinamide (Merck), 1 pM ABT869 (Cayman Chemical), 1 pM ruxolitinib (Cayman Chemical), and 0.5 pM VTP50469 (MedChem Express). Cells were cultured under 5% oxygen, but otherwise standard conditions, in the described supplemented media for 7, 14, and 23 days. Control cultures were cultured in the first medium without supplementation. In a second culturing stage, cultures that had been incubated for 23 days were then cultured in new enriched media without small molecules that was supplemented with RepSox, CHIR99021, TTNPB, Y-27632, SAG, Nicotinamide, ABT869, ruxolitinib, VTP50469 as in the first culturing stage + 10 pM 5-aza-dC (Cayman Chemical), 2 pM Tranylcypromine (Peprotech), 0.5 pM Dorsomorphin (Cayman Chemical), lOOng/ml bFGF (Peprotech), 1 pM JNKIN8 (Cayman Chemical), 1 pM UNC0224 (Cayman Chemical), 2 pM BIRB796 (MedChemExpress), and 2 pM SGC-CBP30 Cayman Chemical. In this second stage, cells were cultured under atmospheric (21%) oxygen, but otherwise standard conditions. After 8 days in the second stage culture, the cells were cultured for 9 days in minimal medium under 5% oxygen, but otherwise standard conditions. Following the third stage of the culturing series, cellular morphology was observed. Epigenetic age of the cells at various stages of the noted method was determined by the Horvath multitissue and blood&skin methods (Horvath et al., Aging 10(7):1758-1775, 2018). Additionally, cellular identity was verified by RNAseq sequencing with CellNet analysis (Cahan et al., Cell 158(4):903-915, 2014).

[0105] As indicated in Fig. 1A and Fig. IB, the measured epigenetic age of fibroblasts from both cell types was not significantly changed in all culture conditions, except for those cells which were cultured in all three stages of the noted method. Fig. 1A shows that there was a reduction in epigenetic age only in Sample 5 in comparison to the other cells tested. Similarly, Fig. IB shows that there was a reduction in epigenetic age in Sample 6 in comparison to the other cells tested. The measured reduction in epigenetic age was consistent using both Horvath epigenetic clock algorithms, and indicates rejuvenation of cells. Significantly, the described method did not reduce the measured epigenetic age to 0, which would have been the expected outcome if the cells had been reprogrammed to iPSC. Following the rejuvenation method, the observed morphology of the rejuvenated cells was of normal fibroblasts and without formation of iPSC (Fig. 1C). This observation was biochemically confirmed by way of RNAseq sequencing and CellNet analysis. The CellNet analysis is an algorithm based on RNAseq, developed to compare similarity between cells by analysis of the expression of key genes. In this way it is possible to give a score to each tested cell as to how similar it is to given cell. As shown in Fig. ID, CellNet analysis verifies that following the described method, the transcriptome of the resultant cells are fibroblasts (predominant bar in right column), similar to the transcriptome of non-treated cells (predominant bar in left column).

[0106] To confirm that enriched media without small molecules or minimal media alone, without small molecules, is insufficient to produce cellular rejuvenation, NHDF 63F, NHDF 68F, and NHDF 78F were cultured as described as follows. The results of this experiment are shown in Fig. 2A for NHDF 63F, Fig. 2B for NHDF 68F, and Fig 2C for NHDF 78F. Fig. 2A shows the results of culturing NHDF 63F fibroblasts in 1: minimal media after a 4-day incubation period; 2: minimal media after a 14-day incubation period, but with a second passage; 3: enriched media after a 4-day incubation period; and 4: The 3-stage rejuvenation method described above (including small molecules). Similarly, but using NHDF 68F fibroblasts, Fig. 2B shows the results of culturing the cells in 1: minimal media after a 4-day incubation period; 2: enriched media without small molecules after a 4 day incubation period; and 3: The 3-stage rejuvenation method described above. , Fig. 2C shows the results of culturing the cells in 1: enriched media without small molecules after a 7 day incubation period; 2: minimal media after a 7 day incubation period; and 3: The 3-stage rejuvenation method described above. As shown in the right panels of Figs. 2A, 2B and 2C, the culture condition that demonstrated a rejuvenating effect on epigenetic age was the described three-stage method. None of the other conditions tested had a significant effect. Therefore, it can be concluded that the rejuvenation observed is the result of the small molecules, as described.

[0107] In previously published experiments, Schoenfeldt et al. (bioRxiv, 10.1101/2022.08.29.505222, available online at biorxiv.org/content/early/2022/08/31/2022.08.29.505222) reported that culturing cells for only 6 days in a small subset of the small molecule additives used in the above methods was sufficient to ameliorate some forms of cellular damage that accompanies aging, yet they did not examine the effect on the epigenetic clock. Schoenfeldt et al. indicated that a combination of DZNep, RepSox, CHIR99021, TTNPB, Tranylcypromine, and Valproic Acid (referred to as "7C" in Figs. 3A and 3B) or RepSox and Tranylcypromine (referred to as "2C" in Figs. 3A and 3B) was sufficient to have the reported effect. To test whether this published protocol can reverse the epigenetic age of somatic cells, NHDF 63F and NHDF 68F fibroblasts were cultured in enriched media without small molecules or minimal media for 6 or 12 days with and without the 2C and 7C small molecule combinations, according to the authors' instructions. The specific conditions are indicated in Figs. 3A and 3B, top panels. As shown in Figs. 3A and 3B, bottom panels, none of the tested conditions had an effect on cellular epigenetic age similar to that demonstrated with the three-stage method described herein.

[0108] Example 2: Verification of Human Somatic Cell Rejuvenation

[0109] As demonstrated in Example 1, the three-stage method described herein is capable of rejuvenating human somatic cells, while not inducing formation of iPSCs or otherwise changing cellular identity. Example 1 demonstrated the reduction in epigenetic age of fibroblasts cultured for defined periods of time and with specific combinations of small molecules. To further or alternatively verify that a cell treated by the described method has been rejuvenated, but not reprogrammed into IPSC or have an alternative cellular identity by the end of the process, cells in culture that have been treated by the described methods are further assayed for morphology (such as shown in Fig. 1C), rates of cell division, and/or gene expression (such as shown in Fig. ID).

[0110] Pluripotent stem cells possess a cellular morphology that is clearly distinguishable from differentiated somatic cells. Accordingly, following the described methods, the morphology of cells that are rejuvenated are verified. Similarly the rate of cell division of a somatic cell prior to, during, and following the described method is assayed. Following the final cell culture step, in media without added small molecules, the rate of cell division will return to that which is typical for the specific somatic cell type.

[0111] Lastly, in addition to assays of the cellular epigenetic clock, rejuvenation is verified by determination of the cellular transcriptomic profile (for example as shown in Fig. ID). Following the third step of the method in which the cells are cultured in medium without small molecule additives (e.g., RPMI + 10% FBS), the cellular transcriptome is verified by the RNA-seq technique. The mRNA of the cells is sequenced and compared to known values of mRNA expression in the particular somatic cell type, for example in fibroblasts. Following the described rejuvenation method, cellular mRNA expression is expected to return to that of the specific somatic cell type.

[0112] Example 3: Confirmation of Cellular Rejuvenation in Fibroblasts by Transcriptomic Profiling

[0113] This example demonstrates that rejuvenation of fibroblasts by the described methods can also be detected and measured by transcriptomic profiling.

[0114] NHDF 63F fibroblast cells were cultured as described in Example 1. In particular, cells were cultured in the first culture media for 22 days, followed by culturing in the second culture media for 8 days. Lastly, the cells were cultured in 10% FBS supplemented RPMI for a further 10 days, before EPIC and RNAseq processing, to determine epigenetic age by the Horvath Skin and Blood method (EPIC) and age by the transcriptomic profile (RNAseq). The results of these analyses are shown in Figs. 4B and 4A. respectively.

[0115] As shown in Fig. 4B, cells which were incubated only in 10% FBS-RPMI for 36 days (NT, left bar) were determined to have an epigenetic age of 73.77 years. In contrast, cells that were rejuvenated by the described methods were determined to have an epigenetic age of 59.04 years. Therefore, the total calculated rejuvenation was 14.73 years. Similarly, and as shown in Fig. 4A, when the transcriptomic clock was calculated from the RNAseq result, the cells were determined to have rejuvenated about 13.5 years compared to NT.

[0116] Based on these results it is possible to conclude that using the described supplemented cell culture method, the rejuvenation of cultured fibroblasts can be detected both through measurement of the epigenetic clock and the transcriptomic clock.

[0117] Example 4: Cellular Rejuvenation of Mesenchymal Stem Cells and Chondrocyte Cells

[0118] This example shows that the described methods of cellular rejuvenation that were demonstrated to be effective for fibroblasts can also effectively rejuvenate mesenchymal stem cells (MSC) and human chondrocyte cells (HCH).

[0119] Two MSC cell types, 63F and 62F were cultured as described in Example 1. In particular, both 63F and 62F cells were cultured in the first culture media for 21 days, followed by culturing in the second culture media for 8 days. Lastly, the cells were cultured in 20% FBS supplemented Mem-Alpha MSC medium for a further 9 days (62F) or 12 days (63F), before EPIC processing to determine epigenetic age by the Horvath Skin and Blood method. Calculations of epigenetic age are shown in Fig. 5A (63F) and Fig. 5B (62F) in comparison to non-treated (NT) cells that were cultured only in 20% Mem-Alpha.

[0120] As shown in Fig. 5A, MSC 63F cells that were cultured according to the described method were determined to have been rejuvenated by 9.79 years in comparison to NT cells. Similarly, as shown in Fig. 5B, MSC 62F cells that were cultured according to the described method were determined to have been rejuvenated by 15.28 years in comparison to NT cells.

[0121] Based on these results it is possible to conclude that using the described supplemented cell culture method, mesenchymal stem cells can be rejuvenated.

[0122] Similarly, two HCH cell types, 66M and 82F were cultured as described in Example 1. In particular, both 66M and 82F cells were cultured in the first culture media for 15 days, followed by culturing in the second culture media for 7 days. Lastly, the cells were cultured in Chondrocyte growth medium (purchased from PromoCell, C-27101) medium for a further 23 days, before EPIC processing to determine epigenetic age by two epigenetic clock algorithms. Calculations of epigenetic age are shown in Fig. 6 as indicated in comparison to non-treated (NT) cells that were cultured only in Chondrocyte growth medium (purchased from PromoCell, C-27101). [0123] As shown in Fig. 6, HCH 66F cells that were cultured according to the described method were determined to have been rejuvenated by about 4 years according to Horvath multitissue algorithm in comparison to non-treated cells. Similarly, HCH 82F cells that were cultured according to the described method were determined to have been rejuvenated about 8 years in comparison to NT cells.

[0124] Based on these results it is possible to conclude that using the described supplemented cell culture method, chondrocytes can be rejuvenated.

[0125] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

Claims

1. A method for rejuvenating human somatic cells without changing cell identity or inducing formation of pluripotent stem cells, the method comprising:

(a) Culturing human somatic cells in a first cell culture media supplemented with effective amounts of a DNA methyltransferase inhibitor, a DOTH inhibitor, a ALK5 inhibitor, a GSK3P inhibitor, retinoic acid receptor ligand, a ROCK1 inhibitor, a Hedgehog signaling activator, a coenzyme or cofactor, an inhibitor of VEGFR and PDGFR, an inhibitor of JAK1 and JAK2 protein kinases, and a Menin-MLL interaction inhibitor;

(b) Culturing the human somatic cells from (a) in a second cell culture media supplemented with effective amounts of a ALK5 inhibitor, a GSK3P inhibitor, a retinoic acid receptor ligand, a ROCK1 inhibitor, a Hedgehog signaling activator, a coenzyme or cofactor, an inhibitor of VEGFR and PDGFR, an inhibitor of JAK1 and JAK2 protein kinases, a Menin-MLL interaction inhibitor, a DNA methyltransferase inhibitor, a Lysine-specific demethylase 1 inhibitor, a BMP signaling inhibitor, fibroblast growth factor, an inhibitor of JNK-1 and JNK-2, a G9a and GLP inhibitor, a p38 MAPK inhibitor, and an inhibitor of the bromodomain-containing transcription factors CREBBP (CBP) and EP300; and

(c) Culturing the human somatic cells from (b) in a third cell culture media without supplementation; thereby rejuvenating human somatic cells, without changing cell identity or inducing formation of pluripotent stem cells.

2. The method of claim 1, wherein the human somatic cells are cultured in the first cell culture medium for 15-30 days.

3. The method of claim 1 or claim 2, wherein the human somatic cells are cultured in the second cell culture medium for 7-13 days.

4. The method of any one of claims 1-3, wherein the human somatic cells are cultured in the third cell culture medium from 2-25 days.

5. The method of any one of claims 1-4, wherein the DNA methyltransferase inhibitor of the first cell culture media is DZNep.

6. The method of any one of claims 1-5, wherein the DOTIL inhibitor of the first cell culture media is selected from EPZ004777 and SGC0946.

7. The method of any one of claims 1-6, wherein the ALK5 inhibitor of the first and second cell culture media is selected from RepSox (E616452) and GW788388.

8. The method of any one of claims 1-7, wherein the GSK3P inhibitor of the first and second cell culture media is selected from CHIR99021 and Kenpaullone.

9. The method of any one of claims 1-8, wherein the retinoic acid receptor ligand of the first and second cell culture media is TTNPB.

10. The method of any one of claims 1-9, wherein the ROCK1 inhibitor of the first and second cell culture media is selected from Y-27632 and Thiazovivin (Tzv).

11. The method of any one of claims 1-10, wherein the Hedgehog signaling activator of the first and second cell culture media is selected from Smoothened agonist (SAG), Hh-Agl.5, and Purmorphamine (Purmo).

12. The method of any one of claims 1-11, wherein the coenzyme or cofactor of the first and second cell culture media is Nicotinamide.

13. The method of any one of claims 1-12, wherein the inhibitor of VEGFR and PDGFR of the first and second cell culture media is ABT869.

14. The method of any one of claims 1-13, wherein the inhibitor of the JAK1 and JAK2 protein kinases of the first and second cell culture media is ruxolitinib.

15. The method of any one of claims 1-14, wherein the Menin-MLL interaction inhibitor of the first and second culture cell media is VTP50469.

16. The method of any one of claims 1-15, wherein the first cell culture medium is supplemented with DZNep, EPZ004777, RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, and VTP50469.

17. The method of any one of claims 1-16, wherein the DNA methyltransferase inhibitor of the second cell culture media is 5-aza-dC.

18. The method of any one of claims 1-17, wherein the Lysine-specific demethylase 1 inhibitor of the second cell culture media is Tranylcypromine.

19. The method of any one of claims 1-18, wherein the BMP signaling inhibitor of the second cell culture media is selected from Dorsomorphin and LDN193189.

20. The method of any one of claims 1-19, wherein the inhibitor of JNK-1 and JNK-2 of the second cell culture media is JNKIN8.

21. The method of any one of claims 1-20, wherein the G9a and GLP inhibitor of the second culture cell media is UNC0224.

22. The method of any one of claims 1-21, wherein the p38 MAPK inhibitor of the second cell culture media is selected from BIRB796 and PD169316.

23. The method of any one of claims 1-22, wherein the inhibitor of the bromodomaincontaining transcription factors CREBBP (CBP) and EP300 of the second cell culture media is SGC-CBP30.

24. The method of any one of claims 1-23, wherein the second cell culture medium is supplemented with RepSox, CHIR99021, TTNPB, Y-27632, SAG, ABT869, ruxolitinib, VTP50469 + 5-aza- dC, Tranylcypromine, Dorsomorphin, bFGF, JNKIN8, UNC0224, BIRB796, and SGC-CBP30.

25. The method of any one of claims 1-24, wherein the human somatic cells are cultured in the third cell culture medium for sufficient time to return the cells to their original morphology, normal replication rate, and normal transcriptomic profile.

26. The method of any one of claims 1-25, wherein rejuvenating human somatic cells, without changing cell identity or inducing formation of pluripotent stem cells is indicated by a reduction in cellular epigenetic age or by cellular transcriptomic profiling.

27. The method of any one of claims 1-26, further comprising (d) determining the epigenetic age or the transcriptomic profiling of the rejuvenated human somatic cells.

28. The method of any one of claims 1-27, wherein the human somatic cells are fibroblast, mesenchymal, or chondrocyte cells.