Transient cellular reprogramming for reversal of cell aging

Abstract

To provide methods and compositions useful in cellular rejuvenation, tissue engineering, and regenerative medicine.SOLUTION: Compositions and methods for rejuvenating aged cells and tissues to restore functionality are disclosed. In particular, cells are rejuvenated by transient exposure to non-integrated mRNAs encoding reprogramming factors to rejuvenate cells while retaining the cells in a differentiated state.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 642,538, filed March 13, 2018, which is hereby incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Aging is characterized by the gradual loss of function that occurs at the molecular, cellular, tissue, and organismal levels. At the chromatin level, aging is accompanied by the progressive accumulation of epigenetic errors that ultimately lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell / tissue homeostasis. Nuclear reprogramming techniques to pluripotency through the overexpression of a small number of transcription factors can revert both the age and identity of any cell to that of an embryonic cell by driving epigenetic reprogramming. The undesired erasure of cell identity is problematic for the development of rejuvenation therapies due to the resulting disruption of structure, function, and cell type distribution in tissues and organs. Summary of the Invention [Problem to be solved by the invention]

[0003] (Summary of the Invention) In view of the foregoing, there is a need for improved methods of rejuvenating cells that avoid dedifferentiation and loss of cellular identity. The present disclosure addresses such a need and provides additional benefits as well. [Means for solving the problem]

[0004] The present disclosure relates generally to cellular rejuvenation, tissue engineering, and regenerative medicine. In particular, the present disclosure relates to compositions and methods for rejuvenating aged cells and tissues and restoring functionality by transient exposure to non-integrated mRNA encoding reprogramming factors that rejuvenate cells while maintaining them in a differentiated state.

[0005] The present disclosure relates to cell-based therapies utilizing rejuvenated cells. In particular, the present disclosure relates to methods for rejuvenating aged cells and tissues and restoring functionality by transient exposure to non-integrated mRNA encoding reprogramming factors that rejuvenate cells while maintaining them in a differentiated state.

[0006] In certain aspects, provided herein are methods of rejuvenating cells, comprising transfecting cells with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for no more than five consecutive days, thereby producing rejuvenated cells.

[0007] In certain aspects, provided herein are methods for treating an age-related disease or condition, a cartilage degenerative disorder, a neurodegenerative disorder, and / or a musculoskeletal dysfunction in a subject, the method comprising administering a therapeutically effective amount of cells comprising one or more unintegrated messenger RNAs encoding one or more cellular reprogramming factors.

[0008] In certain aspects, provided herein are methods for treating an age-related disease or condition, a cartilage degeneration disorder, and / or treating a subject having a musculoskeletal dysfunction in a subject, the methods comprising administering a therapeutically effective amount of one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors.

[0009] In one aspect, provided herein is a method for rejuvenating engineered tissue ex vivo, comprising transfecting the tissue with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors for no more than five consecutive days, thereby producing a rejuvenated engineered tissue.

[0010] In one aspect, provided herein is a pharmaceutical composition comprising rejuvenated cells obtained by transfecting cells with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for not more than five consecutive days.

[0011] Thus, in one aspect, the disclosure includes a method of rejuvenating a cell, the method comprising: a) transfecting a cell with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, wherein the transfecting is performed once per day for at least two days and not more than four days, and b) translating the one or more non-integrated messenger RNAs to produce the one or more cellular reprogramming factors in the cell, resulting in transient reprogramming of the cell, wherein the cell rejuvenates without dedifferentiating into a stem cell. The method can be performed on a cell in vitro, ex vivo, or in vivo.

[0012] In certain embodiments, transfection with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors is performed once per day for 2, 3, or 4 days.

[0013] In certain embodiments, the one or more cellular reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In one embodiment, the one or more cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0014] The method can be performed on any type of cell. In some embodiments, the cell is a mammalian cell (e.g., human, non-human primate, rodent, feline, canine, bovine, equine, porcine, caprine, etc.). For example, the method can be performed on fibroblasts, endothelial cells, chondrocytes, or skeletal muscle stem cells. In another embodiment, the cell is derived from an aged subject.

[0015] In certain embodiments, transient reprogramming results in increased expression of HP1γ, H3K9me3, lamina support proteins LAP2α and SIRT1, decreased expression of GMSCF, IL18 and TNFα, decreased nuclear folding, decreased blebbing, increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, increased mitochondrial membrane potential, or decreased reactive oxygen species (ROS).

[0016] In certain embodiments, the cells are in a tissue or organ. Transient reprogramming according to the methods described herein can restore the function of cells in the tissue or organ, increase the differentiation potential of cells in the tissue or organ, reduce the number of senescent cells in the tissue or organ, enhance the replicative potential of cells in the tissue or organ, or extend the lifespan of cells in the tissue or organ.

[0017] In another aspect, the disclosure includes a method for treating an age-related disease or condition in a subject, the method comprising the steps of: a) transfecting cells of the subject with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, wherein the transfecting is performed once daily for at least two days and not more than four days, and b) expressing the one or more cellular reprogramming factors in cells in the subject, resulting in transient reprogramming of the cells, wherein the cells rejuvenate without dedifferentiating into stem cells. The cells can be transfected ex vivo or in vivo.

[0018] In certain embodiments, the one or more cellular reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In one embodiment, the one or more cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0019] In certain embodiments, the age-related disease or condition is a degenerative disease, a neurodegenerative disease, a cardiovascular disease, a peripheral vascular disease, a skin disease, an eye disease, an autoimmune disease, an endocrine disorder, a metabolic disorder, a musculoskeletal disorder, a gastrointestinal disease, or a respiratory disease.

[0020] In another embodiment, the disclosure includes a method for treating a disease or disorder involving cartilage degeneration in a subject, the method comprising the steps of: a) transfecting chondrocytes of the subject with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, wherein the transfecting is performed once daily for at least two days and not more than four days, and b) expressing the one or more cellular reprogramming factors in the chondrocytes, resulting in transient reprogramming of the chondrocytes without dedifferentiating them into stem cells. The rejuvenated chondrocytes can be transplanted, for example, into an arthritic joint of the subject.

[0021] The method may be performed ex vivo, in vitro or in vivo. In one embodiment, chondrocytes are isolated from a cartilage sample obtained from a subject, transfected ex vivo and then implanted into the subject.

[0022] In certain embodiments, the disease or disorder involving cartilage degeneration is arthritis (eg, osteoarthritis or rheumatoid arthritis).

[0023] In certain embodiments, the treatment reduces inflammation in the subject.

[0024] In certain embodiments, the therapy / treatment reduces the expression of RANKL, iNOS, IL6, IL8, BDNF, IFNα, IFNγ, and LIF, and increases the expression of COL2A1 by chondrocytes.

[0025] In another aspect, the disclosure includes a method for treating a disease or disorder involving muscle degeneration in a subject, the method comprising the steps of: a) transfecting skeletal muscle stem cells of the subject with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, wherein the transfecting is performed once daily for at least two days and not more than four days; and b) expressing the one or more cellular reprogramming factors in the skeletal muscle stem cells, resulting in transient reprogramming of the skeletal muscle stem cells, wherein the skeletal muscle stem cells are rejuvenated without losing their ability to differentiate into muscle cells.

[0026] The method can be performed ex vivo, in vitro, or in vivo. In one embodiment, skeletal muscle stem cells are isolated from a muscle tissue sample obtained from a subject, transfected ex vivo, and then transplanted into a muscle in need of repair or regeneration in the subject.

[0027] In certain embodiments, the one or more cellular reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In one embodiment, the one or more cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0028] In certain embodiments, the therapy / treatment restores the differentiation potential of skeletal muscle stem cells, hi certain embodiments, the therapy / treatment results in the regeneration of muscle fibers.

[0029] The methods of the present disclosure can be performed on any subject. In certain embodiments, the subject is a mammal, such as a human, a non-human primate, a rodent, a cat, a dog, a cow, a horse, a pig, or a goat. In some embodiments, the subject is an elderly person.

[0030] These and other embodiments of the subject disclosure will readily occur to those of ordinary skill in the art in view of the disclosure herein. [Brief explanation of the drawings]

[0031] [Figure 1A] This figure shows that transient reprogramming reverses aging physiology in fibroblasts toward a more youthful state. Representative plots demonstrating the variance of effect (ROS) by duration of treatment: 2 days vs. 4 days of transient reprogramming, both followed by 2 days of relaxation. All box and bar plots are generated after combining data across all individual cell, biological, and technical replicates for ease of visualization. The significance levels shown allow for grace in single pairwise comparisons with regard to inter-patient variability. [Figure 1B] Figure 1 shows that transient reprogramming reverts aged physiology towards a more youthful state in fibroblasts.Figure 2 shows the quantification of single-cell levels of heterochromatin markers H3K9me3 and HP1γ using immunocytochemistry. [Figure 1C] Figure 1 shows that transient reprogramming reverts aged physiology in fibroblasts toward a more youthful state. Figure 2 shows the quantification of the presence of the nuclear lamina-supporting polypeptide LAP2α in single cells using immunocytochemistry, and the percentage of abnormal nuclei (folded or blebbed) in each population. [Figure 1D] We demonstrate that transient reprogramming reverts aged physiology in fibroblasts toward a more youthful state. We show results from live-cell imaging with a florescent-tagged substrate that is cleaved upon autophagosome formation in single cells and chymotrypsin-like 20S proteolytic activity in the total population. [Figure 1E] Figure 1 shows that transient reprogramming reverts aged physiology in fibroblasts toward a more youthful state. Figure 2 shows individual cell mitochondrial membrane potential and ROS levels quantified by a mitochondria-specific dye. [Figure 1F]Figure 1 shows that transient reprogramming reverts aged physiology towards a more youthful state in fibroblasts.Figure 2 shows single cell quantification of immunostaining for SIRT1. [Figure 1G] Figure 1 shows the results of telomere quantitative fluorescence in situ hybridization (QFISH) in single cells, demonstrating that transient reprogramming reverts aged physiology in fibroblasts toward a more youthful state. [Figure 1H] Figure 1 shows that transient reprogramming reverts aged physiology towards a more youthful state in fibroblasts.Figure 2 shows the results of SAβGal staining for senescent populations. [Figure 1I] Figure 1 shows transient reprogramming reverts aged physiology towards a more youthful state in fibroblasts.Figure 2 shows quantification of inflammatory cytokine profiling using a panel of analyte-antibody conjugated beads for multiplex cytometry. [Figure 1J] Figure 1 shows that transient reprogramming reverses aged physiology toward a more youthful state in fibroblasts. Representative plots show the maintenance of the youthful shift during longer periods of relaxation, 4 and 6 days, after 4 days of transient reprogramming. Cells in each cohort were then subjected to 80-base pair paired-end RNA sequencing to obtain transcriptome profiles for each group (young, aged, and treatment—R4X2). [Figure 1K] Figure 1 shows that transient reprogramming reverts aged physiology towards a more youthful state in fibroblasts.Figure 2 shows principal component analysis on the subspace defined by the aging signature. [Figure 1L]This figure shows that transient reprogramming reverses aged physiology in fibroblasts toward a more youthful state. This figure shows a comparison of the log fold change between young and aging (x-axis) and treatment and aging (y-axis). All dark gray dots are genes in the aging signature, while light gray dots are genes that significantly differ between treatment and aging and also overlap with the treatment signature. The majority of genes fall along the y=x line, indicating that the magnitude of change with treatment closely matched the magnitude of the difference between young and aging. Significance was calculated by Student's t-test pairwise between treatment and aging, and group-wise when comparing with young patients. P-values: *<0.05, **<0.01, ***<0.001. The asterisk color matches the population being compared. [Figure 2A] This figure shows that transient reprogramming reverses aged physiology in endothelial cells toward a more youthful state. Representative plots demonstrating the variance in effect (ROS) by duration of treatment: 2 days versus 4 days of transient reprogramming, both followed by 2 days of relaxation. All box and bar plots are generated after combining data across all individual cell, biological, and technical replicates for ease of visualization. The significance levels shown allow for grace in single pairwise comparisons with regard to inter-patient variability. [Figure 2B] Figure 1 shows that transient reprogramming reverts aged physiology towards a more youthful state in endothelial cells.Figure 2 shows the quantification of single-cell levels of heterochromatin markers H3K9me3 and HP1γ using immunocytochemistry. [Figure 2C] Figure 1 shows that transient reprogramming reverts aged physiology in endothelial cells toward a more youthful state. Figure 2 shows the quantification of the presence of the nuclear lamina-supporting polypeptide LAP2α in single cells using immunocytochemistry, and the percentage of abnormal nuclei (folded or blebbed) in each population. [Figure 2D]Figure 1 shows that transient reprogramming reverts aged physiology in endothelial cells toward a more youthful state. Live cell imaging with a florescent-tagged substrate that is cleaved upon autophagosome formation in single cells and chymotrypsin-like 20S proteolytic activity in the total population. [Figure 2E] Figure 1 shows that transient reprogramming reverts aged physiology in endothelial cells toward a more youthful state. Figure 2 shows individual cell mitochondrial membrane potential and ROS levels quantified by a mitochondria-specific dye. [Figure 2F] Figure 1 shows that transient reprogramming reverts aged physiology in endothelial cells toward a more youthful state.Figure 2 shows single-cell quantification of immunostaining for SIRT1. [Figure 2G] Figure 1 shows the results of telomere quantitative fluorescence in situ hybridization (QFISH) in single cells, demonstrating that transient reprogramming reverts aged physiology in endothelial cells toward a more youthful state. [Figure 2H] Figure 1 shows that transient reprogramming reverts aged physiology in endothelial cells towards a more youthful state.Figure 2 shows the results of SAβGal staining for senescent populations. [Figure 2I] Figure 1 shows that transient reprogramming reverses aged physiology in endothelial cells toward a more youthful state. Representative plots show the maintenance of the youthful shift during longer periods of relaxation, 4 and 6 days, after 4 days of transient reprogramming. Cells in each cohort were then subjected to 80-base pair paired-end RNA sequencing to obtain transcriptome profiles for each group (young, aged, and treatment—R4X2). [Figure 2J] Figure 1 shows that transient reprogramming reverts aged physiology in endothelial cells towards a more youthful state.Figure 2 shows principal component analysis in the subspace defined by the aging signature. [Figure 2K] Figure 1 shows that transient reprogramming reverts aged physiology in endothelial cells toward a more youthful state. Figure 1 compares the log fold change between young and aged (x-axis) and treatment and aging (y-axis). All dark gray dots are genes in the aging signature, while light gray dots are genes that differ significantly between treatment and aging and also overlap with the treatment signature. The majority of genes fall along the y=x line, indicating that the magnitude of change with treatment closely matched the magnitude of the difference between young and aging. Significance is calculated by Student's t-test pairwise between treatment and aging, and group-wise when comparing with young patients. P-values: *<0.05, **<0.01, ***<0.001; asterisk color matches the population being compared. [Figure 3A] Figures 3A-3I show that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for ease of visualization. The significance levels shown allow for flexibility in pairwise comparisons due to inter-patient variability. Treatment refers to an optimized 3-day reprogramming and 2-day relaxation. Figure 3A-3I shows population results of cell viability staining. [Figure 3B] Figure 1 shows that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for ease of visualization. The significance levels shown allow for flexibility in pairwise comparisons due to inter-patient variability. Treatment refers to an optimized 3-day reprogramming period and 2-day relaxation period. Figure 1 shows qRT-PCR assessment of RNA levels of the anabolic protein factor COL2A1. [Figure 3C]Figure 1 shows that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for ease of visualization. The significance levels shown allow for flexibility in pairwise comparisons due to inter-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. Quantification of ATP concentrations in each cohort. [Figure 3D] Figure 3D shows that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for clarity. The significance levels shown allow for flexibility in pairwise comparisons given inter-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. Figure 3E shows qRT-PCR assessment of RNA levels of the antioxidant SOD2. Note that the juvenile levels are below OA levels, as elevated SOD2 is only beneficial in the presence of ROS, i.e., OA conditions (Figure 3E). [Figure 3E] Figure 3D shows that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for clarity. The significance levels shown allow for flexibility in pairwise comparisons given inter-patient variability. Treatment refers to optimized 3-day reprogramming and 2-day relaxation. Figure 3E shows qRT-PCR assessment of RNA levels of the antioxidant SOD2. Note that the juvenile levels are below OA levels because SOD2 elevation is only beneficial in the presence of ROS, i.e., OA conditions (Figure 3E). [Figure 3F]Figure 3C shows that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for clarity. The significance levels shown allow for flexibility in pairwise comparisons given inter-patient variability. Treatment refers to an optimized 3-day reprogramming period followed by 2 days of relaxation. Figure 3F shows qRT-PCR assessment of RNA levels of the catabolic factor MMP13 (Figure 3F). [Figure 3G] Figure 3G shows that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for clarity. The significance levels shown allow for flexibility in pairwise comparisons given inter-patient variability. Treatment refers to an optimized 3-day reprogramming period and 2-day relaxation period. qRT-PCR assessment of RNA levels of the catabolic factor MMP3 (Figure 3G). [Figure 3H] Figure 3B shows that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for clarity. The significance levels shown allow for flexibility in pairwise comparisons due to interpatient variability. Treatment refers to an optimized 3-day reprogramming period followed by 2 days of relaxation. Figure 3C shows RT-PCR assessment of RNA levels of the pro-inflammatory factor RANKL (Figure 3H) profiling using a panel of analyte-antibody-conjugated beads for multiplex cytometry analysis. Significance is calculated pairwise between treatments and aging, and group-wise when comparing with young patients, by Student's t-test; P values: *<0.05, **<0.01, ***<0.001. [Figure 3I]Figure 3A shows that transient reprogramming alleviates the osteoarthritic phenotype in affected chondrocytes: all box and bar plots are combined across biological and technical replicates for clarity. The significance levels shown allow for flexibility in pairwise comparisons due to interpatient variability. Treatment refers to an optimized 3-day reprogramming period followed by 2 days of relaxation. Figure 3B shows RT-PCR assessment of RNA levels of the pro-inflammatory factor iNOS (Figure 3I) profiling using a panel of analyte-antibody-conjugated beads for multiplex cytometry analysis. Significance is calculated pairwise between treatments and aging, and group-wise when comparing with young patients, by Student's t-test; P values: *<0.05, **<0.01, ***<0.001. [Figure 4A] Figure 1 shows that transient reprogramming restores the differentiation potential of aged muscle stem cells. Figure 2 shows the measurement of MuSC activation from quiescence. Freshly isolated aged MuSCs were incubated with EdU and fixed after 2 days of treatment and 1 or 2 days of relaxation. All box and bar plots are combined across biological and technical replicates for ease of visualization. The significance levels shown allow for flexibility in pairwise comparisons given inter-patient variability. [Figure 4B] Figure 1 shows transient reprogramming restores aged muscle stem cell differentiation potential. Quantification of bioluminescence measured from mice 11 days after transplantation of treated / untreated + luciferase mouse MuSCs into the TA muscle at different time points after transplantation and injury. [Figure 4C] Figure 4B shows that transient reprogramming restores aged muscle stem cell differentiation potential. Figure 4C shows quantification of immunofluorescent staining of GFP expression in mouse TA muscle cross sections imaged and quantified in Figure 4B. [Figure 4D]Figure 1 shows that transient reprogramming restores aged muscle stem cell differentiation potential.Figure 2 shows quantification of the cross-sectional area of ​​donor-derived GFP+ fibers in TA muscles that were recipients of transplanted MuSCs. [Figure 4E] Transient reprogramming restores the differentiation potential of aged muscle stem cells. Figure 1 shows the results of bioluminescence imaging of TA muscles re-injured (second injury) 60 days after transplantation. A second injury was performed to examine whether the bioluminescence signal increased as a result of activation and expansion of luciferase+ / GFP+ MuSCs that were initially transplanted and engrafted beneath the basal layer. [Figure 4F] Figure 1 shows that transient reprogramming restores aged muscle stem cell differentiation potential. Quantified results of bioluminescence measured from mice 11 days after transplantation of treated luciferase+ human MuSCs into the TA muscle. [Figure 4G] Transient reprogramming restores the differentiation potential of aged muscle stem cells. Figure 1 shows the difference in bioluminescence ratios between treated and untreated MuSCs obtained from healthy donors of different age groups. Significance is calculated by Student's t-test pairwise between treatment and aging, and groupwise when comparing with young patients. P values: *<0.05, **<0.01, ***<0.001. The asterisk color matches the population being compared. [Figure 5A] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows the distribution of epigenetic and nuclear markers for H3K9me3. [Figure 5B] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows the distribution of epigenetic and nuclear markers for HP1γ. [Figure 5C] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows the distribution of epigenetic and nuclear markers for LAP2α. [Figure 5D] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows the distribution of nutrient and energy regulation for SIRT1. [Figure 5E] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows the distribution of nutrient and energy regulation in relation to Mitochondrial membrane potential. [Figure 5F] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows the distribution of nutrient and energy regulation for Mito ROS. [Figure 5G] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows the distribution and bulk waste clearance in autophagosomes and senescence. [Figure 5H] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows proteasome activity in young, aged, and treated cells. [Figure 5I]Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows the senescence activity of young, aged, and treated cells. [Figure 5J] Figure 1 shows that transient reprogramming reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells. Fibroblasts and endothelial cells were obtained from otherwise healthy young and aged individuals. Figure 2 shows secreted cytokines in young, aged, and treated cells. [Figure 6A] Figure 1 shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells. Figure 2 shows the transcriptome profile of young versus aged fibroblasts. The data show that 961 genes (5.85%) (678 upregulated and 289 downregulated) in fibroblasts differed between young and aged cells with a significance criterion of p<0.05 and a log fold change cutoff of + / -0.5. [Figure 6B] Figure 1 shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells.Figure 2 shows PCA analysis of fibroblasts. [Figure 6C] Figure 1 shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells.Figure 2 shows expression analysis of fibroblasts. [Figure 6D] Figure 1 shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells. Figure 2 shows the young vs. aged profile of endothelial cells. Data show that 748 genes (4.80%) (389 up-regulated and 377 down-regulated) in endothelial cells differed between young and aged cells with a significance criterion of p<0.05 and a log fold change cutoff of + / -0.5. [Figure 6E] Figure 1 shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells.Figure 2 shows PCA analysis of endothelial cells. [Figure 6F]Figure 1 shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells.Figure 2 shows expression analysis of endothelial cells. [Figure 6G] Figure 1 shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells. Figure 2 shows a graph of the methylation age of fibroblasts assessed by Horvath clock before and after treatment, with the data showing a general trend of decline. [Figure 6H] Figure 1 shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells. Figure 2 shows a graph of the methylation age of endothelial cells assessed by the Horvath clock before and after treatment, with the data showing a general trend of decline. [Figure 6I] Figure 6I shows transcriptome and methylome analysis of aged fibroblasts and endothelial cells. Figure 6I shows a dendrogram showing unsupervised clustering in methylation patterns separated by treatment condition, sex, patient, and cell type. The clustering demonstrates collective retention of cell identity, at least when comparing fibroblasts and endothelial cells. [Figure 7A] Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data show that transient reprogramming alleviates the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Figure 2 shows the increase in ATP levels in chondrocytes upon treatment, as measured by fluorophore-based glycerol ATP concentration. [Figure 7B]Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data show that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Figure 2 shows that ROS activity, measured by live single-cell imaging of cells incorporating a superoxide-inducible fluorescent dye, shows reduced signal after treatment. [Figure 7C] Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data show that transient reprogramming alleviates the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Figure 2 shows the results of qRT-PCR assessment of treatment-induced elevation of RNA levels of the antioxidant SOD2. [Figure 7D]Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data demonstrate that transient reprogramming alleviates the inflammatory phenotype in diseased chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Figure 2 shows cell proliferation in young, aged, and aged-treated chondrocytes. Aged-treated cells shift toward levels close to those of young cells. [Figure 7E] Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data demonstrate that transient reprogramming attenuates the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Figure 2 shows data from qRT-PCR reflecting elevated RNA levels of the extracellular matrix protein component COL2A1 in young, aged, and aged-treated chondrocytes. Aged and treated cells shift towards levels close to those of young cells. [Figure 7F]Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data show that transient reprogramming alleviates the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Data show that qRT-PCR levels of the chondrogenic identity and function transcription factor SOX9 are preserved after treatment. [Figure 7G] Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data show that transient reprogramming alleviates the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Figure 2 summarizes RT-PCR evaluation showing that treatment reduces intracellular RNA levels of the NF-κB ligand RANKL. [Figure 7H]Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data show that transient reprogramming alleviates the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Figure 2 summarizes RT-PCR evaluation showing that treatment propagates inflammatory stimuli by reducing iNOS levels for nitric oxide production in response, shifting them more closely to those of young chondrocytes. [Figure 7I] Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Data show that transient reprogramming reduces the inflammatory phenotype in affected chondrocytes. Chondrocytes were obtained from cartilage biopsies from patients diagnosed with aged, late-stage osteoarthritis (OA). Aged OA cells and transiently reprogrammed OA cells were evaluated for OA-specific phenotypes. All box and bar plots are combined across biological and technical replicates for ease of visualization. Overall significance ranking is determined by the second most stringent p-value. Significance was calculated by Student's t-test. P values: *<0.05, **<0.01, ***<0.001. Error bars indicate root mean square error (RMSE). Data reflect that cytokine profiling of chondrocyte secretion showed increased pro-inflammatory cytokines that were reduced with treatment. [Figure 7J] Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells.Figure 2 shows a patient-specific distribution shift towards treatment-decreased levels of p16 in mesenchymal stem cells. [Figure 7K]Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells.Figure 2 shows a patient-specific distribution shift towards treatment-decreased levels of p21 in mesenchymal stem cells. [Figure 7L] Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells.Figure 2 shows fold changes corresponding to increased cell proliferation in aged and treated mesenchymal stem cells. [Figure 7M] Figure 1 shows transient reprogramming in osteoarthritic chondrocytes and mesenchymal stem cells. Figure 2 shows the percentage of aged and treated mesenchymal stem cells that have senesced, corresponding to a reduction in cellular senescence. [Figure 8A]

[0023] Figure 1 shows the effect of transient reprogramming of engineered skin tissue.

[0024] Figure 1 shows skin aging parameters for fibroblasts and keratinocytes.

[0025] Figure 1 shows that histology scores, incorporating metrics of morphology, structure, and organization, show improvement with mRNA treatment but not with the commonly available skin treatment retinoic acid. [Figure 8B] 8A and 8B show the effects of transient reprogramming of engineered skin tissue. Skin aging parameters of fibroblasts and keratinocytes are shown. mRNA treatment reduces aging parameters, as shown in FIG. 8B (SaβGal) and FIG. 8C (left panel) (p16), and inflammatory parameters, as shown in FIG. 8C (middle panel) (IL-8) and FIG. 8C (right panel) (MMP-1), with further comparison to the effects of retinoic acid. [Figure 8C] 8A and 8B show the effects of transient reprogramming of engineered skin tissue. Skin aging parameters of fibroblasts and keratinocytes are shown. mRNA treatment reduces aging parameters, as shown in FIG. 8B (SaβGal) and FIG. 8C (left panel) (p16), and inflammatory parameters, as shown in FIG. 8C (middle panel) (IL-8) and FIG. 8C (right panel) (MMP-1), with further comparison to the effects of retinoic acid. [Figure 8D]Figure 1 shows the effect of transient reprogramming of engineered skin tissue, muscle regeneration in satellite cells, and quantified results of bioluminescence measured from mice 11 days after transplantation of treated luciferase+ human MuSCs into the TA muscle. [Figure 8E] Figure 1 shows the effect of transient reprogramming of engineered skin tissue. Figure 2 shows muscle regeneration in satellite cells. Figure 3 shows bioluminescence for cohorts 10-30 days old, 30-55 days old, and 60-80 days old. Differences in bioluminescence ratios between treated and untreated MuSCs obtained from healthy donors of different age groups. Significance is calculated by Student's t-test pairwise between treatment and aging, and groupwise when comparing with young patients (age groups: 10-30: n=5; 30-55: n=7; 60-80: n=5). P values: *<0.05, **<0.01, ***<0.001. The asterisk color matches the population being compared. [Figure 8F] Figure 1 shows the effect of transient reprogramming of engineered skin tissue. Figure 2 shows muscle regeneration in satellite cells. Figure 3 shows tetanic force measurements of injured, aged muscles transplanted with aged MuSCs. TA muscles were dissected and subjected to ex vivo electrophysiological testing for tetanic measurements. Baseline force production of untransplanted muscles was measured in young (4 months, blue dashed line) and aged (27 months, red dashed line) mice. Treated aged MuSCs were transplanted into the TA muscles of aged mice, and force production was measured 30 days later (n=5). [Figure 8G] Figure 1 shows the effect of transient reprogramming of engineered skin tissue, muscle regeneration in satellite cells, and quantified results of bioluminescence of treated, aged, and young cells at different time points after transplantation and injury (n=10). [Figure 8H] Figure 1 shows the effect of transient reprogramming of engineered skin tissue. Figure 2 shows muscle regeneration in satellite cells. Figure 3 shows quantification of immunofluorescence staining in TA muscle cross sections from mice transplanted with aged-treated and aged-untreated cells (n=5). [Figure 8I](a) shows the effect of transient reprogramming of engineered skin tissue; (b) shows muscle regeneration in satellite cells; and (c) shows the quantification of the cross-sectional area of ​​donor-derived GFP+ fibers in TA muscles that were recipients of transplanted MuSCs (n=5). [Figure 8J] Figure 1 shows the effect of transient reprogramming of engineered skin tissue. Figure 2 shows muscle regeneration in satellite cells. Figure 3 shows the results of bioluminescence imaging of TA muscles re-injured (second injury) 60 days after MuSC transplantation (n=6). A second injury was performed to examine whether the bioluminescence signal increased as a result of activation and expansion of luciferase+ / GFP+ MuSCs that were initially transplanted and engrafted below the basal layer. [Figure 9A] Figure 1 shows transfection of corneal epithelial cells with transiently reprogrammed cells.Figure 2 shows aging versus reduced senescence as measured by expression of p16 in treated cells. [Figure 9B] Figure 1 shows transfection of corneal epithelial cells with transiently reprogrammed cells.Figure 2 shows aging versus reduced senescence as measured by p21 expression in treated cells. [Figure 9C] Figure 1 shows transfection of corneal epithelial cells with transiently reprogrammed cells.Figure 2 shows the reduction of the inflammatory factor IL8 in aged versus treated cells. [Figure 9D] Figure 1 shows transfection of corneal epithelial cells with transiently reprogrammed cells.Figure 2 shows increased mitochondrial biogenesis as measured by PGC1α expression. [Figure 10-1] Figure 1 shows a chart showing the P-values ​​of changes in cell-specific markers between treatments and aging cells using RNAseq analysis. None of the 8 fibroblast and 50 endothelial cell markers showed significant changes with treatment in each cell type, suggesting the preservation of cell identity. [Figure 10-2]Figure 1 shows a chart showing the P-values ​​of changes in cell-specific markers between treatments and aging cells using RNAseq analysis. None of the 8 fibroblast and 50 endothelial cell markers showed significant changes with treatment in each cell type, suggesting the preservation of cell identity. [Figure 10-3] Figure 1 shows a chart showing the P-values ​​of changes in cell-specific markers between treatments and aging cells using RNAseq analysis. None of the 8 fibroblast and 50 endothelial cell markers showed significant changes with treatment in each cell type, suggesting the preservation of cell identity. [Figure 11] FIG. 1 shows characteristics of aging analyzed using a panel of 11 established assays. DETAILED DESCRIPTION OF THE INVENTION

[0032] The practice of the techniques described herein will employ, unless otherwise indicated, conventional methods of medicine, cell biology, pharmacology, chemistry, biochemistry, molecular biology, and recombinant DNA techniques and immunology, which are within the skill of those in the art, and such techniques are explained fully in the literature. See, e.g., G. Vunjak-Novakovic and R.I. Freshney, Culture of Cells for Tissue Engineering (Wiley-Liss, 1st ed., 2006); Arthritis Research: Methods and Protocols, Volumes 1 and 2: (Methods in Molecular Medicine, edited by Cope, Humana Press, 2007); Cartilage and Osteoarthritis (Methods in Molecular Medicine, edited by M. Sabatini, P. Pastoureau, and F. De Ceuninck, Humana Press; 2004); Handbook of Experimental Immunology, Volumes I-IV (edited by D.M. Weir and C.C. Blackwell, Blackwell Scientific Publications); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); and Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001).

[0033] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0034] I. Definition In describing this disclosure, the following terms will be employed and are intended to be defined as indicated below.

[0035] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes mixtures of two or more cells, etc.

[0036] References throughout this specification to, for example, "one embodiment," "an embodiment," "another embodiment," "particular embodiment," "related embodiment," "certain particular embodiment," "additional embodiment," or "further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of these phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0037] As used herein, the term "about" refers to a range of values ​​including the specified value that one of ordinary skill in the art would consider to be reasonably similar to the specified value. In embodiments, the term "about" refers to within a standard deviation using measurements generally accepted in the art. In embodiments, about refers to a range extending to + / - 10% of the specified value. In embodiments, about refers to the specified value.

[0038] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to imply the inclusion of the described step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the word "consisting of." Thus, the word "consisting of" indicates that the listed elements are required or essential, and that other elements may not be present. "Consisting essentially of" means including any elements listed after the word, and is limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

[0039] As used herein, the term "biocompatible" generally refers to a material and any metabolites or breakdown products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects in the subject.

[0040] As used herein, the term "cell" refers to an intact, living cell, of natural origin or modified. Cells that are mixed with other cells in culture or within a tissue (partial or intact) or organism may be isolated from other cells. The methods described herein may be performed on samples that include, for example, single cells, populations of cells, or tissues or organs containing cells.

[0041] As used herein, the term "non-integrated" with reference to messenger RNA (mRNA) refers to an mRNA molecule that is not integrated intra- or extra-chromosomally into a host genome, or integrated into a vector.

[0042] As used herein, the term "transfection" refers to the uptake of exogenous DNA or RNA by a cell. A cell is "transfected" when exogenous DNA or RNA is introduced inside the cell membrane. Numerous transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology 52:456; Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd ed., Cold Spring Harbor Laboratories, New York; Davis et al. (1995) Basic Methods in Molecular Biology, 2nd ed., McGraw-Hill; and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA or RNA molecules into a cell. The term refers to both stable and transient uptake of DNA or RNA molecules. For example, transfection can be used for the transient uptake of mRNA encoding cellular reprogramming factors into cells in need of rejuvenation.

[0043] As used herein, the term "transient reprogramming" refers to the exposure of cells to cell reprogramming factors for a period of time sufficient to rejuvenate the cells (i.e., eliminate all or some characteristics of aging), but not long enough to cause dedifferentiation into stem cells. Such transient reprogramming results in rejuvenated cells that retain their identity (i.e., differentiated cell type).

[0044] As used herein, the term "rejuvenated cell(s)" refers to aged cells that have been treated or transiently reprogrammed with one or more cell reprogramming factors such that the cells have the transcriptomic profile of a younger cell while still retaining one or more cell-identifying markers.

[0045] As used herein, the term "mammalian cell" refers to any cell derived from a mammalian subject that is suitable for transplantation into the same or a different subject. The cell may be xenogeneic, autologous, or allogeneic. The cell may be a primary cell obtained directly from the mammalian subject. The cell may be a cell derived from the culture and expansion of cells obtained from the subject. In some embodiments, the cell is genetically engineered to express a recombinant protein and / or nucleic acid.

[0046] As used herein, the term "stem cell" refers to a cell that retains the ability to renew itself through cell division and can differentiate into a diverse range of specialized cell types. Mammalian stem cells can be divided into three broad categories: embryonic stem cells derived from blastocysts, adult stem cells found in adult tissues, and umbilical cord blood stem cells found in the umbilical cord. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem and progenitor cells act as the body's repair system by replenishing specialized cells. Totipotent stem cells are produced from the fusion of egg and sperm cells. Cells produced by the first few divisions of a fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can only produce cells of closely related families of cells (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be obtained from adult somatic cells, such as skin or blood cells.

[0047] As used herein, the term "transcriptome profile" refers to the set of all RNA molecules in a cell or cell population. It may also refer to total RNA or simply mRNA, depending on the specific experiment. It differs from the exome in that it includes only RNA molecules found in a given cell population and typically includes the amount or concentration of each RNA molecule in addition to its molecular identity. Methods for obtaining transcriptome profiles include DNA microarrays and next-generation sequencing technologies, such as RNA-Seq. Transcription can be examined at the level of individual cells through single-cell transcriptomics. There are two general methods for inferring transcriptome sequences. One approach maps sequence reads to a reference genome, either of the organism itself (whose transcriptome is being examined) or of a closely related species. The other approach, de novo transcriptome assembly, uses software to infer transcripts directly from short sequence reads.

[0048] As used herein, the term "root mean square error" or "RMSE" refers to the standard deviation of the residuals (prediction errors). The residuals are a measure of the distance of the data points from the regression line. The RMSE is a measure of the spread of these residuals. In other words, it indicates how concentrated the data is around the line of best fit.

[0049] As used herein, the term "cell viability" refers to a measure of the number of living or dead cells based on a total cell sample. High cell viability, as defined herein, refers to a cell population in which greater than 85% of the total cells are viable, preferably greater than 90-95% are viable, and more preferably, a population characterized by high cell viability containing greater than 99% viable cells.

[0050] As used herein, the term "autophagosome" refers to a spherical structure with a bilayer membrane. It is a key structure in macroautophagy, an intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles) and for invading microorganisms. After formation, autophagosomes deliver cytoplasmic components to lysosomes. The outer membrane of the autophagosome fuses with the lysosome to form an autolysosome. Lysosomal hydrolases degrade the contents delivered to the autophagosome and its inner membrane.

[0051] As used herein, the term "proteasome activity" refers to the breakdown of unnecessary or damaged proteins by the protein complex proteasome through proteolysis, a chemical reaction that cleaves peptide bonds. The term "chymotrypsin-like proteasome activity" refers to a distinct catalytic activity of the proteasome.

[0052] As used herein, the term "mitochondrial membrane potential" refers to the electrical potential and proton gradient resulting from redox transitions associated with the activity of the Krebs cycle and serving as an intermediate form of energy storage for generating ATP. This is generated by proton pumps and is an essential process for energy storage in oxidative phosphorylation. It plays a key role in mitochondrial homeostasis by selectively eliminating dysfunctional mitochondria.

[0053] As used herein, the term "pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the present disclosure and that causes no significant adverse toxicological effects in patients.

[0054] As used herein, the term "reactive oxygen species" or "ROS" refers to chemically reactive species containing oxygen. Examples include peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha-oxygen. In the biological context, ROS are formed as natural by-products of the normal metabolism of oxygen and play important roles in cellular signaling and homeostasis.

[0055] As used herein, the term "senescence-associated secretory phenomenon" or "SASP" refers to the diverse array of cytokines, chemokines, growth factors, and proteases that are characteristic features of senescent cells. Senescent cells are stable, non-dividing cells that remain metabolically active and exhibit upregulation of a wide range of genes, including those encoding secreted proteins such as inflammatory cytokines, chemokines, extracellular matrix remodeling factors, and growth factors. These secreted proteins function physiologically in the tissue microenvironment, whereby they can propagate stress responses and communicate with neighboring cells. This phenotype, termed senescence-associated secretory phenomenon (SASP), reveals the paracrine functions of senescent cells and is an important feature that distinguishes senescent cells from non-senescent, cell cycle-arrested cells, such as quiescent and terminally differentiated cells. "SASP cytokines" specifically refer to cytokines produced by senescent cells that create the senescence-associated secretory phenomenon. Cytokines include, but are not limited to, IL18, IL1A, GROA, IL22, and IL9.

[0056] As used herein, the term "methylation landscape" refers to the DNA methylation pattern of a cell or cell population.

[0057] As used herein, the term "epigenetic clock" refers to a biochemical test that can be used to measure age. This test is based on DNA methylation levels. The first multi-tissue epigenetic clock, the Horvath epigenetic clock or "Horvath clock," was developed by Steve Horvath (Horvath 2013).

[0058] As used herein, the term "cell reprogramming factors" refers to a set of transcription factors that can convert adult or differentiated cells into pluripotent stem cells. In embodiments herein, the factors include OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0059] "Pharmaceutically acceptable salts" include, but are not limited to, salts prepared with inorganic acids such as amino acid salts, chlorides, sulfates, phosphates, diphosphates, bromides, and nitrates, or salts prepared from the corresponding inorganic acid forms of any of the precursors, such as hydrochlorides, or salts prepared with organic acids such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, pamoate, salicylate, and stearate, as well as estolate, gluceptate, and lactobionate. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0060] As used herein, the term "transplantation" refers to the transfer of cells, tissues, or organs from another source to a subject. The term is not limited to a particular mechanism of transfer. Cells can be transplanted by any suitable method, such as by injection or surgical implantation.

[0061] As used herein, the term "arthritis" includes, but is not limited to, osteoarthritis, rheumatoid arthritis, lupus-related arthritis, juvenile idiopathic arthritis, reactive arthritis, enteropathic arthritis, and psoriatic arthritis.

[0062] As used herein, the term "age-related disease or condition" refers to neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia, and stroke), cardiovascular and peripheral vascular diseases (e.g., atherosclerosis, peripheral arterial disease (PAD), hematoma, calcification, thrombosis, embolism, and aneurysm), ophthalmological diseases (e.g., age-related macular degeneration, glaucoma, cataracts, dry eye, diabetic retinopathy, vision loss), skin diseases (e.g., psoriasis, glaucoma, cataracts, dry eye, diabetic retinopathy, vision loss), and the like. Skin disorders (skin atrophy and thinning, elastolysis and wrinkling, sebaceous gland hyperplasia or hypoplasia, senile lentigines and other pigmentation disorders, graying of hair, hair loss or thinning, and chronic skin ulcers), autoimmune diseases (e.g., polymyalgia rheumatica (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthritis and spondyloarthropathy (SPA)), endocrine and metabolic Sexual dysfunction (e.g., adult hypopituitarism, hypothyroidism, asexual thyrotoxicosis, osteoporosis, diabetes mellitus, adrenal insufficiency, various forms of hypogonadism and endocrine malignancies), musculoskeletal disorders (e.g., arthritis, osteoporosis, myeloma, gout, Paget's disease, fractures, bone marrow failure syndromes, ankylosis, diffuse idiopathic osteomyelitis, hematogenous osteomyelitis, muscle atrophy, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS)) ), Duchenne muscular dystrophy, primary lateral sclerosis and myasthenia gravis), diseases of the digestive system (e.g., cirrhosis, liver fibrosis, Barrett's esophagus), respiratory diseases (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer and infections), and any other disease or disorder associated with aging.

[0063] As used herein, the term "disease or disorder involving cartilage degeneration" refers to any disease or disorder involving cartilage and / or joint degeneration. The term "disease or disorder involving cartilage degeneration" includes conditions, disorders, syndromes, diseases and injuries affecting spinal discs or joints (e.g., facet joints) in animals, including humans, including, but not limited to, arthritis, chondrophasia, spondyloarthropathy, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjogren's syndrome.

[0064] As used herein, the term "muscle degenerative disease or disorder" refers to any disease or disorder involving muscle degeneration. This term includes conditions, disorders, syndromes, diseases, and injuries that affect muscle tissue, including, but not limited to, muscle atrophy, disuse muscle, muscle tear, burns, surgery, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis, myasthenia gravis, cancer, AIDS, congestive heart failure, chronic obstructive pulmonary disease (COPD), liver disease, kidney failure, eating disorders, malnutrition, starvation, infection, or treatment with glucocorticoids.

[0065] By "therapeutically effective dose or amount" is intended an amount of rejuvenated cells or non-integrated messenger RNA that results in a positive therapeutic response in a subject in need of tissue repair or regeneration, such as an amount that restores function and / or results in the generation of new tissue at the treatment site. Rejuvenated cells can be produced by in vitro, ex vivo, or in vivo transfection with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors, as described herein. Thus, for example, a "positive therapeutic response" would be an improvement in an age-related disease or condition associated with the therapy, such as restored tissue functionality, reduced pain, improved stamina, increased strength, increased mobility, and / or improved cognitive function, and / or an improvement in one or more symptoms of an age-related disease or condition associated with the therapy. The exact amount (of cells or mRNA) required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the mode of administration, etc. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0066] For example, a therapeutically effective dose or amount of rejuvenated chondrocytes contemplates an amount that, when administered as described herein, results in a positive therapeutic response in a subject with cartilage damage or loss, such as an amount that results in the production of new cartilage at the treatment site (e.g., an injured joint). For example, a therapeutically effective dose or amount may be used to treat cartilage damage or loss resulting from traumatic injury, or degenerative diseases such as arthritis or other diseases involving cartilage degeneration. Preferably, a therapeutically effective amount restores function and / or reduces pain and inflammation associated with cartilage damage or loss.

[0067] In another example, a therapeutically effective dose or amount of rejuvenated skeletal muscle stem cells contemplates an amount that, when administered as described herein, results in a positive therapeutic response in a subject with muscle damage or loss, such as an amount that results in the generation of new muscle fibers at the treatment site (e.g., an injured muscle). For example, a therapeutically effective dose or amount can be used to treat muscle damage or loss resulting from traumatic injury, or a disease or disorder involving muscle degeneration. Preferably, a therapeutically effective amount improves muscle strength and muscle function.

[0068] As used herein, the terms "subject," "individual," and "patient" are used interchangeably herein and refer to any vertebrate subject, including, but not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; livestock, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; rodents, such as mice, rats, rabbits, hamsters, and guinea pigs; and birds, including domestic, wild, and game birds, such as chickens, turkeys, and other poultry birds, ducks, geese, and others. In some cases, the methods of the present disclosure find use in laboratory animals, veterinary applications, and the development of animal models for disease. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be encompassed.

[0069] II. Method Before describing the present disclosure in detail, it is to be understood that the present disclosure is not limited to particular formulations or process parameters, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing only particular embodiments of the present disclosure, and is not intended to be limiting.

[0070] Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, the preferred materials and methods are described herein.

[0071] The present disclosure relates to methods for rejuvenating and restoring functionality to aged cells and tissues by transient overexpression of mRNAs that affect, for example, mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion, or senescence. In particular, the inventors have shown that mRNAs encoding OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG can be used to rejuvenate various cell types, including fibroblasts, endothelial cells, chondrocytes, and skeletal muscle stem cells, while maintaining the cells in a differentiated cellular state.

[0072] To further the understanding of the present disclosure, a more detailed description of methods for rejuvenating cells by transient reprogramming with mRNA and cell-based therapies using such rejuvenated cells is provided below.

[0073] a. Cellular rejuvenation In certain aspects, provided herein are methods of rejuvenating cells, comprising transfecting the cells with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for no more than five consecutive days, thereby producing rejuvenated cells.

[0074] In embodiments, the rejuvenated cells have a phenotypic or activity profile similar to that of young cells, including one or more of the following: transcriptome profile, gene expression of one or more nuclear and / or epigenetic markers, proteolytic activity, mitochondrial health and function, SASP cytokine expression, and methylation landscape.

[0075] In embodiments, the rejuvenated cells have a transcriptomic profile similar to that of young cells. In embodiments, the transcriptomic profile of the rejuvenated cells comprises increased gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1, and Sin3a. In embodiments, the transcriptomic profile of the rejuvenated cells comprises increased gene expression of RPL37. In embodiments, the transcriptomic profile of the rejuvenated cells comprises increased gene expression of RHOA. In embodiments, the transcriptomic profile of the rejuvenated cells comprises increased gene expression of SRSF3. In embodiments, the transcriptomic profile of the rejuvenated cells comprises increased gene expression of EPHB4. In embodiments, the transcriptomic profile of the rejuvenated cells comprises increased gene expression of ARHGAP18. In embodiments, the transcriptome profile of the rejuvenated cell comprises increased gene expression of RPL31. In embodiments, the transcriptome profile of the rejuvenated cell comprises increased gene expression of FKBP2. In embodiments, the transcriptome profile of the rejuvenated cell comprises increased gene expression of MAP1LC3B2. In embodiments, the transcriptome profile of the rejuvenated cell comprises increased gene expression of Elf1. In embodiments, the transcriptome profile of the rejuvenated cell comprises increased gene expression of Phf8. In embodiments, the transcriptome profile of the rejuvenated cell comprises increased gene expression of Pol2s2. In embodiments, the transcriptome profile of the rejuvenated cell comprises increased gene expression of Taf1. In embodiments, the transcriptome profile of the rejuvenated cell comprises increased gene expression of Sin3a. In embodiments, the transcriptome profile of the rejuvenated cells comprises increased gene expression of RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1 and Sin3a.

[0076] In embodiments, rejuvenated cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to a reference value. In embodiments, the one or more nuclear and / or epigenetic markers are selected from HP1 gamma, H3K9me3, the lamina supporting protein LAP2 alpha, and SIRT1 protein. In embodiments, rejuvenated cells exhibit increased gene expression of HP1 gamma. In embodiments, rejuvenated cells exhibit increased gene expression of H3K9me3. In embodiments, rejuvenated cells exhibit increased gene expression of the lamina supporting protein LAP2 alpha. In embodiments, rejuvenated cells exhibit increased gene expression of SIRT1 protein. In embodiments, rejuvenated cells exhibit increased gene expression of HP1 gamma, H3K9me3, the lamina supporting protein LAP2 alpha, and SIRT1 protein.

[0077] In embodiments, rejuvenated cells have proteolytic activity similar to that of young cells. In embodiments, proteolytic activity is measured as increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, or a combination thereof. In embodiments, proteolytic activity is measured as increased cellular autophagosome formation. In embodiments, proteolytic activity is measured as increased chymotrypsin-like proteasome activity. In embodiments, proteolytic activity is measured as increased cellular autophagosome formation and increased chymotrypsin-like proteasome activity.

[0078] In embodiments, the rejuvenated cells exhibit improved mitochondrial health and function compared to a reference value. In embodiments, the improved mitochondrial health and function is measured as increased mitochondrial membrane potential, decreased reactive oxygen species (ROS), or a combination thereof. In embodiments, the improved mitochondrial health and function is measured as increased mitochondrial membrane potential. In embodiments, the improved mitochondrial health and function is measured as decreased reactive oxygen species (ROS). In embodiments, the improved mitochondrial health and function is measured as increased mitochondrial membrane potential and decreased reactive oxygen species (ROS).

[0079] In embodiments, rejuvenated cells exhibit decreased expression of one or more SASP cytokines compared to a reference level. In embodiments, the one or more SASP cytokines include IL18, IL1A, GROA, IL22, and IL9. In embodiments, rejuvenated cells exhibit decreased expression of IL18. In embodiments, rejuvenated cells exhibit decreased expression of IL1A. In embodiments, rejuvenated cells exhibit decreased expression of GROA. In embodiments, rejuvenated cells exhibit decreased expression of IL22. In embodiments, rejuvenated cells exhibit decreased expression of IL9. In embodiments, rejuvenated cells exhibit decreased expression of IL18, IL1A, GROA, IL22, and IL9.

[0080] In embodiments, the rejuvenated cells exhibit a reversal of the methylation landscape, in embodiments, the reversal of the methylation landscape is measured by estimation with the Horvath clock.

[0081] In an embodiment, the reference value is obtained from aged cells.

[0082] In embodiments, cells are rejuvenated by transient reprogramming with mRNA encoding one or more cellular reprogramming factors. Transient reprogramming is achieved by transfecting cells with non-integrated mRNA once per day for at least two days and not more than five days. "Non-integrated" means that the mRNA molecule is not integrated intra- or extra-chromosomally into the host genome or into a vector, such that the reprogramming is transient and does not destroy the identity of the rejuvenated cell (i.e., the cell retains its ability to differentiate into its adult cell type). In embodiments, transient reprogramming of cells eliminates various characteristics of aging while avoiding complete dedifferentiation of the cell into a stem cell.

[0083] In embodiments, transfecting cells with messenger RNA may be accomplished by a transfection method selected from lipofectamine and LT-1-mediated transfection, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, electroporation, encapsulation of mRNA in liposomes, and direct microinjection. In embodiments, transfecting cells with messenger RNA may be accomplished by lipofectamine and LT-1-mediated transfection. In embodiments, transfecting cells with messenger RNA may be accomplished by dextran-mediated transfection. In embodiments, transfecting cells with messenger RNA may be accomplished by calcium phosphate precipitation. In embodiments, transfecting cells with messenger RNA may be accomplished by polybrene-mediated transfection. In embodiments, transfecting cells with messenger RNA may be accomplished by electroporation. In embodiments, transfecting cells with messenger RNA may be accomplished by encapsulation of the mRNA in liposomes. In embodiments, transfecting cells with messenger RNA may be accomplished by direct microinjection.

[0084] Cellular aging reversal or rejuvenation can be achieved by transient overexpression of one or more mRNAs encoding cellular reprogramming factors. Such cellular reprogramming factors can include transcription factors, epigenetic remodelers, or small molecules that affect mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion, or senescence. In some embodiments, the cellular reprogramming factors include one or more of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In other embodiments, the cellular reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In certain embodiments, the cellular reprogramming factors consist of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0085] In embodiments, the methods provided herein can be applied to any type of cell requiring rejuvenation. Cells can be isolated from other cells in culture or mixed with other cells in a tissue (partial or intact) or living organism. The methods described herein can be performed on samples including, for example, single cells, populations of cells, or tissues or organs containing cells. The cells selected for rejuvenation depend on the desired therapeutic effect for the treatment of age-related diseases or conditions.

[0086] In embodiments, the cells are mammalian cells. In embodiments, the cells are human cells. In embodiments, the cells are derived from an elderly subject.

[0087] In embodiments, the methods provided herein may be performed on cells, tissues, or organs of the nervous system, muscular system, respiratory system, cardiovascular system, skeletal system, reproductive system, integumentary system, lymphatic system, excretory system, endocrine system (e.g., endocrine and exocrine), or digestive system. Any type of cell may potentially be rejuvenated as described herein, including, but not limited to, epithelial cells (e.g., squamous, cuboidal, columnar, and pseudostratified epithelial cells), endothelial cells (e.g., venous, arterial, and lymphatic endothelial cells), and cells of connective tissue, muscle, and the nervous system. Such cells include epidermal cells, fibroblasts, chondrocytes, skeletal muscle cells, satellite cells, cardiac myocytes, smooth muscle cells, keratinocytes, basal cells, ameloblasts, exocrine secretory cells, myoepithelial cells, osteoblasts, osteoclasts, neurons (e.g., sensory neurons, motor neurons, and interneurons), glial cells (e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells, and satellite cells), columnar cells, adipocytes, pericytes, astrocytes, lung cells, blood and immune cells. These may include, but are not limited to, immune system cells (e.g., erythrocytes, monocytes, dendritic cells, macrophages, neutrophils, eosinophils, mast cells, T cells, B cells, natural killer cells), hormone-secreting cells, germ cells, interstitial cells, lens cells, photoreceptor cells, taste receptor cells, and olfactory cells; and cells and / or tissues from the kidney, liver, pancreas, stomach, spleen, gallbladder, intestine, bladder, lung, prostate, breast, urogenital tract, pituitary cells, oral cavity, esophagus, skin, hair, nails, thyroid, parathyroid, adrenal gland, eye, nose, or brain.

[0088] In some embodiments, the cell is selected from a fibroblast, an endothelial cell, a chondrocyte, a skeletal muscle stem cell, a keratinocyte, a mesenchymal stem cell, and a corneal epithelial cell. In embodiments, the cell is a fibroblast. In embodiments, the cell is an endothelial cell. In embodiments, the cell is a chondrocyte. In embodiments, the cell is a skeletal muscle stem cell. In embodiments, the cell is a keratinocyte. In embodiments, the cell is a mesenchymal stem cell. In embodiments, the cell is a corneal epithelial cell.

[0089] In embodiments, the rejuvenated fibroblasts exhibit a transcriptome profile similar to that of young fibroblasts. In embodiments, the rejuvenated fibroblasts exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to the reference values ​​described above. In embodiments, the rejuvenated fibroblasts have proteolytic activity similar to that of young cells described above. In embodiments, the rejuvenated fibroblasts exhibit improved mitochondrial health and function compared to the reference values ​​described above. In embodiments, the rejuvenated fibroblasts exhibit a reversal of the methylation landscape.

[0090] In embodiments, the rejuvenated endothelial cells exhibit a transcriptome profile similar to that of young endothelial cells. In embodiments, the rejuvenated endothelial cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to the reference values ​​described above. In embodiments, the rejuvenated endothelial cells have proteolytic activity similar to that of young cells described above. In embodiments, the rejuvenated endothelial cells exhibit improved mitochondrial health and function compared to the reference values ​​described above. In embodiments, the rejuvenated endothelial cells exhibit a reversal of the methylation landscape.

[0091] In embodiments, the rejuvenated chondrocytes exhibit decreased expression of inflammatory factors and / or increased ATP and collagen metabolism. In embodiments, the inflammatory factors include RANKL, iNOS2, IL6, IFNα, MCP3 and MIP1A. In embodiments, the rejuvenated chondrocytes exhibit decreased expression of RANKL. In embodiments, the rejuvenated chondrocytes exhibit decreased expression of iNOS2. In embodiments, the rejuvenated chondrocytes exhibit decreased expression of IL6. In embodiments, the rejuvenated chondrocytes exhibit decreased expression of IFNα. In embodiments, the rejuvenated chondrocytes exhibit decreased expression of MCP3. In embodiments, the rejuvenated chondrocytes exhibit decreased expression of MIP1A. In embodiments, the rejuvenated chondrocytes exhibit decreased expression of RANKL, iNOS2, IL6, IFNα, MCP3 and MIP1A. In embodiments, the rejuvenated chondrocytes exhibit increased ATP and collagen metabolism. In embodiments, ATP and collagen metabolism is measured by one or more of increased ATP levels, decreased ROS and increased SOD2 expression, increased COL2A1 expression, and overall proliferation by chondrocytes. In embodiments, ATP and collagen metabolism is measured by increased ATP levels. In embodiments, ATP and collagen metabolism is measured by decreased ROS and increased SOD2 expression. In embodiments, ATP and collagen metabolism is measured by increased COL2A1 expression and overall proliferation by chondrocytes.

[0092] In embodiments, rejuvenated skeletal muscle stem cells exhibit higher proliferative potential, enhanced ability to differentiate into myoblasts and muscle fibers, lower activation kinetics restored from quiescence, ability to rejuvenate muscle microniches, restoration of youthful force in muscle, or combinations thereof.

[0093] In embodiments, rejuvenated keratinocytes exhibit higher proliferative capacity, a reduced inflammatory phenotype, lower RNAKL and INOS2 expression, reduced expression of cytokines MIP1A, IL6, IFNa, MCP3, increased ATP, and increased levels of SOD2 and COL2A1 expression.

[0094] In embodiments, the rejuvenated mesenchymal stem cells exhibit reduced senescence parameters, increased cell proliferation and / or reduced ROS levels. In embodiments, the rejuvenated mesenchymal stem cells exhibit reduced senescence parameters. In embodiments, the senescence parameters include p16 expression, p21 expression and positive SAβGal staining. In embodiments, the rejuvenated mesenchymal stem cells exhibit increased cell proliferation. In embodiments, the rejuvenated mesenchymal stem cells exhibit reduced ROS levels. In embodiments, the rejuvenated mesenchymal stem cells exhibit reduced senescence parameters, increased cell proliferation and reduced ROS levels.

[0095] In embodiments, the rejuvenated corneal epithelial cells exhibit a decrease in aging parameters. In embodiments, the aging parameters include one or more of expression of p21, expression of p16, mitochondrial biosynthetic PGC1α, and expression of the inflammatory factor IL8. In embodiments, the aging parameter includes p21. In embodiments, the aging parameter includes expression of p16. In embodiments, the aging parameter includes mitochondrial biosynthetic PGC1α. In embodiments, the aging parameter includes expression of the inflammatory factor IL8. In embodiments, the aging parameter includes one or more of expression of p21, expression of p16, mitochondrial biosynthetic PGC1α, and expression of the inflammatory factor IL8.

[0096] The disclosed methods can be used to rejuvenate cells in culture (e.g., ex vivo or in vitro) to improve function and differentiation potential for use in cell therapy. Cells used in patient treatment can be autologous or allogeneic. Preferably, the cells are derived from the patient or a matched donor. For example, in ex vivo therapy, cells are obtained directly from the patient to be treated, transfected with mRNA encoding the cellular reprogramming factors described herein, and reimplanted into the patient. Such cells can be obtained, for example, from a biopsy or surgical procedure performed on the patient. Alternatively, cells requiring rejuvenation can be directly transfected in vivo with mRNA encoding the cellular reprogramming factors.

[0097] Transfection can be performed using any suitable method known in the art that results in the transient uptake of mRNA encoding cellular reprogramming factors into cells in need of rejuvenation (i.e., for transient reprogramming). In embodiments, methods for ex vivo, in vitro, or in vivo delivery of mRNA into cells of interest can include methods selected from lipofectamine and LT-1 mediated transfection, dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, electroporation, encapsulation of mRNA in liposomes, direct microinjection of mRNA into cells, or combinations thereof.

[0098] b. Composition In one aspect, provided herein is a pharmaceutical composition comprising rejuvenated cells obtained by transfecting cells with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for no more than five consecutive days.

[0099] In embodiments, the rejuvenated cells are autologous. In embodiments, the rejuvenated cells are allogeneic.

[0100] In embodiments, the one or more cellular reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In embodiments, the cellular reprogramming factors are OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0101] In embodiments, rejuvenated cells exhibit one or more of the following: increased expression of HP1 gamma, H3K9me3, LAP2 alpha, SIRT1, increased mitochondrial membrane potential and decreased reactive oxygen species, and decreased expression of SASP cytokines, which in embodiments include one or more of IL18, IL1A, GROA, IL22, and IL9.

[0102] In certain embodiments, compositions comprising rejuvenated cells for use in cell therapy can further comprise one or more additional factors to improve cell function or viability, such as nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics, antioxidants, or immunosuppressants. The composition can also further comprise a pharmaceutically acceptable carrier.

[0103] Examples of growth factors include, but are not limited to, fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), transforming growth factor beta (TGF-β), epiregulin, epidermal growth factor ("EGF"), endothelial growth factor ("ECGF"), nerve growth factor ("NGF"), leukemia inhibitory factor ("LIF"), bone morphogenetic protein-4 ("BMP-4"), hepatocyte growth factor ("HGF"), vascular endothelial growth factor-A ("VEGF-A"), and cholecystokinin octapeptide.

[0104] Examples of ECM components include, but are not limited to, proteoglycans (e.g., chondroitin sulfate, heparan sulfate, and keratan sulfate), non-proteoglycan polysaccharides (e.g., hyaluronic acid), fibers (e.g., collagen and elastin), and other ECM components (e.g., fibronectin and laminin).

[0105] Examples of immunosuppressants include, but are not limited to, steroidal (e.g., prednisone) or non-steroidal (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), and anti-IL2R daclizumab (Zenapax, Roche Canada). Other immunosuppressive drugs include 15-deoxyspergualin, cyclosporine, methotrexate, rapamycin, rapamune (sirolimus / rapamycin), FK506, or lisofylline (LSF).

[0106] One or more pharmaceutically acceptable excipients may be included, including, but not limited to, carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

[0107] For example, an antimicrobial agent may be included to prevent or inhibit the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for the present disclosure include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof. Antimicrobial agents also include antibiotics that can be used to prevent bacterial infections. Examples of antibiotics include amoxicillin, penicillin, sulfonamides, cephalosporins, erythromycin, streptomycin, gentamicin, tetracycline, clarithromycin, ciproflozacin, azithromycin, and others. Antifungal agents such as myconazole and terconazole are also included.

[0108] Various antioxidants may be included, such as reduced glutathione (GSH) or its precursors, glutathione or glutathione analogs, glutathione monoesters, and molecules with thiol groups such as N-acetylcysteine. Other suitable antioxidants include superoxide dismutase, catalase, vitamin E, trolox, lipoic acid, lazaroid, butylhydroxyanisole (BHA), vitamin K, and others.

[0109] Suitable excipients for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants. Carbohydrates such as sugars, alditols, derivatized sugars such as aldonic acid, esterified sugars, and / or sugar polymers can be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, myo-inositol, and the like. The excipient may also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0110] An acid or base can also be present as an excipient. Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, but are not limited to, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0111] Typically, the optimal amount of any individual excipient is determined by routine experimentation, i.e., by preparing compositions containing varying amounts of excipient (ranging from low to high amounts), testing stability and other parameters, and then determining the range within which optimal performance is achieved without significant adverse effects. In general, however, the excipient(s) will be present in the composition in an amount of from about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, and more preferably from about 15 to about 95% by weight of the excipient, with concentrations of less than 30% by weight being most preferred. These aforementioned pharmaceutical excipients, along with other excipients, are described in "Remington: The Science & Practice of Pharmacy," 19th Edition, Williams & Williams (1995), "Physician's Desk Reference," 52nd Edition, Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, DC, 2000.

[0112] c. Administration The disclosed methods can be used to treat subjects with age-related diseases or conditions. For example, cell therapy (e.g., in vitro, ex vivo, or in vivo) involving transient reprogramming of cells by transfection with non-integrated mRNA encoding reprogramming factors can be used to treat neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia, and stroke), cardiovascular and peripheral vascular diseases (e.g., atherosclerosis, peripheral arterial disease (PAD), hematoma, calcification, thrombosis, embolism, and stroke), and other conditions. and aneurysms), eye diseases (e.g., age-related macular degeneration, glaucoma, cataracts, dry eye, diabetic retinopathy, vision loss), skin diseases (skin atrophy and thinning, elastolysis and wrinkling, sebaceous gland hyperplasia or hypoplasia, senile lentigines and other pigmentation disorders, gray hair, hair loss or thinning, and chronic skin ulcers), autoimmune diseases (e.g., polymyalgia rheumatica (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthritis and spondyloarthropathy (SPA)), endocrine and metabolic dysfunction (e.g., adult hypopituitarism, hypothyroidism, apathetic thyrotoxicosis, osteoporosis, diabetes mellitus, adrenal insufficiency, various forms of hypogonadism and endocrine malignancies), musculoskeletal disorders (e.g., arthritis, osteoporosis, myeloma, gout, Paget's disease, fractures, bone marrow failure syndromes, ankylosis, diffuse idiopathic osteomyelitis, hematogenous osteomyelitis, muscle atrophy, peripheral neuropathy, multiple sclerosis, The compounds may be used to treat subjects with a variety of age-related diseases and conditions, including, but not limited to, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis, and myasthenia gravis), diseases of the digestive system (e.g., cirrhosis, liver fibrosis, Barrett's esophagus), respiratory diseases (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer, and infectious diseases), and any other diseases and disorders associated with aging.

[0113] In certain aspects, provided herein are methods for treating an age-related disease or condition, a cartilage degenerative disorder, a neurodegenerative disorder, and / or a musculoskeletal dysfunction in a subject, the method comprising administering a therapeutically effective amount of cells comprising one or more unintegrated messenger RNAs encoding one or more cellular reprogramming factors.

[0114] At least one therapeutically effective treatment cycle by transfection with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors can be administered to a subject for the treatment of an age-related disease or condition.

[0115] In embodiments, the age-related disease or condition is selected from ocular, skin, or musculoskeletal dysfunction.

[0116] In embodiments, the subject has a cartilage degenerative disorder. In embodiments, the disorder is selected from arthritis, chondrophasia, spondyloarthropathy, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjogren's syndrome. In embodiments, the disorder is arthritis. In embodiments, the disorder is chondrophasia. In embodiments, the disorder is spondyloarthropathy. In embodiments, the disorder is ankylosing spondylitis. In embodiments, the disorder is lupus erythematosus. In embodiments, the disorder is relapsing polychondritis. In embodiments, the disorder is Sjogren's syndrome.

[0117] In embodiments, the treatment reduces expression of one or more inflammatory factors and / or increases ATP and collagen metabolism. In embodiments, the inflammatory factors are selected from RANKL, iNOS2, IL6, IFNα, MCP3, and MIP1A. In embodiments, ATP and collagen metabolism is measured by one or more of increased ATP levels, decreased ROS and increased SOD2, increased COL2A1, and overall proliferation by chondrocytes.

[0118] In embodiments, treatment of a subject with rejuvenated cells by ex vivo or in vitro transfection in cell culture, compositions for transplanting rejuvenated cells, are typically, but not necessarily, administered by injection or surgical implantation into the area requiring tissue regeneration or repair.

[0119] In embodiments, the therapeutically effective amount of rejuvenated cells is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells. In embodiments, the therapeutically effective amount of rejuvenated cells is fibroblasts. In embodiments, the therapeutically effective amount of rejuvenated cells is endothelial cells. In embodiments, the therapeutically effective amount of rejuvenated cells is chondrocytes. In embodiments, the therapeutically effective amount of rejuvenated cells is skeletal muscle stem cells. In embodiments, the therapeutically effective amount of rejuvenated cells is keratinocytes. In embodiments, the therapeutically effective amount of rejuvenated cells is mesenchymal stem cells. In embodiments, the therapeutically effective amount of rejuvenated cells is corneal epithelial cells.

[0120] In embodiments, the rejuvenated corneal epithelium exhibits reduced aging parameters, including one or more of the expression of p21 and p16, mitochondrial biogenesis PGC1α, and the expression of the inflammatory factor IL8.

[0121] In one embodiment, chondrocytes in the area of ​​cartilage damage or loss are transfected in vivo with an effective amount of one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors sufficient to result in chondrocyte rejuvenation and new cartilage production at the treatment site. Alternatively, rejuvenated chondrocytes produced by ex vivo or in vitro transfection can be administered locally to the area of ​​cartilage damage or loss, such as an injured joint or other suitable treatment site of a subject. A therapeutically effective dose or amount of rejuvenated chondrocytes refers to an amount that results in a positive therapeutic response in a subject with cartilage damage or loss, such as an amount that results in the production of new cartilage at the treatment site (e.g., an injured joint). For example, a therapeutically effective dose or amount can be used to treat cartilage damage or loss due to traumatic injury, or degenerative diseases such as arthritis or other diseases involving cartilage degeneration. Preferably, a therapeutically effective amount restores function and / or reduces pain and inflammation associated with cartilage damage or loss.

[0122] In another embodiment, skeletal muscle stem cells are transfected in vivo with an effective amount of one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors sufficient to rejuvenate (i.e., restore differentiation potential) the skeletal muscle stem cells and generate new muscle fibers at the treatment site (e.g., injured muscle). Alternatively, rejuvenated skeletal muscle stem cells produced by ex vivo or in vitro transfection can be administered locally to injured muscles in need of repair or regeneration. For example, a therapeutically effective dose or amount can be used to treat diseases or disorders involving muscle damage or loss due to traumatic injury, muscle atrophy, or muscle degeneration. A therapeutically effective dose or amount of rejuvenated skeletal muscle stem cells refers to an amount that results in a positive therapeutic response in a subject with muscle damage or loss, such as an amount that results in the generation of new muscle fibers at the treatment site (e.g., injured muscle). Preferably, a therapeutically effective amount improves muscle strength and function, reduces pain, improves stamina, and / or increases mobility.

[0123] In certain aspects, provided herein are methods for treating an age-related disease or condition, a cartilage degenerative disorder, and / or treating a subject having a musculoskeletal dysfunction in a subject, as described herein above, the method comprising administering a therapeutically effective amount of one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, as described herein above.

[0124] In embodiments, cells in a subject can be rejuvenated by in vivo transfection with an effective amount of one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, as described herein.

[0125] In one aspect, provided herein is a method for rejuvenating engineered tissue ex vivo, comprising transfecting the tissue for no more than five consecutive days with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors, thereby producing a rejuvenated engineered tissue.

[0126] In embodiments, the engineered tissue exhibits reduced senescence parameters, reduced pro-inflammatory factors, improved histological score, or a combination thereof. In embodiments, the engineered tissue exhibits reduced one or more senescence parameters. In embodiments, the senescence parameters are selected from p16 expression, positive SAβGal staining, and expression of the pro-inflammatory factors IL8 and MMP1. In embodiments, the engineered tissue exhibits reduced p16 expression. In embodiments, the engineered tissue exhibits reduced positive SAβGal staining. In embodiments, the engineered tissue exhibits reduced expression of the pro-inflammatory factors IL8 and MMP1. In embodiments, the engineered tissue exhibits improved histological score. In embodiments, the histological score includes morphology, organization, and / or quality.

[0127] In embodiments, the engineered tissue is engineered skin tissue and organoids.

[0128] d. Kit The present disclosure also provides kits comprising one or more containers holding a composition comprising one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for transient reprogramming of cells. The kits can further comprise a transfection agent, a medium for culturing the cells, and, optionally, one or more other factors, such as growth factors, ECM components, antibiotics, etc. The mRNA encoding the cellular reprogramming factors and / or other compositions can be in liquid form or lyophilized. Such kits can also contain components that preserve or maintain the mRNA, protecting it from degradation. Such components can be RNAse-free or can protect against RNAses. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. The containers can be made of a variety of materials, including glass or plastic. The containers can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle).

[0129] The kit may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may also contain other materials useful to the end user, including buffers, other pharmaceutically acceptable formulation solutions such as diluents, filters, needles and syringes, or other delivery devices. The delivery device may be pre-filled with the composition.

[0130] The kit may also include a package insert containing written instructions for a method of treating an age-related disease or condition. The package insert may be an unapproved draft package insert or may be a package insert approved by the Food and Drug Administration (FDA) or other regulatory agency.

[0131] In certain embodiments, the kit comprises mRNA encoding one or more cellular reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG, hi one embodiment, the kit comprises mRNA encoding OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG cellular reprogramming factors.

[0132] III. Experiment Below are provided examples of specific embodiments for carrying out the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the disclosure in any way.

[0133] Efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.), but some experimental error and deviation should, of course, be allowed for.

[0134] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [Example]

[0135] Example 1: Transient and non-integrative cell reprogramming promotes multifaceted reversal of aging The experiments described herein illustrate the extent of the aging-reversal effect that can be achieved by transient reprogramming protocols that are stopped before irreversible loss of cellular identity. Recent evidence has also shown that partial transgenic reprogramming can ameliorate age-related features and extend lifespan in progeroid mice. However, it is unclear how this form of "epigenetic rejuvenation" can be broadly applied to natural aging, and importantly, how it can be safely translated to human cells. The data herein demonstrate that transient reprogramming based on mRNA technology reverses physiological aging features, reduces age-related disease phenotypes, and restores the regenerative response diminished with age in somatic cells and stem cells derived from human clinical samples. The non-integrative method of transient cell reprogramming described herein paves the way for novel, more translational strategies for ex vivo cell rejuvenation therapies aimed at regenerative medicine, as well as for in vivo tissue rejuvenation therapies to delay or reverse the physiological attenuation of natural aging and the pathogenesis of age-related diseases.

[0136] To examine whether substantial and measurable reprogramming of aging cells can be achieved before the point of no return and whether this can result in the remission of cellular function and physiology, we evaluated the effect of transient reprogramming on the aging physiology of two distinct cell types, fibroblasts and endothelial cells, derived from otherwise healthy human subjects and compared them with the same cell types obtained from young donors. Fibroblasts were derived from minced arm and abdominal skin biopsies (young control, 25-35 years old, n = 3; aged group, 60-70 years old, n = 3), while endothelial cells were extracted from collagenase digestion of iliac veins and arteries (young control, 15-25 years old, n = 3; aged group, 45-50 years old, n = 3).

[0137] We utilized a non-integrative reprogramming protocol. We optimized the protocol based on a cocktail of mRNAs expressing OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG (OSKMLN). Multiple reprogramming durations were explored, and both cell types exhibited rapid changes in many aging parameters as early as R2X2 (two reprogramming transfections followed by two days of relaxation back to the ground state), with the most pronounced effects observed in R4X2 (Figures 1A and 2A). The protocol consistently produces induced pluripotent stem cell (iPSC) colonies after 12–15 daily transfections, regardless of donor age; based on the observation that the first detectable expression of endogenous pluripotency-associated lncRNAs occurs on day 5, we concluded that PNR in our platform occurs around day 5 of reprogramming. Therefore, we employed a transient reprogramming protocol in which OSKMLN was transfected daily for 4 consecutive days, with gene expression analysis performed 2 days after interruption.

[0138] Paired-end bulk RNA sequencing was performed in both cell types for the same three cohorts: young (Y), untreated vs. aged (UA), and treated vs. aged (TA). We first compared the quantile-normalized transcriptomes of young and untreated vs. aged cells per cell type ("Y vs. UA"). The data show that 961 genes (5.85%) in fibroblasts (678 up-regulated, 289 down-regulated) and 748 genes (4.80%) in endothelial cells (389 up-regulated, 377 down-regulated) differed between young and aged cells at a significance criterion of p<0.05 and a log fold change cutoff of + / -0.5 (Figure 6). These gene sets were enriched for many of the known aging pathways identified in the signature gene set collection of the Molecular Signature Database. When the directionality of above- or below-average expression for each gene was mapped, a clear similarity between treated and young cells, as opposed to aged cells, was observed for both fibroblasts and endothelial cells. Principal component analysis (PCA) on this gene set space determined that the young and aged populations were separable along the first principal component (PC1), which explained 64.8% of the variance in fibroblasts and 60.9% of the variance in endothelial cells. Interestingly, treated cells also clustered closer to the younger population along PC1 (Figures 1K and 2J).

[0139] Using the same significance criteria defined above, we compared untreated and treated aging populations ("UA vs. TA") and found 1042 genes (734 upregulated, 308 downregulated) in fibroblasts and 992 (461 upregulated, 531 downregulated) in endothelial cells that were differentially expressed. Interestingly, even within these gene sets, we found enrichment for aging pathways within the molecular signature database. When comparing young vs. untreated-aged ("Y vs. UA") and untreated-aged vs. treated-aged ("UA vs. TA") profiles in each cell type, a 24.7% overlap in fibroblasts (odds ratio 4.53, p<0.05) and a 16.7% overlap in endothelial cells (odds ratio 3.84, p<0.05) was observed, with the direction of gene expression changes matching that of young (i.e., higher in young is higher in treated-aged); less than 0.5% of either cell type shifted in the opposite direction.

[0140] Next, these transcriptome profiles were used to verify the retention of cell identity after transient reprogramming. To this end, using established cell identity markers, the applicants verified that none were significantly altered by the treatment (Figure 10). In addition, the applicants were unable to detect the expression of any pluripotency-related markers (other than transfected OSKMLN mRNA) (Figure 10). In summary, the analysis of transcriptome signatures revealed that transient reprogramming induces a more youthful gene expression profile while retaining cell identity.

[0141] Epigenetic clocks based on DNA methylation levels are the most accurate molecular biomarkers of age across tissues and cell types and predict many aging conditions, including lifespan. Exogenous expression of canonical reprogramming factors (OSKMs) is known to revert the epigenetic age of primary cells to prenatal states. To examine whether transient expression of OSKMLN can reverse the epigenetic clock, we used two epigenetic clocks applied to human fibroblasts and endothelial cells: Horvath's original pan-tissue epigenetic clock (based on 353 cytosine-phosphate-guanine pairs) and the skin and blood clock (based on 391 CpGs).

[0142] According to the pan-tissue epigenetic clock, transient OSKMLN significantly (two-sided mixed-effects model P = 0.023) reverted DNA methylation age (mean age difference = -3.40 years, standard error 1.17). The rejuvenation effect was more pronounced in endothelial cells (mean age difference = -4.94 years, SE = 1.63, Figure 6H) than in fibroblasts (mean age difference = -1.84, SE = 1.46, Figure 6G). Using the skin and blood epigenetic clocks, qualitatively similar but less significant results were obtained (overall rejuvenation effect -1.35 years, SE = 0.67, one-sided mixed-effects model P = 0.042; mean rejuvenation in endothelial cells and fibroblasts was -1.62 years and -1.07 years, respectively).

[0143] These results prompted us to analyze the effects of transient reprogramming on various hallmarks of cellular physiological aging. Using a panel of 11 established assays spanning the hallmarks of aging (Figure 11), we performed the majority of our analysis using single-cell high-throughput imaging to capture quantitative changes and distribution shifts of single cells across entire cell populations. All analyses were performed separately for each individual cell line (a total of 19 fibroblast cell lines: 3 young, 8 aged, and 8 treatment-aged; a total of 17 endothelial cell lines: 3 young, 7 aged, and 7 treatment-aged). Statistical analyses were performed for each paired sample set; data were subsequently pooled by age category for ease of presentation (see Materials and Methods for a detailed description of the statistical methods used). Control experiments were performed employing the same transfection scheme using mRNA encoding GFP.

[0144] To expand our epigenetic knowledge, we performed experiments to quantitatively measure the epigenetic repressive mark H3K9me3, the heterochromatin-associated protein HP1γ, and the nuclear lamina-supporting protein LAP2α by immunofluorescence (IF) (Figures 1B, 1C, 2B, 2C, 5A-5C). Aged fibroblasts and endothelial cells showed decreased nuclear signals for all three markers compared to young cells. Treatment of aged cells resulted in an increase in these markers in both cell types. Next, we examined pathways involved in cellular proteolytic activity by measuring autophagosome formation and chymotrypsin-like proteasome activity, both of which decrease with age. Treatment increased both pathways to levels similar to or even higher than those in young cells, suggesting that an early step in reprogramming promotes the active clearance of degraded biomolecules (Figures 1D, 2D, 5G-5H).

[0145] From the perspective of energy metabolism, aged cells exhibit decreased mitochondrial activity, reactive oxygen species (ROS) accumulation, and deregulated nutrient sensing. Therefore, we examined the effects of treatment in aged cells by measuring mitochondrial membrane potential, mitochondrial ROS, and sirtuin 1 protein (SIRT1) levels in the cells. Transient reprogramming increased mitochondrial membrane potential in both cell types (Figure 1E, left panel; Figure 2E, left panel; Figure 5E), which, like young cells (Figure 1F, Figure 2F, and Figure 5D), reduced ROS (Figure 1E, right panel; Figure 2E, right panel; Figure 5F) and increased SIRT1 protein levels in fibroblasts. Senescence-associated beta-galactosidase staining showed a significant reduction in the number of senescent cells in aged endothelial cells (Figure 1H, Figure 2H, and Figure 5I). This reduction was accompanied by a decrease in pro-inflammatory senescence-associated secretory phenomenon (SASP) cytokines, also in endothelial cells (Figure 5J). Finally, in both cell types, telomere length, measured by quantitative fluorescence in situ hybridization, did not significantly increase with treatment (Fig. 1G and Fig. 2G), suggesting that the cells did not dedifferentiate to a stem cell-like state in which telomerase activity is reactivated.

[0146] We next assessed the durability of these effects and found that most were significantly retained 4 and 6 days after interruption of reprogramming. We investigated how rapidly these physiological rejuvenation changes manifested by repeating the same set of experiments in fibroblasts and endothelial cells transfected for only 2 consecutive days. Remarkably, the data showed that most of the rejuvenation effects were already visible after 2 days of treatment, although they were mostly more modest.

[0147] Taken together, this data demonstrates that transient expression of OSKMLN can induce rapid and sustained reversal of cellular aging in human cells at the transcriptomic, epigenetic, and cellular levels. Importantly, these data demonstrate that a process of "cellular rejuvenation," termed epigenetic reprogramming of aging or "ERA" herein, is involved very early and rapidly in the iPSC reprogramming process. These epigenetic and transcriptional changes occur before any epigenetic reprogramming of the cell identity takes place.

[0148] Given these indications of ERA's beneficial effects on cellular aging, we conducted experiments to investigate whether ERA could also reverse the inflammatory phenotype associated with aging. After obtaining preliminary evidence of this reversal in endothelial cells (Figure 5J), we extended our analysis to osteoarthritis, a disease strongly associated with aging and characterized by a pronounced inflammatory spectrum affecting chondrocytes within the joint. We isolated chondrocytes from cartilage of 60-70 year-old patients undergoing total joint replacement surgery for advanced OA and compared treatment outcomes with chondrocytes isolated from younger individuals. Transient reprogramming was performed for 2 or 3 days, and analysis was performed 2 days after reprogramming was discontinued, but longer treatments produced more consistent effects across patients. Treatment demonstrated significant reductions in pro-inflammatory cytokines (Figure 7I), intracellular mRNA levels of RANKL and iNOS2, and levels of inflammatory factors secreted by the cells (Figures 3H-3I and 7I). Additionally, ERA promoted cell proliferation (Figures 3A and 7D), increased ATP production (Figures 3C and 7A), and reduced oxidative stress as evidenced by reduced mitochondrial ROS and elevated RNA levels of the antioxidant SOD2, a gene shown to be downregulated in OA (Figures 3D, 3E, 7B, and 7D). ERA did not affect the expression levels of SOX9 (a transcription factor central to chondrocyte identity and function) but significantly increased the expression levels of COL2A1 (a major collagen in articular cartilage) (qRT-PCR in Figures 3B, 7E, and 7F), suggesting the retention of chrondrogenic cell identity. Together, these results indicate that transient expression of OSKMLN can partially reverse gene expression and promote cellular physiology in aged OA chondrocytes toward a healthier state.

[0149] Loss of stem cell function and regenerative capacity represents another important hallmark of aging. Experiments were conducted to evaluate the effect of transient reprogramming on age-related changes in somatic stem cells that impair regeneration. First, the effect of transient reprogramming was examined in mouse-derived skeletal muscle stem cells (MuSCs). MuSCs were treated for two days while maintained in a quiescent state using an artificial niche. Initial experiments were performed using young (3 months) and aged (20-24 months) mouse MuSCs isolated by FACS. Treatment of aged MuSCs reduced both the time to first division and mitochondrial mass, which approximates the faster activation kinetics of quiescent young MuSCs. Furthermore, treatment partially rescued the reduced ability of single MuSCs to form colonies. Further culturing of these cells revealed that treatment did not alter the expression of the myogenic marker MyoD but instead improved their ability to differentiate into myotubes, suggesting that transient reprogramming does not disrupt myogenic fate but may enhance myogenic potential.

[0150] Next, we examined MuSC function and differentiation potential to regenerate new tissue in vivo. To do this, we transduced young, aged, or transiently reprogrammed aged MuSCs with lentivirus expressing luciferase and green fluorescent protein (GFP) and then transplanted these cells into injured tibialis anterior (TA) muscles of immune-compromised mice. Longitudinal bioluminescence imaging (BLI) showed that initially, muscles transplanted with treated and aged MuSCs exhibited the highest signal (day 4, Figure 4B), but by day 11 post-transplantation, this signal became comparable to that of muscles with young MuSCs; conversely, muscles with untreated and aged MuSCs exhibited lower signals at all time points post-transplantation (Figure 4B). Immunofluorescence analysis revealed that TAs transplanted with treated MuSCs exhibited a higher level of GFP (from a greater number of donors) compared with untreated and aged MuSCs. + ) further revealed muscle fibers (Figure 4C). Furthermore, GFP from treated and aged cells +Muscle fibers exhibited increased cross-sectional area compared with their untreated counterparts, and in fact were even larger than those in young controls (Figure 4D). Taken together, these results suggest improved tissue regeneration capacity of transiently reprogrammed aged MuSCs. Three months later, all mice were autopsied, and no neoplastic lesions or teratomas were found.

[0151] To examine the potential long-term benefits of the treatment, a second injury was induced 60 days after cell transplantation, and again, the data showed that TA muscles transplanted with transiently reprogrammed aged MuSCs produced higher BLI signals (Figure 4E).

[0152] Sarcopenia is an age-related condition characterized by a loss of muscle mass and force production. Similarly, in mice, muscle function exhibits progressive degeneration with age. To examine whether transient reprogramming of aged MuSCs improves cell-based treatments in restoring muscle physiological function in older mice, we performed electrophysiological studies to measure tetanic force production in TA muscles isolated from young (4 months) or aged (27 months) immune-compromised mice. Data showed that TA muscles from aged mice had lower tetanic force compared to young mice, suggesting an age-related loss of force production (Figure 8F). Next, we isolated MuSCs from aged mice (20-24 months). After treatment with aged MuSCs, we transplanted the cells into cardiotoxin-injured TA muscles of aged (27 months) immune-compromised mice. Thirty days were allowed for sufficient time for the transplanted muscles to fully regenerate. Electrophysiological studies to measure tetanic force production were performed. Muscles transplanted with untreated and aged MuSCs exhibited force comparable to that of untransplanted muscles from aged control mice (Figure 4h). Conversely, muscles receiving treated and aged MuSCs exhibited tetanic force comparable to that of untransplanted muscles from young control mice. These results support that transient reprogramming combined with MuSC-based therapy can restore the physiological function of aged muscle to that of youthful muscle.

[0153] Finally, we translated these results to human MuSCs. We repeated the study using surgical specimens from patients of different age ranges (10-80 years) transduced with GFP- and luciferase-expressing lentiviral vectors. Similar to mice, transplanted transiently reprogrammed aged human MuSCs resulted in increased BLI signals compared with untreated MuSCs from the same individuals, comparable to those observed in young MuSCs (Figure 8D). Interestingly, the BLI signal ratio between contralateral muscles from treated and untreated MuSCs was higher in older age groups (60-80 years) than in younger age groups (10-30 or 30-55 years), suggesting that ERA restores lost function in aged cells to younger levels (Figure 8E). Taken together, these results suggest that transient reprogramming can partially restore the differentiation potential of aged MuSCs to a degree similar to that of young MuSCs without compromising their fate, and thus has potential as a cell therapy in regenerative medicine.

[0154] Three-dimensional (3D) in vitro engineered skin was reconstituted by combining fibroblasts and keratinocytes from patients over 65 years of age and transfected with a cocktail of reprogramming factors by adding them to the culture medium. Histological analysis was performed to assess quality, and a numerical score was assigned (Figure 8A). Rejuvenation was observed with the reprogramming factors, as measured by increased numerical scores compared to control untreated and retinoic acid-treated samples.

[0155] Retinal epithelial cells were cultured ex vivo and transiently reprogrammed with OSKMN for 2 or 3 days. The results showed a significant decrease in the expression of p16 (Fig. 9A), p21 (Fig. 9B), IL8 (Fig. 9C), and PGC1a (Fig. 9D).

[0156] Nuclear reprogramming into induced pluripotent stem cells (iPSCs) is a multiphasic process involving initiation, maturation, and stabilization. After the completion of this dynamic and complex "epigenetic reprogramming," iPSCs are not only pluripotent but also youthful. The data herein demonstrate that a non-integrative, mRNA-based platform for transient cell reprogramming can very rapidly reverse aging characteristics during the initiation phase, when epigenetic erasure of cell identity has not yet occurred. The data indicate that a rejuvenation process occurs in aged human cells by restoring lost functionality in diseased cells and aged stem cells while preserving cell identity.

[0157] Example 2: Method mRNA transfection Cells were transfected using either mRNA-In (mTI Global Stem) for fibroblasts and chondrocytes, which reduces cytotoxicity, or Lipofectamine MessengerMax (Thermo Fisher) for endothelial cells and MuSCs, which are more difficult to transfect, using the manufacturer's protocols. Culture medium was changed for fibroblasts and endothelial cells 4 hours after transfection, but not for chondrocytes or MuSCs, as overnight incubation is required to produce significant mRNA uptake. Delivery efficiency was confirmed by both GFP mRNA and immunostaining for individual factors in the OSKMNL cocktail. mRNA synthesis and transfection optimization were performed by Jens Durruthy-Durruthy, a member of the Sebastiano Lab, at the facility at ESI BIO, where he serves as a consultant.

[0158] Fibroblast isolation and cultureIsolations were performed in healthy patients, using 2 mm punch biopsies from the mesial surface of the mid-upper arm or abdomen from a mix of male and female patients in their 60s-70s (older) and 30s-40s (younger). Cells from these explants were cultured and maintained in Eagle's minimum essential medium containing Earle's salts, supplemented with nonessential amino acids, 10% fetal bovine serum, and 1% penicillin / streptomycin.

[0159] Endothelial cell isolation and culture At the Coriell Institute, the cells were isolated from iliac arteries and veins removed premortem from otherwise healthy donors in their 40s and 50s (older) and teenage years (younger) who had died from sudden head trauma. The tissue was digested with collagenase, and cells released from the lumen were used to initiate cultures. Cells were maintained in Medium 199 supplemented with 2 mM L-glutamine, 15% fetal bovine serum, 0.02 mg / ml endothelial growth supplement, 0.05 mg / ml heparin, and 1% penicillin / streptomycin.

[0160] nuclear immunocytochemistry Cells were washed with HBSS and then fixed with 15% paraformaldehyde in PBS for 15 minutes. Next, cells were blocked with a blocking solution of 1% BSA and 0.3% Triton X-100 in PBS for 30 minutes. Next, primary antibodies were applied in 1% BSA and 0.3% Triton X-100 in PBS and incubated overnight at 4°C. The next day, cells were washed with HBSS and incubated for 2 hours before switching to the corresponding Alexa Flour-labeled secondary antibody. Next, cells were washed again and then stained with DAPI for 30 minutes. Finally, cells were switched to HBSS for imaging.

[0161] Autophagosome formation stainingCells were washed with HBSS and switched to a staining solution containing an LC3-based fluorescent autophagosome marker (Sigma). Cells were then incubated at 37°C with 5% CO for 20 minutes. Next, cells were washed twice with HBSS / Ca / Mg. Cells were then stained with the cell-labeling dye CellTracker Deep Red for 15 minutes. Cells were then switched to HBSS / Ca / Mg for single-cell imaging with Operetta.

[0162] Proteasome activity measurement Wells were first stained with the cell viability dye PrestoBlue (Thermo) for 10 minutes. Well signals were read using a TECAN fluorescent plate reader. Cells were then washed with HBSS / Ca / Mg before switching to original medium containing LLVY-R110 fluorogenic substrate (Sigma), which is cleaved by chymotrypsin-like 20S proteasome activity. Cells were then incubated at 37°C with 5% CO for 2 hours before being read again in the TECAN fluorescent plate reader.

[0163] Mitochondrial membrane potential staining: Tetramethylrhodamine, methyl ester, perchlorate (Thermo) was added to cell culture medium. This dye is sequestered by mitochondria based on their membrane potential. Cells were then incubated for 30 minutes at 37°C with 5% CO2. Cells were then washed twice with HBSS / Ca / Mg before staining with CellTracker Deep Red for 15 minutes. Finally, cells were imaged in Operetta in fresh HBSS / Ca / Mg.

[0164] Mitochondrial ROS measurementCells were washed with HBSS / Ca / Mg and then switched to HBSS / Ca / Mg containing MitoSOX, a fluorogenic dye oxidized by superoxide in mitochondria. Cells were incubated for 10 minutes at 37°C with 5% CO2. Next, cells were washed twice with HBSS / Ca / Mg and then stained with CellTracker Deep Red for 15 minutes. Finally, cells were imaged in fresh HBSS / Ca / Mg with Operetta.

[0165] SaβGal histochemistry Cells were washed twice with HBSS / Ca / Mg and then fixed with 15% paraformaldehyde in PBS for 6 minutes. Cells were then rinsed three times with HBSS / Ca / Mg before staining with X-gal chromogenic substrate, which is cleaved by endogenous B-galactosidase. Cells were maintained in the staining solution and incubated overnight at 37°C with ambient CO2. The next day, cells were washed again with HBSS / Ca / Mg before switching to a 70% glycerol solution for imaging under a Leica brightfield microscope.

[0166] Cytokine profilingThis work was performed in collaboration with the Human Immune Monitoring Center at Stanford University. Cell culture media was collected and spun at 400 rcf for 10 minutes at room temperature. The supernatant was then snap-frozen in liquid nitrogen until analysis. Analysis was performed using the Human 63-plex kit (eBiosciences / Affymetrix). Beads were added to a 96-well plate and washed in a Biotek ELx405 washer. Samples were added to the plate containing the mixed antibody-conjugated beads and incubated at room temperature for 1 hour, followed by an overnight incubation at 4°C with shaking. Low-temperature and room-temperature incubation steps were performed on an orbital shaker at 500-600 rpm. After the overnight incubation, the plate was washed in a Biotek ELx405 washer, followed by the addition of biotinylated detection antibody for 75 minutes with shaking at room temperature. The plate was washed as described above, and streptavidin-PE was added. After a 30-minute incubation at room temperature, washing was performed as described above, and read buffer was added to the wells. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument with 50 lower binding beads per cytokine per sample. Custom assay control beads from Radix Biosolutions were added to all wells.

[0167] antibody Five primary antibodies were used for nuclear measurements: rabbit anti-histone H3K9me3 histone methylation (1:4000), mouse anti-HP1γ heterochromatin marker (1:200), rabbit anti-LAP2α (1:500) nuclear organizing protein, mouse anti-laminin A / C nuclear envelope marker, and rabbit anti-SIRT1 (1:200).

[0168] RNA sequencing and data analysis Cells were washed and digested with TRIzol (Thermo). Total RNA was isolated using a total RNA purification kit (Norgen Biotek Corp). RNA quality was assessed using an RNA analysis screentape (R6K screentape, Agilent). RNA with an RIN > 9 was reverse transcribed into cDNA. A cDNA library was prepared using 1 μg of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina). RNA quality was assessed using an Agilent Bioanalyzer 2100. RNA with an RIN > 9 was reverse transcribed into cDNA. A cDNA library was prepared using 500 ng of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina), which has the added benefit of molecular indexing. Prior to either PCR amplification step, all cDNA fragment ends were randomly ligated to adapter pairs containing unique 8-bp molecular indexes. The molecularly indexed cDNA library was then PCR amplified (15 cycles) and subsequently QC was performed using Bioanalyzer and Qubit. After successful QC, the reads were sequenced on an Illumina Nextseq platform to obtain 80-bp single-end reads. The reads were trimmed by two nucleotides at each end to remove low-quality segments and improve mapping to the genome. The resulting 78-nucleotide reads were compressed by removing duplicates, but the number of times each sequence occurred in each sample in the database was tracked. Next, unique reads were mapped to the human genome using exact matches. This removed exon-exon boundary crossings and misread reads with errors and SNPs / mutations, but this did not substantially affect the estimation of the expression level of each gene. Each mapped read was then assigned an annotation derived from the underlying genome. In the case of multiple annotations (e.g., miRNAs occurring in the introns of a gene), a heuristic-based hierarchy was used to assign a unique identity to each read.This was then used to identify reads belonging to each transcript and establish the coverage across each position of the transcript. Because this coverage was uneven and peaked, we used the median of this coverage as an estimate of the gene's expression value. Quantile normalization was used to compare expression in different samples. Further data analysis was performed in MATLAB®. Ratios of expression levels were then calculated to estimate the logarithm (base 2) of the fold change. Student's t-test was used to determine significance with a p<0.05 cutoff. ENCODE gene analysis, developed and publicly available by the Butte Lab at the Stanford Center for Biomedical Informatics Research, was used for transcription factor identification.

[0169] mouse C57BL / 6 male and NSG mice were obtained from Jackson Laboratory. NOD / MrkBomTac-Prkdcscid female mice were obtained from Taconic Biosciences. Mice were housed and maintained in the Veterinary Medical Unit at Veterans Affairs Palo Alto Health Care Systems. Animal protocols were approved by the Administrative Panel of Laboratory Animal Care at Stanford University.

[0170] Human skeletal muscle specimen Subjects ranged in age from 10 to 78 years. Human muscle biopsy specimens were collected after obtaining patient informed consent as part of a human testing research protocol approved by the Stanford University Institutional Review Board. All experiments were performed using fresh muscle specimens according to clinical procedure availability. Sample processing for cellular analysis began within 1 to 12 hours of specimen isolation. In all studies, standard deviations reflect variability in data derived from studies using true biological replicates (i.e., unique donors). Data were not correlated with donor identity.

[0171] MuSC isolation and purificationMuscles were collected from the hind limbs and mechanically dissociated to obtain a fragmented muscle suspension. This was followed by a 45-50 min digestion in Collagenase II-Ham's F10 solution (500 units per ml; Invitrogen). After washing, a second digestion was performed with Collagenase II (100 units per ml) and Dispase (2 units per ml; ThermoFisher) for 30 min. The resulting cell suspension was washed, filtered, and stained with 1:100 dilutions of VCAM-biotin (clone 429; BD Bioscience), CD31-FITC (clone MEC13.3; BD Bioscience), CD45-APC (clone 30-F11; BD Bioscience), and Sca-1-Pacific-Blue (clone D7; Biolegend) antibodies. Human MuSCs were purified from fresh surgical specimens. Surgical specimens were carefully dissected free of fat and fibrous tissue, and dissociated muscle suspensions were prepared as described for mouse tissue. The resulting cell suspensions were then washed, filtered, and stained with anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2 / 16; BioLegend; #303008, 1:75), and anti-NCAM-biotin (clone HCD56; BioLegend; #318319, 1:75). Next, unbound primary antibody was washed away, and cells were incubated in streptavidin-PE / Cy7 (BioLegend) for 15 minutes at 4°C to detect NCAM-biotin. MuSC populations were obtained by cell sorting on a calibrated BD-FACS Aria II® or BD FACSAria III flow cytometer equipped with 488-nm, 633-nm, and 405-nm lasers. A small portion of the sorted cells was plated and stained for Pax7 and MyoD to assess the purity of the sorted population. See Supplementary Information for FACS gating strategies.

[0172] Bioluminescence Imaging Bioluminescent imaging was performed using a Xenogen IVIS-Spectrum System (Caliper Life Sciences). Mice were anesthetized using 2% isoflurane at a flow rate of 2.5 l / min (n=4). An intraperitoneal injection of D-luciferin (50 mg / ml, Biosynth International Inc.) dissolved in sterile PBS was administered. Immediately after injection, mice were imaged for 30 seconds at maximum sensitivity (f-stop 1) and highest resolution (small binning). A 30-second exposure was used every minute until the peak intensity of the bioluminescent signal began to diminish. Each image was saved for subsequent analysis. Imaging was performed in a blinded manner; the researchers performing the imaging were unaware of the identity of the experimental conditions of the transplanted cells.

[0173] Bioluminescence image analysis Each image was analyzed using Living Image software, version 4.0 (Caliper Life Sciences). A manually generated circle was placed over the region of interest and resized to completely surround the recipient mouse limb or designated area. Similarly, a background region of interest was placed over the area of ​​the mouse outside the transplanted leg.

[0174] Tissue collection The TA muscles were carefully dissected away from the bone, weighed, and placed in 0.5% PFA solution overnight for fixation. They were then transferred to a 20% sucrose solution for 3 hours, or until the muscle reached its saturation point and began to sink. The tissue was then embedded in Optimal Cutting Temperature (OCT) medium, frozen, and stored at -80°C until sectioning. Sectioning was performed on a Leica CM3050S cryostat set to produce 10 μm sections. Sections were mounted on Fisherbrand Colorfrost slides. These slides were stored at -20°C until immunohistochemistry could be performed.

[0175] histology TA muscles were fixed for 5 hours using 0.5% electron microscopy-grade paraformaldehyde and then transferred to 20% sucrose overnight. The muscles were then frozen in OCT and cryosectioned at 10 μm thickness for staining. Samples were processed for colorimetric staining with hematoxylin and eosin (Sigma) or Gomorri trichrome (Richard-Allan Scientific) according to the manufacturer's recommended protocol.

[0176] MuSC immunostaining A 1-hour blocking step with 20% donkey serum / 0.3% Triton in PBS was used to prevent unwanted primary antibody binding for all samples. Primary antibodies were applied and incubated overnight at 4°C in 20% donkey serum / 0.3% Triton in PBS. After four washes with 0.3% PBST, fluorescently conjugated secondary antibodies were added and incubated in 0.3% PBST for 1 hour at room temperature. After three additional rinses, each slide was mounted using Fluoview mounting medium.

[0177] antibody The following antibodies were used in this study. The source of each antibody is indicated. Mouse: GFP (Invitrogen, #A11122, 1:250); luciferase (Sigma-Aldrich, #L0159, 1:200); collagen I (Cedarlane Labs, #CL50151AP, 1:200); HSP47 (Abcam, #ab77609, 1:200).

[0178] Imaging Samples were imaged using a standard fluorescence microscope and either a 10x or 20x air objective. Volocity imaging software was used to adjust excitation and emission filters, with pre-programmed AlexaFluor filter settings that were used whenever possible. Total exposure times were optimized for the first round of imaging and then kept constant throughout all subsequent imaging.

[0179] Image analysis Using Image J, the percentage of area composed of collagen was calculated using the color threshold plug-in to create a mask of only collagen-positive areas. This area was then divided by the total area of ​​the sample found using the free drawing tool. All other analyses were performed using Volocity software, where fibers were manually counted using the free drawing tool, and the number of nuclei, eMHC+ fibers, neuromuscular junctions, and blood vessels were manually counted.

[0180] Lentiviral transduction Luciferase and GFP protein reporters were subcloned into a third-generation HIV-1 lentiviral vector (CD51X DPS, SystemBio). To transduce freshly isolated MuSCs, cells were plated at a density of 30,000–40,000 cells per well on poly-D-lysine (Millipore Sigma, A-003-E) and ECM-coated 8-well chamber slides (Millipore Sigma, PEZGS0896) and incubated with 5 μl of concentrated virus per well and 8 μg / mL polybrene (Santa Cruz Biotechnology, sc-134220). Plates were spun at 3200 g for 5 min and 2500 g for 1 h at 25°C. Cells were then washed twice with fresh medium, scraped from the plate, and resuspended in the final volume appropriate for the experimental conditions.

[0181] MitoTracker staining and flow cytometry analysis MuSCs undergoing reprogramming and controls were washed twice with pure HamsF10 (no serum or pen / strep). MuSCs were then stained with 0.5 μM MitoTracker Green FM (ThermoFisher, M7514) and DAPI for 30 minutes at 37°C, washed three times with pure HamsF10, and analyzed using a BD FACSAria III flow cytometer.

[0182] statistical analysisUnless otherwise stated, all statistical analyses were performed using MATLAB R2017a (MathWorks Software) or GraphPad Prism 5 (GraphPad Software). For statistical analysis, t-tests were used. All error bars represent s.e.m.; *p<0.05; **p<0.001; ***p<0.0001.

[0183] While preferred embodiments of the present disclosure have been illustrated and described, it will be recognized that various changes can be made therein without departing from the spirit and scope of the disclosure.

[0184] [Table 1] TIFF2025131775000003.tif229164TIFF2025131775000004.tif168155

[0185] P embodiment

[0186] Embodiment P1. A method of rejuvenating cells, comprising: a) transfecting a cell with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, wherein the transfecting is performed once per day for at least two days and not more than four days; and b) translating the one or more non-integrated messenger RNAs to produce the one or more cellular reprogramming factors in the cell, resulting in transient reprogramming of the cell, wherein the cell rejuvenates without dedifferentiation into a stem cell.

[0187] Embodiment P2. The method of embodiment P1, wherein the one or more cellular reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0188] Embodiment P3. The method of embodiment P2, wherein the cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0189] Embodiment P4. The method of embodiment P1, wherein the cell is a mammalian cell.

[0190] Embodiment P5. The method of embodiment P4, wherein the cell is a human cell.

[0191] Embodiment P6. The method of embodiment P1, wherein the cells are derived from an elderly subject.

[0192] Embodiment P7. The method of embodiment P1, wherein the cells are fibroblasts, endothelial cells, chondrocytes, or skeletal muscle stem cells.

[0193] Embodiment P8. The method of embodiment P1, wherein transient reprogramming results in increased expression of HP1 gamma, H3K9me3, lamina support protein LAP2 alpha and SIRT1 protein, decreased nuclear folding, decreased blebbing, increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, increased mitochondrial membrane potential, or decreased reactive oxygen species (ROS).

[0194] Embodiment P9. The method of embodiment P1, wherein the cell is in a tissue or organ.

[0195] Embodiment P10. The method of embodiment P9, wherein transient reprogramming reduces the number of senescent cells in a tissue or organ.

[0196] Embodiment P11. The method of embodiment P9, wherein the transient reprogramming reduces expression of GMSCF, IL18 and TNFα.

[0197] Embodiment P12. The method of embodiment P9, wherein the treatment restores function, increases differentiation potential, enhances survival, or increases replicative capacity or lifespan of cells within the tissue or organ.

[0198] Embodiment P13. The method of embodiment P1, performed in vitro, ex vivo or in vivo.

[0199] Embodiment P14. The method of embodiment P1, wherein the transfecting step is performed once a day for 3 or 4 days.

[0200] Embodiment P15. A method for treating an age-related disease or condition in a subject, comprising the steps of: a) transfecting in vivo or ex vivo a cell in need of rejuvenation with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, wherein the transfecting is performed once daily for at least 2 days and not more than 4 days; and b) expressing the one or more cellular reprogramming factors in the cell, resulting in transient reprogramming of the cell, wherein the cell rejuvenates without dedifferentiation into a stem cell.

[0201] Embodiment P16. The method of embodiment P15, wherein the one or more cellular reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0202] Embodiment P17. The method of embodiment P16, wherein the cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0203] Embodiment P18. The method of embodiment P15, further comprising transplanting the rejuvenated cells into a subject.

[0204] Embodiment P19. The method of embodiment P15, wherein the age-related disease or condition is a degenerative disease.

[0205] Embodiment P20. The method of embodiment P15, wherein the age-related disease or condition is a neurodegenerative disease or a musculoskeletal disorder.

[0206] Embodiment P21. A method for treating a disease or disorder involving cartilage degeneration in a subject, comprising the steps of: a) transfecting chondrocytes in need of rejuvenation in vivo or ex vivo with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, wherein the transfecting is performed once daily for at least 2 days and not more than 4 days; and b) expressing the one or more cellular reprogramming factors in the chondrocytes, resulting in transient reprogramming of the chondrocytes, wherein the chondrocytes rejuvenate without dedifferentiation into stem cells.

[0207] Embodiment P22. The method of embodiment P21, wherein the one or more cellular reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0208] Embodiment P23. The method of embodiment P22, wherein the cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0209] Embodiment P24. The method of embodiment P21 wherein the disease or disorder involving cartilage degeneration is arthritis.

[0210] Embodiment P25. The method of embodiment P24, wherein the arthritis is osteoarthritis or rheumatoid arthritis.

[0211] Embodiment P26. The method of embodiment P21, wherein the treatment reduces inflammation in the subject.

[0212] Embodiment P27. The method of embodiment P21, wherein the transfecting step is performed ex vivo and the rejuvenated chondrocytes are transplanted into the arthritic joint of the subject.

[0213] Embodiment P28. The method of embodiment P27, wherein the chondrocytes are isolated from a cartilage sample obtained from the subject.

[0214] Embodiment P29. The method of embodiment P21 wherein the treatment reduces expression of RANKL, iNOS, IL6, IL8, BDNF, IFNα, IFNγ and LIF, and increases expression of SOX9 and COL2A1 by chondrocytes.

[0215] Embodiment P30. The method of embodiment P21, wherein the subject is an elderly subject.

[0216] Embodiment P31. The method of embodiment P21, wherein the subject is a mammalian subject.

[0217] Embodiment P32. The method of embodiment P31, wherein the mammalian subject is a human subject.

[0218] Embodiment P33. A method for treating a disease or disorder involving muscle degeneration in a subject, the method comprising the steps of: a) transfecting skeletal muscle stem cells in vivo or ex vivo with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, wherein the transfecting is performed once daily for at least 2 days and not more than 4 days; and b) expressing the one or more cellular reprogramming factors in the skeletal muscle stem cells, resulting in transient reprogramming of the skeletal muscle stem cells, wherein the skeletal muscle stem cells are rejuvenated without losing their ability to differentiate into muscle cells.

[0219] Embodiment P34. The method of embodiment P33, wherein the one or more cellular reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0220] Embodiment P35. The method of embodiment P34, wherein the cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0221] Embodiment P36. The method of embodiment P33, wherein the transfecting step is performed ex vivo and the rejuvenated skeletal muscle stem cells are transplanted into a muscle in need of repair or regeneration in the subject.

[0222] Embodiment P37. The method of embodiment P33, wherein the skeletal muscle stem cells are isolated from a muscle sample obtained from the subject.

[0223] Embodiment P38. The method of embodiment P33, wherein the treatment results in regeneration of muscle fibers.

[0224] Embodiment P39. The method of embodiment P33, wherein the treatment restores the differentiation potential of skeletal muscle stem cells.

[0225] Embodiment P40. The method of embodiment P33, wherein the subject is an elderly subject.

[0226] Embodiment P41. The method of embodiment P33, wherein the subject is a mammalian subject.

[0227] Embodiment P42. The method of embodiment P41, wherein the mammalian subject is a human subject.

[0228] Embodiment

[0229] Embodiment 1. A method of rejuvenating a cell, comprising transfecting the cell for no more than five consecutive days with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors, thereby producing a rejuvenated cell.

[0230] Embodiment 2. The method of embodiment 1, wherein the transcriptome profile of the rejuvenated cells becomes similar to the transcriptome profile of young cells.

[0231] Embodiment 3. The method of embodiment 2, wherein the transcriptome profile of the rejuvenated cells comprises increased gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1, and Sin3a.

[0232] Embodiment 4. The method of any one of the preceding embodiments, wherein the rejuvenated cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to a reference value.

[0233] Embodiment 5. The method of embodiment 4, wherein the markers are selected from HP1 gamma, H3K9me3, lamina supporting protein LAP2 alpha, and SIRT1 protein.

[0234] Embodiment 6 The method of any one of the preceding embodiments, wherein the rejuvenated cells exhibit increased proteolytic activity compared to a reference value.

[0235] Embodiment 7. The method of embodiment 6, wherein the increased proteolytic activity is measured as increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, or a combination thereof.

[0236] Embodiment 8 The method of any one of the preceding embodiments, wherein the rejuvenated cells exhibit improved mitochondrial health and function compared to a reference value.

[0237] Embodiment 9. The method of embodiment 8, wherein improved mitochondrial health and function is measured as increased mitochondrial membrane potential, decreased reactive oxygen species (ROS), or a combination thereof.

[0238] Embodiment 10 The method of any one of the preceding embodiments, wherein the rejuvenated cells exhibit decreased expression of one or more SASP cytokines compared to a reference value.

[0239] Embodiment 11. The method of embodiment 10, wherein the SASP cytokines comprise one or more of IL18, IL1A, GROA, IL22, and IL9.

[0240] Embodiment 12 The method of any one of the preceding embodiments, wherein the rejuvenated cells exhibit a reversal of the methylation landscape.

[0241] Embodiment 13. The method of embodiment 12, wherein the reversal of the methylation landscape is measured by estimation with a Horvath clock.

[0242] Embodiment 14. The method according to any one of embodiments 4 to 13, wherein the reference value is obtained from aged cells.

[0243] Embodiment 15. The method of any one of the preceding embodiments, wherein the step of transfecting the cells with messenger RNA comprises a method selected from lipofectamine and LT-1 mediated transfection, dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, electroporation, encapsulation of mRNA in liposomes, and direct microinjection.

[0244] Embodiment 16. The method of any one of the preceding embodiments, wherein the one or more cellular reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0245] Embodiment 17. The method of any one of the preceding embodiments, wherein the one or more cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0246] Embodiment 18 The method of any one of the preceding embodiments, wherein the cell is a mammalian cell.

[0247] Embodiment 19 The method of any one of the preceding embodiments, wherein the cell is a human cell.

[0248] Embodiment 20 The method of any one of the preceding embodiments, wherein the cells are derived from an elderly subject.

[0249] Embodiment 21 The method of any one of the preceding embodiments, wherein the cells are selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0250] Embodiment 22. The method of embodiment 21, wherein the cells are mesenchymal stem cells.

[0251] Embodiment 23. The method of embodiment 22, wherein the rejuvenated mesenchymal stem cells exhibit reduced senescence parameters (p16, p21 and positive SAβGal staining), increased cell proliferation, and / or reduced ROS levels.

[0252] Embodiment 24 The method of any one of the preceding embodiments, which is carried out in vitro, ex vivo, or in vivo.

[0253] Embodiment 25. The method of embodiment 24, which is carried out in vivo.

[0254] Embodiment 26. The method of embodiment 25, wherein the cells are in a tissue or organ.

[0255] Embodiment 27. The method of any one of embodiments 25 to 27, wherein the number of senescent cells in a tissue or organ is reduced.

[0256] Embodiment 28. The method of any one of embodiments 25 to 27, wherein the expression of one or more of IL18, IL1A, GROA, IL22, and IL9 is reduced.

[0257] Embodiment 29. The method of any one of the preceding embodiments, wherein the method restores cell function, increases differentiation potential, enhances survival rate, increases replicative capacity or lifespan, or a combination thereof.

[0258] Embodiment 30. The method of any one of embodiments 1 to 24, wherein the transfecting step is performed once a day for 5 days.

[0259] Embodiment 31 The method of any one of embodiments 1 to 24, wherein the transfecting step is performed once a day for four days.

[0260] Embodiment 32 The method of any one of embodiments 1 to 24, wherein the transfecting step is performed once a day for three days.

[0261] Embodiment 33 The method of any one of embodiments 1 to 24, wherein the transfecting step is performed once a day for two days.

[0262] Embodiment 34. A method for treating an age-related disease or condition, a cartilage degenerative disorder, a neurodegenerative disorder and / or a musculoskeletal dysfunction in a subject, comprising administering a therapeutically effective amount of cells, wherein the cells comprise one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors.

[0263] Embodiment 35. The method of embodiment 34, wherein the one or more cellular reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0264] Embodiment 36. The method of any one of embodiments 34-35, wherein the one or more cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0265] Embodiment 37. The method of any one of embodiments 34-36, wherein the subject has an age-related disease or condition.

[0266] Embodiment 38. The method of embodiment 34, wherein the age-related disease or condition is selected from ocular, skin, or musculoskeletal dysfunction.

[0267] Embodiment 39. The method of any one of embodiments 34-36, wherein the subject has a cartilage degenerative disorder.

[0268] Embodiment 40. The method of embodiment 39, wherein the disorder is selected from arthritis, chondrophasia, spondyloarthropathy, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjogren's syndrome.

[0269] Embodiment 41 The method of any one of embodiments 39 or 40, wherein the treatment reduces the expression of inflammatory factors and / or increases ATP and collagen metabolism.

[0270] Embodiment 42. The method of embodiment 41, wherein the inflammatory factor is selected from RANKL, iNOS2, IL6, IFNα, MCP3, and MIP1A.

[0271] Embodiment 43. The method of embodiment 42, wherein ATP and collagen metabolism is measured by one or more of increased ATP levels, decreased ROS and increased SOD2, increased COL2A1, and overall proliferation by chondrocytes.

[0272] Embodiment 44. The method of any one of embodiments 34 to 36, wherein the subject has a musculoskeletal dysfunction.

[0273] Embodiment 45. The method of any one of embodiments 34 to 44, wherein the step of administering a therapeutically effective amount of cells comprises injection or surgical implantation.

[0274] Embodiment 46. The method of any one of embodiments 34 to 45, wherein the therapeutically effective amount of rejuvenated cells is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0275] Embodiment 47. The method of embodiment 46, wherein the therapeutically effective amount of rejuvenated cells are corneal epithelial cells.

[0276] Embodiment 48. The method of embodiment 47, wherein the rejuvenated corneal epithelium exhibits a decrease in aging parameters.

[0277] Embodiment 49. The method of embodiment 48, wherein the aging parameters include one or more of the expression of p21 and p16, mitochondrial biogenesis PGC1α, and expression of the inflammatory factor IL8.

[0278] Embodiment 50. A method for treating an age-related disease or condition, a cartilage degeneration disorder in a subject, and / or treating a subject having musculoskeletal dysfunction, comprising administering a therapeutically effective amount of one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors.

[0279] Embodiment 51. The method of embodiment 50, wherein the one or more cellular reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0280] Embodiment 52. The method of any one of embodiments 50-51-48, wherein the one or more cellular reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0281] Embodiment 53. The method of any one of embodiments 50-52, wherein the subject has an age-related disease or condition.

[0282] Embodiment 54. The method of embodiment 53, wherein the age-related disease or condition is selected from ocular, skin, or musculoskeletal dysfunction.

[0283] Embodiment 55. The method of any one of embodiments 50-52, wherein the subject has a cartilage degenerative disorder.

[0284] Embodiment 56. The method of embodiment 55, wherein the disorder is selected from arthritis, chondrophasia, spondyloarthropathy, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjogren's syndrome.

[0285] Embodiment 57. The method of any one of embodiments 50-52, wherein the subject has a musculoskeletal dysfunction.

[0286] Embodiment 58 The method of any one of embodiments 50 to 57, wherein administering a therapeutically effective amount of one or more non-integrated messenger RNAs comprises direct injection into the target cells.

[0287] Embodiment 59. The method of embodiment 58, wherein the target cells are selected from epithelial cells, endothelial cells, connective tissue cells, muscle cells, and nervous system cells.

[0288] Embodiment 60. A method of rejuvenating engineered tissue ex vivo, comprising transfecting the tissue with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for no more than five consecutive days, thereby producing rejuvenated engineered tissue.

[0289] Embodiment 61. The method of embodiment 60, wherein the engineered tissue exhibits reduced aging parameters, pro-inflammatory factors, improved histological scores, or a combination thereof.

[0290] Embodiment 62. The method of any one of embodiments 60 or 61, wherein the engineered tissue is engineered skin tissue.

[0291] Embodiment 63. The method according to any one of embodiments 60 to 62, wherein the senescence parameters are selected from p16 and positive SAβGal staining and the pro-inflammatory factors IL8 and MMP1.

[0292] Embodiment 64. The method of any one of embodiments 60 to 63, wherein the histological score includes morphology, organization and / or quality.

[0293] Embodiment 65. A pharmaceutical composition comprising rejuvenated cells, wherein the rejuvenated cells are obtained by transfecting the cells with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for not more than 5 consecutive days.

[0294] Embodiment 66. The method of any one of the preceding embodiments, wherein the one or more cellular reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0295] Embodiment 67. The composition of embodiment 65 or 66, wherein the cells exhibit one or more of increased expression of HP1 gamma, H3K9me3, LAP2 alpha, SIRT1, increased mitochondrial membrane potential, and decreased reactive oxygen species, and decreased expression of SASP cytokines.

[0296] Embodiment 68. The composition of embodiment 67, wherein the SASP cytokines comprise one or more of IL18, IL1A, GROA, IL22 and IL9.

[0297] Embodiment 69. The composition of any one of embodiments 65 to 68, further comprising one or more additional components selected from nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics, antioxidants, and immunosuppressants.

[0298] Embodiment 70. The composition of any one of embodiments 65 to 69, further comprising a pharmaceutically acceptable carrier.

[0299] Embodiment 71. The composition of any one of embodiments 65 to 70, wherein the cells are autologous or allogeneic.

Claims

1. A method for rejuvenating a cell, comprising transfecting the cell with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for not more than five consecutive days, thereby producing a rejuvenated cell.

2. 2. The method of claim 1, wherein the transcriptome profile of the rejuvenated cells becomes similar to the transcriptome profile of young cells.

3. 3. The method of claim 2, wherein the transcriptome profile of the rejuvenated cells comprises increased gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1 and Sin3a.

4. 2. The method of claim 1, wherein the rejuvenated cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers compared to reference values.

5. 5. The method of claim 4, wherein the marker is selected from HP1 gamma, H3K9me3, lamina support protein LAP2 alpha and SIRT1 protein.

6. 10. The method of claim 1, wherein the rejuvenated cells exhibit increased proteolytic activity compared to a reference value.

7. 7. The method of claim 6, wherein the increased proteolytic activity is measured as increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, or a combination thereof.

8. 10. The method of claim 1, wherein the rejuvenated cells exhibit improved mitochondrial health and function compared to a reference value.

9. 9. The method of claim 8, wherein improved mitochondrial health and function is measured as increased mitochondrial membrane potential, decreased reactive oxygen species (ROS), or a combination thereof.

10. 10. The method of claim 1, wherein the rejuvenated cells exhibit decreased expression of one or more SASP cytokines compared to a reference value.

11. 11. The method of claim 10, wherein the SASP cytokines include one or more of IL18, IL1A, GROA, IL22, and IL9.

12. 10. The method of claim 1, wherein the rejuvenated cells exhibit a reversal of the methylation landscape.

13. 13. The method of claim 12, wherein reversals in the methylation landscape are measured by estimation with a Horvath clock.

14. The method of claim 4, wherein the reference value is obtained from aged cells.

15. 10. The method of claim 1, wherein the step of transfecting the cells with messenger RNA comprises a method selected from lipofectamine and LT-1 mediated transfection, dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, electroporation, encapsulation of mRNA in liposomes, and direct microinjection.

16. 2. The method of claim 1, wherein the one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

17. 2. The method of claim 1, wherein the one or more cell reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

18. The method of claim 1 , wherein the cell is a mammalian cell.

19. 19. The method of claim 18, wherein the cell is a human cell.

20. 20. The method of claim 19, wherein the cells are derived from an elderly subject.

21. 19. The method of claim 18, wherein the cells are selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

22. 22. The method of claim 21, wherein the cells are mesenchymal stem cells.

23. 23. The method of claim 22, wherein the rejuvenated mesenchymal stem cells exhibit reduced senescence parameters (p16, p21 and positive SAβGal staining), increased cell proliferation, and / or reduced ROS levels.

24. 10. The method of claim 1, which is carried out in vitro, ex vivo or in vivo.

25. 25. The method of claim 24, which is carried out in vivo.

26. 26. The method of claim 25, wherein the cell is in a tissue or organ.

27. 27. The method of claim 26, wherein the number of senescent cells in a tissue or organ is reduced.

28. 28. The method of claim 27, wherein the expression of one or more of IL18, IL1A, GROA, IL22, and IL9 is reduced.

29. 10. The method of claim 1, wherein the method restores cell function, increases differentiation potential, enhances survival rate, increases replicative capacity or lifespan, or a combination thereof.

30. 10. The method of claim 1, wherein the transfecting step is performed once a day for five days.

31. 10. The method of claim 1, wherein the transfecting step is performed once a day for four days.

32. 10. The method of claim 1, wherein the transfecting step is performed once a day for three days.

33. 10. The method of claim 1, wherein the transfecting step is performed once a day for two days.

34. 1. A method for treating an age-related disease or condition, a cartilage degenerative disorder, a neurodegenerative disorder, and / or a musculoskeletal dysfunction in a subject, comprising administering a therapeutically effective amount of cells, wherein the cells comprise one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors.

35. 35. The method of claim 34, wherein the one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

36. 36. The method of claim 35, wherein the one or more cell reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

37. 35. The method of claim 34, wherein the subject has an age-related disease or condition.

38. 35. The method of claim 34, wherein the age-related disease or condition is selected from ocular, skin, or musculoskeletal dysfunction.

39. 35. The method of claim 34, wherein the subject has a cartilage degenerative disorder.

40. 40. The method of claim 39, wherein the disorder is selected from arthritis, chondrophasia, spondyloarthropathy, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjogren's syndrome.

41. 35. The method of claim 34, wherein the treatment reduces the expression of inflammatory factors and / or increases ATP and collagen metabolism.

42. 42. The method of claim 41, wherein the inflammatory factor is selected from RANKL, iNOS2, IL6, IFNα, MCP3, and MIP1A.

43. 43. The method of claim 42, wherein ATP and collagen metabolism is measured by one or more of increased ATP levels, decreased ROS and increased SOD2, increased COL2A1, and overall proliferation by chondrocytes.

44. 35. The method of claim 34, wherein the subject has a musculoskeletal dysfunction.

45. 35. The method of claim 34, wherein administering a therapeutically effective amount of cells comprises injection or surgical implantation.

46. 35. The method of claim 34, wherein the therapeutically effective amount of rejuvenated cells is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

47. 47. The method of claim 46, wherein the therapeutically effective amount of rejuvenated cells are corneal epithelial cells.

48. 48. The method of claim 47, wherein the rejuvenated corneal epithelium exhibits a reduction in aging parameters.

49. 49. The method of claim 48, wherein the aging parameters include one or more of the expression of p21 and p16, mitochondrial biogenesis PGC1α, and expression of the inflammatory factor IL8.

50. A method for treating an age-related disease or condition, a cartilage degeneration disorder, and / or treating a subject having a musculoskeletal dysfunction, comprising administering a therapeutically effective amount of one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors.

51. 51. The method of claim 50, wherein the one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

52. 52. The method of claim 51, wherein the one or more cell reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

53. 51. The method of claim 50, wherein the subject has an age-related disease or condition.

54. 54. The method of claim 53, wherein the age-related disease or condition is selected from ocular, skin, or musculoskeletal dysfunction.

55. 51. The method of claim 50, wherein the subject has a cartilage degenerative disorder.

56. 56. The method of claim 55, wherein the disorder is selected from arthritis, chondrophasia, spondyloarthropathy, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjogren's syndrome.

57. 51. The method of claim 50, wherein the subject has a musculoskeletal dysfunction.

58. 51. The method of claim 50, wherein administering a therapeutically effective amount of one or more non-integrated messenger RNAs comprises direct injection into the target cells.

59. 59. The method of claim 58, wherein the target cells are selected from epithelial cells, endothelial cells, connective tissue cells, muscle cells, and nervous system cells.

60. A method for rejuvenating engineered tissue ex vivo, comprising transfecting the tissue with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for not more than five consecutive days, thereby producing rejuvenated engineered tissue.

61. 61. The method of claim 60, wherein the engineered tissue exhibits reduced aging parameters, pro-inflammatory factors, improved histological scores, or a combination thereof.

62. 61. The method of claim 60, wherein the engineered tissue is engineered skin tissue.

63. 62. The method of claim 61, wherein the senescence parameters are selected from p16 and positive SAβGal staining and the pro-inflammatory factors IL8 and MMP1.

64. 62. The method of claim 61, wherein the histological score comprises morphology, organization and / or quality.

65. A pharmaceutical composition comprising rejuvenated cells, wherein the rejuvenated cells are obtained by transfecting the cells with one or more non-integrated messenger RNAs encoding one or more cellular reprogramming factors for not more than five consecutive days.

66. 66. The composition of claim 65, wherein the one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

67. The composition of claim 65, wherein the cells exhibit one or more of increased expression of HP1 gamma, H3K9me3, LAP2 alpha, SIRT1, increased mitochondrial membrane potential, and decreased reactive oxygen species, and decreased expression of SASP cytokines.

68. 68. The composition of claim 67, wherein the SASP cytokines include one or more of IL18, IL1A, GROA, IL22, and IL9.

69. 66. The composition of claim 65, further comprising one or more additional components selected from nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics, antioxidants, and immunosuppressants.

70. 66. The composition of claim 65, further comprising a pharmaceutically acceptable carrier.

71. 66. The composition of claim 65, wherein the cells are autologous or allogeneic.

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