CN116334002A

CN116334002A - Younger skeletal muscle cell, preparation method and application thereof

Info

Publication number: CN116334002A
Application number: CN202211260323.5A
Authority: CN
Inventors: 黄仕强; 王瑞琦; 王鹏; 刘旭鹏; 赵赫; 程业倩; 马诗琳; 广璐; 陈煜�; 姚子月
Original assignee: Beijing Institute Of Stem Cell And Regenerative Medicine; Institute of Zoology of CAS
Current assignee: Beijing Institute Of Stem Cell And Regenerative Medicine; Institute of Zoology of CAS
Priority date: 2021-10-14
Filing date: 2022-10-14
Publication date: 2023-06-27
Also published as: CN118318046A; WO2023061485A8; WO2023061485A1; WO2023061485A9

Abstract

The present invention relates to the field of cells. In particular, the invention relates to a younger, skeletal muscle cell, a method of producing the same, and uses of the same.

Description

Younger skeletal muscle cell, preparation method and application thereof

Technical Field

The present invention relates to the field of skeletal muscle cells. In particular, the present invention relates to a method of rejuvenating skeletal muscle cells and the use of said cells in cell transplantation, tissue repair and/or tissue regeneration cell therapy and gene therapy.

Background

Aging is associated with progressive degeneration of tissues, which has a negative impact on the structure and function of vital organs, one of the most important known risk factors for most chronic diseases. Given that the proportion of the world population over 60 years old will double in the next 40 years, the increasing incidence of age-related chronic diseases will place a tremendous burden on medical resources. Aging is characterized by the gradual accumulation of lesions, leading to increased vulnerability to loss of physiological integrity, impaired function and death. The aging process affects the entire organism, including the human germline. After twenty years or more of active metabolism, all human cells, including the human germ line, accumulate molecular damage such as modified long-lived proteins, genetic and epigenetic mutations, metabolic byproducts, and other age-related deleterious changes before re-younger offspring can be produced. Recently, scientists have observed that during early embryogenesis, the biological age of cells has decreased significantly, i.e., a rejuvenation event has occurred, using the concept of an epigenetic clock (kerepeir et al, 2021).

However, the effects of the pathways and factors involved in this process on skeletal muscle cells in vitro remain a mystery. In biology, although many factors that promote growth, proliferation and regeneration have been reported, none of these factors generally have the function of reversing aging, depletion, anergy, apparent clocks. Despite the tremendous advances in our understanding of pluripotency, reprogramming and transdifferentiation, our molecular basis for the rejuvenation and/or prolongation of self-renewal capacity of skeletal muscle cells remains poorly understood. In particular, we have not yet found any factor or factors that can rejuvenate a somatic cell part.

Disclosure of Invention

The inventors of the present application have made extensive experimentation and repeated studies to obtain a younger skeletal muscle cell, methods and reagents for its preparation, and the use of the cell and reagent in cell therapy. Through a surprising discovery, the inventors discovered that certain pathways and genes involved in the above processes are regulated in skeletal muscle cells, which are capable of not only partially reversing senescence, depletion, anergy, apparent clocks, cellular biological age, but also prolonging self-renewal capacity of skeletal muscle cells, and continuing to proliferate in vitro.

Rejuvenating skeletal muscle cells, cell populations and pharmaceutical combinations

The present application provides an isolated, modified skeletal muscle cell that has the following characteristics:

(i) Any one or more genes of Bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4 are increased

(ii) The passaging may be stable for at least 5 times, such as at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more.

In certain embodiments, the isolated modified skeletal muscle cells provided herein may be stably passaged at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more; its network of rejuvenating transcription factors: the expression of any gene or genes of Grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx is increased relative to unmodified skeletal muscle cells. In certain embodiments, in addition to the above-described younger transcription factor network genes, expression of LIN28 (including LIN28A or LIN 28B) is simultaneously increased relative to unmodified skeletal muscle cells. In certain embodiments, the isolated modified skeletal muscle cells provided herein may be stably passaged at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more; its younger epigenetic modification network: the expression of any gene or genes of Bcl11a, bcl11b, dnmt3b, mettl20, arid3c is increased relative to unmodified skeletal muscle cells. In certain embodiments, in addition to the above-described rejuvenating epigenetic modified network genes, the expression of LIN28 (including LIN28A or LIN 28B) is simultaneously increased relative to unmodified skeletal muscle cells. In certain embodiments, the isolated modified skeletal muscle cells provided herein may be stably passaged at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more; its younger signal ligand, receptor and related kinase network: the expression of any gene or genes of Fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4 is increased relative to unmodified skeletal muscle cells. In certain embodiments, the expression of LIN28 (including LIN28A or LIN 28B) is simultaneously increased relative to unmodified skeletal muscle cells, in addition to the genes for the rejuvenating signal ligands, receptors and related kinase networks described above. In certain embodiments, the isolated modified skeletal muscle cells provided herein may be stably passaged at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more; its younger nucleic acid binding factor network: the expression of any gene or genes of Foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28 is increased relative to unmodified skeletal muscle cells. In certain embodiments, in addition to the above-described network of younger nucleic acid binding factors, expression of LIN28 (including LIN28A or LIN 28B) is simultaneously increased relative to unmodified skeletal muscle cells. In certain embodiments, the isolated modified skeletal muscle cells provided herein may be stably passaged at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more; its gene expression of LIN28 (including LIN28A or LIN 28B) is increased relative to unmodified skeletal muscle cells. In certain embodiments, the isolated modified skeletal muscle cells provided herein may be stably passaged at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more; any gene or genes of which LIN28 (including LIN28A or LIN 28B), BCL11A, BCL11B, LMO2, OTX2, PBX1, PABPC4L is/are increased relative to unmodified skeletal muscle cells.

In certain embodiments, the skeletal muscle cells of the invention exhibit at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold increase in the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36) genes selected from the group consisting of: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the skeletal muscle cells of the invention exhibit at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold increase in the expression level of one or more younger transcription factor network genes selected from the group consisting of: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, in addition to the above-described younger transcriptional network genes, the modified skeletal muscle cells of the present invention have a LIN28 (LIN 28A or LIN 28B) gene expression level that is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold greater than that of the unmodified skeletal muscle cells. In certain embodiments, the skeletal muscle cells of the invention exhibit at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold increase in the expression level of the younger epigenetic modification network genes as compared to the expression level of the younger epigenetic modification network genes in unmodified skeletal muscle cells, at least about 10-fold, at least about 100-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 150-fold: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, in addition to the above described younger epigenetic modification networks, the modified skeletal muscle cells of the present invention have a LIN28 (LIN 28A or LIN 28B) gene expression level of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold as compared to the unmodified skeletal muscle cells. In certain embodiments, the skeletal muscle cells of the present invention exhibit at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold increase in the expression level of one or more younger signal ligands, receptors, and related kinase network genes selected from the group consisting of: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, in addition to the above described younger signal ligands, receptors and related kinase network genes, the modified skeletal muscle cells of the present invention have a LIN28 (LIN 28A or LIN 28B) gene expression level of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold as compared to the unmodified skeletal muscle cells. In certain embodiments, the skeletal muscle cells of the invention exhibit at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold increase in the expression level of one or more of the younger nucleic acid binding factor network genes selected from the group consisting of: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, in addition to the above-described network genes for the young ribonucleotide binding factor, the modified skeletal muscle cells of the present invention have a LIN28 (LIN 28A or LIN 28B) gene expression level of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold as compared to unmodified skeletal muscle cells. In certain embodiments, the skeletal muscle cells of the invention exhibit at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold increase in the expression level of LIN28 (LIN 28A or LIN 28B) over the expression level of these young, ribonucleic acid binding factor network genes in unmodified skeletal muscle cells. In certain embodiments, the skeletal muscle cells of the invention exhibit at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold increase in the expression level of BCL11A, BCL11B, LMO2, OTX2, PBX1, PABPC4L over the expression level of any one or more of these rejuvenating factors in the unmodified skeletal muscle cells.

There are many ways to increase the expression or expression level of a certain gene on a cell. The most common approach is to deliver a nucleic acid (e.g., DNA or RNA) encoding a gene to a cell by means of transgenes, which also include elements that regulate the amount of expression of the gene (e.g., promoters, drug-regulated promoters, protein-regulated promoters, tissue-specific promoters, protein introns, transposons, endonucleases (e.g., cre-lox systems), retrotransposons, etc.) to increase the expression of the gene in the cell. These are well known to those skilled in the art. In addition, there are also many ways to deliver genes to cells, such as but not limited to using viruses, transposons, nanoparticles, lipid vesicles, and the like. The non-transgenic mode of increasing gene expression includes the use of CRISPRa to regulate endogenous genes and up-regulate the expression of a gene. The amount of expression of a gene in a cell can be measured by means known in biology, such as but not limited to Western blotting, immunofluorescence, fluorescent quantitative PCR, RNA or DNA sequencing, etc.

In certain embodiments, the skeletal muscle cell is transgenic for at least 12 hours in which expression of any one or more of the following genes is increased: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the skeletal muscle cells are transgenic for at least 12 hours in which expression of the network gene of any one or more of the following rejuvenating transcription factors is increased: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the gene expression level of the skeletal muscle cell LIN28 (LIN 28A or LIN 28B) is also increased by transgenic means for at least 12 hours. In certain embodiments, the skeletal muscle cells have increased expression of the younger apparent modified network gene by transgenic means for at least 12 hours at any one or more of the following: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the gene expression level of the skeletal muscle cell LIN28 (LIN 28A or LIN 28B) is also increased by transgenic means for at least 12 hours. In certain embodiments, the skeletal muscle cells are transgenic for at least 12 hours in which expression of a younger signal ligand, receptor or related kinase network gene is increased in any one or more of the following: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the gene expression level of the skeletal muscle cell LIN28 (LIN 28A or LIN 28B) is also increased by transgenic means for at least 12 hours. In certain embodiments, the skeletal muscle cells are transgenic for at least 12 hours in which expression of the network gene of the younger nucleic acid binding factor is increased in any one or more of the following: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the gene expression level of the skeletal muscle cell LIN28 (LIN 28A or LIN 28B) is also increased by transgenic means for at least 12 hours. In certain embodiments, the skeletal muscle cells of the invention have increased expression in LIN28 (LIN 28A or LIN 28B) by transgenic means for at least 12 hours. In certain embodiments, the skeletal muscle cells are transgenically increased in the expression of any one or more of the following rejuvenation factor genes for at least 12 hours: bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4l. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression.

In certain embodiments, the skeletal muscle cells are capable of sustained expansion in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days, or even more.

Skeletal muscle cells in certain embodiments, the skeletal muscle cells have at least about 5-fold, 10-fold, 20-fold, 30-fold, or even greater expression of MDM4 and TEP1 as compared to skeletal muscle cells.

In certain embodiments, the biological age of the cell is significantly reduced compared to skeletal muscle cells, which biological age can be measured by testing the cells for gene expression or epigenetic modification (e.g., epigenetic clock). The biological age is different from the actual time age, because two animals of the same actual years may have a difference in aging rate, that is, the biological age difference may cause the animals to have different risks of aging-related diseases. In recent years, the epigenetic clock has become a powerful biomarker of the mammalian aging process, including humans, mice, dogs and wolves, and whales. Epigenetic clocks are mathematical models that can be trained on big data to predict years and biological age using epigenetic modifications of a small number of genomic loci in the genome (Horvath and Raj,2018; bell et al, 2019). In 2013, steve Horvath developed the most widely used multi-tissue epigenetic clock for humans (Horvath 2013). Interestingly, deviations in the biological age predicted by the epigenetic clock from the actual time of age (also known as epigenetic age acceleration or EAA) have a strong correlation with the time of death and many premature aging diseases in humans, including hiv infection, down's syndrome, obesity, walner's syndrome and huntington's disease. Epigenetic clock can be understood as a representation for quantifying the change of the epigenetic genome with aging (Martin-Herranz et al, 2019), such as predicting the age of a human organism using the DNA methylation status of CpG sites (Horvath clock; horvath 2013), the age of a mouse organism (stub multi-t.clock; stub et al, 2017), the age of a mouse blood organism (Petkovitch blood clock; petkovitch et al, 2018), the age of a mouse multi-organ organism (Thompson multi-t.en clock; thompson et al, 2018), or predicting the age of a mouse blood organism using the DNA methylation status of ribosomal nucleic acids (Wang blood rDNA clock; wang and Lemos, 2019), or predicting the age of a organism using the methylation status of chromatin histone H3 (Martin-Herranz et al, 2019;Jeffries et al, 2019). In addition, the expression profile of the genome also changes with age-related epigenetic changes (Martin-Herranz et al, 2019), and measuring the expression of these genes also measures the biological age of a species or cell.

In another aspect, the invention provides an isolated population of cells comprising skeletal muscle cells described above, or any combination thereof; preferably, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100%) of the cells in the population of cells are skeletal muscle cells described above.

In another aspect, the invention also provides a pharmaceutical combination comprising a skeletal muscle cell or cell population as described above, and a pharmaceutically acceptable carrier and/or excipient. In certain embodiments, the pharmaceutical combinations of the invention may be used in cell therapy comprising administering the cells of the invention to a patient in combination with a pharmaceutically acceptable carrier and/or a fu-agent. Currently, cell therapy is widely used in the medical field, and the cells produced by the invention can provide high-quality and younger cells for cell therapy and increase the yield of cells.

Method for preparing younger cells or reversing skeletal muscle cell senescence

In another aspect, the invention provides a method of reversing cellular senescence comprising increasing expression of any (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) gene of: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the invention provides a method of reversing cellular senescence comprising increasing expression of any one or more of the following younger transcription factor network genes: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the present invention provides methods of reversing cellular senescence, in addition to increasing expression of the above-described network of rejuvenating transcription factors, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the invention provides a method of reversing cellular senescence comprising increasing expression of any one or more of the following younger epigenetic modified network genes: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the invention provides methods of reversing cellular senescence, in addition to increasing expression of the above-described rejuvenating epigenetic modified network genes, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the invention provides a method of reversing cellular senescence comprising increasing expression of any one or more of the following younger signal ligands, receptors, and related kinase network genes: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the invention provides methods of reversing cellular senescence, in addition to increasing expression of the above-described genes that younger signal ligands, receptors, and related kinase networks, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the invention provides a method of reversing cellular senescence comprising increasing expression of any one or more of the following younger nucleic acid binding factor network genes: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the invention provides methods of reversing cellular senescence, in addition to increasing expression of the above-described network genes of rejuvenating nucleic acid binding factors, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the invention provides a method of reversing cellular senescence comprising increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the invention provides a method of reversing cellular senescence comprising increasing expression of any one or more of Bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l. In certain embodiments, the present invention provides methods of reversing cellular senescence, in addition to increasing expression of the genes described above, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the regimen is by transgenic means to increase expression of the gene for at least 12 hours. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression.

As used herein, the term "cellular senescence" refers to the loss of normal cellular activity, such as proliferation and differentiation, of cells while maintaining a certain activity and metabolic activity. Cellular senescence may be caused by a variety of stimuli or factors, including shortening of telomeres due to DNA end replication, DNA damage, altered activity of tumor suppressor and oncogenes, oxidative stress, inflammation, chemotherapeutics, and exposure to ultraviolet radiation or ionizing radiation (Kuilman et al, genes & development (2010) 24:2463-2479). As used herein, "reversing cellular senescence" or "reversing senescence" refers to restoring the proliferation and/or differentiation capacity of a cell. Biologically, the measurement of cellular senescence can generally be identified using β -galactosidase activity reagents. As used herein, "reverse cell senescence" or "reverse senescence" refers to the restoration of normal cellular activity such as proliferation and/or differentiation of cells, and can be identified by measurement using a β -galactosidase active agent.

In certain embodiments, the invention also provides methods of making the above younger skeletal muscle cells, comprising increasing the expression of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4; the biological age of a cell can be measured by testing the cell for gene expression or genetic modification (e.g., epigenetic clock). In certain embodiments, the present invention provides methods of making the above younger cells comprising increasing expression of any one or more of the following younger transcription factor network genes: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the present invention provides methods of making the above rejuvenating cells, except for increasing expression of the above described rejuvenating transcription factor network genes, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the present invention provides methods of making the above younger cells comprising increasing expression of any one or more of the following younger epigenetic modified network genes: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the present invention provides methods of making the above rejuvenating cells, except for increasing expression of the above described rejuvenating epigenetic modified network genes, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the present invention provides methods of making the above younger cells comprising increasing gene expression of any one or more of the following younger signal ligands, receptors and related kinase networks: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the present invention provides methods of making the above younger cells, except for increasing expression of the above younger signal ligands, receptors and related kinase network genes, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the present invention provides methods of making the above rejuvenating cells, comprising increasing expression of any one or more of the following rejuvenating nucleic acid binding factor network genes: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the present invention provides methods of making the above rejuvenating cells, except for increasing expression of the above described rejuvenating nucleic acid binding factor network genes, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the present invention provides a method of preparing the above younger cells comprising increasing the expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the invention provides a method of making the above younger cells comprising increasing the expression of any one or more of Bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l. In certain embodiments, the present invention provides methods of preparing the above younger cells, except for increasing expression of the above-described genes, while increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the regimen is by transgenic means to increase expression of the gene for at least 12 hours. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression.

As used herein, the term "rejuvenation" refers to a decrease in the biological age of a cell or species or a biological feature possessed by a younger cell such as a more powerful self-renewing capacity, regenerating capacity, growing capacity, gene expression or epigenetic modification profile closer to embryonic stage, or better biological function. The biological age of the cells described above can be measured by testing the cells for gene expression or genetic modification (e.g., epigenetic clock).

Skeletal muscle cells

In certain embodiments, the method is capable of reversing cell depletion in skeletal muscle cells, including increasing expression of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) genes of: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the method is capable of reversing cell depletion in skeletal muscle cells, including increasing expression of any one or more of the following network genes for a rejuvenating transcription factor: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger transcription factor network genes. In certain embodiments, the method is capable of reversing cell depletion in skeletal muscle cells, including increasing expression of any one or more of the following younger epigenetic modification network genes: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger epigenetic modified network genes. In certain embodiments, the method is capable of reversing cell depletion in skeletal muscle cells, including increasing expression of genes for any one or more of the following younger signal ligands, receptors, and related kinase networks: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the methods increase expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described genes that rejuvenate the signal ligand, receptor, and related kinase network. In certain embodiments, the method is capable of reversing cell depletion in skeletal muscle cells, comprising increasing expression of any one or more of the following network genes for a rejuvenating nucleic acid binding factor: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described network genes of the rejuvenating nucleic acid binding factors. In certain embodiments, the method is capable of reversing cell depletion in skeletal muscle cells, including increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the method is capable of reversing cell depletion in skeletal muscle cells, including increasing expression of any one or more of Bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the genes described above. In certain embodiments, the regimen is by transgenic means to increase expression of the gene for at least 12 hours. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression.

As used herein, the term "cell depletion" refers to the loss of a portion of the cell's function during prolonged activation, cell function tests can generally be used to identify, for example, differentiation efficiency of skeletal muscle stem cells, damage repair capacity, degree of muscle fiber hypertrophy, and the like.

As used herein, the term "reverse cell depletion" refers to allowing a cell to resume function, as well as being identified using a functional test.

In certain embodiments, the method is capable of reversing inotropic in skeletal muscle cells, including increasing the expression of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) genes of: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the method is capable of reversing anergy in skeletal muscle cells, including increasing expression of any one or more of the following network genes for a younger transcription factor: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger transcription factor network genes. In certain embodiments, the method is capable of reversing anergy in skeletal muscle cells, including increasing expression of any one or more of the following younger epigenetic modification network genes: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger epigenetic modified network genes. In certain embodiments, the method is capable of reversing disability in skeletal muscle cells, including increasing expression of genes for any one or more of the following younger signal ligands, receptors, and related kinase networks: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the methods provide for, in addition to increasing expression of the above-described genes that rejuvenate signaling ligands, receptors, and related kinase networks, while increasing the expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the method is capable of reversing anergy in skeletal muscle cells, including increasing expression of any one or more of the following younger nucleic acid binding factor network genes: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described network genes of the rejuvenating nucleic acid binding factors. In certain embodiments, the method is capable of reversing anergy in skeletal muscle cells, including increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the method is capable of reversing inotropic in skeletal muscle cells, including increasing the expression of any one or more of Bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the genes described above. In certain embodiments, the regimen is by transgenic means to increase expression of the gene for at least 12 hours. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression.

As used herein, the term "anergy" refers to the inability of a cell to initiate a response to an external signal, such as the inability of a muscle stem cell to respond to FGF2, to proliferate, and generally can be tested by cell proliferation assays.

As used herein, the term "reverse anergy" refers to allowing a cell to resume a response and function that it should have to an external signal, including proliferation.

In certain embodiments, the method is capable of extending the life span of a skeletal muscle cell, including increasing the expression of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the method is capable of extending the life span of skeletal muscle cells, including increasing expression of a gene of a regenerative transcription factor gene of a younger transcription factor of any one or more of the following: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger transcription factor network genes. In certain embodiments, the method is capable of extending the life span of skeletal muscle cells, including increasing expression of any one or more of the following younger epigenetic modification network genes: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger epigenetic modified network genes. In certain embodiments, the methods are capable of extending the life span of skeletal muscle cells, including increasing expression of genes for any one or more of the following younger signal ligands, receptors, and related kinase networks: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the methods increase expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described genes that rejuvenate the signal ligand, receptor, and related kinase network. In certain embodiments, the method is capable of increasing the longevity of skeletal muscle cells, including increasing expression of any one or more of the following younger nucleic acid binding factor network genes: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described network genes of the rejuvenating nucleic acid binding factors. In certain embodiments, the methods are capable of extending the life of skeletal muscle cells, including increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the method is capable of increasing the longevity of skeletal muscle cells, including increasing expression of any one or more of the following genes: bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4l. In certain embodiments, the method, in addition to increasing expression of the genes described above, while increasing the expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the regimen is by transgenic means to increase expression of the gene for at least 12 hours. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression.

In general, the lifetime of a cell can be measured by how long the cell survives, and whether the cell survives can be identified by staining (e.g., propidium iodide) or by observation of the morphology of the cell under a microscope by those skilled in the art.

In certain embodiments, the methods are capable of allowing cells to continue to expand in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days, or even more days, including increasing the expression of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calCr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the methods are capable of allowing cells to continue to expand in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days, or even more, including increasing expression of a younger transcription factor network gene of any one or more of the following: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger transcription factor network genes. In certain embodiments, the methods are capable of allowing cells to continue to expand in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days, or even more, including increasing expression of a younger epigenetic modified network gene of any one or more of the following: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger epigenetic modified network genes. In certain embodiments, the methods are capable of allowing cells to continue to expand in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days, or even more, including increasing expression of genes for any one or more of the following younger signal ligands, receptors, and related kinase networks: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the methods increase expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described genes that rejuvenate the signal ligand, receptor, and related kinase network. In certain embodiments, the methods are capable of allowing cells to continue to expand in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days, or even more, including increasing expression of a network gene of a younger nucleic acid binding factor of any one or more of the following: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described network genes of the rejuvenating nucleic acid binding factors. In certain embodiments, the methods of the methods capable of prolonging skeletal muscle cells allow for continued expansion of the cells in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days, or even more, including increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the methods are capable of allowing cells to continue to expand in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days, or even more, including increasing expression of a rejuvenation factor gene of any one or more of the following: bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4l. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the genes described above. In certain embodiments, the regimen is by transgenic means to increase expression of the gene for at least 12 hours. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression.

In certain embodiments, the methods are capable of stably passaging skeletal muscle cells at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more, including increasing the expression of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the method is capable of stably passaging skeletal muscle cells at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more, including increasing expression of any one or more of the following younger transcription factor network genes: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger transcription factor network genes. In certain embodiments, the methods are capable of stably passaging skeletal muscle cells at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more, including increasing expression of any one or more of the following younger epigenetic modified network genes: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger epigenetic modified network genes. In certain embodiments, the methods are capable of stably passaging skeletal muscle cells at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more, including increasing expression of genes for any one or more of the following younger signal ligands, receptors, and related kinase networks: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the methods increase expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described genes that rejuvenate the signal ligand, receptor, and related kinase network. In certain embodiments, the method is capable of stably passaging skeletal muscle cells at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more, including increasing expression of any one or more of the following younger nucleic acid binding factor network genes: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described network genes of the rejuvenating nucleic acid binding factors. In certain embodiments, the method is capable of stably passaging skeletal muscle cells at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more, including increasing expression of any one or more of the following rejuvenation factor genes: bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4l. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described rejuvenation factor genes. In certain embodiments, the methods are capable of stably passaging skeletal muscle cells at least 5 times, e.g., at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or more, including increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the regimen is by transgenic means to increase expression of the gene for at least 12 hours. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression.

In certain embodiments, the method is capable of rejuvenate aged skeletal muscle cells, including increasing the expression of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) genes of: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. In certain embodiments, the method is capable of rejuvenate senile skeletal muscle cells, including increasing expression of any one or more of the following network genes of younger transcription factors: grhl2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger transcription factor network genes. In certain embodiments, the method is capable of rejuvenate senile skeletal muscle cells, including increasing expression of any one or more of the following younger epigenetic modified network genes: bcl11a, bcl11b, dnmt3b, mettl20, arid3c. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described younger epigenetic modified network genes. In certain embodiments, the method is capable of rejuvenate senile skeletal muscle cells, including increasing expression of genes for any one or more of the following younger signal ligands, receptors, and related kinase networks: fgf5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc4. In certain embodiments, the methods increase expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described genes that rejuvenate the signal ligand, receptor, and related kinase network. In certain embodiments, the method is capable of rejuvenate senile skeletal muscle cells, including increasing expression of any one or more of the following younger nucleic acid binding factor network genes: foxr2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described network genes of the rejuvenating nucleic acid binding factors. In certain embodiments, the method is capable of rejuvenate senile skeletal muscle cells, including increasing expression of LIN28 (LIN 28A or LIN 28B). In certain embodiments, the method is capable of rejuvenate senile skeletal muscle cells, including increasing expression of any one or more of the following rejuvenation factor genes: bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4l. In certain embodiments, the method increases expression of LIN28 (LIN 28A or LIN 28B) in addition to increasing expression of the above-described rejuvenation factor genes. In certain embodiments, the regimen is by transgenic means to increase expression of the gene for at least 12 hours. In certain embodiments, the expression of the gene is transient gene expression. In certain embodiments, the gene expression is constitutive gene expression. In certain embodiments, the skeletal muscle cell is transgenic for at least 12 hours in which expression of any one or more of the following genes is increased: LIN28 (LIN 28A or LIN 28B), bcl11B, arid3c, otx2, lmo2, pabpc4l, mettl20, pbx1.

In certain embodiments, the method is by increasing the expression of one or more of the following genes in a cell by a promoter that is responsive to inflammation or injury: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

In certain embodiments, the methods comprise transgenic means to increase expression of one or more of the following genes in a cell: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

In certain embodiments, the cells are treated (e.g., genetically engineered) to express a higher level of regenerative factor in skeletal muscle cells than would be expressed in the absence of such treatment. In some embodiments, the cells are treated to over-express one or more of the following genes in skeletal muscle cells: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. One method of cell processing is to infect a cell with a virus (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus) or to transfect a cell with a viral vector (e.g., retrovirus, lentivirus, adenovirus) comprising a factor sequence operably linked to appropriate expression control elements to drive expression in the cell after infection or transfection, and optionally integration into the genome as known in the art. In certain embodiments, the methods of treating a cell also include using a transposon or your transposon to deliver the genes described above and a promoter to control the amount of gene expression. In certain embodiments, the protocol of treating cells can utilize electroporation to deliver a vector comprising a transposon or retrotransposon, an element controlling protein expression (promoter, intron (intein), endonuclease (e.g., cre-lox system)), and a sequence encoding one or more proteins of Bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, cef 4, pdzd4, piez 2, shc4. Further details regarding the compositions and methods of the present invention are provided below.

In certain embodiments, the transgenic methods utilized by the methods comprise utilizing any vector known in medicine, such as, but not limited to, viral vectors, transposons, nanoparticles, retrotransposons, endonucleases).

Agent and kit for bone muscle cell rejuvenation or cell aging reversal

The present invention also provides a kit or combination of reagents that can be used to produce the above-described younger skeletal muscle cells comprising:

(i) Nucleic acids (e.g., deoxyribonucleic acid, ribonucleic acid) encoding any one or more of the following: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4

And

(ii) A pharmaceutically acceptable carrier (e.g., viral vector, nanoparticle, lipid vesicle, transposon, retrotransposon, exosome, etc.).

In certain embodiments, the kit or combination of reagents also comprises an element that modulates the amount of expression of the above-described genes or proteins, such as, but not limited to, a promoter, a drug-regulated promoter, a protein-regulated promoter, a tissue-specific promoter, an intron (intein), a transposon, an endonuclease (e.g., cre-lox system), a retrotransposon. In certain embodiments, the kit or combination of reagents comprises a vector or the like comprising any of the above elements that regulate the amount of expression of the above genes or proteins, and nucleic acids encoding one or more of Bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc 4. In certain embodiments, the agent or combination of agents is infection of a cell with a virus (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus) or transfection of a cell with a viral vector (e.g., retrovirus, lentivirus, adenovirus) comprises a factor sequence operably linked to a suitable expression control element to drive expression in the cell after infection or transfection, and optionally integration into the genome as known in the art. In certain embodiments, the agent or combination of agents comprises a promoter that utilizes a transposon or retrotransposon to deliver the genes described above and control the amount of gene expression. In certain embodiments, the agent or combination of agents is delivered by using electroporation to deliver a vector comprising a transposon or retrotransposon, an element controlling protein expression (promoter, intron (intein), endonuclease (e.g., cre-lox system)), and a nucleic acid sequence encoding one or more of Bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, pizo 2, shac 4. Further details regarding the compositions and methods of the present invention are provided below.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, laboratory procedures in the fields of stem cells, biochemistry, nucleic acid chemistry, immunology and the like as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "skeletal muscle cells" refers to cells whose fate is established, which are incapable of differentiating into a plurality of different cells, such as adult cells, germ cells, adult stem cells, and the like, and which are not embryonic stem cells, induced pluripotent stem cells

As used herein, the term "biological age" refers to the determination of the age or youth of a species by its health and aging indicators.

As used herein, the term "epigenetic clock" or "epigenetic clock" refers to the use of epigenetic as a biological indicator to measure the biological age of a species.

As used herein, the term "self-renewing ability" refers to the ability of a cell to self-maintain cellular properties over multiple passages without significant changes in properties such as cell fate. In some embodiments, the number of passages is at least about 5, at least about 10, at least about 20, at least about 30, at least about 50, or at least about 100.

As used herein, the term "expand" or "proliferation" refers to maintaining cells and ultimately cell growth substantially without differentiation, i.e., increasing (e.g., at least 2-fold) the population of cells without concomitant increased differentiation.

As used herein, the term "precursor cell" or "group of cells" refers to cells that have a specific fate and can only differentiate into a particular adult cell or germ layer.

As used herein, the term "in vitro" refers to the artificial environment, and processes and reactions therein. The in vitro environment is exemplified by, but not limited to, test tubes and cell cultures.

As used herein, the term "in vivo" refers to the natural environment (i.e., animal or cell) and processes and reactions therein.

As used herein, the term "basal medium" refers to any medium capable of supporting cell growth, typically comprising inorganic salts, vitamins, glucose, buffer systems, and essential amino acids, and typically having an osmolality of about 280-330 mOsmol.

As used herein, the term "serum replacement" has a meaning well known to those skilled in the art, and refers to a composition or formulation used as a replacement for serum during the culturing of stem cells while maintaining an undifferentiated state. That is, the serum replacement is capable of supporting the growth of undifferentiated stem cells without the need for serum supplementation. In certain exemplary embodiments, the serum replacement comprises: one or more amino acids, one or more vitamins, one or more trace metal elements. In some cases, the serum replacement may further comprise one or more selected from the group consisting of Is characterized by comprising the following components: albumin, reduced glutathione, transferrin, insulin, and the like. Non-limiting examples of serum substitutes include, but are not limited to, knockOut ^TM SR (abbreviated as KSR), N-2, B-27, physiologix ^TM XF SR、StemSure ^TM Serum Substitute Supplement, etc.

As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH adjusters, surfactants, ionic strength enhancers, agents to maintain osmotic pressure, agents to delay absorption, diluents, adjuvants, preservatives, and the like. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvant (e.g., complete Freund's adjuvant), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. In certain embodiments, the pharmaceutically acceptable carrier or excipient is a sterile isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. In certain embodiments, a "pharmaceutically acceptable carrier" also comprises a means of delivering nucleic acids such as, but not limited to, viral vectors, nanoparticles, lipid vesicles, exosomes, and the like.

As used herein, the term "about" refers to a value or composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, when "about" is used to describe a measurable value (e.g., concentration of a substance, mass ratio, etc.), it is meant to encompass a range of + -10%, + -5%, or + -1% of the given value.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

Figure 1 shows that Lin28a+ satellite cells are involved in all types of myofibers during regeneration. Fig. 1 (a) shows a schematic of tamoxifen treatment. Tamoxifen was injected 6 days before the freeze injury, once every 1 week every 7 days after the injury, and samples were collected (B) on Lin28a-T2A-Creer 14 days after the injury; the tibialis anterior and soleus muscles of LSL-tdTO mice were cryogenically injured and harvested on day 14 post-injury. The control was contralateral tibialis anterior or soleus muscle. Scale bar: 200 microns. (C) quantifying Lin28a-T2A-Creer; the images in (B) were quantified by tdTomato+ (tdTO+) muscle fiber number per 100 muscle fibers in tibialis anterior and soleus when LSL-tdTO mice were undamaged and injured for 14 days. For each muscle slice, at least 3 complete fields of view are quantified and averaged. (D) cryogenically damaged Lin28a-T2A-CreER; the tibialis anterior muscle of the LSL-tdTO mice was harvested 14 days after injury and muscle sections were taken. Muscle sections co-stained for laminin (grey), DAPI (blue) and Pax7 (green) or Pax3 (green). The arrow indicates Lin28a+Pax7+ or Lin28a+Pax3+ cells, and the triangular arrow indicates Lin28 a+Pax3-or Lin28 a+Pax7-cells. Scale bar: 20 microns. (E) quantitatively detecting Lin28a-T2A-Creer; pax7+, lin28a+ and pax7+/Lin28a+ biscationic cell numbers in tibialis anterior 14 days after LSL-tdTO mice injury. The image in (D) is quantized. For each muscle slice, at least 5 different regions are quantified and averaged.

(F) Quantitatively detecting Lin28a-T2A-Creer; pax3+, lin28a+ and pax3+/Lin28a+ biscationic cell numbers in tibialis anterior 14 days after LSL-tdTO mice injury. The image in (D) is quantized. For each muscle slice, at least 5 different regions are quantified and averaged.

(G) Lin28a-T2A-Creer 14 days after injury; flatfish muscle and tibialis anterior muscle cross-section immunofluorescent staining of LSL-tdTO mice, myofiber type I (red), type IIA (green), type IIX (blue) and type IIB (black) myofibers were compared to tdto+ fluorescence (arrows). Scale bar: 100 microns.

(H) Lin28a-T2A-Creer at 14 days undamaged; immunofluorescent staining of tibialis anterior muscle cross section of LSL-tdTO mice, myofiber type I (red), type IIA (green), type IIX (blue) and type IIB (black) myofibers were compared to tdto+ fluorescence (arrows). Scale bar: 100 microns.

Figure 2 Lin28a+ cells are muscle satellite cells that exhibit strong myogenic potential in vitro.

FIG. 2A) flow cytometry analysis of damaged or undamaged Lin28a-T2A-Creer; lin28a-tdTO+ cell status in LSL-tdTO mouse muscle. The control group is undamaged Lin28a-T2A-Creer; LSL-tdTO mice. All mice were injected with tamoxifen and harvested 14 days after injury.

(B) Damaged or undamaged Lin28a-T2A-Creer; quantification of tdto+ cell number in LSL-tdTO mice, 6 replicates per group.

(C) Fresh flow cytometry isolated Lin28a-tdTO+ cells were observed with a confocal microscope. Scale bar: 50 microns.

(D) Tdto+ cells were analyzed by flow cytometry. Cells were first labeled with antibodies that bind CD31 (APC), CD45 (BV 421), VCAM1 (PE), sca1 (FITC) fluorochromes. Lin28a-tdTO+ cells were predominantly VCAM1+CD31+Sca1+CD45-cells.

(E) Immunofluorescent staining (green) of Pax7 and MyoD showed that Lin28a+ cells cultured in growth medium expressed mostly Pax7 and MyoD. Scale bar: 100 microns.

(F) Lin28a+ cells begin to fuse, differentiate and form multinucleated myotubes on day 1-2 in differentiation medium. Scale bar: 100 microns.

(G) Myosin Heavy Chain (MHC) (green) and Hoechst (blue) staining showed that most Lin28a-tdTO+ cells expressed MHC after differentiation into myotubes. Scale bar: 50 microns.

(H) Quantitative RT-PCR showed that, after 7d of culture in differentiation medium, the myogenic differentiation related genes of MyoG, ckm, myh1, myh2, myh4, etc. were strongly activated, whereas the myogenic progenitor related genes of Pax3, pax7, myoD, myf5, etc. were significantly reduced, relative to the undifferentiated Lin28a+ cells in the growth medium.

(I) Immunofluorescence images are shown from Lin28a-T2A-Creer; tibialis anterior was isolated from the 14 day old LSL-tdTO mice, and normal (con) MuSCs (CellTrace Violet marker), lin28a+ cells, alone or in 1:1 mix with Lin28a+ cells, were differentiated for 36h into myotubes. Scale bar: 100 microns.

(J) Relative frequency distribution of myotube diameters formed by fusion of Lin28a+ cells and/or con MuSCs. Each group quantified 200 myotubes, p=6.15×10 ^-7 . Right figure: quantification of myotube fusion index. At least 3 different fields per group are quantified.

(K) Western blot detects expression of Pax3, pax7 and MyoD proteins in Lin28a+ cells, and quantitative RT-PCR detects changes in expression levels of Pax3, pax7, myoD, myf5 and Twist1 in Lin28a+ cells relative to con MuSCs when propagated in Growth Medium (GM). Gapdh was used as loading control.

(L) Western blot detection of MHC and MyoG protein expression in Lin28a+ cell-derived myotubes, quantitative RT-PCR detection of Myf5, twist1, ckm, myoG, myh1/2/4/7 expression levels in Lin28a+ and con MuSCs-derived myotubes after differentiation in differentiation culture. Gapdh was used as loading control.

＊：P＜0.05；＊＊：P＜0.01；＊＊＊：P＜0.001.

FIG. 3 transcriptome analysis shows that Lin28a promotes MuSC dedifferentiation.

(A) Clustering analysis of adult Lin28a+MuSCs, adult Pax7+MuSCs, and embryonic Pax7+MuSCs transcriptomes. Each group was repeated 3 times.

(B) GO analysis of cluster 2 in (A).

(C) KEGG analysis of cluster 3 in (a).

(D) (a) GO analysis of cluster 5.

(E) Methylation levels of CpG sites near the Mef2c gene of Lin28a+ MuSCs, pax7+ embryo and Pax7+ adult MuSCs (signal values 0-100%).

(F) Methylation levels of CpG sites near the Myf5 gene of Lin28a+ MuSCs, pax7+ embryo and Pax7+ adult MuSCs (signal values 0-100%).

(G) Clustering analysis of Lin28a+ MuSCs and conventional MuSCs transcriptomes. Each group was repeated 3 times.

(H) Volcanic map analysis of Lin28a+ MuSCs versus con MuSCs differentially expressed genes. Red: up-regulating by more than 2 times and p is less than 0.05; green: drop > 2-fold and p < 0.05.

(1) The expression levels of the three primitive limb mesoderm Zu Zhuailu factors Meis2, six1, eya, tep1 and Mdm4 in Lin28a + MuSCs relative to con MuSCs. (J) The expression levels of the marker factors Peg3 and Pdgfra for other non-traditional PAX7 muscle progenitor cells in Lin28a+ MuSCs compared to con MuSCs. (K) In contrast to con MuSCs, the expression levels of myogenic terminal differentiation markers in Lin28a+ MuSCs include a number of troponins (Tnni 1, tnnt 3), troponin (Casq 1), muscle polysaccharides (Sgcd), and tropomyosin (Tpm 3). (L) Lin28a+ Muscs (black) or con Muscs (gray) enrichment features identified by gene set enrichment analysis. (M) A representative GSEA profile is shown, along with Normalized Enrichment Score (NES) and P values. X: p is less than 0.05; the following materials: p is less than 0.01; the preparation method is as follows: p is less than 0.001

Fig. 4.Lin28a promotes self-renewal of MuSCs. FIG. 4 (A-B) cell proliferation rates of con MuSCs (A) and Lin28a-tdTO+ MuSCs (B) after retroviral infection of P20 (con) or P10 (Lin 28 a+) with empty vector (CTRL) or Lin28 a. (P represents the number of passages). (C-D) quantitative RT-PCR detection of the myogenic differentiation markers of con MuSCs (C) or Lin28a-tdTO+MuSCs (D) overexpressing Lin28a relative to con MuSCs or Lin28a-tdTO+MuSCs of empty vector (CTRL). Data are mean ± SEM,3 independent experiments. Panel (E) shows Pax7-CreERT2 (PC) and Pax7-Creer; photographs of tibial anterior muscle inflammatory regression and muscle regeneration 7 days after NFKB-LSL-Lin28a (PM) mice were injured. Scale bar: 5 mm. (F) Hematoxylin and eosin staining of PC and PM mice tibialis anterior muscle sections on

days

7 and 14 post-frostbite. Scale bar: 300 microns. (G) Immunofluorescent staining of PM and PC mice with compromised tibialis anterior MyoD, pax7 and Ki 67. Scale bar: 20 microns. (H) Quantification of percentage of myopic anterior muscle sections stained MyoD and Pax7 positive cells. For each muscle, at least 7 different regions are quantified. X: p is less than 0.05; the following materials: p is less than 0.01; the preparation method is as follows: p < 0.001.

FIG. 5 Lin28a-T2A-CreERT2 mice were generated and evaluated for lineage follow-up. (A) targeting strategy to generate Lin28a-T2A-CreERT2 mice. The CreERT2 fragment is inserted between the last exon of Lin28a and the 3' UTR. (B) Lin28a-T2A-CreERT2 mouse genotype results. (C) Lin28a-T2A-Creer; LSL-tdTO mouse testes showed a PLZF+ Spermatogonial Stem Cell (SSCs) subpopulation of tdTO+ over 14 days of lineage follow-up. Scale bar: 50 microns. (D) Western blot analysis of Cre protein in conventional (Con) MuSCs and tdTO+MuSCs shortly after in vitro culture. Lin28-Cre cells are positive control cells that overexpress Cre protein. Lin28a expression disappeared shortly after in vitro culture of Con and tdTO+MuSCs (FIG. 7A). (E) Western blot analysis of Wild Type (WT), LSL-tdTO, lin28a-T2A-CreERT2, lin28a-T2A-CreERT; lin28a protein in LSL-tdTO mouse testis. Lin28a-T2A-CreERT2 mice exhibited a low band of endogenous Lin28a and a high band of Lin28 a-T2A. The intensity of the two bands was quantified using the Lin28a/GAPDH ratio. GAPDH protein is a loading control.

FIG. 6 in vitro differentiation of freshly sorted Lin28 a+MuSCs.

(A) Phase contrast and fluorescence microscopy assess changes in vitro differentiated Lin28a+ cells under adipogenic, endothelial or osteogenic conditions. Alkaline phosphatase (ALP) staining showed that tdto+ cells still expressed MyoD after 10 days of differentiation under osteogenic conditions. Yellow arrows indicate some tdto+ cells lightly stained with ALP and co-stained with MyoD. Scale bar: 100 microns.

(B) Ratio of oil red stained positive cells and MyoD positive cells per field after incubation under adipogenic conditions. Data are mean ± SEM, with 3 independent experiments, each with 5 fields averaged.

(C) Proportion of MyoD positive cells per field after culture under endothelial conditions. Data are mean ± SEM, with 3 independent experiments, each with 5 fields averaged.

(D) Ratio of ALP positive cells and MyoD positive cells per field after culture under osteogenic conditions. Data are mean ± SEM. Data are mean ± SEM, with 3 independent experiments, each with 5 fields averaged.

FIG. 7 conventional and Lin28a+ MuSCs gene expression.

(A) Western blot analysis of Lin28a protein in myogenic Growth Medium (GM) and Differentiation Medium (DM) of mouse C2C12 myoblasts, conventional (con) MuSCs and Lin28a+ cells. LTS was a positive control cell line that overexpressed Lin28 a. Once the cells were cultured in medium, either growth or differentiation medium, no loss of Lin28a expression was detected, using GAPDH protein as a loading control.

(B) Quantitative RT-PCR showed the expression levels of Lin28a in conventional (con) MuSCs and Lin28a+ cells in myogenic Growth Medium (GM) and Differentiation Medium (DM). Data are mean ± SEM, with 3 independent experiments.

(C) Volcanic pattern analysis of con MuSCs gene differential expression after Lin28a overexpression compared to conventional MuSCs.

(D) Bisulphite whole genome sequencing (WGBS) analysis was performed on Lin28a + MuSCs of embryo and adult pax7+ MuSCs (3 independent experiments). (E) GO and KEGG analysis of cluster 2 in FIG. 3A. (F) GO and KEGG analysis of cluster 3 in FIG. 3A. (G) GO and KEGG analysis of cluster 5 in FIG. 3A. (H) Quantitative RT-PCR analysis of expression of the Iet-7 target proteins (Hmga 2, igfbp 2), igf2 and Iet-7 pathway-related genes (Zcchc 6, zcchc11, dis3l 2) in freshly screened con and Lin28a+ Muscs in GM. (I) Western blot analysis of expression of the Lin28a and Iet-7 target proteins IGF2BP2 and HMGA2 in con-MuSCs or Lin28a-tdTO+ MuSCs overexpressing Lin28a relative to con-MuSCs or Lin28a-tdTO+ MuSCs of empty vector (CTRL). (J) Quantitative RT-PCR analysis expression of fat, osteogenic and vascular genes in Lin28a-tdTO+ MuSCs overexpressing Lin28a relative to empty vector (CTRL). Data are mean ± SEM, with 3 independent experiments. (K) Enrichment was performed in con MuSCs over-expressed Lin28a (black) or empty vector (gray) by gene set enrichment analysis. (L) shows a representative GSEA profile, and Normalized Enrichment Score (NES) and P values. Ns, no significant difference: p is less than 0.05; the following materials: p is less than 0.01; the preparation method is as follows: p < 0.001.

FIG. 8 Lin28a+ cells in E12.5 embryo limb.

Treatment of Lin28a-T2A-Creer with Tamoxifen (TMX) at E11.5; LSL-tdTO embryos were collected at E12.5. White arrows indicate Lin28a+ limb progenitor cells of the embryonic limb, and yellow arrows indicate Lin28a+ muscle fibers of the embryonic limb. Scale bar: 500 microns.

FIG. 9 shows a classification of rejuvenating genes, including rejuvenating transcription factor network genes, rejuvenating apparent modification network genes, rejuvenating signal ligands, genes for receptors and related kinase networks, rejuvenating nucleic acid binding factor network genes, the expression of which is capable of rejuvenating non-pluripotent cells, reversing senescence, reversing depletion of non-pluripotent cells, reversing non-multipotent cells, prolonging the life of non-multipotent cells, increasing the number of passages of non-multipotent cells.

FIG. 10 shows the extent to which Lin28a-tdTomato+ positive muscle cells were activated 48 hours after infection with Bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4l virus versus empty vector virus.

FIG. 11 shows the extent to which Lin28a-tdTomato+ positive muscle cells were activated 72 hours after infection with Bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc4l virus versus empty vector virus.

FIG. 12 shows the final SA-Bgal+ senescent cell fraction of senescent mouse muscle cells (MuSCs-6G) transfected with 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l) and senescent mouse muscle cells (MuSCs) of empty vector control.

FIG. 13 shows proliferation rate and cell life of senescent mouse muscle cells (MuSCs-6G) transfected with 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l) and senescent mouse muscle cells (MuSCs) of empty vector control 3 days after FGF2 stimulation. Aged mouse muscle cells (MuSCs) apparently failed to proliferate and passaged until the end of life. Aged mouse muscle cells (MuSCs-6G) transfected with 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l) restored FGF2 induction and proliferation capacity, and were passaged > 10 times.

FIG. 14A shows the% senescent cell area of senescent human muscle cells (6 viruses) transfected with 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l) and of the empty vector control (control) after FGF2 stimulation. FIG. 14B shows the% senescent cell numbers of senescent human muscle cells (6 viruses) transfected with 6 genes (Bcl 11a, bcl11B, lmo2, otx2, pbx1, pabpc4 l) and of empty vector controls (controls) after FGF2 stimulation. The 6 genes significantly reversed the senescent cell proportion.

FIG. 15 shows that senescent mouse muscle cells (6 viruses) transfected with 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l) and senescent mouse muscle cells (empty vector control) transfected with empty vector controls all significantly overexpressed 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l). P is less than 0.001

FIG. 16 shows that senescent human muscle cells transfected with 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l) (6 viruses) and senescent human muscle cells of empty vector control (empty control) all significantly overexpressed 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l). P is less than 0.001

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

The following examples relate to the sources of the main reagents as follows:

case 1

For a conservative relationship between young and tissue regeneration, charles-Dart is the first proposed person. Lin28a is a candidate for tissue rejuvenation, known for its function in somatic reprogramming and embryogenesis, but its function in skeletal muscle is unknown. By lineage tracking we found a rare population of Lin28a expressing muscle satellite cells that can respond to acute injury by partially expressing P ax3 or Pax7 during proliferation and contribute to all types of muscle fibers during muscle regeneration. Lin28a+MuSCs expressed more Pax3 than traditional MuSCs (muscle stem cells) and showed enhanced myogenic capacity in vitro. In terms of epigenetic clocks, adult Lin28a+musc is located between adult pax7+musc and embryonic pax7+myoblasts according to DNA methylation profile. We found that MuSCs overexpressing Lin28a+ upregulate several fetal limb bud mesoderm transcription factors and can maintain a stable dedifferentiated young state in vitro and in vivo, enhancing stem cell self-renewal and stress response.

Introduction to the invention

Despite the tremendous advances we have made in understanding multipotency, somatic reprogramming and transdifferentiation, the molecular basis of stem cell self-renewal remains unclear. In particular, no single factor was found to rejuvenate somatic cells. The potential risks of using multiple reprogramming factors of Oct4, sox2, klf4, myc, etc. to achieve somatic rejuvenation are: if the cells rejuvenate thoroughly, a pluripotent stem is obtained, and teratomas are produced. It would be a breakthrough in this field if it could be found which individual stem cell factor could allow the cell part to return or maintain youth and would not be oncogenic in normal expression in adult cells. Lin28 is one of the genes that was originally discovered to be involved in the juvenile development and growth process, a concept that is related to stem cell self-renewal and differentiation ^1-3 And can be formed by the Iin28-Iet-7 pathway ^4-8 A part of the mechanism is explained.

Skeletal muscle has a strong regenerative capacity. Skeletal muscle satellite cells(MuSCs) are a population of resident stem cells, which are embedded between the fibrous membrane and the basement membrane of the muscle, self-renew, and are essential for skeletal muscle regeneration ¹² In injured muscle, muSCs are activated and begin to proliferate into fate-determined myogenic progenitor cells ¹³ These activated myogenic cells fuse with existing muscle fibers, or form muscle fibers from the head, to effect muscle repair and regeneration ¹⁴ Although upregulation of Lin28a in normal adult mammalian tissue was observed only during muscle regeneration ¹⁵ It is not clear what cell types express Lin28a and whether Lin28a plays a role in maintaining MuSC self-renewal. This is in part because no strict lineage follow-up study has been performed on Lin28 a.

In this paper, we consider that Lin28a mRNA is regulated post-transcriptionally ¹⁶ Is described herein, the Lin28a protein is coupled using a "self-cleaving" CreERT2 recombinase to follow the Lin28a+ cell lineage in adult murine muscles. We have surprisingly found that a group of previously unknown and rare MuSCs, which can form all types of muscle fibers in adult muscles, are characterized by fetal limb muscle progenitor cells. Lin28a+MuSCs exhibit higher stress response and self-renewal capacity and have enhanced myogenic capacity. Transgenic mice that can activate Lin28a expression under conditions of damaging stress also show that Lin28a can promote skeletal muscle regeneration in vivo.

Results

Lin28a+ satellite cells can produce all types of myofibers during regeneration

To see if Lin28a is expressed in a specific skeletal muscle cell subset, we have coupled "self-cleaving" T2A-CreeRT2 at the C-terminus of Lin28a protein using homologous recombination, constructed an artificial Lin28a-T2A-CreeRT2 transgenic mouse (FIGS. 5A, B) and hybridized with a Rosa26-Ioxp-stop-Ioxp-tdTomato (tdTO) reporter mouse to track fate of Lin28a+ cells following muscle injury. After approximately 20 days of tracking (fig. 1A), we found that the pre-Tibial (TA) muscle of the uninjured mice had a small amount of Lin28a-tdto+ and Lin28a-tdto+ monocytes in the muscle matrix (fig. 1B), indicating that Lin28a+ cells were present in the muscle matrix and could fuse with about 10% of the existing TA muscle fibers during normal muscle homeostasis (fig. 1B). After injury, the numbers of Lin28a-tdTO+ myofibers and Lin28a-tdTO+ monocytes increased significantly (FIG. 1B, C), indicating that injury stimulated proliferation of Lin28a+ cells and increased their fusion number to existing myofibers by an amount exceeding 3-fold (FIG. 1C). In another more slowly updated muscle group (soleus muscle) 17, the number of Lin28a-tdTO+ muscle fibers also increased significantly after injury (FIG. 1B, C). To ensure that our Lin28a-tdTO+ lineage follow-up can specifically and truly reflect the expression of endogenous Iin28a, we examined the distribution of tdTO+ cells in the testes. As a result, we found that after 20 days of lineage follow-up, only a portion of PLZF+ spermatogonial stem cells located at the periphery of seminiferous tubules were specifically labeled as Lin28a-tdTO+ cells (FIG. 5C). When Lin28a+ cells were cultured in vitro, the expression of Lin28a protein and Cre protein disappeared simultaneously, indicating that the turnover rate of "cleaved" T2A-CreERT2 was sufficiently fast to accurately label Lin28a+ cells with high fidelity (FIG. 5D). Since this manual manipulation can interfere with Lin28a ends and 3' UTR, lin28a activity is likely to be slightly elevated. To ensure that endogenous Lin28a expression was not interfered with by coupled T2A-CreERT2, we examined the total Lin28a protein expression of heterozygous Lin28a-T2A-CreERT2 mice to find wild-type and Lin28a-T2A-CreERT2; although there was some difference between LSL-tdTO mice, the difference was not too great (FIG. 5E).

Pax7 is generally considered a decisive marker for adult muscle stem cells (MuSCs) 19, but it has also been reported that some MuSCs do not express Pax7, but rather Pax 3-22. Therefore, we attempted to determine if Lin28a-tdTO+ monocytes expressed Pax3 or Pax7. Immunofluorescence results showed that all Lin28a-tdto+ monocytes were located between the basal membrane and myofibrillar membrane, similar to the positions of pax7+ muscle satellite cells, but only a small fraction co-expressed Pax7 or Pax3 (fig. 1D). Overall, lin28a-tdto+ cells accounted for only < 30% of the pax7+ or pax3+ MuSC pool (fig. 1E, F), indicating that they accounted for only a small portion of the Pax7/pax3+ MuSC population. Quantification showed that 37.1% of Lin28a-tdTO+ monocytes co-expressed Pax7 (FIG. 1E), while 40.6% of Lin28a-tdTO+ monocytes co-expressed Pax3 (FIG. 1F), with the remaining Lin28a-tdTO+ cells being Pax7-Pax3-. Overall, our western blot and lineage tracking results indicate that at least some satellite cells or MuSCs express Lin28a, but do not express Pax7 and Pax3, and that these Lin28a + MuSCs can respond to injury by transiently proliferating to Lin28a-pax7+/pax3+ MuSCs, thereby contributing to muscle regeneration.

Skeletal muscle fibers exhibit a degree of metabolic and functional diversity upon terminal differentiation, with each muscle group comprising different types of muscle fibers, such as type I, IIa, IIx and type IIb muscle fibers. These muscle fibers can be broadly classified into slow muscle fibers (I) and fast muscle fibers (IIa, IIx, IIb), or oxidative (I, IIa) and glycolysis (IIx, IIb) 23. In view of the differential results of Lin28a-tdTO+ markers in fast and slow myoflatfish muscles, we next identified whether the myofiber types formed by Lin28a-tdTO+ cells were specific. Immunofluorescent staining of uninjured mouse myofibers showed that Lin28a-tdto+ cells could produce type IIa, IIx and IIb myofibers in TA muscle (fig. 1H), but not type I myofibers in soleus muscle (fig. 1B, C) in a 20 day tracking window. However, after injury, lin28a-tdTO+ cells can produce all types of myofibers in the slow and fast muscle soleus muscles (FIG. 1G). These results indicate that Lin28a+ MuSCs can proliferate and differentiate into all types of muscle fibers during skeletal muscle regeneration in vivo.

Lin28a+ cells are MuSCs and exhibit enhanced myogenic potential in vitro

To further determine the characteristics of Lin28a-tdTO+ monocytes relative to traditional Pax7+ MuSC, we decided to analyze this population of cells using flow cytometry. On the tdTomato channel we observed a significant increase in tdTO+ cells only after muscle injury (P < 0.001). To further characterize tdto+ cells, we used different cell surface antibodies: CD31 (endothelial lineage), CD45 (hematopoietic lineage), sca1 (mesoderm), VCAM1 (classical pax7+musc) to identify these cells (Liu et al 2015). Flow cytometry analysis showed that tdTO cells were predominantly CD45 negative (-90%) (fig. 2D) and VCAM1 positive (> 99.99%), similar to pax7+musc (Liu et al 2015). However, it should be noted that Lin28a-tdTO+ cells account for only a small fraction (0.73%) of the total amount of VCAM1+ cells. The difference is that traditional MuSC are only CD 31-and Sca1-, whereas about 60% of Lin28a+ cells are CD31 positive, about 70% are Sca1 positive, while the rest are CD31/CD45-Sca1-, - (fig. 2D) like traditional pax7+musc, these results indicate that most Lin28a+ cells may be more primitive vcam1+cd31+sca1+ mesodermal progenitor cells.

In view of these surface marker profiles, we attempted to differentiate freshly sorted Lin28a+ cells into different cell lineages, such as skeletal muscle, vascular endothelial cells, adipocytes, or osteoblasts, in various differentiation media. The results indicate that after differentiation of Lin28a+ cells in the adipogenic, osteogenic and endothelial cell culture media, most cells senesce or die and cannot differentiate further, although a small fraction (-20%) did differentiate into alkaline phosphatase positive osteoblasts (FIGS. 6B-E). However, all cells retained MyoD expression, indicating that they maintained myogenic differentiation potential even under conditions of other lineage differentiation induction (fig. 6B-E).

To further compare the muscle differentiation potential of Lin28a+ cells with VCAM1+CD31-Sca1-Pax7+ MuSC (hereinafter conventional MuSC), we proliferated and differentiated Lin28a+ cells in muscle stem cell expansion medium and differentiation medium, respectively, followed by immunofluorescent staining. The results indicate that 100% of Lin28a+ cells can express the muscle stem/progenitor cell markers MyoD and Pax7 upon proliferation (fig. 2E), and they can form robustly upon differentiation, polynuclear myotubes expressing Myosin Heavy Chain (MHC) proteins (fig. 2F-L). During myogenic differentiation, the expression of many muscle stem/progenitor markers, such as Pax3, pax7, myoD, myf5, was down-regulated, while the expression heavy chains of many myogenic differentiation-related genes, such as muscle creatine kinase (Ckm) and myosin (Myh 1, myh2, and Myh 4), were significantly up-regulated (FIG. 2H). These results indicate that Lin28a+ cells can proliferate in vitro in the form of skeletal muscle progenitor cells and are capable of myogenic fusion and differentiation into multinuclear myotubes.

To compare the fusion efficiency of Lin28a+ cells with conventional MuSCs, we cultured Lin28a+ cells, conventional MuSCs, and 1:1 mixed cells, and measured their fusion index at myogenic differentiation. Interestingly, we observed that the Lin28a+ cell-derived myotubes were thicker in diameter and therefore more hypertrophic than the traditional MuSC-derived myotubes (fig. 2I, J; p=6.15×10-7). In addition, our results indicate that the fusion index of Lin28a+ cells is higher than that of conventional MuSCs, indicating that Lin28a+ cells have higher myogenic capacity.

To further compare the molecular differences between Lin28a+ cells and conventional MuSCs, we compared the myogenic factor expression of both groups of cells by qRT-PCR and Western blot analysis. We found that Lin28a+ cells expressed more Pax3 protein than traditional MuSC (FIG. 2K), while during proliferation, pax7 protein and MyoD protein were expressed at similar levels (FIG. 2K). After differentiation, myotubes derived from Lin28a+ cells expressed more MHC proteins and MyoG proteins than traditional MuSC-derived myotubes (FIG. 2L). mRNA expression was similar to WB, with Lin28a+ cells expressing significantly higher levels of Pax3 in the amplification medium than conventional MuSC (FIG. 2K). After terminal differentiation, lin28a+ cell-derived myotubes expressed higher levels of Myf5, myoG, ckm, myh1 and Myh4 than traditional MuSC-derived myotubes (FIG. 2L). Notably, even after quenching of Lin28a expression in Lin28a+ cells in vitro, myogenic differences resulting from these early programmes remained because expression of Lin28a protein and mRNA was undetectable both in expansion culture and in differentiation (fig. 7a, b). Taken together, these results indicate that these early programmed Lin28a+ cells differ from conventional MuSCs, both at the functional and molecular level.

Epigenomic and transcriptomic profiles showed Lin28a dedifferentiated MuSCs

Lin28a and Pax3 are typical expressed genes during embryogenesis and fetal development. Whereas previous studies have shown that in mammals, the DNA methylation epigenetic clock can exclusively indicate biological senescence from embryonic stem cells to adult cells to senescent cells, we want to understand the epigenetic profile of Lin28a+ cells at the whole genome level. Thus, we compared Lin28a+MuSCs with embryonic and adult Pax7+MuSCs (N=3 each) by whole genome bisulfite sequencing (WGBS; FIG. 7D). Cluster analysis showed that while adult Lin28a + MuSCs were more similar to adult pax7+ MuSCs, they were also somewhat similar to embryonic pax7+ myoblasts (fig. 3 a). Clusters (DMRs) with 5 differentially methylated regions, instead of 8 randomly accidentally expected, indicate a consistency of random distribution among the MuSC subsets, showing higher information structures than expected (fig. 3A).

Clusters

1 and 4 demonstrate the similarity of two adult MuSCs. Cluster 3 consisted of 6573 DMRs and 5883 genes, suggesting a striking similarity between adult Lin28a + MuSCs and embryonic pax7+ myoblasts. Cluster 2 and cluster 5 demonstrate the epigenetic uniqueness of Lin28a+ cells as a unique subset of MuSCs.

In particular, according to GO gene enrichment analysis, the muscle development and epithelialization genes of cluster 2 were highly enriched (fig. 3B and 7E), indicating that Lin28a+ cells had silenced many skeletal muscle differentiation genes, as well as genes that induced terminal differentiation, indicating that Lin28a+ MuSCs were more readily dedifferentiated than pax7+ MuSCs. According to KEGG analysis, cluster 3 was highly enriched for growth hormone signals involved in adult muscle-related calcium signal contraction, neuromuscular junction axon guidance signals, phospholipase D signals 26, and hypertrophic growth (fig. 3C and 7F). These results confirm that adult Lin28a+ MuSCs are similar to embryonic Pax7+ myoblasts, silencing many muscle maturation-related genes. Cluster 5 is highly enriched for genes involved in cell migration, vascularization or angiogenesis (fig. 3D and 7G), suggesting that Lin28a+ cells are similar to the original somite progenitor cells, which have high migration and angiogenesis capacity. 27 28, for example, the primitive muscle transcriptome factors Mef2c sites 29, 30 in Lin28a+ are relatively demethylated relative to embryonic and adult pax7+ MuSCs (fig. 3E). However, lin28a+ MuSCs still originate from adults like adult Pax7+ MuSCs, e.g., they are in the vicinity of the myogenic transcription factor Myf5 gene, a similar methylation pattern (FIG. 3F). Overall, lin28a + MuSCs are more similar to adult pax7+ MuSCs, but have unique embryoid-like characteristics and dedifferentiate at the epigenomic level.

Then, we performed an RNA-seq analysis to confirm the dissimilarity between adult Lin28a+ and Pax7+ MuSCs. Cluster analysis (n=3 each) showed that Lin28a+ cells were transcriptionally very different from traditional pax7+ MuSCs (fig. 3G) volcanic pattern analysis, showing that 78 genes were significantly up-regulated > 2-fold (P < 0.05) and 83 genes were significantly down-regulated > 2-fold (P < 0.05) (fig. 3H). Among the upregulated genes are 3 primitive limb bud mesodermal progenitor cell transcription factors 31-36: meis2 (-27 fold), six1 (-10 fold) and Eya (-3 fold) indicated that Lin28a+ cells were similar to fetal limb muscle progenitor cells (fig. 3I). In addition, lin28a+ cells also had higher Mdm4 (-25-fold) and Tep1 (-6-fold) (FIG. 3I). Interestingly, significant Iet-7 targets such as Igf2bp2 and Hmga2 were not significantly upregulated (FIG. 7H, I). We also examined the expression of other unconventional, pax7 independent muscle progenitor marker genes Peg3 and Pdgfra 20, 37, and found no significant differences in Iin28a+ cells compared to the traditional pax7+ MuSCs (fig. 3J). In contrast, the marker gene set for myogenic terminal differentiation was one of the most significantly down-regulated gene sets, including troponin and tropomyosin (fig. 3K), confirming that Lin28a + MuSCs were dedifferentiated relative to pax7+ MuSCs. Gene collection enrichment analysis (GSEA) showed that in Lin28a+ cells, some stem cell signals were up-regulated, including Notch pathway, neural Progenitor cells (Meissner_NPC_ICP_H24Me3), and signals of hematopoietic stem and Progenitor cells (Eppert_HSC_R and Eppert_Progenitor) (P < 0.05; FIG. 3L). In Lin28a+ cells, many stress response pathways are also up-regulated: PERK-mediated unfolded protein response, TNF-NF-. Kappa.B and IL6-STAT 3-mediated pro-inflammatory stress, and FoxO-mediated oxidative stress (FIG. 3L). In contrast, mTOR pathway components, ca2+ signaling-related signals, and Myogenic differentiation markers (myogenic_targets_of_pax3) in Lin28a+ cells were down-regulated compared to traditional MuSCs, which all mean that Myogenic terminal differentiation was inhibited (fig. 3l, m). Overall, our analysis results indicate that Lin28a+ cells exhibit higher dedifferentiation and stress responsiveness compared to traditional pax7+ MuSCs. To determine whether these features are associated with only Lin28a expression, or due to Lin28a expression, we overexpressed Lin28a more than 2-fold in conventional MuSCs and repeated the analysis (FIG. 7C). GSEA showed that Lin28a overexpression again resulted in increased dry signaling, such as Wnt, notch and hedgehog signaling pathways, and e2 f-associated mitotic or DNA replication signaling (fig. 7k, l). Interestingly, lin28a also upregulated some of the Hypoxia signaling pathways, i.e., genes downregulated on HIF1A RNAi (Manalo_Hypoxia). In contrast, lin28a significantly down-regulated the Myogenic differentiation trait (myogenic_target_of_Pax3; structured_Muscul_control) (FIG. 7K, L), indicating that Lin28a promotes self-renewal and dedifferentiation of MuSCs.

Lin28a promotes self-renewal of MuSCs, rejuvenate senile muscle progenitor cells.

Considering that expression of Lin28a was lost after in vitro culture of Lin28a+ cells, we re-overexpressed Lin28a 2-fold more in Lin28a-tdTO+ cells and traditional MuSCs with lentiviral vectors with CMV promoter to explore the function of Lin28a in muscle stem cells. Tdto+ cells that overexpressed Lin28a had slightly more proliferative self-renewal capacity within 3 days compared to empty vector controls and conventional MuSCs (fig. 4, A, B). qRT-PCR also confirmed these findings (fig. 4c, d), showing significant decrease in differentiation-related genes such as Ckm, myoG, myh1, myh2, myh4 after Lin28a overexpression (fig. 4C, D). In addition, we tested whether overexpression altered the lineage differentiation potential of tdto+ cells by differentiating cells into adipocytes, osteoblasts and endothelial cells, respectively. Based on qRT-PCR results, we found that over-expression of Lin28a enhanced osteogenic differentiation, inhibited adipogenic differentiation less but significantly, and had little effect on vascular cell differentiation (FIG. 7J). These results indicate that activation of Lin28a in cultured tdto+ cells can partially mimic the characteristics of freshly sorted Lin28a+ cells, such as showing weak osteogenic propensity, but no adipogenesis or vascular differentiation (fig. 6 and 7J). Taken together, these results suggest that the function of Lin28a in Lin28a + MuSCs may be to promote self-renewal while retaining enhanced myogenic and even osteogenic differentiation potential.

Lin28a promotes MuSC dedifferentiation and muscle regeneration in vivo

Our in vitro experiments showed that Lin28a was over-expressed by more than 2-fold, and that it was able to promote self-renewal and dedifferentiation of mouse muscle stem cells without affecting their myogenic capacity in 3 days, so we wanted to know if Lin28a was over-expressed in adult Pax7+ muscle stem cells as well as to enhance muscle regeneration in vivo.

Thus, we constructed NFKB-Ioxp-stop-Ioxp-Lin 28a transgenic mice (mt 190) and hybridized to Pax7-CreERT2 mice to obtain heterozygous PM (Pax 7-CreERT2; mt 190) mice that were able to express Lin28a in Pax7+ MuSCs only during muscle injury and shut it down after the inflammatory signal resolved. Following the same schedule of TMX and injury injections as our lineage follow-up experiment (fig. 1A), we tested PM mice for regenerative capacity on

days

7 and 14 after double-blind freeze injury testing of TA muscles. At 7 days post injury, it was visually apparent that PM mice were better repaired and less inflamed than PC mice (Pax 7-CReERT 2) (FIG. 5K)

Hematoxylin and eosin staining was performed on post-injury muscles, 7-14 days after PM mice injury, with smaller pink necrotic areas in the TA muscles and larger central nuclear fiber regeneration areas in the PM mice (fig. 5L). These results indicate that Lin28a significantly enhances the muscle regeneration capacity of PM mice. To determine which phase of muscle regeneration of PM mice was enhanced, we immunofluorescent stained TA muscle sections. We found that, consistent with our observations, lin28a overexpression promoted self-renewal of MuSCs, since the number of Pax7+ MuSCs and MyoD+ myoblasts proliferated in the TA muscle of PM mice was significantly higher than that of PC mice (FIG. 5N, O). These results indicate that pax7+ MuSCs injury induces Lin28a overexpression to promote their self-renewal and proliferation, thereby enhancing skeletal muscle regeneration following acute injury.

Discussion of the invention

Lin28a is expressed primarily during embryonic development and decreases with development, while ectopic overexpression thereof promotes regeneration of various adult tissues 11, 49. However, its endogenous expression and role in adult tissue regeneration has not been elucidated so far. By lineage tracking we found a group of previously unidentified bone MuSCs expressing Lin28a that can respond to muscle damage and show enhanced regeneration potential. Interestingly, this population of cells was not traditional pax7+ MuSCs, as they had unique epigenomic and transcriptomic profiles, suggesting that they were located between adult and embryonic pax7+ muscle stem cells. Furthermore, we demonstrate that Lin28a+ cells express more Pax3 and shoot mesoderm transcription factors, such as Meis2, six1 and Eya, than traditional Pax7+ MuSCs. Pax3 regulates limb muscle development by modulating families 27, 35, 36, 50-53 of Six1 and Eya, while Lin28a expresses 54, 55 at an early stage of limb bud development during mouse embryogenesis. Consistent with these reports, we also tracked Lin28a+ cells during embryonic limb development (FIG. 8), and found migrating limb muscle progenitor cells, which also produced limb muscle fibers. Lin28a up-regulates Notch signaling, enhances the self-renewal and stress response capabilities of MuSCs, and simultaneously inhibits terminal differentiation, further supporting the notion that Lin28a can reverse the epigenetic clock, maintaining a stable dedifferentiated state. To further confirm these findings in vivo, pax7+ cells following freezing injury expressed inflammation-induced Lin28a, such that Lin28a was activated only in MuSCs that underwent injury and inflammation. We found that injury activated Lin28a enhanced the number of pax7+ and myod+ muscle progenitor cells, thereby accelerating the regression of necrotic areas and improving muscle regeneration after freeze injury. This expands our knowledge of Lin28a, which was previously thought to promote cell proliferation only during reprogramming and regeneration 5, 56, 57. Given the small number of Lin28a+ cells (VCAM1+ cells 0.7%), ablation of Lin28a+ cells is likely to have no effect on steady state adult muscle mass, number, and hypertrophy. While the previous report found that Lin28a itself was not necessary for myogenesis, it did not exclude the importance of Lin28a to maintain a stable dedifferentiated state in a few muscs, as we showed by lineage tracing. Furthermore, previous findings indicate that Lin28a has important physiological roles in the development of mouse limb bud and tail bud mesoderm 55, 58. Given our findings in Lin28a in human muscle progenitor cells, future work may also focus on the role of Lin28+ cells in primate development.

When we sorted Lin28a-tdTO cells by flow, expression of Lin28a was found to be quenched after in vitro culture. To distinguish whether the phenotype still resulted from Lin28a expression is related or only, we re-overexpressed Lin28a in Lin28-tdTO and conventional MuSCs, and as a result, found that Lin28a can promote dedifferentiation and proliferation of adult MuSCs. In addition, after Lin28a reactivation, the myogenic capacity of conventional MuSCs is also significantly improved, which is important in view of the steady decrease in the myogenic capacity of MuSCs with development and aging, perhaps to improve the myogenic capacity of muscle stem cells in the elderly.

Materials and methods

Transgenic mice

The strategy for constructing Lin28a-T2A-CreERT2 transgenic mice is shown in FIG. S1A. Construction of donor vector: the genomic fragment of the third exon of Lin28a extending to the last intron and the genomic fragment in the 3' UTR of Lin28a were used as two homology arms, respectively. The Frt-Neo-Frt-last exon-2A-CreERT2 expression cassette was inserted between the two arms to obtain the donor vector. The donor vector was electroporated into mouse embryonic stem cells. By homologous recombination, the donor vector can insert a Frt-Neo-Frt-2A-CreERT2 fragment between the last exon of Lin28a and the 3' UTR. Clones of target ES cells were selected by G418 selection. Next, the Frt-Neo-Frt expression cassette in the selected ES cell clone was deleted, and the resulting ES cells were injected into C57BL/6 albino embryos. Transgenic mice can be identified by their hair color, and amplified by mating with normal C57BL/6 mice, while genotyping. Construction of donor vector in construction of NF-. Kappa.B-LSL-lin 28a-T2A-luc (No. mt190) transgenic mice: NF-KB response element and its downstream TAp promoter (minimum TA promoter of herpes simplex virus) 65 were inserted upstream of the LSL fragment. The LSL fragment contains 3 SV40 late polyA fragments between 2 LoxP sites and 2 LoxP sites. Downstream of the LSL fragment is the CDS of Lin28a, which is labeled with T2A-Iuc (firefly luciferase reporter). As previously described, targeting vectors were integrated into the H11 site of C57BL/6 mice to ensure the specificity of the NF-. Kappa.B responsive elements. R26-tdTO ((ROSA) 26Sortm14 (CAG-tdTomato), stock No. 007914) was obtained from JAX laboratories. All animal procedures were approved by the institute of animal and the institute of stem cell and regenerative medicine, academy of sciences of China.

Tamoxifen and frozen lesions

Tamoxifen (TMX, sigma-Aldrich) was added at 20mg ml ^-1 Is dissolved in corn oil and 100mg/kg TMX is administered by intraperitoneal injection to 6 week old mice as shown. Prior to freezing injury, all mice were anesthetized with isoflurane. The skin was incised with a scalpel to expose the anterior Tibial (TA) muscle. A 4 mm diameter steel probe was cooled in liquid nitrogen and placed on TA muscle for 10 seconds twice. Then, the skin incision is immediately closed with surgical suture and euiodine is applied to the wound to prevent post-injury infection. TA and soleus muscles were harvested 10 or 14 days after freeze injury.

Immunofluorescence.

After harvesting skeletal muscle, it was fixed in 4% paraformaldehyde at 4 ℃ for 1 hour. The tissues were then converted to 20% sucrose/ddH 2O overnight at 4℃and then frozen for embedding with OCT and sectioned to a slice thickness of 10. Mu.m. Before immunostaining, the frozen slides were fixed in methanol at room temperature for 10 minutes and then washed with PBS. Next, the slide immersed in the citrate antigen retrieval solution (ab 208572) was placed in a pressure cooker for antigen retrieval. Next, the slides were treated with 0.3% Triton X-100/PBS for 15 minutes at room temperature according to the manufacturer's instructions, and then incubated with mouse IgG blocking solution (M.O.M.kit, vector Lab) for 1 hour. Slides were incubated overnight at 4℃with primary antibody diluted in 0.3% Triton X-100, 5% goat serum/PBS. The next morning, the slides were washed with PBS and incubated at room temperature with secondary antibodies diluted in 0.3% Triton X-100, 5% goat serum/PBS, protected from light for 1 hour. The slides were then washed with PBS and blocked with glycerol and DAPI. For immunofluorescence, the following antibodies were used: pax7, pax3, myosin I (BA-D5), myosin IIa (SC-71) (all from Developmental Studies Hybridoma Bank (DSHB), 1:10), IIx, IIb (fastblastin), (Abcam, ab 91506), laminin (Sigma-Aldrich, no. L9393, 1:500), desmin (Abcam, ab32362, 1:200). Alexa Fluor secondary antibodies were used according to the manufacturer's instructions. The images were taken on a Nikon confocal microscope or PerkinElmer Vectra Polaris. For myofiber type recognition, type I and type IIa myofibers were recognized by staining directly with antibodies directed against Myosin I (BA-D5) and Myosin IIa (SC-71), type IIb myofibers were stained lighter with Fast Myosin antibody (Abcam, ab 91506), type IIx myofibers were stained darker with the same antibody, but simultaneously were not stained with Myosin IIa antibody (SC-71).

Fluorescence activated cell sorting analysis.

Conventional pax7+ MuSC was isolated by FACS sorting into VCAM1 positive and CD45/CD31/Sca1 negative populations, 67. Monocytes were isolated as previously described 68. Monocytes (107 cells) from one mouse were resuspended in 500ul PBS/10% fbs/3mM EDTA after isolation, and then incubated on ice for 40 minutes using the following fluorophore-conjugated antibodies: APC-CD31 (clone MEC 13.3), FITC-Sca1 (clone E13-161.7), VCAM 1-biotin (clone 429) (all from Biolegend, 1:100), BV421-CD45 (Becton Dickinson, clone 30-F11, 1:250), and then treated with PE-Cy7 streptavidin (BioL egend, cat.no.405206, 1:100) for 20 minutes. These cells were subjected to FMO control treatment. Cells stained with the same antibody isolated from wild type mice were used as tdTomato FMO controls. Undyed cells isolated from wild-type mice were used as undyed controls. Cells were analyzed on a BD FACSAria fusion flow cytometer and FACS data was analyzed using FlowJo software (treesar). The values of FACS analysis were taken from the average of more than three independent experiments.

In vitro cell culture and myogenesis, adipogenesis and osteoblast differentiation experiments.

Lin28a+ cells and conventional MuSCs cells were cultured in Matrigel coated plates, all cells at 37℃and 5% CO ₂ And a Growth Medium (GM) comprising DMEM/F-12 (Gibco) and 20% Fetal Bovine Serum (FBS) (GE Healthcare), 1% L-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco). At each passage, after 80% confluence, cells were trypsinized and passaged at 1:4 dilution. Differentiation was initiated by replacing the growth medium with Differentiation Medium (DM) including DMEM/F-12, 2% knockout serum replacement (Gibco), 1% L-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco). For fusion comparison experiments, cellTrac was usede Violet (Thermo Fisher, C34557), lin28a+ cells and 1:1 mixed cells were cultured in GM, respectively, and then transformed into DM to induce myotube formation. Freshly sorted Lin28a+ cells were grown in GM for 3 days and subsequently differentiated to myotubes, adipocytes, or osteoblasts. For myotube differentiation, DM was used in place of growth medium for 2-3 days. The cells were then immunostained for MyoG (Santa Cruz Biotechnology, sc-12732,1:100) and MYH1 (clone MF20, DSHB, 5. Mu.g/ml) to observe differentiated myotubes. For adipocyte and osteoblast differentiation, cells were examined following method 69 as previously described, followed by staining treatment.

Immunostaining of cultured cells.

For immunostaining, cells cultured on plates were fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized with 0.3% Triton X-100/PBS. Goat serum at 10% and Triton X-100/PBS at 0.1% were blocked for 1 hour at room temperature. Primary antibodies were diluted in 1% goat serum/0.1% triton X-100 and incubated for 2 hours at room temperature. Cells were washed and secondary antibodies were diluted in 1% goat serum/0.1% triton X-100 and incubated for 1 hour at room temperature. Cells were stained with Hoechst dye (1:2,000 in PBS) for 10 min at room temperature. The antibody comprises: pax3 (DSHB, 1:20), pax7 (DSHB, 1:20), myoD (Santa Cruz Biotechnology, sc-377460,1:100), myoG (Santa Cruz Biotechnology, sc-12732,1:100), MYH1 (clone MF20, DSHB, 5. Mu.g/ml). MYHC-IIb eFluor 660 (50-6503-32;Thermo Fisher;1:100), alpha-actin (sc-7453;Santa Cruz;1:500), 8-oxoguanine (ab 206461; abcam; 1:400). The fusion index is calculated as the ratio of tdto+ or MuSC nuclei number to tdto+ or MuSC nuclei total number within the multi-nuclei myotubes. In DM at 24 and 36 hours, at least 4 independent microscopic fields were used per group in three independent differentiation experiments.

RNA isolation and RNA-seq analysis.

Cells were resuspended in 500ul Trizol and total RNA was isolated according to manufacturer's instructions (Invitrogen). The quality of RNA is verified by an Agilent 2100 biological analyzer, and RIN is more than or equal to 7, and 28S/18S is more than or equal to 1.5:1. And the RNA quantity was verified by QUBIT RNA ASSAY KIT. A cDNA library was constructed using NEBNext Ultra RNA Library Prep Kit for IIIumina. RNA-seq was performed using IIIumina Novaseq-6000.

Real-time quantitative PCR analysis.

Total RNA was extracted from the sorted cells using Trizol (Invitrogen) according to the manufacturer's instructions. cDNA was reverse transcribed from this RNA using PrimeScript RT kit (Takara, RR 047B). The resulting cDNA was diluted 5-fold prior to qPCR using qPCR SYBR Green Mix. QPCR primer sequences were from OriGene website.

Western blot analysis.

Proteins were extracted with RIPA buffer supplemented with protease inhibitor cocktails I and II (Sigma) and phosphatase inhibitor cocktail group III (Calbiochem). Proteins were quantified using the Pierce BCA protein assay kit (Thermo Fisher) and analyzed using a Sunrise Tecan reader. After SDS-PAGE and electrotransfer onto PVDF membrane, western blotting was performed using the following primary antibodies and concentrations: lin28a (1:1000, CST), pax7 (0.28. Mu.g/ml, DSHB), pax3 (0.31. Mu.g/ml, DSHB), myoD (1: 1000,Santa Cruz Biotechnology), MHC (0.23. Mu.g/ml, DSHB), myoG (1: 1000,Santa Cruz Biotechnology), IGF2BP2 (1:1000, proteintech), hmga2 (1:1000, CST), cre (1:1000, millipore), GAPDH (1:1000, CST), vinculin (K106900P, 1:5000, solarbio), tubulin (ab 210797; abcam; 1:1000), P53 (sc-126; santa Cruz; 1:100), IMP1/2/3 (sc-271785;Santa Cruz;1:1000), citrate synthase (G-3) (sc-390693;Santa Cruz;1:1000).

Virus production

The following plasmids were used to produce viruses for various transgenic cell lines: lentiviral vector plasmids (Addgene # 19119), dR8.2 packaging plasmid (Addgene # 8455), VSV-G envelope plasmid (Addgene # 8454), pMSCV-mLin28A (Addgene # 26357), virus supernatants were collected in a 48-96 hour window and filtered with a 0.45 μm filter (Sartorius). Lentiviral vectors possessing a CMV promoter may also overexpress other rejuvenating genes.

Cell growth rate and lifetime assays

Will be 8×10 ⁴ Cells were seeded in one well of a 6-well plate (Falcon), growth medium packComprises DMEM/F-12 (Gibco) and 20% Fetal Bovine Serum (FBS) (GE), 1% L-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco). Nuclei were stained with Hoechst, photographed under a fluorescent microscope, and imageJ counted. This process was repeated for 3 days.

Statistical methods.

All statistical analyses were performed using GraphPad Prism 6 (GraphPad software). Data are expressed as mean ± sem. The statistical significance of the differences between groups was tested using a double sample t-test. P < 0.05 was considered significant. The biological (non-technical) number of replicates for each experiment is shown in the legend.

Case 2: based on our genomic and epigenetic bioinformatic analysis, we found that a range of gene networks have an interactive relationship with Lin28a, playing an important role in mediating Lin28a rejuvenation cells. As shown in FIG. 9, these networks can be classified into younger transcription factor networks (Grhl 2, zic5, zic2, utf1, otx2, snai3, lmo2, hopx), younger epigenetic modification networks (Bcl 11a, bcl11b, dnmt3b, mettl20, arid3 c), younger signal ligands, receptor and related kinase networks (Fgf 5, wnt3, calcr, epha1, epor, galr2, piezo2, ripk4, pak6, map3k15, pdzd4, shc 4), and younger nucleic acid binding factor networks (Foxr 2, hif3a, pbx1, zfp946, batf3, pabpc4l, celf4, lin28a, 28 b). We over-expressed the above rejuvenating genes in skeletal muscle stem cells by using lentiviral vectors with CMV promoters over 2-fold, and found that these rejuvenating genes can achieve cell rejuvenation after 1 week, reducing biological age, especially reversing cell senescence, reversing depletion (differentiation efficiency), reversing inotropic (FGF 2 sensitivity), prolonging cell life and self-renewal capacity (increasing passage number), enhancing cell regeneration capacity. We also calculated biological age using epigenetic histology (Stubbs Multi-t.clock; stubbs et al, 2017) and genomic transcriptomics (Martin-Herranz et al, 2019), found that these younger genes both significantly reversed the epigenetic clock (P < 0.05) and brought the gene expression profile closer to embryo developmental stage (R > 0.6).

Virus production

The following plasmids were used to produce viruses for various transgenic cell lines: lentiviral vector plasmid (Addgene # 19119), dR8.2 packaging plasmid (Addgene # 8455), VSV-G envelope plasmid (Addgene # 8454), viral supernatants were collected over a 48-96 hour window and filtered with a 0.45 μm filter (Sartorius). Lentiviral vectors possessing a CMV promoter can overexpress all younger genes.

Cell growth rate and lifetime assays

Will be 8×10 ⁴ Cells were seeded in one well of a 6-well plate (Falcon), and the growth medium contained DMEM/F-12 (Gibco) and 20% Fetal Bovine Serum (FBS) (GE), 1% l-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco). Nuclei were stained with Hoechst, photographed under a fluorescent microscope, and imageJ counted. This process was repeated for 3 days.

Biological information and statistical methods.

All code for performing epigenetic clock and genome analysis can be found in the Github store (https:// gitub. Com/demh/epigenetic_agejclock)). All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software). Data are expressed as mean ± sem. The statistical significance of the differences between groups was tested using a double sample t-test. P < 0.05 was considered significant. The biological (non-technical) number of replicates for each experiment is shown in the legend.

Case 3:

from the above cell expression profile we obtained a gene sheet co-expressed with Lin28 a: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4. Case 1 shows that Lin28a+ cells have the ability to reverse senescence. We therefore predicted whether muscle cells were life-prolonging using Lin28a-tdTomato as a reporter gene. As a case, we tested the following 6 genes in the monocot: whether Bcl11a, bcl11b, lmo2, otx2, pbx1, pabpc41 is capable of allowing senescent mouse muscle cells to initiate Lin28a expression, reverse senescence, restore FGF2 induction capacity, proliferation capacity, passaging capacity, and extend their life. It is well known that senescent cells generally lose FGF2 sensing capacity, proliferative capacity, passaging capacity, and begin to express SA-Bgal. Therefore, we want to test whether Bcl11a, bcl11b, otx2, lmo2, pbx1, pabpc4l could promote FGF2 induction, proliferation, passaging in senescent cells of mice, thus reversing senescence. We extracted senescent muscle cells from mice and transfected 6 viruses (vectors containing Bcl11a, bcl11b, otx2, lmo2, pbx1, pabpc4l, respectively) and empty vector, respectively, using known methods, and measured Lin28a + specific, SA-Bgal + specific of senescent cells within 72 hours, while measuring proliferation fold of cells at 24 hours, 48 hours, 72 hours, and area% and number% of senescent cells. As shown in FIGS. 10 and 11, we found that aged mouse muscle cells transfected with 6 genes (vectors containing Bcl11a, bcl11b, otx2, lmo2, pbx1, pabpc4l, respectively) were significantly higher in Lin28a+ than in the empty vector control group over 48-72 hours using fluorescence microscopy (Nikon). As shown in FIG. 12, after glutaraldehyde fixation and age-related beta-galactosidase staining (Solarbio #G1580), it was shown that the SA-Bgal+ ratio of aged mouse muscle cells transfected with 6 genes in monocots was also smaller than that of aged mouse muscle cells transfected with empty vector within 48-72 hours, thus confirming that we could reverse aging by transgenic pattern (transfection of viral vector containing 6 genes in monocots) and let aged muscle cells younger. Meanwhile, as shown in fig. 13, we found that the capacity, proliferation capacity, and passaging capacity of an equivalent amount of senescent muscle cell induction medium FGF2 (GeminiBio, 10 ng/ml) can be promoted by a transgenic pattern (transfection of a viral vector containing 6 genes in a single sheet) using an operatta high content microscope (PerkinElmer). The empty vector control senescent muscle cells stopped proliferating within 1 generation, but the 6 viral senescent muscle cells could be passaged more than 10 times. We further tested whether 6 genes in the monocot could reverse senescence in human non-pluripotent cells. We differentiated embryonic pluripotent stem cells into muscle cells using known protocols (Chua et al, 2019) and after senescence of these cells (at the time of stopping proliferation), each transfected with a virus containing 6 genes in monocots and containing empty vector, and measured the percentage of senescent cells using an operaetta high content microscope (PerkinElmer) within 72 hours. As shown in FIG. 14, we can overexpress 6 genes in a single sheet by transgenic means (viral transfection) and successfully reverse the% senescent area and% senescent number of human muscle cells. As can be seen, the genes in both monocots have the ability to initiate Lin28a expression, reverse senescence, reverse anergy, restore proliferation capacity, passaging capacity, and extend their life span.

We further tested the expression of the aged muscle cell Bcl11a, bcl11b, otx2, lmo2, pbx1, pbbpc 4l genes transfected with 6 genes (vectors containing Bcl11a, bcl11b, otx2, lmo2, pbx1, palpc 4l, respectively) using a fluorescent quantitative nucleic acid amplification assay (qPCR). As shown in fig. 15 and 16, senescent muscle cells (6 viruses) transfected with 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l) and senescent muscle cells (empty vector control) transfected with empty vector controls significantly overexpressed 6 genes (Bcl 11a, bcl11b, lmo2, otx2, pbx1, pabpc4 l).

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Reterences

1.Horvitz，H.R.&Sulston，J.E.Isolation and genetic characterization of cell-Iineage mutants of the nematode Caenorhabditis elegans.Genetics 96，435-454(1980).

2.Sulston，J.E.&Horvitz，H.R.Abnormal cell Iineages in mutants of the nematode Caenorhabditis elegans.Developmental biology82，41-55(1981).

3.Ambros，V.&Horvitz，H.R.Heterochronic mutants of the nematode Caenorhabditis elegans.Science(New York，N.Y.)226，409-416(1984).

4.Viswanathan，S.R.，Daley，G.Q.&Gregory，R.I.Selective Blockade of MicroRNA Processing by Lin28.320，97-100(2008).

5.Zhu，H.et al.Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies.Nature Genetics 42，626-630(2010).

6.Tsanov，K.M.et al.LIN28 phosphorylation by MAPK/ERK couples signalling to the post-transcriptional control of pluripotency.Nature cell biology 19，60-67(2017).

7.Yermalovich，A.V.et al.Lin28 and Iet-7 regulate the timing of cessation of murine nephrogenesis.Nature communications 10，168(2019).

8.Osborne，J.K.et al.Lin28 paralogs regulate lung branching morphogenesis.Cell Rep 36，109408-109408(2021).

9.Kerepesi，C.，Zhang，B.，Lee，S.-G.，Trapp，A.&Gladyshev，V.N.Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aqinq.Sci Adv7，eabg6082(2021).

10.Moss，E.G.，Lee，R.C.&Ambros，V.The cold shock domain protein LIN-28controls developmental timing in C.elegans and is regulated by the Iin-4 RNA.Cell88，637-646(1997).

11.Shyh-Chang，N.et al.Lin28 enhances tissue repair by reprogramming cellular metabolism.Cell155，778-792(2013).

12.Mauro，A.Satellite cell of skeletal muscle fibers.The Journal of biophysical and biochemical cytology 9，493-495(1961).

13.Yin，H.，Price，F.&Rudnicki，M.A.Satellite cells and the muscle stem cell niche.Physiological reviews 93，23-67(2013).

14.yon Maltzahn，J.，Chang，N.C.，Bentzinger，C.F.&Rudnicki，M.A.Wnt signaling in myogenesis.Trends in cell biology 22，602-609(2012).

15.Polesskaya，A.et al.Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency.Genes&development 21，1125-1138(2007).

16.West，J.A.et al.A role for Lin28 in primordial germ-cell development and germ-cell malignancy.Nature 460，909-913(2009).

17.Keefe，A.C.et al.MusCle stem cells contribute to myofibres in sedentary adult mice.Nature Communications 6,7087(2015).

18.Zheng，K.，Wu，X.，Kaestner，K.H.&Wang，P.J.The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse.BMC Developmental Biology 9，38(2009).

19.Seale，P.et al.Pax7 Is Required for the Specification of Myogenic Satellite Cells.Cell 102，777-786(2000).

20.Tedesco，F.S.，Dellavalle，A.，Diaz-Manera，J.，Messina，G.&Cossu，G.Repairing skeletal muscle：regenerative potential of skeletal muscle stem cells.The Journal of clinical investigation 120，11-19(2010).

21.Der Vartanian，A.et al.PAX3 Confers Functional Heterogeneity in Skeletal Muscle Stem Cell Responses to Environmental Stress.Cell stem cell 24，958-973.e959(2019).

22.de Morree，A.et al.Alternative polyadenylation of Pax3 controls muscle stem cell fate and muscle function.Science(New York，N.Y.)366，734-738(2019).

23.Schiaffino，S.&Reggiani，C.Fiber Types in Mammalian Skeletal Muscles.91，1447-1531(2011).

24.Horvath，S.DNA methylation age of human tissues and cell types.Genome Biology14，3156(2013).

25.Horvath，S.&Raj，K.DNA methylation-based biomarkers and the epigenetic clock theory of ageing.Nature reviews.Genetics 19，371-384(2018).

26.Jaafar，R.et al.Phospholipase D regulates the size of skeletal muscle cells through the activation of mTOR signaling.Cell Communication and Signaling 11，55(2013).

27.Bober，E.，Franz，T.，Arnold，H.H.，Gruss，P.&Tremblay，P.Pax-3 is required for the development of limb muscles：a possible role for the migration of dermomyotomal muscle progenitor cells.Development(Cambridge，England)120，603-612(1994).

28.Yusuf，F.et al.lnhibitors of CXCR4 affect the migration and fate of CXCR4+progenitors in the developing limb of chick embryos.235，3007-3015(2006).

29.Edmondson，D.G.，Lyons，G.E.，Martin，J.F.&Olson，E.N.Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis.Development(Cambridge，England)120，1251-1263(1994).

30.Subramanian，S.V.&Nadal-Ginard，B.Early expression of the different isoforms of the myocyte enhancer factor-2(MEF2)protein in myogenic as well as non-myogenic cell lineages during mouse embryogenesis.Mechanisms of development 57，103-112(1996).

31.Mercader，N.et al.Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes.Development(Cambridge，England)127，3961-3970(2000).

32.Capdevila，J.，Tsukui，T.，Esteban，C.R.，Zappavigna，V.&Belmonte，J.C.I.Control of Vertebrate Limb Outgrowth by the Proximal Factor Meis2 and Distal Antagonism of BMPs by Gremlin.Molecular Cell4，839-849(1999).

33.Oliver，G.et al.Homeobox genes and connective tissue patterning.121，693-705(1995).

34.Grifone，R.et al.Eya1 and Eya2 proteins are required for hypaxial somitic myogenesis in the mouse embryo.Developmental Biology 302，602-616(2007).

35.Heanue，T.A.et al.Synergistic regulation of vertebrate muscle development by Dach2，Eya2，and Six1，homologs of genes required for Drosophila eye formation.Genes&Development13，3231-3243(1999).

36.Grifone，R.et al.Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo.Development(Cambridge，Eng/and)132，2235-2249(2005).

37.Mitchell，K.J.et al.ldentification and characterization of a non-satellite cell muscle resident progenitor during postnatal development.Nature cellbiology 12，257-266(2010).

38.Scaramozza，A.et al.Lineage Tracing Reveals a Subset of Reserve Muscle Stem Cells Capable of Clonal Expansion under Stress.Cell stem cell 24，944-957.e945(2019).

39.Drummond，M.J.et al.Aging and microRNA expression in human skeletal muscle：a microarray and bioinformatics analysis.Physiological genomics 43，595-603(2011).

40.Chua，M.-W.J.et al.Assessment of different strategies for scalable production and proliferation of human myoblasts.52，e12602(2019).

41.Shyh-Chang，N.&Daley，G.Q.Lin28：primal regulator of growth and metabolism in stem cells.Cell stem cell 12，395-406(2013).

42.De Cecco，M.et al.L1 drives IFN in senescent cells and promotes age-associated inflammation.Nature 566，73-78(2019).

43.Peng，S.et al.Genome-wide studies reveal that Lin28 enhances the translation of genes important for growth and survival of human embryonic stem cells.Stem cells(Dayton，Ohio)29，496-504(2011).

44.Zhang，J.et al.LlN28 Regulates Stem Cell Metabolism and Conversion to Primed Pluripotency.Cell stem cell 19，66-80(2016).

45.Miwa，S.&Brand，M.D.Mitochondrial matrix reactive oxygen species production is very sensitive to mild uncoupling.Biochemical Society transactions 31，1300-1301(2003).

46.Sena，L.A.&Chandel，N.S.Physiological roles of mitochondrial reactive oxygen species.Molecular cell 48，158-167(2012).

47.Gutscher，M.et al.Real-time imaging of the intracellular glutathione redox potential.Nature methods 5，553-559(2008).

48.Emerling，B.M.et al.Mitochondrial reactive oxygen species activation of p38 mitogen-activated protein kinase is required for hypoxia signaling.Molecular and cellular biology 25，4853-4862(2005).

49.Jun-Hao，E.T.，Gupta，R.R.&Shyh-Chang，N.Lin28 and let-7 in the Metabolic Physiology of Aging.Trends in endocrinology and metabolism：TEM 27，132-141(2016).

50.Relaix，F.，Rocancourt，D.，Mansouri，A.&Buckingham，M.A Pax3/Pax7-dependent population of skeletal muscle progenitor cells.Nature 435，948-953(2005).

51.Tremblay，P.et al.A Crucial Role forPax3in the Development of the Hypaxial Musculature and the Long-Range Migration of Muscle Precursors.Developmental Biology 203，49-61(1998).

52.Ridgeway，A.G.&Skerjanc，I.S.Pax3 Is Essential for Skeletal Myogenesis and the Expression of Six1 and Eya2＊.Journal of Biological Chemistry 276，19033-19039(2001).

53.Grifone，R.et al.Eya1 and Eya2 proteins are required for hypaxial somitic myogenesis in the mouse embryo.Dev Biol302，602-616(2007).

54.Yang，D.H.&Moss，E.G.Temporally regulated expression of Lin-28 in diVerse tissues of the developing mouse.Gene expression patterns：GEP 3，719-726(2003).

55.Yokoyama，S.et al.Dynamic gene expression of Lin-28 during embryonic development in mouse and chicken.Gene Expression Patterns 8，155-160(2008).

56.Buganim，Y.et al.Single-cell expression analyses during cellular repragramming reveal an early stachastic and a late hierarchic phase.Cell 150，1209-1222(2012).

57.Yu，J.et al.Induced pluripotent stem cell Iines derived from human somatic cells.Science(New York，N.Y.)318，1917-1920(2007).

58.Robinton，D.A.et al.The Lin28/Iet-7 Pathway Regulates the Mammalian Caudal Body Axis Elongation Program.Developmental cell 48，396-405.e393(2019).

59.

-Cánoves，P.，Neves，J.&Sousa-Victor，P.Understanding muscle regenerative decline with aging：new approaches to bring back youthfulness to aged stem cells.The FEBS Journal287，406-416(2020).

60.Sousa-Victor，P.&

-Cánoves，P.Regenerative decline of stem cells in sarcopenia.Molecular Aspects of Medicine 50，109-117(2016).

61.Francoz，S.et al.Mdm4 and Mdm2 cooperate to inhibit activity in proliferating and quiescent cells＜em＞in vivo＜/em＞.103，3232-3237(2006).

62.Poderycki，M.J.，Rome，L.H.，Harrington，L.&Kickhoefer，V.A.The p80 homology region of TEP1 is sufficient for its association with the vault RNAs，and the vault particle.Nucleic acids research 33，893-902(2005).

63.Majmundar，A.J.，Wong，W.J.&Simon，M.C.Hypoxia-inducible factors and the response to hypoxic stress.Molecular cell40，294-309(2010).

64.Yang，X.，Yang，S.，Wang，C.&Kuang，S.The hypoxia-inducible factors HIF1αand HIF2αare dispensable for embryonic muscle development but essential for postnatal muscle regeneration.The Journal of biological chemistry 292，5981-5991(2017).

65.Wilson，A.A.et al.Lentiviral delivery of RNAi for in vivo lineage-specific modulation of gene expression in mouse lung macrophages.Molecular therapy：the journal of the American Society of Gene Therapy 21，825-833(2013).

66.Tasic，B.et al.Site-specific integrase-mediated transgenesis in mice via pronuclear injection.108，7902-7907(2011).

67.Liu，L.，Cheung，T.H.，Charville，G.W.&Rando，T.A.Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.Nature protocols 10，1612-1624(2015).

68.Feige，P.&Rudnicki，M.A.Isolation of satellite cells and transplantation into mice for lineage tracing in muscle.Nat Protoc 15，1082-1097(2020).

69.Liu，T.M.et al.Ascorbate and lron Are Required for the Specification and Long-Term Self-Renewal of Human Skeletal Mesenchymal Stromal Cells.Stem cell reports14，210-225(2020).

70.Hanson，G.T.et al.Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein IndiCators.Journal of Biological Chemistry 279，13044-13053(2004).

71.S.Horvath，DNA methylation age of human tissues and cell types.Genome Biol.14，3156(2013).

72.S.Horvath，K.Raj，DNA methylation-based biomarkers and the epigenetic clock theory of ageing.Nat.Rev.Genet.19，371-384(2018).

73.C.G.Bell，R.Lowe，P.D.Adams，A.A.Baccarelli，S.Beck，J.T.Bell，B.C.Christensen，V.N.Gladyshev，B.T.Heijmans，S.Horvath，T.ldeker，J.P.J.Issa，K.T.Kelsey，R.E.Marioni，W.Reik，C.L.Relton，L.C.Schalkwyk，A.E.Teschendorff，W.Wagner，K.Zhang，V.K.Rakyan，DNA methylation aging clocks：Challenges and recommendations.Genome Biol.20，249(2019).

74.D.A.Petkovich，D.I.Podolskiy，A.V.Lobanov，S.-G.Lee，R.A.Miller，V.N.Gladyshev.Using DNA methylation profiling to evaluate biological age and Iongevity interventions.Cell Metab.25，954-960.e6(2017).

75.T.Wang，B.Tsui，J.F.Kreisberg，N.A.Robertson，A.M.Gross，M.K.Yu，H.Carter，H.M.Brown-Borg，P.D.Adams，T.Ideker，Epigenetic aging signatures in mice Iivers are slowed by dwarfism，calorie restriction，and rapamycin treatment.Genome Biol.18，57(2017).

76.M.V.Meer，D.I.Podolskiy，A.Tyshkovskiy，V.N.Gladyshev，A whole lifespan mouse multi-tissue DNA methylation clock.eLife 7，e40675(2018).

77.M.J.Thompson，K.

，L.Rubbi，A.J.Lusis，R.C.Davis，A.Srivastava，R.Korstanje，G.A.Churchill，S.Horvath，M.Pellegrini，A multi-tissue full lifespan epigenetic clock for mice.Aging 10，2832(2018).

78.T.M.Stubbs，M.J.Bonder，A.-K.Stark，F.Krueger，F.yon Meyenn，O.Stegle，W.Reik.Multi-tissue DNA methylation age predictor in mouse.Genome Biol.18，68(2017).

79.M.Wang，B.Lemos，Ribosomal DNA harbors an evolutionarily conserved clock of biological aging.Genome Res.29，325-333(2019).

80.Martin-Herranz DE et al.Genome Biol 20：146(2019).

81.Jeffries AR，Maroofian R，Salter CG，Chioza BA，Cross HE，Patton MA，et al.Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging.Genome Res.29：1057-1066(2019).

Claims

1. An isolated modified skeletal muscle cell, which has the following characteristics:

(i) Any one or more of its Bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4 genes are increased relative to the unmodified skeletal muscle cells.

2. The skeletal muscle cell of claim 1, wherein the expression of any one or more of Bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calCr, epha1, epor, gar 2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4 is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 150, 200 or even greater than the unmodified skeletal muscle cell.

3. The skeletal muscle cell of claims 1-2, capable of sustained expansion for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days or even more.

4. A skeletal muscle cell of claims 1-3 having a significantly reduced biological age compared to an unmodified skeletal muscle cell.

5. The skeletal muscle cell of any one of claims 1-4, wherein the expression of MDM4 and TEP1 is at least about 5-fold, 10-fold, 20-fold, 30-fold or even higher compared to an unmodified skeletal muscle cell.

6. An isolated population of cells comprising the skeletal muscle cells of any one of claims 1-5, or any combination thereof;

preferably, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100%) of the cells in the population of cells are skeletal muscle cells of any one of claims 1-5.

7. A pharmaceutical composition comprising the skeletal muscle cell of any one of claims 1-6 or the population of cells of claim 8, and a pharmaceutically acceptable carrier and/or excipient.

8. A method for producing the cell of any one of claims 1-5 or the population of cells of claim 6, wherein the cell is produced by increasing expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

9. A method for rejuvenating or reducing skeletal muscle cells biological age or skeletal muscle cell repair by increasing expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

10. A method capable of reversing senescence in skeletal muscle cells by increasing expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

11. A method capable of reversing cell depletion in skeletal muscle cells by increasing expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4-, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

12. A method capable of reversing cellular inability in skeletal muscle cells by increasing expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4-, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

13. A method capable of extending skeletal muscle cell life by increasing expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

14. A method capable of allowing cells to continue to expand in vitro for at least 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days, 150 days, 200 days, 300 days, 400 days or even more by increasing the expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

15. A method capable of stably passaging skeletal muscle cells at least 5 times, such as at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times or more, by increasing expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

16. An agent capable of rejuvenate skeletal muscle cells of an elderly human by increasing expression of one or more of the following genes: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

17. The method of any one of claims 8-15, comprising transgenically increasing the expression of one or more of the following genes in the cell: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4-, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

18. A reagent or kit for producing the cell or cell population as described in any one of claims 1 to 5 or reversing skeletal muscle cell senescence, comprising

(i) Nucleic acids (e.g., deoxyribonucleic acid, ribonucleic acid) encoding any of the following proteins: bcl11a, fgf5, wnt3, batf3, lin28a, lin28b, dnmt3b, arid3c, bcl11b, lmo2, grhl2, zic5, foxr2, hif3a, zic2, pbx1, snai3, zfp946, mettl20, hopx, utf1, otx2, aadat, mal2, pabpc4l, calcr, epha1, epor, galr2, ripk4, pak6, map3k15, celf4, pdzd4, piezo2, shc4.

And

(ii) A pharmaceutically acceptable carrier (e.g., viral vector, nanoparticle, lipid vesicle, exosome, etc.).

19. The reagent or kit of claim, wherein also includes the regulation of the above genes expression of the original, such as but not limited to, promoter, drug regulation of promoter, protein regulation of promoter, tissue specific promoter, protein intron (intein), transposon, endonuclease (such as cre-lox system), retrotransposon.