New reprogramming method

Abstract

The present invention relates to a method of reprogramming somatic cells, which comprises culturing the somatic cells in the presence of one or more Yamanaka factors and further culturing the somatic cells in the absence of said one or more Yamanaka factors. The present invention also relates to reprogrammed somatic cells produced according to the method defined herein. Cosmetic methods, cosmetic compositions, reprogrammed somatic cells and compositions for treatment or rejuvenation are also provided, as well as methods for screening for age regulators, factors and / or cellular processes, including the method and reprogrammed somatic cells defined herein.

Description

Technical Field

[0001] The present invention relates to a method of reprogramming somatic cells, which comprises culturing the somatic cells in the presence of one or more Yamanaka factors and further culturing the somatic cells in the absence of the one or more Yamanaka factors. The present invention also relates to reprogrammed somatic cells produced according to the method defined herein. Cosmetic methods, cosmetic compositions, reprogrammed somatic cells and compositions for treatment or rejuvenation are also provided, as well as methods for screening for age regulators, factors and / or cellular processes, including the methods and reprogrammed somatic cells defined herein. Background of the Invention

[0003] Aging is characterized by a progressive loss of function that occurs at the molecular, cellular, tissue, and organismal levels. As an individual ages, the DNA methylation pattern at the chromatin level changes as some sites gain and some sites lose this mark. DNA methylation is an epigenetic modification that plays multiple roles in mammalian cells, from transposable element silencing to X-chromosome inactivation. Thus, changes and progressive accumulation of epigenetic marks are associated with aberrant gene expression and regulation, stem cell exhaustion, aging, and disruption of tissue homeostasis. These changes are relatively consistent between individuals and can be used to predict age. Predictors of this type (such as the Horvath epigenetic clock) generate a value called the DNA methylation age (also known as the epigenetic age), which is thought to represent the biological age of an individual or tissue. Lifestyle factors that affect the aging process (such as diet) also affect the DNA methylation age. However, the biological basis of the epigenetic clock and DNA methylation age remains unclear.

[0004] During the reprogramming of induced pluripotent stem (iPS) cells, somatic cells are transformed or de-differentiated into pluripotent stem cells. Gene expression profiling has revealed three stages of reprogramming: initiation, maturation, and stabilization. While the initiation stage is characterized by the direct transformation of mesenchymal to epithelial cells, the expression of a subset of pluripotency-related genes (OCT4, NANOG, and SALL4) is detected during the maturation stage. The acquisition of the final iPS cell state requires a late stabilization stage marked by the expression of the remaining pluripotency-related genes (such as UTF1, LIN28, DPPA2, and DPPA4). The resulting iPS cells are similar to natural pluripotent stem cells (such as embryonic stem (ES) cells) in many respects, including their ability to differentiate into multiple cell types. However, during iPS cell reprogramming, the DNA methylation age is reset to zero years, regardless of the age of the donor tissue from which the somatic cells were obtained. Thus, the process of iPS cell reprogramming resets the epigenetic marks of somatic cells to an embryonic-like state and causes the loss of somatic cell lineage identity.

[0005] Therefore, there is a need to generate reprogrammed somatic cells that have a low DNA methylation age or epigenetic age but retain their lineage identity. Such reprogrammed cells will find applications in many therapeutic and cosmetic applications as well as in treating and / or ameliorating age-related or degenerative diseases and disorders. Summary of the Invention

[0007] According to a first aspect of the present invention, there is provided a method of reprogramming somatic cells to a pluripotent-like or rejuvenated state, comprising:

[0008] i) culturing the somatic cells in the presence of one or more Yamanaka factors for at least 5 days, and / or until expression of a pluripotency marker is detectable on or within the somatic cells, and / or until a somatic lineage-specific marker is no longer detectable on the surface of the somatic cells;

[0009] ii) further culturing the somatic cells in the absence of the one or more Yamanaka factors until expression of the pluripotency marker on or within the somatic cells is reduced, and / or until expression of a somatic lineage-specific marker is detectable on the surface of the somatic cells.

[0010] According to another aspect of the present invention, there is provided a reprogrammed somatic cell produced by the method as defined herein.

[0011] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a reprogrammed somatic cell as defined herein.

[0012] According to another aspect of the present invention, there is provided a reprogrammed somatic cell as defined herein or a pharmaceutical composition as defined herein for treating and / or ameliorating a degenerative or age-related disease or disorder, or for rejuvenating a tissue or organ.

[0013] According to another aspect, there is provided a cosmetic composition comprising a reprogrammed somatic cell as defined herein.

[0014] According to another aspect, there is provided a cosmetic method of skin regeneration or rejuvenation, comprising administering or applying to an individual in need a reprogrammed somatic cell as defined herein or a cosmetic composition as defined herein.

[0015] According to another aspect, there is provided a method of screening for an age modulator, the method comprising:

[0016] (i) performing the method as defined herein in the presence and absence of a test agent to generate reprogrammed somatic cells; and

[0017] (ii) determining a molecular marker of the reprogrammed somatic cell, such as an epigenetic marker,

[0018] Among them, the difference between the molecular markers determined for the reprogrammed somatic cells generated in the presence of the test agent and the molecular markers determined for the reprogrammed somatic cells generated in the absence of the test agent indicates the age-regulating effect of the test agent.

[0019] According to another aspect, there is provided a method for screening an age-regulating factor or a cellular process, the method comprising:

[0020] (i) reprogramming somatic cells from a diseased tissue or organ according to the method defined herein; and

[0021] (ii) determining the molecular markers of the reprogrammed somatic cells from the diseased tissue or organ and the reprogrammed somatic cells defined herein or the un-reprogrammed somatic cells from the diseased tissue or organ, such as epigenetic markers,

[0022] Among them, the difference between the molecular markers determined for the reprogrammed somatic cells from the diseased tissue or organ and the molecular markers determined for the reprogrammed somatic cells defined herein or the un-reprogrammed somatic cells from the diseased tissue or organ indicates an age-regulating factor or a cellular process related to the disease. Brief Description of the Drawings

[0024] Figure 1 : Flow cytometry plots showing the expression of CD13 and SSEA4 on the surface of human fibroblast cells after 13 days of culture in the presence of the expression of Yamanaka factors (plots marked with "+"). The negative control cultures do not express Yamanaka factors (lower plots; marked with "-").

[0025] Figure 2 : Flow cytometry plots showing the expression of CD13 and SSEA4 on the surface of human fibroblast cells after 13 days of culture in the presence of the expression of Yamanaka factors and further cultured for 4 weeks in the absence of the expression of Yamanaka factors ("reversal" as defined herein). The plot marked with "+SSEA4" shows the cells identified as CD13-SSEA4+ on day 13. The plots marked with "+CD13" and "-" show the cells identified as CD13+SSEA4- on day 13 and the cells cultured without the expression of Yamanaka factors (i.e., negative control cultures), respectively.

[0026] Figure 3 : Bright-field phase contrast images of human fibroblast cells identified as CD13-SSEA4+ on day 13 of culture in the presence of the expression of Yamanaka factors after further culturing for 16 days in the absence of the expression of Yamanaka factors ("reversal").

[0027] Figure 4: Bar graph showing the DNA methylation age of human dermal fibroblasts after reprogramming and reversal according to the method section defined herein (as determined using the Horvath epigenetic clock). "+OSKM SSEA4" indicates cells that were identified as SSEA4+ on day 13 of culture in the presence of Yamanaka factor expression and further cultured in the absence of Yamanaka factor expression. "+OSKM CD13" and "-OSKM CD13" represent cells that were identified as CD13+ on day 13 of culture and cells that were cultured without Yamanaka factor expression (i.e., negative control cultures), respectively. Error bars represent two standard deviations.

[0028] Figure 5 : Schematic diagram of the transient reprogramming experiment.

[0029] Figure 6 : Cell morphology during and after transient reprogramming. After doxycycline treatment, the cells became iPSC-like and formed colony structures. After growth in the absence of doxycycline, the cells reverted to a fibroblast-like morphology.

[0030] Figure 7 : Principal component analysis of the methylomes of transiently reprogrammed cells, fibroblasts, reprogrammed cells, and iPSCs. PC1 separates the cells according to the degree of reprogramming and indicates that transiently reprogrammed cells are similar to fibroblasts.

[0031] Figure 8 : DNA methylation level of the Oct4 locus. The gray rectangle represents the promoter element (from the Ensembl regulatory build) near the Oct4 gene (black rectangle). The Oct4 promoter is demethylated in iPSCs but remains highly methylated in transiently reprogrammed cells.

[0032] Figure 9 : DNA methylation level of the FSP1 locus. The gray rectangle represents the promoter element (from the Ensembl regulatory build) near the FSP1 gene (black rectangle). The FSP1 promoter is highly methylated in iPSCs but remains demethylated in transiently reprogrammed cells.

[0033] Figure 10 : Principal component analysis of the transcriptomes of transiently reprogrammed cells, fibroblasts, reprogrammed cells, and iPSCs. PC1 separates the cells according to the degree of reprogramming and indicates that transiently reprogrammed cells are similar to fibroblasts.

[0034] Figure 11: The average expression level of fibroblast-specific protein 1 (FSP1). FSP1 is highly expressed in transiently reprogrammed cells, control groups, and reference fibroblasts, and is lowly expressed in iPSCs. Error bars represent standard deviation.

[0035] Figure 12 : The average Nanog expression level. Nanog is not expressed in transiently reprogrammed cells, control groups, and reference fibroblasts, and is expressed in iPSCs. Error bars represent standard deviation.

[0036] Figure 13 : The average DNA methylation age of the samples. Error bars represent standard deviation. Transient reprogramming rejuvenates the transcriptional age by 30 - 40 years relative to the control group. The maximum rejuvenation was observed with 13 days of doxycycline treatment.

[0037] Figure 14 : Box plot of the H3K9me3 level in individual cells measured by immunofluorescence. The H3K9me3 level decreases with age and is restored by transient reprogramming.

[0038] Figure 15 : The average transcriptional age of the samples. Error bars represent standard deviation. Transient reprogramming rejuvenates the transcriptional age by approximately 30 - 40 years relative to the control group. Rejuvenation was observed for all durations of doxycycline treatment.

[0039] Figure 16 : The average expression of collagen genes. Error bars represent standard deviation. P-values were calculated using DESeq2. *p < 0.05, ***p < 0.001. Transient reprogramming increases the expression of some collagen genes.

[0040] Figure 17 : Box plot of the type I collagen level in individual cells measured by immunofluorescence. Collagen levels decrease with age and are restored by 10 days of transient reprogramming. DETAILED DESCRIPTION OF THE INVENTION

[0042] According to a first aspect of the present invention, there is provided a method for reprogramming somatic cells into a pluripotent-like or rejuvenated state, which comprises:

[0043] i) culturing the somatic cells in the presence of one or more Yamanaka factors for at least 5 days, and / or until the expression of pluripotency markers can be detected on or inside the somatic cells, and / or until the somatic lineage-specific markers can no longer be detected on the surface of the somatic cells;

[0044] ii) Further culture the somatic cells in the absence of said one or more Yamanaka factors until the expression of said pluripotency marker decreases on or within the somatic cells, and / or until the expression of a somatic cell lineage-specific marker is detected on the surface of the somatic cells.

[0045] As will be appreciated from the present disclosure, contrary to what was previously known, it is surprisingly shown herein that reprogramming of somatic cells can be carried out when the somatic cells are cultured for a long time in the presence of Yamanaka factors. For example, Sarkar et al. (2019), bioRxiv 573386 (doi:https: / / doi.org / 10.1101 / 573386) have previously shown that transient reprogramming of somatic cells can be achieved in cultures of up to 4 days using a mixture of mRNAs encoding OCT4, KLF4, c-MYC, SOX2, LIN28, and NANOG. Thus, it has been proposed that culturing for 5 days in the presence of these factors represents a "point of no return" for somatic cell reprogramming. After culturing for 5 days in the presence of Yamanaka factors, such a "point of no return" occurs, indicating that epigenetic marks defining cell lineage identity are erased and reprogramming to an induced pluripotent stem (iPS) cell-like state is irreversible. Thus, according to Sarkar et al., in order to partially reprogram somatic cells to a pluripotent-like or rejuvenated state or a more pluripotent state, the culturing of the somatic cells in the presence of Yamanaka factors must be transient (i.e., less than 5 days) and only carried out at the "initiation" stage of iPS cell reprogramming.

[0046] During iPS cell reprogramming, somatic cells are transformed or de-differentiated into pluripotent stem cells. Such iPS cells are similar in many respects to natural pluripotent stem cells (e.g., embryonic stem (ES) cells), including their ability to differentiate into multiple cell types. However, during iPS cell reprogramming, the DNA methylation age is reset to zero years old, regardless of the age of the donor tissue from which the somatic cells were obtained. Thus, the process of iPS cell reprogramming resets the epigenetic marks of somatic cells to an embryo-like state and causes the loss of somatic cell lineage identity.

[0047] Thus, according to certain embodiments of the present invention, provided herein are methods for reprogramming somatic cells to a pluripotent-like or rejuvenated state (especially a rejuvenated state), wherein the reprogramming is incomplete reprogramming and / or partial reprogramming and / or transient reprogramming. It should be understood that the "incomplete" and / or "partial" and / or "transient" reprogramming mentioned herein is in comparison with cells having a high level of pluripotency (e.g., ES cells or iPS cells). In another embodiment, the reprogramming of somatic cells is incomplete and / or partial and / or transient reprogramming compared to iPS cells.

[0048] As used herein, "somatic cell" refers to any type of cell that makes up an organism, excluding germ cells and undifferentiated stem cells. Thus, somatic cells can include, for example, skin, heart, muscle, nerve, bone, or blood cells. In one embodiment of the invention, the somatic cell is a skin cell. In another embodiment, the somatic cell is a cell from connective tissue, such as a fibroblast. In another embodiment, the somatic cell is a blood cell. In one embodiment, the somatic cell is a bone marrow cell. Thus, it should be understood that in certain embodiments, the somatic cell can form blood or a part of blood. In another embodiment, the somatic cell is a nerve cell, such as a cell from the central and / or peripheral nervous system. Thus, in one embodiment, the cell is a neuron. In another embodiment, the cell is a sensory neuron. In an alternative embodiment, the cell is a motor neuron. In another embodiment, the cell is an interneuron. In another embodiment, the neuron is a brain cell. In another embodiment, the cell is a pancreatic cell. Thus, in one embodiment, the cell is a pancreatic alpha cell. In an alternative embodiment, the cell is a pancreatic beta cell. In another embodiment, the cell is a pancreatic delta cell. In another embodiment, the cell is a pancreatic F cell. In another embodiment, the cell is a heart cell. Thus, in one embodiment, the cell is a cardiac myocyte (also known as a cardiac muscle cell, cardiomyocyte, and myocardiocyte). In another embodiment, the cell is a sinoatrial node or pacemaker cell.

[0049] In one embodiment, the somatic cell is from an animal. In another embodiment, the somatic cell is from a mammal. In another embodiment, the mammal is a human. Thus, in a specific embodiment, the somatic cell is from a human and is a human body cell. In an alternative embodiment, the mammal is a mouse and the somatic cell is a mouse somatic cell. In another alternative embodiment, the somatic cell is from a non-human mammal, such as a cat, dog, or horse. For example, the rejuvenating properties of the somatic cells of the present invention have special uses in extending the lifespan of pets.

[0050] In another embodiment, the incomplete and / or partial and / or transient reprogramming comprises culturing somatic cells in the presence of one or more Yamanaka factors for a period of time that is continuously considered to be within the initiation and / or maturation phase of iPS cell reprogramming. In another embodiment, the incomplete and / or partial and / or transient reprogramming comprises culturing somatic cells in the presence of one or more Yamanaka factors at a time point prior to what is considered to be the stable phase of iPS cell reprogramming. In a specific embodiment, the incomplete and / or partial and / or transient reprogramming comprises culturing somatic cells in the presence of one or more Yamanaka factors at a time point that is considered to be the maturation phase of reprogramming. Thus, in certain embodiments, the culturing in the presence of one or more Yamanaka factors is not carried out during the stable phase of iPS cell reprogramming.

[0051] As used herein, "incomplete reprogramming" and / or "partial reprogramming" and / or "transient reprogramming" refer to one or more processes of reprogramming somatic cells into a pluripotent-like or rejuvenated state (especially a rejuvenated state), which state comprises fewer molecular markers or a DNA methylation age that is younger or less than that of the donor tissue or organism from which the somatic cells were obtained. A DNA methylation age that is younger or less than that of the donor tissue or organism from which the somatic cells were obtained comprises epigenetic markers that correspond to those of somatic cells from an earlier time point in the life cycle of the tissue or organism. Thus, "incomplete" and / or "partial" reprogramming and / or "transient" reprogramming as used herein also refer to those in which the reprogrammed somatic cells contain molecular markers, such as epigenetic markers, that correspond to those of somatic cells from an earlier time point in the life cycle of the tissue or organism from which the somatic cells were obtained.

[0052] Thus, in one embodiment, the reprogrammed somatic cells contain molecular markers, such as epigenetic markers, that correspond to those of somatic cells from an earlier time point in the life cycle of the tissue and / or organism. In another embodiment, the molecular markers, such as epigenetic markers, correspond to those of somatic cells from an earlier time point in the life cycle of the tissue or organism from which the somatic cells were obtained.

[0053] The "incomplete", "partial", or "transient" reprogramming referred to herein also refers to reprogramming somatic cells into a pluripotent-like or rejuvenated state (especially a rejuvenated state), which state includes molecular markers that are younger or less aged than the donor tissue or organism from which the somatic cells were obtained. Molecular markers that are younger or less aged than the donor tissue or organism from which the somatic cells were obtained include epigenetic markers that correspond to the epigenetic markers of somatic cells from an earlier time point in the life cycle of the tissue or organism. Other molecular markers include: transcriptomic signatures, the number of γ-H2AX foci, the concentration of reactive oxygen species, the enrichment of histone marks (such as H3K9me3 and H4K20me3), collagen levels, vimentin and E-cadherin levels, senescence-associated β-galactosidase activity, cell proliferation rate, and / or karyotypic characteristics.

[0054] In certain embodiments, the Horvath epigenetic clock is used to determine molecular markers, such as epigenetic markers, of reprogrammed, non-reprogrammed somatic cells, and / or reference cells (such as iPS cells). In other embodiments, the Horvath epigenetic clock is used to determine the DNA methylation age of reprogrammed somatic cells, non-reprogrammed somatic cells, and / or reference cells. The Horvath epigenetic clock can be used as a method for estimating age based on DNA methylation at CpG dinucleotide motifs in DNA. The DNA methylation age (also referred to as the "predicted age") is characterized by the following properties: it is close to zero for ES and iPS cells; it is related to the number of cell passages; it yields a highly heritable measure of age acceleration; and it can be applied to chimpanzee tissues. The DNA methylation age of blood has been shown to predict all-cause mortality in later life, even after adjustment for known risk factors, indicating its association with the processes underlying aging. Similarly, markers of physical and mental health are also associated with the epigenetic clock. A particular feature of the Horvath epigenetic clock is its high precision and applicability to a wide range of tissues and cell types. Since it allows for the comparison of the ages of different tissues and cells from the same individual (including reprogrammed somatic cells containing non-reprogrammed somatic cells or pluripotent cells such as iPS cells from the same tissue), it can be used to identify tissues and cells that show evidence of accelerated aging caused by disease. In addition, the Horvath epigenetic clock can be used to identify any changes in DNA methylation age caused by a treatment (such as reprogramming).

[0055] In other embodiments, transcriptomic clocks are used to determine the molecular markers defined herein. Thus, in one embodiment, multiple or one gene expression marker is used to determine the molecular markers. In another embodiment, the method described in Fleischer et al. (2018) Genome Biology 19, 221 is used to determine the transcriptomic clock.

[0056] In one embodiment, the molecular markers and / or DNA methylation age of the reprogrammed somatic cells are younger or less than those of the somatic cells or the somatic cells before reprogramming of the same tissue or organism from which the somatic cells are obtained. In another embodiment, the molecular markers and / or DNA methylation age of the reprogrammed somatic cells are in the form of such epigenetic markers that indicate younger or less aged somatic cells or non-reprogrammed somatic cells from the same tissue or organism from which the somatic cells are obtained. In certain embodiments, the DNA methylation age and / or molecular markers of the reprogrammed somatic cells as epigenetic markers are compared with the DNA methylation age and / or molecular markers of somatic cells from another tissue or organism as epigenetic markers (reference). In this case, it should be understood that the DNA methylation age and / or molecular markers of the reprogrammed somatic cells as epigenetic markers can be compared with a reference cell, tissue or organism that is of the same age, older or younger than the tissue or organism from which the somatic cells are obtained. In an alternative embodiment, the DNA methylation age and / or molecular markers of the reprogrammed somatic cells as epigenetic markers are compared with pluripotent cells such as iPS cells.

[0057] In other embodiments, the DNA methylation age and / or molecular markers of the reprogrammed somatic cells calculated by the Horvath epigenetic clock as epigenetic markers indicate an age or DNA methylation age that is at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years or at least 40 years younger or less than that of the non-reprogrammed somatic cells. In other embodiments, the molecular markers of the reprogrammed somatic cells as epigenetic markers and / or DNA methylation age indicate an age that is at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years or at least 40 years younger or less than that of the somatic cells from the tissue or organism from which the reprogrammed somatic cells are obtained. In another embodiment, the molecular markers as epigenetic markers or DNA methylation age indicate an age that is at least 20 years younger or less than that of the non-reprogrammed somatic cells or the somatic cells from the same tissue or organism from which the reprogrammed somatic cells are obtained. In another embodiment, the molecular markers as epigenetic markers or DNA methylation age indicate an age that is at least 30 years younger or less than that of the non-reprogrammed somatic cells or the somatic cells from the same tissue or organism from which the reprogrammed somatic cells are obtained. In another embodiment, the molecular markers as epigenetic markers or DNA methylation age indicate an age that is at least 40 years younger or less than that of the non-reprogrammed somatic cells or the somatic cells from the same tissue or organism from which the reprogrammed somatic cells are obtained.

[0058] In other embodiments, the DNA methylation age and / or molecular markers, such as epigenetic markers, calculated by the Horvath epigenetic clock of the reprogrammed somatic cells indicate an age or DNA methylation age that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% younger or less than that of the non-reprogrammed somatic cells. In other embodiments, the molecular markers, such as epigenetic markers or DNA methylation age, of the reprogrammed somatic cells indicate an age that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% younger or less than that of the somatic cells from the tissue or organism from which the reprogrammed somatic cells were obtained. In another embodiment, the molecular markers, such as epigenetic markers or DNA methylation age, indicate an age that is at least 10% younger or 10% less than that of the non-reprogrammed somatic cells or the somatic cells from the same tissue or organism from which the reprogrammed somatic cells were obtained. In another embodiment, the molecular markers, such as epigenetic markers or DNA methylation age, indicate an age that is at least 40% younger or 40% less than that of the non-reprogrammed somatic cells or the somatic cells from the same tissue or organism from which the reprogrammed somatic cells were obtained. In another embodiment, the molecular markers, such as epigenetic markers or DNA methylation age, indicate an age that is at least 70% younger or 70% less than that of the non-reprogrammed somatic cells or the somatic cells from the same tissue or organism from which the reprogrammed somatic cells were obtained.

[0059] It will be further understood that "incomplete" and / or "partial" and / or "transient" reprogramming as used herein includes cases where the reprogrammed somatic cells retain and / or contain the phenotype of non-reprogrammed somatic cells. Such retention and / or inclusion of the non-reprogrammed somatic cell phenotype includes cases where the expression of surface markers indicating the cell lineage or identity of the somatic cells is retained. In addition, such retention and / or inclusion may also include cases where the epigenetic markers of the non-reprogrammed somatic cell lineage or identity are retained and / or contained by the reprogrammed somatic cells.

[0060] Thus, in one embodiment, the reprogrammed somatic cells retain the phenotype of the non-reprogrammed somatic cells. In another embodiment, the reprogrammed somatic cells contain the phenotype of the non-reprogrammed somatic cells. In another embodiment, the reprogrammed somatic cells retain and / or contain the phenotype of the non-reprogrammed somatic cells of the tissue from which the reprogrammed somatic cells were obtained. In another embodiment, the reprogrammed somatic cells retain and / or contain the phenotype and / or epigenetic markers indicating the cell lineage or identity of the somatic cells.

[0061] One or more of the Yamanaka factors mentioned herein include one or more of OCT4, KLF4, c-MYC, and SOX2. In one embodiment, the one or more Yamanaka factors may further comprise LIN28 and NANOG. In an alternative embodiment, the one or more Yamanaka factors are selected from one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following: OCT4, KLF4, c-MYC, SOX2, LIN28, NANOG, ESSRRB, NR5A2, and / or c / EBPα. In another embodiment, the one or more Yamanaka factors are selected from OCT4, KLF4, c-MYC, and / or SOX2. In another embodiment, the one or more Yamanaka factors are selected from OCT4, KLF4, and / or SOX2. In an alternative embodiment, the one or more Yamanaka factors are selected from OCT4, SOX2, and / or ESSRRB. In an alternative embodiment, the one or more Yamanaka factors are selected from KLF4, SOX2, and / or NR5A2. In an alternative embodiment, the one or more Yamanaka factors are selected from OCT4, SOX2, KLF4, c-MYC, and / or c / EBPα. In an alternative embodiment, the one or more Yamanaka factors are selected from OCT4, KLF4, and / or c-MYC. In another embodiment, the one or more Yamanaka factors are selected from OCT4 and / or KLF4. In an alternative embodiment, the one or more Yamanaka factors are selected from OCT4, SOX2, LIN28, and / or NR5A2. In another embodiment, the one or more Yamanaka factors are selected from OCT4 and / or SOX2. In an alternative embodiment, the one or more Yamanaka factors are selected from OCT4, SOX2, and / or NR5A2. In another embodiment, the one or more Yamanaka factors is OCT4.

[0062] In another embodiment, the method of reprogramming somatic cells as defined herein includes culturing the somatic cells in the presence of one or more Yamanaka factors for at least 5 days. In another embodiment, the somatic cells are cultured in the presence of one or more Yamanaka factors for at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, or at least 16 days. In a specific embodiment, the somatic cells are cultured in the presence of one or more Yamanaka factors for at least 13 days. In another embodiment, the somatic cells are cultured in the presence of one or more Yamanaka factors for no more than 17 days, no more than 16 days, no more than 15 days, or no more than 14 days. In a specific embodiment, the somatic cells are cultured in the presence of one or more Yamanaka factors for 13 days. In an alternative embodiment, the somatic cells are cultured in the presence of one or more Yamanaka factors for 15 days. In another alternative embodiment, the somatic cells are cultured in the presence of one or more Yamanaka factors for 17 days.

[0063] Culturing the somatic cells for 17 days in the presence of one or more Yamanaka factors as mentioned herein will be understood to relate to the period that should not be exceeded when strictly following the procedure of the method described herein. It will be further understood that the period of culturing the somatic cells in the presence of the one or more Yamanaka factors may vary depending on the identity of the somatic cells. For example, if the somatic cells are fibroblasts, they are cultured in the presence of one or more Yamanaka factors for at least 5 days, at least 13 days, at least 15 days, no more than 17 days, no more than 15 days, or for 13, 15, or 17 days. Alternatively, if the cells are not fibroblasts, the number of days of culturing in the presence of one or more Yamanaka factors may be less than or more than the number of days defined herein.

[0064] In one embodiment, the method of reprogramming somatic cells as defined herein includes culturing the somatic cells in the presence of one or more Yamanaka factors until expression of a pluripotency marker is detectable on or within the somatic cells. It should be understood that the "pluripotency marker" referred to herein may include any marker associated with pluripotency or with a pluripotent-like or rejuvenated state (especially a rejuvenated state) expressed by the somatic cells undergoing reprogramming. Such markers may be expressed on the surface of the somatic cells or within the cells (i.e., "in", such as in the case of pluripotency-associated transcription factors). In one embodiment, the pluripotency marker is selected from OCT4, SOX2, NANOG, KLF4, TRA-1-60, TRA-1-81, TRA-1-54, SSEA1, and / or SSEA4. In another embodiment, the pluripotency marker is a transcription factor and expression is detected within the cells, and the pluripotency marker is selected from OCT4, SOX2, NANOG, and / or KLF4. In another embodiment, the pluripotency marker is detected on the surface of the somatic cells and is selected from TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, and / or SSEA4.

[0065] In a specific embodiment, the pluripotency marker detected on the surface of the somatic cells is SSEA4 (stage-specific embryonic antigen-4).

[0066] Stage-specific embryonic antigen-4 (SSEA4) is a glycolipid carbohydrate antigen that is expressed on the surface of human embryonal carcinoma (EC), embryonic germ (EG), undifferentiated ES and iPS cells, a subset of mesenchymal stem cells, and rhesus ES cell lines. Expression of SSEA4 is downregulated upon differentiation of human EC, ES, and iPS cells. Thus, SSEA4 surface expression can be used as a marker for dedifferentiation or reprogramming of somatic cells to a pluripotent-like or rejuvenated state (especially a rejuvenated state).

[0067] In an alternative embodiment, the pluripotency marker detected on the surface of the somatic cells is SSEA1 (stage-specific embryonic antigen-1, also known as CD15).

[0068] Stage-specific embryonic antigen-1 (SSEA1) is a milk-series oligosaccharide that is expressed on the surface of mouse embryonal carcinoma cells, embryonic stem cells, and germ cells but is expressed only on human germ cells. Expression of SSEA1 on human cells increases with differentiation, while differentiation of mouse cells results in decreased expression.

[0069] In an alternative embodiment, the pluripotency marker detected on the surface of the somatic cells is SSEA3 (stage-specific embryonic antigen-3).

[0070] Stage - specific embryonic antigen - 3 (SSEA3) is a glycosphingolipid oligosaccharide that contains five sugar units linked to a sphingolipid. Such sphingolipids play a key role in cell signaling, and SSEA3 has been shown to play a key role in identifying many types of mammalian cells with pluripotent and stem - cell - like characteristics.

[0071] In an alternative embodiment, the pluripotency markers detected on the surface of somatic cells are selected from TRA - 1 - 60, TRA - 1 - 81, and / or TRA - 2 - 54. TRA - 1 - 60, TRA - 1 - 81, and TRA - 2 - 54 are keratin sulfate antigens expressed on the surface of human ES cells.

[0072] In other embodiments, the pluripotency marker is a transcription factor, such as a transcription factor associated with pluripotency or pluripotent - like or rejuvenated states (especially rejuvenated states). Thus, in one embodiment, the pluripotency marker is OCT4.

[0073] Octamer - binding transcription factor 4 (OCT4) is a POU - family homeodomain transcription factor encoded by the human POU5F1 gene. It is critically involved in the self - renewal of undifferentiated embryonic stem cells, is initially active as a maternal factor in oocytes, and remains active in the embryo throughout the pre - implantation stage. Oct4 gene knockout promotes differentiation, demonstrating the role of these factors in the self - renewal of human embryonic stem cells. Mouse embryos with defective Oct4 or low Oct4 expression levels cannot form the inner cell mass, lose pluripotency, and differentiate into trophectoderm. Thus, the expression level of Oct4 in mice is crucial for regulating pluripotency and early cell differentiation.

[0074] In another embodiment, the pluripotency marker is SOX2.

[0075] SRY (sex - determining region Y) - box 2 (SOX2) is a transcription factor essential for maintaining the self - renewal or pluripotency of undifferentiated embryonic stem cells. SOX2 is a member of the Sox transcription factor family and has been shown to play a key role in maintaining embryonic and neural stem cells. SOX2 binds DNA cooperatively with OCT4 at non - palindromic sequences to activate the transcription of key pluripotency factors. Thus, it should be understood that, as described herein, OCT4 and SOX2 can be used interchangeably and / or cooperatively.

[0076] In another embodiment, the pluripotency marker is NANOG.

[0077] NANOG is a homeobox protein, which is a transcription factor that helps ES cells maintain pluripotency by inhibiting cell - determining factors. It is thought that NANOG acts in concert with other factors such as OCT4 and SOX2 to establish ES cell identity.

[0078] In one embodiment, the pluripotency marker is KLF4.

[0079] Kruppel-like factor 4 (KLF4, also known as gut-enriched Krüppel-like factor or GKLF) is a zinc finger transcription factor involved in the regulation of proliferation, differentiation, apoptosis, and somatic cell reprogramming. In ES cells, KLF4 has been shown to be a good indicator of stem cell-like capacity and has also been proposed to be so in mesenchymal stem cells.

[0080] According to certain embodiments, it is to be understood that when the pluripotency marker is a transcription factor (e.g., OCT4, SOX2, NANOG, and / or KLF4), the pluripotency marker does not have the same identity as one or more of the Yamanaka factors for culturing somatic cells in the presence thereof according to the methods defined herein. It will be further understood that when the pluripotency marker is a transcription factor, expression of the pluripotency marker is not detected on the surface of somatic cells and expression of the transcription factor pluripotency marker in somatic cells can be detected by expression and / or activation of a reporter or downstream effector of the transcription factor.

[0081] In another embodiment, the method of reprogramming somatic cells defined herein includes culturing the somatic cells in the presence of one or more Yamanaka factors until expression of a somatic cell lineage-specific marker (e.g., CD13) is no longer detectable on the surface of the somatic cells. In an alternative embodiment, the somatic cells are cultured in the presence of one or more Yamanaka factors until expression of the somatic cell lineage-specific marker on the surface of the somatic cells is downregulated or reduced. It is to be understood that "no longer detected", "downregulated", and "reduced" as referred to herein encompass any change in surface expression of the marker compared to non-reprogrammed somatic cells or compared to the somatic cells prior to reprogramming, including loss, where non-reprogrammed somatic cells contain higher or more marker expression. It is to be further understood that such references herein can also be compared to reference pluripotent cells such as ES or iPS cells.

[0082] "Culturing in the presence of one or more Yamanaka factors" as referred to herein will be understood to include providing the one or more Yamanaka factors as defined herein to somatic cells in culture in any form. In one embodiment, such culturing in the presence of one or more Yamanaka factors can include adding the Yamanaka factors in the form of one or more proteins or peptides to one or more culture media. In another embodiment, culturing in the presence of one or more Yamanaka factors includes culturing somatic cells in the presence of cells that express one or more Yamanaka factors as defined herein. In other embodiments, culturing in the presence of one or more Yamanaka factors includes expressing the one or more Yamanaka factors in the somatic cells. Thus, according to one embodiment, culturing in the presence of one or more Yamanaka factors as defined herein includes expression of the endogenous one or more Yamanaka factor-encoding genes from the somatic cells. According to this embodiment, expression of the one or more Yamanaka factors in the somatic cells does not include transfection, transduction, or introduction of foreign sequences. In another embodiment, expression of the one or more Yamanaka factors in the somatic cells includes stimulation of expression using compounds and / or treatments that upregulate or "turn on" the expression of the one or more Yamanaka factor-encoding genes. Thus, in one embodiment, culturing in the presence of one or more Yamanaka factors includes adding a compound known to cause expression of the one or more Yamanaka factor-encoding genes. In a specific embodiment, the compound is known to cause expression of the one or more Yamanaka factor-encoding genes in the somatic cells.

[0083] In an alternative embodiment, culturing in the presence of one or more Yamanaka factors includes introducing an exogenous sequence encoding the one or more Yamanaka factors as defined herein into the somatic cells. Thus, in one embodiment, culturing in the presence of one or more Yamanaka factors includes expression of the one or more Yamanaka factors from one or more exogenous sequences.

[0084] In one embodiment, the exogenous sequence encoding the one or more Yamanaka factors as defined herein is present in the form of Yamanaka factor-encoding mRNA. Thus, in one embodiment, culturing somatic cells in the presence of one or more Yamanaka factors includes culturing somatic cells in the presence of Yamanaka factor-encoding mRNA. In another embodiment, culturing somatic cells in the presence of Yamanaka factors includes providing Yamanaka factor-encoding mRNA to the somatic cells.

[0085] In one embodiment, an exogenous sequence encoding one or more Yamanaka factors as defined herein is introduced into somatic cells by transfection. In an alternative embodiment, the exogenous sequence is introduced into somatic cells by transduction, such as viral transduction. It should be understood that viral transduction is not limited to any specific virus. However, in one specific non-limiting embodiment, the viral transduction is lentiviral transduction. In an alternative embodiment, the viral transduction is retroviral transduction. In one embodiment, the exogenous one or more Yamanaka factor coding sequences as defined herein can be introduced into somatic cells in the form of a vector capable of transfecting somatic cells. In one embodiment, the vector is a transposon vector. Vectors suitable for introducing the expression of one or more Yamanaka factors as used herein into host cells such as somatic cells are well known in the art. The vector may also contain various regulatory / response sequences or elements that control the transcription and / or translation of the target sequence (such as those response elements that allow inducible expression as defined herein). Examples of vectors include: viral vectors, transposon vectors, plasmid vectors or cosmid vectors. It should also be understood that the Yamanaka factors can be introduced into host cells such as somatic cells by the CRISPR / Cas-9 method. Such a method can be a drug-(i.e., doxycycline (dox)) inducible or non-inducible CRISPR / Cas-9 method and is well known to those skilled in the art.

[0086] Transposon vectors utilize mobile genetic elements called transposons to move target sequences between the vector and the chromosome by a "cut and paste" mechanism. Examples of transposon vectors include PiggyBac vectors (System Biosciences) or EZ-Tn5 TM transposon construction vectors (Illumina, Inc.).

[0087] Viral vectors consist of DNA or RNA within genetically engineered viruses. Viral vectors can be used to integrate target sequences into the host cell genome (i.e., integrative viral vectors). Examples of viral vectors include adenoviral vectors, adeno-associated vectors, retroviral vectors or lentiviral vectors (such as HIV). Viral vectors can be introduced into host cells such as somatic cells by viral transduction. Thus, according to one embodiment, the expression of one or more Yamanaka factors in somatic cells and / or culturing in the presence of one or more Yamanaka factors includes integrating one or more Yamanaka factor coding sequences into the genome of somatic cells. In another embodiment, the expression of one or more Yamanaka factors in somatic cells and / or culturing in the presence of one or more Yamanaka factors includes using a viral vector to integrate one or more Yamanaka factor coding sequences into the somatic cell genome.

[0088] Plasmid vectors usually consist of circular double-stranded DNA. Like most engineered vectors, plasmid vectors have a multiple cloning site (MCS), which is a short region containing several commonly used restriction sites that allows for easy insertion of the DNA fragment of interest.

[0089] As used herein, "transfection" refers to the process of introducing a vector into a host cell (e.g., a somatic cell) so that the target sequence can be expressed. Methods for transfecting host cells with a vector include electroporation, sonoporation, or optical transfection, which are well known in the art.

[0090] In one embodiment, the expression of one or more Yamanaka factors as defined herein can be introduced and / or provided to somatic cells in the form of an expression cassette. In another embodiment, culturing in the presence of one or more Yamanaka factors includes introducing the coding sequences of one or more Yamanaka factors into somatic cells in the form of an expression cassette. Thus, in one embodiment, the expression of one or more Yamanaka factors as defined herein is from an expression cassette. In a specific embodiment, such an expression cassette may contain mRNA-derived sequences encoding one or more Yamanaka factors as defined herein. In another embodiment, the expression cassette further contains a sequence encoding a protein or a marker that allows for identification of the expression of the expression cassette. In a specific embodiment, the protein or marker for identifying the expression is a fluorescent protein. In a certain embodiment, the fluorescent protein is green fluorescent protein (GFP).

[0091] Thus, in one embodiment, somatic cells can be selected based on the expression of the protein or marker contained in the expression cassette. In a specific embodiment, somatic cells are selected based on the expression of a fluorescent protein (e.g., GFP) that allows for identification of the expression. It should be understood that "selection" as used herein may include flow cytometry methods such as fluorescence-activated cell sorting (FACS).

[0092] In another embodiment, a marker that allows for identification of the expression can be selected from drug resistance genes. Examples of drug resistance genes may include: puromycin resistance gene, ampicillin resistance gene, neomycin resistance gene, tetracycline resistance gene, kanamycin resistance gene, or chloramphenicol resistance gene. Cells can be cultured in a medium containing the appropriate drug (i.e., selection medium), and only those cells containing and expressing the resistance gene can survive. Thus, by culturing cells in a selection medium, it is possible to easily select cells containing the drug resistance gene.

[0093] Alternative markers that allow the identification of expression include chromogenic enzyme genes. Examples of chromogenic enzyme genes include: the β-galactosidase gene, the β-glucuronidase gene, the alkaline phosphatase gene, or the secreted alkaline phosphatase SEAP gene. Cells expressing these chromogenic enzyme genes can be detected by applying an appropriate chromogenic substrate (e.g., X-gal for β-galactosidase), such that cells expressing the marker gene produce a detectable color (e.g., blue in the blue-white screening assay).

[0094] In another embodiment, the expression cassette is an inducible expression cassette that allows the expression or co-expression of one or more Yamanaka factor coding sequences upon induction of expression with a suitable compound or treatment. Such inducible expression cassettes will be understood to include response elements that allow the expression of the cassette by promoting transcription and / or translation or removing inhibition of transcription and / or translation. In one embodiment, the response element is a tetracycline response element. Thus, in certain embodiments, the inducible expression cassette allows the expression or co-expression of one or more Yamanaka factor coding sequences (as exemplified in the data shown herein) upon addition of an antibiotic such as tetracycline, especially doxycycline.

[0095] Thus, it should be understood that, according to one embodiment, culturing somatic cells in the presence of one or more Yamanaka factors includes adding a compound or treatment capable of inducing expression from the inducible expression cassette. In certain embodiments, culturing somatic cells in the presence of one or more Yamanaka factors includes adding tetracycline.

[0096] In one embodiment, the exogenous sequence encoding one or more Yamanaka factors as defined herein is present in the form of a protein expressed from the Yamanaka factor coding mRNA. It should be understood that the expressed protein (i.e., the protein expressed from the Yamanaka factor coding mRNA) can be directly transferred (i.e., transfected) into somatic cells using a suitable protein delivery method. It should be understood that such suitable methods for directly transferring proteins into cells are well known to those skilled in the art and include the functional double-arginine translocation (Tat) system. Alternatively, the protein can be directly transferred into somatic cells via a targeted delivery system such as a nanoparticle delivery system. Similarly, such targeted delivery systems are well known to those skilled in the art.

[0097] In another embodiment, the method of reprogramming somatic cells as defined herein includes further culturing the somatic cells for at least 2 weeks in the absence of said one or more Yamanaka factors. In another embodiment, the somatic cells are further cultured for at least 2.5 weeks, at least 3 weeks, at least 3.5 weeks or at least 4 weeks in the absence of said one or more Yamanaka factors. In another embodiment, the somatic cells are further cultured for no more than 5 weeks, no more than 4 weeks, no more than 3 weeks or no more than 2.5 weeks in the absence of said one or more Yamanaka factors. In a specific embodiment, the somatic cells are further cultured for 4 weeks in the absence of said one or more Yamanaka factors. In an alternative embodiment, the somatic cells are further cultured for 3 weeks in the absence of said one or more Yamanaka factors. In another alternative embodiment, the somatic cells are further cultured for 2 weeks in the absence of said one or more Yamanaka factors.

[0098] In one embodiment, the method of reprogramming somatic cells as defined herein includes further culturing the somatic cells in the absence of said one or more Yamanaka factors until the expression of pluripotency markers is downregulated or reduced on or within the surface of the somatic cells. In another embodiment, the somatic cells are further cultured in the absence of said one or more Yamanaka factors until the expression of pluripotency markers is no longer detectable on or within the surface of the somatic cells. According to this embodiment, the terms "downregulated", "reduced" or "no longer detectable" mentioned will be understood relative to the somatic cells before further culturing in the absence of said one or more Yamanaka factors and / or relative to reference pluripotent cells.

[0099] It should be understood that the pluripotency markers according to these embodiments can be the same as or different from the pluripotency markers detected on or within the surface of somatic cells cultured in the presence of one or more Yamanaka factors. Thus, in a specific embodiment, the expression of pluripotency markers that is downregulated or no longer detectable on or within the surface of somatic cells during culture in the absence of one or more Yamanaka factors is the pluripotency markers that can be detected on or within the surface of somatic cells after culture in the presence of one or more Yamanaka factors.

[0100] In another embodiment, the method of reprogramming somatic cells as defined herein includes further culturing the somatic cells in the absence of said one or more Yamanaka factors until expression of a somatic cell lineage-specific marker (such as CD13) is detected on the surface of the somatic cells. In an alternative embodiment, further culturing in the absence of said one or more Yamanaka factors is carried out until the expression of the somatic cell lineage-specific marker is upregulated or increased on the surface of the somatic cells. As used herein, "upregulated" and "increased" encompass any change in the surface expression of the marker, including an increase, as compared to the somatic cells prior to the step of further culturing in the absence of said one or more Yamanaka factors. In such a case, it is understood that the somatic cells prior to the step of further culturing in the absence of said one or more Yamanaka factors express the somatic cell lineage-specific marker at a lower, lesser or non-existent level. As further used herein, "detectable", "upregulated" and "increased" may also be compared to reference pluripotent stem cells such as iPS cells. In an alternative embodiment, further culturing in the absence of said one or more Yamanaka factors is carried out until the expression of the somatic cell lineage-specific marker is restored as compared to the expression of the reprogrammed somatic cells prior to the step of further culturing in the absence of said one or more Yamanaka factors, or as compared to the expression of the somatic cells prior to culturing in the presence of one or more Yamanaka factors, or as compared to the expression of the non-reprogrammed somatic cells.

[0101] Thus, it is understood that further culturing in the absence of the expression of said one or more Yamanaka factors as defined herein may be referred to as "reversal" or "restoration".

[0102] In another embodiment, further culturing the somatic cells in the absence of said one or more Yamanaka factors includes removing a compound or treatment capable of inducing expression from an inducible expression cassette. In certain embodiments, further culturing the somatic cells in the absence of said one or more Yamanaka factors includes removing tetracycline. In an alternative embodiment, further culturing in the absence of said one or more Yamanaka factors includes a compound or treatment capable of preventing or stopping expression from an inducible expression cassette.

[0103] According to another aspect of the invention, there is provided a reprogrammed somatic cell produced by the method as defined herein. It is understood that the "a" or "the" reprogrammed somatic cell as used herein includes a single or a small number of cells, as well as a population of reprogrammed somatic cells, the number of which may be large. Thus, it is understood that any singular as used herein includes the plural and vice versa.

[0104] In one embodiment, the reprogrammed somatic cells generated according to the methods defined herein comprise a DNA methylation age, epigenetic age, or molecular marker that is younger or less than that of the non-reprogrammed somatic cells or the somatic cells of the tissue or organism from which the reprogrammed somatic cells were obtained. In another embodiment, the reprogrammed somatic cells comprise a molecular marker, such as an epigenetic marker, that indicates a younger or smaller epigenetic age than that of the non-reprogrammed somatic cells or the somatic cells of the tissue or organism from which the reprogrammed somatic cells were obtained. In a specific embodiment, the reprogrammed somatic cells generated according to the methods defined herein comprise a molecular marker that is similar to the molecular marker of somatic cells at an earlier time point in the life cycle of the tissue or organism from which the somatic cells were obtained, such as an epigenetic marker. In other embodiments, the reprogrammed somatic cells generated according to the methods defined herein comprise a phenotype and / or molecular marker, such as an epigenetic marker, that is similar to the phenotype and / or molecular marker of the non-reprogrammed somatic cells.

[0105] A composition comprising reprogrammed somatic cells

[0106] According to another aspect of the invention, there is provided a pharmaceutical composition comprising the reprogrammed somatic cells defined herein.

[0107] According to another aspect of the invention, there is provided a cosmetic composition comprising the reprogrammed somatic cells defined herein.

[0108] According to certain embodiments, the pharmaceutical or cosmetic composition further comprises one or more pharmaceutically acceptable excipients in addition to the reprogrammed somatic cells defined herein. In other embodiments, the pharmaceutical or cosmetic composition further comprises one or more pharmaceutically acceptable excipients in addition to the reprogrammed somatic cells generated according to the methods defined herein.

[0109] Generally, the pharmaceutical and cosmetic compositions of the present invention will be used in conjunction with a pharmaceutically suitable excipient or carrier. Generally, these excipients or carriers include water or alcohol / aqueous solutions, emulsions or suspensions, including saline and / or buffer media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's solution. Suitable physiologically acceptable adjuvants, if necessary, for keeping the composition comprising the reprogrammed somatic cells defined herein in discrete locations can be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginate. Intravenous carriers include fluids and nutrient supplements as well as electrolyte supplements such as Ringer's dextrose-based supplements. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition), may also be present.

[0110] The route of administration of the pharmaceutical composition defined herein can be any route known to those of ordinary skill in the art. For example, it can be administered by any suitable means, including parenterally, intravenously, intramuscularly, intraperitoneally, dermally or transdermally. In a specific embodiment, the pharmaceutical composition defined herein can be administered intravenously or transdermally.

[0111] In addition, the route of administration of the cosmetic composition defined herein can also be any route known to those of ordinary skill in the art. For example, it can be administered by any suitable means, including the above-mentioned means. In a specific embodiment, the cosmetic composition defined herein can be administered topically, dermally or transdermally.

[0112] Therapeutic uses and methods

[0113] It can be understood from the disclosure provided herein that the methods and compositions of the present invention will be particularly useful for treating and / or improving age-related or degenerative diseases and / or disorders or rejuvenating tissues or organs.

[0114] Accordingly, in one aspect, there is provided a method for treating and / or improving an age-related or degenerative disease or disorder, comprising administering to an individual in need thereof the reprogrammed somatic cells produced by the method defined herein. In another embodiment, there is provided a method for treating and / or improving an age-related or degenerative disease or disorder, comprising administering to an individual in need thereof the reprogrammed somatic cells defined herein. In one embodiment, the method comprising the reprogrammed somatic cells defined herein is used for treating and / or improving an age-related or degenerative disease or disorder of the skin. In an alternative embodiment, the method comprising the reprogrammed somatic cells defined herein is used for treating or improving an age-related or degenerative disease or disorder of the pancreas, such as for treating or improving type 2 diabetes. In another embodiment, the method comprising the reprogrammed somatic cells defined herein is used for treating and / or improving an age-related disease or disorder, wherein the age-related disease or disorder is a neurodegenerative disease. In another embodiment, the method comprising the reprogrammed somatic cells defined herein is used for treating and / or improving an age-related disease or disorder, wherein the age-related disease or disorder is a disease or disorder of the blood and / or bone marrow. In another embodiment, the method comprising the reprogrammed somatic cells defined herein is used for treating and / or improving an age-related disease or disorder, wherein the age-related disease or disorder is a disease or disorder of the heart. Thus, in one embodiment, the disease or disorder is a cardiovascular disease. In another embodiment, the disease or disorder is cardiomyopathy. In another embodiment, the disease or disorder is ischemic heart disease. In another embodiment, the disease or disorder is arrhythmia. In another embodiment, the disease or disorder is heart failure.

[0115] In another embodiment, there is provided the use of the reprogrammed somatic cells as defined herein and / or reprogrammed somatic cells produced according to the methods defined herein in the treatment and / or amelioration of age-related or degenerative diseases or disorders. In another embodiment, there is provided a method of producing the reprogrammed somatic cells as defined herein for the treatment and / or amelioration of age-related or degenerative diseases or disorders.

[0116] According to another aspect, there is provided a pharmaceutical composition as defined herein for the treatment and / or amelioration of a degenerative or age-related disease or disorder, or for rejuvenating a tissue or organ. In one embodiment, the pharmaceutical composition comprises the reprogrammed somatic cells as defined herein. In an alternative embodiment, the pharmaceutical composition comprises reprogrammed somatic cells produced according to the methods defined herein.

[0117] In one embodiment, a pharmaceutical composition for use comprising the reprogrammed somatic cells as defined herein or the reprogrammed somatic cells as defined herein is for skin treatment or for the treatment and / or amelioration of skin diseases or disorders. Thus, in certain embodiments, the age-related disease or disorder includes skin diseases or disorders. In another embodiment, the treatment of the skin is for preventing, inhibiting, reducing, and / or reversing skin aging. Examples of skin aging include wrinkles, dryness, loss of elasticity, fragility, and / or loss of barrier properties.

[0118] In an alternative embodiment, a pharmaceutical composition for use comprising the reprogrammed somatic cells as defined herein or the reprogrammed somatic cells as defined herein is for the treatment and / or amelioration of pancreatic diseases or disorders. Thus, in certain embodiments, the age-related disease or disorder comprises pancreatic diseases or disorders. In another embodiment, the pancreatic disease or disorder is type 2 diabetes.

[0119] In another embodiment, a pharmaceutical composition for use comprising the reprogrammed somatic cells as defined herein or the reprogrammed somatic cells as defined herein is for the treatment and / or amelioration of neurodegenerative diseases.

[0120] In another embodiment, a pharmaceutical composition for use comprising the reprogrammed somatic cells as defined herein or the reprogrammed somatic cells as defined herein is for the treatment and / or amelioration of diseases or disorders of the blood and / or bone marrow.

[0121] In another embodiment, a pharmaceutical composition for use comprising the reprogrammed somatic cells as defined herein or the reprogrammed somatic cells as defined herein is for the treatment and / or amelioration of heart diseases or disorders.

[0122] In other embodiments, the tissue or organ defined herein is selected from: skin, liver, pancreas, heart, brain, central nervous system, peripheral nervous system, blood, and / or bone marrow. Thus, according to one embodiment, the tissue or organ is selected from blood, and the treatment and / or amelioration includes subjecting the blood or blood cells to one or more of the methods defined herein and providing the blood or blood cells to a patient or subject in need thereof. In another embodiment, the tissue or organ is selected from bone marrow, and the rejuvenation includes subjecting the bone marrow or bone marrow cells to one or more of the methods defined herein and providing the bone marrow or bone marrow cells to a patient or subject in need thereof.

[0123] In a specific embodiment, the tissue or organ is selected from the liver, and the methods and pharmaceutical compositions defined herein are used to rejuvenate the liver. It should be understood that according to this embodiment, the rejuvenation may include rejuvenating only a portion of the liver tissue or organ or somatic cells from the liver tissue or organ and providing the rejuvenated liver tissue or liver tissue cells to a patient or subject in need thereof. In another embodiment, the rejuvenated liver defined herein may continue to rejuvenate or be further rejuvenated in vivo.

[0124] In another embodiment, the tissue or organ is selected from the heart, and the methods and pharmaceutical compositions defined herein are used to rejuvenate the heart or heart tissue. Thus, according to one embodiment, the tissue or organ is selected from the heart, and the treatment and / or amelioration or rejuvenation includes subjecting heart cells (such as cardiomyocytes) to one or more of the methods defined herein and providing the heart cells to a patient or subject in need thereof. In another embodiment, the tissue or organ is selected from the heart, and the rejuvenation includes performing one or more of the methods defined herein on heart cells (such as cardiomyocytes) and providing the heart cells to a patient or subject in need thereof. In another embodiment, the tissue or organ is selected from the heart, and the reprogrammed somatic cells defined herein are heart cells, such as cardiomyocytes, and the treatment and / or amelioration or rejuvenation includes providing the reprogrammed heart somatic cells to a patient or subject in need thereof.

[0125] It should be understood that according to the embodiments described herein, the tissue or organ may be from the patient or subject in need thereof, or alternatively from a donor subject.

[0126] It should be understood that the patient or subject in need mentioned herein equally relates to animals and humans, and the present invention is particularly useful for veterinary treatment of any of the above diseases, disorders, and conditions that also exist in the animals.

[0127] It should be understood that the terms "treatment" and "improvement" as referred to herein include terms such as "prevention", "reversal", and "inhibition". In addition, such references include the administration, prior to the occurrence of a disease or disorder, of the reprogrammed somatic cells defined herein or a composition comprising such reprogrammed somatic cells. It is also contemplated that the reprogrammed somatic cells defined herein or a composition comprising such reprogrammed somatic cells may be administered after an inducing event of a disease or disorder, or before the clinical manifestation of the disease or disorder, or after the appearance of symptoms.

[0128] Cosmetic uses and methods

[0129] According to one aspect of the present invention, there is provided a cosmetic method for rejuvenating or restoring the skin, which comprises administering or applying to a subject in need thereof the reprogrammed somatic cells defined herein or a cosmetic composition defined herein.

[0130] In an alternative aspect, the cosmetic method is for rejuvenating a tissue or organ in need thereof, wherein the tissue or organ is not the skin. In another aspect, the cosmetic composition defined herein can be used for rejuvenating a tissue or organ in need thereof, wherein the tissue or organ is not the skin.

[0131] It should be understood that the cosmetic compositions and methods comprising the reprogrammed somatic cells defined herein can be suitably used for rejuvenating or restoring the skin. In addition, such cosmetic compositions and methods can be used for reducing scar formation or for regenerating connective tissue. Alternatively and / or additionally, the cosmetic composition defined herein can be used in or after cosmetic surgery for the regeneration or rejuvenation of the skin and / or connective tissue. Suitably, the cosmetic composition defined herein can be used for the regeneration or rejuvenation of the skin and / or connective tissue, including reducing the age, such as the DNA methylation age or epigenetic age, or making the skin and / or connective tissue younger.

[0132] In addition, the cosmetic method defined herein can be used for the regeneration of the skin and / or connective tissue after cosmetic surgery. The cosmetic method defined herein is contemplated to be particularly useful for the regeneration of the skin and / or connective tissue used in cosmetic surgery.

[0133] It should be further understood that the cosmetic compositions defined herein can be administered and / or used prophylactically. For example, a cosmetic composition comprising the reprogrammed somatic cells defined herein can be used at a time point before the tissue and / or organism is considered to be aging and / or undergoing senescence, such as at an early time point in the life cycle of the tissue and / or organism.

[0134] Screening methods

[0135] According to another aspect, there is provided a method for screening an age regulator, the method comprising:

[0136] (i) Performing the methods defined herein in the presence and absence of a test agent to generate reprogrammed somatic cells; and

[0137] (ii) Determining molecular markers of the reprogrammed somatic cells, such as epigenetic markers,

[0138] wherein a difference between the molecular markers determined for the reprogrammed somatic cells generated in the presence of the test agent and the molecular markers determined for the reprogrammed somatic cells generated in the absence of the test agent indicates the age-regulating effect of the test agent.

[0139] It should be understood that in this aspect of the invention, the test agent can comprise any compound, treatment, condition or process that can increase, accelerate or speed up the aging of cells, tissues, organs or organisms, or alternatively can reduce, slow down or decelerate the aging of cells, tissues, organs or organisms. Thus, in one embodiment, the test agent accelerates, speeds up or increases the aging of cells, tissues or organisms. In an alternative embodiment, the test agent slows down, decelerates or reduces the aging of cells, tissues or organisms. In another embodiment, the test agent reduces the effect of the reprogramming method defined herein. In another embodiment, the test agent increases the effect of the reprogramming method defined herein. In an alternative embodiment, the test agent prevents the reprogramming effect of the method defined herein.

[0140] In one embodiment, a difference in molecular markers is determined between reprogrammed somatic cells defined herein or generated according to the methods defined herein that have been exposed to a test agent and reprogrammed somatic cells defined herein or generated according to the methods defined herein that have not been exposed to the test agent. Thus, in one embodiment, a difference between the molecular markers determined for the reprogrammed somatic cells reprogrammed in the presence of the test agent and the reprogrammed somatic cells reprogrammed in the absence of the test agent is determined. In another embodiment, a difference in molecular markers is determined between reprogrammed somatic cells reprogrammed according to the methods defined herein or reprogrammed somatic cells defined herein and non-reprogrammed somatic cells exposed to the test agent. In another embodiment, a difference in molecular markers is determined between reprogrammed somatic cells reprogrammed according to the methods defined herein or reprogrammed somatic cells defined herein exposed to the test agent and non-reprogrammed somatic cells.

[0141] According to another aspect, there is provided a method of screening for age-regulating factors or cellular processes, the method comprising:

[0142] (i) Reprogramming somatic cells from a diseased tissue or organ according to the methods defined herein; and

[0143] (ii) Determining molecular markers of the reprogrammed somatic cells from the diseased tissue or organ and the reprogrammed somatic cells defined herein or non-reprogrammed somatic cells from the diseased tissue or organ, such as epigenetic markers,

[0144] Among them, the difference between the molecular markers determined for the reprogrammed somatic cells from diseased tissues or organs and the molecular markers determined for the reprogrammed somatic cells as defined herein or the non-reprogrammed somatic cells from diseased tissues or organs indicates an age regulator or a cell process related to the disease.

[0145] In one embodiment, the age regulator is a factor expressed or present in somatic cells that is involved in or regulates, or is suspected of being involved in or regulating, the age or aging of cells, tissues, organs, or organisms. In another embodiment, the age-regulating cell process is a cell process that is involved in or regulates, or is suspected of being involved in or regulating, the age or aging of cells, tissues, organs, or organisms. In another embodiment, the age regulator or cell process is involved in or regulates, or is suspected of being involved in or regulating, age-related diseases or disorders.

[0146] In one embodiment, the somatic cells are obtained from a diseased tissue or organ, where the disease is an age-related disease or disorder. In certain embodiments, the age-related disease or disorder is selected from the age-related diseases or disorders described herein.

[0147] Thus, in one embodiment, the difference between the molecular markers is determined between the reprogrammed somatic cells obtained from a diseased tissue or organ and the non-reprogrammed somatic cells obtained from a diseased tissue or organ or the same diseased tissue or organ. In another embodiment, the difference between the molecular markers is determined between the reprogrammed somatic cells obtained from a diseased tissue or organ and the non-reprogrammed somatic cells obtained from a non-diseased tissue or organ. In an alternative embodiment, the difference between the molecular markers is determined between the reprogrammed somatic cells obtained from a non-diseased tissue or organ and the non-reprogrammed somatic cells obtained from a diseased tissue or organ. It should be understood that according to these embodiments, the diseased or non-diseased tissue or organ from which the reprogrammed somatic cells and / or non-reprogrammed somatic cells are obtained can be the same tissue or organ, such as different parts of a tissue or organ, or from different tissues or organs. Examples

[0148] Materials and Methods

[0149] Human fibroblasts from three different donors were co-infected with lentiviruses containing the doxycycline-responsive transactivator protein (available from www.addgene.org) and the tetO-GFP-hOKMS construct (an inducible expression cassette encoding the Yamanaka factors as defined herein) in the presence of Polybrene (8 μg / ml). Next, after adding the virus, the cells were centrifuged at 1000 rpm for 1 hour to improve transduction efficiency. Twenty-four hours after infection (day 0), doxycycline (2 μg / ml) was added to the fibroblast medium (DMEM-F12, 10% FBS, 1x Glutamax, 1x MEM-NEAA, 1x β-ME, 0.2x Pen / Strep, 16 ng / ml FGF2). Then, on day 2 of doxycycline treatment, the cells were sorted by fluorescence-activated cell sorting (FACS) for GFP expression and re-plated onto gelatin-coated dishes. On day 7 after infection, the cells were passaged onto dishes containing irradiated mouse embryonic fibroblasts (iMEFs). The next day, the medium was changed to human embryonic stem cell medium (DMEM-F12, 20% KSR, 1x Glutamax, 1x MEM-NEAA, 1x β-ME, 0.2x Pen / Strep, 8 ng / ml FGF2). On days 13, 15, and 17, the cells undergoing reprogramming were sorted by FACS for surface expression of CD13 and SSEA4 using antibodies against these markers (obtained from Biolegend). The CD13+SSEA4- and CD13-SSEA4+ populations were collected and re-plated in fibroblast medium on dishes containing iMEFs. The re-plated cells were grown for four weeks without doxycycline so that they could revert to their original cell type (the "reversion" as defined herein). At the end of the four-week reversion, the cells were harvested for flow cytometry analysis, DNA methylation array, and RNA sequencing.

[0150] DNA methylation array

[0151] Genomic DNA was extracted from cell samples using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions, including an optional RNase digestion step. The genomic DNA samples were further processed at the Barts and The London Genome Centre and run on the Infinium MethylationEPIC array (Illumina).

[0152] RNA-Seq

[0153] RNA was extracted from cell samples using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The RNA samples were treated with DNase (Thermo Scientific) to remove contaminating DNA. RNA-Seq libraries were prepared at the Wellcome Sanger Institute and run on the HiSeq 2500 system (Illumina) for 50bp single-end sequencing.

[0154] DNA methylation analysis

[0155] Array data were processed using the minfi R package and NOOB normalization to generate β values. DNA methylation age was calculated using the Horvath epigenetic clock (Horvath (2013) Genome Biology 14, R115). Reference datasets for reprogrammed fibroblasts and iPSCs were obtained from Ohnuki et al. (2014) Proc. Natl. Acad. Sci. 111, 12426 - 12431 and Banovich et al. (2018) Genome Res 28, 122 - 131. In addition, the reference datasets include unpublished data from intermediate stages of fibroblasts reprogrammed with the CytoTune TM -iPS2.0 Sendai virus reprogramming kit (Invitrogen).

[0156] RNA-seq analysis

[0157] Reads were trimmed using Trim Galore (version 0.6.2) and aligned to the human genome (GRCh38) using Hisat2 (version 2.1.0). Raw counts and log2-transformed counts were generated by Seqmonk. Reference datasets for fibroblasts and iPSCs were obtained from Fleischer et al. (2018) Genome Biol. 19, 221 and Banovich et al. (2018) (see above). In addition, the reference datasets include unpublished data from intermediate stages of fibroblasts reprogrammed with the CytoTune TM -iPS2.0 Sendai virus reprogramming kit (Invitrogen).

[0158] Immunofluorescence and imaging

[0159] After fixation with 2% PFA for 30 minutes at room temperature, antibody staining was performed on cells grown on coverslips or cytospun smears as described previously (Santos et al. (2003) Curr. Biol. 13, 1116 - 1121). Briefly, cells were permeabilized with PBS containing 0.5% Triton X - 100 for 1 h; blocked with PBS containing 1% BSA and 0.05% Tween 20 (BS) for 1 h; incubated with the appropriate primary antibody diluted in BS; and then washed in BS and the secondary antibody. All secondary antibodies were Alexa - Fluor conjugated (Molecular Probes), diluted 1:1000 in BS, and incubated for 30 minutes. Incubations were carried out at room temperature. DNA was counterstained with PBS containing 5 μg / mL DAPI. Optical sections were captured using a Zeiss LSM780 microscope (63x oil immersion objective). Fluorescence semi - quantitative analysis was performed using Volocity 6.3 (Improvision). The antibodies used were as follows:

[0160] Anti - H3K9me3; 07 - 442, Merck / Millipore (1:500);

[0161] Anti - type I collagen; ab254113, Abcam (1:400).

[0162] Example 1: Partially reprogrammed somatic cells are fibroblast - like

[0163] At the intermediate stage of the process, cells expressing Yamanaka factors became heterogeneous. Some cells remained CD13+SSEA4 - and were classified as non - reprogrammed cells, while some cells became CD13 - SSEA4+ and were classified as successfully reprogrammed (see Figure 1 ). Both populations were sorted by fluorescence - activated cell sorting (FACS) and re - cultured for 4 weeks without doxycycline (defined herein as "reversal"). At the end of this stage, successfully reprogrammed cells reverted to a fibroblast phenotype and became CD13+SSEA4 - (see Figure 2 ). These cells were also morphologically similar to fibroblasts (see Figure 3 ). Non - reprogrammed cells (shown in the figure labeled "+CD13") or cells not cultured in the presence of Yamanaka factors (" - " condition) always remained fibroblast - like.

[0164] Similar findings were also observed in longer - term reprogramming (data not shown).

[0165] Example 2: Partially reprogrammed somatic cells show a younger epigenetic age

[0166] After 13 days of culture in the presence of Yamanaka factors, cells that were successfully reprogrammed and then reverted showed a DNA methylation age 30 - 40 years younger than their respective controls. Longer reprogramming (15 or 17 days) before the reversion stage slightly reduced the rejuvenation effect. Unreprogrammed cells expressing Yamanaka factors were the same age as negative control cells that never expressed Yamanaka factors (see Figure 4 ).

[0167] Thus, the data presented herein indicate that expression of Yamanaka factors alone for a short period of time (i.e., within the initial stage of iPS cell reprogramming, or for a period of less than 5 days, before the expression of pluripotency markers such as SSEA4, before somatic cell line - specific marker expression is no longer detectable on the cell surface) is not sufficient to rejuvenate the epigenetic age or to successfully reprogram somatic cells to display a younger DNA methylation age / epigenetic signature. The data also indicate that in order to be considered successfully reprogrammed, cells must also become positive for the reprogramming / pluripotency marker SSEA4.

[0168] Example 3: Transient Reprogramming Experiment

[0169] (a) Experimental Design

[0170] To investigate the possibility of transient reprogramming, we infected fibroblasts from elderly donors with a doxycycline - inducible reprogramming construct containing Oct4, Sox2, Klf4, c - Myc, and GFP. Additionally, we "mock - infected" some fibroblasts without using the construct to generate negative controls. After infection, we performed positive selection on cells expressing GFP by flow sorting, and for the negative controls, we sorted an equal number of live cells by flow sorting. Then, we treated the cells with doxycycline for different durations, and then flow - sorted the successfully reprogrammed cells and the cells that failed to reprogram based on the cell - surface markers CD13 and SSEA4. Finally, we cultured the sorted cells for at least 4 weeks in the absence of doxycycline and then collected the cells for analysis ( Figure 5 ). The cells were morphologically similar to fibroblasts at the end of the process ( Figure 6 ).

[0171] (b) Transiently Reprogrammed Cells Are Epigenetically Similar to Fibroblasts

[0172] We performed principal component analysis on the methylomes of transiently reprogrammed cells together with a reference dataset examining complete fibroblast reprogramming. As expected, principal component 1 separated cells according to the degree of reprogramming and generated a reprogramming trajectory using the reference dataset. Notably, transiently reprogrammed cells (and controls) remained at the start of the reprogramming trajectory, indicating that they resembled fibroblasts rather than reprogramming intermediates or iPSCs( Figure 7 ).

[0173] During iPSC reprogramming, many DNA methylation changes occur at regulatory elements such as promoters. Notably, the Oct4 promoter is demethylated during iPSC reprogramming. However, when we examined the Oct4 promoter in transiently reprogrammed cells, we found that it remained highly methylated at levels similar to controls and reference fibroblasts( Figure 8 ). We also observed that the promoter of FSP1 (a fibroblast marker gene) became highly methylated in iPSCs. But in transiently reprogrammed cells, we found that this promoter remained demethylated( Figure 9 ).

[0174] (c) Transiently reprogrammed cells are transcriptionally similar to fibroblasts

[0175] We also performed principal component analysis on the transcriptomes of transiently reprogrammed cells together with a reference dataset examining complete fibroblast reprogramming. As with the methylome analysis, principal component 1 separated samples according to the degree of reprogramming and generated a reprogramming trajectory using the reference dataset. The transcriptomes of transiently reprogrammed cells were at the start of this trajectory, indicating that these cells were also transcriptionally similar to fibroblasts( Figure 10 ).

[0176] During iPSC reprogramming, marker genes of the starting cell type are downregulated and pluripotency genes are upregulated. We found that fibroblast marker genes such as FSP1( Figure 11 ) were not downregulated in transiently reprogrammed cells and pluripotency marker genes such as Nanog( Figure 12 ) were not upregulated.

[0177] (d) Transient reprogramming rejuvenates many senescence markers

[0178] To investigate the effect of transient reprogramming on epigenomic rejuvenation, we calculated the DNA methylation age of cells after transient reprogramming using the Horvath epigenetic clock. We found that, relative to controls, transient reprogramming rejuvenated DNA methylation by up to 40 years. Notably, transient reprogramming using 13 days of doxycycline treatment produced the strongest rejuvenation, indicating that this is the optimal amount of epigenetic rejuvenation( Figure 13 ).

[0179] Other features of the epigenome change with aging, such as the overall levels of histone modifications. The level of H3K9me3 decreases with aging, and we found that transient reprogramming has the potential to increase H3K9me3 to youthful levels( Figure 14 ).

[0180] To investigate the effect of transient reprogramming on rejuvenating the transcriptome, we used published data from fibroblasts and trained a transcriptional clock using random forest regression ((Fleischer et al. (2018), see above). The median absolute error of this transcriptional clock for predicting age was 13.48 years. Using this clock, we found that transient reprogramming rejuvenated the transcriptional age by approximately 30 - 40 years, which is similar to the degree of rejuvenation observed with the epigenetic clock. In contrast to the epigenetic clock, transcriptional age rejuvenation was observed for all doxycycline treatment durations studied( Figure 15 ).

[0181] Collagen secretion is a key function of fibroblasts. We found that transient reprogramming increased the expression of several collagen genes. Notably, these increases were highly significant for COL4A1 and COL4A2( Figure 16 ). We also studied the protein level of type I collagen by immunofluorescence and found that transient reprogramming (using 10 days of doxycycline treatment) restored collagen to youthful levels( Figure 17 ).

Claims

1. A method for reprogramming somatic cells to a pluripotent-like or rejuvenated state, which comprises: i) culturing the somatic cells in the presence of one or more Yamanaka factors during the maturation stage of reprogramming, and until the expression of pluripotency markers can be detected on or inside the somatic cells, and until the somatic cell lineage-specific markers can no longer be detected on the surface of the somatic cells, wherein the one or more Yamanaka factors are selected from one or more of OCT4, KLF4, c-MYC, and SOX2; ii) further culturing the somatic cells in the absence of the one or more Yamanaka factors until the expression of the pluripotency markers on or inside the somatic cells decreases, and until the expression of the somatic cell lineage-specific markers is detected on the surface of the somatic cells.

2. The method according to claim 1, which comprises: i) culturing the somatic cells in the presence of one or more Yamanaka factors during the maturation stage of reprogramming for at least 5 days, and until the expression of pluripotency markers can be detected on or inside the somatic cells, and until the somatic cell lineage-specific markers can no longer be detected on the surface of the somatic cells, wherein the one or more Yamanaka factors are selected from one or more of OCT4, KLF4, c-MYC, and SOX2; ii) further culturing the somatic cells in the absence of the one or more Yamanaka factors until the expression of the pluripotency markers on or inside the somatic cells decreases, and until the expression of the somatic cell lineage-specific markers is detected on the surface of the somatic cells.

3. The method according to claim 2, wherein the somatic cells are cultured in the presence of the one or more Yamanaka factors for at least 6 days or at least 13 days, and / or wherein the somatic cells are cultured in the presence of the one or more Yamanaka factors for no more than 17 days or no more than 15 days.

4. The method according to claim 1 or claim 2, wherein the pluripotency marker is stage-specific embryonic antigen-4 (SSEA4).

5. The method according to any one of claims 1 to 4, wherein the reprogramming of the somatic cells is incomplete and / or partial reprogramming and / or transient reprogramming.

6. The method according to any one of claims 1 to 5, wherein the reprogrammed somatic cells contain molecular markers corresponding to somatic cells from an earlier time point in the tissue life cycle, and / or wherein the reprogrammed somatic cells retain the phenotype and / or molecular markers of the non-reprogrammed somatic cells.

7. The method according to claim 6, wherein the reprogrammed somatic cells contain molecular markers corresponding to somatic cells from an earlier time point in the tissue life cycle, wherein the molecular markers are epigenetic markers, and / or wherein the reprogrammed somatic cells retain the phenotype and / or molecular markers of the non-reprogrammed somatic cells, wherein the molecular markers are epigenetic markers.

8. The method according to any one of claims 1 to 6, wherein the molecular marker of the reprogrammed and / or non-reprogrammed somatic cells is an epigenetic marker and is determined using the Horvath epigenetic clock.

9. The method according to claim 8, wherein the molecular marker of the reprogrammed and / or non-reprogrammed somatic cells is an epigenetic marker and is determined using the Horvath epigenetic clock, wherein the epigenetic marker of the reprogrammed somatic cells indicates an epigenetic age that is at least 10%, at least 40% or at least 70% younger than that of the non-reprogrammed somatic cells.

10. The method according to any one of claims 1 to 8, wherein the one or more Yamanaka factors are provided from an inducible expression cassette transduced or transfected into the somatic cells.

11. The method according to claim 10, wherein the one or more Yamanaka factors are provided from an inducible expression cassette transduced or transfected into the somatic cells, wherein the culture carried out in the presence of the one or more Yamanaka factors further comprises adding a compound capable of inducing the expression from the inducible expression cassette, and / or wherein the culture carried out in the absence of the one or more Yamanaka factors comprises removing the compound capable of inducing the expression from the inducible expression cassette.

12. The method according to any one of claims 1 to 11, wherein the one or more Yamanaka factors are provided in the form of Yamanaka factor-encoding mRNA, or wherein the one or more Yamanaka factors are provided in the form of a protein expressed from Yamanaka factor-encoding mRNA, or wherein the Yamanaka factor is introduced into the somatic cells by CRISPR / Cas-9.

13. The method according to claim 12, wherein the one or more Yamanaka factors are provided in the form of Yamanaka factor-encoding mRNA, wherein the culture carried out in the absence of the one or more Yamanaka factors comprises removing the Yamanaka factor-encoding mRNA from the culture, or wherein the one or more Yamanaka factors are provided in the form of a protein expressed from Yamanaka factor-encoding mRNA, wherein the protein is directly delivered to the somatic cells by a targeted delivery system, or wherein the Yamanaka factor is introduced into the somatic cells by drug-inducible or non-inducible CRISPR / Cas-9.

14. The method according to claim 13, wherein the one or more Yamanaka factors are provided in the form of Yamanaka factor-encoding mRNA, wherein the culture carried out in the absence of the one or more Yamanaka factors comprises removing the Yamanaka factor-encoding mRNA from the culture, or wherein the one or more Yamanaka factors are provided in the form of a protein expressed from Yamanaka factor-encoding mRNA, wherein the protein is directly delivered to the somatic cell by a targeted delivery system, wherein the targeted delivery system is a functional twin-arginine translocation (Tat) system or a nanoparticle delivery system, or wherein the Yamanaka factor is introduced into the somatic cell by doxycycline (dox)-inducible or non-inducible CRISPR / Cas-9.

15. The method according to any one of claims 1 to 14, wherein the one or more Yamanaka factors additionally comprise LIN28 and / or NANOG.

16. The method according to claim 15, wherein the one or more Yamanaka factors additionally comprise LIN28 and / or NANOG, or one or more, or two or more, or three or more, or all of OCT4, KLF4, c-MYC, and / or SOX2.

17. A reprogrammed somatic cell produced by the method according to any one of claims 1 to 16.

18. A pharmaceutical composition comprising the reprogrammed somatic cell according to claim 17.

19. Use of the reprogrammed somatic cell according to claim 17 or the pharmaceutical composition according to claim 18 for the preparation of a medicament for treating and / or ameliorating a degenerative or age-related disease or disorder, or for the preparation of a medicament for rejuvenating a tissue or organ, wherein the degenerative or age-related disease or disorder comprises: a skin disease or disorder; or a pancreatic disease or disorder; or a neurodegenerative disease.

20. Use of the reprogrammed somatic cell according to claim 17 or the pharmaceutical composition according to claim 18 for the preparation of a medicament for treating and / or ameliorating a degenerative or age-related disease or disorder, or for the preparation of a medicament for rejuvenating a tissue or organ such as skin, blood, bone marrow, liver, or heart, wherein the degenerative or age-related disease or disorder comprises: a skin disease or disorder; or a pancreatic disease or type 2 diabetes; or a neurodegenerative disease.

21. Use of the reprogrammed somatic cell or the pharmaceutical composition according to claim 19 or 20 for the preparation of a medicament for treating and / or ameliorating a degenerative or age-related disease or disorder, or for the preparation of a medicament for rejuvenating a tissue or organ, wherein the reprogrammed somatic cell or the pharmaceutical composition is for administration to a human or animal subject.

22. A method of screening for an aging modulator or an aging modulating factor or a cellular process, the method comprises: (i) performing the method according to any one of claims 1 to 16 in the presence and absence of a test agent to produce reprogrammed somatic cells, or reprogramming somatic cells from a diseased tissue or organ according to the method according to any one of claims 1 to 16; and (ii) determining molecular markers of the reprogrammed somatic cells, or determining molecular markers of reprogrammed somatic cells from a diseased tissue or organ and the reprogrammed somatic cells according to claim 17 or non-reprogrammed somatic cells from the diseased tissue or organ, Wherein, the difference between the molecular markers determined for the reprogrammed somatic cells generated in the presence of the test agent and the molecular markers determined for the reprogrammed somatic cells generated in the absence of the test agent indicates the age-regulating effect of the test agent, or Wherein, the difference between the molecular markers determined for the reprogrammed somatic cells from a diseased tissue or organ and the molecular markers determined for the reprogrammed somatic cells according to claim 17 or the non-reprogrammed somatic cells from a diseased tissue or organ indicates an age-regulating factor or a cell process related to the disease.

23. The method according to claim 22, wherein the molecular marker is an epigenetic marker.

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