New reprogramming method

Abstract

The present invention relates to a method of reprogramming somatic cells comprising 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 invention also relates to reprogrammed somatic cells produced according to the methods defined herein. Also provided are cosmetic methods, cosmetic compositions, reprogrammed somatic cells and compositions for treating or restoring youth, and methods for screening for age modulators, factors, and / or cellular processes, including methods as defined herein and reprogrammed somatic cells.

Description

[0001] This application is a divisional application of Chinese patent application 202080050131.4. The application date of the original application is July 10, 2020, and the name of the invention is “New Reprogramming Method”. Technical Field

[0002] The present invention relates to a method for reprogramming somatic cells, comprising 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 methods defined herein. Also provided are cosmetic methods, cosmetic compositions, reprogrammed somatic cells, and compositions for treating or rejuvenating a condition, as well as methods for screening age-modifying agents, factors, and / or cellular processes, comprising the methods and reprogrammed somatic cells defined herein. Background of the Invention

[0003] Aging is characterized by a gradual loss of function at the molecular, cellular, tissue, and organismal levels. With aging, DNA methylation patterns at the chromatin level change, with some sites gaining and others losing these marks. DNA methylation is an epigenetic modification that plays a variety of roles in mammalian cells, ranging from transposable element silencing to X chromosome inactivation. Consequently, changes and progressive accumulation of epigenetic marks are associated with aberrant gene expression and regulation, stem cell depletion, aging, and dysregulation of tissue homeostasis. These changes are relatively consistent across individuals and can be used to predict age. Such predictors, such as the Horvath epigenetic clock, generate a value called DNA methylation age (also known as epigenetic age), which is believed to represent the biological age of an individual or tissue. Lifestyle factors that influence the aging process, such as diet, can also affect DNA methylation age. However, the biological basis of the epigenetic clock and DNA methylation age remains unclear.

[0004] During induced pluripotent stem (iPS) cell reprogramming, somatic cells transform or dedifferentiate into pluripotent stem cells. Gene expression profiling reveals three phases of reprogramming: initiation, maturation, and stabilization. While the initiation phase is characterized by direct mesenchymal-to-epithelial conversion, expression of a subset of pluripotency-associated genes (OCT4, NANOG, and SALL4) is detected during the maturation phase. Acquisition of the final iPS cell state requires a later stabilization phase, marked by the expression of additional pluripotency-associated genes (such as UTF1, LIN28, DPPA2, and DPPA4). The resulting iPS cells resemble naive pluripotent stem cells (e.g., embryonic stem (ES) cells) in many respects, including their ability to differentiate into a variety of cell types. However, during iPS cell reprogramming, the DNA methylation age is reset to zero, regardless of the age of the donor tissue from which the somatic cells were derived. Thus, the process of iPS cell reprogramming resets the epigenetic marks of somatic cells to an embryonic-like state and causes a loss of their 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 use in many therapeutic and cosmetic applications, as well as in treating and / or ameliorating age-related or degenerative diseases and conditions. SUMMARY OF THE INVENTION

[0006] According to a first aspect of the present invention, there is provided a method for reprogramming somatic cells to a pluripotent-like or youth-rejuvenated state, comprising:

[0007] i) culturing the somatic cells in the presence of one or more Yamanaka factors for at least 5 days, and / or until expression of pluripotency markers is detectable on the surface of or within the somatic cells, and / or until somatic cell lineage-specific markers are no longer detectable on the surface of the somatic cells;

[0008] ii) further culturing the somatic cells in the absence of the one or more Yamanaka factors until the expression of the pluripotency marker on the surface or inside the somatic cells is reduced and / or until the expression of somatic cell lineage-specific markers is detected on the surface of the somatic cells.

[0009] According to another aspect of the present invention, there is provided a reprogrammed somatic cell produced according to the method defined herein.

[0010] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the reprogrammed somatic cells defined herein.

[0011] 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 use in treating and / or ameliorating a degenerative or age-related disease or condition, or for rejuvenating a tissue or organ.

[0012] According to another aspect, there is provided a cosmetic composition comprising the reprogrammed somatic cells as defined herein.

[0013] According to another aspect, there is provided a cosmetic method for regenerating or rejuvenating the skin comprising administering or applying to an individual in need thereof a reprogrammed somatic cell as defined herein or a cosmetic composition as defined herein.

[0014] According to another aspect, there is provided a method for screening for an age-modifying agent, the method comprising:

[0015] (i) performing a method as defined herein in the presence and absence of a test agent to generate reprogrammed somatic cells; and

[0016] (ii) determining molecular markers, such as epigenetic markers, of the reprogrammed somatic cells,

[0017] wherein a difference between the molecular signature determined for reprogrammed somatic cells generated in the presence of the test agent and the molecular signature determined for reprogrammed somatic cells generated in the absence of the test agent indicates an age-modulated effect of the test agent.

[0018] According to another aspect, there is provided a method for screening for an age-regulating factor or cellular process, the method comprising:

[0019] (i) reprogramming somatic cells from a diseased tissue or organ according to a method as defined herein; and

[0020] (ii) determining molecular markers, such as epigenetic markers, of the reprogrammed somatic cells from the diseased tissue or organ and the reprogrammed somatic cells as defined herein or non-reprogrammed somatic cells from the diseased tissue or organ,

[0021] wherein differences between molecular signatures determined for reprogrammed somatic cells from a diseased tissue or organ and molecular signatures determined for reprogrammed somatic cells as defined herein or non-reprogrammed somatic cells from a diseased tissue or organ indicate age-regulatory factors or cellular processes associated with the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Flow cytometry graphs showing the expression of CD13 and SSEA4 on the surface of human fibroblasts after 13 days of culture, accompanied by the expression of Yamanaka factors (graphs marked with "+"). Negative control cultures do not express Yamanaka factors (lower graph; marked with "-").

[0023] Figure 2 : Flow cytometry graphs showing the expression of CD13 and SSEA4 on the surface of human fibroblasts after 13 days of culture with expression of Yamanaka factors and further 4 weeks of culture in the absence of Yamanaka factor expression ("reversal" is defined herein). The graph labeled "+SSEA4" shows cells identified as CD13-SSEA4+ on day 13. The graphs labeled "+CD13" and "-" show cells identified as CD13+SSEA4- on day 13 and cells cultured without expression of Yamanaka factors (i.e., negative control cultures), respectively.

[0024] Figure 3 : Bright field phase contrast images of human fibroblasts identified as CD13-SSEA4+ on day 13 of culture with expression of Yamanaka factors after further culture for 16 days in the absence of Yamanaka factor expression ("reversal").

[0025] Figure 4 : Bar graph showing the DNA methylation age (as determined using the Horvath epigenetic clock) of human somatic fibroblasts after partial reprogramming and reversal according to the methods defined herein. "+OSKM SSEA4" indicates cells identified as SSEA4+ on day 13 of culture with expression of Yamanaka factors and further cultured in the absence of Yamanaka factor expression according to the methods defined herein. "+OSKM CD13" and "-OSKM CD13" indicate cells identified as CD13+ on day 13 of culture and cells cultured without expression of Yamanaka factors (i.e., negative control cultures, error bars represent two standard deviations), respectively.

[0026] Figure 5 : Schematic diagram of transient reprogramming experiment.

[0027] Figure 6 Figure 3: Cell morphology during and after transient reprogramming. After doxycycline treatment, cells became iPSC-like and formed colonies. After growth in the absence of doxycycline, cells regained a fibroblast-like morphology.

[0028] Figure 7 : Principal component analysis of the methylomes of transiently reprogrammed cells, fibroblasts, reprogrammed cells, and iPSCs. PC1 separates cells according to the degree of reprogramming and shows that transiently reprogrammed cells resemble fibroblasts.

[0029] Figure 8: DNA methylation levels at the Oct4 locus. Grey rectangles represent promoter elements (from Ensembl regulatory elements) near the Oct4 gene (black rectangles). The Oct4 promoter is demethylated in iPSCs but remains hypermethylated in transiently reprogrammed cells.

[0030] Figure 9 : DNA methylation levels at the FSP1 locus. Gray rectangles represent promoter elements (from the Ensembl regulatory framework) near the FSP1 gene (black rectangle). The FSP1 promoter is highly methylated in iPSCs but remains demethylated in transiently reprogrammed cells.

[0031] Figure 10 : Principal component analysis of the transcriptomes of transiently reprogrammed cells, fibroblasts, reprogrammed cells, and iPSCs. PC1 separates cells according to the degree of reprogramming and shows that transiently reprogrammed cells resemble fibroblasts.

[0032] Figure 11 : Mean expression levels of fibroblast-specific protein 1 (FSP1). FSP1 is highly expressed in transiently reprogrammed cells, control cells, and reference fibroblasts, and is lowly expressed in iPSCs. Error bars represent standard deviations.

[0033] Figure 12 : Mean Nanog expression levels. Nanog is not expressed in transiently reprogrammed cells, control cells, and reference fibroblasts, but is expressed in iPSCs. Error bars represent standard deviations.

[0034] Figure 13 Mean DNA methylation age of samples. Error bars represent standard deviation. Transient reprogramming rejuvenated transcriptional age by 30–40 years relative to controls. Maximum rejuvenation was observed with 13 days of doxycycline treatment.

[0035] Figure 14 Figure 3: Boxplot of H3K9me3 levels in individual cells measured by immunofluorescence. H3K9me3 levels decrease with age and are restored by transient reprogramming.

[0036] Figure 15 = Mean transcriptional age of samples. Error bars represent standard deviation. Transient reprogramming resulted in a transcriptional rejuvenation of approximately 30–40 years relative to controls. Rejuvenation was observed for all durations of doxycycline treatment.

[0037] Figure 16 Mean expression of collagen genes. Error bars represent standard deviation. P values ​​were calculated using DESeq2. *p < 0.05, ***p < 0.001. Transient reprogramming can increase the expression of some collagen genes.

[0038] Figure 17 Figure 3: Boxplot of type I collagen levels 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

[0039] According to a first aspect of the present invention, there is provided a method for reprogramming somatic cells to a pluripotent-like or youth-rejuvenated state, comprising:

[0040] i) culturing the somatic cells in the presence of one or more Yamanaka factors for at least 5 days, and / or until expression of pluripotency markers is detectable on the surface of or within the somatic cells, and / or until somatic cell lineage-specific markers are no longer detectable on the surface of the somatic cells;

[0041] ii) further culturing the somatic cells in the absence of the one or more Yamanaka factors until the expression of the pluripotency marker on the surface or inside the somatic cells is reduced and / or until the expression of somatic cell lineage-specific markers is detected on the surface of the somatic cells.

[0042] As will be understood from the present disclosure, contrary to what was previously known, it is surprisingly shown herein that when somatic cells are cultured for a long time in the presence of Yamanaka factors, the somatic cells can be reprogrammed. 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 up to 4 days of culture using a mixture of mRNA encoding OCT4, KLF4, c-MYC, SOX2, LIN28, and NANOG. Therefore, it has been proposed that day 5 of culture in the presence of these factors represents a "point of no return" for somatic cell reprogramming. This "point of no return" occurs after 5 days of culture in the presence of Yamanaka factors, indicating that the epigenetic markers that define cell lineage identity are erased and reprogramming to an induced pluripotent stem (iPS) cell-like state is irreversible. Therefore, 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 culture of the somatic cells in the presence of Yamanaka factors must be transient (i.e., less than 5 days) and only during the "initiation" phase of iPS cell reprogramming.

[0043] During iPS cell reprogramming, somatic cells are converted or dedifferentiated into pluripotent stem cells. Such iPS cells are similar to natural pluripotent stem cells (e.g., embryonic stem (ES) cells) in many respects, including their ability to differentiate into various 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. Therefore, the process of iPS cell reprogramming resets the epigenetic markers of somatic cells to an embryonic state and causes the loss of somatic cell lineage identity.

[0044] Therefore, according to certain embodiments of the present invention, provided herein are methods for reprogramming somatic cells to a pluripotent or youthful state (especially a youthful 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 compared to cells with high-level capabilities (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.

[0045] "Somatic cells" as used herein refer to any type of cell that constitutes an organism, excluding germ cells and undifferentiated stem cells. Thus, somatic cells may include, for example, skin, heart, muscle, nerve, bone, or blood cells. In one embodiment of the present 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 may form blood or a portion of blood. In another embodiment, the somatic cell is a neural 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 α cell. In an alternative embodiment, the cell is a pancreatic β cell. In another embodiment, the cell is a pancreatic δ cell. In another embodiment, the cell is a pancreatic F cell. In another embodiment, the cell is a cardiac 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.

[0046] 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 somatic 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 particular utility in extending the lifespan of pets.

[0047] In another embodiment, the incomplete and / or partial and / or transient reprogramming comprises culturing the somatic cells in the presence of one or more Yamanaka factors for a period of time that is considered to be within the initial and / or maturation phase of iPS cell reprogramming. In another embodiment, the incomplete and / or partial and / or transient reprogramming comprises culturing the somatic cells in the presence of one or more Yamanaka factors at a time point prior to being considered to be in the stable phase of iPS cell reprogramming. In a specific embodiment, the incomplete and / or partial and / or transient reprogramming comprises culturing the somatic cells in the presence of one or more Yamanaka factors at a time point that is considered to be in the mature phase of reprogramming. Thus, in certain embodiments, culturing in the presence of one or more Yamanaka factors is not performed during the stable phase of iPS cell reprogramming.

[0048] “Incomplete reprogramming” and / or “partial reprogramming” and / or “transient reprogramming” mentioned herein refers to one or more processes of reprogramming somatic cells to a pluripotent or rejuvenated state (especially a rejuvenated state), which includes molecular markers or DNA methylation age that is younger or less than the donor tissue or organism from which the somatic cell was obtained. The DNA methylation age that is younger or less than the donor tissue or organism from which the somatic cell was obtained includes epigenetic markers that correspond to epigenetic markers of somatic cells from an earlier time point in the life cycle of the tissue or organism. Therefore, “incomplete” and / or “partial” reprogramming and / or “transient” reprogramming mentioned herein also refers to somatic cells in which the reprogrammed cells contain molecular markers, such as epigenetic markers, that correspond to epigenetic markers of somatic cells from an earlier time point in the life cycle of the tissue or organism from which the somatic cell was obtained.

[0049] Thus, in one embodiment, the reprogrammed somatic cell comprises a molecular signature, such as an epigenetic signature, that corresponds to an epigenetic signature of a somatic cell from an earlier point in the tissue and / or organism's life cycle. In another embodiment, the molecular signature, such as an epigenetic signature, corresponds to an epigenetic signature of a somatic cell from an earlier point in the tissue or organism's life cycle from which the somatic cell was obtained.

[0050] "Incomplete", "partial" or "transient" reprogramming mentioned herein also refers to reprogramming somatic cells to a pluripotent or youthful state (especially a youthful state), which includes molecular markers that are younger or younger than the donor tissue or body from which the somatic cells were obtained. Molecular markers that are younger or younger than the donor tissue or body from which the somatic cells were obtained include epigenetic markers that correspond to epigenetic markers of somatic cells from an earlier time point in the life cycle of the tissue or body. Other molecular markers include: transcriptome characteristics, 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, aging-related β-galactosidase activity, cell proliferation rate and / or karyotype characteristics.

[0051] 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 (e.g., 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 an age estimation method based on DNA methylation at CpG dinucleotide motifs in DNA. DNA methylation age (also called "predicted age") is characterized by the following properties: it is close to zero for ES and iPS cells; it is correlated with the number of cell passages; it produces a highly heritable accelerated measurement of age; and it can be applied to chimpanzee tissue. It has been shown that DNA methylation age of blood can predict all-cause mortality in late life, even after adjustment for known risk factors, indicating that it is related to the process that causes aging. Similarly, markers of physical and mental health are also associated with the epigenetic clock. A special feature of the Horvath epigenetic clock is its high accuracy and applicability to a wide range of tissues and cell types. Because it allows comparison of the ages of different tissues and cells from the same individual (including reprogrammed somatic cells containing unreprogrammed 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 manipulations such as reprogramming.

[0052] In other embodiments, the transcriptome clock is used to determine the molecular markers defined herein. Thus, in one embodiment, a plurality of or one gene expression markers are used to determine the molecular markers. In another embodiment, the transcriptome clock is determined using the method described in Fleischer et al. (2018) Genome Biology 19, 221.

[0053] In one embodiment, the molecular markers and / or DNA methylation age of the somatic cells of the reprogramming are younger or less than the molecular markers and / or DNA methylation age of the somatic cells or the same tissue or organism from which the somatic cells are obtained before reprogramming. In another embodiment, the molecular markers and / or DNA methylation age of the somatic cells of the reprogramming are in the form of epigenetic markers that indicate younger or less aged somatic cells or unreprogrammed 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 such as epigenetic markers of the somatic cells of the reprogramming are compared with the DNA methylation age and / or molecular markers such as epigenetic markers of the somatic cells from another tissue or organism (reference). In this case, it should be understood that the DNA methylation age and / or molecular markers such as epigenetic markers of the somatic cells of the reprogramming can be compared with a reference cell, tissue or organism that is the same, older or younger than the age of the tissue or organism from which the somatic cells are obtained. In an alternative embodiment, the DNA methylation age and / or molecular markers such as epigenetic markers of the reprogrammed somatic cells are compared to pluripotent cells such as iPS cells.

[0054] 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 years younger or less than that of the unreprogrammed somatic cells. In other embodiments, the molecular markers such as epigenetic markers and / or DNA methylation age of the reprogrammed somatic cells indicate an age that is at least 10 years younger or less than that of the somatic cells of 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 20 years younger or less than that of the unreprogrammed somatic cells or somatic cells of the same tissue or organism from which the reprogrammed somatic cells were obtained. In another embodiment, the molecular marker, such as an epigenetic marker or DNA methylation age, indicates an age that is at least 30 years younger or less than that of a non-reprogrammed somatic cell or a somatic cell from the same tissue or organism from which the reprogrammed somatic cell was obtained. In another embodiment, the molecular marker, such as an epigenetic marker or DNA methylation age, indicates an age that is at least 40 years younger or less than that of a non-reprogrammed somatic cell or a somatic cell from the same tissue or organism from which the reprogrammed somatic cell was obtained.

[0055] 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 than that of the unreprogrammed 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 than that of 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 unreprogrammed 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 marker, such as an epigenetic marker or DNA methylation age, indicates an age that is at least 40% younger or less than 40% of a non-reprogrammed somatic cell or a somatic cell from the same tissue or organism from which the reprogrammed somatic cell was obtained. In another embodiment, the molecular marker, such as an epigenetic marker or DNA methylation age, indicates an age that is at least 70% younger or less than 70% of a non-reprogrammed somatic cell or a somatic cell from the same tissue or organism from which the reprogrammed somatic cell was obtained.

[0056] It will be further understood that "incomplete" and / or "partial" and / or "transient" reprogramming as used herein includes wherein the reprogrammed somatic cell retains and / or comprises the phenotype of the unreprogrammed somatic cell. Such retention and / or inclusion of the unreprogrammed somatic cell phenotype includes wherein expression of surface markers indicative of the cell lineage or identity of the somatic cell is retained. Furthermore, such retention and / or inclusion may also include wherein epigenetic markers of the unreprogrammed somatic cell lineage or identity are retained and / or included by the reprogrammed somatic cell.

[0057] Thus, in one embodiment, the reprogrammed somatic cells retain the phenotype of the unreprogrammed somatic cells. In another embodiment, the reprogrammed somatic cells comprise the phenotype of the unreprogrammed somatic cells. In another embodiment, the reprogrammed somatic cells retain and / or comprise the phenotype of the unreprogrammed somatic cells of the tissue from which the reprogrammed somatic cells were obtained. In another embodiment, the reprogrammed somatic cells retain and / or comprise phenotypic and / or epigenetic markers indicative of the cell lineage or identity of the somatic cells.

[0058] The one or more 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 also include LIN28 and NANOG. In an alternative embodiment, the one or more Yamanaka factors may be selected from the following: 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: 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 ESRRB. 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 / EBPa. 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 are OCT4.

[0059] In another embodiment, the method of reprogramming somatic cells as defined herein comprises culturing the somatic cells for at least 5 days in the presence of one or more Yamanaka factors. In another embodiment, the somatic cells are cultured 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 the presence of one or more Yamanaka factors. In a specific embodiment, the somatic cells are cultured for at least 13 days in the presence of one or more Yamanaka factors. In another embodiment, the somatic cells are cultured for no more than 17 days, no more than 16 days, no more than 15 days, or no more than 14 days in the presence of one or more Yamanaka factors. In a specific embodiment, the somatic cells are cultured for 13 days in the presence of one or more Yamanaka factors. In an alternative embodiment, the somatic cells are cultured for 15 days in the presence of one or more Yamanaka factors. In another alternative embodiment, the somatic cells are cultured for 17 days in the presence of one or more Yamanaka factors.

[0060] Reference herein to culturing the somatic cells for 17 days in the presence of one or more Yamanaka factors will be understood to relate to a period of time that should not be exceeded when strictly following the procedures of the methods described herein. It will further be understood that the period of time for 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, the cells may be cultured in the presence of the 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 cells may be cultured in the presence of the one or more Yamanaka factors for fewer days than defined herein, or for more days than defined herein.

[0061] In one embodiment, the method for reprogramming somatic cells as defined herein comprises culturing the somatic cells in the presence of one or more Yamanaka factors until expression of a pluripotency marker can be detected on or inside the somatic cell surface. It should be understood that the "pluripotency marker" mentioned 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 somatic cells or expressed intracellularly (i.e., "in," for example, in the case of pluripotency-related 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 intracellularly, 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 a somatic cell and is selected from TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3 and / or SSEA4.

[0062] In a specific embodiment, the pluripotency marker detected on the surface of somatic cells is SSEA4 (stage-specific embryonic antigen-4).

[0063] Stage-specific embryonic antigen-4 (SSEA4) is a glycolipid carbohydrate antigen expressed on the surface of human embryonic carcinoma (EC), embryonic germ (EG), undifferentiated ES and iPS cells, mesenchymal stem cell subsets, and macaque ES cell lines. SSEA4 expression is downregulated upon differentiation of human EC, ES, and iPS cells. Therefore, SSEA4 surface expression can be used as a marker for somatic cell dedifferentiation or reprogramming to a pluripotent or rejuvenated state, particularly a rejuvenated state.

[0064] In an alternative embodiment, the pluripotency marker detected on the surface of somatic cells is SSEA1 (stage-specific embryonic antigen-1, also known as CD15).

[0065] Stage-specific embryonic antigen-1 (SSEA1) is a milk oligosaccharide expressed on the surface of mouse embryonic carcinoma cells, embryonic stem cells, and germ cells, but only on human germ cells. SSEA1 expression increases with differentiation in human cells, whereas differentiation in mouse cells leads to decreased expression.

[0066] In an alternative embodiment, the pluripotency marker detected on the surface of somatic cells is SSEA3 (Stage Specific Embryonic Antigen-3).

[0067] Stage-specific embryonic antigen-3 (SSEA3) is a glycosphingolipid oligosaccharide consisting of five sugar units attached to sphingolipids. This class of sphingolipids plays 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-like characteristics.

[0068] In an alternative embodiment, the pluripotency marker detected on the surface of somatic cells is 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.

[0069] In other embodiments, the pluripotency marker is a transcription factor, such as a transcription factor associated with pluripotency or a pluripotent-like or rejuvenated state (especially a rejuvenated state). Thus, in one embodiment, the pluripotency marker is OCT4.

[0070] Octamer-binding transcription factor 4 (OCT4) is a homeodomain transcription factor of the POU family, encoded by the human POU5F1 gene. It is crucially involved in the self-renewal of undifferentiated embryonic stem cells, initially active in the oocyte as a maternal factor and remaining active in the embryo throughout the preimplantation period. OCT4 gene knockout promotes differentiation, demonstrating a role for these factors in the self-renewal of human embryonic stem cells. Mouse embryos deficient in Oct4 or with low Oct4 expression levels fail to form an inner cell mass, lose pluripotency, and differentiate into the trophectoderm. Therefore, the expression level of Oct4 in mice is crucial for regulating pluripotency and early cell differentiation.

[0071] In another embodiment, the pluripotency marker is SOX2.

[0072] SRY (sex-determining region Y)-box 2 (SOX2) is a transcription factor essential for maintaining self-renewal or pluripotency in undifferentiated embryonic stem cells. SOX2 is a member of the Sox family of transcription factors and has been shown to play a key role in maintaining embryonic and neural stem cells. SOX2 and OCT4 synergistically bind DNA at non-palindromic sequences to activate transcription of key pluripotency factors. Therefore, it should be understood that OCT4 and SOX2 can be used interchangeably and / or synergistically as described herein.

[0073] In another embodiment, the pluripotency marker is NANOG.

[0074] NANOG is a homeobox protein that is a transcription factor that helps ES cells maintain pluripotency by repressing cell-determining factors. NANOG is thought to work in concert with other factors such as OCT4 and SOX2 to establish ES cell identity.

[0075] In one embodiment, the pluripotency marker is KLF4.

[0076] 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 been proposed to be the case in mesenchymal stem cells as well.

[0077] According to certain embodiments, it will 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 Yamanaka factors in the presence of which somatic cells are cultured 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 molecule or downstream effector of the transcription factor.

[0078] In another embodiment, the method for reprogramming somatic cells defined herein includes culturing the somatic cells in the presence of one or more Yamanaka factors until the expression of somatic cell lineage-specific markers (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 the expression of somatic cell lineage-specific markers on the surface of the somatic cells is downregulated or reduced. It should be understood that “no longer detected,” “downregulated,” and “reduced” mentioned herein encompass any changes in the surface expression of markers compared to unreprogrammed somatic cells or compared to somatic cells before reprogramming, including loss, wherein unreprogrammed somatic cells comprise higher or more marker expression. It should be further understood that such references herein may also be compared to reference pluripotent cells such as ES or iPS cells.

[0079] Reference herein to "culturing in the presence of one or more Yamanaka factors" will be understood to include providing the one or more Yamanaka factors defined herein to the somatic cells in culture in any form. In one embodiment, such culturing in the presence of one or more Yamanaka factors may include adding one or more Yamanaka factors in the form of 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 expressing one or more Yamanaka factors defined herein. In other embodiments, culturing in the presence of one or more Yamanaka factors includes expressing the one or more Yamanaka factors in somatic cells. Thus, according to one embodiment, culturing in the presence of one or more Yamanaka factors defined herein includes expression of endogenous one or more Yamanaka factor-encoding genes from somatic cells. According to this embodiment, expression of one or more Yamanaka factors in somatic cells does not include transfection, transduction, or introduction of exogenous sequences. In another embodiment, expression of one or more Yamanaka factors in somatic cells includes stimulating expression using compounds and / or treatments that upregulate or "turn on" expression of one or more Yamanaka factor-encoding genes. Thus, in one embodiment, culturing in the presence of one or more Yamanaka factors comprises adding a compound known to cause expression of 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 cell.

[0080] In an alternative embodiment, culturing in the presence of one or more Yamanaka factors comprises introducing into the somatic cells an exogenous sequence encoding one or more Yamanaka factors as defined herein. Thus, in one embodiment, culturing in the presence of one or more Yamanaka factors comprises expressing one or more Yamanaka factors from one or more exogenous sequences.

[0081] In one embodiment, the exogenous sequence encoding 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 comprises culturing somatic cells in the presence of Yamanaka factor-encoding mRNA. In another embodiment, culturing somatic cells in the presence of a Yamanaka factor comprises providing the somatic cells with Yamanaka factor-encoding mRNA.

[0082] In one embodiment, an exogenous sequence encoding one or more Yamanaka factors defined herein is introduced into somatic cells by transfection. In an alternative embodiment, an 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 a 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 defined herein can be introduced into somatic cells in the form of a vector transfected into somatic cells. In one embodiment, the vector is a transposon vector. Vectors suitable for introducing expression of one or more Yamanaka factors used herein into host cells such as somatic cells are well known in the art. The vector may also contain a variety of regulatory / response sequences or elements that control 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. This approach can be a drug- (i.e., doxycycline (dox)) inducible or non-inducible CRISPR / Cas-9 approach and is well known to those skilled in the art.

[0083] Transposon vectors utilize mobile genetic elements called transposons to move target sequences between the vector and the chromosome via a "cut and paste" mechanism. Examples of transposon vectors include PiggyBac vectors (System Biosciences) or EZ-Tn5 TM Transposon construction vector (Illumina, Inc.).

[0084] Viral vector is made up of the DNA or RNA in genetically engineered viruses. Viral vector can be used for integrating the target sequence into the host cell genome (i.e., integrative viral vector). The example of viral vector includes adenoviral vector, adenovirus-associated vector, retroviral vector or lentiviral vector (e.g., HIV). Viral vector can be introduced into host cells such as somatic cells by means of viral transduction. Therefore, according to one embodiment, one or more Yamanaka factors are expressed in somatic cells and / or cultivated in the presence of one or more Yamanaka factors and include one or more Yamanaka factor encoding sequences being integrated into the somatic genome. In another embodiment, one or more Yamanaka factors are expressed in somatic cells and / or cultivated in the presence of one or more Yamanaka factors and include using viral vectors to integrate one or more Yamanaka factor encoding sequences into the somatic genome.

[0085] Plasmid vectors are typically composed of circular double-stranded DNA. Like most engineered vectors, plasmid vectors have a multiple cloning site (MCS), a short region containing several commonly used restriction sites that allows for easy insertion of the desired DNA fragment.

[0086] "Transfection" mentioned herein refers to the process of introducing a vector into a host cell (eg, 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.

[0087] In one embodiment, expression of one or more Yamanaka factors as defined herein can be introduced into 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 comprises introducing one or more Yamanaka factor coding sequences into somatic cells in the form of an expression cassette. Thus, in one embodiment, expression of one or more Yamanaka factors as defined herein is from an expression cassette. In a specific embodiment, such an expression cassette may comprise an mRNA-derived sequence encoding one or more Yamanaka factors as defined herein. In another embodiment, the expression cassette further comprises a sequence encoding a protein or marker that allows identification of expression of the expression cassette. In a specific embodiment, the protein or marker used to identify expression is a fluorescent protein. In a certain embodiment, the fluorescent protein is green fluorescent protein (GFP).

[0088] Thus, in one embodiment, somatic cells can be selected based on the expression of a protein or marker contained in an expression cassette. In a specific embodiment, somatic cells are selected based on the expression of a fluorescent protein (e.g., GFP), which allows identification of expression. It should be understood that "selection" as used herein can include flow cytometry methods, such as fluorescence activated cell sorting (FACS).

[0089] In another embodiment, the marker that allows identification expression can be selected from drug resistance genes. The example of drug resistance genes can include: puromycin resistance gene, ampicillin resistance gene, neomycin resistance gene, tetracycline resistance gene, kanamycin resistance gene or chloramphenicol resistance gene. Cell can be cultivated in the culture medium (i.e., selection culture medium) containing appropriate drugs, and only those cells containing and expressing resistance genes can survive. Therefore, by using selection culture medium to cultivate cells, it is possible to easily select cells that comprise drug resistance genes.

[0090] Alternative markers that allow identification of expression include chromogenic enzyme genes. Examples of chromogenic enzyme genes include: β-galactosidase gene, β-glucuronidase gene, alkaline phosphatase gene, or 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), so that cells expressing the marker gene produce a detectable color (e.g., blue in a blue-white screening assay).

[0091] In another embodiment, the expression cassette is an inducible expression cassette that allows expression or co-expression of one or more Yamanaka factor coding sequences upon induction of expression with an appropriate compound or treatment. Such inducible expression cassettes will be understood to include a response element that allows 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 expression or co-expression of one or more Yamanaka factor coding sequences upon addition of an antibiotic (such as tetracycline, particularly doxycycline) (as exemplified in the data presented herein).

[0092] Thus, it will be understood that, according to one embodiment, culturing somatic cells in the presence of one or more Yamanaka factors comprises adding a compound or treatment capable of inducing expression from an inducible expression cassette. In certain embodiments, culturing somatic cells in the presence of one or more Yamanaka factors comprises adding tetracycline.

[0093] 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 a Yamanaka factor encoding mRNA. It will be appreciated that the expressed protein (i.e., the protein expressed from the Yamanaka factor encoding mRNA) can be directly transferred (i.e., transfected) into somatic cells using a suitable protein delivery method. It will be appreciated that suitable methods for directly transferring proteins into cells are well known to those skilled in the art and include functional twin-arginine translocation (Tat) systems. Alternatively, the protein can be directly transferred to somatic cells via a targeted delivery system (e.g., a nanoparticle delivery system). Similarly, such targeted delivery systems are well known to those skilled in the art.

[0094] In another embodiment, the method of reprogramming somatic cells as defined herein comprises further culturing the somatic cells in the absence of the one or more Yamanaka factors for at least 2 weeks. In another embodiment, the somatic cells are further cultured in the absence of the one or more Yamanaka factors for at least 2.5 weeks, at least 3 weeks, at least 3.5 weeks, or at least 4 weeks. In another embodiment, the somatic cells are further cultured in the absence of the one or more Yamanaka factors for no more than 5 weeks, no more than 4 weeks, no more than 3 weeks, or no more than 2.5 weeks. In a specific embodiment, the somatic cells are further cultured in the absence of the one or more Yamanaka factors for 4 weeks. In an alternative embodiment, the somatic cells are further cultured in the absence of the one or more Yamanaka factors for 3 weeks. In another alternative embodiment, the somatic cells are further cultured in the absence of the one or more Yamanaka factors for 2 weeks.

[0095] In one embodiment, the method of reprogramming somatic cells as defined herein comprises further culturing the somatic cells in the absence of the one or more Yamanaka factors until the expression of pluripotency markers on the surface or in the somatic cells is downregulated or reduced. In another embodiment, the somatic cells are further cultured in the absence of the one or more Yamanaka factors until the expression of pluripotency markers on the surface or in the somatic cells is no longer detectable. According to this embodiment, the reference to "downregulated," "reduced," or "no longer detectable" will be understood as relative to the somatic cells before further culturing in the absence of the one or more Yamanaka factors and / or relative to a reference pluripotent cell.

[0096] It will be understood that the pluripotency markers according to these embodiments may be the same as or different from the pluripotency markers detected on the surface of or within somatic cells cultured in the presence of one or more Yamanaka factors. Thus, in a specific embodiment, the expression of a pluripotency marker that is downregulated or no longer detectable on the surface of or within somatic cells when cultured in the absence of one or more Yamanaka factors is the pluripotency marker that is detectable on the surface of or within somatic cells after culture in the presence of one or more Yamanaka factors.

[0097] In another embodiment, the method of reprogramming somatic cells defined herein includes further culturing the somatic cells in the absence of the one or more Yamanaka factors until the expression of somatic cell lineage-specific markers (e.g., CD13) is detected on the surface of the somatic cells. In an alternative embodiment, further culturing is performed in the absence of the one or more Yamanaka factors until the expression of somatic cell lineage-specific markers on the surface of the somatic cells is upregulated or increased. "Upregulated" and "increased" mentioned herein encompass any changes in the surface expression of markers compared to somatic cells before the step of further culturing in the absence of the one or more Yamanaka factors, including increases. In this case, it will be understood that the somatic cells before the step of further culturing in the absence of the one or more Yamanaka factors contain lower, less, or no expression of somatic cell lineage-specific markers. "Detectable," "upregulated," and "increased" further mentioned herein may also be compared with reference pluripotent stem cells such as iPS cells. In an alternative embodiment, culturing is further carried out in the absence of the one or more Yamanaka factors until expression of somatic cell lineage-specific markers is restored as compared to expression in the reprogrammed somatic cells before the step of further culturing in the absence of the one or more Yamanaka factors, or as compared to expression in the somatic cells before culturing in the presence of one or more Yamanaka factors, or as compared to expression in non-reprogrammed somatic cells.

[0098] Thus, it will be understood that further culturing in the absence of expression of the one or more Yamanaka factors as defined herein may be referred to as "reversal" or "recovery."

[0099] In another embodiment, further culturing the somatic cells in the absence of the one or more Yamanaka factors comprises removing a compound or treatment capable of inducing expression from the inducible expression cassette. In certain embodiments, further culturing the somatic cells in the absence of the one or more Yamanaka factors comprises removing tetracycline. In an alternative embodiment, further culturing in the absence of the one or more Yamanaka factors comprises removing a compound or treatment capable of preventing or halting expression from the inducible expression cassette.

[0100] According to another aspect of the present invention, there is provided a reprogrammed somatic cell produced according to the method defined herein. It should be understood that the "one" or "the" reprogrammed somatic cell mentioned herein includes a single or a small number of cells, as well as a population of reprogrammed somatic cells, the number of which can be large. Therefore, it should be understood that any singular number mentioned herein includes the plural number, and vice versa.

[0101] In one embodiment, the reprogrammed somatic cells produced according to the methods defined herein include such DNA methylation age, epigenetic age or molecular markers, which are younger or less than somatic cells that are not reprogrammed or the somatic cells of the tissue or body from which the reprogrammed somatic cells are obtained. In another embodiment, the reprogrammed somatic cells include such molecular markers, such as epigenetic markers, which indicate an epigenetic age that is younger or less than somatic cells that are not reprogrammed or the somatic cells of the tissue or body from which the reprogrammed somatic cells are obtained. In a specific embodiment, the reprogrammed somatic cells produced according to the methods defined herein include such molecular markers, which are similar to molecular markers such as epigenetic markers of somatic cells at an earlier time point in the life cycle of the tissue or body from which the somatic cells are obtained. In other embodiments, the reprogrammed somatic cells produced according to the methods defined herein include such phenotypes and / or molecular markers, such as epigenetic markers, which are similar to the phenotypes and / or molecular markers of somatic cells that are not reprogrammed.

[0102] Compositions comprising reprogrammed somatic cells

[0103] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the reprogrammed somatic cells defined herein.

[0104] According to another aspect of the present invention, there is provided a cosmetic composition comprising the reprogrammed somatic cells defined herein.

[0105] According to certain embodiments, the pharmaceutical or cosmetic composition comprises, in addition to the reprogrammed somatic cells defined herein, one or more pharmaceutically acceptable excipients. In other embodiments, the pharmaceutical or cosmetic composition comprises, in addition to the reprogrammed somatic cells produced according to the methods defined herein, one or more pharmaceutically acceptable excipients.

[0106] Typically, this medicine and cosmetic composition will be used together with a pharmacologically suitable excipient or carrier. Typically, these excipients or carriers include water or alcohol / aqueous solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, and lactated Ringer's solution. Suitable physiologically acceptable adjuvants, if necessary, for maintaining the composition of the somatic cells comprising the reprogramming defined herein in discrete locations, can be selected from thickeners such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin, and alginate. Intravenous vehicles include fluid and nutrient supplements and electrolyte supplements, such as supplements based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16th edition) may also be present.

[0107] The route of administration of the pharmaceutical compositions defined herein can be any route known to those of ordinary skill in the art. For example, the compositions can be administered by any suitable means, including parenteral, intravenous, intramuscular, intraperitoneal, cutaneous, or transdermal. In specific embodiments, the pharmaceutical compositions defined herein can be administered intravenously or transdermally.

[0108] In addition, the cosmetic compositions defined herein can be administered by any route known to those of ordinary skill in the art. For example, the cosmetic compositions can be administered by any suitable means, including those described above. In specific embodiments, the cosmetic compositions defined herein can be administered topically, dermally, or transdermally.

[0109] Therapeutic uses and methods

[0110] It will be appreciated from the disclosure provided herein that the methods and compositions of the present invention will be particularly useful in treating and / or ameliorating age-related or degenerative diseases and / or conditions or in rejuvenating tissues or organs.

[0111] Therefore, according to one aspect, there is provided a method for treating and / or improving age-related or degenerative diseases or conditions comprising reprogrammed somatic cells produced according to the methods defined herein, the method further comprising administering the reprogrammed somatic cells to an individual in need. In another embodiment, there is provided a method for treating and / or improving age-related or degenerative diseases or conditions comprising reprogrammed somatic cells as defined herein, the method further comprising administering the reprogrammed somatic cells to an individual in need. In one embodiment, the method comprising reprogrammed somatic cells as defined herein is used to treat and / or improve age-related or degenerative diseases or conditions of the skin. In an alternative embodiment, the method comprising reprogrammed somatic cells as defined herein is used to treat or improve age-related or degenerative diseases or conditions of the pancreas, for example, for treating or improving type 2 diabetes. In another embodiment, the method comprising reprogrammed somatic cells as defined herein is used to treat and / or improve age-related diseases or conditions, wherein the age-related diseases or conditions are neurodegenerative diseases. In another embodiment, the method comprising reprogrammed somatic cells as defined herein is used to treat and / or improve age-related diseases or conditions, wherein the age-related diseases or conditions are diseases or conditions of the blood and / or bone marrow. In another embodiment, the method comprising reprogramming somatic cells as defined herein is used to treat and / or ameliorate an age-related disease or condition, wherein the age-related disease or condition is a disease or condition of the heart. Thus, in one embodiment, the disease or condition is a cardiovascular disease. In another embodiment, the disease or condition is a cardiomyopathy. In another embodiment, the disease or condition is ischemic heart disease. In another embodiment, the disease or condition is arrhythmia. In another embodiment, the disease or condition is heart failure.

[0112] In another embodiment, there is provided a method for producing a reprogrammed somatic cell as defined herein for treating and / or ameliorating an age-related or degenerative disease or condition. In another embodiment, there is provided a method for producing a reprogrammed somatic cell as defined herein for treating and / or ameliorating an age-related or degenerative disease or condition.

[0113] According to another aspect, a pharmaceutical composition as defined herein is provided for treating and / or ameliorating degenerative or age-related diseases or conditions, or for rejuvenating a tissue or organ. In one embodiment, the pharmaceutical composition comprises reprogrammed somatic cells as defined herein. In an alternative embodiment, the pharmaceutical composition comprises reprogrammed somatic cells produced according to the method defined herein.

[0114] In one embodiment, the pharmaceutical composition for use comprising the reprogrammed somatic cells defined herein or the reprogrammed somatic cells defined herein are used for skin treatment or for treating and / or ameliorating a skin disease or condition. Thus, in certain embodiments, the age-related disease or condition comprises a skin disease or condition. In another embodiment, the treatment of the skin is to prevent, inhibit, reduce and / or reverse skin aging. Examples of skin aging include wrinkles, dryness, loss of elasticity, fragility and / or loss of barrier properties.

[0115] In an alternative embodiment, the pharmaceutical composition for use comprising the reprogrammed somatic cells defined herein or the reprogrammed somatic cells defined herein are used to treat and / or improve a pancreatic disease or condition. Therefore, in certain embodiments, the age-related disease or condition comprises a pancreatic disease or condition. In another embodiment, the pancreatic disease or condition is type 2 diabetes.

[0116] In another embodiment, a pharmaceutical composition for use comprising a reprogrammed somatic cell as defined herein or a reprogrammed somatic cell as defined herein is used for treating and / or ameliorating a neurodegenerative disease.

[0117] In another embodiment, a pharmaceutical composition for use comprising a reprogrammed somatic cell as defined herein or a reprogrammed somatic cell as defined herein is used to treat and / or ameliorate a disease or disorder of the blood and / or bone marrow.

[0118] In another embodiment, a pharmaceutical composition for use comprising a reprogrammed somatic cell as defined herein or a reprogrammed somatic cell as defined herein is used to treat and / or ameliorate a cardiac disease or disorder.

[0119] In other embodiments, the tissue or organ as defined herein is selected from the group consisting of: 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 improvement comprises subjecting the blood or blood cells to one or more methods as 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 comprises subjecting the bone marrow or bone marrow cells to one or more methods as defined herein and providing the bone marrow or bone marrow cells to a patient or subject in need thereof.

[0120] 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 will be understood that, according to this embodiment, the rejuvenation may include rejuvenating only a portion of the liver tissue or organ, or somatic cells derived 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 be rejuvenated or further rejuvenated in vivo.

[0121] 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 cardiac tissue. Therefore, according to one embodiment, the tissue or organ is selected from the heart, and the treatment and / or improvement or rejuvenation includes subjecting cardiac cells (such as cardiomyocytes) to one or more methods defined herein, and providing the cardiac cells to patients or subjects in need. In another embodiment, the tissue or organ is selected from the heart, and the rejuvenation includes subjecting cardiac cells (such as cardiomyocytes) to one or more methods defined herein, and providing the cardiac cells to patients or subjects in need. In another embodiment, the tissue or organ is selected from the heart, and the reprogrammed somatic cells defined herein are cardiac cells, such as cardiomyocytes, and the treatment and / or improvement or rejuvenation includes providing the reprogrammed cardiac somatic cells to patients or subjects in need.

[0122] It will be understood that according to the embodiments described herein, the tissue or organ can be from the patient or subject in need thereof, or alternatively from a donor subject.

[0123] It will be understood that references herein to a patient or subject in need thereof relate equally to animals and humans, and that the present invention is particularly useful in the veterinary treatment of any of the above-mentioned diseases, disorders and conditions also present in such animals.

[0124] It should be understood that references to "treating" and "improving" herein include terms such as "preventing," "reversing," and "inhibiting." Furthermore, such references include administering the reprogrammed somatic cells defined herein, or compositions comprising the reprogrammed somatic cells, prior to the onset of a disease or condition. It is also contemplated that administration of the reprogrammed somatic cells defined herein, or compositions comprising the reprogrammed somatic cells, may be administered after the induction event of the disease or condition, prior to clinical manifestation of the disease or condition, or after symptoms have manifested.

[0125] Cosmetic uses and methods

[0126] According to one aspect of the present invention, there is provided a cosmetic method for regenerating or rejuvenating skin, comprising administering or applying the reprogrammed somatic cells defined herein or the cosmetic composition defined herein to a subject in need thereof.

[0127] 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 is for rejuvenating a tissue or organ in need thereof, wherein the tissue or organ is not the skin.

[0128] It will be appreciated that the cosmetic compositions and methods comprising the reprogrammed somatic cells defined herein may be suitably used to regenerate or rejuvenate the skin. In addition, such cosmetic compositions and methods may be used to reduce scarring or to regenerate connective tissue. Alternatively and / or additionally, the cosmetic compositions defined herein may be used to regenerate or rejuvenate the skin and / or connective tissue used during or after cosmetic surgery. Suitably, the cosmetic compositions defined herein may be used to regenerate or rejuvenate the skin and / or connective tissue, including reducing age, such as DNA methylation age or epigenetic age, or making the skin and / or connective tissue younger.

[0129] Furthermore, the cosmetic method defined herein may be used for the regeneration of skin and / or connective tissue following cosmetic surgery.The cosmetic method defined herein is intended in particular for the regeneration of skin and / or connective tissue used in cosmetic surgery.

[0130] It will be further understood that the cosmetic compositions defined herein can be administered and / or used prophylactically. For example, a cosmetic composition comprising reprogrammed somatic cells as defined herein can be used at a time point before a tissue and / or organism is considered senescent and / or aged, such as at an early point in the life cycle of the tissue and / or organism.

[0131] Screening methods

[0132] According to another aspect, there is provided a method for screening for an age-modifying agent, the method comprising:

[0133] (i) performing a method as defined herein in the presence and absence of a test agent to generate reprogrammed somatic cells; and

[0134] (ii) determining molecular markers of reprogrammed somatic cells, such as epigenetic markers,

[0135] wherein a difference between the molecular signature determined for reprogrammed somatic cells generated in the presence of the test agent and the molecular signature determined for reprogrammed somatic cells generated in the absence of the test agent indicates an age-modulated effect of the test agent.

[0136] It should be understood that according to this aspect of the invention, the test agent may comprise any compound, treatment, condition or process that can increase, accelerate or accelerate the aging of cells, tissues, organs or organisms, or alternatively can reduce, slow down or decelerate the aging of cells, tissues, organs or organisms. Therefore, in one embodiment, the test agent accelerates, accelerates or increases the aging of cells, tissues or organisms. In an alternative embodiment, the test agent slows down, slows down 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.

[0137] In one embodiment, the difference between the molecular markers of the somatic cells of the reprogrammed cells as defined herein or produced according to the method defined herein and not exposed to the test agent is determined. Therefore, in one embodiment, the difference between the molecular markers is determined for the somatic cells reprogrammed in the presence of a test agent and the somatic cells reprogrammed in the absence of a test agent. In another embodiment, the difference between the molecular markers of the somatic cells reprogrammed according to the method defined herein or the reprogrammed somatic cells defined herein and the non-reprogrammed somatic cells exposed to the test agent is determined. In another embodiment, the difference between the molecular markers of the somatic cells reprogrammed according to the method defined herein or the reprogrammed somatic cells defined herein and the non-reprogrammed somatic cells exposed to the test agent is determined.

[0138] According to another aspect, there is provided a method for screening for an age-regulating factor or cellular process, the method comprising:

[0139] (i) reprogramming somatic cells from a diseased tissue or organ according to a method as defined herein; and

[0140] (ii) determining molecular markers, such as epigenetic markers, of the reprogrammed somatic cells from the diseased tissue or organ and the reprogrammed somatic cells as defined herein or non-reprogrammed somatic cells from said diseased tissue or organ,

[0141] wherein differences between molecular signatures determined for reprogrammed somatic cells from a diseased tissue or organ and molecular signatures determined for reprogrammed somatic cells as defined herein or non-reprogrammed somatic cells from a diseased tissue or organ indicate age-regulatory factors or cellular processes associated with the disease.

[0142] In one embodiment, the age-regulating factor is a factor expressed or present in somatic cells that participates in or regulates, or is suspected of participating in or regulating, the age or aging of a cell, tissue, organ, or organism. In another embodiment, the age-regulating cellular process is a cellular process that participates in or regulates, or is suspected of participating in or regulating, the age or aging of a cell, tissue, organ, or organism. In another embodiment, the age-regulating factor or cellular process participates in or regulates, or is suspected of participating in or regulating, an age-related disease or condition.

[0143] In one embodiment, the somatic cell is obtained from a diseased tissue or organ, wherein 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.

[0144] Therefore, in one embodiment, the difference between the molecular markers is determined between the somatic cells obtained from the reprogramming of a diseased tissue or organ and the somatic cells obtained from a diseased tissue or organ or the non-reprogrammed somatic cells of the diseased tissue or organ. In another embodiment, the difference between the molecular markers is determined between the somatic cells obtained from the reprogramming of a diseased tissue or organ and the non-reprogrammed somatic cells obtained from non-diseased tissue or organ. In an alternative embodiment, the difference between the molecular markers is determined between the somatic cells obtained from the reprogramming of 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, as different parts of a tissue or organ, or from different tissues or organs. Example

[0145] Materials and methods

[0146] In the presence of polybrene (8 μg / ml), human fibroblasts from three different donors were infected simultaneously with a lentivirus containing a doxycycline-responsive transactivator (available from www.addgene.org) and a tetO-GFP-hOKMS construct (an inducible expression cassette encoding the Yamanaka factors defined herein). Next, after adding the virus, the cells were centrifuged at 1000 rpm for 1 hour to increase transduction efficiency. 24 hours after infection (day 0), doxycycline (2 μg / ml) was added to fibroblast culture medium (DMEM-F12, 10% FBS, 1x Glutamax, 1x MEM-NEAA, 1xβ-ME, 0.2xPen / Strep, 16ng / ml FGF2). The cells were then flow sorted (FACS) for GFP expression on the second day of doxycycline treatment and re-seeded on gelatin-coated culture dishes. At day 7 after infection, cells were passaged onto a culture dish containing irradiated mouse embryonic fibroblasts (iMEFs). The next day, culture medium was replaced with human embryonic stem cell culture medium (DMEM-F12, 20% KSR, 1x Glutamax, 1xMEM-NEAA, 1xβ-ME, 0.2x Pen / Strep, 8ng / mlFGF2). On days 13, 15, and 17, cells undergoing reprogramming were flow-sorted (FACS) for CD13 and SSEA4 surface expression using antibodies against these markers (obtained from Biolegend). CD13+SSEA4- and CD13-SSEA4+ colonies were collected and re-plated in fibroblast culture medium on a culture dish containing iMEFs. Re-plated cells were grown for four weeks in the absence of doxycycline so that they could be reversed to their initial cell type ("reversal" defined herein). At the end of the four-week reversal, cells were harvested for flow cytometry analysis, DNA methylation arrays, and RNA sequencing.

[0147] DNA methylation array

[0148] 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. Genomic DNA samples were further processed at Barts and the London Genome Centre and run on Infinium Methylation EPIC arrays (Illumina).

[0149] RNA-Seq

[0150] RNA was extracted from cell samples using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. 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 a HiSeq 2500 system (Illumina) for 50-bp single-end sequencing.

[0151] DNA methylation analysis

[0152] 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, reference datasets included unpublished checks using CytoTune. TM - Data on the intermediate stage of fibroblasts reprogrammed using the iPS2.0 Sendai virus reprogramming kit (Invitrogen).

[0153] RNA-seq analysis

[0154] 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, reference datasets included unpublished checks using CytoTune. TM - Data on the intermediate stage of fibroblasts reprogrammed using the iPS2.0 Sendai virus reprogramming kit (Invitrogen).

[0155] Immunofluorescence and imaging

[0156] After fixation with 2% PFA for 30 minutes at room temperature, cells grown on coverslips or cytospun slides were stained with antibodies as previously described (Santos et al. (2003) Curr. Biol. 13, 1116-1121). Briefly, cells were permeabilized with 0.5% Triton X-100 in PBS for 1 hour; blocked with 1% BSA in 0.05% Tween 20 in PBS (BS) for 1 hour; incubated with the appropriate primary antibody diluted in BS; then washed in BS and secondary antibodies. All secondary antibodies were Alexa Fluor-conjugated (Molecular Probes), diluted 1:1000 in BS, and incubated for 30 minutes. Incubation was performed at room temperature. DNA was counterstained with 5 μg / mL DAPI in PBS. Optical sections were captured using a Zeiss LSM780 microscope (63x oil immersion objective). Fluorescence semiquantitative analysis was performed using Volocity 6.3 (Improvision). The antibodies used are as follows:

[0157] Anti-H3K9me3; 07-442, Merck / Millipore (1:500);

[0158] Anti-collagen type I; ab254113, Abcam (1:400).

[0159] Example 1: Partially reprogrammed somatic cells are fibroblast-like

[0160] During the intermediate stages of the process, cells expressing the Yamanaka factors become heterogeneous. Some cells remain CD13+SSEA4- and are classified as unreprogrammed cells, while some become CD13-SSEA4+ and are classified as successfully reprogrammed (see Figure 1 Both populations were flow cytometry sorted (FACS) and cultured for an additional 4 weeks in the absence of doxycycline (“reversal” as defined herein). At the end of this period, successfully reprogrammed cells reverted to a fibroblastic phenotype and became CD13+SSEA4- (see Figure 2 These cells are also morphologically similar to fibroblasts (see Figure 3 ). Cells that were not reprogrammed (shown in the graph labeled "+CD13") or cultured without the presence of Yamanaka factors ("-" conditions) remained fibroblast-like throughout.

[0161] Similar findings were observed during longer reprogramming periods (data not shown).

[0162] Example 2: Partially reprogrammed somatic cells display a younger epigenetic age

[0163] Cells that were successfully reprogrammed and then reverted after 13 days of culture in the presence of Yamanaka factors showed a DNA methylation age that was 30-40 years younger than their respective controls. Longer reprogramming times (15 or 17 days) before the reversal phase slightly reduced the rejuvenation effect. Unreprogrammed cells expressing Yamanaka factors were the same age as negative control cells that had never expressed Yamanaka factors (see Figure 4 ).

[0164] Thus, the data presented herein indicate that expression of the Yamanaka factors alone within a short period of time (i.e., a period prior to the expression of pluripotency markers such as SSEA4, before somatic lineage-specific marker expression is no longer detectable on the cell surface, within the initial stages of iPS cell reprogramming, or a period of less than 5 days) is not sufficient to rejuvenate 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.

[0165] Example 3: Transient reprogramming experiment

[0166] (a) Experimental design

[0167] 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. In addition, we “mock-infected” some fibroblasts without the construct to generate a negative control. After infection, we positively selected for cells expressing GFP by flow cytometry, and for the negative control, we sorted an equal number of viable cells by flow cytometry. We then treated the cells with doxycycline for different lengths of time and flow-sorted the cells that successfully reprogrammed and those that failed to reprogram based on the cell surface markers CD13 and SSEA4. Finally, we cultured the sorted cells in the absence of doxycycline for at least 4 weeks before harvesting the cells for analysis ( Figure 5 The cells are morphologically similar to fibroblasts at the end of the process ( Figure 6 ).

[0168] (b) Transiently reprogrammed cells are epigenetically similar to fibroblasts

[0169] We performed principal component analysis on the methylomes of transiently reprogrammed cells along with a reference dataset examining complete fibroblast reprogramming. As expected, principal component 1 separated cells according to the extent of reprogramming and generated reprogramming trajectories using the reference dataset. Notably, transiently reprogrammed cells (and controls) remained at the beginning of the reprogramming trajectory, suggesting that they resemble fibroblasts rather than reprogramming intermediates or iPSCs ( Figure 7 ).

[0170] 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 those in control and reference fibroblasts ( Figure 8 We also observed that the promoter of FSP1 (a fibroblast marker gene) became highly methylated in iPSCs. However, in transiently reprogrammed cells, we found that this promoter remained demethylated ( Figure 9 ).

[0171] (c) Transiently reprogrammed cells are transcriptionally similar to fibroblasts

[0172] We also performed principal component analysis on the transcriptomes of transiently reprogrammed cells along with a reference dataset examining complete fibroblast reprogramming. As with the methylome analysis, principal component 1 separated samples based on the extent of reprogramming, and a reprogramming trajectory was generated using the reference dataset. The transcriptomes of transiently reprogrammed cells were at the beginning of this trajectory, indicating that these cells are also transcriptionally similar to fibroblasts ( Figure 10 ).

[0173] During iPSC reprogramming, marker genes of the starting cell type are downregulated, while 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 ) was not adjusted upwards.

[0174] (d) Transient reprogramming restores many aging markers to youth

[0175] To investigate the effects of transient reprogramming on epigenome rejuvenation, we calculated the DNA methylation age of cells after transient reprogramming using the Horvath epigenetic clock. We found that transient reprogramming rejuvenated DNA methylation by up to 40 years relative to the control group. Notably, transient reprogramming using 13 days of doxycycline treatment produced the strongest rejuvenation, suggesting that this is the optimal amount of epigenetic rejuvenation ( Figure 13 ).

[0176] Other features of the epigenome change with aging, such as the overall levels of histone modifications. H3K9me3 levels decrease with aging, and we found that transient reprogramming could increase H3K9me3 to youthful levels ( Figure 14 ).

[0177] To investigate the transcriptome rejuvenation effects of transient reprogramming, we trained a transcriptional clock using random forest regression using published data from fibroblasts (Fleischer et al. (2018), supra). This transcriptional clock predicted age with a median absolute error of 13.48 years. Using this clock, we found that transient reprogramming rejuvenated transcriptional age by approximately 30–40 years, a similar degree of rejuvenation observed using the epigenetic clock. In contrast to the epigenetic clock, transcriptional rejuvenation was observed for all durations of doxycycline treatment studied ( Figure 15 ).

[0178] 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 investigated the protein levels 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 or youthful state, comprising: i) culturing the somatic cells in the presence of one or more Yamanaka factors for at least 5 days, and / or until expression of pluripotency markers is detectable on the surface of or within the somatic cells, and / or until somatic cell lineage-specific markers are no longer detectable on the surface of the somatic cells; ii) further culturing the somatic cells in the absence of the one or more Yamanaka factors until the expression of the pluripotency marker on the surface or inside the somatic cells is reduced and / or until the expression of somatic cell lineage-specific markers is detected on the surface of the somatic cells.

2. The method of claim 1, wherein the method reprograms somatic cells to a rejuvenated state.

3. The method of claim 1, 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.

4. The method according to any one of claims 1 to 3, 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. 5 . The method according to claim 1 , wherein the somatic cells are cultured in the presence of the one or more Yamanaka factors until expression of pluripotency markers is detectable on the surface of or inside the somatic cells.

6. The method according to any one of claims 1 to 5, wherein the pluripotency marker is stage-specific embryonic antigen-4 (SSEA4).

7. The method according to any one of claims 1 to 6, wherein the somatic cells are cultured in the presence of the one or more Yamanaka factors until expression of somatic cell lineage-specific markers is no longer detected on the surface of the somatic cells.

8. The method according to any one of claims 1 to 7, wherein the somatic cells are cultured in the absence of the one or more Yamanaka factors until expression of the pluripotency marker on the surface of the somatic cells is reduced.

9. The method according to any one of claims 1 to 8, wherein the somatic cells are cultured in the absence of the one or more Yamanaka factors until expression of somatic cell lineage-specific markers is detected on the surface of the somatic cells. 10 . The method according to claim 1 , wherein the reprogramming of the somatic cell is incomplete and / or partial reprogramming and / or transient reprogramming.

11. The method according to any one of claims 1 to 10, wherein the somatic cell is cultured in the presence of the one or more Yamanaka factors during the maturation phase of reprogramming.

12. The method according to any one of claims 1 to 11, wherein the reprogrammed somatic cells comprise molecular markers, such as epigenetic markers, corresponding to somatic cells from an earlier time point in the tissue life cycle.

13. The method according to any one of claims 1 to 12, wherein the reprogrammed somatic cell retains the phenotypic and / or molecular markers, such as epigenetic markers, of the non-reprogrammed somatic cell.

14. The method according to any one of claims 1 to 13, wherein the molecular markers of the reprogrammed and / or non-reprogrammed somatic cells are epigenetic markers and are determined using a Horvath epigenetic clock, such as wherein the epigenetic markers of the reprogrammed somatic cells indicate an epigenetic age that is at least 10% younger, at least 40% younger, or at least 70% younger than that of the non-reprogrammed somatic cells.

15. The method according to any one of claims 1 to 14, wherein the one or more Yamanaka factors are provided from an inducible expression cassette that is transduced or transfected into the somatic cell, such as wherein culturing in the presence of the one or more Yamanaka factors further comprises adding a compound capable of inducing expression from the inducible expression cassette.

16. The method according to any one of claims 1 to 15, wherein the culturing in the absence of the one or more Yamanaka factors comprises removing a compound capable of inducing expression from the inducible expression cassette.

17. The method according to any one of claims 1 to 16, wherein the one or more Yamanaka factors are provided in the form of Yamanaka factor-encoding mRNA.

18. The method of claim 17, wherein culturing in the absence of the one or more Yamanaka factors comprises removing Yamanaka factor-encoding mRNA from the culture.

19. The method according to any one of claims 1 to 16, wherein the one or more Yamanaka factors are provided in the form of proteins expressed from Yamanaka factor-encoding mRNA.

20. The method of claim 19, wherein the protein is delivered directly to the somatic cell via a targeted delivery system, such as a functional twin-arginine translocation (Tat) system or a nanoparticle delivery system.

21. The method according to any one of claims 1 to 16, wherein the Yamanaka factor is introduced into the somatic cell by CRISPR / Cas-9, such as drug (i.e. doxycycline (dox)) inducible or non-inducible CRISPR / Cas-9.

22. The method according to any one of claims 1 to 21, wherein the one or more Yamanaka factors are selected from one or more of OCT4, KLF4, c-MYC, SOX2, LIN28 and / or NANOG, or preferably selected from one or more, or two or more, or three or more or all of OCT4, KLF4, c-MYC and / or SOX2.

23. A reprogrammed somatic cell produced according to the method of any one of claims 1 to 22. 24 . A pharmaceutical composition comprising the reprogrammed somatic cell according to claim 23 .

25. The reprogrammed somatic cell according to claim 23 or the pharmaceutical composition according to claim 24, for use in treating and / or ameliorating a degenerative or age-related disease or condition, or for rejuvenating a tissue or organ such as skin, blood, bone marrow, liver or heart, wherein the degenerative or age-related disease or condition comprises: a skin disease or condition; or a pancreatic disease or condition, such as type 2 diabetes; or neurodegenerative diseases.

26. The reprogrammed somatic cell according to claim 23 or the pharmaceutical composition according to claim 24 for use according to claim 25, for administration to a human or animal subject.

27. A cosmetic composition comprising the reprogrammed somatic cell according to claim 23.

28. A cosmetic method for regenerating or rejuvenating skin, comprising administering or applying the reprogrammed somatic cell of claim 23 or the cosmetic composition of claim 27 to a subject in need thereof.

29. A method for screening an age-modifying agent, the method comprising: (i) performing the method of any one of claims 1 to 22 in the presence and absence of a test agent to produce reprogrammed somatic cells; and (ii) determining molecular markers, such as epigenetic markers, of the reprogrammed somatic cells, wherein a difference between the molecular signature determined for reprogrammed somatic cells generated in the presence of the test agent and the molecular signature determined for reprogrammed somatic cells generated in the absence of the test agent indicates an age-modulated effect of the test agent.

30. A method for screening for age-regulating factors or cellular processes, the method comprising: (i) reprogramming somatic cells from a diseased tissue or organ using the method according to any one of claims 1 to 22; and (ii) determining molecular markers, such as epigenetic markers, of the reprogrammed somatic cells from the diseased tissue or organ and the reprogrammed somatic cells according to claim 23 or the non-reprogrammed somatic cells from the diseased tissue or organ, Wherein, 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 of claim 23 or the non-reprogrammed somatic cells from the diseased tissue or organ indicates age-regulating factors or cellular processes associated with the disease.

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