- Open Access
Age reprogramming and epigenetic rejuvenation
© The Author(s) 2018
- Received: 7 October 2018
- Accepted: 12 December 2018
- Published: 20 December 2018
Age reprogramming represents a novel method for generating patient-specific tissues for transplantation. It bypasses the de-differentiation/redifferentiation cycle that is characteristic of the induced pluripotent stem (iPS) and nuclear transfer-embryonic stem (NT-ES) cell technologies that drive current interest in regenerative medicine. Despite the obvious potential of iPS and NT-ES cell-based therapies, there are several problems that must be overcome before these therapies are safe and routine. As an alternative, age reprogramming aims to rejuvenate the specialized functions of an old cell without de-differentiation; age reprogramming does not require developmental reprogramming through an embryonic stage, unlike the iPS and NT-ES cell-based therapies. Tests of age reprogramming have largely focused on one aspect, the epigenome. Epigenetic rejuvenation has been achieved in vitro in the absence of de-differentiation using iPS cell reprogramming factors. Studies on the dynamics of epigenetic age (eAge) reprogramming have demonstrated that the separation of eAge from developmental reprogramming can be explained largely by their different kinetics. Age reprogramming has also been achieved in vivo and shown to increase lifespan in a premature ageing mouse model. We conclude that age and developmental reprogramming can be disentangled and regulated independently in vitro and in vivo.
- Age reprogramming
- Epigenetic rejuvenation
- Somatic cell nuclear transfer (SCNT)
- iPS cells
- Reprogramming factors
- Epigenetic clock
Terminology of reprogramming
Nuclear reprogramming is the process by which a differentiated cell reacquires developmental and ageing potential.
Developmental reprogramming is the process by which a differentiated cell reacquires developmental potential.
Age reprogramming is the process by which a differentiated cell reacquires ageing potential.
Epigenetic rejuvenation represents one aspect of age reprogramming and is the process by which an aged epigenotype is reprogrammed to a young epigenotype.
Both techniques can reverse molecular hallmarks of ageing . For example, telomere attrition can be reversed by induction of iPS cells whereupon telomerase lengthens the telomeres . Telomeres are also extended in nuclei of reconstructed embryos  although the mechanism(s) involved is likely to be more complicated, using both telomerase and telomere sister chromatid exchange . iPS cells also have reduced DNA damage  and enhanced mitochondrial function . Cells differentiated from iPS cells lose expression of markers of senescence and acquire gene expression profiles of young cells . Invariably, the assays used above to demonstrate reversal of hallmarks of ageing rely upon de-differentiated embryonic cells or cells derived from them. From these data, it would seem that rejuvenation requires passage through an embryonic stage. Notably, embryonic cells and their differentiated derivatives are the substrate for regenerative therapies although their use is associated with several well-documented disadvantages . One of the most serious being the development of teratomas when reprogramming factors are expressed in vivo [16, 17]. To overcome these drawbacks, a new approach has been put forward.
The transient epigenetic rejuvenation of HP1β provides evidence that age-related epigenetic changes can be reversed without de-differentiation. Of the age-related changes that have been described, the most well known is DNA methylation and, in particular, the recently discovered “epigenetic clock” that can measure eAge . It is to the epigenetic clock and its associated eAge we now turn as recent work indicates that eAge provides a robust measure for the degree of epigenetic rejuvenation that takes place during age reprogramming.
The relationship of DNA methylation with ageing is long and well documented [22, 23]. It is now on a firm statistical foundation through the development of an “epigenetic clock” based on the level of cytosine methylation at 353 CpG sites in the human genome . The epigenetic clock can be used to predict the eAge of multiple tissues and has a strikingly accurate correlation with chronological age (r = 0.96) and with a median error of 3.6 years [21, 24]. Its accuracy is greater than other biological markers such as telomere length , and all indications are that eAge may be a measure of biological age. In this context, the foundational study of eAge found that the eAge of ES cells and iPS cells is zero . This confirmed that eAge had been reprogrammed—eAge of iPS cells was considerably less than the cells from which they were derived. However, the question of whether reprogramming of eAge is separable from the developmental reprogramming resulting in iPS cells remained open. An answer was provided recently using the rationale that had furnished evidence for epigenetic rejuvenation of HP1β mobility . Olova et al.  undertook an in silico analysis of a previously published 49-day iPS reprogramming time course on HDFs  which revealed that eAge reprogramming is indeed separable from developmental reprogramming. They observed a decrease in eAge after reprogramming factors were introduced into HDFs (eAge ~ 65 years) that began between days 3 and 7 post-introduction. Thereafter, a steady decrease in eAge was measured at 3.8 years per day, reaching zero by day 20 (Fig. 1b). Notably, the decline in eAge began well in advance of the earliest wave of pluripotency gene expression. Fibroblast-specific expression showed a more complex pattern where two of three clusters of fibroblast-specific genes showed an immediate decline that plateaued from day 7 until day 15, by which time there had been a significant drop in eAge. It was on day 35 that fibroblast-specific gene expression was finally extinguished and marked the loss of fibroblast identity. By that time, eAge had been zero for 15 days. It would seem that age reprogramming as measured by eAge is separable from developmental reprogramming as measured by loss of somatic identity (Fig. 1c).
The decrease in eAge of 3.8 years/day is striking in its regularity. The predictable decrease in eAge may provide a mechanism for choosing a preferred eAge by manipulating the timing, duration and levels of expression of iPS reprogramming factors.
Cyclic expression of OSKM in LAKI mice had a dramatic effect. Not only were age-associated features reversed but there was also a significant increase in both median and maximal life span  (Fig. 2). In physiologically aged wild-type mice, cyclic expression enhanced the regenerative capacity of β cells of the pancreas and satellite cells of the muscle after chemical injury (Fig. 2). There was no increase in formation of teratomas or mortality in vivo. “Partial reprogramming” did not lead to the loss of differentiation markers and expression of pluripotency markers such as Nanog, indicating that age reprogramming in vivo can be achieved in the absence of developmental reprogramming.
Age reprogramming has several advantages over current regenerative therapies  including direct reprogramming where trans-differentiation of fibroblasts into another cell type, without passage through an embryonic stage, has been shown not to reprogram hallmarks of ageing . In short, age reprogramming enables the generation of rejuvenated cells for regenerative therapies, without having to go through a de/redifferentiation cycle . Nevertheless, there is some way to go before age reprogramming can be seen as a viable alternative to the iPS and NT-ES cell therapies currently being developed. Work on “interrupted reprogramming” has shown promise in cell replacement therapy in mice, although the rejuvenated status of the engrafted cells was not determined . A study that addressed this issue using mesenchymal stromal cells (MSCs) concluded that interrupted reprogramming does not rejuvenate MSCs, albeit the indicated caveats for this study included, inter alia, uncontrolled heterogeneous expression of reprogramming factors from episomal vectors . Age reprogramming in vivo will, most probably, be driven by development of efficient methods for delivering reprogramming factors to sites of injury or disease. Small molecules that can substitute for the classical reprogramming gene products  will be in the vanguard of in vivo studies due to the ease of crossing cell membranes. Tailoring the chemical nature, timing and quantity of reprogramming factors could also have the added advantage of avoiding the possibility of developing teratomas that can arise from unfettered expression of classical iPS reprogramming factors in vivo [16, 17]. All these are goals for the future. At the pace we are now advancing it should not be long before there will be signs that they can be achieved.
PBS wrote the first draft. AGN drew the figures. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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This work was supported by a grant from the Ministry of Education and Science of Russian Federation #14.Y26.31.0024; PBS was supported by Nazarbayev University Grant 090118FD5311. AGN was supported by Russian Science Foundation (RSF) Grant 15-14-10021.
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- Briggs R, King TJ. Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. PNAS. 1952;38:455–63.View ArticleGoogle Scholar
- Gurdon JB. Adult frogs derived from the nuclei of single somatic cells. Dev Biol. 1962;4:256–73.View ArticleGoogle Scholar
- Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997;385:810–3.View ArticleGoogle Scholar
- Mizutani E, Ono T, Li C, Maki-Suetsugu R, Wakayama T. Propagation of senescent mice using nuclear transfer embryonic stem cell lines. Genesis. 2008;46:478–83.View ArticleGoogle Scholar
- Wakayama S, Mizutani E, Wakayama T. Production of cloned mice from somatic cells, ES cells, and frozen bodies. In: Wassarman PM, Soriano PM, editors. Methods in enzymology. Cambridge: Academic Press; 2010. p. 151–69. https://doi.org/10.1016/s0076-6879(10)76009-2.View ArticleGoogle Scholar
- Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 2008;321:1218–21.View ArticleGoogle Scholar
- Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–76.View ArticleGoogle Scholar
- Lapasset L, Milhavet O, Prieur A, Besnard E, Babled A, Aït-Hamou N, et al. Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. Genes Dev. 2011;25:2248–53.View ArticleGoogle Scholar
- López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013;153:1194–217.View ArticleGoogle Scholar
- Marión RM, Strati K, Li H, Tejera A, Schoeftner S, Ortega S, et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell. 2009;4:141–54.View ArticleGoogle Scholar
- Schaetzlein S, Lucas-Hahn A, Lemme E, Kues WA, Dorsch M, Manns MP, et al. Telomere length is reset during early mammalian embryogenesis. PNAS. 2004;101:8034–8.View ArticleGoogle Scholar
- Liu L, Bailey SM, Okuka M, Muñoz P, Li C, Zhou L, et al. Telomere lengthening early in development. Nat Cell Biol. 2007;9:1436–41.View ArticleGoogle Scholar
- Marión RM, Strati K, Li H, Murga M, Blanco R, Ortega S, et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature. 2009;460:1149–53.View ArticleGoogle Scholar
- Suhr ST, Chang EA, Tjong J, Alcasid N, Perkins GA, Goissis MD, et al. Mitochondrial rejuvenation after induced pluripotency. PLoS ONE. 2010;5:e14095.View ArticleGoogle Scholar
- Herberts CA, Kwa MSG, Hermsen HPH. Risk factors in the development of stem cell therapy. J Transl Med. 2011;9:29.View ArticleGoogle Scholar
- Abad M, Mosteiro L, Pantoja C, Cañamero M, Rayon T, Ors I, et al. Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature. 2013;502:340–5.View ArticleGoogle Scholar
- Ohnishi K, Semi K, Yamamoto T, Shimizu M, Tanaka A, Mitsunaga K, et al. Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation. Cell. 2014;156:663–77.View ArticleGoogle Scholar
- Manukyan M, Singh PB. Epigenetic rejuvenation. Genes Cells. 2012;17:337–43.View ArticleGoogle Scholar
- Singh PB, Zacouto F. Nuclear reprogramming and epigenetic rejuvenation. J Biosci. 2010;35:315–9.View ArticleGoogle Scholar
- Manukyan M, Singh PB. Epigenome rejuvenation: HP1β mobility as a measure of pluripotent and senescent chromatin ground states. Sci Rep. 2014;4:4789.View ArticleGoogle Scholar
- Horvath S. DNA methylation age of human tissues and cell types. Genome Biol. 2013;14:R115.View ArticleGoogle Scholar
- Jung M, Pfeifer GP. Aging and DNA methylation. BMC Biol. 2015;13:7.View ArticleGoogle Scholar
- Vanyushin BF, Nemirovsky LE, Klimenko VV, Vasiliev VK, Belozersky AN. The 5-methylcytosine in DNA of rats. GER. 1973;19:138–52.Google Scholar
- Hannum G, Guinney J, Zhao L, Zhang L, Hughes G, Sadda S, et al. Genome-wide methylation profiles reveal quantitative views of human aging rates. Mol Cell. 2013;49:359–67.View ArticleGoogle Scholar
- Gibbs WW. The clock-watcher. Nature. 2014;508:168–70.View ArticleGoogle Scholar
- Olova N, Simpson DJ, Marioni R, Chandra T. Partial reprogramming induces a steady decline in epigenetic age before loss of somatic identity. Aging Cell. 2018. https://doi.org/10.1111/acel.12877 View ArticlePubMedGoogle Scholar
- Ohnuki M, Tanabe K, Sutou K, Teramoto I, Sawamura Y, Narita M, et al. Dynamic regulation of human endogenous retroviruses mediates factor-induced reprogramming and differentiation potential. PNAS. 2014;111:12426–31.View ArticleGoogle Scholar
- Ocampo A, Reddy P, Martinez-Redondo P, Platero-Luengo A, Hatanaka F, Hishida T, et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell. 2016;167(1719–1733):e12.Google Scholar
- Scaffidi P, Misteli T. Reversal of the cellular phenotype in the premature aging disease Hutchinson–Gilford progeria syndrome. Nat Med. 2005;11:440–5.View ArticleGoogle Scholar
- Gurdon JB, Melton DA. Nuclear reprogramming in cells. Science. 2008;322:1811–5.View ArticleGoogle Scholar
- Yamanaka S, Blau HM. Nuclear reprogramming to a pluripotent state by three approaches. Nature. 2010;465:704–12.View ArticleGoogle Scholar
- Tang Y, Liu M-L, Zang T, Zhang C-L. Direct reprogramming rather than iPSC-based reprogramming maintains aging hallmarks in human motor neurons. Front Mol Neurosci. 2017;10:359.View ArticleGoogle Scholar
- Guo L, Karoubi G, Duchesneau P, Shutova MV, Sung H-K, Tonge P, et al. Generation of induced progenitor-like cells from mature epithelial cells using interrupted reprogramming. Stem Cell Rep. 2017;9:1780–95.View ArticleGoogle Scholar
- Goebel C, Goetzke R, Eggermann T, Wagner W. Interrupted reprogramming into induced pluripotent stem cells does not rejuvenate human mesenchymal stromal cells. Sci Rep. 2018;8:11676.View ArticleGoogle Scholar
- Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013;341:651–4.View ArticleGoogle Scholar