A Review of the Present State of Epigenetic Reprogramming to Treat Aging
The Yamanaka transcription factors can be used to recreate the transformation of cell type that occurs in early embryonic development, inducing a process of reprogramming that can transform any somatic cell into an induced pluripotent stem cell. Initially, this discovery was applied to the development of cell therapies and tissue engineering, a way to produce cells of a specific type matched to the recipient, or to generate cell banks able to reliably supply cells of specific types, or to chase the grail of universal cell lines that can be used in any patient. After nearly twenty years of development, some of the first therapies to transplant cells derived from induced pluripotent stem cells have reached clinical trials - progress in the highly regulated field of medicine is slow at best.
Separately, researchers have discovered that reprogramming doesn't just change cell type, it also rejuvenates a cell by restoring youthful epigenetic control over gene expression. That in turn restores youthful mitochondrial function and numerous other aspects of cell behavior and performance. It cannot repair DNA damage, and cannot enable cells to break down molecular waste that even youthful cells struggle to handle. Nonetheless, there is a great deal of interest in finding ways to use this phenomenon as a basis for therapy. What is known as partial reprogramming involves exposing cells to the Yamanaka factors for long enough to produce this desirable outcome of epigenetic rejuvenation, but not long enough to turn cells into induced pluripotent stem cells. Cells retain their state, with improved function.
Today's open access review provides a good introduction to the science behind the promise and the challenges of partial reprogramming as a basis for therapy. Positive results have been produced in animal studies, but sizable hurdles are involved in trying to reprogram large portions of the body rather than employing a very narrow, restricted use in isolated tissues such as the retina. Different cell types in different tissues have different requirements and restrictions for partial reprogramming. What is good for lung cells is bad for liver cells. What is good for one type of cell in the liver is bad for its neighbor. "Bad" in this context means cell death, tissue dysfunction, and cancer. There is no good solution at this time that would lead to a simple partial reprogramming therapy that affects the whole body without either (a) watering it down to produce negligible benefits, or (b) causing severe issues in some tissues.
Organ-Specific Dedifferentiation and Epigenetic Remodeling in In Vivo Reprogramming
The advent of in vivo reprogramming through transient expression of the Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC, abbreviated OSKM) holds strong promise for regenerative medicine, despite ongoing concerns about safety and clinical applicability. This review synthesizes recent advances in in vivo reprogramming, focusing on its potential to restore regenerative competence and promote rejuvenation across diverse tissues, including the retina, skeletal muscle, heart, liver, brain, and intestine.
In physiologically aged mice, long-term cyclic induction of OSKM restores youthful multi-omics signatures - including DNA methylation, transcriptomic, and lipidomic profiles - across multiple organs such as the spleen, liver, skin, kidney, lung, and skeletal muscle. Importantly, this regimen also promotes functional regeneration: while short-term reprogramming enhances muscle repair through local niche control, sustained cyclic reprogramming improves wound healing and reduces fibrosis in both muscle and skin. Consistent with these findings, even a single 1-week cycle of OSKM in aged mice (55 weeks old) elicits systemic rejuvenation, evidenced by DNA methylation reprogramming across the pancreas, liver, spleen, and blood.
Nevertheless, significant challenges to its application remain, including tumor formation, intestinal and liver failure, and loss of cellular identity. Achieving precise spatiotemporal control over reprogramming will be essential to minimize these risks while preserving therapeutic benefits. Future efforts should prioritize refining delivery methods and exploring safer alternatives such as small molecules or modified gene sets.
Interest in this field is rapidly growing within the biotech sector, summarized in recent reviews which provide detailed accounts of company pipelines and translational strategies. In this review, we instead focus on mechanistic insights into injury-induced and OSKM-induced reprogramming, offering a framework for understanding how regenerative competence can be harnessed across tissues. With careful modulation, OSKM-based approaches hold strong potential to transform regenerative medicine and the treatment of age-related diseases.
"It cannot repair DNA damage"
And yet every 35 years our cells become "new" again. And those cells (although they are germ cells and so have fewer mutations) pass along their mutations to the next generation.
So.. do cells accumulate dna damage at an increasing rate as they age?
Mesenchymal stem cell technology is further along than IPSC technology. Mesenchymal stem cells are now licensed by the FDA, since 12/24.
I see no reason why partial reprogramming should not also be achieved using MSC's as an anti ageing approach. It is a lot more available.
A segment from an article titled "Scientists Achieve Cellular Age Reversal in Mice, but Fleeting Youth Comes at a Heavy Cost": "A deeper question is whether the epigenetic changes reversed by Izpisúa Belmonte in his lab are the true cause of aging or merely a sign of aging-much like wrinkles on aging skin. If it is the latter, then Izpisúa Belmonte's treatment might be purely cosmetic, akin to removing wrinkles. There is no way to know, and no evidence that DNA methylation causes cellular aging. Furthermore, a fundamental issue looms over Izpisúa Belmonte's research findings: although he successfully rejuvenated mice with progeria, he has not yet achieved this in normal aged animals."
The best measurement for age reversal and if an aging model is correct or wrong is lifespan control experiment on WILDTYPE mice, not progeria model mice or the remaining lifespan of middle-aged and elderly mice.
In the process of cellular aging, the global DNA methylation level gradually decreases, thus it can be used as a quantitative clock to measure the degree of cellular or individual aging. Moreover, many researchers believe that the root cause of cellular aging may be epigenetic. However, there is a clear boundary or threshold in cell reprogramming induced by Yamanaka factors. Prolonged induction causes cells to undergo dedifferentiation, turning into iPS cells with significantly elongated telomeres and methylation age reset to zero. Short-term induction does not lead to dedifferentiation; cell identity remains unchanged, telomeres do not elongate, and upon cessation of induction, the methylation age quickly reverts to its original state.
A correct theory does not allow for contradictory evidence or loopholes, whereas the epigenetic theory of aging has at least ten loopholes, indicating that the theory is essentially incorrect. Therefore, I believe that the fundamental cause of cellular aging does not lie in epigenetics, based on the following:
1. Epigenetics is reversible and unstable. Therefore, the methylation clock, or epigenetic clock, is merely an artificially assigned clock rather than a true clock, because methylation is unstable and lacks timing properties, unable to serve as a stable "reference clock" substance. Thus, the epigenetic clock is calibrated indirectly by more stable "reference clocks" such as telomeres and rDNA through mediators like P53. For example, activating telomerase can reverse the epigenetic clock. But why does the methylation clock more accurately reflect chronological age than the telomere clock? I believe this is because the methylation clock is indirectly calibrated through P53, and the correlation between telomere length and P53 levels is influenced by various factors, such as rDNA copy number and factors affecting P53 degradation. Therefore, telomeres of the same length may correspond to different P53 levels in different tissue cells or individuals.
2. Some organisms, such as *Tribolium castaneum* and *Caenorhabditis elegans*, do not have methylated DNA.
3. Experiments show that during reprogramming, the rejuvenation of cellular physiological state occurs before the rejuvenation of epigenetic characteristics [Zhang, W., Qu, J., Liu, GH. et al. The ageing epigenome and its rejuvenation[J]. Nat Rev Mol Cell Biol ,2020,21:137-150.], indicating that cellular rejuvenation during reprogramming is unrelated to epigenetic reset.
4. iPS cells are reprogrammed cells with fully demethylated DNA. Telomere length can extend from 30 kb before reprogramming to 70 kb, and after several divisions, continue to lengthen to 110 kb, similar to ES cell telomere length. Telomerase-knockout G1 mouse cells can be induced to generate iPS cells, which still have 30 kb telomeres. These iPS cells cannot generate chimeric mice when implanted into blastocysts [Rosa M. Marion, Maria A. Blasco, Han Li, et al. Telomeres Acquire Embryonic Stem Cell Characteristics in Induced Pluripotent Stem Cells[J]. Cell Stem Cell, 2009, 4: 141-154.]. This is because the essence of individual development is a genetic program involving the sequential expression of different genes. However, driving this genetic program requires a timing driver, which in turn needs a clock.
5. Direct reprogramming can also alter epigenetics, but directly reprogramming aged fibroblasts into neural stem cells retains the senescent phenotype (Merten et al., 2015).
6. Although cell experiments show that short-term expression of Yamanaka factors can reverse multiple aging markers, upon cessation of Yamanaka factor expression, these aging symptoms quickly begin to accumulate again, including the methylation age reverting to its original state. This indicates that only cell cycle genes are activated, promoting tissue regeneration, without elongating telomeres and rDNA, because elongating telomeres and rDNA would produce intrinsic and extrinsic lasting rejuvenation. But why can multiple cycles of Yamanaka factor expression extend the lifespan of progeria mice by 30%? This may be related to Yamanaka factors promoting tissue repair and regeneration, similar to how upregulating vascular endothelial growth factor (VEGF) secretion in transgenic mouse livers to promote vascular regeneration can extend mouse lifespan by 48%.
Why can short-term expression of Yamanaka factors reverse multiple aging markers and promote tissue regeneration? And why do these aging symptoms quickly accumulate again after stopping Yamanaka factor expression?
Firstly, P53 is the master control factor of aging, as continuous inhibition of P53 with antibodies allows fibroblasts to proliferate indefinitely. It is known that inhibiting P53 can also increase the efficiency of somatic cell conversion to iPS cells by Yamanaka factors. For example, reducing the expression levels of p53 or its target gene p21 can improve iPS cell induction efficiency. Lowering p53 protein levels allows high-quality iPS cells to be obtained using only Oct4 and Sox2 [Kawamura T, Suzuki J, Wang YV, et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 2009; 460(7259): 1140-1144.]. One mechanism by which vitamin C improves iPS cell induction efficiency is by reducing the expression of p53 and p21 [Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 2010; 6(1): 71-79.]. Yamanaka factors are also highly expressed in many tumor cells, where the P53 protein is either mutated or degraded too quickly, indicating that Yamanaka factors rejuvenate cells by reducing P53.
That is to say, short-term reprogramming uses Yamanaka factors to temporarily reduce P53, resulting in temporary cellular rejuvenation. Once Yamanaka factor expression stops, P53 levels return to pre-induction levels, so these aging symptoms quickly accumulate again, making it impossible to maintain the cells in a persistently young state.
7. Reversing epigenetic age indicators is merely reversing the appearance of aging, or even accelerating aging, because epigenetic age can only represent metabolic level. For example, growth hormone can increase metabolic levels and reverse epigenetic age but actually accelerates aging.
8. The lifespan extension in heterochronic parabiosis mice is less than the reduction in methylation age [Bohan Zhang, James P. White, et al. (2021) Multi-omic rejuvenation and lifespan extension upon exposure to youthful circulation]. This suggests that methylation age can only represent metabolic age, not chronological age, because the underlying mechanism of aging is the reduction of clock substances like telomeres and rDNA. This is also why I oppose the idea that modifying epigenetics, such as DNA methylation or histone acetylation, can extend lifespan. During aging, histone acetylation levels decrease. In theory, inhibiting histone acetyltransferase expression should shorten lifespan, but it actually shows lifespan extension. Growth hormone can make DNA methylation age younger; in theory, increasing growth hormone should extend lifespan, but it actually shortens it.
9. Growth hormone can reverse the DNA methylation clock [Fahy GM, Brooke RT, James P. Watson JP, et al. Reversal of epigenetic aging and immunosenescent trends in humans[J]. Aging Cell, 2019, 18(6): e13028.], but growth hormone shortens mouse lifespan, whereas telomerase elongating telomeres increases the number of cell divisions. This indicates that the clock executing the genetic program of development and aging is not the DNA methylation clock but the telomere DNA clock.
10. During the process of somatic cell induction into iPS cells, DNA undergoes large-scale demethylation, but this is not rejuvenation; rather, it erases the identity of the cell differentiation type, because cancer cells remain very youthful without identity erasure.
11. The titled "Cell Heavyweight Review | The Twelve Hallmarks of Aging (30,000-Word Comprehensive Interpretation)" states that continuous supplementation of alpha-ketoglutarate (AKG) for 7 months also reversed the epigenetic clock by 8 years [Demidenko O, Barardo D, Budovskii V, et al. Rejuvant®, a potential life-extending compound formulation with alpha-ketoglutarate and vitamins, conferred an average 8 year reduction in biological aging, after an average of 7 months of use, in the TruAge DNA methylation test. Aging (Albany NY). 2021 Nov 30;13(22):24485-24499. doi: 10.18632/aging.203736.]. However, AKG does not extend the lifespan of male mice [ASADI SHAHMIRZADI A, EDGAR D, LIAO C Y, et al. Alpha-Ketoglutarate, an Endogenous Metabolite, Extends Lifespan and Compresses Morbidity in Aging Mice [J]. Cell Metab, 2020, 32(3): 447-566.], and only extends the average lifespan of middle-aged female mice by 12%. This indicates that the epigenetic clock can only measure the degree of aging, not the cause of aging.
"Reversing Aging May Soon Be Possible! Chinese Scientists Decipher Part of the Aging Code" - Yet, they haven't truly deciphered the aging code. The KAT7 gene encodes a histone acetyltransferase. Histone acetylation facilitates gene transcription and promotes protein synthesis. Inhibiting KAT7 reduces histone acetylation levels, thereby downregulating gene transcription and protein synthesis. Since the protein synthesis process requires the synthesis of rRNA, which constitutes 82% of total RNA, in essence, the mechanism by which inhibiting KAT7 extends lifespan is equivalent to caloric restriction.
rDNA exists as very fragile tandemly repeated DNA. The transcription process of rDNA is highly susceptible to damage, leading to copy number loss. A reduction in rDNA copies triggers an increase in P53 levels, consequently pushing cells into a senescent state. In other words, the fundamental reason caloric restriction extends lifespan is by reducing the attrition of telomeres and rDNA [Telomere DNA and Ribosomal DNA Co-regulatory Hypothesis of Cellular Senescence].
Regarding the reason for the modest lifespan extension in heterochronic parabiosis mice, discussions back in 2016 suggested it stemmed from young stem cells in the young mouse's blood, such as hematopoietic stem cells and endothelial progenitor cells, engrafting into the bone marrow of the old mouse. This would then lead to the rejuvenation of blood vessels and blood cells in the old mouse.
On May 24, 2022, research groups led by Liu Guanghui and Qu Jing from the Institute of Zoology, Chinese Academy of Sciences (CAS), in collaboration with Zhang Weiqi's group from the Beijing Institute of Genomics, CAS, published a research paper online in Cell Stem Cell titled: "Heterochronic parabiosis induces stem cell revitalization and systemic rejuvenation across aged tissues" [https://cell.com/cell-stem-cell/fulltext/S1934-5909(22)00170-9].
This study found that in the heterochronic parabiosis system, less than 1% of hematopoietic stem/progenitor cells (HSPCs) in the young individual's bone marrow originated from the old individual. Similarly, in the old individual, young-derived HSPCs accounted for less than 5%.