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The discovery of animal cloning and subsequent development of cell reprogramming technology were quantum leaps as they led to the achievement of rejuvenation by cell reprogramming and the emerging view that aging is a reversible epigenetic process. Here, we will first summarize the experimental achievements over the last 7 years in cell and animal rejuvenation. Then, a comparison will be made between the principles of the cumulative DNA damage theory of aging and the basic facts underlying the epigenetic model of aging, including Horvath’s epigenetic clock. The third part will apply both models to two natural processes, namely, the setting of the aging clock in the mammalian zygote and the changes in the aging clock along successive generations in mammals. The first study demonstrating that skin fibroblasts from healthy centenarians can be rejuvenated by cell reprogramming was published in 2011 and will be discussed in some detail. Other cell rejuvenation studies in old humans and rodents published afterwards will be very briefly mentioned. The only in vivo study reporting that a number of organs of old progeric mice can be rejuvenated by cyclic partial reprogramming will also be described in some detail. The cumulative DNA damage theory of aging postulates that as an animal ages, toxic reactive oxygen species generated as byproducts of the mitochondria during respiration induce a random and progressive damage in genes thus leading cells to a progressive functional decline. The epigenetic model of aging postulates that there are epigenetic marks of aging that increase with age, leading to a progressive derepression of DNA which in turn causes deregulated expression of genes that disrupt cell function. The cumulative DNA damage model of aging fails to explain the resetting of the aging clock at the time of conception as well as the continued vitality of species as millenia go by. In contrast, the epigenetic model of aging straightforwardly explains both biologic phenomena. A plausible initial application of rejuvenation in vivo would be preventing adult individuals from aging thus eliminating a major risk factor for end of life pathologies. Further, it may allow the gradual achievement of whole body rejuvenation.
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R E V I E W Open Access
Rejuvenation by cell reprogramming: a new
horizon in gerontology
Rodolfo G. Goya
1*
, Marianne Lehmann
1
, Priscila Chiavellini
1
, Martina Canatelli-Mallat
1
, Claudia B. Hereñú
2
and
Oscar A. Brown
1
Abstract
The discovery of animal cloning and subsequent development of cell reprogramming technology were quantum
leaps as they led to the achievement of rejuvenation by cell reprogramming and the emerging view that aging is a
reversible epigenetic process. Here, we will first summarize the experimental achievements over the last 7 years in
cell and animal rejuvenation. Then, a comparison will be made between the principles of the cumulative DNA
damage theory of aging and the basic facts underlying the epigenetic model of aging, including Horvaths
epigenetic clock. The third part will apply both models to two natural processes, namely, the setting of the aging
clock in the mammalian zygote and the changes in the aging clock along successive generations in mammals. The
first study demonstrating that skin fibroblasts from healthy centenarians can be rejuvenated by cell reprogramming
was published in 2011 and will be discussed in some detail. Other cell rejuvenation studies in old humans and
rodents published afterwards will be very briefly mentioned. The only in vivo study reporting that a number of
organs of old progeric mice can be rejuvenated by cyclic partial reprogramming will also be described in some
detail. The cumulative DNA damage theory of aging postulates that as an animal ages, toxic reactive oxygen
species generated as byproducts of the mitochondria during respiration induce a random and progressive damage
in genes thus leading cells to a progressive functional decline. The epigenetic model of aging postulates that there
are epigenetic marks of aging that increase with age, leading to a progressive derepression of DNA which in turn
causes deregulated expression of genes that disrupt cell function. The cumulative DNA damage model of aging
fails to explain the resetting of the aging clock at the time of conception as well as the continued vitality of species
as millenia go by. In contrast, the epigenetic model of aging straightforwardly explains both biologic phenomena.
A plausible initial application of rejuvenation in vivo would be preventing adult individuals from aging thus
eliminating a major risk factor for end of life pathologies. Further, it may allow the gradual achievement of whole
body rejuvenation.
Keywords: Aging, Epigenetics, Rejuvenation, Cell reprogramming, Therapeutic potential
Rejuvenation: a perennial dream
The longing of man for eternal youth is universal and
time immemorial. Initially sought in religions, during
the Middle Ages, alchemists, a blend of mystics and
proto-chemists, tried to synthesize a mysterious potion,
the elixir of eternal youth, able to confer indefinite youth
to those that dare to drink it. Now, it seems that science
has found the biological fountain of rejuvenationthe
cytoplasm of the oocyte.
The story of biological rejuvenation began in the early
1960s, with the discovery of animal cloning in frogs by
John Gurdon and collaborators [1]. Mammalian cloning
was achieved 30 years later, in 1996, with the birth of
Dolly, the sheep [2]. Cloning of other mammalian spe-
cies followed soon. It was clear that the cytoplasm of a
mature oocyte contained molecules able to turn a som-
atic nucleus into an embryonic one that could direct the
development of a new individual. At the time, it was
assumed that in the oocytes cytoplasm, there should be
a complex constellation of reprogramming factors,
* Correspondence: goya@isis.unlp.edu.ar
Rodolfo G. Goya and Marianne Lehmann contributed equally to this work.
1
Institute for Biochemical Research (INIBIOLP) - Histology B & Pathology B,
School of Medicine, National University of La Plata, CC 455, 1900 La Plata,
Argentina
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Goya et al. Stem Cell Research & Therapy (2018) 9:349
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necessary to reprogram a somatic nucleus. However, 10
years later, Takahashi and Yamanaka [3] demonstrated
that the transfer of only four master genes, namely oct4,
sox2,klf4, and c-myc (OSKM genes), to adult mouse fi-
broblasts was able to reprogram them, taking the cells to
a pluripotency stage in which they behave like embry-
onic stem cells. Cell reprogramming had been born, an
advance that paved the way for the subsequent imple-
mentation of cell rejuvenation. The studies on rejuven-
ation so far published are summarized below.
The achievement of cell and animal rejuvenation
To our knowledge, the first study reporting cell rejuven-
ation was published in 2011 [4]. It was a seminal piece
of work that merits to be described in some detail.
It was known that cells from old individuals display a
typical transcriptional signature, different from that of
young counterparts [5]. It was also known that fibro-
blasts from old donors have shortened telomeres [6]as
well as dysfunctional mitochondria and higher levels of
oxidative stress [7]. The French group first explored the
effect of cell reprogramming on the above features. In
order to efficiently reprogram fibroblasts from healthy
centenarians and very old donors, the authors added the
pluripotency genes NANOG and LIN28 to the OSKM
reprogramming cocktail. This six-factor combination ef-
ficiently reprogrammed fibroblasts from very old donors
into typical induced pluripotent stem cells (iPSCs)
(Fig. 1). These blastocyst-like cells showed a higher
population-doubling (PD) potential than the cells of ori-
gin as well as elongated telomeres and a youthful
mitochondrial metabolism (estimated by measuring
mitochondrial transmembrane potential and clustering
transcriptome subsets involved in mitochondrial metab-
olism). Using an appropriate differentiation cocktail, the
iPSCs were differentiated back to fibroblasts, whose
transcriptional profile, mitochondrial metabolism, oxida-
tive stress levels, telomere length, and PD potential were
indistinguishable from those of fibroblasts from young
counterparts. Taken together, the data revealed that the
cells had been rejuvenated.
After Lapasset et al.s paper, a core of independent stud-
ies followed which confirmed the initial findings. Thus, re-
programming of skin fibroblasts from aged humans to
Fig. 1 Rejuvenation by cell reprogramming of fibroblasts from healthy centenarian individuals. In culture, fibroblasts from old individuals display a
typical transcriptional signature, different from that of young counterparts as well as shortened telomeres, reduced population-doubling (PD)
potential, dysfunctional mitochondria, and higher levels of oxidative stress. When cells were reprogrammed to iPSC with a six-factor cocktail, the
above alterations were reversed to embryonic levels. Then, iPSCs were differentiated back to fibroblasts by culture in the presence of an
appropriate set of differentiation factors. In the resulting cells, all of the above variables had levels typical of fibroblasts taken from young
individuals, see [4] for further details
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iPSC, followed by differentiation to induced neurons
(iNs), was shown to rejuvenate their transcriptome profile
and nucleocytoplasmic compartmentalization (NCC) to
that of wild-type fibroblasts from young donors [5]. When
iNs were generated by transdifferentiation, a procedure
that bypasses the pluripotency stage, the resulting neurons
retained the transcriptome signature of old fibroblasts and
showed a disrupted NCC as old fibroblasts do, which led
to the conclusion that transient dedifferentiation to the
iPSC stage is necessary to rejuvenate cells [5]. It is now
known that induced neurons generated by conventional
reprogramming and pluripotency factor-mediated direct
reprogramming (PDR) are rejuvenated whereas induced
neurons generated by transdifferentiation (also known as
lineage reprogramming (LR)) are not [8].
In an interesting study with skin fibroblasts from
healthy aged volunteers, it was observed that the lower
oxygen consumption typically observed in the mitochon-
dria of cells from old individuals was restored to youth-
ful levels after the old cells were dedifferentiated to
iPSCs and subsequently differentiated back to rejuve-
nated fibroblasts [9].
In 2013, Nishimura et al. [10] reprogrammed clonally
expanded antigen-specific CD8+ T cells from an
HIV-1-infected patient to pluripotency. The T
cell-derived iPSCs were then redifferentiated into CD8+
T cells that had a high proliferative capacity and elon-
gated telomeres.
Recently, rejuvenation of dysfunctional hematopoietic
stem cells (HSCs) from old mice was achieved by repro-
gramming them to the iPSC stage followed by differenti-
ating them back to HSCs. It was found that the
rejuvenated HSCs from old mice had the same func-
tional performance concerning the production of
different immune and erythroid cell lineages (peripheral
B-, T-, and granulocyte/myeloid cells, as well as bone
marrow erythroid progenitors) than HSCs from normal
young mice [11].
Another study reported that overexpression of the
pluripotency factor NANOG in progeroid or senescent
myogenic progenitors reversed cellular aging and fully
restored their ability to generate contractile force. The
effect was mediated by the reactivation of the ROCK
and TGF-βpathways [12]. For a more detailed descrip-
tion of references [511], see [13].
Until late 2016, it was believed that although cells
taken from old individuals could be fully rejuvenated, re-
juvenation in vivo was not possible as a continuous ex-
pression of the Yamanaka genes in animals had been
shown to cause multiple teratomas [14,15]. However, in
December 2016, it was reported for the first time that
cells and organs can be rejuvenated in vivo [16]. Since it
was a quantum leap in rejuvenation technology, the
study will be described in some detail. The authors used
transgenic progeric mice (LAKI mice) harboring a mu-
tated form of the human gene Lmna which causes the
accumulation of a truncated form of the nuclear mem-
brane protein Lamin A (progerin) present in progeric
patients. They also used transgenic wild-type mice (4F
mice) harboring two constructs. On chromosome 11,
there was a construct that expresses the OSKM genes
which are driven by a tetracycline-responsive (TRE) pro-
moter, and on chromosome 6, an expression cassette
was cloned which expresses the rtTA regulatory protein.
When the rtTA protein binds the antibiotic doxycycline
(DOX), a conformational allosteric change takes place so
that rtTA gains affinity for the TRE promoter turning on
the Yamanaka gene tandem [16]; (Fig. 2ac). After re-
peated backcrossings, progeric transgenic mice express-
ing the 4F system (LAKI-4F mice) were generated [16].
Since the LAKI-4F mice age prematurely, by 2 months
of age, they are already senile. Thus, at age 2 months,
LAKI-4F mice began to be submitted to a rejuvenation
strategy based on the addition of DOX to the drinking
water of experimental mice in order to turn on the
Yamanaka genes; 2 days later, the antibiotic was removed
from the drinking water so that the transgenes were si-
lenced. Mice rested for 5 days after which DOX was
added again for 2 days then removed for 5 days and so
on, thus implementing a cyclic partial reprogramming
strategy (Fig. 2df). After 6 weeks of partial reprogram-
ming cycles, the experimenters could observe some im-
provements in the external appearance of experimental
mice, including a reduction in spine curvature as com-
pared with untreated counterparts (controls). A sub-
group of the experimental and control mice was
sacrificed and some of their tissues and organs analyzed
(skin, kidneys, stomach, and spleen). Controls showed a
variety of alterations at an anatomical and histological
level in the above organs whereas some of these aging
signs disappeared or were attenuated in the experimental
mice. Some aging signs remained unchanged by the
treatment. Furthermore, although the experimental ani-
mals kept aging, they showed a 50% increase in mean
survival time as compared with wild-type progeric con-
trols. If the treatment was interrupted, the aging signs
came back.
An important implication of the study by Ocampo
et al. is that cyclic partial reprogramming causes some
epigenetic aging marks to be erased, but spares the dif-
ferentiation marks, which in turn suggest that both types
of epigenetic mark are not necessarily the same.
Theories of aging: cumulative DNA damage
versus epigenetic theories
Cumulative DNA damage
For several decades now, the theory of cumulative DNA
damage has remained widely accepted by mainstream
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gerontologists. Basically, it proposes that aging is the
consequence of the progressive accumulation of oxida-
tive damage to cell macromolecules, particularly DNA
[17,18]. This phenomenon is thought to be especially
relevant in mitochondria where respiration is associated
with a continuous generation of reactive oxygen species
as byproducts of O
2
reduction (Fig. 3a) [19,20]. Accord-
ing to this model, the process is essentially irreversible
and can only be slowed down by appropriate interven-
tions like calorie restriction [21]. Indeed, the idea that a
progressive age-related deterioration of such an import-
ant molecule as DNA should play a central role in the
aging process sounds sensible. However, experimental
data from different sources do not support this model.
For instance, the naked mole rat, a small rodent whose
average lifespan (28.3 years) is nearly eight times longer
than it is in laboratory rodents, has been shown to pos-
sess high levels of oxidative damage in its cells even
when animals are young [22,23]. It is likely that under
extreme environmental conditions such as a highly ion-
izing atmosphere, radioactive zones or high UV light
intensities, DNA damage may become a longevity-
determining factor. This will also be the case under nor-
mal environmental conditions in pathologies associated
with DNA repair defects, like Xeroderma pigmentosum.
However, wherever normal marine or terrestrial earth
conditions prevail, cumulative DNA damage does not
seem to play a relevant role in aging.
Epigenetic model
The growing evidence that reprogramming of somatic
cells from aged individuals rejuvenates them to their
embryonic stage is giving rise to the idea that the epige-
nome is the central driver of aging, at least in meta-
zoans, and that the process is reversible [24].
Studies in model organisms like yeasts, worms, and
flies have shown that aging is associated with progressive
changes in chromatin regulation. In young cells, the gen-
ome is in a relatively high level of repression which is in
part achieved by DNA methylation, and there are rela-
tively high levels of histone H3 trimethylated at lysine 9
(H3K9me3) and at lysine 27 (H3K27me3) and histone
Fig. 2 Rejuvenation of transgenic progeric mice by cyclic partial cell reprogramming. A polycistronic cassette (4F system) harboring the four
Yamanaka genes under the control of a tetracycline-regulatable (Tet On) promoter (a) was transferred to one-cell embryos of C57bl/6 wild-type
mice in order to generate transgenic mice harboring the 4F system (4F mice) that were subsequently backcrossed with transgenic progeric mice
(LAKI mice). This way, progeric LAKI-4F mice were generated. The antibiotic doxycycline (DOX) binds to the regulatory rtTA protein which then
gains affinity for the Tet On promoter and binds to it turning on the Yamanaka genes (b). When DOX is removed from the medium, the rtTA
protein dissociates from the promoter and the transgenes become silent again (c). When DOX was added to the drinking water of 2-month-old
progeric mice, it turned on the Yamanaka genes and partial cell reprogramming began (d). Two days later, DOX was removed and the Yamanaka
genes silenced (e). After a 5-day resting period, DOX was added again for 2 days (f), then removed for 5 days and so on. This cyclic partial
reprogramming process rejuvenated some tissues and organs of the mice which survived 50% longer than the original progeric mice, see [16] for
further details
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H4 trimethylated at lysine 20 (H4K20me3), all of
which are associated with transcriptionally repressed
chromatin, as well as relatively low levels of histone
H3 trimethylated at lysine 4 (H3K4me3) and histone
H4 acetylated at lysine 16 (H4K16ac), both of which
are associated with active chromatin [25]. Aging
seems to be associated with a progressive derepres-
sion of the transcriptional activity of chromatin,
which is effected in part by a reduction in DNA
methylation; a decrease of epigenetic repressor marks
like H3K9me3, H3K27me3, and H4K20me3 as well as
an increase in the levels of activation marks like
H3K4me3 and H4K16ac (Fig. 3b). Cell reprogram-
ming technology shows that this process is reversible
(Fig. 3b). The epigenetic model of aging provides an
elegant explanation for a number of age-related
processes difficult to explain by conventional theories
of aging (see below).
The epigenetic clock theory
An important advance relevant to the epigenetic model
of aging is the relatively recent discovery that the level
of age-related methylation of a set of cytidine-guanosine
dinucleotides (CpG) located at precise positions
throughout the genome constitutes a highly reliable
biomarker of aging. A mathematical algorithm, the
multi-tissue age predictor also known as the epigenetic
clock, devised by Stephen Horvath [26], uses the
age-dependent methylation state (beta value) of 353
CpGs located at precise positions through the human
genome and generates a number, expressed in years, that
represents epigenetic age. In humans, the epigenetic age
Fig. 3 Diagrammatic representation of the cumulative DNA damage theory and the epigenetic model of aging. aProgressive age-related DNA
damage that takes place in the genome of cells with age due to environmental insults. ROS, reactive oxygen species. bThe upper diagram
represents some of the progressive changes in histones H3 and H4 methylation and acetylation during normal aging. Changes in DNA
methylation are represented by stemmed asterisks on DNA. The lower diagram represents the chronologic changes that may occur on the same
epigenetic marks during OSKM gene-induced rejuvenation/dedifferentiation. Red symbols represent chromatin activating marks whereas black
symbols correspond to chromatin-repressor marks. Blue wavy lines represent the gene transcripts
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generated by this predictor shows a correlation of 0.96
to chronological age and an error margin of 3.6 years
[27]. This is an unprecedented accuracy for a biomarker
of age, far superior to all biomarkers of age so far docu-
mented. Remarkably, the epigenetic clock predicts bio-
logical age with comparable high accuracy when applied
to DNA taken from the whole blood, peripheral blood
mononuclear cells, occipital cortex, buccal epithelium,
colon, adipose, liver, lung, saliva, and uterine cervix [27].
The rate of change in DNA methylation at age-
dependent CpGs represents the ticking rate of the epi-
genetic clock. The rate is very high in humans from
birth to 1 year of age; from 1 to 20 years of age, it pro-
gressively decelerates; and from age 20 onwards, it
changes to a much slower rate [27]. It can also be said
that the ticking rate of the epigenetic clock represents
the changing rate of DNA methylation heterogeneity
among cells in tissues. Now, it seems clear that lifespan
is associated not to the number of total tickings of the
epigenetic clock but to the rate of ticking after 20 years
of age when a human has achieved maturity. There is
compelling evidence that genetics is the primary control-
ler of the rate of epigenetic aging. For instance, it is
known that the rate of epigenetic aging is slower in
supercentenarians and their descendants [28]. It should
be pointed out that the epigenetic clock is an accurate
predictor of chronological age mainly in a context of
normal aging; under pathological circumstances, the epi-
genetic age displayed by the clock represents biological
rather than chronological age. For instance, pathologies
like Huntingtons and Parkinsons diseases are associated
with accelerated epigenetic aging [29,30]. Consistent
with the epigenetic model of aging, when 30-year-old
human [26] and 2-month-old mouse [31] somatic cells
are reprogrammed to iPSCs by the Yamanaka factors,
their epigenetic age reverts to zero years or months, re-
spectively. The epigenetic clock theory of aging hypothe-
sizes that biological aging is an unintended consequence
of both developmental and epigenetic maintenance pro-
grams for which the molecular footprints give rise to
DNA methylation (DNAm) age estimators [32]. Indeed,
the epigenetic clock theory and the epigenetic model of
aging go hand in hand and support each other.
The theories at work
The conception of a new individual involves the genesis
of a completely young organism which does not inherit
any aging trait from its parents, after conception life
starts anew. According to the cumulative DNA damage
theory, when a hypothetical human couple, 25 years of
age each member, conceive a new individual, the DNA
damage accumulated in the genome of their germ cells
during 25 years is probably minimal but higher than
zero. Therefore, the new zygote should inherit that
minimal damage (Fig. 4a, enlarged inset), and as a con-
sequence, it would not be fully young. In contrast, ac-
cording to the epigenetic model, the zygote conceived by
the same couple would have its epigenetic clock reset to
zero by the reprogramming factors present in the cyto-
plasm of that cell (Fig. 4b, enlarged inset). This hypoth-
esis is based on the fact that when human or mouse
adult somatic cells are reprogrammed to iPSCs, their
epigenetic age is reset virtually to zero [26,31].
While every individual of a metazoan species ages,
complex animal life has remained viable over hundreds
of million years. According to the cumulative DNA dam-
age theory, germ cells are likely to sustain some accumu-
lation of DNA damage in spite of the fact that DNA is
highly protected in this type of cells. Since complex ani-
mal life emerged during the era of life evolution known
as the Cambrian explosion, some 550 million years ago
[33], the enormous number of generations of germ cells
that took place over the successive millennia during
which many complex animal species evolved should
have accumulated increasingly large amounts of DNA
damage that would be inherited by the zygotes they con-
ceive (Fig. 4a, main panel). Such accumulating DNA
damage should cause complex animal species to pro-
gressively weaken and eventually become extinct, which
obviously is not the case.
According to the epigenetic model, at the time of
fertilization, all the epigenetic marks of parental aging
are erased from the zygotes genome, resetting its aging
clock back to zero. Therefore, in each generation, the
epigenetic clock of zygotes will restart from zero (Fig. 4b,
main panel), thus allowing that complex animal species
flourish and diversify over the millennia.
Therapeutic potential of cyclic partial
reprogramming
Besides showing that the OSKM genes are able to
partially rejuvenate cells and organs in mice, the same
study described above, Ocampo et al. [16], used
12-month-old, transgenic nonprogeroid mice. Cyclic
partial reprogramming enhanced the otherwise poor
regenerative capacity of their pancreas and skeletal
muscle and made these tissues more resilient to a subse-
quent insult.
Specifically, cyclic transient induction of OSKM trig-
gered proliferation of beta cells in the pancreas and sat-
ellite cells in the skeletal muscle, which are critical for
the maintenance of tissue homeostasis, but whose num-
bers typically decrease with age. Therefore, the benefits
of cyclic partial reprogramming may go beyond rejuven-
ation of old animals; it could also constitute an effective
regenerative treatment [34].
Concerning in vivo rejuvenation, the main challenges
that partial reprogramming in vivo presents are the need
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to employ an approach that does not use transgenic ani-
mals and that does not require indefinitely long applica-
tion of the method to prevent the rapid return of the
aging marks after termination of the treatment. Once such
hurdles are overcome, clinical treatment will become po-
tentially feasible. In such case, the first, more amenable,
clinical approach would be a preventive one in which
adult individuals carrying risk factors for late-life diseases
are submitted to a partial cell reprogramming approach
not necessarily to rejuvenate them but to stop or at least
significantly slow down their aging rate. In a more ad-
vanced stage of the partial reprogramming technology,
progressive rejuvenation of old individuals could be
attempted. At the increasing pace of technology advance-
ment, the above therapeutic aims may become available
routines in the not-too-distant future.
The possibility that human rejuvenation strategies may
become routine treatments raises some ethical issues, a
relevant one being a potential overpopulation of the
planet with the consequential impact on the availability
of food, energy, and other commodities. In any case,
these problems lie in the relatively distant future, and it
is to be hoped that more advanced societies will
establish regulatory legislation aimed at preventing a
demographic explosion.
Concluding remarks
The discovery of animal cloning and subsequent devel-
opment of cell reprogramming technology were
quantum leaps as they led to the achievement of reju-
venation by cell reprogramming which in turn
constitutes a paradigm shift in gerontology. A major
consequence of these discoveries is the emerging view
that aging is an epigenetic process and that somewhere
in the genome of somatic cells, there is a viable genetic
program that can be set in motion by a small number of
master genes. The purpose of this putative program
seems to be the reprogramming of somatic cells into
blastocyst-like, pluripotent embryonic stem cells. Al-
though at present we know virtually nothing about the
location of this hypothetic rejuvenation program, let
alone its structure and mechanism of action, we do
know how to turn on the program even in somatic cells
from very old individuals. We have discovered a power-
ful tool designed by nature and perfected during millions
of years of evolution and have learned how to use it to
Fig. 4 The two theories at work. aAccording to the cumulative DNA damage theory, when a hypothetical 25-year-old human couple conceives
a new individual, the zygote they conceived inherits the DNA damage of the parental germ cells. (Left enlarged panel) According to the theory,
after each generation, DNA damage in the successive zygotes would be accumulated through inherited damage, causing species viability to
decline progressively over the centuries, eventually driving them to extinction. (Main diagram) According to the epigenetic model, at the time of
fertilization, all the epigenetic marks of parental aging are erased from the zygotes genome by the reprogramming factors present in the
cytoplasm, thus resetting its aging clock back to zero (b, left enlarged panel). Consequently, in each generation, the epigenetic clock of zygotes
will restart from zero, thus allowing that complex animal species flourish and diversify over time (b, main diagram)
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rejuvenate cells and to a certain extent, animals. The
goal of understanding the molecular mechanisms in-
volved in pluripotency factor-mediated rejuvenation is
not nearly as imperative as the objective of learning how
to implement cell reprogramming technology in nontrans-
genic old animals and humans in order to erase epigenetic
marks of aging without removing epigenetic marks of cell
identity. The rate of progress in biotechnology is increas-
ingly fast, approaching an exponential curve. It is there-
fore reasonable to expect that clinical approaches to stop
aging and even rejuvenate aged humans will be developed
within the next two or three decades.
It has been stated that any sufficiently advanced tech-
nology is indistinguishable from magic[35]. Now, it
seems that the magic of human technology is making
rejuvenation possible.
Abbreviations
CpG: Cytidine-guanosine dinucleotide; DOX: Doxycycline; H3K27me3: Histone
3 trimethylated at lysine 27; H3K4me3: Histone H3 trimethylated at lysine 4;
H3K9me3: Histone H3 trimethylated at lysine 9; H4K16ac: Histone H4
acetylated at lysine 16; H4K20me3: Histone H4 trimethylated at lysine 20;
HSC: Hematopoietic stem cell; iN: Induced neuron; iPSC: Induced pluripotent
stem cell; NCC: Nucleocytoplasmic compartmentalization; OSKM genes: oct4,
sox2,klf4, and c-myc
Acknowledgements
The authors thank Dr. Kenneth Raj, Public Health England, Didcot, UK, for
enlightening discussions on the epigenetic clock and Yuri Deigin, CEO,
Youthereum Genetics, Canada, for critical reading of the manuscript. The
authors are indebted to Mr. Mario R. Ramos for the design of the figures and
to Ms. Yolanda E. Sosa for the editorial assistance. RGG, CBH, and OAB are
Argentine National Research Council (CONICET) researchers. ML, PC, and
MCM are CONICET doctoral fellows.
Funding
The work from our laboratory is supported in part by grant PICT15-0817 from
the National Agency for the Promotion of Science and Technology and from
research grant MRCF 10-10-17 from the Medical Research Charitable Founda-
tion and the Society for Experimental Gerontological Research, New Zealand,
to RGG.
Availability of data and materials
The information reviewed here are public domain.
Authorscontributions
The bibliographic search was equitatively distributed among all authors. Each
author prepared a draft of an assigned section. RGG was in charge of writing
the final version. All authors read and approved the final version.
Ethics approval and consent to participate
All authors agree to publish this article and have accepted to abide the
ethical standards of our institution.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Author details
1
Institute for Biochemical Research (INIBIOLP) - Histology B & Pathology B,
School of Medicine, National University of La Plata, CC 455, 1900 La Plata,
Argentina.
2
Institute for Experimental Pharmacology Cordoba(IFEC), School of
Chemical Sciences, National University of Cordoba, Cordoba, Argentina.
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... The first clues that somatic cells can be rejuvenated back to ES-like cells came from the development of animal cloning in the early ´60s [27] and more recently, of cell reprogramming [28]. These seminal achievements paved the way for the subsequent implementation of cell rejuvenation [29][30][31]. ...
... Despite the promise offered by partial cell reprogramming for safe rejuvenation in vivo, it should be pointed out that the OSKM genes are unlikely to have evolved as a physiological mechanism to regulate the epigenetic clock during adult life, rather their most plausible role seems to be the resetting of epigenetic age to zero in the zygote [31]. AGING ...
... Up to this stage, aging was considered as an essentially irreversible process. However, with the discovery of cell reprogramming, early in this century, a view began to emerge that considers aging as a reversible epigenetic process [29][30][31]. The hypothesis proposing the epigenome as the driver of aging was significantly strengthened by the converging discovery that DNA methylation at specific CpG sites could be used as a highly accurate biomarker of age defined by the Horvath clock [5]. ...
Article
Full-text available
The view of aging has evolved in parallel with the advances in biomedical sciences. Long considered as an irreversible process where interventions were only aimed at slowing down its progression, breakthrough discoveries like animal cloning and cell reprogramming have deeply changed our understanding of postnatal development, giving rise to the emerging view that the epigenome is the driver of aging. The idea was significantly strengthened by the converging discovery that DNA methylation (DNAm) at specific CpG sites could be used as a highly accurate biomarker of age defined by an algorithm known as the Horvath clock. It was at this point where epigenetic rejuvenation came into play as a strategy to reveal to what extent biological age can be set back by making the clock tick backwards. Initial evidence suggests that when the clock is forced to tick backwards in vivo, it is only able to drag the phenotype to a partially rejuvenated condition. In order to explain the results, a bimodular epigenome is proposed, where module A represents the DNAm clock component and module B the remainder of the epigenome. Epigenetic rejuvenation seems to hold the key to arresting or even reversing organismal aging.
... The first clues that somatic cells can be rejuvenated back to ES-like cells came from the development of animal cloning in the early ´60s [27] and more recently, of cell reprogramming [28]. These seminal achievements paved the way for the subsequent implementation of cell rejuvenation [29][30][31]. ...
... Despite the promise offered by partial cell reprogramming for safe rejuvenation in vivo, it should be pointed out that the OSKM genes are unlikely to have evolved as a physiological mechanism to regulate the epigenetic clock during adult life, rather their most plausible role seems to be the resetting of epigenetic age to zero in the zygote [31]. AGING ...
... Up to this stage, aging was considered as an essentially irreversible process. However, with the discovery of cell reprogramming, early in this century, a view began to emerge that considers aging as a reversible epigenetic process [29][30][31]. The hypothesis proposing the epigenome as the driver of aging was significantly strengthened by the converging discovery that DNA methylation at specific CpG sites could be used as a highly accurate biomarker of age defined by the Horvath clock [5]. ...
Preprint
Full-text available
The view of aging has evolved in parallel with the advances in biomedical sciences. Long considered as an irreversible process where interventions were only aimed at slowing down its progression, breakthrough discoveries like animal cloning and cell reprogramming have deeply changed our understanding of postnatal development, giving rise to the emerging view that the epigenome, rather than DNA, is the driver of aging. The idea was significantly strengthened by the converging discovery that DNA methylation at specific CpG sites could be used as a highly accurate biomarker of age defined by an algorithm known as the Horvath clock. The strong correlation between the dynamics of DNA methylation profiles and the rate of biological aging suggests that the epigenetic clock may be intimately related to the epigenetic pacemaker of aging. It was at this point where epigenetic rejuvenation came into play as a strategy to reveal to what extent biological age can be set back by making the clock tick backwards. The few initial results, to be discussed here, seem to suggest that when the clock is forced to tick backwards in vivo, it is only able to drag the phenotype to a partially rejuvenated condition. In order to explain the results, a bimodular epigenome is proposed , where Module A represents the DNAm clock component and Module B represents the remainder of the epigenome, including but not limited to cell identity marks. Although no firm conclusion can be drawn from the scanty results so far documented, what seems to be already clear is that epigenetic rejuvenation holds the key to both understanding the mechanism by which the epigenome drives the aging process and arresting or even reversing organismal aging.
... I like the science of cellular reprogramming, because it implicitly rejects the dogma that aging is caused by the accumulation of molecular damage, such as DNA damage [6]. This is in line with hyperfunction theory of quasi-programmed aging [7,8]. ...
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Some visionaries prefer to dream of immortality rather than to actually live longer. Here I discuss how combining rapamycin with other modalities may let us live long enough to benefit from future discoveries in cellular reprogramming and what needs to be done at Atlos Labs to make this happen.
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Advances in aging studies brought about by heterochronic parabiosis suggest that agingmight be a reversable process that is affected by changes in the systemic milieu of organs andcells. Given the broadness of such a systemic approach, research to date has mainly questioned theinvolvement of “shared organs” versus “circulating factors”. However, in the absence of a clearunderstanding of the chronological development of aging and a unified platform to evaluate thesuccesses claimed by specific rejuvenation methods, current literature on this topic remains scattered.Herein, aging is assessed from an engineering standpoint to isolate possible aging potentiators via ajuxtaposition between biological and mechanical systems. Such a simplification provides a generalframework for future research in the field and examines the involvement of various factors in aging.Based on this simplified overview, the kidney as a filtration organ is clearly implicated, for the firsttime, with the aging phenomenon, necessitating a re-evaluation of current rejuvenation studies tountangle the extent of its involvement and its possible role as a potentiator in aging. Based on thesefindings, the review concludes with potential translatable and long-term therapeutics for aging whileoffering a critical view of rejuvenation methods proposed to date.
... However, we failed to detect the same correlation either in iPSCs or in NPCs, even if in Kang's study there was also a consistent trend in reducing the amount of variants across reprogramming (see Figure 3E in Kang et al., 2016). The reprogramming procedure implies a sort of rejuvenation process (Goya et al., 2018) in which iPSCs derived from old individuals could reset their genetic heritage through multiple passages and, in doing so, erase the mtDNA alterations accumulated during aging in the parental fibroblasts. Our data are consistent with the number of passages of the iPSCs (15-20), whereas those analyzed by Kang had a significantly lower (2-4) number of passages (Kang et al., 2016), when iPSCs display the greatest heterogeneity and variability (Volpato and Webber, 2020). ...
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... However, adult progenitor cells can change epigenetically their identity with rejuvenated capacity by in vivo direct reprogramming [7,8]. More importantly, direct reprogramming technology might lead to rejuvenation of a cell to their embryonic stage, by controlling and resetting the aging clock at different time levels [9,10]. The advances in cell fat engineering ...
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... The latest is especially important since the formation of teratomas presents a significant limitation for the induced pluripotent stem cell (iPSC)-based clinical applications (Gutierrez-Aranda et al., 2010). These findings established partial epigenetic reprogramming as a promising candidate intervention for aging-associated pathological conditions (Goya et al., 2018;Singh and Newman, 2018;Melo Pereira et al., 2019;Olova et al., 2019). Therefore, epigenetic enzymes involved in the heritable modification of DNA and histones represent promising targets for potential pharmacological interventions aimed to prolong lifespan and healthspan. ...
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The development of interventions aimed at improving healthspan is one of the priority tasks for the academic and public health authorities. It is also the main objective of a novel branch in biogerontological research, geroscience. According to the geroscience concept, targeting aging is an effective way to combat age-related disorders. Since aging is an exceptionally complex process, system-oriented integrated approaches seem most appropriate for such an interventional strategy. Given the high plasticity and adaptability of the epigenome, epigenome-targeted interventions appear highly promising in geroscience research. Pharmaceuticals targeted at mechanisms involved in epigenetic control of gene activity are actively developed and implemented to prevent and treat various aging-related conditions such as cardiometabolic, neurodegenerative, inflammatory disorders, and cancer. In this review, we describe the roles of epigenetic mechanisms in aging; characterize enzymes contributing to the regulation of epigenetic processes; particularly focus on epigenetic drugs, such as inhibitors of DNA methyltransferases and histone deacetylases that may potentially affect aging-associated diseases and longevity; and discuss possible caveats associated with the use of epigenetic drugs.
... However, the application of reprogramming techniques to human subjects has raised ethical issues and prompted safety concerns, as the pluripotent state carries a risk of inducing cancer 13 . As such, much effort has been made to propose safer strategies involving interference in the aging-associated molecular alterations of tissue stem cells, such as via pharmaceutical administration and genetic modification 14 . ...
Article
Full-text available
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Chapter
The article covers the main biotechnology tools - cloning, genetic engineering (recombinant technology) technologies, genome editing, producing transgenic animals, and examples of each tool's practical applications. For each method, the characteristics that determine the scope of its applications are described.
Article
Full-text available
Identifying and validating molecular targets of interventions that extend the human health span and lifespan has been difficult, as most clinical biomarkers are not sufficiently representative of the fundamental mechanisms of ageing to serve as their indicators. In a recent breakthrough, biomarkers of ageing based on DNA methylation data have enabled accurate age estimates for any tissue across the entire life course. These 'epigenetic clocks' link developmental and maintenance processes to biological ageing, giving rise to a unified theory of life course. Epigenetic biomarkers may help to address long-standing questions in many fields, including the central question: why do we age?
Chapter
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Article
Full-text available
The achievement of animal cloning and subsequent development of cell reprogramming technology are having a profound impact on our view of the mechanisms of aging in complex organisms. The experimental evidence showing that an adult somatic nucleus implanted into an enucleated oocyte can give rise to a whole new individual strongly suggests that the integrity of the genome of an adult nucleus is fully preserved. Here, we will review recent experimental evidence showing that pluripotency gene-based cell reprogramming can erase the epigenetic marks of aging and rejuvenate cells from old individuals reversing most signs of aging and that when induced pluripotent stem cells (iPSC) are differentiated back to the cell type of origin, the rejuvenated cells share many of the features of wild-type counterparts from young donors. This evidence supports the idea that progressive epigenetic dysregulation may be the key driver of organismal aging and challenges the conventional view of aging as an irreversible process. The model of aging as an epigenetic process provides an elegant explanation of a number of age-related processes difficult to explain by conventional theories of aging.
Article
Full-text available
Reprogramming adult, fully differentiated cells to pluripotency in vivo via Oct3/4, Sox2, Klf4 and c-Myc (OSKM) overexpression has proved feasible in various independent studies and could be used to induce tissue regeneration owing to the proliferative capacity and differentiation potential of the reprogrammed cells. However, a number of these reports have described the generation of teratomas caused by sustained reprogramming, which precludes the therapeutic translation of this technology. A recent study by the Izpisúa-Belmonte laboratory described a cyclic regime for short-term OSKM expression in vivo that prevents complete reprogramming to the pluripotent state as well as tumorigenesis. We comment here on this and other studies that provide evidence that in vivo OSKM induction can enhance tissue regeneration, while avoiding the feared formation of teratomas. These results could inspire more research to explore the potential of in vivo reprogramming in regenerative medicine.
Article
Full-text available
Ageing associates with significant alterations in somatic/adult stem cells and therapies to counteract these might have profound benefits for health. In the blood, haematopoietic stem cell (HSC) ageing is linked to several functional shortcomings. However, besides the recent realization that individual HSCs might be preset differentially already from young age, HSCs might also age asynchronously. Evaluating the prospects for HSC rejuvenation therefore ultimately requires approaching those HSCs that are functionally affected by age. Here we combine genetic barcoding of aged murine HSCs with the generation of induced pluripotent stem (iPS) cells. This allows us to specifically focus on aged HSCs presenting with a pronounced lineage skewing, a hallmark of HSC ageing. Functional and molecular evaluations reveal haematopoiesis from these iPS clones to be indistinguishable from that associating with young mice. Our data thereby provide direct support to the notion that several key functional attributes of HSC ageing can be reversed.
Article
The DNA methylation levels of certain CpG sites are thought to reflect the pace of human aging. Here, we developed a robust predictor of mouse biological age based on 90 CpG sites derived from partial blood DNA methylation profiles. The resulting clock correctly determines the age of mouse cohorts, detects the longevity effects of calorie restriction and gene knockouts, and reports rejuvenation of fibroblast-derived iPSCs. The data show that mammalian DNA methylomes are characterized by CpG sites that may represent the organism’s biological age. They are scattered across the genome, they are distinct in human and mouse, and their methylation gradually changes with age. The clock derived from these sites represents a biomarker of aging and can be used to determine the biological age of organisms and evaluate interventions that alter the rate of aging.
Article
Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mousemodel of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of ageassociated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.