Studies that Shed New Light on Aging.

Abstract

I will first discuss how all aging models that assume that the aged cell has irreversibly lost its youthful capabilities through such mechanisms as accumulated dysfunction, accumulated damage, and/or accumulation of toxic byproducts of metabolism have been shown to be incorrect. I will then briefly discuss models of aging and propose an experiment that would distinguish between those models and provide a basis for organismic rejuvenation.

Full text

By the turn of the 21st century, gerontologists and
biologists reached a consensus “evolutionary theory of
aging” [13], putting aging research into the mainstream
of biological research. This socalled theory, simply put,
states that because continued selective pressure tends to
make the lifespan of a sexuallyreproducing organism
longer – as a longerlived organism produces more off
spring – it can be assumed that mechanisms retarding
aging are insufficient to allow organisms the long lives
evolutionary theory predicts. The theory assumes that an
aged cell is irreversibly damaged as a result of life’s slings
and arrows, leading to the currently accepted paradigm of
“wear and tear” as the cause of aging.
The “wear and tear” paradigm of aging maintains
that as an organism ages, the incidence of cellular damage
and toxic metabolic byproducts eventually exceeds the
organism’s ability to repair or remove them, leading to
their accumulation [4]. This, in turn, results in the
increase in the number of senescent cells and a decrease
in effective stem cell populations, or an alteration of their
potency [5]. So aging is characterized as the accumula
tion of damage leading to dysfunction. Metaphorically,
the aging of an organism is like that of an automobile,
eventually as parts wear out and malfunction more quick
ly than they can be repaired or replaced, the automobile
becomes a jalopy destined for the junkyard. This is
assumed to be the same case regarding cells, even stem
cells, and consequently the tissues, organs, organ sys
tems, and the organism whose functioning is based on
those cells.
CELLULAR REJUVENATION IMPLIES
STOCHASTIC THEORIES OF AGING
CONFUSE CAUSE AND EFFECT
In spite of inconsistencies and improper assump
tions, these current leading “evolutionary” theories of
aging are widely accepted models yet, to call them “theo
ries”, is normally a scientific assessment of their proven
truth, which is not the case [6]. As this paper will show,
the hypothesis that aging at the cellular level is the prod
uct of the accumulation of irreparable damage and/or un
eliminable toxic substances has been tested and rejected
in numerous studies. Simply demonstrating that cells can
be rejuvenated by exogenous factors is logically equivalent
to demonstrating that cells are not irreparably damaged
by aging, in direct contradiction to the assertion that
aging is the result of irreparable damage. If cells are not
irreparably damaged by aging, then what makes them age
and accumulate damage? As we will show, it is becoming
clear that the accumulation of damage is an effect of
aging and not its cause. This confusion of cause and effect
is evinced in many aging “theories” based the many phe
nomena correlated with aging. So mitochondrial dys
function, ROS (reactive oxygen species) production,
telomere shortening, and DNA damage accumulation –
each has been separately regarded as “the cause” of aging
[7], yet they are all more properly regarded as effects of
aging: the demonstration that each of these “causes” of
aging are reversed by cellular rejuvenation shows that
those are all processes that are not its cause but are all
ISSN 00062979, Biochemistry (Moscow), 2013, Vol. 78, No. 9, pp. 10611070. © Pleiades Publishing, Ltd., 2013.
Published in Russian in Biokhimiya, 2013, Vol. 78, No. 9, pp. 13541366.
1061
Studies that Shed New Light on Aging
H. L. Katcher
Collegiate Professor, University of Maryland, University College, USA; Email: hkatcher@earthlink.net; hkatcher@faculty.umuc.edu
Received May 16, 2013
Abstract—I will first discuss how all aging models that assume that the aged cell has irreversibly lost its youthful capabilities
through such mechanisms as accumulated dysfunction, accumulated damage, and/or accumulation of toxic byproducts of
metabolism have been shown to be incorrect. I will then briefly discuss models of aging and propose an experiment that
would distinguish between those models and provide a basis for organismic rejuvenation.
DOI: 10.1134/S0006297913090137
Key words: aging, aging mechanisms, theories of aging, parabiosis, crossage transplantation, rejuvenation, cellular aging,
agedependent transcriptional patterns

1062 KATCHER
BIOCHEMISTRY (Moscow) Vol. 78 No. 9 2013
consequences of aging, the result of cellular decisions, but
decisions that can be, as we shall see, reversed. All sto
chastic models that assume the hypothesis that aging
results from the irreversible loss of information to entropy
are shown to be incorrect by the demonstration of cellu
lar rejuvenation: the hypothesis that information is irre
trievably lost through aging must be rejected as the infor
mation for a total renewal was not lost in the aged or
senescent cell: it was demonstrably recovered. As we will
show, evidence points to aging being a programmed
process controlled, in mammals, through factors present
in their blood. The ultimate control of the process is epi
genetic, but it is coordinated across the body by soluble
factors and juxtacrine interactions. I believe the process is
a continuation of the developmental program, with a
beginning and an end.
Kirkwood’s [8] statement “but, if programmed, the
programming is very loose, because there is a large varia
tion in the rates of senescence of individual cells within
the population”, is close to what we believe to be true, in
that an organism’s lifespan is governed by a very loose
programming with many environmentallyinfluenced
decision pathways that may extend or diminish lifespan,
but inevitably (except for organisms showing negligible
senescence, or replaying earlier developmental stages like
Turritopsis nutricula) drives an individual from zygote
through reproductive adulthood and to subsequent senes
cence and death. To suggest that aging changes are to a
great degree preordained is to take a position that sounds
more hopeless than the various variations of “wear and
tear” (stochastic) theories which, at least, grant the pos
sibility of interfering with damage production and/or
accumulation. However, as we will show, several studies
indicate that the exogenous factors can reset the age phe
notype of body cells, tissues, organs, and possibly organ
isms, this view of aging leads to the possibility of recover
ing youth – that very “fountain of youth” that Dr.
Kirkwood told us was to go the way of the perpetual
motion machine – an impossibility [9].
AGERELATED TRANSCRIPTION PROFILES
AND THE THEORIES OF AGING
The stochastic theories of aging suggest a general
decline [2] in all aspects of the organism – it is suggested
by all the wear and tear theories that the cell ultimately
becomes dysfunctional to the stage where it cannot per
form its duties nor even maintain itself and dies. It is
expected under such a paradigm that the cell deteriorates
with age, much as an automobile does. As opposed to this,
an explanation more consistent with observed biology is
an epigenetically controlled program that is an approxi
mately lifelong extension of the developmental program
that begins with fertilization [7]. If that is the case, then
aging may be evolutionarily selectable as nature may be
selecting a successful program and not a collection of
genes, or for that matter individual, “selfish” genes.
Evidence for this view comes from the technological
innovation that allows the simultaneous analysis of the
rates of transcription of tens of thousands of genes using
DNA microarrays [1012]. These sorts of studies show age
dependent changes in the regulation of thousands of genes.
The study of Stuart Chambers [13] shows that there are
marked agedependent, order of magnitude differences in
the transcriptional rates of many genes, and of these age
regulated genes, the regulation must be seen as anti
homeostatic. An example of this is the downregulation of
DNA repair genes in mice [13] at ages at which DNA
defects show increased frequency and DNA repair
becomes inefficient [14]. Other examples will be consid
ered later. One would certainly suspect that downregulat
ing the transcription rates of repair enzymes at or just prior
to the time of increased DNA damage accumulation and
the documented decreased ability for DNA repair (“ineffi
cient repair” [14]) must have a causal connection. Yet
homeostasis is an active process that at the level of the cell
tries to preserve integrity – turning down DNA repair
activity at the time of an increase in DNA damage (by
ROS, “leaky” mitochondria, etc.) is exactly contrary to the
principle of homeostasis (even failing to upregulate DNA
repair when there is DNA damage is at least nonhomeo
static) and demands an explanation. The explanation of
the hypothesis called “antagonistic pleiotropy” would be
that some pleiotropic protein that once acted towards the
cell’s benefit now operates (at its evolutionarily untouch
able postreproductive stage of deterioration) to its detri
ment – but what we observe is not a change in the charac
ter of genes, but a quantitative change in their appearance
in the cell – and that change is opposite to what the prin
ciple of homeostasis demands! While Chambers attributes
those negative changes, such as genetic dysregulation and
the loss of genetic suppression to entire segments of chro
mosomes, to epigenetic dysregulation, the very specific
timings of agedependent changes in transcription rates of
agingrelated genes, apparently correlated with develop
mental stages in the postadult lifespan, implies a pro
grammatic decision to downregulate cellular mainte
nance and repair systems aging damage rather than a sto
chastic unraveling of epigenetic control. The agecoordi
nated expression of specific genes appears to result in the
very dysfunctions that their lack or excess might reasonably
be expected to give rise to: decreased synthesis of DNA
repair proteins might be expected to give rise to the accu
mulation of DNA defects, excesses of proinflammatory
cytokines should lead to generalized inflammation,
increased production of amyloidogenic proteins ought to
produce amyloidoses (including Alzheimer’s disease) –
processes associated with aging [13].
It may be safely assumed that cellular deficiencies
including the progressive loss of proliferative potential
and potency in stem cell and progenitor cell populations

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[15] and the appearance of “senescent cells” lead to the
deficiencies seen at the higher levels of tissues, organs and
organ systems and the organism itself [16, 17]. This
occurs because, among other disabilities incurred by
aging, stem cell populations lose the ability to replenish
somatic tissues, some mitotic cells become senescent and
actively produce noxious products; both paths ultimately
culminating, at the organismic level, in the diseases of
aging and death.
Seemingly, the increase or decrease in functionalities
provided by agerelated gene transcription are responsible
and the agerelated increase in the number of senescent
cells are responsible also for the diseases of aging (e.g.
cardiovascular disease, cancer, diabetes, dementia, and
pneumonia) [18]. Which basically means that a cell delib
erately diminishes its repair and maintenance capabili
ties, it deliberately increases its ROS production, it delib
erately decreases the efficiency of its DNA repair, it delib
erately decreases the sirtuins it requires for chromatin
maintenance, it deliberately increases the noxious prod
ucts it vents to the surrounding tissues, it deliberately
increases the production of proinflammatory cytokines,
matrix metalloproteases – or hard to clear amyloid form
ing proteins. How can any of this be accounted for by any
theory that assumes homeostasis is a governing principle
in biology – to what perceived threat or lack thereof does
the cell turn down its protective mechanisms at the begin
ning of middle age [13] – what explanation can the “wear
and tear” hypotheses give to the cell turning down repair
systems as damage increases?
Before outlining a new approach to investigating
aging, I will first put to rest those aging models that
assume, for whatever reason, that the aged cell has irre
versibly lost its youthful capabilities through stochastic
mechanisms; that is, that the cell is irreparably damaged
through aging. I will then briefly propose a new model of
aging as a continuation of development, discuss its rela
tionship to diseases of aging and, finally, propose a possi
ble means by which cellular rejuvenation therapy might
be tested. At this time when the proposed costs of medical
services to be provided by health services for seniors is
reaching astronomical levels, a way of delaying those costs
would gain nations much needed time.
REJECTING THE “WEAR AND TEAR”
HYPOTHESIS OF AGING
Let us now debunk this paradigm of “wear and tear”
for biological aging, using experimental evidence.
If aging results from the accumulation of damage or
of toxic metabolic byproducts, then chronologically old
cells should be damaged beyond the cell’s ability to repair
itself. This is not the case, however based on clear results
from decades of experimentation. Aging at the cellular,
tissue, organ, and organismic levels have been reversed by
exposing the tissues of old animals to a young environ
ment [19].
If cells can be rejuvenated by intrinsic means as a
result of extrinsic signaling, then the presumption that
they are damaged beyond their ability to repair them
selves is logically false. Thus, all theories of aging based
on the “wear and tear” paradigm are proven wrong by
experimentation. If a cell can be rejuvenated, it cannot be
damaged beyond repair by aging.
Let us begin with the weaker evidence and move
towards the stronger.
1. The nuclei of cells that have been passed in cell
culture to the point at which they were senescent and
incapable of further cell division have been used to clone
normal animals. In this series of experiments, somatic
cow fibroblasts were passed serially in culture until senes
cence and their nuclei were then extracted and placed
into enucleated bovine ova. These ova were able to pro
duce genetically normal offspring – this demonstrates
that the nuclei of senescent cells are repairable at least.
Apart from showing that there was no irreparable damage
to these cell’s nuclei, the experiment established that the
aged nuclei proved fully capable of producing normal off
spring (although the telomeres of cattle resulting from
this were longer than normal) [20].
Similarly, induced pluripotent cells can be said to
have been rejuvenated by the process used to produce
them, by bringing them back to earlier developmental
stages, it seems that we also reset their age phenotype. The
studies of Lapasset et al. [21] showed that induced pluripo
tent cells derived from centenarians were rejuvenated such
as to be indistinguishable from those derived from youthful
cells using a set of criteria that include telomere length,
mitochondrial function, gene expression profiles, mito
chondrial efficiency, and ROS production. Those charac
teristics represent what we know of the age phenotype, so
we must conclude that the age phenotype was set back as
well as the state of differentiation. It should be noted here
however that heterochronic parabiosis studies and cross
age organ transplant studies (to be discussed) show that the
age phenotype can be affected independently of the state
of differentiation, that is a cell can be rejuvenated without
changing its state of differentiation [22]. The theory that
increasingly dysfunctional, aging, mitochondria produce
reactive oxygen and nitrogen species resulting in a death
spiral of further dysfunction and destruction cannot be
correct if mitochondria are “rejuvenated” by external sig
naling (showing that they are far from being irreparably
impaired). That evidence together with all the other evi
dence indicates that cause and effect have been confused;
mitochondrial dysfunction, ROS production, and telom
ere shortening are the effects of aging, not the causes. Age
specific transcription patterns are in accord with pro
grammed aging, but not with stochastic aging, especially as
the “programming” is exactly opposite to what would be
expected from a homeostatic system.

1064 KATCHER
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2. Crossage transplantation studies have shown that
tissues and organs can be rejuvenated. The transplanta
tion of tissues or organs of old donors into young recipi
ents provides convincing evidence that old cells are fully
capable of regaining youthful functioning when placed in
the bodies of young animals [19]. Furthermore, it has
been demonstrated that when an aged, involuted thymus
gland is placed in a young body, it is rejuvenated and
regains full functionality, even though it was originally in
a senescent state [23]. If irreversible cellular damage is the
cause of cellular aging, then restoring aged cells to youth
ful functioning by merely changing their environment
would not be possible. So the aged cell is not irreversibly
damaged.
3. Heterochronic parabiosis is a technique by which
the circulatory systems of old and young animals are
joined. As might be expected, based on the crossage
transplantation studies, the stem and progenitor cell pop
ulations are “rejuvenated” to the extent that they act
more like younger stem cell populations. The rejuvena
tion taking place within the old animal was shown to be
entirely dependent on factors present in the shared,
hybrid blood supply [5]. Reciprocally, stem cell popula
tions of the younger heterochronic partner have been
shown to behave more like stem cells from older animals.
More recent studies also show that a heterochronic para
biotic pairing between young and old rats resulted in
increased neurogenesis and functional improvement of
cognitive ability in the older parabiotic partner, making it
appear closer to that of a younger animal, while the
younger partner exhibits decreased neurogenesis and cog
nitive abilities, consistent with that of an older rat [24].
If aging at the cellular level were the result of the
accumulation of damage and/or toxic byproducts of
metabolism, cellular rejuvenation would not have been
possible. If “wear and tear” were correct, rejuvenation by
mere signaling would not be possible, but it is and there
fore further adherence to such theories as postulate the
irreversible degradation of a cell by stochastic processes is
incorrect based on multiple lines of experimental evi
dence.
AGE PHENOTYPE OF STEM CELLS
IS CONTROLLED BY SYSTEMIC SIGNALS
Though largely ignored by mainstream biology since
the 1950’s, the belief that lifespan is subject to evolution
ary forces and may be programmed continues to be inves
tigated [7]. Developments, both those contradicting the
view that cellular accumulation of errors (stochastic dam
age) is the cause of senescence and those providing evi
dence supporting programmed aging have challenged the
mainstream view. At this time, the evidence that aging is
an actively programmed process appears to be com
pelling. And it is very much the same evidence that rele
gates “wear and tear” to the dustbin of history. The cross
age transplantation studies confirm that tissues adjust
their cellular age phenotypes to be consistent with the age
phenotype of their host. The heterochronic parabiosis
studies indicating that both partners’ (young and old) tis
sues tended to resemble each other in that the tissues of
the older parabiotic partner appeared not quite as youth
ful as those of a fully young animal, nor the tissues of the
younger partner as aged as those of the older partner. This
hints at a concentration effect of these bloodborne fac
tors.
An experiment to decide whether aging results from
the increasing incompetence of aged cells or the environ
ment of those cells might be done as follows: tissue from
a young animal is transplanted into an old one, and tissue
from an old animal is transplanted into a young one: if
aging causes the increasing incapacity of cells, if old cells
are damaged cells, we would expect the young tissue in
old animals to fare well, while the old tissue placed into
young animals – being defective, would not. However, the
opposite is what actually occurred, transplants of young
tissue into old organisms failed while old tissues trans
planted into young animals produced successful grafts
[17].
The observation that the stem cells from young ani
mals behave as though they were senescent when trans
planted into old animals confirms that it is the cells’ envi
ronment rather than cellintrinsic defects that result in
the phenotype of an aging cell [25, 26]. If an organism’s
cellular age phenotypes were determined by extrinsic fac
tors, the tissues of aging donors might respond to a young
environment by becoming young. The answer was
unequivocal in the case of muscle satellite cell transplants
in rats, the old tissue in young rats did well, becoming like
young muscle in terms of its ability to proliferate and
repair muscle damage; and on the other hand, the young
muscle transplanted into old rats showed both decreased
proliferative ability and wound healing capacity – charac
teristics of “old” cells [25, 27]. Work with hematopoietic
cells was more equivocal, but under proper conditions
Harrison et al. [28] showed that hematopoietic stem cells
were rejuvenated, totally capable of supplying the needs
of the young mouse and regaining youthful regenerative
capacity though they were derived from immunedefi
cient old mice. Careful controls ensured that organ
regeneration was not the result of cells from the host but
was entirely derived from the grafted tissues. At this point,
two conclusions become clear: cellular age phenotype for
some cell types at least is determined by systemic factors
carried in blood, and cellular aging can and has been
reversed, at least in some tissue types.
What happens in an organism’s tissues does not nec
essarily happen to the entire organism. If aging was a
result of systemic signaling and coordination amongst
widely separated tissues, then surely the blood must
transmit those signals [25]. If that is the case, then replac

STUDIES THAT SHED NEW LIGHT ON AGING 1065
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ing an older animal’s blood, at least in part, by that of a
younger animal’s and vice versa should lead to a change
in the age phenotype of the cells of both animals, such
that the younger cells appear to age and the old cells seem
“younger”. These were the actual results obtained from
experiments [5, 25]. If aging at the cellular level were
reversed, would this lead to the rejuvenation of the animal
at the level of the organism? And would it result in life
extension? The unusual technique of “parabiosis” was
used in the first experiments to investigate the possible
rejuvenation of an animal. The technique consists of the
surgical joining of the circulatory systems of two animals
(and the animals themselves – shoulder to shoulder and
hip to hip) so that they share a common blood pool. In
1957 McCay et al. [29] found that when a young rat was
joined in such a manner to an old rat (heterochronic
parabiosis) for a period of time, the old rat appeared
younger. The evidence was equivocal however relying
solely on the visual appearance of tissues (mostly carti
lage) during autopsy. Some 15 years later, Ludwig et al.
[30] used a more robust and quantitative experimental
design to show that the older animals did, indeed, benefit
from sharing the blood supply of the younger animal by
demonstrating life extension. While this is certainly a
confirmation of McCay’s experiments, it could be
argued, however, that during its parabiotic association,
the young rat’s body took over functions diminished by
aging in the old rat, the young organs did doubleduty in
assisting or replacing the diminished functions of the old
animal and so extended its life span. The experiments
undertaken by Conboy et al. in 2005 [5] showed that stem
cell tissues of the older rat were phenotypically younger
than agematched controls as measured by increased pro
liferation in response to woundhealing. The further
experiments of Villeda et al. [24] showed that two organ
ismic criteria of aging, neurogenesis and cognitive ability,
were also reversed by parabiotic joining with younger ani
mals.
BLOODBORNE FACTORS CONTROL
THE CELLULAR AGE PHENOTYPE
Neither the crossage transplantation studies nor the
parabiosis experiments of Conboy et al. [5] yielded exper
imental evidence distinguishing whether positive factors
(those promoting young phenotypes) or negative factors
(“aging” factors) present in the blood of older animals or
some combination of these positive and negative factors
controlled cellular age phenotype. While it might have
been youthinducing factors provided by “young blood”,
which produced the observed rejuvenation of aged cells or
a decrease in the concentration of negative, “aging
inducing factors” which caused the “rejuvenation” of old
cells, this was not decidable through the experiments
done. In their epublication [31], Silva expresses their
belief that aging is caused by the accumulation of negative
factors. The studies of Zhangfa et al. [23] showed that the
systemic environment of telomerasenegative mice
became the major inhibitor of lymphopoiesis as their
telomeres shortened; thus telomere shortening appeared
to give rise to a systemic environment that caused appar
ent aging in terms of decreased lymphopoiesis.
In agreement with the effects of the systemic envi
ronment on aging, aged stem cells were rejuvenated by
young plasma in vitro, and young stem cells were “aged”
by in vitro exposure to plasma from old animals [32].
Stem cells derived from the young partners in hete
rochronic parabiosis lost proliferative potential compared
to agematched controls, while the cells of the older part
ner were physiologically younger than those of age
matched controls. Still, there is no conclusion about
whether positive factors are increased in young blood, or
negative factors decreased by dilution, or whether a com
bination of the two (presence of positive factors in
“young” blood and the decreased concentration of nega
tive factors found in “old” blood) resulted in rejuvena
tion. More recently, the exploration of the effects of para
biosis on the neurological aspects of aging implicated sev
eral cytokines that showed differentially increased con
centration in the cerebrospinal fluid of old mice, and it
revealed that at least one of these, CCL11 or “eotaxin”,
caused a decrease in neurogenesis and a decrease in cog
nitive ability when injected into the systemic circulation
of young mice [24]. The works of Conboy, Carlson, and
Villeda as well as all of the crossage tissue and organ
transplantation studies established that rejuvenation at
the tissue and organ levels (the functioning of the brain)
takes place when these tissues or organs are exposed to a
younger systemic environment. There is now evidence
that at least some compounds that are differentially ele
vated in the cerebrospinal fluid of aged animals (the
“environment” of neural stem cells) can cause aginglike
changes in an organism’s age phenotype; at least one such
chemical can cause aginglike changes when injected into
the blood, identifying at least one substance differentially
accumulating in aged animals that can be said to have a
negative, agingpromoting effect [24]. It has been
demonstrated using heterochronic parabiosis that all stem
cell populations examined (using a small but important
sampling) were rejuvenated by a young systemic environ
ment. The wellcontrolled experiments of Conboy [5],
Conboy and Carlson [32] in 2009, and later Villeda [24]
(2011) established that factors carried in the blood are
sufficient to produce changes in cellular age phenotype.
The experiments described above imply that the
cell’s age phenotype is determined not only by the pres
ence (or absence) of factors in the blood, but also by the
concentration of those factors. As shown in both the
Conboy and Villeda heterochronic parabiotic experi
ments, the actual degree of rejuvenation of old tissue or
the extent of aging of young tissue was intermediate

1066 KATCHER
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between full youthful phenotype expected in the young
partner and the aged phenotype found in agematched
controls for the older partner. This may be because about
half of the blood plasma is contributed by each partner.
Furthermore, the thymus transplantation study of Zangfa
et al. [23] showed that senescent, involuted thymuses
became fully functional in the young animals into which
they were transplanted. In this case, the transplanted
organ was exposed to a fully youthful environment and
assumed a youthful phenotype. It is unfortunate these
studies did not measure changes, if any, in telomere
length, mitochondrial functioning, gene expression pat
terns (although the transcription rates of some genes ana
lyzed showed a youthful pattern), or in the number of
senescent cells present, aging markers of interest, in the
cells of rejuvenated tissues or organs. It should also be
noted that in the case of crossage transplantation the old
organ (which had only a small fraction of the mass of the
young donor into which it was transplanted) was rejuve
nated rather than the young recipient prematurely aging –
implying that the relative differences in mass determined
the age state of the cells of such an asymmetric joining.
One possibility is that rejuvenation requires the pres
ence (or absence or a required concentration) of a num
ber of different factors; eotaxin was not likely the only
contributing factor to the aging of the brain and loss of
cognitive functioning in aged animals. Of the cytokines
found to increase in concentration in the cerebrospinal
fluid of aging animals, others have been shown to have
aging effects [33]. Evidence that the reduced levels of at
least one cytokine, IL15, cause the symptoms of aging
(sarcopenia, immune senescence, and obesity) [34].
It seems that one conclusion that is borne out by
these studies is that if a cell is placed in a “young envi
ronment” or an “old environment” that cell will assume
the age phenotype appropriate to its environment. And
not to be coy, evidence indicates that an aged stem cell
placed in a young environment will rejuvenate if given
sufficient time.
Though the possibility of rejuvenation does not fit
into the present aging paradigm, it has been demonstrat
ed numerous times. The actuality of rejuvenation at the
cellular level represents proof that the present aging para
digm is incorrect. That rejuvenation can occur at the level
of the cell, tissue, and organ has been conclusively
demonstrated, so that the lack of interest in exploring this
phenomenon is surprising as it represents a potential
therapy for the diseases of aging. It seems indisputable
that aged tissues and organs become rejuvenated if bathed
in the circulating blood of young animals for sufficient
time.
So, the demonstrated ability of cells to rejuvenate and
the fact that the circulating blood of young animals is suf
ficient to bring about rejuvenation of cells, tissues, and
organs must make us reconsider any models of aging that
do not allow for this phenomenon. Two possible models
that would account for the observed aging phenomena are:
1) the accumulation, through aging, in the systemic envi
ronment of factors that produce the aging phenotype in
cells and/or the progressive loss of factors that promote
cellular “youthfulness”, or a combination of the two; and
2) a bloodborne messaging system designed to ensure that
the age phenotype of cells is appropriate to the age of the
organism, though these two mechanisms may be the same.
In the case of the accumulation of deleterious factors
or the loss of youthpromoting factors in the blood, one
would have to posit a mechanism for why accumula
tion/reduction occurred. The second model makes an
assumption that can account for both rejuvenation and
aging; it posits that aging is a programmed process and
coordination of cellular age phenotype throughout the
body is achieved through the bloodstream. Whichever is
the case, the experiments discussed give a clear indication
that aged cells, tissues, and organs can be rejuvenated,
and how this can be accomplished.
POTENTIAL FOR ORGANISMIC REJUVENATION
It has been determined that if a stem cell (at least) is
in an “environment” of a certain age (“young” or “old”),
it will assume the age phenotype of the cells of its type at
the age of the environment, given sufficient time. The
questions remaining are: What is meant by “age pheno
type”? What is meant by “environment”? And, how
much time is “sufficient”?
A cell’s “age phenotype” is taken to mean a set of
characteristics that distinguish older cells from younger
ones. It has been defined by various assays for increased
dysfunction and decreased (mostly) proliferation as well
as for various “markers” of aging as patterns of gene
expression and epigenetic “marks” – but only clearly dis
tinguishes old cells from young ones. Are there several
age phenotypes or is there a continuous increase of age
related dysfunction? The work of Chambers et al. [13]
showed that certain agerelated genes were up or down
regulated at particular stages of the mouse life cycle, cor
responding to what we would call young adulthood, early
middle age, late middle age, and old age; do the same
agespecific distinctions exist in other characteristics
associated with cellular aging such as telomere length,
mitochondrial metabolism, redox environments, and
proportion of senescent cells? Are there distinct age phe
notypes apart from “young”, “old”, and senescent? An
important practical question that needs to be answered
directly is what happens to senescent cells in a young
environment. The answer to these questions will have a
profound impact on our understanding of aging – partic
ularly on whether or not aging is a programmed process
and more importantly, whether or not cellular age phe
notype can be manipulated in situ, in an aged body so as
to rejuvenate it.

STUDIES THAT SHED NEW LIGHT ON AGING 1067
BIOCHEMISTRY (Moscow) Vol. 78 No. 9 2013
That the environment found capable of rejuvenating
cells in vivo was the blood or plasma of young animals is
supported both by crossage transplantation studies and
by parabiosis experiments. Parallel in vitro work per
formed in the original Conboy experiments [5] showed
that plasma obtained from the blood of young animals
was sufficient to rejuvenate old stem cells in cell culture,
and that young cells treated with plasma from old animals
experienced accelerated aging. Furthermore, it was noted
that young cells placed in an “old” environment appeared
to age immediately [32].
Whether it is a question of removing deleterious sub
stances from the blood plasma and stem cell niches of
aged animals, or providing youthpromoting factors, or
providing an internal environment of a certain “age”, then
the answer to organismic rejuvenation would be to
exchange as much blood or plasma as possible to reduce
the fraction of the original blood or plasma remaining. It
should be noted that the “environment” in the case of
heterochronic parabiosis consists of a mixture of the blood
of both young and old parabionts so that both partners
share a circulation that is about an equal mixture of young
and old blood, while in crossage transplantation, when
the tissues or organs of an old animal are transplanted into
a young body (or vice versa) – the recipient organ experi
ences blood that is entirely young or old. The near instan
taneous change in a young cell placed in an environment
of old plasma versus the much less profound changes in
the cells of an organism that is the younger heterochronic
parabiotic partner may result from the differences in con
centration of factors found in the aged plasma it is exposed
to. Using plasma exchange to replace parabiosis, it would
appear that the greater the reduction of the original aged
plasma (exchanging 87% of plasma requires two volumes
of donor plasma), can be obtained, and this might have a
faster and more complete rejuvenating affect.
At a recent Society for Neuroscience conference in
New Orleans, Saul Villeda related an experiment where
in the mere injection of young plasma into old mice (at
about 5% of total blood volume of plasma injected into a
mouse for each injection, 18 such injections for a month)
elicited significantly increased neurogenesis and cognitive
functioning – this experiment (not yet published) indi
cates that positive, “youthpromoting” factors are also
present in young serum as dilution of “aging factors”
would not have taken place considering the small quanti
ties of blood given. Indeed the results of the Villeda’s
experiments demonstrate that factors present in old plas
ma both negatively and positively affect neurogenesis,
synaptic plasticity, and cognitive functioning – should
recommend plasma replacement for the treatment of age
related dementias. If it is the presence of factors accumu
lated in an old environment and/or the absence of factors
(or changes in their concentrations), present in a young
environment, plasma from young animals should be suf
ficient to rejuvenate all stem cell types that are rejuvenat
ed by parabiosis or crossage transplantation. The same
holds true if the systemic circulation determines the age
phenotype through a process of signaling. In that case,
the blood or plasma of a young animal donor should con
tain all signaling factors needed to determine the age phe
notype of the recipient’s cells, at least initially, depending
upon the biological halflife of those factors. The evi
dence that heterochronic parabiosis extends the upper
limit of the species lifespan of the older parabiont, taken
together with the evidence that the tissues of the older
parabiont are rejuvenated, leads to the conclusion that
significant, if not total, rejuvenation of the organism may
take place. Hence, the entire organism might be rejuve
nated by substituting the blood/plasma of a younger ani
mal for its own. If the cellular age phenotype is deter
mined by stochastic factors, there should be a single age
phenotype, that of the young cell, but with varying
degrees of impairment. However, if the aging process is
programmed, and age phenotype is determined/coordi
nated by plasmaborne factors, then there will be a con
tinuum of age states marked by particular patterns of gene
transcription and of epigenetic marks. If such a situation
obtains, than bathing cells in the plasma of a particular
age will cause those cells to produce the transcription pat
terns, epigenetic marks, and mitochondrial ROS produc
tion that are specific to that age [1012]. Such a pro
grammed theory might assume that there are many cellu
larage phenotypes, with transcriptional patterns provid
ing age markers such that cellular age phenotypes might
be broadly classifiable as markers of developmental stages
(gastrula, toddler, early middle age, late middle age, etc.).
The problem with this model, therapeutically at least, is
that some or many of the important signaling molecules
associated with age phenotype determination may have
short halflives, and they may be replaced with molecules
made by the recipient’s body that would signal cells to
assume an older age phenotype. This situation can be
solved by multiple or continuous plasma exchange for the
time required for the recipient’s cells to change their age
phenotypes. It would seem that both the relative propor
tions of old vs. young plasma as well as the length of time
of exposure are both variables in determining the cell’s
age phenotype. The demonstration by Villeda of the anti
aging effects of young serum tends to bolster the notion
that whichever factors produce the youthful transforma
tions of old animals remains in the plasma for sufficient
time to have an effect.
HETEROCHRONIC PLASMA EXCHANGE (HPE)
MAY BE AN EFFECTIVE SUBSTITUTE
FOR PARABIOSIS
While it would appear that a series of exchange trans
fusions might be sufficient to rejuvenate a patient, the
risks (http://www.nhlbi.nih.gov/health/healthtopics/

1068 KATCHER
BIOCHEMISTRY (Moscow) Vol. 78 No. 9 2013
topics/bt/risks.html) and costs [35] associated with com
plete whole blood transfusions make it unacceptable for
therapeutic intervention on the massive scale that would
be needed. However, the demonstration, in vitro, of the
ability of young plasma to rejuvenate old stem cells and
even the brain, leads to the possibility that the in vivo
exchange of the plasma of an old animal for that of a
younger animal might be sufficient to bring about rejuve
nation if provided with the proper schedules of
exchanges. Like the blood of young organisms, “young”
plasma would lack those substances that induce aging and
contain those that permit or encourage cellular youthful
ness, assuming that no part of those factors is carried in
the cellular portion of blood. Experiments, using both
whole blood or plasma replacement, or a schedule of such
replacements (see below) between young and old individ
uals of mouse or rat strains could test whether blood or
plasma exchanges will lead to rejuvenation. Much of the
costs and risks of blood exchange transfusions would be
eliminated by using plasma exchange.
Plasma exchange is a form of plasmapheresis where
in blood is removed from the body, separated by either
centrifugation or filtration into a cellular fraction and
plasma – with the cellular portion is either mixed with
donor plasma or a plasma substitute and returned to the
patient; the patient’s plasma is discarded. Plasma
exchange is used to treat many conditions that benefit
from the removal of plasma or substances from plasma
including autoimmune antibodies and toxins [36].
It now seems clear that organismic aging is controlled
by genetic and epigenetic pathways and the exploration of
these pathways is being pursued by a number of laborato
ries. It is also clear that various cytokines and cellular pro
teins are involved in the signaling pathways that cause
aging in the same way that they drive development at all
stages of human life. These include IGF1, Notch, and
WNT signaling pathways, which have all been implicated
as being in pathways that appear to coordinate aging across
tissues and organs. The IGF1 pathway, in particular,
appears to cause the aging of bone marrow cells, while it
retards the aging of muscle satellite cells; hence, whatever
the signaling hierarchy is, IGF1 is not on top. To estab
lish what factors and at what concentrations are required
to reverse or induce aging might take decades, but we do
not need further elucidation of this phenomenon in order to
apply it. It appears that the plasma of young animals is suf
ficient to cause cellular rejuvenation of old stem cell pop
ulations. An analogy with electricity is in order: we used
electricity to light our homes and drive our machines well
before we had any idea of what electricity was; we knew
what its effects were and used those effects to our own
advantage. That said, as we know that the blood plasma of
young animals can rejuvenate the cells of old mammals, it
should be the case that the plasma of young humans will
also rejuvenate human cells. Even if not all stem and pro
genitor cell populations are rejuvenated, rejuvenation of
those stem cell populations that have already been shown
to be capable of being rejuvenated (e.g. muscle satellite
cells, liver progenitor cells, bone stromal cells, and
hematopoietic stem cells) would be sufficient to markedly
increase the quality of life of old people and likely result in
an increase in the duration of youthful health. This “reju
venation therapy” would include the near complete
replacement of an older person’s plasma with that of a
young person (i.e. heterochronic plasma exchange or
HPE) using a schedule sufficient to allow “cleansing” of
stem cell niches in order to rejuvenate stem cell popula
tions. Plasma exchange is an approved medical procedure;
the single, simple variation of exchanging old blood plas
ma for young, could be readily accommodated by existing
protocols and the procedure could be tested on people
tomorrow. Prudence cautions us to try animal experiments
first to test the principles and the extent of rejuvenation as
well as its safety, yet the technology of plasma exchange
has already been shown to be safe and the age of the plas
ma donor has never been material. Also, while the effec
tiveness of plasma exchange in “rejuvenating” cells has yet
to be demonstrated in humans (though it should logically
follow from experiments performed to date), the entrance
into senescence of a significant portion of the populations
of all industrialized nations at this time tells us to hurry
and demonstrate HPE in animals and assess it on human
volunteers as soon as possible.
FURTHER EXPERIMENTAL WORK
Establishing that blood exchange can functionally
replace heterochronic parabiosis would be the first step.
(If it could, that alone would allow researchers to exam
ine the question of environmental effects on cellular age
phenotype in a much easier and more controllable envi
ronment than by parabiosis.) What follows that would
depend on whether or not plasma exchange substituted
for transfusion exchange. If it did not, there would be the
requirement of blood fractionation and “addback”
experiments to determine which cellular components of
blood were required. Assuming that either transfusion
exchange or plasma exchange can replace parabiosis, the
next step would be to develop a timecourse of changes in
cellular age phenotype following either blood or plasma
exchange in order to develop a schedule of exchanges
necessary to produce “rejuvenation”. As cellular aging
and senescence appear to be behind all diseases of
aging – the object of a “rejuvenation therapy” resulting
from plasma or transfusion exchanges could be any or all
of the diseases of aging.
It will be important is to establish the time course of
blood/plasmainduced cellular changes in order to deter
mine how long young blood/plasma needs to be in con
tact with these cells in order to affect a change in their age
phenotypes. The heterochronic parabiosis studies of

STUDIES THAT SHED NEW LIGHT ON AGING 1069
BIOCHEMISTRY (Moscow) Vol. 78 No. 9 2013
Conboy et al. [5], Carlson et al. [27], and Villeda et al.
(2011) [24] all seem to point to an intermediate level of
rejuvenation when organisms are exposed to the com
bined blood of young and older parabionts. We find that
the rate of proliferation of the stem cells of rejuvenated
organs and tissues is somewhat less than that of chrono
logically young animals, though more than those of age
matched controls. It may be that stem cells “rejuvenated”
by this procedure achieve an age phenotype that is some
what between young and old. This should be investigated
in cell culture comparing blood/plasma derived from ani
mals of an intermediate age to mixed plasmas
(young/old); if aging results from a simple change in con
centration of positive or negative factors, then appropri
ate mixing of old and young plasmas should result in cel
lular rejuvenation equivalent to that achieved using the
plasma of an animal of intermediate age. In addition, it
will be most interesting to see whether stem cells from old
or young donors reach the same state when raised in plas
ma obtained from animals of a particular age – whether,
indeed, the cell’s history or environment contributes the
greater share to aging.
It would also be useful to see which cell types, stem
and progenitor cells, as well as other cycling cell types are
rejuvenated. Experiments with plasma fractionation will
begin to tell us about those factors that “cause”
aging/rejuvenation and these experiments can be per
formed in vitro.
Moreover, it would be interesting to note the effects
of “young” plasma (plasma derived from young animals)
on senescent cells. Crossage transplantation studies
show that old tissue is rejuvenated in a young host, but
they give no information on what happens to the senes
cent cells, which are invariably part of the tissues of an
aged organism. Are senescent cells rejuvenated or elimi
nated or do they remain as they were? And if eliminated,
is the elimination accomplished by the young immune
system or is it intrinsic, as in death by apoptosis? And of
course plasma fractionation experiments could determine
what the agechanging factors are.
While the parabiosis experiments are very difficult, in
vitro experiments using stem cells and plasma derived
from animals of particular ages should not be.
It has been demonstrated that aged cells can be reju
venated, so the lack of attention that the aging research
community has devoted to this most important phenom
enon is surprising and must be remedied before millions
more die of agerelated diseases.
REFERENCES
1. Medawar, P. B. (1952) Accumulation at Old Age through
Aging: An Unsolved Problem in Biology, HK Lewis Publisher,
London.
2. Williams, G. C. (1957) Evolution,11, 398411.
3. Kirkwood, T. B. L., and Rose, M. R. (1991) Phil. Trans. R.
Soc. Lond. B, 332, 1524.
4. Hass, R. (2009) Exp. Gerontol., 44, 634638.
5. Conboy, I. M., Conboy, M. J., Wagers, A. J., Girma, E. R.,
Irving, L., Weissman, I. L., and Rando, T. A. (2005)
Nature, 433, 760764.
6. Le Bourg, E. (2001) Demograph. Res., 4, 128.
7. Goldsmith, T. (2011) Aging by Design, Azinet Press,
Annapolis, ISBN 097887093X.
8. Kirkwood, T. B. L. (2005) Cell,120, 437447.
9. Kirkwood, T. B. L. (1977) Nature, 270, 301304.
10. De Boer, J., Andressoo, J. O., de Wit, J., Huijmans, J.,
Beems, R. B., van Steeg, H., Weeda, G., van der Horst, G.
T., van Leeuwen, W., Themmen, A. P., Meradji, M., and
Hoeijmakers, J. H. (2002) Science, 296, 12761279.
11. Wijnhoven, S. W., Beems, R. B., Roodbergen, M., van den
Berg, J., Lohman, P. H., Diderich, K., van der Horst, G. T.,
Vijg, J., Hoeijmakers, J. H., and van Steeg, H. (2005) DNA
Repair,4, 13141324.
12. Van der Pluijm, I., Garinis, G. A., Brandt, R. M. C.,
Gorgels, T. G. M. F., Wijnhoven, S. W., Diderich, K. E. M.,
de Wit, J., Mitchell, J. R., van Oostrom, C., Beems, R.,
Niedernhofer, L. J., Velasco, S., Friedberg, E. C., Tanaka,
K., van Steeg, H., Hoeijmakers, J. H. J., and van der Horst,
G. T. J. (2005) PLOS Biol.,5, 112.
13. Chambers, S. M., Shaw, C. A., Gatza, C., Fisk, C. J.,
Donehower, L. A., and Goodel, L. A. (2007) PLOS Biol.,5,
201213.
14. Gorbunova, V., Seluanov, A., Mao, Z., and Hine, C. (2007)
Nucleic Acids Res., 35, 74667474.
15. Sudo, K., Ema, H., Morita, Y., and Nakauchij, H. (2000)
Exp. Med.,192, 12731280.
16. Campisi, J. (2005) Cell, 120, 513522.
17. Baker, D. J., Wijshake, T., Tchkonia, T., LeBrasseur, N. K.,
Childs, B. G., van de Sluis, B., Kirkland, J. L., and van
Deursen, J. M. (2011) Nature,479, 232236.
18. Rando, T. A. (2006) Nature, 441, 10801086.
19. Conboy, M. (2009) Chap. 10. Methods in Bioengineering:
Stem Cell Bioengineering (The Artech House Methods in
Bioengineering Series) (Parekkadan, B., and Yarmush, M.
L., eds.).
20. Lanza, R. P., Jose, B., Cibelli, J. B., Blackwell, C., Vincent,
J., Cristofalo, V. J., Francis, M. K., Gabriela, M.,
Baerlocher, G. M., Mak, J., Schertzer, M., Chavez, E. A.,
Sawyer, N., Peter, M., Lansdorp, P. M., and West, M. D.
(2000) Science, 288, 665669.
21. Lapasset, L., Milhavet, O., Prieur, A., Besnard, E., Babled,
A., AitHamou, N., Leschik, J., Pellestor, F., Ramirez, J.
M., De Vos, J., Lehmann, S., and Lemaitre, J. M. (2011)
Genes Dev.,25, 22482253.
22. Rando, T. A., and Chang, H. Y. (2012) Cell, 158, 4657.
23. Zhangfa, S., Wang, J., Guachalla, L. M., Terszowski, G.,
Rodewald, H.R., Ju, Z., and Rudolph, K. L. (2010) Blood,
115, 14811489.
24. Villeda, S. A., Luo, J., Mosher, K. I., Zou, B., Britschgi,
M., Bieri, G., Stan, T. M., Fainberg, N., Ding, Z., Eggel,
A., Lucin, K. M., Czirr, E., JeongSoo Park, Couillard
Despres, S., Aigner, L., Li, G., Peskind, E. R., Kaye, J. A.,
Quinn, J. F., Galasko, D. R., Xie, X. S., Rando, T. A., and
WyssCoray, T. (2011) Nature,477, 9094.
25. Carlson, B. M., and Faulkner, J. A. (1989) Am. J. Physiol.
Cell Physiol.,256, 12621266.

1070 KATCHER
BIOCHEMISTRY (Moscow) Vol. 78 No. 9 2013
26. Zacks, S. I., and Sheff, M. F. (1982) Muscle Nerve,5, 152
161.
27. Carlson, B. M., Dedkov, E. I., Borisov, A. B., and Faulkner,
J. A. (2001) Biol. Sci.,56, 224233.
28. Harrison, D., Astle, C., and Doubleday, J. (1977) J.
Immunol.,118, 12231227.
29. McCay, C. M., Pope, F., Lunsford, W., Sperling, G., and
Sambhavaphol, P. (1957) Gerontologia, 1, 717.
30. Ludwig, F. C., and Elashoff, R. M. (1972) Trans. NY Acad.
Sci.,34, 58275835.
31. Silva, H., and Conboy, I. M. (2008) Aging and Stem Cell
Renewal, July 15, in Stem Book [Internet], Cambridge
(MA): Harvard Stem Cell Institute; 2008 (ailable from:
http://www.ncbi.nlm.nih.gov/books/NBK27025/).
32. Carlson, M. E., Suetta, C., Conboy, M. J., Aagaard,
Mackey, P. A., Kjaer, M., and Conboy, I. M. (2009) EMBO
Mol. Med.,1, 381391.
33. Gupta, S., Chiplunkar, S., Kim, C., Yel, L., and Gollapudi,
S. (2003) Mech. Ageing Dev., 124, 503509.
34. Charles, T., Lutz, C. T., and Quinn, L. S. (2012) Aging, 4,
535546.
35. Shander, A., Hofmann, A., Gombotz, H., Theusinger, O.
M., and Spahn, D. R. (2007) Best Pract. Res. Clin.
Anaesthesiol., 21, 271289.
36. Szczepiorkowski, Z. M., Winters, J. L., Bandarenko, N.,
Kim, H. C., Linenberger, M. L., Marques, M. B., Sarode,
R., Schwartz, J., Weinstein, R., and Shaz, B. H. (2010) J.
Clin. Apher., 25, 83177.