[Cell Cycle 4:3, 407-410; March 2005]; 2005 Landes Bioscience

Aging, Stem Cells and Tissue Regeneration

Perspectives

Lessons from Muscle

ABSTRACT

*Correspondence to: Irina M. Conboy; Department of Neurology and Neurological

Sciences; Stanford University School of Medicine; Stanford, California 94305-5235
USA; Email: iconboy@berkeley.edu
Received 01/06/05; Accepted 01/07/05

Previously published online as a Cell Cycle E-publication:

http://www.landesbioscience.com/journals/cc/abstract.php?id=1518

KEY WORDS

UT
E

Among the universal changes that occur with age are the declines in cognitive, motor,
and sensory abilities. The impairment of these complex processes is accompanied by a
myriad of age-related structural and biochemical changes in tissues that subserve those
functions. Cells from aged animals have evidence of cumulative oxidative damage and
impaired mitochondrial functions.1,2 These and other age-dependent changes in cellular
components lead to physiologic changes and to impaired responses of tissues to injury. The
latter is exemplified by poor would healing of skin, reduced angiogenesis in damaged
organs throughout the body, and limited remyelination in the central nervous system in
response to demyelinating conditions.3-5 Such changes can be attributed to an age-related
decline in the ability of resident stem cells to engage in tissue repair. Stem cells have been
found in virtually every tissue in which they have been sought.6 These cells participate not
only in regenerative responses to acute injury, but also in the general maintenance function
of replacing cells that are lost during normal organismal activity. Thus, age-related
reduction of stem cell function can account for the more subtle and gradual effects of the
aging process on various organs in addition to the effects on the acute responses to injury.
This is true for tissues in which the cellular turnover is rapid, such as blood and intestinal
epithelia,7,8 and tissues in which turnover is very slow, such as skeletal muscle, cardiac
muscle, and brain.9-11
Even though skeletal muscle has very low rate of cellular turnover under normal conditions, it has a remarkable capacity to regenerate in response to injury. With age, there is a
gradual decline in the effectiveness of the regenerative response of skeletal muscle to
damage.12 Instead of the rapid formation of new muscle fibers following a necrotic injury,
aged muscle responds with a delayed and only partially effective myogenic response, often
leaving a fibrous scar where normal muscle would have formed in a younger individual. In
addition to a diminished regenerative response, aging results in muscle fiber atrophy and
a general loss of muscle bulk and strength. Age-related atrophy, like the decline in regenerative potential with age, may be also due to an age-related decline in muscle stem cell
function.
It is well established that resident stem cells, termed satellite cells, account for virtually
all of the regenerative potential of skeletal muscle.13 The designation of satellite cell
comes from the fact that they are quiescent cells closely apposed to the mature muscle cell
(myofiber) membrane and underneath its basal lamina. Such cells account for perhaps

20

05

LA

ND

ES

BIO

SC

IEN

CE

aging, muscle, regeneration, satellite cell, serum

factors, parabiosis, Notch, stem cell

RIB

address: Department of Bioengineering, UC Berkeley, Evans 479,

Berkeley, CA 94720-1762, USA

Present

IST

2GRECC and Neurology Service; VA Palo Alto Health Care System; Palo Alto,
California USA

OT
D

of Neurology and Neurological Sciences; Stanford University School

of Medicine; Stanford, California USA

ON

1Department

With age, there is a gradual decline in the regenerative properties of most tissues due
to a combination of age-dependent changes in tissue-specific stem cells and in the environmental cues that promote those cells to participate in tissue maintenance and repair.
In adult skeletal muscle, where the resident dedicated stem cells (satellite cells) are
capable of rapid and highly effective regeneration in response to injury, there is just such
a loss of regenerative potential with age. Satellite cell activation and cell fate determination are controlled by the Notch signaling pathway that is initiated by the rapid increase
in expression of the Notch ligand, Delta, following injury. In old muscle, this upregulation
of Delta is blunted and thus satellite cell activation is markedly diminished. However, by
indirectly inducing Notch activity, the regenerative potential of aged satellite cells can be
restored. Furthermore, exposure of aged satellite cells to serum from young mice, either
in vivo by heterochronic parabiotic pairings or in vitro, rejuvenates the satellite cell
response. This restorative potential suggests that tissue-specific stem cells do not lose their
ability to participate in tissue maintenance and repair. Therefore, it may be that even very
old stem cells may be capable of maintaining and repairing aged tissues if provided with
optimal environmental cues.

.D

Irina M. Conboy1,,*
Thomas A. Rando1,2

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Aging, Stem Cells and Tissue Regeneration

23% of the nuclei in the tissue.14 In adult muscle, injury to the

tissue results in a stereotypical cascade of cellular and molecular
events that ultimately lead to the formation of new multinucleated
myofibers from satellite cells. Although no single biochemical marker
has been found that identifies all satellite cells or that is specific to
those cells, the identification of sensitive markers, such as M-cadherin, CD34, Myf5, Pax7, and syndecan-4,15,-17 has facilitated the
study of satellite cells both in vivo and in vitro. Recently CXCR4
and 1-integrin have been characterized as markers of a highly
myogenic satellite cell subset.18 In response to injury, these quiescent
stem cells activate to become highly proliferative intermediate
progenitor cells and go on to become fusion competent myoblasts.
Myoblasts eventually align and fuse to form new, multinucleated
myofibers to repair or replace the injured muscle. With age, injury
to muscle results in the generation of fewer myoblasts and thus fewer
cells available to form new myofibers.9,19,20
Is the decline in the generation of myoblasts in aged muscle due
to a loss of satellite cells? The estimates of changes in satellite cell
number with age vary depending on the species and the specific
muscle groups examined. These estimates range from a reduction,20,21
to a negligible change,9 to a relative increase in satellite cells in specific muscle groups.22 Although most studies suggest a gradual
decline of satellite cell number with age, direct comparisons are
confounded not only by the use of different muscles from different
species, but also by the different methods used to estimate cell
number. In some studies, satellite cells have been quantified by
assessing the production of fusion-competent myoblasts in culture
and extrapolating back to the number of satellite cells that might
have given rise to those myoblasts. This would tend to underestimate,
and perhaps vastly underestimate, the number of resident satellite
cells if a significant percentage of those cells in aged muscle either
failed to activate or activated but followed different lineage pathways.19,20 Nevertheless, probably the most important point is that
the decrease in the generation of myoblasts from aged muscle is far
greater than can be accounted for by any estimate of any decline
satellite cell number with age.9,19,20
Therefore, it appears that it is primarily an age-related loss of
satellite cell functionality, not satellite cell number, that underlies the
loss of regenerative potential and the generalized atrophy of aged
skeletal muscle. An age-related impairment of satellite cell function
could be due to an intrinsic aging of the resident stem cells rendering
them less responsive to environmental cues, to an age-related decline
in the extrinsic signals that instruct these stem cells to participate in
tissue repair, or to some combination of the two. An understanding
of the age-related decline in satellite cell activity has emerged from
deciphering the cellular and molecular control of adult myogenesis.23 We have recently shown that is through the activation of the
Notch signaling pathway that the proliferative expansion and
myogenic lineage progression of satellite cells is regulated and maintained.24 Among the first biochemical changes associated with satellite
cell activation is the upregulation of the Notch ligand, Delta, in both
the satellite cells and adjacent myofibers.9,24 This leads to an increase
in Notch signaling in satellite cells and their progeny, all of which
express Notch, and promote proliferation. As these cells expand to
form a pool of intermediate progenitor cells, the Notch antagonist
Numb is upregulated and is localized asymmetrically in dividing
myogenic progenitor cells.24 Such an asymmetric segregation would
be expected to result in an asymmetric cell division, with one daughter
inheriting high levels of the protein and the other daughter inheriting
low levels. High levels of Numb promote progression along the
408

myogenic lineage pathway,24 inducing the progenitor cells to become

fusion competent myoblasts and, ultimately, differentiated multinucleated myofibers. The critical role of the Notch signaling pathway
in these different phases of satellite cell activation and muscle regeneration is demonstrate by the dramatic inhibition of these process
when a Notch inhibitor is introduced into injured muscle.9
Inhibition of Notch signaling in injured, adult muscle leads to a
dramatic impairment of muscle regeneration, comparable to the
diminished regenerative response seen in aged muscle.9 Some of the
key issues that remain unresolved are the molecular mechanisms
controlling the upregulation and the asymmetric distribution of
Numb and the upregulation of Delta.
In aged muscle, have satellite cells lost the ability to respond to
extrinsic cues? Or, alternatively, is it that aged satellite cells are
perfectly capable of responding to environmental cues to begin
activating and generating myoblasts, but the failure of activation is
due to inadequate environmental cues? A complex picture emerges
when the molecular signaling involved in the activation of satellite
cells is analyzed in young and old animals. The levels of Notch
receptor is not changed with age,9 demonstrating that at least some
of the key aspects of the Notch signaling pathway remain largely
intact. However, in response to injury, there is a failure of upregulation
of Delta in aged muscle fibers and satellite cells.9 It is at present
unclear if it is the upregulation of Delta on adjacent muscle fibers or
on satellite cells themselves that initiates Notch signaling in myogenic
progenitor cells. However, this is clearly a key feature of aged muscle
that accounts for diminished regenerative potential since the forced
activation of the Notch signaling pathway by a pseudo-ligand can
restore the regenerative potential to aged muscle.9 In these studies,
the direct activation of Notch in injured, aged muscle, even in the
absence of Delta upregulation, leads to activation of aged satellite
cells, the generation of myoblasts, and effective tissue repair, very
similar to what is seen in the younger animal.9 Thus, there appears
to be no irreversible loss of potential of aged satellite cells. In this
context, a modification of the extrinsic cues restores the regenerative
potential to aged muscle.
The results of these studies suggest that failure of skeletal muscle
maintenance and repair in aged animals may be due more to an
age-related decline in environmental cues than an age-related decline
of stem cell function or potential. Such an interpretation was in fact
suggested by earlier heterochronic transplantation studies. In those
studies, minced muscles from young or old donors (rats or mice)
were transplanted into either young or old recipients, and the
potential for those minced muscles to regenerate was evaluated.25,26
Interestingly, it appeared that the host environment was more
important in determining the regenerative efficacy than was the age
of the donor. In other words, both young and old minced muscle led
to effective regeneration in young animals, whereas both young and
old minced muscle led to ineffective regeneration in old animals.
However, these earlier studies had not excluded the possibility that
host cells may have contributed to the effective regeneration in the
young animals, and could not distinguish among the many possible
host factors, including endocrine, immunologic, paracrine, neural,
and angiogenic factors, that enhanced the regenerative potential of
aged progenitor cells.
In order to determine whether the regenerative potential of aged
satellite cells is enhanced in a young environment and to discern
among the various possible contributions, we recently studied muscle regeneration in parabiotically paired mice.27 In this experimental
situation, two mice are connected surgically and develop a shared

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Aging, Stem Cells and Tissue Regeneration

circulatory system, thus exposing the tissues of one mouse to the

circulating factors of the other mice. Under these conditions, the
regeneration of aged muscle was markedly improved when an old
mouse was paired with a young mouse (heterochronic parabiosis)
but not with another old mouse (isochronic parabiosis). This
enhanced regeneration was paralleled by an increase in satellite cell
activation and generation of myoblasts following injury, and the
progenitor cell response had the youthful molecular signature of
increased expression of the Notch ligand Delta.27 The improved
regeneration of the aged muscle was unequivocally due to the
enhanced activation of the aged, resident satellite cells and not to any
contribution from circulating cells from the young pair as determined
by pairing GFP+ young mice GFP- aged mice and finding that the
engraftment of GFP+ cells into the regenerating old muscle was negligible.27 Rather, it appeared to be that the new systemic milieu that
rejuvenated the regenerative capacity of the old cells, and that new
milieu included both an exposure of the aged cells to young systemic
factors and the reduction in their exposure to old systemic factors.
These in vivo results were also confirmed by the ability of young
serum to promote satellite cell activation from old muscle in vitro.27
Importantly, heterochronic parabiosis also enhanced progenitor cell
proliferation in liver where the enhancement was accompanied by a
restoration of the youthful molecular signature of that tissue, namely
with a reduction of the cEBP-/BRM cell cycle regulatory complex.27
Thus, the age-related effects of the systemic factors on the regenerative potential of the organ stem cells are likely to be general, rather
than muscle-specific. Interestingly, the aged systemic milieu
appeared to have an inhibitory effect on the regeneration on young
tissues, the extent of which varied among different tissues.27
Together these data suggest that extrinsic cues regulating stem cell
activation (possibly both positively and negatively) are present in the
circulation, that the composition of these factors becomes altered
with age, and that exposing aged progenitor cells to young serum has
the potential to restore their regenerative potential. Thus, the capacity
of the aged stem cells to repair tissues remains intact, but their regenerative potential is not successfully triggered in the aged environment.

SUMMARY

The biological and clinical implications of these findings for the

field of regenerative medicine are profound. The most important
finding is that aged tissue hasnt irrevocably lost the ability to regenerate as well as younger tissue in response to damage. Rather, there
may remain, in the form of resident progenitor cells, a regenerative
potential that is untapped because of systemic changes with age. If
these systemic factors can be identified, and to the extent that they
can be manipulated without toxicity, it may be that there is a vast
regenerative potential of aged tissues that can be induced as needed.
A corollary of this point is the idea of transplanting stem cells,
including bone marrow and embryonic stem cells, into aged individuals for tissue repair needs to be considered in the context of these
findings. The age of the stem cells may be of less importance than
the age of the host. It is possible that the transplanted cells may show
a diminished response in the aged environment, just as the endogenous
progenitor cells do. Therefore, restoring the systemic environment to
its youthful state might be important for assuring that donor-derived
cells not only successfully compete with endogenous cells for the
niche in the tissue of interest, but also maintain their regenerative
potential in the aged organism and contribute to tissue repair. Thus,
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identifying systemic factors that regulate the behavior of endogenous

stem cells is likely to be critically important to restore the regenerative
potential of aged or diseased tissues.
In addition to the response of tissues to injury, these studies may
also have implications for the basic biology of the aging process.
Nearly all tissues experience turnover of their cellular constituents
over time, ranging from very rapid and complete turnover as occurs
in blood and gut epithelium, to a very slow and limited turnover as
occurs in muscle, liver, and brain. Clearly, tissue-specific stem cells
are responsible for the restorative aspect of these tissue dynamics. As
loss of tissue function is one of the hallmarks of aging, it may be that
a loss of this maintenance function of resident progenitor cells is
tantamount to tissue aging. Understanding the environmental cues
that instruct resident stem cells to participate in the normal maintenance function may suggest interventions that would slow the
age-related decline in tissue structure and function by enhancing the
ability of resident stem cells to maintain the youthful phenotype of
the tissue. To the extent that there are irreversible biochemical
changes with age, the gradual loss of tissue function may be
inevitable but may be slowed, thereby helping to achieve the goal of
healthy aging.
References
1. Sohal RS, Mockett RJ, Orr WC. Mechanisms of aging: An appraisal of the oxidative stress
hypothesis. Free Radic Biol Med 2002; 33:575-86.
2. Ames BN. Delaying the mitochondrial decay of aging. Ann NY Acad Sci 2004; 1019:406-11.
3. Thomas DR. Age-related changes in wound healing. Drugs Aging 2001; 18:607-20.
4. Edelberg JM, Reed MJ. Aging and angiogenesis. Front Biosci 2003; 8:s1199-209.
5. Franklin RJ, Zhao C, Sim FJ. Ageing and CNS remyelination. Neuroreport 2002; 13:923-8.
6. Young HE, Duplaa C, Romero-Ramos M, Chesselet MF, Vourc'h P, Yost MJ, Ericson K,
Terracio L, Asahara T, Masuda H, Tamura-Ninomiya S, Detmer K, Bray RA, Steele TA,
Hixson D, el-Kalay M, Tobin BW, Russ RD, Horst MN, Floyd JA, Henson NL, Hawkins
KC, Groom J, Parikh A, Blake L, Bland LJ, Thompson AJ, Kirincich A, Moreau C,
Hudson J, Bowyer FP 3rd, Lin TJ, Black AC Jr.. Adult reserve stem cells and their potential for tissue engineering. Cell Biochem Biophys 2004; 40:1-80.
7. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL. The aging of
hematopoietic stem cells. Nat Med 1996; 2:1011-6.
8. Martin K, Potten CS, Roberts SA, Kirkwood TB. Altered stem cell regeneration in irradiated intestinal crypts of senescent mice. J Cell Sci 1998; 111:2297-303.
9. Conboy IM, Conboy MJ, Smythe GM, Rando TA. Notch-mediated restoration of regenerative potential to aged muscle. Science 2003; 302:1575-7.
10. Capogrossi MC. Cardiac stem cells fail with aging: A new mechanism for the age-dependent decline in cardiac function. Circ Res 2004; 94:411-3.
11. Kuhn HG, Dickinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult
rat: Age-related decrease of neuronal progenitor proliferation. J Neurosci 1996; 16:2027-33.
12. Grounds MD. Age-associated changes in the response of skeletal muscle cells to exercise
and regeneration. Ann NY Acad Sci 1998; 854:78-91.
13. Zammit PS, Heslop L, Hudon V, Rosenblatt JD, Tajbakhsh S, Buckingham ME,
Beauchamp JR, Partridge TA. Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers. Exp Cell Res 2002;
281:39-49.
14. Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, Beauchamp JR. Muscle satellite cells adopt divergent fates: A mechanism for self-renewal? J Cell Biol 2004; 166:347-57.
15. Beauchamp JR, Heslop L, Yu DS, Tajbakhsh S, Kelly RG, Wernig A, Buckingham ME,
Partridge TA, Zammit PS. Expression of CD34 and Myf5 defines the majority of quiescent
adult skeletal muscle satellite cells. J Cell Biol 2000; 151:1221-34.
16. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Pax7 is
required for the specification of myogenic satellite cells. Cell 2000; 102:777-86.
17. Cornelison DD, Filla MS, Stanley HM, Rapraeger AC, Olwin BB. Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell
maintenance and muscle regeneration. Dev Biol 2001; 239:79-94.
18. Sherwood RI, Christensen JL, Conboy IM, Conboy MJ, Rando TA, Weissman IL, Wagers
AJ. Isolation of adult mouse myogenic progenitors: Functional heterogeneity of cells within and engrafting skeletal muscle. Cell 2004; 119:543-54.
19. Schultz E, Lipton BH. Skeletal muscle satellite cells: Changes in proliferation potential as
a function of age. Mech Ageing Dev 1982; 20:377-83.
20. Bockhold KJ, Rosenblatt JD, Partridge TA. Aging normal and dystrophic mouse muscle:
Analysis of myogenicity in cultures of living single fibers. Muscle Nerve 1998; 21:173-83.
21. Renault V, Thornell LE, Eriksson PO, Butler-Browne G, Mouly V, Thorne LE.
Regenerative potential of human skeletal muscle during aging. Aging Cell 2002; 1:132-9.
22. Gibson MC, Schultz E. Age-related differences in absolute numbers of skeletal muscle
satellite cells. Muscle Nerve 1983; 6:574-80.

Cell Cycle

409

Aging, Stem Cells and Tissue Regeneration

23. Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration.
Physiol Rev 2004; 84:209-38.
24. Conboy IM, Rando TA. The regulation of Notch signaling controls satellite cell activation
and cell fate determination in postnatal myogenesis. Dev Cell 2002; 3:397-409.
25. Zacks SI, Sheff MF. Age-related impeded regeneration of mouse minced anterior tibial
muscle. Muscle Nerve 1982; 5:152-61.
26. Carlson BM, Faulkner JA. Muscle transplantation between young and old rats: Age of host
determines recovery. Am J Physiol 1989; 256:C1262-6.
27. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation
of aged progenitor cells by exposure to a youthful systemic environment. Nature 2005;
433: 760-4.

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