Aging, Rejuvenation, and Epigenetic Reprogramming: Resetting the Aging Clock Thomas A. Rando Howard Y. Chang

The inexorable tolls of aging are evident in almost all living beings. From the onset of reproductive maturity, organismal aging is generally characterized by a decline in fecundity, an increased susceptibility to disease and tissue dysfunction, and increased risk of mortality (, , ). Aging is associated with a gradual loss of homeostatic mechanisms that maintain the structure and function of adult tissues. A major challenge of aging research has been to distinguish the causes of cell and tissue aging from the myriad of changes that accompany it. One of the hallmarks of cellular aging is an accumulation of damaged macromolecules such as DNA, proteins, and lipids. These become chemically modified by reactive molecules, such as free radicals, that are generated during normal cellular metabolism and whose production increases with age (). DNA damage may lead to cellular dysfunction directly by altering the expression of specific genes or indirectly as result of cellular responses to damage that can alter gene expression more globally (, ). Damage to proteins may independently contribute to cellular aging if misfolded or damaged proteins are replaced more slowly than they are generated, especially when they form stable aggregates that are not degraded by the cell (). Such “proteotoxicity” has been postulated to underlie many age-related diseases and may also be an important part of normal cellular aging ().

The consequences of age-related changes to the macromolecular components of a cell, particularly for long-lived postmitotic cells like neurons and myofibers, lead to gradual loss of normal structure and function—so-called “chronological aging,” marked simply by the passage of time. For continuously dividing cells, like those of the epithelia of the skin or gut, there is the added challenge of “replicative aging,” referring to the accumulation of cellular damage, such as telomere shortening and replication-associated DNA mutations, that occurs during the process of cell division (, ). This is particularly relevant for adult stem cells because they divide throughout the life of the individual and therefore experience both chronological and replicative aging (). As the burden of mutations increases with age, the likelihood that a cell will undergo apoptosis, malignant transformation, or senescence, which for diving cells means irreversible cell-cycle arrest (), also increases. Although cellular function invariably declines with age, it may be that some of the changes, for example senescence and apoptosis, are actually adaptive in order to prevent cellular transformations such as metaplasia or neoplasia that may result from age-related genomic instability.

Despite the fact that aging appears to be inexorable, with the ultimate result being the death of the organism, it is incontrovertible that life span itself can be experimentally manipulated. An unlimited number of genetic defects and environmental challenges that may have no relation to the normal drivers of aging can shorten life span, but both genetic and environmental interventions have been shown to extend the life span of model organisms such as the nematode worm Caenorhabditis elegans (C. elegans), the fruit fly Drosophila melanogaster, and laboratory mice (, ). For example, mutations in individual genes in the insulin/insulin-like growth factor signaling pathway, in the pathways of protein translation involving the enzyme mammalian target , , ). In terms of environmental influences that can extend life span, none is better studied or more broadly effective, from C. elegans). However, extending life span is not equivalent to delaying aging. Interventions may prevent common causes of death (for example, improved safety features to prevent automobile accidents as a sociological intervention or treatment of acute infectious illnesses as a medical intervention), without changing the fundamental rate of organismal aging. Nevertheless, it does seem that many so-called “longevity genes,” as well as dietary restriction, appear to extend not only life span, but also “health span” (, ). In that regard, it does appear that it is possible to experimentally slow the rate of aging. Still, in each case, aging does continue on as if there is some clock that is driving individual aging, ticking relentlessly toward old age and death.

Though these examples support the notion that the process of aging can be slowed, there are also clear examples in nature when the aging clock appears to be temporarily arrested. The average life expectancy of C. elegans is about 2 weeks. However, under specific conditions such as food scarcity, the developing larvae can adopt an alternate fate, called a dauer stage, that does not feed and is metabolically relatively inactive (). Dauer larva can survive for months, effectively prolonging the life span of the worm by an order of magnitude. Such diapauses are common in nature and reflect evolutionarily conserved responses to periods of adverse environmental conditions when survival is at stake. Even more dramatic examples of prolonged periods of survival in an arrested state come from the study of seeds and spores. Viable seeds capable of germination and growth have been obtained from excavations and dated as being 2,000 years old (). Viable bacterial spores have been found preserved in amber for 25–30 million years () and preserved in salt crystals for 250 million years (). Such examples reflect the remarkable survival ability of life forms under extreme conditions. These forms of life exist in suspended states that appear to arrest fundamental biological processes, including any aging that may accompany those processes, uncoupling the process of biological aging from chronological aging measured by the passage of time. However, is there reason to hypothesize that it is possible not only to arrest, but to reverse the aging clock?

Despite the apparently unidirectional and inexorable process of aging of individual metazoans, the ability of the aging clock to be not only halted but reversed, or “reset to zero,” is so deeply embedded in the nature of life itself that it should not be surprising. Yet the process appears so mysterious that it is difficult to reconcile with concepts of individual aging. We are referring here to the resetting of the aging clock that comes with every fertilization event, giving rise to a zygote that will ultimately mature into an adult member of the species. For humans, this involves the fusion of two cells, a sperm and an egg, each of whose chronological age is measured in decades, to form a single cell that somehow erases any trace of the age of the parental cells. This resetting, or “reprogramming,” of the zygotic nucleus, rewinding the aging clock to begin anew, is mediated by factors in the oocyte cytoplasm that are at the heart of this mystery of rejuvenation. Granted, though the “biological age” of germ cells may differ from the biological age of other cells in the soma, there is no evidence that germ cells exist in any kind of diapause or metabolic arrest, completely resistant to the myriad of age-related changes that occur in cells over time. Therefore, the erasure of any manifestations of germ cell aging is central to the survival of the species. Were it not for this resetting of the aging clock, species would age with each generation and ultimately fail to propagate as the germline increasingly bore the burden of the effects of aging that occur during maturation to reproductive maturity.

The reprogramming process that is so central to fertilization, even though poorly understood, was exploited in the very earliest cloning experiments using somatic cell nuclear transfer (SCNT), in which the nucleus of a mature somatic cell is transferred to an enucleated oocyte (). The pioneering work of Dr. John Gurdon in this area showed for the first time that differentiated nuclei from tadpole intestinal or muscle cells could be transferred into enucleated Xenopus eggs and give rise to mature and fertile male and female frogs (). Like the fused nucleus of the sperm and egg during fertilization, the somatic nucleus is reprogrammed by the oocyte cytoplasm to allow the development of a new member of the species, resetting any hallmarks of aging that the somatic nucleus bore upon transplantation. Importantly, these studies challenged the dogma at the time that the process of aging and differentiation from a single fertilized egg to a mature adult involved the loss of genetic material, which would in essence be an irreversible process rendering the resulting nuclei incapable of recapitulating the embryological developmental program. This early work demonstrated unequivocally that the full battery of genetic material present at fertilization and necessary to give rise to an adult organism is maintained in cells through development and maturation to adulthood. They also showed that the nuclei of adult somatic cells, just like that of the genetic material in the adult sperm and egg, can be apparently rejuvenated and can have pluripotency restored in the context of the oocyte cytoplasm.

The fact that the nuclei were capable of giving rise to viable embryos that were themselves capable of developing into fertile adults and did not exhibit premature aging is evidence that the chronological age of the donor nuclei had been reset. Thus, just like the example of fertilization, SCNT appears to be capable of resetting the aging clock for propagating a species. Whether there are any age-related alterations of the transplanted nucleus, for example in the genome, that were not erased by the process of SCNT cannot be ruled out and raises the fundamental question of how to define a young or old nucleus on a molecular level (this issue is considered in a later section). SCNT is, of course, also the process that gave rise to the first cloned mammal, Dolly the sheep (), which led to an explosion of research in cloning. In those studies, nuclei from different developmental and adult tissues were used and yielded viable lambs. Dolly actually died young (at 6 years of age as opposed to the typical life span of about 12 years for her breed). But the cause of death was a viral illness, not a premature aging syndrome. Dolly was fertile and gave birth to numerous offspring. Still, whether animals that are cloned from adult nuclei by SCNT are normal in terms of health and longevity remains to be fully determined. The majority of cloned animals often die in early development or display growth defects postnatally, possibly due to incomplete epigenetic reprogramming () but also possibly due to genomic changes in the donor nucleus or technical limitations associated with the SCNT process. Still, SCNT demonstrates the remarkable ability of the oocyte cytoplasm to reprogram the donor nucleus, not only erasing manifestations of differentiation, but also resetting the chronological age.

Recent advances in stem cell biology have begun to unlock the molecular secrets behind these reprogramming events that occur during fertilization and SCNT. Specifically, these advances refer to the discovery of the process to create induced pluripotent stem cells (iPSCs) (), by which a variety of terminally differentiated adult cells, initially from mouse and human and more recently from other species, can be converted to pluripotent stem cells by the introduction of a small number of transcription factors such as Oct4, Sox2, and Klf4 (). Pluripotency is a cellular property defined as the ability to give rise to differentiated cells in all three germ layers of the embryo: the ectoderm, the endoderm, and the mesoderm. In that regard, iPSCs resemble embryonic stem cells (ESCs), pluripotent stem cells derived from the inner cell mass of an early embryo and able to both give rise to any cell type in the body and support complete fetal development (). Likewise, iPSCs can generate an entire mouse embryo, including the germline (). Indeed, the ability of a limited number of transcription factors to effect iPSC reprogramming stems from their key roles in the ESC gene expression program (). Furthermore, detailed analyses of multiple iPSC lines have shown that their global gene expression programs and chromatin states are remarkably similar to those of ESCs, though not equivalent (, ).

One possible rejuvenating consequence of iPSC reprogramming is reactivation of the expression of telomerase (), the enzyme responsible for maintaining telomere length and long-term self-renewal potential in ESCs (). Genetic defects in components of the telomerase complex may prevent the restoration of telomere length and full telomerase activity during iPSC reprogramming in some conditions but not others (, ). Nevertheless, the fact that differentiated adult cells can be directly reprogrammed to iPSCs by known factors rather than by the complex oocyte cytoplasm has generated yet another paradigm whereby the aging clock is reset. However, just like SCNT, the process of generating iPSCs is inefficient, and the vast majority of cells fail to attain the status of a pluripotent stem cell capable of giving rise to a new, fertile organism (). There is clearly an important process of selection of those cells that are most fit and amenable to reprogramming, although the molecular bases of this fitness test are only now beginning to be revealed.

Although we describe each of these processes in the context of resetting of the aging clock, the major emphasis of research in the field of reprogramming is, in fact, reversal of the differentiation program and the attainment of a pluripotent state (), not the reversal of aging. In all of the examples presented, rewinding of the aging clock is coupled to the reversal of the differentiation program. The cell, whether a sperm or egg in the case of fertilization or an adult somatic cell (or its nucleus) in the case of SCNT or iPSC generation, not only rejuvenates, but also completely loses its differentiated characteristics. Indeed, this “dedifferentiation” is at the essence of the process of fertilization, as well as the ability of either SCNT or iPSC generation to create ESC-like cells. In each case, the cell (or its nucleus) ceases to maintain its identity as a particular differentiated cell type and, instead, adopts a pluripotent state coupled to the adoption of a more youthful state. However, to restore youthful properties to aged tissues for therapeutic purposes, for example to improve wound healing in aged skin or to improve cardiac function in the aged heart, the ideal would be to reset the aging clock but to leave the differentiation program untouched. Converting aged cardiomyocytes to pluripotent stem cells might yield no beneficial effect and might, in fact, have profound detrimental consequences. One of the major limitations of ESCs and iPSCs therapeutically is the fact that, upon transplantation, they have the propensity to form teratomas, tumors that have the features of all three germ layers (). By contrast, converting aged cardiomyocytes into young cardiomyocytes, without going through a pluripotent state, could improve cardiac function directly. This raises the fundamental question: Is it possible to uncouple the resetting of the aging clock from the resetting of the differentiation program?

Recent studies have begun to test the potential of different interventions to restore youthfulness to aged cells or tissues. Although not specifically designed to address the question posed above as to whether cell or tissue rejuvenation can be achieved without dedifferentiation, evidence suggests that it may be possible to uncouple the processes that “maintain” the aged state (a concept that we will return to) from those that maintain the differentiated state. The following are three examples of apparently rejuvenating interventions—one environmental, one genetic, and one pharmacologic—that result in apparently more youthful states of aged cells that retain their differentiated states.