The objectives of previous review (Aging is Linked to Stem Cell Ware Out) were to critically review what is known about the effects of aging on stem cells in general. At present, evidence is marshaled in support of the hypothesis that aging stem cells play a critical role in determining the effects of aging on organ function, and ultimately on the lifespan of a mammal.

Aging is a continuous process beginning at the union of the sperm and the egg. The zygote, of course, is the ultimate stem cell with unlimited developmental potency or plasticity. Development in complex multicellular organisms is all about the compartmentalization and specialization of physiological functions into organ systems. With it goes compartmentalization of stem cells that to various degrees retain some multipotency. A number of factors may conspire to alter the overall multipotency of the stem cell population during aging. For example, as a result of damage acquired through replication or toxic metabolic products, a stem cell's repertoire of developmental programs may be restricted, until in old age it may be able to contribute to only one lineage – and perhaps even that not very well, at least in response to stress.

Age and stem cell numbers
If stem cells play a role in the limitation of organism's longevity, the simplest explanation of how they might do so would be a decline in their numbers in old age. The relationship is clearly not that simple. The evidence that the number of human stem cells declines significantly in old age is not compelling, nor is it in mice. The qualitative changes in stem cells and the composition of the stem cell population with respect to qualitatively distinct subclasses is the important factor in stem cell aging. If we are to accept that the total number of stem cells is evolutionarily conserved in mammals, clearly stem cells of a human have a far bigger job cut out for them over a lifetime than their murine counterparts. Not only is a human roughly 3000 times larger, but also we live about 40 times longer. The discrepancy in the output of mature blood cells from the same number of stem cells in the two species is daunting. Human hematopoietic stem cells are either qualitatively much different, possessing greatly enhanced developmental and replicative potency to begin with, or have exceptional restorative and damage-repair mechanisms. An alternative explanation, of course, is that mouse stem cells have the same properties but, for other reasons, don't live long enough for stem cell potential to be fully realized. The literature on aging and stem cell numbers is confusing. The incidence of anemia and neutropenia in geriatric patients is significantly higher than in the young or middle-aged, although the insufficiencies are usually not life threatening. Rather, the overall hematopoietic suppression is a hallmark of the lack of marrow reserves to counter the stress of infection, blood loss, or rebound following chemotherapy. For example, in mice the tempo of recovery from severe bleeding is dramatically slowed in old animals compared to young mice. The amount of active bone marrow in humans steadily diminishes from childhood, during which the cavities of most bones are active, to old age, during which active marrow is restricted to the pelvis, sternum, and vertebrae, and where even there the marrow cellularity is reduced. Moreover, the number of naturally circulating CD34+ cells in the blood of centenarians is reduced, although this could be due to a number of factors, the most likely of which is not necessarily depletion of marrow reserves.

Age and stem cell replication
The rate of stem cell replication during aging has not been well studied. A hallmark of the population of stem cells from normal young and middle-aged mice or humans is its overwhelming quiescence. In contrast, stem cells from very old mice were found to have a significantly increased fraction in cell cycle. Although there were no obvious signs of end-stage disease in these animals, it is possible that the increased stem cell proliferation was secondary to chronic inflammation or infection. There, of course, is precedent for a natural, rapidly replicating stem cell population in the hematopoietic system during development when it is expanding, but similar cell cycle kinetics late in life lack a cogent explanation. Heightened cell cycle kinetics in old stem cells may be related to increased leukemogenesis in old mice. Bromodeoxyuridine (Brdu) administration has been used to label the stem cell compartments of young, but not old, mice. As expected, when a short pulse of Brdu is given, there is a very small labeling index, consistent with a population-wide average quiescence. Surprisingly, however, long-term administration in the drinking water of mice showed that essentially all stem cells replicated at least once about every 2 months. These results are at odds with the concept that stem cells are deeply quiescent in G₀ phase, many of which may not enter cycle during a mammal's lifetime. In this apparently mistaken view, they would enter cycle only in concert with proliferation and differentiation, as proposed in clonal selection theories. It should be noted that the findings with continuous Brdu labeling do not formally rule out clonal selection; all stem cells may replicate on a regular and relatively frequent basis without necessarily embarking on the development of a multilineage clone contributing wholly or in part to hematopoiesis. It is of great interest to the present discussion if the pattern of cell cycle kinetics of stem cells is altered during aging. It has been suggested that an increased proportion of stem cells in cycle might be correlated with the increased incidences of leukemia, lymphoma, and myelodysplastic syndrome in old age. Thus, it may be that enhanced replication may signal the loss of cell cycle controls in a preneoplastic condition.

Age and stem cell homing
Stem cells in all tissues are closely associated with stromal cells, which provide structural support and maintain the position of stem cells in the context of the organ, as in the crypts of villi in the gastrointestinal lining. They also variously secrete cytokines and provide supporting extracellular matrices to protect stem cells from apoptosis, prevent their entry into a replicative cycle, or prevent activation for differentiation. This review has focused on the cell-intrinsic effects of aging on hematopoietic stem cells with the knowledge that the extrinsic environment, including the stromal microenvironment, surely plays an important role in the response of stem cells to aging. Little is known concerning the effects of age on stem cell – supporting stroma in any tissue, partly because our understanding of which cells constitute the important stromal elements is inconclusive. Transplant studies in which the functional properties of young and old hematopoietic stem cells have been compared almost invariably have used young transplant recipients. Thus, stem cells are compared in the context of young stroma. Since old mice tolerate radiation so poorly (a further example of reduced tolerance to stress), there are compelling practical considerations preventing their use as transplant recipients. In a direct approach to the issue, large numbers of bone marrow cells from young donors was transplanted into old hosts either before or after an erythropoietic stress was induced by bleeding. Infusion of young stem cells did not ameliorate the slow recovery of erythropoiesis in old mice relative to young ones. This suggests that stroma may play an important role in the response of stem cells of any age, that old stroma blunts hematopoietic responses, and lastly that the effects of aging on stroma are not reversed by circulating stem cells or other stromal progenitors from a young donor. In a rare study using both young and old bone marrow transplant recipients, increased post transplant autoimmunity occurred in old recipients irrespective of the donor age, suggesting that age-related damage to stroma caused dysregulation of the immune system. Despite evidence that at least some, and perhaps most, stromal elements of the hematopoietic system are derived from hematopoietic stem cells, the issue of whether hematopoietic stroma can be transplanted remains controversial. Successful homing of stem cells clearly involves important interactions between stem cells and appropriate stroma. Old stem cells home to young stroma with about a 75% reduced efficiency compared to young stem cells, apparently reflecting an age-acquired defect in the stem cells themselves.

Effect of age on stem cell telomere regulation
Regulation of telomeres is an increasingly complex issue. Renewed interest in their importance to mammalian aging, replicative stress, and cancer etiology in the last several years has led to their study in stem cells. The tacit assumption has been that telomeres serve as a counting mechanism in stem cells, as in other cell types, in which the length of telomeres shortens with each round of replication until a critical short length is reached that signals cell senescence. Although the length of telomeres at chromosome ends is important in maintaining genomic stability, it is increasingly clear that telomeres are also the substrate for the binding and interaction of multiple proteins that provide stability. Telomerase and an RNA subunit responsible for hybridizing with DNA telomere repeats is now recognized to have a complex role in telomere biology. In cells in which it is synthesized, telomerase not only lengthens telomeres but, through mechanisms not well understood but independent of telomere lengthening, affect a cell's response to genotoxic damage and progression toward neoplasia. Although most of the seminal basic biology of telomeres has been carried out in nonmammalian systems, the study of mice in which one or the other of the telomerase subunits has been mutated has demonstrated the effects on rapidly proliferating tissues and neoplasia in mammals. Despite the fact that hematopoietic (and presumably other) stem cells produce telomerase, telomeres of stem cells have been shown to progressively shorten during aging and following hematopoietic stress, especially that following stem cell transplantation. The paradox between telomerase expressions in stem cells, yet measurable telomere shortening during hematopoietic stress, is most likely explained by insufficient amounts of telomerase to prevent telomere erosion but perhaps sufficient amounts to retard it. There is on one hand a correlation between age and stem cell properties associated with their quality and on the other a correlation between hematopoietic stress and telomere lengths of stem cells. Whether or not telomere length or stability can be mechanistically linked to stem cell quality remains to be shown. The importance of telomeres, telomerase, and chromosomal stability in tumorigenesis suggests that such a link will be found. Neoplastic conversion of cells to a cancerous phenotype often involves the derailing and subordination of a normal control mechanism.

Response of stem cells to extracellular cues during aging
The impaired homing of stem cells to micro environmental niches in bone marrow following transplantation is but one of several examples that the response of stem cells to extrinsic signals is altered during aging. Unfortunately, in experiments in vivo it is often impossible to distinguish between an altered response of stem cells to the environment, or vice versa. There is growing evidence that many of the properties of the stem cell population are determined by cell-intrinsic mechanisms. Although clearly the milieu in which stem cells find themselves can have powerful effects on the developmental programs in which they participate, cell-intrinsic factors may have an overriding influence by determining the receptivity of stem cells to those signals. If one accepts the concept of stem cell plasticity, one could argue that extrinsic cues exclusively determine developmental potency. Irrespective of whether the soil or the seed is more important, the hematopoietic tree doesn't grow without both being present. Clearly, the most important interface between the stem cell and its environment is the plasma membrane and the signaling proteins embedded in it. Such intrinsic regulators include the myriad cytokine and chemokine receptors, cell adhesion molecules, and autocrine regulators produced by stem cells. The stem cell repertoire of such molecules may change, either dramatically or subtly, during aging just as the availability of the complementary ligands in the environment may change with age. Through elaborate and interactive feedback loops, changes in either stem cells or the environment may affect the other.

What mutations that cause premature aging tell us about aging stem cells
The study of the causes of aging and of the factors that limit longevity in mammals is hampered by the long lifespans of the test subjects. For this reason the bulk of these studies have been done in fruit flies, worms, and yeast. Premature aging syndromes in mice and humans have provided model systems of accelerated aging that have yielded important clues into the mechanistic pathways and genes important in aging. The difficulty has been in determining whether or not the progression of a candidate syndrome accurately reflects the normal events in aging. Syndromes such as Bloom's, Werner's, and trichothiodystrophy (TTD) share the common feature of having defective DNA repair processes that in each was traced to a mutation in a single but separate gene. As in normal aging, Bloom's, Werner's, and TTD are associated with a heightened risk of developing cancers. To the extent that these syndromes mimic normal aging, they are consistent with a model in which accumulated genetic damage is not adequately repaired, resulting in defective transcription, decreased chromosomal stability, and increased apoptosis. Perhaps not surprisingly, defects are especially profound in rapidly proliferating tissues, including the hematopoietic system. Specific studies have not yet focused on hematopoietic stem cells in these and other accelerated aging syndromes, but such may yield important insights to the mechanisms of aging in stem cells of the hematopoietic and other systems. An additional mutation that underscores the close ties between tumorigenesis and stem cell regulation is a hypermorphic mutation in the tumor suppressor p53 gene (p53m) that almost completely suppresses carcinogenesis in the mutant mice but, surprisingly, produces a phenotype reminiscent of aging. The mice have a reduced lifespan, osteoporosis, and very low resistance to stress and show hematologic defects at an early age. For example, the mice regenerate their bone marrow and progenitors slowly and incompletely after 5-fluorouracil administration. It remains unclear how heightened activity of a molecule normally associated with the induction of apoptosis, cell cycle arrest, or senescence of neoplastic cells would lead to a premature aging phenotype. It nonetheless may provide mechanistic clues to the effectors of normal aging.

Aging has especially powerful implications for humans. Advances in public health and medicine have greatly increased the average lifespan of people living in societies with access to those benefits – so much so that the reproductive years of most people's lives are but a small fraction of their total lifespan. In these societies the average age of the population is rapidly increasing, leading to new requirements in health care planning and a revamping of economic considerations to accommodate a populace older than the age at which they traditionally have retired from the workplace. With the extension of lifespan, there is an increasing interest in slowing or reversing the deleterious effects, some real and some perceived, of aging. The yearning for eternal youth plays an impressive role in the economies of developed countries; witness the robust cosmetics industry, the demand for cosmetic surgery, and growing interest in being cryopreserved after death. Increasingly, people are asking: where is the limit to longevity? And what can be done to increase it while maintaining quality of life?