February 2010 | Vol 7 | N.º 2 | CNIC-17 [PDF (841K)]

Insights into stem cell aging

Pilar M. Daroca, Antonio Herrera-Merchán and Susana Gonzalez

Correspondence:
Susana Gonzalez
Foundation Spanish National Cardiovascular Research Centre Carlos III (CNIC)
Regenerative Cardiology Department
Melchor Fernandez Almagro 3
Madrid 28029; Spain
Email sgonzalez@cnic.es

Competing interests: The authors declared no competing interests.

Acknowledgements: We thank Simon Bartlett for editing assistance. This work was supported by grants from the Human Frontiers Science Program Organization (CDA026/2006), Spanish Ministry of Health (FIS-PI06/0627), and ProCNIC Foundation.

ABSTRACT
The increase in average life expectancy in many developed countries has generated an aging society and an associated increase in age-related health problems. Mammalian aging occurs, in part, because of a decline in the restorative capacity of tissue stem cells. The use of stem cells in regenerative medicine promises to revolutionise the treatment of acute and chronic degenerative conditions, and stem cell research holds the key to the development of such therapies. The hallmark of adult stem cells is their ability to both self‑renew and differentiate into multiple lineages. This demands a complex and still poorly understood network of molecular interactions between diverse cell-intrinsic regulators of self-renewal, such as certain Polycomb proteins and the tumour suppressor p16^INK4a, both of which are required for the maintenance of certain stem cell populations. Recent studies have begun to elucidate the molecular mechanisms by which stem cells decide between life and death, highlighting the importance of balance in the pathways as the stem cells age.

AGING AND STEM CELLS
Recent advances in medical research programs and better health care planning have had significant influence on people living in many Western countries, increasing both quality of life and average lifespan. With the extension of lifetime comes an increasing interest in slowing or reversing the negative effects of aging. The fascinating discovery of tissue-resident adult stem and progenitor cells in recent years has led to an explosion of interest in the development of novel stem cell-based therapies to improve endogenous regenerative capacity or to repair damaged and diseased tissues.

A major function of stem cells and their differentiation hierarchies may be to preserve the DNA integrity of the whole organism. Despite error-prevention mechanisms, mutations occur, and when they do, potent tumour-suppressor mechanisms such as senescence and apoptosis eliminate the damaged stem cell, preventing its replicative expansion. However, when unrepaired genetic lesions in a stem cell are passed on to its differentiated daughter cells and therefore accumulate with aging, replacement of the dead and non-functional cells requires newly differentiated cells derived from stem and progenitor cells. To date, the best-studied type of adult tissue stem cell is the hematopoietic stem cell (HSC), which gives rise to all of the mature blood cells in an organism throughout its life. Hematopoiesis in mammals occurs in distinct temporal waves, shifting from the extraembryonic yolk sac and fetal liver in embryos to the bone marrow in adults. Primitive HSCs are “true” stem cells, also termed the “long-term repopulating HSCs” (LT-HSCs), because they both maintain the stem cell population and replenish the pool of blood cells by allowing daughter cells to differentiate into the lymphoid, myeloid, and erythroid lineages. The daily replenishment of blood cells is achieved in large part by the division and subsequent stepwise differentiation of cells descended from the LT-HSC pool, namely short term repopulating HSCs (ST-HSCs) and the slightly more committed multipotent progenitor HSCs (MPP-HSCs). The relative quiescence of the LT-HSCs protects their genomic integrity by reducing the rounds of DNA replication and, thus, the probability of acquiring DNA damage that might compromise multilineage differentiation potential and/or render the HSCs malignant over time. Nevertheless, this pool still appears to age with the host¹. The rapid turnover of the hematopoietic system and the availability of advanced methods to study HSCs via different markers have made this system a widely studied model of the effects of aging on stem cell functionality (Figure 1). It is worth mentioning that, although some aspects of aging may be shared by all somatic stem cells, the mechanisms of aging are likely to be tissue-specific (for example, intestine, muscle and bone marrow).

The evidence for stem cell aging
A growing body of evidence shows that the capacity of stem cells to maintain tissue homeostasis declines with age and suggests that this decline may account for many age-related phenotypes and diseases². Significantly, engraftments of HSCs are capable of serial passages through a succession of mouse recipients, outliving the donor mouse^{3, 4}, though it is not possible to exceed five successful passages because foreign HSCs do not completely restore the hematopoietic system of the recipient mouse ⁵. Several years after transplantation, telomere length in the blood cells of the transplanted recipient are 1-2 kb shorter than those in the donor⁶, indicating that the level of telomerase in the HSCs is insufficient to prevent progressive telomere shortening. On the other hand, immunophenotypic characterisation of hematopoietic stem and progenitor cell subsets diverges from function in old animals. The engraftment efficiency of immunophenotypically selected, long-term HSCs from old mice is approximately threefold lower than that of equivalent cells from young mice^{7, 8}. Additionally, age-related changes in stem-cell function include myeloid-biased differentiation and decreased homing ability⁹. In conclusion, it has been proven that the properties of HSCs change in several ways with age, but it is still poorly known which intrinsic or extrinsic changes regulate the self-renewal and multilineage differentiation capacities of these cells¹⁰.

Although stem and progenitor cell proliferation guarantees tissue repair, and thereby regeneration, that proliferation can also lead to hyperproliferative diseases like cancer, a risk that is moderated by tumour-suppressor mechanisms. For example, while the increased expression of tumour suppressors with age (p53, p16^INK4a) inhibits the development of cancer (inducing apoptosis or/and senescence)^{11, 12}, that increase may have a negative effect on stem cell functionality, reducing the capacity for self-renewal or differentiation and ultimately leading to aging phenotypes^{13, 14}. Thus, many of the same mechanisms that contribute to cellular aging may also act as suppressors of neoplastic growth¹⁵ (Figure 2). We can develop a better understanding of age-related changes in stem cell function by genetically altering the expression of tumour suppressors and examining the consequences, an area of research that may improve effective longevity-promoting therapies.

SELF-RENEWAL REGULATORS IN ADULT HSCS
Stem cells are crucial for the homeostatic maintenance of mature, functional cells in many tissues throughout the lifetime of an animal, and this pool of stem cells must itself be maintained. Maintenance is achieved by self-renewal, a specialised type of cell division in which one or both daughter cells remain undifferentiated and retain essentially the same replication potential of the parent. The self-renewal program must involve dedicated regulatory genes¹⁷, but although the phenotypic and functional properties of HSCs have been extensively characterised, we have only just begun to understand how self-renewal is regulated.

Bmi1 as a proto-oncogene
Recent studies have shown that Polycomb group (PcG) proteins play an important role in the regulation of the self-renewal and lineage restriction of HSCs. It is also reported that PcG genes regulate cellular memory by silencing genes through chromatin modifications. These features make PcG genes interesting subjects for stem cell research because it is conceivable that dynamic reprogramming of cells, for instance, during differentiation, requires alterations in the epigenetic state of genes. Two distinct multiprotein PcG complexes have been identified. Polycomb repressive complex 2 (PRC2) is involved in the initiation of silencing and contains histone deacetylases and histone methyltransferases, which can methylate histone H3 lysine 9 and 27 (markers of silenced chromatin) and histone H1 lysine 26. Deletion of PRC2 genes in mice results in early embryonic lethality, underscoring their importance in development. Polycomb repressive complex 1 (PRC1) is implicated in the stable maintenance of gene repression and recognises, by means of a chromodomain, the H3 lysine 27 methyl group set by PRC2. Thus, the two PcG complexes could function in a cooperative manner to maintain gene silencing¹⁸. Mice mutant for most PRC1 members survive until birth as a result of partial functional redundancy provided by homologs, an exception being Rnf2 deficiency mice¹⁹One prominent self-renewal regulator is Bmi-1, a component of PRC1, which is required for the self-renewal of all postnatal stem cell populations examined, including HSCs and neural stem cells^20-22.Moreover, Bmi-1 is considered a proto-oncogene that regulates chromatin structure by recruiting epigenetic regulators to specific loci¹⁹. The capacity of Bmi1 to maintain HSC self-renewal largely depends on the silencing of one of its targets, the locus encoding the p16^INK4a and ARF tumour suppressors²³ .Deletion of p16^INK4a and/or ARF partially rescues the self-renewal defects observed in various stem cell populations from null-mice, including HSCs and neural stem cells^{24 , 25-27}. These results demonstrate that Bmi1 regulates HSCs by acting as a critical failsafe mechanism against the premature loss of HSCs induced by p16^INK4a and ARF-dependent senescence pathways. Therefore, it seems that repression of p16^INK4a and ARF is a fundamental requirement for the maintenance of adult stem cells.

The tumour suppressors p16^INK4a and ARF. Cell-cycle regulators such as the INK4/ARF locus appear to play an important role in the reaction of adult stem cells to stress and aging. The INK4/ARF locus plays a central role in tumour suppression, reflected in its inactivation in almost 50% of human cancers²⁸. Indeed, this locus is regarded as one of the most important anti-oncogenic defences of the mammalian genome, comparable in importance only to p53. The remarkable feature of the INK4/ARF locus is that it encodes three tumour suppressors in a genomic segment of about 50 kb: p16^INK4a, its related family member p15^INK4b, and ARF (called p19^ARF in mice and p14^ARF in humans). The actions of p16^INK4a, p15^INK4b font-family: Times'> and ARF are well understood. lang=EN-GB style='font-family: Times'>Both p16^INK4a and p15^INK4b inhibit the kinase activity of CDK4/6-cycD complexes, thus contributing to the maintenance of the active, growth-suppressive form of the retinoblastoma (Rb) family of proteins. ARF contributes to the stability of p53 by inhibiting the p53-degrading activity of MDM2. Through the activation of Rb and p53, the INK4/ARF locus is able to induce cell senescence and cell death^29,30. These tumour suppressors have recently taken on additional importance because at least one product of the locus, p16^INK4a, also contributes to the decline in the replication potential of self-renewing cells during the aging of stem cells. The expression of p16^INK4a is relatively low in the HSCs of young mice, but is upregulated with age or in response to cellular stresses³¹. Although the number of immunophenotypic HSCs increases with age in wild-type animals, HSC functionality is impaired. In particular, the HSC compartment of old animals is more rapidly exhausted by serial transplantation than that of young animals. In contrast, aging has the opposite effect on p16^INK4a-/- HSCs, with p16^INK4a-/- HSCs from old animals substantially outperforming young p16^INK4a-/- HSCs in serial transplantation assays ³¹. In fact, old p16^INK4a-/- HSCs perform as well as young wild-type HSCs in this assay. Thus, p16^INK4a compromises HSC functionality in older mice. Similar results were obtained in studies of p16^INK4a-/- neuronal stem cells and pancreatic islets^25,32, revealing a general role for p16^INK4a in the regulation of stem cell and progenitor cell aging. Therefore, on one side of the coin, p16^INK4a acts as a potent tumour suppressor that promotes longevity by suppressing the development of cancer, whereas on the other side, the increase in p16^INK4a level with age impairs the proliferation of stem or progenitor cells, ultimately reducing longevity. These observations suggest the provocative, but as yet unproven, notion that mammalian aging results in part from the beneficial effects of tumour suppressor proteins (Figure 2).

The transcription factor p53
p53 is another tumour suppressor protein that influences stem cell self-renewal, tissue regenerative capacity, age-related disease, and cancer. In fact, p53 activity is lost in nearly half of all human cancers³³. The p53 protein is normally inactive, due in part to its rapid degradation by the specific ubiquitin ligase Mdm2. A multitude of stresses converge on p53 through complex and partially understood signalling pathways that stabilise and modify p53. Analysis of the role of p53 in aging has revealed a dual role that seems to depend on the intensity of p53 activity. Mice that overexpress short isoforms of p53 have greater protection against tumour development than wild-type mice, while at the same time show signs of premature aging^{34, 35}. However, mouse models of increased wild-type p53 activity do not demonstrate premature aging. In particular, bacterial artificial chromosome transgenic mice that bear a third copy of the p53 locus show a decreased cancer incidence, but normal longevity and normal onset of aging phenotypes^36-38. Another mouse model, the super-INK4a/ARF mouse, with an extra copy of the entire INK4a/ARF locus (ARF being an activator of p53), shows a significantly reduced incidence of cancer, although the mice age normally³⁸. Synergistically, mice that bear a third copy of the p53 locus and a third copy of the INK4/ARF locus show increased longevity and delayed aging in a manner that cannot be explained by their reduced incidence of cancer³⁷. Therefore, though the effects of p53 and INK4/ARF locus expression in aging are context and dosage dependent, these results suggest that, during physiological aging (identified by a moderate increase of normally-regulated p53 activity), damaged stem cells are eliminated by either self-destruction (apoptosis) or by removing them from the proliferative pool (senescence). In contrast, the presence of uncontrolled p53 activity (e.g., caused by massive DNA damage) results in the excessive elimination of stem cells, which exhausts tissue regeneration capacity, leading to premature aging.

THE INK4/ARF LOCUS AND AGE-ASSOCIATED PHENOTYPES
p16^INK4a and ARF may also be important to diseases of aging beyond their function in stem cells. Specifically, three research consortia that undertook genome-wide association studies across large, carefully annotated patient samples have reported an association between single nucleotide polymorphisms (SNPs) near the INK4a/ARF locus and frailty³⁹, atherosclerotic heart disease (ASHD)^{40 41}, and type-2 diabetes^{42, 43}. However, at least a few of the associated SNPs are not in linkage disequilibrium with each other, suggesting that more than one polymorphism near the locus influences these aging phenotypes. Therefore, although these studies do not pinpoint specific polymorphisms that affect the incidence of age-related diseases, there are only four genes in the vicinity of the mapped polymorphisms: p16^INK4a, ARF, p15^INK4b, and ANRIL (a noncoding RNA). Additional data suggest specific links: p16^INK4a expression increases with age in pancreatic β cells, and p16^INK4a deficiency increases β-cell regenerative capacity³², providing a mechanism by which polymorphisms that affect p16^INK4a expression or activity might increase a person’s risk for type-2 diabetes. It remains unclear whether these polymorphisms influence the risk of frailty and heart disease via their effects on tissue regenerative capacity, via mechanisms that are completely independent of stem/progenitor cells, or by a combination of the two. Nevertheless, in light of the murine genetic studies that link the INK4a/ARF locus and stem cell function, proteins encoded by the locus are the most likely candidates to mediate the effect of these polymorphisms on the incidence of these age-related diseases.

CONCLUSIONS
The regenerative capacity of many stem cells declines functionally with age, and this decline partly triggers the development of many age-related symptoms and diseases. Certain tumour suppressors, like p16^INK4a, suppress the proliferation of stem or progenitor cells in the bone marrow, pancreas and brain. Thus, p16^INK4a seems to mediate an equilibrium between reducing cancer incidence, which promotes longevity, and decreasing stem cell self-renewal and proliferation, compromising tissue regeneration and repair, which is likely to reduce longevity. These observations allow us suggest the provocative but unproven hypothesis that mammalian aging results, in part, from the efforts of tumour suppressor proteins to prevent cancer. Determining how stem cells age, by isolating reliable biomarkers, deregulated signalling pathways, and the mechanisms leading to the loss of self-renewal and the acquisition of defects in stem cells, will contribute to our understanding of age-associated pathophysiological decline. Likewise, it is essential to characterise the cellular and molecular components of stem cell niches, determine how the niche changes during aging, and determine whether senescent stem or support cells alter the niche. The treatment or replacement of aged and dysfunctional adult stem cells may provide novel avenues to treat aging and age-related disorders, including hematopoietic and immune disorders, heart failure and cardiovascular diseases, neurodegenerative, muscular and gastrointestinal diseases, atherosclerosis and cancer.