**1. Introduction**

The increase in average life expectancy in many developed countries is generating 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 revolutionize 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 tumor suppressor p16INK4a, both of which are absolutely required for the maintenance of certain stem cell population. Recent studies have begun to elucidate the molecular mechanisms underlying how stem cells decide between life and death, and highlight the importance of balance in their aging pathways.

### **2. Aging and stem cells**

Recent advances in medicine research programs, and a better health care planning, have great influences in people living in many Western countries, increasing both quality of life and average lifespan. With the extension of lifetime, there is 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. When mutations occur despite certain errorprevention capacities, potent tumor-suppressor mechanisms such as senescence and apoptosis eliminate the damaged stem cell, limiting its replicative expansion. However, when unrepaired genetic lesions in stem cells are passed on to their differentiated daughters, and accumulate with aging, it is required replacement of dead and nonfunctional cells with newly differentiated cells derived from stem- and progenitor-cells. To date, the best-studied adult tissue stem cell type is the hematopoietic stem cell (HSC), which gives rise to all of the mature blood cells, throughout the life of the organism. Hematopoiesis in mammals occurs in distinct temporal waves shifting from the

Insights Into Stem Cell Aging 249

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 agerelated phenotypes and diseases (Kirkwood and Austad, 2000). Significantly, engraftment of HSCs are capable of serial passages through a succession of mouse recipients, outliving the donor mouse (Ross et al., 1982; Siminovitch et al., 1964), though it is not possible to exceed up to five successful passages, and the recipients do not restore the hematopoietic system to the normal state (Gordon and Blackett, 1998). On the other hand, telomere length in blood cells of the transplanted recipient are 1-2 kb shorter than those in the donor, when evaluated several years following transplantation (Allsopp et al., 2003), which indicates that the level of telomerase is insufficient to prevent progressive telomere shortening in HSC. On the other hand, immunophenotypic characterization 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 approximately is threefold lower than that of the equivalent population from young mice (Morrison et al., 1997; Yilmaz et al., 2006). Also, age-related changes in stem-cell function include myeloid-biased differentiation and decreased homing ability (Liang et al., 2005). In conclusion, it has been extensively proved that the properties of HSCs change in several ways as they age, but still is poorly known which are the changes in the intrinsic and extrinsic factors involved that regulate the self-renewal and multilineage differentiation capacities of these regenerative cells

Although the stem- and progenitor-cell proliferation guarantees tissue repair, and thereby regeneration, it can also develop hyperproliferative diseases, like cancer, risk that is moderated by tumor-suppressors mechanisms. For example, while the increased expression of tumor suppressors with age (p53, p16INK4a) inhibits the development of cancer (inducing apoptosis or/and senescence) (Krishnamurthy et al., 2004; Ressler et al., 2006), over time it may have a negative effect on stem cell functionality, reducing capacity for self-renewal or differentiation, and ultimately leading to aging phenotypes (Beausejour and Campisi, 2006; Rodier et al., 2007). Thus, it is thought that many of the same mechanisms that contribute to cellular aging also act as suppressors of neoplastic growth (Campisi, 2005) (Figure 2). We will therefore need a better understanding of age-related changes in stem cell function by altering genetically the expression of tumor suppressors, which may improve effective

Stem cells are crucial for the homeostatic maintenance of mature, functional cells in many tissues throughout the lifetime of the animal, and this pool of stem cells must itself be maintained (Muller-Sieburg and Sieburg, 2008). This is achieved by self-renewal, a specialized 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 the activity of dedicated regulatory genes (Gazit et al., 2008); but although the phenotypic and functional properties of HSCs have been characterized extensively, we have

**3. The evidence for stem cell aging** 

(Huang et al., 2007).

longevity-promoting therapies.

**4. Self-renewal regulators in adult HSCs** 

only just begun to understand how self-renewal is regulated.

extraembryonic yolk sac and fetal liver in embryos to bone marrow in adults. Primitive HSCs are the "true" stem cells, also termed the long-term repopulating HSCs (LT-HSCs), because they replenish the pool of blood cells by both maintaining the stem cells and allowing daughter cells to differentiate into the lymphoid, myeloid, and erythroid lineages. The daily replenishment of blood cells is achieved in large part by divisions and subsequent stepwise differentiation of cells descendants of LT-HSC pool, namely short term repopulating HSC (ST-HSC), and slightly more committed hematopoietic progenitor cells (MPP-HSC). The relative quiescence of LT-HSCs pool 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 them malignant over time, though they appear to age with the host (Orkin and Zon, 2008). The rapid turnover of the hematopoietic system and the availability of advanced methods to study HSCs by different markers have led to this system being widely used as a model of the effects of aging on stem cell functionality (Figure 1). It is worthy to mention that although some aspects of aging may be shared by all somatic stem cell fractions, the mechanisms of aging are likely to differ between stem cell populations located in specific tissues (for example, intestine, muscle and bone marrow).

Fig. 1. The hierarchically primitive cells of the hematopoietic system. Long-Term hematopoietic stem cells (LT-HSC) maintain hematopoiesis by coordinating self-renewal, and production of short-term HSC (ST-HSC), and subsequently, the multipotent progenitors (MPP), which have an incredible capacity to divide and make other types of cells as they mature, although a limited ability to self-renew. Ultimately, this generates an array of mature blood cells with different functions: lymphoid blood cells (the B-cells; T-cells; natural killer or NK cells; plasma cells; dendritic cells and others), and erythroid and myeloid blood cells (the erythrocytes or red blood cells; megakaryocytes or platelet producing cells; granulocytes such as neutrophils, eosinophils, and basophils; and monocytes which make macrophages). The stem and progenitor cells can be purified to near-homogeneity by surface markers. For example, LT-HSCs express low levels of lineage markers, high levels of Sca1 and CD117/c-KIT receptor, and low levels of CD34 (LSK CD34 lo). With limited renewal potential, the ST-HSC pool has a similar surface immunophenotype to LT-HSC except that it has higher levels of CD34 (LSK CD34 hi). As ST-HSC in turn proliferates to form more differentiated MPP, they increase expression of another surface marker, FLK2 (LSK CD34 hi Flk2 hi).

extraembryonic yolk sac and fetal liver in embryos to bone marrow in adults. Primitive HSCs are the "true" stem cells, also termed the long-term repopulating HSCs (LT-HSCs), because they replenish the pool of blood cells by both maintaining the stem cells and allowing daughter cells to differentiate into the lymphoid, myeloid, and erythroid lineages. The daily replenishment of blood cells is achieved in large part by divisions and subsequent stepwise differentiation of cells descendants of LT-HSC pool, namely short term repopulating HSC (ST-HSC), and slightly more committed hematopoietic progenitor cells (MPP-HSC). The relative quiescence of LT-HSCs pool 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 them malignant over time, though they appear to age with the host (Orkin and Zon, 2008). The rapid turnover of the hematopoietic system and the availability of advanced methods to study HSCs by different markers have led to this system being widely used as a model of the effects of aging on stem cell functionality (Figure 1). It is worthy to mention that although some aspects of aging may be shared by all somatic stem cell fractions, the mechanisms of aging are likely to differ between stem cell populations located in specific tissues (for example, intestine, muscle and bone

Fig. 1. The hierarchically primitive cells of the hematopoietic system. Long-Term

hematopoietic stem cells (LT-HSC) maintain hematopoiesis by coordinating self-renewal, and production of short-term HSC (ST-HSC), and subsequently, the multipotent progenitors (MPP), which have an incredible capacity to divide and make other types of cells as they mature, although a limited ability to self-renew. Ultimately, this generates an array of mature blood cells with different functions: lymphoid blood cells (the B-cells; T-cells; natural killer or NK cells; plasma cells; dendritic cells and others), and erythroid and myeloid blood

cells (the erythrocytes or red blood cells; megakaryocytes or platelet producing cells; granulocytes such as neutrophils, eosinophils, and basophils; and monocytes which make macrophages). The stem and progenitor cells can be purified to near-homogeneity by surface markers. For example, LT-HSCs express low levels of lineage markers, high levels of Sca1 and CD117/c-KIT receptor, and low levels of CD34 (LSK CD34 lo). With limited renewal potential, the ST-HSC pool has a similar surface immunophenotype to LT-HSC except that it has higher levels of CD34 (LSK CD34 hi). As ST-HSC in turn proliferates to form more differentiated MPP, they increase expression of another surface marker, FLK2

marrow).

(LSK CD34 hi Flk2 hi).
