Aging and the Blood

Introduction

Age-related changes to normal blood cell development and function remain poorly understood but measurably evident. In 1961, Hayflick and Moorhead described experiments that established the concept that normal somatic cells have a finite number of cell divisions. After completing this limiting number of cell divisions, a resting cellular phase, or senescence, is irreversibly entered. These postmitotic cells do not immediately die, however. They may survive for several years with normal function but with biochemical changes that ultimately affect themselves and potentially neighboring cells. Cellular senescence has long been used as a cellular model for understanding the mechanisms underlying the aging process, and this may be particularly important for age-related blood cell changes. Extensive observations have suggested that DNA damage accumulates with age and may be due to an increase in the production of reactive oxygen species (ROS) and a decline in DNA repair capacity with age. Mutation or disrupted expression of genes that increase DNA damage often result in premature aging. In contrast, interventions that enhance resistance to oxidative stress and attenuate DNA damage contribute toward longevity.

In this chapter, we will update observations that characterize aging blood cells with the hope that these findings will help provide insight into underlying mechanisms associated with aging, particularly those that can be altered by interventions. Overlap with and potential significance for aging of recently discovered genetic and epigenetic changes identified in several hematologic conditions will be explored. Finally, highlights in the area of blood cell immunosenescence will be discussed. In that blood, bone marrow, and lymphoid tissues are among the most accessible of tissues for human experimental study, advances in this area continue to provide insights into our general understanding of the normal and pathologic physiology of aging. Age-related cytopenias, myelodysplastic and myeloproliferative disorders, chronic lymphocytic leukemia, and other clonal lymphoid disorders are increasingly being recognized as ideal model systems to study the intersection of tissue aging, molecular changes, and physiologic effects.

Sites of Blood Cell Development: Bone Marrow and Stroma

Healthy individuals produce billions of red and white blood cells every day under normal conditions. With infection, bleeding, or other stresses, production is increased in response to complex physiologic mechanisms. The process of hematopoiesis begins with a limited number of hematopoietic stem cells (HSCs), which serve as the reservoir for the progenitors that generate mature blood cell production while maintaining the stem cell compartment. The sites of hematopoiesis change during mammalian development. During the first 6 to 8 weeks of human embryonic life, the yolk sac is the site of hematopoiesis, followed by a fetal liver stage. With further development, the bone marrow becomes the major site of hematopoiesis, other than pathologic disorders such as myeloproliferative neoplasms (MPN) and thalassemia, in which extramedullary hematopoiesis in the spleen, liver, and other sites outside of the bone marrow may occur. Elegant murine studies have tracked the migration of HSCs through these various tissues and identified the earliest site of definitive hematopoiesis in the embryo as the aorta-gonad-mesonephros (AGM) region.

The bone marrow is a complex specialized environment. At birth, the bone marrow is a fully hematopoietically active tissue but, with aging, there is replacement with hematopoietically inactive adipose tissue. A transition of approximately 1%/year in the bone marrow is a rough standard when assessing clinical bone marrow sample cellularity in individuals of different ages. Bone marrow is a diverse cellular mix, minimally including fibroblasts, macrophages, mast cells, reticular cells, endothelial cells, osteoid cells, and adipocytes. Conventional histologic and immunohistologic analysis has identified a generally orderly arrangement of developing cells in the bone marrow, including localization of early granulocytic cells along the bony trabecular margins and erythroid islands, megakaryocytes, and occasional lymphoid nodules positioned in the intertrabecular spaces. Examples of special cellular niche relationships include megakaryocyte localization near draining venules to facilitate platelet release into the bloodstream and juxtaposition of central macrophages and surrounding developing erythroid clusters. Age-related histologic findings include marrow necrosis and fibrosis, loss of bone substance, increase in bone marrow iron stores, expansion of adipose tissue, and accumulation of benign lymphoid aggregates. Although analysis of individual cytokines, cellular compositions, and supportive stromal functions can be measured to decrease with aging, underlying mechanisms have been elusive.

Recent advances have identified a specialized component of the bone marrow microenvironment termed the niche . This three-dimensional functional hematopoietic unit has specialized anatomic relationships among bone, blood vessels, and differentiating hematopoietic cells. The HSC niche functions as an anatomically confined regulatory environment governing HSC numbers and fate. Niche cellular relationships include vascular endothelial and perivascular cells and sympathetic innervation and osteoclasts. Several spatially and likely functionally distinct bone marrow microenvironments and niches have been proposed. The endosteal HSC niche contains osteoblasts as the main supportive cell type. The vascular niche has HSCs associated with the sinusoidal endothelium in the bone marrow and spleen. These environments serve as sites for local cytokine production. Factors implicated in HSC function include the Notch ligands Delta and Jagged, involved in the generation, antidifferentiation, and expansion of HSCs. Wnt signaling is involved in HSC generation and expansion and the maintenance of HSCs in a quiescent state. Bone morphogenic proteins (BMPs) and transforming growth factor-β (TGF-β) regulate HSC activity, and BMP appears to regulate the size of the endosteal niche. Many other soluble factors are also under investigation.

Many of these niche components and relationships have been identified so recently that their potential roles in age-related bone marrow functional changes have not yet been investigated. Based on the importance to normal steady-state hematopoiesis, the niche has been investigated in disease pathogenesis, however. The human myeloproliferative neoplasm primary myelofibrosis (PMF), long known as a disorder of abnormal marrow fibrosis leading to so-called wandering stem cells, has been proposed to be a clonal disorder of the stem cell niche deregulation and abnormal stroma. Myelofibrosis is one of the classic myelo­proliferative neoplasms (MPNs) that also include essential thrombocytosis (ET) and polycythemia vera (PV). These and many other myeloid and lymphoid malignancies have been diagnosed at increasing frequency in aging individuals. Niche perturbations have also been observed in a myeloproliferative disorder that develops in retinoic acid gamma receptor microenvironment murine knockouts. Lyer and colleagues have found that the HSC compartment expands significantly when aged in a niche that contains SHIP1 (Src homology 2-domain-containing inositol 5′-phosphatase 1)-deficient mesenchymal stem cells and also provides potential insight into the development of MPN in older adults.

The bone marrow microenvironment and niche abnormalities have been increasingly implicated in other hematopoietic malignancies frequently found in older adults as well. The myelodysplastic syndromes (MDSs), for example, are a diverse group of clonal hematopoietic malignancies characterized by ineffective hematopoiesis, progressive bone marrow failure, cytogenetic and molecular abnormalities, and risk of progression to acute myelogenous leukemia. Using a retroviral model of induced acute myeloid leukemia (AML), Lane and associates have identified a leukemia stem cell (LSC) niche that is physically distinct and independent of the constraints of Wnt signaling that apply to normal HSCs. Donor cell leukemia (DCL), a rare complication of bone marrow transplantation, has been linked to niche damage from inflammation triggered by the primary underlying malignancy, active chemotherapeutic and radiation conditioning, or transplantation-related immune modulatory treatment, all leading to extrinsic leukemic influences on donor HSCs.

To summarize, the discovery of the niche and stromal contributions to hematopoiesis represent major new areas for the investigation of normal physiology and aging. In addition to serving as a primary site of hematopoiesis, the bone marrow has also been identified as a tissue source of cells for nonhematopoietic wound healing or regeneration. Examples of potential bone marrow-derived tissue contributors include mesenchymal stem cells and fibrocytes. Mesenchymal stem cells (MSCs) are multipotent stem cells. Although originally identified in bone marrow and described as marrow stromal cells, they have since been identified in many other anatomic locations. MSCs can be isolated from bone marrow, adipose tissue, umbilical cord, and other tissues but the richest tissue source of MSCs is fat. Because they are adherent to plastic, they may be expanded in vitro. MSCs have a distinct morphology and express a specific set of cell surface molecules. Under appropriate conditions, MSCs can proliferate and give rise to other cell types and are under evaluation as tissue sources for the treatment of systemic inflammatory and autoimmune conditions and as a replacement for injured tissue following injury or trauma. The heart, cornea, and liver are among many other tissues that are being examined as potential target organs for bone marrow–derived regenerating tissue grafts.

Hematopoietic Stem Cells

The stem cell model of hematopoiesis starts with the totipotent HSC that has the capacity for self-renewal to prevent exhaustion of the HSC compartment. The asymmetric proliferation and differentiation produce large numbers of lineage-restricted hematopoietic cells daily and the ability to reconstitute hematopoiesis in a lethally irradiated host. Although intrinsic and extrinsic control of the early developmental steps from self-renewing HSCs and cells committed to differentiation are poorly understood, these represent an excellent general model system to define basic mechanisms of mammalian cell development and differentiation. The ability of transferred HSCs to reconstitute hematopoiesis provides the clinical basis for bone marrow transplantation. The earliest description of stem cell transplantation (SCT) was based on studies showing murine bone marrow transplanted into lethally irradiated mice, rescuing the recipient by reconstituting donor hematopoiesis. Remarkably, intravenous injection is possible because the HSCs are able to home to the bone marrow and identify and interact with the niche. The biology and physiology of the HSC is enormously complex and has been the subject of many reviews that include descriptions of the characterization and developmental origins of HSCs, enumeration of cellular sources, regulation of cell fate decisions, and clinical implications for bone marrow transplantation. Detailed studies of aging hematopoietic stem cells have provided unique insights into the aging process.

Telomeres and telomerase have been specifically investigated as potential components of age-related bone marrow failure, including hematopoietic stem cell dysfunction. Short telomeres have been linked to the cause of degenerative diseases, including idiopathic pulmonary fibrosis, cryptogenic liver cirrhosis, and bone marrow failure. Natural mutations to the core complex were first discovered in the rare bone marrow failure syndrome dyskeratosis congenita (DC). Heterozygous mutations of these genes have been described for patients with DC, bone marrow failure, and idiopathic pulmonary fibrosis. Mutations in the telomerase RNA ( TERC ) or telomerase reverse transcriptase component ( TERT ) apparatus associated with telomerase dysfunction have been identified in sporadic and familial MDS and AML. The spectrum of mutations in TERT and TERC varies for these diseases and appear, at least in part, to explain the clinical differences observed, including bone marrow failure. Environmental insults and genetic modifiers that accelerate telomere shortening and increase cell turnover may exaggerate the effects of telomerase haploinsufficiency, contributing to the variability of age of onset and tissue-specific organ pathology.

Telomere dysfunction in mouse models has been associated with alveolar stem cell failure. Warren and Rossi, in 2008, reviewed the general lack of direct evidence for progressive depletion of the hematopoietic stem cell pool based on telomere shortening with aging. Serial bone marrow transplantation experiments in mice have suggested that that although the replicative potential of HSCs is finite, there is little evidence that replicative senescence causes depletion of the stem cell pool during the normal life span of mice or humans. Evidence has suggested that HSC numbers substantially increase with advancing age in mice. The expansion of the HSC pool is a cell-autonomous property—HSCs from older donors exhibit a greater capacity than younger controls on transplantation into younger recipients. Although there is an increase in the number of HSCs with age, they have functional deficiencies, including altered homing and mobilization properties and decreased competitive repopulation abilities. Remarkably, a skewing of lineage potential from lymphopoiesis to myelopoiesis has been observed with advancing age. There are reduced lymphoid progenitors in older mice and are maintained to increased myeloid progenitors. These HSC cell-autonomous transplantable property findings may explain age-related immune cell senescence and an increase in myelogenous hematologic malignancy with age.

The reproducible finding of altered lymphoid-to-myeloid blood cell ratios with age has been a focus of intensive molecular investigations. Analysis of a single HSC in long-term transplantation assays and genetic differences in HSC behavior in different strains of inbred mice have demonstrated that many HSC behaviors are fixed intrinsically through genetic or epigenetic mechanisms. A striking example of epigenetically fixed heterogeneity among HSCs is found in myeloid-biased HSCs. These HSCs make typical levels of myeloid cells but generate too few lymphocytes. The diminished lymphoid progeny have impaired responses to interleukin-1 (IL-7). Using highly purified HSCs from young and aged mice, Chambers and colleagues have identified functional deficits as well as an increase in stem cell numbers with advancing age. Gene expression analysis has identified approximately 1,500 of more than 14,000 genes that were age-induced and 1,600 that were age-repressed. Genes associated with the stress response, inflammation, and protein aggregation dominated the upregulated profile, whereas the genes involved in chromatin remodeling and preservation of genomic integrity were downregulated. Many chromosomal regions showed a coordinate loss of transcriptional regulation and an overall increase in transcriptional activity with age, and an inappropriate expression of genes normally regulated by epigenetic mechanisms was observed.

Sun and colleagues have recently extended the observations described earlier. They performed an intensive analysis of highly purified HSC populations comparing genomic properties of young and old murine HSCs with coordinate analyses of global changes in the transcriptome, histone modifications, and DNA methylation. Their group reported a significant link between aging-associated changes in the deposition of histone marks with changes in RNA expression, coding, and noncoding. Pathway analysis revealed a high percentage of aging-associated changes in gene expression related to ultimately decreased TGF-β signaling, as well as upregulation of genes encoding ribosomal proteins. The study by Sun and associates has strongly supported emerging evidence that deregulated epigenetic status represents one of the driving forces behind age-related alterations in the functionality of stem cells. Further work is needed to connect the alterations in DNA methylation and histone modifications and associated changes in gene expression related to increased self-renewal and myeloid-skewed differentiation of aging HSCs.

Epigenetic alterations are pharmacologically targetable. Epigenetic chromatin-modifying drugs have been applied to normal HSC cultures with cytokines with the goal of preserving marrow-repopulating activity. Activation of several genes and their products implicated in HSC self-renewal were observed compared with cells exposed to cytokines alone, which lost their marrow-repopulating activity. Previous attempts to expand HSCs resulted in HSC differentiation and stem cell exhaustion or, at best, asymmetric cell division and maintenance of the same numbers of HSCs. These observations suggest that chromatin-modifying agents may allow for the symmetric division of HSCs and expansion of potential therapeutic grafts, with preservation of stem cell function. Molecular analysis of patients with an informative clonal marker and neutrophil response has indicated that restoration of normal nonclonal hematopoiesis may be a significant component of the epigenetic agent 5-aza-2′-deoxycytidine (decitabine, DAC) used in the treatment of MDS and AML.

Additional support for age-related biologic differences in HSCs and how detailed investigations of malignant hematopoietic disorders provide insight into the aging of blood has been illustrated by recent studies comparing the clinical outcomes of stem cell transplantations using younger or older stem cell donors. Kroger and coworkers have investigated whether a young human leukocyte antigen (HLA)–matched unrelated donor (MUD) should be preferred as the donor to an HLA-identical sibling (matched related donor, MRD) for older patients with MDS who underwent allogeneic stem cell transplantation. Transplantation from younger MUDs had a significantly improved 5-year overall survival in comparison with MRDs and older MUDs. In a multivariate analysis, transplantation from young MUDs remained a significant factor for improved survival in comparison with MRDs. These are not definitive results but illustrate one of the clinical issues related to understanding the age-associated function of the HSCs.

Alternative sources of HSCs for stem cell therapy and regenerative medicine have been sought through the use of embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) technologies. These strategies have yet to yield fully functional cells. More recent approaches have also investigated transcription factor (TF) overexpression to reprogram PSCs and various somatic cells. The induction of pluripotency with just four TFs provides the rationale for an approach to convert cell fates and demonstrates the feasibility of using terminally differentiated cells to generate cells with multilineage potential.