The quantitative trait gene latexin influences the size of the hematopoietic stem cell population in mice.

We mapped quantitative trait loci that accounted for the variation in hematopoietic stem cell (HSC) numbers between young adult C57BL/6 (B6) and DBA/2 (D2) mice. In reciprocal chromosome 3 congenic mice, introgressed D2 alleles increased HSC numbers owing to enhanced proliferation and self-renewal and reduced apoptosis, whereas B6 alleles had the opposite effects. Using oligonucleotide arrays, real-time PCR and protein blots, we identified latexin (Lxn), a gene whose differential transcription and expression was associated with the allelic differences. Expression was inversely correlated with the number of HSCs; therefore, ectopic expression of Lxn using a retroviral vector decreased stem cell population size. We identified clusters of SNPs upstream of the Lxn transcriptional start site, at least two of which are associated with potential binding sites for transcription factors regulating stem cells. Thus, promoter polymorphisms between the B6 and D2 alleles may affect Lxn gene expression and consequently influence the population size of hematopoietic stem cells.

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle.

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca(2+) sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.

Congenic interval of CD45/Ly-5 congenic mice contains multiple genes that may influence hematopoietic stem cell engraftment.

The B6.SJL-Ptprc(d)Pep3(b)/BoyJ (B6.SJL) congenic mouse strain, a valuable and widely used tool in murine bone marrow transplantation studies, has long been considered equivalent to the parental C57B/L6 (B6) strain with the exception of a small congenic interval on chromosome 1 harboring an alternative CD45/Ly-5 alloantigen (Ly-5.1). In this study we compared functional properties of stem and stromal cells between the strains, and delineated the boundary of the B6.SJL congenic interval. We identified a 25% reduction in homing efficiency, 3.8-fold reduction in transplantable long-term hematopoietic stem cells (LT-HSCs), a 5-fold reduction in LT-HSCs capable of 24-hour homing, and a cell-intrinsic engraftment defect of 30% to 50% in B6.SJL-derived bone marrow cells relative to B6-derived cells. These functional differences were independent of stem cell number, cycling, or apoptosis. Genotypic analysis revealed a 42.1-mbp congenic interval in B6.SJL including 306 genes, and at least 124 genetic polymorphisms. Moreover, expression profiling revealed 288 genes differentially expressed between nonhematopoietic stromal cells of the 2 strains. These results indicate that polymorphisms between the B6 and SJL genotype within the B6.SJL congenic interval influence HSC engraftment and result in transcriptional variation within bone marrow stroma.

CG dinucleotide clustering is a species-specific property of the genome.

Cytosines at cytosine-guanine (CG) dinucleotides are the near-exclusive target of DNA methyltransferases in mammalian genomes. Spontaneous deamination of methylcytosine to thymine makes methylated cytosines unusually susceptible to mutation and consequent depletion. The loci where CG dinucleotides remain relatively enriched, presumably due to their unmethylated status during the germ cell cycle, have been referred to as CpG islands. Currently, CpG islands are solely defined by base compositional criteria, allowing annotation of any sequenced genome. Using a novel bioinformatic approach, we show that CG clusters can be identified as an inherent property of genomic sequence without imposing a base compositional a priori assumption. We also show that the CG clusters co-localize in the human genome with hypomethylated loci and annotated transcription start sites to a greater extent than annotations produced by prior CpG island definitions. Moreover, this new approach allows CG clusters to be identified in a species-specific manner, revealing a degree of orthologous conservation that is not revealed by current base compositional approaches. Finally, our approach is able to identify methylating genomes (such as Takifugu rubripes) that lack CG clustering entirely, in which it is inappropriate to annotate CpG islands or CG clusters.

Quantitative trait locus (QTL) mapping in aging systems.

Understanding the genetic basis of the effects of aging on the decline in the immune response is an enormous undertaking. The most prominent age-related change in the immune system is thymic involution. This chapter will focus on the use of C57BL/6 J X DBA/2 J (BXD) recombinant inbred (RI) strains of mice to map genetic loci associated with age-related thymic involution in mice. Strategies to improve the power and precision in which complex traits such as the age-related decline in the immune response have been applied to the large set of BXD RI strains to detect quantitative trait loci (QTLs) that underlie thymic involution. More importantly, approaches have been developed to enable higher resolution mapping of these QTLs and, in some cases, may be adequate to carry out direct identification of candidate genes. It is likely that, given the complexity of the immune system development, the number of cells involved in an immune response, and especially the changes in the immune system with aging, multiple genetic loci and genes will contribute to the age-related changes in the immune response. This chapter outlines ongoing and general quantitative genetic linkage strategies that can be used for mapping and identification of the quantitative trait loci that may have a significant impact on age-related alteration of the immune system.

Isolation of neural precursor cells from Alzheimer's disease and aged control postmortem brain.

Recent studies demonstrate that isolated neural precursor cells are capable of generating neurons, astrocytes, and oligodendrocytes from neurogenic regions of adult brain. Because these studies use surgically resected or fresh postmortem specimens from young subjects, it is not clear whether neural precursor cells remain in the brain of normal aged subjects or subjects with Alzheimer's disease (AD). The purpose of this study was to determine if viable precursor cells remain in aged control and AD brain. AD subjects have significantly fewer viable precursor cells in the hippocampus compared with age-matched normal control subjects. Musashi-1 and Ki-67-positive precursor cells from AD self renew, but reach senescence earlier than cells isolated from normal aged control subjects. Precursor cells from AD and aged normal control specimens can differentiate into tubulin- and Tuj-1-positive neurons and GFAP-positive astrocytes. This study demonstrates that viable precursor cells remain in AD and aged normal control brain specimens and can be induced to differentiate. These results raise the possibility of stimulation of inherent precursor cells of aged individuals or AD patients to replace neurons lost in aging and/or neurodegeneration.

Ionizing radiation and busulfan induce premature senescence in murine bone marrow hematopoietic cells.

Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in >95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P < 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P < 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture, 33% and 72% of hematopoietic cells survived IR- and BU-induced damage, respectively, as compared with control cells, but they could not form colony forming units-granulocyte macrophages. Moreover, these surviving cells expressed an increased level of senescence-associated beta-galactosidase, p16(Ink4a), and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis, whereas BU does so mainly by inducing premature senescence. In addition, induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly, the induction of hematopoietic cell senescence by IR, but not by BU, was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner, whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway.

Stem cells, aging, and cancer: inevitabilities and outcomes.

Given the unique abilities of a stem cell to self-renew, differentiate, and proliferate, it is no wonder that they are critically important to an organism during development and to maintain homeostasis. Likewise, when something goes awry within a stem cell, it is likely to have far-reaching effects, since stem cells persist throughout the lifetime of the individual. Two significant biological phenomena that involve stem cells are the inevitable process of aging and a major health issue whose incidence increases with aging: cancer. In this review, we summarize evidence and theories concerning these two stem cell processes. The inability of stem cells to be passaged indefinitely in mice and the data supporting regular replication of the quiescent stem cell pool are discussed. Further, the current evidence indicating a stem cell origin of acute myeloid leukemia, including examples from both experimental mouse models and human clinical samples, is evaluated. Finally, we propose a model in which aging of the stem cell population of the hematopoietic system in particular can create conditions that are permissive to leukemia development; in fact, we suggest that aging is a secondary event in leukemogenesis.

The role of stem cells in aging.

The objectives of this review were first to critically review what is known about the effects of aging on stem cells in general, and hematopoietic stem cells in particular. Secondly, evidence is marshalled 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 has both quantitative and qualitative effects on stem cells. On balance, the qualitative changes are the more important since they affect the self-renewal potential, developmental potential, and interactions with extrinsic signals, including those from stroma. Although hematopoiesis is generally maintained at normal and life-supporting levels during normal aging, diminished function is acutely apparent when old stem cells are subjected to stress. There is ample evidence of diminished self-renewal capacity, restriction of the breadth of developmental potency, and decreased numbers of progeny of old stem cells subjected to hematopoietic demands. The prediction is made that whatever plasticity in developmental potential possessed by a young stem cell is lost during aging. Those parts of the world enjoying an ever-increasing standard of living are also inhabited by an increasingly elderly population. The effects of age on many physiological functions are not well studied or appreciated. A public health challenge to provide increased quality of life for this growing segment of the population requires more attention to the variable of age in experimental studies. Stem cell populations are likely to be a fruitful subject for studies of this type.

Genetic analysis of progenitor cell mobilization by granulocyte colony-stimulating factor: verification and mechanisms for loci on murine chromosomes 2 and 11.

OBJECTIVE: It is notable that there is significant inter-individual variability in humans and inter-strain variability in mice in the ability to mobilize hematopoietic stem and progenitor cells, suggesting that there is genetic regulation of mobilization. In the murine system, loci on chromosomes 2 and 11 have been linked to an inter-strain variation in granulocyte colonystimulating factor (G-CSF)-induced stem cell mobilization proficiency. The aim of this study was to verify this linkage and to gain insight into the function of these loci. METHODS: Animals congenic for the loci on chromosomes 2 and 11 were generated by a speed-congenic approach and the function of the loci were further analyzed in doubly congenic animals and with a competitive transplantation/mobilization protocol. RESULTS: The analysis of congenic animals verified that both loci are linked to mobilization proficiency. Analysis of mobilization in doubly congenic animals suggested that both loci act in the same regulatory pathway. Mobilization experiments conducted with mice that had previously been competitively repopulated with congenic and parental-strain BM revealed that the locus on chromosome 11 operates via a progenitor cell-intrinsic mechanism. CONCLUSION: We confirmed linkage of loci on chromosomes 2 and 11 to G-CSF-induced mobilization and thus validated their role as regulators of hematopoietic progenitor cell mobilization in mice. These findings will be useful for further studies directed at identifying genes that regulate mobilization proficiency.

Ionizing radiation and busulfan inhibit murine bone marrow cell hematopoietic function via apoptosis-dependent and -independent mechanisms.

OBJECTIVE: Ionizing radiation (IR) and busulfan (BU) are commonly used as preconditioning regimens for bone marrow transplantation (BMT). We examined whether induction of apoptosis in murine bone marrow (BM) hematopoietic cells contributes to IR- and BU-induced suppression of their hematopoietic function. METHODS: The hematopoietic functions of hematopoietic stem cells (HSCs) and progenitors were analyzed by the cobblestone area-forming cell (CAFC) assay. Apoptosis was determined by measuring 3,3'-dihexyloxacarbocyanine iodide (DiCO6) uptake, annexin V staining, and/or sub-G(0/1) cells. Four cell types were studied: murine BM mononuclear cells (BM-MNCs), linage-negative hematopoietic cells (Lin-) cells), Lin- Scal+ c-kit+ cells, and Lin- Scal- c-kit+ cells by flow cytometry. RESULTS: Exposure of BM-MNCs to IR (4 Gy) or incubation of the cells with BU (30 microM) resulted in a significant reduction in CAFC frequency (p<0.001). The survival fractions of various day-types of CAFC for the irradiated cells were less than 10%, while that for BU-treated cells was 71.3% on day 7 and progressively declined to 5.3% on day 35. Interestingly, IR significantly induced apoptosis in BM-MNCs, Lin- cells, HSCs, and progenitors, whereas BU failed to increase apoptosis in these cells. In addition, preincubation of BM-MNCs with z-Val-Ala-Asp (OCH3)-fluoromethylketone, methyl ester (z-VAD) attenuated IR-induced reduction in CAFC but not that induced by BU. CONCLUSION: IR and BU differentially suppress the hematopoietic function of HSCs and progenitors by fundamentally different mechanisms. IR inhibits the function primarily by the induction of HSC and progenitor apoptosis. In contrast, BU suppresses HSC and progenitor function via an apoptosis-independent mechanism.

Measuring the aging process in stem cells.

Stem cells persist in replenishing functional mature cells throughout life by self-renewal and multilineage differentiation. Hematopoietic stem cells (HSCs) are among the best-characterized and understood stem cells, and they are responsible for the life-long production of all lineages of blood cells. HSCs are a heterogeneous population containing lymphoid-biased, myeloid-biased, and balanced subsets. HSCs undergo age-associated phenotypic and functional changes, and the composition of the HSC pool alters with aging. HSCs and their lineage-biased subfractions can be identified and analyzed by flow cytometry based on cell surface makers. Fluorescence-activated cell sorting (FACS) enables the isolation and purification of HSCs that greatly facilitates the mechanistic study of HSCs and their aging process at both cellular and molecular levels. The mouse model has been extensively used in HSC aging study. Bone marrow cells are isolated from young and old mice and stained with fluorescence-conjugated antibodies specific for differentiated and stem cells. HSCs are selected based on the negative expression of lineage markers and positive selection for several sets of stem cell markers. Lineage-biased HSCs can be further distinguished by the level of SLAM/CD150 expression and the extent of Hoechst efflux.

Age-related change in thymic T-cell development is associated with genetic loci on mouse chromosomes 1, 3, and 11.

Age-related decline in thymic T-cell development in 22-month-old C57BL/6J X DBA/2J (BXD) recombinant inbred strains of mice was functionally and phenotypically analyzed and genetically mapped. There was a positive correlation of the concanavalin A (Con A)-induced thymocyte proliferative response with the capability of thymocytes to mature to the CD4(+)CD8(+) stage. The accumulation of CD4(-)CD8(-) stage of thymocytes in 22-month-old BXD mice was further identified to be associated with a developmental block between the CD25(-)CD44(+) and the CD25(+)CD44(+) stages. The quantitative trait loci regulating the Con A-induced thymocyte proliferative response were mapped to mouse chromosome 1, 3, and 11, nearest to 32.1 centimorgan (cM), 5.6 cM, and 18.0 cM, respectively. Our results suggest that several genetic loci regulate the intra-thymic T-cell maturation process and play an important role in determining age-related decline in thymic T-cell development.

Genetic control of stem cells: implications for aging.

Stem cells are currently at the center of both controversy and notoriety. The harvest of human embryonic or fetal stem cells, at least with methods available now, necessarily involves the sacrifice of the embryo or fetus. This critical step in the procurement of stem cells has stimulated intense discussion at all levels of academia, government, and society in general. What societal benefits, if any, justify such a strategy for obtaining these stem cells? In other species it has been possible to generate virtually all cell types found in adult organs from embryonic stem cells. This ability has opened endless clinical possibilities for tissue and organ replacement through the transplantation of cells derived from embryonic stem cells. Luckily, there may be an alternative to this ethical dilemma. It is becoming increasingly clear that stem cells exist in many, if not all, adult tissues. Adult stem cells normally replenish tissue cells lost through the wear and tear of aging or damage from injury or disease. With the proper coaxing in tissue culture and when transplanted, these stem cells may regenerate the full repertoire of organotypic cells and thus may therapeutically regenerate tissues in vivo in much the same way as embryonic stem cells do. For several reasons, the best-studied stem cells are those of the blood-forming system. Mature blood cells generally have short functional life spans, usually measured in days, and therefore require replenishment at a steady pace throughout one's lifetime. Stem cells are intimately involved in this renewal and, because of the relative ease of access to the bone marrow, stem cells have been well studied. Second, bone marrow transplantation following radiation or high-dose chemotherapy in the treatment of cancer has fostered research on the basic biology and therapeutic uses of hematopoietic stem cells over the more than 30 years stem cell transplantation has been used clinically. It is my aim to review what is known about the genes controlling hematopoietic stem cell function. Identifying, and ultimately manipulating, the genes that regulate stem cell number, replication rate, and self-renewal capacity may have important clinical benefits. I discuss evidence suggesting that the characterization of least some of these stem cell genes will shed light on mechanisms important in the aging process. I advance the hypothesis that stem cells accumulate cellular damage during aging that diminishes their developmental potency and ability to replenish blood cells, particularly after hematopoietic stress. In this view, the impaired function of stem cells in hematopoietic and in other self-renewing tissues limits the longevity of animals, and perhaps of humans.

Comprehensive hematopoietic stem cell isolation methods.

The use of flow cytometry has been critical in establishing methods to isolate and characterize hematopoietic stem cells (HSCs) and their progenitors. For more than 30 years, researchers have been uncovering novel markers that when used in combination significantly enhance the purification of HSCs from murine and human bone marrow. The complex interface between HSCs, the lymphohematopoietic system, and their niches, has made identification of HSC markers critical to understanding their biological nature, more so than other adult stem cell populations. Here we review the phenotypic markers and strategies used to purify HSCs, the appropriateness of using these markers for comparisons of HSC function at different stages of ontogeny, and their utility in defining the lineage bias in the HSC compartment.

Mouse strain determines cardiac growth potential.

RATIONALE: The extent of heart disease varies from person to person, suggesting that genetic background is important in pathology. Genetic background is also important when selecting appropriate mouse models to study heart disease. This study examines heart growth as a function of strain, specifically C57BL/6 and DBA/2 mouse strains. OBJECTIVE: In this study, we test the hypothesis that two strains of mice, C57BL/6 and DBA/2, will produce varying degrees of heart growth in both physiological and pathological settings. METHODS AND RESULTS: Differences in heart dimensions are detectable by echocardiography at 8 weeks of age. Percentages of cardiac progenitor cells (c-kit+ cells) and mononucleated cells were found to be in a higher percentage in DBA/2 mice, and more tri- and quad-nucleated cells were in C57BL/6 mice. Cardiomyocyte turnover shows no significant changes in mitotic activity, however, there is more apoptotic activity in DBA/2 mice. Cardiomyocyte cell size increased with age, but increased more in DBA/2 mice, although percentages of nucleated cells remained the same in both strains. Two-week isoproterenol stimulation showed an increase in heart growth in DBA/2 mice, both at cardiomyocyte and whole heart level. In isoproterenol-treated DBA/2 mice, there was also a greater expression level of the hypertrophy marker, ANF, compared to C57BL/6 mice. CONCLUSION: We conclude that the DBA/2 mouse strain has a more immature cardiac phenotype, which correlates to a cardiac protective response to hypertrophy in both physiological and pathological stimulations.

Concise review: hematopoietic stem cell aging, life span, and transplantation.

Self-renewal and multilineage differentiation of stem cells are keys to the lifelong homeostatic maintenance of tissues and organs. Hematopoietic aging, characterized by immunosenescence, proinflammation, and anemia, is attributed to age-associated changes in the number and function of hematopoietic stem cells (HSCs) and their microenvironmental niche. Genetic variants and factors regulating stem cell aging are correlatively or causatively associated with overall organismal aging and longevity. Translational use of HSCs for transplantation and gene therapy demands effective methods for stem cell expansion. Targeting the molecular pathways involved in HSC self-renewal, proliferation, and homing has led to enhanced expansion and engraftment of stem cells upon transplantation. HSC transplantation is less effective in elderly people, even though this is the demographic with the greatest need for this form of treatment. Thus, understanding the biological changes in the aging of stem cells as well as local and systematic environments will improve the efficacy of aged stem cells for regenerative medicine and ultimately facilitate improved health and life spans.

Age-related changes in niche cells influence hematopoietic stem cell function.

In a recent issue of Nature, Mayack et al. (2010) use young and old parabiotic mice to show that systemic signals from the young parabiont rejuvenate hematopoietic stem cell function via the microenvironmental niche.