Unraveling the complex regulation of stem cells: implications for aging and cancer.

Substantial progress in embryonic and adult stem cell research in the past several years has yielded a wealth of information regarding the mechanisms regulating self-renewal and differentiation, two processes often used to define stem cells. Recent evidence suggests that epigenetic as well as genetic processes maintain stem cells in a pluripotent state as well as dictate their transition to more restricted stages of development. In this review, we discuss two emerging themes in stem cell biology, epigenetic control of gene expression and post-transcriptional regulation via microRNAs. We summarize how these regulatory mechanisms facilitate various aspects of normal stem cell biology and extend the discussion to their involvement in aging and tumorigeneisis, two biological phenomena intimately tied to stem cells. We speculate that aberrant epigenetic events and altered miRNA expression profiles in aged stem cell populations play important roles in carcinogenesis.

The isoflavone genistein inhibits LPS-stimulated TNFalpha, but not IL-6 expression in monocytes from hemodialysis patients and healthy subjects.

BACKGROUND: Whole blood and peripheral blood mononuclear cells from hemodialysis (HD) patients show increased production and secretion of inflammatory cytokines. We determined the contribution of blood monocytes to the production of inflammatory cytokines in whole blood from HD patients. METHODS: Whole blood and isolated mononuclear cells from HD patients and healthy control subjects were preincubated with the isoflavone genistein and stimulated with LPS. TNFalpha, IL-6 and IL-10 formation in the whole blood was measured with ELISA and intracellular cytokine formation in CD 14-positive monocytes was determined by flow cytometry. RESULTS: Unstimulated blood levels of TNFalpha, IL-6 and IL-10 were significantly elevated in HD patients compared to controls, but intracellular monocyte content of these cytokines was identical between groups. LPS induced a robust TNFalpha response in both whole blood and monocytes, and TNFalpha formation was 2.3-fold higher in blood from HD patients compared to controls. A similar trend was observed in monocytes. Conversely, LPS stimulation increased IL-6 levels >1000-fold in whole blood, albeit without a noticeable difference between groups. Only minor increases in monocyte IL-6 content were observed. The isoflavone genistein did not inhibit IL-6 formation and did not alter basal TNFalpha levels, but genistein selectively blocked LPS-induced TNFalpha formation in whole blood and monocytes from both groups. CONCLUSION: Intracellular levels of TNFalpha, IL-6 and IL-10 in monocytes are indistinguishable between HD patients and healthy controls. However, monocytes from HD patients are selectively primed for enhanced TNFalpha secretion in response to LPS. The selective inhibition of monocyte TNFalpha production by genistein may explain the anti-inflammatory action of this phytochemical observed in vivo.

Age-related thymic involution in C57BL/6J x DBA/2J recombinant-inbred mice maps to mouse chromosomes 9 and 10.

A comprehensive analysis of initial thymus size and involution rate has not been quantitated for different genetic backgrounds of mice, thus genetic linkage analysis of thymic involution has not been possible. Here, we have used a mathematical method to analyze the age-related decline in thymocyte count in C57BL/6 and DBA/2 mice and have observed that thymic involution could be best fit with a negative exponential curve N(t)=beta(0) x exp(-beta(1)t), where t represents the age (day). This regression model was applied to C57BL/6 x DBA/2 (B x D) recombinant inbred strains of mice to identify the genetic loci influencing age-related thymic involution. There was a dramatic genetic effect of B and D alleles on thymocyte count at young age and the age-related thymic involution rate. The strongest quantitative trait loci (QTL) influencing the rate of thymic involution were mapped to mouse chromosome (Chr) 9 (D9Mit20 at 62 cM) and Chr 10 (D10Mit61 at 32 cM). The strongest QTLs influencing the initial thymocyte count were mapped to ChrX (DXMit324 at 26.5 cM) and Chr 3 (D3Mit127 at 70.3 cM). The present study suggests that the initial thymus size and the rate of thymic involution may be influenced by a relatively small number of genetic loci.

Age- and stage-specific regulation patterns in the hematopoietic stem cell hierarchy.

The molecular mechanisms that regulate self-renewal and differentiation of very primitive hematopoietic stem and progenitor cells in vivo are still poorly understood. Despite the clinical relevance, even less is known about the mechanisms that regulate these cells in old animals. In a forward genetic approach, using quantitative trait linkage analysis in the mouse BXD recombinant inbred set, this study identified loci that regulate the genetic variation in the size of primitive hematopoietic cell compartments of young and old C57BL6 and DBA/2 animals. Linked loci were confirmed through the generation and analysis of congenic animals. In addition, a comparative linkage analysis revealed that the number of primitive hematopoietic cells and hematopoietic stem cells are regulated in a stage-specific and an age-specific manner.

Does functional depletion of stem cells drive aging?

The regenerative power of stem cells has raised issues about their relation to aging. We focus on the question of whether a decline in the function of stem cells may itself be a significant feature of aging. The question is implicitly two-fold: does functional depletion of stem cells affect the accumulation of aging-related deficits, and--whether or not depletion is significant--can activation of stem cells alleviate deficits? Two types of system are considered: 1) the exhaustible pool of ovarian follicles. The depletion of follicles leads to the aging-related phenomenon of menopause; and 2) the reserve of hematopoietic stem cells. Substantial numbers are sustained throughout life, but in mouse models, endogenous replicative activity has been shown to decline sharply with age. We discuss the possible implications of these observations for the rate of aging and the prospects for intervention.

Genetic dissection of age-related changes of immune function in mice.

Understanding of the genetic basis of normal and abnormal development of the immune response is an enormous undertaking. The immune response, at the most minimal level, involves interactions of antigen presenting cells (APCs), T and B cells. Each of these cells produce cell surface and soluble factors (cytokines) that affect both autocrine and paracrine functions. A second level of complexity needs to consider the development of the macrophage/monocyte lineage as well as the production of the common lymphoid precursor which undergoes distinct maturation steps in the thymus and periphery to form mature T cells as well as in BM (BM) and lymphoid organs to form mature B cells. A third level of complexity involves the immune response to infectious agents including viruses and also the response to tumour antigens. In addition, there are imbalances that predispose to decreased responses (immunodeficiencies) or increased responses (autoimmunity). A fourth level of complexity involves attempts to understand the differences in the immune response that occurs at a very young age, in adults, and at a very old age. This review 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 the production of lymphoid precursors in the BM, development of T cells in the thymus, and T-cell responses to stimulation in the peripheral lymphoid organs in adult and in aged mice. Strategies to improve the power and precision in which complex traits such as the age-related immune response can be mapped is limited with the current set of 35 strains of BXD mice. Strategies to increase these strains by generating recombinant intercross (RIX) strains of mice are being developed to enable this large set of lines to detect quantitative trait loci (QTLs) with a much higher consistency and statistical power. More importantly, the resolution with which these QTLs can be mapped would be greatly improved and, in many cases, 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 ageing, mapping hundreds of genes will be required to fully understand age-related changes in the immune response. This review outlines ongoing and future strategies that will enable the mapping and identification of these genes.

Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers.

We have previously demonstrated that young adult DBA/2 (DBA) mice have more stem cells than C57BL/6 (B6) mice, as measured in a cobblestone area-forming cell (CAFC) assay using unfractionated marrow. To study the nature of this difference, we have now compared the proliferative fate of single, highly enriched Sca-1(+)c-kit(+)Lin(-) stem cells from these strains. Although equal in frequency, functional comparison revealed that Sca-1(+)c-kit(+)Lin(-) cells from DBA mice contained twice as many cells with CAFC activity. DBA clones persisted much longer in vitro, and developed later in time. To assess whether these differences were of any functional relevance in vivo, we compared engraftment of lethally irradiated mice transplanted with 1000 B6 or DBA Sca-1(+)c-kit(+)Lin(-) cells. Recipients of enriched DBA cells recovered much faster than animals transplanted with B6 cells. We also studied endogenous hematopoietic recovery after 5-fluorouracil (5-FU) treatment in vivo. Progenitors and peripheral blood cells recovered twice as fast in DBA mice. Thus, DBA stem cells have superior proliferative potential compared with phenotypically identical stem cells obtained from B6 mice. Such genetically determined quantitative and qualitative differences in stem cell behavior likely contribute to the dramatically different hematopoietic recovery rates observed in human transplant patients. (Blood. 2000;96:1374-1379)

Autonomous behavior of hematopoietic stem cells.

Mechanisms that affect the function of primitive hematopoietic stem cells with long-term proliferative potential remain largely unknown. Here we assessed whether properties of stem cells are cell-extrinsically or cell-autonomously regulated. We developed a model in which two genetically and phenotypically distinct stem cell populations coexist in a single animal. Chimeric mice were produced by transplanting irradiated B6D2F1 (BDF1) recipients with mixtures of DBA/2 (D2) and C57BL/6 (B6) day-14 fetal liver cells. We determined the mobilization potential, proliferation, and frequency of D2 and B6 stem and progenitor cells in animals with chimeric hematopoiesis. After granulocyte colony-stimulating factor (G-CSF) administration, peripheral blood D2 colony-forming units granulocyte-macrophage were fourfold to eightfold more numerous than B6 progenitors. We determined that D2 and B6 progenitors maintained their genotype-specific cycling activity in BDF1 recipients. Chimeric marrow was harvested and D2 and B6 cell populations were separated by flow cytometry. Cobblestone area-forming cell (CAFC) analysis of sorted marrow showed that the number of late appearing CAFC subsets within the D2 cell population was approximately threefold higher than within the B6 fraction. We performed secondary transplantation using unfractionated chimeric marrow, which was given in limiting doses to lethally irradiated BDF1 recipients. Comparison of the proportion of animals possessing D2 and/or B6 leukocytes 5 months after transplant revealed that the frequency of D2 LTRA was approximately 10-fold higher than B6 LTRA numbers. Our data demonstrate that genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotypes, indicating that stem cell properties are predominantly regulated cell-autonomously.

G-CSF Primed Autologous Marrow Harvest and Transplantation in Cytapheresis "Mobilization Failure" Patients: A Descriptive Analysis; Bone Marrow Transplantation.

Fifteen cancer patients, deemed blood HSC "mobilization failures" due to CD34 + cell yields of < 0.5 x 10(6) /kg from two consecutive daily cytaphereses, underwent G-CSF primed autologous bone marrow harvest in an attempt to obtain adequate hematopoietic support for subsequent autotransplantation. CD34 + cell yields from the primed marrow harvest were variable; however, some patients had > 5-fold increases in CD34 + cell yields in the marrow compared to cytapheresis, and 4 patients had CD34 + cell yields of > 1.0 (i.e., 1.2, 1.44, 1.61 and 2.45) x 10(6) /kg from the primed marrow harvest. None of the five patients previously exposed to stem cell toxins or fludarabine achieved > 0.85 x 10(6) /kg CD34 + cells with the primed marrow harvest. A significant difference was noted between G-CSF primed blood and marrow for CD34 + cells but not for GM-CFU ( p = 0.011 and p = 0.135, respectively, paired t-test). All evaluable patients engrafted; a median ANC > 0.5 x 10(9) /L recovery was achieved on D + 12 (range + 9 to + 17) in 12 of 13 evaluable patients - one died on D + 9 without recovery. The last day of platelet transfusion occurred at a median D + 13 (range + 8 to > + 66); only one patient remained platelet transfusion-dependent beyond D + 34. As anticipated, patients with higher numbers of CD34 + cells transplanted had somewhat more rapid recoveries. Although stem cell damage is obviously a key factor in mobilization failure patients, these findings raise the possibility that poor mobilization, at least in some patients, results from a mechanism other than, or in addition to, simple stem cell damage. Moreover, they raise the issue of the minimum number of marrow CD34 + - or more arguably other - cells needed for adequate short- and long-term reconstitution. The role of G-CSF in this situation, especially regarding dose and/or schedule, is intriguing but remains to be clarified. G-CSF primed marrow harvest is a potential option in certain poor mobilizers but, as fully expected, is frequently inadequate. Whether such is preferable to "steady-state" marrow harvest, continued or repeated G-CSF primed cytapheresis (with or without chemotherapy), or primed marrow with G-CSF in other schedules - or with other cytokines - is unclear and will be the subject of further study.

Dynamic changes in mouse hematopoietic stem cell numbers during aging.

To address the fundamental question of whether or not stem cell populations age, we performed quantitative measurements of the cycling status and frequency of hematopoietic stem cells in long-lived C57BL/6 (B6) and short-lived DBA/2 (DBA) mice at different developmental and aging stages. The frequency of cobblestone area-forming cells (CAFC) day-35 in DBA fetal liver was twofold to threefold higher than in B6 mice, and by late gestation, the total stem cell number was nearly as large as that of young DBA adults. Following a further approximately 50% increase in stem cells between 6 weeks and 1 year of age, numbers in old DBA mice dropped precipitously between 12 and 20 months of age. In marked contrast, this stem cell population in B6 mice increased at a constant rate from late gestation to 20 months of age with no signs of abatement. Throughout development an inverse correlation was observed between stem cell numbers and the percentage of cells in S-phase. Because a strong genetic component contributed to the changes in stem cell numbers during aging, we quantified stem cells of 20-month old BXD recombinant inbred (RI) mice, derived from B6 and DBA progenitor strains, thus permitting detailed interstrain genetic analysis. For each BXD strain we calculated the stem cell increase or decrease as mice aged from 2 to 20 months. Net changes in CAFC-day 35 numbers among BXD strains ranged from an approximately 10-fold decrease to an approximately 10-fold increase. A genome-wide search for loci associated with this quantitative trait was performed. Several loci contribute to the trait-putative loci map to chromosomes X, 2, and 14. We conclude that stem cell numbers fluctuate widely during aging and that this has a strong genetic basis.

Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal life span.

Normal somatic cells undergo replicative senescence in vitro but the significance of this process in organismic aging remains controversial. We have shown previously that hemopoietic stem cells of common inbred strains of mice vary widely in cycling activity and that this parameter is inversely correlated with strain-dependent mean life span. To assess whether cell cycling and life span are causally related, we searched for quantitative trait loci (QTLs) that contributed to variation of these traits in BXH and BXD recombinant inbred mice. Two QTLs, mapping to exactly the same intervals on chromosomes 7 and 11, were identified that were associated with variation of both cell cycling and life span. The locus on chromosome 11 mapped to the cytokine cluster, a segment that shows synteny with human chromosome 5q, in which deletions are strongly associated with myelodysplastic syndrome. These data indicate that steady-state cell turn-over, here measured in hemopoietic progenitor cells, may have a significant effect on the mean life span of mammals.

Organ-selective homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by Ex vivo expansion.

Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells "home" to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26(+) cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26(+) cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of "spleen-homed" cells also contained approximately 50% higher numbers of CFCs per femur than recipients of "BM-homed" cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1(+)c-kit+Lin- cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

Granulocyte-colony stimulating factor (G-CSF)-primed, delayed marrow harvests as a source of hematopoietic stem and progenitor cells for allogeneic transplantation.

We evaluated the ability of G-CSF to increase the number of hematopoietic stem cells obtained by "delayed" BM harvest for allogeneic transplantation. Five normal donors received G-CSF @ 10 mcg/kg/day x 5 followed by repeat PB and BM assays at day 6 and 16, and BM harvest at day 16. Stem cells were not increased in the BM at day 16. Five patients underwent BMT and engrafted at +10 to +19 days. While the tested strategy offers no intrinsic advantages, its potential cannot be evaluated fully without alternative timing and/or additional, "early acting" growth factors.

Genetic control of lifespan: studies from animal models.

The premise that there are genes that wield a strong influence on longevity has, until recently, not been a popular one and there has been no concerted effort to find such genes. However, the finding that single genes can have large effects on the lifespans of yeast, worms and flies raises the possibility that individual genes in mammals may similarly have relatively large effects on longevity. Recent advances in mammalian genetics, many associated with the large-scale efforts to sequence the human and mouse genomes, have accelerated the search for longevity genes, principally in mice. Here, we review results using animal models that have recently shed light on genes regulating longevity and ageing patterns. A large number of genetically defined strains of mice are available and this, together with their established history of use in genetic research and their relatively short lifespans, has made murine models particularly useful. We also review our own work in which genes regulating mouse lifespan and those regulating cell cycling of haematopoietic progenitor cells have been mapped to the same locations in the genome. These results suggest that some of the same genes affect both traits, and further suggest a cause-and-effect relationship between cumulative cell-cycle activity and longevity of an organism.

Bi-phasic CD34+ cell mobilization of a syngeneic donor during prolonged G-CSF delivery.

BACKGROUND: G-CSF administration over 10 days and neutrophil cytapheresis have been reported in the literature, but the kinetics of CD34+ cells in this situation is unclear. CASE: A 42-year-old female underwent syngeneic transplantation for metastatic breast cancer. The recipient was in critical condition peri-transplant, therefore the donor received G-CSF for 13 days, during which eight cytaphereses for both PBPC and neutrophils were performed. Two peaks in CD34+ cells were noted; the first on Day 5 and the second on Days 10-13 of G-CSF administration; a total of 11.6 x 10(6)/kg CD34+ cells and 38.11 x 10(8)/kg neutrophils were infused. The recipient's ANC exceeded 0.1 x 10(9)/L on Day +3. DISCUSSION: To our knowledge, this is the longest reported cytapheresis of CD34+ cells from a normal donor The bi-phasic pattern in the cytapheresis product is also of interest. It is an unusual pattern that suggests a profound and complicated alteration in the marrow progenitor cell pool. If substantiated, this finding may offer an alternative cytapheresis schedule for donors.

Autologous CD34+ cell transplantation for patients with advanced lymphoma: effects of overnight storage on peripheral blood progenitor cell enrichment and engraftment.

In order to demonstrate the feasibility of mobilization, enrichment and engraftment of autologous peripheral blood CD34+ cells in patients with relapsed lymphoma, 59 peripheral blood progenitor cell (PBPC) collections from 21 patients were enriched for CD34+ cells using CEPRATE SC (CellPro, Bothell, WA, USA) immunoaffinity column. Following high-dose chemotherapy, a mean of 17 x 10(8) (range, 3-34) nucleated cells/kg containing 8.7 x 10(6) (0.3-26) CD34+ cells/kg were re-infused. Blood cell recovery in these patients was compared to engraftment capacity of unenriched PBPCs in a cohort of lymphoma patients treated with an identical high-dose chemotherapy regimen. Neutrophil and platelet engraftment was rapid in both groups including five patients who received < or = 1 x 10(6) CD34+ cells/kg. After infusion of CD34+ enriched cells, neutrophils exceeded 0.5 x 10(9)/l in 11 (8-14) days and platelets exceeded 20 x 10(9)/l (untransfused) in 15 (9-39) days. In order to optimize the immunoaffinity column utilization we stored the first PBPC collections overnight at 4 degrees C and combined them with the next day's collection prior to the CD34+ enrichment procedure in 11 patients. This maneuver resulted in a significant decrease in the CD34+ cell recovery (resulting in reinfusion of a mean of 42% less CD34+ cells). Although overnight storage did not affect neutrophil engraftment, platelet engraftment was prolonged in this group of patients even when > 2.0 x 10(6) CD34+ cells/kg were re-infused. The overnight storage procedure should be further evaluated for its effects on the CD34+ immunoaffinity enrichment procedure, megakaryocyte progenitors and platelet engraftment. We conclude that CD34+ cells enriched from peripheral blood result in rapid engraftment after high-dose chemotherapy in patients with advanced lymphoma that is comparable to that of patients receiving unenriched PBPCs.

Recent progress in identifying genes regulating hematopoietic stem cell function and fate.

Significant advances in the use of genetic and molecular biology strategies have recently begun to identify genes that have a major impact on the determination, commitment and developmental potential of hematopoietic stem cells. Using a variety of experimental strategies, genes such as SCL, GATA-2, HoxB4, Flk-2, c-mpl, dlk, and others have been implicated as important regulators of stem cell growth. In addition, genetic mapping has identified several loci that correlate strongly with stem cell numbers and proliferation.

Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene.

We evaluated in vivo interactions between extrinsic (growth factor induced) and intrinsic (genetically determined) effectors of mouse primitive hemopoietic stem cell proliferation and numbers. Accordingly, stem cell frequency and cell cycle kinetics were assessed in eight strains of inbred mice using the cobblestone area-forming cell (CAFC) assay. A strong inverse correlation was observed between mouse lifespan and the number of autonomously cycling progenitors (CAFC day 7) in the femur. The population size of primitive stem cells (CAFC day 35) varied widely (up to sevenfold) among strains, unlike total CAFC day 7 numbers (cycling and quiescent), which were similar. Administration of the early acting cytokine flt-3 ligand to these strains resulted in activation of quiescent primitive stem cells exclusively in strains with high endogenous stem cell numbers (DBA and AKR), but was unrelated to strain-specific progenitor cell cycling. To map loci affecting stem cell frequency, we quantified stem cells in BXD recombinant inbred mice (offspring of C57BL/6 and DBA/2). The resulting strain distribution pattern showed high concordance with a marker that mapped to chromosome 18 (19 cM). Linkage with this genomic interval was associated with a likelihood of odds score of 3.3, surpassing the level required for significance. Interestingly, this segment, containing the EGR-1 gene, shows synteny with human chromosome 5q, a region strongly associated with various hematological malignancies. Our findings indicate that a gene mapping to this region is mutated in either C57BL/6 or DBA/2 (and possibly AKR) mice. These studies in apparently healthy mice may facilitate the identification of a gene implicated in human 5q-syndromes.