Stem Cell Exhaustion Research Articles

Loss of the Cell Cycle Regulator p26INCA1 Induces Exhaustion of Leukemic Stem Cells

AbstractAbstract 96Acute myeloid leukemia is a clonal disease characterized by a malignant proliferation and accumulation of myeloid progenitor cells. Current therapeutic strategies are often not able to eradicate the leukemic cells. Malignancy is associated with deregulation of cell cycle check- points and the deregulation of checkpoints is associated with altered stem cell properties. A better understanding of malignant stem cells and their cell cycle regulation might help to develop new therapies. Recently, we identified p26INCA1 as a novel cell cycle regulator. GST pulldown assays revealed binding of INCA1 predominantly to CDK2- specific Cyclins and we demonstrated an inhibitory effect of INCA1 on CDK2/ CyclinA complexes in kinase activity assays. The loss of INCA1 and its inhibitory effect on the cell cycle regulation led to an increased cell cycling and consequently to an enlarged stem cell pool in vivo. Upon cytotoxic stress, the loss of Inca1 enhanced cell cycling and bone marrow exhaustion. To analyze a potential role of INCA1 in leukemogenesis we retrovirally transduced wildtype and Inca1−/− bone marrow cells with AML1-ETO9a (A1E9a) and transplanted these cells into wildtype recipients. Most of the wildtype cell- transplanted recipients died due to AML. In contrast, only one of the Inca1−/− cell- transplanted mice developed AML. Engraftment was higher upon transplantion of Inca1−/− cells but engraftment was not sustained. To consider the repopulation capacity of the leukemic cells we performed transplantation of primary leukemic cells into secondary recipients. A1E9a induced leukemia in Inca1 wildtype cells was transplantable and lethal. However Inca1−/− leukemic cells were severely impaired in leukemia development in secondary recipients. Colony forming units and replating capacity were significantly reduced in A1E9a Inca1−/− bone marrow cells although these cells showed increased CDK2 activity. Exhaustion of leukemic cells in the absence of Inca1 was confirmed by cloning efficiency assays. Further analyses were performed with c-myc induced leukemias. Interestingly, Inca1 deletion precluded the development of leukemias in secondary recipients. Taken together, these findings identify an important role for p26INCA1 in the maintenance of leukemia and potentially the self-renewal capacity of leukemic stem cells. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Support by Endothelium

AbstractAbstract SCI-45Accumulating evidence suggest that endothelial cells (ECs) are not simply passive conduits for delivering oxygen and nutrients, but are also endowed with the potential for elaborating potent stem and progenitor cell-active trophogens. These EC-derived trophogens, which we refer to as “angiocrine factors”, are essential for organogenesis, tissue repair as well as tumor growth. Within each organ, specialized ECs establish a unique cellular microenvironment, known as “Vascular Niche”, from which they express membrane-bound or soluble angiocrine factors and deposit extracellular matrix that orchestrate organ regeneration. Sinusoidal ECs (SECs) are prototypical specialized ECs that line the capillaries of certain organs, such as bone marrow, liver and spleen. Sinusoidal ECs could be distinguished from other organ-specific capillaries by the expression of VEGFR3, and lack of expression of Sca1. We have devised physiologically relevant angiogenic models to demonstrate that ECs, through release of angiocrine factors, stimulate long-term in vitro self-renewal of long term-hematopoietic cells (LT-HSCs) and in vivo reconstitution of the LT-HSC pool. In serum/cytokine-free co-cultures supplemented only with the Kit-ligand, ECs stimulated incremental expansion of repopulating CD34−Flt3−cKit+Lineage−Sca1+ LT-HSCs, which retained their self-renewal ability, as determined by single cell and serial transplantation assays. Endothelial cells supported several fold expansion of authentic LT-HSCs beyond 21 days without any evidence of stem cell exhaustion or leukemic transformation. One mechanism by which ECs support LT-HSCs self-renewal is mediated through angiocrine expression of Notch-ligands by ECs, which promote proliferation and prevent exhaustion of LT-HSCs. In support of this notion, we show that endothelial cells supported the expansion of HSCs derived from wild type, but not Notch1/Notch2 deficient mice. Employing the transgenic notch reporter (TNR.Gfp) mice, in which stimulation of the Notch signaling pathway results in GFP expression, we demonstrate that ECs support long-term expansion of TNR.Gfp+cKit+Sca1+Lineage− (TNR.Gfp+KLS), but not Notch1−/−Notch2−/− CD34−Flt-3−KLS LT-HSCs. Remarkably, during hematopoietic recovery from sublethal irradiation, every regenerating TNR.Gfp+cKit+Sca1+Lineage− LT-HSC was detected in close cellular proximity of the SECs. Those TNR.Gfp+cKit+Sca1+Lineage− LT-HSCs that were positioned in the vicinity of the osteoblastic cells were also directly attached to SECs. Notably, selective targeting of the SECs by anti-angiogenic factors, resulted in the downregulation of angiocrine expression of the Notch-ligands resulting in the impaired replenishment of TNR.Gfp+KLS cells. In these experiments the perfusion capacity of the SECs remained intact, suggesting that the angiocrine expression of Notch-ligands by SECs, without contribution from non-EC stromal cells, is sufficient to restore LT-HSC repopulation. Collectively, we demonstrate that within the vascular niche, release of soluble Kit-ligand and angiocrine expression of Notch-ligands by structurally intact SECs establish an instructive niche for the restoration of LT-HCS pool. Endothelial cells provide for an ideal vascular model not only to expand LT-HSCs for therapeutic bone marrow transplantation, but also to identify as yet unrecognized factors that collaborate with Notch and c-Kit signaling to balance LT-HSC expansion and lineage-specific differentiation.1) Butler JM et al. Instructive role of the vascular niche in promoting tumor growth and tissue repair by angiocrine factors. Nature Reviews Cancer, 2010, 10:138–146.2) Butler JM et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell, 2010, 6, 251–64.3) Hooper AT et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell, 2009, 4:263–74. Disclosures:No relevant conflicts of interest to declare.

Loss of Foxo3a In FA Mice Leads to Hematopoietic Stem Cell Exhaustion That Can Be Attenuated by Natural Anti-Oxidant Quercetin.

AbstractAbstract 1166Fanconi anemia (FA) is a genetic disorder characterized by genomic instability, bone marrow (BM) failure and predisposition to cancer. However, FA mouse models do not show spontaneous genetic instability. Previous study shows that FOXO3a is associated with the FA pathway through oxidative stress-specific interaction with FANCD2. To address the consequence of loss of FOXO3a function in FA hematopoiesis, we generated Foxo3a-/-Fancd2-/- and Foxo3a-/-Fancc-/- double-knockout (DKO) mice by crossing Foxo3a+/− with Fancd2+/− or Fancc+/−; mice. Reactive oxygen species are increased in low-density BM (LDBM) cells isolated from DKO mice compared to those from single KO (SKO) or wt mice. Analysis of hematologic parameters shows significantly increased number of nucleate cells and high ratio of eosinophils in peripheral blood of DKO mice. CFU assay shows more progenitor cells in peripheral blood isolated from DKO mice. Moreover, BM progenitor cells from DKO mice exhibit lower adhesion but higher migration activity, compared to those from wt or SKO mice. Consistent with this, Cdc42 pull-down assay shows lower Cdc42 activity in DKO LDBM cells than in wt or SKO cells, indicating that decreased Cdc42 may contribute to the observed aberrant adhesion and migration activities. DKO mice show significant decrease in primitive progenitor (Lin-Sca-1+c-kit+; LSK) cells, increase in BrdU+ and G1-phase LSK cells, and impaired repopulating capacity after competitive BM transplantation, which can be attenuated by the anti-oxidant Quercetin. Taken together, loss of Foxo3a in FA mice results in FA-like syndrome, which may be resulted from increased reactive oxygen species accumulation. Disclosures:No relevant conflicts of interest to declare.

Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

AbstractAbstract 2629The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time.To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined.To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function.Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome

By expressing EVI1 in murine bone marrow (BM), we previously described a myelodysplastic syndrome (MDS) model characterized by pancytopenia, dysmegakaryopoiesis, dyserythropoiesis, and BM failure. The mice invariably died 11-14 months after BM transplantation (BMT). Here, we show that a double point mutant EVI1-(1+6Mut), unable to bind Gata1, abrogates the onset of MDS in the mouse and re-establishes normal megakaryopoiesis, erythropoiesis, BM function, and peripheral blood profiles. These normal features were maintained in the reconstituted mice until the study was ended at 21 months after BMT. We also report that EVI1 deregulates several genes that control cell division and cell self-renewal. In striking contrast, these genes are normalized in the presence of the EVI1 mutant. Moreover, EVI1, but not the EVI1 mutant, seemingly deregulates these cellular processes by altering miRNA expression. In particular, the silencing of miRNA-124 by DNA methylation is associated with EVI1 expression, but not that of the EVI1 mutant, and appears to play a key role in the up-regulation of cell division in murine BM cells and in the hematopoietic cell line 32Dcl3. The results presented here demonstrate that EVI1 induces MDS in the mouse through two major pathways, both of which require the interaction of EVI1 with other factors: one, results from EVI1-Gata1 interaction, which deregulates erythropoiesis and leads to fatal anemia, whereas the other occurs by interaction of EVI1 with unidentified factors causing perturbation of the cell cycle and self-renewal, as a consequence of silencing miRNA-124 by EVI1 and, ultimately, ensues in BM failure.

Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemogenesis

AbstractThe RUNX1/AML1 gene is the most frequently mutated gene in human leukemia. Conditional deletion of Runx1 in adult mice results in an increase of hematopoietic stem cells (HSCs), which serve as target cells for leukemia; however, Runx1−/− mice do not develop spontaneous leukemia. Here we show that maintenance of Runx1−/− HSCs is compromised, progressively resulting in HSC exhaustion. In leukemia development, the stem cell exhaustion was rescued by additional genetic changes. Retroviral insertional mutagenesis revealed Evi5 activation as a cooperating genetic alteration and EVI5 overexpression indeed prevented Runx1−/− HSC exhaustion in mice. Moreover, EVI5 was frequently overexpressed in human RUNX1-related leukemias. These results provide insights into the mechanism for maintenance of pre-leukemic stem cells and may provide a novel direction for therapeutic applications.

Ionizing radiation and aging: rejuvenating an old idea.

This paper reviews the contemporary evidence that radiation can accelerate aging, degenerative health effects and mortality. Around the 1960s, the idea that ionizing radiation caused premature aging was dismissed as the radiation-induced health effects appeared to be virtually confined to neoplasms. More recently, radiation has become associated with a much wider spectrum of age-related diseases, including cardiovascular disease; although some diseases of old age, such as diabetes, are notably absent as a radiation risk. On the basis of recent research, is there a stronger case today to be made linking radiation and aging? Comparison is made between the now-known biological mechanisms of aging and those of radiation, including oxidative stress, chromosomal damage, apoptosis, stem cell exhaustion and inflammation. The association between radiation effects and the free-radical theory of aging as the causative hypothesis seems to be more compelling than that between radiation and the nutrient-sensing TOR pathway. Premature aging has been assessed by biomarkers in calorie restriction studies; yet, biomarkers such as telomere erosion and p16(INK4a) are ambiguous for radiation-induced aging. Some animal studies suggest low dose radiation may even demonstrate hormesis health benefits. Regardless, there is virtually no support for a life span extending hypothesis for A-bomb survivors and other exposed subjects.

Growth Signaling at the Nexus of Stem Cell Life and Death

Stress can activate tumor-suppressive mechanisms, causing the loss of adult stem cell function with age. In this issue of Cell Stem Cell and in Nature, Castilho et al. (2009) and Harrison et al. (2009) highlight the importance of mTOR signaling in stem cell exhaustion and mammalian aging, respectively.

MTOR Mediates Wnt-Induced Epidermal Stem Cell Exhaustion and Aging

Epidermal integrity is a complex process established during embryogenesis and maintained throughout the organism lifespan by epithelial stem cells. Although Wnt regulates normal epithelial stem cell renewal, aberrant Wnt signaling can contribute to cancerous growth. Here, we explored the consequences of persistent expressing Wnt1 in an epidermal compartment that includes the epithelial stem cells. Surprisingly, Wnt caused the rapid growth of the hair follicles, but this was followed by epithelial cell senescence, disappearance of the epidermal stem cell compartment, and progressive hair loss. Although Wnt1 induced the activation of beta-catenin and the mTOR pathway, both hair follicle hyperproliferation and stem cell exhaustion were strictly dependent on mTOR function. These findings suggest that whereas activation of beta-catenin contributes to tumor growth, epithelial stem cells may be endowed with a protective mechanism that results in cell senescence upon the persistent stimulation of proliferative pathways that activate mTOR, ultimately suppressing tumor formation.

Stem cell exhaustion and leukemogenesis

Cancer is the result of a combination of genetic alterations, which aid transformation of cells. However, oncogenic alterations also simultaneously induce some detrimental effects on the cells such as apoptosis, senescence, and differentiation. Such negative effects caused by certain oncogenic events are overcome by other cooperating genetic hits. We propose stem cell exhaustion as a novel detrimental effect that is caused by a wide variety of oncogenic alterations. Interestingly, in most cases, the stem cell exhaustion due to oncogenic alterations is preceded by an abnormal expansion of stem/progenitor cells. This preceding stem/progenitor cell expansion may be a key feature that still promotes cancer development, along with cooperating hits that rescue stem cell exhaustion. This review summarizes current knowledge about hematopoietic stem cell exhaustion and the mechanisms to overcome stem cell exhaustion in cancer development.

Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells

Rare cells with the properties of stem cells are integral to the development and perpetuation of leukaemias. A defining characteristic of stem cells is their capacity to self-renew, which is markedly extended in leukaemia stem cells. The underlying molecular mechanisms, however, are largely unknown. Here we demonstrate that expression of the cell-cycle inhibitor p21 is indispensable for maintaining self-renewal of leukaemia stem cells. Expression of leukaemia-associated oncogenes in mouse haematopoietic stem cells (HSCs) induces DNA damage and activates a p21-dependent cellular response, which leads to reversible cell-cycle arrest and DNA repair. Activated p21 is critical in preventing excess DNA-damage accumulation and functional exhaustion of leukaemic stem cells. These data unravel the oncogenic potential of p21 and suggest that inhibition of DNA repair mechanisms might function as potent strategy for the eradication of the slowly proliferating leukaemia stem cells.

Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Rad18 is required for long-term maintenance of spermatogenesis in mouse testes

Maintaining the integrity of spermatogenic stem cells is essential to transfer genetic information to a descendant. However, knowledge of maintenance of genetic stability in stem cells is still limited. RAD18 is critical for postreplication repair through mono- and multi-ubiquitination of proliferating cell nuclear antigen (PCNA) to maintain genomic stability. Mammalian RAD18 is highly expressed in the spermatocytes and the nuclei of a few spermatogonia in adult mice. To elucidate the physiological function of RAD18, we analyzed a phenotype of Rad18−/− mice. The mice were born and appeared to grow normally. Although the mice were fertile, fertility and testis weight decreased with age. Histological examination revealed normal spermatogenesis in almost all seminiferous tubules in Rad18−/− testes at 2 months old, and abnormal sperm could not be detected in the epididymis. However, 25% of the tubules lost almost all germ cells at 12 months. The seminiferous tubules frequently retained only late differentiated phase germ cells, suggesting that the exhaustion of spermatogonial stem cells leads to the loss of all germ cells in the seminiferous tubules. Wild-type germ cells were successfully transplanted into and colonized in the seminiferous tubules of aged Rad18−/− mice, indicating that Sertoli cells have a normal supportive function even in aged testes. We conclude that RAD18 is intrinsically required for the long-term maintenance of spermatogenesis.

Iron Overload and Haematopoiesis in MDS: Does Blood Transfusion Promote Progression to AML?

Introduction: The biology of myelodysplastic syndrome (MDS) is poorly understood, and treatment options are limited. Thus, most MDS patients require chronic red blood cell transfusion, and many develop secondary iron overload. Although the pathophysiological consequences of iron overload to the heart, liver, and endocrine organs have been well characterized, its effects on haematopoiesis have not been studied. However, it has been observed that chelation therapy in iron-overloaded MDS patients may result in reduction of transfusion requirements, and recent studies have suggested a correlation between the use of iron chelation therapy and improvement in leukaemia-free survival in MDS.At the cellular level, iron toxicity is mediated in large part via the generation of reactive oxygen species (ROS). It has been shown in animal models that accumulation of ROS leads to senescence of haematopoietic stem cells, and that ROS cause DNA damage and promote the development of malignancy. These effects of ROS may be particularly important in MDS, in which haematopoiesis is already severely compromised and genetic instability is a striking feature.Hypothesis: We hypothesize that iron overload secondary to transfusion leads to increased levels of intracellular ROS in early haematopoeitic cells in MDS. The increase in intracellular ROS in MDS would be predicted to lead further impairment of haematopoiesis via stem cell exhaustion and while promoting accumulation of DNA damage by myelodysplastic stem cells and early progenitors, thus accelerating progression of MDS to acute leukaemia.Results: To test this hypothesis, we examined the relationship between transfusion-related iron overload and ROS content of CD34+ bone marrow cells in MDS. ROS content was measured in CD34+ cells by flow cytometry in bone marrow aspirates from 34 consecutive MDS patients (CMML=4, MDS/MPD=2, RA=4, RARS=3, RCMD=2, RAEB 1=6, RAEB 2=12, RAEB-t/AML=1). The patients represented a wide range of prior transfusion burden (0->300 units PRBC) and serum ferritin levels (11->10000 μg/L). ROS was strongly correlated with serum ferritin concentration for patients with iron overload (serum ferritin >1000 μg/L; n=14, R=0.733, p<0.005). The correlation between ROS and ferritin level was even stronger in the subset of patients with RAEB 1 or RAEB 2 and iron overload (n=11, R=0.838, p<0.005). In contrast, no correlation between ROS and ferritin level was demonstrated for patients with serum ferritin <1000 μg/L (n=20). Importantly, iron chelation therapy was associated with a reduction in CD34+ cell ROS content in one patient.To assess the effect of iron overload on normal stem cell and progenitor function, we established a mouse model of subacute bone marrow iron overload. B6D2F1 mice were loaded with iron dextran by intraperitoneal injection (150mg total iron load over 21 days), and sacrificed three days after the end of iron loading. Iron staining of tissue sections confirmed iron deposition in the bone marrow, liver, and myocardium. The development of splenomegaly was noted in iron-loaded animals. Flow cytometric analysis revealed increased apoptosis of bone marrow cells in iron loaded mice based on annexin V+/7 AAD-staining (6.26±0.96% versus 3.54±0.99% for control mice, paired student's t-Test p<0.005). However, ROS content in CD117+ progenitors of iron loaded mice was similar to control mice. Thus, subacute iron loading in mice increases apoptosis but does not alter the ROS content of HSCs; we postulate that chronic iron overload is required to achieve this effect.Conclusions: These results establish a relationship between CD34+ cell ROS content and serum ferritin concentration in MDS patients with iron overload, and indicate that iron chelation therapy in this patient population reverses this ROS accumulation. The physiological consequences of this relationship are currently being investigated in this patient set by haematopoietic colony assays and assessment of DNA damage in CD34+ cells. Nonethelesss, these data may have key implications for the deployment of iron chelation therapy in MDS patients, and may explain the association between the use of iron chelation and improved leukaemia-free survival in MDS.

Age-Adjusted Telomere Length Is Not Associated with Variation in Hematological Parameters in Ageing Women

Background. Telomeres play a crucial role in maintaining physical integrity of chromosomes. In the absence of telomerase, telomere length (TL) shortens with each cell division up to a critical threshold where cellular senescence occurs. TL is inversely correlated with age, is longer in women than in men, and demonstrates a strong heritability. Normal blood counts are maintained through out life by an extraordinary number of cell divisions rendering telomere maintenance primordial to prevent stem cell exhaustion. In fact, some cases of bone marrow failure syndromes, such as aplastic anemia and dyskeratosis congenital, have been linked to mutation in the telomerase gene; and stressed hematopoiesis, such at it occurs during the first year following allogeneic bone marrow transplantation induces TL shortening. We hypothesized that individuals with shorter TL may have lower blood counts and a decreased bone marrow reserve. The evaluation of TL as a potential biomarker of ageing hematopoiesis is important in the context of bone marrow transplantation performed with increasingly old donors.Methods. We measured TL in 1583 women, predominantly aged over 60, all originating from 288 French-Canadian families using a real-time quantitative PCR method that measures the number of telomere repeats relatively to the copy number of a single copy number gene. Telomeres were adjusted for age. Pearson or Spearman correlations were used to determine association between age-adjusted TL (aTL) and hematological parameter according to, respectively, whether or not a normal distribution was observed for these data. A Bonferroni correction was further applied to set the statistical significance threshold.Results. aTL varied significantly between individuals of the cohort, but no correlation was detected with hemoglobin levels (−0,001; p=0,978), mean corpuscular volume (−0,031; p=0,403); leucocytes (0,055; p=0,139); neutrophils (0,078; p=0,036), monocytes, (0,059; p=0,113), eosinophils (−0,032; p=0,394) and platelets (0,030; p=0,428) counts.Conclusion. Based on our analysis, TL do not predict blood cells counts in ageing women and may not be a useful biomarker for donor selection. This could also suggest that there is a threshold beyond which TL has an effect on hematopoiesis and that point was not reached in our cohort.

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.

Inhibition of p38 MAPK Reduces Ionizing Radiation (IR)-Induced Hematopoietic Suppression by Preventing Hematopoietic Stem/Progenitor Cell Senescence.

Bone marrow (BM) suppression is the most common dose-limiting side effect of conventional cancer therapy, particularly with certain alkylating agents and/or ionizing radiation (IR). It has been shown that p38 mitogen-activated protein kinase (p38 MAPK) plays a critical role in regulation of hematopoiesis and its activation mediates IR- and chemotherapy-induced cell injury and oxidative stress-induced hematopoietic stem cell (HSC) exhaustion. Therefore, we examined the role of p38 MAPK in radiation-induced BM injury. First, we monitored the activation of p38 MAPK in BM hematopoietic cells at different time after exposure to IR using our well-established long-term BM cell culture (LTBMC) model system. The activation of p38 MAPK was detected within 24 hours after BM hematopoietic cells were exposed to 4 Gy IR and this activation sustained up to 5 weeks after radiation. Inhibition of p38 MAPK activity with a p38 MAPK specific inhibitor (SB202190) attenuated IR-induced suppression of the hematopoietic function of BM hematopoietic cells in an in vitro colony forming cell (CFC) assay. Moreover, the number of hematopoietic progenitor cells produced by SB202190-treated BM cells was significantly greater than that by the cells without SB202190 treatment after exposure to IR and followed by a five-week LTBMC. Interestingly, p38 MAPK inhibition showed no effect on IR-induced apoptosis in both HSCs and hematopoietic progenitor cells (HPCs), whereas the radioprotection effect of SB202190 was associated with a significantly reduction of p16INK4a expression and senescence-associated β-galactosidase (SA-β-Gal) activity in irradiated BM cells after five weeks of LTBMC. These findings suggest that activation of p38 MAPK may mediate IR-induced hematopoietic suppression by induction of hematopoietic stem/progenitor cell senescence and pharmacological inhibition of the p38 MAPK pathway may have the potential to be developed as an innovatively therapeutic strategy to ameliorate IR- and chemotherapy-induced BM toxicity.

Altered mammary gland development in the p53+/m mouse, a model of accelerated aging

The tumor suppressor p53 is important for inhibiting the development of breast carcinomas. However, little is known about the effects of increased p53 activity on mammary gland development. Therefore, the effect of p53 dosage on mammary gland development was examined by utilizing the p53+/m mouse, a p53 mutant which exhibits increased wild-type p53 activity, increased tumor resistance, a shortened longevity, and a variety of accelerated aging phenotypes. Here we report that p53+/m virgin mice exhibit a defect in mammary gland ductal morphogenesis. Transplants of mammary epithelium into p53+/m recipient mice demonstrate decreased outgrowth of wild-type and p53+/m donor epithelium, suggesting systemic or stromal alterations in the p53+/m mouse. Supporting these data, p53+/m mice display decreased levels of serum IGF-1 and reduced IGF-1 signaling in virgin glands. The induction of pregnancy or treatment of p53+/m mice with estrogen, progesterone, estrogen and progesterone in combination, or IGF-1 stimulates ductal outgrowth, rescuing the p53+/m mammary phenotype. Serial mammary epithelium transplants demonstrate that p53+/m epithelium exhibits decreased transplant capabilities, suggesting early stem cell exhaustion. These data indicate that appropriate levels of p53 activity are important in regulating mammary gland ductal morphogenesis, in part through regulation of the IGF-1 pathway.

Relationships between stem cell exhaustion, tumour suppression and ageing

Ageing is a complex process characterised by a variety of disorders associated with general organismal decline and an inability to maintain tissue homoeostasis. As described in this review, recent studies indicate that ageing may be caused, in part, by the depletion of stem and progenitor cells that govern tissue renewal. The potential causes of stem and progenitor cell attrition are numerous; however, a commonly accepted theory is that these cells are lost as a result of naturally occurring DNA damage and the obligate checkpoint responses that follow. Failure to launch appropriate responses to DNA damage is strongly associated with cancer initiation and progression. Therefore, it is at this nexus, the response to DNA damage, that an important organismal fate may be determined: to degrade regenerative potential for the purpose of preventing cancer. According to this viewpoint, ageing may be the unfortunate mark of successful cancer suppression in stem cells and other cell types. In this review, we will describe how degeneration of tissue renewal capacity links ageing and cancer suppression.

Selective Functional Exhaustion of Hematopoietic Progenitor Cells in the Bone Marrow of Patients With Postinfarction Heart Failure

This study investigated whether reduced levels of circulating endothelial progenitors cells (EPCs) in chronic heart failure (CHF) are secondary to an exhaustion of hematopoietic stem cells (HSCs) in the bone marrow or to reduced mobilization. Circulating EPCs presumably originate from bone marrow-derived HSC. Persistent mobilization of EPCs was shown to be associated with favorable left ventricular infarct remodeling processes. We assessed the number and functional capacity of EPCs in 17 healthy controls, 25 patients with ischemic cardiomyopathy (ICM), and 20 patients with dilated cardiomyopathy (DCM). To document an impairment of HSC function in the bone marrow, the colony-forming unit capacity of bone marrow-derived mononuclear cells and the number of CD34+ HSCs were examined in 6 healthy volunteers, 94 ICM patients, and 25 DCM patients. The number of EPCs was reduced in CHF, irrespective of its etiology. In contrast, the migratory capacity was selectively impaired in EPCs of ICM patients (4.8 +/- 4.0 migrated cells; DCM 9.7 +/- 5.8; p = 0.02). On multivariate analysis, ICM, advanced New York Heart Association functional class, and CHF were independent predictors of functional EPC impairment. The number of bone marrow-derived CD34+ cells did not differ between the CHF populations. However, colony-forming units (CFUs) were selectively reduced in ICM patients (54.4 +/- 24.6; DCM 68.1 +/- 26.9; p < 0.02). Ischemic cardiomyopathy was the only independent predictor of impaired CFU capacity. Impaired CFU capacity was associated with reduced matrix metalloproteinase-9 activity in the bone marrow plasma. Ischemic cardiomyopathy is associated with selective impairment of progenitor cell function in the bone marrow and in the peripheral blood, which may contribute to an unfavorable left ventricular (LV) remodeling process.