The elements of stem cell self-renewal: a genetic perspective

Every day, the body produces billions of new blood cells. Each of these is derived from a rare cell in the bone marrow called the hematopoietic stem cell (HSC). Because most mature blood cells have a limited lifespan, the ability of HSCs to self-renew and replenish the mature cell compartment is critical to sustaining life. While great progress has been made in isolating HSCs and defining their functional and phenotypic characteristics, the molecular mechanisms that regulate their self-renewal remain a mystery. Over the last few years, alterations in HSC frequency and self-renewal capacity in transgenic and knock-out mice have led to the identification of novel mediators of HSC homeostasis in vivo. These genetically modified mice have revealed that maintenance of survival, proliferation, quiescence, and normal telomere length all contribute to the self-renewal of HSCs. They also highlight the need to test in context of the normal microenvironment the role of signaling molecules such as Notch and Wnt, which have emerged recently as important regulators of HSC self-renewal. The emerging picture these data provide of the regulation of self-renewal in HSCs has provided a better understanding of the basic biology of stem cells and holds promise for designing strategies to improve bone marrow transplantation.     

p57 is required for quiescence and maintenance of adult hematopoietic stem cells

Quiescence is required for the maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in HSC quiescence, but loss of p21 or p27 in mice affects HSC quiescence or functionality only under conditions of stress. Although p57 is the most abundant family member in quiescent HSCs, its role has remained uncharacterized. Here we show a severe defect in the self-renewal capacity of p57-deficient HSCs and a reduction of the proportion of the cells in G(0) phase. Additional ablation of p21 in a p57-null background resulted in a further decrease in the colony-forming activity of HSCs. Moreover, the HSC abnormalities of p57-deficient mice were corrected by knocking in the p27 gene at the p57 locus. Our results therefore suggest that, among Cip/Kip family CDK inhibitors, p57 plays a predominant role in the quiescence and maintenance of adult HSCs.     

Lifespan Differences in Hematopoietic Stem Cells are Due to Imperfect Repair and Unstable Mean-Reversion

The life-long supply of blood cells depends on the long-term function of hematopoietic stem cells (HSCs). HSCs are functionally defined by their multi-potency and self-renewal capacity. Because of their self-renewal capacity, HSCs were thought to have indefinite lifespans. However, there is increasing evidence that genetically identical HSCs differ in lifespan and that the lifespan of a HSC is predetermined and HSC-intrinsic. Lifespan is here defined as the time a HSC gives rise to all mature blood cells. This raises the intriguing question: what controls the lifespan of HSCs within the same animal, exposed to the same environment? We present here a new model based on reliability theory to account for the diversity of lifespans of HSCs. Using clonal repopulation experiments and computational-mathematical modeling, we tested how small-scale, molecular level, failures are dissipated at the HSC population level. We found that the best fit of the experimental data is provided by a model, where the repopulation failure kinetics of each HSC are largely anti-persistent, or mean-reverting, processes. Thus, failure rates repeatedly increase during population-wide division events and are counteracted and decreased by repair processes. In the long-run, a crossover from anti-persistent to persistent behavior occurs. The cross-over is due to a slow increase in the mean failure rate of self-renewal and leads to rapid clonal extinction. This suggests that the repair capacity of HSCs is self-limiting. Furthermore, we show that the lifespan of each HSC depends on the amplitudes and frequencies of fluctuations in the failure rate kinetics. Shorter and longer lived HSCs differ significantly in their pre-programmed ability to dissipate perturbations. A likely interpretation of these findings is that the lifespan of HSCs is determined by preprogrammed differences in repair capacity.     

Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice

Despite being a hallmark of hematopoietic stem cells (HSCs), HSC self-renewal has never been quantitatively assessed. Establishment of a clonal and quantitative assay for HSC function permitted demonstration that adult mouse HSCs are significantly heterogeneous in degree of multilineage repopulation and that higher repopulating potential reflects higher self-renewal activity. An HSC with high repopulating potential could regenerate approximately 1000 HSCs, whereas the repopulating activity of regenerated HSCs on average was significantly reduced, indicating extensive but limited self-renewal capacity in HSCs. Comparisons of wild-type mice with mutant mice deficient in the signal adaptor molecule Lnk showed that not only HSC numbers but also the self-renewal capacity of some HSCs are markedly increased when Lnk function is lost. Lnk appears to control HSC numbers by negatively regulating HSC self-renewal signaling.     

IFN-gamma negatively modulates self-renewal of repopulating human hemopoietic stem cells

Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-gamma, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN-gamma might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN-gamma on HSC maintenance and function. Although purified cord blood CD34(+)CD38(-) cells underwent cell divisions in the presence of IFN-gamma, cycling HSCs exposed to IFN-gamma in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN-gamma accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN-gamma can negatively affect human HSC self-renewal.     

CD81 is essential for the re-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the Akt pathway

The regulatory mechanisms governing the cell cycle progression of hematopoietic stem cells (HSCs) are well characterized, but those responsible for the return of proliferating HSCs to a quiescent state remain largely unknown. Here, we present evidence that CD81, a tetraspanin molecule acutely responsive to proliferative stress, is essential for the maintenance of long-term repopulating HSCs. Cd81(-/-) HSCs showed a marked engraftment defect when transplanted into secondary recipient mice and a significantly delayed return to quiescence when stimulated to proliferate with 5-fluorouracil (5FU). In addition, we found that CD81 proteins form a polarized patch when HSCs are returning to quiescence. Thus, we propose that the spatial distribution of CD81 during the HSC recovery phase drives proliferative HSC to quiescence, and is important to preserve the self-renewal properties. Here, we show that lack of CD81 leads to loss of HSC self-renewal, and the clustering of CD81 on HSC membrane results in deactivation of Akt, which subsequently leads to nuclear translocation of FoxO1a. Thus, CD81 functions as part of a previously undefined mechanism that prohibits excessive proliferation of HSCs exposed to environmental stress.     

miR-33-mediated downregulation of p53 controls hematopoietic stem cell self-renewal

Hematopoietic stem cells (HSCs) are defined by their exclusive capacity to both self-renew and to give rise to multipotent progenitors (MPPs) that in turn differentiate into the mature blood cell lineages. The tumor suppressor p53, in addition to its role in the regulation of the cell cycle, plays an importatn role in HSC self-renewal, although it has not fully resolved. Here we report that in super-p53 mice (sp53), which carry one extra gene dose of p53, the miR-33 is down-regulated in HSCs and highly expressed in MPPs. Transplantation assays of miR-33-transduced sp53 HSC results in a significant acquisition of repopulating capacity and a decrease of recipients survival. Moreover, high levels of miR-33 represses the endogenous level of p53 protein in murine embryonic fibroblasts (MEFs), leads both to neoplastic transformation and anchorage independent growth of MEFs, and displays a decrease of apoptotic response using tumor-derived cell lines. Accordingly, we demonstrate that miR-33-mediated down-regulation of p53 is dependent on the binding of miR-33 to two conserved motifs in the 3′UTR of p53. Together, these data show that the miR-33 modifies HSC repopulating efficiency of sp53 mice by impairing the p53 function. Defining the role of miR-33 in controlling the HSC self-renewal through p53 may lead to the prevention and treatment of hematopoietic disorders.     

Granulocyte colony-stimulating factor exacerbates hematopoietic stem cell injury after irradiation

Background

Exposure to a moderate to high dose of ionizing radiation (IR) not only causes acute radiation syndrome but also induces long-term (LT) bone marrow (BM) injury. The latter effect of IR is primarily attributed to the induction of hematopoietic stem cell (HSC) senescence. Granulocyte colony-stimulating factor (G-CSF) is the only treatment recommended to be given to radiation victims soon after IR. However, clinical studies have shown that G-CSF used to treat the leukopenia induced by radiotherapy or chemotherapy in patients can cause sustained low white blood cell counts in peripheral blood. It has been suggested that this adverse effect is caused by HSC and hematopoietic progenitor cell (HPC) proliferation and differentiation stimulated by G-CSF, which impairs HSC self-renewal and may exhaust the BM capacity to exacerbate IR-induced LT-BM injury.

Methods

C57BL/6 mice were exposed to 4 Gy γ-rays of total body irradiation (TBI) at a dose-rate of 1.08 Gy per minute, and the mice were treated with G-CSF (1 μg/each by ip) or vehicle at 2 and 6 h after TBI on the first day and then twice every day for 6 days. All mice were killed one month after TBI for analysis of peripheral blood cell counts, bone marrow cellularity and long-term HSC (CD34-lineage-sca1+c-kit+) frequency. The colony-forming unit-granulocyte and macrophage (CFU-GM) ability of HPC was measured by colony-forming cell (CFC) assay, and the HSC self-renewal capacity was analyzed by BM transplantation. The levels of ROS production, the expression of phospho-p38 mitogen-activated protein kinase (p-p38) and p16^INK4a (p16) mRNA in HSCs were measured by flow cytometry and RT-PCR, respectively.

Results

The results of our studies show that G-CSF administration mitigated TBI-induced decreases in WBC and the suppression of HPC function (CFU-GM) (p < 0.05), whereas G-CSF exacerbated the suppression of long-term HSC engraftment after transplantation one month after TBI (p < 0.05); The increase in HSC damage was associated with increased ROS production, activation of p38 mitogen-activated protein kinase (p38), induction of senescence in HSCs.

Conclusion

Our findings suggest that although G-CSF administration can reduce ARS, it can also exacerbate TBI-induced LT-BM injury in part by promoting HSC senescence via the ROS-p38-p16 pathway.

     

Gadd45a deletion aggravates hematopoietic stem cell dysfunction in ATM-deficient mice

Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM^-/-) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM^-/- HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM^-/- mice. Instead, ATM and Gadd45a double knockout (ATM^-/- Gadd45a^-/-) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM^-/- HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM^-/- Gadd45a^-/- HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM^-/- Gadd45a^-/- mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM^-/- mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM^-/- HSCs.     

The axis of mTOR-mitochondria-ROS and stemness of the hematopoietic stem cells

It has been hypothesized that adult hematopoietic stem cells (HSCs) need to remain quiescent to retain their long-term self-renewal activity and multipotency. However, it is still unclear how lack of quiescence is detrimental to HSC. We identified that the mTOR pathway is the key to HSCs quiescence. mTOR overactivation caused increased mitochondrial biogenesis and accumulation of much higher level of reactive oxygen species (ROS). Removal of ROS rescued HSC defects associated with hyperactivated mTOR. We propose susceptibility to ROS as the underlying cause for HSC’s general requirement for quiescence.     

Identification of adiponectin as a novel hemopoietic stem cell growth factor

The hemopoietic microenvironment consists of a diverse repertoire of cells capable of providing signals that influence hemopoietic stem cell function. Although the role of osteoblasts and vascular endothelial cells has recently been characterized, the function of the most abundant cell type in the bone marrow, the adipocyte, is less defined. Given the emergence of a growing number of adipokines, it is possible that these factors may also play a role in regulating hematopoiesis. Here, we investigated the role of adiponectin, a secreted molecule derived from adipocytes, in hemopoietic stem cell (HSC) function. We show that adiponectin is expressed by components of the HSC niche and its receptors AdipoR1 and AdipoR2 are expressed by HSCs. At a functional level, adiponectin influences HSCs by increasing their proliferation, while retaining the cells in a functionally immature state as determined by in vitro and in vivo assays. We also demonstrate that adiponectin signaling is required for optimal HSC proliferation both in vitro and in long term hemopoietic reconstitution in vivo. Finally we show that adiponectin stimulation activates p38 MAPK, and that inhibition of this pathway abrogates adiponectin's proliferative effect on HSCs. These studies collectively identify adiponectin as a novel regulator of HSC function and suggest that it acts through a p38 dependent pathway.     

Metabolic regulation of hematopoietic stem cells in the hypoxic niche

Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) reside in a hypoxic bone marrow environment, and their metabolic status is distinct from that of their differentiated progeny. HSCs generate energy mainly via anaerobic metabolism by maintaining a high rate of glycolysis. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species, but leaves HSCs susceptible to changes in redox status. In this review, we discuss the importance of oxygen homeostasis and energy metabolism for maintenance of HSC function and long-term self-renewal.     

Notch in the niche

Notch signaling is essential for hematopoietic stem cell (HSC) formation during embryogenesis, and hitherto it was also thought to be required for HSC maintenance. However, in this issue of Cell Stem Cell, Maillard et al. (2008) demonstrate rather conclusively that inactivation of the Notch pathway in HSCs does not interfere with their self-renewal.     

Chromosome Instability Underlies Hematopoietic Stem Cell Dysfunction and Lymphoid Neoplasia Associated with Impaired Fbw7-Mediated Cyclin E Regulation

The Fbw7 ubiquitin ligase critically regulates hematopoietic stem cell (HSC) function, though the precise contribution of individual substrate ubiquitination pathways to HSC homeostasis is unknown. In the work reported here, we used a mouse model in which we introduced two knock-in mutations (T74A and T393A [changes of T to A at positions 74 and 393]) to disrupt Fbw7-dependent regulation of cyclin E, its prototypic substrate, and to examine the consequences of cyclin E dysregulation for HSC function. Serial transplantation revealed that cyclin E^{T74A T393A} HSCs self-renewed normally; however, we identified defects in their multilineage reconstituting capacity. By inducing hematologic stress, we exposed an impaired self-renewal phenotype in cyclin E knock-in HSCs that was associated with defective cell cycle exit and the emergence of chromosome instability (CIN). Importantly, p53 deletion induced both defects in self-renewal and multilineage reconstitution in cyclin E knock-in HSCs with serial transplantation and CIN in hematopoietic stem and progenitor cells. Moreover, CIN was a feature of fatal T-cell malignancies that ultimately developed in recipients of cyclin E^{T74A T393A}; p53-null HSCs. Together, our findings demonstrate the importance of Fbw7-dependent cyclin E control to the hematopoietic system and highlight CIN as a characteristic feature of HSC dysfunction and malignancy induced by deregulated cyclin E.     

In vivo divisional tracking of hematopoietic stem cells

Hematopoietic stem cell (HSC) division leads to self-renewal, differentiation, or death of HSCs, and adequate balance of this process results in sustained, lifelong, high-throughput hematopoiesis. Despite their contribution to hematopoietic cell production, the majority of cells within the HSC population are quiescent at any given time. Recent studies have tackled the questions of how often HSCs divide, how divisional history relates to repopulating potential, and how many HSCs contribute to hematopoiesis. Here, we summarize these recent findings on HSC turnover from different experimental systems and discuss hypothetical models for HSC cycling and maintenance in steady-state and upon hematopoietic challenge.