Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells

Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm(-/-) mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease.     

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.     

Bone-marrow haematopoietic-stem-cell niches

Adult stem cells hold many promises for future clinical applications and regenerative medicine. The haematopoietic stem cell (HSC) is the best-characterized somatic stem cell so far, but in vitro expansion has been unsuccessful, limiting the future therapeutic potential of these cells. Here we review recent progress in characterizing the composition of the HSC bone-marrow microenvironment, known as the HSC niche. During homeostasis, HSCs, and therefore putative bone-marrow HSC niches, are located near bone surfaces or are associated with the sinusoidal endothelium. The molecular crosstalk between HSCs and the cellular constituents of these niches is thought to control the balance between HSC self-renewal and differentiation, indicating that future successful expansion of HSCs for therapeutic use will require three-dimensional reconstruction of a stem-cell-niche unit.     

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.     

Enforced expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo

Hoxa5 is preferentially expressed in haematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs), and is more highly expressed in expanding HSCs. To date, little is known regarding the role of Hoxa5 in HSCs and downstream progenitor cells in vivo. In this study, we show that increased expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo. Hoxa5 differentially modifies the cell cycle of HSCs and lineage committed progenitor cells, depending on the cellular context. Hoxa5 drives HSCs, but not MPPs, through the cell cycle and arrests erythroid progenitor cells in G0 phase. Although the HSC pool shrinks after overexpression of Hoxa5, HSCs sustain the abilities of self-renewal and multipotency. In vivo, Hoxa5 has two effects on erythropoiesis: it causes a predominance of mature erythroid lineage cells and the partial apoptosis of erythroid progenitors. RNA-seq indicates that multiple biological processes, including erythrocyte homeostasis, cell metabolism, and apoptosis, are modified by Hoxa5. The results of this study indicate that Hoxa5 is a key regulator of the HSC cell cycle, and the inappropriate expression of Hoxa5 in lineage-committed progenitor cells leads to aberrant erythropoiesis.     

Enforced expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo

Hoxa5 is preferentially expressed in haematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs), and is more highly expressed in expanding HSCs. To date, little is known regarding the role of Hoxa5 in HSCs and downstream progenitor cells in vivo. In this study, we show that increased expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo. Hoxa5 differentially modifies the cell cycle of HSCs and lineage committed progenitor cells, depending on the cellular context. Hoxa5 drives HSCs, but not MPPs, through the cell cycle and arrests erythroid progenitor cells in G0 phase. Although the HSC pool shrinks after overexpression of Hoxa5, HSCs sustain the abilities of self-renewal and multipotency. In vivo, Hoxa5 has two effects on erythropoiesis: it causes a predominance of mature erythroid lineage cells and the partial apoptosis of erythroid progenitors. RNA-seq indicates that multiple biological processes, including erythrocyte homeostasis, cell metabolism, and apoptosis, are modified by Hoxa5. The results of this study indicate that Hoxa5 is a key regulator of the HSC cell cycle, and the inappropriate expression of Hoxa5 in lineage-committed progenitor cells leads to aberrant erythropoiesis.     

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.     

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.     

Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin

Tissue homeostasis demands regulatory feedback, suggesting that hematopoietic stem cell (HSC) activity is controlled in part by HSC progeny. Yet, cell extrinsic HSC regulation has been well characterized only in niche cells of non-hematopoietic origin. Here we identify feedback regulation of HSCs by megakaryocytes (Mks), which are mature hematopoietic cells, through production of thrombopoietin (Thpo), a cytokine pertinent for HSC maintenance. Induced ablation of Mk cell population in mice perturbed quiescent HSCs in bone marrow (BM). The ablation of Mks resulted in decreased intra-BM Thpo concentration presumably due to Thpo production by Mks. Thpo administration Mk ablated mice restored HSC functions. Overall, our study establishes Mk as an essential cellular component of the HSC niche and delineates cytokine-oriented regulation of HSCs by their own progeny.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

Hematopoietic stem cells: generation and self-renewal

Adult stem cells hold great promise for future therapeutic applications. Hematopoietic stem cells (HSCs) are among the best-characterized adult stem cells. As such, these cells provide a conceptual framework for the study of adult stem cells from other organs. Here, we review the current knowledge of HSC generation during embryonic development and HSC maintenance in the bone marrow (BM) during adult life. Recent scientific progress has demonstrated that the development of HSCs involves many anatomical sites in the embryo, but the relative contribution of each of these sites to the adult HSC pool remains controversial. Specialized anatomical sites in the BM have been identified as stem cell niches, and these play essential roles in regulating the self-renewal and differentiation of HSCs through recently identified signaling pathways. Extracellular signaling from stem cell niches must integrate with the intracellular molecular machinery and/or genetic programs to regulate HSC fate choice. The exact cellular and/or molecular mechanisms defining stem cell niche and 'stemness' of HSC is largely unknown although substantial progress has been made recently. Hence, many questions remain to be answered even in this relatively well-defined model of stem cell biology.     

PTEN in the haematopoietic system and its therapeutic indications

A unique feature of the haematopoietic system is its self-renewal ability while maintaining a stable number of pluripotent haematopoietic stem cells (HSCs). Recently, two publications by Yilmaz and colleagues and Zhang and colleagues demonstrated that the loss of the tumour suppressor phosphatase and tensin homolog (PTEN) in mice disturbed the maintenance of quiescent HSCs and promoted leukemogenesis. Mammalian target of rapamycin (mTOR) inhibition with rapamycin distinctly rescued HSC development and depleted leukemic stem cells. Thus, the regulation of HSCs and leukemic cells seems to be governed by cell-context-dependent, PTEN-mediated regulation of mTOR.     

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.     

A Novel Assay to Trace Proliferation History In Vivo Reveals that Enhanced Divisional Kinetics Accompany Loss of Hematopoietic Stem Cell Self-Renewal

Background

The maintenance of lifelong blood cell production ultimately rests on rare hematopoietic stem cells (HSCs) that reside in the bone marrow microenvironment. HSCs are traditionally viewed as mitotically quiescent relative to their committed progeny. However, traditional techniques for assessing proliferation activity in vivo, such as measurement of BrdU uptake, are incompatible with preservation of cellular viability. Previous studies of HSC proliferation kinetics in vivo have therefore precluded direct functional evaluation of multi-potency and self-renewal, the hallmark properties of HSCs.

Methodology/Principal Findings

We developed a non-invasive labeling technique that allowed us to identify and isolate candidate HSCs and early hematopoietic progenitor cells based on their differential in vivo proliferation kinetics. Such cells were functionally evaluated for their abilities to multi-lineage reconstitute myeloablated hosts.

Conclusions

Although at least a few HSC divisions per se did not influence HSC function, enhanced kinetics of divisional activity in steady state preceded the phenotypic changes that accompanied loss of HSC self-renewal. Therefore, mitotic quiescence of HSCs, relative to their committed progeny, is key to maintain the unique functional and molecular properties of HSCs.     

Regulation of hematopoietic stem cells in the niche

Hematopoiesis provides a suitable model for understanding adult stem cells and their niche. Hematopoietic stem cells(HSCs) continuously produce blood cells through orchestrated proliferation, self-renewal, and differentiation in the bone marrow(BM). Within the BM exists a highly organized microenvironment termed "niche" where stem cells reside and are maintained. HSC niche is the first evidence that a microenvironment contributes to protecting stem cell integrity and functionality in mammals. Although multiple models exist, recent progress has principally elucidated the cellular complexity of the HSC niche that maintains and regulates HSCs in BM. Here we introduce the development and summarize the achievements of HSC niche studies.     

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.