RunX3对小鼠骨髓造血干细胞功能的影响

目的研究RunX3基因对造血干细胞自我更新和分化能力的影响。方法流式细胞术测定小鼠骨髓干细胞和外周血单个核细胞的比例;通过竞争性骨髓移植实验检测RunX3转基因小鼠骨髓干细胞的功能。结果移植后来源于RunX3-/-小鼠骨髓干细胞供体的外周血细胞占总外周血细胞的比例与野生对照鼠相比无明显差异,移植后来源于RunX3-/-小鼠骨髓干细胞供体的外周血中髓系细胞占总外周血髓系细胞的比例较野生型对照鼠高。结论RunX3基因缺失对骨髓造血干细胞的自我更新没有影响,但其可能参与了骨髓造血干细胞的分化过程。     

Robo4 Plays a Role in Bone Marrow Homing and Mobilization,but Is Not Essential in the Long-Term Repopulating Capacity of Hematopoietic Stem Cells

Roundabout (Robo) family proteins are immunoglobulin-type surface receptors critical for cellular migration and pathway finding of neuronal axons. We have previously shown that Robo4 was specifically expressed in hematopoietic stem and progenitor cells and its high expression correlated with long-term repopulating (LTR) capacity. To reveal the physiological role of Robo4 in hematopoiesis, we examined the effects of Robo4 disruption on the function of hematopoietic stem cells (HSCs) and progenitors. In Robo4-deficient mice, basic hematological parameters including complete blood cell count and differentiation profile were not affected. In contrast to the previous report, HSC/hematopoietic progenitor (HPC) frequencies in the bone marrow (BM) were perfectly normal in Robo4^−/− mice. Moreover, Robo4^−/− HSCs were equally competitive as wild-type HSCs in transplantation assays and had normal long-term repopulating (LTR) capacity. Of note, the initial engraftment at 4-weeks after transplantation was slightly impaired by Robo4 ablation, suggesting a marginal defect in BM homing of Robo4^−/− HSCs. In fact, homing efficiencies of HSCs/HPCs to the BM was significantly impaired in Robo4-deficient mice. On the other hand, granulocyte-colony stimulating factor-induced peripheral mobilization of HSCs was also impaired by Robo4 disruption. Lastly, marrow recovery from myelosuppressive stress was equally efficient in WT- and Robo4-mutant mice. These results clearly indicate that Robo4 plays a role in HSC trafficking such as BM homing and peripheral mobilization, but is not essential in the LTR and self-renewal capacity of HSCs.     

Homing and mobilization in the stem cell niche.

All mature blood cells are derived from the haemopoietic stem cell (HSC). In common with all other haemopoietic cells, stem cells are mobile, and it is this property of mobility that has allowed bone marrow transplantation to become a routine clinical option. Successful transplantation requires haemopoietic stem cells to home to the bone marrow, leave the peripheral circulation and become stabilized in regulatory niches in the extravascular space of the bone marrow cavity. This homing and tethering process is reversible - haemopoietic stem cells can be released from their bone marrow tethering through changes in molecular interactions, which are also important in homing following transplantation. The molecular mechanisms regulating this two-way flow of stem cells are beginning to be elucidated, and much recent data has emerged that sheds light on the processes and molecules involved in these complex physiological events. This article reviews current knowledge of the adhesive, homing and proliferative influences acting on HSCs and progenitor cells.     

Establishment and regulation of the HSC niche: Roles of osteoblastic and vascular compartments

Hematopoietic stem cells (HSC) are multi-potent cells that function to generate a lifelong supply of all blood cell types. During mammalian embryogenesis, sites of hematopoiesis change over the course of gestation: from extraembryonic yolk sac and placenta, to embryonic aorta-gonad-mesonephros region, fetal liver, and finally fetal bond marrow where HSC reside postnatally. These tissues provide microenviroments for de novo HSC formation, as well as HSC maturation and expansion. Within adult bone marrow, HSC self-renewal and differentiation are thought to be regulated by two major cellular components within their so-called niche: osteoblasts and vascular endothelial cells. This review focuses on HSC generation within, and migration to, different tissues during development, and also provides a summary of major regulatory factors provided by osteoblasts and vascular endothelial cells within the adult bone marrow niche.     

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.     

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.     

Why is an energy metabolic defect the common outcome in BMFS?

Inherited bone marrow failure syndromes (BMFS) are rare, distressing, inherited blood disorders of children. Although the genetic origin of these pathologies involves genes with different functions, all are associated with progressive haematopoietic impairment and an excessive risk of malignancies. Defects in energy metabolism induce oxidative stress, impaired energy production and an unbalanced ratio between ATP and AMP. This assumes an important role in self-renewal and differentiation in haematopoietic stem cells (HSC) and can play an important role in bone marrow failure. Defects in energetic/respiratory metabolism, in particular in FA and SDS cells, have been described recently and seem to be a pertinent argument in the discussion of the haematopoietic defect in BMFS, as an alternative to the hypotheses already established on this subject, which may shed new light on the evolution of these diseases.     

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.     

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.     

Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs

Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.     

Differentiation characteristics of circulating and bone marrow hematopoietic stem cells and the effect of thymus factors

Circulating hemopoietic stem cells (HSC) considerably differ from bone marrow HSC in active erythroid differentiation. After thymectomy of adult animals the number and differentiation of blood HSC remain unchanged, whereas during the cloning of bone marrow cells, a decrease in the number of granulocytic colonies is revealed. In in-vitro experiments, thymalin does not influence the number or differentiation of circulating HSC. On the contrary, in experiments made in vivo, it dramatically lowers erythroid specialization of blood HSC in thymectomized and sham-operated mice, which is followed by the diminution of the total number of circulating HSC. Differentiation of thymectomized mice bone marrow stem cells is completely normalized after thymalin injection. Sham-operated and thymectomized animals' HSC stimulated by thymalin injection become similar to bone marrow cells of normal mice as regards the trend of differentiation. Thymalin injection is likely to change the bone marrow HSC differentiation profile, thereby preventing the release of the cells with erythroid-oriented differentiation from the bone marrow to blood. The influence of thymalin on HSC is mediated by the environmental component which is present in the bone marrow and absent from the peripheral blood.     

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.     

Role of neuropeptide Y in the bone marrow hematopoietic stem cell microenvironment

The sympathetic nervous system (SNS) or neurotransmitters in the bone marrow microenvironment has been known to regulate hematopoietic stem cell (HSC) functions such as self-renewal, proliferation and differentiation. However, the specific role of neuropeptide Y (NPY) in this process remains relatively unexplored. In this study, we demonstrated that NPY deficient mice have significantly reduced HSC numbers and impaired bone marrow regeneration due to apoptotic destruction of SNS fibers and/or endothelial cells. Moreover, NPY treatment prevented bone marrow impairments in a mouse model of chemotherapy-induced SNS injury, while conditional knockout mice lacking the Y1 receptor in macrophages did not restore bone marrow dysfunction in spite of NPY injection. Transforming growth factor-beta (TGF-β) secreted by NPY-mediated Y1 receptor stimulation in macrophages plays a key role in neuroprotection and HSC survival in the bone marrow. Therefore, this study reveals a new role of NPY in bone marrow HSC microenvironment, and provides an insight into the therapeutic application of this neuropeptide. [BMB Reports 2015; 48(12): 645-646]     

造血干细胞生物学——研究与展望

造血干细胞（hematopoietic stem cell,HSC）是目前研究方法最为多样、研究技术手段最为成熟的一类组织干细胞,并且已经被成功运用于临床上对白血病以及先天性免疫缺陷等疾病的治疗。近年来,通过对一系列“转基因”与“基因敲除”小鼠模型的分析,人们对造血干细胞在胚胎早期发育过程中的发生与起源、造血干细胞“自我更新”与“定向分化”的调节机制、骨髓中造血干细胞的微环境（niche）对造血干细胞功能维持的调控,以及造血干细胞与白血病干细胞之间的相互关系等诸多方面都取得了很大的进展。如何实现造血干细胞的体外长期培养与扩增,实现胚胎干细胞（embryonic stem cell,ESC）或诱导多能干细胞（induced pluripotent stem cell,iPS细胞）向造血干细胞进行有效的定向分化,以及探索造血干细胞在病理状态（如癌症、贫血、衰老等）或应激状态下（如炎症与感染、组织损伤、代谢异常等）的功能变化,都将会是今后造血干细胞研究的重要方向。     

The role of apoptosis in regulating hematopoietic stem cell numbers

The importance of apoptosis, in combination with proliferation, in maintaining stable populations has become increasingly clear in the last decade. Perturbation of either of these processes can have serious consequences, and result in a variety of disorders. Moreover, as the players and pathways gradually emerge, it turns out that there are strong connections in the regulation of cell cycle progression and apoptosis. Apoptosis, proliferation, and the disorders resulting from aberrant regulation have been studied in a variety of cell types and systems. Hematopoietic stem cells (HSC) are defined as primitive mesenchymal cells that are capable of both self-renewal and differentiation into the various cell lineages that constitute the functioning hematopoietic system. Many (but certainly not all) mature hematopoietic cells are relatively short-lived, sometimes with a half-life in the order of days. Homeostasis requires the production of 10⁸ (mouse) to 10¹¹ (human) cells each day. All of these cells are ultimately derived from HSC that mostly reside in the bone marrow in adult mammals. The study of the regulation of HSC numbers has focussed mainly on the choice between self-renewal and differentiation, symmetric and asymmetric cell divisions. Recently, however, it has been directly demonstrated that apoptosis plays an important role in the regulation of hematopoietic stem cells in vivo.     

Niche-to-niche migration of bone-marrow-derived cells

During ontogenesis, haematopoietic stem cells (HSCs) relocate between extra-embryonic and embryonic compartments. Similarly, site-specific homing of HSCs is ongoing during adulthood. With the expanding knowledge of HSC physiology, a new paradigm emerges in which HSCs and haematopoietic progenitor cells (HPCs) migrate to defined microenvironments within the bone marrow (BM) and to 'activated' or 'inducible' niches elsewhere. Here, we summarize current understanding of HSC niche characteristics, and the physiological and pathological mechanisms that guide HSC homing both within the BM and to distant niches in the periphery, promoting new vessel growth in tumours and ischaemia. Recent observations suggest that features of the HSC niche might also be recapitulated in pre-metastatic sites. Clusters of BM-derived HPCs promote invasion of disseminating cancer cells. Clear clinical benefits can be foreseen by modulating HSCs and their microenvironments, in promoting tissue regeneration, and inhibiting tumourigenesis and cancer metastasis.