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.     

Kit ligand has a critical role in mouse yolk sac and aorta–gonad–mesonephros hematopoiesis

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.     

Stem Cell Regulation by the Haemopoietic Stem Cell Niche

Both cellular as well as extracellular matrix components of the stem cell microenvironment, or niche, are critical in stem cell regulation. Recent data highlight a central role for osteoblasts and their by product osteopontin as a key part of the hematopoietic stem cell (HSC) niche. Herein we describe a model for the yin and yang of HSC regulation mediated by osteoblasts. In this respect, osteoblasts synthesise proteins with opposing effects on HSC proliferation and differentiation highlighting their pivotal role in adult hematopoiesis. Although osteoblasts play a central role in HSC regulation other stromal and microenvironmental cell types and their extracellular matrix proteins also contribute to this biology. For example, the glycosaminoglycan hyaluronic acid as well as the membrane bound form of stem cell factor are also key regulators of HSC. Osteopontin and these “niche” molecules are not only involved in regulation of HSC quiescence but also effect HSC homing, trans-marrow migration and lodgement. Accordingly this leads us to expand upon Schofield’s niche hypothesis: we propose that the HSC niche is critical for attraction of primitive hematopoietic progenitors to the endosteal region and tightly tethering them within this location, and by doing so placing them into intimate contact with cells such as osteoblasts whose extracellular products are able to exquisitely regulate their fate.     

Defining the hematopoietic stem cell niche: the chicken and the egg conundrum

Understanding the in vivo regulation of hematopoietic stem cells (HSCs) will be critical to identifying key factors involved in the regulation of HSC self‐renewal and differentiation. The niche (microenvironment) in which HSCs reside has recently regained attention accompanied by a dramatic increase in the understanding of the cellular constituents of the bone marrow HSC niche. The use of sophisticated genetic models allowing modulation of specific lineages has demonstrated roles for mesenchymal‐derived elements such as osteoblasts and adipocytes, vasculature, nerves, and a range of hematopoietic progeny of the HSC as being participants in the regulation of the bone marrow microenvironment. Whilst providing significant insight into the cellular composition of the niche, is it possible to manipulate any given cell lineage in vivo without impacting, knowingly or unknowingly, on those that remain? J. Cell. Biochem. 112: 1486–1490, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Gene profiling reveals association between altered Wnt signaling and loss of T‐cell potential with age in human hematopoietic stem cells

Functional decline of the hematopoietic system occurs during aging and contributes to clinical consequences, including reduced competence of adaptive immunity and increased incidence of myeloid diseases. This has been linked to aging of the hematopoietic stem cell (HSC) compartment and has implications for clinical hematopoietic cell transplantation as prolonged periods of T‐cell deficiency follow transplantation of adult mobilized peripheral blood (PB), the primary transplant source. Here, we examined the gene expression profiles of young and aged HSCs from human cord blood and adult mobilized PB, respectively, and found that Wnt signaling genes are differentially expressed between young and aged human HSCs, with less activation of Wnt signaling in aged HSCs. Utilizing the OP9‐DL1 in vitro co‐culture system to promote T‐cell development under stable Notch signaling conditions, we found that Wnt signaling activity is important for T‐lineage differentiation. Examination of Wnt signaling components and target gene activation in young and aged human HSCs during T‐lineage differentiation revealed an association between reduced Wnt signal transduction, increasing age, and impaired or delayed T‐cell differentiation. This defect in Wnt signal activation of aged HSCs appeared to occur in the early T‐progenitor cell subset derived during in vitro T‐lineage differentiation. Our results reveal that reduced Wnt signaling activity may play a role in the age‐related intrinsic defects of aged HSCs and early hematopoietic progenitors and suggest that manipulation of this pathway could contribute to the end goal of improving T‐cell generation and immune reconstitution following clinical transplantation.     

Expression of tumour‐suppressing chemokine BRAK/CXCL14 reduces cell migration rate of HSC‐3 tongue carcinoma cells and stimulates attachment to collagen and formation of elongated focal adhesions in vitro

BRAK/CXCL14 (breast‐ and kidney‐expressed chemokine/CXC chemokine ligand 14) is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues, including HNSCC (head and neck squamous cell carcinoma). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC (HSC‐3 BRAK) cells decreased the rate of tumour formation and size of tumour xenografts compared with mock‐vector‐introduced (HSC‐3 Mock) cells in athymic nude mice, even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that high‐level expression of the gene is important for the suppression of tumour establishment in vivo. For the first step to study the mechanisms of BRAK‐dependent tumour suppression, we compared characteristics between HSC‐3 BRAK and HSC‐3 Mock cells under in vitro culture conditions. The cell migration rate was lower in HSC‐3 BRAK cells than in HSC‐3 Mock cells. Also, HSC‐3 BRAK cells showed more rapid adhesion than HSC‐3 Mock cells when cultured on type I collagen‐coated dishes but not on fibronectin or laminin 1‐coated ones. This adhesion was mediated by α2β1 integrin. Immunofluorescent analysis of the cells cultured on type I collagen showed that HSC‐3 BRAK cells formed much more elongated focal adhesions co‐localized with paxillin and actin stress fibres than did HSC‐3 Mock cells. Treatment of parental HSC‐3 cells with recombinant BRAK stimulated the activation of Rap1, which is a ras family small GTPase, and formation of elongated focal adhesions, indicating that the difference in cell character observed between HSC‐3 Mock and HSC‐3 BRAK was not due to selection of clones of different character but due to expression of BRAK in the cells. The characteristic morphology of focal adhesions in HSC‐3 BRAK cells was perturbed by the introduction of an expression vector of the Rap‐binding domain of the Ral guanine nucleotide dissociation stimulator, a target of Rap1, into HSC‐3 BRAK cells, suggesting that Rap1 regulated the formation of the morphology of the focal adhesions. These data indicate that the expression of BRAK stimulated the formation of elongated focal adhesions of the HSC‐3 cells in an autocrine or paracrine fashion, in which stimulation may be responsible for the reduced migration of the cells.     

Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

During embryonic development, hematopoiesis occurs through primitive and definitive waves, giving rise to distinct blood lineages. Hematopoietic stem cells (HSCs) emerge from hemogenic endothelial (HE) cells, through endothelial‐to‐hematopoietic transition (EHT). In the adult, HSC quiescence, maintenance, and differentiation are closely linked to changes in metabolism. However, metabolic processes underlying the emergence of HSCs from HE cells remain unclear. Here, we show that the emergence of blood is regulated by multiple metabolic pathways that induce or modulate the differentiation toward specific hematopoietic lineages during human EHT. In both in vitro and in vivo settings, steering pyruvate use toward glycolysis or OXPHOS differentially skews the hematopoietic output of HE cells toward either an erythroid fate with primitive phenotype, or a definitive lymphoid fate, respectively. We demonstrate that glycolysis‐mediated differentiation of HE toward primitive erythroid hematopoiesis is dependent on the epigenetic regulator LSD1. In contrast, OXPHOS‐mediated differentiation of HE toward definitive hematopoiesis is dependent on cholesterol metabolism. Our findings reveal that during EHT, metabolism is a major regulator of primitive versus definitive hematopoietic differentiation.     

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.     

Signaling output: it's all about timing and feedbacks

The central questions in understanding signaling pathway specificity are how these pathways encode which stimulus is present and how this stimulus is decoded to yield the correct cell fate decision. In their recent work, Ryu et al (2015) show by stimulation experiments with different ligands how the differential engagement of feedback and feed‐forward regulation leads to different dynamics of pathway activity, which in turn alters cell fate. Moreover, they show that by considering the timescales of the feedback regulations, the different cellular responses can be triggered with pulsed stimulations by a single ligand.     

SABRE is required for stabilization of root hair patterning in Arabidopsis thaliana

Patterned differentiation of distinct cell types is essential for the development of multicellular organisms. The root epidermis of Arabidopsis thaliana is composed of alternating files of root hair and non‐hair cells and represents a model system for studying the control of cell‐fate acquisition. Epidermal cell fate is regulated by a network of genes that translate positional information from the underlying cortical cell layer into a specific pattern of differentiated cells. While much is known about the genes of this network, new players continue to be discovered. Here we show that the SABRE (SAB) gene, known to mediate microtubule organization, anisotropic cell growth and planar polarity, has an effect on root epidermal hair cell patterning. Loss of SAB function results in ectopic root hair formation and destabilizes the expression of cell fate and differentiation markers in the root epidermis, including expression of the WEREWOLF (WER) and GLABRA2 (GL2) genes. Double mutant analysis reveal that wer and caprice (cpc) mutants, defective in core components of the epidermal patterning pathway, genetically interact with sab. This suggests that SAB may act on epidermal patterning upstream of WER and CPC. Hence, we provide evidence for a role of SAB in root epidermal patterning by affecting cell‐fate stabilization. Our work opens the door for future studies addressing SAB‐dependent functions of the cytoskeleton during root epidermal patterning.     

All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells

While it is clear that a single hematopoietic stem cell?(HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports on?the lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways in?vivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.