UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

Genetic defects in hematopoietic transcription factors and predisposition to acute lymphoblastic leukemia.

Recent genome-wide studies have revealed a plethora of germline variants that significantly influence the susceptibility to acute lymphoblastic leukemia (ALL), thus providing compelling evidence for genetic inheritance of this blood cancer. In particular, hematopoietic transcription factors (eg, ETV6, PAX5, IKZF1) are most frequently implicated in familial ALL, and germline variants in these genes confer strong predisposition (albeit with incomplete penetrance). Studies of germline risk factors for ALL provide unique insights into the molecular etiology of this leukemia.

Loss of 2 Akt (Protein Kinase B) Isoforms in Hematopoietic Cells Diminished Monocyte and Macrophage Survival and Reduces Atherosclerosis in Ldl Receptor-Null Mice.

Objective- Macrophages express 3 Akt (protein kinase B) isoforms, Akt1, Akt2, and Akt3, which display isoform-specific functions but may be redundant in terms of Akt survival signaling. We hypothesize that loss of 2 Akt isoforms in macrophages will suppress their ability to survive and modulate the development of atherosclerosis. Approach and Results- To test this hypothesis, we reconstituted male Ldlr-/- mice with double Akt2/Akt3 knockout hematopoietic cells expressing only the Akt1 isoform (Akt1only). There were no differences in body weight and plasma lipid levels between the groups after 8 weeks of the Western diet; however, Akt1only→ Ldlr-/- mice developed smaller (57.6% reduction) atherosclerotic lesions with more apoptotic macrophages than control mice transplanted with WT (wild type) cells. Next, male and female Ldlr-/- mice were reconstituted with double Akt1/Akt2 knockout hematopoietic cells expressing the Akt3 isoform (Akt3only). Female and male Akt3only→ Ldlr-/- recipients had significantly smaller (61% and 41%, respectively) lesions than the control WT→ Ldlr-/- mice. Loss of 2 Akt isoforms in hematopoietic cells resulted in markedly diminished levels of white blood cells, B cells, and monocytes and compromised viability of monocytes and peritoneal macrophages compared with WT cells. In response to lipopolysaccharides, macrophages with a single Akt isoform expressed low levels of inflammatory cytokines; however, Akt1only macrophages were distinct in expressing high levels of antiapoptotic Il10 compared with WT and Akt3only cells. Conclusions- Loss of 2 Akt isoforms in hematopoietic cells, preserving only a single Akt1 or Akt3 isoform, markedly compromises monocyte and macrophage viability and diminishes early atherosclerosis in Ldlr-/- mice.

Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions.

Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and proliferating cells cope with accumulating endogenous DNA lesions and how these ultimately affect the physiology of cells and tissues. Here we have addressed these questions by investigating the hematopoietic system of mice deficient for Rev1, a core factor in DNA translesion synthesis (TLS), the postreplicative bypass of damaged nucleotides. Rev1 hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of Xpc, essential for global-genome nucleotide excision repair (ggNER) of helix-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the Rev1Xpc bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of helix-distorting oxidative nucleotide lesions by ggNER and Rev1-dependent TLS, respectively. The error-prone nature of TLS may provide mechanistic understanding of the accumulation of mutations in the hematopoietic system upon aging.

[How Methods of Molecular Biology Shape Our Understanding of the Hematopoietic System].

Blood is extremely important for a multicellular organism: it connects all organs and tissues, supplies them with nutrients and oxygen, removes carbon dioxide and metabolic products, maintains homeostasis, and provides protection against infections. That is why studies on blood have always drawn a great deal of attention. In ancient times, it was believed that the soul was in the blood and that it sometimes "sank into the stomach." Initially, the study of blood was limited to morphological methods, to which physiological and cellular research were added in the twentieth century. With their help, researchers established that mature blood cells are formed from a rare population of hematopoietic stem cells (HSCs), which are located in the bone marrow. The development of molecular biology methods and their combination with classical physiological ones allowed a breakthrough in understanding the structure of the hematopoietic system, which changed our understanding not only of hematopoiesis but also about the nature of adult stem cells. This review describes the molecular assays used in experimental hematology, and how their application has gradually been expanding our knowledge of blood formation and continues to provide new information about it.

[Exosomes in the hematopoietic system].

The secretion of extracellular vesicles (EVs) from cells has been observed. Recently, because EVs were found to contain functional molecules such as micro RNAs (miRNAs) and possess the ability to transfer them to other cells, its functions were expanded as an "intracellular communicator." The exosome is one such EV that has been extensively investigated, particularly in cancer research because cancer cells abundantly secrete exosomes, suggesting their potential as promising diagnostic markers. Research on exosomes in the hematopoietic system has just begun. We recently reported that the exosome secreted from the EBV-infected lymphoma cells has critical functions in lymphomagenesis and maintenance. Moreover, EVs in HBV infection are now being investigated to generalize their functions.

ETS transcription factor ETV2/ER71/Etsrp in hematopoietic and vascular development, injury, and regeneration.

The major goal in regenerative medicine is to repair and restore injured, diseased or aged tissue function, thereby promoting general health. As such, the field of regenerative medicine has great translational potential in undertaking many of the health concerns and needs that we currently face. In particular, hematopoietic and vascular systems supply oxygen and nutrients and thus play critical roles in tissue development and tissue regeneration. Additionally, tissue vasculature serves as a tissue stem cell niche and thus contributes to tissue homeostasis. Notably, hematopoietic and vascular systems are sensitive to injury and subject to regeneration. As such, successful hematopoietic and vascular regeneration is prerequisite for efficient tissue repair and organismal survival and health. Recent studies have established that the interplay among the ETS transcription factor ETV2, vascular endothelial growth factor, and its receptor VEGFR2/FLK1 is essential for hematopoietic and vascular development. Emerging studies also support the role of these three factors and possible interplay in hematopoietic and vascular regeneration. Comprehensive understanding of the molecular mechanisms involved in the regulation and function of these three factors may lead to more effective approaches in promoting tissue repair and regeneration. Developmental Dynamics 246:318-327, 2017. © 2016 Wiley Periodicals, Inc.

Aging and neoteny in the B lineage.

Aging and the physiologic decline of tissues and cells were once thought to be irreversible. However, recent studies suggest that various tissues, especially parts of the hematopoietic system, can be rejuvenated. Here we review potential mechanisms for this process and how they may be used to reverse age-related disorders and aging in general. We propose the novel hypothesis that altering the homeostatic process during cellular depletion can reverse aging in the hematopoietic system.

Inducible knockout of GRP78/BiP in the hematopoietic system suppresses Pten-null leukemogenesis and AKT oncogenic signaling.

Traditionally, GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors; thus, its role in the development of hematologic malignancies remains to be determined. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null BM cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through regulation of oncogenic PI3K/AKT signaling. In agreement with PI3K/AKT as an effector for cytosine arabinoside resistance in acute myeloid leukemia, overexpression of GRP78 renders human leukemic cells more resistant to cytosine arabinoside-induced apoptosis, whereas knockdown of GRP78 sensitizes them. These, coupled with the emerging association of elevated GRP78 expression in leukemic blasts of adult patients and early relapse in childhood leukemia, suggest that GRP78 is a novel therapeutic target for leukemia.

[Chronic myeloid leukemia stem cells: cross-talk with the niche]. / Leucémie myéloïde chronique - un modèle de dialogue entre la cellule souche leucémique et la niche hématopoïétique.

The physiological hematopoietic niche located in bone marrow is a pluricellular structure whose components are now well identified. Within this microenvironment, hematopoietic stem cells are in direct contact with mesenchymal stromal cells, osteoblasts and sinusoidal endothelial cells. These close relationships drive specialized cellular functions (proliferation/quiescence, differentiation/self-renewal) ensuring an efficient hematopoiesis. Chronic myeloid leukemia (CML) is a major model of leukemic hematopoiesis. The BCR-ABL1 tyrosine kinase, constitutively activated in CML, plays a critical role in the pathogenesis of the disease. An intensive cross-talk between CML progenitors and the components of the hematopoietic niche has recently been demonstrated. Consequently, the occurrence of the so-called leukemic niche promotes both the proliferation of myeloid cells and the maintenance of quiescent leukemic stem cells. This bone marrow niche could also protect CML stem cells from tyrosine kinase inhibitors and probably contribute to their resistance towards targeted therapies.

The transcriptional programme controlled by Runx1 during early embryonic blood development.

Transcription factors have long been recognised as powerful regulators of mammalian development yet it is largely unknown how individual key regulators operate within wider regulatory networks. Here we have used a combination of global gene expression and chromatin-immunoprecipitation approaches during the early stages of haematopoietic development to define the transcriptional programme controlled by Runx1, an essential regulator of blood cell specification. Integrated analysis of these complementary genome-wide datasets allowed us to construct a global regulatory network model, which suggested that key regulators are activated sequentially during blood specification, but will ultimately collaborate to control many haematopoietically expressed genes. Using the CD41/integrin alpha 2b gene as a model, cellular and in vivo studies showed that CD41 is controlled by both Scl/Tal1 and Runx1 in fully specified blood cells, and initiation of CD41 expression in E7.5 embryos is severely compromised in the absence of Runx1. Taken together, this study represents the first global analysis of the transcriptional programme controlled by any key haematopoietic regulator during the process of early blood cell specification. Moreover, the concept of interplay between sequentially deployed core regulators is likely to represent a design principle widely applicable to the transcriptional control of mammalian development.

Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice.

Heterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when p53 is mutated. Mutagenesis screens in zebrafish and mice identified Hspa9 as a del(5q31.2) candidate gene that may contribute to MDS and AML pathogenesis, respectively. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, we knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow-transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors, but not myeloid or megakaryocytic precursors, and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors c-kit(+)/lineage(-)/Sca-1(+) (KLS) and megakaryocyte/erythrocyte progenitor (MEP) were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.

Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

[Toll-like receptors in development and function of the hematopoietic system]. / Los receptores tipo toll en el desarrollo y función del sistema hematopoyéico.

Virus, bacteria, fungi and parasites are pathogens to which individuals are constantly exposed. Pathogen recognition by cells of the immune system is carried out by a growing list of pattern-recognition receptors (PRRs) which are evolutionally conserved and absent in mammals, named pathogen-associated molecular patterns (PAMPs). PRRs can be found in extracellular matrix, within cytoplasm and on cellular membranes. Among the membrane PRRs, Toll-like receptors (TLRs) induce the production of pro-inflammatory cytokines and the expression of co-stimulatory molecules upon stimulation on mature cells, resulting in the triggering of immune danger signals. Recent reports showing the regulation of hematopoiesis by TLRs, suggest that they are involved in the most primitive stages of hematopoietic development and contribute to emergent replenishment of innate immune cells. These data entail TLRs to hematopoiesis and also revolutionize our understanding of the mechanisms governing infection responses. In this review, we focus on the most relevant findings from the TLR discovery to the use of TLR agonists and antagonists in novel therapies for infectious, autoimmune and neoplastic diseases. Of special interest is the research progress in the TLR functional expression by primitive hematopoietic stem and progenitor cells.

Is Nef the elusive cause of HIV-associated hematopoietic dysfunction?

HIV-associated hematological abnormalities involve all lineages of blood cells, thus implying that the virus impairs the function of early HSCs. However, the underlying mechanisms of this defect are unknown, particularly since HSCs are largely resistant to HIV-1 infection. In this issue of the JCI, Prost and colleagues show that the viral accessory protein Negative factor (Nef) plays a potentially critical role in the pathogenesis of HIV/SIV-associated hematopoietic dysfunction by affecting the clonogenic potential of HSCs (see the related article beginning on page 1765). Soluble Nef induces PPARgamma in uninfected HSCs, thereby suppressing the expression of STAT5A and STAT5B, two factors necessary for proper HSC function. The identification of this novel activity of extracellular Nef defines a new mechanism of HIV/SIV pathogenesis and suggests that approaches aimed at increasing STAT5A and STAT5B expression may be considered in HIV-infected individuals with prominent hematological abnormalities. The results also raise the question of whether dysregulation of hematopoiesis by extracellular Nef plays a role in the development of T cell immunodeficiency and the high levels of chronic immune activation associated with AIDS.

Investigating human keratinocyte stem cell identity.

Studies of the regulatory networks controlling intrinsic properties and fate of adult stem cells are in a large part performed in animal models. Epidermis is one of the most accessible human tissues for researchers, which is a critical parameter for conducting programs dedicated to this knowledge in human stem cell systems. Keratinocyte stem cells constitute a particularly valuable model, because of this practical aspect, but more importantly because their existence is for decades validated by the clinical demonstration of their impressive capacity for epidermis regeneration. For the fundamentalist, human keratinocyte stem cells represent a unique system to dissect the genetic and epigenetic controls of "stemness" and self-renewal. For this purpose, a highly limiting point is our current inability of obtaining a cellular material corresponding to keratinocyte stem cells with homogeneous phenotypic and functional characteristics. The search for tools suitable for the prospective selection of keratinocyte stem cells will benefit from studies conducted at the broad level of the global stem cell field, as well as from more specifically targeted approaches. Advances in that direction are tightly linked to the development of functional assays allowing reliable assessment and modeling of the different stem cell-associated functional characteristics.

Haematopoietic ageing through the lens of single-cell technologies.

Human lifespan is now longer than ever and, as a result, modern society is getting older. Despite that, the detailed mechanisms behind the ageing process and its impact on various tissues and organs remain obscure. In general, changes in DNA, RNA and protein structure throughout life impair their function. Haematopoietic ageing refers to the age-related changes affecting a haematopoietic system. Aged blood cells display different functional aberrations depending on their cell type, which might lead to the development of haematologic disorders, including leukaemias, anaemia or declining immunity. In contrast to traditional bulk assays, which are not suitable to dissect cell-to-cell variation, single-cell-level analysis provides unprecedented insight into the dynamics of age-associated changes in blood. In this Review, we summarise recent studies that dissect haematopoietic ageing at the single-cell level. We discuss what cellular changes occur during haematopoietic ageing at the genomic, transcriptomic, epigenomic and metabolomic level, and provide an overview of the benefits of investigating those changes with single-cell precision. We conclude by considering the potential clinical applications of single-cell techniques in geriatric haematology, focusing on the impact on haematopoietic stem cell transplantation in the elderly and infection studies, including recent COVID-19 research.

The hematopoietic system is a source of odorants that distinguish major histocompatibility types.

Radiation chimeras were made by restoring lethally irradiated inbred mice with bone marrow cells of F1 hybrid mice of crosses between that inbred strain and an H-2-congenic strain. The urine of these chimeras was tested by the Y maze method, and shown to have acquired a scent indicative of the reconstituting donors' H-2 type. Thus, cells of the hematopoietic system contribute to the H-2-related odorant properties that enable mice to distinguish one another according to their H-2 types.

Cytological evidence for a relationship between normal hemotopoietic colony-forming cells and cells of the lymphoid system.

The relationship between hematopoietic colony-forming stem cells and cells in the thymus and lymph nodes of unirradiated mice has been investigated using a chromosome-marker technique. It was found that a high proportion of cells in the thymus may belong to the same clone as normal hematopoietic colony-forming cells. It was also found that cells belonging to the same clone as colony-forming cells may reach the lymph nodes, and that nodes containing such cells can participate in an immunological response against sheep red cells. Either the precursors of cells in thymus and lymph node are identical with hematopoietic colony-forming cells, or they are both descendants of a common precursor which has not yet been identified. The results are compatible with the view that cells of the hematopoietic system and the immune system may be derived from the same stem cell.