Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative ¹³ C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Abstract Bone marrow endothelial cells (BMECs) play a key role in bone formation and haematopoiesis. Although recent studies uncovered the cellular taxonomy of stromal compartments in the bone marrow (BM), the complexity of BMECs is not fully characterized. In the present study, using single-cell RNA sequencing, we defined a spatial heterogeneity of BMECs and identified a capillary subtype, termed type S (secondary ossification) endothelial cells (ECs), exclusively existing in the epiphysis. Type S ECs possessed unique phenotypic characteristics in terms of structure, plasticity and gene expression profiles. Genetic experiments showed that type S ECs atypically contributed to the acquisition of bone strength by secreting type I collagen, the most abundant bone matrix component. Moreover, these cells formed a distinct reservoir for haematopoietic stem cells. These findings provide the landscape for the cellular architecture in the BM vasculature and underscore the importance of epiphyseal ECs during bone and haematopoietic development.

Preculture is indispensable for achieving highly efficient non-homologous end joining (NHEJ)-based genome editing. Here, we present a protocol for optimizing genome editing conditions for murine hematopoietic stem cells (HSCs) and evaluating their function following NHEJ-based genome editing. We describe steps for sgRNA preparation, cell sorting, preculture, and electroporation. We then detail post-editing culture and transplanting of bone marrow. This protocol can be used to study genes related to HSC quiescence. For complete details on the use and execution of this protocol, please refer to Shiroshita et al.¹.

Caloric restriction (CR) is a nutritional intervention that reduces the risk of age-related diseases in numerous species, including humans. CR's metabolic effects, including decreased fat mass and improved insulin sensitivity, play an important role in its broader health benefits. However, the extent and basis of sex differences in CR's health benefits are unknown. We found that 30% CR in young (3-month-old) male mice decreased fat mass and improved glucose tolerance and insulin sensitivity, whereas these effects were blunted or absent in young female mice. Females' resistance to fat and weight loss was associated with decreased lipolysis, lower systemic energy expenditure and fatty acid oxidation, and increased postprandial lipogenesis compared to males. Positron emission tomography-computed tomography (PET/CT) with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) showed that peripheral glucose uptake was comparable between sexes. Instead, the sex differences in glucose homeostasis were associated with altered hepatic ceramide content and substrate metabolism: compared to CR males, CR females had lower TCA cycle activity but higher blood ketone concentrations, a marker of hepatic acetyl-CoA content. This suggests that males use hepatic acetyl-CoA for the TCA cycle whereas in females it accumulates, thereby stimulating gluconeogenesis and limiting hypoglycaemia during CR. In aged mice (18-months old), when females are anoestrus, CR decreased fat mass and improved glucose homeostasis to a similar extent in both sexes. Finally, in a cohort of overweight and obese humans CR-induced fat loss was also sex- and age-dependent: younger females (<45 years) resisted fat loss compared to younger males while in older subjects (>45 years) this sex difference was absent. Collectively, these studies identify age-dependent sex differences in the metabolic effects of CR and highlight adipose tissue, the liver and oestrogen as key determinants of CR's metabolic benefits. These findings have important implications for understanding the interplay between diet and health and for maximising the benefits of CR in humans.

Soft tissue sarcomas (STSs) are a heterogeneous group of tumors that originate from mesenchymal cells. p53 is frequently mutated in human STS. In this study, we found that the loss of p53 in mesenchymal stem cells (MSCs) mainly causes adult undifferentiated soft tissue sarcoma (USTS). MSCs lacking p53 show changes in stem cell properties, including differentiation, cell cycle progression, and metabolism. The transcriptomic changes and genetic mutations in murine p53-deficient USTS mimic those seen in human STS. Furthermore, single-cell RNA sequencing revealed that MSCs undergo transcriptomic alterations with aging-a risk factor for certain types of USTS-and that p53 signaling decreases simultaneously. Moreover, we found that human STS can be transcriptomically classified into six clusters with different prognoses, different from the current histopathological classification. This study paves the way for understanding MSC-mediated tumorigenesis and provides an efficient mouse model for sarcoma studies.

Autism spectrum disorder (ASD) is caused by combined genetic and environmental factors. Genetic heritability in ASD is estimated as 60-90%, and genetic investigations have revealed many monogenic factors. We analyzed 405 patients with ASD using family-based exome sequencing to detect disease-causing single-nucleotide variants (SNVs), small insertions and deletions (indels), and copy number variations (CNVs) for molecular diagnoses. All candidate variants were validated by Sanger sequencing or quantitative polymerase chain reaction and were evaluated using the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines for molecular diagnosis. We identified 55 disease-causing SNVs/indels in 53 affected individuals and 13 disease-causing CNVs in 13 affected individuals, achieving a molecular diagnosis in 66 of 405 affected individuals (16.3%). Among the 55 disease-causing SNVs/indels, 51 occurred de novo, 2 were compound heterozygous (in one patient), and 2 were X-linked hemizygous variants inherited from unaffected mothers. The molecular diagnosis rate in females was significantly higher than that in males. We analyzed affected sibling cases of 24 quads and 2 quintets, but only one pair of siblings shared an identical pathogenic variant. Notably, there was a higher molecular diagnostic rate in simplex cases than in multiplex families. Our simulation indicated that the diagnostic yield is increasing by 0.63% (range 0-2.5%) per year. Based on our simple simulation, diagnostic yield is improving over time. Thus, periodical reevaluation of ES data should be strongly encouraged in undiagnosed ASD patients.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

In diabetic pathology, insufficiency in β-cell mass, unable to meet peripheral insulin demand, and functional defects of individual β-cells in production of insulin are often concurrently observed, collectively causing hyperglycemia. Here we show that the phosphorylation of ERK1/2 is significantly decreased in the islets of db/db mice as well as in those of a cohort of subjects with type 2 diabetes. In mice with abrogation of ERK signaling in pancreatic β-cells through deletion of Mek1 and Mek2, glucose intolerance aggravates under high-fat diet-feeding conditions due to insufficient insulin production with lower β-cell proliferation and reduced β-cell mass, while in individual β-cells dampening of the number of insulin exocytosis events is observed, with the molecules involved in insulin exocytosis being less phosphorylated. These data reveal bifunctional roles for MEK/ERK signaling in β-cells for glucose homeostasis, i.e., in regulating β-cell mass as well as in controlling insulin exocytosis in individual β-cells, thus providing not only a novel perspective for the understanding of diabetes pathophysiology but also a potential clue for new drug development for diabetes treatment.

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.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.

Hematopoietic stem cells (HSCs) and their progeny sustain lifetime hematopoiesis. Aging alters HSC function, number, and composition and increases risk of hematological malignancies, but how these changes occur in HSCs remains unclear. Signaling via p38 mitogen-activated kinase (p38MAPK) has been proposed as a candidate mechanism underlying induction of HSC aging. Here, using genetic models of both chronological and premature aging, we describe a multimodal role for p38α, the major p38MAPK isozyme in hematopoiesis, in HSC aging. We report that p38α regulates differentiation bias and sustains transplantation capacity of HSCs in the early phase of chronological aging. However, p38α decreased HSC transplantation capacity in the late progression phase of chronological aging. Furthermore, codeletion of p38α in mice deficient in ataxia-telangiectasia mutated, a model of premature aging, exacerbated aging-related HSC phenotypes seen in ataxia-telangiectasia mutated single-mutant mice. Overall, these studies provide new insight into multiple functions of p38MAPK, which both promotes and suppresses HSC aging context dependently.

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring BCR-ABL1 fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were BCR-ABL1-negative. Moreover, in some patients BCR-ABL1 was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.

Mammalian hematopoietic stem cells (HSCs) maintain life-long hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after bone marrow injury or in vitro culture. We have recently demonstrated the ability to maintain HSC quiescence in vitro by mimicking the bone marrow microenvironment. Here, we provide a detailed protocol for keeping functional HSCs in the quiescent state in vitro. For complete details on the use and execution of this protocol, please refer to Kobayashi et al. (2019).

Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our culture system provides a means to evaluate properties of steady-state HSCs and test effects of defined factors in vitro under near-physiological conditions.

Hematopoietic stem cells (HSCs) are quiescent cells in the bone marrow niche and are relatively dependent on glycolytic ATP production. On the other hand, differentiated cells, including hematopoietic progenitor cells (HPCs), preferentially generate ATP via oxidative phosphorylation. However, it is unclear how cellular differentiation and the cell cycle status affect nutritional requirements and ATP production in HSCs and HPCs. Using a newly developed culture system, we demonstrated that survival of HPCs was strongly dependent on glucose, whereas quiescent HSCs survived for a certain duration without glucose. Among HPCs, granulocyte/monocyte progenitors (GMPs) were particularly dependent on glucose during proliferation. By monitoring the ATP concentration in live cells, we demonstrated that the ATP level was maintained for a short duration without glucose in HSCs, possibly due to their metabolic flexibility. In addition, HSCs exhibited low ATP turnover, whereas HPCs including GMPs demonstrated high ATP turnover and required efficient ATP production from glucose. These findings show that ATP turnover and nutritional requirements differ between HSCs and HPCs according to the cell cycle and differentiation status.

<jats:title>SUMMARY</jats:title><jats:p>Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our novel culture system provides a means to evaluate properties of steady state HSCs and test effects of defined factors<jats:italic>in vitro</jats:italic>under near-physiological conditions.</jats:p>

As leukemic transformation of myeloproliferative neoplasms (MPNs) worsens the clinical outcome, reducing the inherent risk of the critical event in MPN cases could be beneficial. Among genetic alterations concerning the transformation, the frequent one is TP53 mutation. Here we show that retroviral overexpression of Jak2 V617F mutant into wild-type p53 murine bone marrow cells induced polycythemia vera (PV) in the recipient mice, whereas Jak2 V617F-transduced p53-null mice developed lethal leukemia after the preceding PV phase. The leukemic mice had severe anemia, hepatosplenomegaly, pulmonary hemorrhage and expansion of dysplastic erythroid progenitors. Primitive leukemia cells (c-kit⁺Sca1⁺Lin^- (KSL) and CD34^-CD16/32^-c-kit⁺Sca1^-Lin^- (megakaryocyte-erythroid progenitor; MEP)) and erythroid progenitors (CD71⁺) from Jak2 V617F-transduced p53-null leukemic mice had leukemia-initiating capacity, however, myeloid differentiated populations (Mac-1⁺) could not recapitulate the disease. Interestingly, recipients transplanted with CD71⁺ cells rapidly developed erythroid leukemia, which was in sharp contrast to leukemic KSL cells to cause lethal leukemia after the polycythemic state. The leukemic CD71⁺ cells were more sensitive to INCB18424, a potent JAK inhibitor, than KSL cells. p53 restoration could ameliorate Jak2 V617F-transduced p53-null erythroleukemia. Taken together, our results show that p53 loss is sufficient for inducing leukemic transformation in Jak2 V617F-positive MPN.

During steady-state conditions, hematopoietic stem cells (HSCs) maintain a quiescent status in the cell cycle. Upon infection or inflammation, bone marrow HSCs begin proliferating and generating differentiated hematopoietic cells via multi-lineage differentiation and self-renewal; this effect is partially due to the alteration of their surrounding microenvironment or niche. In addition, recent studies have revealed that the bone marrow niche critically contributes to abnormal hematopoiesis, including leukemogenesis. In this review, we discuss the recent advances in our understanding of HSC/niche functions and the regulatory machineries employed during homeostasis, stress hematopoiesis, or disease conditions.

Polycomb repressive complex 2 (PRC2) participates in transcriptional repression through methylation of histone H3K27. The WD-repeat protein embryonic ectoderm development (EED) is a non-catalytic but an essential component of PRC2 and its mutations were identified in hematopoietic malignancies. To clarify the role(s) of EED in adult hematopoiesis and leukemogenesis, we generated Eed conditional knockout (Eed(Δ/Δ)) mice. Eed(Δ/Δ) mice died in a short period with rapid decrease of hematopoietic cells. Hematopoietic stem/progenitor cells (HSPCs) were markedly decreased with impaired bone marrow (BM) repopulation ability. Cell cycle analysis of HSPCs demonstrated increased S-phase fraction coupled with suppressed G0/G1 entry. Genes encoding cell adhesion molecules are significantly enriched in Eed(Δ/Δ) HSPCs, and consistently, Eed(Δ/Δ) HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other, and these conflicting events would lead to abnormal differentiation and functional defect of Eed(Δ/Δ) HSPCs. In addition, Eed haploinsufficiency induced hematopoietic dysplasia, and Eed heterozygous mice were susceptible to malignant transformation and developed leukemia in cooperation with Evi1 overexpression. Our results demonstrated differentiation stage-specific and dose-dependent roles of EED in normal hematopoiesis and leukemogenesis.

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. We do not yet have a clear understanding of how this metabolic activity changes during stress hematopoiesis, such as bone marrow transplantation. Here, we report a critical role for the p38MAPK family isoform p38α in initiating hematopoietic stem and progenitor cell (HSPC) proliferation during stress hematopoiesis in mice. We found that p38MAPK is immediately phosphorylated in HSPCs after a hematological stress, preceding increased HSPC cycling. Conditional deletion of p38α led to defective recovery from hematological stress and a delay in initiation of HSPC proliferation. Mechanistically, p38α signaling increases expression of inosine-5'-monophosphate dehydrogenase 2 in HSPCs, leading to altered levels of amino acids and purine-related metabolites and changes in cell-cycle progression in vitro and in vivo. Our studies have therefore uncovered a p38α-mediated pathway that alters HSPC metabolism to respond to stress and promote recovery.

Despite the clinical impact of DNMT3A mutation on acute myeloid leukaemia, the molecular mechanisms regarding how this mutation causes leukaemogenesis in vivo are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant haematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are downregulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells, representing a DNA methylation-independent role of mutated DNMT3A. DNMT3A R882H also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, the DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), causing transcriptional silencing, revealing a DNA methylation-independent role of DNMT3A mutation. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. From our data, it is shown that DNMT3A mutants can block the differentiation of HSCs and leukaemic cells via PRC1. This interaction could be targetable in DNMT3A-mutated leukaemias.

Hematopoietic stem/progenitor cells (HSPCs) play important roles in fighting systemic infection as they supply immune cells in a demand-adapted manner. Various mechanisms govern HSPC responses to infection, including cytokine signaling, niche function, and direct sensing of pathogen-derived molecules by HSPCs themselves. Here we review recent advances in our understanding of HSPC responses to infection and also consider newly identified STING-mediated machinery recognizing bacteria-derived cyclic dinucleotides.

Hematopoietic stem cells (HSCs) depend on the bone marrow (BM) niche for their maintenance, proliferation, and differentiation. The BM niche is composed of nonhematopoietic and mature hematopoietic cells, including megakaryocytes (Mks). Thrombopoietin (Thpo) is a crucial cytokine produced by BM niche cells. However, the cellular source of Thpo, upon which HSCs primarily depend, is unclear. Moreover, no specific molecular pathway for the regulation of Thpo production in the BM has been identified. Here, we demonstrate that the membrane protein C-type lectin-like receptor-2 (CLEC-2) mediates the production of Thpo and other factors in Mks. Mice conditionally deleted for CLEC-2 in Mks (Clec2(MkΔ/Δ)) produced lower levels of Thpo in Mks. CLEC-2-deficient Mks showed down-regulation of CLEC-2-related signaling molecules Syk, Lcp2, and Plcg2. Knockdown of these molecules in cultured Mks decreased expression of Thpo. Clec2(MkΔ/Δ) mice exhibited reduced BM HSC quiescence and repopulation potential, along with extramedullary hematopoiesis. The low level of Thpo production may account for the decline in HSC potential in Clec2(MkΔ/Δ) mice, as administration of recombinant Thpo to Clec2(MkΔ/Δ) mice restored stem cell potential. Our study identifies CLEC-2 signaling as a novel molecular mechanism mediating the production of Thpo and other factors for the maintenance of HSCs.

Upon systemic bacterial infection, hematopoietic stem and progenitor cells (HSPCs) migrate to the periphery in order to supply a sufficient number of immune cells. Although pathogen-associated molecular patterns reportedly mediate HSPC activation, how HSPCs detect pathogen invasion in vivo remains elusive. Bacteria use the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) for a variety of activities. Here, we report that c-di-GMP comprehensively regulated both HSPCs and their niche cells through an innate immune sensor, STING, thereby inducing entry into the cell cycle and mobilization of HSPCs while decreasing the number and repopulation capacity of long-term hematopoietic stem cells. Furthermore, we show that type I interferon acted as a downstream target of c-di-GMP to inhibit HSPC expansion in the spleen, while transforming growth factor-β was required for c-di-GMP-dependent splenic HSPC expansion. Our results define machinery underlying the dynamic regulation of HSPCs and their niches during bacterial infection through c-di-GMP/STING signaling.

Just as normal stem cells require niche cells for survival, leukemia-initiating cells (LICs) may also require niche cells for their maintenance. Chronic myeloid leukemia (CML) is caused by the activity of BCR-ABL, a constitutively active tyrosine kinase. CML therapy with tyrosine kinase inhibitors is highly effective; however, due to the persistence of residual LICs, it is not curative. Several factors are known to support CML LICs, but purification of LICs and a thorough understanding of their niche signals have not yet been achieved. Using a CML-like mouse model of myeloproliferative disease, we demonstrate that CML LICs can be divided into CD25(+)FcεRIα(-) Lineage marker (Lin)(-) Sca-1(+)c-Kit(+) (F(-)LSK) cells and CD25(-)F(-)LSK cells. The CD25(+)F(-)LSK cells had multilineage differentiation capacity, with a preference toward cytokine-producing mast cell commitment. Although cells interconverted between CD25(-)F(-)LSK and CD25(+)F(-)LSK status, the CD25(+)F(-)LSK cells exhibited higher LIC capacity. Our findings suggest that interleukin-2 derived from the microenvironment and CD25 expressed on CML LICs constitute a novel signaling axis. The high levels of CD25 expression in the CD34(+)CD38(-) fraction of human CML cells indicate that CD25(+) LICs constitute an "LIC-derived niche" that could be preferentially targeted in therapy for CML.

Hematopoietic stem cell (HSC) gene therapy is a potentially curative treatment modality for monogenic hematological diseases and storage disorders. It is necessary, however, to establish pre-bone marrow (BM) transplant conditioning regimens that minimize DNA damage and toxicity. Type I interferon (IFN) signaling activates quiescent HSCs and enables them to be sensitive to 5-fluorouracil (FU)-mediated cytotoxicity, thus implying a molecular basis for improving HSC transplant outcomes. Here we show that type I IFN preconditioning, without irradiation or DNA alkylating agents, significantly enhanced the HSC engraftment efficiency in wild-type (WT) recipient mice. The importance of active type I IFN signaling in HSC recipients was further demonstrated using mice lacking IFN regulatory factor 2 (IRF2), a transcriptional suppressor of type I IFN signaling. In both WT and Irf2(-/-) recipients, active type I IFN signaling greatly enhanced the sensitivity to 5-FU or low-dose irradiation of HSCs. Importantly, IFN-based pre-BM transplant conditioning was also applicable to the treatment of Sly syndrome, a congenital storage disorder with β-glucuronidase deficiency, in which it restored enzyme expression at the HSC level and reciprocally reduced pathological glycosaminoglycan storage. Our findings suggest type I IFN-based preconditioning, combined with HSC transplantation, as a novel nongenotoxic treatment of some congenital diseases.

Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function.

Ecotropic viral integration site 1 (Evi1), a transcription factor of the SET/PR domain protein family, is essential for the maintenance of hematopoietic stem cells (HSCs) in mice and is overexpressed in several myeloid malignancies. Here, we generate reporter mice in which an internal ribosome entry site (IRES)-GFP cassette is knocked-in to the Evi1 locus. Using these mice, we find that Evi1 is predominantly expressed in long-term HSCs (LT-HSCs) in adult bone marrow, and in the hematopoietic stem/progenitor fraction in the aorta-gonad-mesonephros, placenta, and fetal liver of embryos. In both fetal and adult hematopoietic systems, Evi1 expression marks cells with long-term multilineage repopulating activity. When combined with conventional HSC surface markers, sorting according to Evi1 expression markedly enhances purification of cells with HSC activity. Evi1 heterozygosity leads to marked impairment of the self-renewal capacity of LT-HSCs, whereas overexpression of Evi1 suppresses differentiation and boosts self-renewal activity. Reintroduction of Evi1, but not Mds1-Evi1, rescues the HSC defects caused by Evi1 heterozygosity. Thus, in addition to documenting a specific relationship between Evi1 expression and HSC self-renewal activity, these findings highlight the utility of Evi1-IRES-GFP reporter mice for the identification and sorting of functional HSCs.

B cell lymphoma, unclassifiable, with features intermediate between diffuse large B cell lymphoma and Burkitt lymphoma (BL) is a new entity in WHO classification 2008, and includes Burkitt-like transformation of follicular lymphoma (FL). However, the pathological significance of BCL-2/IgH translocation, which is characteristic in FL, in newly diagnosed cases of this disease is not well understood. We report a patient who developed B cell lymphoma with BL-like morphology on the shoulder, whose bone marrow was involved with FL-like cells. BL-like tumor cells harbored amplified c-MYC genes with BCL-2/IgH translocation, but FL-like cells solely had BCL-2/IgH translocation. The sequence analysis of the IgH gene revealed both of cells originated from the same clone, showing that the aggressive BL-like tumor originated from the FL-like clones simultaneously found in the bone marrow. This case suggests that the IgH/BCL-2 translocation found in newly diagnosed Burkitt-like lymphoma indicates a history of FL-like indolent lymphoma, even if such history is not documented.

DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development.