Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.     

Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

Nicaraven Attenuates Radiation-Induced Injury in Hematopoietic Stem/Progenitor Cells in Mice

Nicaraven, a chemically synthesized hydroxyl radical-specific scavenger, has been demonstrated to protect against ischemia-reperfusion injury in various organs. We investigated whether nicaraven can attenuate radiation-induced injury in hematopoietic stem/progenitor cells, which is the conmen complication of radiotherapy and one of the major causes of death in sub-acute phase after accidental exposure to high dose radiation. C57BL/6 mice were exposed to 1 Gy γ-ray radiation daily for 5 days in succession (a total of 5 Gy), and given nicaraven or a placebo after each exposure. The mice were sacrificed 2 days after the last radiation treatment, and the protective effects and relevant mechanisms of nicaraven in hematopoietic stem/progenitor cells with radiation-induced damage were investigated by ex vivo examination. We found that post-radiation administration of nicaraven significantly increased the number, improved the colony-forming capacity, and decreased the DNA damage of hematopoietic stem/progenitor cells. The urinary levels of 8-oxo-2′-deoxyguanosine, a marker of DNA oxidation, were significantly lower in mice that were given nicaraven compared with those that received a placebo treatment, although the levels of intracellular and mitochondrial reactive oxygen species in the bone marrow cells did not differ significantly between the two groups. Interestingly, compared with the placebo treatment, the administration of nicaraven significantly decreased the levels of the inflammatory cytokines IL-6 and TNF-α in the plasma of mice. Our data suggest that nicaraven effectively diminished the effects of radiation-induced injury in hematopoietic stem/progenitor cells, which is likely associated with the anti-oxidative and anti-inflammatory properties of this compound.     

Divergent effects of Wnt5b on IL-3- and GM-CSF-induced myeloid differentiation

The multiple specialized cell types of the hematopoietic system originate from differentiation of hematopoietic stem cells and progenitors (HSPC), which can generate both lymphoid and myeloid lineages. The myeloid lineage is preferentially maintained during ageing, but the mechanisms that contribute to this process are incompletely understood. Here, we studied the roles of Wnt5a and Wnt5b, ligands that have previously been linked to hematopoietic stem cell ageing and that are abundantly expressed by both hematopoietic progenitors and bone-marrow derived niche cells. Whereas Wnt5a had no major effects on primitive cell differentiation, Wnt5b had profound and divergent effects on cytokine-induced myeloid differentiation. Remarkably, while IL-3-mediated myeloid differentiation was largely repressed by Wnt5b, GM-CSF-induced myeloid differentiation was augmented. Furthermore, in the presence of IL-3, Wnt5b enhanced HSPC self-renewal, whereas in the presence of GM-CSF, Wnt5b accelerated differentiation, leading to progenitor cell exhaustion. Our results highlight discrepancies between IL-3 and GM-CSF, and reveal novel effects of Wnt5b on the hematopoietic system.     

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34⁺ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.Subject terms: Cell biology, Cancer     

Comprehensive analysis of myeloid lineage conversion using mice expressing an inducible form of C/EBP alpha

CCAAT/enhancer-binding protein (C/EBP) alpha is a critical regulator for early myeloid differentiation. Although C/EBPalpha has been shown to convert B cells into myeloid lineage, precise roles of C/EBPalpha in various hematopoietic progenitors and stem cells still remain obscure. To examine the consequence of C/EBPalpha activation in various progenitors and to address the underlying mechanism of lineage conversion in detail, we established transgenic mice expressing a conditional form of C/EBPalpha. Using these mice, we show that megakaryocyte/erythroid progenitors (MEPs) and common lymphoid progenitors (CLPs) could be redirected to functional macrophages in vitro by a short-term activation of C/EBPalpha, and the conversion occurred clonally through biphenotypic intermediate cells. Moreover, in vivo activation of C/EBPalpha in mice led to the increase of mature granulocytes and myeloid progenitors with a concomitant decrease of hematopoietic stem cells and nonmyeloid progenitors. Our study reveals that C/EBPalpha can activate the latent myeloid differentiation program of MEP and CLP and shows that its global activation affects multilineage homeostasis in vivo.     

Primitive AML progenitors from most CD34(+) patients lack CD33 expression but progenitors from many CD34(-) AML patients express CD33

BACKGROUND: AML blast populations are heterogeneous in their phenotype and functional properties, and contain a small subset of cells that regenerate leukemia in immunocompromised mice or produce clonogenic progeny in long-term cultures. This suggests the existence of a hierarchy of AML progenitor cells. CD33 is a myeloid marker absent on normal hematopoietic stem cells but expressed in about 75% of AML patients, and has been used for BM purging strategies and Ab-targeted therapies. These CD33 Ab therapies benefit only a minority of AML patients, suggesting that AML stem cells are heterogeneous in their CD33 expression. METHODS: In order to evaluate this question, we determined expression levels of CD34 and CD33 on AML progenitors with long-term in vitro proliferative ability and NOD/SCID engrafting ability. RESULTS: The CD34(+) CD33(-) subfraction contained the majority of progenitors detected in vitro and most often engrafted the mice. This proliferation was leukemic from the CD34(+) AML patients, however from the CD34(-) AML patients only normal progenitors were detected in this fraction in some cases. DISCUSSION: These data suggest that most leukemic progenitors of CD34(+) patients do not express CD33. In contrast, CD34(-) AML primitive leukemic progenitors may be CD33(+). CD34(-) AML patients could potentially benefit most from CD33-targeted therapies or purging.     

Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow.

The target cell specificity of the B19 parvovirus infection was examined by isolating highly enriched hematopoietic progenitor and stem cells from normal human bone marrow. The efficiency of the B19 parvovirus replication in enriched erythroid progenitor cells was approximately 100-fold greater than that in unseparated bone marrow cells. The more-primitive progenitor cells identical to or closely related to the human pluripotent hematopoietic stem cells, on the other hand, did not support viral replication. The B19 progeny virus produced by the enriched erythroid progenitor cells was infectious and strongly suppressed erythropoiesis in vitro. The susceptibility of both the more-primitive erythroid progenitors (burst-forming units-erythroid) and the more-mature erythroid progenitors (CFU-erythroid) to the cytolytic response of the virus and the lack of effect on the myeloid progenitors (CFU-granulocyte-macrophage) further give evidence to the remarkable tropism of the B19 parvovirus for human hematopoietic cells of erythroid lineage.     

T‐lymphoid progenitors – we know what they are,but know not what they may be

An improved understanding of the biology underlying leukemogenesis, including the determination of the cells of leukemia origin, is of great importance as it can have immediate implications on patient treatment and management. The article by Riemke et al ( 2016 ) provides further evidence that a subgroup of acute myeloid leukemia (AML), the most common acute leukemia in adults, might arise from T‐lymphoid progenitor cells. This study not only supports that the lymphoid fate of early T‐cell progenitors is not yet fully stabilized but also shows that under oncogenic conditions, this multilineage plasticity potential of T‐lymphoid progenitors can lead to transdifferentiation into myeloid leukemia. While gene expression profiles suggest that approximately 5% of all AML cases originate from T‐lymphoid progenitors, novel treatment strategies targeting JAK2/STAT3 signaling might open new avenues for this AML cohort.     

Leukomogenic factors downregulate heparanase expression in acute myeloid leukemia cells

Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RARalpha and PLZF-RARalpha fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RARalpha from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells.     

Serum suppresses myeloid progenitor apoptosis by regulating iron homeostasis

The growth and survival of committed hematopoietic progenitors is dependent upon cytokine signaling. However, serum is also required for optimal growth of these progenitors in culture ex vivo. Here we report that serum withdrawal leads to myeloid progenitor cell apoptosis. Although serum deprivation-induced cell death has many hallmarks typical of apoptosis, these cell deaths were not inhibited by hemopoietins, survival factors such as IGF-I, or treatment with a broad-spectrum caspase inhibitor. Rather, apoptosis due to serum withdrawal was associated with damage to mitochondria. Surprisingly the serum factor required for myeloid cell survival was identified as iron, and loss of iron led to marked reductions in ATP production. Furthermore, supplementing serum-deprived myeloid cells with bound or free iron promoted cell survival and prevented mitochondrial damage. Therefore, serum suppresses hematopoietic cell apoptosis by providing an obligate source of iron and iron homeostasis is critical for proper myeloid cell metabolism and survival.     

Use of in vitro assays to assess hematotoxic effects of environmental compounds

The number of chemicals being introduced into the environment increases and many of these substances may pose a health risk to exposed individuals. Many environmental toxicants with a potential toxicity to the hematopoietic system have been identified by animal experiments. Owing to the risks of severe chronic hematopoietic disorders, it is important to screen chemicals for their hematotoxicity. The aim of this work was to identify, through the use ofin vitro techniques, targets for hematotoxic effects. Our study focused on myeloid and erythroid hematopoietic progenitors and stromal stem cells as possible targets. Thein vitro assays showed that various hematotoxic compounds exert different effects on these cell populations. In vitro exposure of murine bone marrow cells to various inorganic (cadmium, lead) and organic (benzene metabolites, lindane, benzo-[a]-pyrene (BaP), PCB (polychlorinated biphenyl) congeners) environmental chemicals indicated that hematopoietic or stromal bone marrow cells were targets for most of the chemicals. Stromal cells were more affected by lead, cadmium, and BaP compared to myeloid cells. Benzene and phenol gave no response, but the metabolites catechol and hydroquinone were equally toxic to the stromal and the myeloid progenitor cells. Among the PCBs tested, PCB126 was most toxic. Human progenitor cells derived from cord blood were exposedin vitro to catechol, hydroquinone, lead nitrate, and PCBs. Human hematopoietic cells were sensitive to the tested compounds. Human erythroid progenitors are more susceptible to lead exposure than are myeloid progenitors. Based on thein vitro tests, humans are more sensitive to lead, catechol, and PCB126 than are mice. In contrast to the murine data, humans responded with individual differences to lead and PCB126. This revised version was published online in July 2006 with corrections to the Cover Date.     

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.     

A two-mutation model of radiation-induced acute myeloid leukemia using historical mouse data

From studies of the atomic bomb survivors, it is well known that ionizing radiation causes several forms of leukemia. However, since the specific mechanism behind this process remains largely unknown, it is difficult to extrapolate carcinogenic effects at acute high-dose exposures to risk estimates for the chronic low-dose exposures that are important for radiation protection purposes. Recently, it has become clear that the induction of acute myeloid leukemia (AML) in CBA/H mice takes place through two key steps, both involving the Sfpi1 gene. A similar mechanism may play a role in human radiation-induced AML. In the present paper, a two-mutation carcinogenesis model is applied to model AML in several data sets of X-ray- and neutron-exposed CBA/H mice. The models obtained provide good fits to the data. A comparison between the predictions for neutron-induced and X-ray-induced AML yields an RBE for neutrons of approximately 3. The model used is considered to be a first step toward a model for human radiation-induced AML, which could be used to estimate risks of exposure to low doses.     

Interleukin-1β inhibits normal hematopoietic expansion and promotes acute myeloid leukemia progression via the bone marrow niche

Enhanced interleukin-1β (IL-1β) signaling is a common event in patients with acute myeloid leukemia (AML). It was previously demonstrated that chronic IL-1β exposure severely impaired hematopoietic stem cell (HSC) self-renewal capability in mice and promoted leukemia cell growth in primary AML cells. However, the role of IL-1β in the murine bone marrow (BM) niche remains unclear. Here, we explored the role of IL-1β in the BM niche in Il-1r1^−/− mice, chronic IL-1β exposure mice and mixed lineage leukemia-AF9 fusion gene (MLL-AF9)–induced AML mice models. We demonstrated that IL-1R1 deficiency did not affect the function of HSCs or niche cells under steady-state conditions or during transplantation. Chronic exposure to IL-1β decreased the expansion of Il-1r1^−/− hematopoietic cells in Il-1r1^+/+ recipient mice. These results indicated that IL-1β exposure impaired the ability of niche cells to support hematopoietic cells. Furthermore, we revealed that IL-1R1 deficiency in niche cells prolonged the survival of MLL-AF9–induced AML mice. The results of our study suggest that inhibition of the IL-1β/IL-1R1 signaling pathway in the niche might be a non–cell-autonomous therapy strategy for AML.