Blood Progenitor Cells Research Articles

Expansion and Hepatic Differentiation of Adult Blood-Derived CD34+ Progenitor Cells and Promotion of Liver Regeneration After Acute Injury

The low availability of functional hepatocytes has been an unmet demand for basic scientific research, new drug development, and cell-based clinical applications for decades. Because of the inability to expand hepatocytes in vitro, alternative sources of hepatocytes are a focus of liver regenerative medicine. We report a new group of blood-derived CD34(+) progenitor cells (BDPCs) that have the ability to expand and differentiate into functional hepatocyte-like cells and promote liver regeneration. BDPCs were obtained from the peripheral blood of an adult mouse with expression of surface markers CD34, CD45, Sca-1, c-kit, and Thy1.1. BDPCs can proliferate in vitro and differentiate into hepatocyte-like cells expressing hepatocyte markers, including CK8, CK18, CK19, α-fetoprotein, integrin-β1, and A6. The differentiated BDPCs (dBDPCs) also display liver-specific functional activities, such as glycogen storage, urea production, and albumin secretion. dBDPCs have cytochrome P450 activity and express specific hepatic transcription factors, such as hepatic nuclear factor 1α. To demonstrate liver regenerative activity, dBDPCs were injected into mice with severe acute liver damage caused by a high-dose injection of carbon tetrachloride (CCl4). dBDPC treatment rescued the mice from severe acute liver injury, increased survival, and induced liver regeneration. Because of their ease of access and application through peripheral blood and their capability of rapid expansion and hepatic differentiation, BDPCs have great potential as a cell-based therapy for liver disease. Hematopoietic stem/progenitor cell expansion and tissue-specific differentiation in vitro are challenges in regenerative medicine, although stem cell therapy has raised hope for the treatment of liver diseases by overcoming the scarcity of hepatocytes. This study identified and characterized a group of blood-derived progenitor cells (BDPCs) from the peripheral blood of an adult mouse. The CD34(+) progenitor-dominant BDPCs were rapidly expanded and hepatically differentiated into functional hepatocyte-like cells with our established coculture system. BDPC treatment increased animal survival and produced full regeneration in a severe liver injury mouse model caused by CCl4. BDPCs could have potential for liver cell therapies.

A study of Analysis of Endothelial Progenitor cells in Peripheral blood in patients with coronary artery disease

Objective: The study is aimed to analyze endothelial progenitor cells (EPCs) in coronary artery disease and also to identify predictive marker to assess EPCs in the same condition. Background: Endothelial progenitor cells (EPC) predict morbidity and mortality in cardiovascular risk. The ideal way to cure atherosclerosis and the subsequent end organ damage is to restore and rejuvenate the dysfunctional vasculature by induction of EPCs. Methods and Results: EPCs characterized by CD34 and KDR or CD133 markers still in progress to standardize. Hence, current study demonstrates the marker to identify the EPC in peripheral blood of patients with CAD. Flow cytometry used to quantify EPCs in 25 coronary artery disease as confirmed on angiography patients and 25 control subjects without CAD were prospectively enrolled in the study. To identify predictive EPCs in the peripheral blood, we used various combinations of markers like CD34+KDR+CD133+CD45-. We found that CAD patients presented significantly lower levels of EPCs, which expresses the markers CD34+CD45-KDR+ and CD133+CD45-KDR. EPCs which express CD34+CD45-KDR+subpopulation shows higher significant change between CAD and control group (p Conclusion: In conclusion, study indicates that there is a progressive decrease in EPCs levels in CAD patients. Patients with low EPC counts had a higher incidence for cardiovascular events. The level of circulating EPCs with CD34+CD45-KDR+ expression predicts the occurrence of cardiovascular events and may help to identify patients at increased cardiovascular risk. Key words: Atherosclerosis, Endothelial progenitor cells, Bone marrow, Vascular endothelial growth factor.

Scanning Electron Microscopy Reveals Two Distinct Classes of Erythroblastic Island Isolated from Adult Mammalian Bone Marrow.

Erythroblastic islands are multicellular clusters in which a central macrophage supports the development and maturation of red blood cell (erythroid) progenitors. These clusters play crucial roles in the pathogenesis observed in animal models of hematological disorders. The precise structure and function of erythroblastic islands is poorly understood. Here, we have combined scanning electron microscopy and immuno-gold labeling of surface proteins to develop a better understanding of the ultrastructure of these multicellular clusters. The erythroid-specific surface antigen Ter-119 and the transferrin receptor CD71 exhibited distinct patterns of protein sorting during erythroid cell maturation as detected by immuno-gold labeling. During electron microscopy analysis we observed two distinct classes of erythroblastic islands. The islands varied in size and morphology, and the number and type of erythroid cells interacting with the central macrophage. Assessment of femoral marrow isolated from a cavid rodent species (guinea pig, Cavis porcellus) and a marsupial carnivore species (fat-tailed dunnarts, Sminthopsis crassicaudata) showed that while the morphology of the central macrophage varied, two different types of erythroblastic islands were consistently identifiable. Our findings suggest that these two classes of erythroblastic islands are conserved in mammalian evolution and may play distinct roles in red blood cell production.

Simultaneous measurement of human hematopoietic stem and progenitor cells in blood using multicolor flow cytometry.

Hematopoietic stem cells are the source of all inflammatory cell types. Discovery of specific cell surface markers unique to human hematopoietic stem (HSC) and progenitor (HSPC) cell populations has facilitated studies of their development from stem cells to mature cells. The specific marker profiles of HSCs and HSPCs can be used to understand their role in human inflammatory diseases. The goal of this study is to simultaneously measure HSCs and HSPCs in normal human venous blood using multicolor flow cytometry. Our secondary aim is to determine how G-CSF mobilization alters the quantity of each HSC and HSPC population. Here we show that cells within the CD34+ fraction of human venous blood contains cells with the same cell surface markers found in human bone marrow samples. Mobilization with G-CSF significantly increases the quantity of total CD34+ cells, blood borne HSCs, multipotent progenitors, common myeloid progenitors, and megakaryocyte erythroid progenitors as a percentage of total MNCs analyzed. The increase in blood borne common lymphoid and granulocyte macrophage progenitors with G-CSF treatment did not reach significance. G-CSF treatment predominantly increased the numbers of HSCs and multipotent progenitors in the total CD34+ cell population; common myeloid progenitors and megakaryocyte erythroid progenitors were enriched relative to total MNCs analyzed, but not relative to total CD34+ cells. Our findings illustrate the utility of multicolor flow cytometry to quantify circulating HSCs and HSPCs in venous blood samples from human subjects. © 2016 International Clinical Cytometry Society.

Taking the Leap: Runx1 in the Formation of Blood from Endothelium.

Blood cell formation in the embryo occurs from multiple anatomic sites and results in the production of hematopoietic stem and progenitor cells that appear in overlapping waves. The transcription factor Runx1 is involved in a dramatic step of this process, for the transition from an endothelial cell that is integrated in a monolayer to a nonadherent circulating blood cell, a process conceptually similar to the epithelial to mesenchymal cell transition. Here we will review the role of Runx1 in the so-called hemogenic endothelium. We will describe the blood cell progenitors for which Runx1 is required, the proximal upstream transcription factors and signaling events that regulate its expression, and some of its important downstream targets in the hemogenic endothelium.

A Mathematical Model of Hematopoietic Stem Cell Transplantation and Analysis of the Effect of Drug Treatments on Transplantation in Patients with Lymphoma

▪Hematopoietic stem cells (HSCs) present in the bone marrow (BM) are primarily responsible for maintaining peripheral blood (PB) cell counts at a steady state. These hematopoietic progenitors are mobilized and used to support high dose therapy in patients with hematological malignancies. There is considerable variability in the HSC yield in patients and normal donors with standard mobilizing regimens. Understanding the quantitative principles at work in hematopoiesis will allow optimization of mobilizing regimens and eventual prediction of secondary hematopoietic malignancies. We created a mathematical model to account for the dynamics of HSCs in the bone marrow (C) and periphery (P) in addition to progenitor, committed, and peripheral blood platelets (T), neutrophils (N), lymphocytes (L), and erythrocytes (R). We explicitly model the egress of progenitor and committed cells from the BM compartment to the PB. In addition to these dynamics, we model the movement of CD34+ cells (P) from the bone marrow into the blood and the collection of CD34+ cells while on filgrastim. We also model transplantation of these collected cells in patients following myeloablative chemotherapy.We create the model with parameters that govern the rate at which the interactions between these cells occur, in addition to the variables defined previously, see Fig. 1 for equations.HSCs (C) and committed cells (Rc, Nc, Tc, and Lc) proliferate at rates sc, src, snc, stc, and slc, respectively. All source rates are inhibited by the concentration of their cell type in the blood, using a 1/(1+Xb/bxinf). Each cell type decays at a rate determined by the μx. HSCs differentiate into each subsequent committed cell type at the rates kcx and these committed cells differentiate into the progenitors of blood cells (Rm, Nm, Tm, and Lm) in the BM at the rates kxm. Progenitor cells move into PB (Rb, Nb, Tb, and Lb) at the rates kxb. Each parameter for movement from the BM to PB is scaled by the local volume to properly account for changes in concentrations.We begin by determining biologically relevant parameter values that correspond to homeostasis values for known cell concentrations in a normal person. The values for the blood are fit to normal blood cell count values, for example 3-4 million cells/μL for Rb. The range for P is broad (1-15 cells/μL) because there is variability in these levels in healthy individuals. Specific cell counts for the BM cells is not as well known, so we fit this compartment such that the dynamics and relative cell concentrations were reasonable and gave rise to correct concentrations in the PB, e.g. the ratio of Rc:Wc is about 1:5. Additionally, we treat this normal individual as a donor and simulate G-CSF treatment (which increases the rates of proliferation for C and NC and movement of CD34+ cells into the PB) and collection on this individual (black curve in Fig. 2). These results fit within the standard deviation bars for collection on each day from healthy donors.We then sample over a range parameter values to create a population of patients that have blood cell levels relevant to the disease and model collection for each of these individuals. Fig. 3 shows collection results from the simulated diseased population. A point is plotted for each patient at a sc versus kcrc values and colored by their total collection count. Poor collectors (<5,000,000 CD34+ cells/kg) are indicated as dark dots. Most poor collectors are seen when both parameter values are normal or elevated. Determining what drives poor collection for each simulated patients gives insight into which types of patient may benefit from additional drug treatments such as Plerixafor. For patients that self donate or use donor cells, we also simulate transplantation of the stem cells. Mathematical modeling such as this is useful in making predictions about physiological processes and can potentially aid in making decisions. Building validated models can lead to predictions about how patient will respond to a treatment and adapting alternative treatment. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Dominant Gain-of-Function Mutation in Universal Tyrosine Kinase SRC Causes Enhanced Podosome Formation in a Syndrome with Thrombocytopenia, Myelofibrosis, Bleeding and Bone Pathologies

For the BRIDGE-Bleeding and Platelet Disorder Consortium.We here present a pedigree with nine cases that present with a new syndrome characterized by early onset myelofibrosis, thrombocytopenia, bleeding and bone pathologies. The syndrome is caused by a dominant gain-of-function mutation in universal tyrosine kinase SRC and it is the first example of a pathogenic germline mutation in this extensively studied kinase. The genetic discovery has been made possible by making use of our collection of 1,115 patients with inherited bleeding and platelet disorders of unknown molecular etiology enrolled by us at 25 tertiary referral centers as part of the NIHR BioResource - Rare Diseases pilot phase of the 100,000 Genomes Project. The analysis of genome sequencing data for 2 affected individuals was empowered by comparison of Human Phenotype Ontology (HPO; Westbury et al., Genome Medicine, 2015) terms of the patients with OMIM and the Mouse Phenotype Ontology terms from 7,541 strains. The selection of the SRC gene as the most likely candidate gene was also informed by the use of our Blueprint blood cell progenitor RNA-seq data (Chen et al., Science 2014).Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr419 phosphorylation in COS7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation and reduced platelet activity. Patients with myelofibrosis have a hypercellular bone marrow with trilineage dysplasia and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. Patient MKs generate platelets which are dysmorphic, low in number, highly variable in size and with a paucity of α-granules. Over-active SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with enhanced tyrosine phosphorylation levels and with reduced ploidy compared to cells transduced with wild-type SRC. Patient-derived and E527K-transduced MKs show Y419 SRC positive stained podosomes that induce a strongly altered organization of filamentous actin. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Our findings reveal an unexpected and wide-ranging pathobiology for this universal kinase Src with podosome formation.In hematopoiesis constitutive active Src primarily affects megakaryopoiesis with ineffective formation of platelets. Its inadequate function is associated with bleeding. The kinase Src is activated frequently in human cancers hence new SFK inhibitors are undergoing clinical trials and side effects with severe bleeding are observed frequently. It remains to be determined whether this new class of SFK inhibitors inadvertently causes inadequate formation of platelets, which do not function properly. Disclosures:No relevant conflicts of interest to declare.

A Novel Feed-Forward Loop Involving the High Mobility Group A1 (HMGA1) Chromatin Remodeling Protein and cMYC in Acute Myeloid Leukemia Is Targeted By JQ1

Acute myeloid leukemia (AML) comprises a heterogeneous group of highly lethal, clonal disorders of blood cell progenitors for which more effective therapies are urgently needed. The underlying genetic abnormalities transform hematopoietic progenitors into AML blasts that are resistant to apoptosis and fail to undergo normal differentiation. While conventional chemotherapy regimens frequently ablate actively cycling leukemic blasts and induce remissions, most patients relapse with abysmal outcomes. Recent studies highlight an important role for stem cell transcriptional networks in supporting survival and resistance to chemotherapy in leukemic blasts, although druggable targets that maintain these networks have remained elusive. We previously discovered that the cMYC oncoprotein activates HMGA1 expression in experimental models of lymphoid leukemia, while HMGA1 directly induces cMYC in embryonic stem cells. Here, we demonstrate for the first time that the HMGA1-cMYC network is a therapeutic target that is critical for leukemic cell survival and resistance to chemotherapy.In primary AML blasts, we found that both HMGA1 and cMYC gene expression are enriched relative to levels in normal CD34+ hematopoietic stem and progenitor cells and their expression is positively correlated. Silencing HMGA1 or cMYC in AML blasts dramatically halts cell growth, leading to rapid apoptotic cell death. HMGA1 transcriptionally activates cMYC in leukemic blasts, while the cMYC oncoprotein, in turn, induces HMGA1, resulting in potent feed-forward loop that maintains survival in AML blasts.We also discovered that AML blasts exposed to AraC (cytosine arabinoside), a standard cytotoxic agent used in AML, become resistant by up-regulating both HMGA1 and cMYC. Moreover, silencing either HMGA1 or cMYC restores sensitivity to AraC, demonstrating that induction of HMGA1 and cMYC is functionally important. Next, we sought to determine whether the inhibitor of the BET family of bromodomain proteins, JQ1, has anti-leukemic effects in AML blasts resistant to AraC because it functions, at least in part, by disrupting cMYC transcriptional complexes. We found that cMYC is repressed within 4 hours, while HMGA1 requires 24 hours for down-regulation following exposure to JQ1. Moreover, JQ1 restores sensitivity to AraC in the resistant AML blasts.Together, these studies reveal a novel role for HMGA1 and cMYC in a feed-forward loop that is critical for AML blast survival and chemoresistance. These findings also suggest that JQ1 will be effective therapy in AML blasts that are resistant to AraC. DisclosuresNo relevant conflicts of interest to declare.

Effects of hemin on quantity and apoptosis of human umbilical cord blood-derived late endothelial progenitor cells

Objective To investigate the effects of hemin on the quantity and apoptosis of human umbilical cord blood-derived late endothelial progenitor cells (EPCs) in vitro. Methods Mononuclear cells were isolated from human cord blood by density gradient centrifugation and were induced to differentiate to late EPCs in vitro. The second to third generation of attached late EPCs in good state were randomly plated for 24 h under different concentrations(0, 5, 10, 15 and 20 μmol/L)of hemin. Cell viability and proliferation were measured with typan blue staining and cell counting kit-8, respectively. Cell adhesion was analyzed by adhesive assay, and cell apoptosis was detected by flow cytometry. Results Compared to control group, hemin promoted viability of late EPCsat lower concentrations(5and 10μmol/L). Meanwhile, proliferation and adhesion were also improved and apoptosis was inhibited when the concentrations of hemin were 5, 10, or 15 μmol/L. All these effects were most prominent when hemin concentration was 10 μmol/L, while the effects above were reversed when hemin concentration was moderated to 20 μmol/L. In addition, hemin showed a time-dependent manner in promoting cell proliferation and adhesion, and inhibiting apoptosis. That effects were most obvious at 24 h. Conclusions Lower concentration of hemin augments the quantity and adhesion of late EPCs, inhibits cell apoptosis, while higher concentration present the reversed effects. Key words: Hemin/PD; Fetal blood/CY; Endothelial cells/DE/PA; Apoptosis

Change of endothelial progenitor cells in the bone marrow and peripheral blood of patients with acute leukemia and its clinical significance

Objectives To evaluate the count of endothelial progenitor cells (EPCs) in peripheral blood (PB) and bone marrow (BM) of acute leukemia (AL) patients and explore its clinical significance. Methods EPCs were detected by flow cytometry procedures in 43 AL patients and in 10 benign hematologic patients as control group. Results The absolute counts of EPCs in AL patients before the treatment [(119.46±72.23)/μl in BM and (13.69±8.26)/μl in PB] were significantly higher than those in control group [(23.21 ±12.59)/μl in BM and (1.86±1.18)/μl in PB] (P 0.05). After the treatment, the absolute counts of EPCs both in BM and in BP of CR group [(26.32±17.44)/μl and (2.54±2.12)/μl, respectively] were significantly lower than those before treatment [(113.18±69.22)/μl and (14.45±10.76)/μl, respectively] (P 0.05). The absolute counts of EPCs whether in PB or in BM were no significant difference between acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) (P > 0.05). The absolute counts of EPCs in PB of AL had a positive correlation with β2-MG and LDH (P < 0.05). Conclusions EPC levels are significantly increased in BM and BP of AL patients and may correlate with disease status , response to treatment and prognosis. Key words: Acute leukemia; Endothelial progenitor cells; Flow cytometry

Abstract 3632: Synergistic interaction of auranofin with PARP inhibitors in ATM-proficient mantle cell lymphoma

Abstract Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound that was used for treating arthritis. Its primary mechanism of action is the inhibition of thioredoxin reductase (TRR) activity. It consequently induces oxidative/ER stress and also inhibits Heat shock protein (Hsp) 90 chaperone function. Ataxia-telangiectasia mutated and Rad3-related (ATR), Brca1, Chk1 and DNA-PK are Hsp90 client proteins that participate in DNA repair pathways. Based on our observation that AF induces DNA damage, we evaluated whether treatment with AF also impairs Hsp90-dependent DNA damage repair pathways in Mantle Cell Lymphoma (MCL) cells. It has been reported that ATM mutations are found in about 50% cases of MCL. Earlier studies have shown that ATM mutant MCL cells are more sensitive to poly-ADP ribose polymerase (PARP) inhibitors in vitro and in vivo. We hypothesized that by inhibiting Hsp90-dependent DNA repair pathways (such as the ATR pathway), AF would sensitize ATM-proficient MCL cells to PARP inhibition by ABT-888. We demonstrate that treatment of ATM-proficient MCL cell lines, JeKo-1, MO2058 and Z138C with AF induced reactive oxygen species (ROS) levels and resulted in a concomitant decrease in TRR activity. Microarray analysis of AF-treated primary MCL cells revealed that AF significantly altered heat shock response genes, ER and oxidative stress-inducible genes and a subset of genes that were regulated by ATM or Brca1. Treatment with AF also induced Hsp90 hyperacetylation and resulted in the depletion of Hsp90 client proteins including ATR, AKT and Cyclin D1 in MCL cells. Exposure to AF induced significantly more apoptosis in primary MCL cells, as compared to CD19+ normal B cells, CD34+ human cord blood and bone marrow progenitor cells (&amp;lt; 15% apoptosis) (p &amp;lt; 0.01). Co-treatment of MCL cells with AF and the PARP inhibitor ABT-888 resulted in synergistic increase in apoptosis of ATM-proficient MCL cells, with an increase in the induction of γH2AX foci and depletion of p-Chk1 (a downstream target of ATR signaling). This was accompanied with a concomitant increase in the levels of the ER stress-induced transcription factor CHOP (C/EBP homologous protein), cleaved Caspase 3 and PARP. Collectively, our data suggests the synthetic lethal effects observed by the simultaneous inhibition of DNA repair pathway(s) and PARP activity can be extended to ATM-proficient MCL cells. These pre-clinical studies create a strong rationale to determine the in vivo activity of the combination ABT-888 with AF in MCL. Citation Format: Siddhartha Ganguly, Trisha Home, Sumedha Gunewardena, Scott Weir, Joseph McGuirk, Rekha M. Rao. Synergistic interaction of auranofin with PARP inhibitors in ATM-proficient mantle cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3632. doi:10.1158/1538-7445.AM2015-3632

Rapid identification of a Leptotrichia trevisanii catheter-related bloodstream infection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Introduction: Leptotrichia trevisanii is a large, fusiform, non-sporulating, motile, Gram-negative rod, and is a member of the family Fusobacteriaceae in the phylum Fusobacteria. Although L. trevisanii bacteraemia is extremely rare, severe infections have been reported in immunocompromised patients. Case presentation: We present a case of a 69-year-old woman diagnosed with diffuse large B-cell lymphoma, who suffered a catheter-related bloodstream infection due to L. trevisanii during the post-autologous peripheral blood progenitor-cell transplantation aplasia phase. In this case, rapid identification of the opportunistic pathogen was achieved using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry directly from a blood culture, with correct identification of the micro-organism within 2 h after the blood culture became positive. Conclusion: Rapid identification of this opportunistic pathogen allowed initiation of appropriate antimicrobial therapy, which contributed to a successful clinical outcome for the patient.

The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche

The maintenance of stem or progenitor cell fate relies on intrinsic factors as well as local cues from the cellular microenvironment and systemic signaling. In the lymph gland, an hematopoietic organ in Drosophila larva, a group of cells called the Posterior Signaling Centre (PSC), whose specification depends on the EBF transcription factor Collier (Col) and the HOX factor Antennapedia (Antp), has been proposed to form a niche required to maintain the pool of hematopoietic progenitors (prohemocytes). In contrast with this model, we show here that genetic ablation of the PSC does not cause an increase in blood cell differentiation or a loss of blood cell progenitors. Furthermore, although both col and Antp mutant larvae are devoid of PSC, the massive prohemocyte differentiation observed in col mutant is not phenocopied in Antp mutant. Interestingly, beside its expression in the PSC, Col is also expressed at low levels in prohemocytes and we show that this expression persists in PSC-ablated and Antp mutant larvae. Moreover, targeted knockdown and rescue experiments indicate that Col expression is required in the prohemocytes to prevent their differentiation. Together, our findings show that the PSC is dispensable for blood cell progenitor maintenance and reveal the key role of the conserved transcription factor Col as an intrinsic regulator of hematopoietic progenitor fate.

A zebrafish model of myelodysplastic syndrome produced through tet2 genomic editing.

The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. tet2(m/m) (homozygous for the mutation) zebrafish exhibited normal embryonic and larval hematopoiesis but developed progressive clonal myelodysplasia as they aged, culminating in myelodysplastic syndromes (MDS) at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultant tet2(m/m) mutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in nonhematopoietic tissues but not in hematopoietic cells. tet2(m/m) zebrafish are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and, for small-molecule screens in embryos, to identify compounds with specific activity against tet2 mutant cells.

Intermittent Hypoxia Impairs Endothelial Function in Early Preatherosclerosis.

Intermittent hypoxia seems to be a major pathomechanism of obstructive sleep apnea-associated progression of atherosclerosis. The goal of the present study was to assess the influence of hypoxia on endothelial function depending on the initial stage of vasculopathy. We used 16 ApoE-/- mice were exposed to a 6-week-intermittent hypoxia either immediately (early preatherosclerosis) or after 5 weeks of high-cholesterol diet (advanced preatherosclerosis). Another 16 ApoE-/- mice under normoxia served as corresponding controls. Endothelial function was measured by an organ bath technique. Blood plasma CD31+/annexin V+ endothelial microparticles as well as sca1/flk1+ endothelial progenitor cells in blood and bone marrow were analyzed by flow cytometry. The findings were that intermittent hypoxia impaired endothelial function (56.6±6.2% of maximal phenylephrine-induced vasoconstriction vs. 35.2±4.1% in control) and integrity (increased percentage of endothelial microparticles: 0.28±0.05% vs. 0.15±0.02% in control) in early preatherosclerosis. Peripheral repair capacity expressed as the number of endothelial progenitor cells in blood was attenuated under hypoxia (2.0±0.5% vs. 5.3±1.9% in control), despite the elevated number of these cells in the bone marrow (2.0±0.4% vs. 1.1±0.2% in control). In contrast, endothelial function, as well as microparticle and endothelial progenitor cell levels were similar under hypoxia vs. control in advanced preatherosclerosis. We conclude that hypoxia aggravates endothelial dysfunction and destruction in early preatherosclerosis.

Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis.

ABSTRACTHematopoietic homeostasis requires the maintenance of a reservoir of undifferentiated blood cell progenitors and the ability to replace or expand differentiated blood cell lineages when necessary. Multiple signaling pathways function in these processes, but how their spatiotemporal control is established and their activity is coordinated in the context of the entire hematopoietic network are still poorly understood. We report here that loss of the gene Rabex-5 in Drosophila causes several hematopoietic abnormalities, including blood cell (hemocyte) overproliferation, increased size of the hematopoietic organ (the lymph gland), lamellocyte differentiation and melanotic mass formation. Hemocyte-specific Rabex-5 knockdown was sufficient to increase hemocyte populations, increase lymph gland size and induce melanotic masses. Rabex-5 negatively regulates Ras, and we show that Ras activity is responsible for specific Rabex-5 hematopoietic phenotypes. Surprisingly, Ras-independent Notch protein accumulation and transcriptional activity in the lymph gland underlie multiple distinct hematopoietic phenotypes of Rabex-5 loss. Thus, Rabex-5 plays an important role in Drosophila hematopoiesis and might serve as an axis coordinating Ras and Notch signaling in the lymph gland.

The Role of Erythropoietin and its Receptor in the Immune, Central Nervous and Cardiovascular Systems

Primary role of erythropoietin (EPO) is to protect red blood cell progenitors from apoptosis and to stimulate their growth and differentiation by binding to its receptor (EPOR). However, EPOR has been identified on other human cells, such as polymorphonuclear leukocytes (PMNLs), monocytes, lymphocytes, endothelial cells and neurons. Its presence on these cells suggests that beyond erythropoietic function EPO might have some other properties. This review presents current knowledge on the role of EPO and its receptor in the immune, central nervous and cardiovascular systems.

Comparison of DNA methylation profiles in human fetal and adult red blood cell progenitors.

BackgroundDNA methylation is an epigenetic modification that plays an important role during mammalian development. Around birth in humans, the main site of red blood cell production moves from the fetal liver to the bone marrow. DNA methylation changes at the β-globin locus and a switch from fetal to adult hemoglobin production characterize this transition. Understanding this globin switch may improve the treatment of patients with sickle cell disease and β-thalassemia, two of the most common Mendelian diseases in the world. The goal of our study was to describe and compare the genome-wide patterns of DNA methylation in fetal and adult human erythroblasts.MethodsWe used the Illumina HumanMethylation 450 k BeadChip to measure DNA methylation at 402,819 CpGs in ex vivo-differentiated erythroblasts from 12 fetal liver and 12 bone marrow CD34+ donors.ResultsWe identified 5,937 differentially methylated CpGs that overlap with erythroid enhancers and binding sites for erythropoiesis-related transcription factors. Combining this information with genome-wide association study results, we show that erythroid enhancers define particularly promising genomic regions to identify new genetic variants associated with fetal hemoglobin (HbF) levels in humans. Many differentially methylated CpGs are located near genes with unanticipated roles in red blood cell differentiation and proliferation. For some of these new candidate genes, we confirm the correlation between DNA methylation and gene expression levels in red blood cell progenitors. We also provide evidence that DNA methylation and genetic variation at the β-globin locus independently control globin gene expression in adult erythroblasts.ConclusionsOur DNA methylome maps confirm the widespread dynamic changes in DNA methylation that occur during human erythropoiesis. These changes tend to happen near erythroid enhancers, further highlighting their importance in erythroid regulation and HbF production. Finally, DNA methylation may act independently of the transcription factor BCL11A to repress fetal hemoglobin production. This provides cues on strategies to more efficiently re-activate HbF production in sickle cell disease and β-thalassemia patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0122-2) contains supplementary material, which is available to authorized users.

Personalized Drug Combinations for the Treatment of Acute Myeloid Leukemia (AML) Patients

Acute myeloid leukemia (AML) is a highly heterogeneous disorder characterized by the rapid clonal proliferation of blasts derived from hematopoietic progenitor cells, leading to failure of normal hematopoiesis. Although standard therapy, usually including idarubicin and cytarabine, has been used to achieve remission, the long-term survival rates remain low.To show that it is possible to improve existing AML standard therapy, we have used experimental algorithms we have recently developed (iterative algorithms that find effective combinations without a full combinatorial screening) to find selective drug combinations for AML cells. The first search was done using the AML cell line KG-1 for the initial steps and cells from an AML patient for the final step. A normal fibroblast cell line (IMR-90) and normal blood progenitor cells were used as controls for toxicity. Drugs were chosen from those already approved or in clinical trials for AML. As shown in Figure 1, this experimental search was able to achieve selective killing of AML patient cells with a ratio close to 100:1 with respect to non-cancer control cells.To refine the choice of drugs to be used in the search, we recently developed a network model of AML intracellular signal transduction (see www.leukemianetworks.org). The present version (AML 2.1) includes 5667 proteins and 22,218 interactions. The network can be used to integrate genomic and gene expression information from individual patients and to provide a shortlist of drugs to be tested further. Using literature data and our network model we identified three compounds as promising for AML drug combinations: the MEK inhibitor PD0325901, the FLT3 inhibitor Quizartinib and the CDK 4/6 inhibitor Palbociclib. For this experiment, normal progenitor blood cells were the controls. Figure 2 shows the results of 64 experimental tests of the response to different combinations of these drugs. The two AML patients clearly exhibited different drug sensitivities, further supporting the necessity of personalized drug combinations.To improve the clinical relevance of these in vitro studies, we investigated the correlation between clinical and in vitro response to standard AML therapy, using a set of media containing 16 cytokines/growth factors in different combinations. These molecules have been selected using three methods: ligands acting on receptors that are part of gene signatures with prognostic significance in AML; cytokines that promote hematopoietic differentiation of stem cells; and ligands of receptors identified as playing an important role in AML in a recent RNAi screen. During several iterations of our combinatorial search algorithm we studied 185 different media. Figure 3 shows the correlation between clinical response (residual blasts after 28 days of therapy) and response to the same therapy in vitro after 72 h achieved in the last iteration of the search, in a study of six patients. The medium with the highest correlation had a correlation coefficient of 0.99 (p=0.0002). The in vitro response in this medium is clearly able to distinguish patients that respond to standard therapy vs those that do not, as can be seen from the inset of Fig. 3. Some cytokines (BMP-4 and IL-4) were always absent in the group of 14 media with lower correlation shown on the right of Figure 3. The remarkable variation in correlation with clinical response of these media, spanning correlation coefficient values from 0.99 to 0.004, clearly show the importance of optimizing the in vitro microenvironment for prediction of patient drug response.In summary, we identified drug combinations that selectively kill AML primary cells and we optimized media that improve the in vitro prediction of clinical drug response. These methods can together assist in the personalization of AML therapy using patientÕs cells. [Display omitted] [Display omitted] [Display omitted] DisclosuresPiermarocchi:Salgomed Inc.: Equity Ownership. Paternostro:Salgomed Inc.: Equity Ownership.

IL-4 and IL-13 regulate TLR expression and eosinophil-basophil differentiation of cord blood CD34+ progenitor cells

Background Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent allergic disease development [1]. We have previously shown that cord blood (CB) progenitor cells of high atopic risk infants have reduced toll-like receptor (TLR) expression and produce fewer lipopolysaccharide (LPS)-stimulated eosinophil-basophil (Eo/B) colonies, compared to lowatopic risk infants. In the present study, we investigated whether a surrogate ex vivo TH2 milieu (i.e., either IL-4 or IL-13), could represent an underlying mechanism to explain our previous findings.