Myeloid Lineage Differentiation Research Articles

Early sensing of Yersinia pestis airway infection by bone marrow cells

Bacterial infection of the lungs triggers a swift innate immune response that involves the production of cytokines and chemokines that promote recruitment of immune cells from the bone marrow (BM) into the infected tissue and limit the ability of the pathogen to replicate. Recent in vivo studies of pneumonic plague in animal models indicate that the pulmonary pro-inflammatory response to airway infection with Yersinia pestis is substantially delayed in comparison to other pathogens. Consequently, uncontrolled proliferation of the pathogen in the lungs is observed, followed by dissemination to internal organs and death. While the lack of an adequate early immune response in the lung is well described, the response of BM-derived cells is poorly understood. In this study, we show that intranasal (i.n.) infection of mice with a fully virulent Y. pestis strain is sensed early by the BM compartment, resulting in a reduction in CXCR4 levels on BM neutrophils and their subsequent release into the blood 12 hours (h) post infection. In addition, increased levels of BM-derived hematopoietic stem and progenitor cells (HSPC) were detected in the blood early after infection. Mobilization of both immature and mature cells was accompanied by the reduction of BM SDF-1 (CXCL-12) levels and the reciprocal elevation of SDF-1 in the blood 24 h post infection. RT-PCR analysis of RNA collected from total BM cells revealed an early induction of myeloid-associated genes, suggesting a prompt commitment to myeloid lineage differentiation. These findings indicate that lung infection by Y. pestis is sensed by BM cells early after infection, although bacterial colonization of the BM occurs at late disease stages, and point on a potential cross-talk between the lung and the BM at early stages of pneumonic plague.

Transient Activation of Hematopoietic Stem and Progenitor Cells by IFNγ during Acute Bacterial Infection

How hematopoietic stem cells (HSCs) respond to inflammatory signals during infections is not well understood. Our studies have used a murine model of ehrlichiosis, an emerging tick-born disease, to address how infection impacts hematopoietic function. Infection of C57BL/6 mice with the intracellular bacterium, Ehrlichia muris, results in anemia and thrombocytopenia, similar to what is observed in human ehrlichiosis patients. In the mouse, infection promotes myelopoiesis, a process that is critically dependent on interferon gamma (IFNγ) signaling. In the present study, we demonstrate that E. muris infection also drives the transient proliferation and expansion of bone marrow Lin-negative Sca-1+ cKit+ (LSK) cells, a population of progenitor cells that contains HSCs. Expansion of the LSK population in the bone marrow was associated with a loss of dormant, long-term repopulating HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation within that population. The reduced engraftment and myeloid bias of the infection-induced LSK cells was transient, and was most pronounced on day 8 post-infection. The infection-induced changes were accompanied by an expansion of more differentiated multipotent progenitor cells, and required IFNγ signaling. Thus, in response to inflammatory signals elicited during acute infection, HSCs can undergo a rapid, IFNγ-dependent, transient shift from dormancy to activity, ostensibly, to provide the host with additional or better-armed innate cells for host defense. Similar changes in hematopoietic function likely underlie many different infections of public health importance.

Human Umbilical Cord Blood-Derived Stromal Cells Are Superior to Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Inducing Myeloid Lineage Differentiation In Vitro

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.

Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age

In the human hematopoietic system, aging is associated with decreased bone marrow cellularity, decreased adaptive immune system function, and increased incidence of anemia and other hematological disorders and malignancies. Recent studies in mice suggest that changes within the hematopoietic stem cell (HSC) population during aging contribute significantly to the manifestation of these age-associated hematopoietic pathologies. Though the mouse HSC population has been shown to change both quantitatively and functionally with age, changes in the human HSC and progenitor cell populations during aging have been incompletely characterized. To elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated immunophenotypic HSC and other hematopoietic progenitor populations from healthy, hematologically normal young and elderly human bone marrow samples. We found that aged immunophenotypic human HSC increase in frequency, are less quiescent, and exhibit myeloid-biased differentiation potential compared with young HSC. Gene expression profiling revealed that aged immunophenotypic human HSC transcriptionally up-regulate genes associated with cell cycle, myeloid lineage specification, and myeloid malignancies. These age-associated alterations in the frequency, developmental potential, and gene expression profile of human HSC are similar to those changes observed in mouse HSC, suggesting that hematopoietic aging is an evolutionarily conserved process.

Selective Accumulation of CD45RO+CCR7- in the Marrow Aspirates in Patients with Essential Thrombocythemia

Abstract Abstract 5155 Patients with newly diagnosed essential thrombocythemia (ET) were investigated for selective accumulation/expansion of CD4+ lymphocytes subpopulations in the marrow, to verifie the hypothesis that the neoplastic process associated factor(s) are recognized by the immune system. Sixteen patients (F/M: 12/4, age 28–72 - median 60 yrs) with ET diagnosed according to the WHO criteria were studied. They had platelets from 496–809 x 10̂3/ul (median 644 × 10̂3 ), (12 heterozygotes, 2 homozygotes). Cytological evaluation revealed normal differentiation of erythroid and myeloid lineages, in contrast an increased number of megakaryocytes often enlarged, hyperlobulated and in clusters were seen in all patients. Trephine biopsy revealed activation of megakaryopoiesis with enlarged numerous megakaryocytes with normal representation of other lineages and a lack of an increase in the reticulin fibers. Marrow cellularity was from 9 – 126 x10̂3/ul (median 42 × 10̂3/ul). The whole populations of marrow and blood cells were labeled for a purpose of this study with the use CD4, CD25, CD45RO and CCR7 MoAb (Becton Dickinson, San Jose, CA, UmarSA) followed by detection and analysis with the use of flow cytometry (FACS Calibur, Becton Dickinson) equipped with PCLysis programe. Immunostaining of trephine biopsies was used for detection of FOXP3 cells. It was found: (i) Lymphocytes in the marrow were in proportions from 4,5%-15,7% (median 7,25%), in numbers from1377/ul to 8245/ul (median 3870/ul) what was significantly higher as compared to blood from 1300/ul to 3900/ul (median 2100/ul) (Wilcoxon Matched Pairs Test, p=0.010). (ii) CD45RO+CCR7- lymphocytes were higher in numbers in the marrow aspirates as compared to blood (median 1281/ul vs 631,2/ul, p=0,04 Wilcoxon Test for pairs). (iii) In contrast numbers in the marrow aspirates of CD45 RO-CCR7+ (median 297,4/ul vs median 238,2/ul), as well as CD45 RO+CCR7+ (median 149,6/ul vs 146,4/ul) did not differ from those in blood. (iv) It was a significant increase of CD4+CD25high+ cells in the marrow aspirates as compared to blood (median 36,2/ul vs 9,1/ul; p=0,02 Wilcoxon Test for pairs). Indeed in 11 out of 14 trephine biopsies investigated FoxP3+ cells were identified. In conclusion an increase, in the marrow of ET patients, of effector/memory CD4 lymphocytes (CD45RO+CCR7-) but not those of naïve and central memory lymphocytes phenotypes points out on the selective accumulation of these cells in the marrows. Concomitant increase of T regulatory cells (CD4+CD25+high) suggests that an active immunological is taking place at the site. It is possible that effector memory cells are attracted to come to the marrows by ET process associated factors and if so the present observation constitute the primary observation building the rationale behind immunotherapy in ET patients Disclosures: No relevant conflicts of interest to declare.

Membrane-Bound Human Stem Cell Factor Promotes Differentiation of Human Granulocytes and Mast Cells In Vivo,

Abstract Abstract 3419 Recently, advances in xenograft models for human hemamtopoietic stem cells (HSCs), or the humanized mice, have begun to allow investigators to examine the differentiation of human hematopoietic and immune cells in vivo. However, lymphoid-skewed human hematopoietic development in the mouse bone marrow is one of the remaining limitations in the humanized mouse models. The inefficient human myeloid development could at least partly be attributed to the mouse microenvironment not fully supporting differentiation and maturation of human myeloid lineage. To overcome this problem, we focused on the role of membrane-bound human stem cell factor in supporting the maintenance of human HSCs and inducing the development of human myeloid cells and created human stem cell factor transgenic NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. Transplantation of 5000–50000 cord blood-derived Lin-CD34+CD38- cells resulted in significantly higher engraftment of human CD45+ leukocytes at 3–6 months post-transplantation in the bone marrow, spleen, and peripheral blood of hSCF Tg NSG recipients compared with those of non-transgenic NSG recipients. The enhanced human CD45+ engraftment was most prominent in the bone marrow (hSCF Tg recipients: 98.0 +/− 1.3%, n= 15, non-Tg NSG controls: 75.3 +/− 7.3%, n=7). In the bone marrow, the frequency of human CD33+ myeloid cells within the total human CD45+ population was significantly higher in the hSCF Tg NSG recipients than in the non-Tg NSG recipients and constituted the majority of human hematopoietic cells (hSCF Tg recipients: 54.6 +/− 4.5%, n=15 and non-Tg NSG controls: 29.3 +/− 4.0%, n=7). Flow cytometric analysis demonstrated that the majority of engrafted human myeloid cells in the hSCF Tg recipient bone marrow were side-scatter high, HLA-DR negative granulocytes. Reflecting the effect of human SCF on the development of human mast cells, human c-Kit+CD203c+ mast cells were identified in the bone marrow, spleen, and gastrointestinal tracts of the hSCF Tg NSG recipients. Altogether, the in vivo humanized mouse model demonstrates the essential role of membrane-bound SCF in human myeloid development. The hSCF Tg NSG humanized mice may facilitate the in vivo investigation of human HSCs, myeloid progenitors and mature myeloid lineage. Disclosures: No relevant conflicts of interest to declare.

Conserved Transcriptional Deregulation Underlies GFI1 and ELANE Mutant Neutropenia

Abstract Abstract 13 Mutations in ELANE, GFI1, G6PC3, and HAX1 account for the genetic defects in many patients with severe congenital neutropenia (SCN). Here we utilized next generation sequencing technology to characterize genome-wide transcriptome activity and chromatin structure states associated with normal and SCN–affected hematopoietic precursor cells. We profiled cells from Gfi1−/−, Gfi1P2A, LysM-Cre Mcl1flox and wild type mice. Growth factor independent-1 (Gfi1) is a zinc finger transcription repressor required for murine and human granulopoiesis. In Gfi1−/− bone marrow cells, the genetic lack of Gfi1 protein blocks granulopoietic programming. In Gfi1P2A knockin mice the Gfi1 protein is made, but the substitution of alanine for proline (P2A) inhibits the function of the SNAG transcriptional repressor domain; resulting in neutropenia. Mcl1 is a Bcl2 family member required for the survival of multiple hematopoietic lineage cells. LysM-Cre Mcl1flox mice are neutropenic due to myeloid-lineage-specific Cre-mediated deletion of Mcl1 and subsequent apoptosis of neutrophil progenitors after the metamyelocyte stage. Other hematopoietic lineages in LysM-Cre-Mcl1flox mice are normal. Thus, all three models are neutropenic; two lack a functional lineage specifying transcription factor while one lacks a survival factor. Bone marrow cells from each of these mouse models were processed through Miltenyi AutoMacs lineage-depletion. Total RNA was extracted and subjected to paired-end deep RNA-sequencing using Illumina Truseq library preparation methods. Reads were aligned to the reference mouse mm9 genome and Refseq and ENSEMBL gene transcript models using Tophat, Bowtie, and Cufflinks analysis suite. Both gene and transcript model specific gene expression level were estimated using the fragments per kilobase of exon model per million mapped reads (FKPM) approach. Using gene-level analyses, a representative group of 2,186 genes were identified that exhibited relatively high level expression (>50th %ile by FPKM in at least one sample type) and differential gene expression relative to WT sample profiles in at least one model. First, our data indicate that the SNAG domain is critical for most Gfi1 associated functions. The SNAG domain associates with the Lysine specific demethylase-1 (Lsd1), an enzyme which removes histone 3 lysine 4 dimethyl (H3K4me2) marks. Chromatin immunoprecipitation revealed increased H3K4me2 marks in many genes deregulated in Gfi1−/− and Gfi1P2A Lin- bone marrow cells. Moreover, the group of genes overexpressed in Gfi1−/− and Gfi1P2A Lin- bone marrow cells was functionally associated with a variety of signal transduction and transcriptional control programs responsible for the development and differentiation of myeloid and lymphoid lineages, whereas genes that were commonly downregulated were associated with erythroid development. Many of the overexpressed immune myeloid genes were the targets of the ETS family of transcription factors and included a rich overrepresentation of genes associated with vesicle/lysosome/ and granule formation. Furthermore, using statistical and clustering-based analyses, approximately 70% of the dysregulated genes exhibited similar over-expression and under-expression in all three models. The large scale similarity of these patterns and the disparate genetic and biological mechanism of neutropenia in the three models suggests an underlying signature of the bone marrow progenitor activation in response to neutropenia. To confirm these findings in a different setting, we next examined the gene expression of human CD34+ cells from several ELANE mutant SCN patients and a single GFI1N382S patient, compared to normal CD34+ cells. Notably, all patients received G-CSF. In fact, a significant number of genes differentially expressed between ELANE and normal CD34+ cells were commonly deregulated in the GFI1 patient sample. Thus, human samples also demonstrate a signature associated with bone marrow progenitor activation in response to neutropenia. Our results reveal not only the transcriptional circuits controlled by Gfi1, but also cell autonomous transcriptional circuits underlying neutropenia. Disclosures: No relevant conflicts of interest to declare.

Loss of p53 Accelerates the Complications of Myelodysplastic Syndromes (MDS) In the NUP98-HOXD13 Transgenic Mouse Model

Abstract Abstract 2804 The myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis leading to blood cytopenias and a high rate of progression to acute myelogenous leukemia (AML) (Nimer, 2008). Recent studies implicated an important role for p53 in 5q−MDS, a subtype where haploinsufficiency of the RPS14 ribosomal protein drives the anemia that accompanies this disease (Ebert et al., 2008; Barlow et al., 2010 and Dutt et al., 2011). The elevated level of p53 activity apparently triggers the excessive apoptosis and the dysplastic morphology seen in the erythroid cells of the “5q-mice” that lack one copy of the chromosomal region syntenic to the human 5q region that contains the RPS14 gene (Barlow et al., 2010). Thus, the cytopenias in some patients with MDS may be the result of defective ribosomal biosynthesis, leading to activation of p53 and excessive apoptosis. The nucleoporin gene NUP98 is fused to a number of different genes including HOXD13 by chromosomal translocations that are found in patients with MDS, AML and CML, blast crisis. Genetically engineered mice that express a NUP98-HOXD13 (NHD13) transgene (Tg) display the phenotypic features of MDS, including cytopenias, bone marrow dysplasia, and transformation to acute leukemia (Lin et al., 2005). We obtained these mice and analyzed the hematopoietic stem cells (HSC) and the erythroid compartment and found significantly decreased rps14 mRNA expression in the NHD13+ CD71+Ter119+ cells compared to the WT controls. Furthermore, flow cytometry analysis revealed increased intracellular p53 levels in the NHD13+ Lin−Sca-1+ c-Kit+ (LSK) and CD71+Ter119+ cells. These data suggest that defective rps14 ribosomal protein production and an increased p53 level may contribute to the anemic phenotype in NHD13+ Tg mice. To examine whether inhibition of p53 function can improve the cytopenias of NHD13+ Tg mice, mice were injected with Pifithrin-α (a reversible inhibitor of p53-mediated apoptosis and p53-dependent gene transcription, 2 mg/kg body weight) daily for five weeks. We observed partial rescue of the myeloid and lymphoid lineage differentiation defects, with no improvement in the hemoglobin level. To further investigate whether the presence or absence of p53 affects the MDS or AML phase of NHD13 driven disease, we generated NHD13+p53+/− and NHD13+p53−/− mice. Deletion of one allele of p53 rescues the myeloid progenitor cell compartment, while deletion of both alleles reversed the deficit in both the LSK and the MPP populations that was observed in the NHD13+ Tg mice. The clinically healthy NHD13+p53−/− mice (aged 3 to 5 months) and the NHD13+p53+/− mice (aged from 3 to 7 months) both had more severe leukopenia and anemia than the NHD13+ Tg mice or the WT controls (aged 4 to 7 months). 60% of the NHD13+p53−/− mice and the NHD13+p53+/− mice developed MDS with a median survival of 138 d and 190 d respectively; in contrast 30% of the NHD13+ Tg mice had MDS with a median survival of 236 d. These data indicate that the relative or absolute lack of p53 hastens the development of MDS, and shortens the median survival. Lack of one or two p53 alleles significantly accelerated the development of AML, which in NHD13+p53+/− mice resulting in a median survival of 278 d. However, AML in the NHD13+p53−/− mice is more undifferentiated with a median survival of 133 d, and the NHD13+ Tg mice showed AML with a median survival of 324 d. Taken together, these data demonstrated that the chronic loss one allele or two alleles of p53 does not rescue the MDS phenotype induced by NHD13 fusion gene. Rather it accelerates the development of NHD13 driven MDS and leukemia. Our studies suggest that targeting p53 transiently may temporally improve hematopoiesis in MDS, but over the long term has detrimental effects on hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Comparative Study of Hematopoietic Lineage Potential of Ips Cells Derived From Two Mesodermal Cell Lineages

Abstract Abstract 1285 Identifying the key regulatory genes that direct human ES/iPS cell differentiation to certain lineages is required before considering these cells for their potential clinical application. It has been proposed that the cellular starting material used to generate novel iPS cell lines imparts an epigenetic memory which in turn influences the iPS lines' potential to differentiate towards certain developmental lineages. To determine novel key regulators that direct ES/iPS cell differentiation to hematopoietic lineages, we decided to compare differentiation capacity of multiple iPS cell lines generated from two neonatal mesodermal tissue derived cell types. For this, we generated multiple iPS cell lines from umbilical cord blood derived endothelial cells and amniotic fluid derived mesenchymal stem cells, using an identical dedifferentiation system/protocol. Despite the similar germ layer origin of these iPS derived cell lines, these lines showed significant differences in blood generation efficiency and lineage specification capacity. From the 8 cord blood derived iPS cell lines we tested, the average efficiency of hematopoietic (CD45+) cell generation was 34.6+/−21.2% (range 9.8 to 65.8%), of which 5.8+/− 4% were CD45+CD34+ hematopoietic progenitors. From the 9 mesenchymal stem cell derived iPS lines, the average efficiency of CD45+ hematopoietic cells was 14.2+/− 9% (range 1.6 to 26.3%), of which 2.9+/− 1.9% were CD45+CD34+ hematopoietic progenitors (Figure 1A). Note, using our standardized iPS-2-Blood differentiation protocol the efficiency of blood differentiation for individual iPS cell lines was highly reproducible. These results indicate a significant increase in the efficiency of blood generation from the endothelial derived iPS cell lines compared to the mesenchymal stem cell derived iPS cell lines, and suggest that the close ontological relationship between hematopoietic cells and endothelial cells results in the increased efficiency of blood generation seen here. We further compared the iPS cell lines for their potential to differentiate into hematopoietic cells of the myeloid, erythroid, and lymphoid lineages. Both mesoderm lineage derived iPS cell lines showed myeloid and lymphoid lineage differentiation potential as evidenced by CFU assay for presence of macrophages and granulocytes, and by 4-week OP9, and OP9-DL1 culture assays showing CD19, CD10, CD34 expression for B cells, CD1a, CD7, CD5, CD4, TCRb, CD3 expression for T cells, and CD56, CD16 for NK cells. However, further comparative analysis revealed a limited erythroid (BFU-E) potential of cord blood derived iPS cell lines compared to amniotic fluid derived iPS cell lines (Figure 1B). Most amniotic fluid derived iPS lines showed significantly erythroid cell potential, where as the cord blood derived iPS lines showed significantly reduced or no erythroid cell potential. These results suggest that the endothelial origin of cord blood derived iPS lines imparts an epigenetic memory that results in the reduced efficiency of erythroid cell generation compared with mesenchymal stem cell derived iPS lines. The difference in the efficiency of hematopoietic cell generation and lineage specification between the cord blood and amniotic fluid derived iPS lines suggests that iPS reprogramming to a pluripotent state is not complete (despite iPS cell line validation of pluripotency via teratocarcinoma assay), and significant gene expression differences exist between the lines. We are currently performing gene expression and epigenetic profile analyses of these closely related mesodermal lines, and expect that the close ontological relationship of the two mesoderm derived cell types will facilitate the deconvolution of the molecular basis of the differences.Figure 1.Differences in blood differentiation efficiency and lineage potential of iPS lines derived from two ontologically related cell types.Figure 1. Differences in blood differentiation efficiency and lineage potential of iPS lines derived from two ontologically related cell types. Disclosures: No relevant conflicts of interest to declare.

GATA1 mutations Outside the Blast Cell Population in Transient Leukemia of Down Syndrome

Abstract Abstract 2562 Background. Transient leukemia (TL), a disorder of fetal hematopoiesis, occurs in approximately 10% of infants with Down syndrome and in one fifth of cases is followed by the development of acute myeloid leukemia. The diagnosis of TL is based on the detection of a spontaneously resolving population of blasts in the peripheral blood of newborns with DS and, more recently, the detection of clone-specific somatic mutations of the hematopoietic transcription factor GATA1. The alternative terms for this condition, transient myeloproliferative disorder (TMD) and transient abnormal myelopoiesis (TAM), reflect the observation that the peripheral blood of affected infants frequently shows an expansion of immature granulocytic precursor cells at various stages of development as well as nucleated erythroid precursors in addition to a population of blast cells. We hypothesized that the immature myeloid precursor cells observed in TL arise from the same hematopoietic cell clone that is defined by the specific GATA1 mutation found in TL blasts. Methods. Cryopreserved mononuclear cells from peripheral blood samples of three male infants with TL of Down syndrome were stained at a density of 106 cells/100ul with the following antibodies: CD45-Alexa Flour 700, CD61-PE, CD11b-PE-Cy5, CD235a-PE-Cy7, CD3-APC, CD34-APC-Cy7, CD15-Pacific Blue. In addition, aliquots of these cell populations were fixed, permeabilized and stained with for intracellular myeloperoxidase (MPO-FITC). Fractions of cells corresponding to different hematopoietic lineages were prepared by fluorescence activated cell sorting (FACSAriaII, BD Biosciences, San Jose, CA) both as bulk populations and single cells (sorted into 96-well plates). Morphology of sorted cell populations was determined by Giemsa staining. Lineage-specific hematopoietic colonies were cultured from primary mononuclear cell samples in a standard CFC assay. DNA of sorted bulk and single cell populations as well as individual lineage-specific hematopoietic colonies was used to detect GATA1 mutations in exon 2 by PCR, restriction enzyme analysis or sequencing of PCR products, or nested PCR. The location of the GATA1 gene on the X chromosome and the use of samples derived from males meant that wild type GATA1 alleles were not derived from TL cells. Results. Primary TL blood samples could be fractionated in a blast cell (CD45+CD34+and/or CD61+), erythroid (CD45-CD235a+), immature myeloid (CD45+CD15+MPO+) and lymphoid (CD45+CD3+) cell population. Cell morphology was consistent with this sorting strategy. The cell fraction designed to enrich TL blasts in all cases showed the presence of cells harboring only the mutant GATA1 allele indicating a high degree of purity of the blast fraction. As expected, the lymphoid cell populations contained in the CD3+ fractions exclusively harbored wild type alleles. Similarly, both sorted erythroid cell populations and individual erythroid colonies (Bfu-E) derived from unsorted input cells harbored only wild type GATA1 alleles. In contrast, CD15+ and/or MPO+ myeloid cell fractions consistently showed the presence of both mutant and wild type GATA1 alleles. Quantification by single cell PCR is in progress. Conclusion. Somatic GATA1 mutations which result in the translation of a N-terminally truncated mutant protein (GATA1s), are pathognomonic for TL and myeloid leukemia of Down syndrome. Published data do not determine if the cell clone harboring GATA1 mutations in TL extends beyond the population of blast cells and megakaryocytic lineage. We confirmed the presence of GATA1 mutations in TL blasts and their expected absence from lymphoid cell populations. The absence of mutations from cells of erythroid lineage suggests that the altered GATA1 function induced by mutations found in TL is incompatible with erythroid differentiation. The detection of clone-specific GATA1 mutations in myeloid cells expressing MPO was unexpected and raises the possibility that either the progenitor cells giving rise to TL blasts or the blasts themselves retain myeloid lineage differentiation potential. The finding lends support to definitions of TL and myeloid leukemia in Down syndrome that are based on the specific mutational and cellular mechanism and transcend a megakaryoblastic blast phenotype. Disclosures: No relevant conflicts of interest to declare.

Cutting Edge: IRF8 Regulates Bax Transcription In Vivo in Primary Myeloid Cells

A prominent phenotype of IRF8 knockout (KO) mice is the uncontrolled expansion of immature myeloid cells. The molecular mechanism underlying this myeloproliferative syndrome is still elusive. In this study, we observed that Bax expression level is low in bone marrow preginitor cells and increases dramatically in primary myeloid cells in wt mice. In contrast, Bax expression level remained at a low level in primarymyeloid cells in IRF8 KO mice. However, in vitro IRF8 KO bone marrow-differentiated myeloid cells expressed Bax at a level as high as that in wild type myeloid cells. Furthermore, we demonstrated that IRF8 specifically binds to the Bax promoter region in primary myeloid cells. Functional analysis indicated that IRF8 deficiency results in increased resistance of the primary myeloid cells to Fas-mediated apoptosis. Our findings show that IRF8 directly regulates Bax transcription in vivo, but not in vitro during myeloid cell lineage differentiation.

MicroRNAs regulatory networks in myeloid lineage development and differentiation: regulators of the regulators

MicroRNAs (miRNAs) are abundant small molecules of ≈ 22 nucleotides that reprogram gene expression by targeting mRNA degradation and translation disruption. An emerging concept implicates miRNA coupling with transcription factors in myeloid-based development of dendritic cells, monocytes and granulocytes, as well as function as mature cells and contributors to host defense and inflammation. Here, we review this new and important interactive circuitry and how it contributes to reprogramming cell phenotypic responses associated with mature immune competency.

Abstract 4330: Endometrial hyperplasia resulting from myeloid-specific targeting of the tumor suppressor APC

Abstract The Adenomatous polyposis coli (APC1) tumor suppressor is a multi-function protein with a central role in Wnt-regulated cell growth and differentiation. Additionally, APC functions to modulate microtubule and microfilament dynamics to establish polarity in epithelia during directed cell migration and asymmetric cell division. APC frequently collaborates with the canonical Diaphanous-related formin, mDia1 (DIAPH1), which also binds microfilaments and microtubules to modulate cytoskeletal dynamics. APC1 and DIAPH1 are genetically linked on chromosome 5q in a region frequently disrupted in myeloproliferative neoplasms (MPNs). mDia1 knockout mice develop an age-dependent outgrowth of myeloid-lineage cells. The physical and functional linkage of APC and mDia1 as 5q tumor suppressors and in cytoskeletal remodeling led us to explore the role of APC in tumor suppression in myeloid-derived cell lineages affected by mDia1 knockout. To test this hypothesis in a genetic model, we created a mouse in which APC was knocked out in myeloid lineage cells by breeding the Apc-flox mice with mice expressing Cre recombinase under the control of lysozyme M (LyzsM) gene promoter. The resulting phenotype was unexpected and 100% penetrant: Female mice developed cystic endometrial hyperplasia and were unable to sustain pregnancy. Embryo implantation occurred, but embryos were resorbed mid-gestation irrespective of progeny genotype. Two hypothesized mechanisms to explain the initiation of endometrial hyperplasia are being pursued. The first hypothesis is that loss of APC impairs the homeostasis between growth and differentiation of myeloid lineage stem/progenitor cells that transdifferentiate into uterine epithelia. An alternate hypothesis is that loss of APC expression affects the inflammatory response of resident uterine macrophages that lead to defects in epithelial morphology. In either case, these genetic studies imply that APC gene defects in myeloid lineage cells may be an initiating event in endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4330. doi:10.1158/1538-7445.AM2011-4330

Hematopoietic stem cells undergo an IFNγ-dependent, MyD88-independent transition from dormancy to activity during acute bacterial infection (153.8)

Abstract How hematopoietic stem cells (HSCs) respond to and recover from immunologic stress is not well-understood. We demonstrate that infection with the intracellular bacteria, Ehrlichia muris, induces a transient expansion of bone marrow Lin-negative Sca-1+ cKit+ (LSK) progenitor cells. As inflammation can modulate the expression of surface markers used to phenotype stem and progenitor cells, we addressed whether the phenotypic changes correlated with functional changes in the progenitor cell population. The expansion of LSK cells was associated with a loss of dormant, long-term repopulating HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation. The infection-induced changes required IFNγ signaling, and were accompanied by an expansion of more differentiated multipotent progenitor cells. IFNγ was required for the infection-induced loss of functionally-defined dormant HSCs in label retaining-cell assays. In competitive reconstitution experiments, LSK cells purified from infected IFNγR-deficient mice preferentially engrafted, relative to LSK cells purified from infected wild type mice. Furthermore, the infection-induced changes in progenitor cells were independent of intrinsic MyD88-signaling. We propose that IFNγ elicited during acute infection, provides a signal to dormant HSCs to undergo a rapid transition from dormancy to activity, ostensibly, to provide the host with additional or better-armed innate cells for host defense.

Toll-Like Receptors 2 and 4 Mediate the Capacity of Human Bone Marrow-Derived Mesenchymal Stem Cells to Support the Proliferation and Differentiation of CD34+ Cells In a Non-Synergistic Way.

Abstract Abstract 3850 Human bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent nonhematopoietic progenitor cells, which can differentiate into osteoblasts, adipocytes, chondrocytes and other tissues. The most important function of BM-MSCs is to support hematopoiesis. Toll-like receptors (TLRs) are a conserved family of receptors that can be activated by both pathogen components and mammalian endogenous molecules such as heat-shock proteins and extracellular matrix breakdown products. In the past a few year, several studies reported that TLRs are expressed in hematopoietic and non-hematopoietic to modulate their biological functions. We hypothesized that MSCs are equipped with TLRs that enable them to dynamically change hematopoiesis-related cytokines expression pattern and level by sensing correspondent agonists, thus efficiently supporting hematopoiesis. In this study, BM-MSCs were analyzed for mRNA expression of TLR 1–9 by reverse transcription-polymerase chain reaction. TLR 1–6, but not TLR 7–9 were expressed by MSCs. The expression of TLR2 and TLR4 was also confirmed by flow cytometic assay. We further explored the role of TLR2 and TLR4 in mediating the capacity of MSC to support the proliferation and differentiation of CD34+ cells. The pre-stimulation with TLR2 agonists (Pam3Cys) or TLR4 agonists (LPS) enable MSCs to enhance CD34+ cells proliferation and promote CD34+ cells differentiation towards the myeloid lineage (CD33+, CD11b+), as well as granulocyte colony formation by those cells. The production of interleukin 8 (IL-8), IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF were also increased by stimulated MSCs. Interestingly, although Pam3Cys and LPS displayed different inductive magnitudes, they have no synergistic effect on MSCs. We hypothesized there may be some antagonistic effect between TLR2 and TLR4 intracellular signal conductive pathway, or they can downregulate the expressive level of the TLRs on MSCs. Together, our findings suggest that TLR2 and TLR4 signalings may indirectly regulate hematopoiesis by modulating MSCs' functions. The increased haemopoietic proliferation and myeloid lineage differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production. Disclosures: Wang: National Natural Science Foundation (30700329): Research Funding; Anhui Provincial Outstanding Young Investigator Program (08040106810): Research Funding; Fund of Anhui Provincial “115” Industrial Innovation Program: Research Funding.

Intermediate Steps In Erythroid, Megakaryocytic and Myeloid Lineage Specification

Abstract Abstract 4778 Normal human CD34 positive precursor cells can be expanded in vitro and converted with high efficiency into maturing erythroid cells or myeloid cells by treating the cells with erythropoietin (EPO) or with G-CSF, respectively. The maturation of megakaryocytic cells can also be induced from human CD34 positive precursor cells by treatment with thrombopoietin (TPO) with lower efficiency. The induction time varies from 9 to 12 days depending on the lineages. Total RNAs were extracted every other day during the induction period for the purpose of gene profiling at intermediate steps of the differentiation. The samples were analyzed with the “Whole Transcriptome Shotgun Sequencing (RNA-seq)” method. Comparison of the gene expression among these three lineages at different stages of differentiation shows complex changes of a substantial number of genes in categories including transcription factors, cytokines etc. Basically, many genes change similarly in both erythroid and megakaryocytic lineage differentiation, the number of this shared genes are much less when comparing myeloid cells with one of these lineages. Initially during erythropoietin treatment, a large number of megakaryocyte mRNAs are upregulated to levels comparable to those of the erythroid specific genes, or to those seen in precursor cells treated with thrombopoietin. After several days of erythropoietin treatment, most of the megakryocyte related genes are down regulated, while there is continued upregulation of erythroid genes. Unlike many other megakaryocytic genes, the level of mRNA for the thrombopoietin receptor MPL is rapidly down-regulated suggesting that erythroid lineage commitment occurs prior to silencing of many megakaryocytic genes. Comparison of the linage specific genes identified groups of cell surface proteins which may be applied as new markers for sorting various cell populations. Some of these include DARC, LEPR, TFRC, BMPR2, TFR2, AXL, EPHB4, IL15RA, TNFRSF19, FZD5, ULBP1, CELSR2, RYK, MRGPRE, CRY1 which are specific for erythroid lineage; FPR1, EVI2A, CD14, and others totaling 50 proteins for myeloid lineage and more than 80 highly expressed surface markers such as GP1BA, F2RL3, GP1BB, ADRA2A, GABRE unique for megakaryocytic lineage. In parallel, proteomic analysis was carried out with an Isotope coded affinity tag (ICAT)-based protein profiling and Multiplexed Isobaric Tagging Technology (iTRAQ™). Statistical analysis shows good correlation between the relative changes of RNA and protein expression levels during the erythropoiesis, although there were large discrepancies in the relative levels of mRNA and protein at any one time point. Only those genes with very low or very high RNA values show the poor correlations. CTCF and its associated factor Rad21 are involved in the establishment of enhancer boundary elements and chromosomal conformation. We have begun genome wide analysis of these factors during erythroid and myeloid cell growth and differentiation. In this project, we also aimed to investigate the modulation mechanisms of CTCF during the CD34 positive cells differentiation into different lineages. Taking the advantage of the ChIP-Sequencing (ChIP-Seq) technology, we found that progressive changes in the sites of binding of the DNA binding protein CTCF occurs, and are not limited to the regions embedding erythroid genes, suggesting that there is a lineage specific global reorganization of chromatin. In addition to transcriptional controls, there are prominent post-transcriptional effects regulating the expression of key transcription factors including CTCF. Through this study, the regulation mechanisms of the chromatin remodeling, signal pathways and other, molecular events during hematopoiesis will be described in detail. Disclosures: No relevant conflicts of interest to declare.

Vitamin K2 Modulates Differentiation and Apoptosis of Both Myeloid and Erythroid Lineages

Abstract Abstract 3993 Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis eventually leading to maturation arrest and leukemic transformation. It is well-known that VK2 induces differentiation and apoptosis in acute myeloid leukemia (AML) cell lines such as HL-60 and U937. Based on the studies of AML cell lines, several clinical trials of VK2 therapy for MDS patients have been conducted, and they showed improvement of cytopenia and reductions in blastic cells. Interestingly, hematological improvement was also observed in MDS patients with low percentage of blasts, and a differentiation/apoptosis-inducing effect on blasts alone could not explain this fact. Thus, the effects of VK2 on primary hematopoietic progenitors were examined from the perspective of differentiation and apoptosis. Mobilized CD34-positive cells from peripheral blood were used for the examination of myeloid lineage cells, and were cultured in IMDM containing 20% FCS, 20 ng/mL rhSCF, 20 ng/mL rhIL-3, with or without VK2. VK2 induced significant increase of CD11b-positive cells on day 4 (35.8% ± 12.3% with 10 μM VK2 vs. 10.7% ± 1.9% without VK2, P=0.0034) and day 6 (42.7% ± 6.3% with 10 μM VK2 vs. 24.1% ± 8.6% without VK2, P=0.0235). CD14-positive cells also increased significantly on day 4 (8.0% ± 0.3% with 10 μM VK2 vs. 4.1% ± 1.5% without VK2, P=0.008). Furthermore, after treatment with VK2, mRNA expression levels of both C/EBPα and PU.1 were elevated in a dose-dependent manner, and a significant increase was shown at 10 μM of VK2 on day 6. These results indicate that VK2 promotes the differentiation of myeloid progenitors through the upregulation of transcriptional factors C/EBPα and PU.1. The effect of VK2 on the apoptosis of myeloid progenitors was also examined. VK2 increased the number of apoptotic cells determined by Annexin V assay transiently on day 4 (58.9% ± 6.3% with 10 μM VK2 vs. 36.1% ± 2.8% without VK2, P<0.0001), but no significant increase was found on day 6. Next, human erythroid colony forming cells (ECFCs) purified from peripheral blood were used for the examination of erythroid lineage cells. ECFCs were cultured in IMDM containing 15% FCS, 15% human AB serum, 2 U/ml rhEPO, 20 ng/mL rhSCF, 10 ng/mL rhIL-3 (depleted on day 3), with or without VK2 (added on day 3). In ECFCs, VK2 did not affect the expressions of transferrin receptor (TfR) and glycophorin A (GPA) or the expression level of β-globin mRNA. However, the expression of GATA-1 mRNA increased significantly on day 7 with 10 μM of VK2. VK2 seems to have the potential to promote the differentiation of ECFCs through the upregulation of transcriptional factor GATA-1, although this differentiating effect on ECFCs was much smaller than that on myeloid progenitors. Furthermore, VK2 exhibited an anti-apoptotic effect on day 7 ECFCs under erythropoietin (EPO) -depletion. The percentage of apoptotic cells after 24 hours of EPO-depletion, which was determined by Annexin V-positivity, was significantly reduced with VK2 at low concentrations (0.5-2 μM) (76.9% ± 4.7% with 1 μM VK2 vs. 88.3% ± 1.7% without VK2, P=0.0019). VK2 lost its anti-apoptotic effect at concentrations greater than 5 μM. This anti-apoptotic effect was not shown in erythroleukemic cell line AS-E2. Finally, the expression of steroid and xenobiotic receptor (SXR), which was recently identified as a receptor of VK2, on primary hematopoietic cells was examined. SXR was expressed on myeloid progenitors, but not on erythroid progenitors. SXR agonist rifampicin also upregulated the expressions of CD11b and CD14 on myeloid progenitors. The differentiation-promoting effect of VK2 on myeloid progenitors seems to be mediated partly through SXR signaling. These results indicate that the effect of VK2 varies by cell type. The major effect on myeloid progenitors was promoting differentiation, whereas its anti-apoptotic effect seemed to be dominant in erythroid progenitors. Although the detailed mechanism of VK2's effect on differentiation or apoptosis of hematopoietic progenitors remains unknown, the effect of VK2 therapy in MDS patients could be partly explained by these mechanisms. Disclosures: No relevant conflicts of interest to declare.

MARROW of Essential Thrombocythemia PATIENTS IS ENRICHED IN Effector Memory CD4+ and CD4+CD25++ Lymphocytes as Compared to BLOOD

Abstract Abstract 5058 The role of T cells residing in the marrow is poorly understood. However, some literature data suggest that the presence of CD8+ lymphocytes in the marrow plays a role in keeping the tumor cells in a dormant state. In chronic myeloid leukemia a significant role of lymphocytes in controlling the malignant clones is well documented. The aim of this study was to define whether there is a specific enrichment of some CD4+ lymphocytes subpopulations in the marrows of newly diagnosed patents with essential thrombocythemia (ET). Seven patients (F/M: 6/1, age 28–72 - median 6 yrs) with ET diagnosed according to the WHO criteria were studied. They had platelets 425–809 ×10∧3/ul (median 676 × 10∧3), NAP 74–138 score (median 112 score), 6 of them were positive for JAK 2 V617F mutation (heterozygotes). Marrow cellularity was from 18 – 64 ×10∧3/ul (median 44 × 10∧3/ul) Cytological evaluation revealed normal differentiation of erythroid and myeloid lineages, in contrast an increased number of megakaryocytes often enlarged, hyperlobulated and in clusters was seen in all patients. Trephine biopsy revealed activation of megakaryopoiesis with enlarged numerous megakaryocytes with normal representation of other lineages and a lack of an increase in the reticulin fibers. Lymphocytes in the marrows were in proportions from 4.5 – 15,7% (median 10,6%) what resulted with lymphocytosis from 2640/ul to 19405/ul (median 5645/ul). The whole populations of marrow and blood cells were labeled for a purpose of this study with the use CD4, CD25, CD45RO and CCR7 MoAb (Becton Dickinson, San Jose, CA, USA) followed by detection and analysis with the use of flow cytometry (FACS Calibur, Becton Dickinson) equipped with PCLysis programe. We found:Proportions of lymphocytes in the marrows were rather in a normal value range but in numbers they prevailed over blood lymphocytes (2640-19405/ul, median 5645/ul vs 1300–3100/ul, median 1800/ul, p= 0,003 Wilcoxon Test for pairs),Among T cells subpopulations effector/memory CD4+ lymphocytes CD45RO+, CCR7-) were significantly higher in numbers in the marrows of ET patients as compared to blood (744-3241/ul, median 1126/ul vs 406–2437/ul, median 592/ul, p=0,04 Wilcoxon Test for pairs). There was no difference when numbers of naïve (CD45Ro-, CCR7+) and central memory (CD45RO+, CCR7+) CD4+ lymphocytes were analyzed.The marrows of ET patients had higher numbers of CD4+CD25++ lymphocytes as compared to blood (8,1-40,7/ul, median 11,4/ul vs 1,2-14,8/ul, median 7,3/ul; p=0,02 Wilcoxon Test for pairs). It appeared that in ET patients there was a selective enrichment of marrow lymphocyte populations in CD4+EM cells and CD4+CD25++ lymphocytes what strongly suggests the presence of active immune response in the marrows affected by this myeloproliferative disorder. Disclosures: No relevant conflicts of interest to declare.

Interleukin-34 is expressed by giant cell tumours of bone and plays a key role in rankl-induced osteoclastogenesis

Interleukin-34 (IL-34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage-colony stimulating factor receptor (M-CSF receptor, c-fms). M-CSF, the main ligand for c-fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M-CSF in osteoclastogenesis and GCTs, the expression of IL-34 in human GCTs was first assessed. Quantitative analysis of IL-34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M-CSF and c-fms. Immunohistochemistry demonstrated that osteoclast-like cells exhibited a huge immunostaining for IL-34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone-resorbing osteoclasts showed very strong staining for IL-34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL-34 in osteoclastogenesis was then studied in murine and human models. IL-34 was able to support RANKL-induced osteoclastogenesis in the absence of M-CSF in all models. Multinucleated cells generated in the presence of IL-34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL-34 induced phosphorylation of ERK 1/2 and Akt through the activation of c-fms, as revealed by the inhibition of signalling by a specific c-fms tyrosine kinase inhibitor. Furthermore, IL-34 stimulated RANKL-induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL-34 can be entirely substituted for M-CSF in RANKL-induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs.

IFNgamma Induces Expansion of Hematopoietic Progenitor Cells (36.9)

Abstract The balance between type 1 and type 2 helper T (Th1 and Th2) cells is critical for homeostasis of the immune system, since their distinct cytokine production profile directly determines the activation and functions of other immune cells. However, as the end-differentiated lineage cells, how these effector T cells influence the hematopoietic progenitor cells, is largely unknown. Here we showed that Th1 cell products, but not those of Th2 cells, caused a remarkable expansion o f lineage-Sca-1+C-kit+ (LSK) cells. IFNgamma was found to play a major role in this expansion by activating the expression of Sca-1 in lineage-Sca-1-C-kit+ cells, thus converting them into LSK cells. This process was dependent on IFNgammaR1 signaling, but only partially mediated by STAT1 pathway. Furthermore, such IFNgamma-induced LSK cells had a significantly higher colony forming units (CFUs), compared to control lineage-Sca-1-C-kit+ cells and control LSK cells, which might compensate for the loss of progenitor cells caused by the IFNgamma-treatment. Moreover, the IFNgamma-induced expanded LSK cells were verified as functional stem/progenitor cells through the competitive repopulation experiments in vivo. Finally, the IFNgamma-induced hematopoiesis was more biased toward the differentiation of myeloid lineages in vitro and in vivo. Therefore, our findings demonstrated a novel role of IFNgamma in hematopoiesis, thus providing a new insight into the clinical application of IFNgamma.