WASH is required for the differentiation commitment of hematopoietic stem cells in a c-Myc–dependent manner

Hematopoiesis is fully dependent on hematopoietic stem cells (HSCs) that possess the capacity to self-renew and differentiate into all blood cell lineages. WASH, Wiskott–Aldrich syndrome protein (WASP) and SCAR homologue (WASH) is involved in endosomal sorting as an actin-nucleating protein. Here, we show that conditional WASH deletion in the hematopoietic system causes defective blood production of the host, leading to severe cytopenia and rapid anemia. WASH deficiency causes the accumulation of long-term (LT)-HSCs in bone marrow and perturbs their differentiation potential to mature blood lineages. Importantly, WASH is located in the nucleus of LT-HSCs and associates with the nucleosome remodeling factor (NURF) complex. WASH assists the NURF complex to the promoter of c-Myc gene through its VCA domain-dependent nuclear actin nucleation. WASH deletion suppresses the transcriptional activation of c-Myc gene and impairs the differentiation of LT-HSCs. WASH acts as an upstream regulator to modulate c-Myc transcription for hematopoietic regulation.

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Inhibition of PDGFRβ by Imatinib Mesylate Suppresses Proliferation and Alters Differentiation of Human Mesenchymal Stem Cells In Vitro.

Blood ◽

10.1182/blood.v108.11.2563.2563 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2563-2563

Author(s):

Fernando Fierro ◽

Thomas Illmer ◽

Duhoui Jing ◽

Philip Le Coutre ◽

Gerhard Ehninger ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Tyrosine Kinase ◽

Hematopoietic Stem Cells ◽

Imatinib Mesylate ◽

Dependent Manner ◽

Differentiation Potential ◽

Hematopoietic Stem

Abstract Recent data show that the tyrosine kinase inhibitor Imatinib mesylate (IM) also affects normal hematopoietic stem cells (HSC), T lymphocyte activation and dendritic cell function not relying on the specific inhibition of bcr-abl activity. Mesenchymal stem cells (MSC) have been identified in the bone marrow (BM) as multipotent non-hematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of visceral mesoderm. MSC interact with HSC, influencing their homing and differentiation through cell-cell contact and the production of factors including chemokines We evaluated possible effects of IM in vitro on human bone marrow-derived MSC. Screening the activity of fourty-two receptor tyrosine kinases by a phospho-receptor tyrosine kinase (RTK)-array revealed an exclusive inhibition of platelet-derived growth factor receptor (PDGFRβ) by IM which consequently affects downstream targets of PDGFRβ as Akt and Erk1/2 signalling pathways in a concentration and time dependent manner. Furthermore, perinuclear multivesicular bodies harbouring PDGFRβ were found within 18–20 hours culture of MSC in the presence of 5 μM IM. Cell proliferation and clonogenicity (evaluated as the capability to form colony forming units - fibroblasts (CFU-F)) of MSC were significantly inhibited by IM in a concentration dependent fashion. IM inhibits significantly the differentiation process of MSC into osteoblasts as evaluated by decreased alkaline phosphatase activity and reduced calcium phosphate precipitates. In contrary, differentiation of MSC into adipocytes was strongly favoured in presence of IM. All these functional deficits described, probably contribute to an observed 50% reduction in the support of clonogenic hematopoietic stem cells, as evaluated by a long term culture-initiating cells (LTC-IC)-based assay. In summary our experiments show that IM inhibits the capacity of human MSC to proliferate and to differentiate into the osteogenic lineage, favouring adipogenesis. This effect is mainly mediated by an inhibition of PDGFRβ autophosphorylation leading to a more pronounced inhibition of PI3K/Akt compared to Erk1/2 signalling. This work confirms the role of PDGFRβ recently described for the proliferation and differentiation potential of MSC and provides a first possible explanation for the altered bone metabolism found in certain patients treated with IM.

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The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells

Genes & Development ◽

10.1101/gad.227496.113 ◽

2014 ◽

Vol 28 (6) ◽

pp. 576-593 ◽

Cited By ~ 35

Author(s):

D. M. Gerhardt ◽

K. V. Pajcini ◽

T. D'altri ◽

L. Tu ◽

R. Jain ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Transcriptional Activation ◽

Activation Domain ◽

Hematopoietic Stem ◽

Transcriptional Activation Domain ◽

Notch1 Signaling

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Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways.

Blood ◽

10.1182/blood.v120.21.2309.2309 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2309-2309

Author(s):

Jian Huang ◽

Peter S. Klein

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Signaling Pathways ◽

Glycogen Synthase ◽

Dependent Manner ◽

Mtor Inhibition ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 2309 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.

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Evidence for a Novel Blood Lineage Commitment Pathway from Lympho-Myeloid Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v106.11.802.802 ◽

2005 ◽

Vol 106 (11) ◽

pp. 802-802 ◽

Cited By ~ 1

Author(s):

Sten Eirik W. Jacobsen ◽

Robert Mansson ◽

Anne Hultquist ◽

Mikael Sigvardsson ◽

Natalija Buza-Vidas ◽

...

Keyword(s):

Stem Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Single Cell ◽

Lineage Commitment ◽

Mouse Bone Marrow ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Stage Of Development ◽

Number Of Genes

Abstract We recently identified a novel Lin−Sca-1+c-kithiCD34+Flt3hi (LSKCD34+Flt3hi) lymphoid-primed multipotent progenitor (LMPP) in adult mouse bone marrow which, although possessing a combined lymphoid (B and T cell) and myeloid (granulocyte-monocyte; GM) differentiation potential, have little or no ability to adopt erythroid (E) and megakaryocyte (MK) lineage fates (Adolfsson et al, Cell121:295, 2005). The identification of this lineage restricted lymphomyeloid progenitor implicates the existence of alternative roadmaps for lineage commitment of pluripotent hematopoietic stem cells (HSCs), distinct from the classical model suggesting that the first HSC commitment step results in a strict separation into common lymphoid and myeloid progenitors. Herein we provide further, genetic evidence for such a model. Affymetrix global gene profiling, quantitative PCR, and multiplex single cell PCR analysis of LSKCD34−Flt3− long-term (LT)-HSCs, LSKCD34+Flt3− short-term (ST)-HSCs and LSKCD34+Flt3hi LMPPs, demonstrate that LMPPs in contrast to LT-HSCs and ST-HSCs down-regulate or turn off a number of genes critically involved in MkE lineage development, including GATA-1 and the receptors for erythropoietin and thrombopoietin. In contrast, a number of genes specific for early lymphoid development, including Rag-1, sterile Ig and IL-7 receptor are upregulated in LMPPs but absent in LT-HSCs and ST-HSCs. Importantly, within the LMPP, these lymphoid genes are typically co-expressed with a number of GM associated genes such as G-CSF receptor and MPO, but virtually never co-expressed with MkE associated genes. Investigating fetal liver day 14.5 we also provide evidence for existence of the LSKCD34+Flt3hi LMPPs at this early stage of development, and using a single cell clonal assay promoting combined B, T and myeloid lineage development, we demonstrate that a large fraction of fetal LMPPs lacking MkE potential possess a combined GM, B and T cell potential. Thus, evaluation at the single cell level of combined lineage potentials and multilineage gene expression provide compelling evidence for lymphoid-priming within the HSC compartment being preceeded by a loss of MkE potential, but occurring prior to loss of GM potential.

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Identification and Characterization of Osteoblastic Niche Cells That Regulate Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v110.11.1412.1412 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1412-1412

Author(s):

Yuka Nakamura ◽

Fumio Arai ◽

Yumiko Gomei ◽

Kentarou Hosokawa ◽

Hiroki Yoshihara ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Expression Profiles ◽

Osteoblastic Cells ◽

Calcium Deposition ◽

Pcr Analysis ◽

Isolated Cells ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Osteoblastic Lineage

Abstract Currently, the niche for long-term hematopoietic stem cells (HSCs) is thought to consist conceptually of two parts: the endosteal surface (the osteoblastic niche) and a sinusoidal endothelium (the vascular niche), and a subset of osteoblasts functions as a key component of the hematopoietic stem cell niche. However, it is still unclear that the precise cellular and molecular contribution of osteoblastic cells on the HSC supportive microenvironment. In this study, we try to characterize the osteoblastic cells and investigate the property of osteoblastic niche cells. For isolation of osteoblastic cells, we treated the bone fragments of femur and tibiae with collagenase following flush-out of the bone marrow (BM). Non-hematopoietic and non-endothelial cells were then enriched by magnetic cell sorting of the CD45-CD31-Ter119- population, and expression of Sca-1 and platelet derived growth factor receptor α (PDGFRα) was analyzed. FACS analysis showed that CD45-CD31-TER119- cells were subdivided into three fractions: Sca-1+PDGFRα+, Sca-1-PDGFRα-, Sca-1-PDGFRα+. First we examined the multilineage differentiation potential of three populations. Although Sca-1- fractions efficiently differentiated into the osteoblastic lineage and showed calcium deposition, these cells hardly differentiated into adipocytes. In contrast to the Sca-1- cells, we found that Sca-1+PDGFRα+ cells can differentiate into osteoblastic and adipocytic lineages, suggesting that Sca-1+ cells have multi-potency. Next we examined the expression of osteoblastic marker expression by quantitative RT-PCR analysis, and found that Sca-1- populations expressed Runx2 and OB-cadherin. Alkaline phosphatase (ALP) staining of freshly isolated cells showed that Sca-1- fractions expressed ALP, while Sca-1+ cells did not express ALP. These data suggest that Sca-1- populations were the cell fractions, which were already committed to osteoblastic lineage. In addition, osteocalcin was expressed in PDGFRα+ fraction in Sca-1- cells, indicating that Sca-1-PDGFRα+ cells are more mature osteoblastic cells than Sca-1-PDGFRα-cells. Furthermore, N-cadherin was specifically expressed in Sca-1-PDGFRα+ cells, suggesting that N-cadherin was up-regulated with the maturation of osteoblastic cells. In addition, N-cadherin expression was up-regulated in Sca-1-PDGFRα+ cells with the postnatal development of BM. Interestingly, in the freshly isolated cells, we found that Sca-1+PDGFRα+ cells showed higher expression of Angiopoietin-1 (Ang-1), compared to Sca-1- fractions. Ang-1 expression was up-regulated in Sca-1-PDGFRα+ cells after over night incubation. Next we investigated the ability of these fractionated cells to support hematopoiesis. We examined the capacity of these fractionated cells on maintenance of colony formation ability of BM linage-Sca-1+c-Kit+ cells after 5 days of co-culture. Although CFU-C formation was supported Sca-1+PDGFRα+ cells, Sca-1-PDGFRα+ cells maintained CFU-Mix formation compared to the Sca-1+PDGFRα+ and Sca-1-PDGFRα-cells. From these data above, we hypothesize that multiple osteoblastic populations form a “niche complex” and collaborate with other supporting cells, such as CXCL12-abundant reticular (CAR) cells, to support HSCs, and that N-cadherin+ osteoblastic cells provide a foothold for anchoring of quiescent HSCs. Now we are investigating the gene expression profiles of these three populations and are tying to clarify the changes of characteristics of osteoblastic cells during postnatal BM development.

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Characterization of Histone Modifications on Lineage-Affiliated Genes During Hematopoietic Stem Cell Differentiation

Blood ◽

10.1182/blood.v116.21.4784.4784 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4784-4784

Author(s):

Chen Fangping ◽

Huarong Tang

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Histone Modifications ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Low Level ◽

Lineage Specific Genes

Abstract Abstract 4784 Hematopoietic stem cells (HSCs) are multipotent stem cells capable of self-renewal and multi-lineage differentiation. Though it has been shown that multiple factors take part in the maintenance of HSCs’ multipotency and differentiation potential, the mechanisms are unclear. Recent studies showed that histone modifications play an important role in maintenance of embryonic stem cells pluripotency and differentiation. To characterize the histone modification patterns of different lineages, HSCs were collected from umbilical cord blood and induced to differentiate to granulocytic, erythroid, and megakarytic in vitro. genes during HSC differentiation. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) technology was adopted to investigate the dynamic changes of histone modifications on lineage specific transcription factors and lineage–affiliated genes. Our results showed a certain level of H4 acetylation and H3 acetylation together with high level of H3K4me2 and low level of H3K4me3, H3K9me3 and H3K27me3 were present in lineage specific genes in CD34+CD38- HSCs. As CD34+CD38- cells differentiated, the modification level of acH3, acH4, H3K4me2, H3K9me3 and H3K27me3 on lineage specific genes remained the same, while H3K4me3 level increased greatly. In non-lineage specific genes, the acH3 and acH4 levels decreased, and H3K4me3 level remain at low level, while H3K9me3 and H3K27me3 levels increased. Thus, our data suggested that histone modifications played an important role in maintenaning the multipotency and differentiation capability of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure

Blood ◽

10.1182/blood.v124.21.sci-20.sci-20 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-20-SCI-20

Author(s):

Margaret A. Goodell

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Stem Cell Differentiation ◽

Primary Myelofibrosis ◽

Differentiation Potential ◽

Hematopoietic Stem

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development. Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Hematopoietic Stem Cells fromFancc−/−Mice Have Lower Growth and Differentiation Potential in Response to Growth Factors

Stem Cells ◽

10.1634/stemcells.20-5-438 ◽

2002 ◽

Vol 20 (5) ◽

pp. 438-447 ◽

Cited By ~ 11

Author(s):

Michel Aubé ◽

Matthieu Lafrance ◽

Chantal Charbonneau ◽

Isabelle Goulet ◽

Madeleine Carreau

Keyword(s):

Stem Cells ◽

Growth Factors ◽

Hematopoietic Stem Cells ◽

Lower Growth ◽

Differentiation Potential ◽

Hematopoietic Stem

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Age-Associated Characteristics of Murine Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.192.9.1273 ◽

2000 ◽

Vol 192 (9) ◽

pp. 1273-1280 ◽

Cited By ~ 477

Author(s):

Kazuhiro Sudo ◽

Hideo Ema ◽

Yohei Morita ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Lymphoid Cells ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Functional Changes

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34−/lowc-Kit+Sca-1+ lineage marker–negative (CD34−KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34−KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34−KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34−KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

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