Genetic barcoding systematically comparing genes in del(5q) MDS reveals a central role for CSNK1A1 in clonal expansion

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSC) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, a systematic comparison was carried out on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a pro-proliferative gene signature and HSCs a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. A crucial role for Csnk1a1 haploinsufficiency in the selective advantage of the 5q- HSC is therefore proposed. It is implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

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IL-6 blocks a discrete early step in lymphopoiesis

Blood ◽

10.1182/blood-2005-02-0456 ◽

2005 ◽

Vol 106 (3) ◽

pp. 879-885 ◽

Cited By ~ 48

Author(s):

Kazuhiko Maeda ◽

Yoshihiro Baba ◽

Yoshinori Nagai ◽

Kozo Miyazaki ◽

Alexander Malykhin ◽

...

Keyword(s):

Target Cells ◽

Hematopoietic Progenitors ◽

Early Step ◽

Hematopoietic Stem ◽

Lymphoid Lineage ◽

Src Homology 2 ◽

Multipotent Progenitors ◽

Src Homology ◽

Src Homology 2 Domain

Abstract Animals lacking Src homology 2 domain-containing inositol 5-phosphatase (SHIP) display a reduction in lymphopoiesis and a corresponding enhancement of myelopoiesis. These effects are mediated at least in part by elevated levels of interleukin 6 (IL-6). Here, we show the lymphopoiesis block in SHIP–/– mice is due to suppression of the lymphoid lineage choice by uncommitted progenitors. The suppression can be reproduced in vitro with recombinant IL-6, and IL-6 acts directly on hematopoietic progenitors. The block is partially overcome in SHIP–/– IL-6–/– double-deficient animals. IL-6 does not suppress but actually enhances proliferation of lymphoid-committed progenitors, indicating the IL-6 target cells are hematopoietic stem cells or multipotent progenitors. The findings suggest a mechanism for the lymphopenia that accompanies proinflammatory diseases.

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CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adult mice

Blood ◽

10.1182/blood-2010-04-277897 ◽

2011 ◽

Vol 117 (2) ◽

pp. 451-458 ◽

Cited By ~ 58

Author(s):

Mamiko Noda ◽

Yoshiki Omatsu ◽

Tatsuki Sugiyama ◽

Shinya Oishi ◽

Nobutaka Fujii ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Nk Cell ◽

Cell Development ◽

Hematopoietic Stem ◽

Adult Mice ◽

Multipotent Progenitors ◽

Chemokine Signaling ◽

Chemokine Ligand

Abstract Natural killer (NK) cells are granular lymphocytes that are generated from hematopoietic stem cells and play vital roles in the innate immune response against tumors and viral infection. Generation of NK cells is known to require several cytokines, including interleukin-15 (IL-15) and Fms-like tyrosine kinase 3 ligand, but not IL-2 or IL-7. Here we investigated the in vivo role of CXC chemokine ligand-12 (CXCL12) and its primary receptor CXCR4 in NK-cell development. The numbers of NK cells appeared normal in embryos lacking CXCL12 or CXCR4; however, the numbers of functional NK cells were severely reduced in the bone marrow, spleen, and peripheral blood from adult CXCR4 conditionally deficient mice compared with control animals, probably resulting from cell-intrinsic CXCR4 deficiency. In culture, CXCL12 enhanced the generation of NK cells from lymphoid-primed multipotent progenitors and immature NK cells. In the bone marrow, expression of IL-15 mRNA was considerably higher in CXCL12-abundant reticular (CAR) cells than in other marrow cells, and most NK cells were in contact with the processes of CAR cells. Thus, CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adults, and CAR cells might function as a niche for NK cells in bone marrow.

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Homeostasis and regeneration of the hematopoietic stem cell pool are altered in SHIP-deficient mice

Blood ◽

10.1182/blood-2002-12-3939 ◽

2003 ◽

Vol 102 (10) ◽

pp. 3541-3547 ◽

Cited By ~ 37

Author(s):

Cheryl D. Helgason ◽

Jennifer Antonchuk ◽

Caroline Bodner ◽

R. Keith Humphries

Keyword(s):

Negative Regulator ◽

Wild Type Littermate ◽

Hematopoietic Stem ◽

Cell Pool ◽

Multipotent Progenitors ◽

Important Negative Regulator ◽

Total Body ◽

Deficient Mice

AbstractSH2-containing inositol 5-phosphatase (SHIP) is an important negative regulator of cytokine and immune receptor signaling. SHIP-deficient mice have a number of hematopoietic perturbations, including enhanced cytokine responsiveness. Because cytokines play an important role in the maintenance/expansion of the primitive hematopoietic cell pool, we investigated the possibility that SHIP also regulates the properties of cells in these compartments. Primitive hematopoietic cells were evaluated in SHIP-deficient mice and wild-type littermate controls using the colony-forming unit-spleen (CFU-S) and competitive repopulating unit (CRU) assays for multipotent progenitors and long-term lympho-myeloid repopulating cells, respectively. Absence of SHIP was found to affect homeostasis of CFU-S and CRU compartments. Numbers of primitive cells were increased in extramedullary sites such as the spleen of SHIP-deficient mice, although total body numbers were not significantly changed. In vivo cell cycle status of the CRU compartment was further evaluated using 5-fluorouracil (5-FU). SHIP-deficient CRUs were more sensitive to 5-FU killing, indicating a higher proliferative cell fraction. More strikingly, SHIP was found to regulate the ability of primitive cells to regenerate in vivo, as CRU recovery was approximately 30-fold lower in mice that received transplants of SHIP-deficient cells compared with controls. These results support a major role for SHIP in modulating pathways important in homeostasis and regeneration of hematopoietic stem cells, and emphasize the importance of negative cytokine regulation at the earliest stages of hematopoiesis. (Blood. 2003;102:3541-3547)

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Discovering the drivers of clonal hematopoiesis

10.1101/2020.10.22.350140 ◽

2020 ◽

Cited By ~ 1

Author(s):

Oriol Pich ◽

Iker Reyes-Salazar ◽

Abel Gonzalez-Perez ◽

Nuria Lopez-Bigas

Keyword(s):

Positive Selection ◽

Molecular Mechanisms ◽

Somatic Mutations ◽

Cancer Genomics ◽

Variant Calling ◽

Selective Advantage ◽

Cancer Genes ◽

Driver Genes ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis

AbstractMutations in genes that confer a selective advantage to hematopoietic stem cells (HSCs) in certain conditions drive clonal hematopoiesis (CH). While some CH drivers have been identified experimentally or through epidemiological studies, the compendium of all genes able to drive CH upon mutations in HSCs is far from complete. We propose that identifying signals of positive selection in blood somatic mutations may be an effective way to identify CH driver genes, similarly as done to identify cancer genes. Using a reverse somatic variant calling approach, we repurposed whole-genome and whole-exome blood/tumor paired samples of more than 12,000 donors from two large cancer genomics cohorts to identify blood somatic mutations. The application of IntOGen, a robust driver discovery pipeline, to blood somatic mutations across both cohorts, and more than 24,000 targeted sequenced samples yielded a list of close to 70 genes with signals of positive selection in CH, available at http://www.intogen.org/ch. This approach recovers all known CH genes, and discovers novel candidates. Generating this compendium is an essential step to understand the molecular mechanisms of CH and to accurately detect individuals with CH to ascertain their risk to develop related diseases.

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Haploinsufficiency of Dnmt1 Impairs Leukemia Stem Cell Function Through Derepression of Bivalent Chromatin Domains,

Blood ◽

10.1182/blood.v118.21.3459.3459 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3459-3459

Author(s):

Jennifer J. Trowbridge ◽

Amit U. Sinha ◽

Scott A. Armstrong ◽

Stuart H. Orkin

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Dna Methyltransferase ◽

Cell Function ◽

Gene Signature ◽

Conditional Knockout ◽

Specific Gene ◽

Epigenetic Mechanisms ◽

Chromatin Domains ◽

Hematopoietic Stem

Abstract Abstract 3459 Leukemia stem cells (LSCs) are an attractive target in treatment of many types of blood cancers. There remains an incomplete understanding of the epigenetic mechanisms driving LSC formation and maintenance, and how this compares to the epigenetic regulation of normal hematopoietic stem cells (HSCs). One of the major epigenetic modifications, DNA methylation, is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a and Dnmt3b. We observed decreased expression of Dnmt3a and Dnmt3b in LSCs isolated from a model of MLL-AF9-induced acute myeloid leukemia (AML) compared to normal HSCs. In contrast, expression of Dnmt1 was maintained in LSCs compared to HSCs, suggesting that Dnmt1 may have a critical function in the formation and maintenance of LSCs. Supporting this hypothesis, we found that conditional knockout of Dnmt1 fully ablates the development of AML. Furthermore, haploinsufficiency of Dnmt1 (Dnmt1fl/+ Mx-Cre) was sufficient to delay progression of leukemogenesis and impair LSC self-renewal. Strikingly, haploinsufficiency of Dnmt1 did not functionally alter normal hematopoiesis or HSCs, suggesting an enhanced dependence of LSCs on DNA methylation. Mechanistically, we observed that haploinsufficiency of Dnmt1 in LSCs resulted in derepression of genes that had been silenced by MLL-AF9-mediated transformation and marked by bivalent H3K27me3/H3K4me3 chromatin domains. These results suggest that the formation and maintenance of LSCs depends not only upon activation of a leukemogenic program, but also upon silencing of a specific gene signature that is active in HSCs through crosstalk between two epigenetic mechanisms, polycomb-mediated repression and DNA methylation-mediated repression. This silenced gene signature includes known and candidate tumor suppressor genes as well as genes involved in lineage restriction. These studies present evidence that distinct epigenetic regulatory mechanisms are dominant in LSCs compared to HSCs and provide novel gene candidates for targeted reactivation in AML therapy. Disclosures: Armstrong: Epizyme: Consultancy.

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Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽

10.1182/blood-2020-137426 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-31

Author(s):

Maria Rosa Lidonnici ◽

Giulia Chianella ◽

Francesca Tiboni ◽

Matteo Barcella ◽

Ivan Merelli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Gene Set Enrichment Analysis ◽

Lineage Commitment ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Hematopoietic Stem ◽

Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

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A highly efficient reporter system for identifying and characterizing in vitro expanded hematopoietic stem cells

10.1101/2021.06.18.448972 ◽

2021 ◽

Author(s):

James Lok Chi Che ◽

Daniel Bode ◽

Iwo Kucinski ◽

Alyssa H Cull ◽

Fiona Bain ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Gene Expression Signature ◽

The Body ◽

Molecular Profile ◽

Functional Screening ◽

Hematopoietic Stem ◽

Wide Range ◽

Single Clone

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

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Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽

10.1182/blood.v96.2.719 ◽

2000 ◽

Vol 96 (2) ◽

pp. 719-726 ◽

Cited By ~ 397

Author(s):

Nicole Faust ◽

Florencio Varas ◽

Louise M. Kelly ◽

Susanne Heck ◽

Thomas Graf

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Time Lapse ◽

Neutrophil Granulocytes ◽

Egfp Gene ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Myelomonocytic Cells ◽

Green Fluorescent

Abstract Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

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Infused wild-type macrophages reside and self-renew in the liver to rescue the hemolysis and anemia of Hmox1-deficient mice

Blood Advances ◽

10.1182/bloodadvances.2018019737 ◽

2018 ◽

Vol 2 (20) ◽

pp. 2732-2743 ◽

Cited By ~ 4

Author(s):

Ki Soon Kim ◽

De-Liang Zhang ◽

Gennadiy Kovtunovych ◽

Manik C. Ghosh ◽

Hayden Ollivierre ◽

...

Keyword(s):

Stress Responses ◽

Tissue Damage ◽

Iron Homeostasis ◽

Microcytic Anemia ◽

Heme Oxygenase 1 ◽

Wild Type ◽

Intravascular Hemolysis ◽

Hematopoietic Stem ◽

Inflammatory Stress

AbstractHeme oxygenase 1 (HMOX1), the inducible enzyme that catabolizes the degradation of heme into biliverdin, iron, and carbon monoxide, plays an essential role in the clearance of senescent and damaged red blood cells, systemic iron homeostasis, erythropoiesis, vascular hemostasis, and oxidative and inflammatory stress responses. In humans, HMOX1 deficiency causes a rare and lethal disease, characterized by severe anemia, intravascular hemolysis, as well as vascular and tissue damage. Hmox1 knockout (KO) mice recapitulated the phenotypes of HMOX1-deficiency patients and could be rescued by bone marrow (BM) transplantation that engrafted donor’s hematopoietic stem cells into the recipient animals after myeloablation. To find better therapy and elucidate the contribution of macrophages to the pathogenesis of HMOX1-deficiency disease, we infused wild-type (WT) macrophages into Hmox1 KO mice. Results showed that WT macrophages engrafted and proliferated in the livers of Hmox1 KO mice, which corrected the microcytic anemia, rescued the intravascular hemolysis, restored iron homeostasis, eliminated kidney iron overload and tissue damage, and provided long-term protection. These results showed that a single macrophage infusion delivered a long-term curative effect in Hmox1 KO mice, obviating the need for BM transplantation, and suggested that the HMOX1 disease stems mainly from the loss of viable reticuloendothelial macrophages. Our work provides new insights into the etiology of HMOX1 deficiency and demonstrates the potential of infusion of WT macrophages to prevent disease in patients with HMOX1 deficiency and potentially other macrophage-related diseases.

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Inflammation as a Driver of Clonal Evolution in Myeloproliferative Neoplasm

Mediators of Inflammation ◽

10.1155/2015/606819 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 12

Author(s):

Angela G. Fleischman

Keyword(s):

Inflammatory Response ◽

Characteristic Feature ◽

Progenitor Cells ◽

Clonal Evolution ◽

Myeloproliferative Neoplasm ◽

Clonal Expansion ◽

Selective Advantage ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

The Impact

Our understanding of inflammation’s role in the pathogenesis of myeloproliferative neoplasm (MPN) is evolving. The impact of chronic inflammation, a characteristic feature of MPN, likely goes far beyond its role as a driver of constitutional symptoms. An inflammatory response to the neoplastic clone may be responsible for some pathologic aspects of MPN. Moreover,JAK2V617Fmutated hematopoietic stem and progenitor cells are resistant to inflammation, and this gives the neoplastic clone a selective advantage allowing for its clonal expansion. Because inflammation plays a central role in MPN inflammation is a logical therapeutic target in MPN.

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