Publications by authors named "Elisa Laurenti"

44 Publications

Somatic mutation landscapes at single-molecule resolution.

Nature 2021 Apr 28. Epub 2021 Apr 28.

Wellcome Sanger Institute, Hinxton, UK.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.
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http://dx.doi.org/10.1038/s41586-021-03477-4DOI Listing
April 2021

Single-cell multi-omics analysis of the immune response in COVID-19.

Nat Med 2021 Apr 20. Epub 2021 Apr 20.

Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, UK.

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16C1QA/B/C) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34 hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8 T cells and an increased ratio of CD8 effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.
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http://dx.doi.org/10.1038/s41591-021-01329-2DOI Listing
April 2021

Transcriptional characterization of human megakaryocyte polyploidization and lineage commitment.

J Thromb Haemost 2021 May 29;19(5):1236-1249. Epub 2021 Mar 29.

Department of Hematology, University of Cambridge, Cambridge, UK.

Background: Megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. They mature within the adult bone marrow and release platelets into the circulation. Until now, there have been no transcriptional studies of primary human bone marrow MKs.

Objectives: To characterize MKs and HSCs from human bone marrow using single-cell RNA sequencing, to investigate MK lineage commitment, maturation steps, and thrombopoiesis.

Results: We show that MKs at different levels of polyploidization exhibit distinct transcriptional states. Although high levels of platelet-specific gene expression occur in the lower ploidy classes, as polyploidization increases, gene expression is redirected toward translation and posttranslational processing transcriptional programs, in preparation for thrombopoiesis. Our findings are in keeping with studies of MK ultrastructure and supersede evidence generated using in vitro cultured MKs. Additionally, by analyzing transcriptional signatures of a single HSC, we identify two MK-biased HSC subpopulations exhibiting unique differentiation kinetics. We show that human bone marrow MKs originate from these HSC subpopulations, supporting the notion that they display priming for MK differentiation. Finally, to investigate transcriptional changes in MKs associated with stress thrombopoiesis, we analyzed bone marrow MKs from individuals with recent myocardial infarction and found a specific gene expression signature. Our data support the modulation of MK differentiation in this thrombotic state.

Conclusions: Here, we use single-cell sequencing for the first time to characterize the human bone marrow MK transcriptome at different levels of polyploidization and investigate their differentiation from the HSC.
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http://dx.doi.org/10.1111/jth.15271DOI Listing
May 2021

Sphingosine-1-phosphate receptor 3 potentiates inflammatory programs in normal and leukemia stem cells to promote differentiation.

Blood Cancer Discov 2021 Jan 1;2(1):32-53. Epub 2020 Dec 1.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis, driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSC by activating stress myelopoiesis, such roles in LSC are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator which drives myeloid differentiation and activates inflammatory programs in both HSC and LSC. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across AML patient cohorts, each with distinct phenotypic and clinical properties. S1PR3 was high in LSC and blasts of mature myeloid samples with linkages to chemosensitivity, while S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116590PMC
January 2021

Blood stem cells SELect quiescence.

Authors:
Elisa Laurenti

Blood 2020 12;136(26):2967-2968

University of Cambridge.

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http://dx.doi.org/10.1182/blood.2020009554DOI Listing
December 2020

Beyond "to divide or not to divide": Kinetics matters in hematopoietic stem cells.

Exp Hematol 2020 12 11;92:1-10.e2. Epub 2020 Nov 11.

Department of Haematology and Wellcome and MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK. Electronic address:

Lifelong blood production is ensured by a population of rare and largely quiescent, long-lived hematopoietic stem cells (HSCs). The advent of single-cell technologies has recently highlighted underlying molecular and functional heterogeneity within the HSC pool. Despite heterogenous HSC behaviors, quiescence remains as the most uncontroversial and unifying property of HSCs. Nonetheless, a multifaceted and complex continuum of states has recently been identified within what was previously described as just "quiescent." Here we review such evidence and discuss how it challenges preconceived ideas on the contribution of cell cycle kinetics to HSC function. Specifically, we detail how both the frequency and kinetics of HSC division, largely determined by a network of molecular regulators linked to early G, influence long-term HSC functionin vivo. In addition, we present data that indicate lengthening the duration of G by inhibiting CDK6 decreases lymphoid differentiation of a subset of lymphoid-primed human HSCs, thus linking cell cycle kinetics to cell fate decisions in HSCs. Finally, we reflect on how these new insights can be helpful to fully harness HSC potential in clinical applications that require ex vivo culture.
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http://dx.doi.org/10.1016/j.exphem.2020.11.003DOI Listing
December 2020

A transcriptomic continuum of differentiation arrest identifies myeloid interface acute leukemias with poor prognosis.

Leukemia 2021 03 13;35(3):724-736. Epub 2020 Jul 13.

Université de Paris, Institut Necker Enfants Malades (INEM), Institut national de la santé et de la recherche médicale (INSERM UMR1151), 75743, Paris, France.

Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a-CD7- subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options.
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http://dx.doi.org/10.1038/s41375-020-0965-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7932917PMC
March 2021

Sphingolipid Modulation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal.

Cell Stem Cell 2019 11 17;25(5):639-653.e7. Epub 2019 Oct 17.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G0A3, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S1A8, Canada. Electronic address:

Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics.
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http://dx.doi.org/10.1016/j.stem.2019.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838675PMC
November 2019

Decoding human fetal liver haematopoiesis.

Nature 2019 10 9;574(7778):365-371. Epub 2019 Oct 9.

Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.
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http://dx.doi.org/10.1038/s41586-019-1652-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861135PMC
October 2019

A track of the clones: new developments in cellular barcoding.

Exp Hematol 2018 12 15;68:15-20. Epub 2018 Nov 15.

Institute Curie, PSL Research Univeristy, CNRS UMR168, Paris, France; Sorbonne Universités, UPMC University. Electronic address:

International experts from multiple disciplines gathered at Homerton College in Cambridge, UK from September 12-14, 2018 to consider recent advances and emerging opportunities in the clonal tracking of hematopoiesis in one of a series of StemCellMathLab workshops. The group included 35 participants with experience in the fields of theoretical and experimental aspects of clonal tracking, and ranged from doctoral students to senior professors. Data from a variety of model systems and from clinical gene therapy trials were discussed, along with strategies for data analysis and sharing and challenges arising due to underlying assumptions in data interpretation and communication. Recognizing the power of this technology underpinned a group consensus of a need for improved mechanisms for sharing data and analytical protocols to maintain reproducibility and rigor in its application to complex tissues.
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http://dx.doi.org/10.1016/j.exphem.2018.11.005DOI Listing
December 2018

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.

Nat Commun 2018 10 5;9(1):4100. Epub 2018 Oct 5.

Department of Haematology and Wellcome and MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19CD34CD38CD45RACD49fCD90 (49f) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9ACD34 cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9ACD34 cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment.
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http://dx.doi.org/10.1038/s41467-018-06442-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173731PMC
October 2018

Population dynamics of normal human blood inferred from somatic mutations.

Nature 2018 09 5;561(7724):473-478. Epub 2018 Sep 5.

Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, UK.

Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000-200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans.
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http://dx.doi.org/10.1038/s41586-018-0497-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6163040PMC
September 2018

From haematopoietic stem cells to complex differentiation landscapes.

Nature 2018 01;553(7689):418-426

Department of Haematology and Wellcome and MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.

The development of mature blood cells from haematopoietic stem cells has long served as a model for stem-cell research, with the haematopoietic differentiation tree being widely used as a model for the maintenance of hierarchically organized tissues. Recent results and new technologies have challenged the demarcations between stem and progenitor cell populations, the timing of cell-fate choices and the contribution of stem and multipotent progenitor cells to the maintenance of steady-state blood production. These evolving views of haematopoiesis have broad implications for our understanding of the functions of adult stem cells, as well as the development of new therapies for malignant and non-malignant haematopoietic diseases.
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http://dx.doi.org/10.1038/nature25022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555401PMC
January 2018

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Nat Cell Biol 2017 Sep 21;19(9):1093-1104. Epub 2017 Aug 21.

Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.
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http://dx.doi.org/10.1038/ncb3597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633079PMC
September 2017

Human megakaryocytes: finding the root.

Authors:
Elisa Laurenti

Blood 2017 06;129(25):3277-3279

UNIVERSITY OF CAMBRIDGE.

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http://dx.doi.org/10.1182/blood-2017-04-776351DOI Listing
June 2017

DNA Methylation Dynamics of Human Hematopoietic Stem Cell Differentiation.

Cell Stem Cell 2016 12 17;19(6):808-822. Epub 2016 Nov 17.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria; Max Planck Institute for Informatics, Saarland Informatics Campus, 66123 Saarbrücken, Germany; Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria; Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, 1090 Vienna, Austria. Electronic address:

Hematopoietic stem cells give rise to all blood cells in a differentiation process that involves widespread epigenome remodeling. Here we present genome-wide reference maps of the associated DNA methylation dynamics. We used a meta-epigenomic approach that combines DNA methylation profiles across many small pools of cells and performed single-cell methylome sequencing to assess cell-to-cell heterogeneity. The resulting dataset identified characteristic differences between HSCs derived from fetal liver, cord blood, bone marrow, and peripheral blood. We also observed lineage-specific DNA methylation between myeloid and lymphoid progenitors, characterized immature multi-lymphoid progenitors, and detected progressive DNA methylation differences in maturing megakaryocytes. We linked these patterns to gene expression, histone modifications, and chromatin accessibility, and we used machine learning to derive a model of human hematopoietic differentiation directly from DNA methylation data. Our results contribute to a better understanding of human hematopoietic stem cell differentiation and provide a framework for studying blood-linked diseases.
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http://dx.doi.org/10.1016/j.stem.2016.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5145815PMC
December 2016

A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

Blood 2016 08 30;128(8):e20-31. Epub 2016 Jun 30.

Department of Haematology and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.

Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice.
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http://dx.doi.org/10.1182/blood-2016-05-716480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305050PMC
August 2016

Molecular landscapes of human hematopoietic stem cells in health and leukemia.

Ann N Y Acad Sci 2016 04 11;1370(1):5-14. Epub 2015 Dec 11.

Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.

Blood cells are organized as a hierarchy with hematopoietic stem cells (HSCs) at the root. The advent of genomic technologies has opened the way for global characterization of the molecular landscape of HSCs and their progeny, both in mouse and human models, at the genetic, transcriptomic, epigenetic, and proteomics levels. Here, we outline our current understanding of the molecular programs that govern human HSCs and how dynamic changes occurring during HSC differentiation are necessary for well-regulated blood formation under homeostasis and upon injury. A large body of evidence is accumulating on how the programs of normal hematopoiesis are modified in acute myeloid leukemia, an aggressive adult malignancy driven by leukemic stem cells. We summarize these findings and their clinical implications.
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http://dx.doi.org/10.1111/nyas.12981DOI Listing
April 2016

Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.

Science 2016 Jan 5;351(6269):aab2116. Epub 2015 Nov 5.

Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada.

In a classical view of hematopoiesis, the various 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. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34(+) cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult "two-tier" hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.
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http://dx.doi.org/10.1126/science.aab2116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816201PMC
January 2016

CDK6 levels regulate quiescence exit in human hematopoietic stem cells.

Cell Stem Cell 2015 Mar 19;16(3):302-13. Epub 2015 Feb 19.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Regulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. The molecular mechanisms underlying this variability in HSC division kinetics are unknown. We report here that quiescence exit kinetics are differentially regulated within human HSC subsets through the expression level of CDK6. LT-HSCs lack CDK6 protein. Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation. Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function. Computational modeling suggests that this independent control of quiescence exit kinetics inherently limits LT-HSC divisions and preserves the HSC pool to ensure lifelong hematopoiesis. Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system.
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http://dx.doi.org/10.1016/j.stem.2015.01.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359055PMC
March 2015

Intercellular network structure and regulatory motifs in the human hematopoietic system.

Mol Syst Biol 2014 Jul 15;10:741. Epub 2014 Jul 15.

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada The Donnelly Centre, University of Toronto, Toronto, ON, Canada Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada McEwen Centre for Regenerative Medicine, University of Health Network, Toronto, ON, Canada Heart & Stroke/Richard Lewar Centre of Excellence, Toronto, ON, Canada

The hematopoietic system is a distributed tissue that consists of functionally distinct cell types continuously produced through hematopoietic stem cell (HSC) differentiation. Combining genomic and phenotypic data with high-content experiments, we have built a directional cell-cell communication network between 12 cell types isolated from human umbilical cord blood. Network structure analysis revealed that ligand production is cell type dependent, whereas ligand binding is promiscuous. Consequently, additional control strategies such as cell frequency modulation and compartmentalization were needed to achieve specificity in HSC fate regulation. Incorporating the in vitro effects (quiescence, self-renewal, proliferation, or differentiation) of 27 HSC binding ligands into the topology of the cell-cell communication network allowed coding of cell type-dependent feedback regulation of HSC fate. Pathway enrichment analysis identified intracellular regulatory motifs enriched in these cell type- and ligand-coupled responses. This study uncovers cellular mechanisms of hematopoietic cell feedback in HSC fate regulation, provides insight into the design principles of the human hematopoietic system, and serves as a foundation for the analysis of intercellular regulation in multicellular systems.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299490PMC
http://dx.doi.org/10.15252/msb.20145141DOI Listing
July 2014

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.

Nature 2014 Jun 28;510(7504):268-72. Epub 2014 Apr 28.

1] Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario M5G 2M9, Canada [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.
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http://dx.doi.org/10.1038/nature13228DOI Listing
June 2014

Improved HSC reconstitution and protection from inflammatory stress and chemotherapy in mice lacking granzyme B.

J Exp Med 2014 May 21;211(5):769-79. Epub 2014 Apr 21.

Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), D-69120 Heidelberg, Germany.

The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress.
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http://dx.doi.org/10.1084/jem.20131072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010905PMC
May 2014

Reduced lymphoid lineage priming promotes human hematopoietic stem cell expansion.

Cell Stem Cell 2014 Jan;14(1):94-106

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5G 1L7, Canada. Electronic address:

The hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.
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http://dx.doi.org/10.1016/j.stem.2013.11.021DOI Listing
January 2014

The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment.

Nat Immunol 2013 Jul 26;14(7):756-63. Epub 2013 May 26.

Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Understanding how differentiation programs originate from the gene-expression 'landscape' of hematopoietic stem cells (HSCs) is crucial for the development of new clinical therapies. We mapped the transcriptional dynamics underlying the first steps of commitment by tracking transcriptome changes in human HSCs and eight early progenitor populations. We found that transcriptional programs were extensively shared, extended across lineage-potential boundaries and were not strictly lineage affiliated. Elements of stem, lymphoid and myeloid programs were retained in multilymphoid progenitors (MLPs), which reflected a hybrid transcriptional state. By functional single cell analysis, we found that the transcription factors Bcl-11A, Sox4 and TEAD1 (TEF1) governed transcriptional networks in MLPs, which led to B cell specification. Overall, we found that integrated transcriptome approaches can be used to identify previously unknown regulators of multipotency and show additional complexity in lymphoid commitment.
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http://dx.doi.org/10.1038/ni.2615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961471PMC
July 2013

A KRAB/KAP1-miRNA cascade regulates erythropoiesis through stage-specific control of mitophagy.

Science 2013 Apr 14;340(6130):350-3. Epub 2013 Mar 14.

School of Life Sciences and Frontiers in Genetics Program, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.

During hematopoiesis, lineage- and stage-specific transcription factors work in concert with chromatin modifiers to direct the differentiation of all blood cells. We explored the role of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor KAP1 in this process. In mice, hematopoietic-restricted deletion of Kap1 resulted in severe hypoproliferative anemia. Kap1-deleted erythroblasts failed to induce mitophagy-associated genes and retained mitochondria. This was due to persistent expression of microRNAs (miRNAs) targeting mitophagy transcripts, itself secondary to a lack of repression by stage-specific KRAB-ZFPs. The KRAB/KAP1-miRNA regulatory cascade is evolutionarily conserved, as it also controls mitophagy during human erythropoiesis. Thus, a multilayered transcription regulatory system is present, in which protein- and RNA-based repressors are superimposed in combinatorial fashion to govern the timely triggering of an important differentiation event.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3678075PMC
http://dx.doi.org/10.1126/science.1232398DOI Listing
April 2013

Molecular and functional characterization of early human hematopoiesis.

Ann N Y Acad Sci 2012 Aug;1266:68-71

Campbell Family Institute for Cancer Research/Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada.

Through improvements in xenograft assay methods and in the identification of novel cell surface markers, significant progress has been made in our understanding of the human hematopoietic stem and progenitor hierarchy. The isolation of clonally pure populations of stem cells and early progenitors opens the way to carry out gene expression profiling studies to uncover the molecular regulators of each developmental step and to gain insight into the process of lineage commitment in human hematopoiesis.
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http://dx.doi.org/10.1111/j.1749-6632.2012.06577.xDOI Listing
August 2012

Hematopoiesis: a human perspective.

Cell Stem Cell 2012 Feb;10(2):120-36

Division of Stem Cell and Developmental Biology, Campbell Family Institute for Cancer Research/Ontario Cancer Institute, Toronto, ON M5G 1L7, Canada.

Despite its complexity, blood is probably the best understood developmental system, largely due to seminal experimentation in the mouse. Clinically, hematopoietic stem cell (HSC) transplantation represents the most widely deployed regenerative therapy, but human HSCs have only been characterized relatively recently. The discovery that immune-deficient mice could be engrafted with human cells provided a powerful approach for studying HSCs. We highlight 2 decades of studies focusing on isolation and molecular regulation of human HSCs, therapeutic applications, and early lineage commitment steps, and compare mouse and humanized models to identify both conserved and species-specific mechanisms that will aid future preclinical research.
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http://dx.doi.org/10.1016/j.stem.2012.01.006DOI Listing
February 2012