ABSTRACT
In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.
Subject(s)
Cell Lineage , Leukemia, Myeloid, Acute/pathology , Neoplasm Recurrence, Local/pathology , Neoplastic Stem Cells/pathology , Animals , Clone Cells/metabolism , Clone Cells/pathology , Female , Humans , Immunophenotyping , Leukemia, Myeloid, Acute/genetics , Mice , Mutation , Myeloid Progenitor Cells/metabolism , Myeloid Progenitor Cells/pathology , Neoplasm Recurrence, Local/genetics , Neoplastic Stem Cells/metabolismABSTRACT
In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.
Subject(s)
Hematopoietic Stem Cells/cytology , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/cytology , Animals , Cell Differentiation , Cell Division , Cell Lineage , Clone Cells/cytology , Clone Cells/metabolism , Clone Cells/pathology , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methyltransferase 3A , Drug Resistance, Neoplasm/drug effects , Female , Hematopoiesis , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Heterografts , Humans , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Mutation/genetics , Neoplasm Transplantation , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Nuclear Proteins/genetics , Nucleophosmin , Remission Induction , T-Lymphocytes/metabolism , T-Lymphocytes/pathologyABSTRACT
The nonobese diabetic/severe combined immune deficiency (NOD-scid) xenotransplantation model is the "gold standard" for assaying human hematopoietic stem cell activity. Systematic advancements, such as depletion of natural killer cell activity with anti-CD122 antibody, direct intrafemoral injection, and deletion or truncation of IL2Rgamma, have improved human cell engraftment; however, questions remain whether these mouse models are equivalent or, if not, which model is superior for assaying hematopoietic stem cell activity. To address this, we compared overall engraftment and multilineage differentiation of near-limiting doses of lineage-depleted human umbilical cord blood cells by direct intrafemoral injection into NOD/Lt-scid, NOD/Shi-scid, NOD/Lt-scid/IL2Rgamma(null) (NSG), and NOD/Shi-scid/IL2Rgamma(null) mice. Transplantation into NSG mice generated moderately higher human engraftment levels in bone marrow compared with other strains. At limiting doses, NSG mice of both sexes were 3.6-fold more sensitive in detecting SCID-repopulating cells compared with NOD/Lt-scid mice. However, NSG females exhibited higher engraftment at limiting cell doses, resulting in an overall increase in SCID-repopulating cell detection of 9-fold. Both NSG and NOD/Shi-scid/IL2Rgamma(null) support significantly improved engraftment in peripheral tissues compared with NOD/Lt-scid and NOD/Shi-scid mice, whereas NSG mice provide greater human engraftment in bone marrow than all other strains, especially at limiting doses.
Subject(s)
Cord Blood Stem Cell Transplantation/methods , Mice, Inbred NOD , Models, Animal , Transplantation, Heterologous/immunology , Transplantation, Heterologous/methods , Animals , Cell Differentiation/immunology , Cell Lineage/immunology , Cell Separation , Female , Fetal Blood/cytology , Fetal Blood/immunology , Flow Cytometry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Humans , Male , MiceABSTRACT
OBJECTIVE: Although increased expression of CD38 on the surface of human CD34(+) cells is associated with differentiation, we reported recently that both lineage-negative (Lin(-)) CD34(+)CD38(-) and Lin(-)CD34(+)CD38(lo) fractions of cord blood contain primitive severe combined immunodeficient (SCID)-repopulating cells (SRC). Thus, it is important to determine if a hierarchical relationship exists between the SRC from these two populations or if CD38 is reversibly expressed. MATERIALS AND METHODS: To determine if SRC from the CD34(+)CD38(-) and CD34(+)CD38(lo) cell fractions could generate SRC of the same and/or alternate CD38 expression, cells from primary nonobese diabetic/SCID mice transplanted with CD34(+)CD38(-) cells were resorted into both CD34(+)CD38(-) and CD34(+)CD38(lo) fractions and injected into separate secondary recipients, which were evaluated for human cell engraftment 7 to 10 weeks later. As primary mice transplanted with CD34(+)CD38(lo) cells also contained cells of both immunophenotype, these cells were also resorted and transplanted into separate secondary recipients. The cell-cycle status of various CD34(+) SRC fractions were evaluated using Hoechst 33342 and Pyronin Y staining in order to determine if CD38 expression was coordinated with divisional activation. RESULTS: Each cell fraction obtained from primary recipients was able to reconstitute secondary mice, indicating that CD38 expression reversibly oscillates between negative and low levels on CD34(+) repopulating cells. CD38 expression on repopulating cells correlated with a transition between the G(0) and G(1) phases of the cell cycle. CONCLUSION: CD38 is reversibly expressed on CD34(+) SRC between negative and low levels and corresponds to a change in the cell-cycle state. These observations establish a foundation to uncover the molecular program of stem cell regulation and underscore the importance of functional assessments when isolating and characterizing human hematopoietic stem cells.
Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Antigens, CD34/biosynthesis , Cell Cycle/drug effects , Hematopoiesis/physiology , Hematopoietic Stem Cells/immunology , Receptors, Cell Surface/metabolism , Animals , Cell Culture Techniques/methods , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Humans , Mice , Mice, SCID , Transplantation, HeterologousABSTRACT
Progress to uncover the molecular and cellular regulators that govern human hematopoietic stem cell (HSC) fate has been impeded by an inability to obtain highly purified fractions of HSCs. We report that the rhodamine 123 (Rho 123) dye effluxing fraction of the Lin-CD34+CD38- population contains SCID-repopulating cells (SRCs) capable of long-term repopulation in primary nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Purification based on Rho uptake led to a 4-fold enrichment of SRCs in the Lin-CD34+CD38- fraction, with a frequency of 1 SRC in 30 Lin-CD34+CD38-Rholo cells. The Lin-CD34+CD38-Rholo fraction also possesses long-term self-renewal capacity as measured by serial transplantation totaling more than 20 weeks. We conclude that Rho dye efflux provides an additional means of purifying human HSCs in the quest to achieve homogeneous populations of primitive cells for both experimental and therapeutic applications.
Subject(s)
ADP-ribosyl Cyclase 1/biosynthesis , Antigens, CD34/biosynthesis , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Rhodamine 123/pharmacokinetics , Animals , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Structure-Activity RelationshipABSTRACT
Hematopoiesis requires tight regulation of the hematopoietic stem cell (HSC) population; however, the dynamics of HSC use at steady state are uncertain. Over 3-7 months, we evaluated the repopulation and self-renewal of more than 600 individual human 'severe combined immunodeficiency mouse-repopulating cells' (SRCs), tracked on the basis of lentiviral integration sites, in serially transplanted immune-deficient mice, as well as of SRC daughter cells that migrated to different marrow locations in a single mouse. Our data demonstrate maintenance by self-renewing SRCs after an initial period of clonal instability, a result inconsistent with the clonal succession model. We found wide variation in proliferation kinetics and self-renewal among SRCs, as well as between SRC daughter cells that repopulated equivalently, suggesting that SRC fate is unpredictable before SRCs enter more rigid 'downstream' developmental programs.
Subject(s)
Cell Differentiation/immunology , Cell Proliferation , Hematopoietic Stem Cells/immunology , ADP-ribosyl Cyclase 1/metabolism , Animals , Antigens, CD34/metabolism , Cells, Cultured , Clone Cells , Fetal Blood/cytology , Fetal Blood/transplantation , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Humans , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCIDABSTRACT
OBJECTIVE: The absence of effective strategies for the ex vivo expansion of human hematopoietic stem cells (HSCs) limits the development of many cell-based therapies. Prior attempts to stimulate HSC expansion have focused on media supplementation using cytokines and growth factors. In these cultures, cellular and microenvironmental compositions change with time. In this study, the impact of controlling these dynamic changes on HSC output is determined. MATERIALS AND METHODS: Cord blood-derived lin(-) cells were cultured for 8 days in serum-free medium supplemented with stem cell factor, Flt3 ligand, and thrombopoietin. Functional, phenotypic, and molecular (gene and protein) analyses were used to characterize dynamic changes in cellular and microenvironmental composition. The effects of these changes and the mechanism behind their effects on HSC expansion were assessed using a selection/media exchange-based global culture manipulation (GCM) technique. RESULTS: We show that the direct secretion of negative regulators by culture-generated lin(+) cells, and the indirect stimulation of cells to secrete negative regulators by culture-conditioned media, limits in vitro HSC generation. The GCM strategy was able to abrogate these effects to produce elevated numbers of LTC-ICs (14.6-fold relative to input), migrating rapid NOD/SCID repopulating cells (12.1-fold), and long-term NOD/SCID repopulating cells (5.2-fold). CONCLUSIONS: Cellular and microenvironmental changes that occur during all in vitro HSC cultures can significantly affect HSC output through the direct or indirect secretion of negative regulators. This study provides insight into the mechanisms regulating HSC fate in vitro and describes a novel methodology to regulate overall in vitro microenvironmental dynamics to enable the generation of clinically relevant numbers of HSCs.
Subject(s)
Cell Proliferation/drug effects , Cytokines/pharmacology , Fetal Blood/physiology , Growth Substances/metabolism , Hematopoietic Stem Cells/physiology , Animals , Cell Culture Techniques , Cell Separation/methods , Cell- and Tissue-Based Therapy/methods , Cells, Cultured , Colony-Forming Units Assay/methods , Culture Media, Serum-Free , DNA-Activated Protein Kinase , DNA-Binding Proteins , Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Humans , Mice , Mice, Inbred NOD , Nuclear ProteinsABSTRACT
The nonobese diabetic/severe combined immune deficiency (NOD/SCID) xenotransplantation model has emerged as a widely used assay for human hematopoietic stem cells; however, barriers still exist that limit engraftment. We previously identified a short-term SCID-repopulating cell (SRC) following direct intrafemoral injection into NOD/SCID mice, whereas others characterized similar SRCs using NOD/SCID mice depleted of natural killer (NK) cell activity. To determine the model that most efficiently detects short-term SRCs, we compared human engraftment in 6 different xenotransplantation models: NOD/SCID-beta2-microglobulin-null mice, anti-CD122 (interleukin-2 receptor beta [IL-2Rbeta])-treated or unmanipulated NOD/SCID mice, each given transplants by intravenous or intrafemoral injection. Human cell engraftment was highest in intrafemorally injected anti-CD122-treated NOD/SCID mice compared to all other groups at 2 and 6 weeks after transplantation. These modifications to the SRC assay provide improved detection of human stem cells and demonstrate that CD122+ cells provide barriers to stem cell engraftment, a finding with potential clinical relevance.
Subject(s)
Hematopoiesis , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/cytology , Receptors, Interleukin-2 , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , Femur , Hematopoietic Stem Cell Transplantation/standards , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Models, Animal , Receptors, Interleukin-2/deficiency , Receptors, Interleukin-2/immunology , Receptors, Interleukin-2/physiology , Transplantation, Heterologous , beta 2-Microglobulin/deficiencyABSTRACT
Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs) that comprise the human HSC pool and the consequence of culture on each class is required for effective therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice revealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to establish whether HSC heterogeneity is intrinsic or whether the culture conditions required for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivectors to transduce HSCs with minimal manipulation during overnight culture without cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were identified on the basis of repopulation dynamics demonstrating that their existence is not an experimental artifact but reflects the state of the HSC pool. However, 4 days of culture in conditions previously used for SRC retroviral transduction significantly reduced SRC number as assessed by clonal analysis. These studies provide a foundation to understand the molecular and cellular determinants of human HSC development and to develop therapies targeted to specific HSC classes.
Subject(s)
Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Lentivirus/genetics , Animals , Cell Culture Techniques/methods , Colony-Forming Units Assay , Flow Cytometry , Genetic Vectors , Humans , Infant, Newborn , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Phosphoglycerate Kinase/genetics , Promoter Regions, Genetic , Stem Cell Transplantation , Stem Cells/cytology , Stem Cells/physiology , TransfectionABSTRACT
A major problem hampering effective stem cell-based therapies is the absence of a clear understanding of the human hematopoietic stem cell (HSC) pool composition. The severe combined immunodeficiency (SCID) repopulating cell (SRC) xenotransplant assay system provides a powerful tool for characterizing the frequency, cell surface markers, cell cycle status, homing and response to cytokine stimulation of human HSCs. Clonal tracking of retrovirally transduced SRCs and transplantation of specific subpopulations revealed SRC classes with distinct repopulation potentials. However, all HSC repopulation assays are based on intravenous injection, a complex process that requires circulation through blood, recognition and extravasation through bone marrow vasculature, and migration to a supportive microenvironment. Thus, some classes of HSCs may remain undetected. By direct intrafemoral injection, we identified rapid SRCs (R-SRCs) within the Lin-CD34+CD38loCD36- subpopulation. R-SRCs rapidly generate high levels of human myeloid and erythroid cells within the injected femur, migrate to the blood and colonize individual bones of non-obese diabetic (NOD)-SCID mice within 2 weeks after transplantation. Lentivector-mediated clonal analysis of individual R-SRCs revealed heterogeneity in their proliferative and migratory properties. The identification of a new HSC class and an effective intrafemoral assay provide the tools required to develop more effective stem cell-based therapies that rely on rapid reconstitution.
Subject(s)
Erythroid Precursor Cells/cytology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Molecular Biology/methods , Myeloid Cells/cytology , ADP-ribosyl Cyclase/metabolism , ADP-ribosyl Cyclase 1 , Animals , Antigens, CD/metabolism , Antigens, CD34/metabolism , Antigens, Surface/metabolism , CD36 Antigens/metabolism , Cell Lineage , Erythroid Precursor Cells/physiology , Female , Femur , Fetal Blood/cytology , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cells/classification , Humans , Injections, Intravenous , Membrane Glycoproteins , Mice , Mice, Inbred NOD , Mice, SCID , Myeloid Cells/physiology , Pregnancy , Transplantation, HeterologousABSTRACT
A major problem hampering the development of effective stem cell-based therapies is the absence of a clear understanding of the composition of the hematopoietic stem cell (HSC) pool in humans and how ex vivo manipulation can differentially affect the various HSC classes. This paper will review recent advances in the use of the NOD/SCID xenotransplant assay to characterize the human stem cell compartment and to determine how ex vivo culture affects stem cells. Using lentivector-mediated clonal tracking we found that only 4 days of culture can significantly reduce the number of SCID-repopulating cells (SRCs) contributing to the human graft. Similar results were seen with a competitive assay strategy where non-cultured cells marked with the RFP-lentivector markedly outcompete cultured cells marked with a EGFP-lentivector both transplanted into the same NOD/SCID mouse. A novel intrafemoral (IF) assay was developed to permit the transplantation of human stem cells that might be difficult to detect using the traditional IV injection method. With the IF assay we identified a novel class of human stem cell with the ability to rapidly generate a large graft of human myeloid and erythroid cells within 2 weeks post transplant.