RESUMO
Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor-stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.
Assuntos
Células-Tronco Hematopoéticas , Mitógenos , Adulto , Humanos , Animais , Camundongos , Integrina alfa6/metabolismo , Mitógenos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Antígenos CD34/metabolismo , Proliferação de Células , Pontos de Checagem do Ciclo Celular , Ciclo Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismoRESUMO
Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses.
Assuntos
Linhagem da Célula/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Plaquetas/metabolismo , Plaquetas/fisiologia , Diferenciação Celular/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Interferons/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Células Mieloides/fisiologiaRESUMO
Identifying new regulators of the stem cell state offers potential for future gains in biomedicine. Evidence that the tumor suppressor p53 is a key regulator of the stem cell state (Cicalese et al., 2009) suggests a broad role for this protein and its pathways in the control of normal tissue homeostasis and tumor formation.
Assuntos
Células-Tronco Neoplásicas/citologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Divisão Celular , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
The mammary gland in adult women consists of biologically distinct cell types that differ in their surface phenotypes. Isolation and molecular characterization of these subpopulations of mammary cells have provided extensive insights into their different transcriptional programs and regulation. This information is now serving as a baseline for interpreting the heterogeneous features of human breast cancers. Examination of breast cancer mutational profiles further indicates that most have undergone a complex evolutionary process even before being detected. The consequent intra-tumoral as well as inter-tumoral heterogeneity of these cancers thus poses major challenges to deriving information from early and hence likely pervasive changes in potential therapeutic interest. Recently described reproducible and efficient methods for generating human breast cancers de novo in immunodeficient mice transplanted with genetically altered primary cells now offer a promising alternative to investigate initial stages of human breast cancer development. In this review, we summarize current knowledge about key transcriptional regulatory processes operative in these partially characterized subpopulations of normal human mammary cells and effects of disrupting these processes in experimentally produced human breast cancers.
Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Glândulas Mamárias Humanas/química , Adulto , Animais , Linhagem Celular Tumoral , Evolução Molecular , Feminino , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Camundongos , Mutação , Transplante de NeoplasiasRESUMO
Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.
Assuntos
Diferenciação Celular , Linhagem da Célula , Rastreamento de Células , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células , Células Clonais/efeitos dos fármacos , Células Clonais/patologia , Epigênese Genética , Feminino , Glioblastoma/tratamento farmacológico , Xenoenxertos , Humanos , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , Processos EstocásticosRESUMO
Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.
Assuntos
Regulação Leucêmica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Transplante de NeoplasiasRESUMO
Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging ("inflammaging") has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a-/- myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a-/- HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a-/- mice. miR-146a-/- HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.
Assuntos
Envelhecimento/genética , Inflamação/genética , Interleucina-6/fisiologia , Leucemia Mieloide Aguda/etiologia , MicroRNAs/genética , Fator de Necrose Tumoral alfa/fisiologia , Adolescente , Adulto , Idoso , Envelhecimento/imunologia , Animais , Diferenciação Celular , Autorrenovação Celular , Senescência Celular , Citocinas/biossíntese , Metilação de DNA , Feminino , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Inflamação/fisiopatologia , Interleucina-6/antagonistas & inibidores , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/biossíntese , Pessoa de Meia-Idade , NF-kappa B/fisiologia , Análise de Célula Única , Transcriptoma , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto JovemRESUMO
Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of microRNAs (miRNAs) in regulating drug resistance and leukemic stem cell (LSC) fate, we performed global transcriptome profiling in treatment-naive chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor: its restored expression impaired survival of drug-resistant cells, sensitized them to TKIs in vitro, and markedly eliminated long-term repopulating LSCs and infiltrating blast cells, conferring a survival advantage in preclinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185, and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity, sensitizing therapy-resistant cells to TKIs. Thus, miR-185 presents as a potential predictive biomarker, and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL1 may provide a valuable strategy for overcoming drug resistance in patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Quinases Ativadas por p21/genética , Animais , Regulação Leucêmica da Expressão Gênica/genética , Xenoenxertos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Camundongos SCID , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/metabolismoRESUMO
Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in â¼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.
Assuntos
Antígenos CD34/metabolismo , Linhagem da Célula , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteoma/análise , Análise de Célula Única/métodos , Diferenciação Celular , Células Cultivadas , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Proteoma/metabolismoRESUMO
Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.
Assuntos
Neoplasias da Mama/fisiopatologia , Carcinoma Ductal de Mama/fisiopatologia , Transformação Celular Neoplásica , Glândulas Mamárias Humanas/fisiopatologia , Animais , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Linhagem da Célula/genética , Células Cultivadas , Código de Barras de DNA Taxonômico , Feminino , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Lentivirus/genética , Glândulas Mamárias Humanas/citologia , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Fatores de Tempo , Transdução Genética , Proteínas ras/genéticaRESUMO
Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution, underpinning important emergent features such as drug resistance and metastasis. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (<5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single-cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies using patient-derived breast cancer xenoengraftment.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células Clonais/metabolismo , Células Clonais/patologia , Genoma Humano/genética , Análise de Célula Única , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias da Mama/secundário , Análise Mutacional de DNA , Genômica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Transplante de Neoplasias , Fatores de Tempo , Transplante Heterólogo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Aberrantly expressed cytokines in the bone marrow (BM) niche are increasingly recognized as critical mediators of survival and expansion of leukemic stem cells. To identify regulators of primitive chronic myeloid leukemia (CML) cells, we performed a high-content cytokine screen using primary CD34+ CD38low chronic phase CML cells. Out of the 313 unique human cytokines evaluated, 11 were found to expand cell numbers ≥2-fold in a 7-day culture. Focusing on novel positive regulators of primitive CML cells, the myostatin antagonist myostatin propeptide gave the largest increase in cell expansion and was chosen for further studies. Herein, we demonstrate that myostatin propeptide expands primitive CML and normal BM cells, as shown by increased colony-forming capacity. For primary CML samples, retention of CD34-expression was also seen after culture. Furthermore, we show expression of MSTN by CML mesenchymal stromal cells, and that myostatin propeptide has a direct and instant effect on CML cells, independent of myostatin, by demonstrating binding of myostatin propeptide to the cell surface and increased phosphorylation of STAT5 and SMAD2/3. In summary, we identify myostatin propeptide as a novel positive regulator of primitive CML cells and corresponding normal hematopoietic cells.
Assuntos
Células-Tronco Hematopoéticas , Leucemia Mielogênica Crônica BCR-ABL Positiva , Antígenos CD34 , Medula Óssea , Células Cultivadas , Citocinas , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Miostatina/genéticaRESUMO
Motivation: High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging. Results: Here, we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers and clarify underlying biology. Availability and implementation: The GateFinder algorithm is implemented as a free and open-source package for BioConductor: https://nalab.stanford.edu/gatefinder. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Biomarcadores/análise , Citometria de Fluxo , SoftwareRESUMO
Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) phenotype (â¼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and ß-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90-CD49f- cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and ß-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Transdução de Sinais/fisiologia , Animais , Proliferação de Células , Sobrevivência Celular , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Fatores de TranscriçãoRESUMO
BACKGROUND: Human prostate cancers display numerous DNA methylation changes compared to normal tissue samples. However, definitive identification of features related to the cells' malignant status has been compromised by the predominance of cells with luminal features in prostate cancers. METHODS: We generated genome-wide DNA methylation profiles of cell subpopulations with basal or luminal features isolated from matched prostate cancer and normal tissue samples. RESULTS: Many frequent DNA methylation changes previously attributed to prostate cancers are here identified as differences between luminal and basal cells in both normal and cancer samples. We also identified changes unique to each of the two cancer subpopulations. Those specific to cancer luminal cells were associated with regulation of metabolic processes, cell proliferation and epithelial development. Within the prostate cancer TCGA dataset, these changes were able to distinguish not only cancers from normal samples, but also organ-confined cancers from those with extraprostatic extensions. Using changes present in both basal and luminal cancer cells, we derived a new 17-CpG prostate cancer signature with high predictive power in the TCGA dataset. CONCLUSIONS: This study demonstrates the importance of comparing phenotypically matched prostate cell populations from normal and cancer tissues to unmask biologically and clinically relevant DNA methylation changes.
Assuntos
Metilação de DNA , Fenótipo , Neoplasias da Próstata/genética , Ilhas de CpG , Humanos , MasculinoRESUMO
Hematopoietic stem cell (HSC) research took hold in the 1950s with the demonstration that intravenously injected bone marrow cells can rescue irradiated mice from lethality by reestablishing blood cell production. Attempts to quantify the cells responsible led to the discovery of serially transplantable, donor-derived, macroscopic, multilineage colonies detectable on the spleen surface 1 to 2 weeks posttransplant. The concept of self-renewing multipotent HSCs was born, but accompanied by perplexing evidence of great variability in the outcomes of HSC self-renewal divisions. The next 60 years saw an explosion in the development and use of more refined tools for assessing the behavior of prospectively purified subsets of hematopoietic cells with blood cell-producing capacity. These developments have led to the formulation of increasingly complex hierarchical models of hematopoiesis and a growing list of intrinsic and extrinsic elements that regulate HSC cycling status, viability, self-renewal, and lineage outputs. More recent examination of these properties in individual, highly purified HSCs and analyses of their perpetuation in clonally generated progeny HSCs have now provided definitive evidence of linearly transmitted heterogeneity in HSC states. These results anticipate the need and use of emerging new technologies to establish models that will accommodate such pluralistic features of HSCs and their control mechanisms.
Assuntos
Células-Tronco Hematopoéticas/citologia , Animais , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Hematopoese , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , HumanosRESUMO
Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.
Assuntos
Basófilos/patologia , Diferenciação Celular/efeitos dos fármacos , Eosinófilos/patologia , Fator de Transcrição Ikaros/antagonistas & inibidores , Leucemia Mieloide de Fase Crônica/patologia , Fator de Transcrição STAT5/metabolismo , Animais , Antígenos CD34/metabolismo , Apoptose/efeitos dos fármacos , Basófilos/efeitos dos fármacos , Basófilos/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Progressão da Doença , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Citometria de Fluxo , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Técnicas Imunoenzimáticas , Leucemia Mieloide de Fase Crônica/genética , Leucemia Mieloide de Fase Crônica/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/genéticaRESUMO
Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.
Assuntos
Células Epiteliais/classificação , Células Epiteliais/metabolismo , Glutationa/metabolismo , Glândulas Mamárias Humanas/citologia , Estresse Oxidativo/fisiologia , Western Blotting , Dano ao DNA/fisiologia , Citometria de Fluxo , Humanos , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/metabolismoRESUMO
Plerixafor (P) together with granulocyte colony-stimulating factor (G) is now recognized as an important strategy for mobilizing hematopoietic cells for use in patients given myelosuppressive therapies. However, quantitative comparisons of their ability to mobilize human cells with different hematopoietic activities in vitro or in vivo (in immunodeficient mice) and their interrelationships have not been investigated. To address these questions, we collected samples from 5 normal adult volunteers before and after administering P alone and from another 5 before and after a 4-day course of G and again after a subsequent injection of P. Measurements of their blood content of CD34+ cells, in vitro myeloid colony-forming cells, 3- and 6-week long-term culture (LTC) cell outputs, and levels of circulating human platelets, as well as myeloid and lymphoid cells obtained in immunodeficient mice that received transplants, showed all activities were maximal 4 hours after P preceded by G, and 3-week LTC outputs showed the highest concordance with the 3-week circulating human neutrophil levels obtained in mice that received transplants. Thus, human cells capable of producing neutrophils rapidly in vivo were optimally mobilized by the G + P protocol, and the 3-week LTC assay appears to offer a more specific predictor of their levels than conventional CD34+ cell or colony-forming cell counts.
Assuntos
Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Mobilização de Células-Tronco Hematopoéticas/métodos , Compostos Heterocíclicos/administração & dosagem , Animais , Antígenos CD34/sangue , Antígenos CD34/efeitos dos fármacos , Benzilaminas , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Contagem de Células , Técnicas de Cultura de Células , Ciclamos , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacosRESUMO
Leukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.