RESUMO
The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.
Assuntos
Linfócitos , Mutação , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Diferenciação Celular , Proliferação de Células , Microambiente Celular , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Memória Imunológica/genética , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Linfócitos/patologia , Neoplasias/genética , Neoplasias/patologiaRESUMO
Age-related change in human haematopoiesis causes reduced regenerative capacity1, cytopenias2, immune dysfunction3 and increased risk of blood cancer4-6, but the reason for such abrupt functional decline after 70 years of age remains unclear. Here we sequenced 3,579 genomes from single cell-derived colonies of haematopoietic cells across 10 human subjects from 0 to 81 years of age. Haematopoietic stem cells or multipotent progenitors (HSC/MPPs) accumulated a mean of 17 mutations per year after birth and lost 30 base pairs per year of telomere length. Haematopoiesis in adults less than 65 years of age was massively polyclonal, with high clonal diversity and a stable population of 20,000-200,000 HSC/MPPs contributing evenly to blood production. By contrast, haematopoiesis in individuals aged over 75 showed profoundly decreased clonal diversity. In each of the older subjects, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before the subject was 40 years old, but only 22% had known driver mutations. Genome-wide selection analysis estimated that between 1 in 34 and 1 in 12 non-synonymous mutations were drivers, accruing at constant rates throughout life, affecting more genes than identified in blood cancers. Loss of the Y chromosome conferred selective benefits in males. Simulations of haematopoiesis, with constant stem cell population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure in the elderly. Rapidly decreasing clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently identified.
Assuntos
Envelhecimento , Hematopoiese Clonal , Células Clonais , Longevidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Criança , Pré-Escolar , Hematopoiese Clonal/genética , Células Clonais/citologia , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/citologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Células-Tronco Multipotentes/citologia , Adulto JovemRESUMO
Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. 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.
Assuntos
Células Sanguíneas/metabolismo , Diferenciação Celular/genética , Análise Mutacional de DNA/métodos , Músculo Liso/metabolismo , Mutação , Neurônios/metabolismo , Imagem Individual de Molécula/métodos , Células-Tronco/metabolismo , Doença de Alzheimer/genética , Células Sanguíneas/citologia , Divisão Celular , Estudos de Coortes , Colo/citologia , Epitélio/metabolismo , Granulócitos/citologia , Granulócitos/metabolismo , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso/citologia , Mutagênese , Taxa de Mutação , Neurônios/citologia , Células-Tronco/citologiaRESUMO
ABSTRACT: Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.
Assuntos
Ciclo Celular , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células Cultivadas , Transdução de Sinais , Diferenciação Celular , Técnicas de Cultura de Células/métodos , Adaptação FisiológicaRESUMO
Lymphopoiesis is the process in which B and T cells, and innate lymphoid cells (ILCs) develop from hematopoietic progenitors that exhibit early lymphoid priming. The branching points where lymphoid-primed human progenitors are further specified to B/T/ILC differentiation trajectories remain unclear. Here, we discuss the emerging role of interferon regulatory factor (IRF)8 as a key factor to bridge human lymphoid and dendritic cell (DC) differentiation, and the current evidence for the existence of circulating and tissue-resident CD123+CD127+ lymphoid progenitors. We propose a model whereby DC/B/T/ILC lineage programs in circulating CD123+CD127+ lymphoid progenitors are expressed in balance. Upon tissue seeding, the tissue microenvironment tilts this molecular balance towards a specific lineage, thereby determining in vivo lineage fates. Finally, we discuss the translational implication of these lymphoid precursors.
Assuntos
Imunidade Inata , Subunidade alfa de Receptor de Interleucina-3 , Humanos , Hematopoese , Fatores Reguladores de Interferon , LinfócitosRESUMO
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.
Assuntos
Linfócitos B/citologia , Redes Reguladoras de Genes , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Diferenciação Celular/genética , Linhagem da Célula , Biologia Computacional , Perfilação da Expressão Gênica/métodos , Humanos , RNA Mensageiro/química , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
Adult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil-induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.
Assuntos
Células-Tronco Hematopoéticas , Megacariócitos , Fator de Transcrição STAT1 , Animais , Proliferação de Células , Fluoruracila/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interferons , Megacariócitos/metabolismo , Camundongos , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismoRESUMO
Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic-skewing of PB is perturbed with age in essential thrombocythemia and ß-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Adulto , Medula Óssea , Células da Medula Óssea/fisiologia , Eritropoese , Humanos , MegacariócitosRESUMO
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.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Adultas/citologia , Animais , Ciclo Celular , Linhagem da Célula , Autorrenovação Celular , Perfilação da Expressão Gênica , Doenças Hematológicas/terapia , Hematopoese/genética , Humanos , Células-Tronco Multipotentes/citologia , Análise de Célula ÚnicaRESUMO
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.
Assuntos
Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Linhagem da Célula/genética , Análise Mutacional de DNA , Mutação , Células-Tronco Adultas/citologia , Teorema de Bayes , Contagem de Células , Divisão Celular , Células Clonais/citologia , Células Clonais/metabolismo , Desenvolvimento Embrionário/genética , Genoma Humano/genética , Granulócitos/citologia , Granulócitos/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Adultas/fisiologia , Animais , Antígenos de Diferenciação/metabolismo , Medula Óssea/fisiologia , Bromouracila/análogos & derivados , Fluoruracila/metabolismo , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas/fisiologia , Homeostase , Camundongos , Camundongos Transgênicos , Uridina/análogos & derivados , Uridina/metabolismoRESUMO
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.
Assuntos
Estresse do Retículo Endoplasmático , Células-Tronco Hematopoéticas/citologia , Resposta a Proteínas não Dobradas/fisiologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Xenoenxertos , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteína Fosfatase 1/metabolismo , Transdução de Sinais , Fator de Transcrição CHOP/metabolismo , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Análise de Célula Única/métodos , Animais , Biomarcadores/metabolismo , Ciclo Celular/genética , Diferenciação Celular/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.
Assuntos
Predisposição Genética para Doença/genética , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Idade de Início , Criança , Variações do Número de Cópias de DNA/genética , Genes ras/genética , Genoma Humano/genética , Genômica , Hematopoese/genética , Histonas/metabolismo , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Dados de Sequência Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores de Interleucina-7/genética , Proteína Reelina , Análise de Sequência de DNA , Transdução de Sinais/genética , Células-Tronco/metabolismo , Células-Tronco/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Translocação Genética/genéticaRESUMO
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.
Assuntos
Comunicação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Algoritmos , Células Cultivadas , Perfilação da Expressão Gênica , Hematopoese , Humanos , LigantesRESUMO
The bone marrow is the main site of blood cell production in adults, however, rare pools of hematopoietic stem and progenitor cells with self-renewal and differentiation potential have been found in extramedullary organs. The lung is primarily known for its role in gas exchange but has recently been described as a site of blood production in mice. Here, we show that functional hematopoietic precursors reside in the extravascular spaces of the human lung, at a frequency similar to the bone marrow, and are capable of proliferation and engraftment. The organ-specific gene signature of pulmonary and medullary CD34+ hematopoietic progenitors indicates greater baseline activation of immune, megakaryocyte/platelet and erythroid-related pathways in lung progenitors. Spatial transcriptomics mapped blood progenitors in the lung to a vascular-rich alveolar interstitium niche. These results identify the lung as a pool for uniquely programmed blood stem and progenitor cells with the potential to support hematopoiesis in humans.
RESUMO
Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.
Assuntos
Movimento Celular/fisiologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Quimiocina CXCL12/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Fator de Crescimento de Hepatócito/metabolismo , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell populations containing HSCs into syngeneic or immunocompromised mice. The size and multilineage composition of the graft is then measured over time, usually by flow cytometry. Classically, a population containing HSCs is injected into the circulation of the animal, after which the HSCs home to the bone marrow, where they lodge and begin blood production. Alternatively, HSCs and/or progenitor cells (HSPCs) can be placed directly in the bone marrow cavity. This paper describes a protocol for intrafemoral injection of human HSPCs into immunodeficient mice. In short, preconditioned mice are anesthetized, and a small hole is drilled through the knee into the femur using a needle. Using a smaller insulin needle, cells are then injected directly into the same conduit created by the first needle. This method of transplantation can be applied in varied experimental designs, using either mouse or human cells as donor cells. It has been most widely used for xenotransplantation, because in this context, it is thought to provide improved engraftment over intravenous injections, therefore improving statistical power and reducing the number of mice to be used.