RESUMO
The adult immune system consists of cells that emerged at various times during ontogeny. We aimed to define the relationship between developmental origin and composition of the adult B cell pool during unperturbed hematopoiesis. Lineage tracing stratified murine adult B cells based on the timing of output, revealing that a substantial portion originated within a restricted neonatal window. In addition to B-1a cells, early-life time-stamped B cells included clonally interrelated IgA plasma cells in the gut and bone marrow. These were actively maintained by B cell memory within gut chronic germinal centers and contained commensal microbiota reactivity. Neonatal rotavirus infection recruited recurrent IgA clones that were distinct from those arising by infection with the same antigen in adults. Finally, gut IgA plasma cells arose from the same hematopoietic progenitors as B-1a cells during ontogeny. Thus, a complex layer of neonatally imprinted B cells confer unique antibody responses later in life.
Assuntos
Imunoglobulina A , Microbiota , Animais , Linfócitos B , Centro Germinativo , Camundongos , PlasmócitosRESUMO
Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigated whether the developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. Using cellular barcoding for in vivo single-cell fate analyses, we found that fetal liver definitive HSCs gave rise to both B-1a and B-2 cells. Whereas B-1a potential diminished in all HSCs with time, B-2 output was maintained. B-1a and B-2 plasticity could be reinitiated in a subset of adult HSCs by ectopic expression of the RNA binding protein LIN28B, a key regulator of fetal hematopoiesis, and this coincided with the clonal reversal to fetal-like elevated self-renewal and repopulation potential. These results anchor the attenuation of B-1a cell output to fetal HSC behavior and demonstrate that the developmental decline in regenerative potential represents a reversible HSC state.
Assuntos
Linfócitos B/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Fígado/fisiologia , Subpopulações de Linfócitos/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Plasticidade Celular , Autorrenovação Celular , Células Clonais , Proteínas de Ligação a DNA/genética , Feminino , Hematopoese/genética , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Ligação a RNA , Análise de Célula ÚnicaRESUMO
In the version of Supplementary Fig. 1 originally published with this paper, some images in panel e were accidental duplicates of images in panel b. This error has been corrected in the online integrated supplementary information and in the Supplementary Information PDF.
RESUMO
The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.
Assuntos
Astrócitos/citologia , Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOX9/metabolismo , Humanos , Fatores de Transcrição NFI/metabolismoRESUMO
The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521/Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521-deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Neoplasias Experimentais/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/patologia , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Fatores de Transcrição/genética , Translocação GenéticaRESUMO
Several monogenic causes of familial myelodysplastic syndrome (MDS) have recently been identified. We studied 2 families with cytopenia, predisposition to MDS with chromosome 7 aberrations, immunodeficiency, and progressive cerebellar dysfunction. Genetic studies uncovered heterozygous missense mutations in SAMD9L, a tumor suppressor gene located on chromosome arm 7q. Consistent with a gain-of-function effect, ectopic expression of the 2 identified SAMD9L mutants decreased cell proliferation relative to wild-type protein. Of the 10 individuals identified who were heterozygous for either SAMD9L mutation, 3 developed MDS upon loss of the mutated SAMD9L allele following intracellular infections associated with myeloid, B-, and natural killer (NK)-cell deficiency. Five other individuals, 3 with spontaneously resolved cytopenic episodes in infancy, harbored hematopoietic revertant mosaicism by uniparental disomy of 7q, with loss of the mutated allele or additional in cisSAMD9L truncating mutations. Examination of 1 individual indicated that somatic reversions were postnatally selected. Somatic mutations were tracked to CD34+ hematopoietic progenitor cell populations, being further enriched in B and NK cells. Stimulation of these cell types with interferon (IFN)-α or IFN-γ induced SAMD9L expression. Clinically, revertant mosaicism was associated with milder disease, yet neurological manifestations persisted in 3 individuals. Two carriers also harbored a rare, in trans germ line SAMD9L missense loss-of-function variant, potentially counteracting the SAMD9L mutation. Our results demonstrate that gain-of-function mutations in the tumor suppressor SAMD9L cause cytopenia, immunodeficiency, variable neurological presentation, and predisposition to MDS with -7/del(7q), whereas hematopoietic revertant mosaicism commonly ameliorated clinical manifestations. The findings suggest a role for SAMD9L in regulating IFN-driven, demand-adapted hematopoiesis.
Assuntos
Disfunção Cognitiva/diagnóstico , Síndromes de Imunodeficiência/diagnóstico , Mutação , Síndromes Mielodisplásicas/diagnóstico , Pancitopenia/diagnóstico , Proteínas Supressoras de Tumor/genética , Adulto , Alelos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Proliferação de Células , Criança , Cromossomos Humanos Par 7/química , Disfunção Cognitiva/complicações , Disfunção Cognitiva/genética , Disfunção Cognitiva/imunologia , Feminino , Expressão Gênica , Hematopoese/imunologia , Heterozigoto , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Imunofenotipagem , Interferon Tipo I/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Masculino , Pessoa de Meia-Idade , Mosaicismo , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/imunologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/patologia , Pancitopenia/complicações , Pancitopenia/genética , Pancitopenia/imunologia , Linhagem , Proteínas Supressoras de Tumor/metabolismoRESUMO
Self-renewal of hematopoietic stem cells (HSCs) and leukemia-initiating cells (LICs) has been proposed to be influenced by low oxygen tension (hypoxia). This signaling, related to the cellular localization inside the bone marrow niche and/or influenced by extrinsic factors, promotes the stabilization of hypoxia-inducible factors (HIFs). Whether HIF-1α can be used as a therapeutic target in the treatment of myeloid malignancies remains unknown. We have used 3 different murine models to investigate the role of HIF-1α in acute myeloid leukemia (AML) initiation/progression and self-renewal of LICs. Unexpectedly, we failed to observe a delay or prevention of disease development from hematopoietic cells lacking Hif-1α. In contrast, deletion of Hif-1α resulted in faster development of the disease and an enhanced leukemia phenotype in some of the investigated models. Our results therefore warrant reconsideration of the role of HIF-1α and, as a consequence, question its generic therapeutic usefulness in AML.
Assuntos
Genes Supressores de Tumor , Células-Tronco Hematopoéticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucemia Mieloide Aguda/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Deleção de Genes , Células-Tronco Hematopoéticas/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Single cell gene expression assays have become a powerful tool with which to dissect heterogeneous populations. While methods and software exist to interrogate such data, what has been lacking is a unified solution combining analysis and visualisation which is also accessible and intuitive for use by non-bioinformaticians, as well as bioinformaticians. RESULTS: We present the Single cell expression visualiser (SCExV), a webtool developed to expedite the analysis of single cell qRT-PCR data. SCExV is able to take any data matrix of Ct values as an input, but can handle files exported by the Fluidigm Biomark platform directly. In addition, SCExV also accepts and automatically integrates cell surface marker intensity values which are measured during index sorting. This allows the user to directly visualise relationships between a single cell gene expression profile and the immunophenotype of the interrogated cell. CONCLUSIONS: SCExV is a freely available webtool created to import, filter, analyse, and visualise single cell gene expression data whilst being able to simultaneously consider cellular immunophenotype. SCExV is designed to be intuitive to use whilst maintaining advanced functionality and flexibility in how analyses are performed.
Assuntos
DNA/análise , Reação em Cadeia da Polimerase em Tempo Real , Interface Usuário-Computador , Animais , Internet , Camundongos , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Aging of hematopoietic stem cells (HSCs) leads to several functional changes, including alterations affecting self-renewal and differentiation. Although it is well established that many of the age-induced changes are intrinsic to HSCs, less is known regarding the stability of this state. Here, we entertained the hypothesis that HSC aging is driven by the acquisition of permanent genetic mutations. To examine this issue at a functional level in vivo, we applied induced pluripotent stem (iPS) cell reprogramming of aged hematopoietic progenitors and allowed the resulting aged-derived iPS cells to reform hematopoiesis via blastocyst complementation. Next, we functionally characterized iPS-derived HSCs in primary chimeras and after the transplantation of re-differentiated HSCs into new hosts, the gold standard to assess HSC function. Our data demonstrate remarkably similar functional properties of iPS-derived and endogenous blastocyst-derived HSCs, despite the extensive chronological and proliferative age of the former. Our results, therefore, favor a model in which an underlying, but reversible, epigenetic component is a hallmark of HSC aging.
Assuntos
Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Epigênese Genética/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/genética , Senescência Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Estudo de Associação Genômica Ampla , Camundongos , Camundongos Endogâmicos C57BL , Telômero/genética , Células-Tronco Totipotentes/citologia , Células-Tronco Totipotentes/fisiologia , Transcrição Gênica/fisiologiaRESUMO
It has become increasingly clear that several age-associated pathologies associate with mutations in the mitochondrial genome. Experimental modeling of such events has revealed that acquisition of mitochondrial DNA (mtDNA) damage can impair respiratory function and, as a consequence, can lead to widespread decline in cellular function. This includes premature aging syndromes. By taking advantage of a mutator mouse model with an error-prone mtDNA polymerase, we here investigated the impact of an established mtDNA mutational load with regards to the generation, maintenance, and differentiation of induced pluripotent stem (iPS) cells. We demonstrate that somatic cells with a heavy mtDNA mutation burden were amenable for reprogramming into iPS cells. However, mutator iPS cells displayed delayed proliferation kinetics and harbored extensive differentiation defects. While mutator iPS cells had normal ATP levels and glycolytic activity, the induction of differentiation coincided with drastic decreases in ATP production and a hyperactive glycolysis. These data demonstrate the differential requirements of mitochondrial integrity for pluripotent stem cell self-renewal versus differentiation and highlight the relevance of assessing the mitochondrial genome when aiming to generate iPS cells with robust differentiation potential.
Assuntos
Diferenciação Celular/genética , DNA Mitocondrial/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Reprogramação Celular/genética , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/genética , Glicólise/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fator 3 de Transcrição de Octâmero/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genéticaRESUMO
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.
Assuntos
Anemia de Diamond-Blackfan/dietoterapia , Suplementos Nutricionais , Modelos Animais de Doenças , Hematínicos/uso terapêutico , Hematopoese , Leucina/uso terapêutico , Regulação para Cima , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/patologia , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Regulação para Baixo , Contagem de Eritrócitos , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Hemoglobinas/análise , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recent studies have identified a number of transcriptional regulators, including E proteins, EBF1, FOXO1, and PAX5, that act together to orchestrate the B-cell fate. However, it still remains unclear as to how they are linked at the earliest stages of B-cell development. Here, we show that lymphocyte development in HEB-ablated mice exhibits a partial developmental arrest, whereas B-cell development in E2A(+/-)HEB(-/-) mice is completely blocked at the LY6D(-) common lymphoid progenitor stage. We show that the transcription signatures of E2A- and HEB-ablated common lymphoid progenitors significantly overlap. Notably, we found that Foxo1 expression was substantially reduced in the LY6D(-) HEB- and E2A-deficient cells. Finally, we show that E2A binds to enhancer elements across the FOXO1 locus to activate Foxo1 expression, linking E2A and FOXO1 directly in a common pathway. In summary, the data indicate that the earliest event in B-cell specification involves the induction of FOXO1 expression and requires the combined activities of E2A and HEB.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Fatores de Transcrição Forkhead/genética , Células Progenitoras Linfoides/imunologia , Animais , Antígenos Ly/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Forkhead Box O1 , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Hematopoese/imunologia , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/imunologia , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/imunologia , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Camundongos , Camundongos KnockoutRESUMO
The maturation of B-lymphoid cells from hematopoietic stem cells in the bone marrow is a complex process under control of interplay between extrinsic and intrinsic factors. In addition to serving as a model for normal cell differentiation, disturbances in this process results in immunodeficiencies and malignancies, such as leukemia and lymphoma, making the subject of high relevance for modern medicine. Although the process of B lymphopoiesis has been under intense investigation, recent methodological developments within the area of cell sorting, genome-wide expression, and DNA-binding analysis has allowed for a rapid development of the understanding of B-lymphocyte differentiation. This has suggested that the path to B-lymphoid cell fate may be initiated by lineage priming reflected in the expression of lymphoid associated genes already in multipotent hematopoietic progenitors. Upon differentiation, the gene expression profile is changed to involve an increasing number of B-lineage-restricted genes linked to loss of alternative developmental potentials and B-lineage commitment. This review focuses on the molecular regulation of early B-lymphoid development and aims to provide an up to date summary of the current status of the research area.
Assuntos
Antígenos de Diferenciação/imunologia , Linfócitos B/imunologia , Linhagem da Célula/imunologia , Animais , Transformação Celular Neoplásica , Regulação da Expressão Gênica/imunologia , Hematopoese/imunologia , Humanos , Transdução de Sinais/imunologiaRESUMO
The interplay between aging and immune system deterioration presents a formidable challenge to human health, especially in the context of a globally aging population. Aging is associated with a decline in the body's ability to combat infections and an increased risk of various diseases, underlining the importance of rejuvenating the immune system as a strategy for promoting healthier aging. In issue 628 of Nature (2024), Ross et al. present a compelling study that introduces a novel strategy for rejuvenating the aged immune system (Ross et al., 2024). By using antibodies to selectively eliminate "aberrant" hematopoietic stem cells (HSCs), this research opens new avenues for addressing age-related immune deterioration.
Assuntos
Envelhecimento , Células-Tronco Hematopoéticas , Sistema Imunitário , Humanos , Envelhecimento/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , AnimaisRESUMO
A decline in hematopoietic stem cell (HSC) function is believed to underlie hematological shortcomings with age; however, a comprehensive molecular understanding of these changes is currently lacking. Here we provide evidence that a transcriptional signature reported in several previous studies on HSC aging is linked to stress-induced changes in gene expression rather than aging. Our findings have strong implications for the design and interpretation of HSC aging studies.
Assuntos
Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Expressão Gênica/genéticaRESUMO
The scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCs ex vivo. After 3 weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential for ex vivo self-renewal, with alternative outcomes revealed by single-cell multimodal RNA and ATAC sequencing profiling. While most HSC progeny offered only transient in vivo reconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations for ex vivo HSC expansion, with major implications also for assessment of normal HSC activity.
Assuntos
Células-Tronco Hematopoéticas , RNA , Animais , Camundongos , Divisão Celular , FenótipoRESUMO
Infant and adult MLL1/KMT2A-rearranged (MLLr) leukemia represents a disease with a dismal prognosis. Here, we present a functional and proteomic characterization of in utero-initiated and adult-onset MLLr leukemia. We reveal that fetal MLL::ENL-expressing lymphomyeloid multipotent progenitors (LMPPs) are intrinsically programmed towards a lymphoid fate but give rise to myeloid leukemia in vivo, highlighting a complex interplay of intra- and extracellular factors in determining disease subtype. We characterize early proteomic events of MLL::ENL-mediated transformation in fetal and adult blood progenitors and reveal that whereas adult pre-leukemic cells are mainly characterized by retained myeloid features and downregulation of ribosomal and metabolic proteins, expression of MLL::ENL in fetal LMPPs leads to enrichment of translation-associated and histone deacetylases signaling proteins, and decreased expression of inflammation and myeloid differentiation proteins. Integrating the proteome of pre-leukemic cells with their secretome and the proteomic composition of the extracellular environment of normal progenitors highlights differential regulation of Igf2 bioavailability, as well as of VLA-4 dimer and its ligandome, upon initiation of fetal- and adult-origin leukemia, with implications for human MLLr leukemia cells' ability to communicate with their environment through granule proteins. Our study has uncovered opportunities for targeting ontogeny-specific proteomic vulnerabilities in in utero-initiated and adult-onset MLLr leukemia.
Assuntos
Proteína de Leucina Linfoide-Mieloide , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Humanos , Camundongos , Animais , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Rearranjo Gênico , Proteômica/métodos , Feto/metabolismo , Adulto , Feminino , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia/genética , Leucemia/patologia , Leucemia/metabolismoRESUMO
ABSTRACT: Natural killer (NK) cells represent the cytotoxic member within the innate lymphoid cell (ILC) family that are important against viral infections and cancer. Although the NK cell emergence from hematopoietic stem and progenitor cells through multiple intermediate stages and the underlying regulatory gene network has been extensively studied in mice, this process is not well characterized in humans. Here, using a temporal in vitro model to reconstruct the developmental trajectory of NK lineage, we identified an ILC-restricted oligopotent stage 3a CD34-CD117+CD161+CD45RA+CD56- progenitor population, that exclusively gave rise to CD56-expressing ILCs in vitro. We also further investigated a previously nonappreciated heterogeneity within the CD56+CD94-NKp44+ subset, phenotypically equivalent to stage 3b population containing both group-1 ILC and RORγt+ ILC3 cells, that could be further separated based on their differential expression of DNAM-1 and CD161 receptors. We confirmed that DNAM-1hi S3b and CD161hiCD117hi ILC3 populations distinctively differed in their expression of effector molecules, cytokine secretion, and cytotoxic activity. Furthermore, analysis of lineage output using DNA-barcode tracing across these stages supported a close developmental relationship between S3b-NK and S4-NK (CD56+CD94+) cells, whereas distant to the ILC3 subset. Cross-referencing gene signatures of culture-derived NK cells and other noncytotoxic ILCs with publicly available data sets validated that these in vitro stages highly resemble transcriptional profiles of respective in vivo ILC counterparts. Finally, by integrating RNA velocity and gene network analysis through single-cell regulatory network inference and clustering we unravel a network of coordinated and highly dynamic regulons driving the cytotoxic NK cell program, as a guide map for future studies on NK cell regulation.
Assuntos
Células Matadoras Naturais , Análise de Célula Única , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Análise de Célula Única/métodos , Linhagem da Célula , Imunidade Inata , Diferenciação CelularRESUMO
Endomucin (EMCN) currently represents the only hematopoietic stem cell (HSC) marker expressed by both murine and human HSCs. Here, we report that EMCN+ long-term repopulating HSCs (LT-HSCs; CD150+CD48-LSK) have a higher long-term multi-lineage repopulating capacity compared to EMCN- LT-HSCs. Cell cycle analyses and transcriptional profiling demonstrated that EMCN+ LT-HSCs were more quiescent compared to EMCN- LT-HSCs. Emcn-/- and Emcn+/+ mice displayed comparable steady-state hematopoiesis, as well as frequencies, transcriptional programs, and long-term multi-lineage repopulating capacity of their LT-HSCs. Complementary functional analyses further revealed increased cell cycle entry upon treatment with 5-fluorouracil and reduced granulocyte colony-stimulating factor (GCSF) mobilization of Emcn-/- LT-HSCs, demonstrating that EMCN expression by LT-HSCs associates with quiescence in response to hematopoietic stress and is indispensable for effective LT-HSC mobilization. Transplantation of wild-type bone marrow cells into Emcn-/- or Emcn+/+ recipients demonstrated that EMCN is essential for endothelial cell-dependent maintenance/self-renewal of the LT-HSC pool and sustained blood cell production post-transplant.
Assuntos
Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Movimento Celular , Fluoruracila/farmacologia , Humanos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Ciclo Celular , Células Endoteliais/metabolismoRESUMO
For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.