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1.
Immunity ; 50(4): 1054-1068.e3, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30926235

RESUMO

Innate lymphoid cell (ILC) development proposes that ILC precursors (ILCPs) segregate along natural killer (NK) cell versus helper cell (ILC1, ILC2, ILC3) pathways, the latter depending on expression of Id2, Zbtb16, and Gata3. We have developed an Id2-reporter strain expressing red fluorescent protein (RFP) in the context of normal Id2 expression to re-examine ILCP phenotype and function. We show that bone-marrow ILCPs were heterogeneous and harbored extensive NK-cell potential in vivo and in vitro. By multiplexing Id2RFP with Zbtb16CreGFP and Bcl11btdTomato strains, we made a single-cell dissection of the ILCP compartment. In contrast with the current model, we have demonstrated that Id2+Zbtb16+ ILCPs included multi-potent ILCPs that retained NK-cell potential. Late-stage ILC2P and ILC3P compartments could be defined by differential Zbtb16 and Bcl11b expression. We suggest a revised model for ILC differentiation that redefines the cell-fate potential of helper-ILC-restricted Zbtb16+ ILCPs.


Assuntos
Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/citologia , Imunidade Inata , Proteína 2 Inibidora de Diferenciação/genética , Linfopoese/genética , Transferência Adotiva , Animais , Linhagem da Célula , Fator de Transcrição GATA3/biossíntese , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/fisiologia , Genes Reporter , Células-Tronco Hematopoéticas/metabolismo , Proteína 2 Inibidora de Diferenciação/biossíntese , Células Matadoras Naturais/citologia , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Proteína com Dedos de Zinco da Leucemia Promielocítica/biossíntese , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/fisiologia , Análise de Célula Única , Linfócitos T Auxiliares-Indutores/citologia , Transcrição Gênica , Proteína Vermelha Fluorescente
2.
EMBO J ; 42(21): e113448, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37737560

RESUMO

The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T-cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)-cell fate in T cells. In addition, T cells upregulate the NK cell-associated receptors and transcription factors, lyse NK-cell targets, and are reprogrammed into NK-like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1-mediated elevated OXPHOS enhances cellular acetyl-CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T-cell fate directly by repressing NK cell-associated transcription and indirectly through a metabolic-epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs.


Assuntos
Histonas , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Animais , Humanos , Camundongos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Dinâmica Mitocondrial , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo
3.
Immunity ; 48(2): 227-242.e8, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29466755

RESUMO

How chromatin reorganization coordinates differentiation and lineage commitment from hematopoietic stem and progenitor cells (HSPCs) to mature immune cells has not been well understood. Here, we carried out an integrative analysis of chromatin accessibility, topologically associating domains, AB compartments, and gene expression from HSPCs to CD4+CD8+ T cells. We found that abrupt genome-wide changes at all three levels of chromatin organization occur during the transition from double-negative stage 2 (DN2) to DN3, accompanying the T lineage commitment. The transcription factor BCL11B, a critical regulator of T cell commitment, is associated with increased chromatin interaction, and Bcl11b deletion compromised chromatin interaction at its target genes. We propose that these large-scale and concerted changes in chromatin organization present an energy barrier to prevent the cell from reversing its fate to earlier stages or redirecting to alternatives and thus lock the cell fate into the T lineages.


Assuntos
Linhagem da Célula , Núcleo Celular/fisiologia , Cromatina/fisiologia , Linfócitos T/fisiologia , Animais , Diferenciação Celular , Humanos , Proteínas Repressoras/fisiologia , Proteínas Supressoras de Tumor/fisiologia
4.
J Biol Chem ; 300(1): 105556, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38097188

RESUMO

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.


Assuntos
Macrófagos Alveolares , Células-Tronco Pluripotentes , Suínos , Animais , Endocitose , Hematopoese/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Mesoderma/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Transdução de Sinais/efeitos dos fármacos , Suínos/virologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fatores de Tempo
5.
J Allergy Clin Immunol ; 153(4): 1125-1139, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38072195

RESUMO

BACKGROUND: Inborn errors of immunity (IEI) often lack specific disease models and personalized management. Signal transducer and activator of transcription (STAT)-1 gain of function (GoF) is such example of an IEI with diverse clinical phenotype with unclear pathomechanisms and unpredictable response to therapy. Limitations in obtaining fresh samples for functional testing and research further highlights the need for patient-specific ex vivo platforms. OBJECTIVE: Using STAT1-GoF as an example IEI, we investigated the potential of patient-derived expanded potential stem cells (EPSC) as an ex vivo platform for disease modeling and personalized treatment. METHODS: We generated EPSC derived from individual STAT1-GoF patients. STAT1 mutations were confirmed with Sanger sequencing. Functional testing including STAT1 phosphorylation/dephosphorylation and gene expression with or without Janus activating kinase inhibitors were performed. Functional tests were repeated on EPSC lines with GoF mutations repaired by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) editing. RESULTS: EPSC were successfully reprogrammed from STAT1-GoF patients and expressed the same pluripotent makers as controls, with distinct morphologic differences. Patient-derived EPSC recapitulated the functional abnormalities of index STAT1-GoF patients with STAT1 hyperphosphorylation and increased expression of STAT1 and its downstream genes (IRF1, APOL6, and OAS1) after IFN-γ stimulation. Addition of ruxolitinib and baricitinib inhibited STAT1 hyperactivation in STAT1-GoF EPSC in a dose-dependent manner, which was not observed with tofacitinib. Corrected STAT1 phosphorylation and downstream gene expression were observed among repaired STAT1-GoF EPSC cell lines. CONCLUSION: This proof-of-concept study demonstrates the potential of our patient-derived EPSC platform to model STAT1-GoF. We propose this platform when researching, recapitulating, and repairing other IEI in the future.


Assuntos
Mutação com Ganho de Função , Fator de Transcrição STAT1 , Células-Tronco , Humanos , Mutação , Fosforilação , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Células-Tronco/imunologia , Células-Tronco/metabolismo
6.
EMBO Rep ; 23(8): e54104, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35766181

RESUMO

Developmental neuron death plays a pivotal role in refining organization and wiring during neocortex formation. Aberrant regulation of this process results in neurodevelopmental disorders including impaired learning and memory. Underlying molecular pathways are incompletely determined. Loss of Bcl11a in cortical projection neurons induces pronounced cell death in upper-layer cortical projection neurons during postnatal corticogenesis. We use this genetic model to explore genetic mechanisms by which developmental neuron death is controlled. Unexpectedly, we find Bcl6, previously shown to be involved in the transition of cortical neurons from progenitor to postmitotic differentiation state to provide a major checkpoint regulating neuron survival during late cortical development. We show that Bcl11a is a direct transcriptional regulator of Bcl6. Deletion of Bcl6 exerts death of cortical projection neurons. In turn, reintroduction of Bcl6 into Bcl11a mutants prevents induction of cell death in these neurons. Together, our data identify a novel Bcl11a/Bcl6-dependent molecular pathway in regulation of developmental cell death during corticogenesis.


Assuntos
Neocórtex , Fatores de Transcrição , Morte Celular/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Neocórtex/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo
7.
EMBO Rep ; 23(6): e54275, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35437924

RESUMO

Our understanding of human hepatocellular carcinoma (HCC) development and progression has been hampered by the lack of in vivo models. We performed a genetic screen of 10 oncogenes and genetic mutations in Fah-ablated immunodeficient mice in which primary human hepatocytes (PHHs) are used to reconstitute a functional human liver. We identified that MYC, TP53R249S , and KRASG12D are highly expressed in induced HCC (iHCC) samples. The overexpression of MYC and TP53R249S transform PHHs into iHCC in situ, though the addition of KRASG12D significantly increases the tumorigenic efficiency. iHCC, which recapitulate the histological architecture and gene expression characteristics of clinical HCC samples, reconstituted HCC after serial transplantations. Transcriptomic analysis of iHCC and PHHs showed that MUC1 and FAP are expressed in iHCC but not in normal livers. Chimeric antigen receptor (CAR) T cells against these two surface markers efficiently lyse iHCC cells. The properties of iHCC model provide a biological basis for several clinical hallmarks of HCC, and iHCC may serve as a model to study HCC initiation and to identify diagnostic biomarkers and targets for cellular immunotherapy.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/genética , Hepatócitos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Proteínas Proto-Oncogênicas p21(ras)
8.
Nature ; 554(7690): 62-68, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29364867

RESUMO

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Evolução Molecular , Dosagem de Genes , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Animais , Carcinogênese/genética , Proteínas de Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Progressão da Doença , Feminino , Genes myc , Genes p53 , Humanos , Masculino , Camundongos , Mutação , Subunidade p52 de NF-kappa B/genética , Metástase Neoplásica/genética , Proteínas Nucleares/genética , Fenótipo , Fosfoproteínas/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Fator de Crescimento Transformador beta1/genética , Proteínas de Sinalização YAP
9.
Proc Natl Acad Sci U S A ; 118(15)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33833056

RESUMO

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.


Assuntos
Bovinos/genética , Células-Tronco Embrionárias/citologia , Técnicas de Transferência Nuclear/veterinária , Cultura Primária de Células/métodos , Animais , Blastocisto/citologia , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Cultura Primária de Células/veterinária , Transcriptoma
10.
Biol Reprod ; 108(6): 887-901, 2023 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-37040346

RESUMO

The mule is the interspecific hybrid of horse and donkey and has hybrid vigor in muscular endurance, disease resistance, and longevity over its parents. Here, we examined adult fibroblasts of mule (MAFs) compared with the cells from their parents (donkey adult fibroblasts and horse adult fibroblasts) (each species has repeated three independent individuals) in proliferation, apoptosis, and glycolysis and found significant differences. We subsequently derived mule, donkey, and horse doxycycline (Dox)-independent induced pluripotent stem cells (miPSCs, diPSCs, and hiPSCs) from three independent individuals of each species and found that the reprogramming efficiency of MAFs was significantly higher than that of cells of donkey and horse. miPSCs, diPSCs, and hiPSCs all expressed the high levels of crucial endogenous pluripotency genes such as POU class 5 homeobox 1 (POU5F1, OCT4), SRY-box 2 (SOX2), and Nanog homeobox (NANOG) and propagated robustly in single-cell passaging. miPSCs exhibited faster proliferation and higher pluripotency and differentiation than diPSCs and hiPSCs, which were reflected in co-cultures and separate-cultures, teratoma formation, and chimera contribution. The establishment of miPSCs provides a unique research material for the investigation of "heterosis" and perhaps is more significant to study hybrid gamete formation.


Assuntos
Células-Tronco Pluripotentes Induzidas , Cavalos , Animais , Reprogramação Celular , Equidae , Células Cultivadas , Diferenciação Celular/genética , Fibroblastos , Fator 3 de Transcrição de Octâmero/genética
11.
Nature ; 550(7676): 393-397, 2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-29019987

RESUMO

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.


Assuntos
Blastômeros/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Blastocisto/citologia , Blastômeros/metabolismo , Linhagem da Célula , Células Cultivadas , Quimera , Embrião de Mamíferos/citologia , Endoderma/citologia , Epigênese Genética , Epigenômica , Feminino , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Placenta/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Gravidez , Análise de Célula Única , Transcriptoma , Trofoblastos/citologia
12.
Proc Natl Acad Sci U S A ; 117(50): 31945-31953, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33268499

RESUMO

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene Prdm16 during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of Prdm16 in adult HSCs remain unclear. To investigate the role of Prdm16 in adult HSCs, we generated a novel conditional knockout mouse model and deleted Prdm16 in adult mouse hematopoietic system using the IFN-inducible Mx1-Cre Our results show that Prdm16 deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. Prdm16 deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of Prdm16 significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without Prdm16 deletion showed that Prdm16 loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which Cdkn1a and Egr1 were identified as direct targets of Prdm16 Our results suggest that Prdm16 preserves the function of adult LT-HSCs by promoting their quiescence.


Assuntos
Células-Tronco Adultas/fisiologia , Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Feminino , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Knockout , RNA-Seq , Fatores de Transcrição/genética
13.
BMC Biol ; 20(1): 14, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35027054

RESUMO

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.


Assuntos
Vírus da Febre Suína Africana , Doenças Transmissíveis , Vírus da Febre Suína Africana/genética , Animais , Interações Hospedeiro-Patógeno/genética , Macrófagos , Células-Tronco , Suínos
14.
Nature ; 539(7627): 102-106, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27749818

RESUMO

Innate lymphoid cells (ILCs) functionally resemble T lymphocytes in cytotoxicity and cytokine production but lack antigen-specific receptors, and they are important regulators of immune responses and tissue homeostasis. ILCs are generated from common lymphoid progenitors, which are subsequently committed to innate lymphoid lineages in the α-lymphoid progenitor, early innate lymphoid progenitor, common helper innate lymphoid progenitor and innate lymphoid cell progenitor compartments. ILCs consist of conventional natural killer cells and helper-like cells (ILC1, ILC2 and ILC3). Despite recent advances, the cellular heterogeneity, developmental trajectory and signalling dependence of ILC progenitors are not fully understood. Here, using single-cell RNA-sequencing (scRNA-seq) of mouse bone marrow progenitors, we reveal ILC precursor subsets, delineate distinct ILC development stages and pathways, and report that high expression of programmed death 1 (PD-1hi) marked a committed ILC progenitor that was essentially identical to an innate lymphoid cell progenitor. Our data defined PD-1hiIL-25Rhi as an early checkpoint in ILC2 development, which was abolished by deficiency in the zinc-finger protein Bcl11b but restored by IL-25R overexpression. Similar to T lymphocytes, PD-1 was upregulated on activated ILCs. Administration of a PD-1 antibody depleted PD-1hi ILCs and reduced cytokine levels in an influenza infection model in mice, and blocked papain-induced acute lung inflammation. These results provide a perspective for exploring PD-1 and its ligand (PD-L1) in immunotherapy, and allow effective manipulation of the immune system for disease prevention and therapy.


Assuntos
Sequência de Bases , Linhagem da Célula , Imunidade Inata , Linfócitos/citologia , Células Progenitoras Linfoides/citologia , Receptor de Morte Celular Programada 1/metabolismo , Análise de Célula Única , Animais , Anticorpos/imunologia , Diferenciação Celular , Linhagem da Célula/genética , Separação Celular , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Imunoterapia/tendências , Influenza Humana/imunologia , Influenza Humana/metabolismo , Células Matadoras Naturais/citologia , Ativação Linfocitária , Linfócitos/imunologia , Linfócitos/metabolismo , Células Progenitoras Linfoides/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptores de Interleucina/metabolismo , Proteínas Repressoras/deficiência , Proteínas Repressoras/metabolismo , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismo
15.
EMBO Rep ; 20(11): e47967, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31566294

RESUMO

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Distroglicanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Distroglicanas/genética , Edição de Genes , Marcação de Genes , Loci Gênicos , Glicosilação/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem Molecular , Distrofias Musculares/tratamento farmacológico , Distrofias Musculares/etiologia , Distrofias Musculares/metabolismo , Mutação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo
16.
Genome Res ; 27(12): 2061-2071, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29146772

RESUMO

Despite being time-consuming and costly, generating genome-edited pigs holds great promise for agricultural, biomedical, and pharmaceutical applications. To further facilitate genome editing in pigs, we report here establishment of a pig line with Cre-inducible Cas9 expression that allows a variety of ex vivo genome editing in fibroblast cells including single- and multigene modifications, chromosome rearrangements, and efficient in vivo genetic modifications. As a proof of principle, we were able to simultaneously inactivate five tumor suppressor genes (TP53, PTEN, APC, BRCA1, and BRCA2) and activate one oncogene (KRAS), achieved by delivering Cre recombinase and sgRNAs, which caused rapid lung tumor development. The efficient genome editing shown here demonstrates that these pigs can serve as a powerful tool for dissecting in vivo gene functions and biological processes in a temporal manner and for streamlining the production of genome-edited pigs for disease modeling.


Assuntos
Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Endonucleases/genética , Edição de Genes/métodos , Genoma , Porco Miniatura/genética , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas/genética , Feminino , Fibroblastos/metabolismo , Rearranjo Gênico , Genes Supressores de Tumor , Humanos , Integrases/metabolismo , Neoplasias Pulmonares/genética , Masculino , Oncogenes , Suínos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Ativação Transcricional
17.
Stem Cells ; 37(5): 599-608, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30353613

RESUMO

Xist is the master regulator of X chromosome inactivation. In order to further understand the Xist locus in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-activator-like effectors-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which caused higher H3K9me3 followed by X chromosome opening and repression of X-linked genes in mouse embryonic fibroblasts, rather than affecting Xist expression, substantially improved the iPSC generation and the SCNT preimplantation embryo development. Conversely, the dTF activator targeting the same genomic region of R6 decreased iPSC formation and blocked SCNT-embryo development. These results thus uncover the critical requirement for the Xist locus in epigenetic resetting, which is not directly related to Xist transcription. This may provide a unique route to improving the reprogramming. Stem Cells 2019;37:599-608.


Assuntos
Reprogramação Celular/genética , RNA Longo não Codificante/genética , Ativação Transcricional , Inativação do Cromossomo X/genética , Animais , Desenvolvimento Embrionário/genética , Epigenômica , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons/genética , Camundongos , Cromossomo X/genética
18.
Proc Natl Acad Sci U S A ; 114(23): 5800-5807, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28584128

RESUMO

T-cell development from hematopoietic progenitors depends on multiple transcription factors, mobilized and modulated by intrathymic Notch signaling. Key aspects of T-cell specification network architecture have been illuminated through recent reports defining roles of transcription factors PU.1, GATA-3, and E2A, their interactions with Notch signaling, and roles of Runx1, TCF-1, and Hes1, providing bases for a comprehensively updated model of the T-cell specification gene regulatory network presented herein. However, the role of lineage commitment factor Bcl11b has been unclear. We use self-organizing maps on 63 RNA-seq datasets from normal and perturbed T-cell development to identify functional targets of Bcl11b during commitment and relate them to other regulomes. We show that both activation and repression target genes can be bound by Bcl11b in vivo, and that Bcl11b effects overlap with E2A-dependent effects. The newly clarified role of Bcl11b distinguishes discrete components of commitment, resolving how innate lymphoid, myeloid, and dendritic, and B-cell fate alternatives are excluded by different mechanisms.


Assuntos
Diferenciação Celular/genética , Redes Reguladoras de Genes , Proteínas Repressoras/fisiologia , Linfócitos T/citologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Notch , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
19.
Am J Hum Genet ; 99(2): 253-74, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27453576

RESUMO

Intellectual disability (ID) is a common condition with considerable genetic heterogeneity. Next-generation sequencing of large cohorts has identified an increasing number of genes implicated in ID, but their roles in neurodevelopment remain largely unexplored. Here we report an ID syndrome caused by de novo heterozygous missense, nonsense, and frameshift mutations in BCL11A, encoding a transcription factor that is a putative member of the BAF swi/snf chromatin-remodeling complex. Using a comprehensive integrated approach to ID disease modeling, involving human cellular analyses coupled to mouse behavioral, neuroanatomical, and molecular phenotyping, we provide multiple lines of functional evidence for phenotypic effects. The etiological missense variants cluster in the amino-terminal region of human BCL11A, and we demonstrate that they all disrupt its localization, dimerization, and transcriptional regulatory activity, consistent with a loss of function. We show that Bcl11a haploinsufficiency in mice causes impaired cognition, abnormal social behavior, and microcephaly in accordance with the human phenotype. Furthermore, we identify shared aberrant transcriptional profiles in the cortex and hippocampus of these mouse models. Thus, our work implicates BCL11A haploinsufficiency in neurodevelopmental disorders and defines additional targets regulated by this gene, with broad relevance for our understanding of ID and related syndromes.


Assuntos
Proteínas de Transporte/genética , Haploinsuficiência/genética , Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Córtex Cerebral/metabolismo , Montagem e Desmontagem da Cromatina/genética , Códon sem Sentido/genética , Transtornos Cognitivos/genética , Mutação da Fase de Leitura/genética , Hipocampo/metabolismo , Humanos , Deficiência Intelectual/patologia , Deficiência Intelectual/psicologia , Masculino , Camundongos , Microcefalia/genética , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/patologia , Transtornos do Neurodesenvolvimento/fisiopatologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Repressoras , Comportamento Social , Síndrome , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcriptoma
20.
Brain ; 141(8): 2299-2311, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29985992

RESUMO

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.


Assuntos
Transtornos do Neurodesenvolvimento/genética , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Adolescente , Animais , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica/genética , Mutação em Linhagem Germinativa , Haploinsuficiência , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Linfócitos/patologia , Linfócitos/fisiologia , Masculino , Camundongos , Mutação , Proteínas Repressoras/metabolismo , Linfócitos T/fisiologia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/metabolismo
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