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Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Ataxia Telangiectasia/prevenção & controle , Dano ao DNA , Reparo do DNA , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Estresse Oxidativo , Ataxia Telangiectasia/etiologia , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fosforilação , Recuperação de Função FisiológicaRESUMO
While there is a body of experimental data concerning dimers formed by an aromatic molecule and its radical cation, information on the corresponding dimer radical anions (DRAs) is scarce. In this work, evidence for the formation of the DRAs of decafluorobiphenyl and 4-aminononafluorobiphenyl has been obtained by the optically detected electron paramagnetic resonance and the time-resolved magnetic field effect techniques. Theoretical investigation (DFT B3LYP-D3/6-31+G*) of these DRAs and the DRAs of octafluoronaphtalene and 1,2,4,5-tetrafluorobenzene previously detected by Werst has been undertaken to gain greater insight into the structure of the polyfluoroarene DRAs. Without substituents different from a fluorine atom, an extra electron is evenly delocalized over two fragments; the bonding interaction is π stacking. On the potential energy surfaces (PES), there are two minima of nearly equal energy corresponding to the structures of perfect and parallel displaced sandwiches. Such a PES structure is due to a conical intersection between two electronic states of different symmetry. The DRA of 4-aminononafluorobiphenyl is an ion-molecular associate stabilized by electrostatic interactions involving NH2 groups. The complex cyclic structure of the PES of this DRA suits the successive electron transfers between the dimer fragments. The calculated hyperfine coupling constants averaged over the PES minima agree well with the experimental ones.
RESUMO
An analysis of gene expression variability can provide an insightful window into how regulatory control is distributed across the transcriptome. In a single cell analysis, the inter-cellular variability of gene expression measures the consistency of transcript copy numbers observed between cells in the same population. Application of these ideas to the study of early human embryonic development may reveal important insights into the transcriptional programs controlling this process, based on which components are most tightly regulated. Using a published single cell RNA-seq data set of human embryos collected at four-cell, eight-cell, morula and blastocyst stages, we identified genes with the most stable, invariant expression across all four developmental stages. Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression. The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes. Genes with low expression variability at early stages of development are involved in regulation of DNA methylation, responses to hypoxia and telomerase activity, whereas by the blastocyst stage, low-variability genes are enriched for metabolic processes as well as telomerase signaling. Based on changes in expression variability, we identified a putative set of gene expression markers of morulae and blastocyst stages. Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells. Collectively our analyses identified new regulators involved in human embryonic development that would have otherwise been missed using methods that focus on assessment of the average expression levels; in doing so, we highlight the value of studying expression variability for single cell RNA-seq data.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células Cultivadas , Desenvolvimento Embrionário , Humanos , TranscriptomaRESUMO
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with a typical 5 year survival rate of <40 %. DNA methylation in tumour-suppressor genes often occurs at an early stage of tumorigenesis, hence DNA methylation can be used as an early tumour biomarker. Saliva is an ideal diagnostic medium to detect early HNSCC tumour activities due to its proximity to tumour site, non-invasiveness and ease of sampling. We test the hypothesis that the surveillance of DNA methylation in five tumour-suppressor genes (RASSF1α, p16 INK4a , TIMP3, PCQAP/MED15) will allow us to diagnose HNSCC patients from a normal healthy control group as well as to discriminate between Human Papillomavirus (HPV)-positive and HPV-negative patients. METHODS: Methylation-specific PCR (MSP) was used to determine the methylation levels of RASSF1α, p16 INK4a , TIMP3 and PCQAP/MED15 in DNA isolated from saliva. Statistical analysis was carried out using non-parametric Mann-Whitney's U-test for individually methylated genes. A logistic regression analysis was carried out to determine the assay sensitivity when combing the five genes. Further, a five-fold cross-validation with a bootstrap procedure was carried out to determine how well the panel will perform in a real clinical scenario. RESULTS: Salivary DNA methylation levels were not affected by age. Salivary DNA methylation levels for RASSF1α, p16 INK4a , TIMP3 and PCQAP/MED15 were higher in HPV-negative HNSCC patients (n = 88) compared with a normal healthy control group (n = 122) (sensitivity of 71 % and specificity of 80 %). Conversely, DNA methylation levels for these genes were lower in HPV-positive HNSCC patients (n = 45) compared with a normal healthy control group (sensitivity of 80 % and specificity of 74 %), consistent with the proposed aetiology of HPV-positive HNSCCs. CONCLUSIONS: Salivary DNA tumour-suppressor methylation gene panel has the potential to detect early-stage tumours in HPV-negative HNSCC patients. HPV infection was found to deregulate the methylation levels in HPV-positive HNSCC patients. Large-scale double-blinded clinical trials are crucial before this panel can potentially be integrated into a clinical setting.
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Radical anions (RAs) are the key intermediates of the selective hydrodefluorination of polyfluoroarenes. We used the techniques of optically detected electron paramagnetic resonance (OD EPR), time-resolved fluorescence, time-resolved magnetic field effect (TR MFE), and the density functional theory to study the possibility of RAs formation from 4-aminononafluorobiphenyl (1) and pentafluoroaniline (2) and estimate their lifetimes and decay channels. To our knowledge, both RAs have not been detected earlier. We have registered the OD EPR spectrum for relatively stable in nonpolar solutions 1(-â¢) but failed to register the spectra for 2(-â¢). However, we have managed to fix the 2(-â¢) by the TR MFE method and obtained its hyperfine coupling constants. The lifetime of 2(-â¢) was found to be only a few nanoseconds. The activation energy of its decay was estimated to be 3.6 ± 0.3 kcal/mol. According to the calculation results, the short lifetime of 2(-â¢) is due to the RA fast fragmentation with the F(-) elimination from ortho-position to the amine group. The calculated energy barrier, 3.2 kcal/mol, is close to the experimental value. The fragmentation of 2(-â¢) in a nonpolar solvent is possible due to the stabilization of the incipient F(-) anion by the binding with the amine group proton.
Assuntos
Compostos de Anilina/química , Compostos de Bifenilo/química , Fluorescência , Teoria Quântica , Ânions/química , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Campos Magnéticos , Estrutura Molecular , Fatores de TempoRESUMO
Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.
Assuntos
Síndrome de Down/etiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Diferenciação Celular/fisiologia , Síndrome de Down/genética , Síndrome de Down/patologia , Feminino , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Neuritos/patologia , Neuritos/fisiologia , Neurogênese , Neurônios/patologia , Neurônios/fisiologia , TranscriptomaRESUMO
Many developmental and epileptic encephalopathies (DEEs) result from variants in cation channel genes. Using mRNA transfection, we generated and characterised an induced pluripotent stem cell (iPSC) line from the fibroblasts of a male late-onset DEE patient carrying a heterozygous missense variant (E1211K) in Nav1.2(SCN2A) protein. The iPSC line displays features characteristic of the human iPSCs, colony morphology and expression of pluripotency-associated marker genes, ability to produce derivatives of all three embryonic germ layers, and normal karyotype without SNP array-detectable abnormalities. We anticipate that this iPSC line will aid in the modelling and development of precision therapies for this debilitating condition.
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Encefalopatias , Células-Tronco Pluripotentes Induzidas , Humanos , Masculino , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação de Sentido Incorreto , Heterozigoto , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2/genéticaRESUMO
We employed a Sendai virus-based reprogramming method to transform human lymphoblastoid cell lines (LCL) derived from two individuals diagnosed with phenylketonuria (PKU) into induced pluripotent stem cells (iPSC). This reprogramming process involved the expression of the four Yamanaka factors: KLF4, OCT4, SOX2, and C-MYC. The resulting patient-specific iPSCs exhibited a normal karyotype and expressed endogenous pluripotent markers NANOG and OCT-4. Notably, these iPSCs demonstrated strong differentiation capabilities, giving rise to cell populations representing the ectoderm, endoderm, and mesoderm germ layers.
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Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Fator 4 Semelhante a Kruppel , Fenilcetonúrias , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fenilcetonúrias/metabolismo , Fenilcetonúrias/patologia , Linhagem Celular , Masculino , Linfócitos/metabolismo , Reprogramação CelularRESUMO
Phenylketonuria is a rare autosomal recessive metabolic disorder mainly due to a significant reduction in the enzyme phenylalanine hydroxylase, resulting in elevation of phenylalanine in the blood. Here, we have established two fibroblast-derived induced pluripotent stem cell lines using Sendai virus-based reprogramming. The established induced pluripotent stem cell lines exhibited a normal karyotype and expressed markers of pluripotency assessed through quantitative PCR, flow cytometry and immunocytochemistry. These cell lines also demonstrated the ability to differentiate into the three primary germ layers of the human body, including ectoderm, endoderm, and mesoderm.
Assuntos
Diferenciação Celular , Fibroblastos , Células-Tronco Pluripotentes Induzidas , Fenilcetonúrias , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fenilcetonúrias/metabolismo , Fenilcetonúrias/patologia , Fibroblastos/metabolismo , Linhagem Celular , Masculino , CriançaRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which the TDP-43 protein is believed to play a central role in disease pathophysiology. Using the CRISPR-Cas9 system, we introduced the heterozygous c.1144G > A (p.A382T) missense mutation in exon 6 of the TARDBP gene into an iPSC line derived from a healthy individual. These edited iPSCs displayed normal cellular morphology, expressed major pluripotency markers, were capable of tri-lineage differentiation, and possessed a normal karyotype.
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Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genéticaRESUMO
Midbrain dopamine (mDA) neurons can be replaced in patients with Parkinson's disease (PD) in order to provide long-term improvement in motor functions. The limited capacity for long-distance axonal growth in the adult brain means that cells are transplanted ectopically, into the striatal target. As a consequence, several mDA pathways are not re-instated, which may underlie the incomplete restoration of motor function in patients. Here, we show that viral delivery of GDNF to the striatum, in conjunction with homotopic transplantation of human pluripotent stem-cell-derived mDA neurons, recapitulates brain-wide mDA target innervation. The grafts provided re-instatement of striatal dopamine levels and correction of motor function and also connectivity with additional mDA target nuclei not well innervated by ectopic grafts. These results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types.
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Dopamina , Células-Tronco Pluripotentes , Adulto , Dopamina/metabolismo , Terapia Genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Mesencéfalo/metabolismo , Células-Tronco Pluripotentes/metabolismo , Substância Negra/metabolismo , Substância Negra/transplanteRESUMO
BACKGROUND: Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers. It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-expressing stem cells, these so-called "tumour-initiating" cells, reminiscent in their properties of the normal intestinal stem cells (ISCs), may explain the apparent heterogeneity of colon cancer cell lines. Also, colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5. Furthermore, in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell. AIM: To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells. METHODS: A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines (Caco-2 and LoVo). These cells were then characterized according to their proliferation capacity, gene expression signatures of ISC markers, and their tumorigenic properties in vivo and in vitro. RESULTS: The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2, but not from the LoVo, cell lines, in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog (+4 crypt) stem cells. This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny. Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein (EYFP) from this cell line exhibit tumorigenic-like properties in vivo and in vitro. In contrast, cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties. Thus, cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells, but not LoVo cells. CONCLUSION: Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines. Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data. We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.
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Neoplasias do Colo , Animais , Células CACO-2 , Neoplasias do Colo/genética , Proteínas de Ligação ao GTP , Humanos , Leucina , Camundongos , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
With a view towards harnessing the therapeutic potential of canine mesenchymal stromal cells (cMSCs) as modulators of inflammation and the immune response, and to avoid the issues of the variable quality and quantity of harvested cMSCs, we examined the immunomodulatory properties of cMSCs derived from canine induced pluripotent stem cells (ciMSCs), and compared them to cMSCs harvested from adipose tissue (cAT-MSC) and bone marrow (cBM-MSC). A combination of deep sequencing and quantitative RT-PCR of the ciMSC transcriptome confirmed that ciMSCs express more genes in common with cBM-MSCs and cAT-MSCs than with the ciPSCs from which they were derived. Both ciMSCs and harvested cMSCs express a range of pluripotency factors in common with the ciPSCs including NANOG, POU5F1 (OCT-4), SOX-2, KLF-4, LIN-28A, MYC, LIF, LIFR, and TERT. However, ESRRB and PRDM-14, both factors associated with naïve, rather than primed, pluripotency were expressed only in the ciPSCs. CXCR-4, which is essential for the homing of MSCs to sites of inflammation, is also detectable in ciMSCs, cAT- and cBM-MSCs, but not ciPSCs. ciMSCs constitutively express the immunomodulatory factors iNOS, GAL-9, TGF-ß1, PTGER-2α and VEGF, and the pro-inflammatory mediators COX-2, IL-1ß and IL-8. When stimulated with the canine pro-inflammatory cytokines tumor necrosis factor-α (cTNF-α), interferon-γ (cIFN-γ), or a combination of both, ciMSCs upregulated their expression of IDO, iNOS, GAL-9, HGF, TGF-ß1, PTGER-2α, VEGF, COX-2, IL-1ß and IL-8. When co-cultured with mitogen-stimulated lymphocytes, ciMSCs downregulated their expression of iNOS, HGF, TGF-ß1 and PTGER-2α, while increasing their expression of COX-2, IDO and IL-1ß. Taken together, these findings suggest that ciMSCs possess similar immunomodulatory capabilities as harvested cMSCs and support further investigation into their potential use for the management of canine immune-mediated and inflammatory disorders.
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Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Mesenquimais/fisiologia , Tecido Adiposo/citologia , Animais , Anti-Inflamatórios/metabolismo , Células da Medula Óssea/fisiologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/imunologia , Citocinas/metabolismo , Cães , Regulação da Expressão Gênica , Fatores Imunológicos/metabolismo , Ativação Linfocitária , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , TranscriptomaRESUMO
Stem Cell Research is pleased to introduce into its publication portfolio a new article type: a template-driven short report on the generation of a novel Genetically Modified Cell Line. This resource type is typically derived from human pluripotent stem cell lines via the introduction of nucleases and/or foreign genetic material leading to stable genomic alterations, maintained in a single cell-derived clonal cell line. Interest in, and demand for, genetically modified cell lines has grown exponentially in the last few years. This overview provides a brief introduction to this incredibly versatile lab resource and marks the beginning of a new and exciting addition to the publication portfolio of Stem Cell Research. A dramatic increase in the accessibility of the human genome in the last decade has given a long-anticipated boost to advanced biomedical studies in human in vitro systems. Pluripotent stem cells represent a particularly attractive gateway into this line of experimentation due to their unique suitability for the isolation of clonal genetically modified cell lines (GMCLs), and the ability to be differentiated into essentially any cell type upon the lines' virtually limitless expansion.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes , Linhagem Celular , Endonucleases/genética , Genoma Humano , Humanos , Células-Tronco Pluripotentes/metabolismoRESUMO
Marsupials have long attracted scientific interest because of their unique biological features and their position in mammalian evolution. Mesenchymal stem cells (MSCs) are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory, and regenerative properties. MSCs have been harvested from various tissues in numerous eutherian species; however, there are no descriptions of MSCs derived from a marsupial. In this study, we have generated Tasmanian devil (Sarcophilus harrisii) MSCs from devil induced pluripotent stem cells (iPSCs), thus providing an unlimited source of devil MSCs and circumventing the need to harvest tissues from live animals. Devil iPSCs were differentiated into MSCs (iMSCs) through both embryoid body formation assays (EB-iMSCs) and through inhibition of the transforming growth factor beta/activin signaling pathway (SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly proliferative and express the MSC-specific surface proteins CD73, CD90, and CD105, in addition to the pluripotency transcription factors OCT4/POU5F1, SOX2, and NANOG. Expression of the marsupial pluripotency factor POU5F3, a paralogue of OCT4/POU5F1, is significantly reduced in association with the transition from pluripotency to multipotency. Devil iMSCs readily differentiate along the adipogenic, osteogenic, and chondrogenic pathways in vitro, confirming their trilineage differentiation potential. Importantly, in vitro teratoma assays confirmed their multipotency, rather than pluripotency, since the iMSCs only formed derivatives of the mesodermal germ layer. Devil iMSCs show a tropism toward medium conditioned by devil facial tumor cells and express a range of immunomodulatory and anti-inflammatory factors. Therefore, devil iMSCs will be a valuable tool for further studies on marsupial biology and may facilitate the development of an MSC-based treatment strategy against Devil Facial Tumor Disease.
Assuntos
Neoplasias Faciais/genética , Fatores Imunológicos/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Marsupiais/genética , Células-Tronco Mesenquimais/metabolismo , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adipogenia/genética , Animais , Condrogênese/genética , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Endoglina/genética , Endoglina/metabolismo , Neoplasias Faciais/metabolismo , Neoplasias Faciais/patologia , Expressão Gênica , Fatores Imunológicos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Marsupiais/metabolismo , Células-Tronco Mesenquimais/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteogênese/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Tropismo/genéticaRESUMO
We generated double-transgenic mice carrying cointegrated tissue-specific Gal4 and Gal4 reporter transgenes to direct transgene overexpression in the mononuclear phagocyte system (MPS). A modified promoter of the Csf1r (c-fms) gene, containing a deletion of the trophoblast-specific promoter, was used to drive the expression of Gal4VP16 transcriptional activator specifically in macrophages. This module was cointegrated with a fluorescent reporter, enhanced cyan fluorescent protein (ECFP), driven by a Gal4-dependent promoter. ECFP fluorescence was first detected in forming blood islands of the yolk sac at 8 dpc, then in macrophages in the yolk sac and the embryo proper. In adult mice ECFP was detected primarily in monocytes, tissue macrophages, microglia, and dendritic cells, including Langerhans cells of the skin. Crossing of these mice to transgenics containing tagged protein under control of a Gal4-dependent promoter directed expression of that protein in mononuclear phagocytes of double-transgenic animals. The new mouse line provides a useful tool for overexpression of transgenes in cells of the myeloid lineage, while simultaneously labeling them by ECFP expression.
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Células Dendríticas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Genes Sintéticos/genética , Genes fms , Proteínas de Fluorescência Verde/genética , Macrófagos/metabolismo , Camundongos Transgênicos/genética , Microglia/metabolismo , Monócitos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transgenes , Animais , Linhagem Celular/metabolismo , Linhagem da Célula , Cruzamentos Genéticos , Proteínas de Ligação a DNA , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos/embriologia , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Ativação Transcricional , Transgenes/genética , Saco Vitelino/citologiaRESUMO
The platypus (Ornithorhynchus anatinus) is an egg-laying monotreme mammal whose ancestors diverged â¼166 million years ago from the evolutionary pathway that eventually gave rise to both marsupial and eutherian mammals. Consequently, its genome is an extraordinary amalgam of both ancestral reptilian and derived mammalian features. To gain insight into the evolution of mammalian pluripotency, we have generated induced pluripotent stem cells from the platypus (piPSCs). Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors POU5F1/OCT4, SOX2, and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 and 166 million years ago, primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. Using the RNAseq data, we were also able to demonstrate that in both fibroblasts and piPSCs, the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1, indicating that there is no upregulation of X-linked genes. Finally, the RNAseq data also allowed us to explore the process of X-linked gene inactivation in the platypus, where we determined that for any given gene, there is no preference for silencing of the maternal or paternal allele; that is, within a population of cells, the silencing of X-linked genes is not imprinted.
Assuntos
Diferenciação Celular , Ornitorrinco , Células-Tronco Pluripotentes/citologia , Transcriptoma , Animais , Células Cultivadas , Receptor Nuclear Órfão DAX-1/genética , Receptor Nuclear Órfão DAX-1/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Impressão Genômica , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOX/metabolismo , Inativação do Cromossomo XRESUMO
Originally recognized as an essential part of the innate and acquired immune systems, macrophages emerged as omnipresent and influential regulators of embryo- and organo-genesis, as well as of tissue and tumor growth. Macrophages are present essentially in all tissues, beginning with embryonic development and, in addition to their role in host defense and in the clearance of apoptotic cells, are being increasingly recognized for their trophic function and role in regeneration. Some tissue macrophages are also found to possess a substantial potential for autonomous self-renewal. Macrophages are associated with a significant proportion of malignant tumors and are widely recognized for their angiogenesis-promoting and trophic roles, making them one of the new promising targets for cancer therapies. Recent expression profiling of embryonic macrophages from different tissues revealed remarkable consistency of their gene expression profiles, independent of their tissue of origin, as well as their similarities with tumor-associated macrophages. Macrophages are also capable of fusion with other cells in tissue repair and metastasizing tumors, as well as with each other in the immune response and osteoclastogenesis.
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Desenvolvimento Embrionário , Macrófagos/metabolismo , Fagócitos , Animais , Diferenciação Celular , Transformação Celular Neoplásica , Humanos , Macrófagos/citologiaRESUMO
We demonstrate the generation of Tasmanian devil (Sarcophilus harrisii) induced pluripotent stem cells (DeviPSCs) from dermal fibroblasts by lentiviral delivery of human transcription factors. DeviPSCs display characteristic pluripotent stem cell colony morphology, with individual cells having a high nuclear-to-cytoplasmic ratio and alkaline phosphatase activity. DeviPSCs are leukemia inhibitory factor dependent and have reactivated endogenous octamer-binding transcription factor 4 [OCT4, POU domain, class 5, transcription factor 1 (POU5F1)], POU2 [POU domain, class 5, transcription factor 3 (POU5F3)], sex determining region Y-box 2 (SOX2), Nanog homeobox (NANOG) and dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) genes, retained a normal karyotype, and concurrently silenced exogenous human transgenes. Notably, co-expression of both OCT4 and POU2 suggests that they are representative of cells of the epiblast, the marsupial equivalent of the inner cell mass. DeviPSCs readily form embryoid bodies and in vitro teratomas containing derivatives of all three embryonic germ layers. To date, DeviPSCs have been stably maintained for more than 45 passages. Our DeviPSCs provide an invaluable resource for studies into marsupial pluripotency and development, and they may also serve as an important tool in efforts to combat the threat of devil facial tumor disease.
Assuntos
Evolução Biológica , Técnicas de Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Marsupiais/metabolismo , Fatores de Transcrição/biossíntese , Transdução Genética , Animais , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Marsupiais/genética , Fatores de Transcrição/genéticaRESUMO
Early-onset Alzheimer disease (AD)-like pathology in Down syndrome is commonly attributed to an increased dosage of the amyloid precursor protein (APP) gene. To test this in an isogenic human model, we deleted the supernumerary copy of the APP gene in trisomic Down syndrome induced pluripotent stem cells or upregulated APP expression in euploid human pluripotent stem cells using CRISPRa. Cortical neuronal differentiation shows that an increased APP gene dosage is responsible for increased ß-amyloid production, altered Aß42/40 ratio, and deposition of the pyroglutamate (E3)-containing amyloid aggregates, but not for several tau-related AD phenotypes or increased apoptosis. Transcriptome comparisons demonstrate that APP has a widespread and temporally modulated impact on neuronal gene expression. Collectively, these data reveal an important role for APP in the amyloidogenic aspects of AD but challenge the idea that increased APP levels are solely responsible for increasing specific phosphorylated forms of tau or enhanced neuronal cell death in Down syndrome-associated AD pathogenesis.