RESUMO
Inhibition of E-cad in mouse embryonic stem cells (mESCs) leads to a switch from LIF-BMP to Activin/Nodal-dependent pluripotency, consistent with transition from a naïve to primed pluripotent phenotype. We have used both genetic ablation and steric inhibition of E-cad function in mESCs to assess alterations to phenotype using quantitative mass spectrometry analysis, network models, and functional assays. Proteomic analyses revealed that one third of detected proteins were altered in E-cad null mESCs (Ecad-/- mESCs) compared to wild type (624 proteins were downregulated and 705 were proteins upregulated). Network pathway analysis and subsequent cellular flux assays confirmed a metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, specifically through mitochondrial complex III downregulation and hypoxia inducible factor 1a target upregulation. Central to this was the transcriptional coactivator EP300. E-cad is a well-known tumor suppressor, its downregulation during cancer initiation and metastasis can be linked to the metabolic switch known as Warburg effect. This study highlights a phenomena found in both primed pluripotent state and cancer stemness and links it to loss of E-cad. Data are available via ProteomeXchange with identifier PXD012679.
Assuntos
Caderinas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Ciclo Celular/genética , Células Cultivadas , Proteína p300 Associada a E1A/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Glicólise , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , Proteoma/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/genética , HIV-1/genética , RNA Viral , Sequências Reguladoras de Ácido Ribonucleico , Transdução Genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Fator IX/genética , Expressão Gênica , Ordem dos Genes , Genes Reporter , Terapia Genética , Genoma Viral , Repetição Terminal Longa de HIV , Hemofilia B/sangue , Hemofilia B/genética , Hemofilia B/terapia , Humanos , Camundongos , Provírus/genética , Recombinação Genética , Transgenes , Replicação Viral/genéticaRESUMO
Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse Bmi-1 + hTERT, but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of human BMI-1 alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. Cystic fibrosis (CF) and non-CF bronchial epithelial cells were transduced by lentivirus with BMI-1 and then their morphology, replication kinetics, and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function, and transepithelial electrophysiology were measured. Finally, shRNA knockdown of DNAH5 in BMI-1 cells was used to model primary ciliary dyskinesia (PCD). BMI-1-transduced basal cells showed normal cell morphology, karyotype, and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics of BMI-1-transduced cells were similar to those of untransduced cells. shRNA knockdown of DNAH5 in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination.
Assuntos
Brônquios/citologia , Diferenciação Celular , Cílios/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Muco/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Animais , Dineínas do Axonema/metabolismo , Proliferação de Células , Forma Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Dineínas/metabolismo , Impedância Elétrica , Fenômenos Eletrofisiológicos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Síndrome de Kartagener/fisiopatologia , Cariotipagem , Camundongos , Microtúbulos/metabolismo , Modelos Biológicos , Fenótipo , Transdução GenéticaRESUMO
Angiogenesis is essential for the development of a normal vasculature, tissue repair and reproduction, and also has roles in the progression of diseases such as cancer and rheumatoid arthritis. The heparan sulphate proteoglycan syndecan-2 is expressed on mesenchymal cells in the vasculature and, like the other members of its family, can be shed from the cell surface resulting in the release of its extracellular core protein. The purpose of this study was to establish whether shed syndecan-2 affects angiogenesis. We demonstrate that shed syndecan-2 regulates angiogenesis by inhibiting endothelial cell migration in human and rodent models and, as a result, reduces tumour growth. Furthermore, our findings show that these effects are mediated by the protein tyrosine phosphatase receptor CD148 (also known as PTPRJ) and this interaction corresponds with a decrease in active ß1 integrin. Collectively, these data demonstrate an unexplored pathway for the regulation of new blood vessel formation and identify syndecan-2 as a therapeutic target in pathologies characterised by angiogenesis.
Assuntos
Neovascularização Patológica/metabolismo , Sindecana-2/metabolismo , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos SCID , Sindecana-2/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics.
Assuntos
Diferenciação Celular , Expressão Gênica , Vetores Genéticos/genética , Plasmídeos/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos , Perfilação da Expressão Gênica , Humanos , Camundongos , TransgenesRESUMO
CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Fosfofrutoquinase-2/metabolismo , Animais , Autofagia , Pré-Escolar , Modelos Animais de Doenças , Feminino , Humanos , Doenças por Armazenamento dos Lisossomos/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Lipofuscinoses Ceroides Neuronais/genética , Neurônios/metabolismo , Fosfofrutoquinase-2/genética , Regulação para CimaRESUMO
The transforming growth factor-ß (TGFß) family plays a critical regulatory role in repair and coordination of remodeling after cutaneous wounding. TGFß1-mediated chemotaxis promotes the recruitment of fibroblasts to the wound site and their resultant myofibroblastic transdifferentiation that is responsible for elastic fiber deposition and wound closure. TGFß3 has been implicated in an antagonistic role regulating overt wound closure and promoting ordered dermal remodeling. We generated a mutant form of TGFß3 (mutTGFß3) by ablating its binding site for the latency-associated TGFß binding protein (LTBP-1) in order to improve bioavailability and activity. The mutated cytokine is secreted as the stable latency-associated peptide (LAP)-associated form and is activated by normal intracellular and extracellular mechanisms including integrin-mediated activation but is not sequestered. We show localized intradermal transduction using a lentiviral vector expressing the mutTGFß3 in a mouse skin wounding model reduced re-epithelialization density and fibroblast/myofibroblast transdifferentiation within the wound area, both indicative of reduced scar tissue formation.
Assuntos
Terapia Genética , Fator de Crescimento Transformador beta3/genética , Cicatrização/fisiologia , Animais , Cicatriz/patologia , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Camundongos , Mutação , Pele/patologia , Cicatrização/genéticaRESUMO
The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.
RESUMO
The Neuronal Ceroid Lipofuscinoses (NCL), otherwise known as Batten disease, are a group of neurodegenerative diseases caused by mutations in 13 known genes. All except one NCL is autosomal recessive in inheritance, with similar aetiology and characterised by the accumulation of autofluorescent storage material in the lysosomes of cells. Age of onset and the rate of progression vary between the NCLs. They are collectively one of the most common lysosomal storage diseases, but the enigma remains of how genetically distinct diseases result in such remarkably similar pathogenesis. Much has been learnt from cellular studies about the function of the proteins encoded by the affected genes. Such research has utilised primitive unicellular models such as yeast and amoeba containing gene orthologues, cells derived from naturally occurring (sheep) and genetically engineered (mouse) animal models or patient-derived cells. Most recently, patient-derived induced pluripotent stem cell (iPSC) lines have been differentiated into neural cell-types to study molecular pathogenesis in the cells most profoundly affected by disease. Here, we review how cell models have informed much of the biochemical understanding of the NCLs and how more complex models are being used to further this understanding and potentially act as platforms for therapeutic efficacy studies in the future.
Assuntos
Modelos Biológicos , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Animais , Modelos Animais de Doenças , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Lipofuscinoses Ceroides Neuronais/genéticaRESUMO
In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice. Further, we describe whole body bioluminescence imaging of conscious mice in a noninvasive manner, thus enforcing the 3R's (replacement, reduction, and refinement) of biomedical animal research.
Assuntos
Expressão Gênica , Genes Reporter , Medições Luminescentes/métodos , Animais , Técnicas Biossensoriais , Ordem dos Genes , Vetores Genéticos/genética , Luciferases de Vaga-Lume/genética , Camundongos , Plasmídeos/genética , Transfecção , TransgenesRESUMO
Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The in vitro whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.
RESUMO
We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Camundongos Transgênicos/genética , Animais , Técnicas Biossensoriais/métodos , Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Inflamação/genética , Luciferases de Vaga-Lume/genética , Camundongos , NF-kappa B/genética , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Vírus Formadores de Foco no Baço/genética , Transcrição Gênica/genéticaRESUMO
The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471-39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.
Assuntos
Condrócitos/fisiologia , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Actinas/metabolismo , Cartilagem Articular/citologia , Células Cultivadas , Colágeno Tipo II/metabolismo , Humanos , Concentração Osmolar , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Fatores de Transcrição SOX9/genéticaRESUMO
We investigated Notch signaling during chondrogenesis in human bone marrow stromal cells (hMSC) in three-dimensional cell aggregate culture. Expression analysis of Notch pathway genes in 14-day chondrogenic cultures showed that the Notch ligand Jagged-1 (Jag-1) sharply increased in expression, peaking at day 2, and then declined. A Notch target gene, HEY-1, was also expressed, with a temporal profile that closely followed the expression of Jag-1, and this preceded the rise in type II collagen expression that characterized chondrogenesis. We demonstrated that the shut-down in Notch signaling was critical for full chondrogenesis, as adenoviral human Jag-1 transduction of hMSC, which caused continuous elevated expression of Jag-1 and sustained Notch signaling over 14 days, completely blocked chondrogenesis. In these cultures, there was inhibited production of extracellular matrix, and the gene expression of aggrecan and type II collagen were strongly suppressed; this may reflect the retention of a prechondrogenic state. The JAG-1-mediated Notch signaling was also shown to be necessary for chondrogenesis, as N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT) added to cultures on days 0-14 or just days 0-5 inhibited chondrogenesis, but DAPT added from day 5 did not. The results thus showed that Jag-1-mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but it must be switched off for chondrogenesis to proceed.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Condrogênese , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Agregação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Dipeptídeos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Ligantes , Proteínas de Membrana/genética , Transporte Proteico/efeitos dos fármacos , Receptores Notch/genética , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Transdução GenéticaRESUMO
The Hippo pathway is an important regulator of cell growth, proliferation, and migration. TEAD transcription factors, which lie at the core of the Hippo pathway, are essential for regulation of organ growth and wound repair. Dysregulation of TEAD and its regulatory cofactor Yes-associated protein (YAP) have been implicated in numerous human cancers and hyperproliferative pathological processes. Hence, the YAP-TEAD complex is a promising therapeutic target. Here, we use in silico molecular docking using Bristol University Docking Engine to screen a library of more than 8 million druglike molecules for novel disrupters of the YAP-TEAD interaction. We report the identification of a novel compound (CPD3.1) with the ability to disrupt YAP-TEAD protein-protein interaction and inhibit TEAD activity, cell proliferation, and cell migration. The YAP-TEAD complex is a viable drug target, and CPD3.1 is a lead compound for the development of more potent TEAD inhibitors for treating cancer and other hyperproliferative pathologies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Simulação de Acoplamento Molecular , Fatores de Transcrição/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Simulação por Computador , Expressão Gênica/efeitos dos fármacos , Humanos , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
Treatment of the neuronal ceroid lipofuscinoses, also known as Batten disease, is at the start of a new era because of diagnostic and therapeutic advances relevant to this group of inherited neurodegenerative and life-limiting disorders that affect children. Diagnosis has improved with the use of comprehensive DNA-based tests that simultaneously screen for many genes. The identification of disease-causing mutations in 13 genes provides a basis for understanding the molecular mechanisms underlying neuronal ceroid lipofuscinoses, and for the development of targeted therapies. These targeted therapies include enzyme replacement therapies, gene therapies targeting the brain and the eye, cell therapies, and pharmacological drugs that could modulate defective molecular pathways. Such therapeutic developments have the potential to enable earlier diagnosis and better targeted therapeutic management. The first approved treatment is an intracerebroventricularly administered enzyme for neuronal ceroid lipofuscinosis type 2 disease that delays symptom progression. Efforts are underway to make similar progress for other forms of the disorder.
Assuntos
Terapia de Reposição de Enzimas , Terapia Genética/métodos , Lipofuscinoses Ceroides Neuronais/terapia , Progressão da Doença , Humanos , Mutação , Lipofuscinoses Ceroides Neuronais/tratamento farmacológico , Lipofuscinoses Ceroides Neuronais/genéticaRESUMO
BACKGROUND: Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. OBJECTIVE: We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. METHODS: Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. RESULTS: Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and ßIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. CONCLUSION: We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy.
RESUMO
Human neural development begins at embryonic day 19 and marks the beginning of organogenesis. Neural stem cells in the neural tube undergo profound functional, morphological, and metabolic changes during neural specification, coordinated by a combination of exogenous and endogenous cues. The temporal cell signaling activities that mediate this process, during development and in the postnatal brain, are incompletely understood. We have applied gene expression studies and transcription factor-activated reporter lentiviruses during in vitro neural specification of human pluripotent stem cells. We show that nuclear factor κB orchestrates a multi-faceted metabolic program necessary for the maturation of neural progenitor cells during neurogenesis.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Metabolismo Energético , NF-kappa B/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Autofagia , Biomarcadores , Ciclo Celular , Diferenciação Celular/genética , Células Cultivadas , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Imuno-Histoquímica , Modelos Biológicos , Neurogênese/genética , Fenótipo , Transdução de SinaisRESUMO
The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-ß subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1-negative lung cancers.
Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Neoplasias Pulmonares/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.