RESUMEN
Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.
Asunto(s)
Encefalopatías Metabólicas Innatas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virología , ARN/química , ARN/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Encefalopatías Metabólicas Innatas/patología , Tronco Encefálico/patología , Encefalitis Viral/genética , Escherichia coli/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Herpesvirus Humano 1 , Humanos , Interferones/metabolismo , Intrones/genética , Masculino , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Sistemas de Lectura Abierta/genética , Linaje , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/deficiencia , ARN Nucleotidiltransferasas/genética , Receptor Toll-Like 3/metabolismo , Replicación ViralRESUMEN
In recent years, the electronic structures of organocuprates in general and the complex [Cu(CF3)4]- in particular have attracted significant interest. A possible key indicator in this context is the reactivity of these species. Nonetheless, this aspect has received only limited attention. Here, we systematically study the series of tetra-alkyl cuprates [MenCu(CF3)4-n]- and their unimolecular reactivity in the gas phase, which includes concerted formal reductive eliminations as well as radical losses. Through computational studies, we characterize the electronic structures of the complexes and show how these are connected to their reactivity. We find that all [MenCu(CF3)4-n]- ions feature inverted ligand fields and that the distinct reactivity patterns of the individual complexes arise from the interplay of different effects.
RESUMEN
The cuprate complexes [Cu(R)(CF3 )3 ]- (R=organyl) offer an efficient synthetic access to valuable trifluoromethylation products RCF3 . Here, electrospray-ionization mass spectrometry is used to analyze the formation of these intermediates in solution and probe their fragmentation pathways in the gas phase. Furthermore, the potential energy surfaces of these systems are explored by quantum chemical calculations. Upon collisional activation, the [Cu(R)(CF3 )3 ]- complexes (R=Me, Et, Bu, s Bu, allyl) afford the product ions [Cu(CF3 )3 ]â - and [Cu(CF3 )2 ]- . The former obviously results from an Râ loss, whereas the latter originates either from the stepwise release of Râ and CF3 â radicals or a concerted reductive elimination of RCF3 . The gas-phase fragmentation experiments as well as the quantum chemical calculations indicate that the preference for the stepwise reaction toward [Cu(CF3 )2 ]- increases with the stability of the formed organyl radical Râ . This finding suggests that the recombination of Râ and CF3 â radicals may possibly contribute to the formation of RCF3 from [Cu(R)(CF3 )3 ]- in synthetic applications. In contrast, the [Cu(R)(CF3 )3 ]- complexes (R=aryl) only yield [Cu(CF3 )2 ]- when subjected to collision-induced dissociation. These species exclusively undergo a concerted reductive elimination because the competing stepwise pathway is disfavored by the low stability of aryl radicals.
RESUMEN
The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord. The ENS has been called the 'second brain' given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrb(s-l/s-l)), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.
Asunto(s)
Linaje de la Célula , Tratamiento Basado en Trasplante de Células y Tejidos , Descubrimiento de Drogas/métodos , Sistema Nervioso Entérico/patología , Enfermedad de Hirschsprung/tratamiento farmacológico , Enfermedad de Hirschsprung/patología , Neuronas/patología , Envejecimiento , Animales , Diferenciación Celular , Línea Celular , Movimiento Celular , Separación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Embrión de Pollo , Colon/efectos de los fármacos , Colon/patología , Modelos Animales de Enfermedad , Femenino , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/patología , Enfermedad de Hirschsprung/terapia , Humanos , Masculino , Ratones , Neuronas/efectos de los fármacos , Pepstatinas/metabolismo , Células Madre Pluripotentes/patología , Receptor de Endotelina B/metabolismo , Transducción de SeñalRESUMEN
Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Some HSE children carry inborn errors of the Toll-like receptor 3 (TLR3)-dependent IFN-α/ß- and -λ-inducing pathway. Induced pluripotent stem cell (iPSC)-derived cortical neurons with TLR3 pathway mutations are highly susceptible to HSV-1, due to impairment of cell-intrinsic TLR3-IFN immunity. In contrast, the contribution of cell-intrinsic immunity of human trigeminal ganglion (TG) neurons remains unclear. Here, we describe efficient in vitro derivation and purification of TG neurons from human iPSCs via a cranial placode intermediate. The resulting TG neurons are of sensory identity and exhibit robust responses to heat (capsaicin), cold (icilin), and inflammatory pain (ATP). Unlike control cortical neurons, both control and TLR3-deficient TG neurons were highly susceptible to HSV-1. However, pretreatment of control TG neurons with poly(I:C) induced the cells into an anti-HSV-1 state. Moreover, both control and TLR3-deficient TG neurons developed resistance to HSV-1 following pretreatment with IFN-ß but not IFN-λ. These data indicate that TG neurons are vulnerable to HSV-1 because they require preemptive stimulation of the TLR3 or IFN-α/ß receptors to induce antiviral immunity, whereas cortical neurons possess a TLR3-dependent constitutive resistance that is sufficient to block incoming HSV-1 in the absence of prior antiviral signals. The lack of constitutive resistance in TG neurons in vitro is consistent with their exploitation as a latent virus reservoir in vivo. Our results incriminate deficiencies in the constitutive TLR3-dependent response of cortical neurons in the pathogenesis of HSE.
Asunto(s)
Inmunidad/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Receptor Toll-Like 3/metabolismo , Antivirales/farmacología , Diferenciación Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Niño , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Humanos , Inmunidad/genética , Células Madre Pluripotentes Inducidas/citología , Interferón beta/farmacología , Mutación , Neuronas/efectos de los fármacos , Neuronas/virología , Poli I-C/farmacología , Receptor Toll-Like 3/genética , Ganglio del Trigémino/citologíaRESUMEN
Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.
Asunto(s)
Anomalías Inducidas por Medicamentos/genética , Células Madre Embrionarias/metabolismo , Epigénesis Genética/efectos de los fármacos , Placa Neural/embriología , Anomalías Inducidas por Medicamentos/patología , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Madre Embrionarias/fisiología , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Metilación , Proteína Homeótica Nanog , Placa Neural/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Células Neuroepiteliales/metabolismo , Células Neuroepiteliales/fisiología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Análisis de Componente Principal , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transcripción Genética , Transcriptoma , Ácido Valproico/efectos adversosRESUMEN
The directed generation of pure astrocyte cultures from pluripotent stem cells has proven difficult. Generation of defined pluripotent-stem-cell derived astrocytes would allow new approaches to the investigation of plasticity and heterogeneity of astrocytes. We here describe a two-step differentiation scheme resulting in the generation of murine embryonic stem cell (mESC) derived astrocytes (MEDA), as characterized by the upregulation of 19 astrocyte-associated mRNAs, and positive staining of most cells for GFAP (glial fibrillary acidic protein), aquaporin-4 or glutamine synthetase. The MEDA cultures could be cryopreserved, and they neither contained neuronal, nor microglial cells. They also did not react to the microglial stimulus lipopolysaccharide, while inflammatory activation by a complete cytokine mix (CCM) or its individual components (TNF-α, IL1-ß, IFN-γ) was readily observed. MEDA, stimulated by CCM, became susceptible to CD95 ligand-induced apoptosis and produced NO and IL-6. This was preceded by NF-kB activation, and up-regulation of relevant mRNAs. Also GFAP-negative astrocytes were fully inflammation-competent. Neurotrophic support by MEDA was found to be independent of GFAP expression. In summary, we described here the generation and functional characterization of microglia-free murine astrocytes, displaying phenotypic heterogeneity as is commonly observed in brain astrocytes.
Asunto(s)
Astrocitos/patología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/genética , Células Madre Embrionarias/patología , Proteína Ácida Fibrilar de la Glía/fisiología , Inflamación/patología , Factores de Crecimiento Nervioso/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Línea Celular , Linaje de la Célula/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteína Ácida Fibrilar de la Glía/deficiencia , Inflamación/metabolismo , Ratones , Ratones Endogámicos BALB C , Fenotipo , Cultivo Primario de CélulasRESUMEN
Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8 minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-state-sustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-ß gene expression, resulting in endogenous ERK inhibition. These features are dependent on lipid-free culture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast in vivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.
Asunto(s)
Blastocisto/citología , Estratos Germinativos/citología , Células Madre Pluripotentes/fisiología , Diferenciación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Células Madre Embrionarias , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Metabolismo de los Lípidos , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.
Asunto(s)
Encefalitis por Herpes Simple/genética , Herpesvirus Humano 1/genética , Neuronas/inmunología , ARN Nucleolar Pequeño/genética , Adulto , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/virología , Preescolar , Encefalitis por Herpes Simple/inmunología , Encefalitis por Herpes Simple/patología , Encefalitis por Herpes Simple/virología , Femenino , Predisposición Genética a la Enfermedad , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Humanos , Inmunidad/genética , Lactante , Masculino , Metagenoma/genética , Metagenoma/inmunología , Persona de Mediana Edad , Neuronas/virología , ARN Nucleolar Pequeño/inmunologíaRESUMEN
Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Mesencéfalo/metabolismo , Técnicas Biosensibles , Proteínas HSP90 de Choque Térmico/fisiología , Mesencéfalo/patología , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Estrés FisiológicoRESUMEN
Directing the fate of human pluripotent stem cells (hPSCs) into different lineages requires variable starting conditions and components with undefined activities, introducing inconsistencies that confound reproducibility and assessment of specific perturbations. Here we introduce a simple, modular protocol for deriving the four main ectodermal lineages from hPSCs. By precisely varying FGF, BMP, WNT, and TGFß pathway activity in a minimal, chemically defined medium, we show parallel, robust, and reproducible derivation of neuroectoderm, neural crest (NC), cranial placode (CP), and non-neural ectoderm in multiple hPSC lines, on different substrates independently of cell density. We highlight the utility of this system by interrogating the role of TFAP2 transcription factors in ectodermal differentiation, revealing the importance of TFAP2A in NC and CP specification, and performing a small-molecule screen that identified compounds that further enhance CP differentiation. This platform provides a simple stage for systematic derivation of the entire range of ectodermal cell types.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Ectodermo/citología , Células Madre Pluripotentes/citología , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Cresta Neural/citología , Placa Neural/citología , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Fenantrolinas/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Transcripción AP-2/metabolismoRESUMEN
There is a paucity of information concerning the developmental neurotoxicity (DNT) hazard posed by industrial and environmental chemicals. New testing approaches will most likely be based on batteries of alternative and complementary (non-animal) tests. As DNT is assumed to result from the modulation of fundamental neurodevelopmental processes (such as neuronal differentiation, precursor cell migration or neuronal network formation) by chemicals, the first generation of alternative DNT tests target these processes. The advantage of such types of assays is that they capture toxicants with multiple targets and modes-of-action. Moreover, the processes modelled by the assays can be linked to toxicity endophenotypes, i.e., alterations in neural connectivity that form the basis for neurofunctional deficits in man. The authors of this review convened in a workshop to define criteria for the selection of positive/negative controls, to prepare recommendations on their use, and to initiate the setup of a directory of reference chemicals. For initial technical optimization of tests, a set of > 50 endpoint-specific control compounds was identified. For further test development, an additional "test" set of 33 chemicals considered to act directly as bona fide DNT toxicants is proposed, and each chemical is annotated to the extent it fulfills these criteria. A tabular compilation of the original literature used to select the test set chemicals provides information on statistical procedures, and toxic/non-toxic doses (both for pups and dams). Suggestions are provided on how to use the > 100 compounds (including negative controls) compiled here to address specificity, adversity and use of alternative test systems.
Asunto(s)
Alternativas a las Pruebas en Animales , Evaluación Preclínica de Medicamentos/métodos , Neuronas/efectos de los fármacos , Neurotoxinas/análisis , Pruebas de Toxicidad/métodos , Animales , Congresos como Asunto , Femenino , Sustancias Peligrosas , Humanos , Masculino , Neuronas/citología , Síndromes de Neurotoxicidad/etiología , Embarazo , Efectos Tardíos de la Exposición PrenatalRESUMEN
Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.
Asunto(s)
Diferenciación Celular/fisiología , Fármacos del Sistema Nervioso Central/administración & dosificación , Neuronas/citología , Neuronas/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Técnicas de Cultivo Celular por Lotes/métodos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Humanos , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Neuronas/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacosRESUMEN
Human pluripotent stem cells (hPSCs) provide an unlimited cell source for regenerative medicine. Hormone-producing cells are particularly suitable for cell therapy, and hypopituitarism, a defect in pituitary gland function, represents a promising therapeutic target. Previous studies have derived pituitary lineages from mouse and human ESCs using 3D organoid cultures that mimic the complex events underlying pituitary gland development in vivo. Instead of relying on unknown cellular signals, we present a simple and efficient strategy to derive human pituitary lineages from hPSCs using monolayer culture conditions suitable for cell manufacturing. We demonstrate that purified placode cells can be directed into pituitary fates using defined signals. hPSC-derived pituitary cells show basal and stimulus-induced hormone release in vitro and engraftment and hormone release in vivo after transplantation into a murine model of hypopituitarism. This work lays the foundation for future cell therapy applications in patients with hypopituitarism.
Asunto(s)
Corticotrofos/metabolismo , Células Madre Embrionarias/metabolismo , Hipopituitarismo/terapia , Células Madre Pluripotentes/metabolismo , Tirotrofos/metabolismo , Hormona Adrenocorticotrópica/biosíntesis , Hormona Adrenocorticotrópica/metabolismo , Animales , Benzamidas/farmacología , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Tratamiento Basado en Trasplante de Células y Tejidos , Corticotrofos/citología , Corticotrofos/efectos de los fármacos , Dioxoles/farmacología , Modelos Animales de Enfermedad , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Hormona Folículo Estimulante/biosíntesis , Hormona Folículo Estimulante/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Expresión Génica , Hormona del Crecimiento/biosíntesis , Hormona del Crecimiento/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hipopituitarismo/genética , Hipopituitarismo/metabolismo , Hipopituitarismo/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Hipófisis/metabolismo , Hipófisis/patología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Tirotrofos/citología , Tirotrofos/efectos de los fármacos , Factor de Transcripción AP-2/genética , Factor de Transcripción AP-2/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Familial dysautonomia (FD) is a debilitating disorder that affects derivatives of the neural crest (NC). For unknown reasons, people with FD show marked differences in disease severity despite carrying an identical, homozygous point mutation in IKBKAP, encoding IκB kinase complex-associated protein. Here we present disease-related phenotypes in human pluripotent stem cells (PSCs) that capture FD severity. Cells from individuals with severe but not mild disease show impaired specification of NC derivatives, including autonomic and sensory neurons. In contrast, cells from individuals with severe and mild FD show defects in peripheral neuron survival, indicating that neurodegeneration is the main culprit for cases of mild FD. Although genetic repair of the FD-associated mutation reversed early developmental NC defects, sensory neuron specification was not restored, indicating that other factors may contribute to disease severity. Whole-exome sequencing identified candidate modifier genes for individuals with severe FD. Our study demonstrates that PSC-based modeling is sensitive in recapitulating disease severity, which presents an important step toward personalized medicine.
Asunto(s)
Sistema Nervioso Autónomo/fisiopatología , Disautonomía Familiar/fisiopatología , Células Madre Pluripotentes Inducidas , Células Receptoras Sensoriales/citología , Adolescente , Adulto , Sistema Nervioso Autónomo/citología , Sistema Nervioso Autónomo/crecimiento & desarrollo , Proteínas Portadoras/genética , Estudios de Casos y Controles , Supervivencia Celular/genética , Niño , Disautonomía Familiar/genética , Femenino , Genotipo , Humanos , Masculino , Modelos Neurológicos , Mutación , Cresta Neural/citología , Neuronas/citología , Fenotipo , Análisis de Secuencia de ADN , Índice de Severidad de la Enfermedad , Factores de Elongación Transcripcional , Adulto JovenRESUMEN
Functional assays, such as the "migration inhibition of neural crest cells" (MINC) developmental toxicity test, can identify toxicants without requiring knowledge on their mode of action (MoA). Here, we were interested, whether (i) inhibition of migration by structurally diverse toxicants resulted in a unified signature of transcriptional changes; (ii) whether statistically-identified transcript patterns would inform on compound grouping even though individual genes were little regulated, and (iii) whether analysis of a small group of biologically-relevant transcripts would allow the grouping of compounds according to their MoA. We analyzed transcripts of 35 'migration genes' after treatment with 16 migration-inhibiting toxicants. Clustering, principal component analysis and correlation analyses of the data showed that mechanistically related compounds (e.g. histone deacetylase inhibitors (HDACi), PCBs) triggered similar transcriptional changes, but groups of structurally diverse toxicants largely differed in their transcriptional effects. Linear discriminant analysis (LDA) confirmed the specific clustering of HDACi across multiple separate experiments. Similarity of the signatures of the HDACi trichostatin A and suberoylanilide hydroxamic acid to the one of valproic acid (VPA), suggested that the latter compound acts as HDACi when impairing neural crest migration. In conclusion, the data suggest that (i) a given functional effect (e.g. inhibition of migration) can be associated with highly diverse signatures of transcript changes; (ii) statistically significant grouping of mechanistically-related compounds can be achieved on the basis of few genes with small regulations. Thus, incorporation of mechanistic markers in functional in vitro tests may support read-across procedures, also for structurally un-related compounds.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Sustancias Peligrosas/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Cresta Neural/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Línea Celular Transformada , Análisis Discriminante , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Embrionarias Humanas , Humanos , Ácidos Hidroxámicos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Tiempo , Pruebas de Toxicidad , Transfección , Regulación hacia Arriba/efectos de los fármacos , VorinostatRESUMEN
Stem cell-derived specialized cell types are of interest as an alternative cell system to identify and research neurotoxic effects and modes of action. Developmental toxicity may be studied during differentiation, while organ-specific toxicity may be assessed in fully functional cells, such as neurons. In this study we tested if fully differentiated neurons derived from murine embryonic stem cells (ESCN) could be used to investigate the effects of the well characterized neurotoxic model compound acrylamide (ACR) and if ESCN behave similar to murine primary cortical neurons (pCN) from 16 days old embryos. We characterized the differentiation process of cryopreserved ESC-derived neural precursor cells (NPC) differentiating to ESCN. During the differentiation process (days 11-20) a strong increase in calcium responses to glutamate, acetylcholine and GABA were observed. Moreover, neuron specific marker proteins, ß-III-tubulin, MAP2, Tau, Rbfox3 and synaptophysin showed similar patterns to pCN. In ESCN and pCN the neuronal structure, e.g. neurites, was not affected by low concentrations of ACR [0.5-1.6mM]. However, 24h incubation periods with 0.5-1.6mM ACR led to a reduction of acetylcholine and glutamate induced calcium responses. In conclusion, we show that non-cytotoxic concentrations of ACR alter neurotransmission in ESCN as well as pCN.
Asunto(s)
Acrilamida/farmacología , Calcio/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotransmisores/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neuritas/efectos de los fármacos , Neuronas/citología , Neuronas/metabolismoRESUMEN
Cranial placodes are embryonic structures essential for sensory and endocrine organ development. Human placode development has remained largely inaccessible despite the serious medical conditions caused by the dysfunction of placode-derived tissues. Here, we demonstrate the efficient derivation of cranial placodes from human pluripotent stem cells. Timed removal of the BMP inhibitor Noggin, a component of the dual-SMAD inhibition strategy of neural induction, triggers placode induction at the expense of CNS fates. Concomitant inhibition of fibroblast growth factor signaling disrupts placode derivation and induces surface ectoderm. Further fate specification at the preplacode stage enables the selective generation of placode-derived trigeminal ganglia capable of in vivo engraftment, mature lens fibers, and anterior pituitary hormone-producing cells that upon transplantation produce human growth hormone and adrenocorticotropic hormone in vivo. Our results establish a powerful experimental platform to study human cranial placode development and set the stage for the development of human cell-based therapies in sensory and endocrine disease.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Embrionarias/citología , Células Endocrinas/citología , Neuronas/citología , Células Madre Pluripotentes/citología , Hormona Adrenocorticotrópica/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Células Endocrinas/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Estratos Germinativos/citología , Hormona del Crecimiento/metabolismo , Humanos , Cristalino/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neuronas/metabolismo , Periferinas/genética , Periferinas/metabolismo , Hipófisis/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante , Ganglio del Trigémino/citologíaRESUMEN
Development of in vitro systems, such as those based on embryonic stem cell differentiation, depends on the selection of adequate test and training compounds. We recommend the use of two classes of positive controls, the "gold standard compounds" for which developmental neurotoxicity (DNT) has been proven in man, and the "pathway compounds" that are known to disrupt signalling pathways and key processes relevant for neuronal differentiation. We introduce the concept of toxicity endophenotypes (TEP) as changes in neuronal connectivity resulting from exposure to developmental toxicants. Thus, TEPs provide the scientific rationale for modeling DNT with simple in vitro models of key neurodevelopmental events. In this context, we discuss scientific and technical aspects of the test compound selection process. We suggest to include compounds with unspecific toxicity, besides negative control compounds, and we recommend tandem approaches to determine relative toxicities instead of absolute measures. Finally, we discuss how to avoid pitfalls by distinguishing between unspecific forms of cytotoxicity and specific developmental neurotoxicity. A compilation of compound lists corresponding to the above-discussed principles supplement this review.
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Evaluación Preclínica de Medicamentos/métodos , Modelos Neurológicos , Neurogénesis/efectos de los fármacos , Neurotoxinas/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Humanos , Técnicas In Vitro , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Neuritas/efectos de los fármacos , FenotipoRESUMEN
BACKGROUND: Information on the potential developmental toxicity (DT) of the majority of chemicals is scarce, and test capacities for further animal-based testing are limited. Therefore, new approaches with higher throughput are required. A screening strategy based on the use of relevant human cell types has been proposed by the U.S. Environmental Protection Agency and others. Because impaired neural crest (NC) function is one of the known causes for teratologic effects, testing of toxicant effects on NC cells is desirable for a DT test battery. OBJECTIVE: We developed a robust and widely applicable human-relevant NC function assay that would allow for sensitive screening of environmental toxicants and defining toxicity pathways. METHODS: We generated NC cells from human embryonic stem cells, and after establishing a migration assay of NC cells (MINC assay), we tested environmental toxicants as well as inhibitors of physiological signal transduction pathways. RESULTS: Methylmercury (50 nM), valproic acid (> 10 µM), and lead-acetate [Pb(CH3CO2)4] (1 µM) affected the migration of NC cells more potently than migration of other cell types. The MINC assay correctly identified the NC toxicants triadimefon and triadimenol. Additionally, it showed different sensitivities to various organic and inorganic mercury compounds. Using the MINC assay and applying classic pharmacologic inhibitors and large-scale microarray gene expression profiling, we found several signaling pathways that are relevant for the migration of NC cells. CONCLUSIONS: The MINC assay faithfully models human NC cell migration, and it reveals impairment of this function by developmental toxicants with good sensitivity and specificity.