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
The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells. SIGNIFICANCE STATEMENT: Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic acid modulates the chromatin state of cells to be permissive for motoneuron generation and directly suppresses the induction of GLI3, a negative regulator of SHH signaling. Therefore, our data point to a novel mechanism by which retinoic acid exposure can bypass the requirement for extrinsic SHH treatment during motoneuron induction.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Hedgehog/farmacología , Factores de Transcripción de Tipo Kruppel/metabolismo , Neuronas Motoras/citología , Proteínas del Tejido Nervioso/metabolismo , Tretinoina/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Masculino , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Tretinoina/farmacología , Proteína Gli3 con Dedos de ZincRESUMEN
While the availability of pluripotent stem cells has opened new prospects for generating neural donor cells for nervous system repair, their capability to integrate with adult brain tissue in a structurally relevant way is still largely unresolved. We addressed the potential of human embryonic stem cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES cells) to establish axonal projections after transplantation into the adult rodent brain. Transgenic and species-specific markers were used to trace the innervation pattern established by transplants in the hippocampus and motor cortex. In vitro, lt-NES cells formed a complex axonal network within several weeks after the initiation of differentiation and expressed a composition of surface receptors known to be instrumental in axonal growth and pathfinding. In vivo, these donor cells adopted projection patterns closely mimicking endogenous projections in two different regions of the adult rodent brain. Hippocampal grafts placed in the dentate gyrus projected to both the ipsilateral and contralateral pyramidal cell layers, while axons of donor neurons placed in the motor cortex extended via the external and internal capsule into the cervical spinal cord and via the corpus callosum into the contralateral cortex. Interestingly, acquisition of these region-specific projection profiles was not correlated with the adoption of a regional phenotype. Upon reaching their destination, human axons established ultrastructural correlates of synaptic connections with host neurons. Together, these data indicate that neurons derived from human pluripotent stem cells are endowed with a remarkable potential to establish orthotopic long-range projections in the adult mammalian brain.
Asunto(s)
Células Madre Embrionarias/citología , Neuronas/citología , Animales , Axones/fisiología , Trasplante de Tejido Encefálico , Células Cultivadas , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/trasplante , Humanos , Neurogénesis , Neuronas/trasplante , Ratas , Ratas Sprague-Dawley , Sinapsis/patología , Sinapsis/ultraestructuraRESUMEN
An intriguing question in human embryonic stem cell (hESC) biology is whether these pluripotent cells can give rise to stably expandable somatic stem cells, which are still amenable to extrinsic fate instruction. Here, we present a pure population of long-term self-renewing rosette-type hESC-derived neural stem cells (lt-hESNSCs), which exhibit extensive self-renewal, clonogenicity, and stable neurogenesis. Although lt-hESNSCs show a restricted expression of regional transcription factors, they retain responsiveness to instructive cues promoting the induction of distinct subpopulations, such as ventral midbrain and spinal cord fates. Using lt-hESNSCs as a donor source for neural transplantation, we provide direct evidence that hESC-derived neurons can establish synaptic connectivity with the mammalian nervous system. Combining long-term stability, maintenance of rosette-properties and phenotypic plasticity, lt-hESNSCs may serve as useful tool to study mechanisms of human NSC self-renewal, lineage segregation, and functional in vivo integration.
Asunto(s)
Diferenciación Celular , Separación Celular/métodos , Embrión de Mamíferos/citología , Neuronas/citología , Células Madre/citología , Sinapsis , Línea Celular , Humanos , Neuronas/metabolismo , Células Madre/clasificación , Células Madre/metabolismo , Sinapsis/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The mechanistic basis of gliogenesis, which occurs late in human development, is poorly understood. Here we identify nuclear factor IA (NFIA) as a molecular switch inducing human glial competency. Transient expression of NFIA is sufficient to trigger glial competency of human pluripotent stem cell-derived neural stem cells within 5 days and to convert these cells into astrocytes in the presence of glial-promoting factors, as compared to 3-6 months using current protocols. NFIA-induced astrocytes promote synaptogenesis, exhibit neuroprotective properties, display calcium transients in response to appropriate stimuli and engraft in the adult mouse brain. Differentiation involves rapid but reversible chromatin remodeling, glial fibrillary acidic protein (GFAP) promoter demethylation and a striking lengthening of the G1 cell cycle phase. Genetic or pharmacological manipulation of G1 length partially mimics NFIA function. We used the approach to generate astrocytes with region-specific or reactive features. Our study defines key mechanisms of the gliogenic switch and enables the rapid production of human astrocytes for disease modeling and regenerative medicine.
Asunto(s)
Diferenciación Celular/genética , Factores de Transcripción NFI/genética , Neurogénesis/genética , Células Madre Pluripotentes/metabolismo , Animales , Astrocitos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Factores de Transcripción NFI/metabolismo , Células-Madre Neurales/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/metabolismo , Regiones Promotoras GenéticasRESUMEN
Epilepsy therapy is largely symptomatic and no effective therapy is available to prevent epileptogenesis. We therefore analysed the potential of stem cell-derived brain implants and of paracrine adenosine release to suppress the progressive development of seizures in the rat kindling-model. Embryonic stem (ES) cells, engineered to release the inhibitory neuromodulator adenosine by biallelic genetic disruption of the adenosine kinase gene (Adk-/-), and respective wild-type (wt) cells, were differentiated into neural precursor cells (NPs) and injected into the hippocampus of rats prior to kindling. Therapeutic effects of NP-derived brain implants were compared with those of wt baby hamster kidney cells (BHK) and adenosine releasing BHK cell implants (BHK-AK2), which were previously shown to suppress seizures by paracrine adenosine release. Wild-type NP-graft recipients were characterized by an initial delay of seizure development, while recipients of adenosine releasing NPs displayed sustained protection from developing generalized seizures. In contrast, recipients of wt BHK cells failed to display any effects on kindling development, while recipients of BHK-AK2 cells were only moderately protected from seizure development. The therapeutic effect of Adk(-/-)-NPs was due to graft-mediated adenosine release, since seizures could transiently be provoked after blocking adenosine A1 receptors. Histological analysis of NP-implants at day 26 revealed cell clusters within the infrahippocampal cleft as well as intrahippocampal location of graft-derived cells expressing mature neuronal markers. In contrast, BHK and BHK-AK2 cell implants only formed cell clusters within the infrahippocampal cleft. We conclude that ES cell-derived adenosine releasing brain implants are superior to paracrine adenosine release from BHK-AK2 cell implants in suppressing seizure progression in the rat kindling-model. These findings may indicate a potential antiepileptogenic function of stem cell-mediated adenosine delivery.
Asunto(s)
Adenosina/metabolismo , Encéfalo/cirugía , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Epilepsia/cirugía , Excitación Neurológica/metabolismo , Adenosina Quinasa/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Epilepsia/metabolismo , Epilepsia/patología , Ingeniería Genética , Excitación Neurológica/patología , Masculino , Modelos Animales , Comunicación Paracrina , Ratas , Ratas Sprague-DawleyRESUMEN
As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system. Stem Cells Translational Medicine 2018;7:477-486.
Asunto(s)
Adenosina/metabolismo , Edición Génica/métodos , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Adenosina/análisis , Adenosina Quinasa/deficiencia , Adenosina Quinasa/genética , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Células Madre Embrionarias Humanas/citología , Humanos , Cariotipificación , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/citología , Células-Madre Neurales/trasplante , Neuronas/citología , Polimorfismo de Nucleótido Simple , Nucleasas con Dedos de Zinc/genéticaRESUMEN
Capturing the full potential of human pluripotent stem cell (PSC)-derived neurons in disease modeling and regenerative medicine requires analysis in complex functional systems. Here we establish optogenetic control in human PSC-derived spinal motorneurons and show that co-culture of these cells with human myoblast-derived skeletal muscle builds a functional all-human neuromuscular junction that can be triggered to twitch upon light stimulation. To model neuromuscular disease we incubated these co-cultures with IgG from myasthenia gravis patients and active complement. Myasthenia gravis is an autoimmune disorder that selectively targets neuromuscular junctions. We saw a reversible reduction in the amplitude of muscle contractions, representing a surrogate marker for the characteristic loss of muscle strength seen in this disease. The ability to recapitulate key aspects of disease pathology and its symptomatic treatment suggests that this neuromuscular junction assay has significant potential for modeling of neuromuscular disease and regeneration.
Asunto(s)
Células Madre Embrionarias/citología , Neuronas Motoras/patología , Miastenia Gravis/inmunología , Enfermedades Neuromusculares/fisiopatología , Unión Neuromuscular/fisiopatología , Optogenética/métodos , Autoinmunidad , Técnicas de Cocultivo , Proteínas del Sistema Complemento , Humanos , Inmunoglobulina G/química , Inmunohistoquímica , Luz , Músculo Esquelético/fisiología , Músculos/fisiología , Miastenia Gravis/fisiopatología , Mioblastos/citología , Células Madre Pluripotentes/citología , Regeneración , Médula Espinal/patología , Sinapsinas/metabolismo , Sinapsinas/fisiologíaRESUMEN
Short episodes of ischemia can protect neuronal cells and tissue against a subsequent lethal ischemia by a phenomenon called ischemic preconditioning. The development of this tolerance depends on protein synthesis and takes at least 1 day. It therefore seems reasonable that preconditioning leads to upregulation and translation of protective genes or posttranslational modification of pro- or antiapoptotic proteins. We recently used suppression subtractive hybridization to identify transcripts upregulated in rat primary neuronal cultures preconditioned by oxygen glucose deprivation. In this contribution, we describe the previously unknown 7-kb full-length sequence of an upregulated expressed sequence tag and show that it constitutes the 3' end of the large untranslated region of the noncatalytic "truncated" growth factor receptor TrkB.T1. TrkB.T1 is expressed most prominently in the adult brain and its mRNA was found to be 2.1-fold upregulated by ischemic preconditioning. At the protein level, however, TrkB.T1 was clearly downregulated, possibly by increased degradation in preconditioned cultures. TrKB.T1 can act as a dominant-negative inhibitor of its catalytic counterpart TrkB, which is the receptor for brain-derived neurotrophic factor (BDNF), a factor induced by ischemia that can protect from ischemia-induced neuron loss. We hypothesize that the downregulation of TrkB.T1 at the protein level can prolong BDNF-mediated protective signaling via the catalytic receptor and thus participates in the development of ischemic preconditioning.
Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Precondicionamiento Isquémico , Neuronas/metabolismo , Biosíntesis de Proteínas , Receptor trkB/metabolismo , Animales , Encéfalo/citología , Isquemia Encefálica/fisiopatología , Factor Neurotrófico Derivado del Encéfalo , Regulación hacia Abajo , Feto , Genes Dominantes , Neuronas/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor trkB/genética , Distribución TisularRESUMEN
Stem cell-based therapies hold considerable promise for many currently devastating neurological disorders. Substantial progress has been made in the derivation of disease-relevant human donor cell populations. Behavioral data in relevant animal models of disease have demonstrated therapeutic efficacy for several cell-based approaches. Consequently, cGMP grade cell products are currently being developed for first in human clinical trials in select disorders. Despite the therapeutic promise, the presumed mechanism of action of donor cell populations often remains insufficiently validated. It depends greatly on the properties of the transplanted cell type and the underlying host pathology. Several new technologies have become available to probe mechanisms of action in real time and to manipulate in vivo cell function and integration to enhance therapeutic efficacy. Results from such studies generate crucial insight into the nature of brain repair that can be achieved today and push the boundaries of what may be possible in the future.
Asunto(s)
Encefalopatías/cirugía , Trasplante de Células Madre/métodos , Células Madre/fisiología , HumanosRESUMEN
Recent studies have shown evidence of behavioral recovery after transplantation of human pluripotent stem cell (PSC)-derived neural cells in animal models of neurological disease. However, little is known about the mechanisms underlying graft function. Here we use optogenetics to modulate in real time electrophysiological and neurochemical properties of mesencephalic dopaminergic (mesDA) neurons derived from human embryonic stem cells (hESCs). In mice that had recovered from lesion-induced Parkinsonian motor deficits, light-induced selective silencing of graft activity rapidly and reversibly re-introduced the motor deficits. The re-introduction of motor deficits was prevented by the dopamine agonist apomorphine. These results suggest that functionality depends on graft neuronal activity and dopamine release. Combining optogenetics, slice electrophysiology and pharmacological approaches, we further show that mesDA-rich grafts modulate host glutamatergic synaptic transmission onto striatal medium spiny neurons in a manner reminiscent of endogenous mesDA neurons. Thus, application of optogenetics in cell therapy can link transplantation, animal behavior and postmortem analysis to enable the identification of mechanisms that drive recovery.
Asunto(s)
Diferenciación Celular/genética , Neuronas Dopaminérgicas/trasplante , Optogenética/métodos , Enfermedad de Parkinson/genética , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/citología , Células Madre Embrionarias Humanas/citología , Humanos , Mesencéfalo/citología , Mesencéfalo/trasplante , Ratones , Células-Madre Neurales/trasplante , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , Transmisión Sináptica/genéticaRESUMEN
Subsets of rodent neurons are reported to express major histocompatibility complex class I (MHC-I), but such expression has not been reported in normal adult human neurons. Here we provide evidence from immunolabel, RNA expression and mass spectrometry analysis of postmortem samples that human catecholaminergic substantia nigra and locus coeruleus neurons express MHC-I, and that this molecule is inducible in human stem cell-derived dopamine (DA) neurons. Catecholamine murine cultured neurons are more responsive to induction of MHC-I by gamma-interferon than other neuronal populations. Neuronal MHC-I is also induced by factors released from microglia activated by neuromelanin or alpha-synuclein, or high cytosolic DA and/or oxidative stress. DA neurons internalize foreign ovalbumin and display antigen derived from this protein by MHC-I, which triggers DA neuronal death in the presence of appropriate cytotoxic T cells. Thus, neuronal MHC-I can trigger antigenic response, and catecholamine neurons may be particularly susceptible to T-cell-mediated cytotoxic attack.
Asunto(s)
Neuronas Adrenérgicas/metabolismo , Neuronas Dopaminérgicas/metabolismo , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I/genética , Locus Coeruleus/metabolismo , Microglía/metabolismo , ARN Mensajero/metabolismo , Sustancia Negra/metabolismo , Neuronas Adrenérgicas/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Animales , Supervivencia Celular , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interferón gamma/farmacología , Masculino , Melaninas/metabolismo , Ratones , Persona de Mediana Edad , Estrés Oxidativo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , ARN Mensajero/efectos de los fármacos , Linfocitos T Citotóxicos , alfa-Sinucleína/metabolismoRESUMEN
Store-operated Ca(2+) entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca(2+) stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca(2+) concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Epilepsia del Lóbulo Temporal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Enfermedad Crónica , Corteza Entorrinal/citología , Corteza Entorrinal/fisiopatología , Epilepsia del Lóbulo Temporal/fisiopatología , Hipocampo/citología , Hipocampo/fisiopatología , Humanos , Proteínas de Neoplasias/metabolismo , Red Nerviosa/metabolismo , Red Nerviosa/fisiopatología , Neuronas/citología , Técnicas de Cultivo de Órganos , Cultivo Primario de Células , Ratas , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2RESUMEN
Totipotency, defined as the ability of a single cell to generate an entire individual, has traditionally served as a cornerstone to frame the moral relevance of nascent human life. This "potentiality principle" has served as an ethical reference point for shaping legal regulations for stem cell research in most Western countries. Based on heterogeneous ethical, religious, and political views, different countries cope with recent advances in mammalian cloning and reprogramming in a remarkably diverse manner. This and related issues were key topics at a recent meeting held in Berlin, Germany, on ethical aspects of stem cell research in Europe. An emerging view from this event is that international heterogeneity in stem cell politics and legislation must be overcome in order to develop this field toward biomedical application.
Asunto(s)
Células Madre Adultas/fisiología , Investigaciones con Embriones/ética , Células Madre Embrionarias/fisiología , Células Madre Adultas/citología , Animales , Células Madre Embrionarias/citología , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Creación de Embriones para Investigación/éticaRESUMEN
Brain ischemia can be studied in vitro by depriving primary neurons of oxygen and glucose by replacing oxygen with argon and glucose with its antimetabolite 2-deoxy-D-glucose. In this contribution, we explain how to construct a reliably functioning ischemia chamber and use it to study neuronal cell death in neuron-enriched fetal primary cortical cultures grown under serum-free conditions. We observed that these cultures exhibited a significant cell death even during exposure to oxygenated balanced salt solution used as control for oxygen-glucose deprivation. We show that addition of only 2% fetal calf serum 24 h prior, during, and after treatment almost abolished this undesirable cell loss and proportionally increased cell death induced by oxygen-glucose deprivation. Western blots and immunocytochemistry showed that these effects were mainly due to an increase in neuronal viability under control conditions accompanied by a limited glial proliferation independent of the treatment condition. Under these modified conditions, the cultures could also still be effectively preconditioned by a short-term oxygen-glucose deprivation. In summary, this modified protocol combines the advantages of serum-free neuronal culture, where potentially toxic antimitotic substances can be omitted, with a serum-mediated protection of neurons against unspecific factors and concomitant sensitization for oxygen-glucose deprivation.