RESUMO
Discovery of therapeutics for neurological diseases is hampered by the lack of predictive in vitro and in vivo models. Traditionally, in vitro assays rely on engineered cell lines grown two-dimensionally (2D) outside a physiological tissue context, which makes them very amenable for large scale drug screening but reduces their relevance to in vivo neurophysiology. In recent years, three-dimensional (3D) neural cell culture models derived from human induced pluripotent stem cells (iPSCs) have been developed as an in vitro assay platform to investigate brain development, neurological diseases, and for drug screening. iPSC-derived neural spheroids or organoids can be developed to include complex neuronal and glial cell populations and display spontaneous, synchronous activity, which is a hallmark of in vivo neural communication. In this report we present a proof-of-concept study evaluating 3D iPSC-derived cortical neural spheroids as a physiologically- and pharmacologically-relevant high-throughput screening (HTS) platform and investigate their potential for use for therapeutic development. To this end, a library of 687 neuroactive compounds were tested in a phenotypic screening paradigm which measured calcium activity as a functional biomarker for neural modulation through fluctuations in calcium fluorescence. Pharmacological responses of cortical neural spheroids were analyzed using a multi-parametric approach, whereby seven peak characteristics from the calcium activity in each well were quantified and incorporated into principal component analysis and Sammon mapping to measure compound response. Here, we describe the implementation of the 687-compound library screen and data analysis demonstrating that iPSC-derived cortical spheroids are a robust and information-rich assay platform for HTS.
Assuntos
Células-Tronco Pluripotentes Induzidas , Cálcio/metabolismo , Técnicas de Cultura de Células/métodos , Humanos , Neurônios/metabolismo , Organoides/metabolismoRESUMO
With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have different susceptibilities than mature dormant cells, which may yet be different than mature active cells. Thus, in vitro test systems should reflect the cellular context of the native cell: developing, nascent, or functionally active. To that end, we have developed high-throughput, two- and three-dimensional human induced pluripotent stem cell (hiPSC)-derived neural screening platforms that reflect different neurodevelopmental stages. As a proof of concept, we implemented this in vitro human system to swiftly identify the potential neurotoxicity profiles of 29 therapeutic compounds that could be repurposed as anti-virals. Interestingly, many compounds displayed high toxicity on early-stage neural tissues but not on later stages. Compounds with the safest overall viability profiles were further evaluated for functional assessment in a high-throughput calcium flux assay. Of the 29 drugs tested, only four did not modulate or have other potentially toxic effects on the developing or mature neurospheroids across all the tested dosages. These results highlight the importance of employing human neural cultures at different stages of development to fully understand the neurotoxicity profile of potential therapeutics across normal ontogeny.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Reposicionamento de Medicamentos/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Neurônios/química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neurônios/efeitos dos fármacosRESUMO
Duplication or deficiency of the X-linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics. Using a strategic series of increasingly complex human stem cell-derived technologies, including human neurons, MECP2-mosaic neurospheres to model RTT female brain mosaicism, and cortical organoids, we identified synaptic dysregulation downstream from knockout of MECP2 and screened select pharmacological compounds for their ability to treat this dysfunction. Two lead compounds, Nefiracetam and PHA 543613, specifically reversed MECP2-knockout cytologic neuropathology. The capacity of these compounds to reverse neuropathologic phenotypes and networks in human models supports clinical studies for neurodevelopmental disorders in which MeCP2 deficiency is the predominant etiology.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Neurônios/efeitos dos fármacos , Organoides , Pirrolidinonas/farmacologia , Quinuclidinas/farmacologia , Síndrome de Rett , Feminino , Técnicas de Inativação de Genes , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Organoides/efeitos dos fármacos , Fenótipo , Síndrome de Rett/genéticaRESUMO
Human induced Pluripotent Stem Cells (iPSCs) are a powerful tool to dissect the biology of complex human cell types such as those of the central nervous system (CNS). However, robust, high-throughput platforms for reliably measuring activity in human iPSC-derived neuronal cultures are lacking. Here, we assessed 3D cultures of cortical neurons and astrocytes displaying spontaneous, rhythmic, and highly synchronized neural activity that can be visualized as calcium oscillations on standard high-throughput fluorescent readers as a platform for CNS-based discovery efforts. Spontaneous activity and spheroid structure were highly consistent from well-to-well, reference compounds such as TTX, 4-AP, AP5, and NBQX, had expected effects on neural spontaneous activity, demonstrating the presence of functionally integrated neuronal circuitry. Neurospheroid biology was challenged by screening the LOPAC®1280 library, a collection of 1280 pharmacologically active small molecules. The primary screen identified 111 compounds (8.7%) that modulated neural network activity across a wide range of neural and cellular processes and 16 of 17 compounds chosen for follow-up confirmed the primary screen results. Together, these data demonstrate the suitability and utility of human iPSC-derived neurospheroids as a screening platform for CNS-based drug discovery.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Astrócitos/citologia , Sinalização do Cálcio/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Sistema Nervoso Central/citologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Programas de Rastreamento/métodos , Células-Tronco Neurais/citologiaRESUMO
Exosomes are thought to be released by all cells in the body and to be involved in intercellular communication. We tested whether neural exosomes can regulate the development of neural circuits. We show that exosome treatment increases proliferation in developing neural cultures and in vivo in dentate gyrus of P4 mouse brain. We compared the protein cargo and signaling bioactivity of exosomes released by hiPSC-derived neural cultures lacking MECP2, a model of the neurodevelopmental disorder Rett syndrome, with exosomes released by isogenic rescue control neural cultures. Quantitative proteomic analysis indicates that control exosomes contain multiple functional signaling networks known to be important for neuronal circuit development. Treating MECP2-knockdown human primary neural cultures with control exosomes rescues deficits in neuronal proliferation, differentiation, synaptogenesis, and synchronized firing, whereas exosomes from MECP2-deficient hiPSC neural cultures lack this capability. These data indicate that exosomes carry signaling information required to regulate neural circuit development.
Assuntos
Exossomos/metabolismo , Rede Nervosa/metabolismo , Neurogênese , Potenciais de Ação , Animais , Contagem de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Giro Denteado/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 2 de Ligação a Metil-CpG/deficiência , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Esferoides Celulares/citologia , Sinapses/metabolismoRESUMO
MeCP2 is an X-linked gene; its mutation causes Rett Syndrome (RTT), a severe neurodevelopmental disability that affects mainly girls. Acting as a transcription factor, the MeCP2 protein is able to regulate several hormone-related genes, such as the thyroid hormones (TH), which are known to play an important role in the development of the central nervous system (CNS). Although only a few studies have associated RTT and TH, TH deficit can lead to neurological deregulation by triggering functional deficiencies during adulthood. Here, we used human-induced pluripotent stem cell (iPSC) to generate MeCP2-knockout neuronal progenitor cells and adult neurons. Using this cellular model, we then investigated the expression of genes associated with TH homeostasis, such as the TH transporters (LAT1, LAT2, MCT8, MCT10, and OATP4A1) and deiodinases (DIO1, 2, and 3). Then, we treated the neural cells with THs and analyzed the expression of several genes related to neurodevelopment and functional maintenance. Our results showed that several TH-related genes, such as deiodinases, are altered in RTT samples when compared to WT cells. Moreover, the treatment of the neural cells with THs increased the amount of MAP2 and synapsin-1 expression in RTT cells. Our work provided evidences that TH homeostasis is compromised in RTT-derived neural cells, which could be an important factor to contribute to the imbalance in the neurodevelopmental phenotype presented in this syndrome and can lead us to better understand other neurodevelopmental diseases.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Iodeto Peroxidase/genética , Proteínas de Membrana Transportadoras/genética , Proteína 2 de Ligação a Metil-CpG/deficiência , Neurônios/metabolismo , Hormônios Tireóideos/metabolismo , Humanos , Iodeto Peroxidase/metabolismo , Cariotipagem , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Síndrome de Rett/enzimologia , Síndrome de Rett/genéticaRESUMO
Rett syndrome (RTT) is a pervasive developmental disorder caused by mutations in MECP2. Complete loss of MECP2 function in males causes congenital encephalopathy, neurodevelopmental arrest, and early lethality. Induced pluripotent stem cell (iPSC) lines from male patients harboring mutations in MECP2, along with control lines from their unaffected fathers, give us an opportunity to identify some of the earliest cellular and molecular changes associated with MECP2 loss-of-function (LOF). We differentiated iPSC-derived neural progenitor cells (NPCs) using retinoic acid (RA) and found that astrocyte differentiation is perturbed in iPSC lines derived from two different patients. Using highly stringent quantitative proteomic analyses, we found that LIN28, a gene important for cell fate regulation and developmental timing, is upregulated in mutant NPCs compared to WT controls. Overexpression of LIN28 protein in control NPCs suppressed astrocyte differentiation and reduced neuronal synapse density, whereas downregulation of LIN28 expression in mutant NPCs partially rescued this synaptic deficiency. These results indicate that the pathophysiology of RTT may be caused in part by misregulation of developmental timing in neural progenitors, and the subsequent consequences of this disruption on neuronal and glial differentiation.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Proteína 2 de Ligação a Metil-CpG/genética , Neuroglia/citologia , Proteínas de Ligação a RNA/genética , Diferenciação Celular , Linhagem Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação com Perda de Função , Masculino , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , ProteômicaRESUMO
Neurological disorders affect millions of people worldwide and appear to be on the rise. Whereas the reason for this increase remains unknown, environmental factors are a suspected contributor. Hence, there is an urgent need to develop more complex, biologically relevant, and predictive in vitro assays to screen larger sets of compounds with the potential for neurotoxicity. Here, we employed a human induced pluripotent stem cell (iPSC)-based 3D neural platform composed of mature cortical neurons and astrocytes as a model for this purpose. The iPSC-derived human 3D cortical neuron/astrocyte co-cultures (3D neural cultures) present spontaneous synchronized, readily detectable calcium oscillations. This advanced neural platform was optimized for high-throughput screening in 384-well plates and displays highly consistent, functional performance across different wells and plates. Characterization of oscillation profiles in 3D neural cultures was performed through multi-parametric analysis that included the calcium oscillation rate and peak width, amplitude, and waveform irregularities. Cellular and mitochondrial toxicity were assessed by high-content imaging. For assay characterization, we used a set of neuromodulators with known mechanisms of action. We then explored the neurotoxic profile of a library of 87 compounds that included pharmaceutical drugs, pesticides, flame retardants, and other chemicals. Our results demonstrated that 57% of the tested compounds exhibited effects in the assay. The compounds were then ranked according to their effective concentrations based on in vitro activity. Our results show that a human iPSC-derived 3D neural culture assay platform is a promising biologically relevant tool to assess the neurotoxic potential of drugs and environmental toxicants.
Assuntos
Astrócitos/efeitos dos fármacos , Substâncias Perigosas/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Testes de Toxicidade/métodos , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/patologia , Bibliotecas de Moléculas Pequenas/toxicidadeRESUMO
The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/transplante , Medula Espinal/transplante , Envelhecimento , Animais , Diferenciação Celular , Reprogramação Celular , Doença Crônica , Fibroblastos/citologia , Regulação da Expressão Gênica , Tolerância Imunológica , Imunidade Humoral , Terapia de Imunossupressão , Neostriado/patologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Ratos , Pele/citologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Análise de Sobrevida , Suínos , Porco Miniatura , Transplante Homólogo , Transplante IsogênicoRESUMO
Therapeutic intervention is an important need in ameliorating the severe consequences of Rett Syndrome (RTT), a neurological disorder caused by mutations in the X-linked gene methyl-CpG-binding protein-2 (MeCP2). Following previously observed morphological defects in induced pluripotent stem cell (iPSC)-derived neurons obtained from female RTT patients, we hypothesized that transfection with the L1 cell adhesion molecule (L1) could contribute to normalizing a pathological male cell system bearing a nonsense mutation of MeCP2. We found a decreased expression of L1 in RTT iPSCs-derived neural precursor cells (RTT NPCs) and decreased neuritogenesis. Expression of wild-type MeCP2 in RTTNPCs revealed a positive correlation between the levels of MeCP2 and L1, and normalization of cell survival. Expression of L1 in RTTNPCs enhanced neuritogenesis and soma size. Knock-down of MeCP2 in wild type NPCs reduced neuritogenesis. L1 expression is regulated by the MeCP2 promoter. These results suggest that a deficiency in L1 may partially account for RTT phenotypes.
Assuntos
Síndrome da Retração Ocular/metabolismo , Síndrome da Retração Ocular/patologia , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese , Crescimento Neuronal , Células Cultivadas , Feminino , Humanos , Masculino , Molécula L1 de Adesão de Célula Nervosa/genéticaRESUMO
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder in which the MECP2 (methyl CpG-binding protein 2) gene is mutated. Recent studies showed that RTT-derived neurons have many cellular deficits when compared to control, such as: less synapses, lower dendritic arborization and reduced spine density. Interestingly, treatment of RTT-derived neurons with Insulin-like Growth Factor 1 (IGF1) could rescue some of these cellular phenotypes. Given the critical role of IGF1 during neurodevelopment, the present study used human induced pluripotent stem cells (iPSCs) from RTT and control individuals to investigate the gene expression profile of IGF1 and IGF1R on different developmental stages of differentiation. We found that the thyroid hormone receptor (TRalpha 3) has a differential expression profile. Thyroid hormone is critical for normal brain development. Our results showed that there is a possible link between IGF1/IGF1R and the TRalpha 3 and that over expression of IGF1R in RTT cells may be the cause of neurites improvement in neural RTT-derived neurons.
Assuntos
Fator de Crescimento Insulin-Like I/genética , Proteína 2 de Ligação a Metil-CpG/genética , Receptores de Somatomedina/genética , Síndrome de Rett/genética , Receptores alfa dos Hormônios Tireóideos/genética , Diferenciação Celular/genética , Corpos Embrioides/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Transtornos do Neurodesenvolvimento , Plasticidade Neuronal/genética , Neurônios/metabolismo , Neurônios/patologia , Receptor IGF Tipo 1 , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologia , Coluna Vertebral/crescimento & desenvolvimento , Coluna Vertebral/patologia , Sinapses/genética , Sinapses/patologia , Transcriptoma/genéticaRESUMO
Rett syndrome is a severe form of autism spectrum disorder, mainly caused by mutations of a single gene methyl CpG binding protein 2 (MeCP2) on the X chromosome. Patients with Rett syndrome exhibit a period of normal development followed by regression of brain function and the emergence of autistic behaviors. However, the mechanism behind the delayed onset of symptoms is largely unknown. Here we demonstrate that neuron-specific K(+)-Cl(-) cotransporter2 (KCC2) is a critical downstream gene target of MeCP2. We found that human neurons differentiated from induced pluripotent stem cells from patients with Rett syndrome showed a significant deficit in KCC2 expression and consequently a delayed GABA functional switch from excitation to inhibition. Interestingly, overexpression of KCC2 in MeCP2-deficient neurons rescued GABA functional deficits, suggesting an important role of KCC2 in Rett syndrome. We further identified that RE1-silencing transcriptional factor, REST, a neuronal gene repressor, mediates the MeCP2 regulation of KCC2. Because KCC2 is a slow onset molecule with expression level reaching maximum later in development, the functional deficit of KCC2 may offer an explanation for the delayed onset of Rett symptoms. Our studies suggest that restoring KCC2 function in Rett neurons may lead to a potential treatment for Rett syndrome.
Assuntos
Neurônios/metabolismo , Síndrome de Rett/metabolismo , Simportadores/metabolismo , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Modelos Biológicos , Mutação/genética , Proteínas Repressoras/metabolismo , Simportadores/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Macroautophagy (hereafter autophagy) is a key pathway in neurodegeneration. Despite protective actions, autophagy may contribute to neuron demise when dysregulated. Here we consider X-linked spinal and bulbar muscular atrophy (SBMA), a repeat disorder caused by polyglutamine-expanded androgen receptor (polyQ-AR). We found that polyQ-AR reduced long-term protein turnover and impaired autophagic flux in motor neuron-like cells. Ultrastructural analysis of SBMA mice revealed a block in autophagy pathway progression. We examined the transcriptional regulation of autophagy and observed a functionally significant physical interaction between transcription factor EB (TFEB) and AR. Normal AR promoted, but polyQ-AR interfered with, TFEB transactivation. To evaluate physiological relevance, we reprogrammed patient fibroblasts to induced pluripotent stem cells and then to neuronal precursor cells (NPCs). We compared multiple SBMA NPC lines and documented the metabolic and autophagic flux defects that could be rescued by TFEB. Our results indicate that polyQ-AR diminishes TFEB function to impair autophagy and promote SBMA pathogenesis.
Assuntos
Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Transtornos Musculares Atróficos/patologia , Peptídeos/metabolismo , Receptores Androgênicos/metabolismo , Animais , Reprogramação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Transtornos Musculares Atróficos/metabolismo , Fagossomos/fisiologiaRESUMO
An important component for successful translation of cell replacement-based therapies into clinical practice is the utilization of large animal models to conduct efficacy and/or safety cell dosing studies. Over the past few decades, several large animal models (dog, cat, nonhuman primate) were developed and employed in cell replacement studies; however, none of these models appears to provide a readily available platform to conduct effective and large-scale preclinical studies. In recent years, numerous pig models of neurodegenerative disorders were developed using both a transgenic approach as well as invasive surgical techniques. The pig model (naïve noninjured animals) was recently used successfully to define the safety and optimal dosing of human spinal stem cells after grafting into the central nervous system (CNS) in immunosuppressed animals. The data from these studies were used in the design of a human clinical protocol used in amyotrophic lateral sclerosis (ALS) patients in a Phase I clinical trial. In addition, a highly inbred (complete major histocompatibility complex [MHC] match) strain of miniature pigs is available which permits the design of comparable MHC combinations between the donor cells and the graft recipient as used in human patients. Jointly, these studies show that the pig model can represent an effective large animal model to be used in preclinical cell replacement modeling. This review summarizes the available pig models of neurodegenerative disorders and the use of some of these models in cell replacement studies. The challenges and potential future directions in more effective use of the pig neurodegenerative models are also discussed.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Modelos Animais de Doenças , Doenças Neurodegenerativas/cirurgia , Animais , Humanos , SuínosRESUMO
OBJECTIVES: Since it has been reported that in humans there is a relationship between human respiratory syncytial virus (hRSV)-specific cytotoxic T lymphocytes and symptom reduction, and that the polymerase (structural L protein) is highly conserved among different strains, this work aimed to identify the CD8 T cell epitopes H-2(d) restricted within the L sequence for immunization purposes. METHODS: We screened the hRSV strain A2 L protein sequence using two independent algorithms, SYFPEITHI and PRED/(BALB/c), to predict CD8 T cell epitopes. The selected peptides were synthesized and used to immunize BALB/c mice for the evaluation of T cell response. The production of IFN-γ from splenocytes of hRSV-infected animals stimulated by these peptides was assayed by ELISPOT. RESULTS: Nine peptides showing the best binding scores to the BALB/c MHC-I molecules (H-2K(d), L(d) and D(d)) were selected. Sequence homology analysis showed that these sequences are conserved among different hRSV strains. Two of these peptides induced significant IFN-γ production by ex vivo-stimulated T cells. CONCLUSIONS: Our results indicate that the hRSV L protein contains H-2(d)-restricted epitopes.
Assuntos
Linfócitos T CD8-Positivos/imunologia , RNA Polimerases Dirigidas por DNA/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito T/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Estruturais Virais/imunologia , Animais , ELISPOT , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB CRESUMO
Human neurons, generated from reprogrammed somatic cells isolated from live patients, bring a new perspective on the understanding of Autism Spectrum Disorders (ASD). The new technology can nicely complement other models for basic research and the development of therapeutic compounds aiming to revert or ameliorate the condition. Here, we discuss recent advances on the use of stem cells and other models to study ASDs, as well as their limitations, implications and future perspectives.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/terapia , Neurônios/citologia , Síndrome de Rett/terapia , Células-Tronco/citologia , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Transtornos Globais do Desenvolvimento Infantil/genética , Modelos Animais de Doenças , Humanos , Neurônios/fisiologia , Síndrome de Rett/genéticaRESUMO
Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific ß3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin-induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less ß3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.
Assuntos
Diferenciação Celular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptor B2 da Bradicinina/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Wistar , Receptor B2 da Bradicinina/genética , Transdução de SinaisRESUMO
The adult mammalian brain contains self-renewable, multipotent neural stem cells (NSCs) that are responsible for neurogenesis and plasticity in specific regions of the adult brain. Extracellular matrix, vasculature, glial cells, and other neurons are components of the niche where NSCs are located. This surrounding environment is the source of extrinsic signals that instruct NSCs to either self-renew or differentiate. Additionally, factors such as the intracellular epigenetics state and retrotransposition events can influence the decision of NSC's fate into neurons or glia. Extrinsic and intrinsic factors form an intricate signaling network, which is not completely understood. These factors altogether reflect a few of the key players characterized so far in the new field of NSC research and are covered in this review.
Assuntos
Células-Tronco Neurais/citologia , Diferenciação Celular , Epigenômica , Humanos , Células-Tronco Neurais/metabolismo , Neurogênese , Transdução de SinaisRESUMO
Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients' fibroblasts. RTT patients' iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Neurogênese , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/patologia , Proliferação de Células , Feminino , Fibroblastos/citologia , Humanos , Síndrome de Rett/genética , Sinapses , Inativação do Cromossomo XRESUMO
Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (P) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines.