RESUMEN
The main critical step in single-cell transcriptomics is sample preparation. Several methods have been developed to preserve cells after dissociation to uncouple sample handling from library preparation. Yet, the suitability of these methods depends on the cell types to be processed. In this project, we perform a systematic comparison of preservation methods for droplet-based single-cell RNA-seq on neural and glial cells derived from induced pluripotent stem cells. Our results show that while DMSO provides the highest cell quality in terms of RNA molecules and genes detected per cell, it strongly affects the cellular composition and induces the expression of stress and apoptosis genes. In contrast, methanol fixed samples display a cellular composition similar to fresh samples and provide a good cell quality and little expression biases. Taken together, our results show that methanol fixation is the method of choice for performing droplet-based single-cell transcriptomics experiments on neural cell populations.
Asunto(s)
Metanol , Transcriptoma , Metanol/farmacología , Perfilación de la Expresión Génica/métodos , Neuronas , NeuroglíaRESUMEN
Semaphorin 7A (sema7A) is classified as an immune semaphorin with dual functions in the immune system and in the central nervous system (CNS). These molecules are of interest due to their potential role in multiple sclerosis (MS), which is a chronic demyelinating and neurodegenerative disease of autoimmune origin. In this study, we elucidated the role of sema7A in neuroinflammation using both in vitro and in vivo experimental models. In an in vitro model of neuroinflammation, using cerebellar organotypic slice cultures, we observed that challenge with lipopolysaccharide (LPS) endotoxin did not affect demyelination or cell death in sema7A-deficient cultures compared to wild-type cultures. Moreover, the in vivo outcome of experimental autoimmune encephalomyelitis (EAE) in sema7A-deficient mice was altered in an antigen- and adjuvant-dose-dependent manner, while no differences were observed in the wild-type counterparts. Altogether, these results indicate that sema7A is involved in peripheral immunity and CNS inflammation in MS pathogenesis. Indeed, these data suggest that sema7A might be a potential therapeutic target to treat MS and autoimmune conditions.
Asunto(s)
Antígenos CD/metabolismo , Terapia Molecular Dirigida , Esclerosis Múltiple/terapia , Semaforinas/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Susceptibilidad a Enfermedades , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Inmunización , Inflamación/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Vaina de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Degeneración Nerviosa/patología , Fragmentos de Péptidos , Semaforinas/deficienciaRESUMEN
We characterised the expression of semaphorin (sema)3A, sema7A and their receptors in the immune and the central nervous system (CNS) at different stages of experimental autoimmune encephalomyelitis (EAE). We also studied their expression in neonatal and adult oligodendrocyte progenitor cell (OPC) and in mature oligodendrocyte cultures. Our results show that sema3A is increased in the CNS and decreased in the immune system upon EAE induction. However, sema7A expression is increased in both the CNS and the immune system during EAE. We also detected sema3A, sema7A and their receptors in neonatal and adult OPCs and in mature oligodendrocytes. These data suggest that sema3A and sema7A are involved in the pathogenesis of EAE, in the modulation of the immune response and in the neurodegeneration that take place in the CNS. Sema7A may represent an intriguing potential therapeutic target for the treatment of both the neurodegenerative and immune-mediated disease processes in MS.
Asunto(s)
Antígenos CD/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Semaforina-3A/inmunología , Semaforinas/inmunología , Animales , Antígenos CD/genética , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Citometría de Flujo , Regulación de la Expresión Génica , Immunoblotting , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Oligodendroglía/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semaforina-3A/genética , Semaforinas/genética , Médula Espinal/inmunología , Médula Espinal/patologíaRESUMEN
Semaphorins have been classically defined as axonal signalling cues involved in central nervous system (CNS) development, but in adults these molecules are expressed in distinct tissues and exert various functions under several physiological and pathological contexts. Semaphorins capable of modulating the immune system are particularly relevant in autoimmune diseases, especially multiple sclerosis (MS), which is a demyelinating, neurodegenerative disease. In this article, we compile recent insights into the specific roles of semaphorin (sema)3A and sema7A to clarify the details of their possible participation in the inflammatory and neurodegenerative phases of MS.