Oligodendrogenesis from neural stem cells: Perspectives for remyelinating strategies

Mobilization of remyelinating cells spontaneously occurs in the adult brain. These cellular resources are specially active after demyelinating episodes in early phases of multiple sclerosis (MS). Indeed, oligodendrocyte precursor cells (OPCs) actively proliferate, migrate to and repopulate the lesioned areas. Ultimately, efficient remyelination is accomplished when new oligodendrocytes reinvest nude neuronal axons, restoring the normal properties of impulse conduction. As the disease progresses this fundamental process fails. Multiple causes seem to contribute to such transient decline, including the failure of OPCs to differentiate and enwrap the vulnerable neuronal axons. Regenerative medicine for MS has been mainly centered on the recruitment of endogenous self-repair mechanisms, or on transplantation approaches. The latter commonly involves grafting of neural precursor cells (NPCs) or neural stem cells (NSCs), with myelinogenic potential, in the injured areas. Both strategies require further understanding of the biology of oligodendrocyte differentiation and remyelination. Indeed, the success of transplantation largely depends on the pre-commitment of transplanted NPCs or NSCs into oligodendroglial cell type, while the endogenous differentiation of OPCs needs to be boosted in chronic stages of the disease. Thus, much effort has been focused on finding molecular targets that drive oligodendrocytes commitment and development. The present review explores several aspects of remyelination that must be considered in the design of a cell-based therapy for MS, and explores more deeply the challenge of fostering oligodendrogenesis. In this regard, we discuss herein a tool developed in our research group useful to search novel oligodendrogenic factors and to study oligodendrocyte differentiation in a time- and cost-saving manner

HACCP numa indústria corticeira: implementação, identificação de pontos críticos e proposta de acções correctivas

Mestrado em Engenharia QuímicaO presente trabalho tem como objectivo a descrição das várias etapas inerentes à implementação de um sistema de segurança alimentar numa empresa de cortiça (Pietec Cortiças, S.A), com base em princípios e conceitos preventivos de modo a garantir a segurança dos seus produtos (rolhas). A metodologia HACCP – Hazards Analysis and Critical Control Points (Análise de Perigos e Pontos Críticos de Controlo) constitui actualmente a referência aceite para a implementação de sistemas de segurança alimentar identificando os perigos específicos e as medidas preventivas para o seu controlo em todas as etapas de produção. Baseia-se numa abordagem sistemática, documentada e verificável cuja aplicação é possível em todos os segmentos e sectores da cadeia alimentar. Antes da aplicação do HACCP, devem estar em pleno funcionamento os pré-requisitos que controlem os perigos associados com a envolvente à empresa (instalações, equipamento, plano de higienização, plano de controlo de pragas, serviço de abastecimento de água, gestão de resíduos, selecção de fornecedores, higiene e saúde pessoal, plano de formação, embalagem, informação sobre a rastreabilidade de produtos). Os princípios do sistema propriamente dito foram também avaliados, nomeadamente a criação de fluxogramas para os diferentes produtos, a análise de perigos, a determinação de medidas preventivas de controlo, a determinação de pontos críticos de controlo e respectivos limites, e sugestão de acções correctivas. Neste estudo foram encontradas duas etapas onde se considera essencial o controlo, de modo a minimizar o perigo, a lavação e a embalagem, constituindo quatro pontos críticos de controlo. Em todas elas, os procedimentos de monitorização baseiam-se essencialmente no cumprimento correcto do controlo. A implementação do sistema HACCP na Pietec Cortiças, S.A., não pode ser considerada como concluída. O alargamento do período de implementação do sistema HACCP para o prazo de um ano deve ser uma possibilidade a considerar, dada a complexidade do processo e a realidade da empresa.This work aims to describe the several stages involved in a food safety system implementation in the cork company Pietec Cortiças, S.A, based on prevention principles and concepts to control the production process and ensure the safety of their products (stoppers). The Hazard Analysis and Critical Control Point methodology (HACCP) is the currently accepted preventive approach to food safety systems implementation, identifying the potential safety hazards and preventive measures for their control at all stages of production. It is based on a systematic, documented and verifiable approach which implementation is possible in all segments and sectors of the food chain. Before the HACCP implementation, operational pre-requisites should be fulfilled to control the hazards associated with the company environment, namely, facilities, equipment, hygiene plan, pest control plan, water supply, waste management, suppliers selection, hygiene and personal health, training plan, packaging, and information on the traceability of products. The system principles were also evaluated including the elaboration of flow diagrams for the different sold products, hazards analysis, determining preventive control actions, critical control points and their critical limits to suggest corrective actions. In this study, two stages were found where control is considered essential in order to minimize or prevent hazards. Washing and packing stages constitute four critical control points. For all of them, monitoring procedures are based essentially on the application of the proper compliance control. The implementation of HACCP in Pietec Cortiças, S.A can not be considered complete, and considering the process complexity, the duration of implementation should be increased to one year

Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures

Tumor necrosis factor (TNF)- has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF- strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1-3 C57BL/6 mice. By using single-cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF- resulted in increased differentiation of cells displaying a neuronal-like profile of [Ca2+]i responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF- receptor, we found that the proneurogenic effect of 1 ng/ml TNF- is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein-positive neurons was increased following exposure to mouse TNF-. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF- induced cell proliferation, whereas 10 and 100 ng/ml TNF- induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF- for 15 minutes or 6 hours caused an increase in the phospho-stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF- induces neurogenesis in neonatal SVZ cell cultures of mice. TNF-, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infectio

Heterocellular Contacts with Mouse Brain Endothelial Cells Via Laminin and alpha 6 beta 1 Integrin Sustain Subventricular Zone (SVZ) Stem/Progenitor Cells Properties

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell-cell contacts. In vivo, SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via (01 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEG) regulate SVZ cells neurogenic capacities, cocultures of SVZ neurospheres and primary BEG, both obtained from C57BL/6 mice, were performed. The involvement of laminin integrin interactions in SVZ homeostasis was tested in three ways. Firstly, SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (GHX) prior to coculture, a treatment expected to decrease membrane proteins. Secondly, SVZ cells were cocultured with BEG in the presence of an anti-alpha 6 integrin neutralizing antibody. Thirdly, BEC were cultured with beta 1(-/-) SVZ cells. We showed that contact with BEC supports, at least in part, proliferation and stemness of SVZ cells, as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEG. These effects are dependent on BEG-derived laminin binding to alpha 6 beta 1 integrin and are decreased in cocultures incubated with anti-alpha 6 integrin neutralizing antibody and in cocultures with SVZ beta 1(-/-) cells. Moreover, BEG-derived laminin sustains sternness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving alpha 6 beta 1 integrin binding to laminin, contribute to SVZ cell proliferation and stemness

Histamine stimulates neurogenesis in the rodent subventricular zone

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potential source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell-based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2, and Ngn1 genes. Biocompatible poly (lactic-co-glycolic acid) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine-loaded microparticles facilitated neuronal differentiation of SVZ-GFP cells grafted in hippocampal slices and in in vivo rodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial-derived vehicles may represent a new tool for brain repair strategies. STEM CELLS 2012; 30:773784Fundacao para a Ciencia e a Tecnologia-Portugal [SFRH/BD/42848/2008, SFRH/BPD/34841/2007, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, PTDC/CTM/099659/2008]; Fundacao Calouste Gulbenkian [96542]info:eu-repo/semantics/publishedVersio

Modulation of subventricular zone oligodendrogenesis: a role for hemopressin?

Neural stem cells (NSCs) from the subventricular zone (SVZ) have been indicated as a source of new oligodendrocytes to use in regenerative medicine for myelin pathologies. Indeed, NSCs are multipotent cells that can self-renew and differentiate into all neural cell types of the central nervous system. In normal conditions, SVZ cells are poorly oligodendrogenic, nevertheless their oligodendrogenic potential is boosted following demyelination. Importantly, progressive restriction into the oligodendrocyte fate is specified by extrinsic and intrinsic factors, endocannabinoids being one of these factors. Although a role for endocannabinoids in oligodendrogenesis has already been foreseen, selective agonists and antagonists of cannabinoids receptors produce severe adverse side effects. Herein, we show that hemopressin (Hp),a modulator of CB1 receptors, increased oligodendroglial differentiation in SVZ neural stem/progenitor cell cultures derived from neonatal mice. The original results presented in this work suggest that Hp and derivates may be of potential interest for the development of future strategies to treat demyelinating diseases

Neuronal replacement therapy: previous achievements and challenges ahead

Abstract Lifelong neurogenesis and incorporation of newborn neurons into mature neuronal circuits operates in specialized niches of the mammalian brain and serves as role model for neuronal replacement strategies. However, to which extent can the remaining brain parenchyma, which never incorporates new neurons during the adulthood, be as plastic and readily accommodate neurons in networks that suffered neuronal loss due to injury or neurological disease? Which microenvironment is permissive for neuronal replacement and synaptic integration and which cells perform best? Can lost function be restored and how adequate is the participation in the pre-existing circuitry? Could aberrant connections cause malfunction especially in networks dominated by excitatory neurons, such as the cerebral cortex? These questions show how important connectivity and circuitry aspects are for regenerative medicine, which is the focus of this review. We will discuss the impressive advances in neuronal replacement strategies and success from exogenous as well as endogenous cell sources. Both have seen key novel technologies, like the groundbreaking discovery of induced pluripotent stem cells and direct neuronal reprogramming, offering alternatives to the transplantation of fetal neurons, and both herald great expectations. For these to become reality, neuronal circuitry analysis is key now. As our understanding of neuronal circuits increases, neuronal replacement therapy should fulfill those prerequisites in network structure and function, in brain-wide input and output. Now is the time to incorporate neural circuitry research into regenerative medicine if we ever want to truly repair brain injury

Estratégias para o uso de células estaminais neurais em reparação cerebral

Tese de doutoramento em Ciências da Saúde, no ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da Universidade de Coimbr

Oligodendrogenesis from neural stem cells: Perspectives for remyelinating strategies

Mobilization of remyelinating cells spontaneously occurs in the adult brain. These cellular resources are specially active after demyelinating episodes in early phases of multiple sclerosis (MS). Indeed, oligodendrocyte precursor cells (OPCs) actively proliferate, migrate to and repopulate the lesioned areas. Ultimately, efficient remyelination is accomplished when new oligodendrocytes reinvest nude neuronal axons, restoring the normal properties of impulse conduction. As the disease progresses this fundamental process fails. Multiple causes seem to contribute to such transient decline, including the failure of OPCs to differentiate and enwrap the vulnerable neuronal axons. Regenerative medicine for MS has been mainly centered on the recruitment of endogenous self-repair mechanisms, or on transplantation approaches. The latter commonly involves grafting of neural precursor cells (NPCs) or neural stem cells (NSCs), with myelinogenic potential, in the injured areas. Both strategies require further understanding of the biology of oligodendrocyte differentiation and remyelination. Indeed, the success of transplantation largely depends on the pre-commitment of transplanted NPCs or NSCs into oligodendroglial cell type, while the endogenous differentiation of OPCs needs to be boosted in chronic stages of the disease. Thus, much effort has been focused on finding molecular targets that drive oligodendrocytes commitment and development. The present review explores several aspects of remyelination that must be considered in the design of a cell-based therapy for MS, and explores more deeply the challenge of fostering oligodendrogenesis. In this regard, we discuss herein a tool developed in our research group useful to search novel oligodendrogenic factors and to study oligodendrocyte differentiation in a time- and cost-saving manner