Translate this page to:
Portuguese
In JoVE (1)
Other Publications (11)
- Investigative Ophthalmology & Visual Science
- Visual Neuroscience
- Neuroreport
- Organic & Biomolecular Chemistry
- Bioorganic & Medicinal Chemistry
- Biochemical and Biophysical Research Communications
- Biomedical Engineering Online
- BMC Cell Biology
- Cellular & Molecular Biology Letters
- Stem Cells and Development
- Neuroscience Letters
Automatic Translation
This translation into Portuguese was automatically generated.
English Version | Other Languages
Articles by Moritz J. Frech in JoVE
JoVE Bioengineering
Cultivo de células progenitoras neurais em um hidrogel Peptide 3-dimensional de auto-montagem
Albrecht-Kossel-Institute for Neuroregeneration, University of Rostock
Aqui nós descrevemos o uso de uma auto-montagem 3-dimensional andaime para a cultura humana células progenitoras neurais. Nós apresentamos um protocolo para liberar as células da scaffolds a serem analisados posteriormente por exemplo, por citometria de fluxo. Este protocolo pode ser adaptado para outros tipos de células para realizar estudos detalhados mecanicamente.
Other articles by Moritz J. Frech on PubMed
Spontaneous Synaptic Activity in an Organotypic Culture of the Mouse Retina
Many strains of mutant mice die at birth, when the retina is still very immature. The retinas of such mice can be studied in organotypic cultures. After a preceding anatomic study of the synaptic development, the electrical activity of the synaptic circuits within such cultures was studied in wild-type and gephyrin-deficient mice.
Characterization of Inhibitory Postsynaptic Currents in Rod Bipolar Cells of the Mouse Retina
The synaptic terminals of mammalian rod bipolar cells are the targets of multiple presynaptic inhibitory inputs arriving from glycinergic and GABAergic amacrine cells. To investigate the contribution of these different inhibitory receptor types, we have applied the patch-clamp technique in acutely isolated slices of the adult mouse retina. By using the whole-cell configuration, we measured and analyzed the spontaneous postsynaptic currents (PSCs) in rod bipolar cells. The spontaneous synaptic activity of rod bipolar cells was very low. However, when amacrine cells were depolarized by AMPA or kainate, the PSC frequency in rod bipolar cells increased significantly. These PSCs comprised several types that could be distinguished by pharmacological and kinetic criteria. Strychnine-sensitive, glycinergic PSCs were characterized by a mean peak amplitude of -43.5 pA and a weighted decay time constant (tauw) of 10.9 ms. PSCs that persisted in the presence of strychnine, but were completely inhibited by bicuculline, were mediated by GABAARs. They had a mean peak amplitude of -20.0 pA and a significantly faster tauw of 5.8 ms. Few PSCs remained in the presence of strychnine and bicuculline, suggesting that they were mediated by GABACRs. These PSCs were characterized by much smaller amplitudes (-6.2 pA) and a significantly slower decay kinetics (tauw=51.0 ms). We conclude that rod bipolar cells express at least three types of functionally different inhibitory receptors, namely GABAARs, GABACRs, and GlyRs that may ultimately regulate the Ca2+ influx into rod bipolar cell terminals, thereby modulating their glutamate release.
Protection of Neurons Derived from Human Neural Progenitor Cells by Veratridine
The survival of developing dopaminergic neurons has been shown to be modulated by voltage-dependent mechanisms. Manipulation of these mechanisms in human neural progenitor cell cultures could improve the survival of immature dopaminergic neurons, and therefore aid research into pharmacological and cell replacement therapies for Parkinson's disease. Here, we examined the effect of the Na+ channel agonist veratridine on the human fetal neural progenitor ReNcell VM cell line. Neuronal differentiation was determined by immunocytochemistry, whereas patch clamp recordings showed the expression of functional voltage-gated sodium channels. Our results show that veratridine is neuroprotective in human fetal neural progenitor cells, which may benefit studies investigating neuronal development by reducing premature death amongst developing neurons.
A New Facile Synthesis of 3-amidoindole Derivatives and Their Evaluation As Potential GSK-3beta Inhibitors
3-Amidoindoles were synthesized from commercially available arylhydrazines and propargylamines over Zn-salt mediated one pot procedure in excellent regioselectivity and up to 94% yield.
Novel Indolylmaleimide Acts As GSK-3beta Inhibitor in Human Neural Progenitor Cells
The Wnt pathway is involved in cellular processes linked to either proliferation or differentiation. Therefore small molecules offer an attractive opportunity to modulate this pathway, whereas the key enzyme GSK-3beta is of special interest. In this study, non-symmetrically substituted indolylmaleimides have been synthesized and their ability to function as GSK-3beta inhibitors has been investigated in a human neural progenitor cell line. Among the newly synthesized compounds, the substance IM-12 showed a significant activity in several biological tests which was comparable or even outplayed the effects of the known GSK-3beta inhibitor SB-216763. Furthermore the treatment of human neural progenitor cells with IM-12 resulted in an increase of neuronal cells. Therefore we conclude that indolylmaleimides act via the canonical Wnt signalling pathway by inhibition of the key enzyme GSK-3beta.
Differentiation of Human Neural Progenitor Cells Regulated by Wnt-3a
Wnt ligands play pivotal roles in the control of cell growth and differentiation during central nervous system development via the Wnt signaling pathway. In this study, we investigated the effects of Wnt-3a and β-catenin on the differentiation of ReNcell VM human neural progenitor cells. After overexpression of Wnt-3a or mutant-stabilized β-catenin in ReNcell VM cells, their effects on TCF-mediated transcription, Wnt target gene expression and differentiation into neuronal and glial cells were investigated. Our results show that activation of Wnt/β-catenin signaling increases TCF-mediated transcription and the expression of the Wnt target genes Axin2, LEF1 and CyclinD1 in ReNcell VM cells. In contrast to mutant-stabilized β-catenin, Wnt-3a increases neurogenesis during the differentiation of ReNcell VM cells. Thus, our data suggest that neurogenesis induced by Wnt-3a is independent of the transcriptional activity of Wnt/β-catenin pathway in ReNcell VM cells.
Effect of 3D-scaffold Formation on Differentiation and Survival in Human Neural Progenitor Cells
ABSTRACT: BACKGROUND: 3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenviroment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them. METHODS: In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay. RESULTS: Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures. CONCLUSIONS: Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.
Erythropoietin and the Effect of Oxygen During Proliferation and Differentiation of Human Neural Progenitor Cells
Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process.
Quantitative and Kinetic Profile of Wnt/β-catenin Signaling Components During Human Neural Progenitor Cell Differentiation
ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/β-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/β-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized β-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and β-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/β-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/β-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.
Human Neural Progenitor Cells Show Functional Neuronal Differentiation and Regional Preference After Engraftment Onto Hippocampal Slice Cultures
The transplantation of stem cells offers potential therapies for many neurodegenerative disorders that currently have limited or no treatment options. However, relatively little is known about how the host environment affects the development and integration of these cells. In this study we have engrafted immortalized human midbrain neural progenitor cells (NPCs) onto rat hippocampal brain slice cultures to examine the influence of a neural environment on differentiation. Patch clamp recordings revealed that the transplanted progenitor cells could express neuronal-type voltage-gated currents and rapidly receive synaptic input from the hippocampal brain slice. The distribution of progenitor cells across the hippocampal slices was strongly influenced by the neural architecture, with most cells located in the fissural regions and sending processes parallel to the laminar structure, while in contrast, cells located in the dentate gyrus showed no organized pattern. Almost no cells were found in the stratum radiatum or pyramidal cell layers. Together, these results demonstrate the potential for the architecture of the host environment to regulate the integration of transplanted cells, and highlight the utility of coculture systems for studying the mechanisms underlying the migration, integration, and differentiation of human NPCs in structured neural environments.
Small Molecule GSK-3 Inhibitors Increase Neurogenesis of Human Neural Progenitor Cells
Human neural progenitor cells provide a source for cell replacement therapy to treat neurodegenerative diseases. Therefore, there is great interest in mechanisms and tools to direct the fate of multipotent progenitor cells during their differentiation to increase the yield of a desired cell type. We tested small molecule inhibitors of glycogen synthase kinase-3 (GSK-3) for their functionality and their influence on neurogenesis using the human neural progenitor cell line ReNcell VM. Here we report the enhancement of neurogenesis of human neural progenitor cells by treatment with GSK-3 inhibitors. We tested different small molecule inhibitors of GSK-3 i.e. LiCl, sodium-valproate, kenpaullone, indirubin-3-monoxime and SB-216763 for their ability to inhibit GSK-3 in human neural progenitor cells. The highest in situ GSK-3 inhibitory effect of the drugs was found for kenpaullone and SB-216763. Accordingly, kenpaullone and SB-216763 were the only drugs tested in this study to stimulate the Wnt/β-catenin pathway that is antagonized by GSK-3. Analysis of human neural progenitor differentiation revealed an augmentation of neurogenesis by SB-216763 and kenpaullone, without changing cell cycle exit or cell survival. Small molecule inhibitors of GSK-3 enhance neurogenesis of human neural progenitor cells and may be used to direct the differentiation of neural stem and progenitor cells in therapeutic applications.
