Recombinant Spider Silk Protein Matrices Facilitate Differentiation of Neural Stem Cells Into Mature and Functional Neurons

Neural stem cells (NSCs) show great promise in drug discovery and clinical application. Yet few efforts have been made to optimize biocompatible materials for such cells to be expanded and used in clinical conditions. We have previously demonstrated that NSCs are readily cultured on substrates of certain recombinant spider silk protein without addition of animal- or human-derived components. The question remains however whether this material allows differentiation into functional neurons, and whether such differentiation can take place also when the NSCs are cultured not only upon but also within the biodegradable material. Here we demonstrate that “foam”-like structures generated from recombinant spider silk protein (4RepCT) provided excellent matrices for the generation and multicellular analysis of functional excitatory neurons from NSCs without addition of animal- or human-derived components. NSCs isolated from the cerebral cortices of rat embryos were cultured at either 4RepCT matrices shaped as foam-like structures without coating, or on conventional polystyrene plates coated with poly-L-ornithine and fibronectin. Upon treatment with recombinant proteins including the extracellular signaling factor BMP4 or a combination of BMP4 and the signaling factor Wnt3a, the cortical NSCs cultured in 4RepCT foam-like structures differentiated efficiently into neurons that responded to glutamate receptor agonists, such as AMPA, to the same extent as control cultures. Matrices derived from recombinant spider silk proteins thus provide a functional microenvironment for neural stem cells with little or no animal- or human-derived components and can be employed in the development of new strategies in stem cell research and tissue engineering.

Glutamatergic Activation of Neuronostatin Neurons in the Periventricular Nucleus of the Hypothalamus

Neuronostatin, a newly identified anorexigenic peptide, is present in the central nervous system. We tested the hypothesis that neuronostatin neurons are activated by feeding as a peripheral factor and that the glutamatergic system has regulatory influences on neuronostatin neurons. The first set of experiments analyzed the activation of neuronostatin neurons by refeeding as a physiological stimulus and the effectiveness of the glutamatergic system on this physiological stimulation. The subjects were randomly divided into three groups: the fasting group, refeeding group, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)+refeeding group. We found that refeeding increased the phosphorylated signal transducers and transcription activator-5 (pSTAT5) expression in neuronostatin-positive neurons and that the CNQX injection significantly suppressed the number of pSTAT5-expressing neuronostatin neurons. The second set of experiments analyzed the activation pathways of neuronostatin neurons and the regulating effects of the glutamatergic system on neuronostatin neurons. The animals received intraperitoneal injections of glutamate receptor agonists (kainic acid, α-amino-3-hydroxy-5methyl-4-isoazepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA)) or 0.9% NaCl. The number of c-Fos-expressing neuronostatin neurons significantly increased following the AMPA and NMDA injections. In conclusion, we found that the neuronostatin neurons were activated by peripheral or central signals, including food intake and/or glutamatergic innervation, and that the glutamate receptors played an important role in this activation.

Deleterious Effect of NMDA Plus Kainate on the Inner Retinal Cells and Ganglion Cell Projection of the Mouse

Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal “b” wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the “a” wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber.

THE EFFECT OF GLUTAMATE ON THE MIGRATION OF T CELLS FROM HEALTHY DONORS AND PATIENTS WITH MULTIPLE SCLEROSIS IN VITRO

Aim of study. To study chemotactic properties of glutamate and glutamate receptor agonists on T cells migration from healthy donors and patients with multiple sclerosis (MS) in vitro. Materials and methods. T cell migration of 15 patients with MS and 15 healthy donors was studied in vitro using transwells. Lymphocytes were activated with PMA (10 ng/mL). T cells were added to transwells with fibronectin (10 μg/mL) pretreated membrane. The lower chamber contained glutamate or AMPA or NMDA (100 μM for each) in complete RPMI medium. Migrated cells were collected and stained with antibodies to CD3-marker for subsequent analysis by cytofluorimetry. Results and conclusion. In presence of glutamate, there is a tendency to a decrease in migration activity in both groups of donors. T-cell chemotaxis of healthy donors, but not MS patients, decreased in concentration gradient of NMDA. The activation of lymphocytes with PMA leads to a decrease in the number of migrated cells by an average of 17% (p < 0.01). In MS patients there is a tendency to an increase in chemotaxis of activated cells in concentration gradient of glutamate, and a decrease with AMPA. Thus, glutamate and glutamate receptors agonists do not possess pronounced chemotactic properties, but rather enhance T-cell migration through synthesis of adhesion molecules on the surface of lymphocytes and endothelium.

Recombinant spider silk protein matrices facilitate multi-analysis of calcium-signaling in neural stem cell-derived AMPA-responsive neurons

Neural progenitors or stem cells (NSCs) show great promise in drug discovery and clinical application. Yet few efforts have been made to optimize biocompatible materials for such cells to be expanded and used in clinical conditions. We have previously demonstrated that NSCs are readily cultured on substrates of certain recombinant spider silk protein without addition of animal- or human-derived components. The question remains however whether this material allows differentiation into functional neurons and glia, and whether such differentiation can take place also when the NSCs are cultured within the material in a pseudo-3D context. Here we demonstrate that “foam”-like structures generated from recombinant spider silk protein (4RepCT) provided excellent matrices for the generation and multicellular analysis of functional excitatory neurons from NSCs without addition of animal- or human-derived components. NSCs isolated from the cerebral cortices of rat embryos were cultured on either 4RepCT matrices shaped as foam-like structures without coating, or on conventional polystyrene plates coated with poly-L-ornithine and fibronectin. Upon treatment with recombinant proteins including the growth factor BMP4 or a combination of BMP4 and the signaling factor Wnt3a, the cortical NSCs cultured in 4RepCT foam-like structures differentiated efficiently into neurons that responded to glutamate receptor agonists, such as AMPA, to at least the same extent as control cultures. Matrices derived from recombinant spider silk proteins thus provide a functional microenvironment for neural stem cells without any animal- or human-derived components, and can be employed in the development of new strategies in stem cell research and tissue engineering.

The Effect of Glutamate Receptor Agonists on Mouse Retinal Astrocyte [Ca2+]i

Calcium-imaging techniques were used to determine if mouse retinal astrocytesin siturespond to agonists of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA; N-methyl-D-aspartate, NMDA) and metabotropic (S-3,5-dihydroxyphenylglycine, DHPG;trans-1-amino-1,3-cyclopentanedicarboxylic acid, ACPD) glutamate receptors. In most cases we found no evidence that retinal astrocyte intracellular calcium ion concentration (Ca2+i) increased in response to these glutamate agonists. The one exception was AMPA that increasedCa2+iin some, but not all, mouse retinal astrocytesin situ. However, AMPA did not increaseCa2+iin mouse retinal astrocytesin vitro, suggesting that the effect of AMPAin situmay be indirect.